losartan-potassium and Disease-Models--Animal

Spinal cord injury (SCI) is one of the most destructive traumatic diseases in human beings. The balance of inflammation in the microenvironment is crucial to the repair process of spinal cord injury. Inflammatory cytokines are direct mediators of local lesion inflammation and affect the prognosis of spinal cord injury to varying degrees. In spinal cord injury models, some inflammatory cytokines are beneficial for spinal cord repair, while others are harmful. A large number of animal studies have shown that local targeted administration can effectively regulate the secretion and delivery of inflammatory cytokines and promote the repair of spinal cord injury. In addition, many clinical studies have shown that drugs can promote the repair of spinal cord injury by regulating the content of inflammatory cytokines. However, topical administration affects only a small portion of inflammatory cytokines. In addition, different individuals have different inflammatory cytokine profiles during spinal cord injury. Therefore, future research should aim to develop a personalized local delivery therapeutic cocktail strategy to effectively and accurately regulate inflammation and obtain substantial functional recovery from spinal cord injury.

Topics: Animals; Chemokine CXCL12; Cytokines; Disease Models, Animal; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Interferon-beta; Interleukin-1; Interleukin-10; Interleukin-33; Mice; Neuroinflammatory Diseases; Neuroprotective Agents; Recovery of Function; Spinal Cord; Spinal Cord Injuries

In addition to its role in erythropoiesis, erythropoietin (Epo) plays a role in tissue protection, which includes cardioprotective, nephroprotective and neuroprotective effects. The presence of Epo and its receptor (Epo-R) in pulmonary tissue also suggests a cytoprotective effect of Epo in the lung. Our project aims to document this role in a murine model under-expressing Epo. The obtained results will lead to a better understanding of the cytoprotective effects of Epo and will also give an appreciation of its beneficial effects in cases of lung injury.

Topics: Acute Lung Injury; Animals; Cytoprotection; Disease Models, Animal; Erythropoietin; Hematopoiesis; Humans; Kidney; Lung; Mice

Anemia is a common complication of chronic kidney disease (CKD) in adult and pediatric patients. It has traditionally been treated with erythropoietin therapy and iron supplementation, with great success. With the discovery of the major transcription factor hypoxia inducible factor (HIF) for the erythropoietin gene in 1992, molecules were created that inhibit the HIF prolyl-hydroxylase enzyme. This new class of drug-called HIF stabilizers, or HIF prolyl-hydroxylase inhibitors-prevents the proteasomal degradation of HIF-α, thereby inducing upregulation of the erythropoietin gene. This new strategy for treating CKD anemia is already in phase III clinical trials in adults, and the potential advantages of this therapy are that it is orally active (thereby avoiding injections), and patients are exposed to lower circulating levels of erythropoietin. The long-term safety of this strategy, however, requires elucidation in these trials, particularly since there are many other hypoxia-sensitive genes, notably, angiogenic factors such as vascular endothelial growth factors (VEGF), as well as glycolytic enzymes. As with all new therapies, it is only once a positive benefit: risk profile has been ascertained in adults that the treatment will translate across into pediatrics. Specific issues in the pediatric CKD population are discussed in this review.

Topics: Adult; Age Factors; Anemia; Animals; Child; Clinical Trials, Phase III as Topic; Disease Models, Animal; Erythropoiesis; Erythropoietin; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Prolyl-Hydroxylase Inhibitors; Proteolysis; Renal Insufficiency, Chronic; Signal Transduction; Treatment Outcome; Up-Regulation; Vascular Endothelial Growth Factor A; Von Hippel-Lindau Tumor Suppressor Protein

Hemoglobins (Hbs) are heme-containing proteins binding oxygen, carbon monoxide, and nitric oxide. While erythrocytes are the most well-known location of Hbs, Hbs also exist in neurons, glia and oligodendroglia and they are primarily localized in the inner mitochondrial membrane of neurons with likely roles in cellular respiration and buffering protons. Recently, studies have suggested links between hypoxia and neurodegenerative disorders such as Alzheimer Disease (AD) and furthermore suggested involvement of Hbs in the pathogenesis of AD. While cellular immunohistochemical studies on AD brains have observed reduced levels of Hb in the cytoplasm of pre-tangle and tangle-bearing neurons, other studies on homogenates of AD brain samples observed increased Hb levels. This potential discrepancy may result from differential presence and function of intracellular versus extracellular Hbs. Intracellular Hbs may protect neurons against hypoxia and hyperoxia. On the other hand, extracellular free Hb and its degradation products may trigger inflammatory immune and oxidative reactions against neural macromolecules and/or damage the blood-brain barrier. Therefore, biological processes leading to reduction of Hb transcription (including clinically silent Hb mutations) may influence intra-erythrocytic and neural Hbs, and reduce the transport of oxygen, carbon monoxide and nitric oxide which may be involved in the (patho)physiology of neurodegenerative disorders such as AD. Agents such as erythropoietin, which stimulate both erythropoiesis, reduce eryptosis and induce intracellular neural Hbs may exert multiple beneficial effects on the onset and course of AD. Thus, evidence accumulates for a role of Hbs in the central nervous system while Hbs deserve more attention as possible candidate molecules involved in AD.

Topics: Alzheimer Disease; Animals; Blood-Brain Barrier; Brain; Cell Hypoxia; Disease Models, Animal; Erythropoietin; Hemoglobins; Humans; Mutation; Neurons; Thalassemia

Hypoxia increases erythropoiesis mediated by hypoxia-inducible transcription factors (HIF), which regulate erythropoietin transcription. Neocytolysis is a physiological mechanism that corrects polycythemia from chronic sustained hypoxemia by transient, preferential destruction of young RBCs after normoxia is restored. We showed that neocytolysis is caused by excessive mitochondrial-derived reactive oxygen species in reticulocytes mediated by downregulation of HIF-controlled BNIP3L regulated mitophagy and a decrease in RBC antioxidant catalase (CAT) in hypoxia-produced erythrocytes. Decreased CAT results from hypoxia-induced miR-21 that downregulates CAT. This correlates with a transient acute decrease of HIF-1 at normoxic return that is associated with normalization of red cell mass.

Topics: Animals; Catalase; Disease Models, Animal; Erythrocytes; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Membrane Proteins; Mice; Mitophagy; Proto-Oncogene Proteins; Reactive Oxygen Species; Tumor Suppressor Proteins

The renoprotection of erythropoietin (EPO) and its derivatives such as helix B surface peptide (HBSP) have attracted a great deal of attention from scientists and clinicians alike. The evolutional achievement in the dissociation of tissue protection and erythropoiesis is obtained through HBSP characterisation and synthesis. We performed a series of studies using EPO, as well as HBSP, in a variety of biological models subjected to transplant-related renal injuries such as ischemia reperfusion injury (IRI) and/or immunosuppressant nephrotoxicity. In this short review, we would like to address the effects of EPO in different formats, and its underlying mechanisms with focuses on apoptosis and inflammation in in vitro, ex vivo and in vivo renal injury models, and to further explore potential applications and challenges in humans.

Topics: Acute Kidney Injury; Animals; Anti-Inflammatory Agents; Apoptosis; Cytokine Receptor Common beta Subunit; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Humans; Kidney; Oxidative Stress; Peptide Fragments; Protective Agents; Receptors, Erythropoietin; Reperfusion Injury

Erythropoietin (EPO), recognized early as a tissue protective agent, can trigger antiinflammatory and anti-apoptotic processes to delimit injury and promote repair by binding tissueprotective receptor (TPR). However, only at a high dosage can EPO exert tissue protective effect, which may elicit severe side-effects at the meantime. Helix B surface peptide (HBSP), a 11-amnio acid sequence derived from the non-erythropoietic helix B of EPO, not only shows higher affinity to TPR but also plays a more specific and powerful role in tissue protection without erythropoietic side-effects. While it has obvious merits, the 2-min plasma half-life of HBSP restricts its application in vivo. Therefore, based on the amino acid sequence of HBSP, we originally designed and synthesized thioethercyclized helix B peptide (CHBP) for an increased resistance to proteolytic degradation as well as an improved tissue protective potency, implying a brighter prospective for translational application. In this review, we will mainly discuss the development from EPO to CHBP, the merits and limitation of CHBP and the probable mechanism mediating tissue protection.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Cyclization; Cytokine Receptor Common beta Subunit; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Half-Life; Humans; Kidney; Oxidative Stress; Peptide Fragments; Peptides, Cyclic; Protective Agents; Receptors, Erythropoietin; Reperfusion Injury

Vitamin A is an essential micronutrient throughout life. Its physiologically active metabolite retinoic acid (RA), acting through nuclear retinoic acid receptors (RARs), is a potent regulator of patterning during embryonic development, as well as being necessary for adult tissue homeostasis. Vitamin A deficiency during pregnancy increases risk of maternal night blindness and anemia and may be a cause of congenital malformations. Childhood Vitamin A deficiency can cause xerophthalmia, lower resistance to infection and increased risk of mortality. RA signaling appears to be essential for expression of genes involved in developmental hematopoiesis, regulating the endothelial/blood cells balance in the yolk sac, promoting the hemogenic program in the aorta-gonad-mesonephros area and stimulating eryrthropoiesis in fetal liver by activating the expression of erythropoietin. In adults, RA signaling regulates differentiation of granulocytes and enhances erythropoiesis. Vitamin A may facilitate iron absorption and metabolism to prevent anemia and plays a key role in mucosal immune responses, modulating the function of regulatory T cells. Furthermore, defective RA/RARα signaling is involved in the pathogenesis of acute promyelocytic leukemia due to a failure in differentiation of promyelocytes. This review focuses on the different roles played by vitamin A/RA signaling in physiological and pathological mouse hematopoiesis duddurring both, embryonic and adult life, and the consequences of vitamin A deficiency for the blood system.

Topics: Anemia, Iron-Deficiency; Animals; Cell Differentiation; Disease Models, Animal; Embryonic Development; Epigenesis, Genetic; Erythropoiesis; Erythropoietin; Female; Granulocytes; Hematopoiesis; Humans; Leukemia, Promyelocytic, Acute; Pregnancy; Receptors, Retinoic Acid; Signal Transduction; Tretinoin; Vitamin A; Vitamin A Deficiency

Trials raising concerns about erythropoiesis-stimulating agents, revisions to their labeling, and changes to practice guidelines and dialysis payment systems have provided strong stimuli to decrease erythropoiesis-stimulating agent use and increase intravenous iron administration in recent years. These factors have been associated with a rise in iron utilization, particularly among hemodialysis patients, and an unprecedented increase in serum ferritin concentrations. The mean serum ferritin concentration among United States dialysis patients in 2013 exceeded 800 ng/ml, with 18% of patients exceeding 1200 ng/ml. Although these changes are broad based, the wisdom of these practices is uncertain. Herein, we examine influences on and trends in intravenous iron utilization and assess the clinical trial, epidemiologic, and experimental evidence relevant to its safety and efficacy in the setting of maintenance dialysis. These data suggest a potential for harm from increasing use of parenteral iron in dialysis-dependent patients. In the absence of well powered, randomized clinical trials, available evidence will remain inadequate for making reliable conclusions about the effect of a ubiquitous therapy on mortality or other outcomes of importance to dialysis patients. Nephrology stakeholders have an urgent obligation to initiate well designed investigations of intravenous iron in order to ensure the safety of the dialysis population.

Topics: Anemia, Iron-Deficiency; Animals; Cross-Sectional Studies; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Erythropoietin; Ferritins; Hematinics; Humans; Infusions, Intravenous; Iron Compounds; Kidney Failure, Chronic; Prognosis; Randomized Controlled Trials as Topic; Renal Dialysis; Risk Assessment; Treatment Outcome

Beneficial effects of short-term whole-molecule erythropoietin (EPO) therapy have been demonstrated on several animal models of diverse central nervous system pathology. However, the increased hematocrit induced by EPO-driven marrow stimulation greatly limits its potential for side effect-free therapy. We created a library of EPO-derived fragments based on the hypothesis that 2 distinct functions, erythropoiesis and tissue protection, reside in different regions of the molecule. Several small EPO-derived peptides within the Aβ loop of whole EPO molecule were screened for tissue protection in EAE mice. The 19-mer JM-4 peptide that contains 2 cysteine molecules consistently demonstrated the most potent clinical beneficial effects without producing hematocrit alterations in animal models of EAE. The JM-4-induced tissue protection was associated with modulation of the immunoregulatory process that drives inflammation and provokes subsequent autoimmune damage. Like the whole EPO molecule, JM-4 effectively modulated immune/inflammatory reaction within both the peripheral lymphatic tissue and central nervous system. The major effects induced by JM-4 include blocked expansion of monocyte/dendritic antigen presenting cell and T helper 17 cell populations, decreased proinflammatory cytokine production, and sharply enhanced expansion of the regulatory T-cell population. JM-4 shows promise for treatment of a broad spectrum of neural and non-neural conditions associated with inflammation.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Erythropoietin; Humans; Lymph Nodes; Lymphocytes; Mice, Inbred C57BL; PC12 Cells; Rats; Spinal Cord

Despite extensive research, no therapeutic interventions have been shown to prevent AKI, accelerate recovery of AKI, or reduce progression of AKI to CKD in patients. This failure in translation has led investigators to speculate that the animal models being used do not predict therapeutic responses in humans. Although this issue continues to be debated, an important concern that has not been addressed is whether improvements in preclinical study design can be identified that might also increase the likelihood of translating basic AKI research into clinical practice using the current models. In this review, we have taken an evidence-based approach to identify common weaknesses in study design and reporting in preclinical AKI research that may contribute to the poor translatability of the findings. We focused on use of N-acetylcysteine or sodium bicarbonate for the prevention of contrast-induced AKI and use of erythropoietin for the prevention of AKI, two therapeutic approaches that have been extensively studied in clinical trials. On the basis of our findings, we identified five areas for improvement in preclinical study design and reporting. These suggested and preliminary guidelines may help improve the quality of preclinical research for AKI drug development.

Topics: Acetylcysteine; Acute Kidney Injury; Animals; Contrast Media; Disease Models, Animal; Erythropoietin; Free Radical Scavengers; Humans; Research Design; Sodium Bicarbonate; Translational Research, Biomedical

Erythropoietin (EPO) has neuroprotective effects in many models of damage and disease of the nervous system where neuroinflammation plays a substantial role, including experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Since the first pioneering studies, in which EPO was shown to protect rats with acute EAE mainly by inhibiting inflammation, many other studies have pointed out other mechanisms of protection, including oligodendrogenesis and inhibition of axonal damage.Here we review the preclinical studies in which EPO has shown therapeutic efficacy in several models of EAE in mice and rats. Moreover, we report in detail the protocol to administer EPO to mice with myelin oligodendrocyte glycoprotein (MOG)-induced chronic progressive EAE, and a representative result. In this model, EPO inihibits the clinical score of the disease when administered according to a preventive but also to a therapeutic schedule, and therefore at disease onset, suggesting that it might not only inhibit inflammation but also actively stimulate repair.

Topics: Animals; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Erythropoietin; Mice; Multiple Sclerosis; Myelin-Oligodendrocyte Glycoprotein; Rats

Recent advances in understanding the cellular and molecular basis of psychiatric illnesses have shed light on the important role played by trophic factors in modulating functional parameters associated with disease causality and drug action. Disease mechanisms are now thought to involve multiple cell types, including neurons and endothelial cells. These functionally distinct but interactively coupled cell types engage in cellular cross talk via shared and common signaling molecules. Dysregulation in their cellular signaling pathways influences brain function and alters behavioral performance. Multifunctional trophic factors such as VEGF and EPO that possess both neurotrophic and angiogenic actions are of particular interest due to their ability to rescue structural and plasticity deficits in neurons and vasculature. Obtaining insight into the behavioral, cellular and molecular actions of multi-functional trophic factors has the potential to open new and transformative therapeutic approaches.

Topics: Animals; Antidepressive Agents; Astrocytes; Behavior, Animal; Biomimetics; Cognition Disorders; Depression; Disease Models, Animal; Endothelial Cells; Erythropoietin; Humans; Neurons; Vascular Endothelial Growth Factors

One of the major disadvantages of mandibular distraction osteogenesis (MDO) is the prolonged time required for consolidation of the regenerate bone. The objective of the present study is to perform a contemporary review of various adjuvant therapies to enhance bone consolidation in MDO.. A PubMed search for articles related to MDO, along with the references of those articles, was performed. Inclusion and exclusion criteria were applied to all experimental studies assessing adjuvant therapies to enhance bone consolidation.. A total of 1414 titles and abstracts were initially reviewed; 61 studies were included for full review. Many studies involved growth factors, hormones, pharmacological agents, gene therapy, and stem cells. Other adjuvant therapies included mechanical stimulation, laser therapy, and hyperbaric oxygen. Majority of the studies demonstrated positive bone healing effects and thus adjuvant therapies remain a viable strategy to enhance and hasten the consolidation period.. Although most studies have demonstrated promising results, many questions still remain, such as optimal amount, timing, and delivery methods required to stimulate the most favorable bone regeneration. As well, further studies comparing various adjuvant therapies and documentation of long-term adverse effects are required prior to clinical application.

Topics: Adjuvants, Pharmaceutic; Alendronate; Animals; Bone Morphogenetic Protein 4; Bone Regeneration; Disease Models, Animal; Dogs; Electric Stimulation Therapy; Erythropoietin; Genetic Therapy; Mandible; Nerve Growth Factor; Osteogenesis; Osteogenesis, Distraction; Rabbits; Rats; Risk Assessment; Stem Cell Transplantation; Treatment Outcome

Erythropoietin (EPO) is the main hormone that regulates erythropoiesis. Beyond its well-known hematopoietic action, EPO has diverse cellular effects in non-hematopoietic tissues. It has been shown to inhibit apoptosis by activating pro-survival pathways in the myocardium, to mobilize endothelial progenitor cells and to inhibit migration of inflammatory cells. EPO has also been shown to have potent pro-angiogenic properties. Numerous experimental data support the cardioprotective effects of EPO in animal models of acute myocardial infarct (AMI). However, these findings are not supported by recent clinical trials designed to investigate the safety and efficacy of EPO in patients with AMI. In this article, we begin by providing a comprehensive review of the cardioprotective effects of EPO in experimental animal models and the molecular mechanisms underlying these effects. We then discuss the EPO data obtained through clinical trials. We compare similarities and differences between the animal and human studies as well as between the different clinical studies in terms of sample size and study design including the dose, the route and the timing of administration as well as confounding factors such as comorbidities and concomitant treatments. Finally, we question the gap between the experimental and the translational clinical data and propose further developments to address these discrepancies and clearly evaluate the role of EPO in the clinical setting of MI.

Topics: Animals; Cardiotonic Agents; Clinical Trials as Topic; Disease Models, Animal; Erythropoietin; Humans; Myocardial Infarction

Discovered as the primary regulator of erythropoiesis, erythropoietin (EPO) is involved in a broad variety of processes that play a major role in cardiovascular diseases. In particular, the antiapoptotic and pro-angiogenic properties of EPO have prompted a growing interest in the use of EPO for the treatment of myocardial infarction and heart failure. In a variety of myocardial ischemic injury animal models, EPO administration has been shown to acutely reduce infarct size, thereby preserving ventricular function. In addition, cardiac long-term effects of EPO, such as prevention of ventricular remodeling and heart failure, have been described. In recent years, several trials have tested the effects of recombinant human erythropoietin (rhEPO) administration in patients with myocardial infarction and chronic heart failure, in the attempt to translate the cardioprotection found in experimental models to human patients. In view of the generally controversial findings, in this updated review we provide an overview of the results of the most recent trials that investigated the role of erythropoiesis-stimulating agents (ESAs), including rhEPO and its analogue darbepoetin, in the treatment of acute myocardial infarction and heart failure. The problems related to safety and tolerability of ESA therapy are also discussed. Our analysis of the available literature demonstrates that the results of clinical studies in patients with cardiac disease are not uniform and the conclusions are contradictory. Further larger prospective studies are required to test clinical efficacy and safety of EPO.

Topics: Animals; Disease Models, Animal; Erythropoietin; Heart Failure; Humans; Myocardial Infarction; Randomized Controlled Trials as Topic; Recombinant Proteins

Erythropoietin (EPO) belongs to a group of pharmacological agents with multifunctional effects. EPO was originally acknowledged as the main regulator of erythropoiesis, but it also exhibits several extra haematopoietic properties, such as promoting the maintenance of homeostasis of cells under stress. These pleiotropic effects have been extensively investigated in preclinical models including models of ischaemic-reperfusions injuries, inflammation, neuroprotection, neovascularisation and wound healing. Promising effects of EPO have especially been reported in models of ischaemic-reperfusions injuries. The mechanisms by which EPO exerts these organ-protective effects are not completely understood, although anti-apoptotic, anti-inflammatory and anti-oxidative properties have been described. Activation of the EPO receptor initiates several intracellular signalling systems, such as, phosphatidylinositol 3-kinase, STAT5, mitogen-activated protein kinase and nuclear factor-kappa B. These pathways are recognized as involved in the cellular response to stress and regulation of apoptosis. Although EPO has been demonstrated to be effective in animal models, the effect has not been clearly demonstrated in clinical trials. This MiniReview gives a brief introduction to the pleiotropic effects of EPO, the evidence of organ protection in animal models, and discusses the disappointing results obtained from recent clinical trials.

Topics: Animals; Apoptosis; Critical Illness; Disease Models, Animal; Erythropoietin; Hematopoiesis; Humans; Immunomodulation; Neuroprotective Agents; Oxidative Stress; Randomized Controlled Trials as Topic; Receptors, Erythropoietin

Erythropoietin (EPO) is a growth hormone and cytokine that plays an important role in erythropoiesis and neuroprotection. However, EPO treatment for neurological diseases requires repeated injections or high-dose systemic administration, which may cause systemic side effects. The lack of any effective treatment of acute and chronic neurodegenerative diseases and the promising outcome by EPO in animal models in vivo demand a critical evaluation of intranasal EPO delivery to the brain as an alternative administration method.. The current use and intranasal administration of EPO and its derivatives in preclinical studies and recent clinical trials with EPO in neurological diseases.. This paper gives an overview of the therapeutic considerations of intranasal EPO and EPO derivatives for neuroprotection.. Intranasal delivery (ID) of neuroprotective drugs is an area of great interest. Among the administration strategies used at present, ID of EPO is the most promising. Further preclinical and clinical studies are needed to evaluate the potential significance of this alternative route for increasing EPO bioavailability and decreasing side effects.

Topics: Administration, Intranasal; Animals; Brain; Central Nervous System Diseases; Disease Models, Animal; Drug Delivery Systems; Erythropoietin; Humans; Neuroprotective Agents

Erythropoietin (EPO) has shown promise as a neuroprotectant in animal models of ischemic stroke. EPO is thought not only to protect neurons from cell death, but also to promote regeneration after stroke. Here, we report a systematic review and meta-analysis of the efficacy of EPO in animal models of focal cerebral ischemia. Primary outcomes were infarct size and neurobehavioral outcome. Nineteen studies involving 346 animals for infarct size and 425 animals for neurobehavioral outcome met our inclusion criteria. Erythropoietin improved infarct size by 30.0% (95% CI: 21.3 to 38.8) and neurobehavioral outcome by 39.8% (33.7 to 45.9). Studies that randomized to treatment group or that blinded assessment of outcome showed lower efficacy. Erythropoietin was tested in animals with hypertension in no studies reporting infarct size and in 7.5% of the animals reporting neurobehavioral outcome. These findings show efficacy for EPO in experimental stroke, but when the impact of common sources of bias are considered, this efficacy falls, suggesting we may be overestimating its potential benefit. As common human co-morbidities may reduce therapeutic efficacy, broader testing to delineate the range of circumstances in which EPO works best would be beneficial.

Topics: Animals; Behavior, Animal; Brain Ischemia; Databases, Factual; Disease Models, Animal; Erythropoietin; Humans; Hypertension; Neuroprotective Agents; Stroke

Environmental heat exposure represents one of the most deadly natural hazards in the United States. Heat stroke is a life-threatening illness that affects all segments of society with few effective treatment strategies to mitigate the long-term debilitating consequences of this syndrome. Although the etiologies of heat stroke are not fully understood, the long-term sequelae are thought to be due to a systemic inflammatory response syndrome (SIRS) that ensues following heat-induced tissue injury. Endotoxin and cytokines have been implicated as key mediators of the heat-induced SIRS, based almost exclusively on correlative data that show high circulating concentrations of these substances in heat stroke patients and animal models. However, endotoxin and cytokine neutralization studies have not consistently supported this hypothesis indicating that the mechanisms of heat stroke morbidity / mortality remain poorly understood. This review discusses the current understanding of the role of endotoxin and cytokines in heat-induced SIRS. Insight is provided into genetic conditions that may predispose to heat stroke and potential therapeutic strategies that may be efficacious against the adverse consequences of this debilitating illness.

Topics: Animals; Cytokines; Disease Models, Animal; Endotoxins; Erythropoietin; Genetic Predisposition to Disease; Glucocorticoids; Heat Stroke; Hot Temperature; Humans; Hypothermia, Induced; Malignant Hyperthermia; Mutation; Polymorphism, Genetic; Prostaglandin Antagonists; Protein C; Recombinant Proteins; Signal Transduction; Systemic Inflammatory Response Syndrome

Pleiotropic substances are characterized by their versatile and complex range of actions which makes them potential new active agents for the therapy of wounds. Besides its known effect to increase red blood cell production, the glycoprotein hormone erythropoietin (EPO) has been found to demonstrate a tissue protective effect in several other organs. The administration of EPO during skin wound healing is most likely essentially based on its cytopotective, proangiogenic, antiapoptotic and antiinflammatory effects. Herein EPO stimulates a coordinated interaction of different types of cells at a low or only a single dose. This review article aims to present the advantages and disadvantages of EPO administration in different experimental models to study the healing and regeneration processes of the skin and discusses possible clinical applications.

Topics: Animals; Apoptosis; Dermatologic Surgical Procedures; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Humans; Injections, Subcutaneous; Recombinant Proteins; Regeneration; Wound Healing

In the United States, TBI remains a major cause of morbidity and mortality in children and young adults. A total of 1.5 million Americans experience head trauma every year, and the yearly economic cost of this exceeds $56 billion. The magnitude of this problem has generated a great deal of interest in elucidating the complex molecular mechanism underlying cell death and dysfunction after TBI and in the development of neuroprotective agents that will reduce morbidity and mortality.. A review of recent literature on EPO, TBI, and apoptosis is conducted with analysis of pathophysiologic mechanisms of TBI. In addition, animal experiments and clinical trials pertaining to mechanisms of cell death in TBI and EPO as a neuroprotective agent are reviewed.. The literature and evidence for EPO as a potent inhibitor of apoptosis and promising therapeutic agent in a variety of neurological insults, including trauma, are mounting. With the recent interest in clinical trials of EPO in human stroke, it is both timely and prudent to consider the use of this pharmaceutical avenue in TBI in man.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Brain Injuries; Cytoprotection; Disease Models, Animal; Erythropoietin; Humans; Nerve Degeneration; Neurons; Neuroprotective Agents

Spinal cord injury (SCI) is a devastating condition for individual patients and costly for health care systems requiring significant long-term expenditures. Cytokine erythropoietin (EPO) is a glycoprotein mediating cytoprotection in a variety of tissues, including spinal cord, through activation of multiple signaling pathways. It has been reported that EPO exerts its beneficial effects by apoptosis blockage, reduction of inflammation, and restoration of vascular integrity. Neuronal regeneration has been also suggested. In the present review, the pathophysiology of SCI and the properties of endogenous or exogenously administered EPO are briefly described. Moreover, an attempt to present the current traumatic, ischemic and inflammatory animal models that mimic SCI is made. Currently, a clearly effective pharmacological treatment is lacking. It is highlighted that administration of EPO or other recently generated EPO analogues such as asialo-EPO and carbamylated-EPO demonstrate exceptional preclinical characteristics, rendering the evaluation of these tissue-protective agents imperative in human clinical trials.

Topics: Animals; Clinical Trials as Topic; Disease Models, Animal; Drug Evaluation, Preclinical; Erythropoietin; Humans; Myelitis; Nerve Degeneration; Neuroprotective Agents; Spinal Cord; Spinal Cord Injuries

Traumatic brain injury is a leading cause of mortality and long-term morbidity, particularly affecting young people. With our best therapies, one half of the patients with severe traumatic brain injury are never capable of living independently. Two interventions, which have real potential to improve neurological outcomes in patients with traumatic brain injury, are (i) very early induction of prophylactic hypothermia and (ii) exogenous erythropoietin therapy. There is substantial experimental evidence, a plausible biological rationale, and supportive clinical evidence from clinical trials to suggest a possible beneficial effect of prophylactic hypothermia and also for exogenous erythropoietin therapy in severe traumatic brain injury. Despite the recent guidelines and publications recommending these interventions, critical care clinicians should be conservative towards implementing these therapies outside clinical trials due to substantial efficacy and safety concerns. Nevertheless the high morbidity and mortality associated with severe traumatic brain injury (TBI) demands that we investigate the safety and efficacy of these promising potential therapies as a matter of urgency.

Topics: Animals; Body Temperature; Brain Injuries; Critical Care; Disease Models, Animal; Erythropoietin; Glasgow Coma Scale; Hematinics; Humans; Hypothermia, Induced; Neuroprotective Agents; Recombinant Proteins; Thrombosis; Treatment Outcome; Ultrasonography

Erythropoietin (EPO) was explored regarding its suitability as a candidate stroke drug in animal experimental studies. We performed a meta-analysis to obtain an overall impression of the efficacy of EPO in published animal experimental stroke studies and for potential guidance of future clinical studies.. By electronic and manual searches of the literature, we identified studies describing the efficacy of EPO in experimental focal cerebral ischemia. Data on study quality, EPO dose, time of administration, and outcome measured as infarct volume or functional deficit were extracted. Data from all studies were pooled by means of a meta-analysis.. Sixteen studies were included in the meta-analysis. When administered after the onset of ischemia, EPO and its analogues reduced infarct size by 32% and improved neurobehavioral deficits significantly. A meta-regression suggests higher doses of EPO to be associated with smaller infarct volumes. When administered earlier than 6 hours EPO was more effective compared to a later treatment initiation. Both hematopoietic and nonhematopoietic EPO analogues showed efficacy in experimental stroke.. In conclusion, this analysis further strengthens confidence in the efficacy of EPO and its analogues in stroke therapy. Nonhematopoietic EPO analogues which are known to have less systemic adverse effects compared to EPO are also promising candidate stroke drugs. Further experimental studies are required that evaluate the safety of a combination of EPO with thrombolysis and whether EPO is also effective in animals with comorbidity.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Gerbillinae; Meta-Analysis as Topic; Mice; Rats; Stroke; Thrombolytic Therapy; Time Factors

Intranasal delivery provides a practical, noninvasive method of bypassing the blood-brain barrier (BBB) in order to deliver therapeutic agents to the brain. This method allows drugs that do not cross the BBB to be delivered to the central nervous system in a few minutes. With this technology, it will be possible to eliminate systemic administration and its potential side effects. Using the intranasal delivery system, researchers have demonstrated neuroprotective effects in different animal models of stroke using erythropoietin (EPO) as a neuroprotector or other different types of EPO without erythropoiesis-stimulating activity. These new molecules retain their ability to protect neural tissue against injury and they include Asialoerythropoietin (asialoEPO) carbamylated EPO (CEPO), and rHu-EPO with low sialic acid content (Neuro-EPO). Contrary to the other EPO variants, Neuro-EPO is not chemically modified, making it biologically similar to endogenous EPO, with the advantage of less adverse reactions when this molecule is applied chronically. This constitutes a potential benefit of Neuro-EPO over other variants of EPO for the chronic treatment of neurodegenerative illnesses. Nasal administration of EPO is a potential, novel, neurotherapeutic approach. However, it will be necessary to initiate clinical trials in stroke patients using intranasal delivery in order to obtain the clinical evidence of its neuroprotectant capacity in the treatment of patients with acute stroke and other neurodegenerative disorders. This new therapeutic approach could revolutionize the treatment of neurodegenerative disorders in the 21st century.

Topics: Administration, Intranasal; Animals; Asialoglycoproteins; Blood-Brain Barrier; Brain Ischemia; Clinical Trials as Topic; Disease Models, Animal; Erythropoietin; Humans; Neurodegenerative Diseases; Neuroprotective Agents; Receptors, Erythropoietin; Recombinant Proteins; Stroke

Divalent metal transporter 1 (DMT1) is the protein that allows elemental iron entry into the duodenal cell. It is expressed ubiquitously and it also allows the iron exit from the endosomes. This protein plays a central role in iron metabolism and it is strictly regulated. Several animal models elucidate its role in physiology. Recently three patients affected with DMT1 deficiency have been described. This recessively inherited condition appears at birth with severe microcytic anemia. Serum markers could be particularly useful to establish a correct diagnosis: high serum iron, normal total iron-binding capacity (TIBC), increased saturation of transferrin (Tf), slightly elevated ferritin, and increased soluble transferrin receptor (sTfR). Increased free erythrocyte protoporphyrins (FEPs) could address the diagnosis to iron-deficient anemia. All patients appeared to respond to erythropoietin (Epo) administration. Because mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) did not change during Epo treatment, it was concluded that Epo did not improve iron utilization of the erythroblasts but likely reduced the degree or intensity of apoptosis, affecting erythropoiesis. Moreover liver iron overload was present and documented in all of the affected patients. In this review we analyze the role of DMT1 in iron metabolism and the major causes of reduction and their consequences in animal models as well in humans, and we attempt to define the correct treatment for human mutants.

Topics: Anemia; Animals; Biomarkers; Cation Transport Proteins; Disease Models, Animal; Erythropoiesis; Erythropoietin; Genotype; Hematinics; Humans; Iron; Iron Overload; Iron-Binding Proteins; Mutation; Phenotype; Treatment Outcome; Up-Regulation

Granulocyte colony-stimulating factor (G-CSF) and erythropoietin are two cytokines that have been demonstrated to improve cardiac function and perfusion in myocardial infarction. G-CSF was initially evaluated as a stem cell mobilizer and erythropoietin as a cytoprotective agent. However, both cytokines have direct cytoprotective effects and stem cell-mobilizing ability. Direct cytoprotective effects of both cytokines are commonly mediated by the Jak-STAT pathway. In preclinical study, G-CSF and erythropoietin improved cardiac function and perfusion by angiomyogenesis and protection of cardiomyocytes in myocardial infarction. However, results from recent clinical trials did not support beneficial effects of cytokine therapy with G-CSF or erythropoietin alone in patients with myocardial infarction. Further studies are required to elucidate the mechanism of action and to improve therapeutic efficacy by employing novel strategies, such as combined cytokines.

Topics: Animals; Cell- and Tissue-Based Therapy; Clinical Trials as Topic; Combined Modality Therapy; Disease Models, Animal; Erythropoietin; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Mobilization; Humans; Myocardial Infarction; Neovascularization, Physiologic; Regeneration; Sensitivity and Specificity; Severity of Illness Index; Treatment Outcome; Ventricular Remodeling

Besides its established function in erythropoiesis, erythropoietin (EPO) is currently also appreciated for its neuroprotective effects. The detrimental sequelae of prolonged cerebral hypoxia and ischemia have been shown to attenuate by EPO treatment. After binding to the EPO receptor, EPO is capable of initiating a cascade of events which--via different pathways--may lead to neuroprotection. The circumstances that determine which specific signalling route(s) are activated by EPO are largely unknown. We aim to provide the reader with a timely overview on the use of EPO in models of stroke and hypoxia-ischemia and to discuss the molecular events that underlie its neuroprotection.

Topics: Animals; Disease Models, Animal; Erythropoietin; Humans; Hypoxia-Ischemia, Brain; Neuroprotective Agents; Signal Transduction; Time Factors

Carbamylated erythropoietin (CEPO) is a modified erythropoietin molecule not affecting hematocrit. It is a potentially important pharmacological agent that may be applied to the treatment of several diseases affecting central and peripheral system neurons.. Carbamylated erythropoietin is a prime candidate for development because of its potent cell survival and function enhancing effects. This article reviews the preclinical development profile of CEPO in animal models to determine whether further clinical development is justified.. The review spans a detailed analysis of patents and scientific publications related to CEPO in animal models.. Use of CEPO in treating diseases such as stroke and traumatic brain and spinal cord injuries is important because these conditions affect millions of patients every year. Extensive preclinical studies support further clinical studies of CEPO for acute ischemic stroke. However, further studies are required for testing CEPO in patients with many other indications.

Topics: Animals; Brain Injuries; Clinical Trials as Topic; Diabetic Neuropathies; Disease Models, Animal; Erythropoietin; Humans; Motor Neuron Disease; Nervous System Diseases; Spinal Cord Injuries; Stroke; Treatment Outcome

The injured brain can be stimulated to amplify its intrinsic restorative processes to improve neurological function. Thus, after stroke, both cell and pharmacological neurorestorative treatments, amplify the induction of brain neurogenesis and angiogenesis, and thereby reduce neurological deficits. In this manuscript, we describe the use of bone marrow mesenchymal cells (MSCs) and erythropoietin (EPO) as examples of cell-based and pharmacological neurorestorative treatments, respectively, for both stroke and a mouse model of experimental autoimmune encephalomyelitis (EAE). We demonstrate that these therapies significantly improve neurological function with treatment initiated after the onset of injury and concomitantly promote brain plasticity. The application of MRI to monitor changes in the injured brain associated with reduction of neurological deficit is also described.

Topics: Animals; Brain Injuries; Disease Models, Animal; Erythropoietin; Hematopoietic Stem Cell Transplantation; Humans; Mice; Neuronal Plasticity; Recombinant Proteins

Neonatal stroke is increasingly recognized in preterm and term infants, and the rate of arterial ischemic infarction occurring around the time of birth is as high as the annual incidence of large-vessel ischemic stroke in adults. Thus, neonatal stroke is a major contributor to perinatal morbidity and mortality, and a considerable number of these children will develop long-term neurodevelopmental disabilities. Our ability to investigate this situation has been limited by the technical challenges in developing suitable animal models. Our objective is to describe recent evidence in relation to animal models of neonatal stroke. In addition, we review and report potential neuroprotective strategies specific to neonatal stroke, with a focus on erythropoietin and cardiotrophin-1 because of their potential role in protection as well as repair.

Topics: Animals; Animals, Newborn; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Humans; Nerve Tissue Proteins; Neuroprotective Agents; Risk Factors; Stroke; Time Factors

Several studies with erythropoiesis-stimulating agents (ESAs) have raised a number of safety issues. Therefore, a discussion of available data in light of the current EORTC guidelines 2006 on the use of ESAs in anemic patients is warranted.. Literature is reviewed with respect to experimental and clinical data on the effect of ESA therapy on tumor growth both in the preclinical setting and on patient survival.. Studies showing an adverse effect of ESA therapy on patient survival generally exhibit considerable methodological deficiencies. Moreover, they investigated treatment situations for which ESAs are not approved and/or did not involve recommended baseline ("intervention") or target hemoglobin levels.. When used as indicated, ESAs are valuable and safe drugs for the treatment of anemia and do not negatively affect patient survival. In particular, the data situation confirms the importance and correctness of the EORTC guidelines 2006 and their recently updated version. It is therefore recommended that these guidelines continue to be strictly followed in the treatment of chemotherapy-induced anemia.

Topics: Adult; Anemia; Animals; Antineoplastic Agents; Blood Transfusion; Clinical Trials as Topic; Clinical Trials, Phase II as Topic; Disease Models, Animal; Disease Progression; Epoetin Alfa; Erythropoietin; Female; Hematinics; Hemoglobins; Humans; Male; Meta-Analysis as Topic; Mice; Neoplasms; Neoplasms, Experimental; Oxygen; Practice Guidelines as Topic; Prospective Studies; Randomized Controlled Trials as Topic; Rats; Receptors, Erythropoietin; Recombinant Proteins; Risk Factors; Safety; Tumor Cells, Cultured

The kidney can achieve a structural and functional recovery after the damage induced by ischemia and reperfusion. This is due to the regeneration of epithelial tubular cells, the intervention of immature cells mainly localized in the medulla, and a small number of bone marrow-derived stem cells. In many instances, however, recovery is delayed or does not occur at all. The mechanisms allowing the renal cells to de-differentiate still need to be clarified in order to find a therapeutic approach that can amplify this ability and then stop the fibroid involution and the progression toward renal failure. Several authors have hypothesized a protective effect of EPO against ischemic and cytotoxic renal damage and observed that patients precociously treated with EPO showed a slower progression of renal failure. EPO has been demonstrated to have proliferative and anti-apoptotic effects in ischemia-reperfusion models in the brain and cell cultures. Moreover, EPO can mobilize stem cells and increase the plasmatic levels and the renal expression of VEGF. These effects seem to be dose-dependent and could be due to the activation of signal transduction systems, like Jak and STAT. In the presence of high doses of exogenous EPO or during the treatment with long-acting EPO-like molecules, non-specific receptors may be activated through a low-affinity link. Further investigations are needed to determine new therapeutic applications for EPO and other analogous hormones. Very long-acting molecules or molecules with cyto-protective but no erythropoietic effect may represent useful tools in the study of the molecular mechanisms underlying EPO's action and may have a rapid and safe therapeutic application.

Topics: Acute Kidney Injury; Animals; Cell Differentiation; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Erythropoietin; Humans; Ischemia; Kidney; Recombinant Proteins; Signal Transduction

Topics: Amyotrophic Lateral Sclerosis; Animals; Cell Death; Clinical Trials as Topic; Disease Models, Animal; Erythropoietin; Humans; Mice; Motor Neurons; Neuroprotective Agents; Rats; Receptors, Cell Surface; Superoxide Dismutase; Superoxide Dismutase-1

Treatments for spontaneous intracerebral, thrombolytic-induced and intraventricular hemorrhages (IVH) are still at the preclinical or early clinical investigational stages. There has been some renewed interest in the use of surgical evacuation surgery or thrombolytics to remove hematomas, but these techniques can be used only for specific types of brain bleeding. The STICH (Surgical Trial in Intracerebral Haemorrhage) clinical trials should provide some insight into the potential for such techniques to counteract hematoma-induced damage and subsequently, morbidity and mortality. More recently, clinical trials (ATACH [Antihypertensive Treatment in Acute Cerebral Hemorrhage] and INTERACT [Intensive Blood Pressure Reduction in Acute Cerebral Hemorrhage Trial]) have begun testing whether or not regulating blood pressure affects the well-being of hemorrhage patients, but the findings thus far have not conclusively demonstrated a positive result. More promising trials, such as the early stage CHANT (Cerebral Hemorrhagic And NXY-059 Treatment) and the late stage FAST (Factor VIIa for Acute Hemorrhagic Stroke Treatment), have addressed whether or not manipulating oxidative stress and components of the blood coagulation cascade can achieve an improved prognosis following spontaneous hemorrhages. However, CHANT was halted prematurely because although it showed that the spin trap agent NXY-059 was safe, it also demonstrated that the drug was ineffective in treating acute ischemic stroke. In addition, the recombinant activated factor VII FAST trial recently concluded with only modestly positive results. Despite a beneficial effect on the primary end point of reducing hemorrhage volume, controlling the coagulation cascade with recombinant factor VIIa did not decrease the mortality rate. Consequently, Novo Nordisk has abandoned further development of the drug for the treatment of intracerebral hemorrhaging. Even though progress in hemorrhage therapy that successfully reduces the escalating morbidity and mortality rate associated with brain bleeding is slow, perseverance and applied translational drug development will eventually be productive. The urgent need for such therapy becomes more evident in light of concerns related to uncontrolled high blood pressure in the general population, increased use of blood thinners by the elderly (e.g., warfarin) and thrombolytics by acute ischemic stroke patients, respectively. The future of drug development for hemorrhage may requi

Topics: Animals; Antihypertensive Agents; Antioxidants; Benzenesulfonates; Blood Pressure; Cardiovascular Agents; Coagulants; Disease Models, Animal; Drug Design; Enzyme Inhibitors; Erythropoietin; Excitatory Amino Acid Antagonists; Factor VIIa; Hemostasis; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intracranial Hemorrhages; Oxidative Stress; Signal Transduction; Stroke; Treatment Outcome

Topics: Anemia; Animals; Disease Models, Animal; Erythropoiesis; Erythropoietin; Female; Heart Failure; Humans; Male; Multicenter Studies as Topic; Myocardial Ischemia; Neovascularization, Physiologic; Oxygen Consumption; Prognosis; Randomized Controlled Trials as Topic; Rats; Recombinant Proteins; Risk Assessment; Stroke Volume; Survival Analysis; Treatment Outcome

Recombinant erythropoietin (EPO) was introduced into clinical practice after the identification of EPO as the major haemopoietic growth factor determining survival and maturation of erythroid precursors. Advances in our understanding of the novel sites of action of EPO in the vasculature, brain, heart and kidney have opened new avenues of therapeutic potential for EPO, and have led to an increased understanding of the biological roles of EPO and its mechanisms of cell protection.

Topics: Animals; Disease Models, Animal; Erythropoietin; Humans; Myocardial Infarction; Recombinant Proteins

This review will focus on the molecular basis of certain polycythemic disorders. Primary polycythemias are characterized by acquired somatic or inherited germ-line mutations expressed within hematopoietic progenitors that cause increased accumulation of red blood cells. Polycythemia vera (PV), an acquired condition, is the most common primary polycythemia; although some progress has been made in the understanding of PV, its molecular basis remains unknown. In contrast, recent advances in delineating the molecular defects of some inherited polycythemias have greatly furthered our knowledge of the regulation of erythropoiesis and hypoxia sensing; however, more work needs to be done.

Topics: Adult; Animals; Child; Cohort Studies; Disease Models, Animal; DNA-Binding Proteins; Erythropoiesis; Erythropoietin; Ethnicity; Female; Founder Effect; Germ-Line Mutation; GPI-Linked Proteins; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Isoantigens; Janus Kinase 2; Male; Membrane Glycoproteins; Mice; Nuclear Proteins; Polycythemia; Polycythemia Vera; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Cell Surface; Receptors, Erythropoietin; Russia; Transcription Factors; Tumor Suppressor Proteins; Ubiquitin; Ubiquitin-Protein Ligases; Von Hippel-Lindau Tumor Suppressor Protein

The incidence of both congestive heart failure (CHF) and end-stage renal disease both are increasing. Anemia is common in both conditions and is associated with a marked increase in mortality and morbidity in both CHF and chronic kidney insufficiency (CKI). Each of these 3 conditions can cause or worsen the other 2. In other words, a vicious circle frequently is present in which CHF can cause or worsen both anemia and CKI, in which CKI can cause or worsen both anemia and CHF, and in which anemia can cause or worsen both CHF and CKI. We have called this vicious circle the cardio renal anemia syndrome. Optimal treatment of CHF with all the recommended CHF medications at their recommended doses will, in our experience, frequently fail to improve the CHF and CKI if anemia is present and is not corrected. On the other hand, correction of the anemia with subcutaneous erythropoietin and intravenous iron has caused a great improvement in the CHF including a marked improvement in patient and cardiac function and a marked reduction in the need for hospitalization and for high-dose diuretics. It also frequently has caused renal function to improve or at least stabilize. In addition, patients' quality of life and exercise capacity also have improved with the correction of the anemia. In CKI patients, anemia also may play an important role in increasing the risk for death, coronary heart disease, stroke, and progression to end-stage renal disease. Erythropoietin may have a direct positive effect on the heart and brain unrelated to correction of the anemia by reducing cell apoptosis and by increasing neovascularization, both of which could prevent tissue damage. This could have profound therapeutic implications not only in CHF but in the future treatment of myocardial infarction, coronary heart disease, strokes, and renal failure.

Topics: Age Factors; Aged; Anemia, Iron-Deficiency; Animals; Cohort Studies; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Drug Administration Schedule; Erythropoietin; Follow-Up Studies; Heart Failure; Humans; Injections, Subcutaneous; Kidney Failure, Chronic; Middle Aged; Recombinant Proteins; Renal Dialysis; Risk Assessment; Severity of Illness Index; Survival Rate; Treatment Outcome

It has been proposed that the basis of severe malarial anaemia, a major cause of morbidity and mortality in endemic areas, is multifactorial. Inappropriately low reticulocytosis is observed in malaria patients suggesting that insufficient erythropoiesis is a major factor. Clinical studies provide conflicting data concerning the production of adequate levels of erythropoietin (EPO) during malaria. Plasmodium chabaudi AS causes non-lethal infection in resistant C57BL/6 mice, and lethal infection in susceptible A/J mice. In P. chabaudi AS infected C57BL/6 and A/J mice, which experience varying degrees of severity of anaemia, kidney EPO production is appropriate to the severity of anaemia and is regulated by haematocrit level. Neutralisation of endogenous EPO during infection leads to lethal anaemia while timely administration of exogenous EPO rescues mice although reticulocytosis is suppressed in proportion to the parasitemia level. Characterisation of alterations in splenic erythroid compartments in naive and P. chabaudi AS infected A/J mice revealed that infection, with or without EPO treatment, leads to sub-optimal increases in TER119+ erythroblasts compared to EPO-treated naive mice. A lower percentage of TER119+ erythroblasts in infected mice undergo terminal differentiation to become mature haemoglobin-producing cells. Furthermore, there is a shift in transferrin receptor (CD71) expression from TER119+ cells to a non-erythroid population. Deficiencies in the number and maturation of TER119+ erythroblasts during infection coincide with blunted proliferation to EPO stimulation in vitro by splenocytes, although a high frequency express EPO receptor (EPOR). Together, these data suggest that during malaria, EPO-induced proliferation of early EPOR+ erythroid progenitors is suppressed, leading to sub-optimal generation of TER119+ erythroblasts. Moreover, a shift in CD71 expression may result in impaired terminal maturation of erythroblasts. Thus, suppressed proliferation, differentiation, and maturation of erythroid precursors in association with inadequate reticulocytosis may be the basis of insufficient erythropoiesis during malaria.

Topics: Anemia; Animals; Disease Models, Animal; Erythropoiesis; Erythropoietin; Humans; Kidney; Malaria; Mice; Reticulocytosis

Within the past decade, clinical trials have shown that the presence of anemia can diminish the physical status, functional abilities, and overall quality of life (QOL) of cancer patients and can negatively influence the outcome of their treatment. However, recent preclinical and clinical studies have also shown that increasing hemoglobin levels by administering recombinant human erythropoietin (rHuEPO, epoetin alfa) may ameliorate anemia and, in doing so, improve QOL and possibly result in better treatment outcomes following radiotherapy, chemotherapy, or a combination of these modalities. Several mechanisms by which rHuEPO may improve treatment outcome have been proposed, including correction of tumor hypoxia, increased sensitivity of tumor cells to radiotherapy and chemotherapy, correction of anemia and its associated symptoms (particularly fatigue), and immune-modulated effects of rHuEPO on tumor growth. Improvement of tumor oxygenation by rHuEPO could affect treatment outcome in two ways. First, correction of hypoxia results in the downregulation of hypoxia-inducible factor 1 (HIF-1), a key regulator of cellular adaptive responses to hypoxia (e.g., angiogenesis), including many pathways that are important for tumor growth and metastasis. Interruption of the HIF-1 pathway not only limits growth of the primary tumor but also reduces the potential for the development of more aggressive tumors and metastatic spread, which could ultimately improve treatment outcome. Second, within the tumor, it is the hypoxic cells that are resistant to oxygen-dependent radiotherapy and chemotherapy, and improvement in their oxygenation would increase their sensitivity to the cytotoxic effects of such treatment. Correction of anemia and its associated symptoms, particularly fatigue, can have a beneficial effect on patient QOL, and this in turn may translate into greater tolerance of radiotherapy and chemotherapy, allowing patients to receive full doses and on-schedule dosing, and thus have an increased likelihood of a therapeutic response. Lastly, results of a study using a murine model of multiple myeloma have indicated that rHuEPO may induce an immune-mediated antitumor effect. Therefore, additional research is warranted to further explore the biologic actions of rHuEPO and to determine their relevance to therapeutic outcome.

Topics: Anemia; Animals; Cell Hypoxia; Clinical Trials as Topic; Combined Modality Therapy; Disease Models, Animal; Drug Interactions; Drug Resistance, Neoplasm; Erythropoietin; Fatigue; Humans; Mice; Multiple Myeloma; Neoplasms; Quality of Life; Radiation Tolerance; Recombinant Proteins; T-Lymphocytes; Treatment Outcome

Practically any stimulus capable of causing injury to a tissue or organ can, when applied close to (but below) the threshold of damage, activate endogenous protective mechanisms--thus potentially lessening the impact of subsequent, more severe stimuli. A sub-threshold ischemic insult applied to the brain, for example, activates certain cellular pathways that can help to reduce damage caused by subsequent ischemic episodes--a phenomenon known as 'ischemic preconditioning' (IP) or 'ischemic tolerance' (IT). Although investigated for some time in model organisms, IP/IT has recently been shown in human brain. This opens a window into endogenous neuroprotection and, potentially, a window of opportunity to utilize these mechanisms in the clinic to treat patients with stroke and other CNS disorders.

Topics: Adaptation, Physiological; Animals; Brain; Brain Ischemia; Disease Models, Animal; Erythropoietin; Humans; Ischemic Preconditioning; Neuroprotective Agents

Chemotherapy-associated cognitive dysfunction occurs in a subset of patients treated with adjuvant chemotherapy. Recent data suggest that development of chemotherapy-related anemia predisposes patients to cognitive dysfunction. Endogenous erythropoietin (EPO) is well recognized for its central role in erythropoiesis, and recombinant human EPO (epoetin alfa) is established as a safe and effective treatment for chemotherapy-related anemia. Treatment with epoetin alfa also improved health-related quality of life in anemic cancer patients undergoing chemotherapy, and several controlled studies have documented increases in quality-of-life scores correlated with increases in hemoglobin. Erythropoietin also plays a role in neuroprotection, presumably by activation of antiapoptotic genes. Erythropoietin and its receptor are expressed in neural cells of the human brain, and their expression is upregulated after hypoxic or ischemic injury. In animal models, systemic administration of epoetin alfa protects against such neural injury. Ongoing and future studies will determine whether epoetin alfa can provide neuroprotection with respect to the development of cognitive dysfunction in patients undergoing adjuvant chemotherapy treatment for breast cancer.

Topics: Anemia; Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cognition Disorders; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Female; Hematinics; Humans; Quality of Life; Rats; Recombinant Proteins

Erythropoietin is the primary physiological regulator of erythropoiesis, and it exerts its effect by binding to cell surface receptors. It has recently been shown that both erythropoietin and its receptor are found in the human cerebral cortex, and that, in vitro, the cytokine is synthesized by astrocytes and neurons, has neuroprotective activity, and is up-regulated following hypoxic stimuli. In animal models, exogenous recombinant human erythropoietin has been reported to be beneficial in treating experimental global and focal cerebral ischemia and reducing nervous system inflammation. These findings suggest that exogenous administration of erythropoietic agents (darbepoetin alfa [Aranesp], epoetin alfa [Epogen, Procrit], and epoetin beta [NeoRecormon]) may be a potential therapeutic tool for central nervous system injury. However, transport of protein therapeutics to the brain's extracellular environment via systemic blood supply generally does not occur due to the negligible permeability of the brain capillary endothelial wall. Therefore, in order to pharmacologically exploit and fully realize the therapeutic benefits of exogenous erythropoietic agents in CNS dysfunction, mechanisms of action and the potential impact of biodistribution barriers need to be elucidated.

Topics: Animals; Blood-Brain Barrier; Brain; Brain Diseases; Clinical Trials as Topic; Darbepoetin alfa; Disease Models, Animal; Epoetin Alfa; Erythropoiesis; Erythropoietin; Humans; Neuroprotective Agents; Receptors, Erythropoietin; Recombinant Proteins

A new field of clinical and scientific interest has recently developed based on the discovery that the hematopoietic cytokine erythropoietin (Epo) has important non-hematopoietic functions in the brain and other organs, particularly during development. The biological effects of Epo in the central nervous system (CNS) involve activation of its specific receptor and corresponding signal transduction pathways. Epo receptor expression is abundant in the developing mammalian brain, and decreases as term approaches. Epo has been identified as a neurotrophic and neuroprotective agent in a wide variety of experimental paradigms, from neuronal cell culture to in vivo models of brain injury. Several mechanisms by which Epo produces neuroprotection are recognized. Epo (i) decreases glutamate toxicity, (ii) induces the generation of neuronal anti-apoptotic factors, (iii) reduces inflammation, (iv) decreases nitric oxide-mediated injury, and (v) has direct antioxidant effects.. Collectively, the evidence suggests that Epo may provide a new approach to the treatment of a variety of CNS disorders in adults and children, especially as a possible therapy for perinatal asphyxia. This review summarizes the current knowledge on the neurotrophic and neuroprotective functions of Epo in the developing and injured brain.

Topics: Animals; Brain; Central Nervous System; Disease Models, Animal; Embryonic and Fetal Development; Erythropoiesis; Erythropoietin; Gerbillinae; Humans; Hypoxia, Brain; Mice; Neuroprotective Agents; Rabbits; Rats; Receptors, Erythropoietin; Recombinant Proteins; Sensitivity and Specificity

Erythropoietin (EPO) is an endogenous cytokine with antiapoptotic, antiinflammatory, and neurotrophic properties. Apart from being produced by the kidney, liver, and spleen in response to hypoxia, EPO is highly expressed in the brain during development and after neuropathological insults. The observation that receptors for EPO are present on brain capillaries and glial capillary end-feet has suggested that circulating (plasma) EPO may be transferred into the brain. This review summarizes the increasing number of studies indicating that peripherally administered recombinant human (rHu) EPO crosses the blood-brain barrier. Moreover, several of these studies have shown that peripherally administered rHuEPO can protect against the damage caused by a diversity of neuropathological conditions such as (a) stroke, (b) head and spinal cord trauma, (c) inflammatory and demyelinating conditions, (d) toxin-induced epileptic seizures, and (e) retinal ischemia. While all these studies are based on experiments in animal models, the effectiveness of rHuEPO in ischemic stroke in human patients has recently been suggested in a proof-of-concept trial, which is also discussed.

Topics: Animals; Blood-Brain Barrier; Brain Injuries; Controlled Clinical Trials as Topic; Cross-Sectional Studies; Demyelinating Diseases; Disease Models, Animal; Erythropoietin; Evidence-Based Medicine; Female; Humans; Injections, Intramuscular; Injections, Intraperitoneal; Male; Mice; Neuroprotective Agents; Rabbits; Rats; Recombinant Proteins; Sensitivity and Specificity; Spinal Cord Injuries; Stroke; Treatment Outcome

Several recently published studies describe moderate to severe cognitive dysfunction in breast cancer survivors who were treated with adjuvant chemotherapy 1-5 years before undergoing extensive neuropsychological testing. While these studies are hypothesis-generating and preliminary given their small size and retrospective nature, they consistently suggest that between approximately 15% and 25% of chemotherapy-treated breast cancer patients will have evidence of cognitive dysfunction some years after chemotherapy, compared to about 10% of breast cancer survivors who did not receive chemotherapy. Recent preclinical data strongly suggest that erythropoetin is a potent, endogenous neuroprotective agent that prevents neuronal apoptosis from a variety of insults including hypoxia, trauma, subarachnoidal hemorrhage, and encephalitis. Erythropoietin also appears to enhance learning in a mouse spatial learning maze model. We have conducted a pilot study of epoetin alfa versus placebo in early-stage breast cancer patients who received standard adjuvant anthracycline-based chemotherapy to determine the feasibility of administering standardized neurocognitive assessment tests in the oncology practice setting in order to understand whether the Executive Interview 25 test can detect the subtle cognitive impairment in verbal fluency, attention, and short-term memory observed with chemotherapy, and to assess whether epoetin alfa-treated patients have less evidence of cognitive dysfunction during and 6 months after chemotherapy compared with control-treated patients. We report here the preliminary results of this pilot clinical trial.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Asthenia; Breast Neoplasms; Chemotherapy, Adjuvant; Cognition Disorders; Controlled Clinical Trials as Topic; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Female; Humans; Mastectomy; Mice; Mood Disorders; Patient Satisfaction; Pilot Projects; Prognosis; Quality of Life; Recombinant Proteins; Severity of Illness Index; Treatment Outcome

Erythropoietin (EPO) primarily is produced in the kidney and acts as a principal mediator of the physiologic response to hypoxia by increasing red blood cell production. Astrocytes and neurons in the central nervous system (CNS) also are known to produce EPO in response to hypoxia/ischemia. EPO appears to play a neuroprotective role based on preclinical data demonstrating the ability of recombinant human erythropoietin (r-HuEPO) to shield neurons from hypoxic/ischemic stress when administered intracerebraventricularly. In CNS models, systemically administered r-HuEPO has not been intensely investigated because large glycosylated molecules generally were deemed incapable of crossing the blood-brain barrier (BBB). A collaborative research effort identified expression of EPO receptors on human brain capillaries and a specific receptor-mediated transport of r-HuEPO across the BBB after a single intraperitoneal (IP) injection in rodents, with subsequent protection against various types of neuronal damage. For example, administration of r-HuEPO 24 hours before or up to 6 hours after focal ischemic stroke significantly reduced the extent of infarction. r-HuEPO also attenuated concussive brain injury, kainate-induced seizure activity, and autoimmune encephalomyelitis. These preclinical findings suggest that r-HuEPO may have therapeutic potential for stroke, head trauma, and epilepsy; additional studies are needed to confirm and extend these encouraging observations in animal models.

Topics: Animals; Central Nervous System Diseases; Disease Models, Animal; Erythropoiesis; Erythropoietin; Humans; Recombinant Proteins

Cushing's syndrome of glucocorticoid excess is named after the eminent Boston neurosurgeon Harvey W. Cushing (1869-1939). The recognition that glucocorticoid excess produces hypertension led to examination of the role of cortisol in essential hypertension, but it is only over the last decade that evidence has emerged to support the concept. Despite the widespread assumption that cortisol raises blood pressure as a consequence of renal sodium retention, there are few data consistent with the notion. Although it has a plethora of actions on brain, heart and blood vessels, kidney, and body fluid compartments, precisely how cortisol elevates blood pressure is unclear. Candidate mechanisms currently being examined include inhibition of the vasodilator nitric oxide system and increases in vasoconstrictor erythropoietin concentration.

Topics: Cardiovascular Diseases; Cushing Syndrome; Disease Models, Animal; Erythropoietin; Glucocorticoids; Humans; Hydrocortisone; Hypertension; Infant, Newborn; Nitric Oxide; Vasoconstriction; Vasodilation

Idiopathic myelofibrosis is the least common and carries the worst prognosis of the chronic myeloproliferative disorders. The primary disease process is a clonal haematopoietic stem cell disorder which results in a chronic myeloproliferation and an atypical megakaryocyte hyperplasia. In contrast, the characteristic stromal proliferation is a reactive phenomenon, resulting from the inappropriate release of megakaryocyte/platelet-derived growth factors, including PDGF, TGF-beta bFGF and calmodulin. The median survival is approximately 4 years, although individual survival varies greatly. A variety of prognostic schema have been developed which enable the identification of high-risk patients, for whom bone marrow transplantation should be considered. Management for the majority of patients, however, is directed towards the alleviation of symptoms and improvement in quality of life. This review summarizes the recent advances in our understanding of the disease's pathogenesis and discusses the limited therapeutic options available to clinicians.

Topics: Anemia; Animals; Bone Marrow; Bone Marrow Transplantation; Cladribine; Combined Modality Therapy; Disease Models, Animal; Erythropoietin; Growth Substances; Hematopoietic Stem Cells; Humans; Hydroxyurea; Interferon-alpha; Karyotyping; Megakaryocytes; Mice; Polyethylene Glycols; Primary Myelofibrosis; Prognosis; Recombinant Proteins; Splenectomy; Stromal Cells; Thrombopoietin; Vitamin D

If one reviews the literature with zeal, it is increasingly apparent that few organs escape recruitment when IBD is chronic or progressive. Insights into mucosal pathophysiology have helped with understanding the more frequent extraintestinal manifestations, but the mechanisms attendant to the development of less common events (e.g. acute pancreatitis, concurrent gluten sensitive enteropathy, or active pulmonary disease) remain either poorly studied or obscure. It is particularly interesting, however, to read reports of abnormal pulmonary function, generally of the obstructive type, correlated to measurements of abnormal intestinal permeability in patients with either active pulmonary sarcoid or pulmonary involvement in Crohn's disease. It has been further speculated that similarities in the mucosal immune system of the lung and intestine are responsible for evidence of bronchial hyperreactivity in patients with active IBD. Finally, it is important to recognize that extensions of the inflammatory process are not restricted to the development of organ-based events but may be responsible for some of the most frequent systemic abnormalities detected in IBD patients. It is now also well confirmed that the cytokine environment in IBD can support activated coagulation and, in some clinical situations, overt vascular thrombosis. The cerebrovascular complications of IBD are well recognized and range from peripheral venous thrombosis to central stroke syndromes and pseudotumor cerebri. Reports of focal white matter lesions in the brains of patients with IBD or an increased incidence of polyneuropathy may be other clinical examples of regional microvascular clotting. Microvascular injury appears to be more ubiquitously present, with reports ranging from a speculated primary causative role (e.g., granulomatous vasculitis in the mesenteric circulation) to the utility of nailbed vasospasm, in Crohn's disease, as a clinical marker for disease activity. It is also reported that IL-6 suppression of erythropoietin production is a major feature of the chronic anemia seen in active IBD. Moreover, the capacity of peripheral monocytes from active IBD patients to secrete TNF and IL-8 is reported predictive for the degree of therapeutic response from recombinant erythropoietin. These collected observations constitute another excellent example of the symmetry between basic science and clinical utility. It is from the context of applied basic science that many future therapies wi

Topics: Anemia; Animals; Brain Diseases; Bronchial Hyperreactivity; Cerebrovascular Disorders; Crohn Disease; Cytokines; Disease Models, Animal; Erythropoietin; Humans; Inflammatory Bowel Diseases; Interleukin-6; Lung Diseases; Lung Diseases, Obstructive; Sarcoidosis; Thrombosis

Topics: Anemia; Animals; Blood Pressure; Disease Models, Animal; Double-Blind Method; Erythropoiesis; Erythropoietin; Hematocrit; Humans; Hypertension, Renal; Kidney; Kidney Failure, Chronic; Rats; Recombination, Genetic

Transgenic animals or animals engrafted with retrovirus-derived expression vectors provide models for studying the in vivo effects of high and continuous serum levels of cytokines. Studies employing these models in order to analyze the biological effects for granulocytes-macrophages colony-stimulating factors (CSFs), granulocytes of interleukin (IL-)2, IL-3, IL-6 and erythropoietin are reviewed. In all of these models, overexpression of the different cytokines achieved by use of these approaches resulted in syndromes that were related to nonneoplastic hyperplasia of the respective target cell population. In some but not all models, these syndromes were lethal, mostly due to hypercellularity. These models allow conclusions about the biological effects of very high and continuous levels of these substances as well as about their regulation in different tissues.

Topics: Animals; Animals, Genetically Modified; Bone Marrow Cells; Cell Division; Cell Line; Cytokines; Disease Models, Animal; Erythropoietin; Interleukin-2; Interleukin-6; Lymphoid Tissue; Mice; Mice, Transgenic; Polycythemia

Topics: Anemia; Anemia, Hypochromic; Anemia, Sideroblastic; Animals; Arthritis, Rheumatoid; Bone Marrow; Chronic Disease; Copper; Disease Models, Animal; Endotoxins; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Freund's Adjuvant; Hematocrit; Humans; Infections; Iron; Mononuclear Phagocyte System; Neoplasms; Porphyrins; Protein Binding; Rats; Transferrin

To evaluate the functional and immunohistochemical effects of ganglioside GM1 and erythropoietin following experimental spinal cord injury.. Thirty-two male BALB/c mice were subjected to experimental spinal cord injury using the NYU Impactor device and were randomly divided into the following groups: GM1 group, receiving standard ganglioside GM1 (30 mg/kg); erythropoietin group, receiving erythropoietin (1000 IU/kg); combination group, receiving both drugs; and control group, receiving saline (0.9%). Animals were evaluated according to the Basso Mouse Scale (BMS) and Hindlimb Mouse Function Score (MFS). After euthanasia, the immunohistochemistry of the medullary tissue of mice was analyzed. All animals received intraperitoneal treatment.. The GM1 group had higher BMS and MFS scores at the end of the experiment when compared to all other groups. The combination group had higher BMS and MFS scores than the erythropoietin and control groups. The erythropoietin group had higher BMS and MFS scores than the control group. Immunohistochemical tissue analysis showed a significant difference among groups. There was a significant increase in myelinated axons and in the myelinated axon length in the erythropoietin group when compared to the other intervention groups (p < 0.01).. Erythropoietin and GM1 have therapeutic effects on axonal regeneration in mice subjected to experimental spinal cord injury, and administration of GM1 alone had the highest scores on the BMS and MFS scales.

Topics: Animals; Disease Models, Animal; Epoetin Alfa; Erythropoietin; G(M1) Ganglioside; Injections, Intraperitoneal; Male; Mice; Spinal Cord; Spinal Cord Injuries

Erythropoietin (EPO) and its covalently modified analogs are neuroprotective in various models of brain damage and disease. We investigated the effect on brain damage and memory performance, of a continuous 3-day intravenous infusion of EPO, starting 20 min after a transient 10 minute period of global cerebral ischemia in the rat.. We found no effect on selective neuronal damage in the CA1 region of the hippocampus, neocortical damage and damage to the striatum assessed at 7 days after ischemia. Also, no differences were observed in sensori-motor scores between EPO treated and saline treated ischemic animals. In contrast, memory performance was significantly improved in the EPO treated group. Saline treated injured animals (n = 7) failed in a test assessing recovery of spatial memory (6/6 and 5/6), while EPO treated animals had few and none failures (0/7 and 1/7).. We conclude that although post-ischemic treatment with EPO is not neuroprotective in a model of cardiac arrest brain ischemia, its markedly positive effect on brain plasticity and recovery of memory function warrants consideration as treatment of cardiac arrest patients.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Erythropoietin; In Situ Nick-End Labeling; Male; Maze Learning; Memory Disorders; Motor Activity; Rats; Rats, Wistar; Rotarod Performance Test

Erythropoietin (EPO) was proven as a promising approach for experimental subarachnoid hemorrhage (SAH). Clinical data are, however, inconclusive so far. A detailed characterization of specific EPO effects could facilitate the design of trials. The aim of the present investigation was, therefore, to characterize these effects on prevention of delayed proximal cerebral vasospasm (CVS), impaired microcirculation and cerebral blood flow (CBF) after experimental SAH.. 27 male Sprague-Dawley rats were randomized in 3 groups: Sham, SAH control, and SAH EPO. SAH was induced by injection of 0.2ml autologous blood into the cisterna magna on days 1 and 2. Animals of the SAH EPO group received 5000iU rh EPO α 6h after the 2nd SAH intravenously. Surviving animals were examined on day 5 by MR perfusion weighted imaging (PWI). Cerebral blood flow (CBF) and volume (CBV) were determined by PWI, proximal CVS by basilar artery (BA) diameter, and neuroprotection by hippocampal cell count (CA1-CA4).. BA diameter was significantly reduced in both SAH groups, but improved significantly after EPO (Sham: 144±3μm, SAH control: 79±6μm, SAH EPO 109±4μm). The rrCBV ratio was 8.78±0.72 Sham, 5.14±1.73 SAH control, and 6.80±0.44 SAH EPO. The improvement by EPO did not reach statistical significance. RrCBF ratio was also significantly reduced in both SAH groups, but was significantly improved by EPO (Sham: 8.78±0.34, SAH control: 4.26±1.05, SAH EPO 5.85±0.46). Surviving neuronal cells were significantly reduced in SAH controls in all areas, but in SAH EPO only in CA1.. The present data suggest that an EPO application in a timely distance to the SAH is sufficient to prevent delayed proximal CVS, but that the doses were insufficient to improve microcirculation or to be directly neuroprotective.

Topics: Analysis of Variance; Animals; Basilar Artery; Brain; Cell Count; Cerebrovascular Circulation; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Hemodynamics; Magnetic Resonance Imaging; Male; Neurologic Examination; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Time Factors; Vasospasm, Intracranial

Several recently published studies describe moderate to severe cognitive dysfunction in breast cancer survivors who were treated with adjuvant chemotherapy 1-5 years before undergoing extensive neuropsychological testing. While these studies are hypothesis-generating and preliminary given their small size and retrospective nature, they consistently suggest that between approximately 15% and 25% of chemotherapy-treated breast cancer patients will have evidence of cognitive dysfunction some years after chemotherapy, compared to about 10% of breast cancer survivors who did not receive chemotherapy. Recent preclinical data strongly suggest that erythropoetin is a potent, endogenous neuroprotective agent that prevents neuronal apoptosis from a variety of insults including hypoxia, trauma, subarachnoidal hemorrhage, and encephalitis. Erythropoietin also appears to enhance learning in a mouse spatial learning maze model. We have conducted a pilot study of epoetin alfa versus placebo in early-stage breast cancer patients who received standard adjuvant anthracycline-based chemotherapy to determine the feasibility of administering standardized neurocognitive assessment tests in the oncology practice setting in order to understand whether the Executive Interview 25 test can detect the subtle cognitive impairment in verbal fluency, attention, and short-term memory observed with chemotherapy, and to assess whether epoetin alfa-treated patients have less evidence of cognitive dysfunction during and 6 months after chemotherapy compared with control-treated patients. We report here the preliminary results of this pilot clinical trial.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Asthenia; Breast Neoplasms; Chemotherapy, Adjuvant; Cognition Disorders; Controlled Clinical Trials as Topic; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Female; Humans; Mastectomy; Mice; Mood Disorders; Patient Satisfaction; Pilot Projects; Prognosis; Quality of Life; Recombinant Proteins; Severity of Illness Index; Treatment Outcome

Topics: Anemia; Animals; Blood Pressure; Disease Models, Animal; Double-Blind Method; Erythropoiesis; Erythropoietin; Hematocrit; Humans; Hypertension, Renal; Kidney; Kidney Failure, Chronic; Rats; Recombination, Genetic

Abdominal aortic aneurysm (AAA) is a potentially fatal vascular disease, but the underlying mechanisms remain obscure. Here, we provide a protocol using erythropoietin (EPO) to induce the formation of AAA in both wild-type (WT) and apolipoprotein E (Apoe

Topics: Animals; Aorta, Abdominal; Aortic Aneurysm, Abdominal; Apolipoproteins E; Disease Models, Animal; Erythropoietin; Mice

In this study, based on the excitatory/inhibitory imbalance theory of autism, the time window of GABA switch, the role of K-Cl co-transporter 2 (KCC2) in adjustment GABA switch, and brain permeability to erythropoietin (EPO), the effects of postnatal -EPO and- nano- erythropoietin (NEPO) have been evaluated in the valproic acid (VPA) rat model of autism. The VPA was administered for animal modeling of autism at gestational day (GD) 12.5 (600 mg/kg). Male offsprings were injected with EPO and NEPO in a clinically proper postnatal dosing regimen on postnatal days (PND) 1-5, and autistic-like behaviors were tested at the end of the first month. Then animals were sacrificed, and neuron morphology and KCC2 expression were examined by Nissl staining and Western blot. According to our findings, high-dose NEPO improved autism-associated phenotypes. Neuroprotective effects of EPO and NEPO have been shown in the hippocampus. Postnatal NEPO treatment reversed KCC2 expression abnormalities induced by prenatal VPA. Our results might support the role of KCC2 in ASD and the excitatory/inhibitory imbalance hypothesis. We suggested Nano- erythropoietin and other KCC2 interventions as a new approach to the early treatment and prevention of autism.

Topics: Animals; Autistic Disorder; Behavior, Animal; Disease Models, Animal; Erythropoietin; Female; gamma-Aminobutyric Acid; Hippocampus; Humans; Male; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Symporters; Valproic Acid

Anemia is a common complication of systemic inflammation. Proinflammatory cytokines both decrease erythroblast sensitivity to erythropoietin (EPO) and increase the levels of the hepatic hormone hepcidin, sequestering iron in stores and causing functional iron deficiency. Anemia of chronic kidney disease (CKD) is a peculiar form of anemia of inflammation, characterized by impaired EPO production paralleling progressive kidney damage. Traditional therapy based on increased EPO (often in combination with iron) may have off-target effects due to EPO interaction with its non-erythroid receptors. Transferrin Receptor 2 (Tfr2) is a mediator of the iron-erythropoiesis crosstalk. Its deletion in the liver hampers hepcidin production, increasing iron absorption, whereas its deletion in the hematopoietic compartment increases erythroid EPO sensitivity and red blood cell production. Here, we show that selective hematopoietic Tfr2 deletion ameliorates anemia in mice with sterile inflammation in the presence of normal kidney function, promoting EPO responsiveness and erythropoiesis without increasing serum EPO levels. In mice with CKD, characterized by absolute rather than functional iron deficiency, Tfr2 hematopoietic deletion had a similar effect on erythropoiesis but anemia improvement was transient because of limited iron availability. Also, increasing iron levels by downregulating only hepatic Tfr2 had a minor effect on anemia. However, simultaneous deletion of hematopoietic and hepatic Tfr2, stimulating erythropoiesis and increased iron supply, was sufficient to ameliorate anemia for the entire protocol. Thus, our results suggest that combined targeting of hematopoietic and hepatic Tfr2 may be a therapeutic option to balance erythropoiesis stimulation and iron increase, without affecting EPO levels.

Topics: Anemia; Animals; Disease Models, Animal; Erythropoiesis; Erythropoietin; Hepcidins; Inflammation; Iron; Iron Deficiencies; Mice; Receptors, Transferrin; Renal Insufficiency, Chronic

Iron deficiency is a potent stimulator of fibroblast growth factor 23 (FGF23), a hormonal regulator of phosphate and vitamin D metabolism, that is classically thought to be produced by bone-embedded osteocytes. Here, we show that iron-deficient transmembrane serine protease 6 knockout (Tmprss6-/-) mice exhibit elevated circulating FGF23 and Fgf23 messenger RNA (mRNA) upregulation in the bone marrow (BM) but not the cortical bone. To clarify sites of Fgf23 promoter activity in Tmprss6-/- mice, we introduced a heterozygous enhanced green fluorescent protein (eGFP) reporter allele at the endogenous Fgf23 locus. Heterozygous Fgf23 disruption did not alter the severity of systemic iron deficiency or anemia in the Tmprss6-/- mice. Tmprss6-/-Fgf23+/eGFP mice showed green fluorescence in the vascular regions of BM sections and showed a subset of BM endothelial cells that were GFPbright by flow cytometry. Mining of transcriptomic data sets from mice with normal iron balance revealed higher Fgf23 mRNA in BM sinusoidal endothelial cells (BM-SECs) than that in other BM endothelial cell populations. Anti-GFP immunohistochemistry of fixed BM sections from Tmprss6-/-Fgf23+/eGFP mice revealed GFP expression in BM-SECs, which was more intense than in nonanemic controls. In addition, in mice with intact Tmprss6 alleles, Fgf23-eGFP reporter expression increased in BM-SECs following large-volume phlebotomy and also following erythropoietin treatment both ex vivo and in vivo. Collectively, our results identified BM-SECs as a novel site for Fgf23 upregulation in both acute and chronic anemia. Given the elevated serum erythropoietin in both anemic models, our findings raise the possibility that erythropoietin may act directly on BM-SECs to promote FGF23 production during anemia.

Topics: Anemia, Iron-Deficiency; Animals; Bone Marrow; Disease Models, Animal; Endothelial Cells; Erythropoietin; Iron; Mice; RNA, Messenger; Up-Regulation

Renal fibrosis is the final manifestation of chronic kidney disease (CKD); its prevention is vital for controlling CKD progression. Indoxyl sulfate (IS), a typical sulfate-conjugated uremic solute, is produced in the liver via the enzyme sulfotransferase (SULT) 1A1 and accumulates significantly during CKD. We investigated the toxicopathological role of IS in renal fibrosis using

Topics: Animals; Disease Models, Animal; Erythropoietin; Fibrosis; Humans; Indican; Inflammation; Kidney; Mice; Mice, Inbred C57BL; Renal Insufficiency, Chronic; Sulfotransferases; Ureteral Obstruction

Patients with chronic pain often complain about memory impairments. Experimental studies have shown neuroprotective effects of Carbamylated erythropoietin (Cepo-Fc) in the treatment of cognitive dysfunctions. However, little is currently known about its precise molecular mechanisms in a model of inflammatory pain. Therefore, this study aimed to investigate neuroprotective effects of Cepo-Fc against cognitive impairment induced by the inflammatory model of Complete Freund's Adjuvant (CFA). Carbamylated erythropoietin was administrated Intraperitoneally (i.p) on the day CFA injection, continued for a 21-days period. After conducting the behavioral tests (thermal hyperalgesia and novel object recognition test), western blot and ELISA were further preformed on days 0, 7, and 21. The results of this study indicate that Cepo-Fc can effectively reverse the CFA induced thermal hyperalgesia and recognition memory impairment. Additionally, Cepo-Fc noticeably decreased the hippocampal microglial expression, production of hippocampal IL-1β, and hippocampal apoptosis and necroptosis induced by the inflammatory pain. Therefore, our findings suggest that neuroprotective effects of Cepo-Fc in the treatment of pain related recognition memory impairment may be mediated through reducing hippocampal microglial expression as well as IL-1β production.

Topics: Animals; Disease Models, Animal; Erythropoietin; Freund's Adjuvant; Hippocampus; Hyperalgesia; Male; Memory; Memory Disorders; Microglia; Neuroprotective Agents; Pain; Rats; Recognition, Psychology

Chronic kidney disease involves disturbances in iron metabolism including anemia caused by insufficient erythropoietin (EPO) production. However, underlying mechanisms responsible for the dysregulation of cellular iron metabolism are incompletely defined. Using the unilateral ureteral obstruction (UUO) model in

Topics: Anemia; Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Hypoxia; Disease Models, Animal; Erythropoietin; Ferritins; Fibrosis; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Iron; Iron Regulatory Protein 1; Kidney; Male; Mice, Inbred C57BL; Mice, Knockout; Procollagen-Proline Dioxygenase; Receptors, Transferrin; Renal Insufficiency; Ureteral Obstruction

Erythropoietin has neuroregenerative effects. Fibrin glue may be used for nerve repair and controlled release of substances. In this study, the authors investigated the effects of erythropoietin-containing fibrin glue on nerve repair, based on the hypothesis that erythropoietin-containing fibrin glue would positively affect nerve regeneration.. Thirty-six Long-Evans rats were used. The animals were divided into six groups. Their left sciatic nerves were isolated, transected, and repaired with saline-containing fibrin glue in group 1, with erythropoietin-containing fibrin glue in group 2, with saline-containing fibrin glue and two sutures in group 3, with erythropoietin-containing fibrin glue and two sutures in group 4, with two sutures in group 5, and with four sutures in group 6. Sciatic Functional Index calculation, pin-prick test, and toe-spread test were performed on days 21, 42, and 63. All animals were killed on day 63. The nerve sections were analyzed histologically.. The Sciatic Functional Index, pin-prick test, and toe-spread test results were the best in group 4 and the worst in group 5. Group 4 showed superior Schwann cell proliferation (p < 0.05). Groups with epineural suture use (groups 3, 4, 5, and 6) had higher endoneurial collagen synthesis scores than the groups without suture use (groups 1 and 2) (p < 0.05). The myelin protein zero immunostaining results were significantly higher in the erythropoietin-treated groups (groups 2 and 4) (p < 0.05).. The combined use of erythropoietin-containing fibrin glue and two epineural sutures (group 4) showed a statistically significant improvement in many parameters.. Fibrin glue is already used in nerve repair. Adding erythropoietin to fibrin glue could be a safe and easy option to improve nerve regeneration.

Topics: Animals; Disease Models, Animal; Erythropoietin; Fibrin Tissue Adhesive; Nerve Regeneration; Peripheral Nerve Injuries; Rats; Rats, Long-Evans; Sciatic Nerve

Fibroblast-like renal erythropoietin (Epo) producing (REP) cells of the corticomedullary border region "sense" a decrease in blood oxygen content following anaemia or hypoxaemia. Burst-like transcription of Epo during tissue hypoxia is transient and is lost during fibrotic tissue remodelling, as observed in chronic kidney disease. The reason for this loss of Epo expression is under debate. Therefore, we tested the hypothesis that REP cell migration, loss and/or differentiation may cause Epo inhibition.. Using a reporter mouse that allows permanent labelling of active REP cells at any given time point, we analysed the spatiotemporal fate of REP cells following their initial hypoxic recruitment in models of hypoxaemia and renal tissue remodelling.. In long-term tracing experiments, tagged REP reporter cells neither died, proliferated, migrated nor transdifferentiated into myofibroblasts. Approximately 60% of tagged cells re-expressed Epo upon a second hypoxic stimulus. In an unilateral model of tissue remodelling, tagged cells proliferated and ceased to produce Epo before a detectable increase in myofibroblast markers. Treatment with a hypoxia-inducible factor (HIF) stabilizing agent (FG-4592/roxadustat) re-induced Epo expression in the previously active REP cells of the damaged kidney to a similar extent as in the contralateral healthy kidney.. Rather than cell death or differentiation, these results suggest cell-intrinsic transient inhibition of Epo transcription: following long-term dormancy, REP cells can repeatedly be recruited by tissue hypoxia, and during myofibrotic tissue remodelling, dormant REP cells are efficiently rescued by a pharmaceutic HIF stabilizer, demonstrating persistent REP cell functionality even during phases of Epo suppression.

Topics: Anemia; Animals; Disease Models, Animal; Erythropoietin; Hypoxia; Kidney; Mice; Renal Insufficiency, Chronic

This study aimed to investigate the efficacy of human-derived umbilical cord mesenchymal stem cells (HDUMSC) and human-derived umbilical cord mesenchymal stem cells expressing erythropoietin (HDUMSC-EPO) to rescue total degenerated retina in a rat model.. The study included four treatment groups, namely negative control using normal saline (HBSS) injection, positive control using sodium iodide 60 mg/kg (SI), SI treated with HDUMSC, and SI treated with HDUMSC-EPO given via subretinal and intravenous routes, to test the efficacy of retinal regeneration following SI-induced retinal degeneration. Retinal function in both phases was tested via electroretinography (ERG) and histological staining examining the outer nuclear layer (ONL).. There was a statistically significant result (P < 0.05) in the SI treated with HDUMSC-EPO only when comparing day 11 (mean = 23.6 μv), day 18 (mean = 25.2 μv), day 26 (mean = 26.3 μv), and day 32 (mean = 28.2 μv) to the b-wave ERG on day 4 rescue injection day (mean = 12.5 μv). The SI treated with HDUMSC-EPO showed significant improvement in b-wave ERG readings in the Sprague-Dawley (SD) rat but did not restore baseline readings prior to degeneration (day 0). Both treated groups' ONL thicknesses did not show significant changes compared to the negative control group (HBSS) following rescue therapy.. Total retinal degeneration following intravenous SI injection was observed at 60 mg/kg. SI treated with HDUMSC and HDUMSC-EPO showed no regenerative potential compared to baseline in SI-induced total retina degeneration on ERG or histology, whereas SI treated with HDUMSC-EPO group showed a substantial increase in b-wave ERG amplitude over time.

Topics: Animals; Disease Models, Animal; Electroretinography; Erythropoietin; Humans; Mesoderm; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Stem Cells

Cancer-related anemia (CRA) negatively influences cancer patients' survival, disease progression, treatment efficacy, and quality of life (QOL). Current treatments such as iron therapy, red cell transfusion, and erythropoietin-stimulating agents (ESAs) may cause severe adverse effects. Therefore, the development of long-lasting and curative therapies is urgently required.. In this study, a cell and gene therapy strategy was developed for in vivo delivery of EPO cDNA by way of genetic engineering of human Wharton's jelly mesenchymal stem cells (hWJMSCs) to produce and secrete human EPO protein for extended periods after transplantation into the mice model of CRA.. To evaluate CRA's treatment in cancer-free and cancerous conditions, first, a recombinant breast cancer cell line 4T1 which expressed herpes simplex virus type 1 thymidine kinase (HSV1-TK) by a lentiviral vector encoding HSV1-TK was developed and injected into mice. After three weeks, all mice developed metastatic breast cancer associated with acute anemia. Then, ganciclovir (GCV) was administered for ten days in half of the mice to clear cancer cells. Meanwhile, another lentiviral vector encoding EPO to transduce hWJMSCs was developed. Following implantation of rhWJMSCs-EPO in the second group of mice, peripheral blood samples were collected once a week for ten weeks from both groups.. Analysis of peripheral blood samples showed that plasma EPO, hemoglobin (Hb), and hematocrit (Hct) concentrations significantly increased and remained at therapeutic for >10 weeks in both treatment groups.. Data indicated that rhWJMSCs-EPO increased the circulating level of EPO, Hb, and Hct in both mouse subject groups and improved the anemia of cancer in both cancer-free and cancerous mice.

Topics: Anemia; Animals; Breast Neoplasms; Disease Models, Animal; DNA, Complementary; Erythropoietin; Female; Ganciclovir; Hemoglobins; Herpesvirus 1, Human; Humans; Iron; Mesenchymal Stem Cells; Mice; Quality of Life; Recombinant Proteins; Thymidine Kinase

In addition to the long-established role in erythropoiesis, erythropoietin (Epo) has protective functions in a variety of tissues, including the heart. This is the most affected organ in chronic Chagas disease, caused by the protozoan Trypanosoma cruzi. Despite seven million people being infected with T. cruzi worldwide, there is no effective treatment preventing the disease progression to the chronic phase when the pathological involvement of the heart is often observed. Chronic chagasic cardiomyopathy has a wide variety of manifestations, like left ventricular systolic dysfunction, dilated cardiomyopathy, and heart failure. Since Epo may help maintain cardiac function by reducing myocardial necrosis, inflammation, and fibrosis, this study aimed to evaluate whether the Epo has positive effects on experimental Chagas disease. For that, we assessed the earlier (acute phase) and also the later (chronic phase) use of Epo in infected C57BL/6 mice. Blood cell count, biochemical parameters, parasitic load, and echocardiography data were evaluated. In addition, histopathological analysis was carried out. Our data showed that Epo had no trypanocide effect nor did it modify the production of anti-T. cruzi antibodies. Epo-treated groups exhibited parasitic burden much lower in the heart compared to blood. No pattern of hematological changes was observed combining infection with treatment with Epo. Chronic Epo administration reduced CK-MB serum activity from d0 to d180, irrespectively of T. cruzi infection. Likewise, echocardiography and histological results indicate that Epo treatment is more effective in the chronic phase of experimental Chagas disease. Since treatment is one of the greatest challenges of Chagas disease, alternative therapies should be investigated, including Epo combined with benznidazole.

Topics: Animals; Cardiovascular Agents; Chagas Cardiomyopathy; Chagas Disease; Disease Models, Animal; Erythropoietin; Humans; Mice; Mice, Inbred C57BL; Parasite Load; Trypanosoma cruzi

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by memory impairment in patients. Erythropoietin (EPO) has been reported to stimulate neurogenesis. This study was conducted to determine the regenerative effects of EPO in an AD model and to assess its underlying mechanism. Recombinant human EPO was intraperitoneally administered to AD mice induced by intracerebroventricular Aβ oligomer injection. Behavioral assessments with novel object recognition test and passive avoidance task showed improvement in memory function of the EPO-treated AD mice compared to that of the saline-treated AD mice (p < 0.0001). An in vivo protein assay for the hippocampus and cortex tissue indicated that EPO treatment modulated neurotransmitters, including dopamine, serotonin, and adrenaline. EPO treatment also restored the activity of serotonin receptors, including 5-HT4R, 5-HT7R, and 5-HT1aR (p < 0.01), at mRNA levels. Furthermore, EPO seemed to exert an anti-inflammatory influence by downregulating TLR4 at mRNA and protein levels (p < 0.05). Finally, an immunohistochemical assay revealed increments of Nestin(+) and NeuN(+) neuronal cells in the CA3 region in the EPO-treated AD mice compared to those in the saline-treated AD mice. The conclusion is that EPO administration might be therapeutic for AD by activating the serotonergic pathway, anti-inflammatory action, and neurogenic characteristics.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Hippocampus; Humans; Mice; Neurodegenerative Diseases; Recombinant Proteins; RNA, Messenger; Serotonin

Impaired clearance of beta-amyloid (Aβ) is a primary cause of sporadic Alzheimer's disease (AD). Aβ clearance in the periphery contributes to reducing brain Aβ levels and preventing Alzheimer's disease pathogenesis. We show here that erythropoietin (EPO) increases phagocytic activity, levels of Aβ-degrading enzymes, and Aβ clearance in peripheral macrophages via PPARγ. Erythropoietin is also shown to suppress Aβ-induced inflammatory responses. Deletion of EPO receptor in peripheral macrophages leads to increased peripheral and brain Aβ levels and exacerbates Alzheimer's-associated brain pathologies and behavioral deficits in AD-model mice. Moreover, erythropoietin signaling is impaired in peripheral macrophages of old AD-model mice. Exogenous erythropoietin normalizes impaired EPO signaling and dysregulated functions of peripheral macrophages in old AD-model mice, promotes systemic Aβ clearance, and alleviates disease progression. Erythropoietin treatment may represent a potential therapeutic approach for Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Disease Models, Animal; Erythropoietin; Macrophages; Mice; Mice, Transgenic

Spinal cord injury (SCI) is a major disabling disorder for which no effective treatment has yet been found. Regenerative incapability of neuronal cells as well as the secondary mechanisms of injury are the major reasons behind this clinical frustration. Thus, here we fabricated an erythropoietin-chitosan/alginate (EPO-CH/AL) hydrogel and investigated its local therapeutic effects on the apoptotic and inflammatory indices of SCI secondary injury.. EPO-CH/AL hydrogels were fabricated by the ionic gelation method, and they were characterized using SEM and FTIR. In vitro drug release profile of EPO-CH/AL hydrogels was evaluated by UV-vis spectroscopy. Experimental SCI was inflicted in rats which were then treated with CH/AL hydrogels containing different doses of EPO (1000, 5000 and 10,000 IU/kg). The relative expression of Bax and Bcl2 (apoptosis index) and active and inactive forms of NF-κB (inflammation index) were assessed using western blot. Total serum levels of TNF-α were also assessed with ELISA, and histopathological and immunohistochemistry studies were carried out to check the overall changes in the injured tissues.. In vitro drug release test indicated that the EPO-CH/AL hydrogels had a sustained- and controlled-release profile for EPO under these conditions. All the fabricated hydrogels dramatically reduced the elevated inflammation and apoptosis indices of the SCI-inflicted rats (p ≤ 0.05). Nevertheless, only EPO-CH/AL hydrogel (1000 IU/kg EPO) significantly improved the tissue repair and histopathological appearance of the spinal cord at the sites of injury.. Based on our findings, EPO-CH/AL hydrogel (1000 IU/kg EPO) can effectively improve experimental SCI in rats via inhibiting apoptosis and inflammation. Further studies are warranted to elucidate the contributing role of the scaffold in the observed effects.

Topics: Alginates; Animals; bcl-2-Associated X Protein; Cell Line; Cell Survival; Chitosan; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression Regulation; Humans; Hydrogels; Male; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Random Allocation; Rats; Spinal Cord Injuries; Tumor Necrosis Factor-alpha

Perinatal hypoxic-ischemic (HI) brain injury, often in conjunction with an inflammatory insult, is the most common cause of death or disability in neonates. Therapeutic hypothermia (TH) is the standard of care for HI encephalopathy in term and near-term infants. However, TH may not always be available or efficacious, creating a need for novel or adjunctive neurotherapeutics. Using a near-term model of inflammation-sensitized HI brain injury in postnatal day (P) 17 ferrets, animals were randomized to either the control group (

Topics: Animals; Disease Models, Animal; Erythropoietin; Ferrets; Hypothermia, Induced; Hypoxia-Ischemia, Brain; Neuroprotection; Neuroprotective Agents; Uridine

Our previous clinical studies demonstrated the synergistic therapeutic effect induced by co-administering recombinant human erythropoietin (rhEPO) in human umbilical cord blood (hUCB) therapy for children with cerebral palsy. However, the cellular mechanism beyond the beneficial effects in this combination therapy still needs to be elucidated. A hypoxic-ischemic encephalopathy (HIE) model of neonates, representing cerebral palsy, was prepared and randomly divided into five groups (hUCB+rhEPO combination, hUCB, and rhEPO treatments over HIE, HIE control, and sham). Seven days after, hUCB was administered intraperitoneally and the rhEPO injections were started. Neurobehavioral tests showed the best outcome in the combination therapy group, while the hUCB and rhEPO alone treatments also showed better outcomes compared with the control (

Topics: Animals; Animals, Newborn; Apoptosis; Brain Injuries; Disease Models, Animal; Erythropoietin; Female; Fetal Blood; Hypoxia-Ischemia, Brain; Male; Mice; Mice, Inbred ICR; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Signal Transduction

Due to (i) the uremia-enhanced sepsis severity, (ii) the high prevalence of sepsis with pre-existing renal injury and (iii) the non-erythropoiesis immunomodulation of erythropoietin (EPO), EPO was tested in sepsis with pre-existing renal injury models with the retrospective exploration in patients. Then, EPO was subcutaneously administered in mice with (i) cecal ligation and puncture (CLP) after renal injury including 5/6 nephrectomy (5/6Nx-CLP) and bilateral nephrectomy (BiNx-CLP) or sham surgery (sham-CLP) and (ii) lipopolysaccharide (LPS) injection, along with testing in macrophages. In patients, the data of EPO administration and the disease characteristics in patients with sepsis-induced acute kidney injury (sepsis-AKI) were evaluated. As such, increased endogenous EPO was demonstrated in all sepsis models, including BiNx-CLP despite the reduced liver erythropoietin receptor (EPOR), using Western blot analysis and gene expression, in liver (partly through hepatocyte apoptosis). A high-dose EPO, but not a low-dose, attenuated sepsis in mouse models as determined by mortality and serum inflammatory cytokines. Furthermore, EPO attenuated inflammatory responses in LPS-activated macrophages as determined by supernatant cytokines and the expression of several inflammatory genes (

Topics: Aged; Animals; Bacteremia; Cytokines; Disease Models, Animal; Drug Repositioning; Erythropoietin; Female; Gene Expression Regulation; Hep G2 Cells; Humans; Inflammation Mediators; Kidney; Lipopolysaccharides; Liver; Macrophage Activation; Male; Mice; Mice, Inbred C57BL; Middle Aged; Proportional Hazards Models; RAW 264.7 Cells; Receptors, Erythropoietin; Sepsis; Treatment Outcome

Although erythropoiesis-stimulating agents (ESAs) exert renoprotective effects in renal disease models, it has not been revealed whether the prolonged duration of action of ESAs contributes to their renoprotective effects.. We examined whether the prolonged duration of ESAs' action contributes to their renoprotective effects by comparing a divided administration of a short-acting ESA, epoetin beta (EPO), or a single administration of a long-acting ESA, epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.), to a single administration of EPO in chronic glomerulonephritis (GN) rats.. Chronic GN was induced by intravenous injection of anti-Thy 1.1 antibody (0.6 mg/kg) into uninephrectomized rats (day 0). Chronic GN rats were intravenously injected once with vehicle (disease control; DC), EPO 5,000 IU/kg (single EPO), or C.E.R.A. 25 μg/kg (single C.E.R.A.) on day 1; or 3 times during the first week with EPO 1,667 IU/kg from day 1 (divided EPO; total 5,000 IU/kg). Hemoglobin (Hb) level and urinary total protein (U-TP) level which are the indexes of hematopoiesis and renoprotective effects, respectively, were measured several times over 8 weeks.. Divided EPO and single C.E.R.A. increased Hb levels more greatly than did single EPO. In all chronic GN rats, elevated U-TP levels decreased transiently 2 weeks after chronic GN induction and then flared again. Single EPO significantly suppressed this exacerbation of U-TP levels compared to DC. Divided EPO and single C.E.R.A. each significantly suppressed the exacerbation of U-TP levels compared to single EPO.. Prolonged duration of ESAs' action contributed significantly to their renoprotective effects.

Topics: Anemia; Animals; Disease Models, Animal; Disease Progression; Drug Administration Schedule; Erythropoiesis; Erythropoietin; Glomerulonephritis; Hematinics; Hemoglobins; Hypoxia; Injections, Intravenous; Iron; Isoantibodies; Kidney; Male; Polyethylene Glycols; Protective Agents; Proteinuria; Rats, Inbred F344; Recombinant Proteins

Silkworm faeces are the dry faeces of the insect Bombyx mori (Linnaeus) and have historically been used in traditional Chinese medicine to treat blood deficiency and rheumatic pain. Silkworm faeces extract (SFE) is derived from silkworm faeces.. Clinical observations of patients in the Department of Nephrology have shown that SFE effectively improves renal anaemia. However, the molecular mechanism remains unclear. This article mainly explores the regulatory effects of SFE on erythropoietin (EPO) and hepcidin to identify the molecular mechanism of SFE.. A rat model of renal anaemia was established by feeding rats food containing 0.75% adenine. SFE was orally administered to the rats, while recombinant human erythropoietin (rhEPO) was used as a positive control drug. Haematological parameters and inflammation levels were compared between rats from each group, and pathological kidney sections from each rat were observed. The serum EPO and hepcidin levels were detected using enzyme-linked immunosorbent assay (ELISA) kits, while Western blot analyses were performed to detect the levels of proteins involved in the EPO-related hypoxia-inducible factor 2α (HIF-2α)/prolyl hydroxylase 2 (PHD2) signalling pathway and hepcidin-related BMP6/SMAD4 and interleukin-6 (IL-6)/STAT3 signalling pathways.. SFE significantly ameliorated haematological parameters, renal function, and inflammation levels in the rats. A mechanistic study showed that SFE promoted EPO expression by upregulating HIF-2α expression and inhibiting the expression of NF-κB and GATA2 both in vivo and in vitro. In particular, SFE inhibited PHD2 expression, resulting in a decrease in the enzymatic reaction of HIF-2α to increase EPO expression. Furthermore, SFE inhibited hepcidin expression by blocking the BMP6/SMAD4 and IL-6/STAT3 pathways.. SFE regulated iron metabolism by inhibiting hepcidin and simultaneously promoted EPO synthesis to improve renal anaemia in rats.

Topics: Adenine; Anemia; Animals; Bombyx; Disease Models, Animal; Erythropoietin; Feces; Hepcidins; Humans; Iron; Kidney Diseases; Male; Rats; Rats, Sprague-Dawley

Apoptosis that occurs after hypoxia/reoxygenation (H/R) has an important role in the pathogenesis of necrotizing enterocolitis (NEC). Telomerase activity, showing the regeneration capacity, may also be important in the recovery process. Therefore, we aimed to investigate the effects of insulin-like growth factor-1 (IGF-1) and erythropoietin (EPO) on apoptosis and telomerase activity in an H/R model.. Young mice were divided into four groups each containing ten Balb/c mice. Group 1 (H/R) were exposed to H/R; group 2 and group 3 were pretreated with IGF-1 and EPO, respectively, for 7 days before H/R. Group 4 served as control. Intestinal injury was evaluated by histological scoring and assessment of apoptosis was performed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) test. Proapoptotic and antiapoptotic gene expressions and telomerase activity were analyzed by real-time PCR.. IGF-1- and EPO-treated animals had decreased histological damage and apoptosis, confirmed by TUNEL test and caspase activity. Telomerase activity was increased in these animals in addition to increased expression of antiapoptotic genes. However, proapoptotic gene expressions were not statistically different.. The protective effects of IGF-1 and EPO in H/R damage may be through increased expression of antiapoptotic genes and increased telomerase activity, especially for IGF-1.. This is a comprehensive study measuring various variables, namely IGF-1, EPO, apoptosis, apoptotic and antiapoptotic genes, and telomerase activity in the NEC model. The intestinal protective effects of IGF-1 and EPO in H/R damage may occur through increased expression of antiapoptotic genes and increased telomerase activity. To the best of our knowledge, telomerase activity has not been investigated in the NEC model before. Regarding our results, novel strategies may be implemented for the early definitive diagnosis, robust preventive measures, and effective treatment modalities for NEC.

Topics: Animals; Apoptosis; Disease Models, Animal; Enterocolitis, Necrotizing; Erythropoietin; Insulin-Like Growth Factor I; Mice; Mice, Inbred BALB C; Telomerase

Hypoxia-inducible factors (HIFs) play important roles in the pathogenesis of erythrocytosis in chronic mountain sickness (CMS). von Hippel-Lindau (VHL) is a key regulator of hypoxia that can direct the poly-ubiquitylation and degradation of HIFs. Epigenetic mechanisms are believed to contribute toward adaption to chronic hypoxia. Here, we investigated the contribution and mechanism of VHL methylation in rats with erythrocytosis in CMS.. The methylation status of VHL was measured via bisulfite sequencing PCR, while VHL, DNMT1, DNMT3α, and DNMT3β expression were assessed using real-time reverse transcription PCR and western blotting. HIF-2α and EPO expression levels in bone marrow were determined via immunohistochemical staining, and erythroid hyperplasia in bone marrow sections were observed with hematoxylin and eosin staining.. We found that chronic hypoxia triggered erythroid hyperplasia in the bone marrow and increased the quantity of peripheral red blood cells in CMS rats. Chronic hypoxia significantly induced methylation at the CpG site in the VHL promoter, decreased VHL expression, and increased HIF-2α and EPO expression. Chronic hypoxia increased DNMT3α and DNMT3β expression, consistent with the decrease in VHL expression. The DNA methyltransferase inhibitor 5-azacytidine reduced chronic hypoxia-induced erythroid proliferation in the bone marrow of rats with CMS by suppressing VHL methylation and DNMTs expression.. Our study suggests that VHL methylation contributes toward excessive erythrocytosis in CMS by upregulating the HIF-2α/EPO pathway in the bone marrow of rats. We demonstrated that the DNMT inhibitor 5-azacytidine can attenuate erythroid hyperplasia in the bone marrow by demethylating the VHL promoter.

Topics: Altitude Sickness; Animals; Basic Helix-Loop-Helix Transcription Factors; Chronic Disease; Disease Models, Animal; DNA Methylation; Erythropoietin; Gene Expression Regulation; Hypoxia; Male; Polycythemia; Rats; Rats, Sprague-Dawley; Von Hippel-Lindau Tumor Suppressor Protein

Dysmotility and postoperative ileus (POI) are major clinical problems after surgical trauma and it is associated with increased intestinal inflammation and oxidative stress. Despite the high occurrence of POI following intra-abdominal surgeries, no effective treatment is currently available. Erythropoietin (EPO) is a multifunctional tissue-protective cytokine with potent anti-inflammatory and anti-oxidative properties, and it is an FDA approved medicine for clinical use. While both EPO and EPO receptors (EPOR) are widely expressed in the gut, the role of EPO in POI is largely unknown. This study was designed to explore the possible beneficial effect of EPO in a mouse model of POI.. Mice were subjected to intestinal manipulation to induce standard POI and intestinal transit time was determined at 24-h post-injury with or without EPO treatment (5000 units/kg, once, IP, immediately after intestinal trauma). Intestinal samples were harvested for histological and immunohistochemical analysis.. Systemic EPO significantly improved intestinal transit time compared with control group and it was associated with significantly increased levels of tissue macrophages and reduced levels of oxidative stress.. This is the first pre-clinical study to document novel beneficial effects of EPO in gut dysmotility and our findings suggest that the beneficial effects of EPO in POI is predominantly mediated by its anti-oxidative and immunomodulatory properties.

Topics: Animals; Disease Models, Animal; Erythropoietin; Gastrointestinal Motility; Intestinal Pseudo-Obstruction; Male; Mice; Mice, Inbred C57BL; Postoperative Complications; Recovery of Function

Prominent activation of microglial immune/inflammatory processes is a characteristic feature of brains of patients with tauopathies including Alzheimer's disease (AD), suggesting that neuroinflammation may be a critical factor in their pathogenesis. Strategies aimed at developing new therapeutics for tauopathies based on anti-inflammation or immunomodulation are likely to be promising avenues of research. We previously developed JM4-a 19'mer cyclic peptide derived from the first loop of human erythropoietin. This peptide possesses beneficial immune modulatory and tissue protective effects while lacking the undesirable side effects of full-length erythropoietin. In this preclinical study, we investigated the effect of chronic JM4 treatment on the PS19 mouse that carries the P301S mutant human tau gene, linked to a form of frontotemporal dementia. This transgenic mouse has been widely used as a model of tauopathies including AD and related dementias.. Daily subcutaneous treatment of female PS19 mice with JM4 was initiated before disease onset and continued on for the animals' lifespan. The progression of neurological deficit and the lifespan of these mice were assessed. To evaluate the effect of JM4 treatment on cognition of these animals, the PS19 mice underwent Barnes maze test and elevated plus maze test. In addition, neuronal loss, phosphorylated tau aggregation, and microglial activation were assessed using immunohistochemistry of PS19 mouse brain sections.. JM4 treatment of PS19 mice initiated before disease onset reduced neurological deficit, prolonged lifespan, and rescued memory impairment. The beneficial effects of JM4 were accompanied by reductions in neuronal loss, phosphorylated tau aggregation, and microglial activation in the PS19 mouse brain.. Use of a single dose of JM4 and female mice only.. JM4 is a potential novel therapeutic agent for the treatment of tauopathies including AD and related dementias.

Topics: Animals; Brain; Disease Models, Animal; Erythropoietin; Female; Humans; Mice; Mice, Transgenic; tau Proteins; Tauopathies

Erythrocyte mass contributes to maintaining systemic oxygen delivery and blood viscosity, with the latter being one of the determinants of blood pressure. However, the physiological response to blood pressure changes under anaemic conditions remain unknown.. We show that anaemia decreases blood pressure in human patients and mouse models. Analyses of pathways related to blood pressure regulation demonstrate that anaemia enhances the expression of the gene encoding the vasopressor substance renin in kidneys. Although kidney juxtaglomerular cells are known to continuously produce renin, renal interstitial fibroblasts are identified in the present study as a novel site of renin induction under anaemic hypotensive conditions in mice and rats. Notably, some renal interstitial fibroblasts are found to simultaneously express renin and the erythroid growth factor erythropoietin in the anaemic mouse kidney. Antihypertensive agents but not hypoxic stimuli induced interstitial renin expression, suggesting that blood pressure reduction triggers interstitial renin induction in anaemic mice. The interstitial renin expression was also detected in injured fibrotic kidneys of the mouse and human, and the renin-expressing interstitial cells in murine fibrotic kidneys were identified as myofibroblasts originating from renal interstitial fibroblasts. Since the elevated expression levels of renin in fibrotic kidneys along with progression of renal fibrosis were well correlated to the systemic blood pressure increase, the renal interstitial renin production seemed to affect systemic blood pressure.. Renal interstitial fibroblasts function as central controllers of systemic oxygen delivery by producing both renin and erythropoietin.. Grants-in-Aid from Japan Society for the Promotion of Science (JSPS) KAKENHI (17K19680, 15H04691, and 26111002) and the Takeda Science Foundation.

Topics: Aged; Anemia; Animals; Biomarkers; Blood Pressure; Chronic Disease; Disease Models, Animal; Erythropoietin; Female; Fibroblasts; Fibrosis; Gene Expression; Humans; Hypotension; Hypoxia; Kidney; Kidney Diseases; Male; Mice; Mice, Knockout; Mice, Transgenic; Middle Aged; Renin; Signal Transduction

Topics: Animals; Animals, Newborn; Area Under Curve; Biomarkers; Chemokines; Cytokines; Disease Models, Animal; Erythropoietin; Female; Hypothermia, Induced; Hypoxia-Ischemia, Brain; Intercellular Signaling Peptides and Proteins; Macaca nemestrina; Monocyte Chemoattractant Proteins; Pregnancy; ROC Curve; Severity of Illness Index

The high mortality rate associated with acute kidney injury (AKI) is commonly due to progressive, inflammatory multiple organ dysfunction, which often involves neurological complications. The AKI-stimulated mechanisms leading to brain dysfunction are not well understood, which hinders development of new therapeutic avenues to minimize AKI-mediated neural effects. The hippocampal CA1 area is a particularly vulnerable region during AKI but the electrophysiological and inflammatory mechanisms involved in this vulnerability remain largely unknown. Here, we used immunohistochemistry to quantitatively investigate the number of astrocytes expressing glial fibrillary acidic protein (GFAP) as an indicator of inflammation, and whole cell patch clamp to evaluate electrophysiological changes in CA1 at different time points following induction of bilateral renal ischemia (BRI) in male Wistar rats. Further we evaluated the effectiveness of erythropoietin (EPO, 1000 U/kg i.p.) in mitigating BRI-associated changes. Plasma concentrations of blood urea nitrogen (BUN) were significantly enhanced at 24 h, 72 h and 1 week, and creatinine (Cr) was increased at 24 h after reperfusion, which were changes reduced by EPO. BRI led to an increase in CA1 GFAP-positive cells 24 h and 72 h, but not 1 week, after reperfusion, and EPO reversed this effect of BRI at 24 h. Additionally, BRI caused an increase in the peak amplitude and coefficient of variation of CA1 pyramidal neuronal action potentials, which were changes not seen in presence of EPO. When taken together, altered neuronal electrophysiological properties and astrogliosis could contribute to the neurological complications induced by AKI, and EPO offers hope as a potential neuroprotective agent.

Topics: Acute Kidney Injury; Animals; Astrocytes; Blood Urea Nitrogen; CA1 Region, Hippocampal; Creatinine; Disease Models, Animal; Erythropoietin; Ischemia; Kidney; Male; Neurons; Neuroprotective Agents; Rats; Rats, Wistar

Macrophages play an important role in the pathogenesis of systemic lupus erythematosus-associated diffuse alveolar hemorrhage (DAH). The immunomodulation of macrophage responses might be a potential approach for the prevention and treatment of DAH. Erythropoietin (EPO) could regulate macrophage bioactivities by binding to the EPO receptor expressing on macrophages. This study assessed the effects of EPO on DAH protection using an immune-mediated DAH murine model with macrophages as the major contributor. A DAH murine model was established in female C57BL/6 mice by an i.p. injection of pristane. We found that EPO administration alleviates DAH by reducing pulmonary macrophages recruitment and promoting phenotype switch toward M2 macrophages in vivo. EPO drove macrophages to the anti-inflammatory phenotype in the primary murine bone marrow-derived macrophages and macrophages cell line RAW 264.7 with LPS, IFN-γ, and IL-4 in vitro. Moreover, EPO treatment increases the expression of EPOR and decreases the expression of miR-494-3p, resulting in increased phosphorylation of JAK2 and STAT3. In conclusion, EPO can be a potential therapeutic agent in DAH by reducing cell apoptosis and regulating macrophage polarization through the EPOR/JAK2/STAT3 axis. Further studies are also needed to validate the direct target of miR-494-3p in regulating JAK2/STAT3 signaling transduction.

Topics: Animals; Cell Differentiation; Disease Models, Animal; Erythropoietin; Female; Hemorrhage; Humans; Janus Kinase 2; Lung Diseases; Macrophages, Alveolar; Mice; Mice, Inbred C57BL; Pulmonary Alveoli; RAW 264.7 Cells; Receptors, Erythropoietin; Signal Transduction; STAT3 Transcription Factor; Terpenes

Endogenous mechanisms underlying bacterial infection resolution are essential for the development of novel therapies for the treatment of inflammation caused by infection without unwanted side effects. Herein, we found that erythropoietin (EPO) promoted the resolution and enhanced antibiotic actions in

Topics: Animals; Anti-Bacterial Agents; Disease Models, Animal; Disease Susceptibility; Drug Resistance, Bacterial; Erythropoietin; Escherichia coli; Escherichia coli Infections; Host-Pathogen Interactions; Macrophages; Mice; Peritonitis; Phagocytosis; PPAR gamma; Signal Transduction; Staphylococcal Infections; Staphylococcus aureus

Antibody-mediated rejection (AMR) represents a major cause of allograft dysfunction and results in allograft failure in solid organ transplantation. Cyclic helix B peptide (CHBP) is a novel erythropoietin-derived peptide that ameliorated renal allograft rejection in a renal transplantation model. However, its effect on AMR remains unknown. This study aimed to investigate the effect of CHBP on AMR using a secondary allogeneic skin transplantation model, which was created by transplanting skin from BALB/c mice to C57BL/6 mice with or without CHBP treatment. A secondary syngeneic skin transplantation model, involving transplantation from C57BL/6 mice to C57BL/6 mice, was also created to act as a control. Skin graft rejection, CD19

Topics: Allografts; Animals; B-Lymphocytes; Disease Models, Animal; Erythropoietin; Germinal Center; Graft Rejection; Graft Survival; Immunohistochemistry; Immunophenotyping; Isoantibodies; Male; Mice; Peptide Fragments; Peptides, Cyclic; Plasma Cells; Skin Transplantation; Spleen; Transplantation, Homologous

The number of patients with end stage kidney disease (ESKD) are increasing world-side. While interstitial fibrosis (IF) is a common step for the progression to ESKD, therapeutic options for IF is still limited in clinical settings. We have reported that bone marrow-derived fibrotic cell, fibrocyte, is involved in the pathogenesis of kidney fibrosis. Also recent studies revealed that erythropoietin has protective effect on kidney diseases. However, it is unknown whether erythropoietin (EPO) inhibits fibrosis in progressive kidney injury. Therefore, we explored the impacts of EPO on kidney fibrosis with focusing on fibrocyte.. Fibrocyte was differentiated from peripheral mononuclear cells of healthy donor. Fibrocyte was stimulated with transforming growth factor beta (TGF)-β with/without EPO treatment. Moreover, the therapeutic effect of EPO was evaluated in murine unilateral ureteral obstruction (UUO) model.. TGF-β stimulation increased the expression of COL1 mRNA in fibrocyte. EPO signal reduced the expression of COL1 mRNA in dose dependent manner. EPO reduced mitochondrial oxidative stress and ameliorated mitochondrial membrane depolarization induced by TGF-β stimulation. Moreover, EPO reduced the mRNA expression of mitochondria related molecules, TRAF6, in fibrocyte. In addition, the count of CD45+/αSMA + double-positive fibrocyte was decreased in the EPO-administered UUO kidneys.. EPO signals function to prevent kidney fibrosis, particularly in fibrocyte. Regulating the renal accumulation of fibrocyte is a part of the anti-fibrotic functions of EPO.

Topics: Animals; Bone Marrow Cells; Cells, Cultured; Collagen; Disease Models, Animal; Disease Progression; Erythropoietin; Fibrosis; Humans; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; RNA, Messenger; Transforming Growth Factor beta

Objective: Roxadustat is a new medication for the treatment of renal anemia. EPO (erythropoietin)-the current treatment standard-has been reported to enhance platelet activation and production. However, to date, the effect of roxadustat on platelets is unclear. To address this deficiency, herein, we have evaluated the effect of roxadustat on platelet production and function.\ \ Approach and Results: We performed several mouse platelet functional assays in the presence/absence of in vitro and in vivo roxadustat treatment. Both healthy and 5/6 nephrectomized mice were utilized. The effect of roxadustat on platelet function of healthy volunteers and chronic kidney disease patients was also evaluated. For platelet production, megakaryocyte maturation and proplatelet formation were assayed in vitro. Peripheral platelet and bone marrow megakaryocyte counts were also determined. We found that roxadustat could not stimulate washed platelets directly, and platelet aggregation, spreading, clot retraction, and P-selectin/JON/A exposure were similar with or without in vitro or in vivo roxadustat treatment among both healthy and 5/6 nephrectomized mice. In vivo mouse thrombosis models were additionally performed, and no differences were detected between the vehicle and roxadustat treatment groups. EPO, which was considered a positive control in the present study, promoted platelet function and production as reported previously. Megakaryocyte maturation and proplatelet formation were also not significantly different between control mice and those treated with roxadustat. After receiving roxadustat for 14 days, no difference in the peripheral platelet count was observed in the mice.\ \ Conclusions: Administration of roxadustat has no significant impact on platelet production and function.

Topics: Animals; Blood Coagulation; Blood Platelets; Case-Control Studies; Disease Models, Animal; Erythropoietin; Glycine; Hematinics; Humans; Isoquinolines; Male; Mice, Inbred C57BL; Platelet Activation; Renal Insufficiency, Chronic; Thrombopoiesis; Thrombosis

Renal ischemia-reperfusion (IR) injury and cyclosporine A (CsA) nephrotoxicity affect allograft function and survival. The prolonged effects and underlying mechanisms of erythropoietin derived cyclic helix B peptide (CHBP) and/or caspase-3 small interfering RNA (CASP-3siRNA) were investigated in mouse kidneys, as well as kidney epithelial cells (TCMK-1), subjected to transplant-related injuries. Bilateral renal pedicles were clamped for 30 min followed by reperfusion for 2 and 8 weeks, with/without 35 mg/kg CsA gavage daily and/or 24 nmol/kg CHBP intraperitoneal injection every 3 days. The ratio of urinary albumin to creatinine was raised by IR injury, further increased by CsA and lowered by CHBP at 2, 4, 6 and 8 weeks, whereas the level of SCr was not significantly affected. Similar change trends were revealed in tubulointerstitial damage and fibrosis, HMGB1 and active CASP-3 protein. Increased apoptotic cells in IR kidneys were decreased by CsA and CHBP at 2 and/or 8 weeks. p70 S6 kinase and mTOR were reduced by CsA with/without CHBP at 2 weeks, so were S6 ribosomal protein and GSK-3β at 8 weeks, with reduced CASP-3 at both time points. CASP-3 was further decreased by CHBP in IR or IR + CsA kidneys at 2 or 8 weeks. Furthermore, in TCMK-1 cells CsA induced apoptosis was decreased by CHBP and/or CASP-3siRNA treatment. Taken together, CHBP predominantly protects kidneys against IR injury at 2 weeks and/or CsA nephrotoxicity at 8 weeks, with different underlying mechanisms. Urinary albumin/creatinine is a good biomarker in monitoring the progression of transplant-related injuries. CsA divergently affects apoptosis in kidneys and cultured kidney epithelial cells, in which CHBP and/or CASP-3siRNA reduces inflammation and apoptosis.

Topics: Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Cell Line; Cyclosporine; Disease Models, Animal; Erythropoietin; Kidney; Macrophages; Male; Mice; Mice, Inbred BALB C; Peptide Fragments; Peptides, Cyclic; Reperfusion Injury; RNA, Messenger; RNA, Small Interfering

Topics: Animals; Cell Culture Techniques; Disease Models, Animal; Erythropoietin; Fetal Growth Retardation; Fetal Weight; Fetus; Hep G2 Cells; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; In Vitro Techniques; Insulin-Like Growth Factor Binding Protein 1; Mechanistic Target of Rapamycin Complex 1; Microscopy, Fluorescence; Organ Size; Papio; Phosphorylation; Protein Kinase C-alpha; Receptors, Erythropoietin; Transcription Factors; Vascular Endothelial Growth Factor A

Our laboratory has demonstrated that captopril, an angiotensin converting enzyme inhibitor, mitigates hematopoietic injury following total body irradiation in mice. Improved survival in mice is correlated with improved recovery of mature blood cells and bone marrow, reduction of radiation-induced inflammation, and suppression of radiation coagulopathy. Here we investigated the effects of captopril treatment against radiation injuries in the Göttingen mini pig model of Hematopoietic-Acute Radiation Syndrome (H-ARS). Minipigs were given captopril orally (0.96 mg/kg) twice daily for 12 days following total body irradiation (60Co 1.79 Gy, 0.42-0.48 Gy/min). Blood was drawn over a time course following irradiation, and tissue samples were collected at euthanasia (32-35 days post-irradiation). We observed improved survival with captopril treatment, with survival rates of 62.5% in vehicle treated and 87.5% in captopril treated group. Additionally, captopril significantly improved recovery of peripheral blood mononuclear cells, and a trend toward improvement in recovery of red blood cells and platelets. Captopril significantly reduced radiation-induced expression of cytokines erythropoietin and granulocyte-macrophage colony-stimulating factor and suppressed radiation-induced acute-phase inflammatory response cytokine serum amyloid protein A. Using quantitative-RT-PCR to monitor bone marrow recovery, we observed significant suppression of radiation-induced expression of redox stress genes and improved hematopoietic cytokine expression. Our findings suggest that captopril activities in the Göttingen minipig model of hematopoietic-acute radiation syndrome reflect findings in the murine model.

Topics: Acute Radiation Syndrome; Animals; Captopril; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic System; Humans; Leukocytes, Mononuclear; Mice; Oxidation-Reduction; Radiation Injuries, Experimental; Swine; Swine, Miniature; Whole-Body Irradiation

Therapeutic neovascularization has some obstacles, such as it requires more than one proangiogenic factor, and these factors have short half-lives. To overcome these obstacles, combined delivery of granulocyte-colony stimulating factor (G-CSF), erythropoietin (EPO) and vascular endothelial growth factor (VEGF) using protein/dextran/poly (lactic-co-glycolic acid) (PLGA) sustained-release microspheres was proposed to promote neovascularization.. Dextran microparticles loaded with G-CSF, EPO or VEGF were prepared and encapsulated in PLGA microspheres to obtain protein-dextran-PLGA microspheres. The release behavior of microspheres was studied in vitro. The protein/dextran/PLGA microspheres were injected into the ischemic hindlimbs of rats. Neovascularization in ischemic muscle was measured.. Microspheres released G-CSF, EPO and VEGF in vitro for more than 4 weeks. Combined therapy with VEGF, EPO and G-CSF promoted the expression of B-cell lymphoma-2 and stromal cell-derived factor 1, cellular proliferation and the incorporation of C-X-C chemokine receptor 4 positive cells. Capillary density and smooth muscle α-actin+ vessel density were higher in the combined treatment of VEGF, EPO and G-CSF than in the single factor treatment.. The combined and sustained delivery of VEGF, EPO and G-CSF using dextran-PLGA microspheres had a more significant neovascularization effect than monotherapy with each factor alone. This combined therapy might be a promising treatment for ischemic vascular diseases.

Topics: Angiogenesis Inducing Agents; Animals; Cell Proliferation; Delayed-Action Preparations; Dextrans; Disease Models, Animal; Drug Carriers; Drug Compounding; Drug Liberation; Erythropoietin; Granulocyte Colony-Stimulating Factor; Hindlimb; Injections, Intramuscular; Ischemia; Kinetics; Male; Microspheres; Muscle, Skeletal; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neovascularization, Physiologic; Particle Size; Polyesters; Rats, Sprague-Dawley; Vascular Endothelial Growth Factor A

In the setting of type-2 diabetes, there are declines of structural stability and functionality of blood capillaries and red blood cells (RBCs), increasing the risk for microcirculatory disturbances. Correcting hyperglycemia is not entirely effective at reestablishing normal cellular metabolism and function. Therefore, identification of pathological changes occurring before the development of overt hyperglycemia may lead to novel therapeutic targets for reducing the risk of microvascular dysfunction. Here we determine whether RBC-capillary interactions are altered by prediabetic hypersecretion of amylin, an amyloid forming hormone co-synthesized with insulin, and is reversed by endothelial cell-secreted epoxyeicosatrienoic acids. In patients, we found amylin deposition in RBCs in association with type-2 diabetes, heart failure, cancer and stroke. Amylin-coated RBCs have altered shape and reduced functional (non-glycated) hemoglobin. Amylin-coated RBCs administered intravenously in control rats upregulated erythropoietin and renal arginase expression and activity. We also found that diabetic rats expressing amyloid-forming human amylin in the pancreas (the HIP rat model) have increased tissue levels of hypoxia-inducible transcription factors, compared to diabetic rats that express non-amyloid forming rat amylin (the UCD rat model). Upregulation of erythropoietin correlated with lower hematocrit in the HIP model indicating pathologic erythropoiesis. In the HIP model, pharmacological upregulation of endogenous epoxyeicosatrienoic acids protected the renal microvasculature against amylin deposition and also reduced renal accumulation of HIFs. Thus, prediabetes induces dysregulation of amylin homeostasis and promotes amylin deposition in RBCs and the microvasculature altering RBC-capillary interaction leading to activation of hypoxia signaling pathways and pathologic erythropoiesis. Hence, dysregulation of amylin homeostasis could be a therapeutic target for ameliorating diabetic vascular complications.

Topics: Adult; Amyloid; Animals; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Disease Models, Animal; Eicosanoids; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Humans; Islet Amyloid Polypeptide; Kidney; Male; Microcirculation; Microvessels; Middle Aged; Rats; Retrospective Studies

Helix B-Surface peptide (HBSP) is the latest discovered erythropoietin (EPO) analogue that can retain the activity of EPO. EPO, which is widely used for treating renal anemia, has recently been proved to have protective effects on ischemia-reperfusion injury of brain, heart and kidney. The protective effects of EPO and HBSP on cardiac function were found in rats with myocardial ischemia. However, the effect of HBSP on sepsis-induced renal injury is still unclear.. Establishment of rat kidney injury model and treated with HBSP and lipoposaccharide. Renal injury in rats was observed by hematoxylin-eosin staining and injury index score. Levels of serum creatinine (SCr), blood urea nitrogen (BUN) and Cystatin C (Cys C) were detected using fully automatic biochemical analyzer, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β were detected by enzyme-linked immunosorbent assay. Western blot analysis was performed to determine the role of HBSP in phosphatidylinositol 3-kinase (PI3K)/Akt pathway.. Acute kidney injury (AKI) appeared after modeling, however, HBSP alleviated the pathological conditions of the kidney injury. In addition, HBSP lowered kidney injury index score in the rats, and decreased the levels of SCr, BUN, Cys C, TNF-α, IL-6 and IL-1β, moreover, HBSP also showed the effect of activating PI3K/Akt pathway.. HBSP alleviated lipoposaccharide-induced AKI and improved kidney function of the rats with sepsis. More importantly, the effects of HBSP on lipoposaccharid-induced AKI were realized via activating PI3K/Akt pathway. The findings in the current study provide new insights into the therapeutic mechanism for treating the disease.

Topics: Acute Kidney Injury; Animals; Disease Models, Animal; Erythropoietin; Kidney; Peptide Fragments; Phosphatidylinositol 3-Kinase; Protective Agents; Proto-Oncogene Proteins c-akt; Rats; Sepsis; Signal Transduction; Treatment Outcome

The present study was designed to investigate the effect of topical erythropoietin on the healing process of induced necrotizing scleritis and to evaluate the ocular side effects of this treatment modality in a rabbit model. Necrotizing scleritis was induced in 8 New Zealand albino rabbits. The animals were then randomly divided into one of two groups: a treated group administered a topical erythropoietin-containing cellulose-based gel every 8 h or a control group treated with a cellulose-based gel without erythropoietin every 8 h. The sizes of the lesions measured at different time points were compared between the groups. After three months, the rabbits' eyes were enucleated and histologically and immunohistochemically evaluated for angiogenesis and apoptosis. The lesions were completely vascularized in all eyes of the treated group and 50% of eyes of the control group. The mean interval from the induction of scleral necrosis to a complete improvement was 28 days in the treated group and 62.5 days in the control group (P = 0.04). Histological examination revealed that erythropoietin enhanced the improvement of necrotizing scleritis by stimulating angiogenesis and reducing apoptosis. Neovascularization of the cornea, iris, or retina was not observed in the treated group. We observed a significantly faster recovery to complete improvement of necrotizing scleritis in rabbit eyes treated with erythropoietin compared to those of the control group. Treated eyes had a higher rate of complete healing and had no ocular safety concerns. This therapeutic modality represents a promising treatment for scleral necrosis following various types of ocular surgery.

Topics: Administration, Ophthalmic; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Apoptosis; Disease Models, Animal; Erythropoietin; Fluorescent Antibody Technique, Indirect; In Situ Nick-End Labeling; Leukocyte Common Antigens; Male; Platelet Endothelial Cell Adhesion Molecule-1; Rabbits; Recombinant Proteins; Sclera; Scleritis; Wound Healing

Clinical studies have shown that treatment with inhibitors of sodium-glucose cotransporter 2 (SGLT2) significantly increases the hematocrit in patients with type 2 diabetes. To investigate whether SGLT2 inhibitors directly promote erythropoietin production independently on blood glucose reduction, the hematopoietic effect of the specific SGLT2 inhibitor, luseogliflozin, was examined in non-diabetic rats with renal anemia.. Renal anemia was induced by treatment with adenine (200 or 600 mg/kg/day, orally for 10 days) in non-diabetic Wistar-Kyoto or Wistar rats, respectively. Luseogliflozin (10 mg/kg bodyweight) or vehicle (0.5% carboxymethyl cellulose) was then administered for 6 weeks. The hematocrit and the hemoglobin (Hb), blood urea nitrogen, plasma creatinine, and plasma erythropoietin levels were monitored.. Treatment with adenine decreased the hematocrit and the Hb level, which were associated with increases in the blood urea nitrogen and plasma creatinine levels. In Wistar-Kyoto rats treated with 200 mg/kg/day adenine, administration of luseogliflozin induced glycosuria, but did not change the blood urea nitrogen, plasma creatinine levels, hematocrit, Hb or plasma erythropoietin levels. Similarly, luseogliflozin treatment failed to change the hematocrit or the Hb levels in Wistar rats with renal anemia induced by 600 mg/kg/day of adenine. Plasma erythropoietin concentrations were also not different between luseogliflozin- and vehicle-treated rats. Similarly, in human erythropoietin-producing cells derived from pluripotent stem cells, luseogliflozin treatment did not change the erythropoietin level in the medium.. These data suggest that SGLT2 inhibitor fails to exert hematopoietic effects in non-diabetic conditions.

Topics: Adenine; Anemia; Animals; Disease Models, Animal; Erythropoietin; Hematocrit; Hematopoietic Stem Cells; Hemoglobins; Humans; Kidney; Male; Rats; Rats, Inbred WKY; Rats, Wistar; Renal Insufficiency; Sodium-Glucose Transporter 2 Inhibitors; Sorbitol

Although it is accepted that prolonged and repeated seizures can cause epileptogenesis, memory deficits and neuronal death, the precise relation between epileptic seizures and neuronal death remains unclear. Erythropoietin (EPO) exhibits neuroprotective and anti-epileptic effects. We investigated the effect of a single pentylentetrazole (PTZ) induced tonic-clonic seizure on the pyramidal neurons of the cornu ammonis 1 (CA1) and CA3 regions of hippocampus. We also investigated the effects of EPO on seizure, memory and on brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase-B, sirtuin-1 (SIRT1), which are important for memory. Forty male rats were divided into four groups: control, saline treated, single 60 mg/kg dose PTZ treated, 3000 IU/kg EPO treated, and 3000 IU/kg EPO treated 24 h before PTZ administration. Seizure latency and severity were assessed following PTZ injection. A passive avoidance test was performed 24 h after seizure. BDNF, TrkB and SIRT1 levels were measured in serum, hippocampus and cortex. The hippocampus was examined histologically, and neuronal nuclear antigen (NeuN) was investigated using immunohistochemistry. EPO pretreatment decreased seizure severity and prolonged seizure latency. Single dose PTZ-induced seizures did not affect memory. Numbers of cells in the CA1 region did not change, although the number of dark stained neuron increased. Both total cell numbers and percentage of dark stained cells were elevated in the CA3 region following PTZ induced seizures. EPO pretreatment decreased the number of dark cells in both CA1 and CA3 regions and the number of cells in the CA3 region. NeuN labeling was unchanged in the CA1 and CA3 regions in the PTZ group; however, EPO pretreatment increased NeuN labeling in the CA3 region. Although EPO exhibited an anticonvulsive effect, single dose EPO pretreatment did not affect memory in either animals not exposed to PTZ or animals that had been subjected to PTZ-induced seizures. EPO pretreatment prolonged seizure latency and reduced seizure severity after PTZ-induced seizures. The anti-seizure and neuroprotective effects of EPO pretreatment may be due to the protection of CA1 and CA3 neurons, possibly owing to SIRT1 and BDNF activity.

Topics: Animals; Disease Models, Animal; Erythropoietin; Hippocampus; Male; Memory; Neurons; Neuroprotective Agents; Pentylenetetrazole; Rats; Seizures

Intracerebroventricular (icv) administration of streptozotocin (STZ) has been used as a metabolic model of sporadic Alzheimer's disease (AD). Erythropoietin (EPO) possesses neuroprotective and memory-improving effects, which might be advantageous in treating different characteristics of AD. Nevertheless, the hematopoietic effect of EPO has hindered its application as a neuroprotective agent. Previous studies have shown that a new Epo derivative called carbamylated Erythropoietin-Fc (CEPO-Fc), yield noticeable neuroprotective effects without affecting hematopoiesis. In this study, the neuroprotective effects of CEPO-Fc on icv-STZ induced memory impairment and hippocampal apoptosis were examined. Adult male Wistar rats weighing 250-300 g were used. STZ was administered on days 1 and 3 (3 mg/kg in divided doses/icv), and CEPO-Fc was administered at the dose of 5000 IU/ip/daily during days 4-14. The animals were trained in Morris water maze during days 15-17, and the memory retention test was performed on the 18th day. Following behavioral studies, the animals were sacrificed and their hippocampi isolated to determine the amounts of cleaved caspase-3 (the landmark of apoptosis). The results showed that CEPO-Fc treatment at the dose of 5000 IU/kg/ip was able to prevent the learning and memory deficit induced by icv-STZ. Western blot analysis revealed that STZ prompted the cleavage of caspase-3 in the hippocampus while pretreatment with CEPO-Fc significantly reduced the cleavage of this protein. Collectively, our findings suggest that CEPO-Fc could restore STZ-induced learning and memory impairment as well as apoptosis in the hippocampal region in a rat model of sporadic AD induced by icv-STZ.

Topics: Alzheimer Disease; Animals; Antibiotics, Antineoplastic; Apoptosis; Behavior, Animal; Brain; Caspase 3; Disease Models, Animal; Erythropoietin; Hippocampus; Immunoglobulin Fc Fragments; Injections, Intraventricular; Learning; Memory; Morris Water Maze Test; Neuroprotective Agents; Rats; Recombinant Fusion Proteins; Streptozocin

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Topics: Animals; Brazil; Disease Models, Animal; Erythropoietin; Euterpe; Hematinics; Hematopoiesis; Hypoxia; Kidney; Mice; Plant Extracts; Up-Regulation

To investigate the role of stromal cell-derived factor 1 (SDF-1) and C-X-C chemokine receptor type 4 (CXCR-4) in the premature brain with white matter damage (WMD) undergoing treatment with human umbilical cord mesenchymal stem cells (hUC-MSCs) and recombinant human erythropoietin (rhEPO).. Three-day-old Sprague-Dawley (SD) rats were randomly divided into sham operation group, hypoxia-ischemia (HI) group, rhEPO treated HI group, hUC-MSCs treated HI group, and rhEPO + hUC-MSCs treated HI group. WMD was established in all groups except the Sham group. SDF-1 and CXCR-4 levels in each group were detected at postnatal day (P) 5, P7, and P14. Pathological changes were assessed via HE staining at P14 and neuroethological tests were performed at P28.. The rhEPO and hUC-MSCs intervention reduced injury area, increased body weight at P7, and improved neurobehavioral scores at P28. Furthermore, their combined use proved even more beneficial. SDF-1 levels in the rhEPO group were higher than those in the other groups and highest in the hUC-MSCs + rhEPO group (all p < .01). SDF-1 levels in the hUC-MSCs + rhEPO and rhEPO groups were increased at P5 and reached a peak at P7. CXCR-4 levels in the hUC-MSCs group were higher than those in the other groups and highest in the hUC-MSCs + rhEPO group (all p < .01). CXCR-4 levels were also increased at P5 and highest at P14.. hUC-MSCs + rhEPO might reduce nerve cell damage and improve neurobehavioral development, in connection with increased SDF-1 and CXCR-4 expression, in premature rats with WMD due to hypoxic-ischemic injury.

Topics: Animals; Behavior, Animal; Body Weight; Chemokine CXCL12; Disease Models, Animal; Erythropoietin; Female; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Pregnancy; Premature Birth; Rats; Rats, Sprague-Dawley; Receptors, CXCR4; Recombinant Proteins; White Matter

Obesity remodels bone and increases bone marrow adipocytes (BMAT), which negatively regulate hematopoiesis and bone. Reduced BMAT could restore altered hematopoiesis and bone features. We analyzed the potential of erythropoietin (EPO), the cytokine required for erythropoiesis, to inhibit BMAT in C57BL6/J mice fed four weeks of a high-fat diet (HFD). Acute EPO administration markedly decreased BMAT in regular chow diet (RCD) and HFD-fed mice, without affecting whole body fat mass. Micro-CT analysis showed EPO reduced trabecular bone in RCD- and HFD-fed mice, but EPO-treated HFD-fed mice maintained cortical bone mineral density and cortical bone volume, which was reduced on RCD. Despite achieving similar increased hematocrits with BMAT loss in RCD- and HFD-fed mice treated with EPO, decreased bone marrow cellularity was only observed in RCD-fed mice concomitant with an increasing percentage of bone marrow erythroid cells. In contrast, in HFD-fed mice, EPO increased endothelial cells and stromal progenitors with a trend toward the normalization of marrow homeostasis. EPO administration increased c-terminal FGF23 and intact serum FGF23 only in HFD-fed mice. These data demonstrate the distinct EPO responses of bone and marrow in normal and obese states, accompanying EPO-induced loss of BMAT.

Topics: Adipose Tissue; Animals; Bone and Bones; Bone Marrow; Cancellous Bone; Diet, High-Fat; Disease Models, Animal; Erythropoietin; Female; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Mice, Inbred C57BL; Obesity; Osteoblasts; Osteoclasts; Osteocytes; Periosteum

Hypoxia‑inducible factor‑1α (HIF‑1α) is a key transcriptional factor in response to hypoxia and is involved in ischemic stroke. In the present study, the potential for HIF‑1α to inhibit neuronal apoptosis through upregulating erythropoietin (EPO) was investigated in a transient middle cerebral artery occlusion (tMCAO) rat stroke model. For this purpose, a recombinant adenovirus expressing HIF‑1α was engineered (Ad‑HIF‑1α). Control adenovirus (Ad group), Ad‑HIF‑1α (Ad‑HIF‑1α group) or Ad‑HIF‑1α in addition to erythropoietin mimetic peptide‑9 (EMP9), an EPO‑receptor (‑R) antagonist (Ad‑HIF‑1α+EMP9 group), were used for an intracranial injection into rat ischemic penumbra 1 h following MCAO. All rats demonstrated functional improvement following tMCAO, while the improvement rate was faster in rats treated by Ad‑HIF‑1α compared with all other groups. The EPO‑R inhibitor partially reversed the benefits of Ad‑HIF‑1α. Apoptosis induced by tMCAO was significantly inhibited by Ad‑HIF‑1α (P<0.05). The expression of HIF‑1α, evaluated by immunohistochemistry either in neurons or astrocytes, was upregulated by Ad‑HIF‑1α. Both EPO mRNA and protein expression were increased by Ad‑HIF‑1α, however, there was no significant change of EPO‑R either on an mRNA level or protein level. Furthermore, EMP9 did not change the EPO expression which was upregulated by Ad‑HIF‑1α. Activated caspase 3 in neurons was suppressed by Ad‑HIF‑1α. Activated caspase 3 downregulated by HIF‑1α was partially blocked by EMP9. Altogether, the present data demonstrated that HIF‑1α attenuates neuronal apoptosis partially through upregulating EPO following cerebral ischemia in rat. Thus, upregulating HIF‑1α subsequent to a stroke may be a potential treatment for ischemic stroke.

Topics: Animals; Apoptosis; Brain Diseases; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Neurons; Rats; Rats, Sprague-Dawley; Reperfusion Injury

We studied the effect of erythropoietin on functional properties of mesenchymal stem cells under conditions of oxidative stress and their therapeutic potential in the treatment of intervertebral disc degeneration in Wistar rats. It was shown that erythropoietin stimulates proliferation under conditions of oxidative stress. Injection of bone marrow mesenchymal stem cells into the damaged intervertebral disc was followed by an increase in the height of the intervertebral disc and activation of repair processes in the nucleus pulposus. The combination of mesenchymal stem cells with erythropoietin provides the best effect of cell therapy in case of intervertebral disc damage.

Topics: Animals; Bone Marrow Cells; Cell Proliferation; Disease Models, Animal; Erythropoietin; Femur; Humans; Hydrogen Peroxide; Injections, Intralesional; Intervertebral Disc; Intervertebral Disc Degeneration; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Oxidative Stress; Rats; Rats, Wistar

Acute kidney injury (AKI) is the most common condition in hospitalized patients. As ischemia/reperfusion-induced AKI (IR-AKI) is as a major contributor to end-stage disease, an effective therapeutic intervention for IR-AKI is imperative. Erythropoietin (EPO) is a potent stimulator of erythroid progenitor cells and is significantly upregulated during hypoxia. Here, we investigated the renoprotective effects of EPO in an IR-AKI mouse model. Mice were assigned to sham, EPO only, and IR only groups, and the IR group was treated with EPO prior to injury. EPO was administered twice at 30 min prior to bilateral renal artery occlusion, and 5 min before reperfusion, with all mice sacrificed 24 h after IR-AKI. The serum was harvested for renal functional measurements. The kidneys were subjected to histological evaluation, and the biochemical changes associated with renal injury were assessed. EPO significantly attenuated the renal dysfunction associated with IR-AKI, as well as tissue injury. Apoptotic cell death and oxidative stress were significantly reduced in EPO-treated mice. Macrophage infiltration and expression of ICAM-1 and MCP-1 were also significantly reduced in EPO-treated mice. Furthermore, the expression of inflammasome-related factors (NLRP1, NLRP3, and caspase-1 cleavage), via the activation of the COX-2 and NF-B signaling pathways were significantly reduced following EPO treatment. To our knowledge, this is the first study to demonstrate that inflammasome-mediated inflammation might be a potential target of EPO as a treatment for ischemic AKI.

Topics: Acute Kidney Injury; Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Apoptosis Regulatory Proteins; Caspase 1; Cell Hypoxia; Cyclooxygenase 2; Disease Models, Animal; Erythroid Precursor Cells; Erythropoietin; Gene Expression Regulation; Humans; Inflammasomes; Kidney; Mice; NLR Family, Pyrin Domain-Containing 3 Protein; Reperfusion Injury

Iron-deficiency anemia is a potent stimulator of the phosphaturic hormone Fibroblast growth factor-23 (FGF23). Anemia, elevated FGF23, and elevated serum phosphate are significant mortality risk factors for patients with chronic kidney disease (CKD). However, the contribution of anemia to overall circulating FGF23 levels in CKD is not understood. Our goal was to investigate the normalization of iron handling in a CKD model using the erythropoiesis stimulating agents (ESAs) Erythropoietin (EPO) and the hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHDi) FG-4592, on the production of, and outcomes associated with, changes in bioactive, intact FGF23 ("iFGF23"). Our hypothesis was that rescuing the prevailing anemia in a model of CKD would reduce circulating FGF23. Wild-type mice were fed an adenine-containing diet to induce CKD, then injected with EPO or FG-4592. The mice with CKD were anemic, and EPO improved red blood cell indices, whereas FG-4592 increased serum EPO and bone marrow erythroferrone (Erfe), and decreased liver ferritin, bone morphogenic protein-6 (Bmp-6), and hepcidin mRNAs. In the mice with CKD, iFGF23 was markedly elevated in control mice but was attenuated by >70% after delivery of either ESA, with no changes in serum phosphate. ESA treatment also reduced renal fibrosis markers, as well as increased Cyp27b1 and reduced Cyp24a1 mRNA expression. Thus, improvement of iron utilization in a CKD model using EPO and a HIF-PHDi significantly reduced iFGF23, demonstrating that anemia is a primary driver of FGF23, and that management of iron utilization in patients with CKD may translate to modifiable outcomes in mineral metabolism.

Topics: Anemia; Animals; Bone Morphogenetic Protein 6; Cytokines; Disease Models, Animal; Erythropoietin; Female; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Hepcidins; Hypoxia-Inducible Factor-Proline Dioxygenases; Mice, Inbred C57BL; Muscle Proteins; Prolyl-Hydroxylase Inhibitors; Renal Insufficiency, Chronic

Aplastic anemia (AA) is characterized as hypoplasia of bone marrow hematopoietic cells and hematopenia of peripheral blood cells. Though the supplement of exogenous erythropoietin (EPO) has been clinically approved for AA treatment, the side-effects hinder its further application. Here a robust treatment for AA induced by chemotherapy drugs is explored using gadofullerene nanoparticles (GFNPs).

Topics: Anemia, Aplastic; Animals; Antineoplastic Agents, Alkylating; Bone Marrow Cells; Busulfan; Cell Differentiation; Cyclophosphamide; Disease Models, Animal; Erythropoiesis; Erythropoietin; Female; Fullerenes; Hematopoietic Stem Cells; Mice; Mice, Inbred ICR; Nanoparticles

The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG-bound epoetin β pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O

Topics: Anemia; Animals; Blotting, Western; Disease Models, Animal; Erythropoietin; Glycosylation; Humans; Hypoxia; Kidney; Liver; Male; Rats; Rats, Sprague-Dawley

Acute lung injury (ALI) is a clinical problem with poor prognosis and high mortality. The purpose of this study was to explore the effects of helix B position peptide (HBSP) on ALI and its mechanism.. C57/BL6 male mice were used to construct ALI models by LPS tracheal injection and detect the effect of HBSP on mouse ALI by subcutaneously injecting HBSP. In addition, normal human lung epithelial cell line (BEAS-2B) were cultured and stimulated with HBSP. Then, the effects of HBSP on oxidative stress and endoplasmic reticulum stress (ERS) in BEAS-2B cells were examined. Finally, the effect of HBSP on the Nrf2/HO-1 signaling pathway was examined, and the mechanism of action of HBSP was verified using the Nrf2/HO-1 signaling pathway inhibitor ML385.. In vitro, HBSP increased the expression of SOD1/2 and decreased the expression of ERS-related molecules such as CHOP, GRP-78, and caspase-12, indicating that HBSP effectively reduces the level of oxidative stress and ERS in BEAS-2B cells. In addition, HBSP also increased the activity of the Nrf2/HO-1 signaling pathway and ML385 reduced the protective effect of HBSP on BEAS-2B cells. In vivo, HBSP significantly reduced LPS-induced mouse ALI. W/D and inflammatory factors in the BALF of the mouse lung were significantly reduced and the level of oxidative stress was also reduced.. HBSP plays an important role in relieving ALI by activating Nrf2/HO-1 signaling pathway, which reduces the level of inflammation in lung tissue and oxidative stress and ERS in lung epithelial cells.

Topics: Acute Lung Injury; Animals; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum Stress; Erythropoietin; Heme Oxygenase-1; Humans; Injections, Subcutaneous; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; NF-E2-Related Factor 2; Oxidative Stress; Peptide Fragments; Signal Transduction

Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a trinucleotide (CAG) repeat expansion in the huntingtin gene (HTT). The R6/2 transgenic mouse model of HD expresses exon 1 of the human HTT gene with approximately 150 CAG repeats. R6/2 mice develop progressive behavioural abnormalities, impaired neurogenesis, and atrophy of several brain regions. In recent years, erythropoietin (EPO) has been shown to confer neuroprotection and enhance neurogenesis, rendering it a promising molecule to attenuate HD symptoms. In this study, the therapeutic potential of EPO was evaluated in female R6/2 transgenic mice. A single bilateral injection of a lentivirus encoding human EPO (LV-hEPO) was performed into the lateral ventricles of R6/2 mice at disease onset (8 weeks of age). Control groups were either untreated or injected with a lentivirus encoding green fluorescent protein (LV-GFP). Thirty days after virus administration, hEPO mRNA and protein were present in injected R6/2 brains. Compared to control R6/2 mice, LV-hEPO-treated R6/2 mice exhibited reduced hippocampal atrophy, increased neuroblast branching towards the dentate granular cell layer, and improved spatial cognition. Our results suggest that LV-hEPO administration may be a promising strategy to reduce cognitive impairment in HD.

Topics: Animals; Atrophy; Cognition; Disease Models, Animal; Erythropoietin; Female; Genetic Therapy; Hippocampus; Huntington Disease; Injections, Intraventricular; Lentivirus; Mice; Mice, Transgenic; Neural Stem Cells; Organ Size; Spatial Navigation; Transfection

We aimed to evaluate the effects of EPO in the lipopolysaccharide (LPS) induced rat model of autism in terms of social deficits, learning and memory impairments, as well as their neurochemical correlates. Sixteen female Sprague Dawley rats randomly distributed into two equel groups, then were caged with fertile males for mating. At the 10th day of pregnancy, 0.5 ml %0,9 NaCl saline was given to first group, 100 μg/kg LPS was given to second group to induce autism. On postnatal 21th day, forty-eight littermates were divided into four groups as; 8 male, 8 female controls, 16 male and 16 female LPS-exposed. Then, LPS groups were also divided in to two groups as saline (1 mg/kg/day) and EPO 600 U/kg/day groups, and animals were treated 45 days. At 50th day, after behavioral evaluations, brain levels of TNF-α, nerve growth factor (NGF) were measured. Histologically, hippocampal neuronal density and GFAP expression were assessed. Three-chamber sociability and social novelty test, passive avoidance learning test were revealed significant differences among the EPO and control groups. Histologically, hippocampal CA1 & CA3 regions displayed significant alterations regarding gliosis (GFAP-positive cells) and regarding frontal cortical thickness in EPO groups compare to controls. Biochemical measurements of the brain levels of TNF-α and NGF levels showed significant differences between controls and EPO groups. According to our findings EPO treatment has beneficial effects on ASD-like symptoms, learning and memory processes, neuronal loss and neuroinflammation in the LPS induced rat model of autism, with some gender differences through inflammatory and neurotrophic pathways.

Topics: Animals; Autistic Disorder; Avoidance Learning; Behavior, Animal; Disease Models, Animal; Erythropoietin; Female; Hippocampus; Inflammation; Lipopolysaccharides; Male; Memory; Memory Disorders; Neurons; Rats; Rats, Sprague-Dawley; Social Behavior; Tumor Necrosis Factor-alpha

Topics: Animals; Apoptosis; Caspase 3; Creatinine; Disease Models, Animal; Drug Therapy, Combination; Erythropoietin; HMGB1 Protein; Injections, Intraperitoneal; Injections, Intravenous; Kidney; Male; Mice; Mice, Inbred C57BL; Peptide Fragments; Reperfusion Injury; RNA, Small Interfering; Transcriptome

The aim of this study was to investigate the effects of mandibular advancement device (MAD) therapy for obstructive sleep apnea-hypopnea syndrome (OSAHS) on hypoxia-inducible factor-1α (HIF-1α), erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in myocardial tissue. New Zealand rabbits were used to develop OSAHS and MAD models. Cone beam computed tomography (CBCT) of the upper airway and polysomnography (PSG) recordings were performed with the animals in the supine position. All of the animals were induced to sleep in a supine position for 4-6 h each day and were observed continuously for 8 weeks. The myocardial tissue of the three groups was dissected to measure the expression of HIF-1α, EPO and VEGF. The results showed that there was higher expression of HIF-1α, EPO and VEGF in the OSAHS group than those in the MAD and control groups. MAD treatment significantly downregulated the expression of HIF-1α, EPO and VEGF in the OSAHS animals. We concluded that MAD treatment could significantly downregulate the increased expression of HIF-1α, EPO and VEGF in OSAHS rabbits, improving their myocardial function.

Topics: Animals; Case-Control Studies; Cone-Beam Computed Tomography; Disease Models, Animal; Erythropoietin; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Myocardium; Occlusal Splints; Polysomnography; Rabbits; Sleep Apnea, Obstructive; Up-Regulation; Vascular Endothelial Growth Factor A

Exogenous erythropoietin (EPO) is being considered for tissue protection and angiogenesis in retinal vascular diseases. However, studies are limited by insufficient tools to address signaling effects through the EPO receptor (EPOR). We used a humanized mouse model of hypoactive EPOR signaling to test the hypothesis that EPOR signaling supports angiogenesis in retinovascular diseases.. Humanized Knockin EPOR mice (hWtEPOR) with hypoactive EPOR signaling were compared to littermate wild-type mice (WT). Postnatal day (p)7 mice of each genotype were exposed to 75% oxygen for five days, followed by 21% oxygen in the oxygen-induced retinopathy model (OIR) and compared to room-air (RA)-raised pups. At time points after OIR, pups were sacrificed, and flat-mounted, lectin-stained retinas were analyzed for central avascular area or intravitreal neovascular area (IVNV). Flash-frozen retinas were analyzed for angiogenic protein (Epo, VEGF, p-Stat3) and gene (Vegfa, Kdr, Epo, Hif1α, Hif2α) expression levels.. In OIR, hWtEPOR mice had increased AVA compared with WT at p8, p12, and p17, but there was no difference in IVNV between hWtEPOR and WT mice at p17. Although VEGF and p-STAT3 proteins were increased in WT at p17 OIR, there were no differences in retinal angiogenic factor expression levels between hWtEPOR and WT OIR at p17 despite similar areas of IVNV.. Our data support the hypothesis that EPOR signaling was associated with regrowth of vascularization following oxygen-induced capillary dropout and played a role in intravitreal angiogenesis. Additional study of EPOR signaling regulation on other angiogenic factor pathways may be considered.

Topics: Animals; Blotting, Western; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Female; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neovascularization, Pathologic; Oxygen; Real-Time Polymerase Chain Reaction; Receptors, Erythropoietin; Retina; Retinal Neovascularization; Signal Transduction; Vascular Endothelial Growth Factor A

Dendritic cells (DC) play a key role in the adaptive immune response due to their ability to present antigens and stimulate naïve T cells. Many bacteria and viruses can efficiently target DC, resulting in impairment of their immunostimulatory function or elimination. Hence, the DC compartment requires replenishment following infection to ensure continued operational readiness of the adaptive immune system. Here, we investigated the molecular and cellular mechanisms of inflammation-induced DC generation. We found that infection with viral and bacterial pathogens as well as Toll-like receptor 9 (TLR9) ligation with CpG-oligodeoxynucleotide (CpG-ODN) expanded an erythropoietin (EPO)-dependent TER119

Topics: Animals; Biomarkers; Blood Group Antigens; CD11c Antigen; Cell Differentiation; Cytokines; Dendritic Cells; Disease Models, Animal; Erythropoietin; Female; Hematopoiesis, Extramedullary; Immunity, Innate; Immunophenotyping; Infections; Inflammation; Mice; Mice, Transgenic; Oligodeoxyribonucleotides; Spleen

Peripheral nerve injury (PNI) often leads to significant functional loss in patients and poses a challenge to physicians since treatment options for improving functional outcomes are limited. Recent studies suggest that erythropoietin and glucocoticoids have beneficial effects as mediators of neuro-regenerative processes. We hypothesized that combination treatment with erythropoietin and glucocoticoids would have a synergistic effect on functional outcome after PNI.. Sciatic nerve crush injury was simulated in ten-week-old male C57BL/6 mice. The mice were divided into four groups according to the type of drugs administered (control, erythropoietin, dexamethasone, and erythropoietin with dexamethasone). Motor functional recovery was monitored by walking track analysis at serial time points up to 28 days after injury. Morphological analysis of the nerve was performed by immunofluorescent staining for neurofilament (NF) heavy chain and myelin protein zero (P0) in cross-sectional and whole-mount nerve preparations. Additionally, morphological analysis of the muscle was performed by Hematoxylin and eosin staining.. Combination treatment with erythropoietin and dexamethasone significantly improved the sciatic functional index at 3, 7, 14, and 28 days after injury. Fluorescence microscopy of cross sectional nerve revealed that the combination treatment increased the ratio of P0/NF-expressing axons. Furthermore, confocal microscopy of the whole-mount nerve revealed that the combination treatment increased the fluorescence intensity of P0 expression. The cross-sectional area and minimum Feret's diameter of the muscle fibers were significantly larger in the mice which received combination treatment than those in the controls.. Our results demonstrated that combination treatment with erythropoietin and dexamethasone accelerates functional recovery and reduces neurogenic muscle atrophy caused by PNI in mice, which may be attributed to the preservation of myelin and Schwann cell re-myelination. These findings may provide practical therapeutic options for patients with acute PNI.

Topics: Acute Disease; Animals; Axons; Dexamethasone; Disease Models, Animal; Drug Therapy, Combination; Erythropoietin; Male; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Muscles; Muscular Atrophy; Myelin P0 Protein; Peripheral Nerve Injuries; Recovery of Function; Remyelination; Schwann Cells; Sciatic Nerve

Tissue-resident macrophages can be educated to tumor-associated macrophages (TAMs) by the tumor microenvironment and many types of macrophages express erythropoietic receptor (EPOR); However, little is known about the expression of EPOR on TAMs and the identity of EPOR. EPOR-eGFPcre mice were used to determine the expression of EPOR on lung tissue-resident macrophages. Flow cytometry, RT-PCR, and Western blot were examined to define the identity of EPOR. We found that a subpopulation of mouse lung tissue-resident macrophages express EPOR and EPO enhances the proliferation of EPOR. We have characterized TAMs expressing EPOR and CD163

Topics: Adolescent; Adult; Animals; Biomarkers, Tumor; Cell Proliferation; Child; Cytokines; Disease Models, Animal; Erythropoietin; Female; Gene Expression; Genes, Reporter; Humans; Immunophenotyping; Lung Neoplasms; Male; Mice; Mice, Knockout; Osteosarcoma; Prognosis; Receptors, Erythropoietin; Tumor Microenvironment; Tumor-Associated Macrophages; Young Adult

Preeclampsia is a severe disease of late pregnancy. Etiological factors and a pathogenetic pattern of events still require significant clarification, but it is now recognized that a large role is played by placentation disorders and emerging endothelial dysfunction. The administration of short-chain peptides mimicking the spatial structure of the B erythropoietin chain may become one of the directions of searching for new drugs for preeclampsia prevention and therapy. Simulation of ADMA-like preeclampsia in Wistar rats was performed by the administration of a non-selective NOS blocker L-NAME from the 14th to 20th day of pregnancy. The administration of the pHBSP at the doses of 10 µg/kg and 250 µg/kg corrected the established morphofunctional disorders. The greatest effect was observed at a dose of 250 µg/kg. There was a decrease in systolic and diastolic blood pressure by 31.2 and 32.8%, respectively (

Topics: Animals; bcl-2-Associated X Protein; Blood Pressure; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Erythropoietin; Female; Microcirculation; NG-Nitroarginine Methyl Ester; Oligopeptides; Placenta; Pre-Eclampsia; Pregnancy; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease associated with cigarette smoking. Alterations in local lung and systemic iron regulation are associated with disease progression and pathogenesis. Hepcidin, an iron regulatory peptide hormone, is altered in subjects with COPD; however, the molecular role of hepcidin in COPD pathogenesis remains to be determined. In this study, using a murine model of smoke-induced COPD, we demonstrate that lung and circulating hepcidin levels are inhibited by cigarette smoke. We show that cigarette smoke exposure increases erythropoietin and bone marrow-derived erythroferrone and leads to expanded but inefficient erythropoiesis in murine bone marrow and an increase in ferroportin on alveolar macrophages (AMs). AMs from smokers and subjects with COPD display increased expression of ferroportin as well as hepcidin. Notably, murine AMs exposed to smoke fail to increase hepcidin in response to Gram-negative or Gram-positive infection. Loss of hepcidin in vivo results in blunted functional responses of AMs and exaggerated responses to

Topics: Animals; Bone Marrow; Cation Transport Proteins; Cigarette Smoking; Disease Models, Animal; Disease Progression; Erythropoietin; Hepcidins; Humans; Iron; Lung; Macrophages, Alveolar; Mice; Mice, Inbred C57BL; Peptides; Pulmonary Disease, Chronic Obstructive; Smoke; Smoking

The kidneys are the major organs for erythropoietin (EPO) production in adults, and thus, kidney damage results in reduced EPO levels and anemia. Inhibitors of Hypoxia-inducible factor-prolyl hydroxylase domain-containing protein (HIF-PHD) are awaited as new therapeutic options for renal anemia. It can be predicted that most patients who receive HIF-PHD inhibitors have renal dysfunction as a cause of anemia. Therefore, in the present study, we investigated the effects of the HIF-PHD inhibitor molidustat on anemia and renal dysfunction when initiated after the onset of renal anemia. Male C57BL/6J mice received adenine orally to induce nephropathy. After the onset of nephropathy, the mice were treated with either vehicle or molidustat. After 4 weeks of administration, vehicle-treated mice displayed significant anemia, and molidustat ameliorated this anemia. Vehicle-treated mice exhibited reduced creatinine clearance and body weight, increased blood urea nitrogen levels, histopathological changes, immune cell infiltration, and dehydration. Molidustat reversed immune cell infiltration, dehydration, and renal fibrosis without improving renal functional parameters. In conclusion, molidustat treatment initiated after the onset of nephropathy and renal anemia reversed anemia in mice. Molidustat improved some parameters of renal abnormality, but it did not restore renal function.

Topics: Acute Kidney Injury; Adenine; Anemia; Animals; Disease Models, Animal; Erythropoietin; Hypoxia-Inducible Factor 1; Male; Mice, Inbred C57BL; Prolyl-Hydroxylase Inhibitors; Pyrazoles; Triazoles

Retinal degeneration (RD) results in photoreceptor loss and irreversible visual impairments. This study sought to alleviate the photoreceptor degeneration via the adeno-associated virus (AAV)-mediated erythropoietin (EPO) therapy. AAV-2/2-mCMV-EPO vectors were constructed and delivered into the subretinal space of a RD model. The retinal morphology, optokinetic behaviour and electrophysiological function of the treated animals were analysed. The subretinal delivery of AAV-2/2 vectors induced robust EPO gene expressions in the retinas. AAV2/2-mediated EPO therapy ameliorated the photoreceptor degeneration and visual impairments of the RD animal model. Furthermore, the multi-electrodes array (MEA) was used to detect the firing activities of retinal ganglion cells. MEA recording showed that the EPO therapy could restrain the spontaneous firing response, enhance the light-induced firing response and preserve the basic configurations of visual signal pathway in RD model. Our MEA assay provided an example to evaluate the potency of pharmacological compounds on retinal plasticity. In conclusion, AAV2/2-mediated EPO therapy can ameliorate the photoreceptor degeneration and rectify the abnormities in visual signal transmission. These beneficial results suggest the AAV vector is a viable therapeutic option for retinopathies with rapidly degenerating kinetics and lay the groundwork for future development of EPO gene therapy.

Topics: Animals; Cytomegalovirus; Dependovirus; Disease Models, Animal; Erythropoietin; Female; Genetic Therapy; Male; Mice; Mice, Inbred C57BL; Neuroprotection; Photoreceptor Cells; Retina; Retinal Degeneration

To evaluate the effects of interleukin-6 (IL-6) and erythropoietin (EPO) in experimental acute spinal cord injury (SCI) in rats.. Using standardized equipment, namely, a New York University (NYU) Impactor, a SCI was produced in 50 Wistar rats using a 10-g weight drop from a 12.5-mm height. The rats were divided into the following 5 groups of 10 animals each: "Group EPO", treated with erythropoietin only; "Group EPO + IL-6", treated with both substances; "Group IL-6", receiving IL-6 administration only; "Group Placebo", receiving a placebo solution; and "Group Sham", submitted to an incomplete procedure (only laminectomy, without SCI). All drugs and the placebo solution were administered intraperitoneally for three weeks. The animals were followed up for 42 days. Functional motor recovery was monitored by the Basso, Beattie, and Bresnahan (BBB) scale on days 2, 7, 14, 21, 28, 35 and 42. Motor-evoked potential tests were performed on the 42nd day. Histological analysis was performed after euthanasia.. The group receiving EPO exhibited superior functional motor results on the BBB scale. IL-6 administration alone was not superior to the placebo treatment, and the IL-6 combination with EPO yielded worse results than did EPO alone.. Using EPO after acute SCI in rats yielded benefits in functional recovery. The combination of EPO and IL-6 showed benefits, but with inferior results compared to those of isolated EPO; moreover, isolated use of IL-6 resulted in no benefit.

Topics: Animals; Disease Models, Animal; Erythropoietin; Evoked Potentials, Motor; Interleukin-6; Male; Neuroprotective Agents; Rats; Rats, Wistar; Recovery of Function; Spinal Cord Injuries

Erythropoietin (EPO) is a cytokine primarily involved in the regulation of erythropoiesis. In response to hypoxia-ischemia, hypoxia-inducible factor 1 induces EPO production, which, in turn, inhibits apoptosis of erythroid progenitor cells. By the same mechanism and acting through other signaling pathways, EPO exerts neuroprotective effects. Increased resistance to hypoxia and decreased apoptosis are thought to be important mechanisms for tumor progression, including malignant glioma. Because recent studies have demonstrated that EPO and its receptor (EPOR) are expressed in several tumors and can promote tumor growth, in the present study, we investigated EPO and EPOR expression in human glioma and the effect of EPO administration in a rat model of glioma implantation.. Using Western blotting and immunohistochemical analysis, we examined the expression of EPO, EPOR, platelet endothelial cell adhesion molecule, and Ki-67 in human glioma specimens and experimentally induced glioma in rats. In the experimental setting, a daily dose of recombinant human EPO (rHuEPO) or saline solution were administered for 21 days in Fischer rats subjected to 9L cell line implantation.. In both human and animal specimens, we found an increase in EPOR expression as long as the lesion presented with an increasing malignant pattern. A significant direct correlation was found between the expression of EPOR and Ki-67 and EPOR and platelet endothelial cell adhesion molecule in low- and high-grade gliomas. The rats treated with rHuEPO presented with significantly larger tumor spread compared with the saline-treated rats.. The results of our study have shown that the EPO/EPOR complex might play a significant role in the aggressive behavior of high-grade gliomas. The larger tumor spread in rHuEPO-treated rats suggests a feasible role for EPO in the aggressiveness and progression of malignant glioma.

Topics: Adult; Aged; Animals; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Disease Models, Animal; Erythropoietin; Female; Glioma; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Neoplasm Transplantation; Platelet Endothelial Cell Adhesion Molecule-1; Rats, Inbred F344; Receptors, Erythropoietin; Recombinant Proteins; Tumor Burden

Dyslipidemia is commonly observed in various kidney diseases, renal specific secreted erythropoietin (EPO) may participate in this process. However, how this process is regulated remains elusive.. Dyslipidemia was evaluated in chronic kidney disease and ischemia kidney injury animal model. Primary cultured adipocytes were harvested to investigate the lipid metabolic effect of EPO. Lipidemia was evaluated in EPO treated animals. Blood samples from cardiac surgery-induced kidney injury patient were collected to assess correlationship between EPO and lipidemia.. We found a decrease in secreted EPO and hypertriglyceridemia in chronic kidney disease (CKD) mice. In contrast, in renal ischemia animal model, increased EPO triggered by hypoxia signaling activation, was accompanied by decreased triglyceride (TG) in serum. Mechanistically, circulating EPO modulated JAK2-STAT5 signaling, which in turn enhanced lipid catabolism in peripheral adipose tissue and contributed to dysregulated lipidemia. Delivering of recombinant EPO into both wild type and CKD mice suppressed TG in serum by accelerating lipid catabolism in adipose tissue. In a cohort of patients diagnosed with acute kidney injury after cardiopulmonary bypass surgery, the decreased TG and cholesterol negatively correlated with increased EPO in serum.. This study depicted a new mechanism by which renal secreted EPO controlled lipidemia in kidney diseases including chronic kidney disease. Circulating EPO stimulated lipid catabolism by targeting JAK2-STATA5 signaling in peripheral adipose tissue, providing new therapeutic target for dyslipidemia treatment.. This work was supported by grants from the National Natural Science Foundation of China (Nos. 81700640 and 81970608).

Topics: Adipose Tissue; Animals; Disease Models, Animal; Erythropoietin; Hyperlipidemias; Hypoxia; Janus Kinase 2; Kidney; Kidney Diseases; Lipid Metabolism; Lipids; Male; Mice; Signal Transduction; STAT5 Transcription Factor

Erythropoietin (EPO) is a clinically available hematopoietic cytokine. EPO has shown beneficial effects in the context of spinal cord injury and other neurological conditions. The aim of this study was to evaluate the effect of EPO on a rat model of spinal cord compression-induced cervical myelopathy and to explore the possibility of its use as a pharmacological treatment.. To develop the compression-induced cervical myelopathy model, an expandable polymer was implanted under the C5-C6 laminae of rats. EPO administration was started 8 weeks after implantation of a polymer. Motor function of rotarod performance and grip strength was measured after surgery, and motor neurons were evaluated with H-E, NeuN and choline acetyltransferase staining. Apoptotic cell death was assessed with TUNEL and Caspase-3 staining. The 5HT, GAP-43 and synaptophysin were evaluated to investigate the protection and plasticity of axons. Amyloid beta precursor protein (APP) was assessed to evaluate axonal injury. To assess transfer of EPO into spinal cord tissue, the EPO levels in spinal cord tissue were measured with an ELISA for each group after subcutaneous injection of EPO.. High-dose EPO maintained motor function in the compression groups. EPO significantly prevented the loss of motor neurons and significantly decreased neuronal apoptotic cells. Expression of 5HT and synaptophysin was significantly preserved in the EPO group. APP expression was partly reduced in the EPO group. The EPO levels in spinal cord tissue were significantly higher in the high-dose EPO group than other groups.. EPO improved motor function in rats with compression-induced cervical myelopathy. EPO suppressed neuronal cell apoptosis, protected motor neurons, and induced axonal protection and plasticity. The neuroprotective effects were produced following transfer of EPO into the spinal cord tissue. These findings suggest that EPO has high potential as a treatment for degenerative cervical myelopathy.

Topics: Animals; Disease Models, Animal; Erythropoietin; Humans; Male; Motor Neurons; Rats; Rats, Wistar; Recombinant Proteins; Recovery of Function; Spinal Cord Compression; Spinal Cord Diseases

To determine the effects of intraurethral erythropoietin (EPO) on an experimentally induced urethral injury in a rat model with respect to wound healing enhancement and the prevention of spongiofibrosis MATERIAL AND METHODS: A urethral injury model was created by traumatizing the urethra of male rats with a tilted-tip insulin injector. Thirty rats were randomly separated into 3 groups of 10; Group 1 (control) received 0.9% saline solution twice a day, Group II received EPO 25 IU/kg once a day and 0.9% saline solution once a day, and Group III received EPO 25 IU/kg twice a day. All applications were made intraurethrally via a 24 ga catheter sheath. To investigate inflammation and spongiofibrosis and congestion of vessels in the lamina propria, the penises of the rats were harvested for histopathologic evaluation after a follow-up period of 14 days.. Histopathologic analysis revealed less fibrosis and inflammation and higher congestion of vessels in Group III that had received high-dose EPO. There was a significant decrease in both spongiofibrosis and inflammation and an increase in congestion in Groups II and III compared to the control group (P = .001, for all). In the comparison of Group II with Group III, no statistically significant differences were found in terms of these 3 parameters (P = .5, P = .6, P = .27, respectively).. The results of this study have shown that EPO has a preventive effect on spongiofibrosis and improve urethral wound healing in a rat model of urethral injury.

Topics: Animals; Disease Models, Animal; Erythropoietin; Fibrosis; Injections, Intralesional; Male; Random Allocation; Rats; Rats, Wistar; Urethra; Wound Healing

Substantial progress was made in acute kidney injury (AKI) over the past 10 years, but no therapeutic interventions have been shown to prevent AKI or accelerate functional recovery after injury. A large number of preclinical studies supports the use of recombinant human erythropoietin (rHuEPO) to prevent AKI, but the clinical trial data are inconclusive. To address concerns about preclinical study design and reporting in AKI, we here presented our rigorous experiments on the use of rHuEPO in a mouse model simulating the most common post-ischemic AKI in patients.. Use of saline vehicle or rHuEPO (100 or 1000 U/KgBW) in mice subjected to AKI induced by ischemia-reperfusion injury of left kidney 2 weeks after right nephrectomy (NX + IRI).. NX + IRI resulted in a reproducible AKI model. Use of rHuEPO as a pretreatment or posttreatment did not affect AKI severity, functional recovery, and mouse survival regardless of gender, injury severity, or doses of rHuEPO. Administering rHuEPO with 1000 U/KgBW did increase hematocrit and modulate AKI kidney macrophages by Nos2 downregulation and Ccl17 upregulation. Active expression of erythropoietin receptor (EPOR) was not identified in renal cells by lineage tracing study, whereas expression of colony-stimulating factor 2 receptor β (CSF2Rβ) was identified in kidney macrophages and upregulated after AKI. Both EPOR and CSF2Rβ were identified in cultured bone marrow derived macrophages, possibly mediated the robust inhibition of cytokine-induced phenotype switching by rHuEPO.. Use of rHuEPO can modulate macrophage function but not the post-ischemic AKI severity, functional recovery and survival in mice.

Topics: Acute Kidney Injury; Animals; Apoptosis; Disease Models, Animal; Erythropoietin; Female; Kidney; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nephrectomy; Recombinant Proteins; Reperfusion Injury; Signal Transduction

Paraplegia remains a significant complication of thoracoabdominal aortic intervention. We previously reported that diazoxide (DZ), enhances the neuroprotective efficacy of erythropoietin (EPO). We hypothesized that DZ and EPO combined treatment attenuates spinal cord ischemic injury through upregulation of nerve growth factor (NGF).. DZ (pretreatment) was given to adult male C57/BL6 mice by oral gavage and EPO (before surgery) was intraperitoneally injected 32 h after administration of DZ. Spinal cords were harvested 0, 2, 4, and 6 h after injection of EPO. NGF expression was analyzed by western blot. After determining the optimal time, NGF expression was compared between DZ (pretreatment) + EPO (before surgery), DZ + PBS, PBS + EPO, and PBS + PBS (ischemic control). Four groups were studied to compare the motor function after ischemia: DZ + EPO (n = 11), ischemic control (n = 9), DZ + EPO + tropomyosin receptor kinase A receptor inhibitor (n = 9), and sham (without cross-clamp, n = 4). Spinal cord ischemia was induced by a 4-min thoracic aortic cross-clamp. Functional scoring (Basso Mouse Score) was done at 12-h intervals until 48 h, and spinal cords were harvested for evaluation of NGF expression and histological changes.. NGF expression was significantly upregulated 4 h after administration of EPO. At 4 h after injection of EPO, NGF expression in the DZ + EPO group was significantly higher than that in the other groups. DZ + EPO significantly preserved motor function compared with all other groups. At 48 h after reperfusion, the level of NGF expression in the DZ + EPO group, was significantly higher than in all other groups.. DZ + EPO attenuates spinal cord ischemic injury through upregulation of NGF. Better understanding of this mechanism may serve to further prevent ischemic complications for aortic intervention.

Topics: Animals; Aortic Aneurysm, Thoracic; Diazoxide; Disease Models, Animal; Drug Synergism; Erythropoietin; Humans; Male; Mice; Nerve Growth Factor; Paraplegia; Recombinant Proteins; Spinal Cord; Spinal Cord Ischemia; Up-Regulation; Vascular Surgical Procedures

The neurological disorders and neural pathology brought about by chronic alcoholism are difficult to be reversed. Increasing evidence highlights the protective roles of erythropoietin (EPO) in neurodegenerative diseases and injuries of the central nervous system. In the present study, we investigated the therapeutic effects of EPO on the neurological function deficits and neural pathology caused by chronic alcoholism and the regulatory mechanisms. Using the canonical mouse model of chronic alcohol exposure designed to mimic the repeated cycles of heavy abuse typical of chronic alcoholism, it was found that EPO delivered via intranasal route effectively restored the alcohol‑impaired motor cooperation in rotarod and beam walk tests, reversed alcoholic cognitive and emotional alterations in the novel location recognition task and open‑filed test, and rescued alcohol‑disrupted nervous conduction in the somatosensory‑evoked potential (SSEP) test. Consistently, the intranasally administered EPO promoted the remyelination and synapse formation in chronic alcohol‑affected neocortex and hippocampus as evidenced by immunofluorescence staining and transmission electron microscopy. Additionally, we discovered that the exogenous rhEPO, which entered the cerebrum through intranasal route, activated the erythropoietin receptor (EPOR) and the downstream ERKs and PI3K/AKT signaling, and suppressed autophagy‑related degradation of nuclear factor, erythroid 2‑like 2 (Nrf2). Furthermore, the intranasal EPO‑exerted neuroprotection was almost abolished when the specific Nrf2 antagonist ATRA was administered intraperitoneally prior to intranasal EPO treatment. Collectively, our data demonstrated the repairing potential of EPO for the neurological disorders and neural pathology caused by chronic alcoholism, and identified the Nrf2 activity as the key mechanism mediating the protective effects of EPO.

Topics: Administration, Intranasal; Alcoholism; Animals; Autophagy; Disease Models, Animal; Erythropoietin; Hippocampus; Male; Mice; Mice, Inbred C57BL; Neurodegenerative Diseases; Neuroprotective Agents; NF-E2-Related Factor 2; Phosphatidylinositol 3-Kinases; Receptors, Erythropoietin; Signal Transduction

Retinal ischemia remains a common sight threatening end-point in blinding diseases such as diabetic retinopathy and retinopathy of prematurity. Endothelial colony forming cells (ECFCs) represent a subpopulation of endothelial progenitors with therapeutic utility for promoting reparative angiogenesis in the ischaemic retina. The current study has investigated the potential of enhancing this cell therapy approach by the dampening of the pro-inflammatory milieu typical of ischemic retina. Based on recent findings that ARA290 (cibinetide), a peptide based on the Helix-B domain of erythropoietin (EPO), is anti-inflammatory and tissue-protective, the effect of this peptide on ECFC-mediated vascular regeneration was studied in the ischemic retina.. The effects of ARA290 on pro-survival signaling and function were assessed in ECFC cultures in vitro. Efficacy of ECFC transplantation therapy to promote retinal vascular repair in the presence and absence of ARA290 was studied in the oxygen induced retinopathy (OIR) model of retinal ischemia. The inflammatory cytokine profile and microglial activation were studied as readouts of inflammation.. ARA290 activated pro-survival signaling and enhanced cell viability in response to H. Regulation of the pro-inflammatory milieu of the ischemic retina can be enhanced by ARA290 and may be a useful adjunct to ECFC-based cell therapy for ischemic retinopathies.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Endothelium, Vascular; Erythropoietin; Humans; Infant, Newborn; Ischemia; Mice; Mice, Inbred C57BL; Oligopeptides; Retinal Diseases; Retinal Vessels; Signal Transduction; Vasodilation

Erythropoietin (EPO) can enhance neurogenesis and fibroblasts can secrete growth factors; together, they may benefit ischemic stroke. We transplanted EPO-producing fibroblasts into the rodent infarcted brain to test their effect on neurogenesis and functional recovery.. The MCAO rats with EPO/EGFP/3T3 treatment showed high EPO expression in the infarcted brain for at least 1 week. The concentration of brain-derived neurotrophic factor was higher in both hemispheres of MCAO rats with either EGFP/3T3 or EPO/EGFP/3T3 treatment at 14 days poststroke compared with untreated MCAO rats. The number of Ki-67-, nestin-, or doublecortin-immunoreactive cells in bilateral subventricular zones was higher in EPO/EGFP/3T3-treated MCAO rats than it was in untreated MCAO control animals, indicating the enhancement of neurogenesis after EPO/EGFP/3T3 treatment. Notably, post-MCAO EPO/EGFP/3T3 treatment significantly reduced infarct size and improved functional recovery.. The intracerebral transplantation of EPO-producing fibroblasts benefited an ischemic stroke model probably via the enhancement of neurogenesis.

Topics: Animals; Brain Ischemia; Cell- and Tissue-Based Therapy; Disease Models, Animal; Doublecortin Protein; Erythropoietin; Fibroblasts; Male; Neurogenesis; Rats; Recovery of Function; Stroke; Treatment Outcome

This study aimed to characterize the pathophysiology, including possible correlations, of clock gene expression and erythropoietin (EPO) production in the acute stage of blood hemorrhage. Specimens of human cortical tissues (right and left kidneys) and cardiac blood were collected at autopsy from 52 cases following mortality due to acute-stage blood hemorrhage following sharp instrument injury. BMAL1 and PER2 mRNA levels were determined by reverse transcription-polymerase chain reaction; BMAL1 and PER2 protein levels were assessed using immunohistochemistry; BMAL1 protein levels were quantitatively measured by western blotting; and serum EPO levels were measured by chemiluminescent enzyme immunoassay. Separately, a rat model of hemorrhagic conditions was generated and used to confirm the results obtained with autopsy-derived specimens. A positive correlation was observed between BMAL1 protein and serum EPO levels, but not between BMAL1 mRNA levels and serum EPO levels. We also noted that Per2 mRNA expression became elevated in humans who survived for > 3 h after acute hemorrhagic events, with subsequent decreases in serum EPO levels. The rat model showed that even short (30-min) intervals of blood loss yielded increases in both Bmal1 mRNA and serum EPO levels; longer (60-min) intervals resulted in increases in Per2 mRNA expression along with decreases in serum EPO. Thus, the acute-stage human hemorrhage cases and the rat hemorrhage model yielded similar tendencies for clock gene expression and EPO secretion. In conclusion, our results indicated that clock genes are involved in the regulation of EPO production during the early stages of hypoxia/ischemia resulting from the acute hemorrhagic events.

Topics: Acute Disease; Animals; ARNTL Transcription Factors; Disease Models, Animal; Erythropoietin; Gene Expression; Hemorrhage; Humans; Male; Period Circadian Proteins; Postmortem Changes; Rats, Sprague-Dawley; RNA, Messenger; Shock, Hemorrhagic; Time Factors

IL-17-producing CD4+ cells (TH17) are pathogenically linked to autoimmunity including to autoimmune kidney disease. Erythropoietin's (EPO) newly recognized immunoregulatory functions and its predominant intra-renal source suggested that EPO physiologically regulates TH17 differentiation, thereby serving as a barrier to the development of autoimmune kidney disease. Using in vitro studies of human and murine cells and in vivo models, we show that EPO ligation of its receptor (EPO-R) on CD4+ T cells directly inhibits TH17 generation and promotes trans-differentiation of TH17 into IL-17-FOXP3+CD4+ T cells. Mechanistically, EPO/EPO-R ligation abrogates upregulation of SGK1 gene expression and blocks p38 activity to prevent SGK1 phosphorylation, thereby inhibiting RORC-mediated transcription of IL-17 and IL-23 receptor genes. In a murine model of TH17-dependent aristolochic acid (ArA)-induced, interstitial kidney disease associated with reduced renal EPO production, we demonstrate that transgenic EPO overexpression or recombinant EPO (rEPO) administration limits TH17 formation and clinical/histological disease expression. EPO/EPO-R ligations on CD4+ T cells abrogate, while absence of T cell-expressed EPO-R augments, TH17 induction and clinical/histological expression of pristane-induced glomerulonephritis (associated with decreased intrarenal EPO). rEPO prevents spontaneous glomerulonephritis and TH17 generation in MRL-lpr mice. Together, our findings indicate that EPO physiologically and therapeutically modulate TH17 cells to limit expression of TH17-associated autoimmune kidney disease.

Topics: Animals; Aristolochic Acids; Cells, Cultured; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Female; Humans; Immediate-Early Proteins; Interleukin-17; Lupus Nephritis; Male; Mice; Mice, Inbred MRL lpr; Mice, Transgenic; Nephritis, Interstitial; Phosphorylation; Primary Cell Culture; Protein Serine-Threonine Kinases; Receptors, Erythropoietin; Receptors, Interleukin; Severity of Illness Index; T-Lymphocytes, Regulatory; Th17 Cells

Topics: Animals; Animals, Newborn; Apoptosis; Bronchopulmonary Dysplasia; Cell Movement; Cell Proliferation; Chemokine CXCL12; Disease Models, Animal; Endothelium, Vascular; Erythropoietin; Lung; Mesenchymal Stem Cells; Mice; Neovascularization, Physiologic; Receptors, CXCR4; Vascular Endothelial Growth Factor A

Hypoxic-ischemic encephalopathy (HIE) is a common disease that occurs during the perinatal period. The primary cause of neonatal HIE is related to fetal intrauterine anoxia. Carbamylated erythropoietin (CEPO), a derivative of erythropoietin (EPO), does not exert any erythropoietic effect; however, the neuroprotective effects resemble those of EPO. Previous studies have shown the potential benefits of CEPO on the central nervous system. The present study aimed to investigate the role of CEPO in neuronal apoptosis during intrauterine HIE and the underlying mechanisms.. To validate our hypothesis, we established an intrauterine HIE model by occluding the bilateral utero-ovarian arteries of pregnant Sprague-Dawley rats. Compared to the I/R group, neuronal apoptosis in the CEPO group was significantly lower at 4, 12, 24, and 48 h (P < 0.05). CEPO significantly inhibited CC3 expression (P < 0.05) during the early-stages after ischemia-reperfusion (0.5, 4, 8, 12 and 24 h), upregulated Bcl-2 expression, and downregulated Bax expression at 4, 8, 12, and 24 h (P < 0.05).. Carbamylated erythropoietin pretreatment inhibited the expression of proapoptotic protein CC3 in brain and regulated the Bcl-2/Bax ratio, resulting in reduced neuronal apoptosis and thus resulting in a protective effect on intrauterine HIE.

Topics: Animals; Apoptosis; Disease Models, Animal; Erythropoietin; Female; Hypoxia-Ischemia, Brain; Neuroprotective Agents; Pregnancy; Rats; Rats, Sprague-Dawley; Time Factors

Since the nature of ischemia/reperfusion (IR)-induced tissue damage is multifactorial and complex, in the current study, the effects of multiple treatment strategies via concomitant administration of erythropoietin (EPO) and N-acetylcysteine (NAC) with an ischemic preconditioning (IPC) regimen on renal IR injury were examined.. Thirty male Wistar rats were subjected to bilateral occlusion of the renal pedicles for 50 min followed by reperfusion. EPO (1000 IU/kg) was administered for 3 days, as well as IPC before the IR and NAC (150 mg/kg) administration for 4 days after IR. The animals were randomly allocated into 6 groups (n = 5): sham, IR, EPO+IR, IPC+IR, NAC+IR, and EPO+IPC+NAC+IR. Kidney tissues and blood samples were obtained for oxidative stress, proinflammatory cytokines, and renal functional evaluations.. IR caused significant inflammatory response, oxidative stress, and reduced renal function. Treatment with EPO, IPC, and NAC or a combination of two of them attenuated renal dysfunction and reduced the oxidative stress and inflammatory markers. Rats treated with the combination of EPO, IPC, and NAC showed a higher degree of protection compared to the other groups.. These results showed that concomitant administration of EPO and IPC along with posttreatment NAC may have additive beneficial effects on kidney IR injury during IR-induced acute renal failure.

Topics: Acetylcysteine; Animals; Blood Urea Nitrogen; Creatinine; Cytokines; Disease Models, Animal; Erythropoietin; Ischemic Preconditioning; Kidney; Kidney Diseases; Male; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury

Using mouse model of regeneration of critical size cranial defects, we studied combined effect of 1 and 10 μg of BMP-2 of prokaryotic origin and recombinant erythropoietin (Epostim) injected subcutaneously in the area of bone defect in a total dose of 6000 U/kg. Erythropoietin considerably improved quantitative and qualitative characteristics of the bone tissue in the site of implantation when used in combination with BMP-2 in both concentrations.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Regeneration; Disease Models, Animal; Erythropoietin; Male; Mice; Mice, Inbred ICR; Skull

The erythroid growth factor erythropoietin (Epo) is produced by renal interstitial fibroblasts, called REP (renal Epo-producing) cells, in a hypoxia-inducible manner. In chronic kidney disease (CKD), REP cells lose their Epo-production ability, leading to renal anaemia. Concurrently, REP cells are suggested to be transformed into myofibroblasts, which are the major player of renal fibrosis. Although establishment of cultured cell lines derived from REP cells has been a long-term challenge, we here successfully established a REP-cell-derived immortalized and cultivable cell line (Replic cells) by using a genetically modified mouse line. Replic cells exhibited myofibroblastic phenotypes and lost their Epo-production ability, reflecting the situation in renal fibrosis. Additionally, we found that cell-autonomous TGFβ signalling contributes to maintenance of the myofibroblastic features of Replic cells. Furthermore, the promoters of genes for Epo and HIF2α, a major activator of Epo gene expression, were highly methylated in Replic cells. Thus, these results strongly support our contention that REP cells are the origin of myofibroblasts in fibrotic kidneys and demonstrate that cell-autonomous TGFβ signalling and epigenetic silencing are involved in renal fibrosis and renal anaemia, respectively, in CKD. The Replic cell line is a useful tool to further investigate the molecular mechanisms underlying renal fibrosis.

Topics: Anemia; Animals; Cell Line; Disease Models, Animal; Embryo, Mammalian; Epigenesis, Genetic; Erythropoietin; Fibroblasts; Fibrosis; Humans; Kidney; Male; Mice; Mice, Transgenic; Myofibroblasts; Renal Insufficiency, Chronic; Signal Transduction; Transforming Growth Factor beta1

Vascular dementia is the second most common cause of dementia in older people and is characterized by the sudden onset of impairments in thinking skills and behavior, which generally occur following a stroke. Unfortunately, effective therapy for vascular dementia remains inadequate. Erythropoietin (EPO) is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. Recently, a prominent role for EPO has been defined in the nervous system, and there is growing interest in the potential therapeutic use of EPO for neuroprotection. However, whether it is protective from memory impairments and the underlying mechanisms of vascular dementia (VD) remains unknown. In the current study, we reported that supplements with exogenous erythropoietin (EPO) for 4 weeks could restore impaired memory in 2-vessel occlusion (2VO) rats, a well-established vascular dementia animal model. EPO also rescued impairments in dendritic spines and cholinergic dysfunctions in the hippocampus. Moreover, EPO suppressed the overactivation of GSK-3β in the hippocampus by stimulating the JAK2/STAT5/PI3K/Akt signal pathway. Furthermore, we found that genetic knockdown of the EPO receptor (EPO-R) by shRNA blocks the neuroprotection conferred by EPO on memory in VD. We hypothesized that EPO treatment is able to rescue the memory impairments in VD by stimulating the EPO-R/JAK2/STAT5/PI3K/Akt/GSK-3β pathway and suggest the potential usage of EPO in the therapy for VD.

Topics: Animals; Brain; Cerebrovascular Disorders; Cholinergic Neurons; Chronic Disease; Dendritic Spines; Disease Models, Animal; Erythropoietin; Fear; Gene Knockout Techniques; Glycogen Synthase Kinase 3 beta; Janus Kinase 2; Male; Memory Disorders; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats, Wistar; Receptors, Erythropoietin; Signal Transduction; Spatial Memory; STAT5 Transcription Factor

There was no effective measures can be obtained at present to reverse or prevent airway remodeling. We investigated the therapeutic effect of Erythropoietin (EPO) gene modified mesenchymal stem cells (MSCs) on asthmatic airway remodeling and the possible underlied molecular mechanisms. EPO gene was transfected into MSCs via lentivirus vector. The transfected cells (EPO-MSCs) were identified by flow cytometry and the EPO secreting function was detected by PCR and Western blot. MSCs or EPO-MSCs were administrated to albumin (OVA)-induced chronic asthmatic mouse model via tail veins. The asthmatic phenotype was analyzed. Number of cells in bronchoalveolar lavage fluid (BALF) was counted using a hemocytometer. Histological findings of airways were evaluated by microscopic examination. The concentrations of interleukin 4(IL-4), interleukin 5(IL-5), and interleukin 13(IL-13) in lung homogenate were determined by ELISA. The activation state of transforming growth factor-β 1 (TGF-β1), Transforming growth factor beta-activated kinase 1 (TAK1), and p38 Mitogen Activated Protein Kinase (p38MAPK) signaling was detected by Real-Time PCR and Western blotting. EPO-MSCs were successfully constructed. EPO-MSCs showed a more potently suppressive effect on local asthmatic airway inflammation and the level of IL-4, IL-5, and IL-13 in lung tissue than MSCs. Moreover, the numbers of goblet cells, the thicknesses of smooth muscle layer, collagen density, percentage of proliferating cell nuclear antigen positive (PCNA

Topics: Airway Remodeling; Animals; Asthma; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Genetic Therapy; Interleukins; Lentivirus; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C

Erythropoietin (Epo) promotes functional recovery following spinal cord injury (SCI); however, the exact underlying mechanisms are yet to be determined. Although endogenous neural stem cells (NSCs) in the adult spinal cord are a therapeutic target in SCI models, the effect of Epo on this NSC population remains unknown. The present study investigated the effects of Epo on endogenous NSCs in the adult spinal cord both in vitro and in vivo. For the in vivo analyses, normal rats (Normal) and SCI contusion model rats (SCI) received either recombinant human Epo or saline treatment for 7 days (5,000 U/kg), and spinal cords were subsequently analyzed at 2, 8, and 14 days. For in vitro analyses, NSCs harvested from adult rat spinal cords were exposed to Epo (10 U/ml). A significant increase in β‑tubulin+ new neurons (P<0.01) was observed at all three time points and O4+ new oligodendrocytes (P<0.05) at days 8 and 14 in the SCI+Epo group compared with the SCI+Saline group. This was concomitant with a prolonged activation of Epo signaling; however, no effect on NSCs proliferation was observed. Similar results were also obtained in vitro. Motor functional recovery was also noted at days 8 and 14 only in the Epo‑treated SCI rats. Although the expression of Epo and EpoR significantly increased in Normal+Epo rats compared with Normal+Saline rats (P<0.05), the cell numbers and phenotype were comparable between the two groups. To the best of the author's knowledge, this is the first study to demonstrate that Epo signaling promotes both neurogenesis and oligodendrogenesis following SCI and that these may represent the underlying mechanisms for the functional recovery and therapeutic effects of Epo following SCI.

Topics: Animals; Cell Differentiation; Disease Models, Animal; Erythropoietin; Female; Humans; Motor Activity; Neural Stem Cells; Neurogenesis; Oligodendroglia; Rats; Receptors, Erythropoietin; Recovery of Function; Regeneration; Spinal Cord Injuries

This study was designed to investigate the protective effects of centrally administered erythropoietin (EPO) on brain oxidative stress and inflammatory markers to protect the kidneys during hemorrhagic shock (HS).. Animals were assigned into three groups (n=6). Sham rats were subjected to cannulation of femoral artery and vein as well as stereotaxic surgery. In HS group, 50% of total blood volume was withdrawn and resuscitation was started 2h later. In EPO group, stereotaxic surgery in lateral ventricle was performed one week before induction of HS for administration of EPO (2IU) just before resuscitation. Plasma samples, kidney and brain tissues were allocated after a further 3h in all animals.. There was a significant increase in survival rate in the EPO group (69.3%) compared to the HS group (35.7%). Brain EPO administration significantly attenuated the rises in BUN, plasma Cr and NGAL, brain and renal MDA content and also increased SOD activity in the kidney and brain compared to the HS group. Brain, plasma and kidney TNF-α and IL-6 levels were significantly reduced by EPO compared to HS group. EPO increased the phosphorylation of Akt on Ser473 and eNOS mRNA expression in the kidney tissue compared to the HS group.. In conclusion, centrally administered EPO reduced pro-inflammatory and oxidative stress indices in the kidney and reduced apoptosis by activation of the Akt/eNOS signaling pathway. Hence, it can be hypothesized that EPO may play a major role in the central regulation of renal system as a neuromodulator.

Topics: Animals; Apoptosis; Brain; Disease Models, Animal; Erythropoietin; Hemorrhage; Kidney; Male; Nitric Oxide Synthase Type III; Oxidative Stress; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Shock, Hemorrhagic; Signal Transduction

The erythropoietin receptor (EpoR) is widely expressed but its renoprotective action is unexplored. To examine the role of EpoR in vivo in the kidney, we induced acute kidney injury (AKI) by ischemia-reperfusion in mice with different EpoR bioactivities in the kidney. EpoR bioactivity was reduced by knockin of wild-type human EpoR, which is hypofunctional relative to murine EpoR, and a renal tubule-specific EpoR knockout. These mice had lower EPO/EpoR activity and lower autophagy flux in renal tubules. Upon AKI induction, they exhibited worse renal function and structural damage, more apoptosis at the acute stage (<7 days), and slower recovery with more tubulointerstitial fibrosis at the subacute stage (14 days). In contrast, mice with hyperactive EpoR signaling from knockin of a constitutively active human EpoR had higher autophagic flux, milder kidney damage, and better renal function at the acute stage but, surprisingly, worse tubulointerstitial fibrosis and renal function at the subacute stage. Either excess or deficient EpoR activity in the kidney was associated with abnormal peritubular capillaries and tubular hypoxia, creating a "U-shaped" relationship. The direct effects of EpoR on tubular cells were confirmed in vitro by a hydrogen peroxide model using primary cultured proximal tubule cells with different EpoR activities. In summary, normal erythropoietin (EPO)/EpoR signaling in renal tubules provides defense against renal tubular injury maintains the autophagy-apoptosis balance and peritubular capillary integrity. High and low EPO/EpoR bioactivities both lead to vascular defect, and high EpoR activity overides the tubular protective effects in AKI recovery.

Topics: Acute Kidney Injury; Animals; Apoptosis; Autophagy; Capillaries; Cell Hypoxia; Cells, Cultured; Disease Models, Animal; Erythropoietin; Fibrosis; Humans; Kidney Tubules, Proximal; Mice, 129 Strain; Mice, Transgenic; Neovascularization, Physiologic; Receptors, Erythropoietin; Signal Transduction

Children who are born preterm are at risk for encephalopathy of prematurity, a leading cause of cerebral palsy, cognitive delay and behavioral disorders. Current interventions are limited and none have been shown to reverse cognitive and behavioral impairments, a primary determinant of poor quality of life for these children. Moreover, the mechanisms of perinatal brain injury that result in functional deficits and imaging abnormalities in the mature brain are poorly defined, limiting the potential to target interventions to those who may benefit most. To determine whether impairments are reversible after a prenatal insult, we investigated a spectrum of functional deficits and diffusion tensor imaging (DTI) abnormalities in young adult animals. We hypothesized that prenatal transient systemic hypoxia-ischemia (TSHI) would induce multiple functional deficits concomitant with reduced microstructural white and gray matter integrity, and tested whether these abnormalities could be ameliorated using postnatal erythropoietin (EPO), an emerging neurorestorative intervention. On embryonic day 18 uterine arteries were transiently occluded for 60min via laparotomy. Shams underwent anesthesia and laparotomy for 60min. Pups were born and TSHI pups were randomized to receive EPO or vehicle via intraperitoneal injection on postnatal days 1 to 5. Gait, social interaction, olfaction and open field testing was performed from postnatal day 25-35 before brains underwent ex vivo DTI to measure fractional anisotropy, axial diffusivity and radial diffusivity. Prenatal TSHI injury causes hyperactivity, impaired gait and poor social interaction in young adult rats that mimic the spectrum of deficits observed in children born preterm. Collectively, these data show for the first time in a model of encephalopathy of prematurity that postnatal EPO treatment mitigates impairments in social interaction, in addition to gait deficits. EPO also normalizes TSHI-induced microstructural abnormalities in fractional anisotropy and radial diffusivity in multiple regions, consistent with improved structural integrity and recovery of myelination. Taken together, these results show behavioral and memory deficits from perinatal brain injury are reversible. Furthermore, resolution of DTI abnormalities may predict responsiveness to emerging interventions, and serve as a biomarker of CNS injury and recovery.

Topics: Animals; Animals, Newborn; Brain; Diffusion Tensor Imaging; Disease Models, Animal; Embryo, Mammalian; Erythropoietin; Exploratory Behavior; Female; Gait Disorders, Neurologic; Gene Expression Regulation, Developmental; Hindlimb; Hypoxia-Ischemia, Brain; Interpersonal Relations; Male; Olfaction Disorders; Pregnancy; Prenatal Injuries; Rats; Rats, Sprague-Dawley; Sex Factors; Social Behavior Disorders

Burn-induced neuromuscular dysfunction may contribute to long-term morbidity; therefore, it is imperative to develop novel treatments. The present study investigated whether erythropoietin (EPO) administration attenuates burn-induced motor neuron apoptosis and neuroinflammatory response. To validate our hypothesis, a third-degree hind paw burn rat model was developed by bringing the paw into contact with a metal surface at 75°C for 10 s. A total of 24 male Sprague-Dawley rats were randomly assigned to four groups: Group A, sham-control; Group B, burn-induced; Group C, burn + single EPO dose (5000 IU/kg i.p. at D0); and Group D, burn + daily EPO dosage (3000 IU/kg/day i.p. at D0-D6). Two treatment regimens were used to evaluate single versus multiple doses treatment effects. Before sacrifice, blood samples were collected for hematological parameter examination. The histological analyses of microglia activation, iNOS, and COX-2 in the spinal cord ventral horn were performed at week 1 post-burn. In addition, we examined autophagy changes by biomarkers of LC3B and ATG5. The expression of BCL-2, BAX, cleaved caspase-3, phospho-AKT, and mTOR was assessed simultaneously through Western blotting. EPO administration after burn injury attenuated neuroinflammation through various mechanisms, including the reduction of microglia activity as well as iNOS and COX-2 expression in the spinal cord ventral horn. In addition, the expression of phospho-AKT, mTOR and apoptotic indicators, such as BAX, BCL-2, and cleaved caspase-3, was modulated. Furthermore, the activity of burn-induced autophagy in the spinal cord ventral horn characterized by the expression of autophagic biomarkers, LC3B and ATG5, was reduced after EPO administration. The present results indicate that EPO inhibits the AKT-mTOR pathway to attenuate burn-induced motor neuron programmed cell death and microglia activation. EPO can modulate neuroinflammation and programmed cell death and may be a therapeutic candidate for neuroprotection.

Topics: Animals; Apoptosis; Burns; Disease Models, Animal; Erythrocyte Count; Erythropoietin; Hematocrit; Immunohistochemistry; Male; Motor Neurons; Rats; Rats, Sprague-Dawley

Erythropoietin (EPO) is recognized for neuroprotective and angiogenic effects and has been associated with aging and neovascular age-related macular degeneration (AMD). We hypothesized that systemic EPO facilitates the development of choroidal neovascularization (CNV). Wild type mice expressed murine EPOR (mWtEPOR) in RPE/choroids at baseline and had significantly increased serum EPO after laser treatment. To test the role of EPO signaling, we used human EPOR knock-in mice with the mWtEPOR gene replaced by either the human EPOR gene (hWtEPOR) or a mutated human EPOR gene (hMtEPOR) in a laser-induced choroidal neovascularization (LCNV) model. Loss-of-function hWtEPOR mice have reduced downstream activation, whereas gain-of-function hMtEPOR mice have increased EPOR signaling. Compared to littermate controls (mWtEPOR), hMtEPOR with increased EPOR signaling developed larger CNV lesions. At baseline, hMtEPOR mice had increased numbers of macrophages, greater expression of macrophage markers F4/80 and CD206, and following laser injury, had greater expression of cytokines CCL2, CXCL10, CCL22, IL-6, and IL-10 than mWtEPOR controls. These data support a hypothesis that injury from age- and AMD-related changes in the RPE/choroid leads to choroidal neovascularization through EPOR-mediated cytokine production.

Topics: Animals; Cells, Cultured; Choroid; Choroidal Neovascularization; Cytokines; Disease Models, Animal; Erythropoietin; Female; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptors, Erythropoietin; Signal Transduction

It is accepted that osteoblasts/osteocytes are the major source for circulating fibroblast growth factor 23 (FGF23). However, erythropoietic cells of bone marrow also express FGF23. The modulation of FGF23 expression in bone marrow and potential contribution to circulating FGF23 has not been well studied. Moreover, recent studies show that plasma FGF23 may increase early during acute kidney injury (AKI). Erythropoietin, a kidney-derived hormone that targets erythropoietic cells, increases in AKI. Here we tested whether an acute increase of plasma erythropoietin induces FGF23 expression in erythropoietic cells of bone marrow thereby contributing to the increase of circulating FGF23 in AKI. We found that erythroid progenitor cells of bone marrow express FGF23. Erythropoietin increased FGF23 expression in vivo and in bone marrow cell cultures via the homodimeric erythropoietin receptor. In experimental AKI secondary to hemorrhagic shock or sepsis in rodents, there was a rapid increase of plasma erythropoietin, and an induction of bone marrow FGF23 expression together with a rapid increase of circulating FGF23. Blockade of the erythropoietin receptor fully prevented the induction of bone marrow FGF23 and partially suppressed the increase of circulating FGF23. Finally, there was an early increase of both circulating FGF23 and erythropoietin in a cohort of patients with severe sepsis who developed AKI within 48 hours of admission. Thus, increases in plasma erythropoietin and erythropoietin receptor activation are mechanisms implicated in the increase of plasma FGF23 in AKI.

Topics: Acute Kidney Injury; Animals; Bone Marrow Cells; Disease Models, Animal; Erythroid Precursor Cells; Erythropoietin; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Humans; Male; Mice, Inbred C57BL; Prospective Studies; Rats, Sprague-Dawley; Receptors, Erythropoietin; Recombinant Proteins; Sepsis; Shock, Hemorrhagic; Time Factors; Up-Regulation

Multiple sclerosis (MS) is a neuro-inflammatory and demyelinating disease. Downregulation of neuronal mitochondrial gene expression and activity have been reported in several studies of MS. We have previously shown that hemoglobin-β (Hbb) signals to the nucleus of neurons and upregulates H3K4me3, a histone mark involved in regulating cellular metabolism and differentiation. The present study was undertaken to evaluate the effect of erythropoietin (EPO) on the upregulation of hemoglobin and mitochondrial-associated neuroprotection. We found that administering EPO (5000 IU/kg intraperitoneally) to mice upregulated brain Hbb expression, levels of H3K4me3, expression of mitochondrial complex III, complex V, and mitochondrial respiration. We also found that the neuronal mitochondrial metabolite N-acetylaspartate (NAA), a marker of neuronal mitochondrial activity, was increased with EPO treatment. Further, we measured the effects of EPO on preventing mitochondrial deficits in the cuprizone toxic demyelinating mouse model of MS. We found that EPO prevented cuprizone-mediated decreases in Hbb, complex III, and NAA. Our data suggest that EPO mediated regulation of Hbb supports neuronal energetics and may provide neuroprotection in MS and other neurodegenerative diseases where a dysfunction of mitochondria contributes to disease.

Topics: Animals; Aspartic Acid; Brain; Cell Respiration; Cuprizone; Disease Models, Animal; Electron Transport Complex III; Erythropoietin; Hemoglobins; Histones; Lysine; Male; Methylation; Mice, Inbred C57BL; Mitochondria; Models, Biological; Multiple Sclerosis; Myelin Basic Protein; Myelin Sheath; Neurons; Up-Regulation

Kidneys are physiologically hypoxic due to huge oxygen consumption for tubular reabsorption. The physiological hypoxia makes the kidney an appropriate organ for sensitively detecting oxygen levels and producing erythropoietin (EPO). In preterm neonates, immature kidneys cannot produce sufficient EPO, which results in anemia of prematurity (AOP). The cause of EPO insufficiency in AOP has been unclear, therefore current therapeutic options are transfusion and injection of recombinant human EPO. This report shows that the cause of insufficient EPO production in AOP is elevated renal oxygen levels due to poor oxygen consumption by immature tubules. Neonatal mice with AOP showed low tubular transporter expression and elevated renal oxygen levels compared with those without AOP. Enhancing transporter expression in AOP mice induced renal hypoxia and EPO production. In preterm neonates, red blood cell counts, hemoglobin levels, and hematocrit levels correlated with tubular function, but not with serum creatinine, gestational age, or birth weight. Furthermore, pharmacological upregulation of hypoxia signaling ameliorated AOP in mice. These data suggest that tubular maturation with increased oxygen consumption is required for renal EPO production.

Topics: Anemia, Neonatal; Animals; Animals, Newborn; Cell Hypoxia; Creatinine; Disease Models, Animal; Erythropoietin; Humans; Infant, Newborn; Kidney; Mice; Oxygen; Oxygen Consumption

Systemic lupus erythematosus (SLE) is an autoimmune disease, which results in various organ pathologies. However, current treatment towards SLE is suboptimal. Erythropoietin (EPO) has been shown to promote SLE recovery, but clinical application can be limited by its haematopoiesis-stimulating effects. EPO-derived helix-B peptide (ARA290) is non-erythrogenic but has been reported to retain the anti-inflammatory and tissue-protective functions of EPO. Therefore, here we investigated the effects and potential mechanisms of ARA290 on SLE. The administration of ARA290 to pristane-induced SLE and MRL/lpr mice significantly suppressed the level of serum antinuclear autoantibodies (ANAs) and anti-dsDNA autoantibodies, reduced the deposition of IgG and C3, and ameliorated the nephritis symptoms. Moreover, the serum concentrations of inflammatory cytokine IL-6, MCP-1 and TNF-α in SLE mice were reduced by ARA290. Further, ARA290 decreased the number of apoptotic cells in kidney. In vitro experiment revealed that ARA290 inhibited the inflammatory activation of macrophages and promoted the phagocytotic function of macrophages to apoptotic cells. Finally, ARA290 did not induce haematopoiesis during treatment. In conclusion, ARA290 ameliorated SLE, which at least could be partly due to its anti-inflammatory and apoptotic cell clearance promoting effects, without stimulating haematopoiesis, suggesting that ARA290 could be a hopeful candidate for SLE treatment.

Topics: Animals; Cytokines; Disease Models, Animal; Erythropoietin; Female; Hematopoiesis; Inflammation; Kidney; Lupus Erythematosus, Systemic; Macrophage Activation; Mice; Mice, Inbred C57BL; Mice, Inbred MRL lpr; Oligopeptides; Phagocytosis; RAW 264.7 Cells; Terpenes

Major hepatic resection of a cirrhotic liver may result in a fatal clinical course. Preoperative erythropoietin (EPO) treatment has been shown to have protective properties and to stimulate liver regeneration. This study aims to investigate the effect of preoperative EPO on survival following major hepatic resection in a cirrhotic rat model.. Cirrhotic liver was induced by intraperitoneal injection of thioacetamide (200mg/kg/mL) in 72 Lewis rats. Each 36 rats received EPO (1IU/g, every second day, 5 times preoperatively) or saline (control) and major hepatectomy (removal of the left and half of the median lobe) was performed. Biochemical and immunohistochemical parameters, cytokines and overall survival were compared following surgery.. Rats that received preoperative EPO had decreased hepatic aspartate aminotransferase, alanine aminotransferase and interleukin (IL)-1β expression, 48hours following surgery. They had increased hepatocyte growth factor and vascular endothelial growth factor expression at 1hour, increased IL-6 expression at 24, 48 and 120hours and increased Ki-67, 120hours following surgery. Overall, survival was significantly improved among EPO-treated rats (P=0.034).. Preoperative EPO treatment has a protective effect and stimulates liver regeneration, leading to improved overall survival following major hepatectomy in a cirrhotic rat model.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Disease Models, Animal; Erythropoietin; Hepatectomy; Hepatocyte Growth Factor; Interleukin-1beta; Interleukin-6; Ki-67 Antigen; Liver Cirrhosis, Experimental; Liver Regeneration; Premedication; Rats, Inbred Lew; RNA, Messenger; Survival Analysis; Vascular Endothelial Growth Factor A

Autophagy has been regarded as a promising therapeutic target for spinal cord injury (SCI). Erythropoietin (EPO) has been demonstrated to exhibit neuroprotective effects in the central nervous system (CNS); however, the molecular mechanisms of its protection against SCI remain unknown. This study aims to investigate whether the neuroprotective effects of EPO on SCI are mediated by autophagy via AMP-activated protein kinase (AMPK) signaling pathways.. Functional assessment and Nissl staining were used to investigate the effects of EPO on SCI. Expressions of proteins were detected by Western blot and immunohistochemistry.. Treatment with EPO significantly reduced the loss of motor neurons and improved the functional recovery following SCI. Erythropoietin significantly enhanced the SCI-induced autophagy through activating AMPK and inactivating mTOR signaling. The inhibitor of AMPK, compound C, could block the EPO-induced autophagy and beneficial action on SCI, whereas the activator of AMPK, metformin, could mimic the effects of EPO. In the in vitro studies, EPO enhanced the hypoxia-induced autophagy in an AMPK-dependent manner.. The AMPK-dependent induction of autophagy contributes to the neuroprotection of EPO on SCI.

Topics: AMP-Activated Protein Kinases; Animals; Autophagy; Cell Hypoxia; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Erythropoietin; Glucose; Locomotion; Male; Microtubule-Associated Proteins; Neuroprostanes; PC12 Cells; Rats; Rats, Sprague-Dawley; Recovery of Function; Signal Transduction; Spinal Cord Injuries

Erythropoietin may have neuroprotective potential after ischemia of the central nervous system. Here, we conducted a study to characterize the protective effects of erythropoietin on retinal ganglion cells and gliotic reactions in an experimentally induced oligemia model.. Rats were subjected to global oligemia by bilateral common carotid artery occlusion and then received either vehicle or erythropoietin via intravitreal injection after 48 h; they were euthanized one week after the injection. The densities of retinal ganglion cells and contents of glial fibrillary acidic protein (astrocytes/Müller cells) and cluster of differentiation 68 clone ED1 (microglia/macrophages), assessed by fluorescence intensity, were evaluated in frozen retinal sections by immunofluorescence and epifluorescence microscopy.. Retinal ganglion cells were nearly undetectable one week after oligemia compared with the sham controls; however, these cells were partially preserved in erythropoietin-treated retinas. The contents of glial fibrillary acidic protein and cluster of differentiation 68 clone ED1, markers for reactive gliosis, were significantly higher in retinas after bilateral common carotid artery occlusion than those in both sham and erythropoietin-treated retinas.. The number of partially preserved retinal ganglion cells in the erythropoietin-treated group suggests that erythropoietin exerts a neuroprotective effect on oligemic/ischemic retinas. This effect could be related to the down-modulation of glial reactivity, usually observed in hypoxic conditions, clinically observed during glaucoma or retinal artery occlusion conditions. Therefore, glial reactivity may enhance neurodegeneration in hypoxic conditions, like normal-tension glaucoma and retinal ischemia, and erythropoietin is thus a candidate to be clinically applied after the detection of decreased retinal blood flow.

Topics: Animals; Carotid Artery Injuries; Carotid Artery, Common; Cell Count; Disease Models, Animal; Ectodysplasins; Erythropoietin; Glial Fibrillary Acidic Protein; Hematopoietic Cell Growth Factors; Male; Neuroprotective Agents; Rats, Wistar; Retinal Diseases; Retinal Ganglion Cells

Anemia is a known driver for hypoxia inducible factor (HIF) which leads to increased renal erythropoietin (EPO) synthesis. Bone marrow (BM) EPO receptor (EPOR) signals are transduced through a JAK2-STAT5 pathway. The origins of anemia of chronic kidney disease (CKD) are multifactorial, including impairment of both renal EPO synthesis as well as intestinal iron absorption. We investigated the HIF- EPO- EPOR axis in kidney, BM and proximal tibia in anemic juvenile CKD rats.. CKD was induced by 5/6 nephrectomy in young (20 days old) male Sprague-Dawley rats while C group was sham operated. Rats were sacrificed 4 weeks after CKD induction and 5 minutes after a single bolus of IV recombinant human EPO. An additional control anemic (C-A) group was daily bled for 7 days.. Hemoglobin levels were similarly reduced in CKD and C-A (11.4 ± 0.3 and 10.8±0.2 Vs 13.5±0.3 g/dL in C, p<0.0001). Liver hepcidin mRNA was decreased in CA but increased in CKD. Serum iron was unchanged while transferrin levels were mildly decreased in CKD. Kidney HIF2α protein was elevated in C-A but unchanged in CKD. Kidney EPO protein and mRNA levels were unchanged between groups. However, BM EPO protein (which reflects circulating EPO) was increased in C-A but remained unchanged in CKD. BM and proximal tibia EPOR were unchanged in C-A but decreased in CKD. Proximal tibial phospho-STAT5 increased after the EPO bolus in C but not in CKD.. Compared to blood loss, anemia in young CKD rats is associated with inappropriate responses in the HIF-EPO-EPO-R axis: kidney HIF2α and renal EPO are not increased, BM and bone EPOR levels, as well as bone pSTAT5 response to EPO are reduced. Thus, anemia of CKD may be treated with additional therapeutic avenues beyond iron and EPO supplementation.

Topics: Anemia; Animals; Basic Helix-Loop-Helix Transcription Factors; Bone Marrow; Disease Models, Animal; Erythropoietin; Hepcidins; Kidney; Liver; Male; Nephrectomy; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Recombinant Proteins; Renal Insufficiency, Chronic; RNA, Messenger; Signal Transduction; STAT5 Transcription Factor

Cognitive deficits are widespread in psychiatric disorders and frequently as debilitating as the affective component. Widely prescribed antidepressants for treating depressive disorders have limited efficacy in normalizing cognitive function. Erythropoietin (Epo) has been shown to improve cognitive function in schizophrenia and treatment resistant depressed patients. However, the potent elevation of red blood cell counts by Epo can cause hematological complications in non-anemic patients. We investigated a chemically engineered, posttranslational modification of Epo, carbamoylation, which renders it non-erythropoietic. We conducted mass-spectrometry-based peptide mapping of carbamoylated Epo (Cepo) and tested its ability to improve cognitive function after social defeat stress. Gene expression analysis in discrete brain regions was performed to obtain mechanistic insight of Cepo action. Cepo reversed stress-induced spatial working memory deficits while affecting long-term (24 h) novel object recognition in these rats. Contextual fear conditioning following defeat was enhanced by Cepo, but attenuated in controls. However, Cepo improved fear extinction in all rats compared to vehicle treatment. Cepo induced differential gene expression of BDNF, VGF, Arc, TH. and neuritin in the mPFC and discrete hippocampal subfields, with strongest induction in the dorsal hippocampus. Analysis of gene-brain region-behavior interactions showed that Cepo-induced neurotrophic mechanisms influence cognitive function. Carbamoylated erythropoietin can be developed as a therapeutic neurotrophic agent to treat cognitive dysfunction in neuropsychiatric diseases. Due to its distinct mechanism of action, it is unlikely to cross react with the activity of currently prescribed small molecule drugs and can be used as an add-on biologic drug.

Topics: Animals; Cognition; Cognitive Dysfunction; Disease Models, Animal; Erythropoietin; Female; Hippocampus; Male; Mass Spectrometry; Memory Disorders; Mice; Mice, Inbred BALB C; Protein Carbamylation; Psychological Tests; Rats; Rats, Sprague-Dawley; Spatial Memory; Stress, Psychological

Lipotoxicity plays an important role in the progression of chronic kidney damage via various mechanisms, such as endoplasmic reticulum stress. Several studies proposed renal lipotoxicity in glomerular and tubular cells but the effect of lipid on renal erythropoietin (EPO)-producing (REP) cells in the interstitium has not been elucidated. Since renal anemia is caused by derangement of EPO production in REP cells, we evaluated the effect of palmitate, a representative long-chain saturated fatty acid, on EPO production and the endoplasmic reticulum stress pathway. EPO production was suppressed by palmitate (palmitate-conjugated bovine serum albumin [BSA]) or a high palmitate diet, but not oleic acid-conjugated BSA or a high oleic acid diet, especially under cobalt-induced pseudo-hypoxia both in vitro and in vivo. Importantly, suppression of EPO production was not induced by a decrease in transcription factor HIF activity, while it was significantly associated with endoplasmic reticulum stress, particularly transcription factor ATF4 activation, which suppresses 3'-enhancer activity of the EPO gene. ATF4 knockdown by siRNA significantly attenuated the suppressive effect of palmitate on EPO production. Studies utilizing inherited super-anemic mice (ISAM) mated with EPO-Cre mice (ISAM-REC mice) for lineage-labeling of REP cells showed that ATF4 activation by palmitate suppressed EPO production in REP cells. Laser capture microdissection confirmed ATF4 activation in the interstitial area of ISAM-REC mice treated with palmitate-conjugated BSA. Thus, endoplasmic reticulum stress induced by palmitate suppressed EPO expression by REP cells in a manner independent of HIF activation. The link between endoplasmic reticulum stress, dyslipidemia, and hypoxia may contribute to development and progression of anemia in CKD.

Topics: Activating Transcription Factor 4; Anemia; Animals; Basic Helix-Loop-Helix Transcription Factors; Diet, High-Fat; Disease Models, Animal; Endoplasmic Reticulum Stress; Erythropoietin; Gene Knockdown Techniques; Hep G2 Cells; Humans; Kidney; Laser Capture Microdissection; Male; Mice; Mice, Inbred C57BL; Palmitates; Renal Insufficiency, Chronic; RNA, Small Interfering; Unfolded Protein Response

Congenital urinary tract obstruction is one of the most frequent malformations in fetuses or neonates, which usually causes profound impairment of renal function including reductions in both glomerular filtration rate (GFR) and tubular handling of water and solutes. Although obstruction can be released by surgical operation, the child will suffer from diuresis for sometime. It has been reported that erythropoietin (EPO) could prevent the down-regulation of aquaporin-2 (AQP2) and urinary-concentrating defects induced by renal ischemia/reperfusion (I/R) injury. However, whether EPO could promote the recovery of renal function and AQP2 expression after releasing of ureteral obstruction has not been reported yet. The purposes of the present study were to investigate the effects of EPO on renal function and AQP2 expression after release of bilateral ureteral obstruction (BUO-R) in rats. The results showed that EPO could promote interleukin (IL) 10 (IL-10) expression; inhibit tumor necrosis factor-α (TNF-α), IL-6, and inducible nitric oxide synthase (iNOS) expressions; reduce the fractional excretion of sodium (FENa) and plasma creatinine (CREA) and urea; and promote the recovery of water and salt handling and AQP2 expression in BUO-R rats, especially in the high dose of EPO-treated group rats. In conclusion, EPO could promote the recovery of renal function and AQP2 expression in BUO-R rats, which may partially associate with its anti-inflammation effect.

Topics: Animals; Aquaporin 2; Disease Models, Animal; Erythropoietin; Glomerular Filtration Rate; Humans; Kidney; Male; Protective Agents; Rats, Sprague-Dawley; Reperfusion Injury; Ureteral Obstruction

In this study, we investigate a neuroprotective agent, erythropoietin (EPO), in animal hydrocephalus model and its potential reversal effects on hydrocephalus by altering the expression of aquaporin-4 (AQP4).. Obstructive hydrocephalus was induced in 2-week-old rat pups by injecting kaolin (50 μl, 10 mg/ml in saline) into the cisterna magna, while the control pups received only saline. Kaolin-injected pups were divided into two groups on the fifth day after kaolin injection; one group received intra-peritoneal (i.p.) EPO (1 μg/pup) for 5 consecutive days, while other group received i.p. saline for 5 days. The effects of EPO on hydrocephalus were investigated by studying cerebral ventricle size and structural ependymal changes. We examined also the EPO effects on AQP4 expression and microRNA expression.. EPO treatment significantly reduced dilation of the cerebral ventricle and denudation of ependymal line in hydrocephalic pups comparing with the control group. Increased expression of AQP4 in periventricular ependymal lining and cultured astrocytes and increased vascular formation were noted after EPO treatment. Additionally, we identified miR-668 as an endogenous regulator of AQP4 in response to EPO. Anti-miR-668 dampened EPO-induced activation of AQP4 expression.. Together, our results show that EPO-mediated upregulation of AQP4 significantly reduces dilation of the cerebral ventricles in obstructive hydrocephalus pups and may lead to potential therapeutic options for hydrocephalus.

Topics: Animals; Aquaporin 4; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Humans; Hydrocephalus; MicroRNAs; Neuroprotective Agents; Rats; Rats, Sprague-Dawley

Oral mucositis (OM) represents a therapeutic challenge frequently encountered in cancer patients undergoing chemotherapy or radiotherapy. Erythropoietin (EPO) has anti-inflammatory, antioxidant, and wound-healing properties and therefore has important roles in the prevention and treatment of OM. In the current study, we developed a thermally sensitive mucoadhesive gel based on trimethyl chitosan (TMC) containing EPO for the treatment of OM. TMCs with various degrees of substitution (DS) were synthesized and mixed with β-glycerophosphate (GP) and characterized for gelation properties by means of rheological analysis. The effects of DS of TMCs and different concentrations of GP on gelation temperature and time were investigated. The mucoadhesive property of the mixtures was also assessed using cattle buccal mucosa. The optimized mixture was loaded with EPO and subjected to in vitro drug release, wash away, in vitro antimicrobial, and wound-healing tests. The effect of EPO-loaded formulation was also investigated in vivo in Sprague-Dawley rats with chemotherapy-induced mucositis. The best properties were obtained with the blend of TMC of 9.8% DS (5%) and GP (20%). EPO was released from the hydrogel during 8 h, and more than 30% of the drug still remained on the mucosa after 3 h of washing the buccal mucosa with phosphate buffer. TMC/GP mixture was characterized by antimicrobial properties. The EPO hydrogel demonstrated in vitro/in vivo wound-healing properties. Therefore, EPO-loaded hydrogel has the potential to be used in the treatment of OM and other oral or subcutaneous wounds.

Topics: Animals; Cattle; Chitosan; Disease Models, Animal; Drug-Related Side Effects and Adverse Reactions; Erythropoietin; Glycerophosphates; Humans; Hydrogels; Rats; Rats, Sprague-Dawley; Stomatitis; Temperature

Steroids such as glucocorticoid have been widely used for their excellent anti-inflammatory, anti-immune, and anti-shock properties. However, the long-term use in high doses has been found to cause necrosis of femoral head and other serious adverse reactions. Thus, it is of great importance to safely use these medications on patients without inducing bone necrosis.. In this preclinical study, we examined the effects of erythropoietin (EPO) to attenuate the induction of steroid-induced femoral bone necrosis using rats to build up the in-vivo models. Rats were randomly divided into three groups: negative control group (group A), disease group (group B), and EPO group (group C). 20 mg/kg methylprednisolone was administrated into group B and group C for 6 weeks with two intramuscular injections per week per rat. Group C was further given daily intraperitoneal injections of rHuEPO during this period. Group A received only injection of saline at the same schedule. 12 weeks after the initial drug administration, the rats' femoral tissues were harvested for HE staining, immunohistochemistry studies for PECAM-1(also CD31) expression and Western Blotting for VEGF expression.. Histology studies showed that compared with the disease group, EPO group had significant improvement and bone morphology being much closer to the negative control group. Immunohistochemical studies revealed that EPO group had statistically much more expression of PECAM-1 than the other groups did. Western Blot demonstrated that the EPO group had significantly higher VEGF expression than the disease group.. Results suggested that simultaneous injection of EPO could partially prevent steroid-induced ANFH.

Topics: Animals; Disease Models, Animal; Erythropoietin; Female; Femur Head; Femur Head Necrosis; Male; Methylprednisolone; Platelet Endothelial Cell Adhesion Molecule-1; Rats, Sprague-Dawley; Vascular Endothelial Growth Factor A

Klotho, which is a life extension factor, and erythropoietin (EPO) have been introduced as effective neuroprotective factors in several neurological disorders. The present study is an attempt to examine the potential role of klotho and EPO in therapeutic effect of curcumin-loaded nanoparticles (NPs) in pentylenetetrazol (PTZ)-induced kindling model. In order to induce the kindling model, PTZ was administrated intraperitoneally (i.p.) at dose of 36.5 mg/kg every other day for 20 days. Male NMRI mice received pre-treatment of free curcumin or curcumin-loaded NPs (12.5 mg/kg, i.p.) 10 days before PTZ injection and this was continued until 1 h before each PTZ injection. Immunostaining against NeuN, as a marker of neuronal maturation, and EPO was performed on hippocampal brain sections. Quantitative real time polymerase chain reaction (qRT-PCR) was conducted to assess the expression levels of tumor necrosis factor-α (TNF-α), klotho and EPO in the hippocampus. Immunostaining data indicated that treatment with curcumin-loaded NPs significantly alleviates the neuronal cell death in PTZ receiving animals. Curcumin-loaded NPs effectively upregulated the levels of EPO and klotho in PTZ receiving animals. Furthermore, mRNA level of TNF-α was considerably reduced in animals undergone curcumin-loaded NPs treatment. Overall, the results of this study suggest that downregulation of TNF-α and consequent upregulation of klotho and EPO might contribute to the neuroprotective effect of curcumin-loaded NPs in experimental model of epilepsy.

Topics: Animals; Chronic Disease; Curcumin; Disease Models, Animal; DNA-Binding Proteins; Drug Carriers; Epilepsy; Erythropoietin; Glucuronidase; Hippocampus; Kindling, Neurologic; Klotho Proteins; Male; Mice; Nanoparticles; Nerve Tissue Proteins; Neurons; Neuroprotection; Neuroprotective Agents; Nuclear Proteins; Pentylenetetrazole; Random Allocation; Tumor Necrosis Factor-alpha; Up-Regulation

The aim of this study is to optimize the timing of erythropoietin gene modified mesenchymal stem cells (EPO-MSCs) transplantation for bronchopulmonary dysplasia (BPD). Three weeks post-operation, the results indicated that the damage of airway structure and apoptosis were significantly decreased, the proliferation was increased in three EPO-MSCs transplantation groups as compared with BPD mice. Moreover, the inflammation cytokines were improvement in early EPO-MSCs injection mice than in BPD mice, but there was no significant difference between late injection and BPD groups. Furthermore, the protein expression ratio of p-p38/p38MAPK was down-regulation in early mice but not in late transplantation mice. Our findings suggest that EPO-MSCs maybe attenuate BPD injury in early than in late administration by inhibiting inflammation response through down-regulation of the p38MAPK signalling pathway.

Topics: Animals; Apoptosis; Bronchopulmonary Dysplasia; Cell Proliferation; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Humans; Infant, Newborn; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; p38 Mitogen-Activated Protein Kinases; Signal Transduction

Topics: Anemia; Animals; Dihydropyridines; Disease Models, Animal; Enzyme Assays; Enzyme Inhibitors; Erythropoietin; Hematinics; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Inhibitory Concentration 50; Macaca fascicularis; Mice; Mice, Inbred BALB C; Pyridines; Rats; Rats, Sprague-Dawley; Renal Insufficiency, Chronic

β-thalassemias are genetic disorders characterized by anemia, ineffective erythropoiesis, and iron overload. Current treatment of severe cases is based on blood transfusion and iron chelation or allogeneic bone marrow (BM) transplantation. Novel approaches are explored for nontransfusion-dependent patients (thalassemia intermedia) who develop anemia and iron overload. Here, we investigated the erythropoietin (EPO) receptor partner, transferrin receptor 2 (TFR2), as a novel potential therapeutic target. We generated a murine model of thalassemia intermedia specifically lacking BM

Topics: Anemia; Animals; beta-Thalassemia; Cells, Cultured; Disease Models, Animal; Erythroid Cells; Erythropoiesis; Erythropoietin; Female; Gene Deletion; Genetic Therapy; Iron Overload; Male; Mice, Inbred C57BL; Receptors, Transferrin

Erythropoietin (EPO), a glycoprotein cytokine essential to hematopoiesis, has neuroprotective effects in rodent models of Alzheimer's disease (AD). However, high therapeutic doses or invasive routes of administration of EPO are required to achieve effective brain concentrations due to low blood-brain barrier (BBB) penetrability, and high EPO doses result in hematopoietic side effects. These obstacles can be overcome by engineering a BBB-penetrable analog of EPO, which is rapidly cleared from the blood, by fusing EPO to a chimeric monoclonal antibody targeting the transferrin receptor (cTfRMAb), which acts as a molecular Trojan horse to ferry the EPO into the brain via the transvascular route. In the current study, we investigated the effects of the BBB-penetrable analog of EPO on AD pathology in a double transgenic mouse model of AD. Five and a half month old male APPswe/PSEN1dE9 (APP/PS1) transgenic mice were treated with saline ( n = 10) or the BBB-penetrable EPO ( n = 10) 3 days/week intraperitoneally for 8 weeks, compared to same-aged C57BL/6J wild-type mice treated with saline ( n = 8) with identical regiment. At 9 weeks following treatment initiation, exploration and spatial memory were assessed with the open-field and Y-maze test, mice were sacrificed, and brains were evaluated for Aβ peptide load, synaptic loss, BBB disruption, microglial activation, and microhemorrhages. APP/PS1 mice treated with the BBB-penetrable cTfRMAb-EPO fusion protein had significantly lower cortical and hippocampal Aβ peptide number ( p < 0.05) and immune-positive area ( p < 0.05), a decrease in hippocampal synaptic loss ( p < 0.05) and cortical microglial activation ( p < 0.001), and improved spatial memory ( p < 0.05) compared with APP/PS1 saline controls. BBB-penetrating EPO was not associated with microhemorrhage development. The cTfRMAb-EPO fusion protein offers therapeutic benefits by targeting multiple targets of AD pathogenesis and progression (Aβ load, synaptic loss, microglial activation) and improving spatial memory in the APP/PS1 mouse model of AD.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Blood-Brain Barrier; CHO Cells; Cricetulus; Disease Models, Animal; Erythropoietin; Humans; Immunoconjugates; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Permeability; Receptors, Transferrin; Recombinant Fusion Proteins; Treatment Outcome

Acute kidney injury (AKI) is a frequent complication of sepsis, with a high mortality. Hallmarks of septic-AKI include inflammation, endothelial injury, and tissue hypoxia. Therefore, it would be of interest to develop therapeutic approaches for improving the microvascular damage in septic-AKI. Erythropoietin (EPO) is a well-known cytoprotective multifunctional hormone. Thus, the aim of this study was to evaluate the protective effects of EPO on microvascular injury in a murine model of endotoxemic AKI. Male Balb/c mice were divided into four groups: control, LPS (8 mg/kg, ip.), EPO (3000 IU / kg, sc.) and LPS + EPO. A time course study (0-48 h) was designed. Experiments include, among others, immunohistochemistry and Western blottings of hypoxia-inducible transcription factor (HIF-1α), erythropoietin receptor (EPO-R), vascular endothelial growth factor system (VEGF/VEGFR-2), platelet and endothelial adhesion molecule-1 (PeCAM-1), inducible nitric oxide synthase (iNOS) and phosphorylated nuclear factor kappa B p65 (NF-κB). Data showed that EPO attenuates renal microvascular damage during septic-AKI progression through a) the decrease of HIF-1 alpha, iNOS, and NF-κB and b) the enhancement of EPO-R, PeCAM-1, VEGF, and VEGFR-2 expression. In summary, EPO renoprotection involves the attenuation of septic-induced renal hypoxia and inflammation as well as ameliorates the endotoxemic microvascular injury.

Topics: Acute Kidney Injury; Animals; Blotting, Western; Disease Models, Animal; Disease Progression; Endotoxemia; Erythropoietin; Immunohistochemistry; Inflammation; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Microvessels; Sepsis; Time Factors

Topics: Adenine; Animals; Autophagosomes; Autophagy; Cell Line; Chemical and Drug Induced Liver Injury; Chromones; Cobalt; Disease Models, Animal; Erythropoietin; Humans; Liver; Liver Function Tests; Mice; Mice, Inbred C57BL; Morpholines; Peptide Fragments; Proto-Oncogene Proteins c-akt; Random Allocation; Reperfusion Injury; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Neuroprotective effects of erythropoietin (EPO) on peripheral nerve injury remain uncertain. This study investigated the efficacy of EPO in attenuating median nerve chronic constriction injury (CCI)-induced neuropathy. Animals received an intraneural injection of EPO at doses of 1,000, 3,000, or 5,000 units/kg 15 min before median nerve CCI. Afterwards, the behavioral and electrophysiological tests were conducted. Immunohistochemistry and immunoblotting were used for qualitative and quantitative analysis of microglial and mitogen-activated protein kinases (MAPKs), including p38, JNK, and ERK, activation. Enzyme-linked immunosorbent assay and microdialysis were applied to measure pro-inflammatory cytokine and glutamate responses, respectively. EPO pre-treatment dose-dependently ameliorated neuropathic pain behavior, decreased microglial and MAPKs activation, and diminished the release of pro-inflammatory cytokines and glutamate in the ipsilateral cuneate nucleus after CCI. Moreover, EPO pre-treatment preserved myelination of the injured median nerve on morphological investigation and suppressed injury-induced discharges. We also observed that EPO receptor (EPOR) expression was up-regulated in the injured nerve after CCI. Double immunofluorescence showed that EPOR was localized to Schwann cells. Furthermore, siRNA-mediated knockdown of EPOR expression eliminated the therapeutic effects of EPO on attenuating the microglial and MAPKs activation, pro-inflammatory cytokine responses, injury discharges, and neuropathic pain behavior in CCI rats. In conclusion, binding of EPO to its receptors on Schwann cells maintains myelin integrity and blocks ectopic discharges in the injured median nerve, that in the end contribute to attenuation of neuropathic pain via reducing glutamate release from primary afferents and inhibiting activation of microglial MAPKs and production of pro-inflammatory cytokines.

Topics: Action Potentials; Animals; Cytokines; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Hyperalgesia; Male; Median Nerve; Microglia; Mitogen-Activated Protein Kinase Kinases; Neuralgia; Pain Threshold; Peripheral Nervous System Diseases; Phosphorylation; Polyradiculoneuropathy; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; RNA, Small Interfering; Schwann Cells; Signal Transduction

Parkinson's disease (PD) is the second most common neurodegenerative disease, presenting with midbrain dopaminergic neurons degeneration. A number of studies suggest that microglial activation may have a role in PD. It has emerged that inflammation-derived oxidative stress and cytokine-dependent toxicity may contribute to nigrostriatal pathway degeneration and exacerbate the progression of the disease in patients with idiopathic PD. Cell therapies have long been considered a feasible regenerative approach to compensate for the loss of specific cell populations such as the one that occurs in PD. We recently demonstrated that erythropoietin-releasing neural precursors cells (Er-NPCs) administered to MPTP-intoxicated animals survive after transplantation in the recipient's damaged brain, differentiate, and rescue degenerating striatal dopaminergic neurons. Here, we aimed to investigate the potential anti-inflammatory actions of Er-NPCs infused in an MPTP experimental model of PD.. The degeneration of dopaminergic neurons was caused by MPTP administration in C57BL/6 male mice. 2.5 × 10. Er-NPC administration promoted a rapid anti-inflammatory effect that was already evident 24 h after transplant with a decrease of pro-inflammatory and increase of anti-inflammatory cytokines mRNA expression levels. This effect was maintained until the end of the observational period, 2 weeks post-transplant. Here, we show that Er-NPCs transplant reduces macrophage infiltration, directly counteracting the M1-like pro-inflammatory response of murine-activated microglia, which corresponds to the decrease of CD68 and CD86 markers, and induces M2-like pro-regeneration traits, as indicated by the increase of CD206 and IL-10 expression. Moreover, we also show that this activity is mediated by Er-NPCs-derived erythropoietin (EPO) since the co-injection of cells with anti-EPO antibodies neutralizes the anti-inflammatory effect of the Er-NPCs treatment.. This study shows the anti-inflammatory actions exerted by Er-NPCs, and we suggest that these cells may represent good candidates for cellular therapy to counteract neuroinflammation in neurodegenerative disorders.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Coculture Techniques; Corpus Striatum; Cytokines; Disease Models, Animal; Dopamine Plasma Membrane Transport Proteins; Encephalitis; Erythropoietin; Green Fluorescent Proteins; Male; Mice; Mice, Inbred C57BL; Muscle Strength; Neural Stem Cells; Parkinsonian Disorders; Recovery of Function; Smell; Tyrosine 3-Monooxygenase

Recombinant human-erythropoietin (rh-EPO) has therapeutic efficacy for premature infants with brain damage during the active rehabilitation and anti-inflammation. In the present study, we found that the rh-EPO was related to the promotion of neovascularization. Our aim was to investigate whether rh-EPO augments neovascularization in the neonatal rat model of premature brain damage through the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway.. Postnatal day 5 (PD5), rats underwent permanent ligation of the right common carotid artery and were exposed to hypoxia for 2 h. All the rat pups were randomized into five groups as follows: (1) control group; (2) hypoxia-ischemic (HI) group; (3) HI + LY294002 group; (4) HI + rh-EPO group; and (5) HI + rh-EPO + LY294002 group. The phospho-Akt protein was tested 90 min after the whole operation, and CD34, vascular endothelial growth factor receptor 2 (VEGFR2), and vascular endothelial growth factor (VEGF) were also tested 2 days after the whole operation.. In the hypoxic and ischemic zone of the premature rat brain, the rh-EPO induced CD34+ cells to immigrate to the HI brain zone (P < 0.05) and also upregulated the VEGFR2 protein expression (P < 0.05) and VEGF mRNA level (P < 0.05) through the PI3K/Akt (P < 0.05) signaling pathway when compared with other groups.. The rh-EPO treatment augments neovascularization responses in the neonatal rat model of premature brain damage through the PI3K/Akt signaling pathway. Besides, the endogenous EPO may exist in the HI zone of rat brain and also has neovascularization function through the PI3K/Akt signaling pathway.

Topics: Animals; Animals, Newborn; Antigens, CD34; Brain; Disease Models, Animal; Erythropoietin; Female; Humans; Hypoxia-Ischemia, Brain; Neovascularization, Physiologic; Phosphatidylinositol 3-Kinase; Pregnancy; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

An extensive literature has shown a powerful neuroprotective action of Erythropoietin (EPO) both in vivo and in vitro. This study shows that EPO, whether ectopically administered or released by neural precursors, does reverse MPTP-induced parkinsonism in mice. Unilateral stereotaxic injection of 2.5 × 10

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Antiparkinson Agents; Arabidopsis Proteins; Corpus Striatum; Disease Models, Animal; Dopamine Plasma Membrane Transport Proteins; Erythropoietin; Green Fluorescent Proteins; Intramolecular Transferases; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Motor Activity; Muscle Strength; Neural Stem Cells; Parkinsonian Disorders; Recovery of Function; Treatment Outcome; Tyrosine 3-Monooxygenase

Erythropoietin (EPO) confers potent neuroprotection against ischemic injury through a variety of mechanisms. However, the protective effect of EPO on axons after cerebral ischemia in adult mice is rarely covered. The purpose of this study was to investigate the potential neuroprotective effects of EPO on axons in mice after cerebral ischemia.. A total of 30 adult male C57 BL/6 mice were treated with EPO (5000 IU/kg) or vehicle after transient middle cerebral artery occlusion (MCAO). The mortality rate of each experimental group was calculated. Neurological function was assessed by Rota-rod test. Frozen sections from each mouse brain at 14 days after reperfusion were used to evaluate the fluorescent intensity of myelin basic protein (MBP) and neurofilament 200 (NF-200). Immunofluorescence staining and Western blotting were used to assess the protein level of β-amyloid precursor protein (β-APP) and glial fibrillary acidic protein (GFAP), a marker of mature astrocytes. The protein levels of the myelin-derived growth inhibitory proteins, neurite growth inhibitor-A (Nogo-A), myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMG) were also examined by Western blot after MCAO.. The survival rate of the vehicle group 14 days after cerebral ischemia-reperfusion was 50%, which increased to 80% after EPO treatment at the start of reperfusion. EPO improved neurobehavioral outcomes at days 3 and 7 after MCAO was compared with the vehicle group (P < 0.05). Furthermore, EPO ameliorated demyelination, demonstrated by upregulation of the MBP/NF-200 ratio. Meanwhile, increased levels of β-APP, GFAP, Nogo-A, and MAG after MCAO were reduced by EPO treatment (P < 0.05).. EPO treatment attenuates axonal injury and improves neurological function after cerebral ischemia in adult mice.

Topics: Animals; Axons; Disease Models, Animal; Erythropoietin; Infarction, Middle Cerebral Artery; Male; Mice, Inbred C57BL; Neuroprotective Agents; Nogo Proteins

Traumatic brain injury (TBI) can be complicated by TBI-triggered acute lung injury (ALI), in which inflammation plays a central role. It has been reported that an Erythropoietin-derived peptide (pHBSP) was able to ameliorate TBI; however, its function in TBI-caused ALI has not been reported yet.. In this study, we studied the effect of pHBSP on TBI-caused ALI by using a weight-drop induced TBI model. At 8 h and 24 h post-TBI, pulmonary edema (PE) and bronchoalveolar lavage fluid (BALF) proteins were measured, and haematoxylin and eosin (H&E) staining of lung sections was carried out. At 24 h following TBI, the lungs were harvested for immunofluorescence staining and qRT-PCR analysis.. At 8 h and 24 h post-TBI, pHBSP treatment significantly decreased wet/dry ratios, decreased total BALF protein, and attenuated the histological signs of pulmonary injury. At 24 h post-TBI, pHBSP treatment decreased the accumulation of CD68+ macrophages in the lung and reduced the mRNA levels of TNF-α, IL-6, IL-1β and iNOS in the lung.. We identified the protective role that pHBSP played in TBI-caused ALI, suggesting that pHBSP is a potent candidate for systemic therapy in TBI patients.

Topics: Acute Lung Injury; Animals; Brain Injuries, Traumatic; Cytokines; Disease Models, Animal; Erythropoietin; Lung; Macrophages; Male; Nitric Oxide Synthase Type II; Peptides; Rats; Rats, Inbred Lew; Time Factors

Intensive care unit (ICU) anemia is an extreme version of anemia of inflammation that occurs commonly in critically ill patients and is associated with increased morbidity and mortality. Currently available therapies for ICU anemia have shown inconsistent efficacies in clinical trials. We conducted a systematic study of the effects of early versus delayed iron (Fe) and/or erythropoietin (EPO) therapy in our previously characterized mouse model of ICU anemia based on an injection of heat-killed Brucella abortus. To study the effects of ongoing inflammation on the response to therapy, inflamed wild-type (WT) and hepcidin knockout (HKO) mice were treated at either early (days 1 and 2) or delayed (days 7 and 8) time points after the inflammatory stimulus. In the early treatment group, Fe and/or EPO therapy did not increase hemoglobin (Hgb) levels or reticulocyte production in either the inflamed WT or HKO groups. In the delayed treatment group, combination Fe + EPO therapy did increase Hgb and reticulocyte production in WT mice (mean ΔHgb in WT saline group -9.2 g/dL vs. Fe/EPO -5.5 g/dL; P < 0.001). The HKO mice in the delayed treatment group did not improve their Hgb, but HKO mice in all treatment groups developed a milder anemia than the WT mice. Our findings indicate that combination Fe + EPO therapy is effective in partially reversing ICU anemia when administered after the phase of acute inflammation. Hepcidin ablation alone was more effective in attenuating ICU anemia than Fe + EPO therapy, which indicates the potential of antihepcidin therapeutics in treating ICU anemia.

Topics: Anemia; Animals; Disease Models, Animal; Erythropoietin; Female; Hepcidins; Iron; Male; Mice; Mice, Knockout; Sepsis

Cerebral palsy (CP) is the most common motor disability in childhood, with a worldwide prevalence of 1.5-4/1,000 live births. Hypoxic-ischemic encephalopathy (HIE) contributes to the burden of CP, but the long-term neuropathological findings of this association remain limited.. Thirty-four term Macaca nemestrina macaques were included in this long-term neuropathological study: 9 control animals delivered by cesarean section and 25 animals with perinatal asphyxia delivered by cesarean section after 15-18 min of umbilical cord occlusion (UCO). UCO animals were randomized to saline (n = 11), therapeutic hypothermia (TH; n = 6), or TH + erythropoietin (Epo; n = 8). Epo was given on days 1, 2, 3, and 7. Animals had serial developmental assessments and underwent magnetic resonance imaging with diffusion tensor imaging at 9 months of age followed by necropsy. Histology and immunohistochemical (IHC) staining of brain and brainstem sections were performed.. All UCO animals demonstrated and met the standard diagnostic criteria for human neonates with moderate-to-severe HIE. Four animals developed moderate-to-severe CP (3 UCO and 1 UCO + TH), 9 had mild CP (2 UCO, 3 UCO + TH, 3 UCO + TH + Epo, and 1 control), and 2 UCO animals died. None of the animals treated with TH + Epo died, had moderate-to-severe CP, or demonstrated signs of long-term neuropathological toxicity. Compared to animals grouped together as having no CP (no-CP; controls and mild CP only), animals with CP (moderate and severe) demonstrated decreased fractional anisotropy of multiple white-matter tracts including the corpus callosum and internal capsule, when using Tract-Based Spatial Statistics (TBSS). Animals with CP had decreased staining for cortical neurons and increased brainstem glial scarring compared to animals without CP. The cerebellar cell density of the internal granular layer and white matter was decreased in CP animals compared to that in control animals without CP.. In this nonhuman primate HIE model, animals treated with TH + Epo had less brain pathology noted on TBSS and IHC staining, which supports the long-term safety of TH + Epo in the setting of HIE. Animals that developed CP showed white-matter changes noted on TBSS, subtle histopathological changes in both the white and gray matter, and brainstem injury that correlated with CP severity. This HIE model may lend itself to further study of the relationship between brainstem injury and CP.

Topics: Animals; Animals, Newborn; Asphyxia Neonatorum; Cerebral Palsy; Disease Models, Animal; Erythropoietin; Hypothermia, Induced; Hypoxia-Ischemia, Brain; Macaca nemestrina; Random Allocation

Cerebral malaria (CM) is a severe neurological syndrome in humans and the main fatal cause of malaria. In malaria epidemic regions, despite appropriate anti-malarial treatment, 10-20% of deaths still occur during the acute phase. This is largely attributable to poor treatment access, therapeutic complexity and drug resistance; thus, developing additional clinical adjunctive therapies is an urgent necessity. In this study, we investigated the effect of artesunate (AST) and recombinant human erythropoietin (rhEPO) using an experimental cerebral malaria (ECM) model-C57BL/6 mice infected with Plasmodium berghei ANKA (PbA). Treatment with the combination of AST and rhEPO reduced endothelial activation and improved the integrity of blood brain barrier, which led to increased survival rate and reduced pathology in the ECM. In addition, this combination treatment down-regulated the Th1 response during PbA infection, which was correlated with the reduction of CCL2, TNF-α, IFN-γ, IL-12, IL-18, CXCL9 and CXCL10 levels, leading to reduced accumulation of pathogenic T cells in the brain. Meanwhile, AST and rhEPO combination led to decreased maturation and activation of splenic dendritic cells, expansion of regulatory T cells, and increased IL-10 and TGF-β production. In conclusion, these data provide a theoretical basis for clinical adjunct therapy with rhEPO and AST in human cerebral malaria patients.

Topics: Animals; Antimalarials; Artemisinins; Artesunate; Brain; Cytokines; Dendritic Cells; Disease Models, Animal; Drug Synergism; Erythropoietin; Female; Intercellular Adhesion Molecule-1; Malaria, Cerebral; Mice, Inbred C57BL; Plasmodium berghei; Spleen; T-Lymphocytes; Vascular Cell Adhesion Molecule-1

This study aimed to investigate the effects of erythropoiesis-stimulating agents (ESAs) on intestinal flora in peritoneal fibrosis.. Twenty-four Wistar albino rats were divided into 3 groups as the control group, which received 0.9% saline (3 mL/d) intraperitoneally; the chlorhexidine gluconate (CH) group, which received 3 mL/d injections of 0.1% CH intraperitoneally, and the ESA group, which received 3 mL/d injections of 0.1% CH intraperitoneally and epoetin beta (3 doses of 20 IU/kg/wk) subcutaneously. On the 21st day, the rats were sacrificed and the visceral peritoneum samples were obtained from left liver bowel. Blood samples were obtained from abdominal aorta and intestinal flora samples were obtained from transverse colon.. Histopathologically, the CH, ESA, and control groups had peritoneal thickness of 135.4 ± 22.2 µm, 48.6 ± 12.8 µm, and 6.0 ± 2.3 µm, respectively. Escherichia coli was the predominant bacterium in the intestinal flora in the control group. Significant changes in microbial composition of intestinal flora towards Proteus species and Enterobacter species was seen among the groups (P < .001). There was no significant difference between the ESA and CH groups regarding the isolates from blood cultures. However, the bacterial isolates from cultures of intestinal flora among these groups were significantly different (P < .05).. Erythropoiesis-stimulating agents change intestinal flora by a clinically significant amount in experimental peritoneal fibrosis. We consider that ESAs achieve this via regulating intestinal peristaltism.

Topics: Animals; Bacteria; Chlorhexidine; Disease Models, Animal; Erythropoietin; Female; Gastrointestinal Microbiome; Hematinics; Intestines; Peristalsis; Peritoneal Fibrosis; Rats, Wistar; Recombinant Proteins

Several studies indicate that erythropoietin (EPO) has remarkable neuroprotective effects in various central nervous system disorders, while little is known about the effects of EPO in diabetes-associated cognitive dysfunction. Therefore, the present study aimed to investigate whether EPO ameliorates diabetes-associated cognitive dysfunction in vivo and in vitro. We investigated the protective effects of EPO on high-glucose (HG)-induced PC12 cell death and oxidative stress. The effects of EPO (300 U/kg administered three times a week for 4 weeks) on diabetes-associated cognitive decline were investigated in diabetic rats. EPO significantly increased cell viability, increased the activity of superoxide dismutase, decreased the production of malondialdehyde and reactive oxygen species, and decreased the apoptosis rate. Additionally, LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, abolished the protective effects of EPO in HG-treated PC12 cells. In diabetic rats, EPO prevented deficits in spatial learning and memory in the Morris water maze test. The results of real-time PCR and Western blotting showed that EPO upregulated EPO receptor, PI3K, and phosphorylated Akt2 relative to unphosphorylated Akt2 (p-Akt2/Akt2) and downregulated glycogen synthase kinase-3β (GSK-3β). These studies demonstrate that EPO is an effective neuroprotective agent in the context of diabetes-associated cognitive dysfunction and show that this effect involves the PI3K/Akt/GSK-3β pathway.

Topics: Animals; Apoptosis; Cell Survival; Cognitive Dysfunction; Diabetes Complications; Diabetes Mellitus, Experimental; Disease Models, Animal; Erythropoietin; Glucose; Glycogen Synthase Kinase 3 beta; Models, Biological; Neuroprotective Agents; Oxidative Stress; PC12 Cells; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Reactive Oxygen Species; Receptors, Erythropoietin; Signal Transduction

Anaemia is a common complication of chronic kidney disease, for which there is presently no adequate treatment. The delivery of human erythropoietin (hEPO) cDNA to salivary glands reportedly increases red blood cell counts, haematocrit (HCT) and haemoglobin concentration, representing a potential new method of renal anaemia treatment. However, no studies have examined the effects of this method in an animal model of renal anaemia. Here we established a miniature pig animal model of renal anaemia through continuous feeding with adenine. In these animals, we delivered the AAV2hEPO gene to the parotid glands through Stensen's duct. As a control, we transferred AAVLacZ. Enzyme-linked immunosorbent assay was used to detect hEPO in serum and saliva. Red blood counts and serum biochemistry were used to evaluate how hEPO gene administration affected renal anaemia. Compared with the control group, we found increased hEPO concentrations in parotid saliva and serum, respectively, at 2 and 6 weeks after AAV2hEPO administration to the anaemic animals. HCT and haemoglobin were also increased after AAV2hEPO was delivered; most serum indicators of renal damage were not changed over the time span of the experiment, suggesting the adenine-induced kidney damage had not been completely reversed. However, blood urea nitrogen and B2 microglobulin levels showed small but significant improvement. Overall, our present findings suggest that adeno-associated virus 2 (AAV2)-mediated gene transduction of hEPO via the parotid gland is a promising potential alternative therapy for renal anaemia.

Topics: Adenoviridae; Anemia; Animals; Dependovirus; Disease Models, Animal; Erythropoietin; Gene Transfer Techniques; Genetic Vectors; Humans; Parotid Gland; Protein Transport; Renal Insufficiency; Saliva; Salivary Glands; Swine; Swine, Miniature; Transduction, Genetic

Erythropoietin (EPO) is the main hormone driving mammalian erythropoiesis, with activity mediated via the surface receptor, EPO-R, on erythroid progenitor cells. Recombinant human EPO is currently used clinically for the treatment of anemia in patients with end-stage renal disease, and in certain cancer patients suffering from anemia induced either by the tumor itself or by chemotherapy. EPO-R expression is also detected in non-erythroid cells, including macrophages present in the peritoneum, spleen, and bone marrow (BM). Here we demonstrate that Kupffer cells (KCs) - the liver-resident macrophages - are EPO targets. We show that, in vitro, EPO initiated intracellular signalling and enhanced phagocytosis in a rat KC line (RKC-2) and in sorted KCs. Moreover, continuous EPO administration in mice, resulted in an increased number of KCs, up-regulation of liver EPO-R expression and elevated production of the monocyte chemoattractant CCL2, with corresponding egress of Ly6C

Topics: Acetaminophen; Animals; Antigens, Ly; Cell Proliferation; Cells, Cultured; Chemical and Drug Induced Liver Injury; Chemokine CCL2; Disease Models, Animal; Erythropoietin; Humans; Kupffer Cells; Liver; Mice; Phagocytosis; Rats; Receptors, Erythropoietin; Recombinant Proteins; Signal Transduction; Up-Regulation

The production of erythropoietin (EPO) by the kidneys, a principal hormone for the hematopoietic system, is reduced in patients with chronic kidney disease (CKD), eventually resulting in severe anemia. Although recombinant human EPO treatment improves anemia in patients with CKD, returning to full red blood cell production without fluctuations does not always occur. We established a method to generate EPO-producing cells from human induced pluripotent stem cells (hiPSCs) by modifying previously reported hepatic differentiation protocols. These cells showed increased EPO expression and secretion in response to low oxygen conditions, prolyl hydroxylase domain-containing enzyme inhibitors, and insulin-like growth factor 1. The EPO protein secreted from hiPSC-derived EPO-producing (hiPSC-EPO) cells induced the erythropoietic differentiation of human umbilical cord blood progenitor cells in vitro. Furthermore, transplantation of hiPSC-EPO cells into mice with CKD induced by adenine treatment improved renal anemia. Thus, hiPSC-EPO cells may be a useful tool for clarifying the mechanisms of EPO production and may be useful as a therapeutic strategy for treating renal anemia.

Topics: Anemia; Animals; Cell Differentiation; Cell Hypoxia; Cell Proliferation; Disease Models, Animal; Erythropoiesis; Erythropoietin; Human Embryonic Stem Cells; Humans; Induced Pluripotent Stem Cells; Insulin-Like Growth Factor I; Kidney; Mice; Mouse Embryonic Stem Cells; Pluripotent Stem Cells; Stem Cell Transplantation

Treatment with erythropoietin (Epo) in experimental autoimmune encephalomyelitis (EAE), the rodent model of multiple sclerosis (MS), has consistently been shown to ameliorate disease progression and improve overall outcome. The effect has been attributed to modulation of the immune response and/or preservation of the central nervous system (CNS) tissue integrity. It remains unclear, however, if (a) Epo acts primarily in the CNS or the periphery and if (b) Epo's beneficial effect in EAE is mainly due to maintaining CNS tissue integrity or to modulation of the immune response. If Epo acts primarily by modulating the immune system, where is this modulation required? In the periphery, the CNS or both?. To address these questions, we used two well-characterized transgenic mouse strains that constitutively overexpress recombinant human Epo (rhEpo) either systemically (tg6) or in CNS only (tg21) in a MOG-induced EAE model. We assessed clinical severity, disease progression, immunomodulation, and CNS tissue integrity, including neuronal survival.. Although disease onset remained unaffected, EAE progression was alleviated in transgenic animals compared to controls with both lines performing equally well showing that expression of Epo in the periphery is not required; Epo expression in the CNS is sufficient. Immunomodulation was observed in both strains but surprisingly the profile of modulation differed substantially between strains. Modulation in the tg21 strain was limited to a reduction in macrophages in the CNS, with no peripheral immunomodulatory effects observed. In contrast, in the tg6 strain, macrophages were upregulated in the CNS, and, in the periphery of this strain, T cells and macrophages were downregulated. The lack of a consistent immunomodulatory profile across both transgenic species suggests that immunomodulation by Epo is unlikely to be the primary mechanism driving amelioration of EAE. Finally, CNS tissue integrity was affected in all strains. Although myelin appeared equally damaged in all strains, neuronal survival was significantly improved in the spinal cord of tg21 mice, indicating that Epo may ameliorate EAE predominantly by protecting neurons.. Our data suggests that moderate elevated brain Epo levels provide clinically significant neuroprotection in EAE without modulation of the immune response making a significant contribution.

Topics: Animals; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Erythropoietin; Female; Gene Expression Regulation; Humans; Lymphocytes; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Monocytes; Myelin-Oligodendrocyte Glycoprotein; Neurons; Neuroprotection; Peptide Fragments; Proto-Oncogene Proteins c-sis; Spleen

Long‑term administration of glucocorticoid hormones is considered one of predominant pathological factors inducing osteonecrosis of the femoral head (ONFH) development and progression, in which reduction of blood supply leads to a progressive bone loss and impairment of bone structure in the majority of cases. In a non‑hematopoietic system, erythropoietin (EPO) can stimulate angiogenesis and bone regeneration. However, the specific mechanism underlying the role of EPO in ONFH remains to be elucidated. Therefore, the purpose of this study was to determine the effect of EPO on the prevention of bone loss in ONFH. Male C57BL/6J mice 3 months old were divided into two groups: EPO group and control groups. ONFH was established by the administration prednisolone (PDS, 100 mg/kg) with co‑treatment of lipopolysaccharide (LPS, 1 mg/kg). ONFH mice received recombinant mouse EPO (500 U/kg/day) or saline intramuscularly. The mice were sacrificed at 2, 4, 6 and 8 weeks following the initiation of treatment. Alterations in the general architecture and histomorphology of the right femoral head were determined by hematoxylin and eosin staining and micro computed tomography (micro‑CT). The expression of runt‑related transcription factor 2 (Runx2), osteocalcin, vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule (CD31) in the femoral head was tested by immunohistochemistry. Terminal deoxynucleotidyl‑transferase‑mediated dUTP nick end labeling (TUNEL) assay was performed to detect apoptosis in femoral heads. Micro‑CT data revealed that EPO significantly improved bone volume/total volume and bone mineral density following 6 and 8 weeks of treatment. Histological analysis further demonstrated that EPO treatment improved the arrangement of trabeculae, thinning of trabeculae and other fractures in femoral heads, especially following 6 and 8 weeks of treatment. Immunohistochemical analysis suggested that EPO treatment up‑regulated the expressions of Runx2, osteocalcin, VEGF and CD31 at 4 and 8 weeks. The TUNEL apoptosis assay suggested that EPO intervention reduced apoptosis in avascular ONFH. Therefore, EPO prevents bone loss in ONFH in mice through enhancing Runx2‑mediated osteogenesis, VEGF‑mediated angiogenesis and inhibition of cell apoptosis.

Topics: Animals; Apoptosis; Biomarkers; Bone Resorption; Disease Models, Animal; Erythropoietin; Femur Head; Immunohistochemistry; Male; Mice; Organ Size; Osteonecrosis; X-Ray Microtomography

Inflammatory modulation mediated by microglial M1/M2 polarization is one of the main pathophysiological processes involved in early brain injury (EBI) after subarachnoid haemorrhage (SAH). Previous studies have shown that recombinant human erythropoietin (rhEPO) alleviates EBI following experimental SAH. However, the mechanisms of this beneficial effect are still poorly understood. Recent research has suggested that EPO shows anti-inflammatory properties. Therefore, we tried to analyse whether rhEPO administration influenced microglial M1/M2 polarization in early brain injury after SAH and to identify the underlying molecular mechanism of any such effect. We found that treatment with rhEPO markedly ameliorated SAH-induced EBI, as shown by reductions in brain cell apoptosis, neuronal necrosis, albumin exudation and brain edema. Moreover, the expression levels of p-JAK2 and p-STAT3 were significantly increased in the cortex after SAH induction and were further increased by EPO treatment; in addition, the p-JAK2 inhibitor AZD1480 impaired the protective effect of EPO against SAH-induced EBI in vivo. Furthermore, EPO promoted the polarization of microglia towards the protective M2 phenotype and alleviated inflammation. In cultured microglia under oxyhemoglobin (OxyHb) treatment, EPO up-regulated the expression of the EPO receptor (EPOR), which did not occur in response to OxyHb treatment alone, and EPO magnified OxyHb-induced increases in p-JAK2 and p-STAT3 and modulated OxyHb-challenged microglial polarization towards M2. Interestingly, the effect of EPO on microglia polarization was cancelled by EPOR knockdown or by p-JAK2 or p-STAT3 inhibition, suggesting a core role of the EPOR/JAK2/STAT3 pathway in modulating microglial function and phenotype. In conclusion, the therapeutic effect of rhEPO on the early brain injury after SAH may relate to its modulation of inflammatory response and microglia M1/M2 polarization, which may be mediated in part by the EPOR/JAK2/STAT3 signalling pathway. These results improved the understanding of the anti-inflammatory effect of EPO on microglia polarization, which might optimize the therapeutic modalities of EPO treatment with SAH.

Topics: Animals; Anti-Inflammatory Agents; Brain Edema; Cell Differentiation; Cell Line; Cerebral Cortex; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Humans; Injections, Intraventricular; Janus Kinase 2; Mice; Mice, Inbred C57BL; Microglia; Neurons; Oxyhemoglobins; Pyrazoles; Pyrimidines; Receptors, Erythropoietin; Recombinant Proteins; Signal Transduction; STAT3 Transcription Factor; Stereotaxic Techniques; Subarachnoid Hemorrhage

Spinal cord injury (SCI) is a debilitating condition characterized by a complex of neurological dysfunctions ranging from loss of sensation to partial or complete limb paralysis. Recently, we reported that intravenous administration of neural precursors physiologically releasing erythropoietin (namely Er-NPCs) enhances functional recovery in animals following contusive spinal cord injury through the counteraction of secondary degeneration. Er-NPCs reached and accumulated at the lesion edges, where they survived throughout the prolonged period of observation and differentiated mostly into cholinergic neuron-like cells.. The aim of this study was to investigate the potential reparative and regenerative properties of Er-NPCs in a mouse experimental model of traumatic spinal cord injury.. We report that Er-NPCs favoured the preservation of axonal myelin and strongly promoted the regrowth across the lesion site of monoaminergic and chatecolaminergic fibers that reached the distal portions of the injured cord. The use of an anterograde tracer transported by the regenerating axons allowed us to assess the extent of such a process. We show that axonal fluoro-ruby labelling was practically absent in saline-treated mice, while it resulted very significant in Er-NPCs transplanted animals.. Our study shows that Er-NPCs promoted recovery of function after spinal cord injury, and that this is accompanied by preservation of myelination and strong re-innervation of the distal cord. Thus, regenerated axons may have contributed to the enhanced recovery of function after SCI.

Topics: Animals; Choline O-Acetyltransferase; Dextrans; Disease Models, Animal; Erythropoietin; Fluorescent Dyes; GAP-43 Protein; Locomotion; Male; Mice; Microtubule-Associated Proteins; Myelin Sheath; Nerve Regeneration; Organic Chemicals; Recovery of Function; Rhodamines; Serotonin; Spinal Cord Injuries; Stem Cell Transplantation; Tubulin; Tyrosine 3-Monooxygenase

Erythropoietin (EPO) plays an essential role in the regulation of erythropoiesis. Its production is under the control of the Hypoxia Inducible Factor (HIF) protein whose stability varies according to the oxygen level. During chronic renal failure, EPO deficiency is the main cause of anemia, but other factors such as iron deficiency and inflammatory syndrome are also involved. More recently, it is hypothesized that other factors such an excess of GDF-11 production may be also involved. Thus, beside Epo treatment HIF and GDF-11 are potentially new therapeutic targets in anemia of chronic kidney disease.

Topics: Anemia; Animals; beta-Thalassemia; Bone Morphogenetic Proteins; Disease Management; Disease Models, Animal; Erythropoiesis; Erythropoietin; Forecasting; Growth Differentiation Factors; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Immunoglobulin A; Intestinal Absorption; Iron Deficiencies; Iron, Dietary; Kidney Failure, Chronic; Mice; Models, Biological; Receptors, Transferrin

To explore the protective effects of Tibetan medicine Zuo-Mu-A Decoction (, ZMAD) on the blood parameters and myocardium of high altitude polycythemia (HAPC) model rats.. Forty male Wistar rats were randomly divided into 4 groups by a random number table, including the normal, model, Rhodiola rosea L. (RRL) and ZMAD groups (10 in each group). Every group was raised in Lhasa to create a HAPC model except the normal group. After modeling, rats in the RRL and the ZMAD groups were administered intragastrically with RRL (20 mL/kg) and ZMAD (7.5 mL/kg) once a day for 2 months, respectively; for the normal and the model groups, 5 mL of distilled water was administered intragastrically instead of decoction. Then routine blood and hematologic rheology parameters were taken, levels of erythropoietin and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were tested, and ultrastructural change in the left ventricular myocardium was observed using transmission electron microscopy.. Compared with the model group, ZMAD significantly reduced the red blood cell count, hemoglobin levels, whole blood viscosity at low/middle shear rates, plasma viscosity, erythrocyte electrophoretic time, erythropoietin and 8-OHdG levels, and also increased the erythrocyte deformation index (P<0.05). There was no difference in all results between the RRL and the ZMAD groups. The cardiac muscle fibers were well-protected, mitochondrial matrix swelled mildly and ultrastructure changes were less prominent in the ZMAD group compared with the model group.. ZMAD has significant protective effects on the blood parameters against HAPC, and also has the beneficial effect in protecting against myocardial injury.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Altitude Sickness; Animals; Deoxyguanosine; Disease Models, Animal; Erythropoietin; Medicine, Tibetan Traditional; Myocardium; Polycythemia; Protective Agents; Rheology

Erythropoietin (EPO) is a promising neuroprotective agent and is currently in Phase III clinical trials for the treatment of traumatic brain injury. The goal of this study was to determine if EPO is also protective in traumatic eye injury.. The left eyes of anesthetized DBA/2J or Balb/c mice were exposed to a single 26 psi overpressure air-wave while the rest of the body was shielded. DBA/2J mice were given intraperitoneal injections of EPO or buffer and analyses were performed at 3 or 7 days post-blast. Balb/c mice were given intramuscular injections of rAAV.EpoR76E or rAAV.eGFP either pre- or post-blast and analyses were performed at 1 month post-blast.. EPO had a bimodal effect on cell death, glial reactivity, and oxidative stress. All measures were increased at 3 days post-blast and decreased at 7-days post-blast. Increased retinal ferritin and NADPH oxygenases were detected in retinas from EPO-treated mice. The gene therapy approach protected against axon degeneration, cell death, and oxidative stress when given after blast, but not before.. Systemic, exogenous EPO and EPO-R76E protects the retina after trauma even when initiation of treatment is delayed by up to 3 weeks. Systemic treatment with EPO or EPO-R76E beginning before or soon after trauma may exacerbate protective effects of EPO within the retina as a result of increased iron levels from erythropoiesis and, thus, increased oxidative stress within the retina. This is likely overcome with time as a result of an increase in levels of antioxidant enzymes. Either intraocular delivery of EPO or treatment with non-erythropoietic forms of EPO may be more efficacious.

Topics: Animals; Blast Injuries; Cell Survival; Dependovirus; Disease Models, Animal; Erythropoietin; Eye Injuries; Ferritins; Genetic Therapy; Genetic Vectors; Green Fluorescent Proteins; In Situ Nick-End Labeling; Injections, Intramuscular; Injections, Intraperitoneal; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; NADPH Oxidases; Oxidative Stress; Polymerase Chain Reaction; Retina; Retinal Diseases; Time Factors; Vision Disorders; Wounds, Nonpenetrating

Secondary polycythemia, a disease characterized by a selective increase in circulating mature erythrocytes, is caused by enhanced erythropoietin (Epo) concentrations triggered by hypoxia-inducible factor-2α (HIF-2α). While mechanisms of hypoxia-dependent stabilization of HIF-2α protein are well established, data regarding oxygen-independent regulation of HIF-2α are sparse. In this study, we generated a novel transgenic mouse model, in which biglycan was constitutively overexpressed and secreted by hepatocytes (BGN

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Biglycan; Disease Models, Animal; Erythrocyte Count; Erythrocytes; Erythropoietin; Gene Expression Regulation; Hematocrit; Hemoglobins; Hepatocytes; Kidney; Liver; Male; Mice; Mice, Transgenic; Polycythemia; RNA, Messenger; Toll-Like Receptor 2

Erythropoietin (EPO) is a cytokine known to have effective cytoprotective action in the brain, particularly in ischemic, traumatic, inflammatory, and neurodegenerative conditions. We previously reported the neuroprotective effect of a low sialic form of EPO, Neuro-EPO, applied intranasally in rodent models of stroke or cerebellar ataxia and in a non-transgenic mouse model of Alzheimer's disease (AD). Here we analyzed the protective effect of Neuro-EPO in APPSwe mice, a reference transgenic mouse model of AD. Mice were administered 3 times a day, 3 days in the week with Neuro-EPO (125, 250 μg/kg) intranasally, between 12 and 14 months of age. Motor responses, general activity, and memory responses were analyzed during and after treatment. The deficits in spontaneous alternation, place learning in the water-maze, and novel object recognition observed in APPSwe mice were alleviated by the low dose of Neuro-EPO. Oxidative stress, neuroinflammation, trophic factor levels, and a synaptic marker were analyzed in the hippocampus or cortex of the animals. The increases in lipid peroxidation or in GFAP and Iba-1 contents in APPSwe mice were significantly reduced after Neuro-EPO. Activation of intrinsic and extrinsic apoptotic pathways was analyzed. The increases in Bax/Bcl-2 ratio, TNFα, or Fas ligand levels observed in APPSwe mice were reduced by Neuro-EPO. Finally, immunohistochemical and ELISA analyses of Aβ1-42 levels in the APPSwe mouse cortex and hippocampus showed a marked reduction in Aβ deposits and in soluble and insoluble Aβ1-42 forms. This study therefore confirmed the neuroprotective activity of EPO, particularly for an intranasally deliverable formulation, devoid of erythropoietic side effects, in a transgenic mouse model of AD. Neuro-EPO alleviated memory alterations, oxidative stress, neuroinflammation, apoptosis induction, and amyloid load in 14-month-old APPSwe mice.

Topics: Administration, Intranasal; Alzheimer Disease; Amyloid beta-Peptides; Animals; Cerebral Cortex; Disease Models, Animal; Erythropoietin; Hippocampus; Maze Learning; Memory; Memory Disorders; Mice, Inbred C57BL; Mice, Transgenic; Motor Activity; N-Acetylneuraminic Acid; Neuroprotective Agents; Nootropic Agents

Erythropoietin (EPO), an essential stimulator of erythropoiesis produced by the fetal liver, is important both in vascular remodeling and modulation of the endothelial response in the pulmonary vasculature. In addition, EPO guides alveolar development, along with retinoic acid (RA). EPO is a direct target of RA, and the retinoid pathway is altered in the nitrofen-induced congenital diaphragmatic hernia (CDH) model. In the present study, we tested the hypothesis that the synthesis of EPO is suppressed in a rat model of CDH.. Pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D19 and D21 and divided into control and CDH groups. Immunohistochemistry and quantitative real-time polymerase chain reaction (RT-PCR) were performed to determine the expression of EPO in the fetal liver and kidney. We also estimated the expression of EPO receptor in the fetal lung.. The relative EPO mRNA expression in the liver on D19 and in the kidney on D21 were significantly lower in the CDH group than in the controls (P = 0.0008 and P = 0.0064, respectively). In addition, the results of immunohistochemistry supported the findings from the RT-PCR analysis. No significant changes were noted in the expression pattern or EPO receptor levels in the fetal lungs of the CDH group compared to the controls.. Our results reveal the suppressed EPO synthesis in the CDH fetus, which may contribute to the pathogenesis of lung hypoplasia and modification of pulmonary vasculature in the CDH rat model. Pediatr Pulmonol. 2017;52:606-615. © 2016 Wiley Periodicals, Inc.

Topics: Animals; Disease Models, Animal; Erythropoietin; Female; Gene Expression Regulation, Developmental; Hernias, Diaphragmatic, Congenital; Lung; Phenyl Ethers; Pregnancy; Rats; Rats, Sprague-Dawley; Tretinoin

Topics: Adenine; Anemia; Animals; Blood Urea Nitrogen; Diet; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Hepcidins; Humans; Iron; Kidney Tubules; Mice; Mice, Inbred C57BL; Mice, Knockout; Renal Insufficiency, Chronic; Serum Amyloid A Protein

Hypoxic-ischemic (HI) injury to the developing brain remains a major cause of morbidity. To date, few therapeutic strategies could provide complete neuroprotection. Erythropoietin (EPO) has been shown to be beneficial in several models of neonatal HI. This study examines the effect of treatment with erythropoietin on postnatal day 2 (P2) rats introduced with HI injury.. Rats at P2 were randomized into four groups: sham, bilateral carotid artery occlusion (BCAO), BCAO + early EPO, and BCAO + late EPO groups. Pups in each group were injected with either saline or EPO (5000U/kg) intraperitoneally once at immediately (early) or 48h (late) after HI induction. Body weight was assessed at P2 before and day 7 after HI. Mortality Rate was assessed at 24h, 48h and 72h after HI and brain water content was assessed at 72h. Brain weight and expression of myelin basic protein (MBP) were assessed at day 7 and day 14. At day 31 to 35 following HI insult, neurological behavior function was assessed via Morris water maze (MWM) test.. HI cause significant higher mortality in male than in female (P=0.0445). Among the surviving animal, HI affect significantly the body growth, brain growth, MBP expression, and neurological behavior. EPO treatments at both early and late time points significantly benefit the rats in injury recovery, in which they promoted weight gains, reduced brain edema, as well as improved spatial learning ability and memory.. We demonstrated a single dose of EPO at 5000U/kg immediately or 48h after HI injury had significant benefit for the P2 rats in injury recovery, and there was no adverse effect associated with either EPO treatment.

Topics: Age Factors; Analysis of Variance; Animals; Animals, Newborn; Body Weight; Brain Edema; Developmental Disabilities; Disease Models, Animal; Erythropoietin; Hypoxia-Ischemia, Brain; Maze Learning; Myelin Basic Protein; Neuroprotective Agents; Rats; Rats, Sprague-Dawley

The erythropoietin (Epo) gene is under tissue-specific inducible regulation. Because the kidney is the primary EPO-producing tissue in adults, impaired EPO production in chronic kidney disorders results in serious renal anemia. The Epo gene contains a liver-specific enhancer in the 3' region, but the kidney-specific enhancer for gene expression in renal EPO-producing (REP) cells remains elusive. Here, we examined a conserved upstream element for renal Epo regulation (CURE) region that spans 17.4 kb to 3.6 kb upstream of the Epo gene and harbors several phylogenetically conserved elements. We prepared various Epo gene-reporter constructs utilizing a bacterial artificial chromosome and generated a number of transgenic-mouse lines. We observed that deletion of the CURE region (δCURE) abrogated Epo gene expression in REP cells. Although transgenic expression of the δCURE construct rescued Epo-deficient mice from embryonic lethality, the rescued mice had severe EPO-dependent anemia. These mouse lines serve as an elaborate model for the search for erythroid stimulatory activity and are referred to as AnRED (anemic model with renal EPO deficiency) mice. We also dissected the CURE region by exploiting a minigene harboring four phylogenetically conserved elements in reporter transgenic-mouse analyses. Our analyses revealed that Epo gene regulation in REP cells is a complex process that utilizes multiple regulatory influences.

Topics: Anemia; Animals; Base Pairing; Brain; Conserved Sequence; Crosses, Genetic; Disease Models, Animal; Embryo Loss; Erythropoietin; Female; Fetus; Genotype; Green Fluorescent Proteins; Homeostasis; Kidney; Male; Mice, Transgenic; Models, Biological; Organ Specificity; Polycythemia; Response Elements; Sequence Deletion; Transgenes

Patients with diabetic nephropathy have a high cardiovascular mortality. Epoetin beta pegol (continuous erythropoietin receptor activator, C.E.R.A.) is a drug for the treatment of renal anemia. In this study, we investigated the effect of C.E.R.A. on vascular endothelial function as evaluated by flow-mediated dilation (FMD) and the relationship between hematopoiesis and FMD in diabetic nephropathy rats.. Male Spontaneously Diabetic Torii rats (SDT, 22 weeks old) were used. C.E.R.A. (0.6, 1.2 μg/kg) was administered subcutaneously once every 2 weeks for 8 weeks. At 1 week after last administration (31 weeks old), we assessed FMD in the femoral arteries of anesthetized rats using a high-resolution ultrasound system. FMD was also measured 1 week after single C.E.R.A. treatment (5.0 μg/kg) to examine the influence of hematopoiesis.. Flow-mediated dilation was significantly decreased in SDT rats before the start of C.E.R.A. treatment (22 weeks old). Repeated administration of C.E.R.A. dose-dependently improved FMD in SDT rats (31 weeks old) without changing blood glucose, nitroglycerin-induced vasodilation, or kidney function. Long-term administration of C.E.R.A. improved the state of endothelial nitric oxide synthase uncoupling in the femoral arteries of SDT rats, which showed a positive correlation with FMD. On the other hand, there was no correlation between FMD and Hb or Hct in SDT rats. Furthermore, at 1 week after single administration of C.E.R.A., FMD was not significantly improved although hemoglobin levels were comparable with levels following long-term C.E.R.A.. Long-term treatment with C.E.R.A. improved FMD in SDT rats even after onset of endothelial dysfunction.

Topics: Animals; Biomarkers; Diabetes Mellitus; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Erythropoietin; Femoral Artery; Hematopoiesis; Hemoglobins; Male; Nitric Oxide Synthase Type III; Polyethylene Glycols; Rats, Inbred Strains; Recovery of Function; Signal Transduction; Time Factors; Vasodilation

Erythropoietin (EPO) is shown to exert protective effects on different tissues in haemorrhagic shock (HS) states. Nitric oxide (NO), as a multifunctional signaling molecule, is implicated in diverse physiologic and pathologic processes. In order to understand the exact mechanism of EPO protection, in this study we evaluated the role of different NOS enzymes in the EPO signaling pathway in male rats.. Rats were randomized to five groups: 1) Sham, 2) HS 3) EPO 4) L-NAME, a non-specific NOS inhibitor 5) 1400W, a specific iNOS inhibitor. HS was induced by withdrawal of 50% of total blood volume. After 2h, resuscitation was performed with the shed blood and Ringer's lactate. In group 3, rats were treated with EPO (300IU/kg, i.v.) over 10min before HS induction. In the L-NAME and 1400W groups, L-NAME (10mg/kg, i.p.) and 1400W (2mg/kg, i.p.) were administered 30min before EPO injection. Blood and kidney tissue samples were obtained 3h after resuscitation.. EPO increased the survival rate and significantly improved kidney function and histology compared to the HS group. There were less renal oxidative stress, apoptosis and systemic inflammatory responses in the EPO group. EPO increased eNOS and more abundantly iNOS mRNA expressions. L-NAME and 1400W significantly abolished all beneficial effects of EPO.. In this in vivo animal model, we showed that EPO administration prior to HS attenuates renal injury and dysfunction in rats. The protective effects of EPO may be mediated by nitric oxide and the expression of different NOS enzymes, especially iNOS isoform.

Topics: Acute Kidney Injury; Animals; Disease Models, Animal; Erythropoietin; Kidney; Male; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidative Stress; Rats; Rats, Wistar; Shock, Hemorrhagic

Iron is crucial for many biological functions, but quantitatively the most important use of iron is in the production of hemoglobin in red blood cell precursors. The amount of iron in the plasma, and hence its availability for hemoglobin synthesis, is determined by the liver-derived iron regulatory hormone hepcidin. When the iron supply to erythroid precursors is limited, as often occurs during stimulated erythropoiesis, these cells produce signals to inhibit hepatic hepcidin production, thereby increasing the amount of iron that enters the plasma. How stimulated erythropoiesis suppresses hepcidin production is incompletely understood, but erythroferrone, Gdf15 and Twsg1 have emerged as candidate regulatory molecules. To further examine the relationship between erythropoiesis and the candidate erythroid regulators, we have studied five mouse models of anemia, including two models of β-thalassemia (Hbbth3/+ and RBC14), the hemoglobin deficit mouse (hbd), dietary iron deficient mice and mice treated with phenylhydrazine to induce acute hemolysis. Hematological parameters, iron status and the expression of Erfe (the gene encoding erythroferrone), Gdf15 and Twsg1 in the bone marrow and spleen were examined. Erfe expression was the most consistently upregulated of the candidate erythroid regulators in all of the mouse models examined. Gene expression was particularly high in the bone marrow and spleen of iron deficient animals, making erythroferrone an ideal candidate erythroid regulator, as its influence is strongest when iron supply to developing erythroid cells is limited. Gdf15 expression was also upregulated in most of the anemia models studied although the magnitude of the increase was generally less than that of Erfe. In contrast, very little regulation of Twsg1 was observed. These results support the prevailing hypothesis that erythroferrone is a promising erythroid regulator and demonstrate that Erfe expression is stimulated most strongly when the iron supply to developing erythroid cells is compromised.

Topics: Anemia; Animals; Disease Models, Animal; Erythroid Precursor Cells; Erythropoietin; Hepcidins; Iron; Iron Deficiencies; Liver; Mice, Inbred C57BL; Models, Genetic; Phenylhydrazines; Receptors, Transferrin

This study compared the hematopoietic capacities of erythropoietin (Epo) and antioxidant drug U-74389G, based on 2 preliminary studies. The provided results on hematocrit levels augmentation were co-evaluated in a hypoxia reoxygenation protocol of an animal model.. Hematocrit levels were evaluated at the 60th reoxygenation min (for groups A, C and E) and at the 120th reoxygenation min (for groups B, D and F) in 60 rats. Groups A and B received no drugs, rats from groups C and D were administered with Epo; whereas rats from groups E and F were administered with U-74389G.. The first preliminary study of Epo non-significantly increased the hematocrit levels by 0.24%+1.38% (p-value=0.8586). The second preliminary study of U-74389G significantly raised the hematocrit levels by 3.16%+1.33% (p-value=0.0196). These 2 studies were co-evaluated since they came from the same experimental setting. The outcome of the co-evaluation was that U-74389G has approximately 12.66-fold higher hematopoietic potency than Epo (p-value=0.0000).. The anti-oxidant capacities of U-74389G provide satisfactory acute hematopoietic properties; presenting approximately 12.66-fold hematocrit level rise than epo (p-value=0.0000).

Topics: Animals; Antioxidants; Disease Models, Animal; Erythropoietin; Female; Hematocrit; Hematopoiesis; Humans; Hypoxia; Male; Pregnatrienes; Rats, Wistar; Reperfusion Injury

Erythropoietin (EPO) has been identified as a neuroregenerative agent. We hypothesize that it may accelerate recovery after crush injury and may vary with crush severity.. Mice were randomized to mild, moderate, or severe crush of the sciatic nerve and were treated with EPO or vehicle control after injury. The sciatic function index (SFI) was monitored over the first week. Microstructural changes were analyzed by immunofluorescence for neurofilament (NF) and myelin (P. In moderate crush injuries, EPO significantly improved SFI at 7 days post-injury, an effect not observed with other severity levels. Increases in the ratio of P. EPO accelerates recovery in moderately crushed nerves, which may be through effects on myelination and vascularization. Injury severity may influence the efficacy of EPO. Muscle Nerve 56: 143-151, 2017.

Topics: Analysis of Variance; Animals; Disease Models, Animal; Erythropoietin; Female; Gene Expression Regulation; Intermediate Filaments; Mice; Mice, Inbred C57BL; Microscopy, Electron; Myelin P0 Protein; Recovery of Function; Sciatic Nerve; Sciatic Neuropathy; Severity of Illness Index

Acute ischemia-reperfusion (I/R) injury is the most common causes of acute renal failure in daily clinical\ practice. It has been recognized that endothelial cell dysfunction and microvascular injury as the pathophysiological changes during I/R injury. Protective effects of erythropoietin (EPO) have been demonstrated in various experimental models of I/R induced injury. Therefore, the aim of the present study was to investigate whether EPO administration has renoprotective effect against acute renal failure I/R injury in rats by promotion of endothelial progenitor cells (EPCs) mobilization and\ neovascularization.. Male Sprague-Dawley rats were pretreated with EPO (1,000 IU/kg/day, ip); or the placebo for 3 days before the induction of I/R procedure. On day 4, the bilateral renal occlusion for 30 min operations to produce renal I/R injury or treatment with EPO 30 min before the initiation of I/R were done. At the end of the reperfusion period at day 1 day 2 and day 4, blood and renal tissues were collected to investigate renal function and pathohistological examination. The expression levels of CAV-1 and CD34 were determined for circulating of EPCs in blood, while CD34, CAV-1 and VEGFR-2 were investigated for mobilized EPCs in kidney, using real time PCR. The expression level of VEGF was also examined to indicate the angiogenesis in kidney using real time PCR and western blotting.. In the I/R group, the significantly increased values of serum urea and creatinine were found on Day 1 after ischemia, as compared to sham group. The development of tubular epithelial cell necrosis, peritubular capillary congestion and mild interstitial infiltration has been observed in this group. Administration of EPO in I/R rat was significantly improved renal function and significantly less the tubular damage. The treatment with EPO significantly increased in expression levels of CD34 and CAV-1 in blood, and also CAV-1, VEGFR-2 and VEGF in kidney tissue in this group, as compared to the I/R group.. These results suggest that treatment with EPO protects the kidney from ischemic acute renal injury via\ increasing the mobilization and recruitment of EPCs, resulting in the induction of expression of VEGF that might play an important role in the repair response.

Topics: Acute Kidney Injury; Animals; Blood Urea Nitrogen; Creatinine; Disease Models, Animal; Erythropoietin; Ischemia; Kidney; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

We investigated the effect of erythropoietin (EPO) posttreatment on survival time and vascular functions in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture. After 20 ± 2 hours of sepsis, thoracic aorta was isolated for assessing its reactivity to norepinephrine (NE) and acetylcholine (ACh). We also measured the tissue nitric oxide (NO) level, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), G protein-coupled receptor kinase 2 (GRK2), and α1D adrenoceptor messenger RNA (mRNA)/protein expression. In septic mice, EPO moderately improved the survival time from 19.68 ± 0.75 to 34.7 ± 3.2 hours. Sepsis significantly decreased the aortic contractile response to NE along with reduced α1D mRNA and protein expression. Erythropoietin significantly preserved the α1D receptor expression and restored NE-induced contractions to control levels in septic mice. Further, it attenuated the aortic α1D receptor desensitization in sepsis which was evident from reduced GRK2 mRNA expression. Accordingly, a selective GRK2 inhibitor markedly restored the contractile responses to NE in sepsis. Erythropoietin treatment attenuated iNOS mRNA expression and iNOS-induced overproduction of NO, but improved endothelium-dependent relaxation to ACh associated with increased eNOS mRNA expression. In conclusion, EPO seems to reverse sepsis-induced vasoplegia to NE through the preservation of α1D adrenoceptor mRNA/protein expression, inhibition of GRK2-mediated desensitization, and attenuation of NO overproduction in the mouse aorta.

Topics: Adrenergic alpha-Agonists; Animals; Aorta, Thoracic; Cecum; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; G-Protein-Coupled Receptor Kinase 2; Ligation; Male; Mice; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Norepinephrine; Protein Kinase Inhibitors; Punctures; Receptors, Adrenergic, alpha-1; RNA, Messenger; Sepsis; Signal Transduction; Time Factors; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents; Vasoplegia

Renal fibrosis is an inevitable outcome of chronic kidney disease (CKD). Erythropoietin (EPO) has been recently reported to be able to mitigate renal fibrosis. The mechanism underlying the protective effect of EPO, however, remains elusive. In the present study, employing a mouse model of renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO), we demonstrated that EPO markedly reduced the disruption of the tubular basement membrane (TBM) through attenuating the activation of tissue plasminogen activator (tPA) and matrix metalloproteinase 9 (MMP9), the major matrix proteolytic network in the obstructed kidney. Instead of acting directly on tPA in the kidney, EPO strongly increased the level of circulating microRNA (miR)-144, which was delivered to the injured renal fibroblasts via extracellular vesicles (EVs) to target the tPA 3'-untranslated region and suppress tPA expression. The protective effect of EPO on mouse TBM was inhibited by miR-144 antagomir. Furthermore, in vitro results confirmed that EPO could stimulate bone marrow-derived Sca-1(+)CD44(+)CD11b(-)CD19(-) cells to secrete miR-144-containing EVs, which markedly suppressed tPA expression, as well as metalloproteinase 9 (MMP9) level and activity, in cultured renal fibroblasts. In conclusion, our study provides the first evidence that EPO protects mouse renal TBM through promoting bone marrow cells to generate and secrete miR-144, which, in turn, is efficiently delivered into the mouse kidney via EVs to inhibit the activation of the tPA/MMP9-mediated proteolytic network. This finding thus suggests that EPO, a hormone widely used to treat anemia in CKD, is a potential therapeutic strategy for renal fibrosis.

Topics: 3' Untranslated Regions; Animals; Binding Sites; Bone Marrow Cells; Cell Line; Cytoprotection; Disease Models, Animal; Enzyme Activation; Enzyme Repression; Erythropoietin; Extracellular Vesicles; Fibroblasts; Fibrosis; Glomerular Basement Membrane; Kidney Diseases; Kidney Tubules; Male; Matrix Metalloproteinase 9; Mice; MicroRNAs; Rats; Recombinant Proteins; Signal Transduction; Tissue Plasminogen Activator; Ureteral Obstruction

Experimental study with rats.. To evaluate functional and histological effects of tacrolimus (FK 506) and erythropoietin (EPO) after experimental spinal cord contusion injury (SCI).. Brazil.. Wistar rats (n=60) were submitted to SCI with the NYU Impactor system. The control group received saline; the EPO group received EPO; the group EPO+FK 506 received EPO associated with tacrolimus and the group FK 506 received tacrolimus only. The Sham group underwent SCI, but did not receive any drug. Locomotor function was evaluated after SCI by BBB (Basso, Beattie and Bresnahan) weekly and by the motor-evoked potential test in 42 days. The spinal cord was histologically evaluated.. There was a significant difference between treated and the control groups from the seventh day on for BBB scores, with no difference between the groups EPO and EPO+FK 506 by the end of the study. There were significant differences between groups for necrosis and bleeding, but not for hiperemia, degeneration and cellular infiltrate. Axon neuron count was different between all groups (P=0.001), between EPO+FK 506 and FK 506 (P=0.011) and between EPO+FK 506 and Sham (P=0.002). Amplitude was significantly different between all groups except between control and sham. For latency, there was no difference.. This study did not reveal significant differences in the recovery of locomotor function, or in the histological and electrophysiological analysis in animals treated with EPO and tacrolimus after thoracic SCI.

Topics: Animals; Disease Models, Animal; Erythropoietin; Evoked Potentials, Motor; Follow-Up Studies; Immunosuppressive Agents; Locomotion; Nervous System Diseases; Rats; Rats, Wistar; Recovery of Function; Spinal Cord Injuries; Statistics, Nonparametric; Tacrolimus; Time Factors

Glaucoma, a common cause of blindness, is currently treated by intraocular pressure (IOP)-lowering interventions. However, this approach is insufficient to completely prevent vision loss. Here, we evaluate an IOP-independent gene therapy strategy using a modified erythropoietin, EPO-R76E, which has reduced erythropoietic function. We used two models of glaucoma, the murine microbead occlusion model and the DBA/2J mouse. Systemic recombinant adeno-associated virus-mediated gene delivery of EpoR76E (rAAV.EpoR76E) was performed concurrent with elevation of IOP. Axon structure and active anterograde transport were preserved in both models. Vision, as determined by the flash visual evoked potential, was preserved in the DBA/2J. These results show that systemic EpoR76E gene therapy protects retinal ganglion cells from glaucomatous degeneration in two different models. This suggests that EPO targets a component of the neurodegenerative pathway that is common to both models. The efficacy of rAAV.EpoR76E delivered at onset of IOP elevation supports clinical relevance of this treatment.

Topics: Animals; Axons; Dependovirus; Disease Models, Animal; Erythropoietin; Genetic Therapy; Genetic Vectors; Glaucoma; Humans; Intraocular Pressure; Mice; Mutation; Optic Nerve

Acute and severe anemia of inflammation (AI) is a common complication of various clinical syndromes, including fulminant infections, critical illness with multiorgan failure, and exacerbations of autoimmune diseases. Building on recent data showing beneficial results with isocitrate treatment for chronic low-grade AI in a rat model, we used a mouse model of acute and severe AI induced by intraperitoneal heat-killed Brucella abortus to determine if isocitrate would be effective in this more stringent application. Inflamed mice treated with isocitrate developed an early but transient improvement in hemoglobin compared to solvent-treated controls, with a robust improvement on day 7, and only a trend towards improvement by day 14. Reticulocyte counts were increased in treated mice transiently, with no significant difference by day 21. Serum erythropoietin (EPO) levels were similar in treated versus control mice, indicating that isocitrate increased sensitivity to EPO. Serum and tissue iron levels showed no significant differences between the treated and control mice, ruling out improved iron availability as the cause of the increased response to endogenous EPO. Compared to the milder rat model, much higher doses of isocitrate were required for a relatively modest benefit.

Topics: Acute Disease; Anemia; Animals; Brucella abortus; Brucellosis; Disease Models, Animal; Erythropoiesis; Erythropoietin; Hepcidins; Inflammation; Iron; Isocitrates; Male; Mice

This study examined effects and functional outcome of recombinant human erythropoietin (rhEPO) and carbamylated erythropoietin fusion protein (cEPO-FC) preconditioning in a rabbit model for spinal cord ischemia and resulting paraplegia. This model was chosen because only a small surgical effect is needed to cause paraplegia in rabbits, which facilitates postoperative observation of animals.. Anesthetized but spontaneously breathing New Zealand White rabbits randomly received cEPO-FC (50 μg/kg; n = 8), rhEPO (5000 IU/kg; n = 10), or vehicle (control; n = 10) 30 minutes before and after infrarenal aortic clamping. Ideal clamping time of 22 minutes was identified from preceding clamping tests (15-25 minutes). Postoperative observation time was 96 hours. Spinal cord function was assessed by neurologic evaluation of hind limb motor function every 12 hours using a modified Tarlov score. Spinal cord tissue damage was evaluated after 96 hours using hematoxylin and eosin, elastica van Gieson, Nissl, Masson-Goldner, and hemosiderin staining. Plasma levels of cell senescence markers stathmin, chitinase 1/3, elongation factor 1-α were determined.. Rabbits that received rhEPO showed significant improvement of spontaneous lower limb movements until 36 hours of reperfusion and improved histologic scores upon examination of the lumbar spinal cord compared with the control group. In contrast, cEPO-FC treatment showed comparable outcome to the control group concerning movements of the lower limbs and histology. Senescence markers were elevated in the control group, but not in the treatment groups, except for chitinase 3 in the rhEPO group. Only stathmin showed no significant effect. Markers for senescence might increase after acute ischemic injury. Attenuation of senescence markers might not come alone from improvement of the spinal cord.. Preconditioning with rhEPO attenuates ischemia/reperfusion injury of the spinal cord, whereas the carbamylated derivative (cEPO-FC) showed no positive effect on spinal cord function.

Topics: Animals; Biomarkers; Cellular Senescence; Chitinases; Disease Models, Animal; Erythropoietin; Male; Motor Activity; Neurologic Examination; Neuroprotective Agents; Paraplegia; Peptide Elongation Factor 1; Rabbits; Recombinant Proteins; Reperfusion Injury; Spinal Cord; Spinal Cord Ischemia; Stathmin; Time Factors

Beneficial effects of erythropoietin (EPO) have been reported in acute kidney injury (AKI) when administered prior to induction of AKI. We studied the effects of EPO administration on renal function shortly after ischemic AKI. For this purpose, rats were subjected to renal ischemia for 30 min and EPO was administered at a concentration of 500 U/kg either i.v. as a single shot directly after ischemia or with an additional i.p. dose until 3 days after surgery. The results were compared with AKI rats without EPO application and a sham-operated group. Renal function was assessed by measurement of serum biochemical markers, histological grading, and using an isolated perfused kidney (IPK) model. Furthermore, we performed flow cytometry to analyze the concentration of endothelial progenitor cells (EPCs) in the peripheral blood and renal vessels. Following EPO application, there was only a statistically non-significant tendency of serum creatinine and urea to improve, particularly after daily EPO application. Renal vascular resistance and the renal perfusion rate were not significantly altered. In the histological analysis, acute tubular necrosis was only marginally ameliorated following EPO administration. In summary, we could not demonstrate a significant improvement in renal function when EPO was applied after AKI. Interestingly, however, EPO treatment resulted in a highly significant increase in CD133- and CD34-positive EPC both in the peripheral blood and renal vessels.

Topics: AC133 Antigen; Acute Kidney Injury; Animals; Blood Vessels; Creatinine; Disease Models, Animal; Endothelial Progenitor Cells; Erythropoietin; Flow Cytometry; Glomerular Filtration Rate; Hematocrit; Ischemia; Kidney; Male; Rats; Rats, Sprague-Dawley; Urea

Aneurysmal subarachnoid hemorrhage (SAH) is a common condition with relatively poor clinical outcome. Pulmonary complication after SAH is an important contributor to poor outcome. Previous studies have shown that labile zinc and inflammatory mediators participate in many pathophysiological processes. The present study investigated the effects of SAH on the levels of labile zinc and certain proinflammatory factors in rat lung and determined the effect of erythropoietin (EPO) on the pulmonary labile zinc and the inflammatory factor after SAH in rats.. Experiment 1 aimed to investigate the time course of increase of pulmonary labile zinc, wet/dry weight ratio, and the expression of inflammatory mediators after SAH. In Experiment 2, we chose the maximum time point which lung injury was maximally severity and assessed the effect of EPO on regulation of the pulmonary labile zinc, inflammatory reaction, and wet/dry weight ratio after SAH.. SAH caused a gradual increase of pulmonary labile zinc as demonstrated by fluorescence staining with Zinpyr-4. The levels of TNF-α and IL-8 and the lung wet/dry weight ratios were higher in the SAH groups compared to the control group and peaked on 3 days following SAH (p < 0.05). EPO significantly reduced the pulmonary labile zinc, the inflammatory mediators, and the lung wet/dry weight ratio compared with SAH group (p < 0.05).. EPO can protect lung from SAH-induced injury by attenuating pulmonary inflammation and labile zinc accumulation in vivo.

Topics: Animals; Disease Models, Animal; Erythropoietin; Inflammation Mediators; Lung; Lung Injury; Male; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Zinc

Increased fragility has been described in humans with polycythemia vera (PV). Herein, we describe an osteoporotic phenotype associated with decreased osteoblast activity in a mouse model of PV and another mouse of polycythemia and elevated circulating erythropoietin (EPO). Our results are important for patients with PV or those treated with recombinant EPO (rEPO).. PV and other myeloproliferative syndromes have been recently associated with an increased risk for fractures. However, the presence of osteoporosis in these patients has not been well documented. EPO, a hormone primarily known to stimulate erythropoiesis, has been shown recently to regulate bone homeostasis in mice. The aim of this study was to examine the bone phenotype of a mouse model of PV and compare it to that of animals with polycythemia caused by elevated circulating EPO.. Bone mass and remodeling were evaluated by micro-computed tomography and histomorphometry. The JAK2(V617F) knock-in mouse, a model of human PV, manifests polycythemia and low circulating EPO levels. Results from this mouse were compared to wild type (wt) controls and the tg6 transgenic mouse that shows polycythemia caused by increased constitutive expression of EPO.. Compared to wt, both JAK2(V617F) and tg6 mice had a decrease in trabecular bone mass. Tg6 mice showed an additional modest decrease in cortical thickness and cortical bone volume per tissue volume (P < 0.01) suggesting a more severe bone phenotype than JAK2(V617F). Decreased osteoblast numbers and bone formation along with normal osteoclast numbers and activity were found in both mice.. This study indicates that PV is associated with low bone mass and decreased osteoblast activity in mice. Our results support future studies of osteoporosis in affected humans. Polycythemia caused by chronically elevated circulating EPO also results in bone loss, and implications on patients treated with rEPO should be evaluated.

Topics: Animals; Bone Density; Bone Diseases, Metabolic; Bone Remodeling; Disease Models, Animal; Erythropoietin; Male; Mice, Knockout; Mice, Transgenic; Osteoblasts; Osteogenesis; Polycythemia; Polycythemia Vera; X-Ray Microtomography

Experimental studies targeting traumatic brain injury (TBI) have reported that erythropoietin (EPO) is an endogenous neuroprotectant in multiple models. In addition to its neuroprotective effects, it has also been shown to enhance reparative processes including angiogenesis and neurogenesis. Based on compelling pre-clinical data, EPO was tested by the Operation Brain Trauma Therapy (OBTT) consortium to evaluate therapeutic potential in multiple TBI models along with biomarker assessments. Based on the pre-clinical TBI literature, two doses of EPO (5000 and 10,000 IU/kg) were tested given at 15 min after moderate fluid percussion brain injury (FPI), controlled cortical impact (CCI), or penetrating ballistic-like brain injury (PBBI) with subsequent behavioral, histopathological, and biomarker outcome assessments. There was a significant benefit on beam walk with the 5000 IU dose in CCI, but no benefit on any other motor task across models in OBTT. Also, no benefit of EPO treatment across the three TBI models was noted using the Morris water maze to assess cognitive deficits. Lesion volume analysis showed no treatment effects after either FPI or CCI; however, with the 5000 IU/kg dose of EPO, a paradoxical increase in lesion volume and percent hemispheric tissue loss was seen after PBBI. Biomarker assessments included measurements of glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase-L1 (UCH-L1) in blood at 4 or 24 h after injury. No treatment effects were seen on biomarker levels after FPI, whereas treatment at either dose exacerbated the increase in GFAP at 24 h in PBBI but attenuated 24-4 h delta UCH-L1 levels at high dose in CCI. Our data indicate a surprising lack of efficacy of EPO across three established TBI models in terms of behavioral, histopathological, and biomarker assessments. Although we cannot rule out the possibility that other doses or more prolonged treatment could show different effects, the lack of efficacy of EPO reduced enthusiasm for its further investigation in OBTT.

Topics: Animals; Biomarkers; Brain Injuries, Traumatic; Disease Models, Animal; Erythropoietin; Glial Fibrillary Acidic Protein; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Recovery of Function; Ubiquitin Thiolesterase

Neurodegenerative disorders such as Alzheimer's disease (AD) are characterized by progressive cognitive dysfunction and memory loss. There is deposition of amyloid plaques in the brain and subsequent neuronal loss. Neuroinflammation plays a key role in the pathogenesis of AD. There is still no effective curative therapy for these patients. One promising strategy involves the stimulation of endogenous stem cells. This study investigated the therapeutic effect of erythropoietin (EPO) in neurogenesis, and proved its manipulation of the endogenous mesenchymal stem cells in model of lipopolysaccharide (LPS)-induced neuroinflammation.. Forty five adult male mice were divided equally into 3 groups: Group I (control), group II (LPS untreated group): mice were injected with single dose of lipopolysaccharide (LPS) 0.8 mg/kg intraperitoneally (ip) to induce neuroinflammation, group III (EPO treated group): in addition to (LPS) mice were further injected with EPO in dose of 40 μg/kg of body weight three times weekly for 5 consecutive weeks. Groups were tested for their locomotor activity and memory using open field test and Y-maze. Cerebral specimens were subjected to histological and morphometric studies. Glial fibrillary acidic protein (GFAP) and mesenchymal stem cell marker CD44 were assessed using immunostaining. Gene expression of brain derived neurotrophic factor (BDNF) was examined in brain tissue.. LPS decreased locomotor activity and percentage of correct choices in Y-maze test. Cerebral sections of LPS treated mice showed increased percentage area of dark nuclei and amyloid plaques. Multiple GFAP positive astrocytes were detected in affected cerebral sections. In addition, decrease BDNF gene expression was noted. On the other hand, EPO treated group, showed improvement in locomotor and cognitive function. Examination of the cerebral sections showed multiple neurons exhibiting less dark nuclei and less amyloid plaques in comparison to the untreated group. GFAP positive astrocytes were also reduced. Cerebral sections of the EPO treated group showed multiple branched and spindle CD44 positive cells inside and around blood vessels more than in LPS group. This immunostaining was negative in the control group. EPO administration increased BDNF gene expression.. This study proved that EPO provides excellent neuroprotective and neurotrophic effects in vivo model of LPS induced neuroinflammation. It enhances brain tissue regeneration via stimulation of endogenous mesenchymal stem cells proliferation and their migration to the site of inflammation. EPO also up regulates cerebral BDNF expression and production, which might contributes to EPO mediated neurogenesis. It also attenuates reactive gliosis thus reduces neuroinflammation. These encouraging results obtained with the use of EPO proved that it may be a promising candidate for future clinical application and treatment of neurodegenerative diseases.

Topics: Analysis of Variance; Animals; Brain-Derived Neurotrophic Factor; Cell Movement; Disease Models, Animal; Encephalitis; Erythropoietin; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Hyaluronan Receptors; Lipopolysaccharides; Locomotion; Male; Maze Learning; Mice; Neuroprotective Agents; RNA, Messenger; Stem Cells

Matriptase-2 (TMPRSS6) is an important negative regulator of hepcidin expression; however, the effects of iron overload or accelerated erythropoiesis on liver TMPRSS6 protein content in vivo are largely unknown. We determined TMPRSS6 protein content in plasma membrane-enriched fractions of liver homogenates by immunoblotting, using a commercial antibody raised against the catalytic domain of TMPRSS6. Plasma membrane-enriched fractions were obtained by centrifugation at 3000 g and washing. TMPRSS6 was detected in the 3000 g fraction as a 120 kDa full-length protein in both mice and rats. Feeding of iron-deficient diet as well as erythropoietin treatment increased TMPRSS6 protein content in rats and mice by a posttranscriptional mechanism; the increase in TMPRSS6 protein by erythropoietin was also observed in Bmp6-mutant mice. Administration of high doses of iron to mice (200, 350 and 700 mg/kg) decreased TMPRSS6 protein content. Hemojuvelin was detected in the plasma membrane-enriched fractions of control animals as a full length protein of approximately 52 kDa; in iron deficient animals, the full length protein was partially cleaved at the N-terminus, resulting in an additional weak band of approximately 47 kDa. In livers from hemojuvelin-mutant mice, TMPRSS6 protein content was strongly decreased, suggesting that intact hemojuvelin is necessary for stable TMPRSS6 expression in the membrane. Overall, the results demonstrate posttranscriptional regulation of liver TMPRSS6 protein by iron status and erythropoietin administration, and provide support for the interaction of TMPRSS6 and hemojuvelin proteins in vivo.

Topics: Anemia, Iron-Deficiency; Animals; Bone Morphogenetic Protein 6; Disease Models, Animal; Erythropoietin; Female; GPI-Linked Proteins; Hemochromatosis Protein; Iron; Iron Deficiencies; Iron Overload; Liver; Male; Membrane Proteins; Mice; Mice, Knockout; Mutation; Rats; Serine Endopeptidases; Sodium-Potassium-Exchanging ATPase

To investigate the healing effects of erythropoietin (EPO) and stem cells (SCs) in traumatic brain injury (TBI).. Twenty-nine Wistar albino rats were used and separated into the following groups: control (C), EPO, SC, and SC+EPO. Group C received a TBI only, with no treatment. In the EPO group, 1000 U/kg EPO was given intraperitoneally at 30 minutes after TBI. In SC group, immediately after formation of TBI, 3 × 10,000 CD34(+) stem cells were injected into the affected area. In the SC+EPO group, half an hour after TBI and the injection of stem cells, 1000 U/kg EPO was injected. Before and after injury, trauma coordination performance was measured by the rotarod and inclined plane tests.. Seven weeks after trauma, rat brains were examined by radiology and histology. Rotarod performance test did not change remarkably, even after the injury. Compared with group C, the SC+EPO group was found to have significant differences in the inclined plane test results.. Separately given, SCs and EPO have a positive effect on TBI, and our findings suggest that their coadministration is even more powerful.

Topics: Animals; Antigens, CD34; Brain; Brain Injuries, Traumatic; Combined Modality Therapy; Cord Blood Stem Cell Transplantation; Disease Models, Animal; Erythropoietin; Female; Humans; Injections, Intraperitoneal; Magnetic Resonance Imaging; Male; Neuroprotective Agents; Rats, Wistar; Rotarod Performance Test; Treatment Outcome

Glaucoma is a complex neurodegeneration and a leading cause of blindness worldwide. Current therapeutic strategies, which are all directed towards lowering the intraocular pressure (IOP), do not stop progression of the disease. We have demonstrated that recombinant adeno-associated virus (rAAV) gene delivery of a form of erythropoietin with attenuated erythropoietic activity (EpoR76E) can preserve retinal ganglion cells, their axons, and vision without decreasing IOP. The goal of this study was to determine if modulation of neuroinflammation or oxidative stress played a role in the neuroprotective activity of EPO.R76E.. Five-month-old DBA/2J mice were treated with either rAAV.EpoR76E or a control vector and collected at 8 months of age. Neuroprotection was assessed by quantification of axon transport and visual evoked potentials. Microglia number and morphology and cytokine and chemokine levels were quantified. Message levels of oxidative stress-related proteins were assessed.. Axon transport and visual evoked potentials were preserved in rAAV.EpoR76E-treated mice. The number of microglia was decreased in retinas from 8-month-old rAAV.EpoR76E-treated mice, but proliferation was unaffected. The blood-retina barrier was also unaffected by treatment. Levels of some pro-inflammatory cytokines were decreased in retinas from rAAV.EpoR76E-treated mice including IL-1, IL-12, IL-13, IL-17, CCL4, and CCL5. TNFα messenger RNA (mRNA) was increased in retinas from 8-month-old mice compared to 3-month-old controls regardless of treatment. Expression of several antioxidant proteins was increased in retinas of rAAV.EpoR76E-treated 8-month-old mice.. Treatment with rAAV.EpoR76E preserves vision in the DBA/2J model of glaucoma at least in part by decreasing infiltration of peripheral immune cells, modulating microglial reactivity, and decreasing oxidative stress.

Topics: Animals; Calcium-Binding Proteins; Cholera Toxin; Cytokines; Dependovirus; Disease Models, Animal; Erythropoietin; Evoked Potentials, Visual; Fluorescein Angiography; Gene Expression Regulation; Genetic Therapy; Glaucoma; Ki-67 Antigen; Mice; Mice, Inbred DBA; Microfilament Proteins; Microglia; Oxidative Stress; Photic Stimulation; Retina; Transduction, Genetic

Renal hypoplasia due to a congenitally reduced number of nephrons progresses to chronic kidney disease and may cause renal anemia, given that the kidneys are a major source of erythropoietin in adults. Hypoplastic kidney (HPK) rats have only about 20% of the normal number of nephrons and develop CKD. This study assessed the renal function and hematologic changes in HPK rats from 70 to 210 d of age. HPK rats demonstrated deterioration of renal excretory function, slightly macrocytic erythropenia at all days examined, age-related increases in splenic hemosiderosis accompanied by a tendency toward increased hemolysis, normal plasma erythropoietin levels associated with increased hepatic and decreased renal erythropoietin production, and maintenance of the response for erythropoietin production to hypoxic conditions, with increased interstitial fibrosis at 140 d of age. These results indicate that increases in splenic hemosiderosis and the membrane fragility of RBC might be associated with erythropenia and that hepatic production of erythropoietin might contribute to maintaining the blood Hgb concentration in HPK rats.

Topics: Age Factors; Anemia; Animals; Biomarkers; Carrier Proteins; Disease Models, Animal; Disease Progression; Erythrocytes; Erythropoietin; Fibrosis; Genetic Predisposition to Disease; Hemolysis; Hemosiderosis; Iron; Kidney; Kidney Diseases; Male; Mutation; Osmotic Fragility; Phenotype; Rats; Rats, Inbred Strains; Renal Insufficiency, Chronic; Spleen

Clinical studies showed that high doses of recombinant human erythropoietin (rHuEPO) used to correct anaemia in chronic kidney disease (CKD) hyporesponsive patients may lead to deleterious effects. The aim of this study was to analyze the effects of rHuEPO in doses usually used to correct CKD-anaemia (100, 200 IU/kg body weight (BW) per week) and in higher doses used in the treatment of hyporesponsive patients (400, 600 IU/kg BW per week), focusing on renal damage, hypoxia, inflammation and fibrosis. Male Wistar rats with chronic renal failure (CRF) induced by 5/6 nephrectomy were treated with rHuEPO or with vehicle, over a 3-week period. Haematological, biochemical and renal function analyses were performed. Kidney and liver mRNA levels were evaluated by quantitative real-time polymerase chain reaction (qPCR) and protein expression by Western blot and immunohistochemistry. Kidney histopathological evaluations were also performed. The CRF group developed anaemia, hypertension and a high score of renal histopathologic lesions. Correction of anaemia was achieved with all rHuEPO doses, with improvement in hypertension, renal function and renal lesions. In addition, the higher rHuEPO doses also improved inflammation. Blood pressure was reduced in all rHuEPO-treated groups, compared to the CRF group, but increased in a dose-dependent manner. The current study showed that rHuEPO treatment corrected anaemia and improved urinary albumin excretion, particularly at lower doses. In addition, it is suggested that a short-term treatment with high doses, used to overcome an episode of hyporesponsiveness to rHuEPO therapy, can present benefits by reducing inflammation, without worsening of renal lesions; however, the pro-hypertensive effect should be considered, and carefully managed to avoid a negative cardiorenal impact.

Topics: Anemia; Animals; Blood Pressure; Body Weight; Cell Hypoxia; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Fibrosis; Gene Expression Regulation; Humans; Hypertension; Inflammation; Kidney; Kidney Failure, Chronic; Male; Organ Size; Rats; Recombinant Proteins; Risk; Risk Assessment

OBJECTIVE Traumatic brain injury (TBI) is a leading cause of death and severe morbidity for otherwise healthy full-term infants around the world. Currently, the primary treatment for infant TBI is supportive, as no targeted therapies exist to actively promote recovery. The developing infant brain, in particular, has a unique response to injury and the potential for repair, both of which vary with maturation. Targeted interventions and objective measures of therapeutic efficacy are needed in this special population. The authors hypothesized that MRI and serum biomarkers can be used to quantify outcomes following infantile TBI in a preclinical rat model and that the potential efficacy of the neuro-reparative agent erythropoietin (EPO) in promoting recovery can be tested using these biomarkers as surrogates for functional outcomes. METHODS With institutional approval, a controlled cortical impact (CCI) was delivered to postnatal Day (P)12 rats of both sexes (76 rats). On postinjury Day (PID)1, the 49 CCI rats designated for chronic studies were randomized to EPO (3000 U/kg/dose, CCI-EPO, 24 rats) or vehicle (CCI-veh, 25 rats) administered intraperitoneally on PID1-4, 6, and 8. Acute injury (PID3) was evaluated with an immunoassay of injured cortex and serum, and chronic injury (PID13-28) was evaluated with digitized gait analyses, MRI, and serum immunoassay. The CCI-veh and CCI-EPO rats were compared with shams (49 rats) primarily using 2-way ANOVA with Bonferroni post hoc correction. RESULTS Following CCI, there was 4.8% mortality and 55% of injured rats exhibited convulsions. Of the injured rats designated for chronic analyses, 8.1% developed leptomeningeal cyst-like lesions verified with MRI and were excluded from further study. On PID3, Western blot showed that EPO receptor expression was increased in the injured cortex (p = 0.008). These Western blots also showed elevated ipsilateral cortex calpain degradation products for αII-spectrin (αII-SDPs; p < 0.001), potassium chloride cotransporter 2 (KCC2-DPs; p = 0.037), and glial fibrillary acidic protein (GFAP-DPs; p = 0.002), as well as serum GFAP (serum GFAP-DPs; p = 0.001). In injured rats multiplex electrochemiluminescence analyses on PID3 revealed elevated serum tumor necrosis factor alpha (TNFα p = 0.01) and chemokine (CXC) ligand 1 (CXCL1). Chronically, that is, in PID13-16 CCI-veh rats, as compared with sham rats, gait deficits were demonstrated (p = 0.033) but then were reversed (p = 0.022) with EP

Topics: Age Factors; Animals; Animals, Newborn; Biomarkers; Brain Injuries, Traumatic; Calpain; Cerebral Cortex; Cytokines; Diffusion Magnetic Resonance Imaging; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Female; Gait Disorders, Neurologic; Gene Expression Regulation, Developmental; Glial Fibrillary Acidic Protein; Image Processing, Computer-Assisted; K Cl- Cotransporters; Male; Rats; Receptors, Erythropoietin; Statistics, Nonparametric; Symporters; Time Factors

Septic conditions contribute to tissue hypoxia, potentially leading to multiple organ failure, including acute kidney injury. The regulation of cellular adaptation to low oxygen levels is regulated by hypoxia-inducible transcription factors (HIFs). While the role of HIFs in ischaemia/reperfusion is more studied, their function in sepsis-induced renal injury is not well characterized. In this study, we investigated whether pharmacological activation of HIFs by suppression of prolyl-hydroxylases (PHDs) protects against septic acute kidney injury.. Two models of sepsis-caecal ligation and punction and peritoneal contamination and infection-were induced on 12-week-old C57BL6/J mice. Pharmacological inhibition of PHDs, leading to HIF activation, was achieved by intraperitoneal application of 3,4-dihydroxybenzoate (3,4-DHB) before sepsis. A quantitative real-time reverse transcription polymerase chain reaction, immunohistology and enzyme-linked immunosorbent assays were utilized to detect gene expression, renal protein levels and renal functional parameters, respectively. Tissue morphology was analysed by periodic acid-Schiff reaction. Early kidney injury was estimated by kidney injury molecule-1 analyses. Apoptosis was detected in situ by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling stain. The systemic effect of 3,4-DHB pretreatment in sepsis was analysed by 72-h survival studies.. Pharmacological activation of HIFs before sepsis induction attenuated sepsis-related vacuolization and dilation of the proximal tubules, reduced tubular apoptosis and correlated to lower T-cell infiltration in renal tissue compared with the non-treated septic animals. PHD suppression elevated the basal renal HIF-1α expression and basal plasma concentrations of HIF targets erythropoietin and vascular endothelial growth factor. Whereas it preserved renal structure in both models, it improved renal function in a model-dependent manner. Moreover, inhibition of PHDs led to increased mortality in both models. Analysis of liver function showed increased organ destruction with massive glycogen loss and hepatocyte's apoptosis due to 3,4-DHB administration before sepsis induction.. In summary, the pharmacological activation of HIFs by 3,4-DHB administration, although it showed renoprotective effects in sepsis-related kidney injury, induced more severe problems in other organs such as the liver during sepsis, leading to increased mortality.

Topics: Acute Kidney Injury; Animals; Apoptosis; Disease Models, Animal; Erythropoietin; Gene Expression; Hydroxybenzoates; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Male; Mice; Mice, Inbred C57BL; Mortality; Prolyl Hydroxylases; Prolyl-Hydroxylase Inhibitors; Sepsis; Vascular Endothelial Growth Factor A

Owing to a lack of effective treatment approaches, sepsis is considered a life-threatening clinical syndrome worldwide. Many therapeutic interventions combating sepsis have been evaluated in animal models and clinical cases over the past few decades. Due to the pleiotropic characteristics of EPO, many studies have shown that erythropoietin (EPO) would be used to alleviate sepsis-induced tissue injury beyond the hemoglobin elevation effect. Nevertheless, the organ protective activity of EPO could not be supported by all of the results. In order to address the unanswered questions, a new methodical approach is necessary to be considered. The latest progress in metabolomics could be helpful to interpret the underlying mechanisms of EPO on sepsis, via metabolite profiling, to bring in some potent predictable fact for clinical application.. Twenty-one male Sprague-Dawley rats were divided into 3 groups of 7 rats each. Sepsis was induced by cecal ligation and puncture (CLP). Rats in the sepsis group were injected with equal volume of saline post-CLP. Rats from the EPO group were treated twice with EPO (immediately and 24 hours after CLP, 3750 IU/kg). The rats in the sham group were subjected to a sham surgery and injected with saline at the same time as the sepsis group. Serum samples were collected for biochemical and metabolomic analysis 72 hours post-CLP.. Biochemistry analysis revealed that erythropoietin improved the condition of multiple organs damaged by sepsis. Fifty-eight serum metabolites, including amino acids and fatty acids, displayed significant differences between the sepsis and sham groups. EPO treatment was found to attenuate the metabolic imbalances induced by CLP.. This study indicated that the metabolomic approach provided a comprehensive insight towards the metabolic targets of EPO treatment of sepsis.

Topics: Animals; Disease Models, Animal; Drug Delivery Systems; Erythropoietin; Male; Metabolomics; Rats; Rats, Sprague-Dawley; Sepsis

The goal of this study was to investigate the protective effect of recombinant human EPO(rhEPO) on cerebral microvascular endothelial cells and the mechanisms by which rhEPO interacts with TJs proteins, claudin-5, Occludin and ZO-1 during the early period following traumatic brain injury.. Rats (n = 81) were randomly divided into sham-operated group, TBI group and rhEPO+TBI group. Traumatic brain injury was induced by the Marmarou method.. Rats were killed at 3, 24, 72 and 168 hours after TBI. The integrity of the blood-brain barrier was investigated by using a spectrophotometer to assess extravasation of Evans blue dye. The expression of Claudin-5, Occludin and ZO-1 were determined by immunohistochemistry and real-time fluorescence quantitative PCR.. From 3 hours to 3 days, rats in the TBI group demonstrated a remarkable increase in Evans blue content in the brain, relative to rats in the sham-operated group (p < 0.05). The expression of Claudin-5 and Occludin was significantly lower than those in the sham-operated group (p < 0.05). In contrast, rats in the TBI+rhEPO group demonstrated a significant decrease in brain levels.. It was found that administration of rhEPO protected cerebral microvascular endothelial cells and reduced permeability of BBB and the mechanisms may be due to increasing the expression of TJs proteins.

Topics: Animals; Blood-Brain Barrier; Brain Injuries, Traumatic; Claudin-5; Disease Models, Animal; Drug Administration Schedule; Endothelial Cells; Erythropoietin; Gene Expression Regulation; Occludin; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tight Junctions; Time Factors; Zonula Occludens-1 Protein

This study explores the effects of helix B surface peptide (HBSP) on myocardial infarct size (IS), cardiac function, cardiomyocyte apoptosis and oxidative stress damage in mouse hearts subjected to myocardial ischemia-reperfusion injury (MIRI) and also the mechanisms underlying the effects.. Male adult mice were subjected to 45 min of ischemia followed by 2 h of reperfusion; 5 min before the reperfusion, they were treated with HBSP or vehicle. MIRI-induced IS, cardiomyocyte apoptosis and cardiac functional impairment were determined and compared. Western blot analysis was then conducted to elucidate the mechanism of HBSP after treatment.. HBSP administration before reperfusion significantly reduced the myocardial IS, decreased cardiomyocyte apoptosis, reduced the activities of superoxide dismutase and malondialdehyde and partially preserved heart function. As demonstrated by the Western blot analysis, HBSP after treatment upregulated Akt/GSK-3β/ERK and STAT-3 phosphorylation; these inhibitors, in turn, weakened the beneficial effects of HBSP.. HBSP plays a protective role in MIRI in mice by inhibiting cardiomyocyte apoptosis, reducing the MIRI-induced IS, oxidative stress and improving the heart function after MIRI. The mechanism underlying these effects of HBSP is related to the activation of the RISK (reperfusion injury salvage kinase, Akt/GSK-3β/ERK) and SAFE (STAT-3) pathways.

Topics: Animals; Apoptosis; Disease Models, Animal; Erythropoietin; Male; Mice; Myocardial Reperfusion Injury; Myocytes, Cardiac; Peptide Fragments; Phosphorylation; Signal Transduction; Ventricular Function

This study aimed at investigating the possible protective effect of erythropoietin beta on experimental diabetic nephropathy (DN) model in rats.. Sprague Dawley rats (n = 32) were allocated into 4 equal groups of 8 each, the control (Group C), diabetes (Group D), erythropoietin beta (Group E), and erythropoietin beta treated DN (Group E + D) groups. Streptozocin (65 mg/kg) was used to induce diabetes in 10-week old rats. Erythropoietin beta was given intraperitoneally at a dose of 500 IU/kg/3 days of a week for 12 weeks. Renal function parameters, intrarenal levels and activities of oxidative stress biomarkers, serum inflammatory parameters and kidney histology were determined.. Group E + D had lower mean albumin-to-creatinine ratio (p < 0.001) as well as higher creatinine clearance (p = 0.035) than the diabetic rats (Group D). Intrarenal malondialdehyde levels were significantly lower (p = 0.004); glutathione (GSH) levels (p = 0.003), GSH peroxidase (p = 0.004) and superoxide dismutase (p < 0.005) activities of renal tissue were significantly higher in Group E + D than in Group D. The mean serum levels of interleukin-4 (p < 0.005), interleukin 1 beta (p = 0.012), interferon gamma (p = 0.018) and tumor necrosis factor alpha (p < 0.005) were significantly lower; serum levels of monocyte chemoattractant protein 1 (p = 0.018) was significantly higher in Group E + D when compared to Group D. The mean scores of tubulointerstitial inflammation (p = 0.004), tubular injury (p = 0.013) and interstitial fibrosis (p = 0.003) were also lower in Group E + D when compared to Group D.. Our data seem to suggest a potential role of erythropoietin beta for reducing the progression of DN in an experimental rat model. This protective effect is, in part, attributable to the suppression of the inflammatory response and oxidative damage.

Topics: Animals; Cytokines; Diabetic Nephropathies; Disease Models, Animal; Erythropoietin; Glutathione; Inflammation Mediators; Male; Oxidative Stress; Rats; Rats, Sprague-Dawley; Superoxide Dismutase

Traumatic brain injury (TBI) is one of the major cause of morbidity and mortality and it affects more than 1.7 million people in the USA. A couple of regenerative pathways including activation of hypoxia-inducible transcription factor 1 alpha (HIF-1α) are initiated to reduce cellular damage following TBI; however endogenous activation of these pathways is not enough to provide neuroprotection after TBI. Thus we aimed to see whether sustained activation of HIF-1α can provide neuroprotection and neurorepair following TBI. We found that chronic treatment with dimethyloxaloylglycine (DMOG) markedly increases the expression level of HIF-1α and mRNA levels of its downstream proteins such as Vascular endothelial growth factor (VEGF), Phosphoinositide-dependent kinase-1 and 4 (PDK1, PDK4) and Erythropoietin (EPO). Treatment of DMOG activates a major cell survival protein kinase Akt and reduces both cell death and lesion volume following TBI. Moreover, administration of DMOG augments cluster of differentiation 31 (CD31) staining in pericontusional cortex after TBI, which suggests that DMOG stimulates angiogenesis after TBI. Treatment with DMOG also improves both memory and motor functions after TBI. Taken together our results suggest that sustained activation of HIF-1α provides significant neuroprotection following TBI.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Amino Acids, Dicarboxylic; Angiogenesis Inducing Agents; Animals; Brain Injuries, Traumatic; Cell Death; Disease Models, Animal; Erythropoietin; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Maze Learning; Memory Disorders; Mice, Inbred C57BL; Motor Activity; Neuroprotective Agents; Nootropic Agents; Protein Serine-Threonine Kinases; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; RNA, Messenger; Vascular Endothelial Growth Factor A

Amikacin (AK) is frequently used on the treatment of Gram-negative infections on neonates, but its usage is restricted because of nephrotoxicity. In this study, on neonatal rats, we aimed to investigate the effects of erythropoietin and vitamin E on AK induced nephrotoxicity. A total of 35 newborn Wistar Albino rats were divided into four groups: (1) injected with saline (serum physiological was administered to placebo controls), (2) injected with AK (1200 mg/kg), (3) injected with AK + vitamin E (150 mg/kg), (4) injected with AK + erythropoietin (EPO) (300 IU/kg/day). In renal tissue, AK levels were significantly high in all groups except the control. Tissue malondialdehyde (MDA) and nitric oxide (NO) levels were statistically higher in AK -treated group than the control. MDA and NO levels were significantly decreased with the administration of vitamin E and EPO. Glutathione peroxidase (GPX) levels were statistically low in AK group compared with the controls. The levels of GPX, in vitamin E group, were increased significantly. However, superoxide dismutase and catalase levels were not significantly different in none of the groups. Insulin-like growth factor-1 values in AK, EPO and vitamin E groups were significantly higher than the control group. Histomorphological changes such as tubular epithelial necrosis were seen in AK treated group. Histopathological improvements observed with EPO and vitamin E administration. AK nephrotoxicity is related to oxidative stress and is supported with biochemical and histopathological findings. Vitamin E and EPO, as antioxidants, can be useful renoprotective agents for ameliorating AK induced nephrotoxicity in neonates.

Topics: Amikacin; Animals; Animals, Newborn; Anti-Bacterial Agents; Antioxidants; Disease Models, Animal; Erythropoietin; Kidney Diseases; Kidney Function Tests; Malondialdehyde; Nitric Oxide; Oxidative Stress; Protective Agents; Rats; Rats, Wistar; Treatment Outcome; Vitamin E

Anemia of chronic kidney disease (CKD) can be corrected by treatment with recombinant human erythropoietin (rHuEPO); however, some patients become hyporesponsive. The molecular mechanisms underlying this resistance remain to be elucidated. Our aim was to study hyporesponsiveness to rHuEPO therapy using the remnant kidney rat model of anemia associated with CKD induced by 5/6 nephrectomy. At starting, male Wistar rats were divided in 3 groups, for a 3-week protocol: Sham, CRF (vehicle) and two rHuEPO (200 k/kg body weight [BW]/week) treated groups; at the end of protocol, the rHuEPO treated rats were subdivided in responders (CRF200) and non-responders (CRF200NR), according to their hematologic response; blood, cellular and tissue studies were performed. The CRF200 group achieved correction of anemia, while the CRF200NR group developed anemia, after an initial response (1st week) to rHuEPO therapy. CRF and CRF200NR groups presented a trend to higher serum CRP levels; CRF200NR showed also high levels of renal inflammatory markers, such as interleukin (IL)-6, IL-1β, nuclear factor kappa B, connective tissue growth factor (CTGF) and transforming growth factor beta 1 (TGF-β1); no changes were found in iron metabolism. Our data suggest that the development of anemia/rHuEPO hyporesponsiveness is associated with a higher systemic and renal inflammatory condition, favoring hypoxia and triggering an increase in renal expression of HIF-1α, TGF-β1 and CTGF that will further aggravate renal fibrosis, which will enhance the inflammatory response, creating a cycle that promotes disease progression. New therapeutic strategies to reduce inflammation in CKD patients could improve the response to rHuEPO therapy and reduce hyporesponsiveness.

Topics: Anemia; Animals; Cytokines; Disease Models, Animal; Drug Resistance; Erythropoietin; Humans; Male; Rats; Rats, Wistar; Recombinant Proteins; Renal Insufficiency, Chronic

The mTOR (mammalian target of rapamycin) signaling pathway is a master regulator of cell growth and proliferation in the nervous system. However, the effects of erythropoietin (EPO) treatment on the mTOR signaling pathway have not been elucidated in neonates with hypoxic/ischemic (H/I) brain injury.. We investigated the mechanism underlying the neuroprotective effect of EPO by analyzing the mTOR signaling pathway after H/I injury in a neonatal rat model.. Seven-day-old rats were subjected to left carotid artery ligation and hypoxic exposure (8%) for 90 min (H/I). EPO at a dose of either 3,000 U/kg or a vehicle (V) was administered by intraperitoneal injection 0, 24 and 48 h after H/I. At 72 h after H/I (postnatal day 10), 2,3,5-triphenyltetrazolium chloride staining, myelin basic protein (MBP) immunofluorescence staining and Western blot analysis of the Akt/mTOR/p70S6K pathway were performed. Neuromotor behavioral tests included Rotarod challenge and cylinder rearing test 1 performed 3 and 6 weeks after H/I.. EPO treatment resulted in significant offsetting of MBP depletion ipsilateral (p = 0.001) and contralateral (p = 0.003) to ligation. Western blot analysis showed that the relative immunoreactivity of phosphorylated (p)-Akt, p-mTOR and p-p70S6K ipsilateral to ligation was significantly decreased in the H/I+V group compared with the sham-operated groups. However, EPO treatment significantly upregulated Akt/mTOR/p70S6K signals ipsilateral to ligation compared to the H/I+V group. The behavior tests showed that EPO attenuates long-term impairment in Rotarod challenge and cylinder test performance from 3-6 weeks.. This study demonstrates an underlying mechanism of the mTOR signaling pathway after EPO treatment, which is a potential target for treating H/I-induced brain injury.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Erythropoietin; Hypoxia-Ischemia, Brain; Male; Neuroprotective Agents; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; TOR Serine-Threonine Kinases

The present study aimed to determine whether the subconjunctival administration of recombinant human erythropoietin (rHuEPO) reached the retina in glaucoma conditions. After subconjunctival rHuEPO administration, in a rat glaucoma model, erythropoietin (EPO) distribution in the rat's retina was studied by immunohistochemistry.. Female Wistar Hannover albino rats (n = 15) were divided into 2 groups, control (n = 3) and treated (n = 12). The animals' unilateral glaucoma was induced by coagulation of episcleral veins, under general anaesthesia. After vein coagulation, 1,000 IU of rHuEPO were administered by the subconjunctival route to the treated group (n = 12). The control group (n = 3) received only a subconjunctival saline injection. The contralateral eye of each animal remained untouched. Treated group animals were euthanized at different time points, i.e. days 1, 3, 7 and 14. Bilateral enucleation was performed, and EPO distribution in the rat's retina was assessed by immunohistochemistry.. Glaucoma was confirmed by results of repeated intraocular pressure measurements over the experimental period. In the test group, EPO was identified in different neuroretinal cells, showing a stronger immunostaining signal during the first 2 time points in the retinal ganglion cell (RGC) layer. EPO protein was still present on day 14 after the subconjunctival injection. EPO was not detected in any of the control eyes or in any contralateral eye of the treated group.. When administered subconjunctivally to glaucomatous eyes, rHuEPO reached the RGC layer and was still present at least 14 days after administration. The subconjunctival route was shown to be a promising alternative for ocular EPO delivery in glaucomatous conditions in a rat animal model.

Topics: Animals; Conjunctiva; Disease Models, Animal; Erythropoietin; Female; Glaucoma; Immunohistochemistry; Injections; Intraocular Pressure; Rats; Rats, Wistar

The aim of this study was to examine the potential biomechanical and histological benefits of systemic erythropoietin administration during the healing of Achilles tendon injury in a rat experimental model.. Eighty Sprague-Dawley female rats were included in this study. Animals were randomly assigned into two groups with 40 animals in each: erythropoietin group and control group. Then each group was further divided into four subgroups corresponding to four time points with 10 animals in each. A full-thickness cut was made on the Achilles tendon of each animal and then the tendon was sutured with modified Kessler method. Erythropoietin groups received intraperitoneal erythropoietin (500 IU/kg/day) every day at same time throughout the study period, and the control groups received saline in a similar manner. Animals were sacrificed at four time points, and tensile test was performed on each tendon sample to assess maximum load for each sample. In addition, histopathological examination and scoring was done.. Both groups had improvement on tensile test (maximum load) over time. However, groups did not differ with regard to maximum load in any of the time points. Similarly, groups did not differ with regard to any of the histopathological scores over time.. The findings of this study do not support the benefit of systemic erythropoietin administration in Achilles tendon healing process. Further evidence from larger experimental studies is required to justify any such potential benefit.

Topics: Achilles Tendon; Animals; Disease Models, Animal; Drug Evaluation, Preclinical; Erythropoietin; Female; Random Allocation; Rats, Sprague-Dawley; Tendon Injuries; Tensile Strength; Wound Healing

To explore the impacts of erythropoietin on vascular endothelial growth factor receptor 2 (VEGFR2) by the extracellular signal-regulated kinase (ERK) signaling pathway in a neonatal rat model of periventricular white matter damage.. All of postnatal day 4 rats were randomized into three groups: the sham group [without hypoxia-ischemia (HI)], the HI group (HI with saline administration), and the erythropoietin (EPO) group [HI with recombinant human erythropoietin (rh-EPO) administration]. Rat pups underwent permanent ligation of the right common carotid artery, followed by 6% O2 for 2 hours or sham operation and normoxic exposure. Immediately after the HI, rats received a single intraventricular injection of rh-EPO (0.6 IU/g body mass) or saline. ERK and phosphorylation-ERK were examined at 60 minutes and 90 minutes after operation, and VEGFR2 were detected at 2 and 4 days after operation by using Western blot.. At 60 minutes and 90 minutes after operation, the proteins of phosphorylation-ERK were significantly higher in HI rats than in the sham rats and significantly higher in HI+EPO rats than in the HI rats (P<0.05). Two days after operation, VEGFR2 was not significantly different between sham and HI rats. However, the proteins of VEGFR2 were increased after administration of rh-EPO (P<0.05). Four days after operation, the proteins of VEGFR2 were significantly higher in HI rats than in the sham rats and significantly higher in HI+EPO rats than in the HI rats (P<0.05).. EPO may regulate VEGFR2 expression by affecting the intracranial ERK signaling pathways.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Erythropoietin; Humans; Hypoxia-Ischemia, Brain; MAP Kinase Signaling System; Phosphorylation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Vascular Endothelial Growth Factor Receptor-2; White Matter

The aim of the present study is to investigate the protection effects of bone marrow mesenchymal stem cells (MSCs) in combination with EPO against hyperoxia-induced bronchopulmonary dysplasia (BPD) injury in neonatal mice. BPD model was prepared by continuous high oxygen exposure, 1×106 bone marrow MSCs and 5000U/kg recombinant human erythropoietin (EPO) were injected respectively. Results showed that administration of MSCs, EPO especially MSCs+EPO significant attenuated hyperoxia-induced lung damage with a decrease of fibrosis, radical alveolar counts and inhibition of the occurrence of epithelial-mesenchymal transition (EMT). Furthermore, MSCs+EPO co-treatment more significantly suppressed the levels of transforming growth factor-β1(TGF-β1) than MSCs or EPO alone. Collectively, these results suggested that MSCs, EPO in particular MSCs+EPO co-treatment could promote lung repair in hyperoxia-induced alveoli dysplasia injury via inhibition of TGF-β1 signaling pathway to further suppress EMT process and may be a promising therapeutic strategy.

Topics: Animals; Bronchopulmonary Dysplasia; Cells, Cultured; Combined Modality Therapy; Disease Models, Animal; Epithelial-Mesenchymal Transition; Erythropoietin; Female; Fibrosis; Humans; Hyperoxia; Mesenchymal Stem Cell Transplantation; Mice; Mice, Inbred C57BL; Pulmonary Alveoli; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta1

Topics: Animals; Disease Models, Animal; Erythropoiesis; Erythropoietin; Hematinics; Mice; Recombinant Proteins

Stroke is associated with neuroinflammation, neuronal loss and blood-brain barrier (BBB) breakdown. Thus far, recombinant tissue-type plasminogen activator (rtPA), the only approved treatment for acute ischemic stroke, increases the risk of intracerebral hemorrhage and is poorly efficient in disaggregating platelet-rich thrombi. Therefore, the development of safer and more efficient therapies is highly awaited. Encouraging neuroprotective effects were reported in mouse models of ischemic stroke following administration of erythropoietin (EPO). However, previous preclinical studies did not investigate the effects of EPO in focal ischemic stroke induced by a platelet-rich thrombus and did not consider the implication of age. Here, we performed middle cerebral artery occlusion by inducing platelet-rich thrombus formation in chimeric 5- (i.e. young) and 20- (i.e. aged) months old C57BL/6 mice, in which hematopoietic stem cells carried the green fluorescent protein (GFP)-tag. Recombinant human EPO (rhEPO) was administered 24 hours post-occlusion and blood-circulating monocyte populations were studied by flow cytometry 3 hours post-rhEPO administration. Twenty-four hours following rhEPO treatment, neuronal loss and BBB integrity were assessed by quantification of Fluoro-Jade B (FJB)-positive cells and extravasated serum immunoglobulins G (IgG), respectively. Neuroinflammation was determined by quantifying infiltration of GFP-positive bone marrow-derived cells (BMDC) and recruitment of microglial cells into brain parenchyma, along with monocyte chemotactic protein-1 (MCP-1) brain protein levels. Here, rhEPO anti-inflammatory properties rescued ischemic injury by reducing neuronal loss and BBB breakdown in young animals, but not in aged littermates. Such age-dependent effects of rhEPO must therefore be taken into consideration in future studies aiming to develop new therapies for ischemic stroke.

Topics: Age Factors; Animals; Blood-Brain Barrier; Brain; Brain Ischemia; Chemokine CCL2; Disease Models, Animal; Erythropoietin; Flow Cytometry; Green Fluorescent Proteins; Humans; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Middle Cerebral Artery; Neuroprotective Agents; Recombinant Proteins; Stroke; Thrombosis

Inflammation is believed to play a key role in the pathophysiology of meconium aspiration syndrome (MAS).. The objective was to determine whether the recombinant human Erythropoietin (rhEPO) pretreatment could attenuate meconium-induced inflammation.. In this study, 24 ventilated adult male rats were studied to examine the effects of recombinant human EPO (rhEPO) on meconium-induced inflammation. Seventeen rats were instilled with human meconium (1.5 mL/kg, 65 mg/mL) intratracheally and ventilated for 3 hours. rhEPO (1000 U/kg) (n = 9) or saline (n = 8) was given to the animals. Seven rats that were ventilated and not instilled with meconium served as a sham-controlled group. Analysis of the blood gases, interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α in blood and bronchoalveolar lavage (BAL) fluid samples, and lung tissue myeloperoxidase levels were performed.. Intrapulmonary instillation of meconium resulted in the increase of TNF-α (p = 0.005 and p < 0.001, respectively) and IL-8 concentrations (p < 0.001 and p < 0.001, respectively) in BAL fluid in the EPO + meconium and saline + meconium groups compared with the sham-controlled group. rhEPO pretreatment prevented the increase of BAL fluid IL-1β, IL-6, and IL-8 levels (p < 0.001, p = 0.021, and p = 0.005, respectively), and serum IL-6 levels (p = 0.036).. rhEPO pretreatment is associated with improved BAL fluid and serum cytokine levels. Pretreatment with rhEPO might reduce the risk of developing of meconium-induced derangements.

Topics: Animals; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Erythropoietin; Humans; Interleukin-6; Interleukin-8; Male; Meconium Aspiration Syndrome; Pneumonia; Premedication; Rats

Bronchopulmonary dysplasia (BPD) is the most common type of chronic lung disease in infancy, for which no effective therapy is currently available. The aim of the present study was to investigate the effect of treatment with bone marrow mesenchymal stem cells (BMSCs) in combination with recombinant human erythropoietin (rHuEPO) on BPD‑induced mouse lung injury, and discuss the underlying mechanism. The BPD model was established by the exposure of neonatal mice to continuous high oxygen exposure for 14 days, following which 1x106 BMSCs and 5,000 U/kg rHuEPO were injected into the mice 1 h prior to and 7 days following exposure to hyperoxia. The animals received four treatments in total (n=10 in each group). After 14 days, the body weights, airway structure, and levels of matrix metalloproteinase‑9 (MMP‑9) and vascular endothelial growth factor (VEGF) were detected using histological and immunohistochemical analyses. The effect on cell differentiation was observed by examining the presence of platelet endothelial cell adhesion molecule (PECAM) and VEGF using immunofluorescence. Compared with the administration of BMSCs alone, the body weight, airway structure, and the levels of MMP‑9 and VEGF were significantly improved in the BMSCs/rHuEPO group. The results of the present study demonstrated that the intravenous injection of BMSCs significantly improved lung damage in the hyperoxia‑exposed neonatal mouse model. Furthermore, the injection of BMSCs in combination with intraperitoneal injection of rHuEPO had a more marked effect, compared with BMSCs alone, and the mechanism may be mediated by the promoting effects of BMSCs and EPO. The results of the present study provided information, which may assist in future clinical trials.

Topics: Animals; Antigens, CD; Bronchopulmonary Dysplasia; Cell Culture Techniques; Combined Modality Therapy; Disease Models, Animal; Erythropoietin; Immunophenotyping; Lung Injury; Matrix Metalloproteinase 9; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Vascular Endothelial Growth Factor A

To test the efficacy of systemic gene delivery of a mutant form of erythropoietin (EPO-R76E) that has attenuated erythropoietic activity, in a mouse model of autosomal dominant retinitis pigmentosa.. Ten-day old mice carrying one copy of human rhodopsin with the P23H mutation and both copies of wild-type mouse rhodopsin (hP23H RHO+/-,mRHO+/+) were injected into the quadriceps with recombinant adeno-associated virus (rAAV) carrying either enhanced green fluorescent protein (eGFP) or EpoR76E. Visual function (electroretinogram) and retina structure (optical coherence tomography, histology, and immunohistochemistry) were assessed at 7 and 12 months of age.. The outer nuclear layer thickness decreased over time at a slower rate in rAAV.EpoR76E treated as compared to the rAAV.eGFP injected mice. There was a statistically significant preservation of the electroretinogram at 7, but not 12 months of age.. Systemic EPO-R76E slows death of the photoreceptors and vision loss in hP23H RHO+/-,mRHO+/+ mice. Treatment with EPO-R76E may widen the therapeutic window for retinal degeneration patients by increasing the number of viable cells. Future studies might investigate if co-treatment with EPO-R76E and gene replacement therapy is more effective than gene replacement therapy alone.

Topics: Animals; Cell Death; Dependovirus; Disease Models, Animal; Erythropoietin; Gene Transfer Techniques; Genetic Therapy; Humans; Mice; Opsins; Point Mutation; Retinal Cone Photoreceptor Cells; Retinitis Pigmentosa; Vision, Ocular

Catalpol is the main active component of the radix from Rehmannia glutinosa Libosch, which has pleiotropic protective effects in neurodegenerative diseases, ischemic stroke, metabolic disorders and others. Catalpol has been shown to have neuroprotective, neurorepair, and angiogenesis effects following ischemic brain injury. However, its molecular mechanisms are still poorly understood. In previous studies, the JAK2/STAT3 signaling pathway was found to play a role in neuroprotection and angiogenesis. This study investigated the role of catalpol in stimulating angiogenesis via the JAK2/STAT3 pathway after permanent focal cerebral ischemia (pMCAO).. Rats were subjected to right middle cerebral artery occlusion through electrocoagulation and were treated with catalpol (5mg/kg), AG490 was also used to inhibit STAT3 phosphorylation (pSTAT3).. Following stroke, Catalpol improved the neuroethology deficit, increased the cerebral blood flow (CBF) of infarcted brain and upregulated EPO and EPOR. AG490 suppressed the phosphorylation of signal transducer and activator of transcription 3 (STAT3), ultimately inhibited VEGF mRNA expression, which reduced VEGF protein expression and inhibited stroke-induced angiogenesis. However, Catalpol enhanced stroke-induced STAT3 activation and subsequently restored STAT3 activity through the recovery of STAT3 binding to VEGF. Moreover, Catalpol reversed the effect of AG490 on STAT3 activation and nuclear translocation, restored the transcriptional activity of the VEGF promoter by recruiting STAT3 to the VEGF promoter, improved VEGF mRNA and protein expression, increased angiogenesis, reduced the difference in CBF between the infarcted and intact brain and ameliorated the neuroethology behaviors after stroke.. Catalpol affects neuroprotection and angiogenesis via the JAK2/STAT3 signaling pathway, which is mediated by STAT3 activation and VEGF expression. Catalpol may be used as a potential therapeutic drug for stroke.

Topics: Angiogenesis Inducing Agents; Animals; Brain; Cerebral Arteries; Cerebrovascular Circulation; Disease Models, Animal; Erythropoietin; Infarction, Middle Cerebral Artery; Iridoid Glucosides; Janus Kinase 2; Male; Neovascularization, Physiologic; Neuroprotective Agents; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Rats, Sprague-Dawley; Receptors, Erythropoietin; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Time Factors; Transcriptional Activation; Up-Regulation; Vascular Endothelial Growth Factor A

To reduce side effects due to non-specific expression, the heme oxygenase-1 (HO-1) gene under control of a hypoxia-inducible erythropoietin (Epo) enhancer (pEpo-SV-HO-1) was developed for site-specific gene therapy of ischemic stroke.. pEpo-SV-HO-1 was constructed by insertion of the Epo enhancer into pSV-HO-1. Dexamethasone-conjugated polyamidoamine (PAMAM-Dexa) was used as a gene carrier. In vitro transfection assays were performed in the Neuro2A cells. In vivo efficacy of pEpo-SV-HO-1 was evaluated in the transient middle cerebral artery occlusion (MCAO) model.. In vitro transfection assay with the PAMAM-Dexa/pEpo-SV-HO-1 complex showed that pEpo-SV-HO-1 had higher HO-1 gene expression than pSV-HO-1 under hypoxia. In addition, pEpo-SV-HO-1 reduced the level of apoptosis more efficiently than pSV-HO-1 in Neuro2A cells under hypoxia. For in vivo evaluation, the PAMAM-Dexa/pEpo-SV-HO-1 complex was injected into the ischemic brain of the transient MCAO model. pEpo-SV-HO-1 increased HO-1 expression and reduced the number of apoptotic cells in the ischemic brain, compared with the pSV-HO-1 injection group. As a result, the infarct volume was more efficiently decreased by pEpo-SV-HO-1 than by pSV-HO-1.. pEpo-SV-HO-1 induced HO-1 gene expression and therapeutic effect in the ischemic brain. Therefore, pEpo-SV-HO-1 may be useful for site-specific gene therapy of ischemic stroke.

Topics: Animals; Brain; Cell Line, Tumor; Dexamethasone; Disease Models, Animal; Erythropoietin; Gene Transfer Techniques; Genetic Therapy; Heme Oxygenase-1; Hypoxia; Infarction, Middle Cerebral Artery; Male; Polyamines; Rats; Rats, Sprague-Dawley; Stroke; Transfection

To investigate the therapeutic effects of erythropoietin sustained-release gelatin hydrogel microspheres (EPO-GHM) on a murine model of hindlimb ischemia and related mechanisms.. Fifty two ten weeks old male C57BL/6J mice were assigned to 5 groups: sham-operated group (the right femoral artery suture was passed through the right femoral artery but not tied, n=8); saline group (right femoral artery ligation and intramuscular injection of saline at a dose of 4 ml/kg into the right hind limb, n=12); EPO group(right femoral artery ligation and intramuscular injection of EPO at a dose of 5 000 IU/kg into the right hind limb, n=12), empty GHM group (right femoral artery ligation and intramuscular injection of empty GHM at a dose of 4 ml/kg into the right hind limb, n=8); EPO-GHM group(right femoral artery ligation and intramuscular injection of EPO-GHM at a dose of 5 000 IU/kg into the right hind limb, n=12). The blood flow ratio of ischemic limb (right)/nonischemic limb (left) was measured using a laser Doppler perfusion imager. After 8 weeks, immunohistochemical analysis were used to evaluate the vessel density (vessel density of CD31 positive), arteriole density(vessel density of α-smooth muscle actin(α-SMA) positive) and muscle area(HHF35 positive area). The proliferating index of vessels was evaluated by double immunofluorescent labeling to evaluate effect of EPO-GHM on angiogenesis of ischemia limb. Western blot was used to evaluate the protein expression of EPO receptor, protein kinase B(AKT), p-AKT, endothelial nitric oxide synthase(eNOS), p-eNOS and matrix metalloproteinase 2(MMP-2).. (1) Eight weeks later, the blood flow ratio of ischemic limb/nonischemic limb was significantly higher in the EPO-GHM group compared with other groups(0.810±0.080, 0.563±0.051, 0.570±0.056 and 0.561±0.052 respectively, all P<0.05). (2) CD31 antibody positive and α-SAM antibody positive densities were higher in the EPO-GHM group compared with other groups(P<0.01 or 0.05). (3)HHF35 positive area in saline group, EPO group, empty GHM group and EPO-GHM group were smaller than that of sham-operated group(all P<0.05). HHF35 positive area in saline group, EPO group, empty GHM group and EPO-GHM group were similar(all P>0.05). (4)The proliferating index of vessels was higher in the EPO-GHM group compared with other groups(P<0.01 or 0.05). (5) Compared with other groups, the protein levels of EPO receptor, AKT, p-AKT, p-eNOS and MMP-2 were significantly higher in EPO-GHM group(P<0.01 or 0.05) and level of eNOS was similar among five groups(P>0.05).. RESULTS from present study suggest EPO-GHM could improve blood perfusion of ischemia limb in mice through increasing capillary and arteriolar densities and these beneficial effects are possibly mediated by EPOR up-regulation and AKT/p-eNOS/MMP-2 signaling pathway activation.

Topics: Animals; Delayed-Action Preparations; Disease Models, Animal; Erythropoietin; Femoral Artery; Gelatin; Hindlimb; Hydrogels; Ischemia; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred C57BL; Microspheres; Neovascularization, Physiologic; Nitric Oxide Synthase Type III; Proto-Oncogene Proteins c-akt; Signal Transduction; Up-Regulation

Background and purpose - Delayed bone healing with non-union is a common problem. Further options to increase bone healing together with surgery are needed. We therefore evaluated a 1-dose single application of erythropoietin (EPO), applied either locally to the defect or systemically during surgery, in a critical-size rabbit long-bone defect. Material and methods - 19 New Zealand White rabbits received a 15-mm defect in the radius diaphysis. An absorbable gelatin sponge was soaked with saline (control group and systemic treatment group) or EPO (local treatment group) and implanted into the gap. The systemic treatment group received EPO subcutaneously. In vivo micro-CT analysis was performed 4, 8, and 12 weeks postoperatively. Vascularization was evaluated histologically. Results - Semiquantitative histomorphometric and radiological evaluation showed increased bone formation (2.3- to 2.5-fold) in both treatment groups after 12 weeks compared to the controls. Quantitative determination of bone volume and tissue volume showed superior bone healing after EPO treatment at all follow-up time points, with the highest values after 12 weeks in locally treated animals (3.0- to 3.4-fold). More vascularization was found in both EPO treatment groups. Interpretation - Initial single dosing with EPO was sufficient to increase bone healing substantially after 12 weeks of follow-up. Local application inside the defect was most effective, and it can be administered directly during surgery. Apart from effects on ossification, systemic and local EPO treatment leads to increased callus vascularization.

Topics: Animals; Bone Regeneration; Disease Models, Animal; Erythropoietin; Female; Fracture Healing; Osteogenesis; Rabbits; Radius Fractures; X-Ray Microtomography

Erythropoietin (EPO) is widely used in diabetic patients receiving hemodialysis. The role of EPO in glucose homeostasis remains unclear. Therefore, we investigated the effect of EPO on hyperglycemia in rats with type 1-like diabetes.. Rats with streptozotocin-induced type 1-like diabetes (STZ rats) were used to estimate the blood glucose-lowering effects of EPO, and changes in the expression levels of glucose transporter 4 (GLUT4) and the hepatic enzyme phosphoenolpyruvate carboxykinase (PEPCK) were identified by Western blot analysis.. EPO attenuated the hyperglycemia in the STZ rats in a dose-dependent manner without altering the hematopoietic parameters, including the hematocrit and number of red blood cells. The involvement of the EPO receptor (EPOR) was identified using EPOR-specific antibodies. In addition, injection of EPO enhanced the glucose utilization, which was assessed using an intravenous glucose tolerance test in rats. However, blood insulin was not changed by EPO in this assay, showing the insulinotropic action of EPO. Moreover, EPO treatment increased the insulin sensitivity. Western blots indicated that the phosphorylation of AMP-activated protein kinase was enhanced by EPO to support the signaling caused by EPOR activation. Furthermore, the decrease in the GLUT4 level in skeletal muscle was reversed by EPO, and the increase in the PEPCK expression in liver was reduced by EPO, as shown in STZ rats.. Taken together, the results show that EPO injection may reduce hyperglycemia in diabetic rats through activation of EPO receptors. Therefore, EPO is useful for managing diabetic disorders, particularly hyperglycemia-associated changes. In addition, EPO receptor will be a good target for the development of antihyperglycemic agent(s) in the future.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; Erythropoietin; Hyperglycemia; Hypoglycemic Agents; Insulin; Male; Rats; Rats, Wistar; Streptozocin

: This study explored a new approach to enhance aneurysm (AN) neck endothelialization via erythropoietin (EPO)-induced endothelial progenitor cell (EPC) stimulation. Results suggest that EPO enhanced the endothelialization of a coiled embolization AN neck by stimulating EPCs via vascular endothelial growth factor modulation. Thus, the promotion of endothelialization with EPO provides an additional therapeutic option for preventing the recurrence of ANs. Endovascular coil embolization is an attractive therapy for cerebral ANs, but recurrence is a main problem affecting long-term outcomes. In this study, we explored a new approach to enhance AN neck endothelialization via EPO-induced EPC stimulation. Ninety adult male Sprague-Dawley rats were selected for an in vivo assay, and 60 of them underwent microsurgery to create a coiled embolization AN model. The animals were treated with EPO, and endothelial repair was assessed via flow cytometry, immunofluorescence, electronic microscopy, cytokine detection, and routine blood work. EPO improved the viability, migration, cytokine modulation, and gene expression of bone marrow-derived EPCs and the results showed that EPO increased the number of circulating EPCs and improved endothelialization compared with untreated rats (p < .05). EPO had no significant effect on the routine blood work parameters. In addition, the immunofluorescence analysis showed that the number of KDR(+) cells in the AN neck was elevated in the EPO-treated group (p < .05). Further study demonstrated that EPO promoted EPC viability and migration in vitro. The effects of EPO may be attributed to the modulation of vascular endothelial growth factor (VEGF). In particular, EPO enhanced the endothelialization of a coiled embolization AN neck by stimulating EPCs via VEGF modulation. Thus, the promotion of endothelialization with EPO provides an additional therapeutic option for preventing the recurrence of ANs.. Erythropoietin (EPO) is involved in erythropoiesis and related conditions and is reported to enhance stem-cell mobilization from bone marrow while elevating stem-cell viability and function. In this study, EPO was also found to stimulate endothelial progenitor cells to induce the endothelialization of a coiled embolic aneurysm neck via vascular endothelial growth factor modulation. Endothelialization induction provides an additional therapeutic opportunity during vascular inner layer repair and remodeling. The results provide important information on the unique role EPO plays during vascular repair and remodeling.

Topics: Animals; Disease Models, Animal; Embolization, Therapeutic; Endothelial Progenitor Cells; Erythropoietin; Flow Cytometry; Fluorescent Antibody Technique; Intracranial Aneurysm; Male; Microscopy, Confocal; Rats; Rats, Sprague-Dawley; Vascular Endothelial Growth Factor A

The cause of persistent injury-associated anemia is multifactorial and includes acute blood loss, an altered erythropoietin (EPO) response, dysregulation of iron homeostasis, and impaired erythropoiesis in the setting of chronic inflammation/stress. Hepcidin plays a key role in iron homeostasis and is regulated by anemia and inflammation. Erythropoietin is a main regulator of erythropoiesis induced by hypoxia. A unique rodent model of combined lung injury (LC)/hemorrhagic shock (HS) (LCHS)/chronic restraint stress (CS) was used to produce persistent injury-associated anemia to further investigate the roles of EPO, hepcidin, iron, ferritin, and the expression of EPO receptors (EPOr).. Male Sprague-Dawley rats were randomly assigned into one of the four groups of rodent models: naive, CS alone, combined LCHS, or LCHS/CS. Plasma was used to evaluate levels of EPO, hepcidin, iron, and ferritin. RNA was isolated from bone marrow and lung tissue to evaluate expression of EPOr. Comparisons between models were performed by t tests followed by one-way analysis of variance.. After 7 days, only LCHS/CS was associated with persistent anemia despite significant elevation of plasma EPO. Combined LCHS and LCHS/CS led to a persistent decrease in EPOr expression in bone marrow on Day 7. The LCHS/CS significantly decreased plasma hepcidin levels by 75% on Day 1 and 84% on Day 7 compared to LCHS alone. Hepcidin plasma levels are inversely proportional to EPO plasma levels (Pearson R = -0.362, p < 0.05).. Tissue injury, hemorrhagic shock, and stress stimulate and maintain high levels of plasma EPO while hepcidin levels are decreased. In addition, bone marrow EPOr and plasma iron availability are significantly reduced following LCHS/CS. The combined deficit of reduced iron availability and reduced bone marrow EPOr expression may play a key role in the ineffective EPO response associated with persistent injury-associated anemia.

Topics: Anemia; Animals; Bone Marrow; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Hepcidins; Lung Injury; Male; Random Allocation; Rats; Rats, Sprague-Dawley; Restraint, Physical; Shock, Hemorrhagic; Stress, Physiological; Wounds and Injuries

The study performed on erythroblastic island cultures and rats with experimental polycythemia showed that recormon and epokrin stimulated erythropoiesis in erythroblastic islands both in vitro and in vivo. In cell cultures, recormon activates the formation of erythroblastic islands de novo and de repeto 1.3 times better than epokrin (p < 0.05). Erythroid cells exbhibited same reaction to epokrin and recormon in vivo: the number of erythroblastic islands in the bone marrow increased 3.4 times (p < 0.05) and the number of reticulocytes in the blood increased 2.2 times (p < 0.05).

Topics: Animals; Animals, Outbred Strains; Bone Marrow; Cell Differentiation; Cell Proliferation; Disease Models, Animal; Erythroblasts; Erythropoiesis; Erythropoietin; Female; Hematinics; Polycythemia; Primary Cell Culture; Protein Isoforms; Rats; Recombinant Proteins

Chronic kidney disease (CKD) is known to increase myocardial infarct size after ischemia/reperfusion. However, a strategy to prevent the CKD-induced myocardial susceptibility to ischemia/reperfusion injury has not been developed. Here, we examined whether epoetin β pegol, a continuous erythropoietin receptor activator (CERA), normalizes myocardial susceptibility to ischemia/reperfusion injury by its effects on protective signaling and metabolomes in CKD. CKD was induced by 5/6 nephrectomy in rats (subtotal nephrectomy, SNx), whereas sham-operated rats served controls (Sham). Infarct size as percentage of area at risk after 20-minutes coronary occlusion/2-hour reperfusion was larger in SNx than in Sham: 60.0±4.0% versus 43.9±2.2%. Administration of CERA (0.6 μg/kg SC every 7 days) for 4 weeks reduced infarct size in SNx (infarct size as percentage of area at risk=36.9±3.9%), although a protective effect was not detected for the acute injection of CERA. Immunoblot analyses revealed that myocardial phospho-Akt-Ser473 levels under baseline conditions and on reperfusion were lower in SNx than in Sham, and CERA restored the Akt phosphorylation on reperfusion. Metabolomic analyses showed that glucose 6-phosphate and glucose 1-phosphate were reduced and malate:aspartate ratio was 1.6-fold higher in SNx than in Sham, suggesting disturbed flux of malate-aspartate shuttle by CKD. The CERA improved the malate:aspartate ratio in SNx to the control level. In H9c2 cells, mitochondrial Akt phosphorylation by insulin-like growth factor-1 was attenuated by malate-aspartate shuttle inhibition. In conclusion, the results suggest that a CERA prevents CKD-induced susceptibility of the myocardium to ischemia/reperfusion injury by restoration of Akt-mediated signaling possibly via normalized malate-aspartate shuttle flux.

Topics: Analysis of Variance; Animals; Disease Models, Animal; Drug Administration Schedule; Erythropoietin; Injections, Subcutaneous; Male; Metabolome; Myocardial Infarction; Myocardial Reperfusion Injury; Nephrectomy; Phosphorylation; Polyethylene Glycols; Proto-Oncogene Proteins c-akt; Random Allocation; Rats; Reference Values; Renal Insufficiency, Chronic; Sensitivity and Specificity

Salmonella infection profoundly affects host erythroid development, but the mechanisms responsible for this effect remain poorly understood. We monitored the impact of Salmonella infection on erythroid development and found that systemic infection induced anemia, splenomegaly, elevated erythropoietin (EPO) levels, and extramedullary erythropoiesis in a process independent of Salmonella pathogenicity island 2 (SPI2) or flagellin. The circulating EPO level was also constitutively higher in mice lacking the expression of signal-regulatory protein α (SIRPα). The expression level of EPO mRNA was elevated in the kidney and liver but not increased in the spleens of infected mice despite the presence of extramedullary erythropoiesis in this tissue. In contrast to data from a previous report, mice lacking EPO receptor (EPOR) expression on nonerythroid cells (EPOR rescued) had bacterial loads similar to those of wild-type mice following Salmonella infection. Indeed, treatment to reduce splenic erythroblasts and mature red blood cells correlated with elevated bacterial burdens, implying that extramedullary erythropoiesis benefits the host. Together, these findings emphasize the profound effect of Salmonella infection on erythroid development and suggest that the modulation of erythroid development has both positive and negative consequences for host immunity.

Topics: Anemia; Animals; Bacterial Load; Disease Models, Animal; Erythropoiesis; Erythropoietin; Flow Cytometry; Kidney; Liver; Mice; Mice, Inbred C57BL; Receptors, Erythropoietin; Receptors, Immunologic; RNA, Messenger; Salmonella Infections; Salmonella typhi; Spleen

Age-related macular degeneration (AMD), affecting the retinal pigment epithelium (RPE), is the leading cause of blindness in middle-aged and older people in developed countries. Genetic and environmental risk factors have been identified, but no effective cure exists. Using a mouse model we show that a transmembrane prolyl 4-hydroxylase (P4H-TM), which participates in the oxygen-dependent regulation of the hypoxia-inducible factor (HIF), is a potential novel candidate gene for AMD. We show that P4h-tm had its highest expression levels in the mouse RPE and brain, heart, lung, skeletal muscle and kidney. P4h-tm

Topics: Animals; Brain; Disease Models, Animal; Erythropoietin; Humans; Hypoxia-Inducible Factor-Proline Dioxygenases; Kidney; Kidney Diseases; Lung; Macular Degeneration; Mice; Muscle, Skeletal; Myocardium; Prolyl Hydroxylases; Retinal Pigment Epithelium; Tissue Distribution

Chronic kidney disease (CKD) is often accompanied by impairment of cardiac function that may lead to major cardiac events. Erythropoietin (EPO), a kidney-produced protein, was shown to be beneficial to heart function. It was suggested that reduced EPO secretion in CKD may play a role in the initiation of heart damage.. To investigate molecular changes in the EPO/ erythropoietin receptor (EPO-R) axis in rat cardiomyocytes using a rat model for CKD.. We established a rat model for CKD by kidney resection. Cardiac tissue sections were stained with Masson's trichrome to assess interstitial fibrosis indicating cardiac damage. To evaluate changes in the EPO/EPO-R signaling cascade in the myocardium we measured cardiac EPO and EPO-R as well as the phosphorylation levels of STAT-5, a downstream element in this cascade.. At 11 weeks after resection, animals presented severe renal failure reflected by reduced creatinine clearance, elevated blood urea nitrogen and presence of anemia. Histological analysis revealed enhanced fibrosis in cardiac sections of CKD animals compared to the sham controls. Parallel to these changes, we found that although cardiac EPO levels were similar in both groups, the expression of EPO-R and the activated form of its downstream protein STAT-5 were significantly lower in CKD animals.. CKD results in molecular changes in the EPO/EPO-R axis. These changes may play a role in early cardiac damage observed in the cardiorenal syndrome.

Topics: Anemia; Animals; Disease Models, Animal; Down-Regulation; Erythropoietin; Fibrosis; Kidney Function Tests; Male; Myocardium; Rats; Receptors, Erythropoietin; Renal Insufficiency, Chronic; Signal Transduction; STAT5 Transcription Factor

Sepsis remains the most important cause of acute kidney injury (AKI) and acute lung injury (ALI) in critically ill patients. The cecal ligation and puncture (CLP) model in experimental mice reproduces most of the clinical features of sepsis. Erythropoietin (EPO) is a well-known cytoprotective multifunctional hormone, which exerts anti-inflammatory, anti-oxidant, anti-apoptotic and pro-angiogenic effects in several tissues. The aim of this study was to evaluate the underlying mechanisms of EPO protection through the expression of the EPO/EPO receptor (EPO-R) and VEGF/VEF-R2 systems in kidneys and lungs of mice undergoing CLP-induced sepsis. Male inbred Balb/c mice were divided in three experimental groups: Sham, CLP, and CLP+EPO (3000IU/kg sc). Assessment of renal functional parameters, survival, histological examination, immunohistochemistry and/or Western blottings of EPO-R, VEGF and VEGF-R2 were performed at 18h post-surgery. Mice demonstrated AKI by elevation of serum creatinine and renal histologic damage. EPO treatment attenuates renal dysfunction and ameliorates kidney histopathologic changes. Additionally, EPO administration attenuates deleterious septic damage in renal cortex through the overexpression of EPO-R in tubular interstitial cells and the overexpression of the pair VEGF/VEGF-R2. Similarly CLP- induced ALI, as evidenced by parenchymal lung histopathologic alterations, was ameliorated through pulmonary EPO-R, VEGF and VEGF-R2 over expression suggesting and improvement in endothelial survival and functionality. This study demonstrates that EPO exerts protective effects in kidneys and lungs in mice with CLP-induced sepsis through the expression of EPO-R and the regulation of the VEGF/VEGF-R2 pair.

Topics: Acute Kidney Injury; Acute Lung Injury; Animals; Blood Urea Nitrogen; Cecum; Creatinine; Disease Models, Animal; Erythropoietin; Kidney; Ligation; Male; Mice, Inbred BALB C; Punctures; Receptors, Erythropoietin; Sepsis; Survival Analysis; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Neurologic sequelae remain a common and destructive problem in patients with acute kidney injury. The objective of this study was to evaluate the possible neuroprotective effect of erythropoietin (EPO) on motor impairments following bilateral renal ischemia (BRI) in two time points after reperfusion: short term (24 h) and long term (1 week). Male Wistar rats underwent BRI or sham surgery. EPO or saline administration was performed 30 min before surgery (1000 U/kg, i.p.). Explorative behaviors and motor function of the rats were evaluated by open field, rotarod, and wire grip tests. Plasma concentrations of blood urea nitrogen (BUN) and creatinine (Cr) were significantly enhanced in BRI rats 24 h after reperfusion. BRI group had only an increased level of BUN but not Cr 1 week after reperfusion. Impairment of balance function by BRI was not reversed by EPO 24 h after reperfusion, but counteracted 7 days after renal ischemia. Muscle strength had no significant differences between the groups. BRI group had a decrease in locomotor activity, and EPO could not reverse this reduction in both time points of the experiment. Although EPO could not be offered as a potential neuroprotective agent in the treatment of motor dysfunctions induced by BRI, it could be effective against balance dysfunction 1 week after renal ischemia.

Topics: Acute Kidney Injury; Animals; Blood Urea Nitrogen; Creatinine; Disease Models, Animal; Erythropoietin; Locomotion; Male; Motor Disorders; Muscle Strength; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury

Cerebral white and grey matter injury is the leading cause of an adverse neurodevelopmental outcome in prematurely born infants. High oxygen concentrations have been shown to contribute to the pathogenesis of neonatal brain damage. Here, we focused on motor-cognitive outcome up to the adolescent and adult age in an experimental model of preterm brain injury. In search of the putative mechanisms of action we evaluated oligodendrocyte degeneration, myelination, and modulation of synaptic plasticity-related molecules. A single dose of erythropoietin (20,000 IU/kg) at the onset of hyperoxia (24 hours, 80% oxygen) in 6-day-old Wistar rats improved long-lasting neurocognitive development up to the adolescent and adult stage. Analysis of white matter structures revealed a reduction of acute oligodendrocyte degeneration. However, erythropoietin did not influence hypomyelination occurring a few days after injury or long-term microstructural white matter abnormalities detected in adult animals. Erythropoietin administration reverted hyperoxia-induced reduction of neuronal plasticity-related mRNA expression up to four months after injury. Thus, our findings highlight the importance of erythropoietin as a neuroregenerative treatment option in neonatal brain injury, leading to improved memory function in adolescent and adult rats which may be linked to increased neuronal network connectivity.

Topics: Animals; Animals, Newborn; Behavior, Animal; Brain Injuries; Cell Survival; Cognition; Diffusion Tensor Imaging; Disease Models, Animal; Down-Regulation; Erythropoietin; Hyperoxia; Immunohistochemistry; Microscopy, Confocal; Mitochondria; Myelin Basic Protein; Neuregulin-1; Neuronal Plasticity; Neuropilin-1; Neuroprotective Agents; Oligodendroglia; Rats; Rats, Wistar; Synaptophysin; White Matter

Aim. To evaluate the effects of erythropoietin administration on the adrenal glands in a swine model of ventricular fibrillation and resuscitation. Methods. Ventricular fibrillation was induced via pacing wire forwarded into the right ventricle in 20 female Landrace/Large White pigs, allocated into 2 groups: experimental group treated with bolus dose of erythropoietin (EPO) and control group which received normal saline. Cardiopulmonary resuscitation (CPR) was performed immediately after drug administration as per the 2010 European Resuscitation Council (ERC) guidelines for Advanced Life Support (ALS) until return of spontaneous circulation (ROSC) or death. Animals who achieved ROSC were monitored, mechanically ventilated, extubated, observed, and euthanized. At necroscopy, adrenal glands samples were formalin-fixed, paraffin-embedded, and routinely processed. Sections were stained with hematoxylin-eosin. Results. Oedema and apoptosis were the most frequent histological changes and were detected in all animals in the adrenal cortex and in the medulla. Mild and focal endothelial lesions were also detected. A marked interindividual variability in the degree of the intensity of apoptosis and oedema at cortical and medullary level was observed within groups. Comparing the two groups, higher levels of pathological changes were detected in the control group. No significant difference between the two groups was observed regarding the endothelial changes. Conclusions. In animals exposed to ventricular fibrillation, EPO treatment has protective effects on the adrenal gland.

Topics: Adrenal Cortex; Adrenal Glands; Adrenal Medulla; Animals; Apoptosis; Cardiopulmonary Resuscitation; Disease Models, Animal; Erythropoietin; Female; Protective Agents; Swine; Ventricular Fibrillation

The aim of this study was to compare the preventive effects of Etanercept, Etomidate, Erythropoietin and their combination in experimentally induced spinal cord trauma by clinical, histopathological, electrophysiological parameters and biochemical examination.. 85 healthy female Wistar-Albino rats were used in this study. Rats were divided 8 trauma groups that consisted of 10 rats for each, and 5 rats for the sham group. Laminectomy was performed under general anesthesia and the spinal cord was exposed with intact dura mater, and injury was created by the clip compression model. After the spinal cord injury, drugs were administered immediately intraperitoneally or subcutaneously. Except the sham group, all groups received drugs and were observed 24 or 72 hours. At the 72nd hour each group was anesthesized and somatosensorial evoked potentials (SEP) were recorded from the interarcuate ligament from the 2 vertebra proximal to the injured spinal cord and spinal cord tissue samples were taken for histopathological and biochemical evaluation.. Etomidate groups showed a lower effect on spinal cord injury than etanercept and erythropoietin in terms of clinical, SEP and TNF-α. Etanercept and erythropoietin's neuroprotective effectiveness was shown alone or in combination treatments. More meaningful results were achieved with the use of erythropoietin and etanercept combination after spinal cord injury (SCI) than using erythropoietin alone. After SCI, highest Basso, Beattie, and Bresnahan (BBB) scores were achieved in the group which Etanercept and Erythropoietin applied together.. The neuroprotective activity of etomidate was suspect. The neuroprotective effect of etanercept and erythropoietin after SCI was shown in individual and combined applications in this study. However, more experimental studies are needed for clinical use.

Topics: Animals; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Erythropoietin; Etanercept; Etomidate; Evoked Potentials, Somatosensory; Female; Neuroprotective Agents; Rats; Spinal Cord Injuries; Tumor Necrosis Factor-alpha

We previously found that administration of erythropoietin (EPO) shortens the course of recovery after experimental crush injury to the mouse sciatic nerve. The course of recovery was more rapid than would be expected if EPO's effects were caused by axonal regeneration, which raised the question of whether recovery was instead the result of promoting remyelination and/or preserving myelin on injured neurons. This study tested the hypothesis that EPO has a direct and local effect on myelination in vivo and in vitro.. Animals were treated with EPO after standard calibrated sciatic nerve crush injury; immunohistochemical analysis was performed to assay for myelinated axons. Combined in vitro neuron-Schwann cell co-cultures were performed to assess EPO-mediated effects directly on myelination and putative protective effects against oxidative stress. In vivo local administration of EPO in a fibrin glue carrier was used to demonstrate early local effects of EPO treatment well in advance of possible neuroregenerative effects.. Systemic Administration of EPO maintained more in vivo myelinated axons at the site of nerve crush injury. In vitro, EPO treatment promoted myelin formation and protected myelin from the effects of nitric oxide exposure in co-cultures of Schwann cells and dorsal root ganglion neurons. In a novel, surgically applicable local treatment using Food and Drug Administration-approved fibrin glue as a vehicle, EPO was as effective as systemic EPO administration at time points earlier than those explainable using standard models of neuroregeneration.. In nerve crush injury, EPO may be exerting a primary influence on myelin status to promote functional recovery.. Mixed injury to myelin and axons may allow the opportunity for the repurposing of EPO for use as a myeloprotective agent in which injuries spare a requisite number of axons to allow early functional recovery.

Topics: Animals; Biopsy, Needle; Crush Injuries; Disease Models, Animal; Erythropoietin; Female; Immunohistochemistry; Infusions, Parenteral; Injections, Intralesional; Mice; Mice, Inbred C57BL; Myelin Sheath; Nerve Regeneration; Random Allocation; Recovery of Function; Sciatic Nerve; Sciatic Neuropathy

The present objective was to investigate endogen erythropoietin (EPO) level and relationship to oxidative stress within the first 24 hours of blunt chest trauma-induced pulmo-nary contusion (PCn) in a rat model.. Thirty-five rats were divided into 3 groups. In the baseline control group (BC, n=7), rats were uninjured and untreated. In the positive control group (PC, n=21) rats were injured but untreated. In the EPO-24 group (n=7), rats were injured and a single dose of intra-peritoneal EPO (5000 IU/kg) was administered immediately after lung injury. The PC group was divided into 3 subgroups: PC-6 (n=7), PC-12 (n=7), and PC-24 (n=7). The BC group was subjected to thoracotomy, and the right lung was harvested. The PC subgroups were eu-thanized at 6, 12, and 24 hours after injury, respectively. The EPO-24 group was euthanized at the 24th hour after injury. Lung samples were obtained, levels of malondialdehyde (MDA) and EPO were analyzed, and activities of superoxide dismutase (SOD) and catalase (CAT) were then measured in homogenized lung tissue samples. Histologic damage to lung tissue in the BC group, the EPO-24 group, and PC subgroup euthanized at the 24th hour after injury were scored by a single pathologist blinded to group assignation.. Mean MDA levels, as well as SOD and CAT activities, of the BC and EPO-24 groups were significantly lower than those of the PC group (p<0.005). Mean EPO concentra-tion of the PC group was significantly higher than that of the BC group (p<0.005). Lung tis-sue damage scores measured at 24 hours after injury were significantly lower in the EPO-24 group than in the PC group (p<0.005).. In the present PCn rat model, EPO concentrations, as well as SOD and CAT levels, were high in lung tissue, when measured at 24 hours after PCn. When administered early after chest trauma, EPO significantly attenuated oxidative damage and tissue damage in the early phase, as assessed by biochemical markers and histologic scoring.

Topics: Animals; Contusions; Disease Models, Animal; Erythropoietin; Infusions, Parenteral; Lung; Lung Injury; Male; Rats; Rats, Sprague-Dawley; Wounds, Nonpenetrating

Erythropoietin (EPO) is important for angiogenesis after hypoxia/ischemia. In this study, we investigated whether recombinant human erythropoietin (rhEPO) can enhance angiogenesis, and promote cognitive function through vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway in a rat model of hypoxic-ischemic encephalopathy (HIE). RhEPO, selective VEGFR2 inhibitor (SU5416) or vehicle was administrated by intraperitoneal injection. The assessment for cognitive function begins on day 60 after anoxia. Vascular density in hippocampus and white matter damage within corpus callosum were examined on day 28 after anoxia. The expression of erythropoietin receptor (EPOR), VEGF, rapidly accelerated fibrosarcoma 1 (Raf1), and extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in hippocampus were evaluated on day 7 after anoxia. RhEPO-treated anoxia rats had better cognitive recovery, higher vascular density, and less white matter damage than in the vehicle anoxia rats. These protective effects associated with increased expression of EPOR, VEGF; and increased phosphorylation of Raf1 and ERK1/2. While this up-regulation, and changes in the histopathologic and functional outcomes were abolished by SU5416. Our data indicate that rhEPO can enhance angiogenesis, reduce white matter damage, and promote cognitive recovery through VEGF/VEGFR2 signaling pathway in anoxia rats.

Topics: Angiogenesis Inducing Agents; Animals; Animals, Newborn; Cognition; Disease Models, Animal; Erythropoietin; Hippocampus; Hypoxia; Hypoxia-Ischemia, Brain; Indoles; Male; Pyrroles; Random Allocation; Rats, Sprague-Dawley; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; White Matter

Mutations in the tumor suppressor gene von Hippel-Lindau (VHL) underlie a hereditary cancer syndrome-VHL disease-and are also frequently observed in sporadic renal cell carcinoma of the clear cell type (ccRCC). VHL disease is characterized by malignant and benign tumors in a few specific tissues, including ccRCC, hemangioblastoma and pheochromocytoma. The etiology of these tumors remains unresolved.. Conditional inactivation of the VHL gene in mouse (Vhlh) was generated to examine the pathophysiological role of the VHL gene function. Specific cell populations were isolated by fluorescence-activated cell sorting (FACS) and bone marrow transplants were performed to identify the Vhlh-inactivated cells responsible for the phenotype.. Previously we showed that inactivation of Vhlh in a subpopulation of kidney distal tubule cells resulted in hyperplastic clear-cell lesions and severe inflammation and fibrosis. Here, we show that this knockout mouse strain also develops Hif-2α-dependent vascular overgrowth (hemangioma) and extramedullary erythropoiesis in the liver. However, Vhlh inactivation was not detected in the liver parenchyma. We instead demonstrate that in these mice, Vhlh is inactivated in liver granulocytes and that hemangiomas are partially rescued in knockout mice reconstituted with wild-type hematopoietic stem cells, indicating the involvement of bone-marrow-derived leukocyte. Interestingly, bone marrow from knockout mice failed to generate the liver phenotype in wild-type recipients, suggesting that an additional cell type that is not derived from the bone marrow is involved in the development of the hemangioma phenotype.. These results support the idea that the development of a full-blown VHL disease phenotype requires inactivation of the VHL gene not only in the tumor proper, but also in the stromal compartment.

Topics: Animals; Biomarkers; Disease Models, Animal; Erythropoietin; Flow Cytometry; Gene Silencing; Granulocytes; Hemangioma; Hematopoiesis, Extramedullary; Hematopoietic Stem Cells; Homeodomain Proteins; Leukocytes; Liver; Liver Neoplasms; Mice; Mice, Knockout; Mutation; Phenotype; Von Hippel-Lindau Tumor Suppressor Protein

Erythropoietin-releasing neural precursor cells (Er-NPCs) are a subclass of subventricular zone-derived neural progenitors, capable of surviving for 6 hr after death of donor. They present higher neural differentiation. Here, Er-NPCs were studied in animal model of Parkinson's disease. Dopaminergic degeneration was caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intraperitoneal administration in C57BL/6 mice. The loss of function was evaluated by specific behavioral tests. Er-NPCs (2.5 × 10

Topics: Animals; Antigens; Antigens, Neoplasm; Cell- and Tissue-Based Therapy; Choline O-Acetyltransferase; Corpus Striatum; Disease Models, Animal; Erythropoietin; gamma-Aminobutyric Acid; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Movement Disorders; MPTP Poisoning; Muscle Strength; Nerve Tissue Proteins; Neural Stem Cells; Proteoglycans; Recovery of Function; Tyrosine 3-Monooxygenase

Persistent alterations of the renal tissue due to maladaptive repair characterize the outcome of acute kidney injury (AKI), despite a clinical recovery. Acute damage may also limit the renal production of erythropoietin, with impairment of the hemopoietic response to ischemia and possible lack of its reno-protective action. We aimed to evaluate the effect of a cell therapy using human CD133

Topics: AC133 Antigen; Acute Kidney Injury; Animals; Disease Models, Animal; Erythropoietin; Fibrosis; Heterografts; Humans; Kidney Tubules; Mice; Mice, SCID; Stem Cell Transplantation; Stem Cells

The leading role in the regulation of erythropoietic activity of adherent bone marrow cells under conditions of post-hemorrhagic anemia is played by classical MAP kinase pathway (ERK pathway). Erythropoietin is not the decisive factor in the formation of erythropoietic activity of adherent cells. PI3K, MAPK/ERK 1/2, and p38-signaling proteins are not the main regulators of local production of erythropoietin after 30% loss of circulating blood volume.

Topics: Anemia; Animals; Bone Marrow Cells; Chromones; Disease Models, Animal; Erythropoiesis; Erythropoietin; Flavonoids; Gene Expression Regulation; Hemorrhage; Imidazoles; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morpholines; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Pyridines; Signal Transduction

The cytokine erythropoietin (EPO) stimulates proliferation and differentiation of erythroid progenitor cells. Moreover, EPO has neuroprotective, anti-inflammatory, and antioxidative effects, but the use of EPO as a neuroprotective agent is hampered by its erythropoietic activity. We have recently designed the synthetic, dendrimeric peptide, Epobis, derived from the sequence of human EPO. This peptide binds the EPO receptor and promotes neuritogenesis and neuronal cell survival. Here we demonstrate that Epobis

Topics: Animals; Anti-Inflammatory Agents; Cells, Cultured; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Erythropoietin; Female; Hematopoiesis; Macrophage Activation; Memory, Short-Term; Microglia; Motor Neurons; Neurites; Neuroprotective Agents; Peptides; Rats; Rats, Wistar

This experimental study was an investigation of the efficacy of erythropoietin and tadalafil in rats with induced spinal cord injury (SCI).. Thirty-five Sprague Dawley rats were distributed into 5 groups. First group was used for normal biochemical values. Spinal cord injury was induced in 4 remaining groups with clip compression technique after laminectomy process to T10 vertebra. Second group was designated solvent group and received 1 cc physiological serum after injury. Third group was medicated with intraperitoneal 2000 u/kg single dose erythropoietin after injury. Orogastric 2 mg/kg single dose tadalafil was administered to fourth group after injury. Fifth group did not receive any treatment and was used for biochemical values with injury. All subjects were sacrificed 48 hours after application. Malondialdehyde (MDA) and total antioxidant capacity (TAOC) values were evaluated using blood and tissue samples.. Lowest serum and tissue MDA values were found in group with erythropoietin intake. While highest serum TAOC values of all groups were seen in tadalafil group, highest tissue TAOC values were observed in group given erythropoietin.. It was concluded that by decreasing oxidative stress, tadalafil and erythropoietin can inhibit secondary damage in SCI.

Topics: Administration, Oral; Animals; Disease Models, Animal; Drug Therapy, Combination; Erythropoietin; Infusions, Parenteral; Male; Neuroprotective Agents; Random Allocation; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Tadalafil; Thoracic Vertebrae

Preterm birth impacts brain development and leads to chronic deficits including cognitive delay, behavioral problems, and epilepsy. Premature loss of the subplate, a transient subcortical layer that guides development of the cerebral cortex and axonal refinement, has been implicated in these neurological disorders. Subplate neurons influence postnatal upregulation of the potassium chloride co-transporter KCC2 and maturation of γ-amino-butyric acid A receptor (GABAAR) subunits. We hypothesized that prenatal transient systemic hypoxia-ischemia (TSHI) in Sprague-Dawley rats that mimic brain injury from extreme prematurity in humans would cause premature subplate loss and affect cortical layer IV development. Further, we predicted that the neuroprotective agent erythropoietin (EPO) could attenuate the injury. Prenatal TSHI induced subplate neuronal loss via apoptosis. TSHI impaired cortical layer IV postnatal upregulation of KCC2 and GABAAR subunits, and postnatal EPO treatment mitigated the loss (n ≥ 8). To specifically address how subplate loss affects cortical development, we used in vitro mechanical subplate ablation in slice cultures (n ≥ 3) and found EPO treatment attenuates KCC2 loss. Together, these results show that subplate loss contributes to impaired cerebral development, and EPO treatment diminishes the damage. Limitation of premature subplate loss and the resultant impaired cortical development may minimize cerebral deficits suffered by extremely preterm infants.

Topics: Age Factors; Animals; Animals, Newborn; Brain Injuries; Cell Death; Cerebral Cortex; Disease Models, Animal; Embryo, Mammalian; Erythropoietin; Fetal Diseases; Gene Expression Regulation, Developmental; Hypoxia-Ischemia, Brain; In Vitro Techniques; K Cl- Cotransporters; Motor Activity; Nuclear Receptor Subfamily 4, Group A, Member 2; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Symporters

Unilateral ureteral obstruction halts ureteral peristalsis, and may cause pain and lead to infection. Ureteral ability to recover after obstruction removal remains unclear. Erythropoietin has protective effects in nonhematopoietic organs and restores peristalsis in hypocontractile intestinal smooth muscle cells. We investigated the role of erythropoietin in ureteral smooth muscle function and its therapeutic value for unilateral ureteral obstruction.. Unilateral ureteral obstruction was created for 24, 48 and 72 hours in 22 mice per group using a nontraumatic microclip via laparotomy. We determined erythropoietin, erythropoietin receptor and β-common receptor expression in obstructed and unobstructed ureters by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Ten mice per group received 20 IU erythropoietin for 4 days and controls received saline. Hydronephrosis regression after obstruction removal was assessed by ultrasound. Peristalsis was determined microscopically before and after obstruction removal.. Erythropoietin, erythropoietin receptor and β-common receptor were expressed in the unobstructed and obstructed ureters of untreated mice. Erythropoietin mRNA was up-regulated in response to obstruction and erythropoietin expression was identified in ureteral smooth muscle. After obstruction removal hydronephrosis and ureteral dysfunction correlated with obstruction duration. Hydronephrosis resolution and ureteral peristalsis restoration were significantly accelerated in erythropoietin treated mice compared to controls.. Erythropoietin treatment significantly promoted functional recovery of the ureter after obstruction removal. Erythropoietin may be a helpful strategy for ureteral motility recovery and hydronephrosis resolution in ureteral obstruction.

Topics: Animals; Disease Models, Animal; Erythropoietin; Hydronephrosis; Male; Mice; Muscle, Smooth; Recovery of Function; Ureteral Obstruction; Urethra

Erythropoietin (EPO) is used to treat anaemia associated with chronic kidney disease (CKD). Hypoxia is associated with anaemia and is known to cause a decrease in cytochrome P450 (P450) expression. As EPO production is regulated by hypoxia, we investigated the role of EPO on P450 expression and function.. Male Wistar rats were subjected to a 0.7% adenine diet for 4 weeks to induce CKD. The diet continued for an additional 2 weeks while rats received EPO by i.p. injection every other day. Following euthanasia, hepatic P450 mRNA and protein expression were determined. Hepatic enzyme activity of selected P450s was determined and chromatin immunoprecipitation was used to characterize binding of nuclear receptors involved in the transcriptional regulation of CYP2C and CYP3A.. EPO administration decreased hepatic mRNA and protein expression of CYP3A2 (P < 0.05), but not CYP2C11. Similarly, EPO administration decreased CYP3A2 protein expression by 81% (P < 0.001). A 32% decrease (P < 0.05) in hepatic CYP3A enzymatic activity (Vmax ) was observed for the formation of 6βOH-testosterone in the EPO-treated group. Decreases in RNA pol II recruitment (P < 0.01), hepatocyte nuclear factor 4α binding (P < 0.05) and pregnane X receptor binding (P < 0.01) to the promoter region of CYP3A were also observed in EPO-treated rats.. Our data show that EPO decreases the expression and function of CYP3A, but not CYP2C in rat liver.

Topics: Adenine; Animals; Constitutive Androstane Receptor; Cytochrome P-450 Enzyme System; Diet; Disease Models, Animal; Erythropoietin; Hepatocyte Nuclear Factor 4; Kidney; Liver; Male; Microsomes, Liver; Pregnane X Receptor; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Recombinant Proteins; Renal Insufficiency, Chronic; RNA Polymerase II; RNA, Messenger

Sepsis continues to be a significant problem for critical care patients.. To evaluate the protective effects of IgM-enriched immunoglobulin and erythropoietin on pulmonary and small intestine tissues in a rat model of intra-abdominal sepsis induced via the cecal ligation and puncture (CLP) method.. Male Sprague-Dawley rats were used. Control group (n = 6): surgical procedure was not performed. Laparotomy was only performed in the sham group (n = 6) and CLP was only performed in the sepsis (CLP) group (n = 30). After erythropoietin (2000 U/kg, intraperitoneal) was given in the sepsis + erythropoietin (CLP + EPO) group (n = 30), IgM-enriched immunoglobulin (600 mg/kg, intraperitoneal) was given in the sepsis + pentaglobin (CLP + PEN) group (n = 30), CLP was created. Intracardiac blood samples were collected for biochemical analysis; lung and small intestine tissue samples were removed for histopathological evaluation.. Plasma TNF-α levels (pg/ml) were similar among CLP, CLP + EPO, and CLP + PEN groups (204.0 ± 52.4, 198.5 ± 17.3, and 214.6 ± 93.6, respectively). The CLP group had higher plasma IL-1β levels (pg/ml) compared with CLP + EPO and CLP + PEN groups (325.1 ± 134.1, 164.3 ± 25.6, and 186.3 ± 26.0, respectively) (p < 0.05). Rats in CLP + EPO and CLP + PEN groups had abolished histopathologic appearance of lung and small intestine tissues compared with rats in the CLP group.. Our findings support the use of EPO and IgM-enriched immunoglobulin in the prevention of lung and small intestine injuries associated with sepsis.

Topics: Animals; Cecum; Disease Models, Animal; Erythropoietin; Immunoglobulin A; Immunoglobulin M; Interleukin-1beta; Intestine, Small; Ligation; Lung; Male; Rats, Sprague-Dawley; Sepsis; Tumor Necrosis Factor-alpha

The mechanism through which the protein kinase Akt (also called PKB), protects the heart against acute ischaemia-reperfusion injury (IRI) is not clear. Here, we investigate whether Akt mediates its cardioprotective effect by modulating mitochondrial morphology. Transfection of HL-1 cardiac cells with constitutively active Akt (caAkt) changed mitochondrial morphology as evidenced by an increase in the proportion of cells displaying predominantly elongated mitochondria (73 ± 5.0 % caAkt vs 49 ± 5.8 % control: N=80 cells/group; p< 0.05). This effect was associated with delayed time taken to induce mitochondrial permeability transition pore (MPTP) opening (by 2.4 ± 0.5 fold; N=80 cells/group: p< 0.05); and reduced cell death following simulated IRI (32.8 ± 1.2 % caAkt vs 63.8 ± 5.6 % control: N=320 cells/group: p< 0.05). Similar effects on mitochondrial morphology, MPTP opening, and cell survival post-IRI, were demonstrated with pharmacological activation of Akt using the known cardioprotective cytokine, erythropoietin (EPO). The effect of Akt on inducing mitochondrial elongation was found to be dependent on the mitochondrial fusion protein, Mitofusin-1 (Mfn1), as ablation of Mfn1 in mouse embryonic fibroblasts (MEFs) abrogated Akt-mediated mitochondrial elongation. Finally, in vivo pre-treatment with EPO reduced myocardial infarct size (as a % of the area at risk) in adult mice subjected to IRI (26.2 ± 2.6 % with EPO vs 46.1 ± 6.5 % in control; N=7/group: p< 0.05), and reduced the proportion of cells displaying myofibrillar disarray and mitochondrial fragmentation observed by electron microscopy in adult murine hearts subjected to ischaemia from 5.8 ± 1.0 % to 2.2 ± 1.0 % (N=5 hearts/group; p< 0.05). In conclusion, we found that either genetic or pharmacological activation of Akt protected the heart against acute ischaemia-reperfusion injury by modulating mitochondrial morphology.

Topics: Animals; Cell Death; Cell Line; Disease Models, Animal; Enzyme Activation; Erythropoietin; GTP Phosphohydrolases; Male; Mice, Inbred C57BL; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Size; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Proto-Oncogene Proteins c-akt; Signal Transduction; Time Factors; Transfection

Approximately 30% of the chronic kidney disease patients using recombinant human erythropoietin (rhuEPO) have an increase in blood pressure (BP). Its mechanism and whether it depends on renal function remain unclear. There is early evidence that acetylsalicylic acid (ASA) prevents the rhuEPO-induced increase in BP. This study aims to verify whether very high doses of rhuEPO can increase BP in nonuremic rats and whether the co-administration of ASA can prevent it.. Forty male Wistar rats were divided into four groups: placebo/placebo; placebo/rhuEPO 200 UI/kg thrice weekly; placebo/ASA 50 mg/kg daily; rhuEPO 200 UI/kg thrice weekly/ASA 50 mg/kg daily. Hematocrit was measured before and after and systolic BP was measured weekly by tail-cuff technique. Direct measurement of the BP was obtained at the end.. The rhuEPO groups had higher final hematocrit (rhuEPO/placebo 56.7 ± 7.6, rhuEPO/ASA 56.7 ± 7.7; p < 0.001 versus placebo/placebo, 42.2 ± 4.7 and ASA/placebo 41.2 ± 4.2); and also increase in systolic BP (rhuEPO/placebo 135.1 ± 15.0, p = 0.01 and rhuEPO/ASA 127.2 ± 6.8, p = 0.02), whereas BP in rats from placebo/placebo (120.9 ± 5.0, p = 0.18) and placebo/ASA (124.6 ± 13.3, p = 0.12) groups remained unchanged. By direct measurement, the final BP was higher in rhuEPO/placebo (DBP 123.1 ± 12.0; SBP 157.4 ± 12.5; MBP 139.8 ± 11.9) than placebo/placebo (DBP 105.1 ± 11.5; SBP 141.0 ± 12.6; MBP 122.1 ± 12.1) and placebo/ASA groups (DBP 106.6 ± 8.1; SBP 141.5 ± 8.4, MBP 122.1 ± 7.2) (p < 0.05 by post hoc Bonferroni test ANOVA). The rhuEPO/ASA group (PAD 115.1 ± 11.4, PAS 147.4 ± 9.1, MBP 130.1 ± 10.3) was not different from other groups.. The administration of very high doses of rhuEPO is associated with an increase in hematocrit and BP in nonuremic rats. The concomitant use of ASA mitigates the rhuEPO-associated BP increase.

Topics: Anemia; Animals; Aspirin; Blood Pressure; Disease Models, Animal; Drug Monitoring; Erythropoietin; Hematinics; Hypertension; Kidney Failure, Chronic; Male; Rats; Rats, Wistar; Treatment Outcome

This study aimed to investigate the impacts of erythropoietin (EPO) on the electroretinogram b‑wave (ERG‑b), and on the mRNA and protein expression levels of hypoxia‑inducible factor‑1α (HIF‑1α), inducible nitric oxide synthase (iNOS), cyclooxygenase‑2 (COX‑2) and caspase‑9 in chronic ocular hypertension rats. Episcleral vein cauterization (EVC) was used to establish the chronic ocular hypertension rat model based on the intraocular pressure (IOP) value. ERG‑b and mRNA and protein expression levels of HIF‑1α, iNOS, COX‑2 and caspase‑9 in normal, EVC‑treated and EVC combined with EPO (EVC+EPO)‑treated rats were measured by electroretinography, RT‑PCR and western blotting, respectively. Moreover, the correlations of HIF‑1α with IOP, ERG‑b, iNOS, COX‑2 and caspase‑9 were evaluated. The mRNA and protein expression levels of HIF‑1α, iNOS, COX‑2 and caspase‑9 in EVC‑treated rats were increased significantly compared with normal rats. The peak expression levels of HIF‑1α, iNOS, COX‑2 and caspase‑9 were respectively obtained 7, 7, 7 and 14 days postoperatively. Compared with EVC‑treated rats, EPO administration weakened the mRNA and protein expression levels of HIF‑1α, iNOS, COX‑2 and caspase‑9. The mRNA expression level of HIF‑1α demonstrated a significant positive correlation with IOP and ERG‑b. HIF‑1α was positively correlated with iNOS, COX‑2 and caspase‑9 at the mRNA and protein levels. The protective effect of EPO on the retina of chronic ocular hypertension rats may be mediated by the HIF‑1 signaling pathway involving iNOS, COX‑2 and caspase‑9.

Topics: Animals; Caspase 9; Chronic Disease; Cyclooxygenase 2; Disease Models, Animal; Electroretinography; Epoetin Alfa; Erythropoietin; Hypoxia-Inducible Factor 1, alpha Subunit; Intraocular Pressure; Male; Neuroprotective Agents; Nitric Oxide Synthase Type II; Ocular Hypertension; Rats; Rats, Wistar; Recombinant Proteins; Retina; RNA, Messenger; Signal Transduction

Paraplegia remains a devastating complication of complex aortic surgery. Erythropoietin (EPO) has been shown to prevent paraplegia after ischemia reperfusion, but the protective mechanism remains poorly described in the spinal cord. We hypothesized that EPO induces the CREB (cAMP [adenosine 3'5' cyclic monophosphate] response element-binding protein) pathway and neurotrophin production in the murine spinal cord, attenuating functional and cellular injury.. Adult male mice were subjected to 4 minutes of spinal cord ischemia via an aortic and left subclavian cross-clamp. Experimental groups included EPO treatment 4 hours before incision (n = 7), ischemic control (n = 7), and shams (n = 4). Hind-limb function was assessed using the Basso motor score for 48 hours after reperfusion. Spinal cords were harvested and analyzed for neuronal viability using histology and staining with a fluorescein derivative. Expression of phosphorylated (p)AKT (a serine/threonine-specific kinase), pCREB, B-cell lymphoma 2, and brain-derived neurotrophic factor were determined using immunoblotting.. By 36 hours of reperfusion, EPO significantly preserved hind-limb function after ischemia-reperfusion injury (P < .01). Histology demonstrated preserved cytoarchitecture in the EPO treatment group. Cords treated with EPO expressed significant increases in pAKT (P = .021) and pCREB (P = .038). Treatment with EPO induced expression of both of the neurotrophins, B-cell lymphoma 2, and brain-derived neurotrophic factor, beginning at 12 hours.. Erythropoietin-mediated induction of the CREB pathway and production of neurotrophins is associated with improved neurologic function and increased neuronal viability following spinal cord ischemia reperfusion. Further elucidation of EPO-derived neuroprotection will allow for expansion of adjunct mechanisms for spinal cord protection in high-risk thoracoabdominal aortic intervention.

Topics: Animals; Brain-Derived Neurotrophic Factor; CREB-Binding Protein; Disease Models, Animal; Erythropoietin; Male; Mice, Inbred C57BL; Motor Activity; Paraplegia; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reperfusion Injury; Signal Transduction; Spinal Cord; Spinal Cord Ischemia; Time Factors

Several studies indicate erythropoietin (Epo) to have remarkable neuroprotection in various central nervous system disorders, including Alzheimer's disease (AD). Amyloid beta (Aβ) is believed to be responsible for the synaptic dysfunction that occurs in AD. Therefore, the present study is aimed to investigate the effects of Epo on the Aβ-induced impairments in learning-memory and hippocampal synaptic plasticity.. Male Sprague-Dawley rats (200-250 g) were used in this study. After the injection of Aβ, they were injected intra-peritoneal with Epo in the Aβ+Epo group or its vehicle in the Aβ+V group every other day for 12 days. A shuttle box apparatus was used for the passive avoidance learning and memory study. Moreover, paired-pulse ratio (PPR) was monitored before and after tetanic stimulation.. Bilateral injection of Aβ decreased step-through latency (STL), whereas the 12 day administration of Epo significantly improved memory performance in Aβ+Epo group. The field potential recording demonstrated that the in vivo administration of Aβ25-35 led to extreme inhibition in long-term potentiation, this inhibition was accompanied by a significant increase of the normalized PPR (PPR after HFS/PPR before HFS) as an index for release probability. However, administration of Epo recovers the magnitude of the LTP and the extent of normalized PPR.. The results of this study demonstrated that the injection of Aβ25-35 resulted in impaired LTP and the memory process, which is likely mediated through increasing the release probability of neurotransmitter vesicles. In addition, treatment with Epo improved the Aβ-induced deficits in memory and LTP induction, probably via recovering the release probability.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Avoidance Learning; Disease Models, Animal; Erythropoietin; Excitatory Postsynaptic Potentials; Glutamic Acid; Hematocrit; Hippocampus; Long-Term Potentiation; Male; Memory Disorders; Neuronal Plasticity; Neuroprotective Agents; Peptide Fragments; Rats

The aim of this study was to investigate the protective effect of erythropoietin (EPO) against acute myocardial injury and its underlying mechanisms. Mice (n=146) were randomly divided in a double‑blind manner into four groups, sham, Rocephin, EPO and sepsis, and mortality was observed on the seventh day after cecal ligation and puncture. In addition, a total of 252 rats were randomly divided into three groups, sham, EPO and sepsis, and indicators of cardiac function, inflammatory mediators and serum creatine kinase levels were assessed. Mitochondrial membrane potential, cell apoptosis and nuclear factor κ‑light‑chain‑enhancer of activated B cells (NF‑κB) p65 expression levels were detected using flow cytometry. Following intervention with EPO, the mortality rate in mice with sepsis was significantly reduced and the cardiac function of septic rats was significantly improved. In addition, the levels of inflammatory mediators, serum creatine kinase and apoptosis and the myocardial mitochondrial membrane potential and expression of NF‑κB p65 in cardiac tissue were all improved following EPO treatment, and the differences between the results for the sepsis and EPO groups were statistically significant (P<0.05). These findings suggest that EPO reduces the myocardial inflammatory response in septic rats, attenuates the reduction in mitochondrial membrane potential and inhibits myocardial cell apoptosis by reducing NF‑κB p65 expression, and therefore exerts a protective effect in the myocardium.

Topics: Animals; Apoptosis; Biomarkers; Cardiomyopathies; Cardiotonic Agents; Cytokines; Disease Models, Animal; Erythropoietin; Hemodynamics; Inflammation Mediators; Male; Membrane Potential, Mitochondrial; Mice; Mortality; Rats; Sepsis; Time Factors; Transcription Factor RelA

Erythropoietin (EPO) is a hematopoietic hormone that protects against renal interstitial fibrosis in animal models; however, the mechanism underlying the anti‑fibrotic activity of EPO has remained elusive. The present study aimed to elucidate this mechanism. Twenty‑four male C57BL6 mice were randomly divided into four groups, each comprising six mice: (i) control group (Sh); (ii) unilateral ureteral obstruction (UUO) plus vehicle group (U+V); (ⅲ) UUO plus 300 U/kg body weight recombinant human (rh)EPO (U+E1) and (ⅳ) UUO plus 1,000 U/kg body weight rhEPO (U+E2). Seven days post‑surgery, the mice were sacrificed for examination. UUO induced significant deposition of extracellular matrix, detected by picro‑sirius red staining, which was decreased following rhEPO treatment. UUO also induced deposition of collagen I and fibronectin, rhEPO treatment was able to attenuate this effect at protein and mRNA levels. Compared with the control groups, UUO resulted in the accumulation of α‑smooth muscle actin‑positive cells in the interstitium, an effect which was ameliorated by rhEPO. Furthermore, rhEPO abrogated the UUO‑induced increase in the number of bone marrow‑derived myofibroblasts. Mechanistically, it was discovered that rhEPO decreased CXC chemokine ligand 16 (CXCL16) expression at protein level. However, treatment with rhEPO did not alter the protein expression of CC chemokine ligand 21 or CXCL12. These results suggested that rhEPO decreased fibrocyte accumulation via the suppression of renal CXCL16, which resulted in the attenuation of renal fibrosis.

Topics: Animals; Chemokine CXCL16; Chemokines, CXC; Disease Models, Animal; Disease Progression; Erythropoietin; Fibrosis; Humans; Kidney Diseases; Male; Mice; Myofibroblasts; Receptors, Scavenger; Recombinant Proteins

Renal ischaemia-reperfusion (I/R) injury, a primary cause of acute renal failure, can induce high morbidity and mortality. This study aimed to explore the effect of erythropoietin on renal I/R injury and its underlying mechanism.. Fifty male Sprague-Dawley rats were randomly allocated to three groups (n = 10): the sham group, the renal ischaemia-reperfusion-saline (IRI) group, and the IRI+-Erythropoietin (EPO) group. Erythropoietin (250, 500, 1000 U/kg) was intraperitoneally injected 30 min before inducing I/R. Renal I/R injury were induced by clamping the left renal artery for 30 min followed by reperfusion, along with a contralateral nephrectomy. Renal function and histological damage were determined after 24 h reperfusion. The expression of pro-inflammatory cytokines interleukin-6 (IL-6), interleukin-1 β (IL-1β), and tumour necrosis factor-α (TNF-α) in the serum and renal tissue were evaluated by enzyme linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR), respectively. Further, the effects of erythropoietin on PI3K/Akt signalling, erythropoietin receptor (EPOR) and nuclear factor (NF)-κB activation were measured by Western blotting.. Erythropoietin pretreatment can significantly decrease the level of renal dysfunction in a dose-dependent manner, attenuated the renal histological changes, the expression of TNF-α, IL-1β, and IL-6, the levels of reactive oxygen species (ROS) production and NF-κB p65 phosphorylation in renal tissue upon IRI. Moreover, erythropoietin pretreatment could further activate the PI3K/Akt signalling and induced EPOR activity.. Erythropoietin pretreatment could attenuate renal I/R injury by suppressing inflammation, which was associated with activating PI3K/Akt signalling though EPOR activation. Our findings suggest that erythropoietin may be a novel practical strategy to prevent renal I/R injury.

Topics: Acute Kidney Injury; Animals; Anti-Inflammatory Agents; Biomarkers; Cytoprotection; Disease Models, Animal; Enzyme Activation; Erythropoietin; Inflammation Mediators; Interleukin-1beta; Interleukin-6; Kidney; Male; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Erythropoietin; Reperfusion Injury; Signal Transduction; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Understanding the mechanisms of protecting the kidneys from injury is of great importance because there are no effective therapies that promote repair and the kidneys frequently do not repair adequately. Evidence has shown that erythropoietin (EPO) has a vital renoprotective role, independent of its erythropoietic effect. However, whether EPO can contribute to kidney repair after injury and the potential mechanisms are not fully understood.. To investigate the renoprotective mechanism of EPO, a kidney ischemia/reperfusion injury (IRI) model was induced in adult male Sprague-Dawley rats. The rats were subsequently randomly treated with EPO or a vehicle 6 hours after the kidney IRI. The rats were sacrificed on Day 3, Day 5, and Day 7 post kidney IRI. Renal function and histological alterations were examined. Renal interstitial macrophage infiltration, cell proliferation, apoptosis, and angiogenesis were evaluated by immunostaining. Furthermore, the effects of EPO on the Wnt/β-catenin pathway and IRI-related micro-RNAs were investigated.. The administration of EPO significantly improved renal function and reduced tubular injury. Furthermore, EPO treatment significantly prevented tubular cell apoptosis and promoted cell proliferation after IRI. Erythropoietin significantly suppressed macrophage infiltration, compared to the vehicle. In addition, treatment with EPO markedly prevented the loss of microvasculature. We have also demonstrated that, compared to the vehicle, EPO administration enhanced the expression of Wnt7b and β-catenin, and downregulated miR-21, -214, -210, and -199a.. Erythropoietin protects the kidneys against IRI by attenuating injury of the renal microvasculature and tubule epithelial cells, by promoting Wnt/β-catenin pathway activation, and by regulating miRNA expression.

Topics: Acute Kidney Injury; Animals; Apoptosis; Disease Models, Animal; Erythropoietin; Kidney; Male; MicroRNAs; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Wnt Signaling Pathway

Anemia is a common comorbidity of patients with heart failure, and iron deficiency is known as one of the causes of anemia in heart failure. Recent studies have shown that iron deficiency alone, without overt anemia, is associated with poor outcomes in patients with heart failure. Thus, to minimize the mortality in patients with heart failure, it is important to understand the link between iron deficiency and cardiac function. Chronic untreated iron deficiency results in cardiac remodeling, and we have previously reported that erythropoietin (Epo) and cardiac Epo receptor (EpoR) signaling may be associated with its remodeling. However, the link between EpoR signaling and its remodeling remains to be elucidated. Herein, we investigated the role of EpoR signaling on cardiac remodeling in response to chronic iron deficiency.. Wild-type mice and transgene-rescued EpoR-null mutant mice, which express EpoR only in the hematopoietic lineage (EpoR-restricted mice), were fed with either a normal or an iron-restricted diet, and the molecular mechanisms were investigated.. Dietary iron restriction gradually induced anemia, Epo secretion, and cardiac hypertrophy in wild-type mice. In contrast, EpoR-restricted mice fed with an iron-restricted diet exhibited anemia, left ventricular dilatation, and cardiac dysfunction compared with wild-type mice. Interestingly, altered cardiac mitochondrial biogenesis was observed in EpoR-restricted mice following iron deficiency. Moreover, cardiac p53 expression was increased in EpoR-restricted mice compared with wild-type mice following iron deficiency.. These data indicate that EpoR signaling is associated with cardiac remodeling following chronic iron deficiency.

Topics: Anemia, Iron-Deficiency; Animals; Chronic Disease; Disease Models, Animal; Erythropoietin; Heart Failure; Iron Deficiencies; Male; Mice; Mice, Knockout; Myocardium; Receptors, Erythropoietin; Signal Transduction

Cisplatin (CDDP) is severely neurotoxic anti-neoplastic drug that causes peripheral neuropathies with clinical signs known as chemotherapy-induced peripheral neurotoxicity. The ameliorating effects of erythropoeitin on cisplatin-induced neuropathy, which seem to be mediated by enhancing the cell resistance to side effects of cisplatin rather than by influencing the formation or repair rates of cisplatin-induced cross-links in the nuclear DNA, had been previously reported. The main objective of our study is to investigate the roles of nitro-oxidative stress, nuclear factor kappa B (NFκB) gene expressions and TNF levels on the previous reported erythropoietin anti-apoptotic neuroprotective effects during cisplatin induced neurotoxicity. The present study compared the effects of erythropoietin (50 μg/kg/d thrice weekly) on cisplatin (2mg/kg/d i.p. twice weekly for 4 weeks) induced neurophysiologic changes and the associated changes in the inflammatory mediators (TNF alpha and NFKB), oxidative stress (malondialdehyde (MDA), superoxide dismutases (SOD) and glutathione) and gene expression of both neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS). In addition, sciatic nerve pro-apoptotic and anti-apoptotic indicators (Bcl, Bax, Caspase 3) were measured. We found that concomitant administration of erythropoietin significantly reversed the cisplatin induced nitro-oxidative stress - with significant increases in sciatic nerve glutathione and superoxide dismutase antioxidant enzyme levels and a significant decrease in iNOS gene expression. We conclude that erythropoietin anti-apoptotic neuro-protective effects could partially contribute to observed antioxidant effects of erthropoietin.

Topics: Animals; Antineoplastic Agents; Antioxidants; Cisplatin; Disease Models, Animal; Electrophysiology; Erythropoietin; Immunohistochemistry; Male; Oxidative Stress; Peripheral Nervous System Diseases; Rats; Real-Time Polymerase Chain Reaction

Biomarkers that indicate the severity of hypoxic-ischemic brain injury and response to treatment and that predict neurodevelopmental outcomes are urgently needed to improve the care of affected neonates. We hypothesize that sequentially obtained plasma metabolomes will provide indicators of brain injury and repair, allowing for the prediction of neurodevelopmental outcomes. A total of 33 Macaca nemestrina underwent 0, 15 or 18 min of in utero umbilical cord occlusion (UCO) to induce hypoxic-ischemic encephalopathy and were then delivered by hysterotomy, resuscitated and stabilized. Serial blood samples were obtained at baseline (cord blood) and at 0.1, 24, 48, and 72 h of age. Treatment groups included nonasphyxiated controls (n = 7), untreated UCO (n = 11), UCO + hypothermia (HT; n = 6), and UCO + HT + erythropoietin (n = 9). Metabolites were extracted and analyzed using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry and quantified by PARAFAC (parallel factor analysis). Using nontargeted discovery-based methods, we identified 63 metabolites as potential biomarkers. The changes in metabolite concentrations were characterized and compared between treatment groups. Further comparison determined that 8 metabolites (arachidonic acid, butanoic acid, citric acid, fumaric acid, lactate, malate, propanoic acid, and succinic acid) correlated with early and/or long-term neurodevelopmental outcomes. The combined outcomes of death or cerebral palsy correlated with citric acid, fumaric acid, lactate, and propanoic acid. This change in circulating metabolome after UCO may reflect cellular metabolism and biochemical changes in response to the severity of brain injury and have potential to predict neurodevelopmental outcomes.

Topics: Animals; Animals, Newborn; Apgar Score; Asphyxia Neonatorum; Biomarkers; Cerebral Palsy; Disease Models, Animal; Erythropoietin; Female; Hypothermia; Hypoxia-Ischemia, Brain; Macaca nemestrina; Male; Metabolome; Umbilical Cord

Neuroprotection of erythropoietin (EPO) following long-term administration is hampered by the associated undesirable effects on hematopoiesis and body weight. For this reason, we tested carbamylated-EPO (CEPO), which has no effect on erythropoiesis, and compared it with EPO in the AβPP/PS1 mouse model of familial Alzheimer’s disease. Groups of 5-month old wild type (WT) and transgenic mice received chronic treatment consisting of CEPO (2,500 or 5,000 UI/kg) or EPO (2,500 U I/kg) 3 days/week for 4 weeks. Memory at the end of treatment was assessed with the object recognition test. Microarray analysis and quantitative-PCR were used for gene expression studies. No alterations in erythropoiesis were observed in CEPO-treated WT and AβPP/PS1 transgenic mice. EPO and CEPO improved memory in AβPP/PS1 animals. However, only EPO decreased amyloid-β (Aβ)plaque burden and soluble Aβ(40). Microarray analysis of gene expression revealed a limited number of common genes modulated by EPO and CEPO. CEPO but not EPO significantly increased gene expression of dopamine receptors 1 and 2, and adenosine receptor 2a, and significantly down-regulated adrenergic receptor 1D and gastrin releasing peptide. CEPO treatment resulted in higher protein levels of dopamine receptors 1 and 2 in WT and AβPP/PS1 animals, whereas the adenosine receptor 2a was reduced in WT animals. The present results suggest that the improved behavior observed in AβPP/PS1 transgenic mice after CEPO treatment may be mediated, at least in part, by the observed modulation of the expression of molecules involved in neurotransmission.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Body Weight; Disease Models, Animal; Erythropoietin; Gastrin-Releasing Peptide; Gene Expression Regulation; Humans; Male; Memory Disorders; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Peptide Fragments; Presenilin-1; Receptors, Catecholamine; Synapses; Time Factors

We have previously shown that glutathione peroxidase (GPx) overexpressing mice (hGPx-tg) have reduced brain injury after neonatal hypoxia-ischemia (HI) as a consequence of reduced hydrogen peroxide accumulation. However, this protection is reversed with hypoxia preconditioning, raising the question of the roles of the genes regulated by hypoxia-inducible factor-1α (HIF-1α) and their transcription products, such as erythropoietin (EPO), in both the initial protection and subsequent reversal of protection. hGPx-tg and their wild-type (WT) littermates underwent the Vannucci procedure of HI brain injury at postnatal day 9 - left carotid artery ligation followed by exposure to 10% oxygen for 50 min. Brain cortices and hippocampi were subsequently collected 0.5, 4 and 24 h later for the determination of protein expression by Western blot for GPx, HIF-1α, HIF-2α, EPO, EPO receptor, ERK1/2, phospho-ERK1/2, spectrin 145/150 (as a marker of calpain-specific necrotic cell death), and spectrin 120 (as a marker of apoptotic cell death mediated via caspase-3). As expected, the GPx overexpressing mouse cortex had approximately 3 times the GPx expression as WT naïve. Also, GPx expression remained higher in the GPx overexpressing brain than WT at all time points after HI (0.5, 4, 24 h). HIF-1α was not significantly changed in hGPx-tg as a consequence of HI but decreased in the WT cortex 4 h after HI. HIF-2α decreased in the WT hippocampus after HI. EPO was higher in the GPx overexpressing cortex and hippocampus 30 min after HI compared to WT, but the EPO receptor was unchanged by HI. ERK1/2 phosphorylation increased in the hippocampus at 4 h after HI and in the cortex at 24 h after HI in both WT and hGPx-tg. Spectrin 145/150 was increased in the WT cortex at 4 and 24 h after HI, and spectrin 120 increased 24 h after HI, perhaps reflecting greater injury in the WT brain, especially at 24 h when brain injury is more evident. The effect of GPx overexpression does not appear to upregulate the HIF pathway, yet EPO was upregulated, perhaps via ERK. This might explain, in part, why cell death takes a necrotic or apoptotic path. This may also be an explanation for why the GPx overexpressing brain cannot be preconditioned. This information may prove valuable in the development of therapies for neonatal HI brain injury.

Topics: Animals; Animals, Newborn; Basic Helix-Loop-Helix Transcription Factors; Cerebral Cortex; Disease Models, Animal; Erythropoietin; Glutathione Peroxidase; Hippocampus; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Ischemia, Brain; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Signal Transduction; Up-Regulation

We tested the hypothesis that endogenous brain Epo is a respiratory stimulant. Adult (3 mo) and newborn (10 days) male and female mice received an intracisternal (cisterna magna) injection of soluble Epo receptor (sEpoR; competes with EpoR to bind Epo; 50 μg/ml) or vehicle (0.1% BSA in PBS). Twenty-four hours after injection, we used whole body plethysmography to record minute ventilation (V̇e) tidal volume (VT), respiratory frequency (fR), O2 consumption (V̇o2), and CO2 production (V̇co2) under normoxia and progressive exposure to hypoxia (12-10-6% O2; 10 min each). In adult male and female mice sEpoR decreased normoxic V̇e (-25%), due to a decrease of VT in males and fR in females. Moreover, sEpoR injection decreased the ventilatory response to 12% O2, assessed as V̇e/V̇o2 or V̇e/V̇co2, in male but not in female mice. In newborn male and female mice sEpoR decreased V̇e (-37% in males, -59% in females) and VT (-38% in males, -47% in females) in normoxia and fR in females. During hypoxia, sEpoR decreased V̇e/V̇o2 and V̇e/V̇co2 in mice of both sexes. Upon extreme hypoxia (6% O2), the newborn mice treated with sEpoR showed respiratory depression, signs of asphyxia (gasping) and a high mortality rate in males and females. We concluded that endogenous brain Epo is a potent respiratory stimulant under normoxia and hypoxia in adult and newborn mice. Because sex-specific effects are different in newborn male and female, sex steroids secreted at different ages mice appear to modulate the effects of Epo on respiratory regulation in normoxia and in response to hypoxia.

Topics: Adaptation, Physiological; Age Factors; Animals; Animals, Newborn; Brain; Carbon Dioxide; Disease Models, Animal; Erythropoietin; Female; Hypoxia; Injections, Intraventricular; Male; Mice, Inbred C57BL; Oxygen Consumption; Pulmonary Ventilation; Receptors, Erythropoietin; Respiration; Respiratory Rate; Sex Factors; Signal Transduction; Tidal Volume; Time Factors

The prevalence of inflammatory bowel disease (IBD) is increasing. Since patients usually need long-term treatment and suffer from reduced quality of life, there is a need to develop new therapeutic strategy. The aim of this study was to investigate the therapeutic potential of erythropoietin (EPO) for the treatment of IBD.. Murine colitis was induced by 3.0% Dextran Sulfate Sodium (DSS). Recombinant human EPO (rhEPO) was given to evaluate the anti-inflammatory and regenerative effects on intestinal inflammation. The effect of rhEPO on human colon epithelial cells was also evaluated. Immunohistochemical analysis of EPO receptor was performed in human IBD tissues.. While about 62% of control mice with severe colitis induced by 5-day DSS died, 85% of mice treated with rhEPO survived. Histological analysis confirmed that EPO treatment reduced the colonic inflammation. Furthermore, EPO treatment significantly downregulated the local expressions of IFN-γ, TNF-α and E-selectin in the colon, suggesting that the effect was associated with inhibiting local immune activation. In a 4-day DSS-induced colitis model, rhEPO significantly improved the recovery of body weight loss compared to controls. Furthermore, proliferating cell nuclear antigen expression was significantly upregulated in the colon tissue from mice treated with rhEPO compared to controls. In addition, rhEPO increased the growth of cultured human colon epithelial cells in a dose-dependent manner. Furthermore, EPO-receptor expression was confirmed in human IBD colon tissues.. Three major functions of EPO, hematopoiesis, anti-inflammation and regeneration, may produce significant effects on intestinal inflammation, therefore suggesting that rhEPO might be useful for IBD.

Topics: Animals; Cell Proliferation; Colitis; Colon; Cytokines; Dextran Sulfate; Disease Models, Animal; Erythropoietin; Humans; Inflammatory Bowel Diseases; Intestinal Mucosa; Male; Mice; Mice, Inbred BALB C; Regeneration

Anemia is a common complication of chronic kidney disease (CKD) that develops early and its severity increases as renal function declines. It is mainly due to a reduced production of erythropoietin (EPO) by the kidneys; however, there are evidences that iron metabolism disturbances increase as CKD progresses. Our aim was to study the mechanisms underlying the development of anemia of CKD, as well as renal damage, in the remnant kidney rat model of CKD induced by 5/6 nephrectomy. This model of CKD presented a sustained degree of renal dysfunction, with mild and advanced glomerular and tubulointerstitial lesions. Anemia developed 3 weeks after nephrectomy and persisted throughout the protocol. The remnant kidney was still able to produce EPO and the liver showed an increased EPO gene expression. In spite of the increased EPO blood levels, anemia persisted and was linked to low serum iron and transferrin levels, while serum interleukin (IL)-6 and high sensitivity C-reactive protein (hs-CRP) levels showed the absence of systemic inflammation. The increased expression of duodenal ferroportin favours iron absorption; however, serum iron is reduced which might be due to iron leakage through advanced kidney lesions, as showed by tubular iron accumulation. Our data suggest that the persistence of anemia may result from disturbances in iron metabolism and by an altered activity/function of EPO as a result of kidney cell damage and a local inflammatory milieu, as showed by the increased gene expression of different inflammatory proteins in the remnant kidney. In addition, this anemia and the associated kidney hypoxia favour the development of fibrosis, angiogenesis and inflammation that may underlie a resistance to EPO stimuli and reduced iron availability. These findings might contribute to open new windows to identify putative therapeutic targets for this condition, as well as for recombinant human EPO (rHuEPO) resistance, which occurs in a considerable percentage of CKD patients.

Topics: Anemia; Animals; Blood Pressure; Body Weight; Disease Models, Animal; Erythropoietin; Fibrosis; Hepcidins; Inflammation; Iron; Kidney; Liver; Male; Organ Size; Rats; Rats, Wistar; Receptors, Erythropoietin; RNA, Messenger

Many acute and chronic anaemias, including haemolysis, sepsis and genetic bone marrow failure diseases such as Diamond-Blackfan anaemia, are not treatable with erythropoietin (Epo), because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production. Treatment of these anaemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently, we showed that glucocorticoids specifically stimulate self-renewal of an early erythroid progenitor, burst-forming unit erythroid (BFU-E), and increase the production of terminally differentiated erythroid cells. Here we show that activation of the peroxisome proliferator-activated receptor α (PPAR-α) by the PPAR-α agonists GW7647 and fenofibrate synergizes with the glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures of both mouse fetal liver BFU-Es and mobilized human adult CD34(+) peripheral blood progenitors, with a new and effective culture system being used for the human cells that generates normal enucleated reticulocytes. Although Ppara(-/-) mice show no haematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis, PPAR-α agonists facilitate recovery of wild-type but not Ppara(-/-) mice from PHZ-induced acute haemolytic anaemia. We also show that PPAR-α alleviates anaemia in a mouse model of chronic anaemia. Finally, both in control and corticosteroid-treated BFU-E cells, PPAR-α co-occupies many chromatin sites with GR; when activated by PPAR-α agonists, additional PPAR-α is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPAR-α agonists in stimulating self-renewal of early erythroid progenitor cells suggests that the clinically tested PPAR-α agonists we used may improve the efficacy of corticosteroids in treating Epo-resistant anaemias.

Topics: Acute Disease; Anemia; Anemia, Hemolytic; Animals; Butyrates; Cell Culture Techniques; Cells, Cultured; Chromatin; Chronic Disease; Disease Models, Animal; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Female; Fenofibrate; Glucocorticoids; Humans; Liver; Mice; Phenylhydrazines; Phenylurea Compounds; PPAR alpha; Receptors, Glucocorticoid; Signal Transduction

Mesenchymal stem cells (MSCs) play a central role in the remediation of cell and tissue damage. Erythropoietin (EPO) may enhance the beneficial influence of MSCs during recovery from tissue and organ injuries. Microvesicles (MVs) released from MSCs contribute to the restoration of kidney damage. We studied the influence of EPO on MVs derived from MSCs, and the protective effects of these factors in subjects with chronic kidney disease (CKD).. The MVs derived from untreated MSCs (MSC-MVs) or from MSCs incubated in different concentrations of EPO (1, 10, 100, and 500 IU/ml EPO-MVs) were used to treat renal injury of unilateral ureteral obstruction (UUO) in vivo, and transforming growth factor-β1 (TGF-β1)-induced fibrosis in a human renal proximal tubular epithelial (HK2) cell line in vitro. Western blot and reverse transcription polymerase chain reaction (RT-PCR) analyses were used to evaluate the expression of epithelial and mesenchymal markers in the renal tissue and HK2 cells. Flow cytometry was used to assess apoptosis within the HK2 cells, and microRNA (miRNA) microarray assays were used to determine the expression profiles of miRNA in the MSC-MVs and EPO-MVs.. Compared to MSC-MVs (untreated), there was a significant increase in the number of EPO-MVs derived from MSCs treated with 1-100 IU/ml EPO, and these EPO-MVs had a greater benefit in UUO mice on days 7 and 14. Moreover, the EPO-MVs had a better restorative effect following TGF-β1-induced fibrosis in HK2 cells at 24 h and 48 h. The flow cytometry results revealed that both types of MVs, especially EPO-MVs, play an important anti-apoptotic role in HK2 cells treated with TGF-β1. The miRNA profiles of the MVs revealed that EPO-MVs changed 212 miRNAs (fold-change ≥ 1.5), including miR-299, miR-499, miR-302, and miRNA-200, and that 70.28 % of these changes involved upregulation. The changed miRNA in EPO-MVs may have contributed to their enhanced protective effects following renal injury compared to MSC-MVs.. There was a dose-dependent increase in the level of EPO-MVs within the range of 1-100 IU/ml EPO. Although both MSC-MVs and EPO-MVs protect the kidney from fibrosis-related damage, there is a superior effect of EPO-MVs.

Topics: Actins; Animals; Apoptosis; Bone Marrow Cells; Cadherins; Cell-Derived Microparticles; Cells, Cultured; Disease Models, Animal; Erythropoietin; Fibrosis; Humans; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Renal Insufficiency, Chronic; Transcriptome; Transforming Growth Factor beta1; Ureteral Obstruction

Recombinant human erythropoietin (rhuEpo) is currently under debate for the treatment of chemotherapy-induced anemia due to clinical trials showing adverse effects in Epo-treated patients and the discovery of the erythropoietin-receptor (EpoR) in tumor and endothelial cells. Here, using Epo-Cy5.5 as theranostic near-infrared fluorescent probe we analyzed the effects of rhuEpo as co-medication to carboplatin in non-small-cell-lung-cancer (NSCLC)-xenografts with different tumor cell EpoR-expression (H838 ~8-fold higher than A549). Nude mice bearing subcutaneous A549 and H838 NSCLC-xenografts received either only carboplatin or carboplatin and co-medication of rhuEpo in two different doses. Tumor sizes and relative blood volumes (rBV) were longitudinally measured by 3D-contrast-enhanced ultrasound (3D-US). Tumoral EpoR-levels were determined by combined fluorescence molecular tomography (FMT)/ micro computed tomography (µCT) hybrid imaging. We found that rhuEpo predominantly acted on the tumor endothelium. In both xenografts, rhuEpo co-medication significantly increased vessel densities, diameters and the amount of perfused vessels. Accordingly, rhuEpo induced EpoR-phoshorylation and stimulated proliferation of endothelial cells. However, compared with solely carboplatin-treated tumors, tumor growth was significantly slower in the groups co-medicated with rhuEpo. This is explained by the Epo-mediated vascular remodeling leading to improved drug delivery as obvious by a more than 2-fold higher carboplatin accumulation and significantly enhanced tumor apoptosis. In addition, co-medication of rhuEpo reduced tumor hypoxia and diminished intratumoral EpoR-levels which continuously increased during carboplatin (Cp) -treatment. These findings suggest that co-medication of rhuEpo in well balanced doses can be used to improve the accumulation of anticancer drugs. Doses and indications may be personalized and refined using theranostic EpoR-probes.

Topics: Animals; Antineoplastic Agents; Carboplatin; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Disease Models, Animal; Erythropoietin; Female; Heterografts; Humans; Mice, Nude; Neovascularization, Pathologic; Perfusion; Therapeutic Uses

Erythropoietin (EPO), a regulator of red blood cell production, is endogenously expressed in the central nervous system. It is mainly produced by astrocytes under hypoxic conditions and has proven to have neuroprotective and neurotrophic effects. In the present study, we investigated the effect of midazolam on EPO expression in primary cultured astrocytes and the mouse brain. Midazolam was administered to 6-week-old BALB/c male mice under hypoxic conditions and pregnant C57BL/6N mice under normoxic conditions. Primary cultured astrocytes were also treated with midazolam under hypoxic conditions. The expression of EPO mRNA in mice brains and cultured astrocytes was studied. In addition, the expression of hypoxia-inducible factor (HIF), known as the main regulator of EPO, was evaluated. Midazolam significantly reduced the hypoxia-induced up-regulation of EPO in BALB/c mice brains and primary cultured astrocytes and suppressed EPO expression in the fetal brain. Midazolam did not affect the total amount of HIF proteins but significantly inhibited the nuclear expression of HIF-1α and HIF-2α proteins. These results demonstrated the suppressive effects of midazolam on the hypoxia-induced up-regulation of EPO both in vivo and in vitro.

Topics: Animals; Astrocytes; Basic Helix-Loop-Helix Transcription Factors; Brain; Cells, Cultured; Disease Models, Animal; Erythropoietin; Female; Fetal Hypoxia; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice, Inbred BALB C; Midazolam; Pregnancy; RNA, Messenger; Up-Regulation

Erythropoietin, Epo, is a 30.4 kDa glycoprotein hormone produced primarily by the fetal liver and the adult kidney. Epo exerts its haematopoietic effects by stimulating the proliferation and differentiation of erythrocytes with subsequent improved tissue oxygenation. Epo receptors are furthermore expressed in non-haematopoietic tissue and today, Epo is recognised as a cytokine with many pleiotropic effects. We hypothesize that hydrodynamic gene therapy with Epo can restore haemoglobin levels in anaemic transgenic mice and that this will attenuate the extracellular matrix accumulation in the kidneys. The experiment is conducted by hydrodynamic gene transfer of a plasmid encoding murine Epo in a transgenic mouse model that overexpresses TGF-β1 locally in the kidneys. This model develops anaemia due to chronic kidney disease characterised by thickening of the glomerular basement membrane, deposition of mesangial matrix and mild interstitial fibrosis. A group of age matched wildtype littermates are treated accordingly. After a single hydrodynamic administration of plasmid DNA containing murine EPO gene, sustained high haemoglobin levels are observed in both transgenic and wildtype mice from 7.5 ± 0.6 mmol/L to 9.4 ± 1.2 mmol/L and 10.7 ± 0.3 mmol/L to 15.5 ± 0.5 mmol/L, respectively. We did not observe any effects in the thickness of glomerular or tubular basement membrane, on the expression of different collagen types in the kidneys or in kidney function after prolonged treatment with Epo. Thus, Epo treatment in this model of chronic kidney disease normalises haemoglobin levels but has no effect on kidney fibrosis or function.

Topics: Anemia; Animals; Collagen; Disease Models, Animal; Disease Progression; Erythropoietin; Fibronectins; Genetic Therapy; Glomerular Basement Membrane; Hemoglobins; Kidney; Liver; Male; Mice; Mice, Transgenic; Plasmids; Renal Insufficiency, Chronic; Transforming Growth Factor beta1

Although ingenol 3,20-dibenzoate (IDB) is known as a selective novel protein kinase C (PKC) agonist, its biologic actions and underlying mechanisms remain incompletely understood. In this study, we identified IDB as a proliferative agent for an erythropoietin (EPO)-dependent cell line, UT-7/EPO, through the screening of a natural compound library. To clarify the underlying mechanism of IDB's EPO-like activities, we thoroughly analyzed the mutual relation between EPO and IDB in terms of in vitro and in vivo activities, signaling molecules, and a cellular receptor. IDB substantially induced the proliferation of UT-7/EPO cells, but not as much as EPO. IDB also lessened the anemia induced by 5-fluorouracil in an in vivo mouse model. Interestingly, IDB showed a synergistic effect on EPO at low concentration, but an antagonistic effect at higher concentration. Physical interaction and activation of PKCs by IDB- and EPO-competitive binding of IDB to EPO receptor (EPOR) explain these synergistic and antagonistic activities, respectively. Importantly, we addressed IDB's mechanism of action by demonstrating the direct binding of IDB to PKCs, and by identifying EPOR as a novel molecular target of IDB. Based on these dual targeting properties, IDB holds promise as a new small molecule modulator of EPO-related pathologic conditions.

Topics: Anemia; Animals; Cell Line; Cell Proliferation; Disease Models, Animal; Diterpenes; Dose-Response Relationship, Drug; Drug Antagonism; Drug Synergism; Erythropoietin; Humans; Mice, Inbred C57BL; Mutation; Protein Kinase C; Receptors, Erythropoietin; Signal Transduction

Amyloid beta (Aβ) is a key molecule in the neurodegenerative progression of Alzheimer׳s disease (AD). It is critical to develop a treatment that can arrest the Aβ-induced pathologic progression of AD. Erythropoietin (EPO) has various protective effects in the nervous system. However, the effect of EPO on Aβ-induced Alzheimer-like cognitive deficits and pathological changes remains unclear. In the present study, we observed that the treatment of mice with EPO (1000 IU/kg) attenuated Aβ42-induced cognitive deficits and tau hyperphosphorylation at multiple AD-related sites through the regulation of glycogen synthase kinase-3β (GSK-3β). We also observed that EPO attenuated the Aβ42-induced mitochondrial dysfunction and apoptosis in brain. These results indicate a potential role for EPO in AD therapy.

Topics: Amyloid beta-Peptides; Animals; Avoidance Learning; Disease Models, Animal; Electron Transport Complex I; Erythropoietin; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; Male; Malondialdehyde; Maze Learning; Membrane Potential, Mitochondrial; Memory Disorders; Mice; Mice, Inbred C57BL; Oxidative Stress; Peptide Fragments; Phosphorylation; Superoxide Dismutase; tau Proteins

Erythropoietin (EPO), regulating erythropoiesis, is used to provide protective and regenerative activity in non-haematopoietic tissues. There is insufficient knowledge about the role of EPO activity in tendon healing. Therefore, we investigated the effect of EPO treatment on healing in rat patellar tendons.. One hundred and twenty-six, four-month-old male Sprague-Dawley rats were randomly assigned to three experimental groups: 1, no treatment; 2, treatment with isotonic saline (NaCl) and 3, treatment with EPO. Each group was randomly subdivided into two groups for sacrifice at three (1a, 2a, 3a) or six weeks (1b, 2b, 3b). Complete incision of the left patellar tendon from the distal patellar pole was performed. We applied body casts for 20 days after the incised edges of the patellar tendon were brought together with a surgical technique. Both legs were harvested and specimens from each group underwent histological, biomechanical, and protein mRNA expression analyses.. There were statistically significant differences in the ultimate breaking force between the EPO group and others at both weeks three and six (p<0.05); significant differences in fibroblast proliferation, capillary vessel formation, and local inflammation were found between groups 1a and 3a, and 2a and 3a (p<0.05). There were statistical differences between 1a, 3a and 2a, 3a for Col III, TGF-β1, and VEGF and between 1b, 3b and 2b, 3b for Col I, Col III, TGF-β1, and VEGF mRNA expressions.. EPO had an additive effect with surgery on the injured tendon healing process in rats compared to the control groups biomechanically, histopathologically and with tissue protein mRNA expression.. This is the first experimental study to analyze the relationship between EPO treatment and the patellar tendon repair process by biomechanical, histopathological, and tendon tissue mRNA expression methodologies.

Topics: Animals; Disease Models, Animal; Erythropoietin; Male; Patellar Ligament; Plastic Surgery Procedures; Rats; Rats, Sprague-Dawley; Tendon Injuries; Wound Healing

In contrast to considerable data demonstrating a decrease in cytochrome P450 (CYP) activity in inflammation and infection, clinically, traumatic brain injury (TBI) results in an increase in CYP and UDP glucuronosyltransferase (UGT) activity. The objective of this study was to determine the effects of TBI alone and with treatment with erythropoietin (EPO) or anakinra on the gene expression of hepatic inflammatory proteins, drug-metabolizing enzymes, and transporters in a cortical contusion impact (CCI) injury model. Microarray-based transcriptional profiling was used to determine the effect on gene expression at 24 h, 72 h, and 7 days post-CCI. Plasma cytokine and liver protein concentrations of CYP2D4, CYP3A1, EPHX1, and UGT2B7 were determined. There was no effect of TBI, TBI + EPO, or TBI + anakinra on gene expression of the inflammatory factors shown to be associated with decreased expression of hepatic metabolic enzymes in models of infection and inflammation. IL-6 plasma concentrations were increased in TBI animals and decreased with EPO and anakinra treatment. There was no significant effect of TBI and/or anakinra on gene expression of enzymes or transporters known to be involved in drug disposition. TBI + EPO treatment decreased the gene expression of Cyp2d4 at 72 h with a corresponding decrease in CYP2D4 protein at 72 h and 7 days. CYP3A1 protein was decreased at 24 h. In conclusion, EPO treatment may result in a significant decrease in the metabolism of Cyp-metabolized drugs. In contrast to clinical TBI, there was not a significant effect of experimental TBI on CYP or UGT metabolic enzymes.

Topics: Animals; Brain Injuries; Cytochrome P-450 Enzyme System; Cytokines; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Glucuronosyltransferase; Inflammation; Interleukin 1 Receptor Antagonist Protein; Interleukin-6; Liver; Male; Membrane Transport Proteins; Rats; Rats, Sprague-Dawley; Time Factors

Previous studies have implicated erythropoietin (EPO) signaling in the regulation of glucose metabolism. Whether EPO can be used treat diabetes and the underlying mechanism remain to be elucidated. The present study aimed to investigate whether EPO affects glucose metabolism, and the underlying mechanisms, in experimental diabetic rats. The effects of EPO (300 U/kg three times a week for 4 weeks) on glucose metabolism, hematopoietic function, blood selenium content and the ultrastructure of pancreatic β‑cells were investigated in low dose (25 mg/kg body weight) streptozotocin‑induced experimental diabetic rats provided with a high‑fat diet. The results demonstrated that EPO significantly decreased the fasting blood glucose, the area under the curve of the oral glucose tolerance and insulin tolerance tests and L‑alanine gluconeogenesis. Ultrastructural examination of the pancreatic islets revealed that EPO prevented the dysfunction of pancreatic β‑cells in experimental diabetic rats, ameliorated cytoplasmic vacuolation and fragmentation of mitochondria, and increased the number of secretory granules. EPO administration increased the activities of superoxide dismutase and glutathione peroxidase, and decreased the level of malondialdehyde. Additionally, EPO increased blood selenium in the diabetic rats and produced a hematopoietic effect. These results indicated that EPO modulated glucose metabolism and improved pancreatic β‑cells damage by increasing anti‑oxidation. The detailed mechanisms underlying these effects require further investigation.

Topics: Animals; Blood Glucose; Carbohydrate Metabolism; Diabetes Mellitus, Experimental; Disease Models, Animal; Erythropoietin; Fasting; Glucose; Glucose Tolerance Test; Glutathione Peroxidase; Hematopoiesis; Insulin; Insulin-Secreting Cells; Male; Malondialdehyde; Rats; Selenium; Superoxide Dismutase

Memory impairment can be progressive in neurodegenerative diseases, and physiological ageing or brain injury, mitochondrial dysfunction and oxidative stress are critical components of these issues. An early clinical study has demonstrated cognitive improvement during erythropoietin treatment in patients with chronic renal failure. As erythropoietin cannot freely cross the blood-brain barrier, we tested EH-201 (2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside, also known as TSG), a low MW inducer of erythropoietin, for its therapeutic effects on memory impairment in models of neurodegenerative diseases, physiological ageing or brain injury.. The effects of EH-201 were investigated in astrocytes and PC12 neuronal-like cells. In vivo, we used sleep-deprived (SD) mice as a stress model, amyloid-β (Aβ)-injected mice as a physiological ageing model and kainic acid (KA)-injected mice as a brain damage model to assess the therapeutic effects of EH-201.. EH-201 induced expression of erythropoietin, PPAR-γ coactivator 1α (PGC-1α) and haemoglobin in astrocytes and PC12 neuronal-like cells. In vivo, EH-201 treatment restored memory impairment, as assessed by the passive avoidance test, in SD, Aβ and KA mouse models. In the hippocampus of mice given EH-201 in their diet, levels of erythropoietin, PGC-1α and haemoglobin were increased. The induction of endogenous erythropoietin in neuronal cells by inducers such as EH-201 might be a therapeutic strategy for memory impairment in neurodegenerative disease, physiological ageing or traumatic brain injury.

Topics: Animals; Astrocytes; Cells, Cultured; Disease Models, Animal; Erythropoietin; Female; Glucosides; Hemoglobins; Hydrogen Peroxide; Kainic Acid; Male; Memory Disorders; Mice, Inbred C57BL; Mitochondria; Neuroprotective Agents; PC12 Cells; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Rats; Reactive Oxygen Species; Stilbenes; Succinate Dehydrogenase; Transcription Factors

Metformin is a widely‑used antidiabetic drug with hypoglycemic activity and previously described anti‑inflammatory properties. Previous studies have demonstrated that metformin attenuates endotoxic hepatitis, however the mechanisms remain unclear. Inflammation and coagulation are closely associated pathological processes, therefore the potential effects of metformin on key steps in activation of the coagulation system were further investigated in endotoxic hepatitis induced by lipopolysaccharide/D‑galactosamine (LPS/D‑Gal). The current study demonstrated that treatment with metformin significantly suppressed the upregulation of tissue factor and plasminogen activator inhibitor‑1 in LPS/D‑Gal‑exposed mice. In addition, a reduction in the expression of interleukin 6 and inhibition of nuclear translocation of nuclear factor‑κB were observed. These data indicate that the LPS/D‑Gal‑induced elevation of the stable protein level of hypoxia inducible factor 1α, the mRNA level of erythropoietin, vascular endothelial growth factor and matrix metalloproteinase‑3, and the hepatic level of lactic acid were also suppressed by metformin. The current study indicates that the suppressive effects of metformin on inflammation‑induced coagulation may be an additional mechanism underlying the hepatoprotective effects of metformin in mice with LPS/D‑Gal‑induced fulminant hepatitis.

Topics: Animals; Anti-Inflammatory Agents; Blood Coagulation; Disease Models, Animal; Erythropoietin; Galactosamine; Hepatitis; Hypoglycemic Agents; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Interleukin-6; Lactic Acid; Lipopolysaccharides; Liver; Matrix Metalloproteinase 3; Metformin; Mice; NF-kappa B; Plasminogen Activator Inhibitor 1; RNA, Messenger; Thromboplastin; Up-Regulation; Vascular Endothelial Growth Factor A

In chronic kidney disease (CKD), anemia and hypertension are significant co-morbidities that contribute to cardiovascular and renal disease progression.. The purpose of the study was to identify correlations between changes in hematologic variables against markers of renal function, blood pressure, and erythropoietin (EPO) in a naturally occurring hypertensive model of CKD, the Lewis polycystic kidney (LPK) rat.. Complete blood count, systolic blood pressure, urea and creatinine concentration, urinary protein to creatinine ratio, and plasma EPO concentration were determined in control Lewis (n = 51) and LPK rats (n = 56) aged 6-24 weeks. Renal EPO gene expression and RBC osmotic fragility were also documented. Hematopoiesis in spleen and bone marrow were assessed.. Lewis polycystic kidney rats had increasing urea and creatinine concentrations, concurrent with the development of a nonregenerative normocytic/normochromic anemia and hypertension, with a significant negative correlation between both HGB and HCT with urea concentration and blood pressure (P < .01). HCT was also significantly negatively correlated with creatinine concentration (P = .014). WBC was significantly negatively correlated with urea (P < .01). Plasma EPO concentration was increased and renal EPO mRNA expression was significantly upregulated in LPK animals. The former was significantly positively correlated with blood pressure and platelet count (P < .05). RBC osmotic fragility was normal in LPK rats and there was no evidence for increased RBC elimination or extramedullary hematopoiesis.. Marked anemia in the LPK CKD rodent model in the presence of elevated EPO suggests inefficient erythropoiesis that is correlated with plasma urea concentration and blood pressure.

Topics: Anemia; Animals; Blood Cell Count; Blood Pressure; Blood Urea Nitrogen; Cohort Studies; Creatinine; Disease Models, Animal; Disease Progression; Erythropoietin; Female; Hypertension; Kidney; Kidney Function Tests; Male; Polycystic Kidney Diseases; Rats; Rats, Inbred Lew; Renal Insufficiency, Chronic

The aim of this study was to investigate the protective effects of erythropoietin, dextran/saline and erythropoietin in combination with dextran/saline on brain edema and lipid peroxidation following traumatic brain injury in rats.. In the study, 40 male 3-month-old albino Wistar rats, weighing 250-340 g, were divided into four groups, each consisting of ten rats. Traumatic brain injury was induced in all rats by the weight-drop method, and erythropoietin (5,000 U/kg) and/or dextran and saline (8 ml/kg) solutions were injected intraperitoneally ten minutes after trauma. Control animals received an equal volume of serum physiologic. All rats were sacrificed 24 hours later. Glutathione peroxidase activity and malondialdehyde levels were measured in the left hemisphere, and edema was quantitated by the wet-dry method.. Brain edema and the levels of malondialdehyde, the last product of lipid peroxidation in tissues, were decreased variably, and the activity of glutathione peroxidase, an antioxidant enzyme, was increased in others compared with the control group.. In this study, it was concluded that the brain edema that developed in rats on which head trauma was induced and the secondary brain damage caused by oxidative stress could be deceased using a combination of erythropoietin, dextran, and saline.

Topics: Animals; Brain Edema; Brain Injuries; Dextrans; Disease Models, Animal; Drug Combinations; Erythropoietin; Glutathione Peroxidase; Injections, Intraperitoneal; Lipid Peroxidation; Male; Malondialdehyde; Neuroprotective Agents; Rats; Rats, Wistar; Sodium Chloride

The rapid death of many spinal motor neurons after nerve root avulsion injury results in limited functional recovery following replantation surgery of avulsed nerves into the spinal cord. Therefore, we investigated the neuroprotective effect of erythropoietin (EPO) on motor neurons after nerve root avulsion injury using a rat model.. After C6 nerve root avulsion injury, EPO (2680 U/kg) was injected subcutaneously once a day for 3 consecutive days with various starting time points. At 28 and 56 days after injury, histological and immunohistological investigations were performed.. EPO-treated rats showed a significant increase in the number of surviving motor neurons at day 28 when the initial dose was started within 96 h after injury. In EPO-treated rats, superoxide formation in the motor neurons and proliferation of microglia were markedly suppressed in the acute phase. GAP-43-positive surviving motor neurons were significantly increased in EPO-treated rats at day 28. However, at 56 days after surgery, EPO-treated rats showed a much greater decrease of surviving motor neurons compared with those at day 28.. The neuroprotective effect of EPO is not long lasting, but may prolong the time before replantation surgery.

Topics: Acute Disease; Animals; Cell Survival; Cervical Vertebrae; Chronic Disease; Disease Models, Animal; Erythropoietin; Injections, Subcutaneous; Male; Microglia; Motor Neurons; Nerve Regeneration; Neuroprotective Agents; Radiculopathy; Random Allocation; Rats, Sprague-Dawley; Receptors, Erythropoietin; Spinal Cord; Superoxides; Treatment Outcome

Chronic kidney disease (CKD) patients have a poor prognosis due to cardiovascular disease. Anemia and endothelial dysfunction are important risk factors for cardiovascular events in CKD patients, and treatment with erythropoiesis-stimulating agent (ESA) has been reported to improve the quality of life in CKD patients. In this study, we evaluated the effect of anemia correcting dose of epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.) on endothelial function in 5/6 nephrectomized rats (Nx rats). C.E.R.A. was subcutaneously administered once a fortnight, 5 times in total, from 1 week after nephrectomy. Twenty-four hours after last administration, endothelial function was evaluated by measuring flow-mediated dilation (FMD) in the femoral arteries of anesthetized Nx rats by ultrasound system. Femoral arteries were harvested for western blot analysis. C.E.R.A. significantly increased FMD of Nx rats. Endothelium-independent vasodilation induced by nitroglycerin injection was not influenced by C.E.R.A treatment. Nox4 expression and nitrotyrosine accumulation were significantly decreased, and phosphorylation of eNOS was significantly enhanced in the femoral arteries of C.E.R.A.-treated rats. C.E.R.A. normalized hemoglobin levels but did not affect body weight, systolic blood pressure, heart rate, urinary protein excretion and plasma creatinine. These results indicate that C.E.R.A. prevented endothelial dysfunction in Nx rats, possibly through reduction of local oxidative stress and enhancement of eNOS phosphorylation in the arteries. This study provides the first evidence that C.E.R.A. prevented endothelial dysfunction in CKD model rats under conditions of amelioration of anemia.

Topics: Animals; Blood Pressure; Body Weight; Creatinine; Disease Models, Animal; Endothelium, Vascular; Erythropoietin; Femoral Artery; Heart Rate; Hemoglobins; Kidney Function Tests; Male; NADPH Oxidase 4; NADPH Oxidases; Nephrectomy; Nitric Oxide Synthase Type III; Nitroglycerin; Polyethylene Glycols; Proteinuria; Rats; Renal Insufficiency, Chronic; Tyrosine; Ultrasonography; Vasodilation

A previous study showed that a 1-h delay in treatment of thoracic spinal cord injury (SCI) with recombinant human erythropoietin (rhEPO) lacked neuroprotective efficacy. The aim of the present study was to reassess delayed administration of different doses of rhEPO on acute spinal cord compressive injury in rats.. The experiment was divided into first and second stages, which SCI rats were observed for 4 and 28 days, respectively. All rats were randomly divided into four groups at both stages: control group, and rhEPO-3,000U (Unit), rhEPO-4,000U and rhEPO-5,000U groups. SCI rats received rhEPO treatment at different time points. The primary indicators were locomotor recovery, histopathology, apoptotic index, inflammatory index, ultrastructural scoring system and volume of areas of demyelination.. The most significant locomotor functional and histopathological improvements and the best myelin protection were observed after administration of 5,000 U/kg rhEPO. rhEPO at 3,000, 4,000 and 5,000 U/kg showed similar ultrastructural neuroprotection, as well as similar inhibition of apoptosis and regulation of inflammation.. Delayed administration of rhEPO can reduce apoptosis and inflammation, and promote myelin repair and functional recovery following spinal cord compressive injury in rats.

Topics: Animals; Apoptosis; Behavior, Animal; Disease Models, Animal; Erythropoietin; Humans; Inflammation; Male; Motor Activity; Myelin Sheath; Random Allocation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Recovery of Function; Spinal Cord Compression; Spinal Cord Injuries; Time Factors

Traumatic brain injury (TBI) is one of the leading causes of disability and death which begins with the formation of edema as the persistent primary causative factor in TBI. Although medical management of cerebral edema by hypothermia, ventriculostomy, mannitol or hypertonic saline have been effective in treating edema, many of these therapies end up with some neurologic deficits, necessitating novel treatment options for treating post-TBI edema. This study investigated edema reducing effects of recombinant human Erythropoietin (rhEPO) in reducing acute brain edema in the CCI mouse model of TBI.. Anti-edema effects of rhEpo in reducing acute brain edema after injury in the CCI mouse model of TBI were assessed by T2 weighted magnetic resonance imaging (T2wMRI) as the accurate detector of brain edema in correlation with Western blot analysis of cerebral aquaporin 4 (AQP4) index as the critical marker of edema.. Results show that rhEpo treatment significantly reduced brain edema with concomitant reduction in AQP4 immunoexpression in the CCI mouse model of TBI.. Current results emphasize clinical utility of rhEpo in treating post-TBI edema.

Topics: Animals; Aquaporin 4; Blotting, Western; Brain; Brain Edema; Brain Injuries; Disease Models, Animal; Drug Evaluation, Preclinical; Erythropoietin; Humans; Imaging, Three-Dimensional; Injections, Intraperitoneal; Magnetic Resonance Imaging; Male; Mice, Inbred C57BL; Neuroprotective Agents; Recombinant Proteins; Treatment Outcome

Cerebral malaria (CM) is associated with excessive host proinflammatory responses and endothelial activation. The hematopoietic hormone erythropoietin (EPO) possesses neuroprotective functions in animal models of ischemic-hypoxic, traumatic, and inflammatory injuries. In the Plasmodium berghei ANKA model of experimental CM (ECM), recombinant human EPO (rhEPO) has shown evident protection against ECM. To elucidate the mechanism of EPO in this ECM model, we investigated the effect of rhEPO on host cellular immune responses. We demonstrated that improved survival of mice with ECM after rhEPO treatment was associated with reduced endothelial activation and improved integrity of the blood-brain barrier. Our results revealed that rhEPO downregulated the inflammatory responses by directly inhibiting the levels and functions of splenic dendritic cells. Conversely, rhEPO treatment led to significant expansion of regulatory T cells and increased expression of the receptor cytotoxic T lymphocyte antigen 4 (CTLA-4). The data presented here provide evidence of the direct effect of rhEPO on host cellular immunity during ECM.

Topics: Analysis of Variance; Animals; Blood-Brain Barrier; CTLA-4 Antigen; Cytokines; Dendritic Cells; Disease Models, Animal; Erythropoietin; Female; Immunity, Cellular; Malaria, Cerebral; Mice; Neuroprotective Agents; Plasmodium berghei; Recombinant Proteins; Spleen; T-Lymphocytes, Regulatory; Th1 Cells

Hypoxia inducible factor (HIF)-1α is the central transcriptional factor for the regulation of oxygen-associated genes in response to hypoxia. Erythropoietin (EPO), a hematopoietic growth factor, increases oxygen availability during hypoxia/ischemia and is associated with neuroprotection following hypoxia-ischemia in laboratory models of stroke. However, EPO has failed to translate in a clinical setting. Thus, it is critical to elucidate the key players in EPO-induced neuroprotection. Our preliminary studies have shown that EPO, as a downstream gene of HIF, inhibits HIF-1α in a dose-dependent manner in an in vitro model of hypoxia-ischemia. This study is designed to elucidate the primary mediator of EPO-induced HIF-1α inhibition and subsequent cell survival/neuroprotection. Oxygen and glucose deprivation (OGD) of nerve growth factor-differentiated rat pheochromocytoma (PC-12) cells were used to model hypoxia-ischemia in an in vitro environment. The profile of HIF-1α, HIF-2α and prolyl hydroxylase domain 2 (PHD-2) expression; HIF-1α and prolyl hydroxylase (PHD-2) mRNA levels; matrix metalloproteinase (MMP)-9; and cell death was evaluated in the presence and absence of either EPO or PHD-2 inhibitor during OGD. Our findings showed that EPO treatment resulted in an increase in PHD-2 transcription and translation, inhibition of HIF-1α expression, reactive oxygen species formation, and MMP-9 activity, resulting in increased cell survival after OGD. We also observed that EPO-induced cell survival/neuroprotection was reversed by siRNA silencing of PHD-2. This led to the conclusion that PHD-2 is a key mediator of EPO-induced HIF-1α inhibition and subsequent neuroprotection in an in vitro model of hypoxia-ischemia.

Topics: Animals; Disease Models, Animal; Erythropoietin; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Hypoxia-Ischemia, Brain; Neuroprotective Agents; PC12 Cells; Procollagen-Proline Dioxygenase; Rats; Reactive Oxygen Species; Transcription, Genetic; Up-Regulation

Erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF) are likely to play broad roles in the brain. We investigated the effects of combination therapy with EPO and G-CSF in hypoxic-ischemic brain injury during the acute, subacute, and chronic phases. A total of 79 C57BL/6 mice with hypoxic-ischemic brain injury were randomly assigned acute (days 1-5), subacute (days 11-15) and chronic (days 28-32) groups. All of them were treated with G-CSF (250 μg/kg) and EPO (5000 U/kg) or saline daily for 5 consecutive days. Behavioral assessments and immunohistochemistry for angiogenesis, neurogenesis, and astrogliosis were performed with an 8-week follow-up. Hypoxia-inducible factor-1 (HIF-1) was also measured by Western blot analysis. The results showed that the combination therapy with EPO and G-CSF in the acute phase significantly improved rotarod performance and forelimb-use symmetry compared to the other groups, while subacute EPO and G-CSF therapy exhibited a modest improvement compared with the chronic saline controls. The acute treatment significantly increased the density of CD31(+) (PECAM-1) and α-smooth muscle actin(+) vessels in the frontal cortex and striatum, increased BrdU(+)/PSA-NCAM(+) neurogenesis in the subventricular zone, and decreased astroglial density in the striatum. Furthermore, acute treatment significantly increased the HIF-1 expression in the cytosol and nucleus, whereas chronic treatment did not change the HIF-1 expression, consistent with the behavioral outcomes. These results indicate that the induction of HIF-1 expression by combination therapy with EPO and G-CSF synergistically enhances not only behavioral function but also neurogenesis and angiogenesis while decreasing the astroglial response in a time-dependent manner.

Topics: Animals; Brain; Disease Models, Animal; Drug Therapy, Combination; Erythropoietin; Granulocyte Colony-Stimulating Factor; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Ischemia, Brain; Male; Mice; Mice, Inbred C57BL; Motor Activity; Neurogenesis; Neuroprotective Agents

Ovarian tissue transplantation is performed to preserve fertility in patients undergoing chemotherapy and radiotherapy. However, ischemia/reperfusion (IR) injury and free radical production occurring during the revascularization of the transplanted tissue are the major limitations of this procedure. The aim of this study was to investigate the effect of erythropoietin (EPO) as an antioxidant on oxidative stress and ovary survival following transplantation. The Naval Medical Research Institute (NMRI) mice (4-5 weeks old) were divided into three groups (six mice per group): control; autograft+saline, and autograft+EPO (500  IU/kg i.p.). After 28 days, ovary compartments were estimated stereologically. DNA fragmentation and plasma malondialdehyde (MDA), progesterone, and estradiol (E2) concentrations were also evaluated. The results were analyzed using one-way ANOVA and Tukey's test, and the means were significantly different at P<0.05. The mean total volume of ovary, cortex, and medulla and the number of follicles increased significantly in the autograft+EPO group (P<0.01). Apoptosis rate in the autograft+EPO group was lower than that in the autograft+saline group. The concentration of MDA decreased significantly in the autografted EPO-treated group than in the autografted saline-treated group (P<0.01). The concentration of E2 increased significantly in the autograft+EPO group than in the autograft+saline group (P<0.01). EPO reduced IR injury, increasing follicle survival and function in grafted ovaries. Free Persian abstract A Persian (Farsi) translation of the abstract is freely available online at http://www.reproduction-online.org/content/147/5/733/suppl/DC1.

Topics: Animals; Apoptosis; Disease Models, Animal; DNA Fragmentation; Erythropoietin; Estradiol; Female; Ischemia; Malondialdehyde; Mice; Mice, Inbred Strains; Ovarian Follicle; Ovary; Oxidative Stress; Progesterone; Transplants

Renal ischemia reperfusion (IR) is an important cause of renal dysfunction. It contributes to the development of acute renal failure. Oxidative damage from reactive oxygen species is considered to be the principal component involved in the pathophysiological tissue alterations observed during IR. The purpose of this study was to evaluate the effect of a combined treatment with erythropoietin (EPO) plus melatonin (MEL), which are known anti-inflammatory and antioxidant agents, in IR-induced renal injury in rats.. Wistar Albino rats were unilaterally nephrectomized and subjected to 45 min of renal pedicle occlusion followed by 24 h of reperfusion. MEL (10 mg/kg, i.p) and EPO (5000 U/kg, i.p) were administered prior to ischemia. After 24 h of reperfusion, blood samples were collected for the determination of superoxide dismutase (SOD), glutathione peroxidase (GPx), plasma levels of total antioxidant capacity (TAC), and malondialdehyde (MDA) and serum urea level. Also, renal samples were taken for histological evaluation.. Ischemia reperfusion significantly increased urea, blood SOD, and GPx levels. Histological findings of the IR group indicated that there was increase in tubular and glomerular hyaline cast, thickening of Bowman capsule basement membrane, and renal impairment in the glomerular epithelium. Treatment with EPO and MEL significantly decreased blood SOD, GPx, and urea levels and increased TAC level. In the EPO + MEL group, while the histopathological changes were lower than those in EPO group, they were the same as MEL group.. EPO and MEL combination treatment exerted more nephroprotective effects than EPO treatment and nearly had protective effects similar to MEL treatment.

Topics: Acute Kidney Injury; Animals; Anti-Inflammatory Agents; Antioxidants; Disease Models, Animal; Drug Therapy, Combination; Erythropoietin; Glutathione Peroxidase; Kidney; Male; Malondialdehyde; Melatonin; Models, Animal; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase

Prolyl hydroxylase domain (PHD) proteins catalyze oxygen-dependent prolyl hydroxylation of hypoxia-inducible factor 1α and 2α, tagging them for pVHL-dependent polyubiquitination and proteasomal degradation. In this study, albumin Cre (Alb(Cre))-mediated, hepatocyte-specific triple disruption of Phd1, Phd2, and Phd3 (Phd(1/2/3)hKO) promoted liver erythropoietin (EPO) expression 1246-fold, whereas renal EPO was down-regulated to 6.7% of normal levels. In Phd(1/2/3)hKO mice, hematocrit levels reached 82.4%, accompanied by severe vascular malformation and steatosis in the liver. In mice double-deficient for hepatic PHD2 and PHD3 (Phd(2/3)hKO), liver EPO increase and renal EPO loss both occurred but were much less dramatic than in Phd(1/2/3)hKO mice. Hematocrit levels, vascular organization, and liver lipid contents all appeared normal in Phd(2/3)hKO mice. In a chronic renal failure model, Phd(2/3)hKO mice maintained normal hematocrit levels throughout the 8-week time course, whereas floxed controls developed severe anemia. Maintenance of normal hematocrit levels in Phd(2/3)hKO mice was accomplished by sensitized induction of liver EPO expression. Consistent with such a mechanism, liver HIF-2α accumulated to higher levels in Phd(2/3)hKO mice in response to conditions causing modest systemic hypoxia. Besides promoting erythropoiesis, EPO is also known to modulate retinal vascular integrity and neovascularization. In Phd(1/2/3)hKO mice, however, neonatal retinas remained sensitive to oxygen-induced retinopathy, suggesting that local EPO may be more important than hepatic and/or renal EPO in mediating protective effects in the retina.

Topics: Anemia; Animals; Blotting, Western; Disease Models, Animal; Erythropoietin; Kidney Failure, Chronic; Liver; Mice; Mice, Knockout; Prolyl Hydroxylases; Real-Time Polymerase Chain Reaction; Retinal Diseases

Acute kidney injury is common, serious with no specific treatment. Ischemia-reperfusion is a common cause of acute kidney injury (AKI). Clinical trials suggest that preoperative erythropoietin (EPO) or remote ischemic preconditioning may have a renoprotective effect. Using a porcine model of warm ischemia-reperfusion-induced AKI (40-min bilateral cross-clamping of renal arteries, 48-h reperfusion), we examined the renoprotective efficacy of EPO (1,000 iu/kg iv.) or remote ischemic preconditioning (3 cycles, 5-min inflation/deflation to 200 mmHg of a hindlimb sphygmomanometer cuff). Ischemia-reperfusion induced significant kidney injury at 24 and 48 h (χ(2), 1 degree of freedom, >10 for 6/7 histopathological features). At 2 h, a panel of biomarkers including plasma creatinine, neutrophil gelatinase-associated lipocalin, and IL-1β, and urinary albumin:creatinine could be used to predict histopathological injury. Ischemia-reperfusion increased cell proliferation and apoptosis in the renal cortex but, for pretreated groups, the apoptotic cells were predominantly intratubular rather than interstitial. At 48-h reperfusion, plasma IL-1β and the number of subcapsular cells in G2-M arrest were reduced after preoperative EPO, but not after remote ischemic preconditioning. These data suggest an intrarenal mechanism acting within cortical cells that may underpin a renoprotective function for preoperative EPO and, to a limited extent, remote ischemic preconditioning. Despite equivocal longer-term outcomes in clinical studies investigating EPO as a renoprotective agent in AKI, optimal clinical dosing and administration have not been established. Our data suggest further clinical studies on the potential renoprotective effect of EPO and remote ischemic preconditioning are justified.

Topics: Acute Kidney Injury; Animals; Creatinine; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Female; Hindlimb; In Situ Nick-End Labeling; Ischemic Preconditioning; Preoperative Care; Recombinant Proteins; Reperfusion Injury; Swine

Sepsis remains the most important cause of acute kidney injury (AKI) in critically ill patients and is an independent predictor of poor outcome. The administration of lipopolysaccharide (LPS) to animals reproduces most of the clinical features of sepsis, including AKI, a condition associated with renal cellular dysfunction and apoptosis. Erythropoietin (EPO) is a well known cytoprotective multifunctional hormone, which exerts anti-inflammatory, anti-oxidant, anti-apoptotic and angiogenic effects in several tissues. The aim of this study was to evaluate the underlying mechanisms of EPO renoprotection through the expression of the EPO receptor (EPO-R) and the modulation of the intrinsic apoptotic pathway in LPS-induced AKI. Male inbred Balb/c mice were divided in four experimental groups: Control, LPS (8 mg/kg i.p.), EPO (3000 IU sc) and LPS+EPO. Assessment of renal function, histological examination, TUNEL in situ assay, immunohistochemistry and Western blottings of caspase-3, Bax, Bcl-xL, EPO-R and Cytochrome c were performed at 24h post treatment. LPS+EPO treatment significantly improved renal function and ameliorated histopathological injury when compared to the LPS treated group. Results showed that EPO treatment attenuates renal tubular apoptosis through: (a) the overexpression of EPO-R in tubular interstitial cells, (b) the reduction of Bax/Bcl-xL ratio, (c) the inhibition Cytochrome c release into the cytosol and (d) the decrease of the active caspase-3 expression. This study suggests that EPO exerts renoprotection on an experimental model of LPS-induced AKI. EPO induced renoprotection involves an anti-apoptotic effect through the expression of EPO-R and the regulation of the mitochondrial apoptotic pathway.

Topics: Acute Kidney Injury; Animals; Apoptosis; Cryoprotective Agents; Disease Models, Animal; Erythropoietin; Humans; Kidney; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Mitochondria; Receptors, Erythropoietin

Erythropoietin (EPO) stimulates proliferation of early-stage erythrocyte precursors and is widely used for the treatment of chronic anemia. However, several types of EPO-resistant anemia are characterized by defects in late-stage erythropoiesis, which is EPO independent. Here we investigated regulation of erythropoiesis using a ligand-trapping fusion protein (ACE-536) containing the extracellular domain of human activin receptor type IIB (ActRIIB) modified to reduce activin binding. ACE-536, or its mouse version RAP-536, produced rapid and robust increases in erythrocyte numbers in multiple species under basal conditions and reduced or prevented anemia in murine models. Unlike EPO, RAP-536 promoted maturation of late-stage erythroid precursors in vivo. Cotreatment with ACE-536 and EPO produced a synergistic erythropoietic response. ACE-536 bound growth differentiation factor-11 (GDF11) and potently inhibited GDF11-mediated Smad2/3 signaling. GDF11 inhibited erythroid maturation in mice in vivo and ex vivo. Expression of GDF11 and ActRIIB in erythroid precursors decreased progressively with maturation, suggesting an inhibitory role for GDF11 in late-stage erythroid differentiation. RAP-536 treatment also reduced Smad2/3 activation, anemia, erythroid hyperplasia and ineffective erythropoiesis in a mouse model of myelodysplastic syndromes (MDS). These findings implicate transforming growth factor-β (TGF-β) superfamily signaling in erythroid maturation and identify ACE-536 as a new potential treatment for anemia, including that caused by ineffective erythropoiesis.

Topics: Activin Receptors, Type II; Anemia; Animals; Bone Morphogenetic Proteins; Disease Models, Animal; Drug Therapy, Combination; Erythrocyte Count; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Growth Differentiation Factors; Haplorhini; Hematinics; Humans; Ligands; Mice; Myelodysplastic Syndromes; Rats; Recombinant Fusion Proteins; Reticulocyte Count; Signal Transduction; Smad2 Protein; Smad3 Protein

Erythropoietin has demonstrated neuroprotective effects against traumatic spinal cord injury (SCI), but the underlying mechanisms remain unclear. The signaling pathway of an antioxidant transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), has been shown to play an important role in protecting SCI-induced secondary spinal cord damage. This study was undertaken to explore the effect of recombinant human erythropoietin (rhEPO) on the activation of Nrf2 signaling pathway and secondary spinal cord damage in rats after SCI.. Adult male Sprague-Dawley rats were subjected to laminectomy at T8-T9 and compression with a vascular clip. Three groups were analyzed: (1) sham group, (2) SCI group, and (3) SCI + rhEPO group (n = 16 per group). In the SCI + rhEPO group, rhEPO was administered at a dose of 5,000 IU/kg at 30 minutes after SCI. Spinal cord samples were extracted at 72 hours after the trauma.. As a result, we found that the treatment with rhEPO markedly up-regulated the messenger RNA expressions and activities of Nrf2 signaling pathway-related agents, including Nrf2, NAD(P)H:quinone oxidoreductase 1(NQO1), and glutathione S-transferase. The administration of rhEPO also significantly ameliorated the secondary spinal cord damage, as shown by a decreased severity of locomotion deficit, spinal cord edema, and apoptosis.. Post-SCI rhEPO administration induces Nrf2-mediated cytoprotective response in the injured spinal cord, and this may be a mechanism whereby rhEPO improves the outcome following SCI.

Topics: Animals; Apoptosis; Biomarkers; Disease Models, Animal; Erythropoietin; Glutathione Transferase; In Situ Nick-End Labeling; Locomotion; Male; Neuroprotective Agents; NF-E2-Related Factor 2; Random Allocation; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Signal Transduction; Spinal Cord Injuries

Huang-Lian-Jie-Du-Tang (HLJDT) is a classical heat-clearing and detoxicating formula of traditional Chinese medicine that is widely used to treat stroke. The present study was designed to investigate the effects of HLJDT preconditioning on neurons under oxygen and glucose deprivation (OGD) and rats subjected to middle cerebral artery occlusion (MCAO).. A stroke model of rats was obtained through MCAO. Following HLJDT preconditioning, the cerebral infarction volume, cerebral water content, and neurological deficient score were determined. Cerebral cortical neurons cultured in vitro were preconditioned with HLJDT and then subjected to OGD treatment. The release of lactate dehydrogenase (LDH) from neurons was detected. The levels of hypoxia-inducible factor-1α (HIF-1α) and PI3K/Akt signaling were analyzed by western blotting, and the levels of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in the supernatant of the neurons and the plasma of MCAO rats were measured through a radioimmunological assay. The apoptosis and proliferation of neurons were analyzed by immunohistochemistry.. HLJDT preconditioning significantly reduced the cerebral infarction volume and cerebral water content and ameliorated the neurological deficient score of MCAO rats. In addition, HLJDT preconditioning protected neurons against OGD. Increased HIF-1α, EPO, and VEGF levels and the activation of PI3K/Akt signaling were observed as a result of HLJDT preconditioning. Furthermore, HLJDT preconditioning was found to inhibit ischemia-induced neuron apoptosis and to promote neuron proliferation under conditions of ischemia/reperfusion.. Both rats and neurons subjected to HLJDT preconditioning were able to resist ischemia/reperfusion or hypoxia injury through the inhibition of apoptosis and the enhancement of proliferation, and these effects were primarily dependent on the activation of the PI3K/Akt signaling pathway and HIF-1α.

Topics: Animals; Brain Ischemia; Cerebral Cortex; Disease Models, Animal; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Erythropoietin; Ethnopharmacology; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemic Preconditioning; Male; Neuroprotective Agents; Rats, Sprague-Dawley; Vascular Endothelial Growth Factor A

Erythropoietin (EPO) has been recognized as a neuroprotective agent. In animal models of neonatal brain injury, exogenous EPO has been shown to reduce lesion size, improve structure and function. Experimental studies have focused on short course treatment after injury. Timing, dose and length of treatment in preterm brain damage remain to be defined. We have evaluated the effects of high dose and long-term EPO treatment in hypoxic-ischemic (HI) injury in 3 days old (P3) rat pups using histopathology, magnetic resonance imaging (MRI) and spectroscopy (MRS) as well as functional assessment with somatosensory-evoked potentials (SEP). After HI, rat pups were assessed by MRI for initial damage and were randomized to receive EPO or vehicle. At the end of treatment period (P25) the size of resulting cortical damage and white matter (WM) microstructure integrity were assessed by MRI and cortical metabolism by MRS. Whisker elicited SEP were recorded to evaluate somatosensory function. Brains were collected for neuropathological assessment. The EPO treated animals did not show significant decrease of the HI induced cortical loss at P25. WM microstructure measured by diffusion tensor imaging was improved and SEP response in the injured cortex was recovered in the EPO treated animals compared to vehicle treated animals. In addition, the metabolic profile was less altered in the EPO group. Long-term treatment with high dose EPO after HI injury in the very immature rat brain induced recovery of WM microstructure and connectivity as well as somatosensory cortical function despite no effects on volume of cortical damage. This indicates that long-term high-dose EPO induces recovery of structural and functional connectivity despite persisting gross anatomical cortical alteration resulting from HI.

Topics: Animals; Animals, Newborn; Astrocytes; Cerebral Cortex; Cicatrix; Diffusion Tensor Imaging; Disease Models, Animal; Erythropoietin; Evoked Potentials, Somatosensory; Female; Hypoxia-Ischemia, Brain; Intermediate Filaments; Male; Metabolome; Metabolomics; Myelin Sheath; Neurons; Neuroprotective Agents; Organ Size; Proton Magnetic Resonance Spectroscopy; Rats; Time Factors

Tissue hypoxia likely contributes to anemia-induced organ injury and mortality. Severe anemia activates hypoxia-inducible factor (HIF) signaling by hypoxic- and neuronal nitric oxide (NO) synthase- (nNOS) dependent mechanisms. However, organ-specific hemoglobin (Hb) thresholds for increased HIF expression have not been defined. To assess organ-specific Hb thresholds for tissue hypoxia, HIF-α (oxygen-dependent degradation domain, ODD) luciferase mice were hemodiluted to mild, moderate, or severe anemia corresponding to Hb levels of 90, 70, and 50 g/l, respectively. HIF luciferase reporter activity, HIF protein, and HIF-dependent RNA levels were assessed. In the brain, HIF-1α was paradoxically decreased at mild anemia, returned to baseline at moderate anemia, and then increased at severe anemia. Brain HIF-2α remained unchanged at all Hb levels. Both kidney HIF-1α and HIF-2α increased earlier (Hb ∼70-90 g/l) in response to anemia. Liver also exhibited an early HIF-α response. Carotid blood flow was increased early (Hb ∼70, g/l), but renal blood flow remained relatively constant, only increased at Hb of 50 g/l. Anemia increased nNOS (brain and kidney) and endothelia NOS (eNOS) (kidney) levels. Whereas anemia-induced increases in brain HIFα were nNOS-dependent, our current data demonstrate that increased renal HIFα was nNOS independent. HIF-dependent RNA levels increased linearly (∼10-fold) in the brain. However, renal HIF-RNA responses (MCT4, EPO) increased exponentially (∼100-fold). Plasma EPO levels increased near Hb threshold of 90 g/l, suggesting that the EPO response is sensitive. Collectively, these observations suggest that each organ expresses a different threshold for cellular HIF/NOS hypoxia responses. This knowledge may help define the mechanism(s) by which the brain and kidney maintain oxygen homeostasis during anemia.

Topics: Acute Disease; Anemia; Animals; Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Brain; Cerebrovascular Circulation; Disease Models, Animal; Erythropoietin; Hemodilution; Hemodynamics; Hemoglobins; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Liver; Luciferases, Firefly; Mice; Mice, Transgenic; Monocarboxylic Acid Transporters; Muscle Proteins; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Recombinant Fusion Proteins; Renal Circulation; Severity of Illness Index

Rehabilitation is the only treatment option for chronic stroke deficits, but unfortunately, it often provides incomplete recovery. In this study, a novel combination of growth factor administration and rehabilitation therapy was used to facilitate functional recovery in a rat model of cortical stroke.. Ischemia was induced via injection of endothelin-1 into the sensorimotor cortex. This was followed by either a 2-week infusion of epidermal growth factor and erythropoietin or artificial cerebrospinal fluid into the ipsilateral lateral ventricle. Two weeks after ischemia, animals began an 8-week enriched rehabilitation program. Functional recovery was assessed after ischemia using the Montoya staircase-reaching task, beam-traversing, and cylinder test of forelimb asymmetry.. The combination of growth factor infusion and rehabilitation led to a significant acceleration in recovery in the staircase task. When compared with controls, animals receiving the combination treatment attained significant recovery of function at 4 weeks after stroke, whereas those receiving rehabilitation alone did not recover until 10 weeks. Significant recovery was also observed on the beam-traversing and cylinder tasks.. Combining behavioral rehabilitation with growth factor infusion accelerates motor recovery. These data suggest a promising new avenue of combination therapies that may have the potential to reduce the rehabilitation time necessary to recover from sensorimotor deficits arising from stroke.

Topics: Animals; Chronic Disease; Disease Models, Animal; Endothelin-1; Epidermal Growth Factor; Erythropoietin; Male; Motor Activity; Rats; Rats, Sprague-Dawley; Stroke; Stroke Rehabilitation

Motoneuron diseases are fatal neurodegenerative disorders characterized by a progressive loss of motoneurons, muscle weakness and premature death. The progressive motor neuronopathy (pmn) mutant mouse has been considered a good model for the autosomal recessive childhood form of spinal muscular atrophy (SMA). Here, we investigated the therapeutic potential of Erythropoietin (Epo) on this mutant mouse. Symptomatic or pre-symptomatic treatment with Epo significantly prolongs lifespan by 84.6% or 87.2% respectively. Epo preserves muscle strength and significantly attenuates behavioural motor deficits of mutant pmn mice. Histological and metabolic changes in the spinal cord evaluated by immunohistochemistry, western blot, and high-resolution (1)H-NMR spectroscopy were also greatly prevented by Epo-treatment. Our results illustrate the efficacy of Epo in improving quality of life of mutant pmn mice and open novel therapeutic pathways for motoneuron diseases.

Topics: Animals; Disease Models, Animal; Erythropoietin; Mice; Mice, Mutant Strains; Motor Activity; Motor Neurons; Receptors, Erythropoietin; Spinal Cord; Spinal Muscular Atrophies of Childhood

Fulminant hepatic failure is a severe clinical syndrome associated with a high rate of patient mortality. Recent studies have shown that in addition to its hematopoietic effect, erythropoietin (EPO) has multiple protective effects and exhibits antiapoptotic, antioxidant and anti-inflammatory activities. The present study aimed to determine the hepatoprotective effect of EPO and to elucidate the underlying mechanisms using a D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced model of acute liver injury. Experimental groups of mice were administered with various doses of EPO (1,000, 3,000 or 10,000 U/kg, intraperitoneal) once per day for 3 days, prior to injection with D-GalN (700 mg/kg)/LPS (10 µg/kg). Mice were sacrificed 8 h after treatment with D‑GalN/LPS. Liver function and histopathology, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH‑Px) activities and EPO receptor (EPOR) and phosphatidylinositol 3-kinase (PI3K) mRNA expression were evaluated. D-GalN/LPS administration markedly induced liver injury, as evidenced by elevated levels of serum aminotransferases, as well as histopathological changes. Compared with the D-GalN/LPS group, pretreatment with EPO significantly decreased the levels of aspartate aminotransferase, alanine aminotransferase and MDA, and increased the activities of SOD and GSH-Px. Furthermore, the protective effects of EPO were paralleled by an upregulation in the mRNA expression of EPOR and PI3K. These data suggest that EPO can ameliorate D-GalN/LPS-induced acute liver injury by reducing oxidative stress and upregulating the mRNA expression of EPOR and PI3K.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Galactosamine; Glutathione Peroxidase; Lipopolysaccharides; Liver; Liver Failure, Acute; Malondialdehyde; Mice; Mice, Inbred BALB C; Phosphatidylinositol 3-Kinase; Protective Agents; Receptors, Erythropoietin; Recombinant Proteins; RNA, Messenger; Superoxide Dismutase

Glomerulonephritis (GN) develops via accumulation of extracellular matrix through macrophage recruitment in glomeruli. It is unclear whether epoetin beta pegol (continuous erythropoietin receptor activator, CERA), a long-acting erythropoiesis-stimulating agent, exerts a renoprotective effect by preventing glomerulosclerosis. We examined the renoprotective effect of CERA in rats with Thy-1 glomerulonephritis (Thy-1-GN), an animal model for mesangial proliferative glomerulonephritis.. Thy-1-GN was induced in F344 rats by injection of anti-Thy1.1 antibody. CERA (25 µg/kg) was intravenously administered 4 h before anti-Thy1.1 antibody injection. After 6 days, blood and urine was collected for biochemical analysis and kidneys harvested for analysis of histopathology and mRNA expression.. In Thy-1-GN rats, CERA suppressed increased urinary total protein, urea nitrogen, and N-acetyl-β-(D)-glucosaminidase. CERA significantly prevented glomerulosclerosis and expression of α-smooth muscle actin, collagen-1, and fibronectin. Increased macrophage infiltration and up-regulated monocyte chemotactic protein-1 were significantly suppressed by CERA. Furthermore, CERA also suppressed up-regulation of arginase-1, a marker of M2 macrophages. Arginase-1 expression levels strongly correlated with levels of collagen-1 and fibronectin mRNA.. These results suggest that CERA has potential to protect kidney function through the prevention of glomerulosclerosis, accompanied by prevention of M2 macrophage recruitment.

Topics: Acetylglucosaminidase; Actins; Animals; Arginase; Blood Urea Nitrogen; Cell Movement; Chemokine CCL2; Collagen Type I; Disease Models, Animal; Erythropoietin; Fibronectins; Glomerulonephritis; Glomerulosclerosis, Focal Segmental; Isoantibodies; Ki-67 Antigen; Macrophages; Male; Polyethylene Glycols; Proteinuria; Rats; Rats, Inbred F344; RNA, Messenger; Tumor Necrosis Factor-alpha

In addition to its role in the endogenous control of erythropoiesis, recombinant human erythropoietin (rh-EPO) has been shown to exert tissue protective properties in various experimental models. However, its role in the cardiac arrest (CA) setting has not yet been adequately investigated.. The aim of this study is to examine the effect of rh-EPO in a pig model of ventricular fibrillation (VF)-induced CA.. Ventricular fibrillation was electrically induced in 20 piglets and maintained untreated for 8 minutes before attempting resuscitation. Animals were randomized to receive rh-EPO (5000 IU/kg, erythropoietin [EPO] group, n = 10) immediately before the initiation of chest compressions or to receive 0.9% Sodium chloride solution instead (control group, n = 10).. Compared with the control, the EPO group had higher rates of return of spontaneous circulation (ROSC) (100% vs 60%, P = .011) and higher 48-hour survival (100% vs 40%, P = .001). Diastolic aortic pressure and coronary perfusion pressure during cardiopulmonary resuscitation were significantly higher in the EPO group compared with the control group. Erythropoietin-treated animals required fewer number of shocks in comparison with animals that received normal saline (P = .04). Furthermore, the neurologic alertness score was higher in the EPO group compared with that of the control group at 24 (P = .004) and 48 hours (P = .021).. Administration of rh-EPO in a pig model of VF-induced CA just before reperfusion facilitates ROSC and improves survival rates as well as hemodynamic variables.

Topics: Animals; Blood Circulation; Blood Pressure; Cardiopulmonary Resuscitation; Disease Models, Animal; Erythropoietin; Female; Heart Arrest; Swine; Treatment Outcome; Ventricular Fibrillation

Recovery from blood loss requires a greatly enhanced supply of iron to support expanded erythropoiesis. After hemorrhage, suppression of the iron-regulatory hormone hepcidin allows increased iron absorption and mobilization from stores. We identified a new hormone, erythroferrone (ERFE), that mediates hepcidin suppression during stress erythropoiesis. ERFE is produced by erythroblasts in response to erythropoietin. ERFE-deficient mice fail to suppress hepcidin rapidly after hemorrhage and exhibit a delay in recovery from blood loss. ERFE expression is greatly increased in Hbb(th3/+) mice with thalassemia intermedia, where it contributes to the suppression of hepcidin and the systemic iron overload characteristic of this disease.

Topics: Anemia; Animals; beta-Thalassemia; Blotting, Western; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Epoetin Alfa; Erythropoiesis; Erythropoietin; Gene Expression Profiling; Hemoglobins; Hemorrhage; Hepcidins; Hormones; Iron; Iron Overload; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Sequence Data; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The purpose of this study was to determine the effectiveness of the systemic administration of high dose erythropoietin (EPO) in a 6‑hydroxydopamine (6‑OHDA)‑ induced rat model. Rats were divided into 7 groups. Groups 1‑4 were administered daily EPO doses of 0; 2,500; 5,000 and 10,000 U/kg via intraperitoneal injection (i.p.) for 5 days. The EPO concentration in cerebrospinal fluid (CSF) was determined by enzyme‑linked immunosorbent assay (ELISA) and western blot analysis. The dose of 10,000 U/kg was then selected for subsequent experiments. In group 5, rats received saline via medial forebrain bundle (MFB). In group 6, rats received 6‑OHDA via MFB. In group 7, an EPO concentration of 10,000 U/kg was constantly administered i.p. for 5 days to rats prior to 6‑OHDA injection via MFB. Behavioral analysis was performed for groups 5‑7 by rat rotation tests. The number of tyrosine hydroxylase (TH)‑immunopositive cells in the substantia nigra (SN) was measured by immuno-cyto-chemistry. The activation of c‑Jun N‑terminal kinase (JNK), extracellular signal‑regulated kinase (ERK), p38 mitogen‑activated protein kinases (MAPKs) and caspase‑3 signaling in rats were analyzed using western blotting. The results showed that there was a significant increase in EPO levels in the CSF in 10,000 U/kg group compared with the 2,500 and 5,000 U/kg groups (P<0.01). Significantly fewer rotational counts were obtained in rats that were pretreated with EPO compared with saline‑pretreated 6‑OHDA‑lesioned rats (P<0.001). The dopaminergic neurons in the 6‑OHDA‑lesioned SN were also increased in the EPO‑pretreated rats when compared with control rats (P<0.01). Western blot analysis revealed that EPO inhibited the 6‑OHDA‑induced activation of JNK, ERK, p38 MAPK and caspase‑3 signaling in the rat model. In conclusion, systemic administration of a high dose of EPO exerted neuroprotective effects in reversing behavioral deficits associated with Parkinson's disease and prevented loss of the dopaminergic neurons through the MAPK pathway.

Topics: Animals; Disease Models, Animal; Dopaminergic Neurons; Erythropoietin; Humans; Hydroxydopamines; Mitogen-Activated Protein Kinase Kinases; Neuroprotective Agents; Parkinson Disease; Rats; Tyrosine 3-Monooxygenase

Recombinant human erythropoietin (rh-EPO) has been used as a drug to treat premature infant anemia for over a decade. In addition to its erythropoietic effect, rh-EPO has also been reported to have protective effects against brain injury.. Our aim was to evaluate the levels of angiogenesis-related cells (CD34+ cells) and angiogenic factors (vascular endothelial growth factor, VEGF, and angiopoietin-1, Ang-1) in a neonatal rat model of cerebral unilateral hypoxia-ischemia (HI) and to identify the effects of rh-EPO on angiogenic responses.. Postnatal day 3 (PD3) rats underwent permanent ligation of the right common carotid artery followed by 6% O2 for 4 h (HI) or sham operation and normoxic exposure (sham). Immediately after HI, the rats received a single intraperitoneal injection of rh-EPO (5 U/g) or saline. Angiogenesis-related cells (CD34+ cells) and angiogenic factors (VEGF and Ang-1) were examined on PD5, 7, 10 and 14.. Compared with the sham rats, the number of CD34+ cells in HI rats increased from PD5 to 7 but decreased from PD10 to 14. VEGF and Ang-1 mRNA levels both increased from PD5 to 14. CD34+ cells, VEGF and Ang-1 were all upregulated in rh-EPO-treated rats compared with HI rats.. In the present study, we show the angiogenic effects of rh-EPO in a rat model of neonatal cerebral unilateral HI. Our results highlight the powerful therapeutic potential of rh-EPO treatment of HI premature brain for the enhancement of angiogenic responses.

Topics: Angiogenesis Inducing Agents; Angiopoietin-1; Animals; Animals, Newborn; Antigens, CD34; Brain; Cytoprotection; Disease Models, Animal; Erythropoietin; Female; Humans; Hypoxia-Ischemia, Brain; Neovascularization, Physiologic; Neuroprotective Agents; Pregnancy; Rats, Sprague-Dawley; Recombinant Proteins; RNA, Messenger; Time Factors; Vascular Endothelial Growth Factor A

States of chronic inflammation such as inflammatory bowel disease are often associated with dysregulated iron metabolism and the consequent development of an anemia that is caused by maldistribution of iron. Abnormally elevated expression of the hormone hepcidin, the central regulator of systemic iron homeostasis, has been implicated in these abnormalities. However, the mechanisms that regulate hepcidin expression in conditions such as inflammatory bowel disease are not completely understood. To clarify this issue, we studied hepcidin expression in mouse models of colitis. We found that dextran sulfate sodium-induced colitis inhibited hepcidin expression in wild-type mice but upregulated it in IL-10-deficient animals. We identified two mechanisms contributing to this difference. Firstly, erythropoietic activity, as indicated by serum erythropoietin concentrations and splenic erythropoiesis, was higher in the wild-type mice, and pharmacologic inhibition of erythropoiesis prevented colitis-associated hepcidin downregulation in these animals. Secondly, the IL-10 knockout mice had higher expression of multiple inflammatory genes in the liver, including several controlled by STAT3, a key regulator of hepcidin. The results of cohousing and fecal transplantation experiments indicated that the microbiota was involved in modulating the expression of hepcidin and other STAT3-dependent hepatic genes in the context of intestinal inflammation. Our observations thus demonstrate the importance of erythropoietic activity and the microbiota in influencing hepcidin expression during colitis and provide insight into the dysregulated iron homeostasis seen in inflammatory diseases.

Topics: Animals; Bacteroides fragilis; Colitis; Dextran Sulfate; Disease Models, Animal; Erythropoiesis; Erythropoietin; Female; Hepcidins; Homeostasis; Inflammation Mediators; Inflammatory Bowel Diseases; Interleukin-10; Intestinal Mucosa; Iron; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microbiota; STAT3 Transcription Factor; Streptococcaceae

Neovascularization and vaso-obliteration are vision-threatening events that develop by interactions between retinal vascular and glial cells. A high-salt diet is causal in cardiovascular and renal disease, which is linked to modulation of the renin-angiotensin-aldosterone system. However, it is not known whether dietary salt influences retinal vasculopathy and if the renin-angiotensin-aldosterone system is involved. We examined whether a low-salt (LS) diet influenced vascular and glial cell injury and the renin-angiotensin-aldosterone system in ischemic retinopathy.. Pregnant Sprague Dawley rats were fed LS (0.03% NaCl) or normal salt (0.3% NaCl) diets, and ischemic retinopathy was induced in the offspring. An LS diet reduced retinal neovascularization and vaso-obliteration, the mRNA and protein levels of the angiogenic factors, vascular endothelial growth factor, and erythropoietin. Microglia, which influence vascular remodeling in ischemic retinopathy, were reduced by LS as was tumor necrosis factor-α. Macroglial Müller cells maintain the integrity of the blood-retinal barrier, and in ischemic retinopathy, LS reduced their gliosis and also vascular leakage. In retina, LS reduced mineralocorticoid receptor, angiotensin type 1 receptor, and renin mRNA levels, whereas, as expected, plasma levels of aldosterone and renin were increased. The aldosterone/mineralocorticoid receptor-sensitive epithelial sodium channel alpha (ENaCα), which is expressed in Müller cells, was increased in ischemic retinopathy and reduced by LS. In cultured Müller cells, high salt increased ENaCα, which was prevented by mineralocorticoid receptor and angiotensin type 1 receptor blockade. Conversely, LS reduced ENaCα, angiotensin type 1 receptor, and mineralocorticoid receptor expression.. An LS diet reduced retinal vasculopathy, by modulating glial cell function and the retinal renin-angiotensin-aldosterone system.

Topics: Adaptor Protein Complex 1; Aldosterone; Animals; Animals, Newborn; Aquaporin 4; Body Weight; Cells, Cultured; Diet, Sodium-Restricted; Disease Models, Animal; Drinking Behavior; Ependymoglial Cells; Epithelial Sodium Channels; Erythropoietin; Gliosis; Hematocrit; Ion Transport; Ischemia; Kidney Glomerulus; MAP Kinase Signaling System; Microglia; Phosphorylation; Potassium Channels, Inwardly Rectifying; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Renin-Angiotensin System; Retinal Ganglion Cells; Retinal Neovascularization; Retinopathy of Prematurity; Sodium; Sodium Chloride, Dietary; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Rats expressing a transgenic polycystic kidney disease (PKD) gene develop photoreceptor degeneration and subsequent vasoregression, as well as activation of retinal microglia and macroglia. To target the whole neuroglialvascular unit, neuro- and vasoprotective Erythropoietin (EPO) was intraperitoneally injected into four-week old male heterozygous PKD rats three times a week at a dose of 256 IU/kg body weight. For comparison EPO-like peptide, lacking unwanted side effects of EPO treatment, was given five times a week at a dose of 10 µg/kg body weight. Matched EPO treated Sprague Dawley and water-injected PKD rats were held as controls. After four weeks of treatment the animals were sacrificed and analysis of the neurovascular morphology, glial cell activity and pAkt localization was performed. The number of endothelial cells and pericytes did not change after treatment with EPO or EPO-like peptide. There was a nonsignificant reduction of migrating pericytes by 23% and 49%, respectively. Formation of acellular capillaries was significantly reduced by 49% (p<0.001) or 40% (p<0.05). EPO-treatment protected against thinning of the central retina by 10% (p<0.05), a composite of an increase of the outer nuclear layer by 12% (p<0.01) and in the outer segments of photoreceptors by 26% (p<0.001). Quantification of cell nuclei revealed no difference. Microglial activity, shown by gene expression of CD74, decreased by 67% (p<0.01) after EPO and 36% (n.s.) after EPO-like peptide treatment. In conclusion, EPO safeguards the neuroglialvascular unit in a model of retinal neurodegeneration and secondary vasoregression. This finding strengthens EPO in its protective capability for the whole neuroglialvascular unit.

Topics: Animals; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Male; Peptides; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Retinal Degeneration

To investigate the effect and mechanism of recombinant human erythropoietin (rhEPO) on angiogenesis in chronic ischemic porcine myocardium.. A ameroid constrictor was placed around the proximal circumflex branch of the left coronary artery in 12 Bama miniatures' swine artery by thoracoscopy. Electrocardiogram and coronary angiography were used to confirm the establishment of myocardial ischemia. The animals were divided into rhEPO treatment group (n = 6) and negative control group (n = 6). Treatment group received subcutaneous injection of rhEPO at 1, 3, 7, 14, 21 days, control group received saline. The expression of vascular endothelial growth factor (VEGF) in serum was assessed by ELISA. Ultrasonography and coronary angiography were assessed 28 days after therapy. Western blot was used to detect the expression of VEGF, phosphorylated protein kinase B (p-Akt) and phosphorylated extracellular signal regulated kinases (p-Erk). The degree of angiogenesis was assessed by immunohistochemical analysis.. Serum VEGF rose significantly in both control and treatment groups, peaking at 3 days and then returning to the near-baseline level at 28 days, but the two groups showed no significant difference at each time point (P > 0.05). Echocardiographic measurements showed that the left ventricular systolic function of animals in treatment group increase significantly after rhEPO therapy. the expression levels of VEGF, p-Akt and p-Erk had markedly increased, which resulted in a 2.5-fold increased of VEGF, 1.1-fold increased of p-Akt, 1.5-fold increased of p-Erk (t = 37.721, 10.907, 12.957, all P = 0.000). there were significant increase in capillary density and arteriole density in the two groups ((944 ± 98) %/mm² vs. (569 ± 102) %/mm², (73 ± 13) %/mm² vs. (45 ± 10) %/mm², t = 4.214, 2.869, P = 0.016, 0.023).. rhEPO can promote angiogenesis and arteriogenesis and improve the left ventricular systolic function in porcine model of chronic myocardial ischemia. The potential mechanism is to up-regulated the expression of p-Akt and p-Erk.

Topics: Animals; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Extracellular Signal-Regulated MAP Kinases; Humans; Male; Myocardial Ischemia; Neovascularization, Physiologic; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Swine; Swine, Miniature; Vascular Endothelial Growth Factor A

Donation after circulatory death (DCD) offers a potential additional source of cardiac allografts. We used a porcine asphyxia model to evaluate viability of DCD hearts subjected to warm ischemic times (WIT) of 20–40 min prior to flushing with Celsior (C) solution. We then assessed potential benefits of supplementing C with erythropoietin, glyceryl trinitrate and zoniporide (Cs), a combination that we have shown previously to activate ischemic postconditioning pathways. Hearts flushed with C/Cs were assessed for functional, biochemical and metabolic recovery on an ex vivo working heart apparatus. Hearts exposed to 20-min WIT showed full recovery of functional and metabolic profiles compared with control hearts (no WIT). Hearts subjected to 30- or 40-min WIT prior to C solution showed partial and no recovery, respectively. Hearts exposed to 30-min WIT and Cs solution displayed complete recovery, while hearts exposed to 40-min WIT and Cs solution demonstrated partial recovery. We conclude that DCD hearts flushed with C solution demonstrate complete recovery up to 20-min WIT after which there is rapid loss of viability. Cs extends the limit of WIT tolerability to 30 min. DCD hearts with ≤30-min WIT may be suitable for transplantation and warrant assessment in a transplant model.

Topics: Animals; Death; Disease Models, Animal; Edema; Erythropoietin; Guanidines; Heart; Heart Failure; Heart Transplantation; Ischemic Preconditioning; Lactates; Myocardium; Nitroglycerin; Oxygen Consumption; Perfusion; Pyrazoles; Swine; Time Factors; Transplantation, Homologous; Troponin; Warm Ischemia

Infants born preterm commonly suffer from a combination of hypoxia-ischemia (HI) and infectious perinatal inflammatory insults that lead to cerebral palsy, cognitive delay, behavioral issues and epilepsy. Using a novel rat model of combined late gestation HI and lipopolysaccharide (LPS)-induced inflammation, we tested our hypothesis that inflammation from HI and LPS differentially affects gliosis, white matter development and motor impairment during the first postnatal month.. Pregnant rats underwent laparotomy on embryonic day 18 and transient systemic HI (TSHI) and/or intra-amniotic LPS injection. Shams received laparotomy and anesthesia only. Pups were born at term. Immunohistochemistry with stereological estimates was performed to assess regional glial loads, and western blots were performed for protein expression. Erythropoietin ligand and receptor levels were quantified using quantitative PCR. Digigait analysis detected gait deficits. Statistical analysis was performed with one-way analysis of variance and post-hoc Bonferonni correction.. Microglial and astroglial immunolabeling are elevated in TSHI + LPS fimbria at postnatal day 2 compared to sham (both P < 0.03). At postnatal day 15, myelin basic protein expression is reduced by 31% in TSHI + LPS pups compared to shams (P < 0.05). By postnatal day 28, white matter injury shifts from the acute injury pattern to a chronic injury pattern in TSHI pups only. Both myelin basic protein expression (P < 0.01) and the phosphoneurofilament/neurofilament ratio, a marker of axonal dysfunction, are reduced in postnatal day 28 TSHI pups (P < 0.001). Erythropoietin ligand to receptor ratios differ between brains exposed to TSHI and LPS. Gait analyses reveal that all groups (TSHI, LPS and TSHI + LPS) are ataxic with deficits in stride, paw placement, gait consistency and coordination (all P < 0.001).. Prenatal TSHI and TSHI + LPS lead to different patterns of injury with respect to myelination, axon integrity and gait deficits. Dual injury leads to acute alterations in glial response and cellular inflammation, while TSHI alone causes more prominent chronic white matter and axonal injury. Both injuries cause significant gait deficits. Further study will contribute to stratification of injury mechanisms in preterm infants, and guide the use of promising therapeutic interventions.

Topics: Animals; Animals, Newborn; Axons; Brain; Calcium-Binding Proteins; Disease Models, Animal; Embryo, Mammalian; Erythropoietin; Female; Gene Expression Regulation, Developmental; Glial Fibrillary Acidic Protein; Hypoxia-Ischemia, Brain; Inflammation; Leukoencephalopathies; Lipopolysaccharides; Microfilament Proteins; Myelin Basic Protein; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin

From our previous clinical trials, intracoronary infusion of granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells ((mob)PBMCs) proved to be effective in improving myocardial contractility and reducing infarct volume in acute myocardial infarction. We tested the effect of priming (mob)PBMCs with erythropoietin (EPO) to augment its therapeutic efficacy.. (mob)PBMCs were obtained from healthy volunteers after a 3-day subcutaneous injection of G-CSF (10 μg/kg). About 40% of (mob)PBMCs were EPO receptor (EPOR) (+) and responded to 6 h EPO-priming (10 IU/mL) by increasing the expression of vasculogenic factors (i.e. IL8, IL10, bFGF, PDGF, MMP9) and adhesion molecules (i.e. integrin αV, β1, β2, β8) through the JAK2 and Akt pathway. These responses were also observed in PBMCs from elderly patients with coronary disease. The conditioned media from EPO-primed (mob)PBMCs contained various cytokines such as IL8, IL10, TNFα, and PDGF, which enhanced the migration and tube formation capability of endothelial cells. EPO-primed (mob)PBMCs also showed increased adhesion on endothelial cells or fibronectin. Augmented vasculogenic potential of EPO-primed (mob)PBMCs was confirmed in a Matrigel plug assay, ischaemic hindlimb, and myocardial infarction models of athymic nude mice. There were two action mechanisms: (i) cellular effects confirmed by direct incorporation of human (mob)PBSCs into mouse vasculature and (ii) indirect humoral effects confirmed by the therapeutic effect of the supernatant of EPO-primed (mob)PBMCs.. Brief ex vivo EPO-priming is a novel method to augment the vasculogenic potential of human (mob)PBMCs, which would help to achieve better results after intracoronary infusion in myocardial infarction patients.

Topics: Angiogenic Proteins; Animals; Bone Marrow Cells; Case-Control Studies; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Coronary Artery Disease; Culture Media, Conditioned; Cytokines; Disease Models, Animal; Erythropoietin; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Mobilization; Hindlimb; Humans; Injections, Subcutaneous; Ischemia; Janus Kinase 2; Leukocytes, Mononuclear; Male; Mice, Nude; Muscle, Skeletal; Myocardial Infarction; Neovascularization, Physiologic; Peripheral Blood Stem Cell Transplantation; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Regeneration; Signal Transduction; Time Factors

To compare the neuroprotection of erythropoietin (EPO) and EPO fusion protein containing transduction domain derived from HIV TAT (EPO-TAT) against ischemic brain injury, inclusive of the side effect, and explore the mechanism underlying the role of EPO-TAT in a transient focal cerebral ischemia model in rats.. Transient focal ischemia was induced by middle cerebral artery occlusion (MCAO) in rats. Rats were treated, respectively, with following regimens: saline, 1000 U/kg EPO, 5000 U/kg EPO, 1000 U/kg EPO-TAT, 1000 U/kg EPOTAT+5 µl of 10 mM LY294002 (or/plus 5 µl of 5 mM PD98059). Neurological deficit scores, infarct volume, and hematologic side effect were assessed at 72 hours after MCAO. Apoptotic cells were determined with TUNEL staining. The expression and localization of phosphorylated AKT (pAKT) and phosphorylated ERK (pERK) were detected with Western blot, immunohistochemistry, and immunofluorescence, respectively.. 1000 U/kg EPO-TAT exhibited a comparable neuroprotection to 5000 U/kg EPO, as evidenced by a comparable attenuation in neurological deficit, infarct volume, and number of apoptotic cells in the rat ischemic cortex after MCAO. The pAKT and pERK levels were significantly elevated solely in neurons of rodents receiving EPO or EPO-TAT treatments, suggesting the concurrent activation of these two pathways. Specific inhibition of either AKT or ERK pathway partially abolished EPO-TAT protection, but exhibited no influence on the activation status of its counterpart, suggesting no cross-modulation between these two protective pathways.. Our study indicates that EPO-TAT at 1000 U/kg displays neuroprotection with no detectable side effects. The mechanism for neuroprotection may be attributable to the simultaneous activation of the AKT and ERK pathways, which preserve neuronal cell viability and attenuate behavioral deficits.

Topics: Animals; Cell Death; Disease Models, Animal; Enzyme Inhibitors; Erythropoietin; Extracellular Signal-Regulated MAP Kinases; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Male; Neurodegenerative Diseases; Neurologic Examination; Neuroprotective Agents; Oncogene Protein v-akt; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Statistics, Nonparametric; tat Gene Products, Human Immunodeficiency Virus

Different murine species differ in their susceptibility to systemic infection with Candida albicans, giving rise to varied host immune responses, and this is compounded by variations in virulence of the different yeast strains used. Hence, this study was aimed at elucidating the pathogenesis of a clinical C. albicans isolate (HVS6360) in a murine intravenous challenge model by examining the different parameters which included the counts of red blood cells and associated components as well as the organ-specific expression profiles of cytokines and chemokines. Kidneys and brains of infected mice have higher fungal recovery rates as compared to other organs and there were extensive yeast infiltration with moderate to severe inflammation seen in kidney and brain tissues. Red blood cells (RBCs) and haemoglobin (Hb) counts were reduced throughout the infection period. Pattern recognition receptors (PRRs), chemokines and cytokine transcription profiles were varied among the different organs (kidney, spleen and brain) over 72 h post infections. Transcription of most of the PRRs, cytokines and chemokines were suppressed at 72 h post infection in spleen while continuous expression of PRRs, cytokines and chemokines genes were seen in brain and kidney. Reduction in red blood cells and haemoglobin counts might be associated with the action of extracellular haemolysin enzyme and haeme oxygenase of C. albicans in conjunction with iron scavenging for the fungal growth. Renal cells responsible for erythropoietin production may be injured by the infection and hence the combined effect of haemolysis plus lack of erythropoietin-induced RBC replenishment leads to aggravated reduction in RBC numbers. The varied local host immune profiles among target organs during systemic C. albicans infection could be of importance for future work in designing targeted immunotherapy through immunomodulatory approaches.

Topics: Animals; Candida albicans; Candidiasis; Disease Models, Animal; Erythropoietin; Female; Mice; Mice, Inbred BALB C

This study aimed to investigate the effect and potential mechanisms of exogenous administration of recombinant human erythropoietin (rhEPO) on retinal angiogenesis in a mouse model of oxygen-induced retinopathy (OIR). Postnatal day 7 (P7) mice (n=132) were randomly assigned to one of six groups: Control group (n=22), OIR group (n=22), OIR + vehicle control group (n=22), OIR + rhEPO 10 IU group (n=22), OIR + rhEPO 50 IU group (n=22), and OIR + rhEPO 100 IU group (n=22). OIR was induced by exposing mice to 75±2% O2 for five days, followed by exposure to room air for a further five days. Animals in groups 3-6 (the OIR + vehicle control group and OIR + rhEPO 10 IU, 50 IU, and 100 IU groups) received an intraperitoneal injection of saline, or rhEPO 10 IU, 50 IU and 100 IU, respectively, which were administered daily from P7-P12. Immunofluorescent and hematoxylin-eosin staining were used to detect retinal neovascularization (RNV) in retinal whole mounts. Quantitative polymerase chain reaction and western blot analysis were used to detect the expression levels of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS). RNV occurred in the OIR groups and was accompanied by dilated, twisted and occluded blood vessels. rhEPO treatment resulted in an increase in the number of newly formed and severely dilated vessels. rhEPO increased RNV in a dose-dependent manner, which was accompanied by an increase in the messenger RNA and protein expression of VEGF, eNOS and nNOS. Thus, exogenous use of rhEPO promotes the RNV in a mouse model of OIR and is accompanied by increased expression levels of VEGF, eNOS and nNOS.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Erythropoietin; Humans; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Oxygen; Recombinant Proteins; Retina; Retinal Neovascularization; RNA, Messenger; Vascular Endothelial Growth Factor A

Erythropoietin (EPO) has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. In this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO) in dystrophic muscles in mdx mice. Total strength was measured using a force transducer coupled to a computer. Gene expression for myostatin, transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) was determined by quantitative real time polymerase chain reaction. Myostatin expression was significantly decreased in quadriceps from mdx mice treated with rhEPO (rhEPO = 0.60 ± 0.11, control = 1.07 ± 0.11). On the other hand, rhEPO had no significant effect on the expression of TGF-β1 (rhEPO = 0.95 ± 0.14, control = 1.05 ± 0.16) and TNF-α (rhEPO = 0.73 ± 0.20, control = 1.01 ± 0.09). These results may help to clarify some of the direct actions of EPO on skeletal muscle.

Topics: Animals; Disease Models, Animal; Down-Regulation; Dystrophin; Erythropoietin; Gene Expression; Male; Mice, Inbred mdx; Muscle Strength; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Myostatin; Phenotype; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Retinal neuronal cell dysfunction and even cell death are associated with increased excitotoxic glutamate (Glu) level in the retina. Our aim was to study a causative mechanism of Glu on retinal cell death and explore the neuroprotective role of erythropoietin (EPO) against Glu neurotoxicity in the diabetic retina.. Male Sprague-Dawley (SD) rats and R28 cell line were employed in this study. Diabetes was induced with intraperitoneal injection of streptozotocin (STZ) in SD rats. Two weeks after diabetes onset, the intravitreal injection was performed; 4 days later, the retinas were harvested for testing. R28 cells were treated with Glu, Glu+EPO, or Glu+EPO+soluble EPO receptor (sEPOR), respectively, for 24 hours, and then the cells were collected for the following studies. Glutamate level in the retina was measured with a glutamate assay kit. Cell death was determined with TUNEL staining. The changes in glutamine synthetase (GS), glutamate-aspartate transporter (GLAST), ionotropic glutamate receptors (iGluRs), apoptosis-inducing factor (AIF), and poly(ADP-ribose) (PAR) polymer were studied with RT-PCR, Western blot, and immunofluorescence.. In 2-week diabetic rat retinas, Glu concentration was approximately 1.21-fold that in normal control. TUNEL staining demonstrated that retinal cell death was increased. Retinal GS and GLAST expressions were decreased, while the iGluRs, for example, KA1 and NR1, and PAR polymer expression was increased. In R28 cells, 24 hours after Glu (10 mM) treatment, the cell viability was decreased by 52.7%; KA1, NR1, PAR polymer, and nuclear AIF all increased in expression. The above conditions could be largely reversed by EPO both in vivo and in vitro. The protective effect of EPO was abolished by sEPOR.. Erythropoietin showed a neuroprotective function against Glu-mediated neurotoxicity both in diabetic rat retina and in Glu-treated R28 cells. The neuroprotective mechanisms were largely through maintaining the normal expression of glutamate-glutamine cycle-related proteins and inhibiting AIF translocation and PAR polymer formation.

Topics: Amino Acid Transport System X-AG; Animals; Apoptosis Inducing Factor; Cell Death; Cell Line; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Disease Models, Animal; Erythropoietin; Excitatory Amino Acid Antagonists; Glutamate-Ammonia Ligase; Glutamic Acid; Intravitreal Injections; Male; Poly Adenosine Diphosphate Ribose; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Receptors, Glutamate; Retina

Samul-tang (Si-Wu-Tang, SMT), a kind of herbal medicines, has been used for the hemato-deficient disease for hundreds of years. In this work, investigate the anti-anemia activity of the H2O extracts from constituent herbal medicines of Samul-tang in an anemia model induced by intravenous infection of phenylhydrazine-HCL (PHZ) at 10 mg/kg for 4 days. After PHZ injection, female Sparague-Dawley rats were administrated extracts from constituent herbal medicines of SMT (300 mg/kg/day, p.o.) daily for 1 week. Results showed that sever hemolysis was induced by PHZ. For Paeonia lactiflora (PL2) H2O extract treated groups, the concentration of hemoglobin, hematocrit and red blood cells number increased much more significantly than PHZ-treated group. Moreover, Angelica gigas (AG), Angelica. acutiloba (AA), Paeonia lactiflora (PL2) and Rehmannia glutinosa (RG) extract administration significantly improved serum erythropoietin concentration. The activity of aminolevulinic acid dehydrates (ALDL) in liver homegenate was increased in Angelica gigas(AA), Paeonia lactiflora (PL2) and Rehmannia glutinosa (RG) treated group.

Topics: Anemia, Hemolytic; Animals; Biomarkers; Disease Models, Animal; Drugs, Chinese Herbal; Erythrocyte Count; Erythrocytes; Erythropoietin; Female; Hematinics; Hematocrit; Hematopoiesis; Hemoglobins; Humans; Liver; Phenylhydrazines; Phytotherapy; Plants, Medicinal; Porphobilinogen Synthase; Rats, Sprague-Dawley; Time Factors

To determine whether erythropoietin given during hemorrhagic shock (HS) ameliorates organ injury while improving resuscitation and survival.. Three series of 24 pigs each were studied. In an initial series, 50% of the blood volume (BV) was removed in 30 minutes and normal saline (threefold the blood removed) started at minute 90 infusing each third in 30, 60, and 150 minutes with shed blood reinfused at minute 330 (HS-50BV). In a second series, the same HS-50BV protocol was used but removing an additional 15% of BV from minute 30 to 60 (HS-65BV). In a final series, blood was removed as in HS-65BV and intraosseous vasopressin given from minute 30 (0.04 U/kg min(-1)) until start of shed blood reinfusion at minute 150 (HS-65BV+VP). Normal saline was reduced to half the blood removed and given from minute 90 to 120 in half of the animals. In each series, animals were randomized 1:1 to receive erythropoietin (1,200 U/kg) or control solution intraosseously after removing 10% of the BV.. In HS-50BV, O2 consumption remained near baseline yielding minimal lactate increases, 88% resuscitability, and 60% survival at 72 hours. In HS-65BV, O2 consumption was reduced and lactate increased yielding 25% resuscitability. In HS-65BV+VP, vasopressin promoted hemodynamic stability yielding 92% resuscitability and 83% survival at 72 hours. Erythropoietin did not affect resuscitability or subsequent survival in any of the series but increased interleukin-10, attenuated lactate increases, and ameliorated organ injury based on lesser troponin I, AST, and ALT increases and lesser neurological deficits in the HS-65BV+VP series.. Erythropoietin given during HS in swine failed to alter resuscitability and 72 hour survival regardless of HS severity and concomitant treatment with fluids and vasopressin but attenuated acute organ injury. The studies also showed the efficacy of vasopressin and restrictive fluid resuscitation for hemodynamic stabilization and survival.

Topics: Animals; Cytokines; Disease Models, Animal; Erythropoietin; Heart; Hemodynamics; Infusions, Intraosseous; Lactic Acid; Male; Myocardium; Oxygen Consumption; Resuscitation; Shock, Hemorrhagic; Swine

An increased risk of skin pressure ulcers (PUs) is common in patients with sensory neuropathies, including those caused by diabetes mellitus. Recombinant human erythropoietin (rhEPO) has been shown to protect the skin against PUs developed in animal models of long-term diabetes. The aim of this work was to determine whether rhEPO could prevent PU formation in a mouse model of drug-induced SFN. Functional SFN was induced by systemic injection of resiniferatoxin (RTX, 50 µg/kg, i.p.). RhEPO (3000 UI/kg, i.p.) was given the day before RTX injection and then every other day. Seven days after RTX administration, PUs were induced by applying two magnetic plates on the dorsal skin. RTX-treated mice expressed thermal and mechanical hypoalgesia and showed calcitonin gene-related peptide (CGRP) and substance P (SP) depletion without nerve degeneration or vascular dysfunction. RTX mice developed significantly larger stage 2 PUs than Vehicle mice. RhEPO prevented thermal and mechanical hypoalgesia and neuropeptide depletion in small nerve fibers. RhEPO increased hematocrit and altered endothelium-dependent vasodilatation without any effect on PU formation in Vehicle mice. The characteristics of PUs in RTX mice treated with rhEPO and Vehicle mice were found similar. In conclusion, RTX appeared to increased PU development through depletion of CGRP and SP in small nerve fibers, whereas systemic rhEPO treatment had beneficial effect on peptidergic nerve fibers and restored skin protective capacities against ischemic pressure. Our findings support the evaluation of rhEPO and/or its non-hematopoietic analogs in preventing to prevent PUs in patients with SFN.

Topics: Animals; Disease Models, Animal; Diterpenes; Erythromelalgia; Erythropoietin; Mice; Neuroprotective Agents; Pressure Ulcer

Recombinant human erythropoietin (EPO) has been successfully tested as neuroprotectant in brain injury models. The first large clinical trial with stroke patients, however, revealed negative results. Reasons are manifold and may include side-effects such as thrombotic complications or interactions with other medication, EPO concentration, penetration of the blood-brain-barrier and/or route of application. The latter is restricted to systemic application. Here we hypothesize that EPO is neuroprotective in a rat model of acute subdural hemorrhage (ASDH) and that direct cortical application is a feasible route of application in this injury type. The subdural hematoma was surgically evacuated and EPO was applied directly onto the surface of the brain. We injected NaCl, 200, 2000 or 20,000IU EPO per rat i.v. at 15min post-ASDH (400μl autologous venous blood) or NaCl, 0.02, 0.2 or 2IU per rat onto the cortical surface after removal of the subdurally infused blood t at 70min post-ASDH. Arterial blood pressure (MAP), blood chemistry, intracranial pressure (ICP), cerebral blood flow (CBF) and brain tissue oxygen (ptiO2) were assessed during the first hour and lesion volume at 2days after ASDH. EPO 20,000IU/rat (i.v.) elevated ICP significantly. EPO at 200 and 2000IU reduced lesion volume from 38.2±0.6mm(3) (NaCl-treated group) to 28.5±0.9 and 22.2±1.3mm(3) (all p<0.05 vs. NaCl). Cortical application of 0.02IU EPO after ASDH evacuation reduced injury from 36.0±5.2 to 11.2±2.1mm(3) (p=0.007), whereas 0.2IU had no effect (38.0±9.0mm(3)). The highest dose of both application routes (i.v. 20,000IU; cortical 2IU) enlarged the ASDH-induced damage significantly to 46.5±1.7 and 67.9±10.4mm(3) (all p<0.05 vs. NaCl). In order to test whether Tween-20, a solvent of EPO formulation 'NeoRecomon®' was responsible for adverse effects two groups were treated with NaCl or Tween-20 after the evacuation of ASDH, but no difference in lesion volume was detected. In conclusion, EPO is neuroprotective in a model of ASDH in rats and was most efficacious at a very low dose in combination with subdural blood removal. High systemic and topically applied concentrations caused adverse effects on lesion size which were partially due to increased ICP. Thus, patients with traumatic ASDH could be treated with cortically applied EPO but with caution concerning concentration.

Topics: Animals; Cerebral Cortex; Combined Modality Therapy; Disease Models, Animal; Erythropoietin; Hematoma, Subdural, Acute; Intracranial Pressure; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley

The aim of this study is to test the hypotheses that central auditory pathology as well as inner ear pathology is contributing mechanisms to observed hypoxic-ischemic encephalopathy (HIE) induced hearing loss and that recombinant erythropoietin (rhEPO) will reduce this cellular pathology and attenuate hearing loss.. Twenty-eight 7-day Wistar albino rat pups were divided into four groups: Control group (n=8) was given only intraperitoneal saline solution. Sham group (n=5) had only a midline neck incisions without carotid ligation under general anesthesia and administration of intraperitoneal saline solution. HIE group (n=8) and rhEPO treated group (n=7) were subjected to left common carotid artery ligation followed by 2.5h hypoxia exposure to a mixture of 8% oxygen and 92% pure nitrogen. HIE group was injected with intraperitoneal saline solution, while the rhEPO treated group received rhEPO 100 U/kg within the same volume as the saline-alone solution. At the end of the seventh week of age hearing (ABRs) was evaluated in response to clicks, 6 kHz and 8 kHz tone burst stimuli. Animals were sacrificed and both temporal lobes, cochleas and brainstems of the animals were collected. Tissue samples were evaluated with light microscopy, immunohistochemical studies, including TUNEL and caspase-3 stainings, and electron microscopy.. Hearing thresholds were elevated in HIE animals. In rhEPO treated animals, ABR values were similar to controls. HIE caused apoptotic changes in brainstem structures as shown by light microscopy and immunohistochemical methods. Apoptotic changes also were found within the organ of Corti, spiral ganglion cells and neurons of temporal lobe by electron microscopic investigation. In rhEPO animals many of these apoptotic changes were observed, but reduced compared to untreated animals.. Mechanisms underlying HIE-induced hearing loss are based on apoptosis in inner ear; however central auditory pathway pathology occurs as well, likely contributing to changes in auditory processing and perception of complex signals not reflected by the ABR threshold shifts. For both clinical and basic significance 'rhRPO' is found to reduce those effects.

Topics: Animals; Apoptosis; Caspase 3; Disease Models, Animal; Erythropoietin; Evoked Potentials, Auditory, Brain Stem; Female; Hearing Loss; Humans; Hypoxia-Ischemia, Brain; Immunohistochemistry; In Situ Nick-End Labeling; Infant, Newborn; Male; Rats; Rats, Wistar; Recombinant Proteins; Spiral Ganglion

Inhibiting VEGF improves adult retino/choroido-vascular diseases, but can lead to recurrent intravitreous neovascularization (IVNV), avascular retina (AVA), and retinal detachment in preterm infants with retinopathy of prematurity (ROP). We sought to understand causes of late-onset IVNV and AVA following anti-VEGF using an ROP model.. In the Penn model of ROP, postnatal day (p)12 pups received 1 μL intravitreal VEGFA164 antibody (anti-VEGF; 25-100 ng) or IgG control in each eye. Analyses included lectin-stained percent IVNV and AVA; VEGF protein, erythropoietin, phosphorylated extracellular signal-related kinases and signal transducer and activator of transcription-3 (p-STAT3); and immunohistochemistry of retinal sections for p-VEGFR2. Western blots of human retinal microvascular endothelial cells (hRMVECs) stimulated with VEGF or erythropoietin were analyzed for p-STAT3. Statistical analysis was performed with one-way ANOVA or two-tailed t-tests.. At p18, 50 ng anti-VEGF reduced IVNV, and at p25, caused increased IVNV and AVA compared with controls. VEGF and p-VEGFR2 labeling increased following 100 ng anti-VEGF. Following 50 ng anti-VEGF, reduced p-STAT3 and increased erythropoietin occurred at p18. Erythropoietin or VEGF stimulated hRMVEC proliferation and STAT3 activation. In vivo, anti-VEGF reduced pup growth.. Increases in erythropoietin and angiogenic signaling following anti-VEGF may account for recurrent IVNV. Anti-VEGF reduced pup growth. Research is needed regarding safety, dose, and type of antiangiogenic treatment for ROP.

Topics: Animals; Antibodies; Biomarkers; Blotting, Western; Body Weight; Case-Control Studies; Disease Models, Animal; Erythropoietin; Humans; Infant, Newborn; Neovascularization, Pathologic; Rats; Rats, Sprague-Dawley; Retina; Retinopathy of Prematurity; STAT3 Transcription Factor; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Prematurity and perinatal hypoxia-ischemia are common problems that result in significant neurodevelopmental morbidity and high mortality worldwide. The Vannucci model of unilateral brain injury was developed to model perinatal brain injury due to hypoxia-ischemia. Because the rodent brain is altricial, i.e., it develops postnatally, investigators can model either preterm or term brain injury by varying the age at which injury is induced. This model has allowed investigators to better understand developmental changes that occur in susceptibility of the brain to injury, evolution of brain injury over time, and response to potential neuroprotective treatments. The Vannucci model combines unilateral common carotid artery ligation with a hypoxic insult. This produces injury of the cerebral cortex, basal ganglia, hippocampus, and periventricular white matter ipsilateral to the ligated artery. Varying degrees of injury can be obtained by varying the depth and duration of the hypoxic insult. This chapter details one approach to the Vannucci model and also reviews the neuroprotective effects of erythropoietin (Epo), a neuroprotective treatment that has been extensively investigated using this model and others.

Topics: Animals; Brain Injuries; Disease Models, Animal; Erythropoietin; Humans; Hypoxia-Ischemia, Brain; Neuroprotective Agents

The cytokine erythropoietin (EPO) is an important regulator of hematopoesis and has well-known tissue protective properties. Neurotrophic action is implicated as mechanistically important in the treatment of depression, and neurotrophic actions of EPO suggest potential therapeutic utility of an EPO-like mechanism in depressive disorder. Rodent behavioral models that are responsive to clinically used antidepressants as well as to neurotrophic compounds can be used to assess potential antidepressant properties of EPO and EPO-like compounds. Rodent models described here are the forced-swim test (FST), a hyponeophagia test and the novel object recognition test. Each of these models provides different information and relevance to depression and each can be tested with EPO and similar compounds.

Topics: Animals; Antidepressive Agents; Depression; Disease Models, Animal; Erythropoietin; Mice; Rats

The various biochemical cascades that follow primary brain injury result in secondary brain injury which can adversely affect the clinical outcome. Over the last few years it has been well established that molecules like erythropoietin (Epo) have a neuroprotective role in experimental traumatic brain injury (TBI). Epo is shown to produce this effect by modulating multiple cellular processes, including apoptosis, inflammation, and regulation of cerebral blood flow. Derivatives of Epo, including asialo Epo and carbamylated Epo, have been developed to separate the neuroprotective properties from the erythropoiesis-stimulating activities of Epo which may have adverse effects in clinical situations. Peptides that mimic a portion of the Epo molecule, including Helix B surface peptide and Epotris, have also been developed to isolate the neuroprotective activities. The TBI model in rodents most commonly used to study the effect of Epo and these derivatives in TBI is controlled cortical impact injury, which is a model of focal contusion following a high velocity impact to the parietal cortex. Following TBI, rodents are given Epo or an Epo derivative vs. placebo and the outcome is evaluated in terms of physiological parameters (cerebral blood flow, intracranial pressure, cerebral perfusion pressure), behavioral parameters (motor and memory), and histological parameters (contusion volumes, hippocampus cell counts).

Topics: Animals; Brain Injuries; Cytokines; Disease Models, Animal; Erythropoietin

The exact mechanism by which erythropoietin protects the liver from ischemia reperfusion (I/R) injury is not yet known. In the present study, we examined the role of protein kinase B (PKB/AKT) and endothelial nitric oxide synthase (eNOS) in the protective effect of recombinant human erythropoietin (rHuEPO) on I/R injury of the liver.. We used a liver in situ I/R model. One hundred twenty adult male Sprague-Dawley rats were divided randomly into six groups. rHuEPO and (or) LY294002 were injected in the tail vein before the operation, and its effect was assessed by measuring the serum levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, nitric oxide (NO), and endothelin-1 (ET-1) and by histologic analysis. The expression of erythropoietin receptor (EPOR) and eNOS was measured by real-time polymerase chain reaction. Total AKT and eNOS and phosphorylated AKT and eNOS were examined by western blot.. rHuEPO dramatically attenuated the functional and morphologic injuries. The serum levels of alanine aminotransferase and lactate dehydrogenase were significantly decreased, but the amount of NO in the serum was increased in the I/R + rHuEPO group. Accordingly, rHuEPO administration significantly ameliorated the histologic damages at 6 h after reperfusion. rHuEPO significantly stimulated the phosphorylation of AKT and eNOS in the rats after liver I/R.. The protective effect of rHuEPO in I/R injury is mediated via the activation of the phosphatidylinositol-3 kinase/AKT/eNOS signaling pathway, at least in part, by increasing p-AKT and p-eNOS and leads to the maintenance of an elevated level of NO.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Chromones; Disease Models, Animal; Enzyme Inhibitors; Erythropoietin; Humans; Ischemic Preconditioning; L-Lactate Dehydrogenase; Liver; Male; Morpholines; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury; Signal Transduction

Severe malaria, a leading cause of mortality among children and nonimmune adults, is a multisystemic disorder characterized by complex clinical syndromes that are mechanistically poorly understood. The interplay of various parasite and host factors is critical in the pathophysiology of severe malaria. However, knowledge regarding the pathophysiological mechanisms and pathways leading to the multisystemic disorders of severe malaria in humans is limited. Here, we systematically investigate infections with Plasmodium coatneyi, a simian malaria parasite that closely mimics the biological characteristics of P. falciparum, and develop baseline data and protocols for studying erythrocyte turnover and severe malaria in greater depth. We show that rhesus macaques (Macaca mulatta) experimentally infected with P. coatneyi develop anemia, coagulopathy, and renal and metabolic dysfunction. The clinical course of acute infections required suppressive antimalaria chemotherapy, fluid support, and whole-blood transfusion, mimicking the standard of care for the management of severe malaria cases in humans. Subsequent infections in the same animals progressed with a mild illness in comparison, suggesting that immunity played a role in reducing the severity of the disease. Our results demonstrate that P. coatneyi infection in rhesus macaques can serve as a highly relevant model to investigate the physiological pathways and molecular mechanisms of malaria pathogenesis in naïve and immune individuals. Together with high-throughput postgenomic technologies, such investigations hold promise for the identification of new clinical interventions and adjunctive therapies.

Topics: Anemia; Animals; Blood Coagulation; Bone Marrow Diseases; Disease Models, Animal; Erythropoiesis; Erythropoietin; Macaca mulatta; Malaria; Male; Monkey Diseases; Plasmodium; Random Allocation; Time Factors

Topics: Alcoholic Intoxication; Animals; Disease Models, Animal; Erythropoietin; Ethanol; Male; Neurons

Evaluation of neuroprotective effects of hypothermia, erythropoietin and their simultaneous use after perinatal asphyxia in newborn rats.. Hysterectomy was performed to Wistar female rats on the last day of gestation. Perinatal asphyxia was induced by submersion of uterus containing pups in saline for 15 min. After resuscitation, pups were randomized into 4 groups, 15 animals in each: G1 - asphyxia; G2 - asphyxia + hypothermia (rectal temperature 33 °C for 1 h); G3 - asphyxia + erythropoietin (Darbepoetin-α 2.5 μg, intraperitoneally) and G4 - asphyxia + erythropoietin + hypothermia. Pups were sacrificed on 7th day of life and histopathological analysis of hippocampus was performed.. Measure of damage to dorsal, ventral and entire hippocampus was significantly lower in groups G2, G3 and G4 than in group G1 (p ~ 0.00; respectively). Measure of damage to hippocampus in group G4 was significantly lower than in group G2 (p = 0.029).. This study demonstrates that simultaneous use of hypothermia and erythropoietin has more expressed neuroprotective effects than sole use of hypothermia after perinatal asphyxia in newborn rats.

Topics: Animals; Animals, Newborn; Asphyxia Neonatorum; Brain Diseases; Combined Modality Therapy; Disease Models, Animal; Erythropoietin; Female; Hippocampus; Hypothermia, Induced; Neurons; Neuroprotective Agents; Pregnancy; Rats; Rats, Wistar

Acute renal injury after cardiopulmonary bypass is common and associated with high mortality. We aimed to demonstrate the glomerular protective effects of recombinant human erythropoietin using an in vivo rat cardiopulmonary bypass model and to explore the possible mechanism.. Dose-related renal protective effects of recombinant human erythropoietin were studied in phase I. Male Sprague Dawley rats were randomly divided into 5 groups: sham group, cardiopulmonary bypass group, and 3 recombinant human erythropoietin-treated cardiopulmonary bypass groups (bolus doses of 500, 3000, and 5000 U/kg 24 hours before surgery). Blood and urine samples were collected just before surgery and at 2, 4, 24, 48, and 72 hours after surgery. In phase II, rats were divided into 3 groups: sham group, cardiopulmonary bypass group, and 5000 U/kg recombinant human erythropoietin group. Kidneys were harvested at 4, 24, 48, and 72 hours after surgery. Ultra-organization of glomeruli was observed. Glomerular transient receptor potential channel 6 (TRPC6) expression was studied by immunofluorescence and Western blot. Nuclei nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) activity was analyzed by enzyme-linked immunosorbent assays and electrophoretic mobility shift assay.. Pretreatment of 5000 U/kg recombinant human erythropoietin decreased the urine protein (72 hours: 7.82 ± 1.13 g/L vs 11.28 ± 1.73 g/L), serum creatinine (72 hours: 35.0 ± 3.5 μmol/L vs 60.7 ± 7.6 μmol/L), and cystatin-C (2 hours: 336.5 ± 28.2 μg/L vs 452.6 ± 63.8 μg/L) compared with the control group (P < .01). Cardiopulmonary bypass induced morphologic abnormalities of podocyte foot processes and slit diaphragms, which was improved by recombinant human erythropoietin. Furthermore, recombinant human erythropoietin significantly relieved glomerular TRPC6 increase and NFATc1 activation induced by cardiopulmonary bypass.. Pretreatment of 5000 U/kg recombinant human erythropoietin elicited potent glomerular protection against cardiopulmonary bypass. This protection may be partly due to downregulation of glomerular TRPC6-NFATc1 pathway.

Topics: Acute Kidney Injury; Animals; Biomarkers; Cardiopulmonary Bypass; Creatinine; Cystatin C; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Humans; Kidney Glomerulus; Male; NFATC Transcription Factors; Podocytes; Protective Agents; Proteinuria; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Signal Transduction; Time Factors; Transcription Factors; TRPC Cation Channels

Approximately one-half of patients with inflammatory bowel disease (IBD) suffer from anemia, with the most prevalent cause being iron deficiency. Accompanying the anemia are increases in erythropoietin, a plasma protein that can initiate the feedback production of new red blood cells. Anemia also occurs in animal models that are used to investigate the mechanisms of IBD; however, the extent to which iron deficiency produces the anemia in these animal models is unknown. Also unknown in the different animal models of IBD is whether the anemia upregulates the production of erythropoietin or, alternatively, whether a decrease in erythropoietin contributes to the induction of anemia.. Two mouse models of colitis were used in this study: (1) acute 6-day ingestion of dextran sodium sulfate and (2) T-cell transfer into lymphopenic recipient mice. Measurements included indices of colitis severity, hematocrit, blood hemoglobin, plasma erythropoietin, serum iron concentration, plasma iron-binding capacities, transferrin saturation, and tissue iron concentrations.. Both models of colitis induced significant decreases in hematocrit, blood hemoglobin, and transferrin saturation, with the spleen and liver showing a decrease in iron content in the T-cell transfer model. Additionally, both models of colitis demonstrated significant increases in plasma erythropoietin and plasma iron-binding capacities.. The measurements of iron, whether in acute (dextran sodium sulfate) or chronic (T-cell transfer) models of colitis, were generally consistent with iron-deficient anemia, with large increases in erythropoietin indicative of tissue hypoxia. These changes in animal models of colitis are similar to those found in human IBD.

Topics: Acute Disease; Anemia; Animals; Chronic Disease; Colitis; Dextran Sulfate; Disease Models, Animal; Erythropoietin; Homeodomain Proteins; Humans; Interleukin-10; Iron; Mice; Mice, Inbred C57BL; Mice, Knockout; T-Lymphocytes

After global cerebral hypoxia, many patients are severely disabled even after intensive neurorehabilitation. Secondary mechanisms of brain injury as a result of biochemical and physiological events occur within a period of hours to months, and provide a window of opportunity for therapeutic intervention. Erythropoietin (EPO) has been shown to be neuroprotective in the brain subjected to a variety of injuries. Fifty-nine 3-month-old male Wistar rats were randomly distributed to experimental groups with respect to the housing (enriched environment - EE, standard housing - SH), to hypoxia exposure, and to EPO treatment. An acute mountain sickness model was used as a hypobaric hypoxia simulating an altitude of 8000 m. One half of the animals received erythropoietin injections, while the others were injected saline. Spatial memory was tested in a Morris water maze (MWM). The escape latency and the path length were measured. Better spatial learning in MWM was only seen in the group that received erythropoietin together with enriched environment. EPO administration itself had no influence on spatial memory. The results were very similar for both latencies and path lengths. These results support the idea that after brain injuries, the recovery can be potentiated by EPO administration combined with neurorehabilitation.

Topics: Altitude Sickness; Animals; Behavior, Animal; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Escape Reaction; Housing, Animal; Hypoxia, Brain; Male; Maze Learning; Memory; Neuroprotective Agents; Rats; Rats, Wistar; Reaction Time; Recombinant Proteins; Time Factors

To investigate the effects of recombinant human erythropoietin on brain oxygenation in a model of diffuse traumatic brain injury.. Adult male Wistar rats.. Neurosciences and physiology laboratories.. Thirty minutes after diffuse traumatic brain injury (impact-acceleration model), rats were intravenously administered with either a saline solution or a recombinant human erythropoietin (5000 IU/kg). A third group received no traumatic brain injury insult (sham-operated).. Three series of experiments were conducted 2 hours after traumatic brain injury to investigate: 1) the effect of recombinant human erythropoietin on brain edema using diffusion-weighted magnetic resonance imaging and measurements of apparent diffusion coefficient (n = 11 rats per group); local brain oxygen saturation, mean transit time, and blood volume fraction were subsequently measured using a multiparametric magnetic resonance-based approach to estimate brain oxygenation and brain perfusion in the neocortex and caudoputamen; 2) the effect of recombinant human erythropoietin on brain tissue PO₂ in similar experiments (n = 5 rats per group); and 3) the cortical ultrastructural changes after treatment (n = 1 rat per group). Compared with the sham-operated group, traumatic brain injury saline rats showed a significant decrease in local brain oxygen saturation and in brain tissue PO₂ alongside brain edema formation and microvascular lumen collapse at H2. Treatment with recombinant human erythropoietin reversed all of these traumatic brain injury-induced changes. Brain perfusion (mean transit time and blood volume fraction) was comparable between the three groups of animals.. Our findings indicate that brain hypoxia can be related to microcirculatory derangements and cell edema without evidence of brain ischemia. These changes were reversed with post-traumatic administration of recombinant human erythropoietin, thus offering new perspectives in the use of this drug in brain injury.

Topics: Animals; Brain; Brain Edema; Brain Injuries; Brain Ischemia; Cerebrovascular Circulation; Diffusion Magnetic Resonance Imaging; Disease Models, Animal; Erythropoietin; Humans; Infusions, Intravenous; Male; Microcirculation; Oxygen Consumption; Random Allocation; Rats; Rats, Wistar; Reference Values; Risk Factors

The β-common receptor (βcR) plays a pivotal role in the nonhematopoietic tissue-protective effects of erythropoietin (EPO). Here we determined whether EPO reduces the acute kidney injury (AKI) caused by sepsis and whether this effect is mediated by the βcR. In young (2 months old) C57BL/6 wild-type and βcR knockout mice, lipopolysaccharide caused a significant increase in serum urea and creatinine, hence AKI. This AKI was not associated with any overt morphological alterations in the kidney and was attenuated by EPO given 1 h after lipopolysaccharide in wild-type but not in βcR knockout mice. In the kidneys of endotoxemic wild-type mice, EPO enhanced the phosphorylation of Akt, glycogen synthase kinase-3β, and endothelial nitric oxide synthase, and inhibited the activation of nuclear factor-κB. All these effects of EPO were lost in βcR knockout mice. Since sepsis is more severe in older animals or patients, we tested whether EPO was renoprotective in 8-month-old wild-type and βcR knockout mice that underwent cecal ligation and puncture. These older mice developed AKI at 24 h, which was attenuated by EPO treatment 1 h post cecal ligation and puncture in wild-type mice but not in βcR knockout mice. Thus, activation of the βcR by EPO is essential for the observed reduction in AKI in either endotoxemic young mice or older mice with polymicrobial sepsis, and for the activation of well-known signaling pathways by EPO.

Topics: Acute Kidney Injury; Animals; Caspase 3; Cecum; Cytokine Receptor Common beta Subunit; Disease Models, Animal; Erythropoietin; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hepatitis A Virus Cellular Receptor 1; Kidney; Ligation; Lipopolysaccharides; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Proto-Oncogene Proteins c-akt; Sepsis; Signal Transduction

Hypoxia-inducible factor 1α (HIF-1α) is a master regulator of cellular adaptation to hypoxia and has been proposed as a potent therapeutic target for cerebral ischemia. However, research on the expression and effects of HIF-1α in subarachnoid hemorrhage (SAH) is limited. The aim of the present study was to investigate the expression of HIF-1α in the hippocampus and its possible protective effect against hippocampal apoptosis and cognitive dysfunction in a rat model of SAH. Seventy-two Sprague-Dawley (SD) rats were randomly divided into the sham group, the SAH+vehicle group, and the SAH+YC-1 group. Immunohistochemical staining and western blotting analyses revealed that the expression of HIF-1α and its downstream effectors, vascular endothelial growth factor (VEGF), erythropoietin (EPO), and glucose transporter 1 (GLUT1), increased in the hippocampus 48h after the induction of SAH. YC-1 blocked this upregulation. The number of active caspase-3-positive cells and the expression of active caspase-3 in the hippocampus significantly increased in the YC-1 group relative to the vehicle group. A cell death assay further revealed that DNA fragmentation was significantly increased at 48h in the YC-1 group compared with the vehicle group. In Morris water maze (MWM) tests, the YC-1 group showed increased escape latency times and distances as well as reduced time spent and distance traveled in the target quadrant. These results indicate that hippocampal apoptosis increased and cognitive function deteriorated when HIF-1α was inhibited, suggesting that HIF-1α has a neuroprotective effect in SAH and may represent an effective therapeutic target.

Topics: Animals; Apoptosis; Blood Pressure; Caspase 3; Cognition Disorders; Disease Models, Animal; DNA Fragmentation; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Excitatory Amino Acid Transporter 2; Hippocampus; Hypoxia-Inducible Factor 1, alpha Subunit; Indazoles; Male; Maze Learning; Platelet Aggregation Inhibitors; Rats; Rats, Sprague-Dawley; Reaction Time; Subarachnoid Hemorrhage; Vascular Endothelial Growth Factor A

The central pathway for oxygen-dependent control of red cell mass is the prolyl hydroxylase domain protein (PHD):hypoxia inducible factor (HIF) pathway. PHD site specifically prolyl hydroxylates the transcription factor HIF-α, thereby targeting the latter for degradation. Under hypoxia, this modification is attenuated, allowing stabilized HIF-α to activate target genes, including that for erythropoietin (EPO). Studies employing genetically modified mice point to Hif-2α, one of two main Hif-α isoforms, as being the critical regulator of Epo in the adult mouse. More recently, erythrocytosis patients with heterozygous point mutations in the HIF2A gene have been identified; whether these mutations were polymorphisms unrelated to the phenotype could not be ruled out. In the present report, we characterize a mouse line bearing a G536W missense mutation in the Hif2a gene that corresponds to the first such human mutation identified (G537W). We obtained mice bearing both heterozygous and homozygous mutations at this locus. We find that these mice display, in a mutation dose-dependent manner, erythrocytosis and pulmonary hypertension with a high degree of penetrance. These findings firmly establish missense mutations in HIF-2α as a cause of erythrocytosis, highlight the importance of this HIF-α isoform in erythropoiesis, and point to physiologic consequences of HIF-2α dysregulation.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Blood Gas Analysis; Cells, Cultured; Disease Models, Animal; Endothelin-1; Erythropoiesis; Erythropoietin; Gene Expression; Gene Knock-In Techniques; Genetic Association Studies; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Kidney; Lung; Mice; Mice, Inbred C57BL; Mutagenesis; Mutation, Missense; Polycythemia; Proto-Oncogene Proteins c-sis; Respiratory Rate; RNA, Messenger; Up-Regulation; Vascular Endothelial Growth Factor A

Erythropoietin (EPO), a type I cytokine originally identified for its critical role in hematopoiesis, has been shown to have nonhematopoietic, tissue-protective effects, including suppression of atherosclerosis. However, prothrombotic effects of EPO hinder its potential clinical use in nonanemic patients. In the present study, we investigated the antiatherosclerotic effects of helix B surface peptide (HBSP), a nonerythropoietic, tissue-protective compound derived from EPO, by using human umbilical vein endothelial cells (HUVECs) and human monocytic THP-1 cells in vitro and Watanabe heritable hyperlipidemic spontaneous myocardial infarction (WHHLMI) rabbits in vivo. In HUVECs, HBSP inhibited apoptosis (≈70%) induced by C-reactive protein (CRP), a direct mediator of atherosclerosis. By using a small interfering RNA approach, Akt was shown to be a key molecule in HBSP-mediated prevention of apoptosis. HBSP also attenuated CRP-induced production of tumor necrosis factor (TNF)-α and matrix metalloproteinase-9 in THP-1 cells. In the WHHLMI rabbit, HBSP significantly suppressed progression of coronary atherosclerotic lesions as assessed by mean cross-sectional stenosis (HBSP 21.3 ± 2.2% versus control peptide 38.0 ± 2.7%) and inhibited coronary artery endothelial cell apoptosis with increased activation of Akt. Furthermore, TNF-α expression and the number of M1 macrophages and M1/M2 macrophage ratio in coronary atherosclerotic lesions were markedly reduced in HBSP-treated animals. In conclusion, these data demonstrate that HBSP suppresses coronary atherosclerosis, in part by inhibiting endothelial cell apoptosis through activation of Akt and in association with decreased TNF-α production and modified macrophage polarization in coronary atherosclerotic lesions. Because HBSP does not have the prothrombotic effects of EPO, our study may provide a novel therapeutic strategy that prevents progression of coronary artery disease.

Topics: Animals; Apoptosis; Cardiovascular Agents; Cell Line; Coronary Artery Disease; Disease Models, Animal; Erythropoietin; Female; Human Umbilical Vein Endothelial Cells; Humans; Male; Matrix Metalloproteinase 9; Peptide Fragments; Proto-Oncogene Proteins c-akt; Rabbits; Tumor Necrosis Factor-alpha

Long noncoding RNA (lncRNA) within mRNA sequences of Alzheimer's disease genes, namely, APP, APOE, PSEN1, and PSEN2, has been analyzed using fractal dimension (FD) computation and correlation analysis. We examined lncRNA by comparing mRNA FD to corresponding coding DNA sequences (CDSs) FD. APP, APOE, and PSEN1 CDSs select slightly higher FDs compared to the mRNA, while PSEN2 CDSs FDs are lower. The correlation coefficient for these sequences is 0.969. A comparative study of differentially expressed MAPK signaling pathway lncRNAs in pancreatic cancer cells shows a correlation of 0.771. Selection of higher FD CDSs could indicate interaction of Alzheimer's gene products APP, APOE, and PSEN1. Including hypocretin sequences (where all CDSs have higher fractal dimensions than mRNA) in the APP, APOE, and PSEN1 sequence analyses improves correlation, but the inclusion of erythropoietin (where all CDSs have higher FD than mRNA) would suppress correlation, suggesting that HCRT, a hypothalamus neurotransmitter related to the wake/sleep cycle, might be better when compared to EPO, a glycoprotein hormone, for targeting Alzheimer's disease drug development. Fractal dimension and entropy correlation have provided supporting evidence, consistent with evolutionary studies, for using a zebrafish model together with a mouse model, in HCRT drug development.

Topics: 5'-Nucleotidase; Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Apolipoproteins E; Computational Biology; Disease Models, Animal; Drug Discovery; Erythropoietin; Fractals; Glycoproteins; Humans; Intracellular Signaling Peptides and Proteins; Mice; Neuropeptides; Orexin Receptors; Orexins; Presenilin-2; Receptors, Erythropoietin; RNA, Long Noncoding; RNA, Messenger; Sequence Analysis, RNA; Zebrafish

Erythropoietin regulates erythropoiesis in a hypoxia-inducible manner. Here we generate inherited super-anaemic mice (ISAM) as a mouse model of adult-onset anaemia caused by erythropoietin deficiency. ISAM express erythropoietin in the liver but lack erythropoietin production in the kidney. Around weaning age, when the major erythropoietin-producing organ switches from the liver to the kidney, ISAM develop anaemia due to erythropoietin deficiency, which is curable by administration of recombinant erythropoietin. In ISAM severe chronic anaemia enhances transgenic green fluorescent protein and Cre expression driven by the complete erythropoietin-gene regulatory regions, which facilitates efficient labelling of renal erythropoietin-producing cells. We show that the majority of cortical and outer medullary fibroblasts have the innate potential to produce erythropoietin, and also reveal a new set of erythropoietin target genes. ISAM are a useful tool for the evaluation of erythropoiesis-stimulating agents and to trace the dynamics of erythropoietin-producing cells.

Topics: Age of Onset; Aging; Alleles; Anemia; Animals; Bone Marrow; Chronic Disease; Disease Models, Animal; Embryo Loss; Erythroblasts; Erythropoietin; Gene Expression Regulation; Green Fluorescent Proteins; Hematopoiesis; Humans; Integrases; Kidney; Liver; Mice; Mice, Knockout; RNA, Messenger; Staining and Labeling; Transgenes

To study the effect of systemic hypoxia-inducible factor prolyl hydroxylase inhibition (HIF PHDi) in the rat 50/10 oxygen-induced retinopathy (OIR) model.. Oxygen-induced retinopathy was created with the rat 50/10 OIR model. OIR animals received intraperitoneal injections of dimethyloxalylglycine (DMOG, 200 μg/g), an antagonist of α-ketoglutarate cofactor and inhibitor for HIF PHD, on postnatal day (P)3, P5, and P7. Control animals received intraperitoneal injections of PBS. On P14 and P21, animals were humanely killed and the effect on vascular obliteration, tortuosity, and neovascularization quantified. To analyze HIF and erythropoietin, rats at P5 were injected with DMOG (200 μg/g). Western blot or ELISA measured the levels of HIF-1 and Epo protein. Epo mRNA was measured by quantitative PCR.. Alternating hyperoxia and hypoxia in untreated rats led to peripheral vascular obliteration on day P14 and P21. Rats that were treated with systemic DMOG by intraperitoneal injections had 3 times less ischemia and greater peripheral vascularity (P = 0.001) than control animals treated with PBS injections. Neovascularization similarly decreased by a factor of 3 (P = 0.0002). Intraperitoneal DMOG administration increased the levels of HIF and Epo in the liver and brain. Serum Epo also increased 6-fold (P = 0.0016). Systemic DMOG had no adverse effect on growth of rats treated with oxygen.. One of the many controversies in the study of retinopathy of prematurity is whether hyperoxia or alternating hyperoxia and hypoxia creates the disease phenotype in humans. We have previously demonstrated that PHDi prevents OIR in mice exposed to 5 days of sustained 75% oxygen followed by 5 days of 21% oxygen. The 50/10 rat experiments demonstrate that PHDi is also effective in a 24-hour alternating hyperoxia-hypoxia model. The rat OIR model further validates the therapeutic value of HIF PHDi to prevent retinopathy of prematurity because it reduces oxygen-induced vascular obliteration and retinovascular growth attenuation in prolonged and/or alternating hyperoxia.

Topics: Amino Acids, Dicarboxylic; Animals; Animals, Newborn; Blotting, Western; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Hyperoxia; Hypoxia-Inducible Factor 1; Injections, Intraperitoneal; Neovascularization, Pathologic; Oxygen; Procollagen-Proline Dioxygenase; Rats; Retinal Diseases; Retinal Vessels

We investigated the metabolic and functional myocardial effects of erythropoietin (EPO) administered during resuscitation from cardiac arrest using an open-chest pig model of ventricular fibrillation and resuscitation by extracorporeal circulation, after having reported in rats a reversal of postresuscitation myocardial dysfunction associated with activation of mitochondrial protective pathways. Ventricular fibrillation was induced in 16 male domestic pigs and left untreated for 8 minutes, after which extracorporeal circulation was started and maintained for 10 additional minutes, adjusting the extracorporeal flow to provide a coronary perfusion pressure of 10 mmHg. Defibrillation was accomplished and the extracorporeal flow was adjusted to secure a mean aortic pressure of 40 mmHg or greater during spontaneous circulation for up to 120 minutes. Pigs were randomized 1:1 to receive EPO (1200 U/kg) or 0.9% NaCl before starting extracorporeal circulation. Severe postresuscitation myocardial dysfunction developed in both groups. However, recovery of myocardial function-comparing baseline with 120 minutes postresuscitation-was better in pigs treated with EPO than NaCl, as shown for left ventricular ejection fraction (from 45 ± 8% to 36 ± 9% in EPO, not significant; and from 46 ± 8% to 26 ± 8% in NaCl, P < 0.001) and for peak systolic pressure/end-systolic volume (from 2.7 ± 0.8 mmHg/mL to 2.4 ± 0.7 mmHg/mL in EPO, not significant; and from 3.0 ± 1.1 mmHg/mL to 1.8 ± 0.6 mmHg/mL, P < 0.001 in NaCl). The EPO effect was associated with significantly higher myocardial O2 consumption (12 ± 6 mL/min/unit of tissue vs 6 ± 2 mL/min/unit of tissue, P < 0.017) without effects on myocardial lactate consumption. Thus, EPO administered during resuscitation from ventricular fibrillation lessened postresuscitation myocardial stunning-an effect that could be useful clinically to help promote postresuscitation hemodynamic stability.

Topics: Animals; Cardiopulmonary Resuscitation; Disease Models, Animal; Dose-Response Relationship, Drug; Echocardiography; Electric Countershock; Erythropoietin; Extracorporeal Circulation; Hematinics; Male; Myocardial Stunning; Swine; Time Factors; Treatment Outcome; Ventricular Fibrillation; Ventricular Function, Left

Erythropoietin (EPO) promotes neurogenesis and neuroprotection. We here compared the protection induced by two EPO formulations in a rodent model of Alzheimer's disease (AD): rHu-EPO and a low sialic form, Neuro-EPO. We used the intracerebroventricular administration of aggregated Aβ₂₅₋₃₅ peptide, a non-transgenic AD model. rHu-EPO was tested at 125-500 µg/kg intraperitoneally and Neuro-EPO at 62-250 µg/kg intranasally (IN). Behavioural procedures included spontaneous alternation, passive avoidance, water-maze and object recognition, to address spatial and non-spatial, short- and long-term memories. Biochemical markers of Aβ₂₅₋₃₅ toxicity in the mouse hippocampus were examined and cell loss in the CA1 layer was determined. rHu-EPO and Neuro-EPO led to a significant prevention of Aβ₂₅₋₃₅-induced learning deficits. Both EPO formulations prevented the induction of lipid peroxidation in the hippocampus, showing an antioxidant activity. rHu-EPO (250 µg/kg) or Neuro-EPO (125 µg/kg) prevented the Aβ₂₅₋₃₅-induced increase in Bax level, TNFα and IL-1β production and decrease in Akt activation. A significant prevention of the Aβ₂₅₋₃₅-induced cell loss in CA1 was also observed. EPO is neuroprotective in the Aβ₂₅₋₃₅ AD model, confirming its potential as an endogenous neuroprotection system that could be boosted for therapeutic efficacy. We here identified a new IN formulation of EPO showing high neuroprotective activity. Considering its efficacy, ease and safety, IN Neuro-EPO is a new promising therapeutic agent in AD.

Topics: Administration, Intranasal; Alzheimer Disease; Amyloid beta-Peptides; Animals; Cell Count; Chemistry, Pharmaceutical; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Hippocampus; Humans; Interleukin-1beta; Lipid Peroxidation; Male; Maze Learning; Mice; Neuroprotective Agents; Peptide Fragments; Receptors, Erythropoietin; Recognition, Psychology; Recombinant Proteins; Retention, Psychology; Signal Transduction; Tumor Necrosis Factor-alpha

Bone-marrow mesenchymal stem cells (BMSCs) transplantation is effective for acute kidney injury (AKI) repair but with limited efficiency. In the present study, BMSCs marked by bromodeoxyuridine (BrdU) were transplanted to the AKI mouse model with erythropoietin (EPO) being subcutaneously injected. The blood urea nitrogen (BUN) and serum creatinine (Scr) levels, pathological changes, distribution of BMSCs, expressions of the cytokeratin 18 (CK18) and the stromal cell-derived factor 1 (SDF-1) in the nephridial tissues were measured. The directional migration of BMSCs to the AKI microenvironment in vitro was also tested. The results showed that BMSCs transplantation or EPO injection alone decreased the BUN and Scr levels and the acute tubular necrosis (ATN) scoring in varied degrees. The combination of these decreased the above indicators' levels significantly. BrdU(+) cells (BMSCs) were observed in the AKI nephridial tissues, and CK18 expressed in the cytoplasm of these cells. EPO injection increased the proportion of BrdU(+) cells with the enhanced expression of SDF-1 in the AKI nephridial tissues. EPO increased the migrating number of BMSCs to the AKI microenvironment in vitro, and additional anti-SDF-1 treatment with SDF-1 antibody neutralized this effect. Our results showed that EPO increased the number of the transplanted BMSCs in the injured nephridial tissues and enhanced the AKI repair effect of BMSCs transplantation. The enhanced kidney-homing efficiency for BMSCs mediated by the SDF-1/CXCR4 pathway is one of the possible mechanisms for EPO performance.

Topics: Acute Kidney Injury; Animals; Bone Marrow; Cell Movement; Cells, Cultured; Disease Models, Animal; Erythropoietin; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL

Neonatal necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of preterm infants. Increased intestinal epithelium permeability is an early event in NEC pathogenesis. Autophagy and apoptosis are induced by multiple stress pathways which may impact the intestinal barrier, and they have been associated with pathogenesis of diverse gastrointestinal diseases including inflammatory bowel disease. Using both in vitro and in vivo models, this study investigates autophagy and apoptosis under experimental NEC stresses. Furthermore this study evaluates the effect of erythropoietin (Epo), a component of breast milk previously shown to decrease the incidence of NEC and to preserve intestinal barrier function, on intestinal autophagy and apoptosis. It was found that autophagy and apoptosis are both rapidly up regulated in NEC in vivo as indicated by increased expression of the autophagy markers Beclin 1 and LC3II, and by evidence of apoptosis by TUNEL and cleaved caspase-3 staining. In the rat NEC experimental model, autophagy preceded the onset of apoptosis in intestine. In vitro studies suggested that Epo supplementation significantly decreased both autophagy and apoptosis via the Akt/mTOR signaling pathway and the MAPK/ERK pathway respectively. These results suggest that Epo protects intestinal epithelium from excessive autophagy and apoptosis in experimental NEC.

Topics: Animals; Animals, Newborn; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Caspase 3; Disease Models, Animal; Enterocolitis, Necrotizing; Epithelial Cells; Erythropoietin; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Humans; Infant, Newborn; Intestinal Mucosa; Intestines; Membrane Proteins; Microtubule-Associated Proteins; Milk, Human; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; TOR Serine-Threonine Kinases

Corneal epithelial defects may heal slowly in patients with diabetes, limbal stem cell deficiency, extensive chemical burns or anesthetized corneas. Studies have shown that erythropoietin, a glycoprotein hormone that promotes red blood cell proliferation and inhibits apoptosis of erythroid progenitors, may also exert a cytoprotective, antiapoptotic effect on nonhematopoietic cells. The aim of the study was to examine the effect of erythropoietin on the healing process of corneal epithelial erosions in rabbit eyes.. Fifteen New Zealand albino rabbits were divided into 3 groups following induction of unilateral uniform corneal epithelial erosions. The first group received local treatment with erythropoietin-containing cellulose-based gel 4 times daily; the second group received treatment with cellulose-based gel without erythropoietin 4 times daily, and the third group received no treatment. The healing process was monitored twice daily using cobalt-blue-filtered slit lamp photography and digital images of fluorescein-stained corneas until complete re-epithelization was achieved. Following re-epithelization, corneas were removed for histologic processing. One-way analysis of variance and Mann-Whitney tests were used for statistical analysis.. Mean ± SD time to complete re-epithelization was 55 ± 2.19 h in the group treated with erythropoietin-containing cellulose-based gel, 66.5 ± 14.25 h in the group treated with gel only and 62.2 ± 9.09 h in the untreated group (p = 0.16, not significant). There was no significant difference among the groups in the time to complete re-epithelization or the rate of epithelial healing. Histologic corneal evaluation revealed stromal vascularization in 2 of the 6 erythropoietin-treated rabbits and in neither of the control groups.. Erythropoietin has no beneficial effect on the rate of healing of corneal epithelial erosions in rabbit eyes, and corneal stroma neovascularization seems to be a significant adverse effect.

Topics: Animals; Corneal Diseases; Corneal Neovascularization; Corneal Stroma; Disease Models, Animal; Epithelium, Corneal; Erythropoietin; Fluorophotometry; Rabbits; Recombinant Proteins; Time Factors; Wound Healing

Cancer-associated cachexia is characterized, among other symptoms, by a dramatic loss of both muscle and fat. In addition, the cachectic syndrome is often associated with anemia. The object of the present investigation was to assess the effects of erythropoietin (EPO) treatment on experimental cancer cachexia models. The results clearly show that, in addition to the improvement of the hematocrit, EPO treatment promoted a partial preservation of adipose tissue while exerting negligible effects on muscle loss. Administration of EPO to tumor-bearing animals resulted in a significant increase of lipoprotein lipase (LPL) activity in adipose tissue, suggesting that the treatment favored triacylglycerol (TAG) accumulation in the adipose tissue. In vitro experiments using both adipose tissue slices and 3T3-L1 adipocytes suggests that EPO is able to increase the lipogenic rate through the activation of its specific receptor (EPOR). This metabolic pathway, in addition to TAG uptake by LPL, may contribute to the beneficial effects of EPO on fat preservation in cancer cachexia.

Topics: 3T3-L1 Cells; Adipose Tissue; Animals; Cachexia; Cell Differentiation; Cell Proliferation; Disease Models, Animal; Erythropoietin; Male; Mice; Muscle, Skeletal; Neoplasms; Rats; Receptors, Erythropoietin

Malaria anaemia is still a major public health problem and its pathogenesis still unclear. Interestingly, the progression of anaemia is at relatively low parasitaemia with some mortality in the semi-immune individuals in the endemic areas despite adequate erythropoietin (EPO) synthesis. A recent study has shown that treatment with exogenous anti-erythropoietin (anti-EPO) antibodies (Ab) of infected mice gives protection against malaria infection, suggesting an important role for anti-EPO Ab in malaria. The objective of the study was to evaluate anti-EPO antibody levels in anaemic condition of different strains of semi-immune mice with malaria.. Semi-immune status was attained in four mice strains (Balb/c, B6, CBA and NZW) by repeated infections with 10⁴Plasmodium berghei ANKA, and treatment with chloroquine/pyrimethamine. ELISA was used to measure anti-EPO Ab, transferrin and EPO while inflammatory cytokines measurement was done using bead-based multiplex assay kit.. The mean anti-EPO Ab levels in the mice strains [Optical Density (OD) values at 450 nm: Balb/c (2.1); B6 (1.3); CBA (1.4) and NZW (1.7)] differed (p = 0.045), and were significantly higher when compared with uninfected controls, p < 0.0001, and mean anti-EPO Ab levels in the mice strains at recovery [OD values at 450 nm: Balb/c (1.8); B6 (1.1); CBA (1.5) and NZW (1.0) also differed (p = 0.0004). Interestingly, EPO levels were significantly high in NZW and low in Balb/c mice (p < 0.05), with those of B6 and CBA of intermediary values. Again, NZW were highly parasitaemic (20.7%) and the other strains (Balb/c, B6 and CBA) ranged between 2.2-2.8% (p = 0.015). Anti-EPO Ab correlated positively with extent of Hb loss (r = 0.5861; p = 0.003). Correlation of anti-EPO antibody with EPO was significant only in Balb/c mice (r = -0.83; p = 0.01). Significant levels of IL6 and IFNγ (p < 0.0001), both known to be associated with erythropoiesis suppression were observed in the Balb/c. Transferrin was significantly lower in Balb/c (p < 0.0001) when compared with the other mice strains (B6, CBA and NZW).. This is the first ever report in estimating endogenous anti-EPO antibodies in malaria anaemia. The data presented here suggest that anti-EPO Ab is produced at infection and is associated with Hb loss. Host factors appear to influence anti-EPO antibody levels in the different strains of mice.

Topics: Anemia; Animals; Autoantibodies; Disease Models, Animal; Erythropoietin; Malaria; Mice; Plasmodium berghei

Erythropoietin (EPO) has been shown to be neuroprotective in various models of neuronal injury. The aim of the present study was to investigate the beneficial effect of recombinant human EPO (rhEPO) following intracerebral hemorrhage (ICH) and the underlying molecular and cellular mechanisms. ICH was induced using autologous blood injection in adult rats. rhEPO (5000 IU/kg) or vehicle was administered to rats with ICH 2 h following surgery and every 24 h for 1 or 3 days. To study the involvement of the PI3K signaling pathway in the rhEPO‑mediated effect, the PI3K inhibitor wortmannin (15 µg/kg), was intravenously administered to rats with ICH 90 min prior to rhEPO treatment. Brain edema was measured 3 days following ICH and behavioral outcomes were measured at 1, 7, 14, 21 and 28 days following ICH using the modified neurological severity score (mNSS) and the corner turn test. Proinflammatory cytokines, including tumor necrosis factor (TNF)‑α, interleukin (IL)-1β and IL-6, in the ipsilateral striatum were analyzed using an enzyme-linked immunosorbent assay 24 h following ICH. Neuronal apoptosis in the perihematomal area was determined by NeuN and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) double-staining. The results showed that rhEPO treatment reversed ICH, increased brain water content, upregulated proinflammatory cytokines, neuronal loss and apoptosis in the perihematomal area and rescued behavioral deficits in injured rats. Inhibiting the PI3K pathway with wortmannin abolished the rhEPO‑mediated neuroprotective effects. Moreover, western blot analysis showed that rhEPO induced the upregulation of Akt phosphorylation and downregulation of glycogen synthase kinase (GSK)‑3β phosphorylation, which were reversed by pretreatment with wortmannin, indicating the involvement of PI3K signaling in rhEPO-mediated anti-apoptotic and anti-inflammatory effects following ICH. In conclusion, these results suggested that rhEPO may exert its beneficial effects in ICH through the activation of the PI3K signaling pathway.

Topics: Animals; Apoptosis; Behavior, Animal; Blotting, Western; Brain Injuries; Cells, Cultured; Cerebral Hemorrhage; Cytokines; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Fluorescent Antibody Technique; Male; Neuroprotective Agents; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Signal Transduction; Stroke

The protective role of recombinant human erythropoietin (RHE) against cisplatin-induced nephrotoxicity has been reported, but the role of sex differences is not clearly known. The aim of this study was to determine the sex-based difference in the protective effect of RHE against cisplatin-induced nephrotoxicity.. Thirty-three Wistar rats were divided into 6 groups. According to protocol l, male and female rats were treated with RHE (100 IU/kg/d) for 3 days and then received a single dose of cisplatin (7 mg/kg). According to protocol 2, the rats received the same single dose of cisplatin and then were treated with RHE for 7 days. Two other groups of male and female rats received a similar regimen of protocol 2 except for saline instead of RHE. All the animals were sacrificed 1 week after cisplatin administration.. All of the experimental animals experienced weight loss. The percentage change of weight in male rats with protocol 1 was significantly less than that in male rats in protocol 2 and control groups. However, in female groups, the percentage of change in weights was slightly higher with protocol 2 than with protocol 1 and control treatment. Administration of RHE significantly decreased changes in serum creatinine, BUN, and malondialdehyde levels in male rats, but not in females. No significant difference was observed in serum nitrite level, kidney weight, and kidney damage score between the groups.. This study suggested that erythropoietin may lead to different responses against cisplatin-induced nephrotoxicity in male and female rats.

Topics: Acute Kidney Injury; Animals; Antineoplastic Agents; Blood Urea Nitrogen; Body Weight; Cisplatin; Creatinine; Disease Models, Animal; Erythropoietin; Female; Kidney; Male; Malondialdehyde; Nitrites; Organ Size; Random Allocation; Rats; Rats, Wistar; Sex Characteristics; Sex Factors

Up to 65% of untreated infants suffering from moderate to severe hypoxic-ischemic encephalopathy (HIE) are at risk of death or major disability. Therapeutic hypothermia (HT) reduces this risk to approximately 50% (number needed to treat: 7-9). Erythropoietin (Epo) is a neuroprotective treatment that is promising as an adjunctive therapy to decrease HIE-induced injury because Epo decreases apoptosis, inflammation, and oxidative injury and promotes glial cell survival and angiogenesis. We hypothesized that HT and concurrent Epo will be safe and effective, improve survival, and reduce moderate-severe cerebral palsy (CP) in a term nonhuman primate model of perinatal asphyxia.. Thirty-five Macaca nemestrina were delivered after 15-18 min of umbilical cord occlusion (UCO) and randomized to saline (n = 14), HT only (n = 9), or HT+Epo (n = 12). There were 12 unasphyxiated controls. Epo (3,500 U/kg × 1 dose followed by 3 doses of 2,500 U/kg, or Epo 1,000 U/kg/day × 4 doses) was given on days 1, 2, 3, and 7. Timed blood samples were collected to measure plasma Epo concentrations. Animals underwent MRI/MRS and diffusion tensor imaging (DTI) at <72 h of age and again at 9 months. A battery of weekly developmental assessments was performed.. UCO resulted in death or moderate-severe CP in 43% of saline-, 44% of HT-, and 0% of HT+Epo-treated animals. Compared to non-UCO control animals, UCO animals exhibit poor weight gain, behavioral impairment, poor cerebellar growth, and abnormal brain DTI. Compared to UCO saline, UCO HT+Epo improved motor and cognitive responses, cerebellar growth, and DTI measures and produced a death/disability relative risk reduction of 0.911 (95% CI -0.429 to 0.994), an absolute risk reduction of 0.395 (95% CI 0.072-0.635), and a number needed to treat of 2 (95% CI 2-14). The effects of HT+Epo on DTI included an improved mode of anisotropy, fractional anisotropy, relative anisotropy, and volume ratio as compared to UCO saline-treated infants. No adverse drug reactions were noted in animals receiving Epo, and there were no hematology, liver, or kidney laboratory effects.. HT+Epo treatment improved outcomes in nonhuman primates exposed to UCO. Adjunctive use of Epo combined with HT may improve the outcomes of term human infants with HIE, and clinical trials are warranted.

Topics: Animals; Asphyxia; Brain; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Humans; Hypothermia; Hypoxia-Ischemia, Brain; Infant; Macaca nemestrina; Recombinant Proteins; Treatment Outcome

Topics: Angiogenesis Inducing Agents; Animals; Disease Models, Animal; Erythropoietin; Hematopoietic Stem Cell Transplantation; Hindlimb; Injections, Intramuscular; Ischemia; Male; Mice; Mice, Inbred C57BL; Random Allocation; Treatment Outcome

Fatty liver disease increases the risk in major liver resection for patients. As previous studies suggested that vascular endothelial growth factor (VEGF) and erythropoietin (EPO) might improve liver regeneration in nonobese animals, we investigated their effect after subtotal hepatectomy (SH) in rats with diet-induced steatosis.. Male Wistar rats were fed with fatty liver-inducing diet (FLD) or normal diet (control) for 11-12 weeks followed by 90% SH. Animals were treated either with EPO, VEGF or NaCl on postoperative days 0, 1 and 3 and sacrificed 24 h or 7 days after SH. Survival rate, liver regeneration and biochemical markers were assessed. Expression of inflammatory cytokines (TNF-α, IL-6) and apoptosis-related genes (PUMA, Bcl-2) was measured by qRT-PCR.. Seven-day survival after SH was significantly decreased in the FLD group compared to controls (50 vs. 100%, p < 0.05). In FLD animals, treatment with VEGF increased 7-day survival to 90% compared to only 40% in the EPO group. After surgery, blood glucose levels of VEGF but not EPO- or NaCl-treated animals remained normal. Inflammatory genes were markedly upregulated in the EPO group 24 h after SH.. Steatosis severely impairs survival and regeneration after extensive liver resection, which can be counteracted at least in part by perioperative treatment with VEGF.

Topics: Animals; Apoptosis Regulatory Proteins; Blood Glucose; Diet; Disease Models, Animal; Erythropoietin; Fatty Liver; Gene Expression; Genes, bcl-2; Gluconeogenesis; Hepatectomy; Interleukin-6; Liver Regeneration; Male; Rats; Rats, Wistar; Survival Rate; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

To identify the mutation responsible for an abnormal electroretinogram (ERG) in a transgenic mouse line (tg21) overexpressing erythropoietin (Epo). The tg21 line was generated on a mixed (C3H; C57BL/6) background and lacked the b-wave component of the ERG. This no-b-wave (nob) ERG is seen in other mouse models with depolarizing bipolar cell (DBC) dysfunction and in patients with the complete form of congenital stationary night blindness (cCSNB). We determined the basis for the nob ERG phenotype and screened C3H mice for the mutation to evaluate whether this finding is important for the vision research community.. ERGs were used to examine retinal function. The retinal structure of the transgenic mice was investigated using histology and immunohistochemistry. Inverse PCR was performed to identify the insertion site of the Epo transgene in the mouse genome. Affected mice were backcrossed to follow the inheritance pattern of the nob ERG phenotype. Quantitative real-time PCR (qRT PCR), Sanger sequencing, and immunohistochemistry were used to identify the mutation causing the defect. Additional C3H sublines were screened for the detected mutation.. Retinal histology and blood vessel structure were not disturbed, and no loss of DBCs was observed in the tg21 nob mice. The mutation causing the nob ERG phenotype is inherited independently of the tg21 transgene. The qRT PCR experiments revealed that the nob ERG phenotype reflected a mutation in Gpr179, a gene involved in DBC signal transduction. PCR analysis confirmed the presence of the Gpr179(nob5) insertional mutation in intron 1 of Gpr179. Screening for mutations in other C3H-derived lines revealed that C3H.Pde6b(+) mice carry the Gpr179 (nob5) allele whereas C3H/HeH mice do not.. We identified the presence of the Gpr179(nob5) mutation causing DBC dysfunction in a C3H-derived transgenic mouse line. The nob phenotype is not related to the presence of the transgene. The Gpr179(nob5) allele can be added to the list of background alleles that impact retinal function in commonly used mouse lines. By providing primers to distinguish between Gpr179 mutant and wild-type alleles, this study allows investigators to monitor for the presence of the Gpr179(nob5) mutation in other mouse lines derived from C3H.

Topics: Alleles; Animals; Crosses, Genetic; Disease Models, Animal; Electroretinography; Erythropoietin; Female; Founder Effect; Gene Expression; Humans; Male; Mice; Mice, Inbred C3H; Mice, Transgenic; Mutagenesis, Insertional; Night Blindness; Receptors, G-Protein-Coupled; Retinal Bipolar Cells; Signal Transduction; Transgenes

Inflammatory bowel disease has been linked to elevated T cells. Excessive production of reactive oxygen species and apoptosis are known to be accompanied by intestinal inflammation. This study was designed to investigate the effects of melatonin (MEL) and erythropoietin (EPO), which is a known anti-inflammatory and antiapoptotic agent, in dinitrobenzene sulfonic acid (DNBS)-induced colitis in pinealectomized (Px) rats. In microscopically results, epithelial and goblet cell loss, absence of crypts, and increased colonic caspase-3 activity were observed in the DNBS group. Also, in flow cytometric analysis, the percentage of CD4+ T cells was highest in the DNBS group. Treatment with MEL or EPO had a curative effect on DNBS-induced colitis. The MEL + EPO groups showed significantly greater improvement when compared with the other treatment groups. Our results indicate that the combination of EPO and MEL may exert more beneficial effects than either agent used alone.

Topics: Animals; Anti-Inflammatory Agents; Benzenesulfonates; Caspase 3; CD4-Positive T-Lymphocytes; Colitis; Disease Models, Animal; Drug Therapy, Combination; Epithelial Cells; Erythropoietin; Flow Cytometry; Goblet Cells; Male; Melatonin; Rats; Rats, Wistar

Mild traumatic brain injury (mTBI) results in an estimated 75-90% of the 1.7 million TBI-related emergency room visits each year. Post-concussion symptoms, which can include impaired memory problems, may persist for prolonged periods of time in a fraction of these cases. The purpose of this study was to determine if an erythropoietin-mimetic peptide, pyroglutamate helix B surface peptide (pHBSP), would improve neurological outcomes following mTBI. Sixty-four rats were randomly assigned to pHBSP or control (inactive peptide) 30 μg/kg IP every 12 h for 3 days, starting at either 1 hour (early treatment) or 24 h (delayed treatment), after mTBI (cortical impact injury 3 m/sec, 2.5 mm deformation). Treatment with pHBSP resulted in significantly improved performance on the Morris water maze task. Rats that received pHBSP required 22.3±1.3 sec to find the platform, compared to 26.3±1.3 sec in control rats (p=0.022). The rats that received pHBSP also traveled a significantly shorter distance to get to the platform, 5.0±0.3 meters, compared to 6.1±0.3 meters in control rats (p=0.019). Motor tasks were only transiently impaired in this mTBI model, and no treatment effect on motor performance was observed with pHBSP. Despite the minimal tissue injury with this mTBI model, there was significant activation of inflammatory cells identified by labeling with CD68, which was reduced in the pHBSP-treated animals. The results suggest that pHBSP may improve cognitive function following mTBI.

Topics: Animals; Brain; Brain Concussion; Disease Models, Animal; Erythropoietin; Maze Learning; Motor Activity; Neuroprotective Agents; Oligopeptides; Rats; Rats, Long-Evans; Recovery of Function

Cerebral malaria (CM) is a life-threatening complication of Plasmodium falciparum infection. The pathogenesis of CM is complex. Cerebral metabolic dysfunction is implicated in CM, which may be caused by both an impaired cerebral microcirculation and a dysregulated inflammatory response affecting cellular respiration of mitochondria. Recombinant human erythropoietin (rhEPO) is a promising new therapy that has been shown to reduce mortality in a mouse model of CM. In order to further elucidate the metabolic dysfunction in CM the objective of the present study was to assess brain mitochondrial respiratory function in CM with and without rhEPO treatment. The P. berghei ANKA - C57BL/6 murine model of CM was used. Mitochondrial respiration was analyzed in brain homogenates using high-resolution respirometry and a multiple substrate and inhibitor protocol. The animals were divided into four groups; infected injected with saline or with rhEPO, non-infected injected with saline or with rhEPO. Infected mice developed CM and treatment with rhEPO attenuated clinical signs of disease. There were no differences in respiratory parameters of brain mitochondria between infected and non-infected mice and no connection between disease severity and mitochondrial respiratory function. Treatment with rhEPO similarly had no effect on respiratory function. Thus cerebral metabolic dysfunction in CM does not seem to be directly linked to altered mitochondrial respiratory capacity as analyzed in brain homogenates ex vivo. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Topics: Animals; Disease Models, Animal; Erythropoietin; Female; Humans; Malaria, Cerebral; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Plasmodium falciparum; Recombinant Proteins

Topics: Animals; Disease Models, Animal; Erythropoietin; Hindlimb; Injections, Intramuscular; Mice; Neovascularization, Pathologic

To study the effect of erythropoietin (EPO) treatment on renal and lung injury following renal ischemia/reperfusion (I/R).. Thirty male Wistar rats were assigned to three groups of 10 rats each. The first group was sham-operated, the second was subjected to renal I/R (30 min of ischemia followed by 24 h of reperfusion). The third group was subjected to renal I/R and treated with EPO in two doses: the first dose 1 h prior to ischemia (1,000 U/kg) and the second dose 6 h after ischemia (1,000 U/kg).. The renal and lung tissue injury index, tissue serum blood urea nitrogen and creatinine (Cr) were higher in the renal I/R group compared to the renal I/R + EPO group; the difference was statistically significant (p < 0.05). Kidney and lung tissue glutathione peroxidase and superoxide dismutase levels were higher in the renal I/R + EPO group than the renal I/R group; the difference was also statistically significant (p < 0.05).. The data showed that EPO pretreatment could be effective in reducing renal and lung injury following renal I/R and could improve the cellular antioxidant defense system. Hence EPO pretreatment may be effective for attenuating renal and lung injury after renal I/R-induced injury during surgical procedures, hypotension, renal transplantation and other conditions inducing renal I/R.

Topics: Animals; Blood Urea Nitrogen; Creatinine; Disease Models, Animal; Erythropoietin; Glutathione Peroxidase; Kidney Diseases; Lung Diseases; Male; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase

It has been recently shown that the biological effects of erythropoietin (EPO) are not limited to the hematopoietic compartment but, as pleiotropic glycoprotein, this hormone can exert pro-angiogenic and tissue-protective functions also in a wide range of non-hematopoietic organs. The role of EPO and the effective functionality of its receptor in solid tumors are still a controversial point of debate. In the present work we analyzed the gene expression of EPO and its cognate receptor (EpoR) in a rat model of thioacetamide-induced damage and tumor. An analysis of the EPO/EpoR axis was also performed on human cholangiocarcinoma (CC) cell lines. A progressive increase of EPO and EpoR mRNA can already be observed during the fibrotic-cirrhotic development with a peak of expression rising at tumor formation (24.7 ± 9.9-fold increase and 15.5 ± 1.1-fold increase, respectively, for the two genes). Co-localization studies by immunofluorescence revealed hepatocytes in the regenerative cirrhotic nodules (Hep Par-1(+)) and in the dysplastic bile duct cells (CK19(+)) as the major EPO producers in this specific condition. The same cell populations, together with endothelial cells, exhibited an increased expression of EpoR, although all the non-parenchymal cell populations in the liver exhibited modest basal mRNA levels. Challenging human CC cells, Mz-Cha-2, with a combination of EPO and SCF resulted in a synergistic effect on the gene expression of EPO, CyclinD1 and PCNA. This study suggests that the autocrine and paracrine release of endogenous EPO in the microenvironment may contribute to the development and maintenance of the CC possibly in cooperation with other signaling pathways.

Topics: Animals; Bile Duct Neoplasms; Cholangiocarcinoma; Disease Models, Animal; Erythropoietin; Humans; Liver; Male; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; RNA, Messenger; Thioacetamide; Tumor Cells, Cultured

Acute ethanol intoxication has been shown to cause oxidative damage in many organ systems including the brain. Erythropoietin has antioxidant effects and prevents neuronal damage in the animal model of ischemic brain injury. In this study, we aimed to investigate the effects of darbepoetin alpha, an analog of erythropoietin with a longer half-life and higher in vivo activity, on ethanol-induced acute brain injury.. Forty-eight Wistar albino rats were allocated to four groups. The first group received ethanol treatment (E), the second group was treated with ethanol and darbepoetin (ED), the third group received only saline treatment (S), and the fourth group received both saline and darbepoetin treatment (SD). Plasma S100-β and neuron-specific enolase (NSE) levels were measured. Histopathological evaluation of the brains was performed.. The plasma S100-β and NSE levels were significantly lower in group ED compared with group E. In group E, we have observed focal red-neuron formation at the granular layer of the dentate gyrus. We did not observe any histopathological changes in the other groups (ED, S, and SD).. Our findings suggest that darbepoetin alpha has neuroprotective effect in acute ethanol intoxication, possibly through its antioxidant effect.

Topics: Alcoholic Intoxication; Animals; Darbepoetin alfa; Disease Models, Animal; Erythropoietin; Ethanol; Male; Neurons; Rats; Rats, Wistar

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of β-thalassemia and sickle cell anemia (Berkeley mice). β- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human β-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in β- thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of β-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies.

Topics: Anemia, Sickle Cell; Animals; beta-Thalassemia; Darbepoetin alfa; Disease Models, Animal; Erythrocyte Count; Erythropoietin; Female; Hematinics; Hemoglobins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Receptors, Erythropoietin; Recombinant Fusion Proteins

The liver shows remarkable regeneration ability after damage or resection. The main stimulant for hepatic regeneration is resection. Erythropoietin (EPO), which was initially used for anemia therapy, is today known as a general tissue protector owing to its anti-inflammatory, anti-oxidant, anti-apoptotic, and angiogenic properties. This study aims to investigate the effect of systemically administered EPO on liver regeneration after partial hepatectomy.. Forty-eight male Wistar albino rats were randomly split in two groups A and B consisting of 24 rats each. Standard 70% hepatectomy was performed on the rats in group A. The same surgical procedure was performed on the rats in group B, and they were additionally administered 3000 U/kg/subcutaneous EPO. The rats were sacrificed 24, 48, and 72 h after resection. The groups were compared in terms of biochemical, morphological, and histopathological parameters.. The biochemical results showed that the administration of EPO decreased aspartate aminotransferase levels significantly (p < 0.05) at 24 h after hepatectomy. A comparison of the groups in terms of relative liver weight showed that EPO-treated groups exhibited a statistically significant increase (p < 0.05) for all three time periods. Histopathology results showed that in the EPO-treated groups, the mitosis index at 48 and 72 h, double nuclei cell number at 72 h, and proliferating cell nuclear antigen ratio at 48 h showed a significant increase (p < 0.05).. Our study showed that systemically administering high-dose EPO increases regeneration by affecting the biochemical, morphological, and histopathological parameters after liver resection.

Topics: Animals; Cell Nucleus; Disease Models, Animal; Erythropoietin; Hepatectomy; Histocytochemistry; Liver; Liver Regeneration; Male; Organ Size; Random Allocation; Rats; Rats, Wistar

Septic encephalopathy is characterized by changes in mental status and an increase in neuronal apoptosis. Accumulating evidence has shown that recombinant human erythropoietin (rhEPO) protects brain against ischemia and hypoxia injury. However, whether rhEPO exerts neuroprotective effects on septic encephalopathy remains unclear. We designed the current study to evaluate possible neuroprotection of rhEPO in a model of sepsis.. For this in vitro study, we determined hippocampal neuronal apoptosis by lactate dehydrogenase release, cell counting kit-8 assay, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining after treatment with lipopolysaccharide. We transfected the signal transducer and activator of transcription 3 (STAT3) short hairpin RNA at 14 d in vitro for 48 h. For the in vivo study, we performed cecal ligation and peroration surgery. We detected the expression of phospho-Janus-activated kinase 2 (JAK2), total JAK2, phospho-STAT3, total STAT3, Bax and Bcl-XL by Western blot, and examined behavior using the Morris water maze.. Treatment with rhEPO reduces apoptosis and increases cell viability in lipopolysaccharide-treated neuronal cultures. In cecal ligation and peroration rats, rhEPO attenuated the inhibition of phospho-JAK2 and phospho-STAT3. In addition, rhEPO enhanced the expression of Bcl-XL, but depressed Bax, which was abolished by additional administration of inhibitor of JAK2/STAT3 signaling 2-cyano-3-(3,4-dihydroxyphenyl)-N-(benzyl)-2-propenamide,2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-2-propenamide or (E)-3(6-bromopyridin-2-yl)-2-cyano-N-([S0-1-phenylethyl]acrylamide)in vivo, and was ameliorated by STAT3 short hairpin RNA transfection in vitro. Alternatively, we confirmed the neuronal protective effect of rhEPO by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelingstaining. For the Morris water maze study, rhEPO improved learning and memory disorders without an alternation in locomotor activity.. These results indicated that rhEPO improves brain dysfunction by reducing neuronal apoptosis, and JAK2/STAT3 signaling is likely to be involved. Application of rhEPO may serve as a potential therapy for the treatment of septic encephalopathy.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Brain Diseases; Cecum; Cell Survival; Cognition Disorders; Disease Models, Animal; Endotoxemia; Erythropoietin; Humans; Janus Kinase 2; Learning Disabilities; Ligation; Lipopolysaccharides; Male; Memory Disorders; Neurons; Rats; Rats, Sprague-Dawley; Recombinant Proteins; STAT3 Transcription Factor

Studies on the neuritis model suggest that in many patients with neuropathic pain, symptoms may be due to nerve inflammation rather than frank nerve injury. Treatments for these patients are often ineffective. The neuroprotective and hematopoietic agent erythropoietin (EPO) has been shown to reverse pain behaviors in nerve injury models and therefore may be of therapeutic benefit. However, EPO can cause thrombosis. ARA290 is an analog of EPO that has the neuroprotective activities of EPO without stimulating hematopoiesis. The present study has examined the effects of ARA290 on pain behavior in the neuritis model. Following neuritis induction, 30 or 120 μg/kg ARA290 or saline vehicle was injected intraperitoneally into rats daily from day 1 post surgery. Animals were assessed for mechanical allodynia and heat hyperalgesia. Levels of the cytokine tumor necrosis factor-α (TNF-α) and chemokine (CC motif) ligand 2 (CCL2) mRNA were also assessed using polymerase chain reaction. Vehicle-treated neuritis animals (n=20) developed signs of mechanical allodynia and heat hyperalgesia that reached a maximum on day 4 and 3 of testing, respectively. Treatment with either 30 (n=11) or 120 μg/kg ARA290 (n=9) prevented the development of mechanical allodynia. However, ARA290 did not significantly affect heat hyperalgesia. There was no significant difference between the effects of each drug dose (p<0.05, unpaired t test comparing area under the curve for mechanical allodynia). The levels of CCL2 and TNF-α mRNA in the nerve and Gelfoam were not significantly different following 120 μg/kg ARA290 treatment (n=3-7) compared to vehicle-treated animals (n=3-7; p=0.24; unpaired t tests). In summary, ARA290 may be beneficial in the treatment of neuropathic pain symptoms where signs of nerve injury are absent on clinical assessment. The mechanisms of action do not appear to involve the inhibition of TNF-α or CCL2 production.

Topics: Animals; Chemokine CCL2; Disease Models, Animal; Erythropoietin; Hyperalgesia; Male; Neuralgia; Neuritis; Neuroprotective Agents; Oligopeptides; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sciatic Nerve; Tumor Necrosis Factor-alpha

Pre-treatment with erythropoietin (EPO) has been demonstrated to exert tissue-protective effects against 'ischemia-reperfusion'-type injuries. This protection might be mediated by mobilization of bone marrow endothelial progenitor cells (EPCs), which are thought to secrete paracrine factors. These effects could be exploited to protect against tissue injury induced in cases where hemorrhage is foreseeable, for example, prior to major surgery. Here, we investigate the effects of EPO pre-treatment on the organ injury and dysfunction induced by hemorrhagic shock (HS). Recombinant human EPO (1000 IU/kg/day i.p.) was administered to rats for 3 days. Rats were subjected to HS on day 4 (pre-treatment protocol). Mean arterial pressure was reduced to 35 ± 5 mmHg for 90 minutes, followed by resuscitation with 20 ml/kg Ringer's lactate for 10 minutes and 50% of the shed blood for 50 minutes. Rats were sacrificed 4 hours after the onset of resuscitation. EPC (CD34(+)/flk-1(+) cell) mobilization was measured following the 3-day pre-treatment with EPO and was significantly increased compared with rats pre-treated with phosphate-buffered saline. EPO pre-treatment significantly attenuated organ injury and dysfunction (renal, hepatic and neuromuscular) caused by HS. In livers from rats subjected to HS, EPO enhanced the phosphorylation of Akt (activation), glycogen synthase kinase-3β (GSK-3β; inhibition) and endothelial nitric oxide synthase (eNOS; activation). In the liver, HS also caused an increase in nuclear translocation of p65 (activation of NF-κB), which was attenuated by EPO. This data suggests that repetitive dosing with EPO prior to injury might protect against the organ injury and dysfunction induced by HS, by a mechanism that might involve mobilization of CD34(+)/flk-1(+) cells, resulting in the activation of the Akt-eNOS survival pathway and inhibition of activation of GSK-3β and NF-κB.

Topics: Animals; Antigens, CD34; Blood Circulation; Cell Nucleus; Disease Models, Animal; Erythropoietin; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hematocrit; Humans; Liver; Male; Nitric Oxide Synthase Type III; Organ Specificity; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Shock, Hemorrhagic; Transcription Factor RelA; Vascular Endothelial Growth Factor Receptor-2

To investigate whether systemic erythropoietin administration can prevent secondary burn progression in an experimental model and to elucidate the underlying mechanisms.. Prospective study.. University-based laboratory research.. Twenty-one male Wistar rats.. The burn comb model creates four rectangular burned surfaces that are intercalated by three unburned zones (interspaces) prone to secondary necrosis. Twenty-one animals were randomized to three experimental groups: 1) Local cooling with water for 20 minutes (control, 17°C); 2) and 3) local cooling with water and intraperitoneal erythropoietin once a day for five days starting 45 minutes after burn injury (500 IU/kg body weight: EPO 500 or 2500 IU/kg body weight: EPO 2500).. Secondary burn progression-both in depth (histology) and in surface (planimetry)-as well as interspace perfusion (laser Doppler flowmetry) and hematocrit were analyzed. Further, dilatory response (inducible nitric oxide synthase expression), inflammation (leukocyte count), and angiogenesis (CD31 expression) were assessed. Finally, wound healing time and contracture rate were reported. Burn progression resulted in complete dermal destruction as well as in important interspace necrosis in control animals, whereas burn progression was significantly reduced in a dose-dependent manner in animals treated with erythropoietin. Tissue protection was associated with an increased interspace perfusion with EPO 500, but not with EPO 2500, and was paralleled by a significant increase in inducible nitric oxide synthase expression and decreased inflammation, independent of the erythropoietin dosage. EPO 2500 led to a significant increase of hematocrit at day 4. Finally, faster wound healing and less contracture were observed in animals treated with EPO 500 only.. Erythropoietin represents an easy-to-use therapeutic approach to prevent secondary burn progression, i.e., to control damage after burn injury. It preserves microcirculatory perfusion within the endangered areas in a dose-dependent manner.

Topics: Animals; Burns; Disease Models, Animal; Disease Progression; Erythropoietin; Male; Necrosis; Neovascularization, Physiologic; Oxidative Stress; Prospective Studies; Random Allocation; Rats; Rats, Wistar; Wound Healing

The involvement of erythropoietin in cardiac adaptation to acute and chronic (CHx) hypoxia was investigated in erythropoietin deficient transgenic (Epo-TAg(h)) and wild-type (WT) mice. Left (LV) and right ventricular functions were assessed by echocardiography and hemodynamics. HIF-1α, VEGF and Epo pathways were explored through RT-PCR, ELISA, Western blot and immunocytochemistry. Epo gene and protein were expressed in cardiomyocytes of WT mice in normoxia and hypoxia. Increase in blood hemoglobin, angiogenesis and functional cardiac adaptation occurred in CHx in WT mice, allowing a normal oxygen delivery (O2T). Epo deficiency induced LV hypertrophy, increased cardiac output (CO) and angiogenesis, but O2T remained lower than in WT mice. In CHx Epo-TAg(h) mice, LV hypertrophy, CO and O2T decreased. HIF-1α and Epo receptor pathways were depressed, suggesting that Epo-TAg(h) mice could not adapt to CHx despite activation of cardioprotective pathways (increased P-STAT-5/STAT-5). HIF/Epo pathway is activated in the heart of WT mice in hypoxia. Chronic hypoxia induced cardiac adaptive responses that were altered with Epo deficiency, failing to maintain oxygen delivery to tissues.

Topics: Adaptation, Physiological; Animals; Blotting, Western; Chronic Disease; Disease Models, Animal; Echocardiography; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Heart; Hemodynamics; Hypoxia; Male; Mice; Mice, Transgenic; Myocytes, Cardiac; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Parkinson's disease is characterized by progressive degeneration of dopaminergic neurons. Thus the development of therapeutic neuroprotection and neurorescue strategies to mitigate disease progression is important. In this study we evaluated the neuroprotective/rescue effects of erythropoietin Fc fusion protein (EPO-Fc) and carbamylated erythropoietin Fc fusion protein (CEPO-Fc) in a rat model of Parkinson's disease. Adult female Sprague-Dawley rats received intraperitoneal injection of EPO-Fc, CEPO-Fc or PBS. Behavioral evaluations consisted of rota-rod, cylinder and amphetamine-induced rotation tests. In the neuroprotection experiment, the CEPO-Fc group demonstrated significant improvement compared with the EPO-Fc group on the amphetamine-induced rotation test throughout the four-week follow-up period. Histologically, significantly more tyrosine hydroxylase (TH)-positive neurons were recognized in the substantia nigra (SN) pars compacta in the CEPO-Fc group than in the PBS and EPO-Fc groups. In the neurorescue experiment, rats receiving CEPO-Fc showed significantly better behavioural scores than those receiving PBS. The histological data concerning striatum also showed that the CEPO-Fc group had significantly better preservation of TH-positive fibers compared to the PBS and EPO-Fc groups. Importantly, there were no increases in hematocrit or hemoglobin levels in the CEPO-Fc group in either the neuroprotection or the neurorescue experiments. In conclusion, the newly developed CEPO-Fc might confer neuroprotective and neurorescue benefits in a rat model of Parkinson's disease without the side effects associated with polycythemia. CEPO-Fc might be a therapeutic tool for patients with Parkinson's disease.

Topics: Adrenergic Agents; Amphetamine; Analysis of Variance; Animals; Corpus Striatum; Disease Models, Animal; Drug Administration Schedule; Erythropoietin; Female; Hematocrit; Hemoglobins; Neuroprotective Agents; Oxidopamine; Parkinson Disease; Psychomotor Performance; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Rotarod Performance Test; Stereotyped Behavior; Substantia Nigra; Time Factors; Tyrosine 3-Monooxygenase

The Cdh23(erl/erl) mice are a novel mouse model for DFNB12 and are characterized by progressive hearing loss. In this study, erythropoietin (EPO) was given to the Cdh23(erl/erl) mice by intraperitoneal injection every other day from P7 for 7 weeks. Phosphate-buffered saline-treated or untreated Cdh23(erl/erl) mice were used as controls. Auditory-evoked brainstem response (ABR) thresholds and distortion product oto-acoustic emission (DPOAE) were measured in the mouse groups at the age of 4, 6 and 8 weeks. The results show that EPO can significantly decrease the ABR thresholds in the Cdh23(erl/erl) mice as compared with those of the untreated mice at stimulus frequencies of click, 8-, 16- and 32-kHz at three time points. Meanwhile, DPOAE amplitudes in the EPO-treated Cdh23(erl/erl) mouse group were significantly higher than those of the untreated groups at f2 frequency of 15383 Hz at the three time points. Furthermore, the mean percentage of outer hair cell loss at middle through basal turns of cochleae was significantly lower in EPO-treated Cdh23(erl/erl) mice than in the untreated mice (P<0.05). This is the first report that EPO acts as an otoprotectant in a DFNB12 mouse model with progressive hearing loss.

Topics: 3,4-Dihydroxyphenylacetic Acid; Acoustic Stimulation; Analysis of Variance; Animals; Auditory Threshold; Cadherins; Cell Count; Chi-Square Distribution; Cochlea; Disease Models, Animal; Erythropoietin; Evoked Potentials, Auditory, Brain Stem; Hair Cells, Auditory; Hearing Loss, Sensorineural; Mice; Mice, Transgenic; Mutation; Otoacoustic Emissions, Spontaneous; Time Factors

Organ shortage in liver transplantation has justified usage of marginal donor livers to expand the donor organ pool. The particular susceptibility of steatotic livers to I/R injury necessitates optimal preservation conditions in order to minimize preservation-reperfusion injury for successful transplantation.. The effect of erythropoietin (EPO) as additive to HTK preservation solution was studied in a mouse model. Lean and steatotic livers were harvested, stored for 24 h in 4°C HTK solution containing either EPO or saline and reperfused for 2 h with 37°C Krebs-Henseleit buffer. Livers without cold storage served as sham controls.. Flushing of livers upon cold storage revealed a transaminase release, which was 2- to 10-fold higher in steatotic versus lean livers. EPO was effective in reducing the enzyme release to 50% in steatotic but not in lean livers. EPO prevented cold storage-induced denudation of the endothelial lining in steatotic livers, but aggravated it in lean livers. During reperfusion, steatotic livers presented with lower oxygen consumption and higher enzyme release than lean livers. In all livers, parameters of reperfusion injury remained unaffected by EPO. Expression of UCP2 was found markedly higher in steatotic livers. After I/R, steatotic livers revealed a significant drop of UCP2, whereas expression in lean livers was only slightly affected. EPO diminished Erk phosphorylation to almost the same extent in both mouse strains.. Fortification of the preservation solution by EPO ameliorates cold ischemic injury of steatotic livers and may thus be considered for use as an adjunctive agent to increase the success of transplanting steatotic livers.

Topics: Animals; Cold Temperature; Disease Models, Animal; Erythropoietin; Fatty Liver; Female; Glucose; Ion Channels; Liver; Liver Transplantation; Male; Mannitol; Mice; Mice, Inbred Strains; Mice, Obese; Mitochondrial Proteins; Mitogen-Activated Protein Kinase Kinases; Obesity; Organ Preservation Solutions; Potassium Chloride; Procaine; Receptors, Erythropoietin; Reperfusion Injury; Signal Transduction; Thinness; Uncoupling Protein 2

Pyroglutamate helix B surface peptide (pHBSP) is an 11 amino acid peptide, designed to interact with a novel cell surface receptor, composed of the classical erythropoietin (EPO) receptor disulfide linked to the beta common receptor. pHBSP has the cytoprotective effects of EPO without stimulating erythropoiesis. Effects on early cerebral hemodynamics and neurological outcome at 2 weeks post-injury were compared in a rat model of mild cortical impact injury (3m/sec, 2.5 mm deformation) followed by 50 min of hemorrhagic hypotension (MAP 40 mm Hg for 50 min). Rats were randomly assigned to receive 5000 U/kg of EPO, 30 μg/kg of pHBSP, or an inactive substance every 12 h for 3 days, starting at the end of resuscitation from the hemorrhagic hypotension, which was 110 min post-injury. Both treatments reduced contusion volume at 2 weeks post-injury, from 20.8±2.8 mm(3) in the control groups to 7.7±2.0 mm(3) in the EPO-treated group and 5.9±1.5 mm(3) in the pHBSP-treated group (p=0.001). Both agents improved recovery of cerebral blood flow in the injured brain following resuscitation, and resulted in more rapid recovery of performance on beam balancing and beam walking tests. These studies suggest that pHBSP has neuroprotective effects similar to EPO in this model of combined brain injury and hypotension. pHBSP may be more useful in the clinical situation because there is less risk of thrombotic adverse effects.

Topics: Animals; Brain Injuries; Cerebrovascular Circulation; Disease Models, Animal; Erythropoietin; Hemodynamics; Neuroprotective Agents; Oligopeptides; Rats; Rats, Long-Evans; Recovery of Function; Shock, Hemorrhagic

The aim of this study was to determine the effects of erythropoietin (EPO), moderate hypothermia, and a combination thereof on the kidneys of newborn rats damaged during perinatal asphyxia. An animal model of perinatal asphyxia (Wistar rats) was used in which after birth, newborn rats were divided into four groups of 15 animals each: G1, rats exposed only to asphyxia; G2, rats exposed to asphyxia and hypothermia (rectal temperature 32°C) and which received EPO (darbepoetin alpha) intraperitoneally; G3, rats exposed to asphyxia and hypothermia; G4, rats exposed to asphyxia and which received EPO. The rats were sacrificed on the 7th day of life and histopathological evaluation of kidneys was performed. Damage to the proximal tubules was significantly higher in group G1 rats than in groups G2, G3, and G4 rats (p < 0.01). Damage to the distal tubules was found only in group G1 rats. Histological changes in the proximal tubules were more prominent than in the distal tubules (p < 0.01). The immature glomeruli zone was less expressed in group G4 rats than in groups G1, G2, and G3 rats (p < 0.01). Based on these results, we conclude that EPO and hypothermia, as well as the combination thereof, have a protective effect on rats' kidneys damaged during perinatal asphyxia.

Topics: Acute Kidney Injury; Animals; Animals, Newborn; Asphyxia Neonatorum; Combined Modality Therapy; Cytoprotection; Darbepoetin alfa; Disease Models, Animal; Erythropoietin; Female; Humans; Hypothermia, Induced; Infant, Newborn; Kidney; Kidney Glomerulus; Kidney Tubules; Male; Protective Agents; Rats; Rats, Wistar; Time Factors

Hypoxia-inducible factor (HIF) activity during the course of chronic kidney disease (CKD) development is poorly defined, and the effect of HIF activation on CKD is still controversial. The purpose of the present study was to characterize HIF expression during the course of CKD development, and to investigate the effect of HIF activation on CKD by using prolyl hydroxylase (PHD) inhibitor L-mimosine.. Rats with remnant kidneys (RK) were killed at week 1, 2, 4, 6, 8, 12 after subtotal nephrectomy. An additional group of RK rats was treated with L-mimosine to study the effect of HIF-α activation.. Tubulointerstitial hypoxia in the remnant kidney began at week 1 and continued, albeit attenuated, until week 12, the last time point examined. The nuclear expression of HIF-1α and HIF-2α, as well as typical HIF target genes VEGF (vascular endothelial growth factor), HO-1 (heme oxygenase-1), GLUT-1 (glucose transporter-1) and EPO (erythropoietin), were all upregulated in the early stage of RK when renal function was stable, and returned to the basal level later, accompanied by impaired renal function and interstitial fibrosis. L-mimosine administered from week 5 to week 12 led to accumulation of HIF-1α and HIF-2α proteins, increased expression of VEGF, HO-1 and GLUT-1, and improved renal function. Furthermore, fibrosis markers α-smooth muscle actin (α-SMA) and Collagen III, as well as peritubular capillary rarefaction index, were all significantly decreased after L-mimosine treatment.. There was a transient HIF-α activation in the remnant kidney of rats at the early stage following subtotal nephrectomy. L-mimosine administered in later stages re-activated HIF-α and reduced tubulointerstitial fibrosis.

Topics: Albuminuria; Animals; Blood Pressure; Disease Models, Animal; Erythropoietin; Glucose Transporter Type 1; Heme Oxygenase-1; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Kidney Function Tests; Mimosine; Nephrectomy; Procollagen-Proline Dioxygenase; Rats; Renal Insufficiency; Up-Regulation; Vascular Endothelial Growth Factor A

Graft dysfunction of steatotic livers (SL) still remains a major challenge in liver transplantation. Different mechanisms are thought to be involved in the impaired tolerance of SL to ischemia-reperfusion injury. Thus, different pharmacologic strategies may need to be combined to effectively protect SL and to reduce graft dysfunction after transplantation. Therefore, we analyzed the effectiveness of a multidrug donor preconditioning (MDDP) procedure to protect SL from cold ischemia-reperfusion injury.. Liver steatosis was induced by a high-carbohydrate, fat-free diet. A total of 24 Sprague-Dawley rats were divided into 3 groups (n = 8 each), including a control group with nonsteatotic livers (Con), a vehicle-treated SL group (SL-Con), and a SL group undergoing MDDP (SL-MDDP), including pentoxyphylline, glycine, deferoxamine, N-acetylcysteine, erythropoietin, melatonin, and simvastatin. MDDP was applied before liver perfusion with 4°C histidine-tryptophan-ketoglutarate (HTK) solution and organ harvest. After 24 hours of cold storage in HTK, postischemic reperfusion was performed in an isolated liver reperfusion model using 37°C Krebs-Henseleit bicarbonate buffer.. After 60 minutes of reperfusion, SL showed a significant reduction of bile flow as well as a marked increase of liver enzyme levels and apoptotic cell death compared with Con. This was associated with an increased malondialdehyde formation, interleukin-1 production, and leukocytic tissue infiltration. MDDP completely abolished the inflammatory response and was capable of significantly reducing parenchymal dysfunction and injury.. MDDP decreases SL injury after cold storage and reperfusion. The concept of MDDP as a simple and safe preoperative regime, thus may be of interest in clinical use, expanding the donor pool from marginal donors.

Topics: Animals; Antioxidants; Cold Ischemia; Disease Models, Animal; Drug Therapy, Combination; Erythropoietin; Fatty Liver; Female; Glycine Agents; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Ischemic Preconditioning; Liver; Liver Transplantation; Male; Primary Graft Dysfunction; Rats; Rats, Sprague-Dawley; Siderophores; Tissue Donors

To elucidate the safety and efficacy of exogenous erythropoietin (EPO) for the protection of photoreceptor cells in a rat model of retinal detachment (RD).. Recombinant rat EPO (400 ng) was injected into the vitreous cavity of normal rats to observe the eye manifestations. Retinal function was assessed by flash electroretinograms. Histopathological examination of retinal tissue was performed at 14 days and 2 months after injection, respectively. To investigate the inhibitory effect of EPO on photoreceptor cell apoptosis in RD rats, 100, 200, or 400 ng EPO was injected into the vitreous cavity immediately after RD model establishment. Apoptosis of photoreceptor cells was determined at 3 days after injection. Caspase-3 activation was measured by western blot analysis and immunofluorescence, respectively, and the level of Bcl-X(L) expression was analyzed by western blot.. Intravitreal injection of EPO 400 ng into normal rats had no significant impact on retinal function, morphology, or structure. Apoptosis of retinal photoreceptor cells apparently increased after RD and was significantly reduced following EPO treatment. The thickness of the outer nuclear layer in the RD + 400 ng group was significantly thicker than that in other experimental RD groups both at 14 days and at 2 months after RD (P < 0.05). Western blot and immunofluorescence analyses showed decreased caspase-3 activation and increased Bcl-X(L) expression following EPO treatment.. Intravitreal injection of EPO 400 ng is safe, and EPO may suppress caspase-3 activation and enhance Bcl-X(L) expression, resulting in inhibition of apoptosis and protection of photoreceptor cells.

Topics: Animals; Apoptosis; bcl-X Protein; Blotting, Western; Caspase 3; Disease Models, Animal; Enzyme Activation; Erythropoietin; Fluorescent Antibody Technique; Intravitreal Injections; Male; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Retinal Detachment

Erythropoietin (EPO) has recently been shown to confer cardioprotective effects via angiogenesis and antiapoptosis. The administration of EPO after myocardial infarction (MI) reduces infarct size and improves cardiac function in small animals. The purpose of this study is to investigate the protective effects of EPO in porcine MI. Each animal in the EPO group received four injections of recombinant human EPO (rhEPO; 6000 U per injection) at 2-day intervals, starting after coronary occlusion. Animals in the control group received saline. Left ventriculography was performed just after coronary occlusion and at 28 days. Time-course changes in serum levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and fibroblast growth factor (FGF) were measured. The number of vessels was calculated, and the mRNA expressions of VEGF and insulin-like growth factor (IGF) were examined. Left ventricular function was similar between the groups. The numbers of cells positive for anti-α-smooth muscle actin, von Willebrand factor, and c-kit were significantly higher in the EPO group than in the controls (P < 0.05). The EPO group exhibited significantly higher HGF and FGF concentrations (P < 0.05) and higher expression of VEGF and IGF mRNA (P < 0.05) compared with the controls. In conclusion, EPO accelerates angiogenesis via the upregulation of systemic levels such as HGF and FGF, and the local expression of VEGF and IGF, in porcine MI.

Topics: Angiogenesis Inducing Agents; Animals; Disease Models, Animal; Erythropoietin; Female; Fibroblast Growth Factors; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Recombinant Proteins; Recovery of Function; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Somatomedins; Stroke Volume; Sus scrofa; Time Factors; Vascular Endothelial Growth Factor A; Ventricular Function, Left

In this study, a single intraperitoneal injection of erythropoietin (EPO) loaded oligochitosan nanoparticles (epo-NPs) (average diameter 266 nm) was investigated as a treatment for periventricular leukomalacia (PVL). Nanoparticles were fabricated using a gelation technology process. PVL rats models were prepared to examine the therapeutic efficacy of epo-NPs and analyze the mechanism by which epo-NPs protect white matter. The metabolization of epo-NPs in the liver was also investigated. The pathology and behavioral data show that this single injection of a low quantity of epo-NPs had an excellent therapeutic effect on the rat model of PVL. The EPO release curve in phosphate buffered saline solution was a good fit with the zero-order kinetics distribution and was maintained at around 25% in 48 h. In vivo experiments demonstrated that 50 IU/kg epo-NPs had the same effect as a 5000 IU/kg direct injection of free EPO. Nanoparticles prolonged the time course of EPO metabolization in the liver and the stable release of EPO from the nanoparticles kept the plasma concentration of EPO at around 100 IU/ml during the 8-12h post-injection. Therefore, we suggest that oligochitosan based nanoparticles are an effective vehicle for drug delivery.

Topics: Animals; Animals, Newborn; Behavior, Animal; Brain; Chemistry, Pharmaceutical; Chitin; Chitosan; Disease Models, Animal; Drug Carriers; Drug Compounding; Erythropoietin; GAP-43 Protein; Humans; Infant, Newborn; Injections, Intraperitoneal; Leukomalacia, Periventricular; Liver; Magnetic Resonance Imaging; Nanotechnology; Oligosaccharides; Particle Size; Rats; Solubility; Technology, Pharmaceutical

Retinal angiogenesis is a major cause of blindness in ischemic retinopathies including diabetic retinopathy and retinopathy of prematurity. Integrin αvβ3 is a promising therapeutic target for ocular angiogenesis, modulating the pro-angiogenic actions of multiple growth factors. In this study, we sought to determine the effects of the integrin αvβ3 antagonist tetra-iodothyroacetic acid (tetrac) on the angiogenic actions of VEGF and erythropoietin (EPO) in cultured human retinal endothelial cells. In addition, we investigated the effect of tetrac and a nanoparticulate formulation of tetrac on retinal angiogenesis in vivo, in the mouse oxygen-induced retinopathy (OIR) model. Tetrac inhibitory activity was evaluated in human retinal endothelial cells treated with VEGF and/or EPO. Endothelial cell proliferation, migration, and tube formation were assessed, in addition to phosphorylation of ERK1/2. For the studies of the oxygen-induced retinopathy model, C57BL/6 mice were exposed to 75% oxygen from postnatal day (P)7 to P12, and then returned to room air. Tetrac and tetrac-nanoparticle (tetrac-NP) were administered at P12 and P15 by either intraperitoneal or intravitreal injection. Retinal neovascularization was quantitated at P18. Tetrac significantly inhibited pro-angiogenic effects of VEGF and/or EPO on retinal endothelial cells, indicating that the angiogenic effects of both growth factors are dependent on integrin αvβ3. Retinal neovascularization in the OIR model was significantly inhibited by both tetrac and tetrac-NP. These results indicate that the integrin αvβ3 antagonist, tetrac, is an effective inhibitor of retinal angiogenesis. The ability of tetrac to inhibit the pro-angiogenic effect of both VEGF and EPO on retinal endothelial cells suggests that tetrac (and antagonism of integrin αvβ3) is a viable therapeutic strategy for proliferative diabetic retinopathy.

Topics: Angiogenesis Inhibitors; Animals; Blotting, Western; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Disease Models, Animal; Endothelium, Vascular; Erythropoietin; Humans; Integrin alphaVbeta3; Intravitreal Injections; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oxygen; Phosphorylation; Retinal Neovascularization; Thyroxine; Vascular Endothelial Growth Factor A

Experimental study.. To investigate the effect of erythropoietin (EPO) pretreatment on spinal cord ischemic injury.. Experimental Research Center at Seoul National University Bundang Hospital, Korea.. Rats were treated with either 1000 IU kg(-1) of EPO (EPO group, n=8) or saline (control group, n=8) 24 h before ischemia. Spinal cord ischemia was induced using a balloon-tipped catheter placed on the proximal descending aorta in the control group and the EPO group, but not in the sham group (n=8). Neurological function was assessed using the motor deficit index (MDI; 0=normal, 6=complete paralysis) until 7 days after reperfusion, and histological examination of spinal cord was performed.. At the first day after reperfusion, the EPO group demonstrated a significantly lower MDI compared with the control group (2.0 (0.3-2.0) vs 4.0 (3.0-4.8), median (interquartile range); EPO group vs control group, respectively; P=0.002). This trend was sustained until 7 days after reperfusion (1.0 (1.0-1.8) vs 4.5 (3.3-5.0); EPO group vs control group, respectively; P=0.001), and more normal motor neurons (29.9±3.1 vs 21.4±3.4, mean±s.d.; EPO group vs control group, respectively; P<0.001) were observed. However, compared with the sham group, the EPO group displayed a significantly higher MDI (0.0, sham group) and fewer intact motor neurons (37.8±5.5, sham group; P<0.001, sham vs control group).. Pretreatment with EPO significantly attenuates neurological injury following spinal cord ischemia, although it cannot completely abolish the ischemic injury.

Topics: Animals; Disease Models, Animal; Erythropoietin; Male; Motor Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury; Spinal Cord Ischemia

The erythropoietin-analogue darbepoetin-α (DPO) improves liver function and regeneration after hepatectomy (Phx), however, also enhances Phx-induced tumor cell engraftment and neovascularization. Because it is unknown whether DPO also enhances the growth of established tumors, we herein studied the effect of DPO on established colorectal liver metastases after Phx.. CT26.WT cells were implanted into the liver of BALB/c mice. Five days after tumor establishment, animals underwent 50% Phx and received 10 μg/kgBW DPO or saline. Non-Phx animals with DPO or saline-treatment served as controls. Seven days after Phx tumors were analyzed regarding blood vessel formation, leukocyte adhesion, cell proliferation, apoptotic cell death, and growth using intravital fluorescence microscopy, histology, and immunohistochemistry.. The growth of established colorectal liver metastases was slightly stimulated after DPO-treatment in hepatectomized and non-hepatectomized animals. However, tumor vessel formation and tumor cell proliferation were significantly enhanced after DPO-treatment in hepatectomized and non-hepatectomized mice compared with controls. Apoptotic cell death and leukocyte-endothelial cell interaction were significantly reduced after DPO-treatment.. Our study indicates that DPO-treatment promotes neovascularization and cell proliferation in established colorectal liver metastases of hepatectomized and non-hepatectomized mice. DPO-application in patients with colorectal liver metastases might promote tumor progression and should therefore be avoided.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Contraindications; Darbepoetin alfa; Disease Models, Animal; Erythropoietin; Female; Hematinics; Hepatectomy; Leukocytes; Liver Circulation; Liver Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Microcirculation; Neoplasm Transplantation; Neovascularization, Pathologic

Erythropoietin has been shown to promote tissue regeneration after ischaemic injury in various organs. Here, we investigated whether Erythropoietin could ameliorate ischaemic spinal cord injury in the mouse and sought an underlying mechanism. Spinal cord ischaemia was developed by cross-clamping the descending thoracic aorta for 7 or 9 min. in mice. Erythropoietin (5000 IU/kg) or saline was administrated 30 min. before aortic cross-clamping. Neurological function was assessed using the paralysis score for 7 days after the operation. Spinal cords were histologically evaluated 2 and 7 days after the operation. Immunohistochemistry was used to detect CD34(+) cells and the expression of brain-derived neurotrophic factor and vascular endothelial growth factor. Each mouse exhibited either mildly impaired function or complete paralysis at day 2. Erythropoietin-treated mice with complete paralysis demonstrated significant improvement of neurological function between day 2 and 7, compared to saline-treated mice with complete paralysis. Motor neurons in erythropoietin-treated mice were more preserved at day 7 than those in saline-treated mice with complete paralysis. CD34(+) cells in the lumbar spinal cord of erythropoietin-treated mice were more abundant at day 2 than those of saline-treated mice. Brain-derived neurotrophic factor and vascular endothelial growth factor were markedly expressed in lumbar spinal cords in erythropoietin-treated mice at day 7. Erythropoietin demonstrated neuroprotective effects in the ischaemic spinal cord, improving neurological function and attenuating motor neuron loss. These effects may have been mediated by recruited CD34(+) cells, and enhanced expression of brain-derived neurotrophic factor and vascular endothelial growth factor.

Topics: Animals; Antigens, CD34; Disease Models, Animal; Erythropoietin; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Spinal Cord Ischemia; Survival Analysis; Treatment Outcome

Previous studies report that erythropoietin (EPO) has a neuroprotective role in some neurodegenerative diseases, but the mechanisms are not completely elucidated. The aim of this study was to investigate whether EPO exerts neuroprotective role in experimental autoimmune encephalomyelitis (EAE) via the routes of anti-inflammation. We established an EAE mice model treated intraperitoneally with EPO at the dose of 5,000 IU/kg on schedule, and recorded the clinical score and weight fluctuation. The infiltration of inflammatory cells in the spinal cord of EAE mice was observed with hemotoxylin and eosin (HE) staining, and the levels of IL-10, IFN-γ, IL-17, and MHC-II in central nervous system (CNS)-infiltrating cells and peripheral mononuclear cells were detected by flow cytometry or ELISA. EPO therapy ameliorates clinical signs of EAE mice, inhibits the body weight loss, and decreases the infiltration of inflammatory cells in spinal cords. IL-17 and IFN-γ are reduced, while IL-10 is not increased significantly, in both CNS-infiltrating cells and peripheral mononuclear cells of EPO-treated EAE mice, as compared with EAE control group. EPO also reduces the expression of MHC-II on peripheral antigen presentation cells. Our results indicate that EPO exerts a beneficial role in EAE by inhibiting the levels of IL-17 and IFN-γ in peripheral splenic cells and CNS-infiltrating cells.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Erythropoietin; Female; Inflammation Mediators; Mice; Mice, Inbred C57BL; Spinal Cord; Treatment Outcome

Erythropoietin (EPO) is a variety of tissue-protective functions, including spinal cord. This study aimed to determine the neuron protective effect of erythropoietin on spinal cord injury (SCI) by assessing C/EBP-homologous protein (CHOP) in the development of a rat model of SCI.. Sixty Sprague-Dawley rats were randomly assigned to three groups: sham-operation control group, SCI group, and EPO treatment group. By using a weight-drop contusion SCI model, the rats in the SCI group and EPO treatment group were killed at 1 and 7 days subsequently. The Basso, Beattie, and Bresnahan (BBB) scores were examined for locomotor function. Pathological changes were observed after hematoxylin-eosin (H&E) staining. The expression of CHOP was determined by immunohistochemical staining and RT-PCR analysis.. BBB scores showed more quick recovery in the erythropoietin treatment group than that in the SCI group (P < 0.01). Pathological changes also revealed a reduction in the volume of cavitations and more neurons regeneration in the EPO treatment rats than that of the SCI rats. The number of CHOP positive cells in the SCI group on day 1 and 7 days after SCI increased compared with the erythropoietin treatment group and sham-operation control group (P < 0.01). CHOP mRNA folds in sham-operation control rat from 1 to 7 days showed the same trend.. Endoplasmic reticulum (ER) stress was triggered at the early stage of SCI. Increased expression of CHOP can be found in the injured segment of the spinal cord after injury. EPO treatment could prevent pathological alterations from severe spinal cord injury by reducing expression of CHOP.

Topics: Animals; Disease Models, Animal; Endoplasmic Reticulum Stress; Erythropoietin; Male; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Transcription Factor CHOP

The aim of this study was to investigate the effects of supplemental erythropoietin (EPO) on its receptor (EPOR) and signal transduction pathways in rat model of retinal detachment (RD).. To investigate the effect of EPO on EPOR expression in RD rats 100, 200 or 400 ng EPO was injected into the vitreous cavity immediately after RD model was induced. Western blot and immunohistochemistry analyses were performed to measure EPOR expression. To investigate the effect of EPO on signal transduction pathways in RD rats single dose of 400 ng EPO was injected into the vitreous cavity immediately after RD model was induced. The total and phosphorylated levels of JAK2, Akt, ERK-1/2, STAT5 and NF-κB were assessed by western blot.. Western blot analysis showed that, compared with the normal control group, EPOR expression in the neurosensory retina was significantly increased in experimental RD groups (P < 0.05), but the differences were not significant between experimental RD groups (P > 0.05). Immunohistochemical examination indicated that EPOR staining on retinas became strongly positive 3 days after RD, with no significant difference in staining intensities between the treatment groups. Phosphorylated levels of JAK2, Akt, ERK-1/2, STAT5, and NF-κB were enhanced 3 days after RD, but only JAK2, Akt, and ERK-1/2 phosphorylation was further enhanced by 400 ng EPO treatment (P < 0.05).. Supplementary EPO cannot affect EPOR expression in detached retina, but EPO may activate both PI-3K/Akt and MAPK/ERK-1/2 signal transduction pathways in RD model.

Topics: Animals; Blotting, Western; Disease Models, Animal; Erythropoietin; Immunohistochemistry; Intravitreal Injections; Janus Kinase 2; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Retinal Detachment; Signal Transduction; STAT5 Transcription Factor; Up-Regulation

Erytropoietin (EPO) has cytoprotective and angiogenic properties and has a beneficial effect in ischaemic conditions. Since the development of renal interstitial abnormalities are often associated with ischaemia, we studied the effects of the long-acting EPO analogue darbepoetin alpha (DA) on kidney damage in TGR(mRen2)27 (Ren2) rats.. Ren2 rats were randomised to DA or vehicle (VEH) or to DA + angiotensin converting enzyme inhibitor (ACEi) or VEH + ACEi. Sprague Dawley (SD) rats served as controls. Blood pressure was measured weekly and 24-h urine was collected to measure proteinuria. Blood samples were collected for creatinine and haematocrit. Kidneys were studied for inflammation and pre-fibrosis. Renal mRNA expression was studied for EPO, EPO-receptor, collagen-3α1 and kidney injury molecule-1 (KIM-1).. DA had no effect on SBP, serum creatinine and proteinuria. Interstitial and glomerular α-SMA expression was significantly increased in Ren2. ACEi but not DA improved the increased renal inflammatory and pro-fibrotic profile in Ren2 rats. DA on top of ACEi further reduced glomerular α-SMA and KIM-1 expression.. Long-term DA treatment has no beneficial effects on renal structural and functional changes in TGR(mRen2)27 rats in the time frame studied and the dose provided.

Topics: Actins; Animals; Cell Adhesion Molecules; Cell Count; Collagen Type III; Darbepoetin alfa; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Hypertension; Kidney; Kidney Glomerulus; Macrophages; Male; Rats; Receptors, Erythropoietin; RNA, Messenger; Time Factors

Pyruvate is known to be cytoprotective through antioxidant and anti-inflammatory mechanisms. We tested the hypothesis that pyruvate protects the brain against ischemia-reperfusion injury by inducing endogenous erythropoietin (EPO) expression.. Pyruvate's protective effect was evaluated in C6 glioma cells and HT22 neuronal cells subjected to transient oxygen glucose deprivation. Cell viability (calcein AM assay) and expression of hypoxia-inducible factor-1α, EPO, Akt and Erk (immunoblot), and EPO receptor (reverse transcription-polymerase chain reaction) were analyzed. Transient focal cerebral ischemia in rats was induced by 2 hours middle cerebral artery occlusion followed by 24 hours reperfusion. Pyruvate or saline was infused from 60 minutes occlusion until 30 minutes reperfusion. Lesion volume and DNA fragmentation were assessed by 2,3,5-triphenyltetrazolium staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, respectively. Immunoblots were conducted to determine cerebral EPO contents.. Pyruvate increased cell viability, hypoxia-inducible factor-1α, EPO, and Akt phosphorylation. Small interfering RNA suppression of hypoxia-inducible factor-1α and EPO abolished pyruvate-induced cytoprotection. In the rat stroke model, pyruvate reduced lesion volume by 84% and DNA fragmentation by 77% versus controls; increased EPO content paralleled these cerebroprotective actions of pyruvate.. Pyruvate activation of the hypoxia-inducible factor-1α-EPO signaling cascade in neurons and glia could protect the brain from ischemia-reperfusion injury.

Topics: Animals; Brain; Cell Line, Tumor; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Proto-Oncogene Proteins c-akt; Pyruvic Acid; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Reperfusion Injury; Signal Transduction; Stroke

The goal of the present study was to determine the minimum concentration of systemic erythropoietin-R76E required for neuroprotection in the retina. Erythropoietin (EPO) exhibits neuroprotective effects in both in vitro and in vivo models of neuronal cell death although its classical function is the regulation of red blood cell production. It can cross the blood brain barrier and therefore can be delivered systemically to affect the retina. However, long-term treatment with exogenous erythropoietin causes polycythemia. To decrease this potentially lethal effect, we generated and tested a modified form that contains a single arginine to glutamate mutation at the 76th position (EPO-R76E). In previous studies, this mutant protected retinal neurons in mouse models of retinal degeneration and glaucoma with similar efficacy as wild-type EPO. However, EPO-R76E has attenuated erythropoietic activity, therefore, neuroprotection can be achieved without causing a significant rise in hematocrit. BALB/cByJ mice received a single intramuscular injection of recombinant adeno-associated virus carrying enhanced green fluorescent protein, Epo, or Epo-R76E. To result in continuous production of four different doses of EPO-R76E, two doses of two different serotypes (2/5 and 2/8) were used. Mice were subjected to optic nerve crush and analysis was performed thirty days later. EPO-R76E showed dose-dependent protection of the retinal ganglion cell bodies, but was unable to prevent axonal degeneration. Furthermore, EPO-R76E induced a dose-dependent rise in the hematocrit that was still attenuated as compared to wild-type EPO.

Topics: Animals; Cell Count; Cytomegalovirus; Dependovirus; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Genetic Vectors; Glaucoma; Green Fluorescent Proteins; Hematocrit; Injections, Intramuscular; Mice; Mice, Inbred BALB C; Nerve Crush; Optic Nerve Injuries; Recombinant Fusion Proteins; Retinal Ganglion Cells

Cerebral ischemia-reperfusion (I/R) injury induces secondary cerebral damage. As drugs for treating this type of injury have shown poor efficacy and adverse side effects in clinical trials, a novel therapeutic strategy has been long awaited. In this study, we focused on the disruption of the blood-brain barrier after stroke, and applied a liposomal drug delivery system (DDS) designed to enhance the pharmacological effect of the neuroprotectant and to avoid side effects. PEGylated liposomes were injected at varying time after the start of reperfusion in transient middle cerebral artery occlusion (t-MCAO) model rats. The results showed PEGylated liposomes accumulated in the ischemic hemisphere at an early stage after reperfusion and were retained in the lesion for at least 24h after injection. We also investigated the effectiveness of asialo-erythropoietin (AEPO)-modified PEGylated liposomes (AEPO-liposomes) in treating the cerebral I/R injury. AEPO-liposome treatment significantly reduced TTC-defined cerebral legion following cerebral I/R injury, and ameliorated motor function compared with vehicle and AEPO treatment. In conclusion, these results indicate that AEPO-liposomes are a promising liposomal formulation for protecting the brain from I/R injury, and that this liposomal DDS has potential as a novel strategy for the treatment of cerebral I/R injury.

Topics: Animals; Apoptosis; Asialoglycoproteins; Blood-Brain Barrier; Cell Culture Techniques; Cell Survival; Disease Models, Animal; Drug Delivery Systems; Erythropoietin; In Situ Nick-End Labeling; Ischemic Attack, Transient; Liposomes; Male; Neuroprotective Agents; PC12 Cells; Rats; Rats, Wistar; Reperfusion Injury; Tissue Distribution

Because retinal ischemia is a common cause of vision loss, we sought to determine the effects of ischemia on neuroretinal function and survival in murine oxygen-induced retinopathy (OIR) and to define the role of endogenous erythropoietin (EPO) in this model. OIR is a reproducible model of ischemia-induced retinal neovascularization; it is used commonly to develop antiangiogenic strategies. We investigated the effects of ischemia in murine OIR on retinal function and neurodegeneration by electroretinography and detailed morphology. OIR was associated with significant neuroretinal dysfunction, with reduced photopic and scotopic ERG responses and reduced b-wave/a-wave ratios consistent with specific inner-retinal dysfunction. OIR resulted in significantly increased apoptosis and atrophy of the inner retina in areas of ischemia. EPO deficiency in heterozygous Epo-Tag transgenic mice was associated with more profound retinal dysfunction after OIR, indicated by a significantly greater suppression of ERG amplitudes, but had no measurable effect on the extent of retinal ischemia, preretinal neovascularization, or neuroretinal degeneration in OIR. Systemic administration of recombinant EPO protected EPO-deficient mice against this additional suppression, but EPO supplementation in wild-type animals with OIR did not rescue neuroretinal dysfunction or degeneration. Murine OIR offers a valuable model of ischemic neuroretinal dysfunction and degeneration in which to investigate adaptive tissue responses and evaluate novel therapeutic approaches. Endogenous EPO can protect neuroretinal function in ischemic retinopathy.

Topics: Animals; Apoptosis; Cell Hypoxia; Disease Models, Animal; Electroretinography; Erythropoietin; Ischemia; Kidney; Mice; Mice, Transgenic; Oxygen; Recombinant Proteins; Retina; Retinal Neovascularization; Retinal Neurons; Retinal Vessels; Up-Regulation

We investigated the effects of asialoerythropoietin (asialoEPO), a nonerythrogenic erythropoietin derivative, on 3 murine models of heart failure with different etiologies.. Doxorubicin (15 mg/kg) induced heart failure within 2 weeks (toxic cardiomyopathy). Treatment with asialoEPO (6.9 μg/kg) for 2 weeks thereafter attenuated the associated left ventricular dysfunction and dilatation. In addition, the asialoEPO-treated heart showed less myocardial fibrosis, inflammation, and oxidative damage, and diminished atrophic cardiomyocyte degeneration, which was accompanied by restored expression of GATA-4 and sarcomeric proteins. Mice with large 6-week-old myocardial infarctions exhibited marked left ventricular dysfunction with adverse remodeling (ischemic cardiomyopathy). AsialoEPO treatment for 4 weeks significantly mitigated progression of the dysfunction and remodeling and reduced myocardial fibrosis, inflammation, and oxidative damage. Finally, 25-week-old δ-sarcoglycan-deficient mice (genetic cardiomyopathy) were treated with asialoEPO for 5 weeks. AsialoEPO mitigated the progressive cardiac remodeling and dysfunction through cardiomyocyte hypertrophy, and upregulated expression of GATA-4 and sarcomeric proteins. AsialoEPO appears to act by altering the activity of the downstream erythropoietin receptor signals extracellular signal-regulated protein kinase, Akt, signal transducer, and activator of transcription 3 and 5 in a model-specific manner.. The findings suggest that asialoEPO exerts broad cardioprotective effects through distinct mechanisms depending on the model, which are independent of the erythrogenic action. This compound may be promising for the treatment of heart failure of various etiologies.

Topics: Animals; Asialoglycoproteins; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Follow-Up Studies; Heart Failure; Mice; Mice, Inbred C57BL; Treatment Outcome; Ventricular Function, Left; Ventricular Remodeling

Sepsis is characterized by systemic biochemical alterations including the central nervous system in the early times and cognitive impairment at later times after sepsis induction in the animal model. Recent studies have shown that, besides its hematological activity, erythropoietin (EPO) has cytoprotective effects on various cells and tissues. In order to corroborate elucidating the effects of alternative drugs for sepsis treatment, we evaluated the effects of both acute and chronic EPO treatment on oxidative stress and energetic metabolism in the hippocampus, and cognitive impairment, respectively, after sepsis induction by cecal ligation and perforation (CLP). To this aim, male Wistar rats underwent CLP with "basic support" or sham operation. In the acute treatment, EPO was administered once immediately after CLP induction. The rats were then killed after 6 and 24 h, and the hippocampus was removed for analysis of oxidative stress and energetic metabolism, respectively. Regarding the chronic treatment, EPO was administered once daily until the 4th day after induction. Aversive memory was tested on the 10th day after surgery. It was observed that the acute use of EPO (a single dose) alters the oxidative parameters and energetic metabolism. Chronic use (4 days) reversed cognitive impairment in the sepsis animal model. Mortality rates were attenuated only during chronic treatment.

Topics: Analysis of Variance; Animals; Avoidance Learning; Citrate (si)-Synthase; Cognition Disorders; Creatine Kinase; Disease Models, Animal; Electron Transport; Electron Transport Chain Complex Proteins; Energy Metabolism; Erythropoietin; Inhibition, Psychological; Ligation; Male; Oxidative Stress; Rats; Rats, Wistar; Sepsis; Statistics, Nonparametric; Time Factors

Darbepoetin-alpha (DA) is a novel erythropoiesis-stimulating agent developed for treating anemia. In animal models, recombinant human erythropoietin has been reported to be beneficial for neuroprotection. In this study, we determined whether DA would protect the spinal cord against ischemia-reperfusion injury in a rabbit model.. Forty rabbits were randomized into five groups of eight animals each: group 1 (sham), group 2 (ischemia), group 3 (vehicle), group 4 (30 mg/kg methylprednisolone), group 5 (30 μg/kg DA). Only laparotomy was performed in the sham group. In all the other groups, the spinal cord ischemia model was created by a 20-min occlusion of the aorta just caudal to renal artery with an aneurysm clip. The drugs were administered immediately after the clamp was removed. The animals were killed 24 h later. Spinal cord segments between L2 and L5 were harvested for analysis. Neurological evaluation was performed with the Tarlov scoring system just before the animals were killed. Level of tissue malondialdehyde was analyzed as a marker of lipid peroxidation and tissue caspase-3 activity as a marker of apoptosis. Also, histopathological evaluation of the tissues was performed.. Both malondialdehyde and caspase-3 levels were significantly decreased by DA administration. Histopathological evaluation of the tissues also demonstrated decrease in neuronal degeneration and infiltration parameters after DA administration. In the DA group, neurological outcome scores were statistically significantly better compared with the ischemia and the vehicle groups.. Although further studies considering different dose regimens and time intervals are required, DA was shown to be at least as effective as methylprednisolone in spinal cord ischemia/reperfusion model.

Topics: Anemia; Animals; Darbepoetin alfa; Disease Models, Animal; Erythropoietin; Hematinics; Male; Neuroprotective Agents; Rabbits; Reperfusion Injury; Spinal Cord Ischemia

The introduction of cyclosporine (CsA) has improved graft survival, but it causes nephropathy, which limits its clinical utility. Recently, we reported that carbamylated erythropoietin (CEPO) protected kidneys from ischemia reperfusion injury as well as EPO. To investigate the clinical applications of CEPO, we next evaluated the long-term therapeutic effect of CEPO using a CsA-induced nephropathy model. CsA caused renal dysfunction, while EPO/CEPO administration significantly improved renal function. EPO treatment significantly increased Hb concentration, while CEPO treatment neither enhanced nor reduced Hb concentration. CsA treatment induced tubular apoptosis, while EPO/CEPO administration inhibited it and increased PI3 kinase activation and Akt phosphorylation. In parallel, morphological assessment revealed that EPO/CEPO significantly reduced CsA-induced interstitial fibrosis and inhibited interstitial macrophage infiltration. In addition, real-time RT-PCR demonstrated that cortical mRNA levels of TGF-β1 and type I collagen were suppressed in the EPO/CEPO group. These results suggest a new therapeutic approach using CEPO to protect kidneys from CsA-induced nephropathy.

Topics: Animals; Apoptosis; Collagen Type I; Cyclosporine; Disease Models, Animal; Dogs; Erythropoiesis; Erythropoietin; Fibrosis; Hemoglobins; Humans; Immunosuppressive Agents; Kidney; Kidney Diseases; Macrophages; Male; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Transforming Growth Factor beta1

Topics: Allergy and Immunology; Animals; Arthritis, Rheumatoid; Disease Models, Animal; Erythropoietin; History, 20th Century; Humans; Immunity, Innate; Tumor Necrosis Factor-alpha

Retinopathy of prematurity (ROP) is a leading cause of blindness in children and is, in its most severe form, characterized by uncontrolled growth of vision-threatening pathologic vessels. Propranolol, a nonselective β-adrenergic receptor blocker, was reported to protect against pathologic retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Based on this single animal study using nonstandard evaluation of retinopathy, clinical trials are currently ongoing to evaluate propranolol treatment in stage 2 ROP patients who tend to experience spontaneous disease regression and are at low risk of blindness. Because these ROP patients are vulnerable premature infants who are still in a fragile state of incomplete development, the efficacy of propranolol treatment in retinopathy needs to be evaluated thoroughly in preclinical animal models of retinopathy and potential benefits weighed against potential adverse effects.. Retinopathy was induced by exposing neonatal mice to 75% oxygen from postnatal day (P) 7 to P12. Three routes of propranolol treatment were assessed from P12 to P16: oral gavage, intraperitoneal injection, or subcutaneous injection, with doses varying between 2 and 60 mg/kg/day. At P17, retinal flatmounts were stained with isolectin and quantified with a standard protocol to measure vasoobliteration and pathologic neovascularization. Retinal gene expression was analyzed with qRT-PCR using RNA isolated from retinas of control and propranolol-treated pups.. None of the treatment approaches at any dose of propranolol (up to 60 mg/kg/day) were effective in preventing the development of retinopathy in a mouse model of OIR, evaluated using standard techniques. Propranolol treatment also did not change retinal expression of angiogenic factors including vascular endothelial growth factor.. Propranolol treatment via three routes and up to 30 times the standard human dose failed to suppress retinopathy development in mice. These data bring into question whether propranolol through inhibition of β-adrenergic receptors is an appropriate therapeutic approach for treating ROP.

Topics: Administration, Oral; Adrenergic beta-Antagonists; Angiopoietin-1; Angiopoietin-2; Animals; Animals, Newborn; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Gene Expression Profiling; Humans; In Vitro Techniques; Infant, Newborn; Injections, Intraperitoneal; Injections, Subcutaneous; Mice; Oxygen; Propranolol; Receptors, Adrenergic, beta; Receptors, Vascular Endothelial Growth Factor; Retina; Retinal Neovascularization; Retinopathy of Prematurity; Reverse Transcriptase Polymerase Chain Reaction; RNA; Up-Regulation; Vascular Endothelial Growth Factor A

Erythropoietin (EPO) has long been utilized for the treatment of renal anemia. The erythropoietin receptor (EPOR) is also expressed in the cardiovascular and central nervous systems in addition to an erythroid lineage, to provide an organoprotective role against several types of cellular stress. Pulmonary hypertension (PH) is a poor prognostic disease caused by primary and secondary pulmonary vascular injury. We observed the effects of EPO derivatives on monocrotaline-induced PH in rats on the supposition that EPO may protect small arteries from injury. Asialoerythropoietin (AEPO) lacks sialic acids in the termini of carbohydrate chains that results in rapid clearance from blood. Carbamyl-erythropoietin (CEPO) interacts with EPOR/βc heterodimers, but not with EPOR homodimers expressed in erythroid cells. Monocrotaline-injected rats were treated with continuous intravenous injection of 2500 ng/kg/day of EPO, AEPO, or CEPO for 21 days, and lung histology, cardiac function, and mRNA expression in the lungs were examined. Wall thickening of small arteries in the lungs and PH were improved by administration of EPO, but not by its non-hematopoietic derivatives, AEPO, or CEPO. Erythropoietin administration increased mRNA expression of the anti-apoptotic molecule, Bcl-xL, and maintained expression of the CD31 antigen. We conclude that lungs may express EPOR homoreceptors, but not heteroreceptors. Adequate serum erythropoietin levels may be essential for pulmonary protective effects.

Topics: Animals; Asialoglycoproteins; Disease Models, Animal; Erythropoietin; Hypertension, Pulmonary; Male; Monocrotaline; Neuroprotective Agents; Rats; Rats, Wistar; Receptors, Erythropoietin; RNA, Messenger; Treatment Outcome

To investigate intestinal motility changes due to uremia, and the effect of pretreatment with erythropoietin.. This randomized control study was conducted in the Department of Physiology, Faculty of Medicine, Ain Shams University, Cairo, Egypt from September 2010 to July 2011. Forty adult female Wistar albino rats were allocated into 3 groups: control group, gentamicin-treated group, receiving intraperitoneal gentamicin sulphate (100 mg/kg for 5 days), and erythropoietin-gentamicin-treated group, receiving subcutaneous erythropoietin (1000 IU/kg for 3 days) prior to gentamicin injection. Isolated segments of duodenum and descending colon was subjected to in vitro motility study. Plasma creatinine and urea were assayed.. Induction of acute renal failure by gentamicin treatment resulted in a significant decrease in frequency of contraction of the duodenum and descending colon, an increase in the average duration of contraction of the duodenum, and a significant decrease in the average force of contraction in the descending colon. Moreover, the average force of contraction in response to acetylcholine was significantly decreased in the duodenum. The erythropoietin-gentamicin-treated group revealed a significant decrease in plasma creatinine and urea, and a significant increase in the duodenal average force of contraction and motility index, and colonic frequency. The duodenal absolute and average forces of contraction after acetylcholine increased significantly.. Acute uremia impairs small and large intestinal motility, probably due to uremic toxins and autonomic dysfunction. Erythropoietin pretreatment protected against intestinal dysmotility through the improvement of renal function and its neurotropic action.

Topics: Acute Disease; Animals; Colon; Creatinine; Disease Models, Animal; Duodenum; Erythropoietin; Female; Gastrointestinal Motility; Gentamicins; Random Allocation; Rats; Rats, Wistar; Reference Values; Sensitivity and Specificity; Uremia

Spinal cord injury (SCI) is a debilitating clinical condition, characterized by a complex of neurological dysfunctions. It has been shown in rats that the acute administration of recombinant human erythropoietin (rhEPO) following a contusive SCI improves the recovery of hindlimb motor function, as measured with the locomotor BBB (Basso, Beattie, Bresnahan) scale. This scale evaluates overall locomotor activity, without testing whether the rhEPO-induced motor recovery is due to a parallel recovery of sensory and/or motor pathways. Aim of the present study was to utilize an electrophysiological test to evaluate, in a rat model of contusive SCI, the transmission of both ascending and descending pathways across the damaged cord at 2, 5, 7, 11, and 30 days after lesion, in animals treated with rhEPO (n=25) vs saline solution (n=25). Motor potentials evoked by epicortical stimulation were recorded in the spinal cord, and sensory-evoked potentials evoked by spinal stimulation were recorded at the cortical level. In the same animals BBB score and immunocytochemical evaluation of the spinal segments caudal to the lesion were performed. In rhEPO-treated animals results show a better general improvement both in sensory and motor transmission through spared spinal pathways, supposedly via the reticulo-spinal system, with respect to saline controls. This improvement is most prominent at relatively early times. Overall these features show a parallel time course to the changes observed in BBB score, suggesting that EPO-mediated spared spinal cord pathways might contribute to the improvement in transmission which, in turn, might be responsible for the recovery of locomotor function.

Topics: Animals; Contusions; Disease Models, Animal; Electrophysiology; Erythropoietin; Evoked Potentials, Motor; Evoked Potentials, Somatosensory; Female; Humans; Motor Activity; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Recovery of Function; Spinal Cord Injuries

The tissue-protective effects of erythropoietin (EPO) have been extensively investigated, and EPO administration can raise the hemoglobin (Hb) concentration. Recently, we reported that carbamylated erythropoietin (CEPO) protected kidneys from ischemia-reperfusion injury as well as EPO.. To investigate the clinical applications of CEPO, we next evaluated the long-term therapeutic effect of CEPO using a tubulointerstitial model rat. We randomized remnant kidney model rats to receive saline, EPO, or CEPO for 8 weeks.. CEPO- and EPO-treated rats had improved serum creatinine levels compared with saline-treated remnant kidney model rats, although the Hb level was significantly increased in EPO-treated rats. Two-photon microscopy revealed that EPO/CEPO significantly ameliorated tubular epithelial cell damage assessed by endocytosis. In addition, CEPO or EPO protected endothelial cells with a sustained blood flow rate. EPO or CEPO suppressed the number of TUNEL-positive apoptotic cells with weak αSMA staining. Furthermore, PCR analysis demonstrated that TGF-β and type I collagen expression was attenuated in EPO- or CEPO-treated rats, accompanied by a significant decrease in interstitial fibrosis.. We established a long-term therapeutic approach to protect tubulointerstitial injury with CEPO, and thus, the therapeutic value of this approach warrants further attention and preclinical studies.

Topics: Animals; Collagen Type I; Creatinine; Disease Models, Animal; Erythropoietin; Fibrosis; Hemoglobins; Kidney; Kidney Diseases; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Transforming Growth Factor beta

Acute renal failure (ARF) can be caused by injuries that induce tissue hypoxia, which in turn can trigger adaptive or inflammatory responses. We previously showed the participation of basic fibroblast growth factor (FGF-2) in renal repair. Based on this, the aim of this study was to analyze the effect of FGF-2 signaling pathway manipulation at hypoxia-induced protein levels, as well as in key proteins from the vasoactive systems of the kidney. We injected rat kidneys with FGF-2 recombinant protein (r-FGF) or FGF-2 receptor antisense oligonucleotide (FGFR2-ASO) after bilateral ischemia, and evaluated the presence of iNOS, EPO and HO-1, in representation of hypoxia-induced proteins, as well as COX-2, renin, kallikrein, and B2KR, in representation of the vasoactive systems of the kidney. A reduction in iNOS, HO-1, EPO, renin, kallikrein, B2KR, and in renal damage was observed in animals treated with r-FGF. The opposite effect was found with FGF-2 receptor down-regulation. In contrast, COX-2 protein levels were higher in kidneys treated with r-FGF and lower in those that received FGFR2-ASO, as compared to saline treated kidneys. These results suggest that the protective role of FGF-2 in the pathogenesis of ARF induced by I/R is a complex process, through which a differential regulation of metabolic pathways takes place.

Topics: Acute Kidney Injury; Animals; Cell Hypoxia; Cyclooxygenase 2; Disease Models, Animal; Erythropoietin; Fibroblast Growth Factor 2; Heme Oxygenase-1; Kallikreins; Kidney; Male; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Receptor, Bradykinin B2; Reperfusion Injury

Erythropoietin (EPO) is a pleiotropic cytokine with neuroprotective, anti-inflammatory, and muscle regenerative properties. The purpose of our study was to analyze the regenerative capacity of systemically applied EPO in a combined muscle-nerve injury model.. We performed a crush injury to the left soleus muscle in 84 male Wistar rats. Using an instrumented clamp, the muscle was crushed over its complete length. Simultaneously, the ipsilateral sciatic nerve was sham manipulated or crushed. Upon induction of the trauma, animals received either EPO (E) (single application of 5,000 IU/kg body weight intraperitonial) or vehicle solution (K). After in vivo assessment of mechanical pain according to Frey, thermal hyperalgesia, latency of nerve conduction velocity, and strength of the soleus muscle were analyzed at days 1, 7, and 42 postinjury (n = 7 per group). Cell proliferation and apoptosis were assessed by means of histology and immunohistochemistry.. Combined muscle-nerve injury showed a significant loss of muscle strength, which incompletely recovered within 42 days. Rats treated with EPO showed an increased muscle strength after 7 days and 42 days compared with the control group. Pain behavior was highest in the muscle-nerve injured animals at day 7. EPO decreased the pain and increased nerve conduction velocity. Nerve injury diminished proliferation of muscle cells, whereas simultaneous therapy with EPO resulted in a boost of bromdesoxyuridine-positive cells.. EPO promoted muscle restoration and enhanced nerve recovery after combined muscle-nerve injury. Thus, EPO might represent an attractive therapeutic option to optimize the posttraumatic course after injury.

Topics: Animals; Crush Syndrome; Disease Models, Animal; Erythropoietin; Hindlimb; Immunohistochemistry; Injections, Intraperitoneal; Male; Muscle Strength; Muscle, Skeletal; Nerve Regeneration; Neural Conduction; Peripheral Nerve Injuries; Random Allocation; Rats; Rats, Wistar; Reference Values; Regeneration; Sciatic Nerve; Sensitivity and Specificity

Cerebrovascular disease is the third leading cause of death and the leading cause of disability in Cuba and in several developed countries. A possible neuroprotective agent is the rHu-EPO, whose effects have been demonstrated in models of brain ischemia. The Neuro-EPO is a derivative of the rHu-EPO that avoids the stimulation of erythropoiesis. The aim of this study was to determine the Neuro-EPO delivery into the central nervous system (CNS) to exert a neuroprotective effect in cerebral ischemia model of the Mongolian gerbil. The Neuro-EPO in a rate of 249.4 UI every 8 hours for 4 days showed 25% higher viability efficacy (P > 0.01), improving neurological score and behavior of the spontaneous exploratory activity, the preservation of CA3 areas of the hippocampus, the cortex, and thalamic nuclei in the focal ischemia model of the Mongolian gerbil. In summary, this study, the average dose-used Neuro-EPO (249.4 UI/10 μL/every 8 hours for 4 days), proved to be valid indicators of viability, neurological status, and spontaneous exploratory activity, being significantly lower than that reported for the systemically use of the rHu-EPO as a neuroprotectant. Indeed, up to 12 h after brain ischemia is very positive Neuro-EPO administration by the nasal route as a candidate for neuroprotection.

Topics: Administration, Intranasal; Animals; Brain Ischemia; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Gerbillinae; Humans; Neuroprotective Agents; Treatment Outcome

 Endothelial colony-forming cells (ECFCs) are promising candidates for cell therapy of ischemic diseases. Erythropoietin (EPO) is a cytokine that promotes angiogenesis after ischemic injury. EPO receptors (EPORs) classically include two EPOR subunits, but may also associate with the β-common chain (CD131) in a newly identified receptor involved in EPO cytoprotective effects.. The aim was to take advantage of the proangiogenic properties of EPO to enhance ECFC graft efficiency. We postulated that priming ECFCs by adding epoietin α in culture medium prior to experiments might increase their angiogenic properties. We also explored the role of the CD131 subunit in EPO priming of ECFCs.. By western blotting on cord blood ECFC lysates, we showed that EPOR and CD131 expression increased significantly after EPO priming. These proteins coimmunoprecipitated and colocalized, suggesting that they are covalently bound in ECFCs. EPO at 5 IU mL(-1) significantly stimulated proliferation, wound healing, migration and tube formation of ECFCs. EPO priming also increased ECFC resistance to H2 O2-induced apoptosis and survival in vivo. Similarly, in vivo studies showed that, as compared with non-primed ECFC injection, 5 IU mL(-1) EPO-primed ECFCs, injected intravenously 24 h after hindlimb ischemia in athymic nude mice, increased the ischemic/non-ischemic ratios of hindlimb blood flow and capillary density. These effects were all prevented by CD131 small interfering RNA transfection, and involved the phosphoinositide 3-kinase-Akt pathway.. These results highlight the potential role of EPO-primed ECFCs for cell-based therapy in hindlimb ischemia, and underline the critical role of CD131 as an EPO coreceptor.

Topics: Animals; Cells, Cultured; Cytokine Receptor Common beta Subunit; Disease Models, Animal; Endothelium, Vascular; Erythropoietin; Humans; Mice; Neovascularization, Physiologic; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Stem Cells; Up-Regulation

In rodents, exposure to intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), is associated with neurobehavioral impairments, increased apoptosis in the hippocampus and cortex, as well as increased oxidant stress and inflammation. Such findings are markedly attenuated in rodents exposed to sustained hypoxia 9SH) of similar magnitude. The hypoxia-sensitive gene erythropoietin (EPO) has emerged as a major endogenous neuroprotectant, and could be involved in IH-induced neuronal dysfunction.. IH induced only transiently increased expression of EPO mRNA in hippocampus, which was continued in (SH)-exposed mice. IH, but not SH, adversely affected two forms of spatial learning in the water maze, and increased markers of oxidative stress. However, on a standard place training task, mice treated with exogenously administered EPO displayed normal learning, and were protected from the spatial learning deficits observed in vehicle-treated (C) littermates exposed to IH. Moreover, anxiety levels were increased in IH as compared to normoxia, while no changes in anxiety emerged in EPO-treated mice. Additionally, C mice, but not EPO-treated IH-exposed mice had significantly elevated levels of NADPH oxidase expression, as well as increased MDA and 8-OHDG levels in cortical and hippocampal lysates.. The oxidative stress responses and neurobehavioral impairments induced by IH during sleep are mediated, at least in part, by imbalances between EPO expression and increased NADPH oxidase activity, and thus pharmacological agents targeting EPO expression in CNS may provide a therapeutic strategy in sleep-disordered breathing.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Analysis of Variance; Animals; Cells, Cultured; Cerebral Cortex; Cognition Disorders; Deoxyguanosine; Disease Models, Animal; Embryo, Mammalian; Erythropoietin; Escape Reaction; Gene Expression Regulation; Humans; Hypoxia; Injections, Intraperitoneal; Lipid Peroxidation; Male; Malondialdehyde; Maze Learning; Memory; Mice; Mice, Inbred C57BL; NADPH Oxidases; Neurons; Phosphopyruvate Hydratase; Sleep Apnea Syndromes; Swimming; Time Factors

Normal brain function is dependent on continuous and controlled oxygen delivery. Chronic moderate hypoxia leads to angiogenesis, suggesting a modulatory role for oxygen in determining capillary density. The objective of this study was to determine physiologic and brain angiogenic adaptational changes during chronic moderate normobaric hyperoxia in mice. Four-month old C56BL/6J mice were kept in a normobaric chamber at 50% O(2) for up to 3 weeks. Normoxic littermates were kept in the same room outside the chamber. Freshly collected or fixed brain specimens were analyzed by RT-PCR, Western blot analysis and immunohistochemistry. Results show accumulation of hypoxia inducible factors 1 and 2α (HIF-1 and 2α), and increased expression of erythropoietin (EPO), cyclooxygenase-2 (COX-2) and angiopoietin-2 (Ang-2). Conversely, vascular endothelial growth factor (VEGF), and VEGF receptor-2 (KDR/Flk-1), Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and prolylhydroxylase-2 (PHD-2) expressions were decreased. VEGF mRNA level was diminished but there was no change in HIF-1α mRNA and von Hippel Lindau E3 ubiquitin ligase (VHL) protein expression. Microvascular density was significantly diminished by the end of the 3rd week of hyperoxia. Overall, our results are: (1) increased expression of the potent neuroprotective molecule, EPO; (2) diminished expression of the potent angiogenic factor, VEGF; and (3) decreased microvascular density. We can, therefore, conclude that brain microvascular density can be controlled by HIF-independent mechanisms, and that brain capillary density is a continuously adjusted variable with tissue oxygen availability as one of the controlling modulators.

Topics: Angiopoietin-2; Animals; Basic Helix-Loop-Helix Transcription Factors; Body Weight; Cerebral Cortex; Cyclooxygenase 2; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Hematocrit; Hyperoxia; Hypoxia-Inducible Factor 1; Male; Mice; Mice, Inbred C57BL; Microvessels; Oxygen; RNA, Messenger; Time Factors; Vascular Endothelial Growth Factor A

We have recently reported spinal blood flow-metabolism uncoupling in an amyotrophic lateral sclerosis (ALS) animal model using Cu/Zn-superoxide dismutase 1 (SOD1)-transgenic (Tg) mice, suggesting a relative hypoxia in the spinal cord. However, the hypoxic stress sensor pathway has not been well studied in ALS. Here, we examined temporal and spatial changes of the hypoxic stress sensor proteins HIF-1α and its downstream proteins (VEGF, HO-1, and EPO) during the normoxiccourse of motor neuron (MN) degeneration in the spinal cord of these ALS model mice. We found that HIF-1α protein expression progressively increased both in the anterior large MNs and the surrounding glial cells in Tg mice from early symptomatic 14 week (W) and end stage 18 W. Double immunofluorescence analysis revealed that HIF-1α, plus GFAP and Iba-1 double-positive surrounding glial cells, progressively increased from 14 W to 18 W, although the immunohistochemistry in large MNs did not change. Expression levels of VEGF and HO-1 also showed a progressive increase but were significant only in the surrounding glial cells at 18 W. In contrast, EPO protein expression was decreased in the surrounding glial cells of Tg mice at 18 W. Because HIF1-α serves as an important mediator of the hypoxic response, these findings indicate that MNs lack the neuroprotective response to hypoxic stress through the HIF-1α system, which could be an important mechanism of neurodegeneration in ALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Blotting, Western; Cell Hypoxia; Disease Models, Animal; Erythropoietin; Fluorescent Antibody Technique; Heme Oxygenase-1; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Transgenic; Motor Neurons; Neuroglia; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1; Vascular Endothelial Growth Factor A

We explored the neuroprotective effects of erythropoietin (EPO)-loaded dextran microparticle-based Poly(DL-lactide-co-glycolide)/Poly(DL-lactide) (PLGA/PLA) microspheres (EPO-dextran PLGA/PLA microspheres) on retinal ganglion cells (RGCs) in optic nerve crush rats for a prolonged period of time.. EPO-dextran PLGA/PLA microspheres were prepared first by a novel solid-in-oil-in-water (S/O/W) technique. Then, the in vitro EPO release profile was assessed. Afterward, the bioactive effect of EPO released from EPO-dextran PLGA/PLA microspheres was explored in vitro on the retinal explants. Lastly, the neuroprotective effects of EPO-dextran PLGA/PLA microspheres on RGCs were evaluated in optic nerve crush rats with TUNEL staining for apoptotic RGCs. The level of glial fibrillary acidic protein (GFAP) expressed in retina was explored by immunohistochemistry staining. Survival RGCs were observed by DiI retrograde labeling using a DiI fluorescent tracer (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate).. The results demonstrated that a sustained release of EPO from PLGA/PLA microspheres could last for at least 60 days. EPO released from the microspheres showed as efficaciously neuroregenerative as EPO protein solution on retinal explants (P = 0.2554 for neurite density, P = 0.1004 for neurite length). TUNEL staining revealed that EPO-dextran PLGA/PLA microspheres remarkably reduced RGCs death when compared to the control (untreated) group (P < 0.01 at five days and one week post-crush, P < 0.05 at two weeks post-crush). Increased GFAP expression in retina was reduced greatly in EPO-dextran PLGA/PLA microspheres-administrated rats two weeks post optic nerve crush. DiI retrograde labeling revealed that a single injection of EPO-dextran PLGA/PLA microspheres significantly promoted RGCs survival (P < 0.01 at four and eight weeks post-crush).. A single intravitreal injection of EPO-dextran PLGA/PLA microspheres appeared to have a prolonged protective effect on RGCs in optic nerve crush rats. The PLGA/PLA microspheres may be a feasible protein delivery system, such as EPO, to intravitreal injection for retinal degeneration diseases.

Topics: Animals; Apoptosis; Biocompatible Materials; Cell Survival; Cells, Cultured; Delayed-Action Preparations; Dextrans; Disease Models, Animal; Drug Carriers; Drug Compounding; Erythropoietin; Glial Fibrillary Acidic Protein; Immunohistochemistry; In Situ Nick-End Labeling; Lactic Acid; Male; Microspheres; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Rats, Sprague-Dawley; Retinal Degeneration; Retinal Ganglion Cells

Erythropoietin (EPO) confers potent neuroprotection against ischemic injury. However, treatment for stroke requires high doses and multiple administrations of EPO, which may cause deleterious side effects due to its erythropoietic activity. This study identifies a novel nonerythropoietic mutant EPO and investigates its potential neuroprotective effects and underlying mechanism in an animal model of cerebral ischemia.. We constructed a series of mutant EPOs, each containing a single amino acid mutation within the erythropoietic motif, and tested their erythropoietic activity. Using cortical neuronal cultures exposed to N-methyl-d-aspartate neurotoxicity and a murine model of transient middle cerebral artery occlusion, neuroprotection and neurofunctional outcomes were assessed as well as activation of intracellular signaling pathways.. The serine to isoleucine mutation at position 104 (S104I-EPO) completely abolished the erythropoietic and platelet-stimulating activity of EPO. Administration of S104I-EPO significantly inhibited N-methyl-d-aspartate-induced neuronal death in primary cultures and protected against cerebral infarction and neurological deficits with an efficacy similar to that of wild-type EPO. Both S104-I-EPO and wild-type EPO activated similar prosurvival signaling pathways such as phosphatidylinositol 3-kinase/AKT, mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2, and STAT5. Inhibition of phosphatidylinositol 3-kinase/AKT or mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 signaling pathways significantly attenuated the neuroprotective effects of S104-I-EPO, indicating that activation of these pathways underlies the neuroprotective mechanism of mutant EPO against cerebral ischemia.. S104-I-EPO confers neuroprotective effects comparable to those of wild-type EPO against ischemic brain injury with the added benefit of lacking erythropoietic and platelet-stimulating side effects. Our novel findings suggest that the nonerythropoietic mutant EPO is a legitimate candidate for ischemic stroke intervention.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Erythropoietin; Mice; Mice, Inbred C57BL; Mutagenesis, Site-Directed; Mutation; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Signal Transduction

The neuroprotective activity of recombinant human erythropoietin (rhEPO) loaded poly(lactic-co-glycolic) acid (PLGA) nanoparticles has been observed in rats with model intracerebral post-traumatic hematoma (hemorrhagic stroke). It is established that rhEPO-loaded PLGA nanoparticles produce a neuroprotective effect in rats with hemorrhagic stroke, which is manifested by reduced number of lethal outcomes and animals with neurological disorders. Treatment with rhEPO-loaded PLGA prevented amnesia of passive avoidance reflex (PAR), which was produced by the hemorrhagic stroke, and reduced the area of brain damage caused by the intracerebral hematoma. These effects were recorded during one-week observation period. Native rhEPO exhibited a similar, but much less pronounced effect on the major disorders caused by the model hemorrhagic stroke in rats.

Topics: Amnesia; Animals; Avoidance Learning; Cerebral Hemorrhage, Traumatic; Disease Models, Animal; Drug Carriers; Erythropoietin; Humans; Lactic Acid; Male; Nanoparticles; Neuroprotective Agents; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Recombinant Proteins; Survival Rate

Seabuckthorn (Hippophae rhamnoides L.) has been used to treat high altitude diseases. The effects of five-week treatment with total flavonoids of seabuckthorn (35, 70, 140 mg/kg, ig) on cobalt chloride (5.5 mg/kg, ip)- and hypobaric chamber (simulating 5,000 m)-induced high-altitude polycythemia in rats were measured. Total flavonoids decreased red blood cell number, hemoglobin, hematocrit, mean corpuscular hemoglobin levels, span of red blood cell electrophoretic mobility, aggregation index of red blood cell, plasma viscosity, whole blood viscosity, and increased deformation index of red blood cell, erythropoietin level in serum. Total flavonoids increased pH, pO₂, Sp(O₂), pCO₂ levels in arterial blood, and increased Na⁺, HCO₃⁻, Cl⁻, but decreased K⁺ concentrations. Total flavonoids increased mean arterial pressure, left ventricular systolic pressure, end-diastolic pressure, maximal rate of rise and decrease, decreased heart rate and protected right ventricle morphology. Changes in hemodynamic, hematologic parameters, and erythropoietin content suggest that administration of total flavonoids from seabuckthorn may be useful in the prevention of high altitude polycythaemia in rats.

Topics: Altitude; Altitude Sickness; Animals; Blood Gas Analysis; Disease Models, Animal; Erythrocyte Indices; Erythropoietin; Flavonoids; Heart Ventricles; Hemodynamics; Hippophae; Male; Polycythemia; Protective Agents; Rats; Rats, Wistar

Our recent studies indicated that up-regulation of calcineurin activity and unfolded protein responses (UPRs) disrupt cytoprotective Akt- and ERK-signaling in OLETF, a model of obese type 2 diabetes (T2DM). To determine whether the mechanisms can be generalized, we used Goto-Kakizaki rats (GK), a model of non-obese T2DM, in this study. Infarct sizes after 20-min ischemia/2-h reperfusion were similar in GK and non-diabetic controls, Wistar rats (Wistar). However, erythropoietin (EPO) limited infarct size in Wistar (64.0±5.3% vs. 45.7±4.4%, p<0.05) but not in GK (56.2±2.2% vs. 52.6±2.3%). Levels of calcineurin activity and EPO-induced phosphorylation of Akt and ERK were similar in GK and Wistar, though cytosolic HSP70 level was 50% lower and mitochondrial HSP60 level was 60% higher in GK. EPO preserved mitochondrial calcium retention capacity (CRC), an index of the threshold for opening of the mitochondrial permeability transition pore (mPTP), after ischemia/reperfusion in Wistar but not in GK. Interaction of cyclophilin D (CypD) with mitochondrial inorganic phosphate carrier (PiC), which sensitizes the mPTP, was enhanced in GK. There was a negative exponential relationship between CypD-PiC interaction and CRC upon reperfusion, indicating that increase in CRC by reduction of CypD-PiC interaction is smaller when CypD-PiC interaction level is at a higher range. A chemical chaperone, 4-phenylbutyric acid, attenuated the changes in HSPs and CypD-PiC interaction and restored responses of CRC and infarct size to EPO in GK. These results suggest that cytoprotective regulation of the mPTP is impaired in GK by enhanced CypD-PiC interaction in which UPRs are involved.

Topics: Animals; Calcineurin; Calcium; Cardiotonic Agents; Cyclophilins; Diabetes Mellitus, Type 2; Disease Models, Animal; Erythropoietin; Heart; In Vitro Techniques; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Reperfusion Injury; Myocardium; Peptidyl-Prolyl Isomerase F; Rats; Rats, Wistar; Signal Transduction

Hypoxia-ischemia (HI) and associated brain injuries are seen in premature as well as term infants with birth complications. The resulting impairments involve deficits in many cognitive domains, including language development. Poor rapid auditory processing is hypothesized to be one possible underlying factor leading to subsequent language delays. Mild hypothermia treatment for HI injuries in term infants is widely used as an intervention but can be costly and time consuming. Data suggest that the effectiveness of hypothermia treatment following HI injury declines beyond 6 h following injury. Consequently, the availability of a therapeutic alternative without these limitations could allow doctors to treat HI-injured infants more effectively and thus reduce deleterious cognitive and language outcomes. Evidence from both human studies and animal models of neonatal HI suggests that erythropoietin (Epo), an endogenous cytokine hormone, may be a therapeutic agent that can ameliorate HI brain injury and preserve subsequent cognitive development and function. The current study sought to investigate the therapeutic effectiveness of Epo when administered immediately after HI injury, or delayed at intervals following the injury, in neonatal rodents. Rat pups received an induced HI injury on postnatal day 7, followed by an intraperitoneal injection of Epo (1,000 U/kg) immediately, 60 min, or 180 min following induction of injury. Subjects were tested on rapid auditory processing tasks in juvenile (P38-42) and adult periods (P80-85). Ventricular and cortical size was also measured from post mortem tissue. Results from the current study show a therapeutic benefit of Epo when given immediately following induction of HI injury, with diminished benefit from a 60-min-delayed injection of Epo and no protection following a 180-min-delayed injection. The current data thus show that the effectiveness of a single dose of Epo in ameliorating auditory processing deficits following HI injury decreases precipitously as treatment is delayed following injury. These data may have important implications for experimental human neonatal intervention with Epo.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Erythropoietin; Hypoxia-Ischemia, Brain; Male; Neuroprotective Agents; Rats; Rats, Wistar

The renoprotective effect of erythropoietin (Epo) against ischaemia-reperfusion injury (IR/I) was evaluated in a non-human primate model.. Crab-eating macaques were divided into two groups: Control (n = 10), treated with saline, and EPO (n = 10), treated with Epo. Epo was injected intravenously at a dose of 12,000 units, 5 min before clamping the renal pedicle and 5 min before declamping. Renal IR/I was created by clamping the left renal artery for 90 min following right nephrectomy. Haemoglobin (Hb), haematocrit (Ht), creatinine (Cr), blood urea nitrogen (BUN), cystatin C and interleukin-6 (IL-6) were measured before (Pre) and after (Day 0) the operation, and on post-operative days: Day 1, Day 3, Day 5 and Day 7. Apoptotic cells were counted on Day 1.. There were no differences in Hb and Ht between the two groups. Cr, BUN, cystatin C and IL-6 levels in the EPO group were lower than those in the Control group at most of the observation points. The number of apoptotic cells in the Control was significantly higher than that of and EPO group.. Epo significantly ameliorates renal IR/I in this non-human primate model. Our findings justify the clinical application of Epo, not only for acute renal failure, but also in transplantation.

Topics: Animals; Apoptosis; Creatinine; Disease Models, Animal; Erythropoietin; Humans; Kidney Function Tests; Macaca fascicularis; Male; Monkey Diseases; Reperfusion Injury

Hypoxia at high altitudes can lead to increased production of red blood cells through the hormone erythropoietin (EPO). In this study, we observed how the EPO-unresponsive hematopoietic stem cell (HSC) compartment responds to high-altitude hypoxic environments and contributes to erythropoiesis.. Using a mouse model at simulated high altitude, the bone marrow (BM) and spleen lineage marker(-)Sca-1(+)c-Kit(+) (LSK) HSC compartment were observed in detail. Normal LSK cells were then cultured under different conditions (varying EPO levels, oxygen concentrations, and BM supernatants) to investigate the causes of the HSC responses.. Hypoxic mice exhibited a marked expansion in BM and spleen LSK compartments, which were associated with enhanced proliferation. BM HSCs seemed to play a more important role in erythropoiesis at high altitude than spleen HSCs. There was also a lineage fate change of BM HSCs in hypoxic mice that was manifested in increased megakaryocyte-erythrocyte progenitors and periodically reduced granulocyte-macrophage progenitors in the BM. The LSK cells in hypoxic mice displayed upregulated erythroid-specific GATA-1 and downregulated granulocyte-macrophage-specific PU.1 messenger RNA expression, as well as the capacity to differentiate into more erythroid precursors after culture. BM culture supernatant from hypoxic mice (but not elevated EPO or varying O(2) tension) could induce expansion and erythroid-priority differentiation of the HSC population, a phenomenon partially caused by increasing interleukin-3 and interleukin-6 secretion in the BM.. The present study suggests a new EPO-independent HSC mechanism of high-altitude erythrocytosis.

Topics: Altitude; Animals; Base Sequence; Cells, Cultured; Cytokines; Disease Models, Animal; DNA Primers; Erythropoietin; Flow Cytometry; Hematopoietic Stem Cells; Hypoxia; Male; Mice; Mice, Inbred BALB C; Oxygen; Polycythemia; Reverse Transcriptase Polymerase Chain Reaction

Cerebral malaria (CM) causes substantial mortality and neurological sequelae in survivors, and no neuroprotective regimens are currently available for this condition. Erythropoietin (EPO) reduces neuropathology and improves survival in murine CM. Using the Plasmodium berghei model of CM, we investigated if EPO's neuroprotective effects include activation of endogenous neural stem cells (NSC). By using immunohistochemical markers of different NSC maturation stages, we show that EPO increased the number of nestin(+) cells in the dentate gyrus and in the sub-ventricular zone of the lateral ventricles, relative to control-treatment. 75% of the EPO-treated CM mice displayed migration as nestin(+) NSC. The NSC showed differentiation towards a neural cell lineage as shown by PSA-NCAM binding and NSC maturation and lineage commitment was significantly affected by exogenous EPO and by CM in the sub ventricular zone. These results indicate a rapid, EPO-dependent activation of NSC during CM pathology.

Topics: Analysis of Variance; Animals; Disease Models, Animal; Erythropoietin; Female; Immunohistochemistry; Intermediate Filament Proteins; Malaria, Cerebral; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Nestin; Neural Cell Adhesion Molecule L1; Neural Stem Cells; Neurites; Neuroprotective Agents; Plasmodium berghei; Sialic Acids; Specific Pathogen-Free Organisms

Recent observations have demonstrated neuroprotective role of erythropoietin (Epo) and Epo receptor in the central nervous system. Here we examined Epo function in the murine spinal cord after transplantation of pluripotent mouse embryonic stem (ES) cells pre-differentiated towards neuronal type following spinal cord injury. Expression of Epo was measured at both mRNA and protein levels in the ES cells as well as in the spinal cords after 1 and 7 days. Our data demonstrated that expression of Epo mRNA, as well as its protein content, in ES cells was significantly decreased after differentiation procedure. In the spinal cords, analysis showed that Epo mRNA level was significantly decreased after 1 day of ES cell injections in comparison to media-injected control. Epo protein level detected by Western blot was diminished as well. Examination of Epo production in the injured spinal cords after media or ES cells injections by indirect immunofluorescence showed increased Epo-immunopositive staining after media injections 1 day after injection. In contrast, ES cell transplantation did not induce Epo expression. Seven days after ES cell injections, Epo-immunopositive cells' distribution in the ipsilateral side was not changed, while the intensity of immunostaining on the contralateral side was increased, approaching levels in control media-injected tissues. Our data let us to presume that previously described immediate positive effects of ES cells injected into the injured zone of spinal cord are not based on Epo, but on other factors or hormones, which should be elucidated further.

Topics: Animals; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Embryonic Stem Cells; Erythropoietin; Flow Cytometry; Gene Expression Regulation; Green Fluorescent Proteins; Mice; Nuclear Receptor Subfamily 4, Group A, Member 2; RNA, Messenger; Spinal Cord Injuries; Time Factors; Transfection

We examined the preventive effect of human recombinant erythropoietin (HrEPO) on nitric oxide (NO)-mediated toxicity to neurons and cysteine protease release into cytoplasm, which is attributed to neuronal death in brain ischemia. Focal cerebral ischemia was induced by permanent occlusion of middle cerebral artery in two sets of rat. The first set was used to monitor NO concentration and cathepsin activity, while the second was used for histological examination with hematoxylin and eosin, and TUNEL staining. A group in both set was administered human recombinant erythropoietin (HrEPO). NO content, cathepsins B and L activity increased significantly in the post-ischemic cerebral tissue (p<0.05). HrEPO treatment reduced NO concentration and cathepsin activity to control level (p>0.05). A significant increase in the number of necrotic and apoptotic neurons was observed in the post-ischemic cerebral cortex (p<0.05). HrEPO treatment was markedly lowered both of these (p<0.05). It is concluded that HrEPO prevents neuronal death by protecting neuronal liposomes from NO-mediated toxicity and suppressing the release of cathepsins.

Topics: Animals; Brain Infarction; Brain Ischemia; Cathepsin B; Cathepsin L; Cell Death; Disease Models, Animal; Erythropoietin; Humans; Nerve Degeneration; Neuroprotective Agents; Rats; Rats, Sprague-Dawley

Stroke is a major human health problem inducing long-term disability without any efficient therapeutic option being currently available. Under hypoxia, hypoxia-inducible factor-1α (HIF-1α) activates several genes as erythropoietin receptor (Epo-R) related with O(2) supply, and the multidrug-resistance gene (MDR-1) related with drug-refractory phenotype. Brain cortical injection of CoCl(2) produces focal hypoxia-like lesion with neuronal and glial alterations, as well as HIF-1α stabilization and MDR-1 overexpression. Intranasal (IN) drug delivery can by-pass blood-brain barrier (BBB) where MDR-1 is normally expressed. We evaluated the effects of IN-rHu-Epo administration on spontaneous motor activity (SMA) and the brain pattern expression of HIF-1α, MDR-1, and Epo-R in our cobalt-induced hypoxia model. Adult male Wistar rats were injected by stereotaxic surgery in frontoparietal cortex, with CoCl(2) (2 μl-50 mM; n = 20) or saline (controls; n = 20). Ten rats of each group were treated with IN-rHu-Epo 24 U or IN-saline. In addition, erythropoietic stimulation was evaluated by reticulocytes (Ret) account during three consecutive days, after intraperitoneal (i.p.)-recombinant-human Epo (rHu-Epo) (950 U; n = 6) or IN-rHu-Epo (24 U; n = 6) administration. SMA was evaluated by open field and rotarod tests, before and after surgical procedures during five consecutive days. Histological and immunostaining studies of HIF-1α, MDR-1, and Epo-R were performed on brain slides. A significant difference in SMA was observed in the hypoxic rats of IN-rHu-Epo-administered group as compared with Co-Saline-treated subjects and controls (p < 0.001). HIF-1α, EPO-R, and MDR-1 were overexpressed in the hypoxic cortex areas, while in contralateral hemisphere or controls, they were negatives. Reticulocytes were only increased in intraperitoneal (i.p.)-rHu-Epo-administered group. In spite of MDR-1 overexpression being detected in neurons, the coexpression of Epo-R could explain the positive effects observed on SMA of IN-rHu-Epo-administered group.

Topics: Administration, Intranasal; Analysis of Variance; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cobalt; Disease Models, Animal; Erythropoietin; Exploratory Behavior; Gene Expression Regulation; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia, Brain; Male; Motor Activity; Rats; Rats, Wistar; Receptors, Erythropoietin; Recombinant Proteins; Recovery of Function; Reticulocytes; Rotarod Performance Test

The hurtful feelings associated with failing can be devastating especially if the failure occurs after the investment of a considerable effort. The reflection of a lifetime of work in translational medicine has revealed that the study of failures can give birth to new insights that can be explored with important consequences. This article discusses the analysis of two failures that have led to remarkable discoveries. The first led to the discovery of TNF as an important mediator of inflammation that can, if unchecked, cause severe damage in mammals. The second is the identification of erythropoietin as the natural inhibitor of the production and biological activity of TNF. I hope that this paper will help give students the courage to persist in looking for the insights that are the by-products of failure, and to understand the long time lines in the path of discoveries.

Topics: Animals; Disease Models, Animal; Erythropoietin; History, 20th Century; Humans; Incidental Findings; Inflammation; Tumor Necrosis Factor-alpha

The objective of the study was to investigate the effects of recombinant human erythropoietin (rhEPO) in a rat model of cervical sub-acute spinal cord compression. 80 Wistar rats were randomly divided into 4 groups. Rats in the sham group (Group A, n=5) underwent surgical procedures without cervical spinal cord compression; while rats in other groups were subjected to the spinal compression process. In the control group (Group B, n=25), rats received an i.v. injection of 1 mL saline at day 7 post-surgery. Rats in the low-dose group (Group C, n=25) and the high-dose group (Group D, n=25) were treated with rhEPO at 500 units/kg body-weight and 5000 units/kg, respectively, via intravenous injection at day 7 post surgery. Limb motor function was scored by Basso-Beattie-Bresnahan (BBB) standards at 3, 7, 14, 21 and 28 days post-surgery. The distribution and quantities of EPO and its receptor (EPO-R) in the compressed segment of the spinal cord were detected by immunohistochemistry. Motor neuron apoptosis in the spinal cord was evaluated using TUNEL staining and flow cytometry at the indicated time points. Finally, IL-8, TNF-α, IL-6, and IL-1β levels in the compressed cervical spinal cord were determined by ELISA within the lesion epicenter at each time point post-surgery. The data suggest that expression of EPO-R was significantly increased following sub-acute cervical spinal cord compression; Groups C and D exhibited better BBB scores at all observed time points compared with the control group (p<0.01). Using TUNEL staining and FCM, we observed that rhEPO profoundly inhibited motor neuron apoptosis in the spinal cord at day 21 (p<0.01). Additionally, treatment with rhEPO halted the elevation of inflammatory cytokines. rhEPO administration decreased motor neuron apoptosis in the cervical spinal cord, improved motor functions and reduced the inflammatory response in a sub-acute cervical spinal cord compression model. Moreover, sustained treatment with low doses of rhEPO revealed a positive therapeutic effect.

Topics: Analysis of Variance; Animals; Annexin A5; Apoptosis; Aquaporins; Cervical Vertebrae; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Eye Proteins; Female; Flow Cytometry; Humans; In Situ Nick-End Labeling; Motor Neurons; Neuroprotective Agents; Random Allocation; Rats; Rats, Wistar; Recombinant Proteins; Spinal Cord Compression; Time Factors

Paraplegia remains a devastating complication for patients undergoing thoracic aortic procedures. Although surgical adjuncts have evolved to reduce the risk of paraplegia, no pharmacologic therapies have proven efficacious in attenuating spinal cord ischemia-reperfusion injury. Effects of erythropoietin in spinal cord ischemia-reperfusion injury, however, have not yet been elucidated. We hypothesized that pretreatment with erythropoietin would attenuate functional and cytoarchitectural spinal cord injury related to high-risk aortic procedures.. Adult male mice were subjected to ischemia-reperfusion. Aortic arch and proximal left subclavian arteries were clamped for 5 minutes; animals were observed for 48 hours. Neurologic scores of hind limb function were assessed every 12 hours. Experimental groups consisted of treatment with erythropoietin 4 hours before crossclamping (n = 7), ischemic controls (n = 7), and sham ischemia (operation without crossclamping, n = 6). Thoracolumbar sections of spinal cord were removed after 48 hours and preserved for cytoarchitectural analysis.. Mice pretreated with erythropoietin exhibited significant preservation of hind limb motor function. All mice without pretreatment were paralyzed at 48 hours. Mice with erythropoietin pretreatment had improved motor function; 3 had no measurable neurologic deficit at 48 hours. Histologic analysis in mice treated with erythropoietin showed markedly reduced neuronal cell injury.. Erythropoeitin preserves both function and histologic appearance in mice undergoing spinal cord ischemia-reperfusion. With further elucidation of mechanisms of protection and optimal administration, erythropoietin could become an important adjunct in reducing the incidence and severity of spinal cord injury related to aortic interventions.

Topics: Animals; Aorta, Thoracic; Constriction; Disease Models, Animal; Erythropoietin; Hindlimb; Male; Mice; Mice, Inbred C57BL; Motor Activity; Muscle, Skeletal; Neuroprotective Agents; Paraplegia; Recombinant Proteins; Reperfusion Injury; Spinal Cord; Spinal Cord Ischemia; Subclavian Artery; Time Factors

Erythropoietin promotes myoblast proliferation and inhibits fibrosis and thus it could impede the pathogenesis of muscle degenerative diseases. However, its stimulation of erythropoiesis limits its use as a therapeutic agent. An erythropoietin analog, carbamylated erythropoietin (C-EPO), retains these protective actions, yet it does not interact with the erythropoietin receptor. To determine whether treatment with C-EPO alleviates the signs of muscular dystrophy in an animal model of Duchenne muscular dystrophy, we treated mdx mice with intraperitoneal injections of 50 μg/kg and 100 μg/kg C-EPO for 4 and 12 weeks, and we monitored weight, serum creatine kinase levels, and changes in muscle histology. Moderate histological improvement was observed at 4 weeks, which did not translate into a significantly decreased level of serum creatine kinase. At the doses tested, C-EPO is not an effective therapeutic for the treatment of a mouse model of Duchenne muscular dystrophy.

Topics: Animals; Cell Proliferation; Creatine Kinase; Disease Models, Animal; Erythropoietin; Female; Injections, Intraperitoneal; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Muscle, Skeletal; Muscular Dystrophy, Animal; Myoblasts; Neuroprotective Agents; Treatment Failure

Erythropoietin (EPO) has been shown to have anti-inflammatory, antiapoptotic, and proangiogenic effects. This study investigated whether early EPO treatment effectively preserves left ventricular (LV) function in porcine acute myocardial infarction (AMI). Eighteen male mini-pigs divided into groups 1 (sham), 2 (AMI), and 3 (AMI with 2 consecutive EPO doses [7500 IU per animal each time] at 30 minutes and 24 hours after AMI induction) underwent echocardiography before and 14 days after AMI induction through left anterior descending artery (LAD) ligation with myocardium harvested for analysis. Larger infarcted areas (IA) were noted in group 2 than in group 3. In both IA and peri-IA, percentage of apoptotic nuclei and CD40-positive cells, messenger RNA expressions of IL-8, matrix metalloproteinase-9, caspase-3, and Bcl-2 associated x protein were highest, whereas proliferator-activated receptor-γ coactivator-1α, endothelial nitric oxide synthase and Bcl-2 were lowest in group 2. Oxidative stress and cytosolic cytochrome c in IA were increased (P < 0.001), whereas protein expression of connexin43, cytochrome c, and protein kinase C-ε; in mitochondria were reduced in group 2 than in other groups (P < 0.045). The fibrosis in IA was notably decreased in group 3 compared with that in group 2. The number of small arterioles and capillary density in IA was highest in group 3, whereas LV performance was lowest in group 2 (P < 0.045). In conclusion, the results demonstrated that early EPO administration in a porcine AMI model effectively limits infarct size, attenuates LV remodeling, and preserves LV function.

Topics: Animals; Disease Models, Animal; Erythropoietin; Humans; Male; Myocardial Infarction; Recombinant Proteins; Swine; Swine, Miniature; Time Factors; Ventricular Function, Left; Ventricular Remodeling

Intrauterine infection is a common antecedent of preterm birth. Infants born very preterm are at increased risk for neurologic dysfunction, including visual deficits. With increasing survival of very preterm infants, there is a need for therapies that prevent adverse neurologic outcomes. Using an ovine model, the authors investigated the neuroprotective potential of recombinant human erythropoietin (rhEPO) on retinal injury induced by intrauterine inflammation.. At 107 ± 1 days of gestational age (DGA), chronically catheterized fetal sheep received either of the following on 3 consecutive days: intravenous (IV) bolus dose of lipopolysaccharide (LPS; ∼0.9 μg/kg; n = 8); IV bolus dose of LPS, followed at 1 hour by 5000 IU/kg rhEPO (LPS + rhEPO; n = 8); rhEPO alone (n = 5). Untreated fetuses (n = 8) were used for comparison with the three treatment groups. Fetal physiological parameters were monitored. At 116 ± 1 DGA, fetal retinas were assessed quantitatively for morphologic and neurochemical alterations.. Exposure to LPS alone, but not to rhEPO alone, resulted in fetal hypoxemia and hypotension (P < 0.05). Exposure to LPS alone caused retinal changes, including reductions in thickness of the inner nuclear layer (INL), somal areas of INL neurons, process growth in the plexiform layers, and numbers of ganglion and tyrosine hydroxylase immunoreactive (TH-IR) dopaminergic amacrine cells. Treatment of LPS-exposed fetuses with rhEPO did not alter the physiological effects of LPS but significantly reduced alterations in retinal layers and ganglion and TH-IR cell numbers.. rhEPO treatment was beneficial in protecting the developing retina after LPS-induced inflammation. Retinal protection could occur by the antiapoptotic or anti-inflammatory actions of EPO.

Topics: Amacrine Cells; Animals; Cell Count; Disease Models, Animal; Erythropoietin; Escherichia coli; Female; Fetal Hypoxia; Fluorescent Antibody Technique, Indirect; Gestational Age; Immunoenzyme Techniques; Lipopolysaccharides; Macrophages; Microglia; Pregnancy; Receptors, Erythropoietin; Recombinant Proteins; Retina; Retinal Diseases; Retinal Ganglion Cells; Sheep, Domestic; Tyrosine 3-Monooxygenase

The impermeant nature of the intestinal barrier is maintained by tight junctions (TJs) formed between adjacent intestinal epithelial cells. Disruption of TJs and loss of barrier function are associated with a number of gastrointestinal diseases, including neonatal necrotizing enterocolitis (NEC), the leading cause of death from gastrointestinal diseases in preterm infants. Human milk is protective against NEC, and the human milk factor erythropoietin (Epo) has been shown to protect endothelial cell-cell and blood-brain barriers. We hypothesized that Epo may also protect intestinal epithelial barriers, thereby lowering the incidence of NEC. Our data demonstrate that Epo protects enterocyte barrier function by supporting expression of the TJ protein ZO-1. As immaturity is a key factor in NEC, Epo regulation of ZO-1 in the human fetal immature H4 intestinal epithelial cell line was examined and demonstrated Epo-stimulated ZO-1 expression in a dose-dependent manner through the PI3K/Akt pathway. In a rat NEC model, oral administration of Epo lowered the incidence of NEC from 45 to 23% with statistical significance. In addition, Epo treatment protected intestinal barrier function and prevented loss of ZO-1 at the TJs in vivo. These effects were associated with elevated Akt phosphorylation in the intestine. This study reveals a novel role of Epo in the regulation of intestinal epithelial TJs and barrier function and suggests the possible use of enteral Epo as a therapeutic agent for gut diseases.

Topics: Animals; Animals, Newborn; Cell Line, Tumor; Disease Models, Animal; Electric Impedance; Enterocolitis, Necrotizing; Enterocytes; Erythropoietin; Fluorescent Antibody Technique; Humans; Immunoblotting; Intestinal Mucosa; Intestines; Membrane Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Proto-Oncogene Proteins c-akt; Rats; RNA Interference; Tight Junctions; Zonula Occludens-1 Protein

Children who experience complex febrile seizures are at a higher risk of subsequent epileptic episodes, and they may require therapy. This issue can be resolved by interventional studies using molecular targets identified and defined in animal models. In the current study, the molecular changes in the rat brain after febrile seizures were examined throughout the latent period, and erythropoietin was administered as a potentially antiepileptogenic intervention.. The changes in the expressions of genes that were differentially regulated during the latent period after febrile seizures were categorized into the following four patterns: (1) continuously high (CH); (2) continuously low (CL); (3) rise and fall (RF); and (4) going-up (GU). Erythropoietin was administered immediately after seizure cessation and then once daily for at most 7 days, and spontaneous recurrent seizures and cellular and molecular changes were investigated.. The CH genes were associated with cell cycle and adhesion, whereas the CL genes were related to energy metabolism. Within the category of RF, the largest changes were for genes involved in inflammation, apoptosis, and γ-aminobutyric acid (GABA) signaling. The GU category included genes involved in ion transport and synaptogenesis. Along with an early rise in inflammatory genes, there were substantial increases in brain edema and activated microglia during the early latent period. Erythropoietin reduced the early inflammatory responses and modulated the molecular alterations after febrile seizures, thereby reducing the risk of subsequent spontaneous seizures.. Erythropoietin treatment may provide a new strategy for preventing epilepsy in susceptible individuals with atypical febrile seizures.

Topics: Animals; Animals, Newborn; Anticonvulsants; Apoptosis; Blood-Brain Barrier; Brain; CD11b Antigen; Cell Adhesion; Cell Cycle; Disease Models, Animal; Electroencephalography; Energy Metabolism; Erythropoietin; gamma-Aminobutyric Acid; Gene Expression Regulation; Ion Transport; Rats; Rats, Sprague-Dawley; Seizures, Febrile; Signal Processing, Computer-Assisted; Signal Transduction; Synapses

Parkinson's disease (PD) is caused by oxidative stress, and erythropoietin (EPO) reduces oxidative stress in the brain. However, EPO cannot be developed as a treatment for PD, because EPO does not cross the blood-brain barrier (BBB). A brain penetrating form of human EPO has been developed wherein EPO is fused to a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), which is designated as the cTfRMAb-EPO fusion protein. The TfRMAb acts as a molecular Trojan horse to transport the fused EPO into brain via transport on the BBB TfR. Experimental PD was induced in adult mice by the intra-striatal injection of 6-hydroxydopamine, and PD mice were treated with 1mg/kg of the cTfRMAb-EPO fusion protein intravenously (IV) every other day starting 1 h after toxin injection. Following 3weeks of treatment mice were euthanized for measurement of striatal tyrosine hydroxylase (TH) enzyme activity. Mice treated with the cTfRMAb-EPO fusion protein showed a 306% increase in striatal TH enzyme activity, which correlated with improvement in three assays of neurobehavior. The blood hematocrit increased 10% at 2weeks, with no further changes at 3weeks of treatment. A sandwich ELISA showed the immune reaction against the cTfRMAb-EPO fusion protein was variable and low titer. In conclusion, the present study demonstrates that a brain penetrating form of EPO is neuroprotective in PD following IV administration with minimal effects on erythropoiesis.

Topics: Animals; Blood-Brain Barrier; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Erythropoietin; Humans; Immunoglobulin G; Injections, Intravenous; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Parkinsonian Disorders; Recombinant Fusion Proteins

Erythropoietin (EPO) improves functional recovery after traumatic brain injury (TBI). This study was designed to investigate long-term (3 months) effects of EPO on brain remodeling and functional recovery in rats after TBI. Young male Wistar rats were subjected to unilateral controlled cortical impact injury. TBI rats were divided into the following groups: (1) saline group (n=7); (2) EPO-6h group (n=8); and (3) EPO-24h group (n=8). EPO (5000 U/kg in saline) was administered intraperitoneally at 6h, and 1 and 2 days (EPO-6h group) or at 1, 2, and 3 days (EPO-24h group) postinjury. Neurological function was assessed using a modified neurological severity score, footfault and Morris water maze tests. Animals were sacrificed at 3 months after injury and brain sections were stained for immunohistochemical analyses. Compared to the saline, EPO-6h treatment significantly reduced cortical lesion volume, while EPO-24h therapy did not affect the lesion volume (P<0.05). Both the EPO-6h and EPO-24h treatments significantly reduced hippocampal cell loss (P<0.05), promoted angiogenesis (P<0.05) and increased endogenous cellular proliferation (BrdU-positive cells) in the injury boundary zone and hippocampus (P<0.05) compared to saline controls. Significantly enhanced neurogenesis (BrdU/NeuN-positive cells) was seen in the dentate gyrus of both EPO groups compared to the saline group. Both EPO treatments significantly improved long-term sensorimotor and cognitive functional recovery after TBI. In conclusion, the beneficial effects of posttraumatic EPO treatment on injured brain persisted for at least 3 months. The long-term improvement in functional outcome may in part be related to the neurovascular remodeling induced by EPO.

Topics: Analysis of Variance; Animals; Body Weight; Brain Injuries; Bromodeoxyuridine; Cell Count; Cell Proliferation; Disease Models, Animal; Erythropoietin; Gait Disorders, Neurologic; Male; Maze Learning; Neovascularization, Pathologic; Phosphopyruvate Hydratase; Rats; Rats, Wistar; Recovery of Function; Severity of Illness Index; von Willebrand Factor

Endothelial dysfunction triggers early pathological changes in vessel walls, potentially leading to the formation of cerebral aneurysm (CA). Endothelial progenitor cells (EPCs) are critical in repairing damaged endothelium and could prevent or slow CA formation. We hypothesize that erythropoietin (EPO) stimulates EPCs mobilization, could alter the rate of CA formation and progression. The hypothesis was tested in a rat model of CA. CAs were induced in male Sprague-Dawley rats and treated with s.c. administration of EPO. Circulating EPCs and serum vascular endothelial grow factor (VEGF) were measured be flow cytometry and ELISA, respectively. mRNAs for inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), matrix metalloproteinase-2 (MMP-2), and MMP-9 in aneurysm tissue were quantified by Real-time PCR. The size, internal elastic lamina (IEL), and media thickness of CAs were evaluated 1 and 3 months after aneurysm induction. Circulating EPCs were significantly lower in CA rats as compared to non-surgical controls. EPO increased levels of circulating EPCs and VEGF. It also decreased iNOS, MMP-2, and MMP-9 mRNA levels, while increased eNOS mRNA in aneurysm tissue. The changes in EPCs and biochemical markers are associated with suppression of new CA formation and prevention of preexisting CA progression. We have shown a close association among circulating EPCs, biochemical markers related to vascular remodeling, and the rate of CA formation and progression. Changes in patterns of cerebral blood flow and hypertension induced by surgical ligations of selected arteries exert significant hemodynamic stress to weaken vessel walls, primarily at sites of basilar bifurcation. The surgical stress also reduced circulating EPCs and slowed vascular repairs. EPO mobilizes EPCs from the bone marrow and promotes their homing. These results suggest that EPCs may serve as a marker for CA progression and EPO a promising candidate for the clinical management of CA.

Topics: Animals; Disease Models, Animal; Endothelial Cells; Erythropoietin; Intracranial Aneurysm; Male; Rats; Rats, Sprague-Dawley; Stem Cells

Erythropoietin (EPO) is a cytokine hormone with cytoprotective effects in many tissues including the brain. Although the benefits of administration of recombinant human EPO (rhEPO) for neonatal hypoxic brain injury have been demonstrated in neuronal tissue, the effect on non-neuronal cell populations is unclear. We tested the hypothesis that rhEPO would not only protect neuronal cells but also glial cells at a stage of brain development where their maturation was particularly sensitive, and also protect the vasculature. This was evaluated in a rat model of hypoxic injury. 1000 IU/kg rhEPO was delivered intraperitoneally at the start of 4 h hypoxia or normoxia. Treatment groups of neonatal rats (day of birth, at least N = 10 per group) were as follows: normoxia; normoxia plus rhEPO; hypoxia (8% FiO(2) delivered in temperature-controlled chambers); and hypoxia plus rhEPO. Day of birth in rats is equivalent to human gestation of 28-32 weeks. The effects of rhEPO administration, especially to non-neuronal cell populations, and the associated molecular pathways, were investigated. Apoptosis was increased with hypoxia and this was significantly reduced with rhEPO (p < 0.05). The neuronal marker, microtubule-associated protein-2, increased in expression (p < 0.05) when apoptosis was significantly reduced by rhEPO. In addition, compared with hypoxia alone, rhEPO-treated hypoxia had the following significant protein expression increases (p < 0.05): the intermediate filament structural protein nestin; myelin basic protein (oligodendrocytes); and glial fibrillary acidic protein (astrocytes). In conclusion, rhEPO protects the developing brain via anti-apoptotic mechanisms and promotes the health of non-neuronal as well as neuronal cell populations at a time when loss of these cells would have long-lasting effects on brain function.

Topics: Animals; Animals, Newborn; Apoptosis; Astrocytes; Disease Models, Animal; Drug Evaluation, Preclinical; Erythropoietin; Hypoxia-Ischemia, Brain; Intermediate Filament Proteins; Nerve Tissue Proteins; Nestin; Neurons; Oligodendroglia; Oxidative Stress; Rats; Receptors, Erythropoietin; Recombinant Proteins; Signal Transduction

Multiple complications can ensue in the cardiovascular, renal, and nervous systems during diabetes mellitus (DM). Given that endothelial cells (ECs) are susceptible targets to elevated serum D-glucose, identification of novel cellular mechanisms that can protect ECs may foster the development of unique strategies for the prevention and treatment of DM complications. Erythropoietin (EPO) represents one of these novel strategies but the dependence of EPO upon Wnt1 and its downstream signaling in a clinically relevant model of DM with elevated D-glucose has not been elucidated. Here we show that EPO can not only maintain the integrity of EC membranes, but also prevent apoptotic nuclear DNA degradation and the externalization of membrane phosphatidylserine (PS) residues during elevated D-glucose over a 48-hour period. EPO modulates the expression of Wnt1 and utilizes Wnt1 to confer EC protection during elevated D-glucose exposure, since application of a Wnt1 neutralizing antibody, treatment with the Wnt1 antagonist DKK-1, or gene silencing of Wnt1 with Wnt1 siRNA transfection abrogates the protective capability of EPO. EPO through a novel Wnt1 dependent mechanism controls the post-translational phosphorylation of the "pro-apoptotic" forkhead member FoxO3a and blocks the trafficking of FoxO3a to the cell nucleus to prevent apoptotic demise. EPO also employs the activation of protein kinase B (Akt1) to foster phosphorylation of GSK-3β that appears required for EPO vascular protection. Through this inhibition of GSK-3β, EPO maintains β-catenin activity, allows the translocation of β-catenin from the EC cytoplasm to the nucleus through a Wnt1 pathway, and requires β-catenin for protection against elevated D-glucose since gene silencing of β-catenin eliminates the ability of EPO as well as Wnt1 to increase EC survival. Subsequently, we show that EPO requires modulation of both Wnt1 and FoxO3a to oversee mitochondrial membrane depolarization, cytochrome c release, and caspase activation during elevated D-glucose. Our studies identify critical elements of the protective cascade for EPO that rely upon modulation of Wnt1, Akt1, FoxO3a, GSK-3β, β-catenin, and mitochondrial apoptotic pathways for the development of new strategies against DM vascular complications.

Topics: Animals; beta Catenin; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Disease Models, Animal; Endothelium, Vascular; Erythropoietin; Forkhead Box Protein O3; Forkhead Transcription Factors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Wnt1 Protein

Multiple system atrophy is a rapidly progressive neurodegenerative disorder with a markedly reduced life expectancy. Failure of symptomatic treatment raises an urgent need for disease-modifying strategies. We have investigated the neuroprotective potential of erythropoietin in (proteolipid protein)-α-synuclein transgenic mice exposed to 3-nitropropionic acid featuring multiple system atrophy-like pathology including oligodendroglial α-synuclein inclusions and selective neuronal degeneration. Mice were treated with erythropoietin starting before (early erythropoietin) and after (late erythropoietin) intoxication with 3-nitropropionic acid. Nonintoxicated animals receiving erythropoietin and intoxicated animals treated with saline served as control groups. Behavioral tests included pole test, open field activity, and motor behavior scale. Immunohistochemistry for tyrosine hydroxylase and dopamine and cyclic adenosine monophosphate-regulated phosphoprotein (DARPP-32) was analyzed stereologically. Animals receiving erythropoietin before and after 3-nitropropionic acid intoxication scored significantly lower on the motor behavior scale and they performed better in the pole test than controls with no significant difference between early and late erythropoietin administration. Similarly, rearing scores were worse in 3-nitropropionic acid-treated animals with no difference between the erythropoietin subgroups. Immunohistochemistry revealed significant attenuation of 3-nitropropionic acid-induced loss of tyrosine hydroxylase and DARPP-32 positive neurons in substantia nigra pars compacta and striatum, respectively, in both erythropoietin-treated groups without significant group difference in the substantia nigra. However, at striatal level, a significant difference between early and late erythropoietin administration was observed. In the combined (proteolipid protein)-α-synuclein 3-nitropropionic acid multiple system atrophy mouse model, erythropoietin appears to rescue dopaminergic and striatal gabaergic projection neurons. This effect is associated with improved motor function. Further studies are warranted to develop erythropoietin as a potential interventional therapy in multiple system atrophy.

Topics: alpha-Synuclein; Animals; Cell Death; Convulsants; Corpus Striatum; Disease Models, Animal; Dopamine and cAMP-Regulated Phosphoprotein 32; Drug Administration Schedule; Erythropoietin; Exploratory Behavior; Humans; Mice; Mice, Transgenic; Motor Activity; Multiple System Atrophy; Myelin Proteolipid Protein; Nitro Compounds; Propionates; Substantia Nigra; Tyrosine 3-Monooxygenase

Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), d-galactosamine (GalN)-induced FHF is a well-established model of liver injury in mice. Erythropoietin has a powerful tissue-protective effect in animal models. The aim of this study was to investigate the effect and mechanism of recombinant human erythropoietin (rhEPO) administration in FHF mice.. C57BL/6 (n=42) mice were studied in vivo in a fulminant model induced by GalN/LPS. rhEPO was administered 30 min after the induction of FHF. Serum liver enzymes and hepatic tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels were determined. Histologic analysis was performed, and apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor (NF)-κB and c-Jun-N-terminal kinase (JNK) activation were studied using Western blot analysis.. After the induction of FHF, all control mice died within 12 hr of GalN/LPS administration. However, 83% of mice that were administered rhEPO were alive 2 weeks later, and overall survival improved (Kaplan-Meier, P<0.001). The serum liver enzymes, hepatic TNF-α and IL-1β levels, liver histologic injury, and apoptotic hepatocytes were significantly reduced in FHF mice that were administered rhEPO compared with untreated mice. A significant decrease in hepatic NF-κB and JNK activation was noted in FHF rhEPO-treated mice compared with FHF untreated mice.. The administration of rhEPO brought about increased survival and attenuation of the hepatic injury. This was associated with decreased hepatic NF-κB and JNK activation and thus TNF-α and IL-1β levels. These findings have important implications for the potential use of rhEPO in FHF.

Topics: Alanine Transaminase; Animals; Apoptosis; Aspartate Aminotransferases; Disease Models, Animal; Erythropoietin; Galactosamine; Humans; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Kaplan-Meier Estimate; Lipopolysaccharides; Liver; Liver Failure, Acute; Mice; Mice, Inbred C57BL; NF-kappa B; Recombinant Proteins; Tumor Necrosis Factor-alpha

Perioperative renal dysfunction is associated with a high mortality. The aim of this study was to investigate whether isoflurane preconditioning provides a protection against renal ischemic-reperfusion injury and whether hypoxia inducible factor 1 α (HIF-1 α) is responsible for the protection afforded by isoflurane in mice.. Adult male C57BL/6 mice received vehicle (PBS), scrambled siRNA or HIF-1 α siRNA via hydrodynamic injection through tail vein. Twenty-four hours after injection, they were exposed to 1.5% isoflurane in oxygen enriched air for 2h while controls without injection were exposed to oxygen enriched air. Twenty-four hours after gas exposure, mice were sacrificed and their kidney were harvested for western blot while other cohorts underwent renal ischemia-reperfusion injury induced by bilateral renal pedicle clamping for 25 min for renal histological or functional analysis 24h after reperfusion or by unilateral clamping for 40 min for survival rate analysis.. Survival rate and the expression of HIF-1 α and erythropoietin were significantly increased while apoptosis, renal tubule score, blood plasma creatinine and urea were decreased by isoflurane preconditioning. HIF-1 α siRNA but not scrambled siRNA injection abrogated the protective effect of isoflurane preconditioning.. Our data suggested that isoflurane preconditioning provided a protection against renal ischemic-reperfusion injury which is very likely due to hypoxia inducible factor-1 α upregulation.

Topics: Animals; Apoptosis; Blotting, Western; Creatinine; Disease Models, Animal; Erythropoietin; Hypoxia-Inducible Factor 1, alpha Subunit; In Situ Nick-End Labeling; Isoflurane; Kidney; Male; Mice; Mice, Inbred C57BL; Protective Agents; Reperfusion Injury; RNA, Small Interfering; Time Factors; Urea

Periventricular leukomalacia (PVL) is the predominant pathology in premature infants, characterized by prominent cerebral white matter injury, and commonly caused by hypoxia-ischemia and inflammation. Activated microglia trigger white matter damage and play a major role in the development of PVL. Erythropoietin (EPO) and its derivative carbamylated erythropoietin (CEPO) have been shown to be neuroprotective in several brain disease models. Here we investigated whether EPO and CEPO could provide protection in mouse models of PVL induced by hypoxia-ischemia or hypoxia-ischemia-inflammation. We administered EPO or CEPO to mice with PVL, and found that both EPO and CEPO treatments decreased microglia activation, oligodendrocyte damage and myelin depletion. We also noted improved performance in neurological function assays. Inhibited disease progression in PVL mice by EPO or CEPO treatment was associated with decreased poly-(ADP-ribose) polymerase-1 (PARP-1) activity. PARP-1 activity was increased dramatically in activated microglia in untreated mice with PVL. Furthermore, we demonstrated that the neuroprotective properties of EPO and CEPO were diminished after PARP-1 gene depletion. The therapeutic doses of EPO and CEPO used in this study did not interfere with normal oligodendrocyte maturation and myelination. Together, our data demonstrate that EPO and CEPO are neuroprotective in cerebral white matter injury via a novel microglial PARP-1 dependent mechanism, and hold promise as a future treatment for PVL and other hypoxic-ischemic/inflammatory white matter diseases.

Topics: Animals; Brain; Brain Ischemia; Disease Models, Animal; Erythropoietin; Humans; Hypoxia; Infant, Newborn; Inflammation; Leukomalacia, Periventricular; Mice; Microglia; Myelin Sheath; Neurons; Neuroprotective Agents; Oligodendroglia

The neuroprotective effect of erythropoietin has been demonstrated by ischemia and reperfusion models in adult and neonatal rodents. However, administration of high-dose erythropoietin has potential complications. The goal of this study was to determine whether local infusion of low dose erythropoietin offers neuroprotective effects after ischemia and reperfusion injury.. Adult male Sprague-Dawley rats subject to middle cerebral artery occlusion were randomly divided into three groups: (1) sham group: the rats received the same procedure as the other two groups except that no suture was inserted; (2) vehicle group: intra-artery local infusion of saline was administered via middle cerebral artery after reperfusion; and (3) treatment group: 50 U/kg intra-artery local infusion of erythropoietin was administered via middle cerebral artery after reperfusion. Neurological deficit scores and infarct volume (determined by hematoxylin-eosin staining) were evaluated 48 hours after reperfusion. Apoptosis was measured through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expression of vascular endothelial growth factor and phosphorylated extracellular signal-regulated kinase were investigated by immunohistochemistry method.. The results show that intra-artery local infusion of erythropoietin, via the middle cerebral artery, significantly reduced neurological deficit scores, foot fault number, and the infarct volume at 48 hours after reperfusion. Significant reductions were also found in the number of positive cells stained by TUNEL assay within the ischemic core and penumbra. Furthermore, local infusion of erythropoietin increased the expression of phosphorylated extracellular signal-regulated kinase and vascular endothelial growth factor.. Local infusion of low-dose erythropoietin via the middle cerebral artery is shown to be neuroprotective against cerebral ischemia and reperfusion injury. The mechanism of neuroprotection may be associated with the increased expression of phosphorylated extracellular signal-regulated kinase and vascular endothelial growth factor.

Topics: Animals; Brain Ischemia; Disease Models, Animal; Erythropoietin; Infarction, Middle Cerebral Artery; Infusions, Intra-Arterial; Male; Neuroprotective Agents; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Stroke affects infants at a rate of 26/100,000 live births each year. Of these strokes, approximately 6.7 are hemorrhagic strokes. Erythropoietin (EPO) is an anti-apoptotic and neuroprotective hormone. In adult rodents, EPO attenuates inflammatory factor expression and blood-brain barrier damage after intracerebral hemorrhage (ICH). However, the effect of EPO in neonatal ICH stroke remains unexplored. This investigation aimed to elucidate the underpinnings of inflammation after ICH in postnatal day 7 (P7) rats and the effect of human recombinant EPO (hrEPO) treatment on ICH-induced inflammation. The P7 rat pups were pretreated with hrEPO (5,000 U/kg i.p.) or saline vehicle 4 h prior to the induction of ICH by blood injection into the right cerebral cortex and basal ganglia. Supplemental half doses of hrEPO treatment or saline injections were subsequently given 16 h after ICH induction. Real-time PCR done 24 h after ICH showed reductions in interleukin1-β (IL1-β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) mRNA expression in the basal ganglia of the hrEPO-treated rats compared to saline-treated rats. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining indicated fewer dying cells in the hrEPO-treated brain. Our data suggest that hrEPO has an anti-inflammatory action in neonates after ICH. The suppression of inflammatory cascades likely contributes to hrEPO's neuroprotective effect, which may be explored as a therapeutic treatment for ICH.

Topics: Analysis of Variance; Animals; Animals, Newborn; Cell Death; Cerebral Hemorrhage; Cytokines; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; In Situ Nick-End Labeling; Inflammation; Rats; Rats, Wistar; RNA, Messenger

Patients treated with recombinant human Epo demonstrate an improvement in insulin sensitivity. We aimed to investigate whether CNTO 530, a novel Epo receptor agonist, could affect glucose tolerance and insulin sensitivity. A single administration of CNTO 530 significantly and dose-dependently reduced the area under the curve in a glucose tolerance test in diet-induced obese and diabetic mice after 14, 21, and 28 days. HOMA analysis suggested an improvement in insulin sensitivity, and this effect was confirmed by a hyperinsulinemic-euglycemic clamp. Uptake of (14)C-2-deoxy-D-glucose indicated that animals dosed with CNTO 530 transported more glucose into skeletal muscle and heart relative to control animals. In conclusion, CNTO530 has a profound effect on glucose tolerance in insulin-resistant rodents likely because of improving peripheral insulin sensitivity. This effect was observed with epoetin-α and darbepoetin-α, suggesting this is a class effect, but the effect with these compounds relative to CNTO530 was decreased in duration and magnitude.

Topics: Animals; Darbepoetin alfa; Diabetes Mellitus, Experimental; Dietary Fats; Disease Models, Animal; Dose-Response Relationship, Drug; Epoetin Alfa; Erythropoietin; Glucose; Glucose Clamp Technique; Glucose Tolerance Test; Insulin Resistance; Male; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Obesity; Receptors, Erythropoietin; Recombinant Fusion Proteins; Recombinant Proteins; Time Factors

To examine whether recombinant erythropoietin (rEPO) attenuates neurodegeneration and the learning disability induced by isoflurane with the postnatal day 7 (P7) mice.. Some of general anesthetic agents induce neurodegeneration in developing brain. Several drugs, but not rEPO, were reported as candidates for the prevention of or treatment for neurodegeneration.. We divided P7 mice into three groups at random. One group (IE group) was exposed to 6-h isoflurane (1.0%) after 50,000 IU·kg(-1) rEPO administered subcutaneously. The second group (I) was exposed to isoflurane in the same manner as IE group except saline instead of rEPO. The third group (E) was exposed to air after rEPO administered. The mice were assigned to the radial arm maze on four consecutive days from P56 (day 1) to P59 (day 4). We divided the number of errors each day by that of day 1 to establish each-day performance ratio. After the test, neurodegenerative change in the hilus of dentate gyrus was assessed using Nissl staining.. In radial maze test, the performance ratios of day 3 (mean ± sd) were 0.3 ± 0.2 (P < 0.05, vs I group), 0.8 ± 0.5, and 0.6 ± 0.2 in IE, I, and E groups, respectively, while those of day 4 were 0.3 ± 0.1 (P < 0.05), 0.8 ± 0.5, and 0.3 ± 0.2 (P < 0.05), respectively. The histopathological study revealed that in IE group the degenerative neuronal change was attenuated compared with I group.. These results suggested that rEPO attenuated isoflurane-induced neurodegeneration.

Topics: Air; Analysis of Variance; Anesthetics, Inhalation; Animals; Animals, Newborn; Apoptosis; Brain; Disease Models, Animal; Erythropoietin; Isoflurane; Maze Learning; Mice; Mice, Inbred C57BL; Nerve Degeneration; Sodium Chloride

Painful stimuli during neonatal stage may affect brain development and contribute to abnormal behaviors in adulthood. Very few specific therapies are available for this developmental disorder. A better understanding of the mechanisms and consequences of painful stimuli during the neonatal period is essential for the development of effective therapies. In this study, we examined brain reactions in a neonatal rat model of peripheral inflammatory pain. We focused on the inflammatory insult-induced brain responses and delayed changes in behavior and pain sensation. Postnatal day 3 pups received formalin injections into the paws once a day for 3 days. The insult induced dysregulation of several inflammatory factors in the brain and caused selective neuronal cell death in the cortex, hippocampus and hypothalamus. On postnatal day 21, rats that received the inflammatory nociceptive insult exhibited increased local cerebral blood flow in the somatosensory cortex, hyperalgesia, and decreased exploratory behaviors. Based on these observations, we tested recombinant human erythropoietin (rhEPO) as a potential treatment to prevent the inflammatory pain-induced changes. rhEPO treatment (5,000 U/kg/day, i.p.), coupled to formalin injections, ameliorated neuronal cell death and normalized the inflammatory response. Rats that received formalin plus rhEPO exhibited normal levels of cerebral blood flow, pain sensitivity and exploratory behavior. Treatment with rhEPO also restored normal brain and body weights that were reduced in the formalin group. These data suggest that severe inflammatory pain has adverse effects on brain development and rhEPO may be a possible therapy for the prevention and treatment of this developmental disorder.

Topics: Animals; Animals, Newborn; Behavior, Animal; Body Weight; Brain; Cell Death; Cerebrovascular Circulation; Cytokines; Disease Models, Animal; Erythropoietin; Exploratory Behavior; Formaldehyde; Humans; Hyperalgesia; Inflammation; Inflammation Mediators; Neurons; Neuroprotective Agents; Organ Size; Pain; Rats

Hypoxia-inducible factor-1 alpha (HIF-1α) is a master transcription factor that regulates the response to hypoxia and ischemia and induces the expression of various genes, including vascular endothelial growth factor (VEGF) and erythropoietin (EPO). This study shows the systemic response of increased HIF-1α, EPO, and VEGF mRNA and protein. In addition, VEGF expression was increased in neurons and over-expressed in glial cells in a model of neuroexcitotoxicity in the hippocampus, in which rats were neonatally exposed to high glutamate concentrations. Simultaneous increases in HIF-1α, EPO and VEGF mRNA in peritoneal macrophages were also observed. Our study is consistent with the hypothesis that these genes exert a protective effect in response to neurotoxicity.

Topics: Age Factors; Animals; Animals, Newborn; Disease Models, Animal; Erythropoietin; Female; Gene Expression Regulation, Developmental; Glial Fibrillary Acidic Protein; Glutamic Acid; Hippocampus; Hypoxia-Inducible Factor 1, alpha Subunit; Macrophages; Male; Neurons; Neurotoxicity Syndromes; Neurotoxins; Pregnancy; Rats; Rats, Wistar; RNA, Messenger; Vascular Endothelial Growth Factor A

Topics: Animals; Disease Models, Animal; Erythropoietin; Humans; Male; Monkey Diseases; Reperfusion Injury

To observe the effects of tonifying kidney,tonifying spleen,invigorating the circulation of blood on the expression of hematopooietic cytokines of bone marrow suppression induced by chemotherapy.. Automated blood analyzer was used to detect the level of RBC and HGB, 14th and 28 days, while real time quantitative RT-PCR was used to detect EPO, EPOR mRNA expression.. Liuwei Dihuang Tang, Buzhong Yiqi Tang and Compound Danshen Decoction group could increase the level of RBC and HGB significantly. Liuwei Dihuang Tang and Buzhong Yiqi Tang groups could increase the mRNA expression level of EPO and EPOR significantly. However, there was no significantly difference when Compound Danshen Decoction group compared with control group on EPO, EPOR mRNA expression level.. The tonifying kidney, tonifying spleen, invigorate the circulation of blood are stable and reliable as to enhance the role of peripheral blood; tonifying kidney, tonifying spleen can improve EPO, EPOR mRNA expression levels, and promote the proliferation of bone marrow hematopoietic stem cells, and promote the differentiation of erythroid blood cells to increase red blood cell line; And invigorate the circulation of blood promote hematopoietic mechanisms have to be further studied.

Topics: Anemia; Animals; Antineoplastic Agents; Bone Marrow; Cell Proliferation; Cyclophosphamide; Disease Models, Animal; Drug Combinations; Drugs, Chinese Herbal; Erythropoietin; Hematopoiesis; Hematopoietic Stem Cells; Male; Medicine, Chinese Traditional; Mice; Mice, Inbred Strains; Plants, Medicinal; Random Allocation; Receptors, Erythropoietin; RNA, Messenger; Spleen

Pressure-induced vasodilatation (PIV), a cutaneous physiological neurovascular (C-fiber/endothelium) mechanism, is altered in diabetes and could possibly contribute to pressure ulcer development. We wanted to determine whether recombinant human erythropoietin (rhEPO), which has protective neurovascular effects, could prevent PIV alteration and pressure ulcer formation. We developed a skin pressure ulcer model in mice by applying two magnetic plates to the dorsal skin. This induced significant stage 2 ulcers (assessed visually and histologically) in streptozotocin-treated mice with 8 weeks of diabetes compared with very few in controls. Control and streptozotocin mice received either no treatment or systematic rhEPO (3,000 UI kg(-1) intraperitoneally, twice a week) during the last 2 weeks of diabetes. After 8 weeks of diabetes, we assessed ulcer development, PIV, endothelium-dependent vasodilation, C-fiber-mediated nociception threshold, and skin innervation density. Pretreatment with rhEPO fully prevented ulcer development in streptozotocin mice and also fully restored C-fiber nociception, skin innervation density, and significantly improved PIV, but had no effect on endothelium-dependent vasodilation. Our finding that rhEPO treatment protects the skin against pressure-induced ulcers in diabetic mice encourages evaluation of the therapeutic potential for non-hematopoietic analogs of EPO in preventing neuropathic diabetic ulcers.

Topics: Animals; Diabetes Mellitus, Experimental; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Injections, Intraperitoneal; Magnetics; Male; Mice; Nerve Fibers, Unmyelinated; Pressure Ulcer; Recombinant Proteins; Skin; Streptozocin; Treatment Outcome

Cerebral malaria (CM) is associated with high mortality and risk of sequelae, and development of adjunct therapies is hampered by limited knowledge of its pathogenesis. To assess the role of cerebral hypoxia, we used two experimental models of CM, Plasmodium berghei ANKA in CBA and C57BL/6 mice, and two models of malaria without neurologic signs, P. berghei K173 in CBA mice and P. berghei ANKA in BALB/c mice. Hypoxia was demonstrated in brain sections using intravenous pimonidazole and staining with hypoxia-inducible factor-1α-specific antibody. Cytopathic hypoxia was studied using poly (ADP-ribose) polymerase-1 (PARP-1) gene knockout mice. The effect of erythropoietin, an oxygen-sensitive cytokine that mediates protection against CM, on cerebral hypoxia was studied in C57BL/6 mice. Numerous hypoxic foci of neurons and glial cells were observed in mice with CM. Substantially fewer and smaller foci were observed in mice without CM, and hypoxia seemed to be confined to neuronal cell somas. PARP-1-deficient mice were not protected against CM, which argues against a role for cytopathic hypoxia. Erythropoietin therapy reversed the development of CM and substantially reduced the degree of neural hypoxia. These findings demonstrate cerebral hypoxia in malaria, strongly associated with cerebral dysfunction and a possible target for adjunctive therapy.

Topics: Animals; Brain; Disease Models, Animal; Erythropoietin; Female; Fluorescent Dyes; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Malaria, Cerebral; Mice; Nitroimidazoles; Parasitemia; Plasmodium berghei; Poly(ADP-ribose) Polymerases; Survival Analysis; Treatment Outcome

Tn order to set up a mouse model of myelofibrosis (MF) induced with high dose recombinant human erythropoietin (rhEPO). 60 mice were collected and divided into EPO and control groups, the former was injected with rhEPO and the latter with normal saline intraperitoneally. 5 mice from each group were executed on day 6, 30, 60, 90, 120 and 150 respectively. Their WBC count, Hb level, MCV, RDW and platelet amount were measured by automatic blood cell analyzer; CD34(+) cell ratio in bone marrow were analyzed by flow cytometry; liver and spleen coefficients were measured; pathological changes of liver, spleen, femur were observed by HE staining and reticular fibers staining; cortex thickness, femoral canal diameter and lumbar spine density were determined by computerized tomography (CT). The results indicated that as compared with normal control group in EPO induced group, WBC count was increased slightly in whole period, but without statistic significance (p > 0.05), Hb level and RDW increased at day 6 and 30 significantly (p < 0.05), MCV increased at day 6 significantly (p < 0.05), but platelet amount decreased significantly at all time points (p < 0.05). Most mice in EPO-induced group had hepatomegalia and their liver and spleen coefficient increased significantly at day 60 (p < 0.05), while most mice had splenomegaly and its coefficient was increased significantly at all time-points (p < 0.05). CD43(+) cell ratio of EPO group increased significantly in whole period (p < 0.05). CT scanning displayed femoral cortical thickening, medulla canal narrowing and lumbar spine density increasing at day 150, meanwhile, HE staining and reticular fiber staining showed the fatty degeneration or vacuolization in liver, splenomegaly with megakaryocytic proliferation, femur bone marrow fibrosis and osteosclerosis. It is concluded that the mouse induced by high dose of rhEPO displays the myelofibrosis associated with splenic extramedullary hemopoiesis, and this study is useful to establish a practical MF model, and to explore its pathological mechanism.

Topics: Animals; Disease Models, Animal; Erythropoietin; Female; Humans; Mice; Mice, Inbred Strains; Primary Myelofibrosis; Recombinant Proteins

The Akita mouse is a robust model of diabetic autonomic neuropathy which develops severe diabetes following beta cell death, which occurs reproducibly at 3-4 weeks of age, and maintains the diabetic state without therapy for as long as 11 additional months. Neuritic dystrophy and neuronopathy involving prevertebral sympathetic superior mesenteric and celiac ganglia begin to develop within the first two months of onset of diabetes and are progressive with time. We have examined the effect of insulin implants resulting in normoglycemia and injections of ARA290, a small erythropoietin peptide which has no effect on glycemic parameters, on the reversal of established neuritic dystrophy and neuronopathy. We have found that 4 weeks of insulin therapy beginning at 2 months of diabetes resulted in normalization of blood glucose, body weight and HbA1c. Insulin therapy successfully reversed established neuritic dystrophy and neuronopathy to control levels. Numbers of sympathetic neurons were not significantly changed in either 3 month diabetic or insulin-treated Akita mice. Treatment with ARA290 for 7 weeks beginning at 4 months of diabetes did not result in altered metabolic severity of diabetes as measured by blood glucose, body weight or HbA1c levels. ARA290 treatment was able to decrease neuritic dystrophy by 55-74% compared to untreated diabetics or in comparison to a separate group of diabetic animals representing the 4 month treatment onset point. Surprisingly, there was no effect of ARA290 on ganglionic neuron number or ongoing neuronopathy (pale/degenerating neurons) in diabetic Akita mice during this time period. The development of neuroprotective EPO-like peptides may provide a possible future therapy for this debilitating complication of diabetes; however, it appears that discrete elements may be differentially targeted by the diabetic state and may require selective therapy.

Topics: Animals; Blood Glucose; Body Weight; Diabetes Mellitus, Type 1; Diabetic Neuropathies; Disease Models, Animal; Erythropoietin; Ganglia, Sympathetic; Hypoglycemic Agents; Insulin; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Microscopy, Electron; Nerve Degeneration; Neurites; Peptides

Sleep disordered breathing (SDB), which is characterized by intermittent hypoxia (IH) during sleep, causes substantial cardiovascular and neurocognitive complications and has become a growing public health problem. SDB is associated with suppression of growth hormone (GH) secretion, the latter being integrally involved in the growth, development, and function of the CNS. Since GH treatment is able to attenuate neurocognitive deficits in a hypoxic-ischemic stroke model, GH, GH receptor (GHR) mRNA expression, and GH protein expression were assessed in rat hippocampus after exposures to chronic sustained hypoxia (CH, 10% O(2)) or IH (10% O(2) alternating with 21% O(2) every 90 s). In addition, the effect of GH treatment (50 μg/kg daily s.c. injection) on erythropoietin (EPO), vascular endothelial growth factor (VEGF), heme oxygenase-1 (HO-1), and GLUT-1 mRNA expression and neurobehavioral function was assessed. CH significantly increased GH mRNA and protein expression, as well as insulin-like growth factor-1 (IGF-1). In contrast, IH only induced a moderate increase in GH mRNA and a slight elevation in GH protein at day 1, but no increases in IGF-1. CH, but not IH, up-regulated GHR mRNA in the hippocampus. IH induced marked neurocognitive deficits compared with CH or room air (RA). Furthermore, exogenous GH administration increased hippocampal mRNA expression of IGF-1, EPO, and VEGF, and not only reduced IH-induced hippocampal injury, but also attenuated IH-induced cognitive deficits. Thus, exogenous GH may provide a viable therapeutic intervention to protect IH-vulnerable brain regions from SDB-associated neuronal loss and associated neurocognitive dysfunction.

Topics: Animals; Caspase 3; Cognition Disorders; Disease Models, Animal; Erythropoietin; Glucose Transporter Type 1; Growth Hormone; Heme Oxygenase-1; Hippocampus; Humans; Hypoxia; Insulin-Like Growth Factor I; Male; Maze Learning; Rats; Rats, Sprague-Dawley; Receptors, Somatotropin; Vascular Endothelial Growth Factor A

In chronic kidney disease, fibroblast dysfunction causes renal fibrosis and renal anemia. Renal fibrosis is mediated by the accumulation of myofibroblasts, whereas renal anemia is mediated by the reduced production of fibroblast-derived erythropoietin, a hormone that stimulates erythropoiesis. Despite their importance in chronic kidney disease, the origin and regulatory mechanism of fibroblasts remain unclear. Here, we have demonstrated that the majority of erythropoietin-producing fibroblasts in the healthy kidney originate from myelin protein zero-Cre (P0-Cre) lineage-labeled extrarenal cells, which enter the embryonic kidney at E13.5. In the diseased kidney, P0-Cre lineage-labeled fibroblasts, but not fibroblasts derived from injured tubular epithelial cells through epithelial-mesenchymal transition, transdifferentiated into myofibroblasts and predominantly contributed to fibrosis, with concomitant loss of erythropoietin production. We further demonstrated that attenuated erythropoietin production in transdifferentiated myofibroblasts was restored by the administration of neuroprotective agents, such as dexamethasone and neurotrophins. Moreover, the in vivo administration of tamoxifen, a selective estrogen receptor modulator, restored attenuated erythropoietin production as well as fibrosis in a mouse model of kidney fibrosis. These findings reveal the pathophysiological roles of P0-Cre lineage-labeled fibroblasts in the kidney and clarify the link between renal fibrosis and renal anemia.

Topics: Anemia; Animals; Benserazide; Cell Differentiation; Cell Lineage; Disease Models, Animal; Drug Combinations; Erythropoietin; Female; Fibroblasts; Fibrosis; Kidney; Levodopa; Mice; Mice, Transgenic; Myofibroblasts; Pregnancy

For decades, autologous ex vivo gene therapy has been postulated as a potential alternative to parenteral administration of recombinant proteins. However, achieving effective cellular engraftment of previously retrieved patient cells is challenging. Recently, our ability to engineer vasculature in vivo has allowed for the introduction of instructions into tissues by genetically modifying the vascular cells that build these blood vessels. In the present study, we genetically engineered human blood-derived endothelial colony-forming cells (ECFCs) to express erythropoietin (EPO) under the control of a tetracycline-regulated system, and generated subcutaneous vascular networks capable of systemic EPO release in immunodeficient mice. These ECFC-lined vascular networks formed functional anastomoses with the mouse vasculature, allowing direct delivery of recombinant human EPO into the bloodstream. After activation of EPO expression, erythropoiesis was induced in both normal and anemic mice, a process that was completely reversible. This approach could relieve patients from frequent EPO injections, reducing the medical costs associated with the management of anemia. We propose this ECFC-based gene-delivery strategy as a viable alternative technology when routine administration of recombinant proteins is needed.

Topics: Anemia; Animals; Blood Vessels; Cells, Cultured; Disease Models, Animal; Erythropoiesis; Erythropoietin; Feasibility Studies; Gene Expression Regulation; Genetic Engineering; Genetic Therapy; Humans; Mesenchymal Stem Cell Transplantation; Mice; Mice, Nude; Radiation Injuries; Renal Insufficiency; Subcutaneous Tissue; Transfection; Transplantation, Autologous; Transplantation, Heterologous

Chronic left ventricular (LV) pressure overload induced by hypertension is one of the most common causes of heart failure. Earlier reports have shown the cardioprotective effects of erythropoietin (EPO). In the present study, we tested the hypothesis that recombinant human EPO exerts a protective effect against pressure-overload induced LV remodeling. Mice subjected to transverse aortic constriction (TAC) (n = 70) were randomly assigned to the treatment with phosphate buffer solution (PBS) (TAC-PBS) or EPO (2,000 U/kg twice a week) (TAC-EPO). At 8 weeks after TAC, LV weight was comparably increased in both TAC groups compared with sham-operated mice (Sham) (both P < 0.001). The treatment with EPO improved the survival of TAC mice as compared with treatment with PBS (80 vs. 47%, P < 0.01), which was associated with reductions in the extent of myocardial fibrosis and the number of TUNEL positive cardiomyocytes (both P < 0.05). Echocardiography revealed that TAC increased LV chamber diameter and decreased LV fractional shortening compared with Sham (P < 0.05), which was ameliorated by the treatment with EPO (P < 0.05). In TAC-EPO as compared to TAC-PBS, phosphorylation of STAT3, Akt and eNOS was all increased, while phosphorylation of p38 was decreased (all P < 0.05). Importantly, the expression level of VEGF and the capillary density in LV myocardium were similar among the 3 groups. These results suggest that recombinant human EPO ameliorates the cardiac remodeling and the premature death associated with chronic LV pressure overload through the mechanisms independent of angiogenesis.

Topics: Animals; Apoptosis; Biomarkers; Blood Pressure; Disease Models, Animal; Echocardiography; Erythropoietin; Heart Ventricles; Hemodynamics; Longevity; Male; Mice; Mice, Inbred C57BL; Mortality, Premature; Myocytes, Cardiac; Organ Size; Recombinant Proteins; Survival Rate; Ventricular Remodeling

The use of Cyclosporine A (CsA) as rejection prophylaxis following organ transplantation is limited by its nephrotoxicity. CsA induces renal damage that is associated with tubulo-interstitial injury and parenchymal sequestration of macrophages, perpetuating pro-inflammatory processes. Furthermore, CsA exerts a diabetogenic effect by damaging pancreatic islet cell integrity. Continuous Erythropoietin Receptor Activator (CERA) was shown to mediate tissue-protective and anti-inflammatory effects in various settings of organ injury. Here, we investigated the effect of low dose CERA in a model of CsA-induced renal and pancreatic injury. Rats were exposed to medium-dose CsA for 28 days. Low-dose CERA was given to the treatment group (CERA) (n=6) once per week vs. a CsA-treated control group (CONTROL) (n=6). The effect of CERA on renal and pancreatic injuries was analyzed by organ function, histology, immunohistochemistry (CD68(+)-macrophages, insulin), ELISA (TGF-β1) and RT-PCR (TGF-β1, Osteopontin, IL-10). CsA induced functional kidney damage. Low dose CERA did not lead to improved kidney function in the treatment group. However, low dose CERA showed a trend toward upregulation of osteopontin accompanied by increased renal macrophage-infiltration and enhanced parenchymal TGF-β1 and IL-10 when compared to controls. Moreover, CERA treated animals showed amelioration of pancreatic islet cell injury. In this model of acute CsA-mediated renal injury, low dose CERA administration was associated with anti-inflammatory effects and preservation of pancreatic islet cell viability.

Topics: Acute Kidney Injury; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Body Weight; Cell Survival; Cyclosporine; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression Regulation; Glucose; Hematologic Tests; Interleukin-10; Islets of Langerhans; Kidney; Macrophages; Male; Osteopontin; Polyethylene Glycols; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta1

The hormone erythropoietin (EPO) has been demonstrated to have cardioprotective properties. The present study investigates the role of EPO to prevent heart failure following cancer treatment with doxorubicin [adriamycin (AD)]. Male Wistar rats (150 ± 10 g) were treated with saline (vehicle control group); with EPO, subcutaneously at 1,000 IU/kg body wt, three times per week for 4 wk (EPO group); with adriamycin, intraperitoneally at 2.5 mg/kg body wt, three times per week for 2 wk (AD group); and with adriamycin and EPO (EPO-AD group). Echocardiographic measurements showed that EPO-AD treatment prevented the AD-induced decline in cardiac function. Each of the hearts was then exposed to ischemia and reperfusion during Langendorff perfusion. The percentage of recovery after ischemia-reperfusion was significantly greater in EPO-AD than the AD-treated group for left ventricular developed pressure, maximal increase in pressure, and rate pressure product. The level of oxidative stress was significantly higher in AD (5 μM for 24 h)-exposed isolated cardiomyocytes; EPO (5 U/ml for 48 h) treatment prevented this. EPO treatment also decreased AD-induced cardiomyocyte apoptosis, which was associated with the decrease in the Bax-to-Bcl2 ratio and caspase-3 activation. Immunostaining of myocardial tissue for CD31 showed a significant decrease in the number of capillaries in AD-treated animals. EPO-AD treatment restored the number of capillaries. In conclusion, EPO treatment effectively prevented AD-induced heart failure. The protective effect of EPO was associated with a decreased level of oxidative stress and apoptosis in cardiomyocytes as well as improved myocardial angiogenesis.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Capillaries; Cardiotonic Agents; Caspase 3; Cells, Cultured; Disease Models, Animal; Doxorubicin; Enzyme Activation; Erythropoietin; Heart Failure; Immunohistochemistry; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Neovascularization, Physiologic; Oxidative Stress; Perfusion; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Recovery of Function; Time Factors; Ultrasonography; Ventricular Function, Left; Ventricular Pressure

The selection of a minimal active sequence of erythropoietin allowed the design of peptide mimetics that exert beneficial effects in the central nervous system but lack an erythropoietic effect. Erythropoietin has been suggested as a promising therapeutic and prophylactic for epilepsies based on its neuroprotective, neuroregenerative, and antiinflammatory potency. Therefore, it is of particular interest to evaluate whether the nonerythropoietic erythropoietin-derived peptide pHBSP can affect epileptogenesis.. In a post-status epilepticus model in rats, we determined the effects of pHBSP and of recombinant human erythropoietin with short-term administration following status epilepticus.. Both pHBSP and erythropoietin further enhanced the status epilepticus-associated increase in hippocampal cell proliferation. Thereby, pHBSP seemed to promote neuronal differentiation and survival resulting in a significant increase in neurogenesis. Neither pHBSP nor erythropoietin affected the number of animals exhibiting spontaneous recurrent seizures as well as the seizure frequency in the chronic phase. In the Morris water maze, pHBSP attenuated cognitive deficits in epileptic animals.. In conclusion, the helix B-derived erythropoietin peptide pHBSP can modulate the cellular and cognitive consequences of a status epilepticus. The impact of pHBSP on spatial learning might indicate that the peptide allows beneficial effects on epileptogenesis-associated cognitive deficits. However, it needs to be considered that learning deficits were not abolished by pHBSP and that the effects were not observed consistently until the end of the study. Therefore, adjustment of timing, duration, and dose of peptide administration might be necessary to further evaluate the efficacy of pHBSP.

Topics: Adaptation, Physiological; Analysis of Variance; Animals; Bromodeoxyuridine; Cell Proliferation; Cognition Disorders; Disease Models, Animal; Electric Stimulation; Erythropoietin; Exploratory Behavior; Female; Humans; Maze Learning; Microglia; Neurogenesis; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Serine Endopeptidases; Status Epilepticus

Gut injury and bacterial translocation develop and persist after limited periods of hemorrhagic shock. Erythropoietin (EPO) can exert hemodynamic, anti-inflammatory, and tissue protective effects. We tested the hypothesis that EPO given at the time of resuscitation with saline will reduce functional ileal injury 24 hours after shock.. Sprague-Dawley rats (n = 6 per group) were randomized to sham surgery or hemorrhagic shock maintained at mean arterial pressure 40 mm Hg for 60 minutes and then treated with either saline resuscitation (three times the volume of shed blood) or saline + recombinant human EPO (rHuEPO) resuscitation. Intravenous rHuEPO (1,000 U/kg) was given at the start of saline resuscitation, and at 24 hours ileal function was evaluated using quantitative cultures of mesenteric lymph nodes to assess for bacterial translocation (colony-forming units per gram of tissue [CFU/g]), determination of portal vein plasma endotoxin levels and histopathological evaluation using semi-thin plastic sections of the distal ileum. In a second series of animals, fluorescein isothiocyanate-dextran 4000 (FD-4) was used to assess mucosal permeability of the distal ileum to macromolecules.. At 24 hours, the saline group had morphologic evidence of intestinal injury when compared with the sham group, and the degree of mucosal injury was less in the saline + rHuEPO when compared with the saline group, which demonstrated significantly reduced bacterial translocation to the mesenteric lymph nodes (383 CFU/g ± 111 CFU/g vs. 1130 CFU/g ± 297 CFU/g; p < 0.05) and decreased terminal ileum permeability to FD-4 (3.08 μg/mL ± 0.31 μg/mL vs. 5.14 μg/mL ± 0.88 μg/mL; p < 0.05). No significant difference was found in the portal vein endotoxin levels between the two groups. Histopathological evaluation demonstrated a trend for decreased enterocyte disarray or disruption and vacuolization in the saline + rHuEPO versus saline group.. Using rHuEPO at time of saline resuscitation resulted in decreased bacterial translocation and permeability to macromolecules 24 hours after shock. These observations suggest that rHuEPO can mediate a protective effect on intestinal mucosal barrier function during ischemic injury.

Topics: Animals; Cell Membrane Permeability; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Follow-Up Studies; Ileum; Infusions, Intravenous; Peptide Fragments; Rats; Rats, Sprague-Dawley; Resuscitation; Shock, Hemorrhagic; Treatment Outcome

Sepsis and septic shock remain the major causes of morbidity and mortality in intensive care units. One mechanism that leads to organ failure is microcirculatory dysfunction. Erythropoietin (EPO) is a glycoprotein produced by the kidney that primarily regulates erythropoiesis, but it also can exert hemodynamic, anti-inflammatory, and tissue protective effects. We previously reported that administration of EPO to septic mice improves mouse skeletal muscle capillary perfusion and tissue bioenergetics. The objective of this study was to explore the potential mechanism(s) involved.. Sepsis was induced by intraperitoneal (i.p.) injection of a fecal suspension (12.5 g in 0.5 saline/mouse) in mice. At 18 hours after sepsis induction, a single dose of rHuEPO (400 U/kg) was given to the mice. Mouse capillary perfusion density and nicotinamide adenine dinucleotide (NADH) fluorescence in skeletal muscle were observed using intravital microscopy. Endothelial cells derived from the skeletal muscle were treated with rHuEPO (5 U/mL) and endothelial nitric oxide synthase (eNOS) activation and activity were assessed.. Septic mice had decreased capillary perfusion density and increased tissue NADH fluorescence indicating impaired tissue bioenergetics, whereas animals treated with rHuEPO demonstrated an improvement in capillary perfusion density and decreased skeletal muscle NADH fluorescence. The beneficial effect of rHuEPO did not occur in septic mice treated with l-NAME (an NOS inhibitor, 20 mg/kg) or mice genetically deficient in eNOS. Treatment of endothelial cells with rHuEPO resulted in activation of eNOS as indicated by increased eNOS phosphorylation and NO production.. Our results suggest that eNOS plays an important role in mediating the beneficial effect of rHuEPO on microcirculation in this septic mouse model.

Topics: Animals; Disease Models, Animal; Enzyme Activation; Erythropoietin; Mice; Mice, Inbred C57BL; Microcirculation; Muscle, Skeletal; Nitric Oxide Synthase Type III; Sepsis; Treatment Outcome

The platelet-derived growth factor (PDGF) signaling system contributes to tumor angiogenesis and vascular remodeling. Here we show in mouse tumor models that PDGF-BB induces erythropoietin (EPO) mRNA and protein expression by targeting stromal and perivascular cells that express PDGF receptor-β (PDGFR-β). Tumor-derived PDGF-BB promoted tumor growth, angiogenesis and extramedullary hematopoiesis at least in part through modulation of EPO expression. Moreover, adenoviral delivery of PDGF-BB to tumor-free mice increased both EPO production and erythropoiesis, as well as protecting from irradiation-induced anemia. At the molecular level, we show that the PDGF-BB-PDGFR-bβ signaling system activates the EPO promoter, acting in part through transcriptional regulation by the transcription factor Atf3, possibly through its association with two additional transcription factors, c-Jun and Sp1. Our findings suggest that PDGF-BB-induced EPO promotes tumor growth through two mechanisms: first, paracrine stimulation of tumor angiogenesis by direct induction of endothelial cell proliferation, migration, sprouting and tube formation, and second, endocrine stimulation of extramedullary hematopoiesis leading to increased oxygen perfusion and protection against tumor-associated anemia.

Topics: Anemia; Animals; Becaplermin; Carcinoma, Lewis Lung; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Erythropoietin; Gene Expression Regulation, Neoplastic; Hematopoiesis; Mice; Mice, Inbred C57BL; Mice, SCID; Neovascularization, Pathologic; Proto-Oncogene Proteins c-sis; Signal Transduction; Stromal Cells

Hyperglycemia is associated with a decreased tolerance to ischemia and an increased severity of renal ischemia reperfusion (I/R) injury. It has been suggested that erythropoietin (EPO) attenuates this effect in normoglycemic animals. This study sought to examine the effects of EPO on treatment renal I/R injury (IRI) in transiently hyperglycemic rats.. Twenty-eight male Wister rats anesthetized with isoflurane received glucose (2.5 g.kg(-1) intraperitoneally) before right nephrectomy. They were randomly assigned to four groups: sham operation (S); IRI (ISO); IRI+EPO, (600 UI kg(-1) low-dose EPO [EL]); and IRI+EPO 5000 UI kg(-1) (high-dose EPO [EH]). IRI was induced by a 25-minute period of left renal ischemia followed by reperfusion for 24 hours. Serum creatinine and glucose levels were measure at baseline (M1), immediately after the ischemic period (M2), and at 24 hours after reperfusion (M3). After sacrificing the animals, left kidney specimens were submitted for histological analysis including flow cytometry to estimate tubular necrosis and the percentages of apoptotic, dead or intact cells.. Scr in the ISO group was significantly higher at M3 than among the other groups. Percentages of early apoptotic cells in ISO group were significantly higher than the other groups. Percentages of late apoptotic cells in S and ISO groups were significantly greater than EL and EH groups. However, no significant intergroup differences were observed regarding the incidence of tubular necrosis.. Our results suggested that, although not preventing the occurrence of tubular necrosis, EPO attenuated apoptosis and glomerular functional impairment among transiently hyperglycemic rats undergoing an ischemia/reperfusion insult.

Topics: Animals; Apoptosis; Blood Glucose; Creatinine; Cytoprotection; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Flow Cytometry; Hyperglycemia; Kidney; Kidney Glomerulus; Kidney Tubules; Male; Necrosis; Nephrectomy; Rats; Rats, Wistar; Recombinant Proteins; Reperfusion Injury; Time Factors

An animal model of cochlear implantation has been developed, and the hearing threshold was evaluated after different surgical procedures. The effect of perioperative systemic administration of erythropoietin on the hearing loss induced by cochlear implantation was tested.. Twenty-nine guinea pigs with normal hearing underwent implantation of a 254-microm-diameter array through a cochleostomy. The effects on hearing of cochleostomy and transient and long-term array implantation (21 days) were assessed by testing of the auditory brain stem responses and compound action potentials. Eleven implanted animals received intraperitoneal administration of erythropoietin. Selected computed tomographic scans and cochlear histologic studies were performed 1 month after implantation to confirm proper placement of the array. The erythropoietin concentration at the time of surgery was assessed in samples of perilymph, cerebrospinal fluid, and blood.. The cochleostomy and transient array insertion had no effect on hearing thresholds. Long-term array implantation induced a stable decrease of hearing threshold (30 dB), a decrease that was reduced by 12 dB in erythropoietin-treated animals. The erythropoietin-treated animals had better hearing preservation at higher frequencies. Fibrosis surrounding the array was seen in both groups.. The hearing loss observed was probably due to the presence of the array in the cochlea. The intraperitoneal injection of erythropoietin improved the hearing threshold shift induced by implantation.

Topics: Action Potentials; Animals; Auditory Threshold; Cochlear Implantation; Disease Models, Animal; Erythropoietin; Evoked Potentials, Auditory, Brain Stem; Guinea Pigs; Hearing Loss; Injections, Intraperitoneal; Male; Treatment Outcome

The neuroprotective activity of recombinant human erythropoietin (r-HuEpo) sorbed on poly(butyl)cyanoacrilate nanoparticles (EPO-PBCA) and on polylactic-co-glycolic acid nanoparticles (EPO-PLGA) has been studied on Wistar rats with intracerebral post-traumatic hematoma (model of hemorrhagic stroke) (IPH-HS) in comparison to native r-HuEpo. It is established that EPO-PBCA produced a protective effect in rats after IPH-HS that was manifested by a decrease in the number of animals with neurological disorders such as circus movement, paresis, and paralysis of hind limbs; the drug also improved coordination (rotating rod test), reduced the number of lost animals, and decreased the loss weight among survived rats. In addition, EPO-PBCA optimized the research behavior of rats with IPH-HS in the open field test and prevented amnesia of passive avoidance reflex (PAR), which was caused by the IPH-HS. These effects were manifested during a two-week observation period. EPO-PLGA has a similar but much less pronounced effect on the major disorders caused by IPH-HS. The efficiency of native r-HuEpo as a neuropotective agent was insignificant and only manifested by decrease in the number of lost animals with IPH-HS.

Topics: Adsorption; Amnesia; Animals; Cerebral Hemorrhage; Disease Models, Animal; Enbucrilate; Erythropoietin; Humans; Lactic Acid; Male; Motor Activity; Nanoparticles; Neuroprotective Agents; Paralysis; Paresis; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Rats, Wistar; Recombinant Proteins; Stroke; Survival Rate; Weight Loss

To increase the survival rate of patients with acute superior mesenteric artery thromboembolism (ASMAT) treated by catheter thrombolysis, we examined the effects of delivering edaravone and asialoerythropoietin, agents with tissue-protective activities, using a rabbit autologous fibrin clot ASMAT model. Japanese white rabbits (n=32) were randomly separated into four equal groups. 45 min after introducing autologous fibrin clot, Group U received urokinase and heparin; Group E received urokinase and heparin plus edaravone; Group A received urokinase and heparin plus asialoerythropoietin; and Group EA received urokinase, heparin and edaravone plus asialoerythropoietin via a catheter. The intestines were removed 6 h later and intestinal mucosal damage was scored using the Park's injury score. Survival time was assessed. Average mucosal injury was 5.78+/-1.52 (Group U), 2.88+/-0.72 (Group E), 1.90+/-1.23 (Group A) and 1.18+/-1.25 (Group EA). The degree of mucosal injury was significantly lower in Group EA than in Groups U and E (p<0.05). Conversely, there was no significant difference between Group A and Group EA, or between Group A and Group E. The survival times were 31.50+/-13.30 h (Group U), 51.00+/-24.74 h (Group E), 48.00+/-16.97 h (Group A) and 82+/-51.07 h (Group EA); the difference among the four groups was not significant. In conclusion, the concomitant administration of asialoerythropoietin and edaravone reduced mucosal membrane injury significantly compared with edaravone alone. However, to improve the survival of ASMAT rabbit models, the delivery of an appropriate dose of asialoerythropoietin is required, together with the development of methods to assess peripheral recanalisation.

Topics: Animals; Antipyrine; Asialoglycoproteins; Catheterization; Disease Models, Animal; Drug Combinations; Edaravone; Erythropoietin; Fibrin; Fibrinolytic Agents; Free Radical Scavengers; Heparin; Injections, Intra-Arterial; Intestinal Mucosa; Mesenteric Artery, Superior; Mesenteric Vascular Occlusion; Rabbits; Random Allocation; Reperfusion Injury; Survival Rate; Thromboembolism; Urokinase-Type Plasminogen Activator

Erythropoietin (EPO), essential for erythropoiesis, provides neuroprotection. The EPO receptor (EPOR) is expressed in both neural and non-neural cells in the brain. This study was designed to test the hypothesis that EPO provides beneficial therapeutic effects, even in the absence of the neural EPOR. In this study, EPOR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue, but not in the neural cells. Anesthetized young adult female EPOR-null and wild-type mice were subjected to traumatic brain injury (TBI) induced by controlled cortical impact. EPO (5000 U/kg) or saline was intraperitoneally administered at 6 h and 3 and 7 days post-injury. Sensorimotor and spatial learning functions were assessed. Expression of EPOR and its downstream signal proteins were evaluated by Western blot analysis. Our data demonstrated that EPO treatment significantly reduced cortical tissue damage and hippocampal cell loss, and improved spatial learning following TBI in both the wild-type and EPOR-null mice. EPO treatment significantly improved sensorimotor functional recovery, with better outcomes in the wild-type mice. EPO treatment upregulated anti-apoptotic proteins (p-Akt and Bcl-XL) in the ipsilateral hippocampus and cortex of the injured wild-type and EPOR-null mice. These data demonstrate that EPO significantly provides neuroprotection following TBI, even in the absence of EPOR in the neural cells, suggesting that its therapeutic benefits may be mediated through vascular protection.

Topics: Animals; Apoptosis Regulatory Proteins; Brain; Brain Injuries; Cerebral Cortex; Disease Models, Animal; Erythropoietin; Female; Hippocampus; Humans; Memory Disorders; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nerve Degeneration; Neuroprotective Agents; Promoter Regions, Genetic; Receptors, Erythropoietin; Treatment Outcome

To determine expression, cellular distribution, and regulation of hemoglobin (Hb) in normal and glaucomatous tissues.. Proteomic analysis of Hb expression was conducted on protein samples from ocular hypertensive and control rat eyes and human donor eyes with or without glaucoma. Proteomic findings were validated by quantitative (q)RT-PCR, Western blot analysis, immunohistochemistry, and the analysis of new Hb synthesis in culture. Hypoxic regulation of Hb expression was also studied in primary cultures of rat RGCs and macroglia and after transfer of the glia-conditioned medium to RGCs. The role of erythropoietin (EPO) signaling in Hb induction and cell survival was determined by applying recombinant (r)EPO treatment and performing EPO neutralization experiments by using soluble EPO receptor treatment of hypoxic cultures.. In vivo findings revealed Hb expression in the retina and optic nerve head macroglia and RGCs, suggesting an approximately two-fold upregulation in ocular hypertensive rat eyes and glaucomatous human donor eyes relative to the control eyes. In vitro findings collectively supported that hypoxia boosts glial Hb expression through hypoxia-inducible EPO signaling in an autocrine manner. Based on passive transfer experiments, hypoxia-induced production of glial EPO was also found to upregulate Hb expression in RGCs in a paracrine manner, thereby increasing the hypoxic survival of these neurons.. Findings of this study provide new insights into tissue oxygen transport in the inner retina and optic nerve head through the regulated expression of Hb in macroglia and RGCs. Upregulation of Hb expression appears to be an intrinsic protective mechanism to facilitate cellular oxygenation and may also provide free radical scavenging.

Topics: Aged; Aged, 80 and over; alpha-Globins; Animals; beta-Globins; Biological Transport; Blotting, Western; Cell Survival; Cells, Cultured; Disease Models, Animal; Erythropoietin; Eye Proteins; Glaucoma; Hemoglobins; Humans; Immunohistochemistry; Mass Spectrometry; Ocular Hypertension; Optic Disk; Oxygen; Rats; Rats, Inbred BN; Recombinant Proteins; Retina; Retinal Ganglion Cells; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

An experimental study was conducted to investigate the effects of erythropoietin on the acute phase of esophageal burn damage induced by sodium hydroxide.. A standard esophageal alkaline burn was produced by the application of 10% sodium hydroxide to the distal esophagus in an in vivo rat model. Fifty-six female rats were allocated into three groups: Group BC (baseline control, n = 8) rats were uninjured and untreated, Group PC (positive control, n = 24) rats were injured but untreated and Group EPO (erythropoietin-treated, n = 24) rats were injured and given subcutaneous erythropoietin (1,000 IU/kg per day), 15 min, 24, and 48 h after administration of the NaOH solution. Six animals from Group PC and six from Group EPO were killed at 4, 24, 48, and 72 h after application of NaOH to the esophagus. All of animals in Group BC were killed 4 h after exposure to 0.9% NaCl. Oxidative damage was assessed by measuring levels of malondialdehyde (MDA) and nitric oxide (NO), and activities of superoxide dismutase (SOD) and catalase (CAT) in homogenized samples of esophageal tissue. Histologic damage to esophageal tissue was scored by a single pathologist blind to groups.. MDA levels in the BC and EPO groups were significantly lower than those in the PC group (p < 0.05). CAT and SOD activities, and NO levels in the BC and EPO groups were significantly higher than in the PC group (p < 0.05). Esophageal tissue damage measured at 4, 24, 48, and 72 h after NaOH application was significantly less in the EPO group than in the PC group (p < 0.05).. When administered early after an esophageal burn induced by 10% sodium hydroxide in this rat model, erythropoietin significantly attenuated oxidative damage, as measured by biochemical markers and histologic scoring.

Topics: Acute-Phase Reaction; Animals; Burns, Chemical; Catalase; Caustics; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Esophagus; Female; Injections, Subcutaneous; Malondialdehyde; Nitric Oxide; Oxidative Stress; Rats; Rats, Sprague-Dawley; Sodium Hydroxide; Superoxide Dismutase; Trauma Severity Indices; Treatment Outcome

Permanent mild-to-severe brain injury with neurologic sequelae remains a significant source of postoperative morbidity in cardiovascular surgery. There is increasing evidence that erythropoietin confers neuroprotective effects in various conditions of neuronal damage, such as hypoxia and cerebral ischaemia. Using a surviving porcine model, this study evaluates whether systemic treatment with erythropoietin induces brain protection in deep hypothermic circulatory arrest (DHCA).. Sixteen pigs (42+/-3 kg) randomly assigned into two groups (n=8) were subjected to 60 min of DHCA at an intracerebral temperature of 20 degrees C. The animals of the erythropietin group were treated perioperatively with 500 IU kg(-1) of recombinant human erythropoietin on 3 consecutive days beginning the day before surgery. Intracerebral monitoring was performed by subcortical microdialysis, brain tissue oxygenation, measurement of brain temperature and intracranial pressure. Neurologic recovery was evaluated daily. Perioperative S100 beta protein serum level was determined. The brains were harvested on the postoperative day 6 after perfusion fixation. Multiple brain regions were investigated histologically for hypoxic-ischaemic damage.. The subcortical brain microdialysis detected significant increase of glycerol and lactate concentrations in both groups (P=0.0001) with considerably higher concentrations in the brain of control animals (P=0.011). There were no significant differences in neurological outcome (P=0.15). Erythropoietin-treated animals tended to a more complete and rapid neurological recovery. By contrast, none of the animals in the control group achieved complete neurological recovery. S100 beta protein as a putative marker of cerebral injury tended to be higher in the control group. Brain infarction was detectable in all control animals but only in two erythropoietin-treated animals.. These results suggest some beneficial neuroprotective effects of erythropoietin in this model of global brain ischaemia induced by 1h of hypothermic circulatory arrest.

Topics: Animals; Brain; Disease Models, Animal; Drug Evaluation, Preclinical; Erythropoietin; Heart Arrest, Induced; Hemodynamics; Hypothermia, Induced; Hypoxia-Ischemia, Brain; Intracranial Pressure; Neuroprotective Agents; Oxygen Consumption; Perioperative Care; Random Allocation; Recombinant Proteins; Sus scrofa

During the early post transplant period, the tubular epithelium is the main target of injuries including ischemia reperfusion and toxicity effects from calcineurin inhibitors. Taking into account the tissue protective effects of erythropoietin mediated through its antiapoptotic properties, we tested whether administration of recombinant human erythropoietin protects against acute cyclosporine nephrotoxicity. Four groups of five rats were intraperitoneally treated over 28 days with 100UI/Kg/48h Epoetin beta (15mg/kg/day CsA diluted in olive oil, 100UI/Kg/48h Epoetin beta+15mg/kg/day CsA, or olive oil. Histological changes due to tubular necrosis were evaluated with Masson'Trichrome staining. Apoptotic nuclei in kidneys were detected using the Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) method. Phospho-Akt, Akt, cleaved caspase 3 and non cleaved caspase 3 expression were evaluated using immunblotting. We demonstrate that recombinant human erythropoietin (epoetin beta) improves renal function and protects against acute tubular injury. Our data suggest that this nephroprotective effect is mediated by Akt activation and inhibition of tubular apoptosis. Indeed, western blotting analysis of caspase 3 cleavage and Akt phosphorylation demonstrates that rhEPO activate Akt signaling and inhibits caspase 3 cleavage induced by CsA. TUNEL staining confirms that rhEPO inhibits CsA-induced tubular apoptosis. In conclusion, we describe here a new potential target of recombinant human erythropoietin and our results provide an interesting framework for further nephroprotective therapies based on recombinant human erythropoietin.

Topics: Acute Disease; Animals; Apoptosis; Biomarkers; Blotting, Western; Caspase 3; Creatinine; Cyclosporine; Cytoprotection; Disease Models, Animal; Erythropoietin; Humans; In Situ Nick-End Labeling; Kidney Cortex Necrosis; Kidney Tubules; Male; Necrosis; Phosphorylation; Protective Agents; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Signal Transduction

We found that recombinant human erythropoietin (rhEPO) reduced significantly the development of brain edema in a rat model of diffuse traumatic brain injury (TBI) (impact-acceleration model). In this study, we investigated the molecular and intracellular changes potentially involved in these immediate effects. Brain tissue nitric oxide (NO) synthesis, phosphorylation level of two protein kinases (extracellular-regulated kinase (ERK)-1/-2 and Akt), and brain water content were measured 1 (H1) and 2 h (H2) after insult. Posttraumatic administration of rhEPO (5,000 IU/kg body weight, intravenously, 30 mins after injury) reduced TBI-induced upregulation of ERK phosphorylation, although it increased Akt phosphorylation at H1. These early molecular changes were associated with a reduction in brain NO synthesis at H1 and with an attenuation of brain edema at H2. Intraventricular administration of the ERK-1/-2 inhibitor, U0126, or the Akt inhibitor, LY294002, before injury showed that ERK was required for brain edema formation, and that rhEPO-induced reduction of edema could involve the ERK pathway. These results were obtained in the absence of any evidence of blood-brain barrier damage on contrast-enhanced magnetic resonance images. The findings of our study indicate that the anti edematous effect of rhEPO could be mediated through an early inhibition of ERK phosphorylation after diffuse TBI.

Topics: Animals; Blood-Brain Barrier; Blotting, Western; Brain Edema; Brain Injuries; Disease Models, Animal; Erythropoietin; Humans; Male; Phosphorylation; Protein Kinases; Rats; Rats, Wistar; Recombinant Proteins

To understand the molecular mechanisms underlying compound-induced hemangiosarcomas in mice, and therefore, their human relevance, a systems biology approach was undertaken using transcriptomics and Causal Network Modeling from mice treated with 2-butoxyethanol (2-BE). 2-BE is a hemolytic agent that induces hemangiosarcomas in mice. We hypothesized that the hemolysis induced by 2-BE would result in local tissue hypoxia, a well-documented trigger for endothelial cell proliferation leading to hemangiosarcoma. Gene expression data from bone marrow (BM), liver, and spleen of mice exposed to a single dose (4 h) or seven daily doses of 2-BE were used to develop a mechanistic model of hemangiosarcoma. The resulting mechanistic model confirms previous work proposing that 2-BE induces macrophage activation and inflammation in the liver. In addition, the model supports local tissue hypoxia in the liver and spleen, coupled with increased erythropoeitin signaling and erythropoiesis in the spleen and BM, and suppression of mechanisms that contribute to genomic stability, events that could be contributing factors to hemangiosarcoma formation. Finally, an immunohistochemistry method (Hypoxyprobe) demonstrated that tissue hypoxia was present in the spleen and BM. Together, the results of this study identify molecular mechanisms that initiate hemangiosarcoma, a key step in understanding safety concerns that can impact drug decision processes, and identified hypoxia as a possible contributing factor for 2-BE-induced hemangiosarcoma in mice.

Topics: Animals; Bone Marrow; Cell Cycle; Cell Differentiation; Cell Hypoxia; Cell Proliferation; Cell Transformation, Neoplastic; Disease Models, Animal; Endothelial Cells; Erythropoiesis; Erythropoietin; Ethylene Glycols; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genomic Instability; Hemangiosarcoma; Hematopoietic Stem Cells; Hemolysis; Hepatitis; Immunohistochemistry; Liver; Macrophage Activation; Male; Mice; Models, Biological; Signal Transduction; Spleen; Systems Biology; Time Factors

This efficacy study was designed to investigate traumatic brain injury (TBI) in rats treated with delayed erythropoietin (EPO) administered in a single dose compared with a triple dose.. Young adult male Wistar rats were randomly divided into the following groups: 1) sham group (6 animals); 2) TBI/saline group (6 animals); 3) TBI/EPOx1 group (6 animals); and 4) TBI/EPOx3 group (7 animals). Traumatic brain injury was induced by controlled cortical impact over the left parietal cortex. Erythropoietin (5000 U/kg) or saline was administered intraperitoneally on Day 1 (EPOx1 group) or on Days 1, 2, and 3 (EPOx3 group) postinjury. Neurological function was assessed using a modified neurological severity score, foot-fault, and Morris water maze tests. Animals were killed 35 days after injury and brain sections were stained for immunohistochemistry.. Compared with the saline treatment, EPO treatment in both the EPOx1 and EPOx3 groups significantly reduced hippocampal cell loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved neurological functional outcome. The EPOx3 group exhibited significantly improved functional and histological outcomes compared with the EPOx1 group.. These data demonstrate that delayed posttraumatic administration of EPO significantly improved histological and long-term functional outcomes in rats after TBI. The triple doses of delayed EPO treatment produced better histological and functional outcomes in rats, although a single dose provided substantial benefits compared with saline treatment.

Topics: Animals; Brain Injuries; Cell Death; Disease Models, Animal; Erythropoietin; Hippocampus; Male; Neovascularization, Physiologic; Neurogenesis; Neurons; Neuroprotective Agents; Parietal Lobe; Random Allocation; Rats; Rats, Wistar; Recovery of Function; Time Factors; Treatment Outcome

An acute-phase response is the systemic reaction of an organism to insult (e.g. infection, trauma and burning). It represents the 'first line' of defence of the body to tissue-damaging attacks. In the present work, we used a rat model of an intra-muscular turpentine oil (TO) injection to analyse erythropoietin (EPO) gene expression changes in the liver, one of the main target organs of acute-phase cytokines. EPO began to increase in the serum of TO-treated animals 6 h after injection and reached a maximum at 24 h (125+/-20 pg/ml). The detection of total RNA by polymerase chain reaction analysis showed that the levels of EPO gene expression in the liver were considerably increased between 2 and 12 h by up to 20-fold at the peak after TO administration, followed by a gradual decrease over the next 48 h, although the values remained significantly higher compared with the control group. In the kidney, after a sudden slight increase, the values declined progressively to 3.5-fold decrease at 12 h after the injection. In the liver, a parallel upregulation of the hypoxia-inducible factor-1 (HIF-1) alpha gene was observed (up to 4.7-fold increase), while HIF-2 alpha gene expression remained unaltered. On the other hand, the protein of both genes became detectable after the injection and increased progressively over 24 h, with a subsequent decline. These results suggest that EPO may be added to the increasing group of positive acute-phase proteins and the liver might represent the major source of the hormone under these conditions in the rat.

Topics: Acute-Phase Proteins; Acute-Phase Reaction; Animals; Basic Helix-Loop-Helix Transcription Factors; Disease Models, Animal; Erythropoietin; Gene Expression; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Liver; Rats; RNA, Messenger; Solvents; Turpentine; Up-Regulation

Aristolochic acid (AA) nephropathy, first reported as Chinese herbs nephropathy, is a rapidly progressive tubulointerstitial nephropathy that results in severe anemia, interstitial fibrosis and end-stage renal disease. Tubulointerstitial injury was studied in a mouse model of AA nephropathy to determine whether low-dose darbepoetin alpha (DPO) treatment prevents acute tubular necrosis and interstitial fibrosis.. AA was administered to C3H/He mice intraperitoneally and some mice were also treated with 0.1 microg/kg of DPO weekly starting on the day of AA administration or on day 28. At 28, 56 or 84 days, blood and urine samples were collected and mice were sacrificed for histological assessment of the kidneys.. AA-treated mice developed anemia, elevation of serum creatinine, severe tubular injury similar to acute tubular necrosis and progressive interstitial fibrosis. Although early treatment with low-dose DPO had minimal effects on the hematocrit, it significantly ameliorated acute tubular injury and interstitial inflammation through increasing the survival of tubular cells. As a result, it contributed to preservation of peritubular capillaries and reduction of interstitial fibrosis.. Low-dose DPO treatment conferred protection against acute tubular damage and attenuated interstitial fibrosis in a mouse model of AA nephropathy. Early administration of low-dose DPO may prevent the progression of acute tubular necrosis and the subsequent renal fibrosis in human AA nephropathy.

Topics: Animals; Apoptosis; Aristolochic Acids; Cell Proliferation; Darbepoetin alfa; Disease Models, Animal; Erythropoietin; Fibrosis; Kidney Tubular Necrosis, Acute; Kidney Tubules; Male; Mice; Mice, Inbred C3H

Topics: Animals; Apoptosis; Disease Models, Animal; Erythropoietin; Hypoxia; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitric Oxide Synthase; Oxidative Stress; Oxygen Consumption; Shock, Septic; Survival Rate

Erythropoietin (EPO) has pleiotropic cytoprotective actions. We investigated the effects of EPO on the physiopathology and cytokine levels after haemorrhagic shock (HS) in conscious rats.. Rats received an intravenous injection of 300 U/kg EPO over 10 min followed by HS via withdrawal of 60% of total blood volume from a femoral arterial catheter (6 ml/100 g body weight) over 30 min. Mean arterial pressure (MAP) and heart rate (HR) were monitored continuously for 18 h after the start of blood withdrawal. Levels of biochemical parameters, including haemoglobin, GOT, GPT, BUN, creatinine (Cr), LDH, CPK, and lactate were measured at 30 min before the induction of HS and 0, 1, 3, 6, 9, 12, and 18 h after HS. Cytokine levels, including TNF-alpha and IL-6, in serum were measured at 1, 9, and 18 h after HS. The kidneys, liver, lungs, and small intestine were removed for pathology assessment at 48 h after HS.. HS significantly increased HR, blood GOT, GPT, BUN, Cr, LDH, CPK, lactate, TNF-alpha, and IL-6 levels and decreased haemoglobin and MAP in rats. Pre-treatment with EPO improved survival rate, preserved the MAP, decreased the tachycardia and markers of organ injury, suppressed the release of TNF-alpha and IL-6 after HS in rats.. Pre-treatment with EPO suppresses the release of serum TNF-alpha and IL-6, along with decreasing the levels of markers of organ injury associated with HS, with such actions ameliorating HS-induced organ damage in rats.

Topics: Animals; Consciousness; Disease Models, Animal; Erythropoietin; Heart Rate; Kidney; Liver; Lung; Male; Random Allocation; Rats; Rats, Inbred WKY; Shock, Hemorrhagic; Time Factors; Tumor Necrosis Factor-alpha

The aims of this study were (1) to evaluate the effect of high-dose erythropoietin (EPO; 5000 U/kg) on expression of inducible nitric oxide synthase (iNOS) in the facial nucleus after facial nerve transection; and (2) to explore whether this effect is relevant to facial motor neuron survival. Forty-two Wistar rats (250-300 g) of both sexes were used in this study. The right facial nerves of 40 rats were transected at the level of the stylomastoid foramen, with the left sides left untreated. The rats were randomly divided into 2 groups: (1) EPO group (treated with EPO twice per week at a dose of 5000 U/kg bodyweight); (2) saline group (treated with saline). The 2 rats that did not undergo axotomy served as the control group. After axotomy, expression of iNOS in the facial nucleus was detected by iNOS immunohistochemistry at various time points, and the number of surviving motor neurons was counted in coronal paraffin sections of the facial nucleus. In both the EPO and saline groups, axotomy caused a significant increase in iNOS expression in the facial nucleus at 1, 2, 3, and 4 weeks after axotomy. iNOS expression was lower in the EPO group than in the saline group. At 2, 3 and 4 weeks after axotomy, a significantly greater proportion of facial motor neurons survived in the EPO group than in the saline group. These results indicate that a high dose of EPO attenuates the increase in iNOS expression in the facial nucleus after facial nerve transection, and thus may enhance the survival of facial motor neurons.

Topics: Analysis of Variance; Animals; Axotomy; Cell Count; Cell Survival; Disease Models, Animal; Erythropoietin; Facial Nerve Diseases; Female; Gene Expression Regulation; Humans; Male; Motor Neurons; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Recombinant Proteins; Time Factors

Individuals with beta-thalassemia develop progressive systemic iron overload, resulting in high morbidity and mortality. These complications are caused by labile plasma iron, which is taken up by parenchymal cells in a dysregulated manner; in contrast, erythropoiesis depends on transferrin-bound iron uptake via the transferrin receptor. We hypothesized that the ineffective erythropoiesis and anemia observed in beta-thalassemia might be ameliorated by increasing the amount of circulating transferrin. We tested the ability of transferrin injections to modulate iron metabolism and erythropoiesis in Hbb(th1/th1) mice, an experimental model of beta-thalassemia. Injected transferrin reversed or markedly improved the thalassemia phenotype in these mice. Specifically, transferrin injections normalized labile plasma iron concentrations, increased hepcidin expression, normalized red blood cell survival and increased hemoglobin production; this treatment concomitantly decreased reticulocytosis, erythropoietin abundance and splenomegaly. These results indicate that transferrin is a limiting factor contributing to anemia in these mice and suggest that transferrin therapy might be beneficial in human beta-thalassemia.

Topics: Animals; Apoptosis; beta-Thalassemia; Cell Survival; Disease Models, Animal; Erythropoietin; Hematopoiesis, Extramedullary; Mice; Splenomegaly; Transferrin

Erythropoiesis is regulated such that a sufficient number of mature erythrocytes is produced. Down-regulation of erythropoiesis causes various types of anemia. Although some anemia-related genes have been identified, there are several types of anemic disease for which the molecular mechanisms are yet unclear, suggesting that unidentified genes in addition to the classical cytokine pathways play important roles in anemia. To address this issue, a new animal model for anemia is required. We established a reversible anemic model in zebrafish by keeping fish at 17 degrees C, a low water temperature. In zebrafish kidney marrow, expression of several genes encoding hematopoietic transcription factors (Runx1, scl, c-myb and GATA-2) and particularly erythropoiesis-related factors (klfd, hbaa1, ba1, GATA-1, EPO, and EPOr) was down-regulated, whereas myelopoiesis-related factors (csf1a and csf3) was up-regulated in low temperature conditions. We propose that this zebrafish model is useful to identify novel genes for hematopoiesis, particularly erythropoiesis.

Topics: Anemia; Animals; Cold Temperature; Disease Models, Animal; Down-Regulation; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Hematopoiesis; Kidney; Myelopoiesis; Zebrafish; Zebrafish Proteins

We hypothesized that donor treatment of deceased brain dead donors would lead to a decrease in inflammatory responses seen in brain death and lead to a restoration of kidney function.. A standardized slow-induction rat brain death model followed by evaluation of kidney function in an isolated perfused kidney model.. Surgery Research Laboratory, University Medical Center Groningen, the Netherlands.. Male Fisher rats.. Donor treatment with erythropoietin, carbamylated erythropoietin, which lacks erythropoietic activity, or vehicle.. In brain death, carbamylated erythropoietin and, to a lesser extent, erythropoietin were able to decrease the expression of several proinflammatory genes and to decrease the infiltration of polymorphonuclear cells in the kidney. No effect on tubular injury parameters was seen. Kidney function decreased almost by 50% after brain death but was fully restored after treatment with both carbamylated erythropoietin and erythropoietin.. Carbamylated erythropoietin can inhibit the inflammatory response caused by brain death more effectively than erythropoietin, whereas both substances can restore kidney function after brain death.

Topics: Animals; Brain Death; Creatinine; Disease Models, Animal; Erythropoietin; Glomerular Filtration Rate; Inflammation; Kidney; Male; Rats; Rats, Inbred F344; Tissue Donors

Erythropoietin (EPO) has been shown to enhance angiogenesis, but its precise mechanisms of enhancement during ischemia are not fully elucidated. We examined the effect of EPO on blood flow recovery from acute hind-limb ischemia induced by ligation of the femoral artery in male C57Bl/6 mice. The density of microvessels with platelet adhesion in ischemic tissues was assessed by intravital microscopy. Treatment with EPO (100 and 1000 IU/kg, i.p.) restored blood flow in a dose-dependent manner and increased plasma levels of soluble-P-selectin, vascular endothelial growth factor (VEGF), and stromal cell-derived factor (SDF-1). Flow cytometric analysis revealed increased P-selectin expression on platelets in EPO-treated mice compared to PBS-treated mice. Intravital microscopic studies showed that EPO increased density of microvessels with platelet adhesion selectively in the ischemic tissues. Neutralizing antibody against P-selectin reduced the density of microvessels with platelet adhesion enhanced with EPO and impaired blood flow recovery with reductions in VEGF and SDF-1 levels. These results suggest that EPO administration enhances recovery from hind-limb ischemia, and platelet adhesion to the microvessels is a key event to enhance the angiogenesis in the ischemic tissues.

Topics: Animals; Chemokine CXCL12; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Flow Cytometry; Hindlimb; Humans; Ischemia; Male; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Microvessels; Neovascularization, Physiologic; P-Selectin; Platelet Adhesiveness; Recombinant Proteins; Vascular Endothelial Growth Factor A

We intended to delineate the mechanisms of erythropoietin (EPO)-induced cardiac vascular endothelial growth factor (VEGF) production and to establish if VEGF is crucial for EPO-induced improvement of cardiac performance.. The effects of EPO on VEGF expression were studied in cultured cardiac cells and EPO-treated hearts. The role of VEGF in EPO-induced neovascularization was studied with two distinct VEGF-neutralizing antibodies or irrelevant control IgG in an aortic sprouting assay and in rats with heart failure (HF) after myocardial infarction (MI) treated with EPO. EPO-alfa (10 IU/mL) was used in vitro and darbepoetin alfa (40 microg/kg/3 weeks, starting 3 weeks after MI) in vivo. EPO stimulated VEGF mRNA expression through the signal transducers and activators of transcription-3 (STAT-3) pathway in neonatal rat cardiomyocytes, but not in endothelial cells or fibroblasts. Similarly, the direct effects of EPO on endothelial sprouting were modest and VEGF independent. In rats with HF, EPO increased VEGF protein expression predominantly in cardiomyocytes, associated with a 37% increase in capillary density and improved cardiac performance. Administration of VEGF-neutralizing antibodies abrogated the salutary effects of EPO on cardiac microvascularization and function. VEGF neutralization attenuated EPO-induced proliferation of myocardial endothelial cells and reduced myocardial incorporation of endothelial progenitor cells (EPCs) in rats with alkaline phosphatase-labelled bone marrow cells.. VEGF is crucial for EPO-induced improvement of cardiac function in HF. EPO fosters VEGF expression predominantly in cardiomyocytes, which in turn stimulates myocardial endothelial proliferation and incorporation of EPCs.

Topics: Animals; Animals, Newborn; Cardiotonic Agents; Cell Movement; Cell Proliferation; Cells, Cultured; Darbepoetin alfa; Disease Models, Animal; Endothelial Cells; Epoetin Alfa; Erythropoietin; Heart Failure; Humans; Male; Myocytes, Cardiac; Neovascularization, Physiologic; Paracrine Communication; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Recombinant Proteins; Recovery of Function; RNA, Messenger; STAT3 Transcription Factor; Stem Cell Transplantation; Stem Cells; Time Factors; Transcription, Genetic; Up-Regulation; Vascular Endothelial Growth Factor A; Ventricular Function, Left; Ventricular Remodeling

Intrauterine infection and inflammation have been linked to preterm birth and brain damage. We hypothesized that recombinant human erythropoietin (rhEPO) would ameliorate brain damage in anovine model of fetal inflammation. At 107 +/- 1 day of gestational age (DGA), chronically catheterized fetal sheep received on 3 consecutive days 1) an intravenous bolus dose of lipopolysaccharide ([LPS] approximately 0.9 microg/kg; n = 8); 2) an intravenous bolus dose of LPS, followed at 1 hour by 5,000 IU/kg of rhEPO (LPS + rhEPO, n = 8); or 3) rhEPO (n = 5). Untreated fetuses (n = 8) served as controls. Fetal physiological parameters were monitored, and fetal brains and optic nerves were histologically examined at 116 +/- 1 DGA. Exposure to LPS, but not to rhEPO alone or saline, resulted in fetal hypoxemia, hypotension (p < 0.05), brain damage, including white matter injury, and reductions in numbers of myelinating oligodendrocytes in the corticospinal tract and myelinated axons in the optic nerve (p < 0.05 for both). Treatment of LPS-exposed fetuses with rhEPO did not alter the physiological effects of LPS but reduced brain injury and was beneficial to myelination in the corticospinal tract and the optic nerve. This is the first study in a long-gestation species to demonstrate the neuroprotective potential of rhEPO in reducing fetal brain and optic nerve injury after LPS exposure.

Topics: Animals; Brain; Brain Damage, Chronic; Demyelinating Diseases; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalitis; Endotoxins; Erythropoietin; Female; Fetal Diseases; Fetal Hypoxia; Injections, Intravenous; Lipopolysaccharides; Nerve Fibers, Myelinated; Neuroprotective Agents; Optic Nerve; Pregnancy; Sheep, Domestic; Treatment Outcome

Several modifications to an esophageal replacement approach have been described, using the left, the right, or the transverse colon as an interposition flap. Interposition of the left colon has become the most popular procedure. Intraoperative clamping of the arterial blood supply and venous drainage of the flap is a possible reason for ischemic flap failure. Thus, we designed a novel model to investigate whether erythropoietin (EPO), which has a tissue-protective effect in ischemia, would have any protective effect on prepared colon flaps in rats.. A total of 56 rats were randomly divided into four main groups, consisting of sham, sham + EPO, colon flap, and colon flap + EPO, and each main group was divided into two sub-groups. In the colon flap and colon flap + EPO groups, the colon flap was prepared and the pediculated free flap fixed tautly to the anterior abdominal wall. The sub-groups were subjected to post-reoperative histopathological investigation on the first and the seventh days, respectively.. Our model was reliable for research related to colon interposition techniques. There was significant histopathological damage in the colon flap group both for the long and short limbs of the flap. On the other hand, EPO administration prevented the mucosal damage seen in the colon flap group.. This study suggests that a colon flap attached tautly to the abdominal side wall simulates colon transposition techniques and also shows that intraperitoneal EPO markedly decreases flap damage in rats with prepared colon flaps.

Topics: Angiogenesis Inducing Agents; Animals; Antioxidants; Colon; Disease Models, Animal; Erythropoietin; Intestinal Mucosa; Ischemia; Male; Rats; Rats, Wistar; Surgical Flaps

Topics: Animals; Brain Ischemia; Clinical Trials, Phase II as Topic; Disease Models, Animal; Erythropoietin; Humans; Neuroprotective Agents; Randomized Controlled Trials as Topic; Recombinant Proteins; Subarachnoid Hemorrhage; Time Factors

Erythropoietin (EPO) is a cytokine with erythropoietic and tissue protective activities. Its action as a tissue protective agent requires, however, high dosage that results in limiting side effects associated with abnormally augmented erythropoiesis. Elimination of the erythropoietic activity of EPO while preserving its tissue protective properties was nevertheless achieved in carbamoylated EPO (CEPO), whose therapeutic activity and apparent safety were documented in experimental models of nervous, heart, kidney and other tissue damage, justifying ongoing clinical trials. Here, we review patent application WO2009094172 by Araim Pharmaceuticals, which describes novel EPO-derived peptides having tissue protective but no erythropoietic activity. The preferred peptide, UEQLERALNSS, which mimics the external 3D structure of the helix B of EPO, was shown to exhibit the same spectrum of tissue protective activity as CEPO in several in vivo models. In addition, it could reduce radiation-induced mortality when administered 24 h after irradiation in mice, suggesting its possible utility in emergency situations after mass irradiation casualties. Owing to their low manufacturing cost, high stability and low immunogenicity, such peptides might well offer a superior alternative to CEPO for therapeutic tissue protection in human pathologies and are likely to provide valuable probes to study the molecular mechanisms of EPO-mediated cytoprotection.

Topics: Animals; Cytoprotection; Disease Models, Animal; Erythropoietin; Humans; Mice; Patents as Topic; Peptides; Protective Agents; Protein Stability; Radiation Injuries; Wounds and Injuries

A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in a majority of patients with polycythemia vera (PV), essential thrombocythemia, and primary myelofibrosis. However, contribution of the JAK2V617F mutation in these 3 clinically distinct myeloproliferative neoplasms (MPNs) remained unclear. To investigate the role of JAK2V617F in the pathogenesis of these MPNs, we generated an inducible Jak2V617F knock-in mouse, in which the expression of Jak2V617F is under control of the endogenous Jak2 promoter. Expression of heterozygous mouse Jak2V617F evoked all major features of human polycythemia vera (PV), which included marked increase in hemoglobin and hematocrit, increased red blood cells, leukocytosis, thrombocytosis, splenomegaly, reduced serum erythropoietin (Epo) levels and Epo-independent erythroid colonies. Homozygous Jak2V617F expression also resulted in a PV-like disease associated with significantly greater reticulocytosis, leukocytosis, neutrophilia and thrombocytosis, marked expansion of erythroid progenitors and Epo-independent erythroid colonies, larger spleen size, and accelerated bone marrow fibrosis compared with heterozygous Jak2V617F expression. Biochemical analyses revealed Jak2V617F gene dosage-dependent activation of Stat5, Akt, and Erk signaling pathways. Our conditional Jak2V617F knock-in mice provide an excellent model that can be used to further understand the molecular pathogenesis of MPNs and to identify additional genetic events that cooperate with Jak2V617F in different MPNs.

Topics: Amino Acid Substitution; Animals; Disease Models, Animal; Erythropoietin; Extracellular Signal-Regulated MAP Kinases; Gene Dosage; Gene Expression Regulation; Homozygote; Humans; Janus Kinase 2; Mice; Mice, Transgenic; Mutation, Missense; Polycythemia Vera; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Signal Transduction; STAT5 Transcription Factor

Pre-operative treatment with recombinant human erythropoietin may improve aortic stenosis patients' condition, including anemia and/or cardiac dysfunction, for subjecting to aortic valve replacement. In this study, we tested this hypothesis in a mouse model of aortic stenosis. Adult male mice were subjected to either aortic stenosis created by aortic ligature or sham operation. Aortic stenosis for 4 weeks caused cardiac hypertrophy, pulmonary congestion and left ventricular dysfunction. It was associated with increased levels of tumor necrosis factor-alpha in serum and myocardium, and reduced levels of interleukin-10 in myocardium but not in serum. Myocyte apoptosis rate, level of cleaved caspase 3, activity of nuclear factor-kappaB and expression of p38-MAPK pathway were also elevated. Erythropoietin treatment increased hematocrit but did not prevent the development of cardiac hypertrophy. It, however, reduced the apoptosis, prevented the increases in tumor necrosis factor-alpha, nuclear factor-kappaB activation and phosphorylation of p38, and attenuated the increases in lung weight, the decreases in LVEF and LVFS, and the increases in LVDd and LVDs. In conclusion recombinant human erythropoietin has cardioprotective effects in maladaptive cardiac hypertrophy by inhibiting nuclear factor-kappaB activation, phosphorylation of p38-MAPK pathway, and production of tumor necrosis factor-alpha, together leading to a reduced apoptosis.

Topics: Animals; Aortic Valve Stenosis; Apoptosis; Apoptosis Regulatory Proteins; Cardiomegaly; Cardiotonic Agents; Disease Models, Animal; Erythropoietin; Ligation; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Recombinant Proteins; Tumor Necrosis Factor-alpha

Primary and/or secondary injury of the renal microvascular endothelium is a common finding in various renal diseases. Besides well-known endothelial repair mechanisms, including endothelial cell (EC) proliferation and migration, homing of extrinsic cells such as endothelial progenitor cells (EPC) and hematopoietic stem cells (HSC) has been shown in various organs and may contribute to microvascular repair. However, these mechanisms have so far not been studied after selective microvascular injury in the kidney. The present study investigated the time course of EPC and HSC stimulation and homing following induction of selective EC injury in the mouse kidney along with various angiogenic factors potentially involved in EC repair and progenitor cell stimulation. Erythropoietin was used to stimulate progenitor cells in a therapeutic approach. We found that selective EC injury leads to a marked stimulation of EPCs, HSCs, and various angiogenic factors to orchestrate microvascular repair. Angiogenic factors started to increase as early as 30 min after disease induction. Progenitor cells could be first detected in the circulation and the spleen before they selectively homed to the diseased kidney. Injection of a high dose of erythropoietin 2 h after disease induction markedly attenuated vascular injury through nonhemodynamic mechanisms, possibly involving vascular endothelial growth factor release.

Topics: Angiogenic Proteins; Animals; Cell Movement; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Erythrocyte Count; Erythropoietin; Green Fluorescent Proteins; Hematopoietic Stem Cells; Injections, Intraperitoneal; Kidney; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microcirculation; Promoter Regions, Genetic; Receptor, TIE-2; Renal Circulation; Spleen; Stem Cells; Thrombotic Microangiopathies; Time Factors

Topics: Animals; Brain Death; Disease Models, Animal; Erythropoietin; Humans; Inflammation; Kidney; Rats; Tissue Donors

STOP (stable tubule only polypeptide) null mice display neurochemical and behavioral abnormalities that resemble several well-recognized features of schizophrenia. Recent evidence suggests that the hematopoietic growth factor erythropoietin improves the cognitive performance of schizophrenics. The mechanism, however, by which erythropoietin is able to improve the cognition of schizophrenics is unclear. To address this question, we first determined whether acute administration of the erythropoietin analog known as darbepoetin alpha (D. alpha) improved performance deficits of STOP null mice in the novel objective recognition task (NORT). NORT performance of STOP null mice, but not wild-type littermates, was enhanced 3 h after a single injection of D. alpha (25 microg/kg, i.p.). Improved NORT performance was accompanied by elevated NADPH diaphorase staining in the ventral hippocampus as well as medial and cortical aspects of the amygdala, indicative of increased nitric oxide synthase (NOS) activity in these structures. NOS generates the intracellular messenger nitric oxide (NO) implicated in learning and memory. In keeping with this hypothesis, D. alpha significantly increased NO metabolite levels (nitrate and nitrite, NOx) in the hippocampus of both wild-type and STOP null mice. The NOS inhibitor, N (G)-nitro-L- arginine methyl ester (L-NAME; 25 mg/kg, i.p.), completely reversed the increase in hippocampal NOx levels produced by D. alpha. Moreover, L-NAME also inhibited the ability of D. alpha to improve the NORT performance of STOP null mice. Taken together, these observations suggest D. alpha enhances the NORT performance of STOP null mice by increasing production of NO.

Topics: Animals; Behavior, Animal; Cognition Disorders; Darbepoetin alfa; Discrimination Learning; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Erythropoietin; Female; Hematinics; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Microtubule-Associated Proteins; NADPH Dehydrogenase; Neuropsychological Tests; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Prosencephalon; Recognition, Psychology

The glycoprotein erythropoietin (EPO) is a multifunctional cytokine involved in erythropoiesis. Recent data have suggested that EPO and EPO receptors are expressed in the central nervous system, where EPO exerts neuroprotective effects. However, peripheral nervous system (PNS) EPO and EPO receptor expression has not been widely studied. EPO and EPO receptor expression was examined in the PNS in an experimental autoimmune neuritis (EAN) rat model in the present study to elucidate EPO/EPO-receptor binding pathway involvement in injured PNS tissue. Western blot analysis demonstrated that EPO was significantly increased in the PNS at the paralytic stage on day 14 post-immunization (PI); levels were significantly decreased at day 30 PI. EPO was identified in PNS-derived vascular endothelial cells, Schwann cells, and axons in normal control rats. Most inflammatory cells in EAN lesions were EPO immunopositive at day 14 PI. In addition, the intensity of EPO immunoreactivity in both Schwann and vascular endothelial cells was greater than that of normal controls at this stage; intensity declined at day 30 PI. These findings suggest that EPO is transiently upregulated in EAN lesions and that the EPO/EPO-receptor binding pathway is associated with neuroprotection in EAN-affected PNS tissues.

Topics: Animals; Disease Models, Animal; Ecdysone; Erythropoietin; Female; Myelin P2 Protein; Neuritis, Autoimmune, Experimental; Neuroglia; Neurons; Peripheral Nervous System; Rats; Rats, Inbred Lew; Receptors, Erythropoietin; S100 Proteins; Time Factors; Up-Regulation

Erythropoiesis-stimulating agents (ESAs) may have therapeutic benefits beyond ameliorating anemia. Although ESAs have renoprotective effects in acute/chronic renal injury models, their effects on blood pressure could also worsen chronic renal failure (CRF). The development of human cell-derived erythropoietin analogue epoetin delta prompted us (1) to investigate whether in a 5/6th nephrectomy-induced CRF rat model, epoetin delta-mediated renoprotective effects occur independently of its hematopoietic effects and (2) to unravel the involvement of particular factors herein.. After induction of CRF in Wistar rats, epoetin delta was administered for 8 weeks at different doses: 0 IU/kg (uremic control); 48, 100 or 300 IU/kg 1x/week, and 16 or 100 IU/kg 3x/week. During this period hematopoietic and renal functional parameters as well as systolic blood pressure (SBP) were monitored.. After 8 weeks, control CRF rats showed reduced hematocrit (Hct)/hemoglobin (Hb) levels and increased SBP. Epoetin delta dose-dependently attenuated the reduction in Hct/Hb. Furthermore, epoetin delta treatment resulted in reduced deterioration of renal function in CRF rats after 8 weeks which was accompanied by decreased collagen deposition, renal fibrosis and interstitial macrophage infiltration. Remarkably, these renoprotective effects did not show the same dose dependency as compared to that seen for the hematopoietic response and were also seen at subhematopoietic doses. Interestingly, epoetin delta treatment resulted in a dose-dependent decrease of profibrotic (TGF-beta) and proapoptotic (Bcl-2-associated X protein) genes together with a significant dose-dependent increase of antifibrotic (hepatocyte growth factor) and antiapoptotic (Bcl-2) genes. Epoetin delta treatment had no effect on VEGF expression.. Epoetin delta treatment could delay the progression of CRF through antiapoptotic and antifibrotic mechanisms. This protective action of epoetin delta on the kidney probably is not directly related to its hematopoietic effects.

Topics: Anemia; Animals; Apoptosis; bcl-2-Associated X Protein; Blood Pressure; Disease Models, Animal; Disease Progression; Erythropoietin; Fibrosis; Hematinics; Hepatocyte Growth Factor; Humans; Kidney; Kidney Failure, Chronic; Male; Nephrectomy; Rats; Rats, Wistar; Recombinant Proteins; Transforming Growth Factor beta

Systemic administration of high-dose recombinant human erythropoietin (rhEPO) is known to attenuate ischemic injury. However, high-dose rhEPO might aggravate ischemic lesions by increasing blood viscosity because of its erythropoietic effects. Asialoerythropoietin (asialoEPO), an EPO derivative with an extremely short plasma half-life, has considerably lesser erythropoietic effect than that of naive EPO. We attempted to determine whether asialoEPO exerts the same neuroprotective effect as naive EPO in a gerbil transient forebrain ischemia model.. Transient occlusion of both the common carotid arteries was performed in 23 adult gerbils. The drugs (asialoEPO or rhEPO, 10 U/g bodyweight) or phosphate-buffered saline (PBS) were injected intraperitoneally at three times (3 hours before, immediately after, and 24 hours after the ischemic insult). Learning and retention tests were performed on days 6 and 7, respectively, and histological analyses were performed on day 7.. Animals treated with asialoEPO and rhEPO showed significant neurological improvement compared to the PBS-treated animals. The number of viable neurons in the CA1 field of the rhEPO-treated (103.57 ± 27.90 cells/mm) and asialoEPO-treated (144.99 ± 34.87 cells/mm) animals was higher than that of the PBS-treated animals (19.53 ± 3.79 cells/mm). Terminal dinucleotidyltransferase-mediated UTP end labeling-positive cells were significantly lower in the rhEPO-treated (33.40 ± 8.13 cells/mm) and asialoEPO-treated (29.28 ± 14.91 cells/mm) animals than in the PBS-treated animals (76.67 ± 8.14 cells/mm). AsialoEPO treatment did not have any effect on erythropoiesis.. Multiple dosing of asialoEPO, like EPO, could protect the hippocampal CA1 neurons from ischemic damage without affecting erythropoiesis.

Topics: Animals; Asialoglycoproteins; Avoidance Learning; CA1 Region, Hippocampal; Carotid Artery Diseases; Cell Count; Cell Death; Chromatography, High Pressure Liquid; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Erythropoietin; Gerbillinae; In Situ Nick-End Labeling; Ischemic Attack, Transient; Neurons; Neuroprotective Agents; Prosencephalon

Erythropoietin (Epo) is an endogenous cytokine that regulates hematopoiesis and is widely used to treat anemia. In addition, it has recently increased interest in the neurosciences since the new concept of Epo as a neuroprotective agent has emerged. The potential protective effect of human recombinant Epo (r-hu-Epo) on a hypoxic-ischemic (HI) pup rat model was studied. Cerebral HI was obtained by permanent left carotid artery ligature of pups followed by a 2-h hypoxia. Three hours after carotid occlusion, brain lesions were assessed by magnetic resonance diffusion weighted imaging. Intraperitoneal administration of r-hu-Epo (30,000 U/kg dose) limited both the HI-induced brain lesion area and the decrease in apparent diffusion coefficient (ADC) in the lesion. To identify potential mechanisms underlying the effects of Epo, immunohistochemical detection of caspase-3 and water channel protein aquaporin-4 (AQP4) were performed. No early apoptosis was detected, but up-regulation of AQP4 expression was observed in HI pups that received r-hu-Epo compared with HI animals without treatment. This study demonstrates an early neuroprotective effect of Epo with regard to brain lesion area and ADC values. One possible mechanism of Epo for decreasing brain edema and cellular swelling could be a better clearance of water excess in brain tissue, a process possibly mediated by AQP4.

Topics: Animals; Animals, Newborn; Aquaporin 4; Brain; Disease Models, Animal; Erythropoietin; Humans; Hypoxia-Ischemia, Brain; Immunohistochemistry; Magnetic Resonance Imaging; Rats; Rats, Wistar; Recombinant Proteins

Anemia may accelerate angiogenesis in ischemic organs through its ability to augment tissue hypoxia-induced generation of several known angiogenic factors and to increase erythropoietin levels, which are also potently angiogenic. We examined the effect of controlled phlebotomy (bloodletting) on blood flow in a mouse ischemic leg model. We ligated the right femoral artery of BALB/c mice. In the phlebotomy group, 200 microl of blood were drawn from the tail vein once a week. After 4 wk, blood flow in the ischemic leg was significantly better in the phlebotomy group (flow ratio of the ischemic to nonischemic leg, 0.87 + or - 0.04) than the control group (0.59 + or - 0.05, P < 0.05), and capillary density was significantly higher. Repeated phlebotomy increased serum erythropoietin levels as well as the expression of hypoxia-inducible transcription factor-1alpha and vascular endothelial growth factor and both the expression and activity of Akt and endothelial nitric oxide synthase (eNOS) in ischemic legs. Treatment with wortmannin or N(omega)-nitro-l-arginine methyl ester significantly attenuated the phlebotomy-induced improvement of blood flow. In addition, fluorescence-activated cell sorting analysis revealed an increase in circulating peripheral endothelial progenitor cells in the phlebotomy group, and treatment with AMD3100, a specific inhibitor of the chemokine receptor CXCR4, blocked the beneficial effect of phlebotomy. These findings suggest that repeated phlebotomy improves blood flow in ischemic legs through an angiogenic action that involves the Akt/eNOS pathway, endothelial progenitor cell mobilization, and their complicated cross talk. An adequately controlled phlebotomy might be one method by which to induce therapeutic angiogenesis.

Topics: Androstadienes; Animals; Benzylamines; Capillaries; Cyclams; Disease Models, Animal; Endothelial Cells; Enzyme Inhibitors; Erythropoietin; Femoral Artery; Heterocyclic Compounds; Hindlimb; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemia; Ligation; Male; Mice; Mice, Inbred BALB C; Muscle, Skeletal; Neovascularization, Physiologic; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type III; Phlebotomy; Proto-Oncogene Proteins c-akt; Receptors, CXCR4; Regional Blood Flow; Signal Transduction; Stem Cells; Time Factors; Vascular Endothelial Growth Factor A; Wortmannin

We report a Jak2V617F knockin mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild-type HSCs. In contrast, myeloid progenitor populations are expanded and skewed toward the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F-positive MPN.

Topics: Amino Acid Substitution; Animals; Antigens, CD; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Cell Count; Cell Differentiation; Disease Models, Animal; Erythroid Precursor Cells; Erythropoietin; Gene Expression; Gene Expression Profiling; Hematocrit; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Heterozygote; Humans; Janus Kinase 2; Megakaryocyte Progenitor Cells; Megakaryocyte-Erythroid Progenitor Cells; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myeloid Progenitor Cells; Myeloproliferative Disorders; Polycythemia Vera; Protein Kinase Inhibitors; Pyrrolidines; Spleen; Sulfonamides; Survival Analysis

Erythropoietin (EPO) exhibits diverse cellular functions, including neurotrophic, anti-oxidant, anti-apoptotic, and anti-inflammatory effects in non-hematopoietic tissues. This study evaluated whether bone marrow mesenchymal stromal cells (MSCs) transduced with the EPO gene (EPO-MSCs) promoted neural cell survival and improved neurological deficits caused by ischemic stroke. EPO-MSCs stably produced high levels of EPO (10IU/ml) without any alteration of their mesenchymal phenotype. Both EPO transduction and treatment with 10 international units (IU) of recombinant human EPO (rhEPO) provided protection from H(2)O(2)-induced oxidative injury in human bone marrow mesenchymal stromal cells and in SH-SY5Y cells. EPO-MSCs were more protected than were MSCs treated with 10IU rhEPO (10U-MSCs). We also found that the expression of the neurotrophic factors BDNF, PD-ECGF, HGF, SDF-1alpha, and TGF-1beta increased in EPO-MSCs, while only BDNF and TGF-1beta increased in 10U-MSCs. Implantation of EPO-MSCs in an animal model of ischemic stroke significantly improved neurological function and decreased infarct volumes without affecting hematocrit level. An evaluation of the brain tissue 21days after implantation showed that EPO and phosphorylated Akt (a downstream mediator of EPO) increased only in brains implanted with EPO-MSCs. Transduction of the EPO gene into MSCs induced secretion of EPO and various trophic factors that may provide excellent neuroprotective effects in both in vitro and in vivo models of ischemic stroke.

Topics: Animals; Antigens, CD; Brain Infarction; Caspase 3; Cell Death; Cell Line; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Flow Cytometry; Gene Expression Regulation; Green Fluorescent Proteins; Humans; Hydrogen Peroxide; Infarction, Middle Cerebral Artery; Lentivirus; Magnetic Resonance Imaging; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Nerve Growth Factors; Nervous System Diseases; Rats; Statistics, Nonparametric; Tetrazolium Salts; Thiazoles; Transduction, Genetic

Biliary obstruction and cholestasis result in hepatocellular necro-inflammation and lead to the development of liver fibrosis. The objective of this study was to analyze whether the multiple tissue-protective properties of erythropoietin are salutary in an experimental model of liver fibrosis. For this purpose, C57BL/6J mice underwent common bile duct ligation (BDL) and were treated with either darbepoetin-α (10 μg/kg i.p.) or physiological saline every third day, beginning 24 h after BDL. Mice were killed at 2, 5, 14, and 28 days after BDL. Beside hematological parameters, markers of inflammation and fibrosis were assessed histomorphometrically and immunohistochemically as well as by quantitative real-time PCR. In addition, a 7-week survival study was performed. BDL provoked cholestatic hepatitis characterized by biliary infarcts with accumulation of macrophages followed by marked collagen deposition and increased expression of profibrotic gene transcripts. Darbepoetin-α treatment significantly diminished the area of focal necrosis, reduced the infiltration of macrophages, decreased levels of profibrotic genes, and lowered collagen deposition. Moreover, darbepoetin-α significantly reduced systemic anemia caused by BDL. Finally, darbepoetin-α treatment significantly prolonged the survival time after BDL. This study suggests that darbepoetin-α, which is a clinically well-established substance, might be used as an efficient therapeutic option for patients with chronic cholestatic liver disease.

Topics: Alanine Transaminase; Anemia; Animals; Cholestasis, Extrahepatic; Collagen; Common Bile Duct; Cytophotometry; Darbepoetin alfa; Disease Models, Animal; Erythropoietin; Hematinics; Immunohistochemistry; Inflammation; Liver Cirrhosis; Mice; Mice, Inbred C57BL; Necrosis

The impact of epoetin beta (recombinant human erythropoietin) on brain infarction area (BIA) and neurological status in a rat model of acute ischemic stroke (IS) induced by distal left internal carotid artery occlusion was investigated.. Adult male Sprague-Dawley rats (n = 30) were categorized into group 2 (IS only) and group 3 (IS plus intraperitoneal erythropoietin 5000 IU/kg at 0, 12, and 24 hours after IS). Healthy Sprague-Dawley rats (n = 10) served as group 1.. Analysis of brain tissues showed larger BIA in group 2 than in group 3 (P < 0.001). Corner test identified highest frequency of left turn in group 2 (P < 0.05). The mRNA expressions of Bax, caspase 3, interleukin 18, toll-like receptor 4, and plasminogen activator inhibitor 1 were highest, whereas Bcl-2 was lowest in group 2 (P < 0.05). Lower CXCR4 and stromal cell-derived factor 1 expressions were noted in group 2 than in group 3 (P < 0.01). Immunohistofluorescence staining showed lower expressions of CXCR4, stromal cell-derived factor 1, von Willebrand factor, and doublecortin with higher number of apoptotic nuclei in group 2 than in group 3 (P < 0.001). Immunohistochemical staining demonstrated lower cellular proliferation and number of small vessels with higher glial fibrillary acid protein expression in group 2 than in group 3 (P < 0.01).. Erythropoietin significantly limited BIA and improved sensorimotor dysfunction after acute IS.

Topics: Acute Disease; Animals; Apoptosis; Brain Infarction; Brain Ischemia; Disease Models, Animal; Doublecortin Protein; Encephalitis; Erythropoietin; Hematinics; Humans; Male; Neovascularization, Physiologic; Neurogenesis; Oxidative Stress; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Recovery of Function

Erythropoietin (EPO) and Klotho expression have both been detected in the kidney. Since a recent study suggested that both EPO and Klotho mitigate kidney damage, we explored the relation between EPO and Klotho in a doxorubicin hydrochloride (DXR)-induced rat nephropathy model treated with recombinant human erythropoietin (rhEPO).. Male Sprague-Dawley rats were subjected to DXR-induced nephropathy. The rhEPO group was intracutaneously injected with rhEPO twice weekly at 4-16 weeks after the DXR injection. The rats were sacrificed at 16 weeks after the DXR administration. Expression of renal Klotho, HSP70, alpha-smooth-muscle actin and E-cadherin were assessed using real-time PCR or western blotting. The hematocrit, plasma creatinine and phosphate levels were also determined. Immunohistochemical studies and Masson-trichrome staining were performed.. The renal Klotho mRNA and Klotho protein expressions were significantly reduced in the DXR nephropathy group. Treatment with rhEPO improved the serum creatinine, phosphate level and histological changes observed in the DXR nephropathy group. The reduction in Klotho expression induced by DXR nephropathy was mitigated by rhEPO administration.. rhEPO is involved in the pathophysiology of DXR nephropathy. rhEPO mitigated elevated plasma phosphate concentrations in an experimental model of chronic kidney disease via the expression of Klotho.

Topics: Animals; Disease Models, Animal; Doxorubicin; Erythropoietin; Gene Expression; Glucuronidase; Humans; Kidney; Kidney Failure, Chronic; Klotho Proteins; Male; Rats; Rats, Sprague-Dawley; Recombinant Proteins; RNA, Messenger

A hypoxia-inducible VEGF expression system with the oxygen-dependent degradation (ODD) domain was constructed and tested to be used in gene therapy for ischemic myocardial disease.. Luciferase and VEGF expression vector systems were constructed with or without the ODD domain: pEpo-SV-Luc (or pEpo-SV-VEGF) and pEpo-SV-Luc-ODD (or pEpo-SV-VEGF-ODD). In vitro gene expression efficiency of each vector type was evaluated in HEK 293 cells under both hypoxic and normoxic conditions. The amount of VEGF protein was estimated by ELISA. The VEGF expression vectors with or without the ODD domain were injected into ischemic rat myocardium. Fibrosis, neovascularization, and cardiomyocyte apoptosis were assessed using Masson's trichrome staining, α-smooth muscle actin (α-SMA) immunostaining, and the TUNEL assay, respectively.. The plasmid vectors containing ODD significantly improved the expression level of VEGF protein in hypoxic conditions. The enhancement of VEGF protein production was attributed to increased protein stability due to oxygen deficiency. In a rat model of myocardial ischemia, the pEpo-SV-VEGF-ODD group exhibited less myocardial fibrosis, higher microvessel density, and less cardiomyocyte apoptosis compared to the control groups (saline and pEpo-SV-VEGF treatments).. An ODD-mediated VEGF expression system that facilitates VEGF-production under hypoxia may be useful in the treatment of ischemic heart disease.

Topics: Animals; Apoptosis; Cell Line; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Genetic Therapy; Genetic Vectors; Humans; Hypoxia; Immunohistochemistry; In Situ Nick-End Labeling; Luciferases; Male; Myocardial Ischemia; Myocardium; Oxygen; Plasmids; Protein Stability; Rats; Rats, Sprague-Dawley; Simian virus 40; Transfection; Vascular Endothelial Growth Factor A

Erythropoietin (EPO) has generated interest as a novel therapy after myocardial infarction (MI), but the safety and efficacy of prolonged therapy have not been studied in a large animal model of reperfused MI.. MI was induced in pigs by a 90-minute balloon occlusion of the left anterior descending coronary artery. Sixteen animals were randomized to either EPO or saline (control group). Inflammatory markers, bone marrow cell mobilization, and left ventricular function (by both echocardiography and pressure-volume measurements) were assessed at baseline, 1 and 6 weeks post-MI. EPO therapy was associated with a significant increase in hemoglobin and mononuclear counts. D-dimer and C-reactive protein levels did not differ between groups. At week 6, EPO therapy prevented further deterioration of left ventricular ejection fraction (39 +/- 2% vs. 33 +/- 1%, P < .01) and improved wall motion score index (P < .02). Histopathology revealed increased areas of viable myocardium, vascular density, and capillary-to-myocyte ratio in the EPO therapy compared with the control (all P < .05).. Prolonged EPO therapy after MI in a large animal model is safe and leads to an increase in viable myocardium, increased vascular density, and prevents further deterioration of left ventricular function. These results support future clinical studies in post-MI patients.

Topics: Animals; Disease Models, Animal; Erythropoietin; Myocardial Infarction; Random Allocation; Swine; Time Factors; Ventricular Dysfunction, Left; Ventricular Function, Left

Erythropoietin is produced by the kidney and stimulates erythropoiesis; however, in chronic renal disease its levels are reduced and patients develop anemia that is treatable with iron and recombinant hormone. The mechanism by which erythropoietin improves iron homeostasis is still unclear, but it may involve suppression of the iron regulatory peptide hepcidin and/or a direct effect on intestinal iron absorption. To investigate these possibilities, we used the well-established 5/6th nephrectomy rat model of chronic renal failure with or without human recombinant erythropoietin treatment. Monolayers of human intestinal Caco-2 cells were also treated with erythropoietin to measure any direct effects of this hormone on intestinal iron transport. Nephrectomy increased hepatic hepcidin expression and decreased intestinal iron absorption; these effects were restored to levels found in sham-operated rats on erythropoietin treatment of the rats with renal failure. In Caco-2 cells, the addition of erythropoietin significantly increased the expression of apical divalent metal transporter 1 (DMT1) and basolateral ferroportin and, consequently, iron transport across the monolayer. Taken together, our results show that erythropoietin not only exerts a powerful inhibitory action on the expression of hepcidin, thus permitting the release of iron from reticuloendothelial macrophages and intestinal enterocytes, but also acts directly on enterocytes to increase iron absorption.

Topics: Animals; Antimicrobial Cationic Peptides; Caco-2 Cells; Cation Transport Proteins; Disease Models, Animal; Duodenum; Erythropoietin; Hepcidins; Humans; Intestinal Absorption; Iron; Kidney Failure, Chronic; Male; Nephrectomy; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; RNA, Messenger; Signal Transduction

Darbepoetin, a long-acting erythropoietin derivative, attenuates cardiomyocyte apoptosis and improves short-term (3 days) cardiac function, but the mechanisms responsible are unknown. We investigated potential mechanisms by which darbepoetin exerts cardioprotection following myocardial infarction in mice and the significance of the erythropoietin receptor (EPOR)-common beta-chain (c-beta-chain) heteroreceptor.. Mice underwent 60 min coronary occlusion followed by treatment with vehicle or a single dose of darbepoetin. Effects on gene expression, apoptosis and neutrophil accumulation in infarcted left ventricle were assessed 24 h later. Cardiac function, effects on vascularization and fibrosis were assessed 28 days later. The significance of EPOR-c-beta-chain heteroreceptor was examined 28 days after infarction using mice deficient in c-beta-chain.. Twenty-four hours after darbepoetin, mRNAs encoding haeme oxygenase-1 (HO-1), iNOS and brain natriuretic peptide (BNP) were markedly elevated only in infarcted regions, and the frequency of apoptotic cells attenuated. Inflammation was also attenuated with reductions in neutrophil numbers. Darbepoetin also elevated mRNAs encoding angiogenic factors: placental growth factor, monocyte chemoattractant protein-1 and interleukin-1beta. Twenty-eight days after treatment, CD31+ vessels in the infarct zone doubled and fibrosis reduced. Cardiac haemodynamics were improved. Darbepoetin also improved cardiac haemodynamics in c-beta-chain-deficient mice, increased HO-1 and iNOS expression and vessel numbers and attenuated fibrosis.. Darbepoetin stimulates expression of haeme oxygenase, iNOS, BNP and angiogenic factors specifically in infarcted left ventricle that attenuates inflammation, apoptosis and fibrosis; elevate vessel numbers; and improve cardiac function. The EPOR-c-beta-chain heteroreceptor is not essential for these effects.

Topics: Animals; Apoptosis; Cardiotonic Agents; Darbepoetin alfa; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Fibrosis; Gene Expression Regulation; Heme Oxygenase-1; Hemodynamics; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardium; Natriuretic Peptide, Brain; Neovascularization, Physiologic; Neutrophil Infiltration; Nitric Oxide Synthase Type II; Receptors, Erythropoietin; Time Factors; Ventricular Function, Left

Erythropoietin (EPO) promotes functional recovery after traumatic brain injury (TBI). This study was designed to investigate whether EPO treatment promotes contralateral corticospinal tract (CST) plasticity in the spinal cord in rats after TBI. Biotinylated dextran amine (BDA) was injected into the right sensorimotor cortex to anterogradely label the CST. TBI was induced by controlled cortical impact over the left parietal cortex immediately after BDA injections. EPO (5000 U/kg) or saline was administered intraperitoneally at Days 1, 2, and 3 post-injury. Neurological function was assessed using a modified neurological severity score (mNSS) and footfault tests. Animals were sacrificed 35 days after injury and brain sections stained for histological analysis. Compared to the saline treatment, EPO treatment significantly improved sensorimotor functional outcome (lower mNSS and reduced footfaults) from Days 7 to 35 post-injury. TBI alone significantly stimulated contralateral CST axon sprouting toward the denervated gray matter of the cervical and lumbar spinal cord; however, EPO treatment further significantly increased the axon sprouting in TBI rats although EPO treatment did not significantly affect axon sprouting in sham animals. The contralesional CST sprouting was highly and positively correlated with sensorimotor recovery after TBI. These data demonstrate that CST fibers originating from the contralesional intact cerebral hemisphere are capable of sprouting into the denervated spinal cord after TBI and EPO treatment, which may at least partially contribute to functional recovery.

Topics: Animals; Axons; Biotin; Brain Injuries; Dextrans; Disease Models, Animal; Erythropoietin; Functional Laterality; Hematocrit; Hindlimb; Male; Neurologic Examination; Psychomotor Performance; Pyramidal Tracts; Rats; Rats, Wistar; Recovery of Function; Spinal Cord; Statistics as Topic; Time Factors

Cisplatin exerts its cytotoxic effect through distinct DNA lesions, leading to peripheral neuropathy. The risk of sensory neuropathy is a common problem during cancer treatment with cisplatin, leading to somatic hyperalgesia. Yet, data focussing on cisplatin-induced impairment of the autonomic nervous system are limited. The present study was aimed to investigate the effect of recombinant human erythropoietin (rhEPO) on cisplatin-induced visceral hyperalgesia.. C57BL/6 mice were treated either with cisplatin (2 mg/kg, once per week) or with cisplatin (2 mg/kg, once per week) plus rhEPO (40 microg/kg, 3 times per week) for 8 weeks. Controls were treated with saline. To quantify the visceromotor response (VMR) at week 9, standardized electrodes were implanted into the external oblique musculature for electromyographic recordings. After that, animals were decapitated and dorsal root ganglia (DRG) was removed for transmission electron microscopy studies.. Cisplatin-treated mice showed a significant increase of VMR compared to the controls [(7080 +/- 969) vs (2864 +/- 279); P< 0.001], while rhEPO dramatically counteracted this effect [(2962 +/- 336) vs (7080 +/- 969); P< 0.001)]. Transmission electron microscopy revealed cisplatin-induced structural lesions of nuclear membrane in DRG cells, which could be ameliorated by rhEPO.. Erythropoietin can significantly ameliorate the cisplatin-induced visceral hyperplasia and DRG nuclear membrane structure damage in mice, indicating a neuroprotective role of erythropoietin.

Topics: Analysis of Variance; Animals; Behavior, Animal; Cisplatin; Colon; Disease Models, Animal; Electromyography; Erythropoietin; Ganglia, Spinal; Humans; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Physical Stimulation; Random Allocation; Recombinant Proteins; Rectum; Sensory Receptor Cells; Viscera

Erythropoietin (EPO) and granulocyte colony stimulating factor (GCSF) are potential novel therapies after myocardial infarction (MI). We first established the optimal and clinically applicable dosages of these drugs in mobilizing hematopoietic stem cells (HSC), and then tested the efficacy of monotherapy and combination therapy post-MI.. Optimal doses were established in enhanced green fluorescent protein (eGFP) + chimeric mice (n = 30). Next, mice underwent MI and randomized into 4 groups (n = 18/group): 1) GCSF; 2) EPO; 3) EPO+GCSF; and 4) control. Left ventricular (LV) function was analyzed pre-MI, at 4 hours and at 28 days post-MI. Histological assessment of infarct size, blood vessels, apoptotic cardiomyocytes, and engraftment of eGFP+ mobilized cells were analyzed at day 28. LV function in the control group continued to deteriorate, whereas all treatments showed stabilization. The treatment groups resulted in less scarring, increased numbers of mobilized cells to the infarct border zone (BZ), and a reduction in the number of apoptotic cardiomyocytes. Both EPO groups had significantly more capillaries and arterioles at the BZ.. We have established the optimal doses for EPO and GCSF in mobilizing HSC from the bone marrow and demonstrated that therapy with these agents, either as monotherapy or combination therapy, led to improvement of cardiac function post-MI. Combination therapy does not seem to have additive benefit over monotherapy in this model.

Topics: Animals; Cytokines; Delayed-Action Preparations; Disease Models, Animal; Drug Therapy, Combination; Erythropoietin; Granulocyte Colony-Stimulating Factor; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myocardial Infarction; Myocytes, Cardiac; Radiation Chimera; Random Allocation; Ventricular Function, Left

Erythropoiesis stimulating agents (ESAs) are widely used to treat anaemia but concerns exist about their potential to promote pathological angiogenesis in some clinical scenarios. In the current study we have assessed the angiogenic potential of three ESAs; epoetin delta, darbepoetin alfa and epoetin beta using in vitro and in vivo models.. The epoetins induced angiogenesis in human microvascular endothelial cells at high doses, although darbepoetin alfa was pro-angiogenic at low-doses (1-20 IU/ml). ESA-induced angiogenesis was VEGF-mediated. In a mouse model of ischaemia-induced retinopathy, all ESAs induced generation of reticulocytes but only epoetin beta exacerbated pathological (pre-retinal) neovascularisation in comparison to controls (p<0.05). Only epoetin delta induced a significant revascularisation response which enhanced normality of the vasculature (p<0.05). This was associated with mobilisation of haematopoietic stem cells and their localisation to the retinal vasculature. Darbepoetin alfa also increased the number of active microglia in the ischaemic retina relative to other ESAs (p<0.05). Darbepoetin alfa induced retinal TNFalpha and VEGF mRNA expression which were up to 4 fold higher than with epoetin delta (p<0.001).. This study has implications for treatment of patients as there are clear differences in the angiogenic potential of the different ESAs.

Topics: Animals; Cells, Cultured; Chromatography, High Pressure Liquid; Disease Models, Animal; Erythropoietin; Hematinics; Ischemia; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Neovascularization, Pathologic; Recombinant Proteins; Retina; Retinal Diseases; Reverse Transcriptase Polymerase Chain Reaction

Using magnetic resonance imaging (MRI) protocols of T(2)-, T(2)*-, diffusion- and susceptibility-weighted imaging (T2WI, T2*WI, DWI, and SWI, respectively) with a 7T system, we tested the hypothesis that treatment of embolic stroke with erythropoietin (EPO) initiated at 24 hr and administered daily for 7 days after stroke onset has benefit in repairing ischemic cerebral tissue. Adult Wistar rats were subjected to embolic stroke by means of middle cerebral artery occlusion (MCAO) and were randomly assigned to a treatment (n = 11) or a control (n = 11) group. The treated group was given EPO intraperitoneally at a dose of 5,000 IU/kg daily for 7 days starting 24 hr after MCAO. Controls were given an equal volume of saline. MRI was performed at 24 hr and then weekly for 6 weeks. MRI and histological measurements were compared between groups. Serial T2WI demonstrated that expansion of the ipsilateral ventricle was significantly reduced in the EPO-treated rats. The volume ratio of ipsilateral parenchymal tissue relative to the contralateral hemisphere was significantly increased after EPO treatment compared with control animals, indicating that EPO significantly reduces atrophy of the ipsilateral hemisphere, although no significant differences in ischemic lesion volume were observed between the two groups. Angiogenesis and white matter remodeling were significantly increased and occurred earlier in EPO-treated animals than in the controls, as evident from T2*WI and diffusion anisotropy maps, respectively. These data indicate that EPO treatment initiated 24 hr poststroke promotes angiogenesis and axonal remodeling in the ischemic boundary, which may potentially reduce atrophy of the ipsilateral hemisphere.

Topics: Animals; Atrophy; Disease Models, Animal; Erythropoietin; Intracranial Embolism; Magnetic Resonance Imaging; Male; Nerve Degeneration; Neuroprotective Agents; Random Allocation; Rats; Rats, Wistar; Recombinant Proteins; Stroke

Executive function impairment, as classically assessed using the Wisconsin Card Sort Test or intradimensional/extradimensional tests, is a key feature of schizophrenia but remains inadequately treated by existing therapies. Recently, however, erythropoietin has been shown to improve attentional set-shifting performance in schizophrenic patients.. The present study utilized the rat intradimensional/extradimensional task to investigate the potential of erythropoietin to reverse a phencyclidine-induced extradimensional shift impairment when given alone or in combination with subchronic haloperidol treatment.. Rats were subjected to a subchronic systemic administration (7 days, b.i.d) of either saline vehicle or phencyclidine (5 mg/kg) followed by a 7-day washout period during which haloperidol was given. Subsequently, rats were trained to dig in baited bowls for a food reward and to discriminate on the basis of digging media or bowl odor. In experiment 1, rats performed a series of discriminations following acute administration of vehicle, erythropoietin, or modafinil. In a second experiment, rats receiving either haloperidol in the drinking water or just normal drinking water were run in the attentional set-shifting task after acute administration of erythropoietin (1,000 or 10,000 IU/ml  i.p., selected from experiment 1).. The subchronic phencyclidine-induced extradimensional deficit was ameliorated by both erythropoietin and modafinil. When combined with subchronic haloperidol, the higher dose of erythropoietin tested was able to reverse the extradimensional shift impairment.. Overall, these findings further support the use of erythropoietin as an adjunct to antipsychotic therapy in order to address, at least part of, the cognitive dysfunction associated with schizophrenia.

Topics: Animals; Antipsychotic Agents; Attention; Behavior, Animal; Benzhydryl Compounds; Discrimination, Psychological; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Therapy, Combination; Epoetin Alfa; Erythropoietin; Executive Function; Haloperidol; Male; Modafinil; Neuropsychological Tests; Phencyclidine; Rats; Recombinant Proteins; Reward; Schizophrenia; Schizophrenic Psychology

Cerebral vasospasm is the common cause of poor outcome after aneurysmal subarachnoid hemorrhage (aSAH). Although many agents are experimentally and clinicaly used to protect or recover from vasospasm, an effective neurotherapeutic drug is still missing. Erythropoietin (EPO) is recently a promising candidate. The aim of this study is to investigate the dose-dependent effects of recombinant human EPO (rhEPO) on arterial wall in a rat femoral artery vasospasm model.. Thirty two animals were divided into four groups: vasospasm without any treatment (group A), vasospasm +250 IU/kg rhEPO group (group B), vasospasm +500 IU/kg rhEPO group (group C), and control group (group D). Rat femoral artery vasospasm model was used. For groups B and C, 7 days of 250 IU/kg and 500 IU/kg intraperitoneal rhEPO in 0.3 ml saline were administered respectively; and for groups A and D, 0.3 ml saline were administered intraperitoneally without any treatment. After 7 days, histological and morphometric analyses were carried out.. Vasospasm alone group demonstrated the highest vessel wall thicknesses, comparing to other groups (p < 0.001). While for groups B and C, vessel wall thickness values were significantly higher than the control group (p < 0.001), between these two groups, there was no significant difference achieved (p > 0.05).. In our study, there was no significant difference between the two rhEPO treatment groups, but rhEPO treatment was shown to be histologically and morphometrically effective in vasospasm. However, if dosage of EPO treatment is augmented, successful results may be achieved.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Erythropoietin; Femoral Artery; Injections, Intraperitoneal; Male; Microscopy, Electron, Transmission; Rats; Rats, Sprague-Dawley; Tunica Intima; Tunica Media; Vasoconstriction; Vasospasm, Intracranial

Erythropoietin (EPO) and granulocyte colony stimulating factor (GCSF) have generated interest as novel therapies after myocardial infarction (MI), but the effect of combination therapy has not been studied in the large animal model. We investigated the impact of prolonged combination therapy with EPO and GCSF on cardiac function, infarct size, and vascular density after MI in a porcine model.. MI was induced in pigs by a 90 min balloon occlusion of the left anterior descending coronary artery. 16 animals were treated with EPO+GCSF, or saline (control group). Cardiac function was assessed by echocardiography and pressure-volume measurements at baseline, 1 and 6 weeks post-MI. Histopathology was performed 6 weeks post-MI.. At week 6, EPO+GCSF therapy stabilized left ventricular ejection fraction, (41 ± 1% vs. 33 ± 1%, p < 0.01) and improved diastolic function compared to the control group. Histopathology revealed increased areas of viable myocardium and vascular density in the EPO+GCSF therapy, compared to the control. Despite these encouraging results, in a historical analysis comparing combination therapy with monotherapy with EPO or GCSF, there were no significant additive benefits in the LVEF and volumes overtime using the combination therapy.. Our findings indicate that EPO+GCSF combination therapy promotes stabilization of cardiac function after acute MI. However, combination therapy does not seem to be superior to monotherapy with either EPO or GCSF.

Topics: Animals; Bone Marrow Cells; Disease Models, Animal; Drug Therapy, Combination; Echocardiography; Erythropoietin; Granulocyte Colony-Stimulating Factor; Heart; Heart Ventricles; Myocardial Infarction; Myocardium; Stroke Volume; Swine

Prolonged human immunodeficiency virus-1 (HIV-1) infection leads to neurological debilitation, including motor dysfunction and frank dementia. Although pharmacological control of HIV infection is now possible, HIV-associated neurocognitive disorders (HAND) remain intractable. Here, we report that chronic treatment with erythropoietin (EPO) and insulin-like growth factor-I (IGF-I) protects against HIV/gp120-mediated neuronal damage in culture and in vivo.. Initially, we tested the neuroprotective effects of various concentrations of EPO, IGF-I, or EPO+IGF-I from gp120-induced damage in vitro. To assess the chronic effects of EPO+IGF-I administration in vivo, we treated HIV/gp120-transgenic or wild-type mice transnasally once a week for 4 months and subsequently conducted immunohistochemical analyses.. Low concentrations of EPO+IGF-I provided neuroprotection from gp120 in vitro in a synergistic fashion. In vivo, EPO+IGF-I treatment prevented gp120-mediated neuronal loss, but did not alter microgliosis or astrocytosis. Strikingly, in the brains of both humans with HAND and gp120-transgenic mice, we found evidence for hyperphosphorylated tau protein (paired helical filament-I tau), which has been associated with neuronal damage and loss. In the mouse brain following transnasal treatment with EPO+IGF-I, in addition to neuroprotection we observed increased phosphorylation/activation of Akt (protein kinase B) and increased phosphorylation/inhibition of glycogen synthase kinase (GSK)-3beta, dramatically decreasing downstream hyperphosphorylation of tau. These results indicate that the peptides affected their cognate signaling pathways within the brain parenchyma.. Our findings suggest that chronic combination therapy with EPO+IGF-I provides neuroprotection in a mouse model of HAND, in part, through cooperative activation of phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling. This combination peptide therapy should therefore be tested in humans with HAND.

Topics: Administration, Intranasal; Adult; Animals; Apoptosis; Cells, Cultured; Cerebral Cortex; Chromones; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Embryo, Mammalian; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HIV Envelope Protein gp120; HIV Infections; Humans; Immunoprecipitation; Insulin-Like Growth Factor I; Male; Mice; Mice, Transgenic; Middle Aged; Morpholines; Nerve Tissue Proteins; Neuroglia; Neurons; Neuroprotective Agents; Olfactory Bulb; Phosphorylation; Rats; tau Proteins

Recent studies have demonstrated that erythropoietin (EPO) and its analogs induce cytoprotective effects on many nonerythroid cells. In this study, we examined whether darbepoetin-α might prevent glomerular lesions in the Thy-1.1 model of glomerulonephritis (Thy-1-GN). GN was induced in Wistar rats by a single injection of monoclonal anti-Thy-1.1 antibody. Rats were killed at 24 h, 72 h, 7 days, 10 days, or 15 days after antibody injection. Kidneys were removed for histological analysis, and proteinuria was measured. Because at day 7 the maximal degree of renal damage and proteinuria was found, the effect of darbepoetin-α was tested at day 7 and two different protocols of administration were used; After anti-Thy-1.1 injection, rats received two doses of darbepoetin-α or vehicle at days 0 and 4 or at days 4 and 6. At day 7, proteinuria, plasma creatinine concentration, and renal morphology analysis were performed. Also, α-actin, desmin, caspase-3, and Ki67 protein expression were evaluated by immunohistochemistry. Our results showed that in both protocols of administration, darbepoetin-α treatment decreased proteinuria in Thy-1-GN rats and this effect correlated with the improvement in renal morphology. Glomerular lesions, α-actin, and caspase-3 protein expression, observed in most glomeruli of Thy-1-GN rats, were significantly reduced in darbepoetin-α-treated rats, while cell proliferation was significantly enhanced. The results indicate that darbepoetin-α treatment promotes glomerular recovery.

Topics: Animals; Caspase 3; Darbepoetin alfa; Disease Models, Animal; Erythropoietin; Glomerulonephritis; Isoantibodies; Kidney Glomerulus; Male; Proteinuria; Rats; Rats, Wistar; Regeneration

Mouse bone marrow erythropoiesis is homeostatic, whereas after acute anemia, bone morphogenetic protein 4 (BMP4)-dependent stress erythropoiesis develops in the spleen. The aim of this work was to compare spleen stress erythropoiesis and bone marrow erythropoiesis in a mouse model of zymosan-induced generalized inflammation, which induces long-lasting anemia and to evaluate the ability of erythropoietin (Epo) injections to correct anemia in this setting. The effects of zymosan and/or Epo injections on erythroid precursor maturation and apoptosis, serum interferon-γ levels, hematologic parameters, and spleen BMP4 expression were analyzed, as well as the effect of zymosan on red blood cell half-life. We found that bone marrow erythropoiesis is suppressed by inflammation and does not respond to Epo administration, despite repression of erythroblast apoptosis. On the contrary, a robust erythropoietic response takes place in the spleen after Epo injections in both control and zymosan-induced generalized inflammation mice. This specific response implies Epo-mediated induction of BMP4 expression by F4/80(+) spleen macrophages, proliferation of stress burst-forming units-erythroid, and increased number of spleen erythroblasts. It allows only partial recovery of anemia, probably because of peripheral destruction of mature red cells. It is not clear whether similar BMP4-dependent stress erythropoiesis can occur in human bone marrow after Epo injections.

Topics: Anemia; Animals; Apoptosis; Blotting, Western; Bone Marrow; Bone Morphogenetic Protein 4; Disease Models, Animal; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Humans; Inflammation; Male; Mice; Mice, Inbred C57BL; Receptors, Erythropoietin; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Spleen; Zymosan

Expanded endothelial progenitor cells (eEPC) improve global left ventricular function in experimental myocardial infarction (MI). Erythropoietin beta (EPO) applied together with eEPC may improve regional myocardial function even further by anti-apoptotic and cardioprotective effects. Aim of this study was to evaluate intramyocardial application of eEPCs and EPO as compared to eEPCs or EPO alone in experimental MI.. In vitro experiments revealed that EPO dosed-dependently decreased eEPC and leukocyte apoptosis. Moreover, in the presence of EPO mRNA expression in eEPC of proangiogenic and proinflammatory mediators measured by TaqMan PCR was enhanced. Experimental MI was induced by ligation and reperfusion of the left anterior descending coronary artery of nude rats (n = 8-9). After myocardial transplantation of eEPC and EPO CD68+ leukocyte count and vessel density were enhanced in the border zone of the infarct area. Moreover, apoptosis of transplanted CD31 + TUNEL + eEPC was decreased as compared to transplantation of eEPCs alone. Regional wall motion of the left ventricle was measured using Magnetic Resonance Imaging. After injection of eEPC in the presence of EPO regional wall motion significantly improved as compared to injection of eEPCs or EPO alone.. Intramyocardial transplantation of eEPC in the presence of EPO during experimental MI improves regional wall motion. This was associated with an increased local inflammation, vasculogenesis and survival of the transplanted cells. Local application of EPO in addition to cell therapy may prove beneficial in myocardial remodeling.

Topics: Animals; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Endothelium; Erythropoietin; Humans; Inflammation; Male; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Rats; Rats, Nude; Recovery of Function; Stem Cell Transplantation; Stem Cells

Chronic kidney disease (CKD) patients develop anemia because of the low kidney erythropoietin (EPO) production, thus promoting cardiovascular complications. The degree of renal insufficiency might determine the moment to start recombinant human erythropoietin (rhEPO) therapy, but the molecular basis for these options deserves better elucidation. This study aimed to clarify the cardio-renal effects of earlier rhEPO therapy in rats with moderate chronic renal failure (CRF). Four groups of rats were evaluated for 15 weeks (control; rhEPO - 50 IU/kg/week; CRF - 3/4 nephrectomy; CRF + rhEPO) to assess renal and hematology data, EPO levels, blood pressure, heart rate, peripheral catecholamines contents, serum-transforming growth factor-β1 (TGF-β1), kidney gene expression of EPO, Caspase 9 (Casp9), and vascular endothelial growth factor (Vegf). This model of moderate CRF showed moderate and corrected anemia, hypertension, tachycardia, sympathetic overactivity, and increased serum TGF-β1 content. The remnant kidney showed a proliferative profile, with hypertrophy, downregulated gene expression of EPO, and upregulated gene expression of Vegf and Casp9. rhEPO treatment promoted erythrocytosis and prevented tachycardia and catecholamines increment, with a rise of serum TGF-β1. Furthermore, the decreased kidney gene expression of EPO and the overexpression of Casp9 were prevented, demonstrating a renoprotective action on the remnant kidney. In conclusion, rhEPO therapy promotes a protective effect on the cardio-renal axis, which might be mainly attributed to its pro-proliferative and anti-apoptotic properties. These findings might recommend its use in earlier stages of CRF, acting as an erythropoiesis stimulating agent, to efficiently correct not only the anemia, one of the major complications in these patients, but also the succeeding adverse cardio-renal effects.

Topics: Animals; Blood Pressure; Caspase 9; Disease Models, Animal; Erythropoietin; Heart Diseases; Heart Rate; Humans; Kidney; Kidney Function Tests; Male; Peripheral Nervous System; Rats; Rats, Wistar; Recombinant Proteins; Renal Insufficiency, Chronic; Sympathetic Nervous System; Vascular Endothelial Growth Factor A

We have previously demonstrated that endothelium-derived hydrogen peroxide (H(2)O(2)) plays an important role in the canine coronary microcirculation as an endothelium-derived hyperpolarizing factor in vivo. However, it remains to be examined whether endogenous H(2)O(2) is involved in the dilatation of coronary collaterals during myocardial ischemia in vivo and, if so, whether erythropoietin (EPO) enhances the responses. Canine subepicardial native collateral small arteries (CSA; ≥ 100 μm) and arterioles (CA; <100 μm) were observed using an intravital microscope. Experiments were performed after left anterior descending coronary artery ischemia (90 min) under the following eight conditions (n = 5 each): control, EPO, EPO+catalase, EPO+N-monomethyl-l-arginine (l-NMMA), EPO+l-NMMA+catalase, EPO+l-NMMA+iberiotoxin [Ca(2+)-activated K(+) (K(Ca)) channel blocker], EPO+l-NMMA+apamin+charybdotoxin (K(Ca) channel blocker), and EPO+wortmannin (phosphatidylinositol 3-kinase inhibitor). Myocardial ischemia caused significant vasodilatation in CA but not in CSA under control conditions, which was significantly decreased by catalase in CA. After EPO, the vasodilatation was significantly increased in both sizes of arteries and was significantly decreased by catalase. The enhancing effect of EPO was reduced by l-NMMA but not by catalase in CSA and was reduced by l-NMMA+catalase in CA, where the greater inhibitory effects were noted with l-NMMA+catalase, l-NMMA+iberiotoxin, L-NMMA+apamin+charybdotoxin, or wortmannin. EPO significantly ameliorated ischemia-induced impairment of myocardial Akt phosphorylation, which was abolished by l-NMMA+catalase or wortmannin. EPO also ameliorated oxidative stress and myocardial injury, as assessed by plasma 8-hydroxydeoxyguanosine and troponin-T, respectively. These results indicate that EPO enhances H(2)O(2)-mediated dilatation of coronary collateral arterioles during myocardial ischemia in dogs in vivo.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Arterioles; Carbon Dioxide; Collateral Circulation; Coronary Circulation; Coronary Vessels; Deoxyguanosine; Disease Models, Animal; Dogs; Enzyme Inhibitors; Erythropoietin; Female; Hydrogen Peroxide; Male; Myocardial Ischemia; Nitric Oxide Synthase Type III; Oxygen; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Potassium Channel Blockers; Protein Kinase Inhibitors; Troponin T; Vasodilation; Vasodilator Agents

Topics: Animals; Disease Models, Animal; Drug Therapy, Combination; Erythropoietin; Granulocyte Colony-Stimulating Factor; Myocardial Infarction; Swine

Neuroprotective, but not hematopoietic, variants of erythropoietin (EPO), such as Neuro-EPO, are promising candidates for treatment in the acute and subacute stroke phase. Characterized by its low sialic acid content and therefore exhibiting a very short plasma half-life, Neuro-EPO can probably not be administered systemically via the blood. As such, alternate routes of delivery are required. In their paper that now appears in TheScientificWorldJOURNAL, Rodríguez Cruz and colleagues provide evidence that Neuro-EPO promotes neurological recovery in the ischemic gerbil brain in a way that is similarly potent, if not superior, to systemically administered EPO. In view of the potential clinical use of Neuro-EPO, stringent proof-of-concept studies are urgently needed to define (1) how intranasally delivered Neuro-EPO reaches the brain, (2) which concentrations are achieved in the ischemic and nonischemic brain tissue of rodents and nonhuman primates, and (3) which are the mechanisms via which Neuro-EPO protects from injury. Only with such information should decisions be made whether intranasal Neuro-EPO may be evaluated in human patients.

Topics: Administration, Intranasal; Animals; Brain Ischemia; Cognition; Disease Models, Animal; Erythropoietin; Gerbillinae; Humans; Neuroprotective Agents; Recombinant Proteins; Stroke; Treatment Outcome

Vascular illness of the brain constitutes the third cause of death and the first cause of disability in Cuba and many other countries. Presently, no medication has been registered as a neuroprotector. Neuroprotection with intranasal Neuro-EPO (EPO, erythropoietin) has emerged as a multifunctional therapy that plays a significant role in neural survival and functional recovery in an animal model of stroke. On the other hand, there is limited access to the brain through the blood brain barrier (BBB) for intravenously applied EPO, and the high EPO dosages needed to obtain a protective effect increase the danger of elevated hematocrit levels and practically exclude chronic or subchronic treatment with EPO. A promising approach has been recently developed with a nonerythropoietic variant of EPO, Neuro-EPO, with low sialic acid content, a very short plasma half-life, and without erythropoietic activity, probably similar to endogenous brain EPO. The objective of this work was to determine the neuroprotective effect of intranasal Neuro-EPO in comparison with the human recombinant EPO injected intraperitoneally in the acute phase of cerebral ischemia, employing the common carotid artery occlusion model in gerbils. Neuro-EPO has demonstrated a better neuroprotective effect, evidenced through increased viability, improvements of the neurological state and cognitive functions, as well as protection of the CA3 region of the hippocampus, temporal cortex, and the thalamus. In conclusion, the intranasal application of Neuro-EPO has a better neuroprotective effect than intraperitoneal EPO, evidenced by the significant improvement of neurological, cognitive, and histological status in the animal model of stroke employed.

Topics: Administration, Intranasal; Animals; Brain; Brain Ischemia; Cognition; Disease Models, Animal; Erythropoietin; Gerbillinae; Humans; Male; Neuroprotective Agents; Random Allocation; Recombinant Proteins; Treatment Outcome

We examined the effect of asialoerythropoietin (asialoEPO), a nonerythrogenic derivative of erythropoietin (EPO), on renal dysfunction-associated heart failure.. Although EPO is known to exert beneficial effects on cardiac function, the clinical benefits in patients with chronic kidney disease are controversial. It remains to be addressed whether previously reported outcomes were the result of relief of the anemia, adverse effects of EPO, or direct cardiovascular effects.. Mice underwent 5/6 nephrectomy to cause renal dysfunction. Eight weeks later, when renal dysfunction was established, anemia and cardiac dysfunction and remodeling were apparent. Mice were then assigned to receive saline (control), recombinant human erythropoietin (rhEPO) at 5,000 IU (714 pmol)/kg, or asialoEPO at 714 pmol/kg, twice/week for 4 weeks.. Although only rhEPO relieved the nephrectomy-induced anemia, both rhEPO and asialoEPO significantly and similarly mitigated left ventricular dilation and dysfunction. The hearts of rhEPO- or asialoEPO-treated mice showed less hypertrophy, reflecting decreases in cardiomyocyte hypertrophy and degenerative subcellular changes, as well as significant attenuation of fibrosis, leukocyte infiltration, and oxidative deoxyribonucleic acid damage. These phenotypes were accompanied by restored expression of GATA-4, sarcomeric proteins, and vascular endothelial growth factor and decreased inflammatory cytokines and lipid peroxidation. Finally, myocardial activation was observed of extracellular signal-regulated protein kinase and signal transducer and activator of transcription pathways in the treated mice.. EPO receptor signaling exerts direct cardioprotection in an animal model of renal dysfunction-associated heart failure, probably by mitigating degenerative, pro-fibrosis, inflammatory, and oxidative processes but not through relief of anemia.

Topics: Anemia; Animals; Asialoglycoproteins; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Heart Failure; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Receptors, Erythropoietin; Recombinant Proteins; Renal Insufficiency; Signal Transduction

Whereas administration of erythropoietin (EPO) acutely after myocardial infarction (MI) reduces infarct size and chronic EPO therapy attenuates post-MI remodeling, the safety of chronic EPO therapy following MI is unknown. Therefore, we examined the thrombogenic effects of a chronic EPO therapy after MI.. Rats underwent coronary occlusion followed by reperfusion. They were assigned to one of the following groups: EPO-A, single injection of EPO 5,000 U/kg at the time of reperfusion; EPO-C, injection of EPO 5,000 U/kg at the time of reperfusion followed by 300 U/kg/week; PBS-C, injection of vehicle only. After eight weeks of treatment they were exposed to a validated prethrombotic test based on partial stenosis of the inferior vena cava.. As compared to the rats receiving vehicle only, the rats treated with EPO exhibited a significant reduction in MI size (28.7 +/- 2.1% and 25.8 +/- 1.9 vs. 39.8 +/- 3.0% in EPO-A, EPO-C and PBS-C, respectively; p < 0.05). Whereas the hematocrit was significantly increased in EPO-C (59.7 +/- 2.0% vs. 44.7 +/- 0.9% in EPO-A, p < 0.001), the proportion of rats in which a thrombus occurred was similar in all groups (p = 0.52).. Chronic EPO therapy added to the single high dose of EPO injected acutely did not induce venous pro-thrombotic effect in rats.

Topics: Animals; Disease Models, Animal; Erythropoietin; Hematocrit; Male; Myocardial Infarction; Myocardium; Rats; Rats, Sprague-Dawley; Venous Thrombosis; Ventricular Remodeling

In humans elective spine surgery can cause iatrogenic spinal cord injury (SCI). Efforts for neuroprotection have been directed to avoid mechanical injury by using intraoperative monitoring and improving surgical techniques. There is, however, uncertainty regarding the efficacy of neuroprotective drugs.. Experimental study on the effectiveness of pharmacological neuroprotection in an animal model of spine surgery simulating anticipated mechanically induced neurological damage.. To compare the efficacy of four drugs to protect against the neurological effects of iatrogenic SCI.. Research Unit for Neurological Diseases, IMSS-Proyecto Camina, Mexico City, Mexico.. Erythropoietin, melatonin, cyclosporine-A and methylprednisolone were administered to rats before, during and after controlled spinal cord contusion of mild intensity. Dosage was in accordance with their pharmacokinetic properties and experience gained with experimental SCI. Drug efficacy was assessed by motor function recovery over a period of 6 weeks and by spinal cord morphometry.. Compared with animals treated with saline, the drug-treated groups showed no differences in their locomotor performance, nor in the amount of spared cord tissue. Notably, spontaneous activity was significantly reduced in rats treated with cyclosporine-A.. The neuroprotectant drugs used here perioperatively did not reduce the extent of neurological damage in a model simulating iatrogenic SCI. Therefore, for now, the only protection in elective spine surgery is avoidance of primary injury altogether.

Topics: Analysis of Variance; Animals; Cyclosporine; Disease Models, Animal; Erythropoietin; Female; Locomotion; Melatonin; Methylprednisolone; Neuroprotective Agents; Postoperative Complications; Rats; Rats, Long-Evans; Recombinant Proteins; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Time Factors

Erythropoietin (EPO), an oxygen-regulated hormone stimulating erythrocyte production, was recently found to be critical for retinal angiogenesis. EPO mRNA expression levels in retina are highly elevated during the hypoxia-induced proliferation phase of retinopathy. The authors investigated the inhibition of retinal EPO mRNA expression with RNA interference as a potential strategy to suppress retinal neovascularization and to prevent proliferative retinopathy.. The authors used a mouse model of oxygen-induced retinopathy. Retinal EPO and Epo receptor (EpoR) expression during retinopathy development were quantified with real-time RT-PCR in whole retina and on laser-captured retinal vessels and neuronal layers. Retinal hypoxia was assessed with an oxygen-sensitive hypoxyprobe. A small interference RNA (siRNA) targeting EPO or control negative siRNA was injected intravitreally at postnatal (P) day 12, P14, and P15 during the hypoxic phase, and the effect on neovascularization was evaluated in retinal flatmounts at P17.. Retinal EPO mRNA expression in total retina was suppressed during the initial phase of vessel loss in retinopathy and was significantly elevated during the hypoxia-induced proliferative phase in all three neuronal layers in the retina, corresponding to an increased level of retinal hypoxia. EpoR mRNA expression levels also increased during the second neovascular phase, specifically in hypoxia-induced neovascular vessels. Intravitreous injection of EPO siRNA effectively inhibited approximately 60% of retinal EPO mRNA expression and significantly suppressed retinal neovascularization by approximately 40%.. Inhibiting EPO mRNA expression with siRNA is effective in suppressing retinal neovascularization, suggesting EPO siRNA is a potentially useful pharmaceutical intervention for treating proliferative retinopathy.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Erythropoietin; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Humans; Infant, Newborn; Injections; Mice; Microscopy, Confocal; Oxygen; Receptors, Erythropoietin; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; RNA, Small Interfering; Vitreous Body

A small experimental cryolesion to the right parietal cortex of juvenile mice causes late-onset global brain atrophy with memory impairments, reminiscent of cognitive decline, and progressive brain matter loss in schizophrenia. However, the cellular events underlying this global neurodegeneration are not understood. Here we show, based on comprehensive stereological analysis, that early unilateral lesion causes immediate and lasting bilateral increase in the number of microglia in cingulate cortex and hippocampus, consistent with a chronic low-grade inflammatory process. Whereas the total number of neurons and astrocytes in these brain regions remain unaltered, pointing to a non- gliotic neurodegeneration (as seen in schizophrenia), the subgroup of parvalbumin-positive inhibitory GABAergic interneurons is increased bilaterally in the hippocampus, as is the expression of the GABA-synthesizing enzyme GAD67. Moreover, unilateral parietal lesion causes a decrease in the expression of synapsin1, suggesting impairment of presynaptic functions/neuroplasticity. Reduced expression of the myelin protein cyclic nucleotide phosphodiesterase, reflecting a reduction of oligodendrocytes, may further contribute to the observed brain atrophy. Remarkably, early intervention with recombinant human erythropoietin (EPO), a hematopoietic growth factor with multifaceted neuroprotective properties (intraperitoneal injection of 5000 IU/kg body weight every other day for 3 weeks), prevented all these neurodegenerative changes. To conclude, unilateral parietal lesion of juvenile mice induces a non- gliotic neurodegenerative process, susceptible to early EPO treatment. Although the detailed mechanisms remain to be defined, these profound EPO effects open new ways for prophylaxis and therapy of neuropsychiatric diseases, e.g. schizophrenia.

Topics: Animals; Astrocytes; Atrophy; Brain; Brain Injuries; Cold Temperature; Cyclic Nucleotide Phosphodiesterases, Type 3; Disease Models, Animal; Erythropoietin; gamma-Aminobutyric Acid; Gliosis; Glutamate Decarboxylase; Humans; Male; Mice; Mice, Inbred C57BL; Microglia; Neurodegenerative Diseases; Neurons; Parvalbumins; Synapsins

Peritoneal dialysis (PD) is an alternative treatment of patients with end-stage renal disease. Unfortunately, long term peritoneal dialysis causes injury of the peritoneum associated with ultra filtration failure. Erythropoietin (EPO) is a potent stimulator of elytroid progenitor cells and its expression is enhanced by hypoxia. The aim of the present study was to evaluate the effect of EPO on the development of experimental peritoneal fibrosis induced by chlorhexidine gluconate (CG). A peritoneal fibrosis model was established using rats treated intraperitoneally with injection of CG. EPO was administered at the dose of 5000 U/kg i.p. three time per week for three weeks. When compared to CG-treated rats, EPO (5000 U/kg i.p. three time per week for three weeks) treated rats subjected to GC-induced peritoneal fibrosis experienced a significantly lower rate in the extent and severity of the histological signs of peritoneal injury. EPO also caused a substantial reduction of (i) the rise in myeloperoxidase activity (mucosa), (ii) the expression in the tissue of TNF-alpha, TGFbeta and VEGF (iii) the increase in staining (immunohistochemistry) for nitrotyrosine and for PAR, as well as (iv) the NF-kappaB activation caused by CG in the peritoneum. Thus, EPO treatment reduces the degree of peritoneal fibrosis caused by CG. We propose that this evidence may help to clarify the potential therapeutic actions of EPO in patients with peritoneal fibrosis.

Topics: Animals; Blotting, Western; Chlorhexidine; Disease Models, Animal; Erythropoietin; Fibrosis; Peritoneal Dialysis; Peritoneal Diseases; Peritoneum; Rats; Rats, Inbred Strains; Thiobarbituric Acid Reactive Substances

Mitochondria play a central role in cellular energetics, calcium homeostasis and apoptosis. Our previous study demonstrates traumatic brain injury induces brain mitochondrial dysfunction after injury. Preservation and/or restoration of mitochondrial function may be one of the strategies for neuroprotection. Erythropoietin, a hormone for erythropoiesis, also provides tissue protection against traumatic brain injury and stroke. The present study was undertaken to evaluate the effect of erythropoietin on traumatic brain injury-induced brain mitochondrial dysfunction. Traumatic brain injury decreased rates of respiration at the active state (state 3), increased that at the resting state (state 4) and consequently decreased respiratory control index (state 3/state 4 ratio) and the efficiency of ATP synthesis (the amount of ADP phosphorylated by inorganic phosphate divided by the amount of oxygen consumed during state 3 respiration). Erythropoietin administered intraperitoneally 30 minutes post-injury at 1000 U/kg partially improved mitochondrial function at day 1 post-injury. However, erythropoietin-induced improvement was not sustained at day 7 post-injury. Erythropoietin at 2000 or 5000 U/kg restored states 3 and 4 examined at day 1 post-injury to the sham levels. Consequently, the energy coupling capacities, such as respiratory control index and/or the efficiency of ATP synthesis, were also improved. The beneficial effect of erythropoietin at these doses persisted for at least 7 days post-injury. The beneficial effect of erythropoietin on brain mitochondrial function was observed with a wide therapeutic window from 5 minutes to 6 hours post-injury. Our data, for the first time, demonstrate that erythropoietin treatment restores brain mitochondrial function after traumatic brain injury, which will enhance cellular energy generation and reduce oxidative stress, strongly supporting erythropoietin as a promising agent for the therapeutic treatment of traumatic brain injury.

Topics: Animals; Brain Injuries; Disease Models, Animal; Erythropoietin; Male; Mitochondria; Neuroprotective Agents; Prosencephalon; Rats; Rats, Sprague-Dawley

Parkinson's disease (PD) is characterized by degeneration of nigrostriatal dopaminergic neuronal systems. Several therapeutic tools for PD include medication using L-DOPA and surgeries such as deep brain stimulation are established. However, the therapies are considered as symptomatic therapy, but not basic remedy for PD and a new regenerative therapy would be desired to explore. In this study, the neuroprotective/rescue effects of erythropoietin (EPO), a well known hematopoietic hormone, on dopaminergic neurons were explored with neurogeneic potencies of EPO. EPO (100 IU/day) was continuously administered with micro-osmotic pump for a week to PD model of rats induced by intrastriatal 6-hydroxydopamine (6-OHDA) injection with subsequent behavioral and immunohistochemical investigations. The number of amphetamine-induced rotations of EPO-treated rats significantly decreased, compared to the control rats. The preservation of dopaminergic neurons of EPO-treated rats were confirmed by tyrosine hydroxylase staining and Fluoro-Gold staining. The number of bromodeoxyuridine (BrdU)/polysialic acid-neural cell adhesion molecule (PSA-NCAM) double positive cells in the subventricular zone of EPO treated rats significantly increased with migratory potencies to the damaged striatum,compared to the control rats. Furthermore, TUNEL staining and phosphorylated Akt staining revealed that the neuroprotective/rescue effects of EPO might be mediated by anti-apoptotic effects through the increase of phosphorylated Akt. These results suggest that continuous low dose infusion of EPO exerts neuroprotective/rescue effects with neurogeneic potentials. EPO might be a strong tool for PD therapy, although the further experiments should be added.

Topics: Animals; Behavior, Animal; Brain; Bromodeoxyuridine; Disease Models, Animal; Dopamine; Erythropoietin; Female; Neural Cell Adhesion Molecules; Neurogenesis; Neurons; Neuroprotective Agents; Oxidopamine; Parkinsonian Disorders; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Sialic Acids; Stilbamidines; Tyrosine 3-Monooxygenase

Deferoxamine (DFO) and erythropoietin (EPO) have each been shown to provide neuroprotection in neonatal rodent models of brain injury. In view of the described anti-oxidative actions of DFO and the anti-apoptotic and anti-inflammatory effects of EPO, we hypothesized that the combination of DFO and EPO would increase neuroprotection after neonatal hypoxic-ischemic brain injury as compared to single DFO or EPO treatment. At postnatal day 7 rats underwent right common carotid artery occlusion followed by a 90-min exposure to 8% oxygen. Rats were treated intraperitoneally with DFO (200mg/kg), recombinant human EPO (1 kU/kg), a combination of DFO-EPO or vehicle at 0, 24 and 48 h after hypoxia-ischemia (HI) and were sacrificed at 72 h. DFO-EPO administration reduced the number of cleaved caspase 3-positive cells in the ipsilateral cerebral cortex. Early neuronal damage was assessed by staining for microtubuli-associated protein (MAP)-2. In our model 63+/-9% loss of ipsilateral MAP-2 was observed after HI, indicating extensive brain injury. DFO, EPO or DFO-EPO treatment did not improve neuronal integrity as defined by MAP-2. Cerebral white matter tracts were stained for myelin basic protein (MBP), a constituent of myelin. Hypoxia-ischemia strongly reduced MBP staining which suggests white matter damage. However, DFO, EPO and DFO-EPO treatment had no effect on the loss of MBP staining. Finally, HI-induced loss of striatal tyrosine hydroxylase staining was not attenuated by DFO, EPO or DFO-EPO. Although DFO-EPO treatment reduced the number of cleaved caspase 3(+) cells, treatment with DFO, EPO, or with the combination of DFO and EPO did not protect against gray or white matter damage in the experimental setting applied.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents; Antioxidants; Brain Infarction; Caspase 3; Cerebral Cortex; Cytoprotection; Deferoxamine; Disease Models, Animal; Drug Combinations; Drug Synergism; Erythropoietin; Hypoxia-Ischemia, Brain; Microtubule-Associated Proteins; Myelin Basic Protein; Nerve Degeneration; Nerve Fibers, Myelinated; Neurons; Rats; Rats, Wistar

Anemia is common in patients with chronic heart failure and an independent predictor of poor prognosis. Chronic anemia leads to left ventricular (LV) hypertrophy and heart failure, but its molecular mechanisms remain largely unknown. We investigated the mechanisms, including the molecular signaling pathway, of cardiac remodeling induced by iron deficiency anemia (IDA). Weanling Sprague-Dawley rats were fed an iron-deficient diet for 20 wk to induce IDA, and the molecular mechanisms of cardiac remodeling were evaluated. The iron-deficient diet initially induced severe anemia, which resulted in LV hypertrophy and dilation with preserved systolic function associated with increased serum erythropoietin (Epo) concentration. Cardiac STAT3 phosphorylation and VEGF gene expression increased by 12 wk of IDA, causing angiogenesis in the heart. Thereafter, sustained IDA induced upregulation of cardiac hypoxia inducible factor-1alpha gene expression and maintained upregulation of cardiac VEGF gene expression and cardiac angiogenesis; however, sustained IDA promoted cardiac fibrosis and lung congestion, with decreased serum Epo concentration and cardiac STAT3 phosphorylation after 20 wk of IDA compared with 12 wk. Upregulation of serum Epo concentration and cardiac STAT3 phosphorylation is associated with a beneficial adaptive mechanism of anemia-induced cardiac hypertrophy, and later decreased levels of these molecules may be critical for the transition from adaptive cardiac hypertrophy to cardiac dysfunction in long-term anemia. Understanding the mechanism of cardiac maladaptation to anemia may lead to a new strategy for treatment of chronic heart failure with anemia.

Topics: Adaptation, Physiological; Anemia, Iron-Deficiency; Animals; Blood Pressure; Body Weight; Disease Models, Animal; Erythropoietin; Heart; Heart Failure; Heart Rate; Hypertrophy, Left Ventricular; Hypoxia-Inducible Factor 1, alpha Subunit; Iron; Kidney; Male; Myocardial Contraction; Myocardium; Neovascularization, Physiologic; Phosphorylation; Pulmonary Edema; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; STAT3 Transcription Factor; Time Factors; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Ventricular Function, Left; Ventricular Pressure; Ventricular Remodeling

Acetazolamide has been recognized as an effective treatment for acute mountain sickness. The efficacy of acetazolamide is related to metabolic acidosis, which promotes chemoreceptors to respond to hypoxic stimuli at altitude. In this study, adult male Sprague-Dawley rats were treated with acetazolamide (100mg/kg or 50mg/kg, I.P.) for 3 days. Primary cultured cortical neurons and PC12 cell lines were exposed to acidosis-permissive (pH 6.5) or standard (pH 7.2) media for 20h. HIF-1alpha and its target genes were assayed by Western blot, real-time PCR, HIF-1 DNA-binding assay and chloramphenicol acetyltransferase reporter gene assay. HIF-1alpha protein level and HIF-1 DNA-binding activities were increased in cerebral cortices of rats treated with acetazolamide. Moreover, the mRNA levels of erythropoietin, vascular endothelial growth factor, and glucose transporter-1 also increased. The HIF-1alpha protein level and activity of HIF-driven chloramphenicol acetyltransferase reporters of cortical neurons and PC12 cells treated with acidosis media were significantly enhanced. We conclude that the normoxic induction of HIF-1alpha and HIF-1 mediated genes by acetazolamide may mediate the effect of acetazolamide in the reduction of symptoms of acute mountain sickness.

Topics: Acetazolamide; Acidosis, Respiratory; Altitude Sickness; Animals; Carbonic Anhydrase Inhibitors; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; DNA-Binding Proteins; Erythropoietin; Glucose Transporter Type 1; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Oxygen; PC12 Cells; Rats; Rats, Sprague-Dawley; RNA, Messenger; Up-Regulation; Vascular Endothelial Growth Factor A

Erythropoietin was recently shown to exert important cytoprotective and anti-apoptotic effects in injury models of the brain, heart and kidney. We examined whether erythropoietin also attenuates renal injury in a rat model of unilateral ureteral obstruction via anti-apoptotic and anti-inflammatory actions.. We divided Sprague-Dawley rats (Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea) into 4 groups, including 1-vehicle treated with sham operation, 2-vehicle treated with unilateral ureteral obstruction for 3 days, 3-erythropoietin treatment with sham operation and 4-erythropoietin treatment for unilateral ureteral obstruction for 3 days. The erythropoietin treatment dose was 3,000 IU/kg per day intraperitoneally, administered daily. We compared competitive reverse transcriptase-polymerase chain reaction data on transforming growth factor-beta, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, osteopontin, Fas and Bcl-2. Furthermore, we examined Western blots for caspase-3 and light microscopy findings with hematoxylin and eosin staining. We applied immunohistochemistry for transforming growth factor-beta, ED-1 and caspase-3, and TUNEL in each group.. Transforming growth factor-beta, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, osteopontin and Fas mRNA levels in the erythropoietin treated, unilateral ureteral obstruction group were significantly lower than in the obstruction only group. The Bcl-2 mRNA level in the erythropoietin treated obstruction group was significantly higher than in the obstruction only group. Caspase-3 activity in the erythropoietin treated obstruction group was significantly lower than in the obstruction only group. On light microscopy interstitially infiltrated inflammatory cells were significantly decreased in the erythropoietin treated obstruction group compared to the obstruction only group. On immunohistochemistry the erythropoietin treated obstruction group showed significantly fewer reactions for transforming growth factor-beta, ED-1 and caspase-3 compared to the obstruction only group. Erythropoietin treatment in rats with unilateral ureteral obstruction significantly decreased the number of TUNEL positive cells.. Erythropoietin exerts renoprotective effects in an experimental unilateral ureteral obstruction rat model via anti-apoptotic and anti-inflammatory actions.

Topics: Animals; Apoptosis; Disease Models, Animal; Erythropoietin; Inflammation; Kidney Diseases; Male; Rats; Rats, Sprague-Dawley; Ureteral Obstruction

The neuroprotective and trophic actions of erythropoietin (EPO) have been tested in several animal models of insult, injury, and neurodegeneration. Recent studies in human volunteers demonstrated that EPO improves cognition and also elicits antidepressant effects. It is believed that the behavioral effects are mediated by EPO's trophic effect on neuronal systems. We therefore tested whether EPO is able to alter behavior and brain gene expression in rats.. The expression of EPO and EPO receptor (EPOR) in multiple brain regions was examined by quantitative polymerase chain reaction, in situ hybridization, and immunohistochemistry. The regulation of EPO and the transcription factor hypoxia-induced factor-alpha (HIF1alpha) after electroconvulsive seizure (ECS) was investigated. Behavioral effects of EPO were tested in the rodent forced swimming and novelty-induced hypophagia (NIH) models. EPO gene profiles were obtained by microarray analysis of the hippocampus after intracerebroventricular infusion.. EPO and EPOR were widely expressed in the brain albeit at low levels. Highest level of EPO and EPOR were in the choroid plexus and striatum, respectively. Peripheral administration of EPO was sufficient to produce a robust antidepressant-like effect in the forced swim and NIH tests. Gene expression profiles revealed that EPO induces the expression of neurotrophic genes such as brain-derived neurotrophic factor, VGF (nonacronymic), and neuritin.. EPO is induced by ECS and independently exhibits antidepressant-like efficacy in the forced swim and NIH tests. EPO regulates the expression of genes implicated in antidepressant action and appears to be a candidate molecule for further testing in neuropsychiatry.

Topics: Animals; Antidepressive Agents; Behavior, Animal; Brain-Derived Neurotrophic Factor; Depression; Disease Models, Animal; Electroshock; Erythropoietin; Exploratory Behavior; Gene Expression Profiling; Gene Expression Regulation; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Locomotion; Male; Oligonucleotide Array Sequence Analysis; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Seizures; Swimming

Recombinant human erythropoietin (rEpo) is neuroprotective in neonatal models of brain injury. Proposed mechanisms of neuroprotection include activation of gene pathways that decrease oxidative injury, inflammation, and apoptosis, while increasing vasculogenesis and neurogenesis. To determine the effects of rEpo on gene expression in 10-d-old BALB-c mice with unilateral brain injury, we compared microarrays from the hippocampi of brain-injured pups treated with saline or rEpo to similarly treated sham animals. Total RNA was extracted 24 h after brain injury and analyzed using Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. We identified sex-specific differences in hippocampal gene expression after brain injury and after high-dose rEpo treatment using single-gene and gene set analysis. Although high-dose rEpo had minimal effects on hippocampal gene expression in shams, at 24-h post brain injury, high-dose rEpo treatment significantly decreased the proinflammatory and antiapoptotic response noted in saline-treated brain-injured comparison animals.

Topics: Animals; Animals, Newborn; Apoptosis; Disease Models, Animal; Erythropoietin; Female; Gene Expression Profiling; Gene Expression Regulation; Hippocampus; Humans; Hypoxia-Ischemia, Brain; Inflammation; Male; Mice; Mice, Inbred BALB C; Neuroprotective Agents; Oligonucleotide Array Sequence Analysis; Recombinant Proteins; Sex Factors; Time Factors

Topics: Anemia; Animals; Biological Assay; Disease Models, Animal; Erythropoietin; European Union; Hematinics; Humans; Mice; Recombinant Proteins; Treatment Outcome

Erythropoietin (Epo) has neuroprotective activity in a variety of settings. Thus, we investigated whether Epo has a role in the functional recovery of rats after facial nerve injury. The right facial nerve of 24 Wistar rats (6 wks old) was crushed twice at the level of the stylomastoid foramen, for 30 s each time, using jeweler's forceps held perpendicular to the nerve. The left facial nerve did not undergo the surgical lesion. The rats were randomly divided into 4 groups: (group 1) the control group (placebo, treated with saline); and groups treated with Epo at a dose of 1,000 U/kg body weight (group 2), 5,000 U/kg body weight (group 3), and 10,000 U/kg body weight (group 4). The Epo and saline were administered subcutaneously pre-operatively and treatment was repeated every 24 h for the first 2 weeks after the operation. Behavioral recovery from facial paralysis was measured daily, beginning 1 day after surgery, until full recovery of the eye blink reflex and whisker movements were observed. The average recovery times for the full blink reflex and whisker movements were significantly shorter (about 2-3 days) in rats treated with a high dose Epo (5,000, 10,000 U/kg body weight) compared to the placebo-treated rats (p<0.05). There was no significant difference between low dose Epo-treated rats (1,000 U/kg body weight) and the placebo-treated rats. These results suggest that high dose Epo can promote the functional recovery of rats following facial nerve injury. Further studies are warranted to probe alternative treatment schedules (dose, mode of administration), underlying histological mechanisms and combination treatment with additional neuroprotective factors.

Topics: Analysis of Variance; Animals; Behavior, Animal; Blinking; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Erythropoietin; Facial Nerve Injuries; Humans; Neuroprotective Agents; Rats; Rats, Wistar; Recovery of Function; Time Factors; Vibrissae

Cardioprotective properties of recombinant human Erythropoietin (rhEpo) have been shown in in vivo regional or ex vivo global models of ischemia-reperfusion (I/R) injury. The aim of this study was to characterize the cardioprotective potential of rhEPO in an in vivo experimental model of global I/R approximating the clinical cardiac surgical setting and to gain insights into the myocardial binding sites of rhEpo and the mechanism involved in its cardioprotective effect.. Hearts of donor Lewis rats were arrested with cold crystalloid cardioplegia and after 45 min of cold global ischemia grafted heterotopically into the abdomen of recipient Lewis rats. Recipients were randomly assigned to control non-treated or Epo-treated group receiving 5000 U/kg of rhEpo intravenously 20 min prior to reperfusion. At 5 time points 5-1440 min after reperfusion, the recipients (n=6-8 at each point) were sacrificed, blood and native and grafted hearts harvested for subsequent analysis.. Treatment with rhEpo resulted in a significant reduction in myocardial I/R injury (plasma troponin T) in correlation with preservation of the myocardial redox state (reduced glutathione). The extent of apoptosis (activity of caspase 3 and caspase 9, TUNEL test) in our model was very modest and not significantly affected by rhEpo. Immunostaining of the heart tissue with anti-Epo antibodies showed an exclusive binding of rhEpo to the coronary endothelium with no binding of rhEpo to cardiomyocytes. Administration of rhEpo resulted in a significant increase in nitric oxide (NO) production assessed by plasma nitrite levels. Immunostaining of heart tissue with anti-phospho-eNOS antibodies showed that after binding to the coronary endothelium, rhEpo increased the phosphorylation and thus activation of endothelial nitric oxide synthase (eNOS) in coronary vessels. There was no activation of eNOS in cardiomyocytes.. Intravenous administration of rhEpo protects the heart against cold global I/R. Apoptosis does not seem to play a major role in the process of tissue injury in this model. After binding to the coronary endothelium, rhEpo enhances NO production by phosphorylation and thus activation of eNOS in coronary vessels. Our results suggest that cardioprotective properties of rhEpo are at least partially mediated by NO released by the coronary endothelium.

Topics: Animals; Apoptosis; Atrial Natriuretic Factor; Body Water; Cardiotonic Agents; Coronary Vessels; Disease Models, Animal; Drug Evaluation, Preclinical; Endothelium, Vascular; Erythropoietin; Heart Transplantation; Male; Myocardial Reperfusion Injury; Nitric Oxide; Oxidative Stress; Rats; Rats, Inbred Lew; Recombinant Proteins; Systemic Inflammatory Response Syndrome; Troponin T

Erythropoietin has recently emerged as a cytoprotective cytokine, which possesses the ability to protect many tissues, including the brain, heart, and kidneys, against ischemia or traumatic injury. We investigated the therapeutic effects of erythropoietin in a murine model of endotoxin shock.. Prospective, randomized study.. University-based research laboratory.. Male BALB/c mice.. Mice intraperitoneally received either lipopolysaccharide (LPS) from Escherichia coli or vehicle. Erythropoietin was administered at a dose of 1000 IU/kg subcutaneously at different time points after LPS administration. We also investigated the effect of erythropoietin on the development of septic shock caused by cecal perforation.. Treatment of mice with erythropoietin, within 2 hours after LPS administration, improved the mortality rate. Treatment of cecal perforated mice with erythropoietin extended survival by 12 hours, but all animals died by 72 hours in both groups. Erythropoietin attenuated apoptosis in the lungs, liver, small intestine, thymus, and spleen, as assessed by terminal deoxynucleotidyl transferase-mediated nucleotide nick-end labeling staining, active caspase-3 immunostaining and immunoblotting, and measurements of caspase-3/7 activity. Erythropoietin also reduced inducible nitric oxide synthase expression, nitric oxide production, peroxynitrite formation, and tissue hypoxia. In contrast, erythropoietin did not affect the degree of LPS-induced inflammation, as assessed by measurements of blood levels of interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, growth-related oncogene/keratinocyte-derived cytokine, and high mobility group box 1, the phosphorylation levels of nuclear factor kappaB, and the number of neutrophils infiltrating the lungs and the liver.. The results of the study demonstrate that administration of a large dose of erythropoietin after induction of experimental endotoxemia improved survival and that the beneficial effects of erythropoietin were associated with inhibition of apoptosis, nitric oxide production, and tissue hypoxia, without alterations in inflammatory responses.

Topics: Animals; Disease Models, Animal; Erythropoietin; Male; Mice; Mice, Inbred BALB C; Shock, Septic

We established models of cancer-related anemia in mice from subcutaneous inoculation of two IL-6-producing cancer cell lines, human lung cancer cell line LC-06-JCK and murine colon26 clone 5 colon cancer cells. In both models, elevated levels of IL-6 were detected in sera and hemoglobin levels significantly decreased compared with non-tumor-bearing mice. In the LC-06-JCK model, serum albumin levels also decreased with elevated levels of human IL-6 in sera. On the other hand, serum levels of EPO increased, although anemia developed and did not improve. The development of cancer-related anemia was prevented by the administration of a rat anti-mouse IL-6 receptor antibody, MR16-1, in the LC-06-JCK model. It is therefore suggested that IL-6 causes anemia independent of a reduction in EPO levels. Our preclinical models should be useful for exploring new modalities for the treatment of cancer-related anemia.

Topics: Anemia; Animals; Antibodies, Monoclonal; Cell Line, Tumor; Cytokines; Disease Models, Animal; Erythropoietin; Humans; Interleukin-6; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Rats

Individually, the cytokines erythropoietin (EPO) and insulin-like growth factor-I (IGF-I) have both been shown to reduce neuronal damage significantly in rodent models of cerebral ischemia. The authors have previously shown that EPO and IGF-I, when administered together, provide acute and prolonged neuroprotection in cerebrocortical cultures against N-methyl-D-aspartate-induced apoptosis. The aim of this study was to determine whether intranasally applied EPO plus IGF-I can provide acute neuroprotection in an animal stroke model and to show that intranasal administration is more efficient at delivering EPO plus IGF-I to the brain when compared with intravenous, subcutaneous, or intraperitoneal administration.. The EPO and IGF-I were administered intranasally to mice that underwent transient middle cerebral artery occlusion (MCAO). Stroke volumes were measured after 1 hour of MCAO and 24 hours of reperfusion. To evaluate the long-term effects of this treatment, behavioral outcomes were assessed at 3, 30, 60, and 90 days following MCAO. Radiography and liquid scintillation were used to visualize and quantify the uptake of radiolabeled 125I-EPO and 125I-IGF-I into the mouse brain after intranasal, intravenous, subcutaneous, or intraperitoneal administration.. Intranasal administration of EPO plus IGF-I reduced stroke volumes within 24 hours and improved neurological function in mice up to 90 days after MCAO. The 125I-EPO and 125I-IGF-I were found in the brain within 20 minutes after intranasal administration and accumulated within the injured areas of the brain. In addition, intranasal administration delivered significantly higher levels of the applied 125I-EPO and 125I-IGF-I to the brain compared with intravenous, subcutaneous, or intraperitoneal administration.. The data demonstrate that intranasal EPO plus IGF-I penetrates into the brain more efficiently than other drug delivery methods and could potentially provide a fast and efficient treatment to prevent chronic effects of stroke.

Topics: Acute Disease; Administration, Intranasal; Animals; Disease Models, Animal; Drug Delivery Systems; Drug Therapy, Combination; Erythropoietin; Infarction, Middle Cerebral Artery; Insulin-Like Growth Factor I; Iodine Radioisotopes; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents

Erythropoietin (EPO) stimulates red blood cell production, in part by inhibiting apoptosis of the red blood cell precursors. The erythropoietic effects of EPO are circadian stage dependent. Retinal injury due to light occurs through oxidative mechanisms and is manifest by retinal and retinal pigment epithelium (RPE) cells apoptosis. The visual cycle might be circadian coordinated as a means of effectively protecting the retina from the detrimental effects of light-induced, oxygen-dependent, free radical-mediated damage, especially at the times of day when light is more intense. We show that the retinal expression of EPO and its receptor (EPOR), as well as subsequent Janus kinase 2 (Jak2) phosphorylations, are each tightly linked to a specific time after oxidative stress and in anticipation of daily light onset. This is consistent with physiological protection against daily light-induced, oxidatively mediated retinal apoptosis. In vitro, we verify that EPO protects RPE cells from light, hyperoxia, and hydrogen peroxide-induced retinal cell apoptosis, and that these stimuli increase EPO and EPOR expression in cultured RPE cells. Together, these data support the premise that EPO and its EPOR interactions represent an important retinal shield from physiologic and pathologic light-induced oxidative injury.

Topics: Animals; Animals, Newborn; Apoptosis; bcl-X Protein; Caspase 3; Cell Survival; Cells, Cultured; Disease Models, Animal; Erythropoietin; Female; Gene Expression Regulation; Heme Oxygenase-1; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Janus Kinase 2; Light; Mice; Mice, Inbred C57BL; Neurons; Proto-Oncogene Proteins c-fos; Receptors, Erythropoietin; Retinal Diseases; Retinal Pigment Epithelium; Rhodopsin; Thioredoxins

Acclimatization to hypoxic exposure relies on an elevated ventilation and erythropoietic activity. We recently proposed that erythropoietin (Epo) links both responses: apart from red blood cell production, cerebral and plasma Epo interact with the central and peripheral respiratory centers. Knowing that women cope better than men with reduced oxygen supply (as observed at high altitude), we analyzed the hypoxic ventilatory response in Epo-overexpressing transgenic male and female mice with high Epo levels in brain and plasma (Tg6) or in wild-type animals injected with recombinant human Epo (rhEpo). Exposure to moderate and severe hypoxia as well as to hyperoxia and injection of domperidone, a potent peripheral ventilatory stimulant, revealed that the presence of transgenic or rhEpo extensively increased the hypoxic ventilatory response in female mice compared with their corresponding male siblings. Alterations of catecholamines in the brain stem's respiratory centers were also sex dependent. In a proof-of-concept study, human volunteers were intravenously injected with 5,000 units rhEpo and subsequently exposed to 10% oxygen. Compared with men, the hypoxic ventilatory response was significantly increased in women. We conclude that Epo exerts a sex-dependent impact on hypoxic ventilation improving the response in female mice and in women that most probably involves sexual hormones. Our data provides an explanation as to why women are less susceptible to hypoxia-associated syndromes than men.

Topics: Adaptation, Physiological; Adult; Animals; Brain Stem; Catecholamines; Disease Models, Animal; Domperidone; Dopamine Antagonists; Erythropoietin; Female; Humans; Hyperoxia; Hypoxia; Injections, Intravenous; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pulmonary Ventilation; Recombinant Proteins; Respiratory Mechanics; Sex Factors; Young Adult

It has been shown that bone marrow (BM)-derived cells are involved in repaired endothelium induced by a model such as neointima-produced wire injury in mice. This has not been shown in a less invasive model that results in simple reendothelialization. A new wire-induced simple endothelial denudation model of the common carotid artery (CCA) of mice, which did not form neointima at 14 days after the operation, was established. At 7 days after operation, the CCAs were reendothelialized from the aortic arch and the carotid bifurcation but not completely, shown by whole-mount CD31 immunohistochemical staining. Scanning electron microscopy revealed that unendothelialized area was covered with platelets. To determine the involvement of BM-derived cells in the repaired endothelium, the wild-type (WT) C57BL/6 mice, in which BM cells derived from strain-matched green fluorescent protein (GFP)-transgenic mice were transplanted, were operated upon. As a result, there was no GFP-positive endothelial cell (EC) in reendothelialized endothelium, otherwise GFP-positive 'dendritic'-like cells were recruited under the repaired endothelial layer. Administration of recombinant human erythropoietin [1,000 IU/(kg day) at 0-3 days after operation subcutaneously], which has been shown to increase endothelial progenitor cells in peripheral blood, also could not recruit BM-derived cells as ECs in BM-transplanted mice despite accelerating reendothelialization in WT mice [%reendothelialized area of the administrated group 78.0 +/- 9.4% (mean +/- SD) vs. the control group 63.0 +/- 4.4%, P < 0.05]. These results suggest that BM-derived cells may not be involved in reendothelialization as ECs after simple endothelial denudation in mice.

Topics: Animals; Bone Marrow Cells; Bone Marrow Transplantation; Carotid Artery Injuries; Cell Movement; Cell Proliferation; Disease Models, Animal; Endothelial Cells; Erythropoietin; Green Fluorescent Proteins; Humans; Injections, Subcutaneous; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Recombinant Proteins; Stem Cells; Time Factors

To investigate the effect of exogenous erythropoietin (EPO) on the denervated muscle atrophy.. Twenty-four SD male rats, weighting 200-220 g were made the models of denervated gastrocnemius muscle after sciatic nerves were transected under the piriform muscle at the right lower leg, and were randomly divided into two groups (n=12). rhEPO (2500 U/kg) was injected daily into the denervated gastrocnemius muscle in EPO group, and normal saline was injected into the denervated gastrocnemius muscle in control group. To observe the general state of health of the experimental animal, the muscle wet weight, the muscle cell diameter, the cross section area, the protein amount, the percentage of the apoptotic muscle cells, and the Na+-K -ATPase and Ca2+ -ATPase activities were measured 2 and 4 weeks after operation.. All experimental animals were survived during experiment without cut infection, and all animals could walk with pulling the right knee. At 4 weeks after operation, 7 cases showed ulcer in the right heel, including 5 in the control group and 2 in the EPO group. At 2 and 4 weeks after operation, the muscle wet weight in EPO group was (885.59 +/- 112.35) and (697.62 +/- 94.74) g, respectively; in control group, it was (760.63 +/- 109.05) and (458.71 +/- 58.76) g, respectively; indicating significant differences between two groups (P < 0.01). The protein amount in EPO group was (77.37 +/- 5.24) and (66.37 +/- 4.87) mg/mL, respectively; in control group, it was (65.39 +/- 4.97) and (54.62 +/- 6.32) mg/mL: indicating significant differences between two groups (P < 0.01). At 2 and 4 weeks after operation, the myofibrillar shapes were nearly normal in EPO group while there were muscle fiber atrophy, some collapse and obviously hyperelastosis between muscle bundle. There were significant differences in the muscle cell diameter and the cross section between two groups (P < 0.01). However, the percentage of the apoptotic muscle cells was 11.80% +/- 1.74% and 28.47% +/- 1.81% in control group, respectively, which was significantly smaller than that in EPO group (21.48% +/- 2.21% and 55.89% +/- 2.88%, P < 0.01). At 2 and 4 weeks after operation, Na+-K+ -ATPase and Ca2+ -ATPase activities in EPO group were higher than those in control group (P < 0.01).. EPO can delay the denervated muscle atrophy.

Topics: Animals; Calcium-Transporting ATPases; Disease Models, Animal; Erythropoietin; Male; Muscular Atrophy; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Sciatic Nerve; Sodium-Potassium-Exchanging ATPase

Various studies demonstrate erythropoietin (EPO) as a cardioprotective growth hormone. Recent findings reveal EPO in addition might induce proliferation cascades inside myocardium. We aimed to evaluate whether a single high-dose intramyocardial EPO administration safely elevates early intracardiac cell proliferation after myocardial infarction (MI). Following permanent MI in rats EPO (3000 U/kg) in MI EPO-treatment group (n=99) or saline in MI control group (n=95) was injected along the infarction border. Intramyocardial EPO injection activated the genes of cyclin D1 and cell division cycle 2 kinase (cdc2) at 24 h after MI (n=6, P<0.05) evaluated by real time-PCR. The number of Ki-67+ intracardiac cells analyzed following immunohistochemistry was significantly enhanced by 45% in the peri-infarction zone at 48 h after EPO treatment (n=6, P<0.001). Capillary density was significantly enhanced by 17% as early as seven days (n=6, P<0.001). After six weeks, left ventricular performance assessed by conductance catheters was restored under baseline and dobutamine induced stress conditions (n=11-14, P<0.05). No thrombus formation was observed in the heart and in distant organs. No deleterious systemic adverse effects were apparent. Single high-dose intramyocardial EPO delivery proved safety and promoted early intracardiac cell proliferation, which might in part have contributed to an attenuated myocardial functional decline.

Topics: Animals; Capillaries; Cardiotonic Agents; CDC2 Protein Kinase; Cell Proliferation; Cyclin D1; Disease Models, Animal; Erythropoietin; Injections, Intralesional; Ki-67 Antigen; Male; Myocardial Contraction; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Rats; Rats, Inbred Lew; Recovery of Function; Time Factors; Ventricular Function, Left

Recent data have demonstrated potent cardioprotective and neuroprotective effects of the application of growth hormones like erythropoietin (EPO) after focal cardiac or cerebral ischemia. In order to assess possible benefits regarding survival and resuscitation conditions, EPO was tested against placebo in a model of cardiac arrest in the rat.. Thirty-four male Wistar rats were randomized into two groups (EPO versus control; n=17 per group). Under anesthesia, cardiac arrest was induced by asphyxia after neuromuscular blockade. After 6 min of global ischemia, animals were resuscitated by external chest compression combined with epinephrine administration. An intravenous bolus of recombinant human EPO (rhEPO, 3000 UIkg(-1) body weight, i.v.) or saline (in control group) was performed 15 min before cardiac arrest, by a blinded investigator. Restoration of spontaneous circulation (ROSC), survival at 1, 24, 48 and 72 h and hemodynamic changes after cardiac arrest were studied.. Survival to 72 h was significantly improved in the EPO group (n=15/17) compared to the control group (n=7/17). All the EPO-treated rats were successfully resuscitated whereas only 13 of 17 control animals resuscitated. EPO-treated animals required a significantly smaller dose of epinephrine before resuscitation, compared to control rats. Time course of systolic arterial blood pressure after resuscitation revealed no significant differences between both groups.. EPO, when administrated before cardiac arrest, improved initial resuscitation and increased the duration of post-resuscitation survival.

Topics: Advanced Cardiac Life Support; Animals; Cardiovascular Agents; Disease Models, Animal; Erythropoietin; Heart Arrest; Male; Rats; Rats, Wistar

To observe the effect of inhibition of retinal neovascularization by small-interference RNA (siRNA) targeting erythropoietin (EPO).. Three siRNAs against EPO were designed and synthesized. Then they were transfected to NIH/3T3 cells by liposomes. RT-PCR and Western blot were used to evaluate the efficacy of siRNA in attenuating EPO expression in NIH/3T3 cells. One-week-old C57BL/6J mice were exposed to 75 +/- 2% oxygen for 5 days, then they were returned to room air to induce retinal neovascularization. The siRNA type shown as most powerful in reducing EPO expression in vitro was intravitreally injected in the treatment group. Retinal neovascularization was evaluated by angiography with injection of fluorescein-dextran and quantification of neovascular proliferative retinopathy after 5 days in room air. Moreover, RT-PCR and immunoblot analysis were used to determine whether local administration of siRNA could affect the expression of EPO in murine retinas.. Among the 3 designed siRNAs (named siEPO1-3), siEPO2 is the most efficient in inhibiting EPO expression. In this murine model of oxygen-induced retinopathy, retinal neovascularization in the eyes with siEPO2 injection was significantly reduced compared with that of the contralateral control eyes. Similarly, histological analysis indicates that the number of neovascular nuclei protruding into the vitreous cavity was decreased compared to the control eyes. Furthermore, the expression of EPO in the retinas injected with siEPO2 was dramatically decreased.. siRNA against EPO could inhibit experimental retinal neovascularization by reducing EPO expression in the retinas of mice. It may provide a powerful and novel therapeutic tool for ischemia-induced retinal diseases.

Topics: Animals; Animals, Newborn; Base Sequence; Blotting, Western; Cell Culture Techniques; Dextrans; Disease Models, Animal; Erythropoietin; Fluorescein Angiography; Fluoresceins; Gene Expression Regulation; Humans; Infant, Newborn; Injections; Mice; Mice, Inbred C57BL; Molecular Sequence Data; NIH 3T3 Cells; Oxygen; Retinal Neovascularization; Retinopathy of Prematurity; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Transfection; Vitreous Body

Erythropoietin (EPO) has been thought to be capable of potentiating protection of jeopardized myocardium by reperfusion in evolving myocardial infarction. However, diversity in study design and measurements of infarct size in studies evaluating EPO has led to inconsistent results. We sought to characterize the effect of EPO on infarct size after myocardial ischemia and reperfusion with the use of assessment of left-ventricular (LV) creatine kinase (CK) depletion and echocardiography.. Acute coronary occlusion was induced in 10-week-old C57BL6 mice by left anterior descending coronary artery ligation for 3 h followed by 72 h of reperfusion. EPO (10,000 U/kg) or an equivalent amount of saline vehicle alone was injected intraperitoneally before ligation or immediately after the onset of reperfusion. Assays of residual LV CK activity and calculation of LV CK depletion were performed on LV homogenates harvested 72 h after onset of reperfusion for measurement of infarct size, and echocardiography was performed immediately before harvest of tissue for measurement of function.. Mice administered EPO before ligation had similar infarct size (37.1+/-4.1%) and echo scores (22.9+/-0.4) compared with those in corresponding control mice administered saline (35.29+/-1.9 and 21.3+/-1.1%, respectively). Mice administered EPO after reperfusion had similar infarct size (39.1+/-4.8%) and echo scores (19.5+/-1.0) compared with those in corresponding control mice administered saline (40.3+/-4.9 and 21.5+/-1.9%, respectively).. EPO does not protect ischemic myocardium such that reperfusion after 3 h can yield additional salvage.

Topics: Animals; Biomarkers; Creatine Kinase; Disease Models, Animal; Echocardiography, Doppler; Epoetin Alfa; Erythropoietin; Injections, Intraperitoneal; Male; Mice; Mice, Inbred C57BL; Myocardial Contraction; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Recombinant Proteins; Time Factors; Ventricular Function, Left

The effect of recombinant human erythropoietin (rhEPO) on neuropathic pain remains unclear. This study aimed to determine the effects of preemptive administration of rhEPO on the behavioural changes and neuroinflammatory responses in a rat model of neuropathic pain.. Fifty rats were randomly allocated into five groups, sham-operation treated with saline and L5 spinal nerve transection treated with different doses of rhEPO (0 [saline], 1000, 3000, or 5000 U x kg(-1), respectively). The rats were intraperitoneally treated from 1 day before surgery to post-surgery day 7. The mechanical (paw pressure thresholds, PPT) and thermal thresholds (paw withdrawal latencies, PWL) were measured on post-surgery days 1, 3, and 7. The contralateral brain was obtained on post-surgery day 7 to determine the expressions of tumour necrosis factor (TNF-alpha), interleukin (IL)-1beta, IL-6, L-10, and nuclear factor-kappa B (NF-kappaB) activity.. There were significant decreases in PPT and PWL after L5 spinal nerve transection (P < 0.001). Compared with the saline group, the rhEPO 3000 and 5000 U x kg(-1) groups resulted in significant increases in PPT and PWL (P < 0.001) and reduced the cerebral expressions of TNF-alpha, IL-1beta, IL-6, and NF-kappaB activity associated with the increase in IL-10 (rhEPO3000 group, P < 0.05, and rhEPO5000 group, P < 0.001, respectively). Administration of rhEPO 1000 U x kg(-1) had no significant effects on these variables.. Preemptive rhEPO dose-dependently attenuated the mechanical and thermal hyperalgesia in L5 spinal nerve transection rats, which correlated with the decreased cerebral expressions of TNF-alpha, IL-1beta, and IL-6 via downregulating NF-kappaB activity and the increased expression of IL-10.

Topics: Animals; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Gene Expression; Interleukin-10; Interleukin-1beta; Interleukin-6; Male; Neuralgia; NF-kappa B; Pain Measurement; Pain Threshold; Random Allocation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Tumor Necrosis Factor-alpha

The matrix protein thrombospondin-4 has an acidic amphipathic C-terminal peptide (C21) which stimulates erythroid cell proliferation. Here we show that C21 stimulates red cell formation in anemic mice in vivo. In vitro experiments indicated that the peptide-mediated increase of erythroid colony formation in cultures of human CD34+ hematopoietic progenitor cells was possible only under continuous presence of erythropoietin. In the absence of this cytokine, C21 stimulated exclusively myeloid colony formation. Therefore, the peptide is not a specific erythroid differentiation factor. In fact, it is mitogenic in non-erythroid cells, such as skin fibroblasts and kidney epithelial cells. In erythroleukemic TF-1 cells, it actually decreased the production of the erythroid differentiation marker glycophorin A. C21-affinity chromatography revealed regulator of differentiation 1 (ROD1) as a major C21-binding protein. ROD1 is the hematopoietic cell paralog of polypyrimidine tract binding proteins (PTBs), RNA splice regulators which regulate differentiation by repressing tissue-specific exons. ROD1 binding to C21 was strongly inhibited by synthetic RNAs in the order poly A > poly U > poly G = poly C and was weakly inhibited by a synthetic phosphorylated peptide mimicking the C-terminal domain of RNA polymerase II. Cellular overexpression or knockdown experiments of ROD1 suggest a role for this protein in the mitogenic activity of C21. Since the nuclear proteins ROD1 and PTBs regulate differentiation at a posttranscriptional level and there is a fast nuclear uptake of C21, we put forward the idea that the peptide is internalized, goes to the nucleus and maintains cells in a proliferative state by supporting ROD1-mediated inhibition of differentiation.

Topics: Active Transport, Cell Nucleus; Anemia; Animals; Binding Sites; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Epithelial Cells; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Fibroblasts; Glycophorins; Humans; Kidney; Leukemia, Erythroblastic, Acute; Male; Mice; Mice, Inbred C57BL; Peptide Fragments; Polypyrimidine Tract-Binding Protein; Protein Structure, Tertiary; Recombinant Proteins; RNA Interference; RNA-Binding Proteins; Skin; Thrombospondins; Time Factors; Transduction, Genetic; Zidovudine

Erythropoietin (EPO) protects the myocardium from ischaemic injury and promotes beneficial remodelling. We assessed the therapeutic efficacy of intracardiac EPO injection and EPO-mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, EPO (3000 U/kg) or saline was delivered by intracardiac injection. Compared to myocardial infarction control group (MIC), EPO significantly improved left ventricular function (n =11-14, P < 0.05) and decreased right ventricular wall stress (n = 8, P < 0.05) assessed by pressure-volume loops after 6 weeks. MI-EPO hearts exhibited smaller infarction size (20.1 +/- 1.1% versus 27.8 +/- 1.2%; n = 6-8, P < 0.001) and greater capillary density (338.5 +/- 14.7 versus 259.8 +/- 9.2 vessels per mm2; n = 6-8, P < 0.001) than MIC hearts. Direct EPO injection reduced post-MI myocardial apoptosis by approximately 41% (0.27 +/- 0.03% versus 0.42 +/- 0.03%; n = 6, P= 0.005). The chemoattractant SDF-1 was up-regulated significantly assessed by quantitative realtime PCR and immunohistology. c-Kit(+) and CD34(+) stem cells were significantly more numerous in MI-EPO than in MIC at 24 hrs in peripheral blood (n = 7, P < 0.05) and 48 hrs in the infarcted hearts (n = 6, P < 0.001). Further, the mRNAs of Akt, eNOS and EPO receptor were significantly enhanced in MI-EPO hearts (n = 7, P < 0.05). Intracardiac EPO injection restores myocardial functions following MI, which may attribute to the improved early recruitment of c-Kit(+) and CD34(+) stem cells via the enhanced expression of chemoattractant SDF-1.

Topics: Animals; Apoptosis; Cell Survival; Chemokine CXCL12; Disease Models, Animal; Erythropoietin; Heart Function Tests; Hematocrit; Hematopoietic Stem Cell Mobilization; Humans; Injections; Matrix Metalloproteinase 2; Myocardial Infarction; Myocytes, Cardiac; Neovascularization, Physiologic; Rats; Receptors, CXCR4; Receptors, Erythropoietin; Recombinant Proteins; Troponin T; Up-Regulation

Selective neuronal loss is closely associated with cognitive impairments that occur following status epilepticus (SE). Our previous study suggested that erythropoietin (Epo) pre-treatment suppressed hippocampal neuronal death in rats after 1 h of SE convulsions. However, the underlying protective mechanism remained unclear. In the present study, we investigated the anti-apoptotic mechanism of Epo pre-treatment in the hippocampus using Li-pilocarpine-induced SE in rats. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed to detect apoptosis and the Morris water maze was employed to assess spatial learning ability and to analyze the protective effects of Epo. Levels of Bcl-2 family (Bid, Bcl-2 and Bax) markers were examined via Western blot and immunofluorescence. We found that Epo pre-treatment prevented SE-induced cognitive impairments. The protection and cognitive effects were associated with higher levels of Bcl-2 and lower levels of Bax. The present results suggest that systemic Epo pre-treatment can confer neuroprotection following SE, and may provide novel insights into pathogenesis and treatment following SE injury.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Cognition Disorders; Disease Models, Animal; Erythropoietin; Hippocampus; In Situ Nick-End Labeling; Male; Maze Learning; Muscarinic Agonists; Neuroprotective Agents; Pilocarpine; Rats; Rats, Sprague-Dawley; Status Epilepticus

This study examines the efficacy of puerarin, a drug used in traditional Chinese medicine, in attenuating ischemic brain injury after cerebral ischemia and reperfusion, and explores possible mechanisms underlying neuroprotective effects.. The animal model of ischemia/reperfusion injury was induced by middle cerebral artery occlusion for 2 hours followed by up to 72 hour reperfusion. The rats were randomly assigned into four groups (n=6/group): puerarin at 100, 200 and 400 mg/kg or saline, administered intraperitoneally. Neurological outcome and infarct volume by 2% triphenyl tetrazolium chloride staining were determined 72 hours after reperfusion. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining was used to detect the cell damage of brains (n=5/group). Erythropoietin activation was detected by enzyme-linked immunosorbent assay (n=5/group).. Compared with the vehicle saline group, puerarin decreased infarction volume at doses of 200 mg/kg (p=0.045) and 400 mg/kg (p=0.0002), but not at 100 mg/kg (p=0.387). Functional neurological outcome was improved with puerarin at 400 mg/kg (p=0.015), but not at 100 mg/kg (p=0.68) or 200 mg/kg (p=0.056). Puerarin significantly decreased the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling staining cells compared with the vehicle group 4, 24 and 72 hours after reperfusion. The erythropoietin activity was higher in puerarin treated group compared with the vehicle group.. Puerarin has neuroprotection effects in rats at doses of 200 and 400 mg/kg, administered intraperitoneally after transient middle cerebral artery occlusion which may be partly due to activation of erythropoietin activity.

Topics: Analysis of Variance; Animals; Apoptosis; Behavior, Animal; Brain Injuries; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Gene Expression Regulation; In Situ Nick-End Labeling; Ischemic Attack, Transient; Isoflavones; Male; Neurologic Examination; Rats; Rats, Sprague-Dawley; Time Factors; Vasodilator Agents

To evaluate the time-dependent effects and pathophysiological mechanism of erythropoietin (Epo) on oxidative stress and liver injury resulting from obstructive jaundice in common bile duct-ligated rats.. Wistar Albino rats were divided into 5 groups, each including 8 rats. The sham group underwent laparotomy only, while the Non-Epo-3 and Non-Epo-7 groups underwent common bile duct ligation and were sacrificed 3 and 7 days, respectively, after the operation. The Epo-3 and Epo-7 groups underwent common bile duct ligation and Epo treatment and were sacrificed 3 and 7 days, respectively, after the operation. Blood and tissue samples were collected from all groups for the determination of oxidative injury and hepatocellular damage. Serum total and direct bilirubin levels, alkaline phosphatase, reduced glutathione (GSH), nitric oxide (NO), malondialdehyde (MDA) and white blood cell counts were measured.. Significantly higher NO and MDA levels were found in Non-Epo groups than Epo groups. Significantly lower GSH levels were found in the Non-Epo-7 group than the Epo-7 and sham groups. Hepatocellular damage was also found to be reduced in Epo groups.. In the present model, while common bile duct ligation increased oxidative injury and hepatocellular damage, treatment with Epo attenuated oxidative injury and hepatocellular damage by decreasing NO and increasing GSH.

Topics: Alkaline Phosphatase; Animals; Bilirubin; Disease Models, Animal; Erythropoietin; Glutathione; Jaundice, Obstructive; Liver; Male; Malondialdehyde; Nitric Oxide; Oxidative Stress; Rats; Rats, Wistar; Recombinant Proteins

Mathematical modelling has proven an important tool in elucidating and quantifying mechanisms that govern the age structure and population dynamics of red blood cells (RBCs). Here we synthesise ideas from previous experimental data and the mathematical modelling literature with new data in order to test hypotheses and generate new predictions about these mechanisms. The result is a set of competing hypotheses about three intrinsic mechanisms: the feedback from circulating RBC concentration to production rate of immature RBCs (reticulocytes) in bone marrow, the release of reticulocytes from bone marrow into the circulation, and their subsequent ageing and clearance. In addition we examine two mechanisms specific to our experimental system: the effect of phenylhydrazine (PHZ) and blood sampling on RBC dynamics. We performed a set of experiments to quantify the dynamics of reticulocyte proportion, RBC concentration, and erythropoietin concentration in PHZ-induced anaemic mice. By quantifying experimental error we are able to fit and assess each hypothesis against our data and recover parameter estimates using Markov chain Monte Carlo based Bayesian inference. We find that, under normal conditions, about 3% of reticulocytes are released early from bone marrow and upon maturation all cells are released immediately. In the circulation, RBCs undergo random clearance but have a maximum lifespan of about 50 days. Under anaemic conditions reticulocyte production rate is linearly correlated with the difference between normal and anaemic RBC concentrations, and their release rate is exponentially correlated with the same. PHZ appears to age rather than kill RBCs, and younger RBCs are affected more than older RBCs. Blood sampling caused short aperiodic spikes in the proportion of reticulocytes which appear to have a different developmental pathway than normal reticulocytes. We also provide evidence of large diurnal oscillations in serum erythropoietin levels during anaemia.

Topics: Anemia; Animals; Bayes Theorem; Bone Marrow Cells; Disease Models, Animal; Erythrocyte Aging; Erythrocyte Count; Erythrocytes; Erythropoietin; Feedback, Physiological; Markov Chains; Mice; Monte Carlo Method; Phenylhydrazines; Reticulocyte Count; Reticulocytes

The primary objectives of this study were to determine if erythropoietin (EPO) is neuroprotective to the photoreceptors in the retinal degeneration slow (rds) mouse in the absence of an increase in hematocrit and to determine if deglycosylated EPO (DEPO) is less neuroprotective. We performed subretinal injections of 10U EPO, DEPO or hyperglycosylated EPO (HEPO) in postnatal day 7 rds mice. Whole eye EPO levels were quantified by ELISA at specified time points post-injection. TUNEL analysis, hematocrit, and immunohistochemistry were performed at postnatal day 20. Half of the amount of EPO measured immediately after injection was detected less than 1 h later. Twenty four hours later, EPO levels were 1000 times lower than the amount originally detected. Uninjected rds mice contained 36 +/- 2 TUNEL-positive cells/mm retina and PBS injected mice contained 17 +/- 3 TUNEL-positive cells/mm retina. EPO, DEPO, and HEPO treated rds retinas contained 5 +/- 2, 9 +/- 2, and 3 +/- 1 TUNEL-positive cells/mm retina, respectively. The hematocrit was 43% in control and 41% in treated rds mice Previous studies have shown neuroprotection of the retina by treatment with as little as 24-39 mU EPO/mg total protein in the eye. In this study, we detected 40 mU/mg EPO in the eye 11 h after injection of 10 U EPO. Treatment with all forms of EPO tested was neuroprotective to the photoreceptors without a concomitant increase in hematocrit.

Topics: Animals; Animals, Newborn; Apoptosis; Darbepoetin alfa; Dependovirus; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Glycosylation; Green Fluorescent Proteins; Hematocrit; Immunohistochemistry; In Situ Nick-End Labeling; Injections; Intermediate Filament Proteins; Membrane Glycoproteins; Mice; Mice, Mutant Strains; Nerve Tissue Proteins; Neuroprotective Agents; Peripherins; Photoreceptor Cells, Vertebrate; Protein Processing, Post-Translational; Retinal Degeneration; Time Factors; Transduction, Genetic

Ischemic postconditioning (IPost) and erythropoietin (EPO) have been shown to attenuate myocardial reperfusion injury using similar signaling pathways. The aim of this study was to examine whether EPO is as effective as IPost in decreasing postischemic myocardial injury in both Langendorff-isolated-heart and in vivo ischemia-reperfusion rat models. Rat hearts were subjected to 25 min ischemia, followed by 30 min or 2 h of reperfusion in the isolated-heart study. Rats underwent 45 min ischemia, followed by 24 h of reperfusion in the in vivo study. In both studies, the control group (n=12; ischemia-reperfusion only) was compared with IPost (n=16; 3 cycles of 10 s reperfusion/10 s ischemia) and EPO (n=12; 1,000 IU/kg) at the onset of reperfusion. The following resulted. First, in the isolated hearts, IPost or EPO significantly improved postischemic recovery of left ventricular developed pressure. EPO induced better left ventricular developed pressure than IPost at 30 min of reperfusion (73.18+/-10.23 vs. 48.11+/-7.92 mmHg, P<0.05). After 2 h of reperfusion, the infarct size was significantly lower in EPO-treated hearts compared with IPost and control hearts (14.36+/-0.60%, 19.11+/-0.84%, and 36.21+/-4.20% of the left ventricle, respectively; P<0.05). GSK-3beta phosphorylation, at 30 min of reperfusion, was significantly higher with EPO compared with IPost hearts. Phosphatidylinositol 3-kinase and ERK1/2 inhibitors abolished both EPO- and IPost-mediated cardioprotection. Second, in vivo, IPost and EPO induced an infarct size reduction compared with control (40.5+/-3.6% and 28.9+/-3.1%, respectively, vs. 53.7+/-4.3% of the area at risk; P<0.05). Again, EPO decreased significantly more infarct size and transmurality than IPost (P<0.05). In conclusion, with the use of our protocols, EPO showed better protective effects than IPost against reperfusion injury through higher phosphorylation of GSK-3beta.

Topics: Animals; Cardiotonic Agents; Coronary Circulation; Darbepoetin alfa; Disease Models, Animal; Erythropoietin; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Heart Rate; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Time Factors; Ventricular Function, Left; Ventricular Pressure

Inspired from preconditioning studies, ischemic postconditioning, consisting of the application of intermittent interruptions of blood flow shortly after reperfusion, has been described in cardiac ischemia and recently in stroke. It is well known that ischemic tolerance can be achieved in the brain not only by ischemic preconditioning, but also by hypoxic preconditioning. However, the existence of hypoxic postconditioning has never been reported in cerebral ischemia.. Adult mice subjected to transient middle cerebral artery occlusion underwent chronic intermittent hypoxia starting either 1 or 5 days after ischemia and brain damage was assessed by T2-weighted MRI at 43 days. In addition, we investigated the potential neuroprotective effect of hypoxia applied after oxygen glucose deprivation in primary neuronal cultures.. The present study shows for the first time that a late application of hypoxia (5 days) after ischemia reduced delayed thalamic atrophy. Furthermore, hypoxia performed 14 hours after oxygen glucose deprivation induced neuroprotection in primary neuronal cultures. We found that hypoxia-inducible factor-1alpha expression as well as those of its target genes erythropoietin and adrenomedullin is increased by hypoxic postconditioning. Further studies with pharmacological inhibitors or recombinant proteins for erythropoietin and adrenomedullin revealed that these molecules participate in this hypoxia postconditioning-induced neuroprotection.. Altogether, this study demonstrates for the first time the existence of a delayed hypoxic postconditioning in cerebral ischemia and in vitro studies highlight hypoxia-inducible factor-1alpha and its target genes, erythropoietin and adrenomedullin, as potential effectors of postconditioning.

Topics: Adrenomedullin; Animals; Atrophy; Brain; Cells, Cultured; Cytoprotection; Disease Models, Animal; Energy Metabolism; Erythropoietin; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Ischemia, Brain; Hypoxia, Brain; Infarction, Middle Cerebral Artery; Male; Mice; Nerve Degeneration; Oxidative Stress; Time Factors

We explored the effects of exogenous and endogenous erythropoietin (EPO) in a seizure model of rat. Adult male Fischer 344 rats received continuous intraventricular infusion of EPO dissolved in saline containing 1mg/ml of rat serum albumin, anti-EPO antibody, saline containing 1mg/ml of rat serum albumin or combined EPO and neuropeptide Y (NPY) Y2-receptor antagonist. Animals were behaviorally evaluated for seizure development over 6h after kainic acid injection followed by immunohistochemical assays. Mortality rate, seizure severity, apoptotic cell death and abnormal cell proliferation in the hippocampus of EPO-treated epileptic rats were significantly attenuated, compared to control rats. Anti-EPO antibody in non-EPO-treated animals worsened seizures and CA1 neuronal cell death, while NPY Y2-receptor antagonist cancelled the therapeutic effects of exogenous EPO. Both exogenous and endogenous EPO might modulate seizure severity and protect the hippocampal neurons in epileptic rats, via novel mechanistic pathways involving blockade of epileptogenic cell formation coupled with NPY receptor modulation in the hippocampus.

Topics: Animals; Apoptosis; Autoantibodies; CA1 Region, Hippocampal; Cell Proliferation; Dentate Gyrus; Disease Models, Animal; Erythropoietin; Hematopoiesis; Kainic Acid; Male; Neurons; Neuropeptide Y; Rats; Rats, Inbred F344; Receptors, Neuropeptide Y; Seizures; Severity of Illness Index; Signal Transduction; Up-Regulation

Erythropoietin (EPO) functions as a tissue-protective cytokine in addition to its crucial hormonal role in red cell production and neuron protection. This study aimed to determine the neuron protective effect of erythropoietin on experimental rats enduring spinal cord injury (SCI) by assessing thrombospondin-1 (TSP-1) level and transforming growth factor-beta (TGF-beta) in the development of a rat model of SCI.. Sixty Sprague-Dawley rats were randomly assigned to three groups: sham operation control group, SCI group and EPO treatment group. By using a weight-drop contusion SCI model, the rats in the SCI group and EPO treatment group were sacrificed at 24 hours and 7 days subsequently. The Basso, Beattie, and Bresnahan (BBB) scores were examined for locomotor function. Pathological changes were observed after HE staining. The expressions of thrombospondin-2 (TSP-1) and TGF-beta were determined by immunohistochemical staining and Western blotting.. Slighter locomotor dysfunction was discovered and it was recovered abruptly as higher BBB scores were found in the EPO treatment group than in the SCI group (P < 0.01). Pathologically, progressive disruption of the dorsal white matter and regeneration of a few neurons were also observed in SCI rats. TSP-1 and TGF-beta expression increased at 24 hours and 7 days after SCI in the injured segment, and it was higher in the SCI group than in the EPO treatment group. Spinal cord samples from the animals demonstrated a TSP-1 optical density of 112.2 +/- 6.8 and TSP-1 positive cells of 5.7 +/- 1.3 respectively. After injury, the TSP-1 optical density and cell number increased to 287.2 +/- 14.3/mm(2) and 23.2 +/- 2.6/mm(2) at 24 hours and to 232.1 +/- 13.2/mm(2) and 15.2 +/- 2.3/mm(2) at 7 days respectively. When EPO treated rats compared with the SCI rats, the TSP-1 optical density and cell number decreased to 213.1 +/- 11.6/mm(2) and 11.9 +/- 1.6/mm(2) at 24 hours and to 189.9 +/- 10.5/mm(2) and 9.3 +/- 1.5/mm(2) at 7 days, respectively (P < 0.01). In the SCI rats, the TGF-beta optical density and positive neuron number were 291.4 +/- 15.2/mm(2) and 28.8 +/- 4.9/mm(2) at 24 hours and 259.1 +/- 12.3/mm(2) and 23.9 +/- 4.1/mm(2) at 7 days respectively. They decreased in the EPO treated rats to 222.8 +/- 11.9/mm(2) and 13.7 +/- 2.1/mm(2) at 24 hours and to 196.5 +/- 9.7/mm(2) and 8.7 +/- 2.2/mm(2) at 7 days (P < 0.01).. Increased expression of TSP-1 and TGF-beta can be found in the injured segment of the spinal cord at 24 hours and 7 days after injury. EPO treatment can effectively prevent pathological alterations from severe spinal cord injury by reduced expression of TSP-1 and TGF-beta.

Topics: Animals; Blotting, Western; Disease Models, Animal; Erythropoietin; Female; Immunohistochemistry; Neuroprotective Agents; Random Allocation; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Thrombospondin 1; Transforming Growth Factor beta

The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2(V617F)) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish ortholog of human JAK2(V617F) (referred herewith jak2a(V581F)) by site-directed mutagenesis and examined its relevance as a model of human PV.. Zebrafish embryos at one-cell stage were injected with jak2a(V581F) mRNA (200pg/embryo). In some experiments, the embryos were treated with a specific JAK2 inhibitor, TG101209. The effects of jak2a stimulation on hematopoiesis, jak/stat signaling, and erythropoietin signaling were evaluated at 18-somites.. Injection with jak2a(V581F) mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp(+) population in dissociated Tg(gata1:gfp) embryos. The response was reduced by stat5.1 morpholino coinjection (control: 4.37% +/- 0.08%; jak2a(V581F) injected: 5.71% +/- 0.07%, coinjecting jak2a(V581F) mRNA and stat5.1 morpholino: 4.66% +/- 0.13%; p<0.01). jak2a(V581F) mRNA also upregulated gata1 (1.83 +/- 0.08 fold; p=0.005), embryonic alpha-hemoglobin (1.61 +/- 0.12 fold; p=0.049), and beta-hemoglobin gene expression (1.65 +/- 0.13-fold; p=0.026) and increased stat5 phosphorylation. These responses were also ameliorated by stat5.1 morpholino coinjection or treatment with a specific JAK2 inhibitor, TG101209. jak2a(V581F) mRNA significantly reduced erythropoietin gene (0.24 +/- 0.03 fold; p=0.006) and protein expression (control: 0.633+/-0.11; jak2a(V581F) mRNA: 0.222+/-0.07 mIU/mL; p=0.019).. The zebrafish jak2a(V581F) model shared many features with human PV and might provide us with mechanistic insights of this disease.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Base Sequence; Blotting, Western; Disease Models, Animal; Embryo, Nonmammalian; Erythropoiesis; Erythropoietin; Gene Expression Regulation, Developmental; Humans; Janus Kinase 2; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Polycythemia Vera; Protein-Tyrosine Kinases; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; STAT5 Transcription Factor; Sulfonamides; Zebrafish; Zebrafish Proteins

Intracerebral hemorrhage (ICH) is a devastating clinical syndrome for which no truly efficacious therapy has yet been identified. In preclinical studies, erythropoietin (EPO) and its long-lasting analog, darbepoetin alfa, have been demonstrated to be neuroprotective in several models of neuronal insult. The objectives of this study were to analyze whether the systemic administration of recombinant human EPO (rHuEPO) and its long-lasting derivative darbepoetin alfa expedited functional recovery and brain damage in a rat model of ICH.. Experimental ICH was induced in rats by injecting autologous blood into the right striatum under stereotactic guidance. Subsequently, animals underwent placebo treatment, daily injections of rHuEPO, or weekly injections of darbepoetin alfa. Animals were killed 14 days after injury.. Both rHuEPO and darbepoetin alfa were effective in reducing neurological impairment after injury, as assessed by the neurological tasks performed. rHuEPO- and darbepoetin alfa-treated animals exhibited a restricted brain injury with nearly normal parenchymal architecture. In contrast, the saline-treated group exhibited extensive cerebral cytoarchitectural disruption and edema. The number of surviving NeuN-positive neurons was significantly higher in the rats treated with rHuEPO and darbepoetin alfa compared with those that received saline (P < 0.05).. These results demonstrate that weekly administered darbepoetin alfa confers behavioral and histological neuroprotection after ICH in rats similar to that of daily EPO administration. Administration of EPO and its long-lasting recombinant forms affords significant neuroprotection in an ICH model and may hold promise for future clinical applications.

Topics: Animals; Basal Ganglia Hemorrhage; Blood Transfusion, Autologous; Brain; Brain Edema; Brain Infarction; Cerebral Hemorrhage; Corpus Striatum; Darbepoetin alfa; Disease Models, Animal; Drug Administration Schedule; Erythropoietin; Hematinics; Humans; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Treatment Outcome

The aim of the study is to investigate the effects of erythropoietin on torsion/detorsion injury in rats.. Forty rats were divided randomly into 5 groups: group I (sham, S), sham operation; group II (torsion/detorsion 1, T/D(1)), 3 hours ischemia and 1 hour reperfusion; group III (torsion/detorsion 2, T/D(2)), 3 hours ischemia and 48 hours reperfusion; group IV (erythropoietin 1, EPO(1)), 3 hours ischemia, 1 hour reperfusion, and a single dose of EPO; and group V (erythropoietin 2, EPO(2)), 3 hours ischemia, 48 hours reperfusion, and 2 doses of EPO. Malondialdehyde (MDA) and nitric oxide (NO) levels and activities of superoxide dismutase and catalase were measured. Tissue damage to ovarian tissue was scored by histologic examination. Data were compared among groups with parametric tests.. The MDA levels in the S and EPO groups were significantly lower than the T/D groups (P < .001). Catalase and superoxide dismutase activities, and NO levels in the S and EPO groups were significantly higher than in the T/D groups (P < .05). Ovarian tissue damage in the S and EPO groups was significantly less than in the T/D groups (P < .05). Levels of all biochemical markers and ovarian tissue damage scores were similar among the S, EPO(1), and EPO(2) groups (P > .05).. Erythropoietin attenuates ischemia-reperfusion injury when given during the acute phase of ovarian torsion-detorsion in a rat model.

Topics: Animals; Antioxidants; Catalase; Disease Models, Animal; Erythropoietin; Female; Malondialdehyde; Nitric Oxide; Ovarian Diseases; Ovary; Random Allocation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury; Superoxide Dismutase; Torsion Abnormality

Bronchopulmonary dysplasia (BPD) is a multifactorial disease resulting from the impact of injury (including oxygen toxicity, barotrauma, volutrauma, and infection) on the immature lung. Oxygen toxicity is thought to be a major contributing factor in the pathogenesis in BPD. Previous animal studies have shown that exposure to hyperoxia in the neonatal period causes lung structural changes that are similar to the histology seen in human infants with BPD. Erythropoietin (EPO) has pleiotropic actions including antioxidant, anti-apoptotic, anti-inflammatory and angiogenic effects. Animal experiments reveal that EPO may have protective effects on hyperoxic lung injury, but the mechanisms remain unknown. The aim of the study was to evaluate the anti-inflammatory effects and understand mechanism of action of EPO on the hyperoxia-induced BPD in newborn rats.. Several litters of Wistar pups were pooled together within 12 hours after birth and randomly divided into four groups: I. air-exposed control group, II. air-exposed human recombinant erythropoietin (rhEPO)-treated group, III. hyperoxia-exposed placebo group and IV. hyperoxia-exposed rhEPO-treated group . Group III and IV rats were exposed to 85% oxygen. Group II and IV rats received rhEPO (1200 IU/kg) subcutaneously on postnatal days 0 and 2. Group I and III received 0.9% saline in the same way. Pups from each group were sacrificed on days 3, 7, and 14. Blood hemoglobin concentration, hematocrit and platelet count were determined by blood cell analyzer. Total protein content in bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) were measured by biochemical assay. Changes of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) mRNA expressions were measured by RT-PCR.. In group III, there were a few inflammatory cells infiltrations in interstitium on day 3 and inflammatory response worsened on day 7. Alveolar and capillary hypoplasia and interstitial fibrosis were evident on day 14. The pathological changes were ameliorated greatly in group IV and the survival was prolonged. There were no abnormal raises of hemoglobin concentration, hematocrit and platelet count in group IV compared with group I. Total protein concentration in BALF was measured as a marker for capillary leakage. MPO is a major constituent of neutrophil cytoplasmic granules and its activity therefore is a direct measure of neutrophil presence and an indirect indicator of lung injury. Total protein concentration and MPO in BALF were greatly depressed in group IV compared with group III, P<0.05, P<0.001. The upregulation of genes of CINC-1 and MCP-1 was closely related with lung inflammation caused by oxidative stress. MCP-1 and CINC-1 mRNA expression were detected and it was found that their changes were in line with the degree of lung inflammation. MCP-1 and CINC-1 mRNA expression increased in group III compared with group I especially on day 7, P<0.01 or <0.001. The changes of MCP-1 and CINC-1 mRNA were positively correlated with changes of MPO in BALF covering all groups on days 3, 7 and 14, respectively (r = 0.391, P<0.05; r = 0.701, P<0.01; r = 0.600, P<0.01; r = 0.471, P<0.01; r = 0.789, P<0.01; r = 0.588, P<0.01).. EPO could significantly reduce the lung inflammatory cell infiltration, and capillary endothelial cell injury in hyperoxic lung injury in newborn rats. The mechanism may be related with the inhibition of MCP-1 and CINC-1 gene expression by EPO.

Topics: Animals; Animals, Newborn; Chemokine CCL2; Disease Models, Animal; Erythropoietin; Female; Hyperoxia; Lung; Lung Injury; Male; Rats; Rats, Wistar; Recombinant Proteins

Erythropoietin (Epo) is a hormone which regulates erythrocyte production. It has recently become known that Epo enhances angiogenesis. However, since shear stress is an initiator of arteriogenesis, this increase with Epo may be due to increased shear stress from erythrocytosis. To clarify this, we compared the effects of Epo on both angiogenesis and arteriogenesis. Myocardial infarction was induced by LAD ligation in Wistar rats (Epo, G-CSF and control). Epo (1,000 IU/kg) was administered immediately after ligation of the LAD. G-CSF was administered at 100 microg/kg/day for 5 days after the coronary ligation. Four weeks later, coronary angiography was performed using synchrotron radiation coronary micro-angiography with a Langendorff apparatus. The number of vessels was investigated by microscopy. The numbers of capillaries and arterioles (> 100 microm in diameter) were measured. Microscopical examination: Capillary density in the twilight zone was 95 +/- 19 in the control group, 126 +/- 24 in the G-CSF group, and 142 +/- 32 in the EPO group (control versus Epo: P < 0.005, control versus G-CSF: P < 0.05). Arteriole numbers were 4.3 +/- 0.2 in the control group, 6.9 +/- 1.0 in the G-CSF, and 11.8 +/- 0.6 in the Epo group (control versus Epo: P < 0.00001, G-CSF versus Epo: P < 0.00001, control versus G-CSF: P < 0.00001). The ratios of arterioles and capillaries were 0.048 +/- 0.013 in the control group, 0.057 +/- 0.016 in the G-CSF group, and 0.088 +/- 0.019 in the Epo group (control versus Epo: P < 0.0005, G-CSF versus Epo: P < 0.05). Angiography: The number of crossing arterioles in the 2 mm lattice was 5.4 +/- 1.7 in the Epo group and 3.8 +/-0.4 in the control group (P < 0.05). The gray scale values for the evaluation of capillaries was 128 +/- 3.7 and 119 +/- 2.1 in the Epo and control groups, respectively (P < 0.00005). Epo enhanced arterioles more significantly than it did capillaries in this infarcted rat heart model.

Topics: Angiography; Animals; Arterioles; Capillaries; Coronary Angiography; Disease Models, Animal; Erythropoietin; Male; Myocardial Infarction; Rats; Rats, Wistar; Synchrotrons

The accumulated knowledge of erythropoietin (EPO) interaction in neural injury has led to potentially novel therapeutic strategies. Previous experimental studies of recombinant human EPO (rhEPO) administration have shown favorable results after central and peripheral neural injury. In the present study we used the aneurysmal clip model to evaluate the efficacy of two different regimes of rhEPO administration on the functional outcome after severe acute spinal cord injury (ASCI).. Thirty rats were operated on with posterior laminectomy at thoracic 10th vertebra. Spinal cord trauma produced by extradural placement of the aneurysm clip, for 1 min. Animals were divided into three groups; the first group received a low total EPO dose (EPO-L), (2 doses of 1,000 IU each s.c.). The second group was administered the high total EPO dose (EPO-H), (14 doses of 1,000 IU each s.c.), and the third was the Control group, which received normal saline in the same time fashion with EPO-H group. Follow-up was for 6 weeks. Estimation of the functional progress of each rat was calculated using the locomotor rating scale of Basso et al, with a range from 0 to 21.. After surgery the animals suffered paraplegia with urinary disturbances. Rats that received EPO demonstrated statistically significant functional improvement compared to the Control group, throughout study interval. On the last follow-up at 6 weeks the EPO-L rats achieved a mean score 17.3 +/- 1.15, the EPO-H 14.7 +/- 1.82, and the control group 8.2 +/- 0.78. Comparison between the two EPO groups reveals superior final outcome of the group treated with lower total dose.. Our study supports current knowledge, that EPO administration has a positive effect on functional recovery after experimental ASCI. These data reflect the positive impact of EPO on the pathophysiologic cascade of secondary neural damage. However, we observed a dose-related effect on functional recovery. Interestingly, large doses do not seem to favor the neurological recovery as lower doses do.

Topics: Animals; Central Nervous System Agents; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Female; Rats; Rats, Wistar; Recombinant Proteins; Spinal Cord Injuries; Treatment Outcome

We previously demonstrated that erythropoietin (EPO)-secreting mesenchymal stromal cells (MSC) can be used for the long-term correction of renal failure-induced anemia. The present study provides evidence that coimplantation of insulin-like growth factor I (IGF-I)-overexpressing MSC (MSC-IGF) improves MSC-based gene therapy of anemia by providing paracrine support to EPO-secreting MSC (MSC-EPO) within a subcutaneous implant. IGF-I receptor RNA expression in murine MSC was demonstrated by RT-PCR. Functional protein expression was confirmed by immunoblots and MSC responsiveness to IGF-I stimulation in vitro. IGF-I was also shown to improve MSC survival following staurosporin-induced apoptosis in vitro. A cohort of C57Bl/6 mice was rendered anemic by right kidney electrocoagulation and left nephrectomy. MSC-EPO were subsequently admixed in a bovine collagen matrix and implanted, in combination with MSC-IGF or MSC null, by subcutaneous injection in renal failure mice. In mice receiving MSC-EPO coimplanted with MSC-IGF, hematocrit elevation was greater and enhanced compared with control mice; heart function was also improved. MSC-IGF coimplantation, therefore, represents a promising new strategy for enhancing MSC survival within implanted matrices and for improving cell-based gene therapy of renal anemia.

Topics: Anemia; Animals; Apoptosis; Cells, Cultured; Disease Models, Animal; Erythropoietin; Female; Genetic Engineering; Genetic Therapy; Hematocrit; Insulin-Like Growth Factor I; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Receptor, IGF Type 1; Renal Insufficiency; RNA, Messenger

Percutaneous vascular interventions lead inevitably to a destruction of the endothelial lining at the site of injury. There are conflicting data on the therapeutic benefit of hematopoietic growth factors aiming at mobilisation of circulating endothelial cells to accelerate the reendothelialisation process. Aim of our study was to evaluate the impact of a maximised 7 day-combination therapy with G-CSF plus EPO on the healing process following balloon injury of the rat carotid artery.. Osmotic pumps to systemically deliver G-CSF and EPO at saturating doses during the first seven days post injury were implanted into the peritoneal cavity of splenectomised male Sprague-Dawley rats. Cytokine treatment resulted in significantly elevated numbers of white blood cells, hematocrit levels, circulating hematopoietic stem cells, and-temporarily-circulating endothelial precursors. Functional reendothelialisation as assessed by Evan's blue staining on day 14 post injury was not affected by the cytokine treatment. The neointimal response was analysed on day 7 and 28 post injury, and was significantly higher at the day 7 timepoint (Cytokines: I/M-ratio 1.10+/-0.09 vs. Saline: 0.36+/-0.06). Cytokine treated rats also displayed higher rates of thrombotic occlusion (Cytokines: 25-33% vs. Saline: 0%). Serum levels of PAI-1, TGFbeta1, and PDGF-BB were not elevated in the cytokine treated rats. The proliferative rates both in the injured vessel wall and the surrounding adventitia as assessed by BrdU incorporation were significantly higher in the cytokine treated animals. The number of CD45(pos) hematopoietic cells was significantly higher in the adventitia of the cytokine treated rats. Vasa vasorum were not found to be significantly different. The increased neointimal response was not due to expression of G-CSF- or EPO-receptors on VSMCs within the vessel wall or myofibroblasts in the adventitia.. The systemic administration of G-CSF plus EPO during the first week after balloon-injury impairs the vascular healing process by increasing the neointimal response and the risk for thrombotic occlusion.

Topics: Animals; Becaplermin; Carotid Artery Injuries; Catheterization; Disease Models, Animal; Drug Therapy, Combination; Endothelial Cells; Erythropoietin; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Mobilization; Hyperplasia; Immunohistochemistry; Leukocyte Common Antigens; Male; Plasminogen Activator Inhibitor 1; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Rats; Rats, Sprague-Dawley; Thrombosis; Transforming Growth Factor beta1; Tunica Intima; Wound Healing

We investigated the effect of an erythropoietin (EPO)-gelatin hydrogel drug delivery system (DDS) applied to the heart on myocardial infarct (MI) size, left ventricular (LV) remodelling and function.. Experiments were performed in a rabbit model of MI. The infarct size was reduced, and LV remodelling and function were improved 14 days and 2 months after MI but not at 2 days after MI in the EPO-DDS group. The number of cluster of differentiation 31(CD31)-positive microvessels and the expression of erythropoietin receptor (EPO-R), phosphorylated-Akt (p-Akt), phosphorylated glycogen synthase kinase 3beta (p-GSK-3beta), phosphorylated extracellular signal-regulated protein kinase (p-ERK), phosphorylated signal transducer and activator of transcription 3 (p-Stat3), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-1 (MMP-1) were significantly increased in the myocardium of the EPO-DDS group.. Post-MI treatment with an EPO-DDS improves LV remodelling and function by activating prosurvival signalling, antifibrosis, and angiogenesis without causing any side effect.

Topics: Animals; Blotting, Western; Cardiovascular Agents; Cell Survival; Chemistry, Pharmaceutical; Disease Models, Animal; Dosage Forms; Drug Carriers; Erythropoietin; Fibrosis; Gelatin; Humans; Hydrogels; Immunohistochemistry; Injections, Subcutaneous; Intracellular Signaling Peptides and Proteins; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Rabbits; Recombinant Proteins; Signal Transduction; Time Factors; Ventricular Function, Left; Ventricular Remodeling

Topics: Animals; Autocrine Communication; Cell Differentiation; Cell Proliferation; Cell Survival; Disease Models, Animal; Erythropoietin; Genetic Therapy; Humans; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Paracrine Communication; Regeneration; Stromal Cells; Ventricular Function, Left

Aortic occlusion causes ischemia/reperfusion injury, kidney and spinal cord being the most vulnerable organs. Erythropoietin improved ischemia/reperfusion injury in rodents, which, however, better tolerate ischemia/reperfusion than larger species. Therefore, we investigated whether erythropoietin attenuates porcine aortic occlusion ischemia/reperfusion injury.. Before occluding the aorta for 45 mins by inflating intravascular balloons, we randomly infused either erythropoietin (n = 8; 300 IU/kg each over 30 mins before and during the first 4 hrs of reperfusion) or vehicle (n = 6). During aortic occlusion, mean arterial pressure was maintained at 80% to 120% of baseline by esmolol, nitroglycerine, and adenosine 5'-triphosphate. During reperfusion, noradrenaline was titrated to keep mean arterial pressure >80% of baseline. Kidney perfusion and function were assessed by fractional Na-excretion, p-aminohippuric acid and creatinine clearance, spinal cord function by lower extremity reflexes and motor evoked potentials. Blood isoprostane levels as well as blood and tissue catalase and superoxide dismutase activities allowed evaluation of oxidative stress. After 8 hrs of reperfusion, kidney and spinal cord specimens were taken for histology (hematoxylin-eosin, Nissl staining) and immunohistochemistry (TUNEL assay for apoptosis).. Parameters of oxidative stress and antioxidative activity were comparable. Erythropoietin reduced the noradrenaline requirements to achieve the hemodynamic targets and may improve kidney function despite similar organ blood flow, histology, and TUNEL staining. Neuronal damage and apoptosis was attenuated in the thoracic spinal cord segments without improvement of its function.. During porcine aortic occlusion-induced ischemia/reperfusion erythropoietin improved kidney function and spinal cord integrity. The lacking effect on spinal cord function was most likely the result of the pronounced neuronal damage associated with the longlasting ischemia.

Topics: Animals; Arterial Occlusive Diseases; Balloon Occlusion; Disease Models, Animal; Erythropoietin; Evoked Potentials, Motor; Female; Hemodynamics; Kidney Function Tests; Male; Oxidative Stress; Reperfusion Injury; Spinal Cord Injuries; Swine

Erythrocyte production is regulated by balancing precursor cell apoptosis and survival signaling. Previously, we found that BH3-only proapoptotic factor, Nix, opposed erythroblast-survival signaling by erythropoietin-induced Bcl-xl during normal erythrocyte formation. Since erythropoietin treatment of human anemia has limitations, we explored the therapeutic potential of abrogating Nix-mediated erythroblast apoptosis to enhance erythrocyte production. Nix gene ablation blunted the phenylhydrazine-induced fall in blood count, enhanced hematocrit recovery, and reduced erythroblast apoptosis, despite lower endogenous erythropoietin levels. Similar to erythropoietin, Nix ablation increased early splenic erythroblasts and circulating reticulocytes, while maintaining a pool of mature erythroblasts as erythropoietic reserve. Erythrocytes in Nix-deficient mice showed morphological abnormalities, suggesting that apoptosis during erythropoiesis not only controls red blood cell number, but also serves a "triage" function, preferentially eliminating abnormal erythrocytes. These results support the concept of targeting erythroblast apoptosis to maximize erythrocyte production in acute anemia, which may be of value in erythropoietin resistance.

Topics: Anemia; Animals; Apoptosis; Bone Marrow; Cell Differentiation; Cell Proliferation; Cell Survival; Disease Models, Animal; Down-Regulation; Erythroblasts; Erythrocyte Count; Erythrocytes; Erythropoietin; Gene Targeting; Membrane Proteins; Mice; Mice, Knockout; Mitochondrial Proteins; Oxidants; Phenylhydrazines; Recovery of Function

In this study, we attempt to determine whether lycopene regulates inflammatory mediators in the ovalbumin (OVA)-induced murine asthma model. To address this, mice were sensitized and challenged with OVA, and then treated with lycopene before the last OVA challenge. Administration of lycopene significantly alleviated the OVA-induced airway hyperresponsiveness to inhaled methacholine. Administration of lycopene also resulted in a significant inhibition of the infiltration of inflammatory immunocytes into the bronchoalveolar lavage, and attenuated the gelatinolytic activity of matrix metalloproteinase-9 and the expression of eosinophil peroxidase. Additionally, lycopene reduced the increased levels of GATA-3 mRNA level and IL-4 expression in OVA-challenged mice. However, it increased T-bet mRNA level and IFN-gamma expression in lycopene-challenged mice. These findings provide new insight into the immunopharmacological role of lycopene in terms of its effects in a murine model of asthma.

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Carotenoids; Disease Models, Animal; Eosinophils; Erythropoietin; Female; GATA3 Transcription Factor; Inflammation Mediators; Interleukin-4; Lycopene; Lymphocytes; Macrophages; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred BALB C; Neutrophils; Ovalbumin; RNA, Messenger

Anemia in critically ill patients is frequent and consists of chronic disease associated with blood losses. These two mechanisms have opposite effects on iron homeostasis, especially on the expression of the iron regulatory hormone hepcidin. We developed a mouse model mimicking the intensive care anemia to explore iron homeostasis.. Experimental study.. University-based research laboratory.. C57BL/6 mice.. Mice received either a single intraperitoneal injection of lipopolysaccharide followed 1 week later by zymosan, or were subjected to repeated phlebotomies by retro-orbital punctures, or both. Several subsets of mice were analyzed over a 14-day period to describe the mouse model of intensive care anemia. Additional mice received erythropoietin injections with or without the zymosan treatment and were killed at day 5, to perform a more detailed analysis.. We observed anemia as soon as 5 days after zymosan injection, together with increased messenger RNA (mRNA) levels for interleukin-6 and hepcidin. Phlebotomies alone fully suppressed hepcidin mRNA expression. Interestingly, in mice treated with zymosan and phlebotomies, hepcidin expression was suppressed, despite the persistent increase in interleukin-6. Stimulation of erythropoiesis by erythropoietin injections also led to a decrease in hepcidin mRNA in zymosan-treated mice. In these situations combining inflammation and erythropoiesis stimulation, there was no change in ferroportin, the membrane iron exporter, at the mRNA level, whereas ferroportin protein increased. Macrophage iron stores (assessed by histology using diaminobenzidine staining, or by quantification of nonheme iron and ferritin concentrations) were depleted in the spleen.. These results suggest that the erythroid factor dominates over inflammation for hepcidin regulation, and that iron could be mobilized in these situations combining inflammation and erythropoiesis stimulation.

Topics: Anemia; Animals; Antimicrobial Cationic Peptides; Cation Transport Proteins; Cell Membrane; Disease Models, Animal; Erythropoietin; Hepcidins; Homeostasis; Interleukin-6; Iron; Male; Mice; Mice, Inbred C57BL; Microsomes, Liver; Phlebotomy; RNA, Messenger

Topics: Animals; Disease Models, Animal; Erythropoietin; Humans; Neuroprotective Agents; Reperfusion Injury; Species Specificity; Spinal Cord Ischemia

Topics: Animals; Awards and Prizes; Child; Child, Preschool; Disease Models, Animal; Erythropoietin; Humans; Infant, Newborn; Insulin-Like Growth Factor I; Ophthalmology; Retinal Vessels; Retinopathy of Prematurity; Societies, Medical; United States; Vascular Endothelial Growth Factor A

Erythropoietin (EPO) has recently been demonstrated to have a tissue protective role by acting as an anti-apoptotic agent in various tissues, such as brain, spinal cord, heart and kidney. The purpose of this study was to determine whether human recombinant EPO reduces epithelial cell apoptosis and attenuates bleomycin-induced pneumonitis in mice.. Bleomycin was instilled intratracheally into C57BL/6 mice. Recombinant human EPO or saline was injected intraperitoneally, daily from day 5 to day 13 after bleomycin instillation.. EPO receptor was expressed in bronchiolar and alveolar type II cells. At 14 days after instillation, the number of terminal uridine deoxynucleotidyl transferase nick end-labelled positive cells in the lung was decreased, and the histological degree of inflammation and fibrosis was attenuated in mice injected with EPO compared with controls. Expression of phosphorylated Akt and Erk, which are thought to mediate the survival signalling pathway induced by EPO, tended to be increased in lung tissues from mice treated with EPO compared with those from mice treated with saline after bleomycin instillation.. As it is likely that EPO protects epithelial cells from injury and apoptosis, EPO administration could be a potential therapeutic strategy for the prevention of lung injury.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Bleomycin; Disease Models, Animal; Epithelial Cells; Erythropoietin; Extracellular Signal-Regulated MAP Kinases; Humans; Male; Mice; Mice, Inbred C57BL; Pneumonia; Proto-Oncogene Proteins c-akt; Pulmonary Alveoli; Receptors, Erythropoietin; Recombinant Proteins

This study was designed to investigate the beneficial effects of recombinant human erythropoietin (rhEPO) treatment of traumatic brain injury (TBI) in mice.. Adult male C57BL/6 mice were divided into 3 groups: 1) the saline group (TBI and saline [13 mice]); 2) EPO group (TBI and rhEPO [12]); and 3) sham group (sham and rhEPO [8]). Traumatic brain injury was induced by controlled cortical impact. Bromodeoxyuridine (100 mg/kg) was injected daily for 10 days, starting 1 day after injury, for labeling proliferating cells. Recombinant human erythropoietin was administered intraperitoneally at 6 hours and at 3 and 7 days post-TBI (5000 U/kg body weight, total dosage 15,000 U/kg). Neurological function was assessed using the Morris water maze and footfault tests. Animals were killed 35 days after injury, and brain sections were stained for immunohistochemical evaluation.. Traumatic brain injury caused tissue loss in the cortex and cell loss in the dentate gyrus (DG) as well as impairment of sensorimotor function (footfault testing) and spatial learning (Morris water maze). Traumatic brain injury alone stimulated cell proliferation and angiogenesis. Compared with saline treatment, rhEPO significantly reduced lesion volume in the cortex and cell loss in the DG after TBI and substantially improved recovery of sensorimotor function and spatial learning performance. It enhanced neurogenesis in the injured cortex and the DG.. Recombinant human erythropoietin initiated 6 hours post-TBI provided neuroprotection by decreasing lesion volume and cell loss as well as neurorestoration by enhancing neurogenesis, subsequently improving sensorimotor and spatial learning function. It is a promising neuroprotective and neurorestorative agent for TBI and warrants further investigation.

Topics: Animals; Brain Injuries; Disease Models, Animal; Drug Administration Schedule; Epoetin Alfa; Erythropoietin; Hematinics; Male; Maze Learning; Mice; Mice, Inbred C57BL; Psychomotor Performance; Recombinant Proteins; Recovery of Function

One of the pathological hallmarks of periventricular leukomalacia (PVL) is the selective vulnerability of late oligodendrocyte progenitors (preoligodendrocytes; preOLs) to hypoxia-ischemia (H-I). It is unknown whether recombinant human erythropoietin (rhEPO) protects preOLs in vivo.. To develop a rat PVL model in which preOLs are selectively damaged and exhibit similar pathological changes to diffuse-type human PVL, various conditions of H-I were compared in P2-P7 rats (P2 = postnatal day 2). To evaluate the effect of rhEPO on oligoprotection (preOLs), rhEPO was administered to P3 PVL rats.. After counts of NG2-positive and O4-positive cells were performed in P2-P7 rats, right common carotid artery occlusion followed by 6% O(2) for 0-120 min was performed in P2-P4 rats. The mortality and histological alterations after hematoxylin/eosin staining and ED1 immunostaining were assessed 2 days after H-I. Various doses of rhEPO (1-30,000 U/kg i.p.) were administered to PVL rats 15 min before administration of 6% O(2).. Double-positive cells for NG2 and O4 were detected from P2, and their number gradually increased until P7. Although right common carotid artery occlusion with 6% O(2) for 60 min resulted in a relatively high proportion of deaths in P2-P4 rats, typical histological changes in the PVL diffuse component were found in most surviving P3 animals. With 50-100 U/kg rhEPO, the histological damage was attenuated.. Histological changes similar to those seen in the PVL diffuse component were induced by H-I in P3 rats, in which preOLs were gradually developing, and a low dose of rhEPO was effective in the treatment of brain damage induced by H-I.

Topics: Animals; Animals, Newborn; Antigens; Disease Models, Animal; Erythropoietin; Female; Humans; Hypoxia-Ischemia, Brain; Hypoxia, Brain; Immunohistochemistry; Infant, Newborn; Leukomalacia, Periventricular; Oligodendroglia; Pregnancy; Proteoglycans; Rats; Rats, Wistar; Stem Cells

Head trauma is a dynamic process characterized by a cascade of metabolic and molecular events. Erythropoietin (EPO) has been shown to have neuroprotective effects in animal models of traumatic brain injury (TBI). Acute in vivo mechanisms and pathological changes associated with EPO following TBI are unknown. In this study the authors compare acute metabolic and pathological changes following TBI with and without systemically administered EPO.. Right frontal lobe microdialysis cannulae and right parietal lobe percussion hubs were inserted into 16 Sprague-Dawley rats. After a 4- to 5-day recovery, TBI was induced via a DragonFly fluid-percussion device at 2.5-2.8 atm. Rats were randomized into 2 groups, which received 5000 U/kg EPO or normal saline intraperitoneally 30 minutes after TBI. Microdialysis samples for glucose, lactate, pyruvate, and glutamate were obtained every 25 minutes for 10 hours. Rats were killed, their brains processed for light microscopy, and sections stained with H & E.. Erythropoietin administered 30 minutes after TBI directly affects acute brain metabolism. Brains treated with EPO maintain higher levels of glucose 4-10 hours after TBI (p<0.01), lower levels of lactate 6-10 hours after TBI (p<0.01), and lower levels of pyruvate 7.5-10 hours after TBI (p<0.01) compared with saline-treated controls. Erythropoietin maintains aerobic metabolism after TBI. Systemic EPO administration reduces acute TBI-induced lesion volume (p<0.05).. Following TBI, neuron use initially increases, with subsequent depletion of extracellular glucose, resulting in increased levels of extracellular lactate and pyruvate. This energy requirement can result in cell death due to increased metabolic demands. These data suggest that the neuroprotective effect of EPO may be partially due to improved energy metabolism in the acute phase in this rat model of TBI.

Topics: Acute Disease; Animals; Brain Injuries; Disease Models, Animal; Energy Metabolism; Erythropoietin; Glucose; Glutamic Acid; Lactic Acid; Microdialysis; Neuroprotective Agents; Pyruvic Acid; Rats; Rats, Sprague-Dawley

This study sought to determine the bio-availability of recombinant human erythropoietin (EPO) in the brain and blood and its effects on the cerebral concentrations of the inflammatory mediators interleukin-1beta (IL-1beta) and macrophage-inflammation protein-2 (MIP-2) following lateral fluid percussion brain injury (FPI) in the rat. After induction of moderate FPI (1.6-1.8 atm), EPO was injected intraperitoneally (IP) or intravenously (IV) at doses of 1000-5000 U/kg in a randomized and blinded manner. Animals were then sacrificed at time points (4, 8, 12, 24 h) post-trauma, and the brain concentrations of EPO, IL-1beta, and MIP-2 were determined. EPO administration leads to a dose-dependent increase in the brain concentration of the drug; however, this could only be detected at doses of 3000 and 5000 U/kg. The cerebral concentration peaked in the first 4 h following trauma. EPO concentrations were significantly higher and decreased more slowly in the traumatized cortex compared to the contralateral side (p<0.0125). IV EPO (5000 U/kg) produced slightly higher concentrations of EPO than same doses injected IP; however, this was not significant. At a dose of 5000 U/kg, EPO significantly reduced the increase in IL-1beta at 8 and 12 h in both cortical sides. It also reduced the increase in MIP-2 but only after 8 h, on the contralateral side and after 12 h on the ipsilateral side. Our results suggest that EPO crosses the blood-brain barrier (BBB) by 4 h after trauma and is localized primarily in the traumatized cortex. Further, it has biological efficacy at 8 h on several inflammatory proteins, yet must be employed at high doses to cross the BBB.

Topics: Animals; Blood-Brain Barrier; Brain; Brain Injuries; Chemokine CXCL2; Disease Models, Animal; Dose-Response Relationship, Drug; Down-Regulation; Drug Administration Schedule; Encephalitis; Erythropoietin; Functional Laterality; Humans; Injections, Intraperitoneal; Injections, Intravenous; Interleukin-1beta; Male; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Time Factors

In anemic patients with heart failure (HF), erythropoietin-type drugs can elicit clinical improvement. This study examined the effects of chronic monotherapy with darbepoetin-alpha (DARB) on left ventricular (LV) function and remodeling in nonanemic dogs with advanced HF. HF [LV ejection fraction (EF) approximately 25%] was produced in 14 dogs by intracoronary microembolizations. Dogs were randomized to once a week subcutaneous injection of DARB (1.0 microg/kg, n=7) or to no therapy (HF, n=7). All procedures were performed during cardiac catheterization under general anesthesia and under sterile conditions. LV end-diastolic volume (EDV), end-systolic volume (ESV), and EF were measured before the initiation of therapy and at the end of 3 mo of therapy. mRNA and protein expression of caspase-3, hypoxia inducible factor-1alpha, and the bone marrow-derived stem cell marker c-Kit were determined in LV tissue. In HF dogs, EDV and ESV increased and EF decreased after 3 mo of followup. Treatment with DARB prevented the increase in EDV, decreased ESV, and increased EF. DARB therapy also normalized the expression of HIF-1alpha and active caspase-3 and enhanced the expression of c-Kit. We conclude that chronic monotherapy with DARB prevents progressive LV dysfunction and dilation in nonanemic dogs with advanced HF. These results suggest that DARB elicits beneficial effects in HF that are independent of the presence of anemia.

Topics: Anemia; Animals; Bone Marrow Cells; Cardiotonic Agents; Caspase 3; Darbepoetin alfa; Disease Models, Animal; Disease Progression; Dogs; Erythropoietin; Heart Failure; Hematinics; Hematopoietic Stem Cells; Hypoxia-Inducible Factor 1, alpha Subunit; Injections, Subcutaneous; Myocardium; Proto-Oncogene Proteins c-kit; RNA, Messenger; Stroke Volume; Ventricular Dysfunction, Left; Ventricular Remodeling

Oxygen-induced retinopathy (OIR) in the mouse, like the analogous human disease retinopathy of prematurity, is an ischemic retinopathy dependent on oxygen-induced vascular obliteration. We tested the hypothesis that chemically overriding the oxygen-induced downregulation of hypoxia-inducible factor (HIF) activity would prevent vascular obliteration and subsequent pathologic neovascularization in the OIR model. Because the degradation of HIF-1alpha is regulated by prolyl hydroxylases, we examined the effect of systemic administration of a prolyl hydroxylase inhibitor, dimethyloxalylglycine, in the OIR model. Our results determine that stabilizing HIF activity in the early phase of OIR prevents the oxygen-induced central vessel loss and subsequent vascular tortuosity and tufting that is characteristic of OIR. Overall, these findings imply that simulating hypoxia chemically by stabilizing HIF activity during the causative ischemia phase (hyperoxia) of retinopathy of prematurity may be of therapeutic value in preventing progression to the proliferative stage of the disease.

Topics: Aerobiosis; Amino Acids, Dicarboxylic; Animals; Basic Helix-Loop-Helix Transcription Factors; Disease Models, Animal; Enzyme Inhibitors; Erythropoietin; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Infant, Newborn; Kidney; Liver; Mice; Oxygen; Procollagen-Proline Dioxygenase; Retina; Retinopathy of Prematurity; Vascular Endothelial Growth Factor A

The protective mechanism of recombinant human erythropoietin (rhEPO) on blood-brain barrier (BBB) after brain injury is associated with the attenuation of neuro-inflammation. We hypothesize that rhEPO treatment after intracerebral hemorrhage (ICH) modulates matrix metalloproteinase (MMP) activity, maintains BBB integrity, and reduces BBB breakdown-associated inflammation. Adult male 129S2/sv mice were subjected to autologous whole blood-induced ICH. rhEPO or saline was administered intraperitoneally immediately after surgery and for 3 more days until day of sacrifice. BBB permeability was measured by Evans blue leakage, and edema was assessed by brain water content. Immunofluorescence and Western blotting were performed to detect expression of tight junction marker occludin, type IV collagen, MMPs, tissue inhibitor of metalloproteinase (TIMP), and glial fibrillary acidic protein, rhEPO prevented Evans blue leakage, reduced brain edema, and preserved expression of occludin and collagen IV. rhEPO treatment decreased MMP-2 expression, increased TIMP-2 expression, and reduced the number of reactive astrocytes in the brain compared to saline control. We conclude that rhEPO reduces MMP activity, BBB disruption, and the glial cell inflammatory reaction 3 days after ICH. Our study provides additional evidence for the mechanism of rhEPO's neurovascular protective effects and a potential clinical application in the treatment of ICH.

Topics: Animals; Blood-Brain Barrier; Brain Edema; Cerebral Hemorrhage; Collagen Type IV; Disease Models, Animal; Erythropoietin; Evans Blue; Glial Fibrillary Acidic Protein; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinases; Membrane Proteins; Mice; Occludin; Permeability; Recombinant Proteins; Time Factors; Tissue Inhibitor of Metalloproteinases

Oxygen toxicity has been identified as a risk factor for adverse neurological outcome in survivors of preterm birth. In infant rodent brains, hyperoxia induces disseminated apoptotic neurodegeneration. Because a tissue-protective effect has been observed for recombinant erythropoietin (rEpo), widely used in neonatal medicine for its hematopoietic effect, we examined the effect of rEpo on hyperoxia-induced brain damage.. Six-day-old C57Bl/6 mice or Wistar rats were exposed to hyperoxia (80% O(2)) or normoxia for 24 hours and received rEpo or normal saline injections intraperitoneally. The amount of degenerating cells in their brains was determined by DeOlmos cupric silver staining. Changes of their brain proteome were determined through two-dimensional electrophoresis and mass spectrometry. Western blot, enzyme activity assays and real-time polymerase chain reaction were performed for further analysis of candidate proteins.. Systemic treatment with 20,000 IE/kg rEpo significantly reduced hyperoxia-induced apoptosis and caspase-2, -3, and -8 activity in the brains of infant rodents. In parallel, rEpo inhibited most brain proteome changes observed in infant mice when hyperoxia was applied exclusively. Furthermore, brain proteome changes after a single systemic rEpo treatment point toward a number of mechanisms through which rEpo may generate its protective effect against oxygen toxicity. These include reduction of oxidative stress and restoration of hyperoxia-induced increased levels of proapoptotic factors, as well as decreased levels of neurotrophins.. These findings are highly relevant from a clinical perspective because oxygen administration to neonates is often inevitable, and rEpo may serve as an adjunctive neuroprotective therapy.

Topics: Animals; Apoptosis; Asphyxia Neonatorum; Brain; Caspases; Cell Death; Disease Models, Animal; Erythropoietin; Humans; Hyperoxia; Infant, Newborn; Mice; Mice, Inbred C57BL; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidative Stress; Oxygen; Oxygen Inhalation Therapy; Proteome; Rats; Rats, Wistar

To investigate the effects of erythropoietin (EPO) on the expression of aquaporin 2 (AQP(2)) after renal ischemia/reperfusion (IR).. Twenty-four Wistar rats were randomly divided into 3 equal groups: IR group undergoing resection of the right kidney, closuring of the left renal artery, vein, and ureter, and un-closuring 40 min later; IR + EPO group undergoing the above mentioned procedures and then intraperitoneal injection of EPO 3000 U/kg on days 1 and 2 after the treatment; and control group undergoing resection of the right kidney only without IR of the left kidney. Urine volume and urine osmotic pressure were measured. Blood samples were collected to detect the serum blood urea nitrogen (BUN) and creatinine (Cr). Three days after the treatment the kidneys were taken out. RT-PCR and Western blotting were used to detect the mRNA and protein expression of AQP(2).. The urine volume of the IR + EPO group was (26.0 +/- 2.3) microl .min(-1).kg(-1), significantly lower than that of the IR group [(59.1 +/- 1.3) microl .min(-1) . kg(-1), P < 0.01]. The urine osmotic pressure of the IR + EPO group was (1508 +/- 121) mOsm/kg H(2)O, significantly higher than that of the IR group [(235 +/- 99) mOsm/kg H(2)O, P < 0.01]. The serum BUN and Cr levels of the IR + EPO group were (12.3 +/- 6.0) mmol/L and (51 +/- 5) micromol/L respectively, both significantly lower than those of the IR group [(29.9 +/- 3.7) mmol/L and (141 +/- 5) micromol/L respectively, both P < 0.01]. The mRNA and protein expression of AQP(2) were highly positive in the control group. The protein expression levels of AQP(2) of the IR + EPO group were not significantly different from those of the control group (both P > 0.05), and the protein expression levels of AQP(2) of the IR group were significantly lower than those of the control group (both P < 0.01).. EPO can inhibit the down-regulation of AQP(2) in response to IR and this may take part in the EPO protective mechanism of renal ischemia/reperfusion injury.

Topics: Animals; Aquaporin 2; Disease Models, Animal; Erythropoietin; Female; Kidney; Kidney Diseases; Rats; Rats, Wistar; Reperfusion Injury

So far, several treatment modalities have been attempted to brain protection in cases such as brain trauma, stroke or brain hemorrhage. However, a treatment method that the effect begins immediately and definitely helpful has not been discovered yet. In this study, we aimed to compare the effects of propofol and erythropoietin (Epo) on brain injury caused by oxidative stress and antioxidant properties of these agents after closed head injury (CHI) in rats. For this study, female Wistar Albino rats were divided into five groups: non-traumatic control group, trauma performed group CHI, trauma with propofol (100 mg/kg) intraperitoneally (i.p.), trauma with Epo (5000 U/kg) i.p. and trauma with propofol and Epo performed study groups. Twenty-four hours after CHI, rats were sacrificed and the brains were removed. Superoxide dismutase (SOD), catalase (CAT), xanthine oxidase (XO), nitric oxide (NO), and malondialdehyde (MDA) levels were measured in brain tissue. MDA and NO levels were decreased significantly in Groups Epo, Propofol and Epo+Propofol than Group CHI (p<0.01). XO activity was significantly lower in Group Epo than Group CHI (p<0.05). Epo and propofol decreased oxidative stress by decreasing MDA and NO level in brain tissue after CHI. However, combination of Epo and propofol has no significant beneficial advantage than Epo or propofol alone.

Topics: Analysis of Variance; Animals; Antioxidants; Brain Chemistry; Catalase; Disease Models, Animal; Erythropoietin; Female; Head Injuries, Closed; Malondialdehyde; Nitric Oxide; Propofol; Rats; Rats, Wistar; Superoxide Dismutase; Xanthine Oxidase

Anticancer agents often cause bone marrow toxicity resulting in progressive anemia which may influence the therapeutic effects of erythropoietic-stimulating agents. The objective of this study was to develop a pharmacodynamic (PD) model to describe chemotherapy-induced anemia in rats. Anemia was induced in male Wistar rats with a single intravenous (i.v.) injection of 60 mg/kg carboplatin. Hematological responses including reticulocytes, red blood cells (RBC), hemoglobin, and endogenous rat erythropoietin (EPO) were measured for up to 4 weeks. A catenary, lifespan-based, indirect response model served as a basic PD model to represent erythroid cellular populations in the bone marrow and blood involved in erythropoiesis. The model assumed that actively proliferating progenitor cells in the bone marrow are sensitive to anti-cancer agents and subject to an irreversible removal process. The removal rate of the target cells is proportional to drug activity concentrations and the cell numbers. An additional RBC loss from the circulation resulting from thrombocytopenia was described by a first-order process. The turnover process of rat EPO and EPO-mediated feedback inhibition mechanism regulated by hemoglobin changes were incorporated. Reticulocyte counts decreased rapidly and reached a nadir by day 3 after administration of carboplatin and returned to the baseline by day 13. This was followed by a gradual increase and the rebound peak occurred at about day 15. The hemoglobin nadir was approximately 9 g/dl observed at about 11-13 days compared to its normal value of 13 g/dl and hemoglobin returned to the baseline by day 30. The increase in endogenous rat EPO mirrored inversely hemoglobin changes and the maximum increase was observed soon after the hemoglobin nadir. The carboplatin-treated rats exhibited progressive anemia. The proposed model adequately described the time course of hematological changes after carboplatin in rats and can be a useful tool to explore potential strategies for the management of anemia caused by chemotherapy.

Topics: Algorithms; Anemia; Animals; Antineoplastic Agents; Blood Cell Count; Carboplatin; Cell Count; Computer Simulation; Disease Models, Animal; Disease Progression; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Hematopoiesis; Hemoglobins; Leukocyte Count; Male; Models, Statistical; Platelet Count; Rats; Stem Cells

To identify neuroprotective changes in gene expression, we developed a neonatal mouse model of moderate to severe oxidative brain injury and hypothesized that recombinant erythropoietin (rEpo) would decrease the expression of proapoptotic and proinflammatory genes 24 and 48 h, respectively, after injury and increase the expression of neurogenic and angiogenic genes 168 h after injury. Postnatal day 10 BALB-c mice underwent sham surgery or right common carotid artery occlusion followed by alternating hypoxia and hyperoxia and were then treated with rEpo (5,000 U/kg s.c.) or saline (vehicle) daily for up to three doses. At death, gross brain injury was assessed, then hippocampus, cortex, and thalamus were isolated for RNA or protein extraction. Microarray analysis, real-time polymerase chain reaction, and Bio-Plex suspension array system validation were performed. rEpo decreased both incidence and severity of brain injury (median injury score 3 vs. 0, p < 0.0001) and reduced the injury-induced increases in interleukin-1alpha and interleukin-6 gene expression (p < 0.001), with corresponding effects on protein translation. Similarly, the expression of caspase-1, caspase-4, and caspase-6 and of p53 was increased by brain injury at 24 h, but mitigated by rEpo (p < 0.01). The interleukin-10 expression was higher in the rEpo-treated animals. Apoptotic and proinflammatory gene expression persisted for 168 h. There was no increase in angiogenic gene expression at the time points studied.

Topics: Animals; Caspase 1; Caspase 6; Caspases; Caspases, Initiator; Disease Models, Animal; Erythropoietin; Gene Expression; Hypoxia, Brain; Interleukin-10; Interleukin-1alpha; Interleukin-6; Mice; Mice, Inbred BALB C; Neuroprotective Agents; Oligonucleotide Array Sequence Analysis; Oxidative Stress; Recombinant Proteins; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

Carnitine improves erythropoetin (EPO) response and anaemia in haemodialysis patients (HD); however, the mechanism(s) responsible remain unidentified. We have reported that carnitine induces haem oxygenase (HO)-1, which is an antioxidant and antiapoptotic that acts via pathways shared with EPO. Therefore carnitine's effect on these pathways may account for the improved EPO response. This study evaluates carnitine's effect on protein expression of HO-1 in unexplained EPO resistant HD. Carnitine's effect was assessed by HO-1 expression in patients and compared to its antiapoptotic effect via HO-1 induction in a rat model of carnitine-treated heart failure.. Unexplained EPO resistant HD mononuclear cell HO-1 and rat gastrocnemious muscle HO-1 and Bcl-2 protein expression were evaluated by western blot.. HD's haemoglobin (Hb) and haematocrit (Ht) were not different before carnitine treatment: 8.8 +/- 0.4 mg/dl versus 8.98 +/- 0.13 and 30.20% +/- 0.84 versus 30.72 +/- 1.14, respectively. Carnitine increased HO-1, Hb and Ht compared with patients not treated with carnitine: 2.40 +/- 0.58 versus 1.49 +/- 0.41, P = 0.02; 11.22 +/- 0.54 versus 8.90 +/- 0.15, P < 0.0001; 32.72 +/- 1.77 versus 30.66 +/- 0.43, P = 0.035, respectively. Carnitine-treated HD's HO-1 significantly correlated with haemoglobin. HO-1 and Bcl-2 protein levels in untreated heart failure rat's gastrocnemious muscle were reduced when compared with controls: 3.41 +/- 0.49 versus 5.32 +/- 0.38 and 0.69 +/- 0.11 versus 1.65 +/- 0.37, respectively, but were higher in carnitine-treated heart failure rats: 4.8 +/- 0.32 versus 3.41 +/- 0.49, P < 0.0002 and 1.09 +/- 0.08 versus 0.69 +/- 0.11, P = 0.0007, respectively.. These results are consistent with an involvement of HO-1 in carnitine's effect on erythropoiesis. The initial signals or effectors responsible for carnitine's effect remain to be identified.

Topics: Animals; Apoptosis; Carnitine; Disease Models, Animal; Erythropoiesis; Erythropoietin; Heart Failure; Hematocrit; Heme Oxygenase-1; Hemoglobins; Humans; Leukocytes, Mononuclear; Male; Monocrotaline; Muscle, Skeletal; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Rats; Renal Dialysis; Signal Transduction

Erythropoietin (EPO) may improve cardiac function and induce neovascularisation in experimental models of chronic heart failure (CHF). However, the increased haematocrit associated with EPO treatment might exert concomitant deleterious effects.. To investigate the haematocrit independent effects of EPO on cardiac function.. Rats underwent permanent coronary artery ligation to induce myocardial infarction (MI) or sham surgery. Three weeks after MI, rats were randomly allocated to treatment with vehicle (MI) or the long-acting EPO analogue darbepoetin alfa administered in a high (40 microg/kg/3 weeks, MI-EPO-high) or a low-dose (0.4 microg/kg/3 weeks, MI-EPO-low). After 9 weeks, haemodynamic parameters, myocardial histology and Myosin Heavy Chain (MHC) isoforms were determined. High-dose EPO resulted in a significant increase in haematocrit (p<0.01) while low-dose EPO had no effect on haematocrit levels. EPO significantly improved cardiac function in both EPO groups, reflected by increased left ventricular (LV)-developed pressure and improved contractility (dP/dt(max)) and relaxation (dP/dt(min)) indices of the LV at 9-weeks (all p<0.05 compared to MI). The improved cardiac function was associated with increased capillary growth (38% in MI-EPO-high (p<0.01) and 27% in MI-EPO-low (p<0.05)) and an attenuated switch to slow beta-MHC isoforms in both EPO groups.. EPO improves cardiac function and induces neovascularisation at a dose that does not increase haematocrit, thereby circumventing the possible deleterious effects of increased erythropoiesis.

Topics: Animals; Apoptosis; Capillaries; Coronary Vessels; Darbepoetin alfa; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Heart Failure; Hematinics; Hematocrit; Male; Myocardial Contraction; Neovascularization, Physiologic; Random Allocation; Rats; Rats, Sprague-Dawley; Treatment Outcome

Apoptosis is a central mechanism of cell death following reperfusion of the ischemic liver. Recombinant human erythropoietin (rhEPO) have an important role in the treatment of myocardial ischemia/reperfusion (I/R) injury, by preventing apoptosis. The aim of the study was to investigate the effect of different regimens of rhEPO in preventing apoptosis following I/R-induced hepatic injury.. Isolated mouse livers were randomly divided into five groups: (1) control group, perfused for the whole study period (105 min); (2) 30-min perfusion followed by 90 min of ischemia and 15 min of reperfusion; (3), (4) and (5) like group 2, but with administration of rhEPO 5,000 units/kg i.p. at 30 min, 24 h, or both 30 min and 24 h respectively, before induction of ischemia. Perfusate liver enzyme levels and intrahepatic caspase-3 activity were measured, and apoptotic cells were identified by morphological criteria, TUNEL assay, and immunohistochemistry for caspase-3. Using immunoblot the expression of the proapoptotic JNK and inhibitor of NFkappaB (IkappaBalpha) were also evaluated. von Willebrand factor (vWF) immunohistochemistry was used as a marker of endothelial cells.. Compared to the I/R livers, all 3 rhEPO pretreated groups showed: a significant reduction in liver enzyme levels (P < 0.05) and intrahepatic caspase-3 activity (P < 0.05), fewer apoptotic hepatocytes (P < 0.05) and positive vWF staining in numerous endothelial cells lining the sinusoids. EPO decreased JNK phosphorylation and the degradation of the inhibitor of NFkappaB (IkappaBalpha) during I/R. There was no added benefit of the multiple- over the single-dose rhEPO regimen.. Pretreatment with one dose of rhEPO can attenuate post-I/R hepatocyte apoptotic liver damage. NFkappaB and JNK activation is likely to play a pivotal role in the pathophysiology of I/R hepatic injury and might have a key role in EPO-mediated protective effects. This effect is associated with the increase in sinusoidal vWF immunostaining suggests an additional effect of rhEPO in liver angiogenesis recovery. These findings have important implications for the potential use of rhEPO in I/R injury during liver transplantation.

Topics: Animals; Apoptosis; Caspase 3; Disease Models, Animal; Erythropoietin; Humans; Ischemia; Liver; Male; MAP Kinase Kinase 4; Mice; NF-kappaB-Inducing Kinase; Protein Serine-Threonine Kinases; Random Allocation; Recombinant Proteins; Reperfusion; Reperfusion Injury; von Willebrand Factor

Treatment of human brain disease with erythropoietin (EPO) in order to achieve neuroprotection and/or neuroregeneration represents a totally new frontier in translational neuroscience. Rather than specifically targeting the cause of a particular disease entity, EPO nonspecifically influences components of the "final common pathway" that determine disease severity and progression in a number of entirely different brain diseases. EPO acts in an antiapoptotic, anti-inflammatory, antioxidant, neurotrophic, angiogenetic, stem cell-modulatory fashion. Importantly, it appears to influence neural plasticity. Most likely due to these properties, EPO has been found by many investigators to be protective or regenerative and to improve cognitive performance in various rodent models of neurological and psychiatric disease. The "Göttingen-EPO-stroke trial" has provided first promising data on humans for a neuroprotective therapy of an acute brain disease. Experimental EPO treatment to improve cognitive function in patients with schizophrenia represents a novel neuroregenerative strategy for a chronic brain disease. An exploratory trial in chronic progressive multiple sclerosis as an example of an inflammatory disease of the nervous system yielded first positive results of EPO treatment on both motor function and cognition. These promising results are just the beginning and will hopefully stimulate further work along these lines.

Topics: Anemia; Animals; Brain; Brain Diseases; Cognition; Disease Models, Animal; Erythropoietin; Humans; Mammals; Mental Disorders; Recombinant Proteins; Spinal Cord

Recombinant human erythropoietin (rHuEpo) is a definitive treatment for anaemia in chronic kidney disease (CKD). During long-term rHuEpo treatment most patients develop and show persistent iron deficiency in spite of oral iron supplementation. Abnormalities of iron absorption and transport in the duodenum may contribute to this deficiency.. To investigate changes in iron absorption and transport in CKD and iron deficiency against the background of rHuEpo treatment, we used severely anaemic rats with adenine-induced renal failure (adenine rats) and sham-treated control rats given only the vehicle. After 4 weeks on adenine or the vehicle, the rats were divided into four groups according to whether or not they received rHuEpo for the next 4 weeks: rHuEpo(-)-adenine, rHuEpo(-)-control, rHuEpo(+)-adenine and rHuEpo(+)-control. We evaluated the effects of rHuEpo treatment on iron balance, duodenal mRNA expression of molecules related to iron absorption and transport and hepatic mRNA expression of hepcidin.. Treatment with rHuEpo improved anaemia and induced iron deficiency only in the adenine rats, in whom the expression of mRNAs for ferroportin 1 and hephaestin 1 increased and for divalent metal transporter 1 (DMT1) was unchanged. In contrast, control rats treated with rHuEpo showed no changes. Hepcidin mRNA expression was greater in adenine rats than in control rats.. In the adenine rats, rHuEpo treatment improved renal anaemia and induced persistent iron deficiency. An alteration of mRNA expression of molecules related to iron metabolism in renal insufficiency may be one of the reasons for this iron deficiency.

Topics: Adenine; Analysis of Variance; Anemia, Iron-Deficiency; Animals; Antimicrobial Cationic Peptides; Disease Models, Animal; Erythrocytes; Erythropoietin; Hepcidins; Humans; Iron; Kidney Failure, Chronic; Kidney Function Tests; Male; Polymerase Chain Reaction; Probability; Random Allocation; Rats; Rats, Wistar; Recombinant Proteins; Renal Insufficiency; RNA, Messenger

Cerebral malaria (CM) is an acute encephalopathy with increased pro-inflammatory cytokines, sequestration of parasitized erythrocytes and localized ischaemia. In children CM induces cognitive impairment in about 10% of the survivors. Erythropoietin (Epo) has - besides of its well known haematopoietic properties - significant anti-inflammatory, antioxidant and anti-apoptotic effects in various brain disorders. The neurobiological responses to exogenously injected Epo during murine CM were examined.. Female C57BL/6j mice (4-6 weeks), infected with Plasmodium berghei ANKA, were treated with recombinant human Epo (rhEpo; 50-5000 U/kg/OD, i.p.) at different time points. The effect on survival was measured. Brain pathology was investigated by TUNEL (Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labelling), as a marker of apoptosis. Gene expression in brain tissue was measured by real time PCR.. Treatment with rhEpo increased survival in mice with CM in a dose- and time-dependent manner and reduced apoptotic cell death of neurons as well as the expression of pro-inflammatory cytokines in the brain. This neuroprotective effect appeared to be independent of the haematopoietic effect.. These results and its excellent safety profile in humans makes rhEpo a potential candidate for adjunct treatment of CM.

Topics: Animals; Apoptosis; Brain; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Female; Gene Expression Regulation; Humans; In Situ Nick-End Labeling; Injections, Intraperitoneal; Malaria, Cerebral; Mice; Mice, Inbred C57BL; Neurons; Plasmodium berghei; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Survival Rate; Time Factors

Many patients with anemia fail to respond to treatment with erythropoietin (Epo), a commonly used hormone that stimulates erythroid progenitor production and maturation by human BM or by murine spleen. The protein product of growth arrest-specific gene 6 (Gas6) is important for cell survival across several cell types, but its precise physiological role remains largely enigmatic. Here, we report that murine erythroblasts released Gas6 in response to Epo and that Gas6 enhanced Epo receptor signaling by activating the serine-threonine kinase Akt in these cells. In the absence of Gas6, erythroid progenitors and erythroblasts were hyporesponsive to the survival activity of Epo and failed to restore hematocrit levels in response to anemia. In addition, Gas6 may influence erythropoiesis via paracrine erythroblast-independent mechanisms involving macrophages. When mice with acute anemia were treated with Gas6, the protein normalized hematocrit levels without causing undesired erythrocytosis. In a transgenic mouse model of chronic anemia caused by insufficient Epo production, Gas6 synergized with Epo in restoring hematocrit levels. These findings may have implications for the treatment of patients with anemia who fail to adequately respond to Epo.

Topics: Anemia; Animals; Axl Receptor Tyrosine Kinase; c-Mer Tyrosine Kinase; Cell Adhesion; Cell Survival; Disease Models, Animal; Drug Resistance; Erythroblasts; Erythropoiesis; Erythropoietin; Humans; Intercellular Signaling Peptides and Proteins; Mice; Mice, Mutant Strains; Oncogene Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor Protein-Tyrosine Kinases; Receptors, Erythropoietin; Recombinant Proteins

Topics: Animals; Disease Models, Animal; Electroretinography; Erythropoietin; Fluorescein Angiography; Fundus Oculi; Humans; Mice; Mice, Transgenic; Ophthalmoscopy; Oxygen; Retina; Retinal Vessels

Erythropoietin (EPO), a member of the cytokine type I superfamily, acts to increase circulating erythrocytes primarily by preventing apoptosis of erythroid progenitors, is known to protect tissues and can raise haemoglobin (Hb) concentrations. Recently, a second receptor for EPO comprising the EPO receptor and beta-common receptor has been reported to mediate EPO-induced tissue protection. EPO modified by carbamylation (CEPO) only signals through this second receptor. Accordingly, we hypothesized that treatment with CEPO, which would not increase Hb concentrations, would protect against tubular damage and thereby inhibit tubulointerstitial injuries.. We evaluated therapeutic effects of CEPO using a rat unilateral ureteral obstruction model.. CEPO decreased tubular apoptosis and alpha-smooth muscle actin (alphaSMA) expression in the absence of polycythaemia, while the untreated obstructed kidneys exhibited increased tubular apoptosis with expanded (alphaSMA) expression. While EPO treatment similarly inhibited tubular apoptosis and alphaSMA expression, EPO treatment increased Hb concentrations and induced a wedge-shaped infarction.. We established a therapeutic approach using CEPO to protect against tubulointerstitial injury. The therapeutic value of this approach warrants further attention and preclinical studies.

Topics: Actins; Animals; Apoptosis; Disease Models, Animal; Epoetin Alfa; Erythropoiesis; Erythropoietin; Hemoglobins; Infarction; Kidney; Kidney Tubules; Male; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Recombinant Proteins; Signal Transduction; Ureteral Obstruction

Stroke remains one of the most urgent medical problems of our times. The failure of most neuroprotective drugs in clinical trials led to the initiation of the Stroke Therapy Academic Industry Roundtable guidelines. Due to this improvement, the positive clinical trial results with the free radical scavenger NXY-059 (SAINT I) was encouraging. However, the subsequent SAINT II trial did not confirm these results. In this article we critically review the history of preclinical and clinical trials based on experience of NXY-059 development and present recommendations for potential future preclinical and clinical development of neuroprotective stroke therapy.

Topics: Animals; Antioxidants; Benzenesulfonates; Brain; Clinical Trials as Topic; Disease Models, Animal; Drug Evaluation; Erythropoietin; Granulocyte Colony-Stimulating Factor; Humans; Magnetic Resonance Imaging; Neuroprotective Agents; Practice Guidelines as Topic; Stroke

A primary objective was to evaluate whether addition of enteral iron supplementation will facilitate a systemic erythropoietic effect when feeding erythropoietin (Epo) to suckling rats. A secondary objective was to confirm that iron does not alter the previous finding that enteral Epo exerts local trophic effects on the small intestine.. Four-day-old Sprague-Dawley rats underwent gastrostomy and were fed a cow's milk-based rat milk substitute for 8 days. We studied rats fed rat milk substitute alone (control), enteral Epo 425 U x kg(-1) x day(-1), and enteral Epo 1700 U x kg(-1) x day(-1), and the effects of oral iron sulfate (Fe) therapy (6 mg x kg(-1) x day(-1)). Blood was collected to measure hemoglobin (Hb), reticulocytes, red cell indices, and zinc protoporphyrin/heme. To confirm previous work describing trophic effects of enteral Epo on the intestine, duodenal villous height was measured.. Hb levels in control (84 +/- 1 g/L) were similar to Epo 425 (87 +/- 1 g/L). Hb levels in control+Fe (97 +/- 1 g/L), Epo 425+Fe (97 +/- 1 g/L), and Epo 1700 (94 +/- 1 g/L) were higher than control, P < 0.001, but mean Hb level in Epo 1700+Fe was higher (105 +/- 1 g/L) than the other groups, P < 0.003. Mean cell volume was higher in rats receiving iron supplementation, compared with those without iron, P < 0.005. Duodenal villous height was taller in Epo 1700+Fe compared with control + Fe, P < 0.01.. If combined with sufficient iron supplementation, high-dose Epo artificially fed to suckling rats exerted a systemic erythropoietic effect in addition to the previously reported local trophic effects.

Topics: Animals; Animals, Suckling; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Therapy, Combination; Duodenum; Enteral Nutrition; Erythrocyte Indices; Erythropoiesis; Erythropoietin; Gastrostomy; Hemoglobins; Humans; Infant, Newborn; Infant, Premature; Infant, Premature, Diseases; Iron, Dietary; Random Allocation; Rats; Rats, Sprague-Dawley; Recombinant Proteins

The aims of this study were to evaluate the efficiency of EPO in the treatment of cold injury-induced brain edema, apoptosis, and inflammation and to compare its effectiveness with DSP.. One hundred fifteen adult male Sprague-Dawley rats weighing between 280 and 300 g were used for the study. Rats were divided into 5 groups. Controls received craniotomy only. The injury group underwent cold injury and had no medication. In the EPO group, a single dose of 1000 IU/kg body weight of EPO was administered. The DSP group received 0.2 mg/kg body weight of DSP. The vehicle group received a vehicle solution containing human serum albumin, which is the solvent for EPO. Brain edema was formed by cold injury using metal sterile rods with a diameter of 4 mm that were previously cooled at -80 degrees C. Twenty-four hours after the injury, animals were decapitated and brain tissues were investigated for brain edema, tissue MPO and caspase-3 levels, and ultrastructure.. A significant increase in brain water content was revealed in injury group of rats at 24 hours after cold injury. Injury significantly increased tissue MPO and caspase-3 levels and resulted in ultrastructural damage. Both EPO and DSP markedly decreased tissue MPO and caspase-3 levels and preserved ultrastructure of the injured brain cortex.. Erythropoietin and DSP were found to be neuroprotective in cold injury-induced brain edema model in rats via anti-apoptotic and anti-inflammatory actions.

Topics: Animals; Blood-Brain Barrier; Brain Edema; Brain Injuries; Caspase 3; Cold Temperature; Dexamethasone; Disease Models, Animal; Erythropoietin; Glucocorticoids; Male; Peroxidase; Rats; Rats, Sprague-Dawley

A number of drugs commonly used for a variety of clinical indications have been found recently to have substantial neuroprotective properties, raising the potential for rapid translation into human clinical trials of spinal cord injury (SCI). In this study we compared the neuroprotective efficacy of erythropoietin and a derivative of it, darbepoetin, in an acute model of thoracic SCI. Sprague-Dawley rats were randomized to receive erythropoietin (5000 IU/kg), darbepoetin (10 mug/kg), or saline, as a single intravenous injection 1 h after a thoracic contusion SCI. The animals were evaluated for behavioral recovery over 6 weeks, which included BBB locomotor testing, horizontal ladder testing, video-analysis of gait, and hindlimb monofilament sensory testing. At 6 week post-injury, the spinal cords were evaluated histologically to measure white and grey matter sparing at and around the epicenter of injury. We found that neither erythropoietin nor darbepoetin led to improved behavioral recovery over saline controls, with no significant differences observed in BBB scores, BBB subscores, footfall errors on horizontal ladder testing, width of hindlimb base of support, or threshold for paw withdrawal on sensory testing. Furthermore, no differences were observed in grey or white matter sparing between the three experimental groups. Using doses of erythropoietin and darbepoetin that other investigators have reported to be beneficial in SCI and stroke models, we were unable to demonstrate a neuroprotective effect when administered 1 h after injury. Further preclinical investigation is necessary to refine the treatment strategy of using erythropoietin or darbepoetin in acute spinal cord injury.

Topics: Analysis of Variance; Animals; Behavior, Animal; Darbepoetin alfa; Disease Models, Animal; Erythropoietin; Locomotion; Male; Neuroprotective Agents; Psychomotor Performance; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Time Factors

We aimed to evaluate whether ischemia is required for erythropoietin (EPO) induced stimulation of endothelial progenitor cells (EPCs) and their related effects on endothelial and cardiac function.. Bone marrow of rats was replaced by transgenic cells to allow tracking of EPCs. Ischemic heart failure was induced by left coronary artery ligation to induce myocardial infarction (MI) and control rats received a sham procedure. Three weeks after surgery, rats were randomized to receive EPO (darbepoetin alfa 40 microg/kg per 3 weeks) or vehicle and were sacrificed 9 weeks after surgery.. In all treated groups, EPO significantly increased circulating EPCs and their incorporation into the endothelium of the ischemic and non-ischemic hearts as well as in the control organs; kidney and liver. This was associated with significantly improved endothelial function, which was strongly correlated with circulating EPCs (R = 0.7, p < 0.01). However, additional EPCs preferentially homed to the ischemic MI borderzone (p < 0.01) resulting in specific EPO-induced improvement of cardiac microvascularization and performance only in ischemic hearts (all p < 0.05). The differential stimulation of neovascularization by EPO was associated with increased EPO-receptor and VEGF expression in ischemic hearts only.. In general, EPO stimulates normal endothelial progenitor cell-mediated endothelial turnover, but improves cardiac microvascularization and function only in the presence of ischemia.

Topics: Alkaline Phosphatase; Angiogenesis Inducing Agents; Animals; Bone Marrow Transplantation; Capillaries; Cell Movement; Coronary Vessels; Darbepoetin alfa; Disease Models, Animal; Endothelial Cells; Erythropoietin; GPI-Linked Proteins; Heart Failure; Humans; Isoenzymes; Male; Myocardial Contraction; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Rats; Rats, Inbred F344; Rats, Transgenic; Stem Cells; Vasodilation; Ventricular Function, Left; Ventricular Pressure

Numerous studies using erythropoietin (EPO) gene delivery vectors, either viral or nonviral, have shown uncontrolled EPO expression leading to transient or sustained erythrocytosis and, more recently, severe autoimmune anemia. Therefore, there is a need to develop other EPO gene delivery systems that allow sustained and adjustable expression of EPO. We have examined a new approach of delivering plasmid encoding mouse EPO cDNA into mouse skeletal muscle, using an amphiphilic block copolymer. Repeated injections of low doses of block copolymer-EPOcDNA formulations increased hematocrit in a dose-dependent manner for more than 9 months, without any initial overshoot. Low doses of block copolymer-EPOcDNA formulations prevented autoimmune anemia in immunocompetent Swiss mice and prevented or reversed chronic anemia in an acquired mouse model of renal failure. We conclude that repeated injections of low doses of block copolymer-DNA formulations that do not induce (1) inflammation at the injection site, (2) overexpression of EPO, or (3) the production of anti-EPO neutralizing auto-antibodies hold promise for in vivo expression of therapeutic proteins, in particular for systemic delivery.

Topics: Anemia; Animals; Creatinine; Disease Models, Animal; Erythropoietin; Female; Genetic Therapy; Inflammation; Injections, Intramuscular; Kidney; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Plasmids; Polyethylene Glycols; Renal Insufficiency; Reticulocyte Count; Urea

Organ toxicity induced by chemotherapeutic drugs is a serious obstacle in the effective treatment of patients suffering from cancer and autoimmune disease. A strong association exists between pulmonary toxicity, particularly fibrosis, and chemotherapeutic drugs. Attempts have been made to identify compounds capable of suppressing fibrosis. In addition to its erythropoietic activity, erythropoietin (EPO) has been shown to have effects on nonhemopoietic cells. Therefore, we postulated that EPO may exert beneficial effects on lung tissue during chemotherapy. To test our hypothesis, we investigated pulmonary changes caused by bleomycin, a fibrosis-inducing agent, in animals treated with the drug alone and in combination with EPO. Fibrosis, cellular alterations and structural changes were assayed by blind analysis of the lung sections. A 6-fold decrease in the number of prominent endothelial cells--suspected to be indicative of cellular activation and inflammatory response--was observed in lung sections derived from mice treated with bleomycin and EPO compared to animals injected with bleomycin alone (p < 0.008). Additionally, there was twice the number of ICAM1-positive endothelial cells in animals treated with bleomycin alone compared with the number in the bleomycin and EPO-treated group (p < 0.05). Alveolar mononuclear phagocytic hyperplasia was reduced by as much as 100% in animals treated with bleomycin and EPO compared to animals treated with bleomycin alone (p < 0.03). Finally, a 5-fold decrease in interstitial fibrosis was observed in lung sections obtained from animals treated with bleomycin and EPO (p < 0.02). We conclude that EPO can ameliorate drug-induced fibrosis and endothelial damage caused by chemotherapeutic agents.

Topics: Animals; Antineoplastic Agents; Bleomycin; Disease Models, Animal; Erythropoietin; Female; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis

The neuroprotective effects of exogenous erythropoietin (EPO) in animals and humans after brain injury may be afforded, in part, by the influence of EPO on cerebral arteries. We tested (1) if EPO itself is vasoactive and (2) if EPO enhances endothelium-mediated dilations, specifically those mediated by endothelium-derived hyperpolarizing factor (EDHF). Immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect EPO receptor. Rat middle cerebral arteries (MCAs) were isolated, pressurized, and perfused in vitro. EPO was directly applied to MCAs to test its vasoactivity. Endothelium-mediated dilations were elicited by UTP, whereas EDHF-mediated dilations were elicited by UTP after inhibition of endothelial nitric oxide synthase and cyclooxygenase. mRNA and protein for EPO receptor was found in rat MCA. Abluminal application of 0.001-10 U/mL EPO, which is selective for vascular smooth muscle, did not alter vessel diameter. In contrast, luminal application of EPO, which is selective for endothelium, resulted in concentration-dependent dilations of up to 39 +/- 16% at 10 U/mL (p = 0.0018), though responses were variable. A single dose of EPO (1,000 U/kg) administered to rats 24 h prior to examining vascular function potentiated dilations to UTP 2.6-fold (p < 0.0001). EDHF-mediated dilations were potentiated 2.1-fold following in vivo EPO treatment (p = 0.0034). This study demonstrates that EPO can directly dilate rat MCAs via the endothelium, though not all vessels are responsive. Additionally, pre-treatment with EPO for 24 h in vivo potentiates endothelium-mediated dilations, specifically those mediated by EDHF. Thus, enhanced endothelium-mediated dilations may partially underlie the neuroprotective effects of EPO after brain injury.

Topics: Animals; Biological Factors; Brain Injuries; Cerebrovascular Circulation; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Endothelial Cells; Erythropoietin; Male; Middle Cerebral Artery; Rats; Rats, Long-Evans; Receptors, Erythropoietin; Treatment Outcome; Uridine Triphosphate; Vasodilation; Vasodilator Agents

To investigate whether desferoxamine (DFO) preconditioning can induce tolerance against cerebral ischemia and its effect on the expression of hypoxia inducible factor 1alpha (HIF-1alpha) and erythropoietin (EPO) in vivo and in vitro.. Rat model of cerebral ischemia was established by middle cerebral artery occlusion with or without DFO administration. Infarct size was examined by TTC staining, and the neurological severity score was evaluated according to published method. Cortical neurons were cultured under ischemia stress which was mimicked by oxygen-glucose deprivation (OGD), and the neuron damage was assessed by MTT assay. Immunofluorescent staining was employed to detect the expressions of HIF-1alpha and EPO.. The protective effect induced by DFO (decreasing the infarction volume and ameliorating the neurological function) appeared at 2 d after administration of DFO (post-DFO), lasted until 7 d and disappeared at 14 d (P < 0.05); the most effective action was observed at 3 d post-DFO. DFO induced tolerance of cultured neurons against OGD: neuronal viability was increased 23%, 34%, 40%, 48% and 56% at 8 h, 12 h, 24 h, 36 h, and 48 h, respectively, post-DFO (P < 0.05). Immunofluorescent staining found that HIF-1alpha and EPO were upregulated in the neurons of rat brain at 3 d and 7 d post-DFO; increase of HIF-1alpha and EPO appeared in cultured cortex neurons at 36 h and 48 h post-DFO.. DFO induced tolerance against focal cerebral ischemia in rats, and exerted protective effect on OGD cultured cortical neurons. DFO significant induced the expression of HIF-1alpha and EPO both in vivo and in vitro. DFO preconditioning can protect against cerebral ischemia, which may be associated with the synthesis of HIF-1alpha and EPO.

Topics: Animals; Brain Ischemia; Cells, Cultured; Cerebral Infarction; Deferoxamine; Disease Models, Animal; Erythropoietin; Fluorescent Antibody Technique; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Ischemia, Brain; Infarction, Middle Cerebral Artery; Iron; Ischemic Preconditioning; Nerve Degeneration; Neurons; Rats; Rats, Sprague-Dawley; Siderophores; Time Factors; Treatment Outcome; Up-Regulation

To investigate the protective effect of erythropoietin (EPO) and its nonhematopoietic derivative (asialoEPO) against intestinal ischemia/reperfusion (I/R) injury in a rat model.. The superior mesenteric artery of Wistar rats was clamped for 60 minutes and then released. The rats were divided into 4 groups (n = 15 in each group): sham operation (Sham), vehicle treatment (Vehicle), EPO treatment (EPO), and asialoEPO treatment (AsialoEPO). EPO and asialoEPO were administered subcutaneously at 1000 units/kg for 10 minutes before clamping, 30 minutes after the start of clamping, and just before declamping. This treatment was followed by determination of 72-hour survival rates, serum TNF-alpha and IL-6 levels, histologic evaluation of the small intestine, quantification of the number of apoptotic cells, and analysis of the antiapoptotic molecules Bcl-xL and XIAP by Western blotting.. The survival rates at 72 hours after I/R injury in the Sham, Vehicle, EPO, and AsialoEPO groups were 100%, 33%, 75%, and 83%, respectively (P < .05). Blood TNF-alpha and IL-6 were significantly more suppressed in the EPO and AsialoEPO groups than in the Vehicle group at 6 hours after I/R injury. Histologically, injury to villi in the EPO and AsialoEPO groups was significantly less than in the Vehicle group. The number of apoptotic cells in the EPO and AsialoEPO groups was significantly less than in the Vehicle group. Western blotting revealed that EPO and asialoEPO constitutively increased the expression of Bcl-xL.. EPO and asialoEPO exert a strong protective effect against intestinal I/R injury, possibly by inhibiting release of TNF-alpha and IL-6 and decreasing apoptosis.

Topics: Animals; Apoptosis; Asialoglycoproteins; Disease Models, Animal; Erythropoietin; Gastrointestinal Agents; Intestinal Diseases; Intestines; Male; Rats; Rats, Wistar; Reperfusion Injury; Survival Analysis

Erythropoietin (Epo) induces erythrocytosis by suppressing erythroid progenitor cell apoptosis through the Janus-activated kinase-signal transducers and activators of transcription (JAK-STAT) pathway. Since apoptosis contributes to cisplatin (CP)-induced nephrotoxicity and Epo receptors (EpoR) are expressed in the kidney, we examined the role of antiapoptosis in recombinant human erythropoietin (rHuEpo)-mediated renal protection. In human renal proximal tubular epithelial (RPTE) cells in culture, rHuEpo, but not inactive rHuEpo (I-rHuEpo), the receptor-binding sites of which are mutated, caused a significant reduction in CP-induced apoptosis at > or = 100 U/ml. rHuEpo, but not I-rHuEpo, increased STAT5 and Akt/PKB phosphorylation, demonstrating functional EpoR expression on RPTE cells. Furthermore, the JAK2 inhibitor tyrphostin AG-490 attenuated rHuEpo protection, suggesting a role of the JAK-STAT pathway in rHuEpo-mediated antiapoptosis. In rats, intravenous administration of 5,000 U/kg rHuEpo, but not an equivalent peptide mass of I-rHuEpo, before a single 5.5 mg/kg iv injection of CP, significantly increased hematocrit (Hct) and reduced the CP-induced increase in serum creatinine. Serum creatinine on day 4 was 3.4 +/- 0.3, 1.9 +/- 0.3, and 3.5 +/- 0.4 mg/dl in the CP, CP + rHuEpo, and CP + I-rHuEpo groups, respectively. Similarly, darbepoietin-alpha (DA), a hyperglycosylated analog of rHuEpo with prolonged in vivo activity when injected at 25 microg/kg iv before CP, significantly increased Hct and reduced serum creatinine. Renal clearance studies based on glomerular filtration rate and renal blood flow confirmed the significant renal protection by DA against CP. Tubular apoptosis and necrosis were significantly reduced in the kidneys of the CP + DA vs. the CP + saline group. Moreover, the equalization of Hct by venesection did not abrogate the DA-mediated renal protection. Administration of DA 48 h after CP injection also conferred significant renal protection. Thus our experiments confirm a role for erythropoiesis-stimulating proteins, including the new analog DA, in limiting CP-induced nephrotoxicity and suggest that antiapoptosis via the Epo-EpoR interaction is an important mechanism for renal protection.

Topics: Acute Kidney Injury; Animals; Antineoplastic Agents; Apoptosis; Cells, Cultured; Cisplatin; Darbepoetin alfa; Disease Models, Animal; Enzyme Inhibitors; Erythropoietin; Hematinics; Hematocrit; Humans; Janus Kinase 2; Kidney; Male; Necrosis; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Recombinant Proteins; STAT5 Transcription Factor; Tyrphostins

Mesenchymal stromal cells (MSCs) possess intrinsic features that identify them as useful for treating ischaemic syndromes. Poor in vivo survival/engraftment of MSCs, however, limits their overall effectiveness. In this work, we tested whether genetically engineering MSCs to secrete erythropoietin (Epo) could represent a better therapeutic platform than MSCs in their native form.. MSCs from C57Bl/6 mice were retrovirally transduced with either an empty vector or one that causes the production of Epo and were then analysed for the alterations in angiogenic and survival potential. Using a mouse model of myocardial infarction (MI), the regenerative potential of null MSCs and Epo-overexpressing MSCs (Epo+MSCs) was assessed using serial echocardiogram and invasive haemodynamic measurements. Infarct size, capillary density and neutrophil influx were assessed using histologic techniques. Using in vitro assays coupled with an in vivo Matrigel plug assay, we demonstrate that engineering MSCs to express Epo does not alter their immunophenotype or plasticity. However, relative to mock-modified MSCs [wild-type (WT)-MSCs], Epo+MSCs are more resilient to apoptotic stimuli and initiate a more robust host-derived angiogenic response. We also identify and characterize the autocrine loop established on MSCs by having them secrete Epo. Furthermore, in a murine model of MI, animals receiving intracardiac injections of Epo+MSCs exhibited significantly enhanced cardiac function compared with WT-MSCs and saline-injected control animals post-MI, owing to the increased myocardial capillary density and the reduced neutrophilia.. Epo overexpression enhances the cellular regenerative properties of MSCs by both autocrine and paracrine pathways.

Topics: Animals; Apoptosis; Autocrine Communication; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Disease Models, Animal; Erythropoietin; Female; Genetic Therapy; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Neutrophil Infiltration; Paracrine Communication; Regeneration; Stromal Cells; Transduction, Genetic; Ventricular Function, Left; Ventricular Remodeling

Stem cell transplantation is a potential treatment strategy for neurodegenerative diseases such as glaucoma. The Müller stem cell line MIO-M1 can be differentiated to produce retinal neurons and glia. The survival, migration, differentiation, and integration of MIO-M1 cells were investigated in a rat model of glaucoma. The effect of modulating the retinal environment with either chondroitinase ABC or erythropoietin was also studied.. Intraocular pressure was chronically increased unilaterally by using a laser glaucoma model in adult rats. EGFP-transduced MIO-M1 cells were transplanted into the vitreous or subretinal space of glaucomatous or untreated eyes. Oral immune suppressants were administered to reduce xenograft rejection. Survival, migration, differentiation, and integration of grafted cells were assessed by immunohistochemistry.. Transplanted cells survived for 2 to 3 weeks in vivo, although microglia/macrophage infiltration and a reduction in graft survival were seen by 4 weeks. Grafted cells displayed a migratory phenotype with an elongated bipolar shape often oriented toward the retina. Transplanted cells expressed markers such as PSA-NCAM, GFAP, and beta-III-tubulin. The host retina was resistant to MIO-M1 migration, but modification of the local environment with erythropoietin or chondroitinase ABC facilitated retinal infiltration by MIO-M1 cells.. The results demonstrate that differentiating MIO-M1 cells within the glaucomatous eye produced cells that expressed neuronal and glial cell markers. The retina was relatively resistant to transplant integration, and long-term xenograft survival was limited. However, local modulation of the retinal environment enhanced the integration of MIO-M1 cells into the glaucomatous retina.

Topics: Adult Stem Cells; Animals; Aqueous Humor; Cell Differentiation; Cell Movement; Cell Survival; Chondroitin ABC Lyase; Disease Models, Animal; Erythropoietin; Fluorescent Antibody Technique, Indirect; Glaucoma; Glial Fibrillary Acidic Protein; Graft Survival; Green Fluorescent Proteins; Humans; Immunosuppressive Agents; Intraocular Pressure; Male; Neural Cell Adhesion Molecule L1; Neuroglia; Rats; Rats, Wistar; Recombinant Proteins; Retina; Sialic Acids; Stem Cell Transplantation; Transfection; Tubulin

Multiply traumatised patients often suffer from blood loss and from subsequent therapy-resistant anaemia, possibly mediated by apoptosis, necrosis, or humoral factors. Therefore, the underlying mechanisms were investigated in bone marrow (BM) and peripheral blood in a murine resuscitated haemorrhagic shock (HS) model.. In healthy male mice, pressure-controlled HS was induced for 60 min. The BM was analysed for Annexin-V, 7-amino-actinomycin D, apoptotic enzymes (caspases-3/7, -8, and -9), expression of death receptors (CD120a, CD95), mitochondrial proteins (Bax, Bcl-2, Bcl-x), as well as erythropoietin (EPO) receptor (EPO-R). Blood cell count, peripheral EPO, and tumour necrosis factor-alpha response were additionally monitored.. Twenty-four and 72 h after HS, EPO and EPO-R were strongly up-regulated in peripheral blood and BM, respectively. Decreasing numbers of erythroid progenitors in BM after HS correlated with significant apoptotic changes confirmed by increased caspases-3/7, -8, -9 activity in total BM, death receptor CD95 and CD120a expression on erythroid progenitors, and down-regulated mitochondrial Bcl-2 expression in total BM. Erythroid progenitors in peripheral blood were found to be increased after 72 h.. Despite the massive EPO response and up-regulation of EPO-R, BM erythroblasts (EBs) decreased. This could be due to deficient maturation of erythroid progenitors. Furthermore, the increased intrinsic and extrinsic apoptosis activation suggests programmed death of erythroid progenitors. We propose that both apoptosis and negatively regulated erythropoiesis contribute to BM dysfunction, while erythroid progenitor egress plays an additional role.

Topics: Analysis of Variance; Animals; Annexin A5; Apoptosis; Caspase Inhibitors; Caspases; Cell Proliferation; Disease Models, Animal; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Male; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins c-bcl-2; Receptors, Erythropoietin; Shock, Hemorrhagic; Time Factors; Tumor Necrosis Factor-alpha

Hyperbaric oxygen preconditioning (HBO-PC) has been shown to be effective in preventing hypoxic injuries in many animal models. The aim of the present study was to examine the hypoxic tolerance induced by HBO-PC and to explore the role of hypoxia-inducible factor-1alpha (HIF-1alpha) in a global hypoxia model. Male mice received HBO-PC before hypoxia exposure and swimming. HBO-PC significantly prolonged the survival time and the tolerance time of swimming under normobaric hypoxia. HBO-PC increased the protein content of HIF-1alpha and erythropoietin (EPO) in the cerebral cortex and hippocampus and prevented the changes of blood brain barrier (BBB) permeability and brain edema caused by hypoxia exposure. The results suggested that HBO-PC induced hypoxic tolerance in mice via up-regulation of HIF-1alpha and its downstream genes.

Topics: Analysis of Variance; Animals; Behavior, Animal; Brain Edema; Cerebral Cortex; Disease Models, Animal; Erythropoietin; Hippocampus; Hyperbaric Oxygenation; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemic Preconditioning; Male; Mice; Mice, Inbred Strains; Survival Analysis; Time Factors; Up-Regulation

Erythropoietin (EPO) has neuron and astroglial protective effects via reduction of tissue-injuring molecules such as reactive oxygen species, glutamate, inflammatory cytokines, and other damaging molecules. Although EPO may constitute an effective therapeutic modality in cases of epileptic insult, no study has been performed on the effects of exogenous EPO on the chronic seizure formation. In this study, we attempted to investigate if EPO could modulate the altered microenvironment in the epileptic rat brain.. Morphological changes in the hippocampi of rats subjected to lithium-pilocarpine-induced status epilepticus (SE) were examined with respect to neuronal loss, inflammation, blood-brain barrier (BBB) leakage, and cell genesis. Spontaneous recurrent seizures (SRSs) were investigated by long-term video-EEG monitoring.. EPO receptor (EPOR) was found to be increased in the hippocampus after SE. Administered EPO prevented, during the latent period following SE, BBB leakage, neuronal death, and microglia activation in the dentate hilus, CA1, and CA3, and inhibited the generation of ectopic granule cells in the hilus and new glia in CA1. Moreover, EPO reduced the risk of SRS development.. These findings suggest that EPO has a potential therapeutic role in the setting of acute epileptic insults.

Topics: Analysis of Variance; Animals; Brain; Bromodeoxyuridine; Capillary Permeability; CD11b Antigen; Cell Death; Disease Models, Animal; Electroencephalography; Erythropoietin; Fluorescein; Gene Expression Regulation; Homeodomain Proteins; Male; Neurons; Phosphopyruvate Hydratase; Pilocarpine; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Statistics, Nonparametric; Status Epilepticus; Tumor Suppressor Proteins

Losartan, the potassium salt of 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazol, is an efficient antihypertensive drug. The vibrational FTIR and Raman spectra of Losartan (its anionic and protonated forms) are discussed. In addition, the copper(II) complex of Losartan was obtained and characterized as a microcrystalline powder. The metal center is bound to the ligand through the nitrogen atoms of the tetrazolate moiety as determined by vibrational spectroscopy. The compound is a dimer with the metal centers in a tetragonal distorted environment but the presence of a monomeric impurity has been determined by EPR spectroscopy. The antioxidant properties of the complex (superoxide dismutase mimetic activity) and its effect on the proliferation and morphology of two osteoblast-like cells in culture are reported. The new compound exerted more toxic effects on tumoral cells than the copper(II) ion and Losartan.

Topics: Animals; Antihypertensive Agents; Antioxidants; Binding, Competitive; Cations, Divalent; Cell Proliferation; Copper; Disease Models, Animal; Electron Spin Resonance Spectroscopy; Ligands; Losartan; Osteoblasts; Rats; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Raman; Structure-Activity Relationship; Superoxide Dismutase; Tetrazoles; Tumor Cells, Cultured

Transplantation of marrow stromal cells (MSC) has been shown to improve heart perfusion and cardiac function after ischemia. Erythropoietin (EPO) is capable of inducing angiogenesis and inhibiting cell apoptosis. The aim of this study was to investigate the effect of EPO on the therapeutic potency of MSC transplantation in a rat model of myocardial infarction.. MSC viability was detected by MTT and flow cytometry following culture in serum-free medium for 24 h with or without EPO. Release of vascular endothelial growth factor (VEGF) by MSC incubated with different doses of EPO was assayed using ELISA. Immediately after coronary ligation, autologous MSC (3 x 10(6) cells) were injected into the ischemic myocardium (MSC and MSC-EPO groups). EPO (3,000 U/kg body weight) was injected daily for 3 consecutive days starting 1 day prior to ligation. The same EPO dose was also injected for consecutive 3 days starting 15 days after surgery (EPO and MSC-EPO groups). Control animals were injected saline solution for the same time period. Cardiac function was assessed by echocardiography 2 and 21 days after surgery, respectively. Western blot and immunohistological assessments were performed to examine the effects of treatments.. In vitro, EPO inhibited MSC apoptosis induced by serum-free medium and increased vascular endothelial growth factor (VEGF) release by MSC. In vivo, cardiac infarct size was significantly smaller, cardiac function significantly improved, and capillary density obviously higher in the MSC and EPO groups than in the control group. Combined treatment with EPO infusion and MSC transplantation demonstrated a further decrease in infarct size, a further improvement in cardiac function, and a further increase in capillary density compared with MSC or EPO alone. Furthermore, a higher ratio of phosphorylated Akt to total Akt was measured by Western blot; Bcl-2 was upregulated and Bax was downregulated by immunohistochemistry in the MSC-EPO group compared to the other three groups.. Transplantation of MSC combined with EPO infusion is superior to MSC monotherapy for angiogenesis and cardiac function recovery.

Topics: Angiogenesis Inducing Agents; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Disease Models, Animal; Erythropoietin; Male; Myocardial Infarction; Rats; Rats, Wistar; Stromal Cells; Transplantation, Autologous

Many neurologic disorders are accompanied by ischemic injury during the pathologic process. To develop a controllable and injury-specific gene therapy system for the neurologic disorders, we constructed a hypoxia inducible plasmid with the erythropoietin (Epo) 3' untranslated region (UTR), which can enhance the stability of target mRNAs in response to hypoxia. The Epo 3' UTR was inserted at the 3' flanking region of luciferase gene in pSV-Luc, resulting in the construction of pSV-Luc-EpoUTR. In pEpo-SV-Luc-EpoUTR, the Epo enhancer was inserted into the upstream of the SV40 promoter to increase the hypoxia inducibility. The plasmids were evaluated in N2a mouse neuroblastoma cells under hypoxic conditions and in a rat spinal cord injury (SCI) model. The results showed that the Epo 3' UTR alone showed a three-fold increase in luciferase activity in hypoxic N2a cells as well as in the rat SCI model when compared to the sham control. In contrast, the Epo 3' UTR showed no effect on the luciferase activity in the presence of the Epo enhancer, probably because the Epo enhancer was more sensitive to hypoxia and showed a dominant effect. However, the Epo enhancer itself showed high level of luciferase activity even in normoxia (about five to eight-folds increase), while the Epo 3' UTR did not show enhanced background activity. Immunohistochemical staining showed expression of luciferase from pSV-Luc-EpoUTR both in neurons and astrocytes around the injured spinal cord of rat. These results suggest that the Epo 3' UTR could provide a specific and safe system for the hypoxia-inducible gene therapy of the neurologic disorders including SCI.

Topics: 3' Untranslated Regions; Animals; Astrocytes; Cell Hypoxia; Cell Line, Tumor; Disease Models, Animal; Enhancer Elements, Genetic; Enzyme Activation; Erythropoietin; Gene Expression; Genetic Therapy; Genetic Vectors; Luciferases; Male; Mice; Neurons; Plasmids; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Simian virus 40; Spinal Cord Injuries; Spinal Cord Ischemia; Up-Regulation

The usefulness of sustained-release erythropoietin for improving left ventricular (LV) function without polycythemia was evaluated in a rat chronic myocardial infarction model.. Four weeks after left coronary artery ligation, 50 Sprague-Dawley rats were assigned to 5 groups (n=10, each). Control group had a gelatin sheet (20x20 mm) containing saline applied to the infarct area, whereas the 4 treatment groups had gelatin sheets incorporating erythropoietin 0.1 U, 1 U, 10 U and 100 U, respectively. Endpoint measurements performed at 8 weeks after the coronary ligation revealed that the fractional area change was larger for erythropoietin 1 U and 10 U than in the other 3 groups. The LV end-systolic elastance and the time constant of isovolumic relaxation were better for erythropoietin 1 U and 10 U than in the other 3 groups. The density of vessels larger than 50 microm in diameter was the highest in the erythropoietin 1 U group. The number of red blood cells was significantly increased in groups receiving erythropoietin 10 U and 100 U.. Gelatin hydrogel sheets incorporating 1 U erythropoietin improved LV function without inducing polycythemia in a rat chronic myocardial infarction model.

Topics: Animals; Delayed-Action Preparations; Disease Models, Animal; Dose-Response Relationship, Drug; Echocardiography; Erythropoietin; Gelatin; Heart; Male; Myocardial Infarction; Neovascularization, Physiologic; Polycythemia; Rats; Rats, Sprague-Dawley; Ventricular Dysfunction, Left; Ventricular Remodeling

Hypoxia-ischemia (HI) is a common injury arising from prematurity/complications at birth and is associated with later language, auditory, and learning impairments.. To investigate the efficacy of two doses (300 or 1000 U/kg) of Erythropoietin (Epo) in protecting against neuropathological and behavioral impairments associated with HI injury in rats.. HI injury (right carotid artery cauterization and 120 min of 8% O(2)) was induced on postnatal day 7 (P7) and Epo or saline was administered i.p. immediately following the procedure. Auditory processing and learning/memory were assessed throughout development.. Both doses of Epo provided behavioral protection following HI injury. Rats given 300 or 1000 U/kg of Epo performed significantly better than HI animals on a short duration complex auditory processing procedure, on a spatial Morris water maze assessing spatial learning/reference memory, and a non-spatial water maze assessing associative learning/reference memory.. Given Epo's extant clinical use (FDA approved for pediatric patients with anemia secondary to prematurity), the current results add to a growing body of literature supporting the use of Epo as a potential protective agent for neurological and behavioral impairments following early HI injury in infants.

Topics: Animals; Animals, Newborn; Atmosphere Exposure Chambers; Auditory Perception; Brain; Brain Infarction; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Hearing Loss, Central; Hypoxia-Ischemia, Brain; Learning Disabilities; Male; Maze Learning; Memory Disorders; Neuroprotective Agents; Rats; Rats, Wistar

The anemia of inflammation is an acquired disorder affecting patients with a variety of medical conditions, and it is characterized by changes in iron homeostasis and erythropoiesis. Mounting evidence suggests that hepcidin antimicrobial peptide plays a primary role in the pathogenesis of the anemia of inflammation. To evaluate which features of this anemia can be attributed to hepcidin, we have generated mice carrying a tetracycline-regulated hepcidin transgene. Expression of the hepcidin transgene resulted in down-regulation of endogenous hepcidin mRNA. The transgenic mice developed a mild-to-moderate anemia associated with iron deficiency and iron-restricted erythropoiesis. Similar to the anemia of inflammation, iron accumulated in tissue macrophages, whereas a relative paucity of iron was found in the liver. Circulating erythrocytes in transgenic animals had normal survival rates, but transgenic animals had an impaired response to erythropoietin. Thus, hepcidin transgenic mice recapitulate each of the key features of anemia of inflammation in human patients and serve as a useful model of this prevalent disorder.

Topics: Anemia, Iron-Deficiency; Animals; Antimicrobial Cationic Peptides; Cell Survival; Disease Models, Animal; Erythrocytes; Erythropoiesis; Erythropoietin; Hepcidins; Inflammation; Iron; Macrophages; Mice; Mice, Transgenic

To determine if posthypoxia treatment with erythropoietin (EPO) has protective effects against subsequent susceptibility to seizure related neuronal injury in rat pups subjected to acute hypoxia at P10.. Four groups of rats were manipulated at P10, as described below, then all received kainic acid (KA) (10 mg/kg i.p.) at P29: Hypoxia-NS-KA group (n = 11): subjected to acute hypoxia (down to 4% O2), and then immediately received saline i.p. Hypoxia-EPO-KA group (n = 10): subjected to acute hypoxia and then immediately received EPO (1,000 U/Kg i.p.). Normoxia-NS-KA group (n = 11): sham manipulated and injected with saline. Normoxia-EPO-KA group (n = 10): sham manipulated then immediately injected with EPO (1000 U/Kg i.p.). After receiving KA at P29, all rats were monitored using videotape techniques, and were sacrificed at P31. TUNEL and Hoechst stains to assess for apoptosis, and regular histology for hippocampal cell counts were performed.. Administration of the single dose of erythropoietin directly after an acute hypoxic event at P10 resulted at P29 in increased latency to forelimb clonus seizures, reduced duration of these seizures, protection against hippocampal cell loss, and decreased hippocampal apoptosis in the Hypoxia-EPO-KA group as compared to the Hypoxia-NS-KA group.. These data support the presence of favorable protective effects of erythropoietin against the long-term consequences of acute hypoxia in the developing brain and raise the possibility of its investigation as a potential neuroprotective agent after human neonatal hypoxic encephalopathy.

Topics: Animals; Animals, Newborn; Cell Count; Disease Models, Animal; Disease Susceptibility; Dose-Response Relationship, Drug; Erythropoietin; Excitatory Amino Acid Agonists; Hippocampus; Humans; Hypoxia; Hypoxia, Brain; Kainic Acid; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Seizures

Protein-energy malnutrition is a syndrome in which anaemia together with multivitamin and mineral deficiency may be present. The pathophysiological mechanisms involved have not, however, yet been completely elucidated. The aim of the present study was to evaluate the pathophysiological processes that occur in this anaemia in animals that were submitted to protein-energy malnutrition, in particular with respect to Fe concentration and the proliferative activity of haemopoietic cells. For this, histological, histochemical, cell culture and immunophenotyping techniques were used. Two-month-old male Swiss mice were submitted to protein-energy malnutrition with a low-protein diet (20 g/kg) compared with control diet (400 g/kg). When the experimental group had attained a 20 % loss of their original body weight, the animals from both groups received, intravenously, 20 IU erythropoietin every other day for 14 d. Malnourished animals showed a decrease in red blood cells, Hb concentration and reticulocytopenia, as well as severe bone marrow and splenic atrophy. The results for serum Fe, total Fe-binding capacity, transferrin and erythropoietin in malnourished animals were no different from those of the control animals. Fe reserves in the spleen, liver and bone marrow were found to be greater in the malnourished animals. The mixed colony-forming unit assays revealed a smaller production of granulocyte-macrophage colony-forming units, erythroid burst-forming units, erythroid colony-forming units and CD45, CD117, CD119 and CD71 expression in the bone marrow and spleen cells of malnourished animals. These findings suggest that, in this protein-energy malnutrition model, anaemia is not caused by Fe deficiency or erythropoietin deficiency, but is a result of ineffective erythropoiesis.

Topics: Anemia; Animals; Blood Proteins; Body Weight; Bone Marrow Cells; Colony-Forming Units Assay; Dietary Proteins; Disease Models, Animal; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Flow Cytometry; Immunophenotyping; Iron; Male; Mice; Protein-Energy Malnutrition; Spleen; Transferrin

Blood-brain barrier (BBB) leakage plays a role in the pathogenesis of many pathological states of the brain including ischemia and some neurodegenerative disorders. In recent years, erythropoietin (EPO) has been shown to exert neuroprotection in many pathological conditions including ischemia in the brain. This study aimed to investigate the effects of EPO on BBB integrity, infarct size and lipid peroxidation following global brain ischemia/reperfusion in rats. Wistar male rats were divided into four groups (each group n=8); Group I; control group (sham-operated), Group II; ischemia/reperfusion group, Group III; EPO treated group (24 h before decapitation--000 U/kg r-Hu EPO i.p.), Group IV; EPO+ ischemia/reperfusion group (24 h before ischemia/reperfusion--3000 U/kg r-Hu EPO i.p.). Global brain ischemia was produced by the combination of bilateral common carotid arteries occlusion and hemorrhagic hypotension. Macroscopical and spectrophotometrical measurement of Evans Blue (EB) leakage was observed for BBB integrity. Infarct size was calculated based on 2,3,5-triphenyltetrazolium chloride (TTC) staining. Lipid peroxidation in the brain tissue was determined as the concentration of thiobarbituric acid-reactive substances (TBARS) for each group. Ischemic insult caused bilateral and regional BBB breakdown (hippocampus, cortex, corpus striatum, midbrain, brain stem and thalamus). EPO pretreatment reduced BBB disruption, infarct size and lipid peroxide levels in brain tissue with 20 min ischemia and 20 min reperfusion. These results suggest that EPO plays an important role in protecting against brain ischemia/reperfusion through inhibiting lipid peroxidation and decreasing BBB disruption.

Topics: Animals; Blood-Brain Barrier; Brain Infarction; Brain Ischemia; Carotid Arteries; Carotid Stenosis; Disease Models, Animal; Drug Administration Schedule; Epoetin Alfa; Erythropoietin; Evans Blue; Lipid Peroxidation; Male; Neuroprotective Agents; Rats; Rats, Wistar; Recombinant Proteins; Reperfusion Injury; Thiobarbituric Acid Reactive Substances

Retinal ganglion cell (RGC) loss occurs in response to increased intraocular pressure (IOP) and/or retinal ischemia in glaucoma and leads to impairment of vision. This study was undertaken to test the efficacy of erythropoietin (EPO) in providing neuroprotection to RGCs in vivo.. The neuroprotective effects of EPO were studied in the DBA/2J mouse model of glaucoma. Mice were intraperitoneally injected with control substances or various doses of EPO, starting at the age of 6 months and continuing for an additional 2, 4, or 6 months. RGCs were labeled retrogradely by a gold tracer. IOP was measured with a microelectric-mechanical system, and EPO receptor (EPOR) expression was detected by immunohistochemistry. Axonal death in the optic nerve was quantified by para-phenylenediamine staining, and a complete blood count system was used to measure the number of erythrocytes.. In DBA/2J mice, the average number of viable RGCs significantly decreased from 4 months to 10 months, with an inverse correlation between the number of dead optic nerve axons and viable RGCs. Treatment with EPO at doses of 3000, 6000, and 12,000 U/kg body weight per week all prevented significant RGC loss, compared with untreated DBA/2J control animals. EPO effects were similar to those of memantine, a known neuroprotective agent. IOP, in contrast, was unchanged by both EPO and memantine. Finally, EPOR was expressed in the RGC layer in both DBA/2J and C57BL/6J mice.. EPO promoted RGC survival in DBA/2J glaucomatous mice without affecting IOP. These results suggest that EPO may be a potential therapeutic neuroprotectant in glaucoma.

Topics: Animals; Axons; Cell Survival; Disease Models, Animal; Erythropoietin; Fluorescent Antibody Technique, Indirect; Glaucoma; Intraocular Pressure; Memantine; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Microscopy, Fluorescence; Neuroprotective Agents; Optic Nerve; Optic Nerve Diseases; Receptors, Erythropoietin; Recombinant Proteins; Retinal Ganglion Cells

Erythropoietin (EPO) treatment has become the standard treatment of renal anemia. Though a link between hematopoiesis-stimulating drugs and thrombosis has not been proven, it is generally assumed that systemic application of EPO and its analogues increases the risk for thrombotic events.. Here we show in C57BL/6J mice that 4-week treatment with the long-lasting EPO analogue darbepoetin-alpha (DPO) at a dose of 10 microg/kg/week induces a reduction of platelet reactivity using flow cytometry and Western blot analysis of tyrosine-specific platelet phosphorylation. Additionally, immunohistochemistry of endothelial adhesion molecule expression and ELISA of circulating endothelial activation markers demonstrated a reduced endothelial activation. Immunohistochemistry and RT-PCR analysis revealed a significant (P<0.05) increase of eNOS expression. Further, DPO did not exert prothrombogenic effects in a murine intravital microscopic thrombosis model of the cremaster muscle. The role of eNOS in prevention of DPO-mediated microvascular thrombosis is further underlined by a significantly accelerated thrombus formation on DPO treatment in eNOS (-/-) mice.. Thus, DPO-related erythropoiesis with a raised hematocrit is not associated with an increased risk for thrombosis as long as endothelial NO production serves as compensatory mechanism.

Topics: Anemia; Animals; Blood Platelets; Blotting, Western; Capillaries; Darbepoetin alfa; Disease Models, Animal; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Flow Cytometry; Gene Expression; Hematinics; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type III; P-Selectin; Prognosis; RNA; Thrombelastography; Thrombosis

Recombinant human EPO (r-Hu-EPO) protects cultured motor neurons from kainate- and serum deprivation-induced toxicity and readily enters the CNS after systemic injection. We examined the effect of rHuEPO in transgenic mice expressing the human Cu/Zn dependent-superoxide dismutase with G93A mutation (SOD1G93A), a model of familial amyotrophic lateral sclerosis. rHuEPO (4 unit/g BW s.c. three times/week), increased the haematocrit and induced a slight delay in impairment of motor function as measured by the rotating bar test. However, it did not prolong life span or reduce motor neuron loss in lumbar spinal cord. The effect on motor function may be due to the improvement of skeletal muscle oxygenation induced by chronic EPO administration.

Topics: Age Factors; Amyotrophic Lateral Sclerosis; Analysis of Variance; Animals; Body Weight; Choline O-Acetyltransferase; Disease Models, Animal; Erythropoietin; Female; Hematocrit; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Motor Neurons; Psychomotor Performance; Recombinant Proteins; Superoxide Dismutase

Anemia frequently accompanies chronic diseases such as progressive renal failure, acquired immunodeficiency syndrome, and cancer. Patients are currently treated with erythropoietin (EPO) replacement therapy, using various recombinant human EPO protein formulations. Although this treatment is effective, gene therapy could be more economical and more convenient for the long-term management of the disease. The objective of this study was to develop a naked DNA-based gene therapy protocol that could fill this need. Hydrodynamic limb vein technology has been shown to be an effective and safe procedure for delivering naked plasmid DNA (pDNA) into the skeletal muscles of limbs. Using this method, we addressed the major challenge of an EPO-based gene therapy of anemia: maintaining stable, long-term expression at a level that sufficiently promotes erythropoiesis without leading to polycythemia. The results of our study, using a rat anemia model, provide proof of principle that repeated delivery of small pDNA doses has an additive effect and can gradually lead to the correction of anemia without triggering excessive hematopoiesis. This simple method provides an alternative approach for regulating EPO expression. EPO expression was also proportional to the injected pDNA dose in nonhuman primates. In addition, long-term (more than 450 days) expression was obtained after delivering rhesus EPO cDNA under the transcriptional control of the muscle-specific creatine kinase (MCK) promoter. In conclusion, these data suggest that the repeated delivery of small doses of EPO expressing pDNA into skeletal muscle is a promising, clinically viable approach to alleviate the symptoms of anemia.

Topics: Anemia; Animals; Creatine Kinase, MM Form; Disease Models, Animal; DNA; Erythropoietin; Genetic Therapy; Injections, Intravenous; Macaca mulatta; Male; Mice; Muscle, Skeletal; Plasmids; Promoter Regions, Genetic; Rats; Rats, Inbred Lew

Recent studies from our lab and others have shown that the hematopoietic cytokine erythropoietin (EPO) can protect the heart from ischemic damage in a red blood cell-independent manner. Here we examined any protective effects of the long-acting EPO analog darbepoetin alfa (DA) in a rat model of ischemia-reperfusion (I/R) injury. Rats were subjected to 30-min ischemia followed by 72-h reperfusion. In a dose-response study, DA (2, 7, 11, and 30 mug/kg) or vehicle was administered as a single bolus at the start of ischemia. To determine the time window of potential cardioprotection, a single high dose of DA (30 mug/kg) was given at either the initiation or the end of ischemia or at 1 or 24 h after reperfusion. After 3 days, cardiac function and infarct size were assessed. Acute myocyte apoptosis was quantified by TUNEL staining on myocardial sections and by caspase-3 activity assays. DA significantly reduced infarct size from 32.8 +/- 3.5% (vehicle) to 11.0 +/- 3.3% in a dose-dependent manner, while there was no difference in ischemic area between groups. Treatment with DA as late as 24 h after the beginning of reperfusion still demonstrated a significant reduction in infarct size (17.0 +/- 1.6%). Consistent with infarction data, DA improved in vivo cardiac reserve compared with vehicle. Finally, DA significantly decreased myocyte apoptosis and caspase-3 activity after I/R. These data indicate that DA protects the heart against I/R injury and improves cardiac function, apparently through a reduction of myocyte apoptosis. Of clinical importance pointing toward a relevant therapeutic utility, we report that even if given 24 h after I/R injury, DA can significantly protect the myocardium.

Topics: Animals; Cardiotonic Agents; Darbepoetin alfa; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Hematinics; Humans; Male; Myocardial Ischemia; Rats; Rats, Sprague-Dawley; Treatment Outcome

To investigate the inheritance of susceptibility to oxygen-induced retinopathy in the rat with the use of formal backcross analysis.. Neonatal offspring of inbred albino Fischer 344 (F344) and pigmented Dark Agouti (DA) crosses and F1xF344 and F1xDA backcrosses were exposed to alternating 24-hour cycles of hyperoxia (80% oxygen in air) and normoxia (21% oxygen in air) for 14 days. Retinal avascular area was analyzed by staining with Griffonia simplicifolia isolectin B4, a marker of vascular endothelial cells. Expression of erythropoietin (EPO) mRNA in retinas was quantified by real-time reverse-transcription polymerase chain reaction.. Oxygen-exposed offspring of two F344xDA F1 crosses showed retinal avascular areas and ocular and coat pigmentation that were similar to those of the DA strain. Mean retinal avascular area was 73%. Offspring of two DAxF1 backcrosses were similar to F344xDA F1 pups, with pigmented eyes and coats and a mean retinal avascular area of 76%. In contrast, offspring of two F344xF1 backcrosses exhibited a range of eye and coat pigmentation. Mean retinal avascular area of pigmented offspring of the F344xF1 backcrosses was 71% (P < 0.001 compared with F344 rats). Mean avascular area of albino offspring of the F344xF1 backcrosses was 27% (P > 0.05 compared with F344 rats). The normalized expression of EPO mRNA was 3.01 +/- 1.00 in retinas from pigmented F344xF1 backcross offspring compared with 1.31 +/- 0.69 for albino offspring (P < 0.001).. Segregation of the susceptibility trait to oxygen-induced retinopathy in the DA and F344 rat strains is associated with pigmentation and erythropoietin expression and can be modeled using an autosomal dominant pattern of inheritance.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Endothelium, Vascular; Erythropoietin; Female; Genetic Predisposition to Disease; Humans; Hyperoxia; Inbreeding; Infant, Newborn; Oxygen; Pedigree; Plant Lectins; Rats; Rats, Inbred F344; Retina; Retinal Vessels; Retinopathy of Prematurity; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin Pigmentation

Adaptive mechanisms to hematocrit levels of 0.9 in our erythropoietin-overexpressing mice (tg6) include increased plasma nitric oxide levels and erythrocyte flexibility. Doubled reticulocyte counts in tg6 suggest an increased erythrocyte turnover. Here we show that compared with wild-type (wt) animals, erythrocyte lifespan in tg6 is 70% lower in tg6 mice. Transgenic mice have a younger erythrocyte population as indicated by higher intercellular water and potassium content, higher flexibility, decreased density, increased surface to volume ratio, and decreased osmotic fragility. Interestingly, despite being younger, the tg6 erythrocyte population also harbors characteristics of accelerated aging such as an increased band 4.1a to 4.1b ratio, signs of oxidative stress, or decreased surface CD47 and sialic acids. In tg6, in vivo tracking of PKH26-labeled erythrocytes revealed dramatically increased erythrocyte incorporation by their liver macrophages. In vitro experiments showed that tg6 macrophages are more active than wt macrophages and that tg6 erythrocytes are more attractive for macrophages than wt ones. In conclusion, in tg6 mice erythrocyte aging is accelerated, which results, together with an increased number and activity of their macrophages, in enhanced erythrocyte clearance. Our data points toward a new mechanism down-regulating red cell mass in excessive erythrocytosis in mice.

Topics: Animals; Disease Models, Animal; Erythrocytes; Erythropoietin; Flow Cytometry; Gene Expression Regulation; Humans; Macrophages; Mice; Mice, Transgenic; Phagocytosis; Polycythemia Vera; Potassium; Reticulocytes

Topics: Animals; Apoptosis; Brain; Disease Models, Animal; Erythropoietin; Humans; Malaria, Cerebral; Mice; Plasmodium berghei; Recombinant Proteins

Erythropoiesis is critically dependent on erythropoietin (EPO), a glycoprotein hormone that is regulated by hypoxia-inducible factor (HIF). Hepatocytes are the primary source of extrarenal EPO in the adult and express HIF-1 and HIF-2, whose roles in the hypoxic induction of EPO remain controversial. In order to define the role of HIF-1 and HIF-2 in the regulation of hepatic EPO expression, we have generated mice with conditional inactivation of Hif-1alpha and/or Hif-2alpha (Epas1) in hepatocytes. We have previously shown that inactivation of the von Hippel-Lindau tumor suppressor pVHL, which targets both HIFs for proteasomal degradation, results in increased hepatic Epo production and polycythemia independent of Hif-1alpha. Here we show that conditional inactivation of Hif-2alpha in pVHL-deficient mice suppressed hepatic Epo and the development of polycythemia. Furthermore, we found that physiological Epo expression in infant livers required Hif-2alpha but not Hif-1alpha and that the hypoxic induction of liver Epo in anemic adults was Hif-2alpha dependent. Since other Hif target genes such phosphoglycerate kinase 1 (Pgk) were Hif-1alpha dependent, we provide genetic evidence that HIF-1 and HIF-2 have distinct roles in the regulation of hypoxia-inducible genes and that EPO is preferentially regulated by HIF-2 in the liver.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Hepatocytes; Liver; Mice; Mice, Knockout; Polycythemia Vera; von Hippel-Lindau Disease; Von Hippel-Lindau Tumor Suppressor Protein

Multifactorial pathogenic mechanisms, including inflammation, attenuated survival signals and enhanced death signals, are involved in amyotrophic lateral sclerosis (ALS). Erythropoietin (EPO) has recently been highlighted as a cytokine with various potent neuroprotective effects, including reduction of inflammation, enhancement of survival signals and prevention of neuronal cell death. This study was undertaken to evaluate the effect of recombinant human EPO (rhEPO) on ALS model mice. We treated 96 ALS model mice with vehicle only, or 1, 2.5 or 5 imu of rhEPO/g of mouse once every other week after they were 60 days old. The treatment significantly prolonged symptom onset and life span, preserved more motor neurons, enhanced survival signals, and attenuated inflammatory signals in a dose-dependent manner. These data suggest that treatment with rhEPO represents a potential therapeutic strategy for ALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Dinoprostone; Disease Models, Animal; Disease Progression; Erythropoietin; Humans; Inflammation; Mice; Mice, Transgenic; Motor Neurons; Recombinant Proteins; Rotarod Performance Test; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1; Survival Rate

Podocyte injury is a significant contributor to proteinuria and glomerulosclerosis. Recent studies have shown a renoprotective effect of erythropoietin (EPO) during ischemic kidney disease. In this study, we examine mechanisms by which a long acting recombinant EPO analog, darbepoetin, may confer renoprotection in the puromycin aminonucleoside-induced model of nephrotic syndrome. Darbepoetin decreased the proteinuria of rats treated with puromycin. This protective effect was correlated with the immunohistochemical disappearance of the podocyte injury markers desmin and the immune costimulator molecule B7.1 with the reappearance of nephrin expression in the slit diaphragm. Podocyte foot process retraction and effacement along with actin filament rearrangement, determined by electron microscopy, were all reversed by darbepoetin treatment. The protective effects were confirmed in puromycin-induced nephrotic rats that had been hemodiluted to normal hematocrit levels. Furthermore, puromycin treatment of rat podocytes in culture caused actin cytoskeletal reorganization along with deranged nephrin distribution. All these effects in vitro were reversed by darbepoetin. Our study demonstrates that darbepoetin treatment ameliorates podocyte injury and decreases proteinuria by a direct effect on podocytes. This may be accomplished by maintenance of the actin cytoskeleton and nephrin expression.

Topics: Actins; Animals; Apoptosis; B7-1 Antigen; Cells, Cultured; Cytoskeleton; Darbepoetin alfa; Desmin; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; In Situ Nick-End Labeling; Male; Membrane Proteins; Nephrotic Syndrome; Podocytes; Protective Agents; Proteinuria; Puromycin Aminonucleoside; Rats; Rats, Wistar; Receptor Protein-Tyrosine Kinases; Receptors, Erythropoietin; RNA, Messenger; Time Factors

Nucleated red blood cells (NRBCs) in fetal circulation have been proposed as a marker of chronic hypoxia in fetuses with intrauterine growth restriction (IUGR). We sought to determine the effects of chronic hypoxia, chronic nitric oxide inhibition with N(G)-nitro-L-arginine methyl ester (L-NAME), or both on NRBC counts, erythropoietin levels, and pathologic changes in an animal model of IUGR.. We assigned timed pregnant adult Sprague Dawley rats to the following groups: (1) 21% oxygen + saline solution (n = 7); (2) 21% oxygen + L-NAME (n = 8); (3) 10% oxygen + saline solution (n = 6); and (4) 10% oxygen + L-NAME (n = 6). We inserted osmotic pumps that were prefilled with saline solution or L-NAME subcutaneously on day 17 of gestation. The animals were placed in a Plexiglas hypoxic chamber, which ensured a constant hypoxic environment. The animals were killed on day 21 of gestation before the onset of spontaneous labor. We collected maternal and fetal blood for measurement of NRBC and erythropoietin levels. The results were interpreted in relationship to maternal arterial blood gases and hemoglobin and hematocrit levels. Fetuses were examined for gross abnormalities and histological abnormalities that are characteristic of vascular disruptions by a blind examiner to experimental manipulation.. Nitric oxide inhibition induced IUGR with maximal effect when both L-NAME and hypoxia treatments were combined. Inhibition of nitric oxide synthesis, but not chronic hypoxia, increased the number of fetal NRBCs and generalized hemorrhagic diathesis in utero. These features were aggravated significantly when the treatments were combined. Moreover, chronic hypoxia induced significant maternal metabolic acidosis and increased hematocrit and erythropoietin levels in maternal and fetal blood. Nitric oxide inhibition increased maternal hematocrit levels while decreasing maternal erythropoietin levels without significantly altering the maternal acid-base status. In contrast with chronic hypoxia, nitric oxide inhibition increased fetal NRBCs without affecting erythropoietin levels.. Our findings indicate that the number of NRBCs in fetal circulation does not serve as a specific marker of chronic hypoxia that accompanies IUGR or of elevated erythropoietin levels but are an epiphenomenon that is related to the inhibition of nitric oxide.

Topics: Animals; Chronic Disease; Disease Models, Animal; Erythroblasts; Erythrocyte Count; Erythropoietin; Female; Fetal Blood; Fetal Growth Retardation; Hypoxia; Nitric Oxide Synthase; Placental Insufficiency; Pregnancy; Rats; Rats, Sprague-Dawley

To investigate the effect of erythropoietin for the management of postresuscitation myocardial dysfunction following asphyxia-induced cardiac arrest. Male adult Wistar rats were used for the prospective controlled animal study. Asphyxia-induced cardiac arrest was performed by turning-off the ventilator and clamping the endotracheal tube. Cardiopulmonary resuscitation with an intravenous injection of 0.01 mg/kg epinephrine and mechanical ventilation were started after 6.5 minutes of asphyxia. The resuscitated animals received either erythropoietin (5000 U/kg) or equivalent volume of 0.9% saline as placebo intravenously 3 minutes after return of spontaneous circulation. The erythropoietin treatment produced better left ventricular dP/dt40 and -dP/dt in the invasive hemodynamic measurements, and left ventricular fraction shortening by echocardiography. Administration of erythropoietin also improved three days survival among those successfully resuscitated. The molecular effects of erythropoietin were shown by activation of its down streaming Akt and ERK 42/44 signaling pathways. EPO has the potential to improve postresuscitation myocardial dysfunction and short term survival in rats after asphyxia-induced cardiac arrest.

Topics: Animals; Asphyxia; Cardiopulmonary Resuscitation; Cardiotonic Agents; Disease Models, Animal; Erythropoietin; Heart; Heart Arrest; Male; Rats; Rats, Wistar; Recombinant Proteins; Signal Transduction; Ventricular Dysfunction, Left

The relationship between oxygen delivery and consumption in sepsis is impaired, suggesting a microcirculatory perfusion defect. Recombinant human erythropoietin (rHuEPO) regulates erythropoiesis and also exerts complex actions promoting the maintenance of homeostasis of the organism under stress. The objective of this study was to test the hypothesis that rHuEPO could improve skeletal muscle capillary perfusion and tissue oxygenation in sepsis.. Septic mice in three experiments received rHu-EPO 400 U/kg subcutaneously 18 hours after cecal ligation and perforation (CLP). The first experiment measured the acute effects of rHuEPO on hemodynamics, blood counts, and arterial lactate level. The next two sets of experiments used intravital microscopy to observe capillary perfusion and nicotinamide adenine dinucleotide (NADH) fluorescence post-CLP after treatment with rHuEPO every 10 minutes for 40 minutes and at 6 hours. Perfused capillary density during a three-minute observation period and NADH fluorescence were measured.. rHuEPO did not have any effects on blood pressure, lactate level, or blood cell numbers. CLP mice demonstrated a 22% decrease in perfused capillary density compared to the sham group (28.5 versus 36.6 capillaries per millimeter; p < 0.001). Treatment of CLP mice with rHuEPO resulted in an immediate and significant increase in perfused capillaries in the CLP group at all time points compared to baseline from 28.5 to 33.6 capillaries per millimeter at 40 minutes; p < 0.001. A significant increase in baseline NADH, suggesting tissue hypoxia, was noted in the CLP mice compared to the sham group (48.3 versus 43.9 fluorescence units [FU]; p = 0.03) and improved with rHuEPO from 48.3 to 44.4 FU at 40 minutes (p = 0.02). Six hours after treatment with rHuEPO, CLP mice demonstrated a higher mean perfused capillary density (39.4 versus 31.7 capillaries per millimeter; p < 0.001) and a lower mean NADH fluorescence as compared to CLP+normal saline mice (49.4 versus 52.7 FU; p = 0.03).. rHuEPO produced an immediate increase in capillary perfusion and decrease in NADH fluorescence in skeletal muscle. Thus, it appears that rHuEPO improves tissue bioenergetics, which is sustained for at least six hours in this murine sepsis model.

Topics: Animals; Disease Models, Animal; Energy Metabolism; Erythropoietin; Mice; Mice, Inbred C57BL; Microcirculation; Muscle, Skeletal; Sepsis; Treatment Outcome

Erythropoietin (EPO) is a pleiotropic cytokine with a therapeutic potential that goes well beyond the treatment of anaemia. The study by Wang et al (2007b) examined the protective effects of EPO in a rat model of embolic stroke. The efficacy and haematological side effects of EPO were compared to those of a carbamylated EPO variant (CEPO). Treatment with EPO dose-dependently reduced infarct volume and improved long-term functional outcome. However, an increase in hematocrit was seen even for doses of EPO that did not offer neuroprotection. These data do not suggest the existence of a therapeutic window between effect and side effect for treatment with EPO. Treatment with CEPO was without haematological side effects.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Hematocrit; Neuroprotective Agents; Rats; Stroke

Erythropoietin protects many organs against the tissue injury and dysfunction caused by ischaemia/reperfusion and excessive inflammation. This editorial comment discusses the effects of erythropoietin in preclinical models of septic shock, endotoxemia, hemorrhagic shock, spinal cord trauma and zymosan-induced multiple organ failure.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Endotoxemia; Erythropoietin; Mice; Multiple Organ Failure; Shock; Shock, Hemorrhagic; Shock, Septic; Spinal Cord Injuries; Treatment Outcome; Zymosan

Hypertension is the most significant complication from treatment with erythropoietin (Epo). Can Epo-induced hypertension be eliminated? We examined systemic and local effects of our genetically engineered products, Epo-binding protein (Epo-bp) and anti-Epo-bp antibodies, on randomly assigned Sprague-Dawley rats at midnight, 4 am, 8 am, noon, 4 pm, and 8 pm. Blood pressure, hematocrit, and body weight were measured immediately before and after the completion of a 4-week, twice-weekly course of Epo (50 U/kg), Epo-bp, anti-Epo-bp antibodies, or physiological saline injections. Epo treatment increased hematocrit markedly overall as compared with the saline, Epo-bp, and anti-Epo-bp antibody groups (0.616 versus 0.427, 0.439, and 0.441, respectively) and at each of the 6 test times (all P<0.0001). Epo-bp and anti-Epo-bp antibody treatment with Epo had almost no effect on the Epo-induced hematocrit increase (0.616 versus 0.580 or 0.591, respectively). Circadian blood pressures for Epo versus saline, Epo-bp, and anti-Epo-bp antibody groups were 136.2+/-2.3 versus 116.2+/-1.7, 118.4+/-2.1, and 116.6+/-2.1 mm Hg, respectively (each P<0.0001). Significantly increased blood pressure was detected at noon, 4 pm, 8 pm, and midnight in Epo treatment. When Epo was given with Epo-bp or anti-Epo-bp antibodies, blood pressure was maintained at similar levels as in saline treatment (each P<0.0001) as compared with Epo treatment alone. Overall, body, brain, and heart weights were significantly lower in Epo treatment than those of other groups. Thus, Epo-bp and anti-Epo-bp antibodies eliminate Epo-induced hypertension without affecting hematocrit and blood volume.

Topics: Animals; Blood Pressure Determination; Body Weight; Circadian Rhythm; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Epoetin Alfa; Erythropoiesis; Erythropoietin; Hematocrit; Hypertension; Probability; Protein Binding; Random Allocation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reference Values; Risk Factors; Sensitivity and Specificity

Detachment or apoptosis of podocytes leads to proteinuria and glomerulosclerosis. There are no current interventions for diabetic or non-diabetic glomerular diseases specifically preventing podocyte apoptosis. Binding of erythropoiesis stimulating proteins (ESPs) to receptors on non-hematopoietic cells has been shown to have anti-apoptotic effects in vitro, in vivo, and in preliminary human studies. Recently, erythropoietin receptors were identified on podocytes; therefore, we tested effects of darbepoetin alfa in preventing podocyte apoptosis. Cultured immortalized mouse podocytes were treated with low-dose ultraviolet-C (uv-C) irradiation to induce apoptosis in the absence or presence of darbepoetin alfa. Apoptosis was quantified by Hoechst staining and by caspase 3 cleavage assessed by Western blots. Pretreatment with darbepoetin alfa significantly reduced podocyte apoptosis with this effect involving intact Janus family protein kinase-2 (JAK2) and AKT signaling pathways. Additionally, darbepoetin alfa was found protective against transforming growth factor-beta1 but not puromycin aminonucleoside induced apoptosis. Mice with anti-glomerular antibody induced glomerulonephritis had significantly less proteinuria, glomerulosclerosis, and podocyte apoptosis when treated with darbepoetin alfa. Our studies show that treatment of progressive renal diseases characterized by podocyte apoptosis with ESPs may be beneficial in slowing progression of chronic kidney disease.

Topics: Animals; Antibodies; Apoptosis; Autoantibodies; Cell Proliferation; Cells, Cultured; Darbepoetin alfa; Disease Models, Animal; Disease Progression; Erythropoietin; Glomerulonephritis; Glomerulosclerosis, Focal Segmental; Janus Kinase 2; Mice; Podocytes; Protective Agents; Proteinuria; Proto-Oncogene Proteins c-akt; Puromycin Aminonucleoside; Receptors, Erythropoietin; Signal Transduction; Transforming Growth Factor beta1; Ultraviolet Rays

Erythropoietin is known to stimulate red cell production and has recently been shown to protect the heart against injury from ischemia/reperfusion. However, it is unknown whether darbepoetin alfa (Dpa), a long-acting analog of erythropoietin, can play a protective role against myocardial infarction. We assessed the potential protective role of Dpa in an in vivo rat model of myocardial ischemia/reperfusion and the underlying mechanisms. We found that a single intravenous Dpa treatment immediately before 30 minutes of regional ischemia reduced myocardial necrosis following 120 minutes of reperfusion in a dose-dependent manner. Optimal protection with Dpa against myocardial infarction was manifest at a dose of 2.5 microg/kg. Dpa conferred cardioprotection when administered after the onset of ischemia and at the start of reperfusion. Dpa (2.5 microg/kg) also reduced infarct size and Troponin I leakage 24 hours after reperfusion. Inhibition of p42/44 MAPK (PD98059), p38 MAPK (SB203580), mitochondrial ATP-dependent potassium (KATP) channels (5-HD), sarcolemmal KATP channels (HMR 1098), but not phosphatidylinositol-3 (PI3) kinase/Akt (Wortmannin and LY 294002) abolished Dpa-induced cardioprotection. Dpa confers immediate and sustained cardioprotection in rats, suggesting a potential therapeutic role of this long-acting erythropoietin analog for the treatment of acute myocardial infarction.

Topics: Animals; Cardiotonic Agents; Darbepoetin alfa; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Erythropoietin; Heart; Male; Mitogen-Activated Protein Kinase Kinases; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Potassium Channels; Rats; Rats, Sprague-Dawley

Erythropoietin (Epo) has anti-apoptotic and pro-angiogenic effects in rodent models of myocardial infarction (MI). We tested the hypothesis that a long-acting Epo derivative (darbepoetin) has a beneficial effect on infarct size and peri-infarct remodeling in a clinically relevant large animal model of ischemia-reperfusion. A human acute MI scenario was simulated in 16 domestic pigs by inflating an angioplasty balloon in the proximal left circumflex (LCx) artery for 60 min. The animals were randomized to darbepoetin 30 microg/kg i.v. or placebo (saline) at the time of reperfusion. Treatment with darbepoetin did not lead to a reduction in the infarct size at 2 weeks as assessed by histology (30.3+/-1.8% of the volume at risk for placebo vs. 33.2+/-2.5% for darbepoetin). However, significant effects were seen in the peri-infarct region. Histological evaluation revealed decreased interstitial fibrosis (6.8+/-0.7% of myocardial sections area vs. 9.6+/-0.7%, p=0.02) and increased average capillary area (106+/-3% of the non-infarcted myocardium vs. 89+/-4%, p=0.003) in the treatment arm in the absence of significant cardiac hypertrophy. This resulted in preserved regional wall motion as assessed by tissue Doppler-derived radial strain (subepicardial radial strain 90.1+/-21.2% for darbepoetin vs. 20.3+/-10.1% for placebo, p<0.05). However, this did not translate to improved wall thickening (126.5+/-6.0% of diastolic thickness for darbepoetin vs. 119.8+/-5.4% for placebo, p=NS). Beneficial effects of darbepoetin to peri-infarct remodeling were observed in a clinically relevant model of ischemia-reperfusion. Although the infarct size was not reduced, there was a limited decrease in interstitial fibrosis, increased capillary area and regional functional improvement in darbepoetin-treated animals.

Topics: Animals; Capillaries; Darbepoetin alfa; Disease Models, Animal; Echocardiography; Erythropoietin; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Receptors, Erythropoietin; Swine; Ventricular Remodeling

Recombinant human erythropoietin (rhEPO; Epoetin-alpha; PROCRITtrade mark) has been shown to exert neuroprotective and restorative effects in a variety of CNS injury models. However, limited information is available regarding the dose levels required for these beneficial effects or the neuronal responses that may underlie them. Here we have investigated the dose-response to rhEPO and compared the effects of rhEPO with those of carbamylated rhEPO (CEPO) in a model of cerebral stroke in rats.. Rats subjected to embolic middle cerebral artery occlusion (MCAo) were treated with rhEPO or CEPO, starting at 6 h and repeated at 24 and 48 h, after MCAo. Cerebral infarct volumes were assessed at 28 days and neurological impairment at 7, 14, 21 and 28 days, post-MCAo.. rhEPO at dose levels of 500, 1150 or 5000 IU kg(-1) or CEPO at a dose level of 50 microg kg(-1) significantly reduced cortical infarct volume and reduced neurologic impairment. All doses of rhEPO, but not CEPO, produced a transient increase in haematocrit, while rhEPO and CEPO substantially reduced the number of apoptotic cells and activated microglia in the ischemic boundary region.. These data indicate that rhEPO and CEPO have anti-inflammatory and anti-apoptotic effects, even with administration at 6 h following embolic MCAo in rats. Taken together, these actions of rhEPO and CEPO are likely to contribute to their reduction of neurologic impairment following cerebral ischemia.

Topics: Animals; Apoptosis; Blood-Brain Barrier; Brain Ischemia; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Epoetin Alfa; Erythropoietin; Hematinics; Hematocrit; Infarction, Middle Cerebral Artery; Male; Microglia; Neuroprotective Agents; Rats; Rats, Wistar; Recombinant Proteins; Stroke

Periventricular leukomalacia (PVL), a common neonatal brain white matter (WM) lesion, is frequently associated with cerebral palsy. Growing evidence has indicated that in addition to ischemia/reperfusion injury, cytokine-induced brain injury associated with maternal or fetal infection may also play an important role in the pathogenesis of PVL. Recent studies have shown that administration of lipopolysaccharide (LPS) to pregnant rats causes enhanced expression of the cytokines, i.e., IL-1 beta, TNF-alpha, and IL-6, in fetal brains. In recent years, it has been shown that erythropoietin (EPO) has a critical role in the development, maintenance, protection and repair of the nervous system. In the present study we investigated the effect of EPO on LPS-induced WM injury in Sprague-Dawley rats. LPS (500 microg/kg) suspension in pyrogen-free saline was administered intraperitoneally to pregnant rats at 18 and 19 days of gestation. The control group was treated with pyrogen-free saline. They were given 5,000 U/kg recombinant human EPO. Seven-day-old Sprague-Dawley rat pups were divided into four groups: control group, LPS-treated group, prenatal maternal EPO-treated group (5,000 U/kg, intraperitoneally given to pregnant rats at 18 and 19 days of gestation), and postnatal EPO-treated group (5,000 U/kg, intraperitoneally given to 1-day-old rat pups). Cytokine induction in the postnatal 7-day-old (P7) rat brain after maternal administration of LPS was determined by the ELISA method. The proinflammatory cytokine levels (IL-1 beta, TNF-alpha, and IL-6) in P7 rat pup brains were significantly increased in the LPS-treated group as compared with the control group. Prenatal maternal EPO treatment significantly reduced the concentration of TNF-alpha and IL-6 in the newborn rat brain following LPS injection. The concentration of IL-1 beta was decreased in the intrauterine EPO treatment group. Postnatal EPO treatment significantly decreased only the IL-6 concentration in the newborn rat brain following LPS injection. The concentration of cytokines, IL-1 beta and TNF-alpha, was reduced in the postnatal EPO treatment group. We demonstrated here that LPS administration in pregnant rats at gestational day 18 and 19 induced WM injury in P7 progeny characterized by apoptosis. Prenatal maternal and postnatal EPO treatment significantly reduced the number of apoptotic cells in the periventricular WM. Using immunohistochemistry techniques, we investigated the effects of maternal admi

Topics: Animals; Animals, Newborn; Apoptosis; Bacterial Infections; Brain; Brain Diseases; Disease Models, Animal; Erythropoietin; Female; Humans; Infant, Newborn; Interleukin-1beta; Interleukin-6; Leukomalacia, Periventricular; Lipopolysaccharides; Myelin Basic Protein; Pregnancy; Random Allocation; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

1. Erythropoietin (EPO) is a hormone regulating the proliferation and differentiation of erythroid precursor cells. The hypothesis that haematopoietic and endothelial cells share a common haemanglioblast progenitor among others is based on the finding that both cell lineages express cell surface antigens, such as CD31 and CD34. 2. In the present study, we investigated the angiogenic potential of recombinant human erythropoietin on cyclosporine A (CsA)-induced nephrotoxicity in the rat kidney and compared it with the effect of basic fibroblast growth factor (bFGF), a well-known angiogenic factor. 3. Rats were divided into five groups: A (control), B (EPO treated), C (CsA treated), D (CsA + EPO treated) and E (CsA + bFGF treated). Mouse anti-human CD31 and CD34 antibodies were used to evaluate the kidney vessels present in histological preparations. 4. Glomerular and peritubular capillaries in Group B (EPO) were increased compared with the control (Group A; P < 0.05). Reduction of the same kidney vessels (glomerular and peritubular capillaries) in Group C (CsA; P < 0.05) compared with controls was observed, whereas in Groups D (CsA + EPO treated) and E (CsA + bFGF treated), capillaries were increased compared with Group C (CsA; P < 0.05). 5. Erythropoietin has a significant angiogenic effect in rat kidney with CsA-induced nephrotoxicity, similar to the effect of the other angiogenic factor bFGF.

Topics: Angiogenic Proteins; Animals; Cyclosporine; Disease Models, Animal; Erythropoietin; Fibroblast Growth Factor 2; Humans; Kidney; Kidney Diseases; Male; Neovascularization, Physiologic; Rats; Rats, Sprague-Dawley; Recombinant Proteins

To evaluate the potential of Hematide, a PEGylated, synthetic peptide-based erythropoiesis-stimulating agent that is in clinical development for the treatment of anemia associated with chronic kidney disease and cancer, to correct antierythropoietin antibody-associated pure red cell aplasia (PRCA).. The binding of anti-Hematide antibodies (mouse, rabbit, and monkey) to recombinant human erythropoietin (rHuEPO) and of anti-rHuEPO antibodies (mouse, goat, rat, and human) to Hematide were evaluated. An anti-EPO antibody-mediated anemia rat model was developed by subcutaneously administering rHuEPO to rats three times weekly for 4 weeks. Sixty percent of the animals developed PRCA as characterized by severe anemia, reduced reticulocytes, anti-EPO antibodies, and limited bone marrow erythroid precursors. The effect of Hematide administration on the PRCA rats was evaluated.. Antibodies to EPO do not cross react with Hematide and, conversely, antibodies to Hematide do not cross react with EPO. Hematide corrected antibody-induced anemia in a rat PRCA model.. The data support the potential of Hematide to correct anti-EPO antibody-associated PRCA in humans. In addition, the data suggest a negligible risk for development of anti-EPO antibody-induced PRCA secondary to Hematide administration.

Topics: Anemia; Animals; Antibodies; Bone Marrow; Colony-Forming Units Assay; Disease Models, Animal; Erythropoietin; Humans; Peptides; Polyethylene Glycols; Rabbits; Rats; Red-Cell Aplasia, Pure

Testicular torsion is an important clinical urgency. Similar mechanisms occurred after detorsion of the affected testis as in the ischemia reperfusion (I/R) damage. This study was designed to investigate the effects of erythropoietin (EPO) treatment after unilateral testicular torsion. Fifty male Sprague-Dawley rats were divided into five groups. Group 1 underwent a sham operation of the right testis under general anesthesia. Group 2 was same as sham, and EPO (3,000 IU/kg) infused i.p., group 3 underwent a similar operation but the right testis was rotated 720 degrees clockwise for 1 h, maintained by fixing the testis to the scrotum, and saline infused during the procedure. Group 4 underwent similar torsion but EPO was infused half an hour before the detorsion procedure, and in group 5, EPO was infused after detorsion procedure. Four hours after detorsion, ipsilateral and contralateral testes were taken out for evaluation. Treatment with EPO improved testicular structures in the ipsilateral testis but improvement was less in the contralateral testis histologically, but EPO treatment decreased germ cell apoptosis in both testes following testicular IR. TNF-alpha, IL-1beta, IL-6 and nitrite levels decreased after EPO treatment especially in the ipsilateral testis. We conclude that testicular I/R causes an increase in germ cell apoptosis both in the ipsilateral and contralateral testes. Erythropoietin has antiapoptotic and anti-inflammatory effects following testicular torsion.

Topics: Animals; Apoptosis; Disease Models, Animal; Erythropoietin; Hematinics; Inflammation; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Seminiferous Tubules; Spermatic Cord Torsion

Status epilepticus (SE) is a grave condition in which the brain undergoes lasting seizures which can lead to neuronal loss. Our previous study suggested that preconditioning with erythropoietin (Epo) suppressed neuronal apoptosis in hippocampus of rats following SE in vivo by inhibiting caspase-3. In this study, we investigated the mechanisms by which Epo preconditioning may exert its anti-apoptotic effects using a lithium-pilocarpine induced SE model in rats. The effects of Epo on neuronal cell death were evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and the role of the Bcl-2 protein family, which have been shown to be anti- (Bcl-2, Bcl-w) or pro- (Bid, Bim) apoptotic, was examined with immunofluorescence. We found Epo preconditioning decreased the total number of TUNEL, Bim and Bid positive cells, but increased the total number of Bcl-w and Bcl-2 positive cells. These results suggest that systemic Epo pretreatment protects neurons in an acute phase of SE and may result in further suppression of neuronal apoptosis in hippocampus by regulating the balance between pro- and anti-apoptotic Bcl-2 family proteins.

Topics: Analysis of Variance; Animals; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; BH3 Interacting Domain Death Agonist Protein; Cell Count; Cell Death; Disease Models, Animal; Drug Interactions; Erythropoietin; In Situ Nick-End Labeling; Indoles; Male; Membrane Proteins; Neurons; Neuroprotective Agents; Pilocarpine; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Status Epilepticus

The renoprotective effect of erythropoietin (EPO) and the nonhematopoietic EPO, asialoEPO was investigated in a murine ischemia-reperfusion injury (I/R) model.. I/R was created by clamping the right renal pedicle for 60 min after left nephrectomy. Balb/c mice were divided into four groups (n=15 in each group): sham operation (Sham), vehicle treatment (Vehicle), EPO treatment (EPO), and asialoEPO treatment (AsialoEPO). EPO and asialoEPO were given at a dose of 500 IU/kg 30 min before I/R. Plasma creatinine (Cr), survival, and the number of apoptotic cells were analyzed. Protein expression was analyzed by Western blotting.. Plasma Cr level was not significantly different at 6 hr after I/R. At 24 hr after I/R, the Cr (mg/dL) levels in Sham, Vehicle, EPO, and asialoEPO were 0.13+/-0.01, 1.24+/-0.70, 0.24+/-0.08, and 0.25+/-0.13, respectively (P<0.05). The numbers of apoptotic cells in these groups were 0.1+/-0.1, 98.9+/-42.6, 3.3+/-0.7, and 2.9+/-1.6, respectively (P<0.05). Western blotting revealed that in kidney tissue of mice treated with EPO and asialoEPO, p38-MAPK and the proapoptotic molecule Bad was decreased, and the antiapoptotic molecules Bcl-xL and XIAP were increased. Survival rates at 7 days after I/R injury in the Sham, Vehicle, EPO, and AsialoEPO groups were 100%, 21.4%, 23.1%, and 53.8%, respectively (P=0.05).. EPO and asialoEPO attenuated renal dysfunction caused by I/R in mouse kidney at the same level, but only asialoEPO improved survival.

Topics: Animals; Apoptosis; Asialoglycoproteins; bcl-Associated Death Protein; bcl-X Protein; Creatinine; Disease Models, Animal; Erythropoietin; Female; Kidney; Mice; Mice, Inbred BALB C; p38 Mitogen-Activated Protein Kinases; Random Allocation; Reperfusion Injury; Survival Rate; X-Linked Inhibitor of Apoptosis Protein

Our objective was to establish a nonhuman primate model of perinatal asphyxia appropriate for preclinical evaluation of neuroprotective treatment strategies under conditions that closely resemble human neonatal emergencies, and to begin testing the safety and efficacy of erythropoietin neuroprotective treatment. Prior to delivery by hysterotomy, the umbilical cords of near term Macaca nemestrina (n = 8) were clamped for times ranging between 12 and 15 min. Animals received erythropoietin (5,000 U/kg/dose x 2 i.v., n = 3), or vehicle (n = 5) after resuscitation. We assessed physiologic parameters, continuous electroencephalogram, magnetic resonance imaging/spectroscopy, safety parameters and behavior. Animals were euthanized at 4 months of age. Mean birth weight was 507 +/- 62 g. Initial arterial pH ranged from 6.75 to 7.12, with base deficits of 17-25 mEq. Animals were flaccid at birth, with attenuated electroencephalograms, and seizures occurred in 3 of 8 animals. We demonstrated magnetic resonance imaging/spectroscopy changes consistent with hypoxia (elevated lactate levels were present in some animals), significant motor and behavioral abnormalities (particularly with 15 min of cord clamping), and evidence of gliosis at the time of death. We have established a reproducible model of moderate to severe perinatal hypoxic-ischemic injury in M. nemestrina newborns. This model, which combines structural, biochemical, and behavioral assessments over time can be used to assess the safety and efficacy of neuroprotective strategies.

Topics: Animals; Animals, Newborn; Asphyxia; Brain; Disease Models, Animal; Electroencephalography; Erythropoietin; Female; Fetal Monitoring; Gliosis; Hypoxia-Ischemia, Brain; Lactic Acid; Macaca nemestrina; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Neuroprotective Agents; Placental Circulation; Pregnancy; Seizures; Umbilical Cord

Erythropoietin (EPO) has recently been shown to have a neuroprotective effect in animal models of traumatic brain injury (TBI). However, the precise mechanisms remain unclear. Cerebral inflammation plays an important role in the pathogenesis of secondary brain injury after TBI. We, therefore, tried to analyze how recombinant human erythropoietin (rhEPO) might effect the inflammation-related factors common to TBI: nuclear factor kappa B (NF-kappaB), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) in a rat TBI model. Male rats were given 0 or 5000 units/kg injections of rhEPO 1h post-injury and on days 1, 2 and 3 after surgery. Brain samples were extracted at 3 days after trauma. We measured NF-kappaB by electrophoretic mobility shift assay (EMSA); IL-1beta, TNF-alpha and IL-6 by enzyme-linked immunosorbent assay (ELISA); ICAM-1 by immunohistochemistry; brain edema by wet/dry method; blood-brain barrier (BBB) permeability by Evans blue extravasation and cortical apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. We found that NF-kappaB, pro-inflammatory cytokines and ICAM-1 were increased in all injured animals. In animals given rhEPO post-TBI, NF-kappaB, IL-1beta, TNF-alpha and ICAM-1 were decreased in comparison to vehicle-treated animals. Measures of IL-6 showed no change after rhEPO treatment. Administration of rhEPO reduced brain edema, BBB permeability and apoptotic cells in the injured brain. In conclusion, post-TBI rhEPO administration may attenuate inflammatory response in the injured rat brain, and this may be one mechanism by which rhEPO improves outcome following TBI.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Blood-Brain Barrier; Brain Edema; Brain Injuries; Cerebral Cortex; Cytoprotection; Disease Models, Animal; Encephalitis; Erythropoietin; Humans; Inflammation Mediators; Intercellular Adhesion Molecule-1; Interleukin-1beta; Interleukin-6; Male; Neuroprotective Agents; NF-kappa B; Rats; Rats, Wistar; Recombinant Proteins; Treatment Outcome; Tumor Necrosis Factor-alpha

A therapeutic strategy that would mitigate the events leading to hyperplasia and facilitate re-endothelialization of an injured artery after balloon angioplasty could be effective for a long-term patency of the artery. It is hypothesized that erythropoietin (EPO), which has both anti-inflammatory and antiapoptotic properties, will prevent hyperplasia, and its ability to proliferate and mobilize endothelial progenitor cells will re-endothelialize the injured artery. To test this hypothesis, EPO (5000 IU/kg) in solution was injected intraperitoneally 6 hours before vascular injury and then on every alternate day for a week or as a single dose (5000 IU/kg) in a sustained release gel formulation 1 week before the vascular injury. Morphometric analysis revealed nearly continuous re-endothelialization of the injured artery in EPO solution-treated animals (90% vs less than 20% in saline control); however, the treatment also caused excessive neointima formation (intima/media ratio, 2.10 +/- 0.09 vs 1.60 +/- 0.02 saline control, n = 5, P < .001). The EPO gel also induced similar excessive neointima formation. Immunohistochemical analysis of the injured arteries from the animals treated with EPO solution demonstrated a significant angiogenic response in adventitia and media, thus explaining the formation of excessive neointima. Although the results are in contrast to expectation, they explain a greater degree of stenosis seen in hemodialysis access fistulas in patients who are on EPO therapy for anemic condition. The results also caution the use of EPO, particularly in patients who are at a risk of vascular injury or are suffering from an atherosclerotic condition.

Topics: Animals; Carotid Arteries; Carotid Artery Injuries; Cell Movement; Cell Proliferation; Delayed-Action Preparations; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Erythropoietin; Gels; Hyperplasia; Immunohistochemistry; Injections, Intraperitoneal; Neovascularization, Pathologic; Rats; Rats, Sprague-Dawley; Stem Cells; Tunica Intima

1. The aim of the present study was to investigate the role of oxidative stress in renal injury and to determine whether erythropoietin (EPO) acts as an anti-oxidant in vancomycin (VCM)-induced renal impairment. 2. Twenty-four rats were divided into three groups as follows: (i) control (Group 1); (ii) VCM treated (Group 2); and (iii) VCM + EPO treated (Group 3). Vancomycin (200 mg/kg, i.p.) was administered to Groups 2 and 3 for 7 days. Erythropoietin (150 IU/kg, i.p.) treatment was started 24 h before VCM and lasted for 7 days. On Day 8, renal tissues were excised and blood samples were collected. Serum creatinine and blood urea nitrogen were measured, along with renal malondialdehyde (MDA) levels, superoxide dismutase (SOD) and catalase (CAT) activity and tissue VCM levels. The kidneys were examined for any histopathological changes. 3. Renal MDA levels were found to be increased, whereas SOD and CAT activity was decreased, in the VCM-treated group compared with the control group. There was a marked decrease in MDA levels and an increase in SOD activity, but not CAT activity, after VCM + EPO treatment. Marked histopathological alterations, including interstitial oedema, tubular dilatation, tubular epithelial cell desquamation and vacuolization, were observed in VCM-treated rats. Histopathological changes were significantly improved after EPO administration. 4. In conclusion, the present data suggest that oxidative stress plays an important role in VCM-induced nephrotoxicity. Erythropoietin seems to act as an anti-oxidant, diminishing the toxic oxidative effects of VCM on renal tissues.

Topics: Animals; Anti-Bacterial Agents; Antioxidants; Blood Urea Nitrogen; Catalase; Creatinine; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Kidney; Kidney Diseases; Male; Malondialdehyde; Oxidative Stress; Rats; Rats, Wistar; Recombinant Proteins; Superoxide Dismutase; Vancomycin

To evaluate the potential protective affects of Epo on left ventricular (LV) function and remodeling after acute myocardial infarction (MI).. Epo was injected into the peritoneum of male Wistar rats (250 g) during 6 weeks post induction of MI. Rats were divided into five groups: MI treated with single high dose (MT1, 5,000 U/kg, n=10), single high dose (5,000 U/kg) and repeated high doses (MTHi, 1,000 U/kg twice a week; n=8), or single high dose (5,000 U/kg) and repeated low doses (MTLo, 750 U/kg once a week, n=10), MI non-treated (MNT, n=10), sham (S, n=5). Echocardiography was performed 3.6+/-1.5 days and 43.7+/-2.3 days post MI. Collagen deposition and infarct size were measured on histological sections using computerized image analysis. Apoptosis was assessed by ApopTag staining.. Baseline fractional shortening (FS) was similar between groups. Six weeks after MI the FS of MTLo (26.9%) was significantly higher compared to MNT (17.8%), MT1 (19.5%) and MTH (22.3%) (p=0.01). However, remodeling indices (end diastolic and end systolic areas, LV circumference) did not improve in the Epo groups, and even worsened in the MTHi group. There was significantly less collagen staining in non-infarct areas in MT1 and MTHi groups compared to MNT and MTLo (0.38+/-0.3%, 0.49+/-0.34%, vs 0.89+/-0.41%, 0.95+/-0.33%, respectively, p<0.001). The number of ApopTag positive nucleus was significantly higher in the MNT group compared to the MT1, MTHi, MTLo groups (14.4+/-8, 7.6+/-4, 5.8+/-7, 4.8+/-5, respectively, p=0.01 for trend).. Repeated low doses of Epo after MI improved LV function, but the role of Epo on remodeling is not clear. It did not reduce left ventricular indices, but reduces fibrosis and apoptosis. High Epo doses reduced LV function and aggravated remodeling.

Topics: Acute Disease; Animals; Apoptosis; C-Reactive Protein; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Electrocardiography; Erythropoietin; Hemoglobins; Male; Myocardial Infarction; Rats; Rats, Wistar; Survival Rate; Ventricular Function, Left; Ventricular Remodeling

Doxorubicin (DOX) is a highly effective chemotherapeutic agent related to dose-dependent cardiomyopathy. Recent evidence suggests that erythropoietin (EPO) can play a protective role in cardiovascular diseases by non-erythropoietic effects. In the present study, we tested the hypothesis that EPO may protect against DOX-induced cardiomyopathy through anti-apoptotic mechanisms both in vitro and in vivo.. Isolated neonatal Wistar rat cardiomyocytes were treated with vehicle, DOX with or without EPO, or EPO. Twenty-four hours later, the cells were used to determine cardiomyocyte apoptosis (TUNEL assay). Cardiomyopathy was induced in Wistar rats by intraperitoneal injections (IP) of DOX (2.5 mg/kg, six times for 2 weeks). EPO (2,500 U/kg, six times for 2 weeks) was administered simultaneously in the DOX+EPO group and the EPO group. Two weeks after the last administration, cardiac function was evaluated by echocardiography and invasive haemodynamic measurements. Rats were then sacrificed for histological and TUNEL analyses, with immunological detection for cardiac Troponin-T (cTnT), alpha-actinin, Bax and Bcl-2.. EPO significantly ameliorated DOX-induced apoptosis of cultured cardiomyocytes as demonstrated by TUNEL assay. In the rat model, cardiac function significantly decreased in the DOX group. In contrast, the DOX+EPO group showed considerable improvement in cardiac function, inhibition of cardiomyocyte apoptosis, reduction of fibrosis, as well as up-regulation of Bcl-2 protein expression.. Our results suggest that EPO exerts preventive cardioprotective effects on DOX-induced cardiomyopathy via anti-apoptotic pathways. The up-regulation of Bcl-2 protein expression may contribute to this.

Topics: Actinin; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cardiomyopathies; Cardiotonic Agents; Disease Models, Animal; Doxorubicin; Echocardiography; Erythropoietin; Immunohistochemistry; In Situ Nick-End Labeling; Male; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Troponin T

Recently we found that the level of anti-infarct tolerance afforded by ischemic preconditioning (IPC) and erythropoietin (EPO) infusion was closely correlated with the level of Ser9-phospho-GSK-3beta upon reperfusion in the heart. To get an insight into the mechanism by which phospho-GSK-3beta protects the myocardium from ischemia/reperfusion injury, we examined the effects of IPC and EPO on interactions between GSK-3beta and subunits of the mitochondrial permeability transition pore (mPTP) in this study. Rat hearts were subjected to 25-min global ischemia and 5-min reperfusion in vitro with or without IPC plus EPO infusion (5 units/ml) before ischemia. Ventricular tissues were sampled before or after ischemia/reperfusion to separate subcellular fractions for immunoblotting and immunoprecipitation. Reperfusion increased mitochondrial GSK-3beta by 2-fold and increased phospho-GSK-3beta level in all fractions examined. Major subunits of mPTP, adenine nucleotide translocase (ANT) and voltage-dependent anion channel (VDAC), were co-immunoprecipitated with GSK-3beta after reperfusion. Phospho-GSK-3beta was co-immunoprecipitated with ANT but not with VDAC. IPC+EPO significantly increased the levels of GSK-3beta and phospho-GSK-3beta that were co-immunoprecipitated with ANT to 145+/-8% and 143+/-16%, respectively, of baseline but did not induce phospho-GSK-3beta-VDAC binding. A PKC inhibitor and a PI3 kinase inhibitor suppressed the IPC+EPO-induced increase in the level of phospho-GSK-3beta-ANT complex. The level of cyclophilin D co-immunoprecipitated with ANT after reperfusion was significantly reduced to 39+/-10% of the control by IPC+EPO. These results suggest that reduction in affinity of ANT to cyclophilin D by increased phospho-GSK-3beta binding to ANT may be responsible for suppression of mPTP opening and myocardial protection afforded by IPC+EPO.

Topics: Animals; Disease Models, Animal; Erythropoietin; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; In Vitro Techniques; Intracellular Membranes; Kinetics; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Ischemia; Phosphorylation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Ventricular Function

A large body of evidence indicates that the hormone erythropoietin (EPO) exerts beneficial effects in the central nervous system (CNS). To date, EPO's effect has been assessed in several experimental models of brain and spinal cord injury. This study was conducted to validate whether treatment with recombinant human EPO (rHuEPO) would limit the extent of injury following experimental TBI. Experimental TBI was induced in rats by a cryogenic injury model. rHuEPO or placebo was injected intraperitoneally immediately after the injury and then every 8 h until 2 or 14 days. Forty-eight hours after injury brain water content, an indicator of brain edema, was measured with the wet-dry method and blood-brain barrier (BBB) breakdown was evaluated by assay of Evans blue extravasation. Furthermore, extent of cerebral damage was assessed. Administration of rHuEPO markedly improved recovery from motor dysfunction compared with placebo group (P<0.05). Brain edema was significantly reduced in the cortex of the EPO-treated group relative to that in the placebo-treated group (80.6+/-0.3% versus 91.8%+/-0.8% respectively, P<0.05). BBB breakdown was significantly lower in EPO-treated group than in the placebo-treated group (66.2+/-18.7 mug/g versus 181.3+/-21 mug/g, respectively, P<0.05). EPO treatment reduced injury volume significantly compared with placebo group (17.4+/-5.4 mm3 versus 37.1+/-5.3 mm3, P<0.05). EPO, administered in its recombinant form, affords significant neuroprotection in experimental TBI model and may hold promise for future clinical applications.

Topics: Analysis of Variance; Animals; Blood-Brain Barrier; Brain Edema; Brain Infarction; Brain Injuries; Disease Models, Animal; Erythropoietin; Evans Blue; Functional Laterality; Humans; Male; Neurologic Examination; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Reaction Time; Recombinant Proteins; Time Factors

Despite effective antibiotic treatment, neuronal injury is frequent among children and adults with bacterial meningitis resulting in a high rate of death and neurologic sequelae. The hematopoietic cytokine erythropoietin (EPO) provides neuroprotection in models of acute and chronic neurologic diseases. We studied whether recombinant EPO (rEPO) reduces neuronal damage in a rabbit model of Escherichia coli meningitis. Inflammation within the central nervous system (CNS) was monitored by measurement of bacterial load, pleocytosis, protein, and lactate in the cerebrospinal fluid (CSF). Neuronal damage was measured by quantification of the density of apoptotic neurons in the hippocampal dentate gyrus and the concentration of the global neuronal destruction marker neuron-specific enolase (NSE) in CSF. To increase clinical relevance, rEPO was applied as adjunctive therapy from the beginning of antibiotic therapy 12 h after infection. EPO treatment applied as an intravenous injection at a dose of 1000 IU/kg body weight resulted in plasma concentrations of 6993 +/- 1406 mIU/mL, CSF concentrations of 1291 +/- 568 mIU/mL, and a CSF-to-plasma ratio of 0.18 +/- 0.07 (mean +/- SD) 6 h after injection. Under these treatment conditions, no anti-inflammatory or neuroprotective effect of EPO was observed. "

Topics: Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents; Apoptosis; Dentate Gyrus; Disease Models, Animal; Drug Therapy, Combination; Erythropoietin; Injections, Intravenous; Meningitis, Escherichia coli; Nervous System Diseases; Neurons; Neuroprotective Agents; Phosphopyruvate Hydratase; Rabbits; Recombinant Proteins; Severity of Illness Index; Time Factors

The aim of this study was to investigate the role of gender in histological and functional outcome, angiogenesis, neurogenesis and therapeutic effects of recombinant human erythropoietin (rhEPO) in mice after traumatic brain injury (TBI). TBI caused both tissue loss in the cortex and cell loss in the dentate gyrus (DG) in the injured hemisphere at day 35 post TBI without a significant gender difference. After TBI, sensorimotor deficits were significantly larger in male mice compared to females, while similar spatial learning deficits were present in both genders. TBI alone significantly stimulated angiogenesis and neurogenesis in the cortex and in the DG of injured hemispheres in both genders. rhEPO at a dose of 5000 units/kg body weight administered intraperitoneally at 6 h, and 3 and 7 days after injury significantly reduced lesion volume and DG cell loss examined at day 35 after TBI as well as dramatically improved sensorimotor and spatial learning performance without an obvious gender proclivity. rhEPO significantly enhanced neurogenesis in the cortex and the DG of the ipsilateral hemisphere in male TBI mice. rhEPO did not affect angiogenesis in the ipsilateral cortex and DG in both genders after TBI. The present data demonstrate that posttraumatic administration of rhEPO improves histological and functional outcome in both genders, which may be mediated by reducing cortical tissue damage and DG cell loss in the ipsilateral hemisphere. In addition, the major gender propensity observed in the present study with mice after TBI without treatment is limited to sensorimotor deficits and cell proliferation.

Topics: Animals; Behavior, Animal; Brain Injuries; Bromodeoxyuridine; Cell Count; Cell Proliferation; Cerebral Cortex; Dentate Gyrus; Disease Models, Animal; Erythropoietin; Female; Gait Disorders, Neurologic; Learning Disabilities; Male; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Neuroprotective Agents; Phosphopyruvate Hydratase; Recombinant Proteins; Sex Characteristics; Time Factors

The R200W mutation in the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL) is unique in that it is not associated with tumor development, but rather with Chuvash polycythemia, a heritable disease characterized by elevated hematocrit and increased serum levels of erythropoietin and VEGF. Previous studies have implicated hypoxia-inducible factor-1alpha (HIF-1alpha) signaling in this disorder, although the effects of this mutation on pVHL function are not fully understood. In order to explore the mechanisms underlying the development of this polycythemia, we generated mice homozygous for the R200W mutation (Vhl(R/R)). Vhl(R/R) mice developed polycythemia highly similar to the human disease. The activity of HIF proteins, specifically the HIF-2alpha isoform, was upregulated in ES cells and tissues from Vhl(R/R) mice. Furthermore, we observed a striking phenotype in Vhl(R/R) spleens, with greater numbers of erythroid progenitors and megakaryocytes and increased erythroid differentiation of Vhl(R/R) splenic cells in vitro. These findings suggest that enhanced expression of key HIF-2alpha genes promotes splenic erythropoiesis, resulting in the development of polycythemia in Vhl(R/R) mice. This mouse model is a faithful recapitulation of this VHL-associated syndrome and represents a useful tool for studying polycythemias and investigating potential therapeutics.

Topics: Amino Acid Substitution; Animals; Basic Helix-Loop-Helix Transcription Factors; Disease Models, Animal; Erythropoiesis; Erythropoietin; Genetic Diseases, Inborn; Hematopoiesis, Extramedullary; Humans; Megakaryocytes; Mice; Mice, Mutant Strains; Mutation, Missense; Polycythemia; Signal Transduction; Spleen; Vascular Endothelial Growth Factors; Von Hippel-Lindau Tumor Suppressor Protein

In view of the emerging role of recombinant human erythropoietin (rhEPO) as a novel therapeutical approach in myocardial ischemia, we performed the first two-way parallel comparison to test the effects of rhEPO pretreatment (1000 U/kg, 12h before surgery) versus EPO transgenic overexpression in a mouse model of myocardial infarction. Unlike EPO transgenic mice who doubled their hematocrit, rhEPO pretreated mice maintained an unaltered hematocrit, thereby offering the possibility to discern erythropoietic-dependent from erythropoietic-independent protective effects of EPO. Animals pretreated with rhEPO as well as EPO transgenic mice underwent permanent left anterior descending (LAD) coronary artery ligation. Resulting infarct size was determined 24h after LAD ligation by hematoxylin/eosin staining, and morphometrical analysis was performed by computerized planimetry. A large reduction in infarction size was observed in rhEPO-treated mice (-74% +/- 14.51; P = 0.0002) and an even more pronounced reduction in the EPO transgenic group (-87% +/- 6.31; P < 0.0001) when compared to wild-type controls. Moreover, while searching for novel early ischemic markers, we analyzed expression of hypoxia-sensitive Wilms' tumor suppressor gene (WT1) in infarcted hearts. We found that its expression correlated with the infarct area, thereby providing the first demonstration that WT1 is a useful early marker of myocardial infarction. This study demonstrates for the first time that, despite high hematocrit levels, endogenously overexpressed EPO provides protection against myocardial infarction in a murine model of permanent LAD ligation.

Topics: Animals; Biomarkers; Coronary Vessels; Disease Models, Animal; Erythropoietin; Hematocrit; Humans; Mice; Mice, Transgenic; Myocardial Infarction; Recombinant Proteins; WT1 Proteins

To investigate the effect of exogenous erythro-poietin (EPO) administration on acute lung injury (ALI) in an experimental model of sodium taurodeoxycholate- induced acute necrotizing pancreatitis (ANP).. Forty-seven male Wistar albino rats were randomly divided into 7 groups: sham group (n = 5), 3 ANP groups (n = 7 each) and 3 EPO groups (n = 7 each). ANP was induced by retrograde infusion of 5% sodium taurodeoxycholate into the common bile duct. Rats in EPO groups received 1000 U/kg intramuscular EPO immediately after induction of ANP. Rats in ANP groups were given 1 mL normal saline instead. All animals were sacrificed at postoperative 24 h, 48 h and 72 h. Serum amilase, IL-2, IL-6 and lung tissue malondialdehyde (MDA) were measured. Pleural effusion volume and lung/body weight (LW/BW) ratios were calculated. Tissue levels of TNF-alpha, IL-2 and IL-6 were screened immunohistochemically. Additionally, ox-LDL accumulation was assessed with immune-fluorescent staining. Histopathological alterations in the lungs were also scored.. The mean pleural effusion volume, calculated LW/BW ratio, serum IL-6 and lung tissue MDA levels were significantly lower in EPO groups than in ANP groups. No statistically significant difference was observed in either serum or tissue values of IL-2 among the groups. The level of tumor necrosis factor-alpha (TNF-alpha) and IL-6 and accumulation of ox-LDL were evident in the lung tissues of ANP groups when compared to EPO groups, particularly at 72 h. Histopathological evaluation confirmed the improvement in lung injury parameters after exogenous EPO administration, particularly at 48 h and 72 h.. EPO administration leads to a significant decrease in ALI parameters by inhibiting polymorphonuclear leukocyte (PMNL) accumulation, decreasing the levels of proinflammatory cytokines in circulation, preserving microvascular endothelial cell integrity and reducing oxidative stress-associated lipid peroxidation and therefore, can be regarded as a cytoprotective agent in ANP-induced ALI.

Topics: Amylases; Animals; Body Weight; Disease Models, Animal; Erythropoietin; Interleukin-2; Interleukin-6; Lipoproteins, LDL; Lung; Male; Malondialdehyde; Neutrophils; Pancreatitis, Acute Necrotizing; Pleural Effusion; Pulmonary Alveoli; Rats; Rats, Wistar; Respiratory Distress Syndrome; Taurodeoxycholic Acid

This study was done to determine whether recombinant human erythropoietin (rhEPO) treatment could attenuate hyperoxia-induced lung injury, and if so, whether this protective effect is mediated by the down-modulation of inflammation in neonatal rats. Newborn Sprague Dawley rat pups were subjected to 14 days of hyperoxia (>95% oxygen) within 10 hr after birth. Treatment with rhEPO significantly attenuated the mortality and reduced body weight gain caused by hyperoxia. With rhEPO treatment, given 3 unit/gm intraperitoneally at 4th, 5th, and 6th postnatal day, hyperoxia- induced alterations in lung pathology such as decreased radial alveolar count, increased mean linear intercept, and fibrosis were significantly improved, and the inflammatory changes such as myeloperoxidase activity and tumor necrosis factor-alpha expression were also significantly attenuated. In summary, rhEPO treatment significantly attenuated hyperoxia-induced lung injury by down-modulating the inflammatory responses in neonatal rats.

Topics: Animals; Animals, Newborn; Cytoprotection; Disease Models, Animal; Erythropoietin; Female; Hyperoxia; Inflammation; Lung; Peroxidase; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Survival Rate; Tumor Necrosis Factor-alpha

Erythropoietin (EPO), originally identified for its critical role in promoting erythrocyte survival and differentiation, has been shown to exert multiple paracrine/autocrine functions. Protective effects of EPO have been demonstrated in various tissues and experimental models of ischaemia-induced injury. In the present study, we investigated the effect of EPO on an in vivo rat model of renal ischaemia/reperfusion (I/R) injury and the possible mechanisms implicated in the EPO-mediated anti-apoptotic action.. Male Wistar rats, subjected to renal ischaemia for 45 min, were administered either saline or EPO (500 U/kg, i.p.) 20 min prior to I/R. A sham-operated group served as the control. At 48 h of reperfusion, the renal dysfunction and injury was assessed by measurement of serum biochemical markers (urea, creatinine) and histological grading. Apoptosis was assessed by the TUNEL method and morphological criteria. Expression of Bax and NF-kappaB (p65) was also evaluated.. High levels of serum urea and creatinine were identified at 48 h after ischaemia. The EPO-treated group had significantly lower serum and creatinine levels. Semi-quantitative assessment of the histological lesions showed that rats subjected to I/R developed marked structural damage, whereas significantly less tubular damage was observed in the EPO-treated group. I/R caused an increase in TUNEL-positive cells that was accompanied by morphological evidence of apoptosis. In the EPO-treated rats only a few scattered TUNEL-positive cells were observed. Up-regulation of Bax in the tubular epithelial cells and increased expression of NF-kappaB was observed in the I/R-treated rats, while diminished expression of Bax and positive immunostaining of NF-kappaB was observed in the EPO-treated rats.. Administration of EPO as a single dose before the onset of ischaemia produced a significant reduction in tubular injury, which was accompanied by a marked amelioration of renal functional impairment. The cytoprotective action of EPO against I/R injury seems to be associated with its anti-apoptotic action. Moreover, transcription factor NF-kappaB is likely to play a pivotal role in the pathophysiology of I/R renal injury and might have a key role in EPO-mediated protective effects.

Topics: Acute Kidney Injury; Animals; Apoptosis; Disease Models, Animal; Erythropoietin; Male; NF-kappa B; Rats; Rats, Wistar; Reperfusion Injury

Erythropoietin (EPO), originally known for its role in stimulation of erythropoiesis, has recently been shown to have a dramatic protective effect in animal models of myocardial ischemia-reperfusion (I-R) injury. However, the precise mechanisms remain unclear. We tried to study the anti-inflammatory properties of recombinant human erythropoietin (rhEPO) using an in vivo myocardial I-R rat model, which was established by 30 min ligation of left descending coronary and 3 h reperfusion. rhEPO or saline solution was intraperitoneally injected 24 h before I-R insult. The infarct size was measured by triphenyltetrazolium chloride (TTC)-Evans blue technique. Myeloperoxidase (MPO) activity and tissue neutrophil infiltration were studied. Ultrastructural organizations were observed and semiquantitatively evaluated. Tumor necrosis-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-10 concentrations of left ventricle were analyzed by enzyme-linked immunosorbance assays; intercellular adhesion molecule-1 (ICAM-1) by reverse-transcription polymerase chain reaction; and nuclear factor-kappa B (NF-kappaB) and activator protein 1 (AP-1) by electrophoretic mobility shift assay, respectively. We found that a single bolus injection of 5000 units/kg of rhEPO 24 h before insult remarkably reduced infarct size and neutrophil infiltration. It greatly attenuated I-R-induced NF-kappaB and AP-1 activation with decreased TNF-alpha, IL-6, and ICAM-1 production, but enhanced IL-10 production. In conclusion, the cardioprotection of EPO may be due in part to the suppression of the inflammatory response via down-regulation of NF-kappaB and AP-1 induced by I-R. IL-10 was also suggested to play a protective role through another independent mechanism involved in cardioprotection of rhEPO.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cardiotonic Agents; Disease Models, Animal; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Humans; Male; Mitochondria, Heart; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Neutrophil Infiltration; Nuclear Proteins; Peroxidase; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction

Erythropoietin (EPO) mediates a wide range of neuroprotective activities, including amelioration of disease and neuroinflammation in rat models of EAE. However, optimum dosing parameters are currently unknown. In the present study, we used a chronic EAE model induced in mice by immunization with the myelin oligodendrocyte glycoprotein peptide (MOG35-55) to compare the effect of EPO given with different treatment schedules. EPO was administered intraperitoneally at 0.5, 5.0 or 50 microg/kg three times weekly starting from day 3 after immunization (preventive schedule), at the onset of clinical disease (therapeutic schedule) or 15 days after the onset of symptoms (late therapeutic schedule). The results show that EPO is effective even when given after the appearance of clinical signs of EAE, but with a reduced efficacy compared to the preventative schedule. To determine whether this effect requires the homodimeric EPO receptor (EPOR2)-mediated hematopoietic effect of EPO, we studied the effect of carbamylated EPO (CEPO) that does not bind EPOR2. CEPO, ameliorated EAE without changing the hemoglobin concentration. Another non-erythropoietic derivative, asialoEPO was also effective. Both EPO and CEPO equivalently decreased the EAE-associated production of TNF-alpha, IL-1beta and IL-1Ra in the spinal cord, and IFN-gamma by peripheral lymphocytes, indicating that their action involves targeting neuroinflammation. The lowest dosage tested appeared fully effective. The possibility to dissociate the anti-neuroinflammatory action of EPO from its hematopoietic action, which may cause undesired side effects in non-anemic patients, present new avenues to the therapy of multiple sclerosis.

Topics: Analysis of Variance; Animals; Body Weight; Chronic Disease; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Encephalomyelitis, Autoimmune, Experimental; Erythropoietin; Female; Glycoproteins; Hematocrit; Humans; Immunohistochemistry; Mice; Mice, Inbred C57BL; Myelin-Oligodendrocyte Glycoprotein; Neuroprotective Agents; Peptide Fragments; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Severity of Illness Index; Spinal Cord; Spleen; Statistics, Nonparametric; Time Factors; Treatment Outcome; Tumor Necrosis Factor-alpha

Recombinant human erythropoietin (r-Hu EPO) has been shown to exert neuroprotection in ischemic, excitotoxicity, trauma, convulsions and neurodegenerative disorders. Blood-brain barrier (BBB) leakage plays a role in the pathogenesis of many pathological states of the brain including neurodegenerative disorders. This study aimed to investigate the effects of r-Hu EPO on BBB integrity in pentylentetrazol (PTZ) induced seizures in rats. Seizures were observed and evaluated regard to latency and intensity for an hour. Macroscopical and spectrophotometrical measurement of Evans Blue (EB) leakage were observed for BBB integrity. r-Hu EPO was given intraperitoneally 24 h prior to seizure induction. Total seizure duration of 720+/-50 s after single PTZ administration (80 mg/kg i.p.) was declined to 190+/-40 s in r-Hu EPO pretreatment. A typical BBB breakdown pattern (i.e. staining in cerebellum, cerebral cortex, midbrain, hippocampus, thalamus and corpus striatum) was observed in rat brains with PTZ induced seizures; whereas, EPO pretreatment confined BBB leakage to cerebellum and cortical areas, and lessened the intensity of tonic-clonic seizures observed in PTZ seizures. The protective effect of r-Hu EPO on BBB permeability in seizures is a new and original finding. The protective action of r-Hu EPO in seizures and some of CNS pathologies warrant further investigations.

Topics: Animals; Blood-Brain Barrier; Capillary Permeability; Convulsants; Disease Models, Animal; Drug Antagonism; Erythropoietin; Hematinics; Injections, Intraperitoneal; Male; Pentylenetetrazole; Rats; Rats, Wistar; Reaction Time; Recombinant Proteins; Seizures

Postinfarct remodeling impairs mechanisms of ischemic preconditioning. We examined whether myocardial response to activation of the erythropoietin (EPO) receptor is modified by postinfarct remodeling. Four weeks after induction of myocardial infarction (MI) by coronary ligation in post-MI group (post-MI) or a sham operation in sham group (sham), rat hearts were isolated and subjected to 25-min global ischemia/2-h reperfusion. Infarct size was expressed as a percentage of risk area (i.e., left ventricle) from which scarred infarct was excluded (%I/R). The heart weight was 15% larger in post-MI, but there was no intergroup difference in plasma EPO levels or myocardial EPO receptor levels. EPO infusion (5 U/ml) significantly reduced %I/R from 59.9 +/- 4.1 to 36.2 +/- 4.2 in sham and from 58.1 +/- 5.0 to 35.2 +/- 4.0 in post-MI. This EPO-induced protection was sensitive to a phosphatidylinositol 3-kinase (PI3K) inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), in sham. However, neither LY294002 nor wortmannin inhibited the EPO-induced protection in post-MI. Phosphorylation of Janus kinase 2 by EPO was attenuated and phosphorylation of Akt was not detected in post-MI. A guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, and a mitochondrial ATP-sensitive K(+) channel (mitoK(ATP) channel) blocker, 5-hydroxydecanoate, inhibited EPO-induced protection in both sham and post-MI. Suppressor of cytokine signaling (SOCS)-1 protein level was higher by 50% in post-MI than in sham, although SOCS-3 levels were similar. These findings suggest that postinfarct remodeling disrupts cellular signaling from the EPO receptor to PI3K, presumably by increased SOCS-1. However, in the remodeled myocardium, lack of PI3K/Akt activation by the EPO receptor seems to be compensated by a mechanism upstream of the guanylyl cyclase-mitoK(ATP) channel pathway to achieve EPO-induced protection.

Topics: Animals; Blood Cell Count; Cardiotonic Agents; Disease Models, Animal; Erythropoietin; Hemodynamics; Humans; Male; Myocardial Infarction; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Recombinant Proteins; Ventricular Remodeling

Several previous studies have demonstrated a beneficial effect of the adenosine receptor (AdoR) antagonist theophylline in different forms of acute renal failure in laboratory animals and in humans. Therefore, we wanted to test whether theophylline can also improve impaired allograft function following ischemia reperfusion injury in experimental kidney transplantation (KT). Orthotopic transplantation of the left kidney was performed from Fisher 344 into Lewis rats. All transplanted rats received daily cyclosporine (5 mg/kg). The effect of theophylline treatment (10 mg/kg) on graft function was compared with appropriate controls on day 5 after KT by assessment of glomerular filtration rate (GFR) (inulin clearance). On day 5, GFR of allografts in control rats was 0.23 +/- 0.05 ml/min/g kidney weight (n = 10) compared with 0.50 +/- 0.09 ml/min/g in rats receiving theophylline (n = 9, p < 0.01), representing a 2-fold increase in GFR. Renal AdoR A(1) mRNA content was significantly increased in both KT groups compared with their respective control groups, whereas mRNA of AdoR A(2a), A(2b), and A(3) were found to be unchanged. Theophylline did not affect significantly interstitial infiltration of the graft by monocytes/macrophages and T-cells. Likewise, serum cytokines [interleukin (IL)-2, IL-6, IL-10, tumor necrosis factor-alpha] and erythropoietin plasma levels were not different among the allograft groups. The present study demonstrates that theophylline remarkably improved early renal allograft function in rats undergoing KT without influencing cytokine serum patterns or tissue inflammation. Since theophylline is a commonly used medication in humans, clinical studies in patients undergoing KT are warranted.

Topics: Animals; Cytokines; Disease Models, Animal; Erythropoietin; Glomerular Filtration Rate; Immunohistochemistry; Kidney; Kidney Cortex; Kidney Transplantation; Male; Purinergic P1 Receptor Antagonists; Rats; Rats, Inbred F344; Rats, Inbred Lew; Receptors, Purinergic P1; Reperfusion Injury; Theophylline

Hypoxia is a common cause of cell death and is implicated in many disease processes including stroke and chronic degenerative disorders. In response to hypoxia, cells express a variety of genes which allow adaptation to altered metabolic demands, decreased oxygen demands, and the removal of irreversibly damaged cells. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates the adaptive response to hypoxia in cells. In this study, we reported an early, time-related, gradual up-regulation of HIF-1alpha, and a moderate increase in vascular endothelial growth factor (VEGF)- and erythropoietin (Epo)-levels following transient focal ischemia. Moreover, we demonstrated, for the first time a specific localization of the pro-apoptotic regulator BNIP3 in striatal and cortical neurons after transient focal ischemia in rats. Prolonged intranuclear BNIP3 immunoreactivity was associated with delayed neuronal death. Experiments showed protein increases on Western blots of brain tissue with peaks at 48h after ischemia. Epo responds to ischemia in an early stage, whereas VEGF and BNIP3 accumulate in cells at later times after ischemia. This suggests the possibility that BH3-only proteins might be one of the major downstream effectors of HIF-1alpha in hypoxic cell death. These findings open the possibility that the hypoxia-regulated pro-apoptotic protein BNIP3 enters the nucleus and could interact with other proteins involved in DNA structure, transcription or mRNA splicing after focal brain ischemia.

Topics: Active Transport, Cell Nucleus; Animals; Apoptosis; Brain Ischemia; Cell Nucleus; Cerebral Cortex; Cerebral Infarction; Corpus Striatum; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Hypoxia-Inducible Factor 1, alpha Subunit; Infarction, Middle Cerebral Artery; Male; Membrane Proteins; Mitochondrial Proteins; Nerve Degeneration; Neurons; Proto-Oncogene Proteins; Rats; Rats, Wistar; Signal Transduction; Time Factors; Up-Regulation; Vascular Endothelial Growth Factor A

Apart from its well-known stimulation of erythropoiesis, erythropoietin (EPO) exhibits angiogenic and anti-apoptotic effects. These cellular protective effects have also been described in experimental acute myocardial infarction models. We investigated the effects of EPO in a porcine model of chronic progressive myocardial ischaemia.. At weeks 2 and 6 after implantation of a circumflex ameroid constrictor, endocardial electromechanical NOGA system (Biosense Webster, Inc., California, USA) mapping of the left ventricle, coronary and ventricular angiography, as well as echocardiography were performed. Two weeks after ameroid placement, 13 pigs were randomized with 7 pigs receiving 10.000 U EPO and 6 pigs receiving placebo into the ischaemic region using a NOGA guided percutaneous transendocardial injection catheter, MYOSTAR. After 6 weeks, histology (Masson's Trichrome) was analyzed.. Endocardial electromechanical mapping showed an increase of mean unipolar voltage (UV) amplitude in the ischaemic myocardial segments in the EPO-treated animals (8.5 mV pre and 10.6 mV post treatment) and a significantly reduced ischaemic surface area compared to the control group (19% vs. 41%) suggesting a decline in ischaemic injury. Echocardiography revealed 2,2 hypokinetic segments of the lateral wall in the EPO group vs. 3,3 in the control groups. The mean ejection fraction was 64% in the EPO group and 55% in the placebo group. Quantitative histological analysis of the ischaemic regions revealed a reduction of myocardial fibrosis (8% vs. 28%) in the EPO group.. Endocardial EPO injection may induce cardioprotective effects in hibernating myocardium and may attenuate the progression of ischaemic tissue damage.

Topics: Animals; Chronic Disease; Coronary Circulation; Disease Models, Animal; Disease Progression; Electrophysiologic Techniques, Cardiac; Endocardium; Erythropoietin; Image Processing, Computer-Assisted; Myocardial Contraction; Myocardial Ischemia; Myocardial Stunning; Swine; Ultrasonography

Mammalian auditory hair cells that are unable to regenerate and various agents, including gentamicin, can irreversibly damage the hair cells. Erythropoietin, known as the primary regulator of erythropoiesis, exerts also neuroprotective effects by binding to its receptor. We tested whether erythropoietin can protect the hair cells from gentamicin-induced damage.. This study localized the erythropoietin receptor in the cochlea and analyzed the effect of erythropoietin on gentamicin-damaged hair cells in vitro.. Expression of erythropoietin receptor in the rat cochlea was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Protection of auditory hair cells from gentamicin was tested in vitro by exposing cultured rat organs of Corti with increasing concentrations of erythropoietin (0.1 U/mL, 1 U/mL, and 10 U/mL).. We detected erythropoietin and erythropoietin receptor mRNA expression in the organ of Corti, spiral ganglion, and stria vascularis by RT-PCR. Immunohistochemistry revealed that the erythropoietin receptor localizes to the outer and inner hair cells and supporting cells of the organ of Corti, as well as to the spiral ganglion cells and the stria vascularis. Significantly less hair cell loss occurred in the organs of Corti that were pretreated with 0.1 U/mL erythropoietin as compared with samples treated with gentamicin only.. Decreased hair cell loss in erythropoietin-treated organs of Corti that had been exposed to gentamicin provides evidence for a protective effect of erythropoietin in aminoglycoside-induced hair cell death.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Cells, Cultured; Disease Models, Animal; Erythropoietin; Gentamicins; Hair Cells, Auditory; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Recombinant Proteins

Chronic sciatic nerve constriction injury (CCI) induces Wallerian degeneration and exaggerated pain-like behaviors. These effects are mediated in large part by pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). In this study, we demonstrate that systemically administered recombinant human erythropoietin (rhEpo) facilitates recovery from chronic neuropathic pain associated with CCI in rats. Because TNF-alpha has been implicated in the development of pain-related behaviors, we measured TNF-alpha mRNA at the nerve injury site. Systemically or locally administered rhEpo decreased TNF-alpha mRNA, compared with that observed in untreated animals. RhEpo also significantly (P < 0.05) decreased axonal degeneration. Immunohistochemistry of CCI nerve showed abundant TNF-alpha in Schwann cells, axoplasm and macrophages. In rhEpo-treated animals, TNF-alpha immunopositivity was decreased selectively in Schwann cells. These results suggest a model in which rhEpo counteracts the effects of TNF-alpha in CCI by blocking expression of TNF-alpha in Schwann cells. To further test this model, we studied primary Schwann cell cultures. RhEpo inhibited TNF-alpha expression in response to lipopolysaccharide, supporting the conclusions of our in vivo CCI experiments. In addition, rhEpo directly counteracted Schwann cell death induced by exogenously added TNF-alphain vitro. These results indicated that rhEpo regulates TNF-alpha by multiple mechanisms; rhEpo regulates TNF-alpha mRNA expression by Schwann cells but also may directly counteract TNF-alpha signaling pathways that lead to injury, chronic pain and/or death.

Topics: Analysis of Variance; Animals; Animals, Newborn; Behavior, Animal; Blood Pressure; Cell Count; Cell Death; Disease Models, Animal; Ectodysplasins; Erythropoietin; Female; Hematocrit; Hyperalgesia; Immunohistochemistry; Lipopolysaccharides; Membrane Proteins; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Schwann Cells; Sciatic Neuropathy; Time Factors; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Wallerian Degeneration

Erythropoietin (EPO) is a hormone that is neuroprotective in models of neurodegenerative diseases. This study examined whether EPO can protect against neuronal death in the CA1 region of the rat hippocampus following global cerebral ischemia. Recombinant human EPO was infused into the intracerebral ventricle either before or after the induction of ischemia produced by using the four-vessel-occlusion model in rat. Hippocampal CA1 neuron damage was ameliorated by infusion of 50 U EPO. Administration of EPO was neuroprotective if given 20 hr before or 20 min after ischemia, but not 1 hr following ischemia. Coinjection of the phosphoinositide 3 kinase inhibitor LY294002 with EPO inhibited the protective effects of EPO. Treatment with EPO induced phosphorylation of both AKT and its substrate, glycogen synthase kinase-3beta, in the CA1 region. EPO also enhanced the CA1 level of brain-derived neurotrophic factor. Finally, we determined that ERK activation played minor roles in EPO-mediated neuroprotection. These studies demonstrate that a single injection of EPO ICV up to 20 min after global ischemia is an effective neuroprotective agent and suggest that EPO is a viable candidate for treating global ischemic brain injury.

Topics: Animals; Brain Ischemia; Brain-Derived Neurotrophic Factor; Cerebral Infarction; Disease Models, Animal; Drug Administration Schedule; Enzyme Inhibitors; Erythropoietin; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; Injections, Intraventricular; Male; Neurons; Neuroprotective Agents; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Signal Transduction; Time Factors

Cerebral involvement during malaria is a complication that leads to seizure, coma, and death. The effect of new neuroprotective therapies has not yet been investigated, although cerebral malaria shares some features with neurological stroke. Erythropoietin (EPO) is one of the more promising drugs in this area. We measured the effect of EPO on the survival of mice infected with Plasmodium berghei ANKA and demonstrated that inoculations of recombinant human EPO at the beginning of the clinical manifestations of cerebral malaria protect >90% of mice from death. This drug has no effect on the course of parasitemia. The effect of EPO was not related to either the inhibition of apoptosis in the brain or the regulation of the increase and decrease of nitric oxide production in the brain and blood, respectively. Tumor necrosis factor-alpha and interferon-gamma mRNA overexpression was inhibited by EPO, and treated mice had fewer brain hemorrhages. EPO has been used in patients with chronic diseases for years, and more recently it has been used to treat acute ischemic stroke. The data presented here provide the first evidence indicating that this cytokine could be useful for the symptomatic prevention of mortality during the acute stage of cerebral malaria.

Topics: Animals; Apoptosis; Brain; Disease Models, Animal; Erythropoietin; Female; Gene Expression; Interferon-gamma; Malaria; Malaria, Cerebral; Mice; Neuroprotective Agents; Nitric Oxide; Parasitemia; Plasmodium berghei; Recombinant Proteins; RNA, Messenger; Survival Analysis; Tumor Necrosis Factor-alpha

Erythropoietin (Epo) is the principal regulator of the erythropoietic response to hypoxic stress, through its receptor, EpoR. The EpoR signals mediating the stress response are largely unknown, and the spectrum of progenitors that are stress responsive is not fully defined. Here, we used flow cytometry to identify stress-responsive Ter119+CD71highFSChigh early erythroblast subsets in vivo. In the mouse spleen, an erythropoietic reserve organ, early erythroblasts were present at lower frequencies and were undergoing higher rates of apoptosis than equivalent cells in bone marrow. A high proportion of splenic early erythroblasts coexpressed the death receptor Fas, and its ligand, FasL. Fas-positive early erythroblasts were significantly more likely to coexpress annexin V than equivalent, Fas-negative cells, suggesting that Fas mediates early erythroblast apoptosis in vivo. We examined several mouse models of erythropoietic stress, including erythrocytosis and beta-thalassemia. We found a dramatic increase in the frequency of splenic early erythroblasts that correlated with down-regulation of Fas and FasL from their cell surface. Further, a single injection of Epo specifically suppressed early erythroblast Fas and FasL mRNA and cell-surface expression. Therefore, Fas and FasL are negative regulators of erythropoiesis. Epo-mediated suppression of erythroblast Fas and FasL is a novel stress response pathway that facilitates erythroblast expansion in vivo.

Topics: Animals; Cell Survival; Disease Models, Animal; Down-Regulation; Erythroblasts; Erythropoiesis; Erythropoietin; Fas Ligand Protein; fas Receptor; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred MRL lpr; Mice, Transgenic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Erythropoietin (EPO), a pleiotropic cytokine involved in erythropoiesis, is tissue-protective in ischemic, traumatic, toxic and inflammatory injuries. In this study, we investigated the effect of EPO in experimental intracerebral hemorrhage (ICH). Two hours after inducing ICH via the stereotaxic infusion of collagenase, recombinant human EPO (500 or 5000 IU/kg, ICH + EPO group) or PBS (ICH + vehicle group) was administered intraperitoneally, then once daily afterwards for 1 or 3 days. ICH + EPO showed the better functional recovery in both rotarod and modified limb placing tests. The brain water content was decreased in ICH + EPO dose-dependently, as compared with ICH + vehicle. The effect of EPO on the brain water content was inhibited by N(omega)-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 10 mg/kg). Mean hemorrhage volume was also decreased in ICH + EPO. EPO reduced the numbers of TUNEL +, myeloperoxidase + or OX-42 + cells in the perihematomal area. In addition, EPO reduced the mRNA level of TNF-alpha, Fas and Fas-L, as well as the activities of caspase-8, 9 and 3. EPO treatment showed up-regulations of endothelial nitric oxide synthase (eNOS) and p-eNOS, pAkt, pSTAT3 and pERK levels. These data suggests that EPO treatment in ICH induces better functional recovery with reducing perihematomal inflammation and apoptosis, coupled with activations of eNOS, STAT3 and ERK.

Topics: Animals; Apoptosis; Biomarkers; Body Water; Brain; Brain Edema; Cell Death; Cerebral Hemorrhage; Disease Models, Animal; Dose-Response Relationship, Drug; Encephalitis; Enzyme Activation; Enzyme Inhibitors; Erythropoietin; Male; Nerve Degeneration; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type III; Rats; Rats, Sprague-Dawley; Recovery of Function; Signal Transduction; STAT3 Transcription Factor; Treatment Outcome

Administration of human recombinant erythropoietin (EPO) at time of acute ischemic renal injury (IRI) inhibits apoptosis, enhances tubular epithelial regeneration, and promotes renal functional recovery. The present study aimed to determine whether darbepoetin-alfa (DPO) exhibits comparable renoprotection to that afforded by EPO, whether pro or antiapoptotic Bcl-2 proteins are involved, and whether delayed administration of EPO or DPO 6 h following IRI ameliorates renal dysfunction. The model of IRI involved bilateral renal artery occlusion for 45 min in rats (N = 4 per group), followed by reperfusion for 1-7 days. Controls were sham-operated. Rats were treated at time of ischemia or sham operation (T0), or post-treated (6 h after the onset of reperfusion, T6) with EPO (5000 IU/kg), DPO (25 mug/kg), or appropriate vehicle by intraperitoneal injection. Renal function, structure, and immunohistochemistry for Bcl-2, Bcl-XL, and Bax were analyzed. DPO or EPO at T0 significantly abrogated renal dysfunction in IRI animals (serum creatinine for IRI 0.17 +/- 0.05 mmol/l vs DPO-IRI 0.08 +/- 0.03 mmol/l vs EPO-IRI 0.04 +/- 0.01 mmol/l, P = 0.01). Delayed administration of DPO or EPO (T6) also significantly abrogated subsequent renal dysfunction (serum creatinine for IRI 0.17 +/- 0.05 mmol/l vs DPO-IRI 0.06 +/- 0.01 mmol/l vs EPO-IRI 0.03 +/- 0.03 mmol/l, P = 0.01). There was also significantly decreased tissue injury (apoptosis, P < 0.05), decreased proapoptotic Bax, and increased regenerative capacity, especially in the outer stripe of the outer medulla, with DPO or EPO at T0 or T6. These results reaffirm the potential clinical application of DPO and EPO as novel renoprotective agents for patients at risk of ischemic acute renal failure or after having sustained an ischemic renal insult.

Topics: Acute Kidney Injury; Animals; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Cell Division; Creatinine; Darbepoetin alfa; Disease Models, Animal; Erythropoietin; Immunohistochemistry; Injections, Intraperitoneal; Kidney Tubular Necrosis, Acute; Kidney Tubules, Proximal; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Regeneration; Reperfusion Injury; Time Factors

Bone marrow cell implantation (BMI) has been utilized to treat patients with limb and heart ischemia. BMI provides angiogenic precursors and angiogenic cytokine-producing cells, especially erythroid cells. In this study, we induced in vitro angiogenesis cultures and in vivo BMI simulation using a murine limb ischemia model to examine the role of erythroid cells and the effect of erythropoietin (EPO). Human erythroid colonies (BFU-e) induced capillary networks around the colonies in vitro. Erythroid cells in human bone marrow produced vascular endothelial growth factor and placental growth factor. The angiogenic effects of erythroid cells were further amplified in the presence of EPO. Limb-ischemic mice were treated with BMI +/- EPO, and limb survival, blood flow recovery, and muscle histology were analyzed. Treatment with whole bone marrow cells + EPO significantly improved limb survival and blood flow. The cumulative effects of EPO on BMI induced and increase in capillary number and artery enlargement. Erythroid cells were essential for the in vivo effects of BMI, and CD14-positive cells supported the biological effects. In addition to the direct effect of EPO on angiogenesis, EPO showed indirect effect on angiogenesis through amplifying the angiogenic effects by erythroid cells supported by CD14-positive cells.

Topics: Adult; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Disease Models, Animal; Endothelium, Vascular; Erythroid Cells; Erythroid Precursor Cells; Erythropoietin; Female; Humans; Lipopolysaccharide Receptors; Male; Mice; Mice, Inbred ICR; Myocardial Ischemia; Neovascularization, Physiologic; Platelet Endothelial Cell Adhesion Molecule-1

Spinal cord injury (SCI) is a devastating clinical syndrome for which no truly efficacious therapy has yet been identified. In preclinical studies, erythropoietin (EPO) and its nonerythropoietic derivatives asialoEPO and carbamylated EPO have markedly improved functional outcome when administered after compressive SCI. However, an optimum treatment paradigm is currently unknown. Because the uninjured spinal cord expresses a high density of EPO receptor (EPOR) in the basal state, signaling through these existing receptors in advance of injury (pharmacological preconditioning) might confer neuroprotection and therefore be potentially useful in situations of anticipated damage.. The authors compared asialoEPO, a molecule that binds to the EPOR with high affinity but with a brief serum half-life (t1/2 < 2 minutes), to EPO to determine whether a single dose (10 microg/kg of body weight) administered by intravenous injection 24 hours before 1 minute of spinal cord compression provides benefit as determined by a 6-week assessment of neurological outcome and by histopathological analysis. Rats pretreated with asialoEPO or EPO and then subjected to a compressive injury exhibited improved motor function over 42 days, compared with animals treated with saline solution. However, pretreatment efficacy was substantially poorer than efficacy of treatment initiated at the time of injury. Serum samples drawn immediately before compression confirmed that no detectable asialoEPO remained within the systemic circulation. Western blot and immunohistochemical analyses performed using uninjured spinal cord 24 hours after a dose of asialoEPO exhibited a marked increase in glial fibrillary acidic protein, suggesting a glial response to EPO administration.. These results demonstrate that EPO and its analog do not need to be present at the time of injury to provide tissue protection and that tissue protection is markedly effective when either agent is administered immediately after injury. Furthermore, the findings suggest that asialoEPO is a useful reagent with which to study the dynamics of EPO-mediated neuroprotection. In addition, the findings support the concept of using a nonerythropoietic EPO derivative to provide tissue protection without activating the undesirable effects of EPO.

Topics: Analysis of Variance; Animals; Asialoglycoproteins; Blotting, Western; Disease Models, Animal; Erythropoietin; Immunoenzyme Techniques; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Spinal Cord Injuries

The goal of this research was to develop a strategy to couple stem cell and gene therapy for in vivo delivery of erythropoietin (Epo) for treatment of anemia of ESRD. It was shown previously that autologous bone marrow stromal cells (MSCs) can be genetically engineered to secrete pharmacologic amounts of Epo in normal mice. Therefore, whether anemia in mice with mild to moderate chronic renal failure (CRF) can be improved with Epo gene-modified MSCs (Epo+MSCs) within a subcutaneous implant was examined. A cohort of C57BL/6 mice were rendered anemic by right kidney electrocoagulation and left nephrectomy. In these CRF mice, the hematocrit (Hct) dropped from a prenephrectomy baseline of approximately 55% to 40% after induction of renal failure. MSCs from C57BL/6 donor mice were genetically engineered to secrete murine Epo at a rate of 3 to 4 units of Epo/10(6) cells per 24 h, embedded in a collagen-based matrix, and implanted subcutaneously in anemic CRF mice. It was observed that Hct increased after administration of Epo+MSCs, according to cell dose. Implants of 3 million Epo+MSCs per mouse had no effect on Hct, whereas 10 million led to a supraphysiologic effect. The Hct of CRF mice that received 4.5 or 7.5 million Epo+MSCs rose to a peak 54+/-4.0 or 63+/-5.5%, respectively, at 3 wk after implantation and remained above 48 or 54% for >19 wk. Moreover, mice that had CRF and received Epo+MSCs showed significantly greater swimming exercise capacity. In conclusion, these results demonstrate that subcutaneous implantation of Epo-secreting genetically engineered MSCs can correct anemia that occurs in a murine model of CRF.

Topics: Anemia; Animals; Bone Marrow Cells; Disease Models, Animal; Erythropoietin; Female; Genetic Engineering; Mice; Mice, Inbred C57BL; Renal Insufficiency; Retroviridae; Stem Cells; Stromal Cells

Selective neuronal loss following status epilepticus (SE) was first described just under 100 years ago. The acute pathology following SE was shown to be 'ischemic cell change' and was assumed to arise through hypoxia/ischemia. Recently, erythropoietin (Epo) has been shown to have potent anti-apoptosis activity in central nervous system neurons in animal models of ischaemic injury.. In this report, in order to determine Epo preconditioning on hippocampus neuronal apoptosis, we examined caspase-3 expression following SE caused by Li-pilocarpine in rats.. Animals were classified into three groups: EP group (pilocarpine group), rhEpo-pilocarpine group and control group. Four hours after preconditioning with Epo intraperitoneally, pilocarpine hydrochloride was administered intraperitoneally and observed for behavioral manifestations of SE. The animals were sacrificed at one hour after SE onset.. At the above-mentioned time point, animals were deeply anesthetized and were perfused through the left ventricle. Detection of hippocampus neuronal apoptosis was performed with caspase-3 immunohistochemical technique on three groups. To further confirm which cell population upregulates caspase-3, brain sections were stained for NeuN (green) and caspase-3 (red).. ANOVA and Fisher's post hoc test was used.. Quatification of hippocampus neurons revealed that the number of caspase-3-positive cells in the CA1/CA3 area and dentate gyrus(DG) of three groups had a significant difference. In comparison with control group, there was an increase by 74% and 534%, 42% and 272% in the CA1/CA3 area and DG of EP group and rhEpo-treated group respectively. There was a decrease by 18% and 26% in the CA1/CA3 area and DG of rhEpo-treated group compared with those in EP group. In addition, colocalization of caspase-3 with NeuN was shown.. Systemic rhEpo therapy reduced caspase-3 expression in SE induced by Li-pilocarpine.

Topics: Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Disease Models, Animal; Erythropoietin; Hippocampus; Male; Neurons; Pilocarpine; Rats; Rats, Sprague-Dawley; Status Epilepticus

To investigate the consequences of inborn excessive erythrocytosis, we made use of our transgenic mouse line (tg6) that constitutively overexpresses erythropoietin (Epo) in a hypoxia-independent manner, thereby reaching hematocrit levels of up to 0.89. We detected expression of human Epo in the brain and, to a lesser extent, in the lung but not in the heart, kidney, or liver of tg6 mice. Although no acute cardiovascular complications are observed, tg6 animals have a reduced lifespan. Decreased swim performance was observed in 5-mo-old tg6 mice. At about 7 mo, several tg6 animals developed spastic contractions of the hindlimbs followed by paralysis. Morphological analysis by light and electron microscopy showed degenerative processes in liver and kidney characterized by increased vascular permeability, chronic progressive inflammation, hemosiderin deposition, and general vasodilatation. Moreover, most of the animals showed severe nerve fiber degeneration of the sciatic nerve, decreased number of neuromuscular junctions, and degeneration of skeletal muscle fibers. Most probably, the developing demyelinating neuropathy resulted in muscular degeneration demonstrated in the extensor digitorum longus muscle. Taken together, chronically increased Epo levels inducing excessive erythrocytosis leads to multiple organ degeneration and reduced life expectancy. This model allows investigation of the impact of excessive erythrocytosis in individuals suffering from polycythemia vera, chronic mountain sickness, or in subjects tempted to abuse Epo by means of gene doping.

Topics: Altitude Sickness; Animals; Disease Models, Animal; Doping in Sports; Erythropoietin; Female; Hematocrit; Humans; Kidney; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Skeletal; Nerve Degeneration; Nervous System; Physical Endurance; Polycythemia

Topics: Animals; Disease Models, Animal; Endothelium, Vascular; Erythropoietin; Gene Expression Regulation; Mice; Mice, Knockout; Neuroprotective Agents; NF-E2-Related Factor 2; Nitric Oxide; Subarachnoid Hemorrhage

Erythropoietin (EPO) has been suggested to have a cardioprotective effect against ischemia. The purpose of this study was to examine the effects of EPO on cardiac remodeling after myocardial infarction (MI). MI was induced by ligation of the coronary artery in Wistar rats. The rats with MI were randomly divided into untreated MI and two EPO-treated MI groups. EPO was administered subcutaneously by injection once a day for 4 days after MI at 5000 U/kg or 3 times a week for 4 weeks at 1000 U/kg. Five days after MI, EPO prevented the increase in activated caspase 3, matrix metalloproteinase-2, and transcriptional activation of activator protein-1 in non-infarcted myocardium. Four weeks after MI, left ventricular weight, left ventricular end-diastolic pressure, and left ventricular dimension were increased, and ejection fraction and E wave deceleration time were decreased. EPO significantly attenuated this ventricular remodeling and systolic and diastolic dysfunction. In addition, EPO significantly attenuated the interstitial fibrosis and remodeling-related gene expression in non-infarcted myocardium. Furthermore, EPO significantly enhanced angiogenesis and reduced apoptotic cell death in peri-infarcted myocardium. In conclusion, when administered after MI, EPO prevents cardiac remodeling and improves ventricular function with enhanced angiogenesis and reduced apoptosis.

Topics: Animals; Apoptosis; Blotting, Northern; Blotting, Western; Caspase 3; Caspases; Disease Models, Animal; Echocardiography, Doppler; Erythropoietin; In Situ Nick-End Labeling; Male; Matrix Metalloproteinase 2; Myocardial Infarction; Myocardium; Rats; Rats, Wistar; Recombinant Proteins; Transcription Factor AP-1; Ventricular Function, Left; Ventricular Pressure; Ventricular Remodeling

Radiocontrast-induced nephropathy (CIN) remains an important iatrogenic cause of acute renal failure in high-risk patients, despite the development of safer contrast media, the improvement of hydration protocols, and the introduction of additional preventive strategies. Erythropoietin (EPO) pretreatment may confer protection against acute renal failure through the induction of stress response genes.. The effect of EPO has been evaluated in a rat model of CIN, induced by iothalamate, following the inhibition of nitric oxide- and prostaglandin-synthesis with indomethacin and N(omega) nitro-L-arginine methyl ester (L-NAME). Twenty-two male Sprague-Dawley rats were subjected to saline (CTR) or EPO injections (3000 U/kg and 600 U/kg, 24 and 2 h before the induction of CIN, respectively).. The decline in creatinine clearance in CTR animals from 0.38 +/- 0.03 to 0.28 +/- 0.03 mL/min/100 g (p < 0.005), was prevented by EPO pretreatment (from 0.34 +/- 0.02 to 0.32 +/- 0.03 mL/min/100 g, NS). The extent of medullary thick ascending limb- and S3-tubular damage in the outer medulla, however, was comparable in the two experimental groups.. EPO pretreatment prevents renal dysfunction in a rat model of CIN. Further experimental and clinical studies are required to confirm these preliminary conclusions regarding a potential protective potency of EPO against CIN.

Topics: Acute Kidney Injury; Animals; Contrast Media; Creatinine; Disease Models, Animal; Erythropoietin; Kidney Medulla; Kidney Tubules; Male; Potassium; Random Allocation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Sodium; Stress, Physiological; Urea

Previous studies in rodents from different kinds of shock models and isolated vessel models indicate that erythropoietin (EPO) has haemodynamic effects through interaction with sympathetic stimuli. This has relevance to the recently described non-haematopoietic effects of EPO, e.g. tissue protective effects. Studies describing the acute effects on integrated physiological haemodynamic variables in larger animal models are scarce though.. To examine the acute effects of EPO on standard physiological haemodynamic parameters as well as a possible synergistic effect of a sympathetic agonist (dopamine) and EPO on these parameters. RESULTS AND DESIGN: A porcine model was applied. Invasive haemodynamic variables were recorded at baseline, during 2 h after EPO injection and with addition of dopamine. Significant changes were only seen with addition of dopamine. Thus cardiac output increased only significantly in control group (21% versus 4%, P<0.05), and accordingly a decline in systemic vascular resistance was only seen in the control group (19% versus 5%, P< 0.05) with addition of dopamine. Pulmonary vascular resistance increased in EPO group (42% versus unchanged, P<0.05). There was a trend towards increase in left ventricular contractility as measured by slope of the pressure-volume relation (E(max)) in EPO group with addition of dopamine.. Erythropoietin has small but significant haemodynamic effects on the response to a sympathetic agonist in the present minimal invasive porcine model.

Topics: Animals; Cardiac Output; Disease Models, Animal; Dopamine; Erythropoietin; Humans; Models, Cardiovascular; Models, Genetic; Models, Statistical; Oxygen; Oxygen Consumption; Shock; Swine; Systole; Time Factors

Using an established mouse model of human periventricular leukomalacia, we investigated whether EPO could reduce excitotoxic damage. When administered 1 h following intracerebral injection of 10 microg ibotenic acid at day 5 of life, both a single injection of EPO (5000 IU/kg bw) and repetitive administrations of EPO reduced white and gray matter lesion size. The therapeutic window for protection was small as the protective effect of EPO was lost when EPO administration was delayed to 4 h post-insult. EPO-mediated upregulation of EPO-R, but not EPO, mRNA was observed within 4 h of the excitotoxic insult. The EPO effect was gender independent. Minor hematopoetic effects were observed following EPO treatment. We conclude that a single dose of EPO is sufficient to reduce excitotoxic brain injury and may therefore possess therapeutic relevance in the clinical setting.

Topics: Animals; Animals, Newborn; Brain; Cytoprotection; Disease Models, Animal; Drug Administration Schedule; Erythropoietin; Female; Glutamic Acid; Humans; Ibotenic Acid; Infant, Newborn; Injections, Intraventricular; Leukomalacia, Periventricular; Male; Mice; Nerve Degeneration; Neuroprotective Agents; Neurotoxins; Receptors, Erythropoietin; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Time Factors

Treatment with erythropoietin and AT1 blockers is protective in experimental acute cerebral ischemia, with promising results in pilot clinical studies in human stroke. This paper examines the effects of using both agents as combination therapy in acute ischemic stroke.. We used the single carotid ligation stroke model in the gerbil. Six groups of 50 gerbils were treated either with placebo, erythropoietin (intraperitoneally, 5000 IU/kg, 2 and 48 h after stroke), olmesartan (10 mg/kg per day in drinking water started 36 h after stroke), ramipril (2.5 mg/kg per day in drinking water started 36 h after stroke), erythropoietin + olmesartan, or erythropoietin + ramipril. Long-term (1 month) Kaplan-Meyer survival curves were obtained, and survivors were submitted at day 30 to immediate (object recognition test) and spatial (Morris water maze) memory function tests.. Erythropoietin alone and olmesartan alone, but not ramipril, significantly increased survival at day 30 compared with untreated controls (38, 30 and 6% versus 12%, respectively). Combined treatment with erythropoietin and olmesartan further increased the survival rate to 56%, whereas combined therapy with erythropoietin and ramipril decreased 30-day survival to 24% (P < 0.0001, erythropoietin + olmesartan versus erythropoietin + ramipril). Untreated stroke survivors had markedly altered performances in both the object recognition test (P = 0.0007) and the Morris water maze (P < 0.0001) tests at day 30 compared with normal gerbils. In erythropoietin-treated animals, ramipril therapy had no beneficial effect whereas olmesartan fully restored normal response to the memory tests.. Post-infarct treatment with olmesartan combined with early erythropoietin therapy has a protective effect on survival, and markedly improves long-term memory dysfunction in this experimental model.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Disease Models, Animal; Drug Therapy, Combination; Erythropoietin; Gerbillinae; Imidazoles; Kaplan-Meier Estimate; Male; Memory Disorders; Neuroprotective Agents; Ramipril; Recombinant Proteins; Stroke; Tetrazoles

Correcting anemia with recombinant human erythropoietin (rhEPO) in chronic renal failure has been associated with an increased blood pressure (BP), which may accelerate the decline in renal function. This has been attributed, in part, to the activation of the renin-angiotensin system. The present study was designed to investigate the protective effect of the angiotensin II-receptor blocker losartan compared with the angiotensin-converting enzyme inhibitor captopril and conventional triple therapy (TRx) in uremic rats receiving rhEPO therapy.. Renal failure was induced by renal mass ablation followed by a 3-week stabilization period. Uremic rats were then divided into five groups with similar systolic BP: vehicle; rhEPO (100 U/kg, subcutaneously, three times per week); rhEPO + losartan (20 mg/kg/d); rhEPO + captopril (20 mg/kg/d); and rhEPO + TRx (reserpine 5 mg/L, hydralazine 80 mg/L, hydrochlorothiazide 20 mg/L). Systolic BP as well as blood and renal parameters were assessed before and after a 3-week treatment period. Renal histology was evaluated at the end of the study.. The uremic rats developed hypertension, anemia, proteinuria, and increased urinary endothelin-1 (ET-1) excretion. The rhEPO corrected the anemia but aggravated the hypertension (P < .01), glomerular sclerosis, tubular atrophy, and interstitial fibrosis. Treatment with losartan, captopril, and the TRx prevented the rhEPO-induced increased in systolic BP. The TRx was less effective in preventing histologic injuries despite similar systolic BP reduction.. Blockade of the renin-angiotensin system is highly effective in preventing both hypertension and renal histologic damage in rhEPO-treated uremic rats and this benefit seems to extend beyond the antihypertensive effect.

Topics: Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Blood Pressure; Body Weight; Captopril; Disease Models, Animal; Drug Therapy, Combination; Erythropoietin; Hydralazine; Hydrochlorothiazide; Hypertension; Kidney; Kidney Failure, Chronic; Losartan; Nephrectomy; Rats; Rats, Wistar; Recombinant Proteins; Renin-Angiotensin System; Reserpine; Time Factors; Uremia

To observe the treatment and it's mechanisms of rHu-EPO on acute myocardial infarction of SD rats in vitro and vivo.. Cardiomyocytes were isolated from neonatal Sprague-Dawley rats. Hypoxia condition and oxidative stress were used to induce apoptosis. rHu-EPO was added to the culture system. Apoptosis was assessed by using Hoechst 33258 dyeing. Apoptosis index (AI) was then calculated. Thirty two rats were divided into three groups including sham operation group (Sham), acute myocardial infarction group (MI) and treated group (MI + EPO). Acute myocardial infarction model was made by ligating the anterior descending coronary artery. rHu-EPO was administered i.p. in MI + EPO group at the dose of 5000 units/kg of body weight immediately after the ligation and the next six days. At the fourteenth day all animals underwent hemodynamic measurements and then executed, the samples were examined with hematoxylin and eosin (HE) stain, immunohistochemistry technique (Bcl-2, Bax) and TdT-mediated dUTP nick end labeling (TUNEL) dyeing.. rHu-EPO significantly down-regulated the apoptosis of cardiomyocytes which underwent hypoxia or oxidative stress. In vivo experiment rHu-EPO protected the hemodynamic function of the rats from myocardial infarction and down-regulated the ratio of the positive cells for TUNEL and Bax. The ratio of the positive cells for Bcl-2 was up-regulated by rHu-EPO.. These findings suggested rHu-EPO improve myocardial infarction by attenuating apoptosis. Potential mechanism is to up-regulated Bcl-2 expression and down-regulated Bax expression.

Topics: Animals; Animals, Newborn; Apoptosis; Cell Hypoxia; Cells, Cultured; Disease Models, Animal; Erythropoietin; Humans; In Situ Nick-End Labeling; Injections, Intraperitoneal; Male; Myocardial Infarction; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Recombinant Proteins

Darbepoietin alfa (DA) is a long-acting analogue of erythropoietin that has reduced receptor affinity and enhanced biological activity. Experiments were done to test the hypothesis that correction of anemia in tumor-bearing mice by DA would increase tumor oxygenation and potentiate radiation-induced tumor cell killing. A SCC VII tumor model was used to study tumor responses to fractionated radiation therapy in mice with anemia induced by total body irradiation. Administration of DA reduced the extent and duration of anemia and associated tumor hypoxia, protected the bone marrow cells and prevented the body weight loss from the effect of irradiation, and facilitated the recovery in a time-dependent manner, with the administration of DA prior to total body irradiation having the greatest protective effect. When combined with fractionated radiation therapy, DA increased the tumor growth delay time from 2.7 days for irradiation alone to 7.3 to 10.6 days for combination of DA and irradiation. The effect of DA on tumor responses to fractionated radiation therapy was observed when DA was given 18 to 4 days before starting radiation therapy, but DA was also equally effective as a radiosensitizer when given only 2 hours before fractionated irradiation therapy. Weekly dosing of DA was as efficacious for the enhancement of radiation responses of tumors as biweekly dosing. Similar results were obtained in the RIF-1 fibrosarcoma tumor model. These studies show that DA can effectively correct anemia in tumor-bearing mice and sensitize tumor cells to fractionated radiation therapy. Importantly, DA was also able to sensitize tumors to radiation in mice with uncorrected anemia and hypoxia, suggesting that the effect of DA on radiosensitivity was independent of these factors and a different mechanism of action may be responsible for this effect.

Topics: Anemia; Animals; Carcinoma, Squamous Cell; Darbepoetin alfa; Disease Models, Animal; Erythropoietin; Kinetics; Male; Mice; Mice, Inbred C3H; Radiotherapy; Whole-Body Irradiation

Erythropoietin (EPO), originally recognized for its central role in erythropoiesis, has been shown to improve neurological outcome after stroke. Here, we investigated the treatment of experimental autoimmune encephalomyelitis (EAE) in mice with EPO. Mice were treated with recombinant human EPO (rhEPO) upon onset of paresis. Neurological functional tests were scored daily by grading of clinical signs (score 0-5). Hematoxylin and eosin (HE) staining of cerebral tissue was performed to detect inflammatory infiltrates. Double staining for Luxol fast blue and Bielshowsky was used to demonstrate myelin and axons, respectively. Immunohistochemistry was performed to measure the expression of bromodeoxyuridine (BrdU, a marker for cell proliferation), NG2 (a marker for oligodendrocyte progenitor cells) and brain-derived neurotrophic factor (BDNF). Treatment with rhEPO significantly improved neurological functional recovery, reduced inflammatory infiltrates and demyelination, and increased oligodendrocyte progenitor cell proliferation and BDNF+ cells compared to the EAE controls. These data indicate that rhEPO treatment improved functional recovery after EAE in mice, possibly, via stimulating oligodendrogenesis, downregulating proinflammatory infiltrates and by elevating BDNF expression.

Topics: Animals; Antigens; Brain-Derived Neurotrophic Factor; Bromodeoxyuridine; Cell Differentiation; Cell Proliferation; Central Nervous System; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Erythropoietin; Female; Immunohistochemistry; Mice; Nerve Fibers, Myelinated; Proteoglycans; Recovery of Function; Treatment Outcome; Up-Regulation

Erythropoietin (EPO) is a potent stimulator of erythroid progenitor cells, and its expression is enhanced by hypoxia. The aim of this study was to investigate the effect of EPO on collagen-induced arthritis (CIA) in the mouse.. CIA was induced by intradermal injection of bovine type II collagen (CII) and Freund's complete adjuvant. Starting on day 25, some of the mice with CIA received daily subcutaneous injections of EPO (1,000 units/kg). Two other groups of mice received sham treatment alone or sham treatment followed by EPO treatment, respectively. Arthritis was assessed clinically, radiologically, and histologically. Cytokine and chemokine levels were measured, and neutrophil infiltration into inflamed joints was quantitated. Immunohistochemistry studies were performed to measure protein nitrosylation. Chondrocyte apoptosis was assessed by TUNEL assay.. Macroscopic clinical evidence of CIA first appeared as periarticular erythema and edema in the hind paws. The incidence of CIA was 100% by day 27 in the CII-challenged mice, and the severity of CIA progressed over a 35-day period, with radiographic evaluation revealing focal resorption of bone. Histopathologic features of CIA included erosion of the cartilage at the joint margins. Treatment with EPO starting at the onset of arthritis (day 25) ameliorated the clinical signs on days 26-35 and improved histologic status in the joints and paws. The degree of oxidative and nitrosative damage was significantly reduced in EPO-treated mice as indicated by decreased nitrotyrosine formation and poly(ADP-ribose) polymerase activation. Plasma levels of the proinflammatory cytokine tumor necrosis factor alpha were also significantly reduced by EPO treatment. In addition, EPO reduced the levels of apoptosis in chondrocytes in articular cartilage, as indicated by decreased TUNEL staining.. These findings demonstrate that EPO exerts an antiinflammatory effect during chronic inflammation and is able to ameliorate the tissue damage associated with CIA.

Topics: Animals; Apoptosis; Arthritis, Experimental; Cartilage, Articular; Chemokines; Chondrocytes; Cytokines; Disease Models, Animal; Erythropoietin; Hematinics; Injections, Subcutaneous; Mice; Mice, Inbred DBA; Neutrophil Infiltration; Poly(ADP-ribose) Polymerases; Tyrosine

Pregnant women often develop anemia concomitant with the increase in serum erythropoietin levels, which are actually lower than those of nonpregnant anemic women due to the possible suppressive effect of endogenous estradiol on erythropoietin induction. The anemia, derived from hemodilution, does not act as a drive for erythropoietin induction, but iron deficiency, often observed during pregnancy, might. In order to demonstrate this, we investigated the effects of iron deficiency on estradiol-induced suppression of erythropoietin induction in rats. Single doses of estradiol suppressed hypoxia-, cobalt-, and bleeding-stimulated elevation of plasma erythropoietin levels and renal erythropoietin mRNA expression. Repeated administration of estradiol at 0.1 and 1 mg/kg for 2 months induced a slight anemic trend without elevation of plasma erythropoietin. Feeding an iron-deficient diet for 2 months induced plasma erythropoietin elevation without obvious anemia, but the simultaneous repeated administration of estradiol suppressed it and reversed the iron deficiency. Plasma erythropoietin levels had distinct negative correlations with plasma iron, plasma ferritin, and iron concentrations in the organs, but not with plasma hemoglobin level. These results suggest that iron deficiency would significantly stimulate erythropoietin induction during pregnancy, although estradiol might suppress it through iron restoration.

Topics: Anemia, Iron-Deficiency; Animal Feed; Animals; Disease Models, Animal; Erythropoiesis; Erythropoietin; Estradiol; Female; Iron; Kidney; Liver; Organ Size; Pregnancy; Pregnancy Complications, Hematologic; Rats; Rats, Wistar

Erythropoietin (EPO) is a pleiotropic cytokine originally identified for its role in erythropoiesis. Recent studies have demonstrated that EPO and its receptor (EPO-R) are expressed in the central nervous system, where EPO exerts neuroprotective functions. Because the expression of the EPO and EPO-R network is poorly investigated in the central nervous system, the aim of the present study was to investigate whether the resident EPO and EPO-R network is activated in the injured nervous system.. A well-standardized model of compressive spinal cord injury in rats was used. EPO and EPO-R expression was determined by immunohistochemical analysis at 8 hours and at 2, 8, and 14 days in the spinal cord of injured and noninjured rats.. In noninjured spinal cord, weak immunohistochemical expression of EPO and EPO-R was observed in neuronal and glial cells as well as in endothelial and ependymal cells. In injured rats, a marked increase of expression of EPO and EPO-R was observed in neurons, vascular endothelium, and glial cells at 8 hours after injury, peaking at 8 days, after which it gradually decreased. Two weeks after injury, EPO immunoreactivity was scarcely detected in neurons, whereas glial cells and vascular endothelium expressed strong EPO-R immunoreactivity.. These observations suggest that the local EPO and EPO-R system is markedly engaged in the early stages after nervous tissue injury. The reduction in EPO immunoexpression and the increase in EPO-R staining strongly support the possible usefulness of a therapeutic approach based on exogenous EPO administration.

Topics: Aneurysm; Animals; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Male; Neurons; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Recombinant Proteins; Spinal Cord Compression; Spinal Cord Injuries

Erythropoietin (Epo) expresses potent neuroprotective activity in the peripheral nervous system; however, the underlying mechanism remains incompletely understood. In this study, we demonstrate that Epo is upregulated in sciatic nerve after chronic constriction injury (CCI) and crush injury in rats, largely due to local Schwann cell production. In uninjured and injured nerves, Schwann cells also express Epo receptor (EpoR), and its expression is increased during Wallerian degeneration. CCI increased the number of Schwann cells at the injury site and the number was further increased by exogenously administered recombinant human Epo (rhEpo). To explore the activity of Epo in Schwann cells, primary cultures were established. These cells expressed cell-surface Epo receptors, with masses of 71 and 62 kDa, as determined by surface protein biotinylation and affinity precipitation. The 71-kDa species was rapidly but transiently tyrosine-phosphorylated in response to rhEpo. ERK/MAP kinase was also activated in rhEpo-treated Schwann cells; this response was blocked by pharmacologic antagonism of JAK-2. RhEpo promoted Schwann cell proliferation, as determined by BrdU incorporation. Cell proliferation was ERK/MAP kinase-dependent. These results support a model in which Schwann cells are a major target for Epo in injured peripheral nerves, perhaps within the context of an autocrine signaling pathway. EpoR-induced cell signaling and Schwann cell proliferation may protect injured peripheral nerves and promote regeneration.

Topics: Animals; Autocrine Communication; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Erythropoietin; Extracellular Signal-Regulated MAP Kinases; Female; Janus Kinase 2; Male; Nerve Regeneration; Peripheral Nerve Injuries; Peripheral Nerves; Phosphorylation; Protein Isoforms; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Recombinant Proteins; RNA, Messenger; Schwann Cells; Sciatic Neuropathy; Up-Regulation

Topics: Anemia; Animals; Disease Models, Animal; Erythropoietin; Humans; Hypoxia; Kidney; Kidney Failure, Chronic; Neovascularization, Physiologic; Rats; Recombinant Proteins

Although recent studies suggest that erythropoietin (EPO) may reduce multiple features of the myocardial ischemia/reperfusion injury, the cellular mechanisms and the clinical implications of EPO-induced cardioprotection are still unclear. Thus, in this study, we clarified dose-dependent effects of EPO administered just before reperfusion on infarct size and the incidence of ventricular fibrillation and evaluated the involvement of the phosphatidylinositol-3 (PI3) kinase in the in vivo canine model. The canine left anterior descending coronary artery was occluded for 90 min followed by 6 h of reperfusion. A single intravenous administration of EPO just before reperfusion significantly reduced infarct size (high dose (1,000 IU/kg): 7.7 +/- 1.6%, low dose (100 IU/kg): 22.1 +/- 2.4%, control: 40.0 +/- 3.6%) in a dose-dependent manner. Furthermore, the high, but not low, dose of EPO administered as a single injection significantly reduced the incidence of ventricular fibrillation during reperfusion (high dose: 0%, low dose: 40.0%, control: 50.0%). An intracoronary administration of a PI3 kinase inhibitor, wortmannin, blunted the infarct size-limiting and anti-arrhythmic effects of EPO. Low and high doses of EPO equally induced Akt phosphorylation and decreased the equivalent number of TUNEL-positive cells in the ischemic myocardium of dogs. These effects of EPO were abolished by the treatment with wortmannin. In conclusion, EPO administered just before reperfusion reduced infarct size and the incidence of ventricular fibrillation via the PI3 kinase-dependent pathway in canine hearts. EPO administration can be a realistic strategy for the treatment of acute myocardial infarction.

Topics: Animals; Apoptosis; Blood Cell Count; Cardiotonic Agents; Coronary Circulation; Disease Models, Animal; Dogs; Dose-Response Relationship, Drug; Erythropoietin; Humans; In Situ Nick-End Labeling; Myocardial Infarction; Myocardial Reperfusion; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Ventricular Fibrillation

ICR-derived glomerulonephritis (ICGN) mice are a novel inbred strain with hereditary nephrotic syndrome and are thus considered a good animal model of human idiopathic nephrotic syndrome. In the present study, we investigated the effect to erythrocyte production by human erythropoietin (hEPO) treatment in ICGN mice during the early nephrotic stage. Erythrocyte count, hemoglobin concentration and hematocrit value in hEPO-treated (5 U/body/day, for 5 days) ICGN mice were recovered to the levels found in normal ICR mice. In addition, there was no correlation between plasma creatinine level, a marker of renal function, and erythrocyte count after hEPO treatment. Therefore, anemia in ICGN mice may be caused by decreased production of EPO in the kidney following progressive parenchymal damage.

Topics: Anemia; Animals; Blood Chemical Analysis; Disease Models, Animal; Erythropoiesis; Erythropoietin; Female; Hematologic Tests; Humans; Kidney; Male; Mice; Mice, Inbred ICR; Nephrotic Syndrome; Recombinant Proteins

Perinatal asphyxia accounts for behavioral dysfunctions that often manifest as sensorimotor, learning or memory disabilities throughout development and into maturity. Erythropoietin (Epo) has been shown to exert neuroprotective effects in different models of brain injury including experimental models of perinatal asphyxia. However, the effect of Epo on functional abilities following cerebral hypoxia-ischemia (HI) in neonatal rats is not known. The aim of the present study is to investigate the effect of Epo on sensorimotor deficits and brain injury induced by hypoxia-ischemia. Seven-day-old rats underwent unilateral, permanent carotid artery ligation followed by 1 h of hypoxia. Epo was administered as a single dose immediately after the hypoxic insult (2000 U/kg). The neuroprotective effect of Epo was evaluated at postnatal day 42 by using a battery of behavioral tests and histological analysis. The results of the present study suggest that Epo treatment immediately after HI insult significantly facilitated recovery of sensorimotor function. Consistently, histopathological evaluation demonstrated that Epo significantly attenuated brain injury and preserved the integrity of cerebral cortex. These findings indicate that long-term neuroprotective effect of Epo on neonatal HI-induced brain injury might be associated with the preservation of sensorimotor functions.

Topics: Animals; Animals, Newborn; Cerebral Cortex; Cerebral Infarction; Disease Models, Animal; Erythropoietin; Female; Humans; Hypoxia-Ischemia, Brain; Ligation; Male; Movement Disorders; Neuroprotective Agents; Rats; Rats, Wistar; Recovery of Function; Sensation Disorders; Treatment Outcome

We assessed the effects of erythropoietin (EPO) treatment in a rat model of post-myocardial infarction (MI) heart failure.. Erythropoietin, traditionally known as a hematopoietic hormone, has been linked to neovascularization. Whereas administration of EPO acutely after MI reduces infarct size and improves cardiac function, its role in the failing heart is unknown.. Rats underwent coronary ligation or sham surgery. Rats with MI were randomly assigned to: untreated (MI), a single bolus of EPO immediately after MI induction (MI-EPO-early), EPO treatment immediately after MI and once every three weeks (MI-EPO-early+late), and EPO treatment starting three weeks after induction of MI, once every three weeks (MI-EPO-late). After nine weeks, hemodynamics, infarct size, myosin heavy chain (MHC) isoforms, myocyte hypertrophy, and capillary density were measured.. Erythropoietin treatment started immediately after MI (MI-EPO-early and MI-EPO-early+late) resulted in a 23% to 30% reduction in infarct size (p < 0.01) and, accordingly, hemodynamic improvement. Erythropoietin treatment, started three weeks after MI (MI-EPO-late), did not affect infarct size, but resulted in an improved cardiac performance, reflected by a 34% reduction in left ventricular end-diastolic pressure (p < 0.01), and 46% decrease in atrial natriuretic peptide levels (p < 0.05). The improved cardiac function was accompanied by an increased capillary density (p < 0.01), an increased capillary-to-myocyte ratio (p < 0.05), and a partial reversal of beta-MHC (p < 0.05) in all treated groups.. In addition to its effect on infarct size reduction, EPO treatment improves cardiac function in a rat model of post-MI heart failure. This observation may be explained by neovascularization, associated with an increased alpha-MHC expression.

Topics: Animals; Atrial Natriuretic Factor; Cardiac Output; Cardiomyopathy, Dilated; Coronary Vessels; Darbepoetin alfa; Disease Models, Animal; Drug Administration Schedule; Erythropoietin; Male; Myocardial Infarction; Myosin Heavy Chains; Neovascularization, Physiologic; Rats; Rats, Sprague-Dawley

Darbepoetin alfa (Aranesp), Amgen) is a novel erythropoiesis-stimulating protein with a serum half-life longer than recombinant human erythropoietin (Epo), used in the treatment of cancer-associated anaemia. Anaemia is known to adversely affect prognosis and response to treatment in cancer patients. Solid tumours contain regions of hypoxia due to poor vascular supply and cellular compaction. Although hypoxic stress usually results in cell death, hypoxia-resistant tumour cells are genetically unstable and often acquire a drug-resistant phenotype. Increasing tumour oxygenation and perfusion during treatment could have the doubly beneficial outcome of reducing the fraction of treatment-resistant cells, while increasing drug delivery to previously hypoxic tissue. In this study, we examined the effect of darbepoetin alfa on chemotherapy sensitivity and delivery in an in vivo model of Lewis lung carcinoma, shown here to express the Epo receptor (EpoR). We identified that weekly darbepoetin alfa treatment, commencing 10 days before chemotherapy, resulted in a significant reduction in tumour volume compared to chemotherapy alone. This was mediated by the prevention of anaemia, a reduction in tumour hypoxia and a concomitant increase in drug delivery. Darbepoetin alfa treatment alone did not modulate the growth of the EpoR-expressing tumour cells. This study identifies an important role for darbepoetin alfa in increasing the therapeutic index of chemotherapy.

Topics: Anemia; Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Lewis Lung; Darbepoetin alfa; Disease Models, Animal; Erythropoietin; Female; Mice; Mice, Inbred C57BL; Receptors, Erythropoietin

Treatment with deferoxamine (DFO) is protective against focal ischemia with global hypoxia when given as a preconditioning stimulus in neonatal rodents. DFO acts as an iron chelator and may stabilize HIF1alpha. Therefore, we hypothesized that DFO would protect against pure ischemia-reperfusion injury when given after the insult and that the protection would be associated with expression of hypoxia-inducible factor 1alpha (HIF1alpha) and downstream target genes such as erythropoietin (Epo). To test these hypotheses, we performed middle cerebral artery (MCA) occlusion in postnatal day 10 (P10) rats for 1.5 h followed by treatment with DFO or vehicle upon reperfusion. Preserved brain volumes were measured with cresyl violet staining 1 week after the insult. HIF1alpha and Epo expression were determined by Western blot and immunocytochemical analyses at different time points after injury. We found that DFO treatment preserved brain volumes when compared to vehicle (P < 0.05). In DFO-treated ischemic cortices, HIF1alpha expression peaked early, while Epo expression was seen in two phases and in different cell populations. Epo immunoreactivity colocalized with neuronal markers at 8 h but with astrocytic markers at 1 week. These results suggest that DFO is protective when administered after neonatal ischemic stroke and that this protection may be like that afforded by preconditioning through the upregulation of similar downstream pathways.

Topics: Animals; Animals, Newborn; Blotting, Western; Deferoxamine; Disease Models, Animal; Erythropoietin; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Immunohistochemistry; Infarction, Middle Cerebral Artery; Iron Chelating Agents; Phosphopyruvate Hydratase; Rats; Time Factors; Up-Regulation

Erythropoietin (epo), initially recognized and used clinically to increase erythropoiesis, has been shown to have beneficial effects on various other tissues in the setting of hypoxia and ischemia. Epo has been shown to reduce apoptosis after myocardial infarction, but few studies have evaluated the long-term effects of epo treatment on left ventricular (LV) remodeling, cardiac function, and blood flow after healing of a permanent coronary artery occlusion. The aim of this study was to assess the effects of epo treatment on the healed heart 6 weeks after myocardial infarction. Anesthetized rats underwent coronary artery occlusion and were treated with erythropoietin (5000 units/kg/day, n=21) or saline (n=20) the day before surgery, the day of, then for 5 days. At 6 weeks LV ventriculography to assess LV volumes and ejection fractions and histologic assessment of infarct size and LV cavity and wall dimensions were performed. Overall epo had no effect on LV remodeling or cardiac function. There were no significant differences in infarct morphology, infarct size (44+/-3% of the LV circumference versus 39+/-3%), LV cavity area, scar thickness, LV systolic volume, or ejection fraction (44+/-3% versus 39+/-3%) between the epo and saline groups, respectively. However, for any given size of myocardial infarct, LV ejection fraction was significantly higher in erythropoietin hearts and LV systolic volumes lower. Thus, in our model, treatment with epo had no long-term beneficial effect on LV remodeling after myocardial infarction but may have exerted some positive effect on LV function.

Topics: Animals; Coronary Circulation; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Female; Myocardial Infarction; Myocardium; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Time Factors; Treatment Failure; Ventricular Function, Left; Ventricular Remodeling

We previously reported that thromboxane (TX)A2 synthesis and receptor blockade prevented recombinant human erythropoietin (rhEPO)-induced hypertension in chronic renal failure rats. The present study was designed to investigate the effect of a cyclooxygenase inhibitor, acetylsalicylic acid (ASA), on blood pressure, renal function, and the concentration of eicosanoïds and endothelin-1 (ET-1) in vascular and renal tissues of rhEPO-treated or rhEPO-untreated uremic rats. Renal failure was induced by a 2-stage 5/6 renal mass ablation. Rats were divided into 4 groups: vehicle, rhEPO (100 U/kg, s.c., 3 times per week), ASA (100 mg x kg(-1) x day(-1), and rhEPO + ASA; all animals were administered drugs for 3 weeks. The TXA2- and prostacyclin (PGI2)-stable metabolites (TXB2 and 6-keto-PGF1alpha, respectively), as well as ET-1, were measured in renal cortex and either the thoracic aorta or mesenteric arterial bed. The uremic rats developed anemia, uremia, and hypertension. They also exhibited a significant increase in vascular and renal TXB2 (p < 0.01) and 6-keto-PGF1alpha (p < 0.01) concentrations. rhEPO therapy corrected the anemia but aggravated hypertension (p < 0.05). TXB2 and ET-1 tissue levels further increased (p < 0.05) whereas 6-keto-PGF1alpha was unchanged in rhEPO-treated rats compared with uremic rats receiving the vehicle. ASA therapy did not prevent the increase in systolic blood pressure nor the progression of renal disease in rhEPO-treated or rhEPO-untreated uremic rats, but suppressed both TXB2 and 6-keto-PGF1alpha tissue concentrations (p < 0.05). ASA had no effect on vascular and renal ET-1 levels. Cyclooxygenase inhibition had no effect on rhEPO-induced hypertension owing, in part, to simultaneous inhibition of both TXA2 and its vasodilatory counterpart PGI2 synthesis, whereas the vascular ET-1 overproduction was maintained. These results stress the importance of preserving PGI2 production when treating rhEPO-induced hypertension under uremic conditions.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Aorta, Thoracic; Aspirin; Blood Pressure; Cyclooxygenase Inhibitors; Disease Models, Animal; Endothelin-1; Epoprostenol; Erythropoietin; Humans; Hypertension; Kidney Cortex; Kidney Function Tests; Male; Mesenteric Arteries; Rats; Rats, Wistar; Recombinant Proteins; Thromboxane B2; Uremia

Erythropoietin (EPO) is a hematopoietic growth factor with tissue-protective properties, and can protect animals from cerebral ischemic injury. However, the central nervous effects of EPO as a glycoprotein is limited by the potential complication resulted from its erythropoietic activity and the problem of the penetration through blood-brain barrier (BBB). To avoid these limitations, in this study we administered recombinant human EPO (rhEPO) intranasally (i.n.) to evaluate its neuroprotective effect in the rats with focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO). We found that rhEPO i.n. at doses of 4.8, 12 and 24 U (administered 10 min after MCAO and 1h after reperfusion) reduced infarct volume, brain swelling and cell damage in the ischemic hemispheres, and improved behavioral dysfunction 24 h after cerebral ischemia. Intraperitoneal rhEPO (5000 U/kg) also showed the protective effect, but the heat-inactivated rhEPO did not show any effect. Thus, intranasal administration of relatively small doses of rhEPO protects rats from acute injury after focal cerebral ischemia, suggesting that intranasal rhEPO may be a more effective and safer administration route for treatments of ischemic or other brain diseases.

Topics: Administration, Intranasal; Animals; Brain Edema; Brain Ischemia; Cerebral Infarction; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Humans; Infarction, Middle Cerebral Artery; Male; Nerve Degeneration; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Recovery of Function; Treatment Outcome

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality in young people in industrialized countries. Although various anti-inflammatory and antiapoptotic modalities have shown neuroprotective effects in experimental models of TBI, to date, no specific pharmacological agent aimed at blocking the progression of secondary brain damage has been approved for clinical use. Erythropoietin (Epo) belongs to the cytokine superfamily and has traditionally been viewed as a hematopoiesis-regulating hormone. The newly discovered neuroprotective properties of Epo lead us to investigate its effect in TBI in a mouse model of closed head injury. Recombinant human erythropoietin (rhEpo) was injected at 1 and 24 h after TBI, and the effect on recovery of motor and cognitive functions, tissue inflammation, axonal degeneration, and apoptosis was evaluated up to 14 days. Motor deficits were lower, cognitive function was restored faster, and less apoptotic neurons and caspase-3 expression were found in rhEpo-treated as compared with vehicle-treated animals (P<0.05). Axons at the trauma area in rhEpo-treated mice were relatively well preserved compared with controls (shown by their density; P<0.01). Immunohistochemical analysis revealed a reduced activation of glial cells by staining for GFAP and complement receptor type 3 (CD11b/CD18) in the injured hemisphere of Epo- vs. vehicle-treated animals. We propose that further studies on Epo in TBI should be conducted in order to consider it as a novel therapy for TBI.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Axons; Brain; Caspase 3; Caspases; CD11b Antigen; CD18 Antigens; Cytokines; Disease Models, Animal; Erythropoietin; Glial Fibrillary Acidic Protein; Head Injuries, Closed; Hematopoiesis; Immunohistochemistry; In Situ Nick-End Labeling; Inflammation; Male; Mice; Neurons; Rats; Recombinant Proteins; Time Factors

Topics: Aged; Animals; Chronic Disease; Disease Models, Animal; Erythropoietin; Humans; Injections, Intravenous; Male; Mice; Mice, Transgenic; Pulmonary Circulation; Pulmonary Heart Disease; Pulmonary Wedge Pressure; Recombinant Proteins

Recombinant human erythropoietin (rhEPO) has been proposed to possess important tissue protective, apart from haematopoietic, effects. Cardioprotective effects have thus been reported in rodents exposed to myocardial ischaemia. Pathways common to the mediation of ischaemic preconditioning may be involved. Before clinical testing such possible cardioprotective effects needs assessment in an experimental large animal model with closer similarity to human ischaemic pathophysiology.. A control group and two rhEPO groups were studied. EPO1 pigs were given EPO corresponding to the early window and EPO2 pigs to the early and late window of ischaemic preconditioning in a closed chest, catheter-based, porcine coronary occlusion model (45 min of occlusion of the left anterior descending artery). Infarct size as a proportion of the ischaemic area (IS/AAR) was measured in vivo by myocardial perfusion imaging (MPI) and postmortem by a histochemical procedure (at 150 min of reperfusion).. IS/AAR did not differ significantly between control (C), EPO1 and EPO2 groups, neither measured by MPI (mean+/-SD for C: 0.87+/-0.13; EPO1: 0.92+/-0.08; EPO2: 0.87+/-0.11), nor histochemically (mean+/-SD for C: 0.64+/-0.20; EPO1: 0.75+/-0.17; EPO2: 0.80+/-0.07). In the EPO2 group mean arterial pulmonary pressure and dP/dtmax were increased compared with control group.. Despite promising results from studies in rodents, rhEPO did not reduce infarct size measured after 2.5 h of reperfusion in our porcine model.

Topics: Animals; Blood Pressure; Cardiac Output; Cardiotonic Agents; Coronary Stenosis; Disease Models, Animal; Erythropoietin; Myocardial Infarction; Myocardial Ischemia; Recombinant Proteins; Swine; Tomography, Emission-Computed, Single-Photon

Recombinant human erythropoietin (rhEPO) protects tissue from ischemic damage, but translation of this finding into useful guidelines with respect to human trials for myocardial infarction (MI) requires a determination of the minimum effective rhEPO dose and the therapeutic window following MI.. Serial echocardiography revealed that during four weeks following MI, induced by a permanent coronary ligation in rats, the LV end-diastolic and end-systolic volumes in untreated rats expanded from 0.35 +/- 0.01 and 0.14 +/- 0.01 ml to 0.84 +/- 0.04 and 0.61 +/- 0.06 ml, respectively, and ejection fraction (EF) reduced by 50%. A single i.v. injection of rhEPO immediately following MI in a dose of 150 IU/kg was as effective as 3,000 IU/kg in causing a 2-fold reduction of the number of apoptotic nuclei in the AAR 24-h later, a 2-fold reduction of the MI size measured 4 weeks later, attenuation of progressive LV dilatation and fall in EF. A 3000 IU/kg dose had similar therapeutic effects when delayed by 4, 8, or 12 h following MI, but was not effective after a 24-h delay. A single dose of 150 IU/kg was effective within 4 h post-MI, but was without effect if administered after an 8-h delay.. Cell death, final MI size, myocardial remodeling and functional decline are significantly reduced in rats by a single injection of rhEPO in a dose as low as 150 IU/kg if administered during the first 4 h after the ischemic event. Higher doses extend the therapeutic window up to 12 h.

Topics: Animals; Apoptosis; Cardiotonic Agents; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Echocardiography; Erythropoietin; Humans; Male; Myocardial Infarction; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Recombinant Proteins

Brain injury evolves over time, often taking days or even weeks to fully develop. It is a dynamic process that involves immediate oxidative stress and excitotoxicity followed by inflammation and preprogrammed cell death. This article presents a brief overview of mechanisms of neuroprotection in the developing brain. Although the focus is on ischemic injury, the conclusions drawn apply to any type of brain insult-epileptic seizures, trauma, or ischemia. Strategies of neuroprotection include salvaging neurons through the use of targeted pharmacotherapies, protecting neurons through preconditioning, and repairing neurons by enhancing neurogenesis. Drug therapies that dampen the impact of immediate and downstream postinjury events are only modestly effective in protecting the brain from ischemic injury. In experimental models, complete or true protection can be achieved only through preconditioning, a process during which an animal develops tolerance to an otherwise lethal stressor. Although of no clinical use, preconditioning models have provided valuable insight into how repair systems work in the brain. Cumulative evidence indicates that the same genes that are upregulated during preconditioning, those mediating true protection, are also upregulated during injury and repair. Specifically, hypoxic preconditioning and hypoxic-ischemic insult have been shown to induce hypoxia inducible factor-1 (HIF-1) and its target survival genes, vascular endothelial growth factor (VEGF), and erythropoietin (Epo) in rodents. Of particular interest is the upregulation of Epo, a growth factor that may have therapeutic potential in the treatment of ischemic stroke. At this time, however, the postinjury enhancement of neurogenesis appears to offer the best hope for long-lasting functional recovery following brain injury.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Disease Models, Animal; Epilepsy; Erythropoietin; Humans; Hypoxia-Ischemia, Brain; Ischemic Preconditioning; Neuronal Plasticity; Neurons; Neuroprotective Agents; Oxidative Stress; Stroke; Up-Regulation

We investigated the effects of recombinant human (rh) erythropoietin (EPO) on erectile function recovery in a rat model of cavernous nerve (CN) injury.. Male rats underwent unilateral CN transection and excision of a 5 mm segment of the contralateral CN. One group received rhEPO (5,000 U/kg) subcutaneously daily for 14 days, while another received rhEPO 1 day and 1 hour prior to nerve injury. An additional group of animals was pretreated with 1 dose of darbepoetin (25 microg/kg). At 14 days following CN injury rats underwent erection physiology studies. Axonal regeneration was evaluated by electron microscopy. EPO receptor expression in the penis and major pelvic ganglion was evaluated immunohistochemically and by real-time polymerase chain reaction.. Daily rhEPO effectively recovered erections after CN injury compared with saline treatment. Maximal intracavernous pressure area under the curve normalized to mean arterial pressure was significantly greater in EPO treated vs saline treated animals (p < 0.05). rhEPO and darbepoetin pretreatment was as effective as continuous 14-day therapy. EPO receptor expression was localized to neuronal cell bodies of the major pelvic ganglion, penile nerves and endothelial cells in the penis. Electron microscopy revealed significant improvement in axonal regeneration in rhEPO treated animals 14 days following injury compared to controls.. EPO receptors are expressed in local neuronal and vascular tissues. Exogenous administration of rhEPO or darbepoetin in the setting of CN injury promotes erectile function recovery. This occurs through axonal regeneration of the injured nerve and possible penile protection.

Topics: Analysis of Variance; Animals; Base Sequence; Biopsy, Needle; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Erectile Dysfunction; Erythropoietin; Immunohistochemistry; Male; Microscopy, Electron; Molecular Sequence Data; Nerve Regeneration; Penis; Probability; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Female; Leukemia, B-Cell; Lymphoma, B-Cell; Lymphoproliferative Disorders; Mice; Mice, Inbred BALB C; Plasmacytoma; Recombinant Proteins

Changes in the reticulocyte subtype distribution (high, medium and low reticulocytes count (HR, MR, LR)) measured by flow cytometry following phlebotomy-induced stress erythropoiesis (abruptly dropping hemoglobin to 3-4 g/dl over 4-5 hr) and the pharmacodynamic (PD) relationship to the stimulated erythropoietin (EPO) was investigated in sheep. A PD model was developed that describes the relationship between EPO and the reticulocyte maturity distribution fractions (r=0.95+/-0.02, mean +/- SD). The lag-time between EPO activation of erythroid progenitor cells and the subsequent increase in the least mature HR fraction in the peripheral circulation was 0.72 +/- 0.08 days. The mean transition times (in days) for all three reticulocyte fractions changed at baseline from, T(HR) : 0.09 +/- 0.06, T(MR) : 0.06 +/- 0.04, and T(LR) : 0.46 +/- 0.24 to T(HR) : 0.13 +/- 0.08, T(MR) : 0.29 +/- 0.15, and T(LR) : 2.3 +/- 0.24 under stress erythropoiesis. The total mean residence time for a reticulocyte in the peripheral circulation, T(total) (T(HR) + T(MR) + T(LR)), increased from 0.60 +/- 0.33 days under basal to 2.8 +/- 0.09 days during stress erythropoiesis. The statistically significant increase observed for T(LR) and T(total) supports the hypothesis that stress erythropoiesis perturbs the mean reticulocyte transition times. A correlation analysis between various new, proposed metrics involving the HR, MR and LR fractions and the total reticulocyte count, with the latter indicative of stress erythropoiesis at higher total counts, revealed a highly significant correlation indicating these new metrics may be a valuable adjunct to the reticulocyte maturation index (RMI) and the immature reticulocyte fractions index (IRF) previously used in assessing erythropoietic activity in response to anemia.

Topics: Anemia; Animals; Cell Differentiation; Disease Models, Animal; Erythropoiesis; Erythropoietin; Hemoglobins; Phlebotomy; Reticulocyte Count; Reticulocytes; Sheep; Time Factors

In cancer cachexia, erythropoietin often yields beneficial therapeutic effects by improving patient's metabolic and exercise capacity via an increased erythrocyte count. However, erythropoietin also has counter-regulatory effects against pro-inflammatory cytokines, which are postulated to be mediators of cancer cachexia. We investigated the mechanisms by which erythropoietin improves the cachectic condition. In this study, 100 Units/day of erythropoietin were administered intraperitoneally to BALB/c male mice, carrying a subclone of colon 26 adenocarcinoma, beginning on the day after tumor inoculation and continuing until they died. Erythropoietin administration attenuated the decline in body weight, as well as the decline in fat and muscle weights, of tumor-bearing mice, but improved the survival of cachectic mice. Mice receiving erythropoietin had increased erythrocyte and platelet counts, but significantly decreased white blood cell count. In addition, erythropoietin administration significantly decreased interleukin-6 levels, not only in serum but also in the inoculated tumor. These results indicate that the positive therapeutic effects of erythropoietin on cancer cachexia are due, not only to improving metabolic and exercise capacity via an increased erythrocyte count, but also to attenuation of cachectic manifestations by decreased production of the cachexia-inducing cytokine, interleukin-6.

Topics: Adenocarcinoma; Animals; Cachexia; Colonic Neoplasms; Cytokines; Disease Models, Animal; Down-Regulation; Eating; Erythropoietin; Interleukin-6; Liver; Male; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Spleen; Survival Rate

Erythropoietin is a hematopoietic growth factor. It is also involved in neurodevelopment and neuroprotection. The purpose of this study was to evaluate the effectiveness of erythropoietin in enhancing the neurological recovery following experimental spinal cord injury.. Rats were randomly divided into 4 groups. Group 1 received only laminectomy. Group 2, 3, and 4 have undergone a spinal contusion injury of 50 gr/cm. Group 2 received no medication. Group 3 received 30 mg/kg methylprednisolone. Group 4 received 5000 IU/kg recombinant human erythropoietin. Following injury, neurological recovery was evaluated for 14 days, using a swimming test.. At day 1, there was no difference between mean motor scores of group 2, 3, and 4. Following day 1, rats in group 4 exhibited a marked improvement in motor score, and this was maintained throughout the study. When compared to group 2, mean motor score of group 4 was significantly higher at day 4, 7, 10, and 14. When compared to group 3, mean motor score of group 4 was significantly higher at day 7, 10, and 14.. These findings suggest that erythropoietin enhances neurological recovery following spinal cord injury and it seems to be more effective than methylprednisolone at the given doses.

Topics: Animals; Disease Models, Animal; Erythropoietin; Humans; Male; Methylprednisolone; Motor Activity; Neurologic Examination; Neuroprotective Agents; Rats; Rats, Wistar; Recombinant Proteins; Recovery of Function; Spinal Cord Injuries; Time Factors

Lipid peroxidation has been reported to play an important role in spinal cord injury (SCI). Erythropoietin (EPO) is a hematopoietic growth factor that stimulates proliferation and differentiation of erythroid precursor cells and is also known to exert neurotrophic activity in the central nervous system. The purpose of this study was to investigate the effectiveness of recombinant human EPO in attenuating the severity of experimental SCI. Rats were divided into seven groups. Controls (1) received only laminectomy. The trauma-only group (2) underwent 50-g/cm contusion injury and had no medication. In group 3, 30 mg/kg of methylprednisolone was introduced. The vehicle group (4) received vehicle solution containing human serum albumin, which is a solvent of EPO. Groups 5, 6, and 7 received 100 IU/kg, 1,000 IU/kg, and 5,000 IU/kg of EPO, respectively. All treatments were given as single doses, intraperitoneally, immediately after injury. Thiobarbituric acid-reactive substances were estimated to demonstrate lipid peroxidation, and ultrastructure was evaluated by electron microscopy. The results showed that lipid peroxidation by-products increased after injury. Administration of EPO and methylprednisolone sodium succinate (MPSS) reduced thiobarbituric acid-reactive substances after trauma. The best biochemical results were obtained with 5,000 IU/kg of EPO. Electron microscopic findings showed that EPO protected the spinal cord from injury. Although 1,000 IU/kg and 5,000 IU/kg of EPO inhibited lipid peroxidation better than MPSS, ultrastructural neuroprotection was similar.

Topics: Acute Disease; Animals; Disease Models, Animal; Erythropoietin; Lipid Peroxidation; Male; Methylprednisolone; Neuroprotective Agents; Rats; Rats, Wistar; Spinal Cord; Spinal Cord Injuries; Thiobarbituric Acid Reactive Substances; Time Factors; Trauma Severity Indices

Erythropoietin (Epo) exerts neuroprotection against neuronal death induced by ischemia and hypoxia in vitro and in vivo. Recent studies suggest that the neuroprotective effects of Epo may depend upon different mechanisms, including the inhibition of nitric oxide (NO). We recently demonstrated that Epo exerts neuroprotection in a model of neonatal hypoxic-ischemic brain damage. In the present study, we directly determined whether systemic administration of recombinant Epo modulates cerebral NO production in a neonatal rat model of hypoxic-ischemic brain injury. Seven-day-old Wistar rat pups were subjected to left carotid artery occlusion followed by 2.5 h of hypoxic exposure. Brain nitrite levels were evaluated in both hemispheres (carotid ligated or nonligated) by Griess reagent 72 h after the hypoxic-ischemic insult. Our results show that hypoxic-ischemic insult results a significant increase in NO production as compared with NO levels in hypoxic hemispheres and control animals. A single dose of Epo treatment (1,000 U/kg i.p.) significantly decreased NO overproduction in the hypoxic-ischemic hemisphere, whereas no significant change appeared in hypoxia alone or in controls. These data suggest that the selective inhibitory effect of Epo on NO overproduction could have a neuroprotective effect in neonatal hypoxic-ischemic brain injury.

Topics: Animals; Brain Chemistry; Brain Diseases; Carotid Arteries; Disease Models, Animal; Erythropoietin; Hypoxia; Hypoxia-Ischemia, Brain; Ligation; Nitric Oxide; Nitrites; Rats; Rats, Wistar; Recombinant Proteins

The interaction of blood with the arterial tree may play an important role in the development of atherosclerotic lesions. The aims of this study were (1) to determine how anemia or increased hematocrit affect the development of atherosclerosis and (2) to find relationships between hematologic and hemorrheologic variables in apolipoprotein (apo) E-deficient mice. Forty-two mice were randomly divided into 3 groups of 14 mice each. There was no further manipulation in the control group. To induce anemia, the mice from one of the groups were repeatedly bled, drawing approximately 250 microL blood from each mouse twice a week. To increase the hematocrit levels in another group of mice, we injected 20 U recombinant human erythropoietin every other day. The development of lesions and the main variables involved in atherogenesis were compared among groups. Our results show that atherosclerosis was attenuated in the mice that were bled, and this was not accounted for by changes in plasma lipid levels, the distribution of lipoprotein particles, the body iron distribution, or oxidation parameters. Moreover, atherosclerosis was enhanced in the mice treated with the continuous administration of erythropoietin. To ascertain the relationship between hematocrit and whole blood viscosity, we measured both variables in pooled blood from 24 additional mice, which were manipulated to ensure a wide range of values. We found a direct and significant correlation between hematocrit and blood viscosity and between hematocrit and lesion size. Our data support in vivo the idea that hemorrheology has an important role in atherogenesis in this particular animal model.

Topics: Anemia; Animals; Aorta; Apolipoproteins E; Arteriosclerosis; Blood Cells; Blood Viscosity; Body Weight; Disease Models, Animal; Erythropoietin; Ferritins; Hematocrit; Hemorheology; Humans; Iron; Lipids; Lipoproteins; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; Nutritional Status; Oxidation-Reduction; Phlebotomy; Recombinant Proteins; Spleen

Erythropoietin (EPO) has recently been shown to exert important cytoprotective and anti-apoptotic effects in experimental brain injury and cisplatin-induced nephrotoxicity. The aim of the present study was to determine whether EPO administration is also renoprotective in both in vitro and in vivo models of ischaemic acute renal failure.. Primary cultures of human proximal tubule cells (PTCs) were exposed to either vehicle or EPO (6.25-400 IU/ml) in the presence of hypoxia (1% O(2)), normoxia (21% O(2)) or hypoxia followed by normoxia for up to 24 h. The end-points evaluated included cell apoptosis (morphology and in situ end labelling [ISEL], viability [lactate dehydrogenase (LDH release)], cell proliferation [proliferating cell nuclear antigen (PCNA)] and DNA synthesis (thymidine incorporation). The effects of EPO pre-treatment (5000 U/kg) on renal morphology and function were also studied in rat models of unilateral and bilateral ischaemia-reperfusion (IR) injury.. In the in vitro model, hypoxia (1% O(2)) induced a significant degree of PTC apoptosis, which was substantially reduced by co-incubation with EPO at 24 h (vehicle 2.5+/-0.5% vs 25 IU/ml EPO 1.8+/-0.4% vs 200 IU/ml EPO 0.9+/-0.2%, n = 9, P<0.05). At high concentrations (400 IU/ml), EPO also stimulated thymidine incorporation in cells exposed to hypoxia with or without subsequent normoxia. LDH release was not significantly affected. In the unilateral IR model, EPO pre-treatment significantly attenuated outer medullary thick ascending limb (TAL) apoptosis (EPO 2.2+/-1.0% of cells vs vehicle 6.5+/-2.2%, P<0.05, n = 5) and potentiated mitosis (EPO 1.1+/-0.3% vs vehicle 0.5+/-0.3%, respectively, P<0.05) within 24 h. EPO-treated rats exhibited enhanced PCNA staining within the proximal straight tubule (6.9+/-0.7% vs vehicle 2.4+/-0.5% vs sham 0.3+/-0.2%, P<0.05), proximal convoluted tubule (2.3+/-0.6% vs vehicle 1.1+/-0.3% vs sham 1.2+/-0.3%, P<0.05) and TAL (4.7+/-0.9% vs vehicle 0.6+/-0.3% vs sham 0.3+/-0.2%, P<0.05). The frequency of tubular profiles with luminal cast material was also reduced (32.0+/-1.6 vs vehicle 37.0+/-1.3%, P = 0.05). EPO-treated rats subjected to bilateral IR injury exhibited similar histological improvements to the unilateral IR injury model, as well as significantly lower peak plasma creatinine concentrations than their vehicle-treated controls (0.04+/-0.01 vs 0.21+/-0.08 mmol/l, respectively, P<0.05). EPO had no effect on renal function in sham-operated controls.. The results suggest that, in addition to its well-known erythropoietic effects, EPO inhibits apoptotic cell death, enhances tubular epithelial regeneration and promotes renal functional recovery in hypoxic or ischaemic acute renal injury.

Topics: Animals; Apoptosis; Biopsy, Needle; Cell Survival; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Female; Humans; Immunohistochemistry; In Vitro Techniques; Kidney Function Tests; Kidney Tubular Necrosis, Acute; Kidney Tubules, Proximal; Male; Probability; Rats; Rats, Sprague-Dawley; Reference Values; Regeneration

Within hypoxic tumor regions anaerobic dissimilation of glucose is the sole source of energy generation. It yields only 5% of the ATP that is normally gained by means of oxidative glucose catabolism. The increased need for glucose may aggravate cancer cachexia. We investigated the impact of recombinant human erythropoietin (RhEPO) and increased inspiratory oxygen concentrations on weight loss in tumor-bearing mice.. Fragments of the murine C26-B adenocarcinoma were implanted in 60 BALB/c-mice. The mice were divided into four groups and assigned to: (A) no treatment; (B) RhEPO- administration (25 IU daily from day 1-11, three times per week from day 12); (C) RhEPO and 25% oxygen; and (D) RhEPO and 35% oxygen. Three control groups of four healthy mice each received the same treatment as groups A, B, and D, respectively. Hematocrit and hemoglobin levels, tumor volume, and body weight were monitored. At day 17 the experiment was terminated and the serum lactate concentration was measured. The tumors were excised and weighed and, for each mouse, the percentage weight loss was calculated. The impact of tumor weight and the treatments on lactate concentration and weight loss was evaluated.. Significant positive correlations were found between tumor weight and lactate concentration and between tumor weight and percentage weight loss. In the mice with the largest tumors, RhEPO displayed a significant weight loss-reducing effect, and a significant negative correlation was found between hemoglobin concentration and weight loss. An oxygen-rich environment did not appear to influence weight loss.. Anaerobic glycolysis in a growing C26-B tumor is related to weight loss. RhEPO administration results in a reduction of the percentage weight loss; this effect is probably mediated by an increased hemoglobin concentration.

Topics: Adenocarcinoma; Animals; Cachexia; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Glycolysis; Hematocrit; Hemoglobins; Inhalation; Mice; Mice, Inbred BALB C; Oxygen; Recombinant Proteins; Weight Loss

Erythropoietin, a hemotopoietic growth factor, has brain protective actions. This study investigated the mechanisms of Recombinant Human EPO (rhEPO)-induced brain protection in neonates. An established rat hypoxia-ischemia model was used by ligation of the right common carotid artery of 7-day-old pups, followed by 90 minute of hypoxia (8% 02 and 92% N2) at 37 degrees C. Animals were divided into three groups: control, hypoxia-ischemia, and hypoxia-ischemia plus rhEPO treatment. In rhEPO treated pups, 300 units rhEPO was administered intraperitoneally 24 hours before hypoxia. rhEPO treatment (300 units) was administered daily for an additional 2 days. ELISA and immunohistochemistry examined the expression of EPO and EPOR. Brain weight, morphology, TUNEL assay, and DNA laddering evaluated brain protection. rhEPO abolished mortality (from 19% to 0%) during hypoxia insult, increased brain weight from 52% to 88%, reduced DNA fragmentation, and decreased TUNEL-positive cells. Real-time RT-PCR, Western blot, and immunohistochemistry revealed an enhanced expression of heat shock protein 27 (HSP27) in ischemic brain hemisphere. Double labeling of TUNEL with HSP27 showed most HSP27 positive cells were negative to TUNEL staining. rhEPO reduces brain injury, especially apoptotic cell death after neonatal hypoxia-ischemia, partially mediated by the activation of HSP27.

Topics: Animals; Animals, Newborn; Brain; Brain Ischemia; Disease Models, Animal; DNA Fragmentation; Erythropoietin; Heat-Shock Proteins; Humans; Hypoxia, Brain; In Situ Nick-End Labeling; Neuroprotective Agents; Organ Size; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Recombinant Proteins

Severe anemia is a major life-threatening complication of malaria. The roles of erythropoietin (Epo) and erythropoiesis during blood-stage malaria were investigated. By treating Plasmodium chabaudi AS-infected C57BL/6 (B6) mice, which are resistant to malaria, with polyclonal anti-human Epo neutralizing antibody, we demonstrated that Epo-induced reticulocytosis was important for alleviating malarial anemia and for host survival. By inducing erythropoiesis in A/J mice, which are susceptible to malaria, and in B6 mice at various periods during infection, by use of exogenous recombinant murine Epo, untimely onset of reticulocytosis was shown to augment multiplication of parasites and result in lethal infection. However, timely inducement of reticulocytosis with Epo treatment alleviated malarial anemia and increased survival. Our data reveal the important role of Epo-induced reticulocytosis in modulating the course and outcome of blood-stage malaria. However, the mechanisms underlying the increased mortality associated with untimely treatment with Epo and the increased protection associated with timely treatment with Epo remain to be investigated.

Topics: Animals; Disease Models, Animal; Disease Susceptibility; Epoetin Alfa; Erythropoietin; Malaria; Male; Mice; Mice, Inbred A; Mice, Inbred C57BL; Plasmodium chabaudi; Recombinant Proteins; Reticulocytosis; Treatment Outcome

Recombinant human erythropoietin (rHuEpo) was introduced into clinical practice more than a decade ago, and has been found to be effective in the treatment of several types of anemia, including anemia of end-stage renal failure and cancer-related anemia. No study has suggested that Epo might have an effect on the biology of the disease, nor any survival advantage to cancer patients treated with Epo for anemia has been reported. Here we report six patients with advanced multiple myeloma (MM) with very poor prognostic features, whose expected survival was <6 months. All six patients were treated with rHuEpo for their anemia, either without any chemotherapy or very mild chemotherapy for a short time. Yet, surprisingly they lived for 45-133 months totally from MM diagnosis and 38-94 months with rHuEpo (with a good quality of life). In fact, one patient, is still alive and well, more than 8 yr after chemotherapy was discontinued because of a resistant aggressive disease. The course in these six MM patients led us to hypothesize that Epo might have an antineoplastic or antimyeloma effect. We proceeded and tested that hypothesis in mouse models of myeloma (Mittelman M et al., Proc Natl Acad Sci USA 98:5181,2001). In these models we confirmed that rHuEpo induced tumor regression in about 50% of the BALB/c mice inoculated with MOPC-315 myeloma cells. We then presented evidence that the mechanism is a new immune-mediated phenomenon, via activation of CD8+ T cells. Furthermore, evidence from the literature supports the antineoplastic effect of Epo. Epo might be used as an adjunct immune treatment in various malignant diseases, in addition to the current regimens and chemotherapeutic protocols. Future trials should determine the role of Epo in myeloma and cancer treatment, besides clarifying concerns about the presence of Epo receptors on some tumor cells.

Topics: Adult; Aged; Anemia; Animals; Antineoplastic Agents; CD8-Positive T-Lymphocytes; Disease Models, Animal; Disease Progression; Erythropoietin; Female; Humans; Male; Mice; Mice, Inbred BALB C; Middle Aged; Multiple Myeloma; Prognosis; Recombinant Proteins; Remission Induction; Survival Rate; Treatment Outcome

In vivo selection may provide a means to increase the relative number of cells of donor origin in recipients with hemopoietic chimerism. We have tested whether in vivo selection using chemical inducers of dimerization (CIDs) can direct the expansion of transduced normal donor erythrocytes in recipients with chimerism using a mouse model of pyruvate kinase deficiency. Marrow cells from normal CBA/N mice were transduced with a vector (F36Vmpl(GFP)) that promotes cell growth in the presence of CIDs. Transduced cells were then transplanted into minimally conditioned, pyruvate kinase-deficient recipients (CBA-Pk-1(slc)/Pk-1(slc)) to establish stable chimerism. CID administration resulted in expansion of normal donor erythrocytes and improvement of the anemia. The preferential expansion of normal erythrocytes also resulted in a decrease in erythropoietin levels, reducing the drive for production of pyruvate kinase-deficient red blood cells. CID-mediated expansion of genetically modified erythrocytes could prove a useful adjunct to transplantation methods that achieve erythroid chimerism.

Topics: Anemia; Animals; Disease Models, Animal; Erythrocytes; Erythropoietin; Female; Gene Transfer Techniques; Genes, Reporter; Green Fluorescent Proteins; Luminescent Proteins; Male; Mice; Mice, Inbred CBA; Pyruvate Kinase; Stem Cell Transplantation; Time Factors; Transplantation Chimera

Topics: Anemia; Cell Hypoxia; Clinical Trials, Phase II as Topic; Clinical Trials, Phase III as Topic; Disease Models, Animal; Dose Fractionation, Radiation; Epoetin Alfa; Erythropoietin; Female; Head and Neck Neoplasms; Hematinics; Hemoglobins; Humans; Male; Meta-Analysis as Topic; Neoplasm Recurrence, Local; Neoplasms; Pelvic Neoplasms; Placebos; Prognosis; Radiotherapy Dosage; Recombinant Proteins; Retrospective Studies; Risk; Risk Factors; Time Factors; Treatment Outcome; Uterine Cervical Neoplasms

Consorcial projects focused on 5 cancer types, breast-, colorectal-, head and neck- and pediatric cancers, and malignant melanoma. Breast cancer studies revealed unique splicing mechanisms concerning BRCA1. In sporadic breast cancers the involvement of DNA-repair genes was proved to be dependent on the histological type. Bone-metastatic tumors have been characterized by decreased NM23 and increased c-met and p53 expressions. C-erbB2 genotype of the primary tumor was not maintained frequently in bone metastases. Application of DNA-microarray and quantitative PCR technologies improved the prediction of therapeutic sensitivity of breast cancers. Colorectal cancer studies revealed regional inhomogenities (clusters) in various geographical regions of Hungary, which were distinct in the case of colonic and rectal cancers. To increase the sensitivity of fecal blood test of colorectal cancer screening, a new double-antibody test was developed and tested in a large cohort of patients. Genetic analysis revealed that hypermethylation is a significant factor in microsatellite instability which, and plays a role in silencing of APC and E-cadherin genes as well. The Hungarian pattern of TS polymorphism was also determined and was correlated not only with the efficacy of 5-FU treatment but with the progression of the disease as well. Population-based studies have been carried out in head and neck cancer patients (HNC) and smokers as well to reveal the genetic background of increasing tumor incidence. These studies revealed polymorphism in XRCC1/3 methylation enzyme gene which has preventive role. Other studies found frequent local immunosuppression in HNC patients. Studies indicated that the success of irradiation in this cancer type is dependent on the anti-vascular effects. Pediatric cancer studies determined the parameters of neuroblastoma screening based on VMA measurements. New splice variants of the WT1 gene involved in the monitoring of MRD of ALL patients was also described this year. We also obtained positive experimental data for the retinoic acid therapy of ALL. Melanoma studies extensively used DNA-microarray technology which identified 4 melanoma-specific and 2 melanoma progression-specific genes. In experimental human melanoma xenograft models we have identified 3 anti-metastatic agents: low molecular weight heparin, 2-methoxyestradiol and erythropoietin-alpha, where the later was characterized by specific effects on tumor vasculature.

Topics: 2-Methoxyestradiol; Adult; Animals; Antineoplastic Agents; Biomarkers, Tumor; Biomedical Research; Bone Neoplasms; Breast Neoplasms; Child; Colorectal Neoplasms; Disease Models, Animal; Disease Progression; DNA Methylation; Epoetin Alfa; Erythropoietin; Estradiol; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genetic Markers; Head and Neck Neoplasms; Heparin, Low-Molecular-Weight; Humans; Hungary; Incidence; Male; Melanoma; Microsatellite Repeats; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Polymorphism, Genetic; Predictive Value of Tests; Recombinant Proteins; Transplantation, Heterologous

The ICR-derived glomerulonephritis (ICGN) mouse, a new inbred mouse strain with a hereditary nephrotic syndrome, is considered to be a good model of human idiopathic nephrotic syndrome and notably exhibits proteinuria and hypoproteinemia from the neonatal stage. In chronic renal disorder (CRD), anemia is a major subsequent symptom (renal anemia). The precise cause of renal anemia remains unclear, primarily owing to the lack of appropriate spontaneous animal models for CRD. To establish adequate animal models for anemia with CRD, we examined the hematological-biochemical properties and histopathological characteristics. With the deterioration of renal function, ICGN mice developed a normochromic and normocytic anemia, and exhibited normochromic and microcytic at the terminal stage. The expression of erythropoietin (EPO) mRNA both in the kidneys and liver and the EPO leak into the urine were observed in ICGN mice, indicating a disrupted metabolism of EPO in ICGN mice. In addition, a lack of iron induced by the hemolysis in the spleen and the leak of transferrin into urine as proteinuria aggravated the anemic condition. In conclusion, the ICGN mouse is a good model for anemia with CRD.

Topics: Analysis of Variance; Anemia; Animals; Crosses, Genetic; Disease Models, Animal; Erythropoietin; Glomerulonephritis; Hematologic Tests; Histological Techniques; Kidney; Kidney Failure, Chronic; Liver; Mice; Mice, Inbred ICR; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by an expanded CAG repeat in the HD gene. Both excitotoxicity and oxidative stress have been proposed to play important roles in the pathogenesis of HD. Since no effective treatment is available, this study was designed to explore the therapeutic potential of erythropoietin (EPO), a cytokine that has been found to prevent excitotoxicity, and to promote neurogenesis. To avoid the side effects of a raised hematocrit, we used asialoerythropoietin (asialoEPO), a neuroprotective variant of EPO that lacks erythropoietic effects in mice. R6/2 transgenic HD mice were treated with this cytokine from five to twelve weeks of age.. We provide new evidence that cell proliferation in the dentate gyrus of the R6/2 hippocampus is reduced by 50% compared to wild-type littermate controls. However, we found that the asialoEPO treatment did not affect the progression of motor symptoms, weight loss or the neuropathological changes. Furthermore, cell proliferation was not enhanced.. We conclude that the chosen protocol of asialoEPO treatment is ineffective in the R6/2 model of HD. We suggest that reduced hippocampal cell proliferation may be an important and novel neuropathological feature in R6 HD mice that could be assessed when evaluating potential therapies.

Topics: Animals; Asialoglycoproteins; Behavior, Animal; Cell Division; Cytokines; Dentate Gyrus; Disease Models, Animal; Disease Progression; Erythropoietin; Female; Huntington Disease; Male; Mice; Mice, Transgenic; Neuroprotective Agents; Phenotype; Treatment Failure; Weight Loss

Here we investigate the effects of erythropoietin (EPO) on the tissue/organ injury caused by hemorrhagic shock (HS), endotoxic shock, and regional myocardial ischemia and reperfusion in anesthetized rats. Male Wistar rats were anesthetized with thiopental sodium (85 mg/kg i.p.) and subjected to hemorrhagic shock (HS; i.e., mean arterial blood pressure reduced to 45 mmHg for 90 min, followed by resuscitation with shed blood for 4 h), endotoxemia (for 6 h), or left anterior descending coronary artery occlusion (25 min) and reperfusion (2 h). HS and endotoxemia resulted in renal dysfunction and liver injury. Administration of EPO (300 IU/kg i.v., n = 10) before resuscitation abolished the renal dysfunction and liver injury in hemorrhagic, but not endotoxic, shock. HS also resulted in significant increases in the kidney of the activities of caspases 3, 8, and 9. This increase in caspase activity was not seen in HS rats treated with EPO. In cultured human proximal tubule cells, EPO concentration-dependently reduced the cell death and increase in caspase-3 activity caused by either ATP depletion (simulated ischemia) or hydrogen peroxide (oxidative stress). In the heart, administration of EPO (300 IU/kg i.v., n = 10) before reperfusion also caused a significant reduction in infarct size. In cultured rat cardiac myoblasts (H9C2 cells), EPO also reduced the increase in DNA fragmentation caused by either serum deprivation (simulated ischemia) or hydrogen peroxide (oxidative stress). We propose that the acute administration of EPO on reperfusion and/or resuscitation will reduce the tissue injury caused by ischemia-reperfusion of the heart (and other organs) and hemorrhagic shock.

Topics: Animals; Blood Pressure; Disease Models, Animal; Erythropoietin; Heart Rate; Male; Myocardial Ischemia; Rats; Reperfusion Injury; Shock, Hemorrhagic

Systemic hypoxia is a common complication in stroke patients and may exacerbate ischemic brain damage. Expression of the hypoxia-inducible cytokine erythropoietin (Epo) is upregulated in the brain in both stroke patients and in animal stroke models and exerts local neuroprotective effects in the ischemic brain. Epo is also well known to stimulate red blood cell (RBC) production. The purpose of the present study was to evaluate whether poststroke systemic hypoxia is present in the rat model and whether it is associated with increased peripheral Epo and RBC production.. Wistar rats underwent 1-hour transient middle cerebral artery occlusion (MCAO) under mechanical ventilation, followed by reperfusion without further ventilation. Groups of MCAO and sham-operated animals were evaluated at extended times after reperfusion for assessment of arterial blood gases, plasma Epo, and complete blood count.. Arterial oxygen saturation was significantly lower in the infarct group between 6 and 24 hours after reperfusion (P=0.0005), and plasma Epo levels were increased 6 hours after reperfusion (P<0.05). RBC counts and hematocrit were transiently increased 2 to 7 days after reperfusion in animals with MCAO compared with sham. Maximal increases were seen at day 7 (22% and 16% increases of RBC count and hematocrit, respectively; P<0.001). In contrast, the white blood cell counts in animals with MCAO decreased by >30% in the same time period.. Plasma Epo levels, RBC counts, and hematocrit are all increased in response to systemic hypoxia after cerebral ischemia in rats.

Topics: Animals; Disease Models, Animal; Erythrocyte Count; Erythropoiesis; Erythropoietin; Hematocrit; Homeostasis; Hypoxia; Infarction, Middle Cerebral Artery; Male; Oxygen; Rats; Rats, Wistar; Stroke

Erythropoietin (EPO) prevents neuronal damage following ischemic, metabolic and excitotoxic stress. Recent studies have shown that EPO plays a significant role in the developing brain. The present study investigates the effect of EPO administration on hypoxic-ischemic brain injury and the possibility that its neuroprotective action may be associated with anti-apoptotic activity. Seven-day-old rats were treated with EPO (2000 U/kg) and subjected to a modified Levine procedure. EPO administration before the hypoxic-ischemic insult significantly reduces the severity of brain damage and improved the short-term functional brain recovery. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and DNA electrophoresis displayed no evidence of DNA fragmentation in EPO-treated animals. These results suggest that EPO might protect the neonatal rat brain by anti-apoptotic mechanisms.

Topics: Animals; Animals, Newborn; Asphyxia Neonatorum; Brain; Disease Models, Animal; DNA Fragmentation; Erythropoietin; Female; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Male; Neuroprotective Agents; Rats; Rats, Wistar; Reflex

Erythropoietin (EPO) is upregulated by hypoxia and causes proliferation and differentiation of erythroid progenitors in the bone marrow through inhibition of apoptosis. EPO receptors are expressed in many tissues, including the kidney. Here it is shown that a single systemic administration of EPO either preischemia or just before reperfusion prevents ischemia-reperfusion injury in the rat kidney. Specifically, EPO (300 U/kg) reduced glomerular dysfunction and tubular injury (biochemical and histologic assessment) and prevented caspase-3, -8, and -9 activation in vivo and reduced apoptotic cell death. In human (HK-2) proximal tubule epithelial cells, EPO attenuated cell death in response to oxidative stress and serum starvation. EPO reduced DNA fragmentation and prevented caspase-3 activation, with upregulation of Bcl-X(L) and XIAP. The antiapoptotic effects of EPO were dependent on JAK2 signaling and the phosphorylation of Akt by phosphatidylinositol 3-kinase. These findings may have major implications in the treatment of acute renal tubular damage.

Topics: Animals; Apoptosis; Blood Proteins; Caspase 3; Caspase Inhibitors; Caspases; Cell Line, Transformed; Creatinine; Disease Models, Animal; Erythropoietin; Humans; Kidney Diseases; Kidney Tubules; Male; Oxidative Stress; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Reperfusion Injury

The ICR-derived glomerulonephritis (ICGN) mouse, a novel inbred mouse strain with a hereditary nephrotic syndrome, develops severe anemia associated with chronic renal failure. To reveal the pathogenic mechanism of anemia in ICGN mice, we subcutaneously administered recombinant human erythropoietin (rhEPO; 5 IU/mouse/day) or saline for 5 days to ICGN mice. In terminal-stage ICGN mice with severe anemia, rhEPO significantly increased hematocrit (Ht), red blood cells (RBC) and hemoglobin levels. Endogenous EPO levels in peripheral blood were reduced by rhEPO injection. No histopathological changes in bone marrow and kidneys were induced by rhEPO injection. Insufficiency of EPO may cause anemia in ICGN mice.

Topics: Anemia; Animals; Bone Marrow; Disease Models, Animal; Erythrocyte Count; Erythropoietin; Female; Glomerulonephritis; Hematocrit; Hemoglobins; Injections, Subcutaneous; Kidney; Kidney Failure, Chronic; Male; Mice; Mice, Inbred ICR; Recombinant Proteins

The hematopoietic cytokine erythropoietin has cytoprotective effects in endothelial cells in vitro that are mediated through direct activation of the pro-survival Akt tyrosine kinase signaling pathway. We tested the hypothesis that low-dose therapy with the long-acting recombinant human erythropoietin analogue darbepoetin alpha protects vascular endothelium in vivo in a classic remnant kidney rat model characterized by severe endothelial damage, progressive vascular sclerosis, and ischemia-induced tissue fibrosis.. Using a parallel group study design, we randomly assigned animals after 5/6 nephrectomy to treatment with either saline (n=36) or 0.1 microg/kg body wt darbepoetin (n=24) subcutaneously once weekly. We monitored hematocrit, blood pressure, and serum creatinine regularly and obtained renal tissue 6 weeks after nephrectomy for morphological and immunohistochemical analysis. Darbepoetin-treated animals had significantly improved survival compared with saline-treated controls (63% versus 33%; P<0.05), although hematocrit levels were similar in both groups. Darbepoetin treatment ameliorated endothelial damage; attenuated the composite tissue injury score (saline 1.9+/-0.4; darbepoetin 0.4+/-0.2; P<0.001), which included vascular sclerosis, glomerulosclerosis, and tubulointerstitial damage; and preserved renal function. We found persistent activation of the pro-survival Akt signaling pathway in endothelial and epithelial glomerular cells in darbepoetin-treated animals, accompanied by a significant reduction of apoptotic cell death in renal tissue.. Low-dose darbepoetin treatment confers vascular and tissue protection that is associated with persistent stimulation of the pro-survival Akt signaling pathway. The use of recombinant human erythropoietin or analogues may have utility in preventing ischemia-related progressive vascular injury and organ failure.

Topics: Animals; Apoptosis; Darbepoetin alfa; Disease Models, Animal; Disease Progression; Drug Evaluation, Preclinical; Endothelium, Vascular; Erythropoietin; Glomerulosclerosis, Focal Segmental; Hematocrit; Hematopoietic Stem Cell Mobilization; Hypertension, Renal; Ischemia; Kidney; Life Tables; Male; Multiple Organ Failure; Nephrectomy; Nephritis, Interstitial; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Random Allocation; Rats; Rats, Sprague-Dawley; Signal Transduction

In this study, we assessed the ability of erythropoietin (EPO) to synergize with various chemotherapeutic agents and suppress the growth and metastasis of solid tumors. Animals were inoculated with Lewis lung carcinoma (LLC) cells and treated with EPO alone, the designated chemotherapeutic drug (cisplatin, mitomycin C or cyclophoshamide) alone, or EPO and the drug. Tumor volume was monitored daily. Thirteen days following cell injection, tumor mass was determined. In addition, the number of the metastatic foci in the lungs was determined. Cisplatin alone was capable of inducing a 7-fold decrease in final tumor volume compared to tumor-bearing animals injected with saline. However, when EPO was combined with cisplatin, the animals experienced an 11-fold reduction in final tumor volume compared to saline-injected animals (P<0.001). A 2.5-fold reduction in tumor mass was observed in animals treated with cisplatin, compared to the saline-injected groups. Furthermore, injections of EPO and cisplatin induced a 4-fold reduction in tumor mass (P<0.001). Blood analysis indicated that a significant increase of more than 30% in WBC was found in animals injected concurrently with cisplatin and EPO, as compared to saline-injected mice (P<0.03). When EPO and mitomycin C were injected together, tumor mass was further reduced by 14% compared to that seen in mice treated with mitomycin C alone. However, this difference was not statistically significant. We conclude from this study that EPO can synergize with chemotherapeutic agents to further suppress the growth of tumors. The level of synergism is drug related.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Carcinoma, Lewis Lung; Cisplatin; Cyclophosphamide; Disease Models, Animal; Drug Interactions; Erythropoietin; Female; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Mitomycin; Neoplasm Metastasis

In multiple sclerosis (MS), long-term disability is primarily caused by axonal and neuronal damage. We demonstrated in a previous study that neuronal apoptosis occurs early during experimental autoimmune encephalomyelitis, a common animal model of MS. In the present study, we show that, in rats suffering from myelin oligodendrocyte glycoprotein (MOG)-induced optic neuritis, systemic application of erythropoietin (Epo) significantly increased survival and function of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve. We identified three independent intracellular signaling pathways involved in Epo-induced neuroprotection in vivo: Protein levels of phospho-Akt, phospho-MAPK 1 and 2, and Bcl-2 were increased under Epo application. Using a combined treatment of Epo together with a selective inhibitor of phosphatidylinositol 3-kinase (PI3-K) prevented upregulation of phospho-Akt and consecutive RGC rescue. We conclude that in MOG-EAE the PI3-K/Akt pathway has an important influence on RGC survival under systemic treatment with Epo.

Topics: Animals; Apoptosis; Caspases; Cell Count; Cell Survival; Disease Models, Animal; Electroretinography; Erythropoietin; Female; In Situ Nick-End Labeling; Multiple Sclerosis; Myelin Proteins; Myelin-Associated Glycoprotein; Myelin-Oligodendrocyte Glycoprotein; Neuroprotective Agents; Optic Nerve; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Inbred BN; Recombinant Proteins; Retinal Ganglion Cells; Signal Transduction

In addition to its well-known erythropoetic effect, erythropoietin (EPO) has also been shown to be neuroprotective in various animal models. In contrast to EPO, carbamylated EPO (CEPO) does not bind to the EPO receptor on UT7 cells or have any haematopoietic/proliferative activity on these cells. In vivo studies in mice and rats showed that even high doses of CEPO for long periods are not erythropoietic. However, in common with EPO, CEPO does inhibit the apoptosis associated with glutamate toxicity in hippocampal cells. Like EPO, CEPO is neuroprotective in a wide range of animal models of neurotoxicity: middle cerebral artery occlusion model of ischaemic stroke, sciatic nerve compression, spinal cord depression, experimental autoimmune encephalomyelitis and peripheral diabetic neuropathy. To date, EPO and CEPO have been exciting developments in the quest for the treatment of various types of neurotoxicity. The development of CEPO should continue.

Topics: Animals; Apoptosis; Brain Ischemia; Cell Line, Tumor; Disease Models, Animal; Erythropoietin; Humans; Mice; Neuroprotective Agents; Rats

The extract from the root of Angelica acutiloba Kitagawa (AR), which is used as herbal medicine in Japan, has been reported to be clinically effective for postmenstrual blood loss and erythropoietin (EPO)-resistant anemia in chronic renal failure, although the pharmacological mechanisms underlying its clinical efficacy are unknown. We prepared an animal model of anemia by bolus injection of 5-fluorouracil (5FU) at 150 mg/kg to mice (8- to 12-week-old female C57BL/6J), and then administered orally the water-soluble fraction of AR to the anemic mice for 10 days. After confirming the anti-anemic effect of the water-soluble fraction of AR (AR-3) containing polysaccharides, we examined the effects of AR-3 on immature erythroid cell activity, EPO production, and plasma cytokine levels. AR-3 administration at 50 mg/kg activated erythroid progenitor cells in bone marrow on day 10, increased the percentage of peripheral reticulocytes in red blood cells on day 15, and led to the recovery of red blood cell count to a value that was almost equal to the basal level on day 20. Although EPO production, which was determined by examining EPO mRNA expression in kidney and liver, remained unaltered by AR-3 administration, this treatment significantly lowered plasma interferon-gamma level, which may suppress the activity of erythroid progenitor cells. These results suggest that the polysaccharides in AR promote hematopoiesis by activating immature erythroid cells, in part, by suppressing cytokine secretion. Since the hematopoietic effect was achieved by high-dose AR-3, identification of specific polysaccharides is still required for the development of a novel medicine for anemia caused by a malignancy or chemotherapy.

Topics: Anemia; Angelica; Animals; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Erythrocyte Count; Erythroid Precursor Cells; Erythropoietin; Female; Fluorouracil; Hematopoiesis; Mice; Mice, Inbred C57BL; Phytotherapy; Plant Extracts; Polysaccharides; Water

The purpose of this study was to test for the presence of liver hypoxia and recovery after reperfusion when blood alcohol levels (BAL) are high. Male rats were fed ethanol intragastrically at a constant rate for 1 month. The pO(2) levels were then measured on the liver surface of these rats, in vivo during laparatomy under isoflurane anesthesia. To measure the response to acute hypoxia, the hepatic blood flow was clamped off at the porta hepatis. When the clamp was released, recovery from hypoxia was measured. A number of hypoxic-inducible genes in the liver were analyzed by means of quantitative RT-PCR as a measure of increased activation of hypoxia initiated transcription. The mRNA levels of genes for adrenomedullin, adrenergic receptor alpha, 1a and 1d, CDK inhibitor 1a, and erythropoietin were all significantly higher at the peaks than troughs. Expression of these same genes in the livers of control rats fed dextrose was lower than at the troughs. Although the mRNA level of the hypoxia-inducible factor (HIF-1alpha) was higher at the trough than at the peak, its protein concentration in the nuclear fraction was not increased at the troughs compared with the peaks. In fact, the nuclear protein level of HIF-1alpha at the peak was significantly higher than in control samples, which is consistent with the presence of hypoxia at the peaks. Further analysis of the HIF-alpha degradation regulation revealed that prolyl 4-hydroxylase (P4ha1) and von Hippel-Lindau syndrome homolog (Vhl) were both up-regulated at the troughs compared with the peaks. The liver surface oxygen levels at the peaks were reduced compared with the control samples. The pO(2) levels fell abruptly when the vessels at the porta hepatis were clamped. When the clamp was removed, allowing reperfusion of the liver, pO(2) returned to baseline levels in the control, and at the troughs but not at the peaks. These results support the hypothesis that hypoxia occurs at the peaks of the BAL cycle and recovery from ischemia is impaired at the peaks.

Topics: Administration, Oral; Adrenomedullin; Animals; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Erythropoietin; Ethanol; Hypoxia; Liver; Liver Diseases, Alcoholic; Male; Oligonucleotide Array Sequence Analysis; Oxygen; Peptides; Rats; Rats, Wistar; Receptors, Adrenergic; Reperfusion Injury; RNA, Messenger

Recently, erythropoietin (EPO) and the nonerythropoietic derivative asialoEPO have been linked to tissue protection in the nervous system. In this study, we tested their effects in a model of neonatal hypoxia-ischemia (HI) in 7-day-old rats (unilateral carotid ligation and exposure to 7.7% O(2) for 50 min). EPO (10 U/g body weight = 80 ng/g; n = 24), asialoEPO (80 ng/g; n = 23) or vehicle (phosphate-buffered saline with 0.1% human serum albumin; n = 24) was injected intraperitoneally 4 h before HI. Both drugs were protective, as judged by measuring the infarct volumes, neuropathological score and gross morphological score. The infarct volumes were significantly reduced by both EPO (52%) and asialoEPO (55%) treatment, even though the plasma levels of asialoEPO had dropped below the detection limit (1 pm) at the onset of HI, while those of EPO were in the nanomolar range. Thus, a brief trigger by asialoEPO before the insult appears to be sufficient for protection. Proteomics analysis after asialoEPO treatment alone (no HI) revealed at least one differentially up-regulated protein, synaptosome-associated protein of 25 kDa (SNAP-25). Activation (phosphorylation) of ERK was significantly reduced in asialoEPO-treated animals after HI. EPO and the nonerythropoietic asialoEPO both provided significant and equal neuroprotection when administered 4 h prior to HI in 7-day-old rats. The protection might be related to reduced ERK activation and up-regulation of SNAP-25.

Topics: Animals; Animals, Newborn; Asialoglycoproteins; Brain Infarction; Disease Models, Animal; Erythropoietin; Female; Hypoxia-Ischemia, Brain; Male; Membrane Proteins; Nerve Tissue Proteins; Proteomics; Rats; Rats, Wistar; Signal Transduction; Synaptosomal-Associated Protein 25; Treatment Outcome

Recent studies show that erythropoietin (EPO) plays a protective role in brain ischemia. In this condition, administration of EPO protects neurons from ischemic damage. Recently, it has been shown that in patients with chronic heart failure (CHF), EPO treatment improved cardiac function. In the present study we assessed the role of EPO and EPO-receptor (EPO-R) in the heart.. We studied the presence and functionality of the EPO-R in isolated rat hearts in the Langendorff set-up. Hearts were perfused for 20 min with 10 U/ml EPO or vehicle. Immunohistochemistry revealed the presence of the EPO-R on endothelial cells, fibroblasts and to a lesser extent cardiomyocytes. Furthermore, perfusion with EPO resulted in a 50% increase in the phosphorylated MAP kinases p42/p44. To evaluate the protective role of EPO in cardiac ischemia, we performed low-flow (0.6 ml/min) ischemia/reperfusion experiments in isolated rat hearts. Administration of EPO (10 U/ml) reduced the cellular damage by 56% (P<0.05) during reperfusion, diminished apoptosis by 15% (P<0.05) and resulted in a significantly improved recovery of left ventricular pressure (P=0.02) and coronary flow (P=0.01).. The present data suggest that a functional EPO-R is present in rat adult cardiac tissue and that exogenous EPO administration improves cardiac function after ischemia/reperfusion injury.

Topics: Animals; Coronary Circulation; Disease Models, Animal; Endothelial Cells; Erythropoietin; Fibroblasts; Humans; Immunohistochemistry; Mitogen-Activated Protein Kinases; Models, Cardiovascular; Phosphorylation; Rats; Receptors, Erythropoietin; Reperfusion Injury; Signal Transduction; Ventricular Function, Left

The presence of hypoxic areas is a common feature of solid tumors and has been associated with decreased sensitivity of the tumors to radiation therapy and oxygen-dependent chemotherapeutic agents, as well as worsened outcomes, including survival. Anemia is also common in cancer patients and is believed to contribute to tumor hypoxia. Thus, the rationale exists for administering recombinant human erythropoietin (rHuEPO, epoetin alfa) in an effort to increase hemoglobin levels, correct anemia, and thereby possibly increase the sensitivity of tumors to standard cancer treatment and improve patient outcomes. The results of several preclinical studies that examined the impact of anemia prevention by rHuEPO on tumor sensitivity to radiation therapy in rodent models of cancer showed that induction of anemia increased hypoxia in tumor cells and that correction of anemia with rHuEPO could improve tumor oxygenation. Further studies in rodent models showed significantly delayed tumor growth in both irradiated mice and irradiated rats treated with rHuEPO. In those studies, the increased radiosensitivity observed was believed to be due to improved tumor oxygenation following the correction of anemia. Similarly, enhancements in chemosensitivity were found in rHuEPO-treated rodent models. In the chemosensitivity studies, as in the radiosensitivity studies, the therapeutic benefit obtained was believed to reflect improved tumor oxygenation subsequent to an rHuEPO-related increase in oxygen availability. One study evaluated the potential biologic effects of epoetin alfa on tumor progression using murine myeloma models (MOPC-315 and 5T33 MM). Treatment of MOPC-315 tumor-bearing mice with epoetin alfa induced complete tumor regression in 30%-60% of mice. Regression was found to be tumor specific, and the effect of epoetin alfa was shown to be T-cell mediated. Additionally, epoetin alfa administration prolonged survival and reduced morbidity and mortality in the 5T33 MM tumor model. Those investigators suggested that epoetin alfa may have antitumor activity in addition to its hematopoietic effects. Overall, these preclinical findings suggest that correction of anemia by rHuEPO can increase tumor sensitivity to both radiation therapy and chemotherapy and that epoetin alfa may exert an immunomodulatory effect in multiple myeloma.

Topics: Anemia; Animals; Antineoplastic Combined Chemotherapy Protocols; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Hypoxia; Disease Models, Animal; Drug Interactions; Erythropoietin; Female; Mice; Mice, Inbred BALB C; Multiple Myeloma; Radiation Tolerance; Recombinant Proteins; Survival Analysis; Treatment Outcome

Topics: Anemia; Animals; Cell Hypoxia; Clinical Trials as Topic; Disease Models, Animal; Epoetin Alfa; Erythropoietin; Hematinics; Humans; Neoplasms; Quality of Life; Recombinant Proteins

The purpose of this study was to investigate whether erythropoietin (EPO) has an effect on the expression of bcl-2 in rat cardiac myocytes following experimental isolated traumatic brain injury (TBI). Forty-eight Wistar-Albino female rats were randomly allocated into eight groups. Groups AC and BC were controls; groups AS and BS were sham-operated animals. Groups A1 and B1 underwent head trauma without treatment. Groups A2 and B2, head traumas plus EPO intraperitoneally (1000 IU/kg); groups A3 and B3, the vehicle groups, head traumas and intraperitoneal albumin (0.4 ml/rat). The method of weight drop was used to produce impact trauma at 24 hours after injury. Samples obtained from the left ventricle were assayed for lipid peroxidation and bcl-2 gene expression using real-time quantitative polymerase chain reactions. Lipid peroxidation in the heart tissue was determined by the concentration of thiobarbituric acid reactive substances (TBARs). The results showed that administration of EPO significantly reduced the increase in lipid peroxidation by-products after moderate or severe trauma. The bcl-2 expression was significantly higher in EPO (A2 and B2) compared to trauma groups (A1 and B1) suggesting a protective effect. These findings suggest that EPO may play an important role in the expression of bcl-2 and decrease in TBARs-the end product of lipid peroxidation in myocytes-after moderate or severe TBI.

Topics: Animals; Brain Injuries; Disease Models, Animal; Erythropoietin; Female; Gene Expression Regulation; Genes, bcl-2; Heart; Lipid Peroxidation; Muscle Cells; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Thiobarbituric Acid Reactive Substances

To evaluate the therapeutic effects of recombinant human erythropoietin (rhEPO) and recombinant human granulocyte colony stimulating factor (rhG-CSF) on sulfur mustard (SM) induced toxicity in dogs.. Ten dogs were used as the animal model. Control group had four dogs while treatment group had six. Half an hour after subcutaneously injected with 12 mg/kg (body weight) of SM, the dogs of the treatment group were treated by rhEPO, rhG-CSF and other medicines. General examinations of blood were measured before injecting SM and were continually watched for a week after poisoning.. Three dogs in control group died in three days after poisoning, dogs in treatment group all survived. WBC of the control group decreased significantly 3 days after poisoning. RBC of the control group had a slight increase at first, then came down. While WBC and RBC of treatment group remained constant. LYM of both groups descended notably right after poisoning, especially in control group. Two days after poisoning, RC in treatment group began to rise and remained at high level for about a week. No obvious changes of RC in control group were found.. The use of rhEPO and rhG-CSF after SM exposure can stimulate the growth of the erythrocyte, reticulocyte and leucocyte in dogs. Therefore, rhEPO and rhG-CSF may be significant assistant drugs in future experiment of SM.

Topics: Animals; Blood Cell Count; Disease Models, Animal; Dogs; Drug Therapy, Combination; Erythropoietin; Granulocyte Colony-Stimulating Factor; Mustard Gas; Poisoning; Recombinant Proteins

The use of perioperative erythropoietin (EPO) therapy is gaining popularity to avoid blood transfusion and correct anemia in head and neck cancer surgery. The purpose of the study was to determine the effect of various doses and durations of EPO treatment on random flap survival.. A McFarlane type random and musculocutaneous (3 x 10 cm) flap were elevated on the dorsum of each rat.. Eighty-four male Albino rats were randomly assigned into seven groups (2 animals in each group): group I, control animals receiving placebo; group II, chronic EPO injections (50 U/kg); group III, chronic EPO injections (100 mg/kg); group IV, chronic EPO injections (150 mg/kg); group V, short-term EPO injections (50 mg/kg); group VI, short-term EPO injections (100 mg/kg); and group VII, short-term EPO injections (150 mg/kg). Rats in groups II to IV began to receive EPO 3 weeks (thrice weekly) before the construction of flaps, and rats in groups V to VII received EPO after flap elevation for 1 week (thrice) subcutaneously. Following 7 days of recovery, the area of flap survival was measured. Hematocrit and systolic blood pressure were followed weekly in all groups.. Erythropoietin increased the hematocrit levels and systolic blood pressure in all groups, but significant increases were noted only in the long-term treatment groups. There was a significant increase in distal necrosis of random skin flaps after long-term EPO treatment (P <.05). However, short-term low and therapeutic doses of EPO improved flap survival significantly (P <.05).. Long-term EPO treatment might have impaired flap survival because of direct or prostaglandin-mediated vasoconstriction, endothelin-induced hypertension, increased peripheral vascular resistance, hyperviscosity, and increased thrombosis. However, EPO might have enhanced flap survival because of its antioxidant effect and modulation of nitric oxide levels. Effects of EPO are controversial, and further research is necessary to delineate the dose and duration relationship and the exact mechanism of action on flap viability.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Erythropoietin; Female; Graft Rejection; Graft Survival; Injections, Subcutaneous; Male; Postoperative Care; Probability; Random Allocation; Rats; Rats, Inbred Strains; Reference Values; Sensitivity and Specificity; Statistics, Nonparametric; Surgical Flaps

An HLF (HIF-1alpha-like factor)/HIF-2alpha-knockout mouse is embryonic lethal, preventing investigation of HLF function in adult mice. To investigate the role of HLF in adult pathological angiogenesis, we generated HLF-knockdown (HLF(kd/kd)) mice by inserting a neomycin gene sandwiched between two loxP sequences into exon 1 of the HLF gene. HLF(kd/kd) mice expressing 80-20% reduction, depending on the tissue, in wild-type HLF mRNA were fertile and apparently normal. Hyperoxia-normoxia treatment, used as a murine model of retinopathy of prematurity (ROP), induced neovascularization in wild-type mice, but not in HLF(kd/kd) mice, whereas prolonged normoxia following hyperoxic treatment caused degeneration of retinal neural layers in HLF(kd/kd) mice due to poor vascularization. Cre-mediated removal of the inserted gene recovered normal HLF expression and retinal neovascularization in HLF(kd/kd) mice. Expression levels of various angiogenic factors revealed that only erythropoietin (Epo) gene expression was significantly affected, in parallel with HLF expression. Together with the results from intraperitoneal injection of Epo into HLF(kd/kd) mouse, this suggests that Epo is one of the target genes of HLF responsible for experimental ROP.

Topics: Animals; Antibodies; Basic Helix-Loop-Helix Transcription Factors; Disease Models, Animal; Electroretinography; Erythropoietin; Female; Gene Expression Regulation; Gene Targeting; Genetic Vectors; Helix-Loop-Helix Motifs; Humans; Hyperoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; In Situ Nick-End Labeling; Infant, Newborn; Integrases; Mice; Mice, Knockout; Retina; Retinal Neovascularization; Retinopathy of Prematurity; RNA, Messenger; Tissue Distribution; Trans-Activators; Transcription Factors; Vimentin; Viral Proteins

Quinolinic acid (QA) is a potent endogenous excitotoxin; elevation of its concentration in an organism has been implicated in the pathogenesis of various disorders. The purpose of this study was the assessment of QA impact on the process of erythropoiesis. Marked increase of QA concentration was observed in plasma and peripheral tissues of uremic rats. These changes were proportional to the amount of the removed renal tissue and positively correlated with the concentration of creatinine but negatively correlated with hematological parameters, i.e., hematocrit and Hb red blood cells count. The changes were accompanied by a slight decrease in the concentration of endogenic erythropoietin (EPO) in the plasma of animals with uremia. Chronic treatment with QA diminished the increase in EPO concentration after introduction of cobalt in rats. These changes were associated with the decrease in all hematological parameters after QA administration. The in vitro study in the conditions of hypoxia showed that QA inhibited the EPO release from HepG2 cells to the culture base. Additionally, in HepG2 cells QA had a dose-dependent inhibitory effect on hypoxia- and cobalt-induced EPO gene expression without any cell toxicity. In conclusion, the erythropoiesis in chronic renal failure could be attributed to the influence of QA on EPO synthesis. Thus we propose that QA can be a uremic toxin responsible for anemia in animals or patients with renal failure.

Topics: Anemia; Animals; Carcinoma, Hepatocellular; Cell Hypoxia; Cell Survival; Cobalt; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Humans; Kidney Failure, Chronic; Male; Neurotoxins; Organ Specificity; Quinolinic Acid; Rats; Rats, Wistar; RNA, Messenger; Tumor Cells, Cultured

Cellular response to hypoxia is mainly controlled by hypoxia-inducible factor 1 (HIF-1). The HIF-1 target gene erythropoietin (EPO) has been described as neuroprotective. Thus, we hypothesize EPO to be an essential mediator of protection in hypoxic preconditioning.. We randomized Sv129 mice into groups for different pretreatments, different hypoxia-ischemia intervals, or different durations of ischemia. For hypoxic preconditioning, the animals were exposed to a hypoxic gas mixture (8% O2 and 92% N2) for 30, 60, 180, 300, or 360 minutes. At 0, 24, 48, 72, or 144 hours later, we performed middle cerebral artery occlusion and allowed reperfusion after 30, 45, 60, or 120 minutes, or occlusion was left to be permanent. We studied EPO gene expression in brain tissue with a real-time reverse transcriptase-polymerase chain reaction and measured HIF-1 DNA-binding activity with an electrophoretic mobility shift assay. To block endogenously produced EPO, we instilled soluble EPO receptor into the cerebral ventricle.. Hypoxic preconditioning for 180 or 300 minutes induced relative tolerance to transient focal cerebral ischemia, as evidenced by a reduction of infarct volumes to 75% or 54% of the control, respectively. Hypoxic pretreatment was effective only when applied 48 or 72 hours before middle cerebral artery occlusion. Sixty minutes after hypoxia, we found a marked activation of HIF-1 DNA-binding activity and a 7-fold induction of EPO transcription. Infusion of soluble EPO receptor significantly reduced the protective effect of hypoxic pretreatment by 40%.. Endogenously produced EPO is an essential mediator of ischemic preconditioning.

Topics: Animals; Brain; Cerebral Infarction; Disease Models, Animal; DNA; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Erythropoietin; Female; Hippocampus; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Infarction, Middle Cerebral Artery; Injections, Intraventricular; Ischemic Preconditioning; Male; Mice; Mice, Inbred Strains; Nuclear Proteins; Receptors, Erythropoietin; Reperfusion; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stroke; Time Factors; Transcription Factors

Despite its frequency and impact on clinical outcomes, anaemia in cancer patients remains poorly understood and suboptimally treated. The definition of optimum treatment schedules with erythropoietic agents requires a suitable model of chemotherapy-induced progressive anaemia. This study investigated novel strategies such as once-per-chemotherapy-cycle dosing, synchronization between erythroid supportive care and chemotherapy, and definition of the optimum timing of erythroid support. A murine model of carboplatin chemotherapy/radiotherapy (CRT)-induced anaemia was used, which caused progressive anaemia across multiple cycles. Weekly administration of recombinant human erythropoietin (rHuEPO) was effective, but the longer-acting darbepoetin alpha resulted in superior responses. In all animals, anaemia became progressive and more refractory across cycles because of accumulated bone marrow damage. Exploiting a specific enzyme-linked immunosorbent assay, which could distinguish between darbepoetin alpha and endogenous erythropoietin, the effect of CRT upon the pharmacokinetics of darbepoetin alpha showed that clearance of darbepoetin alpha, and presumably erythropoietin, was at least partially dependent on a chemotherapy-sensitive pathway. Scheduling data suggested that administration of erythropoietic agents prior to chemotherapy was more effective than administration after chemotherapy. There was no evidence that erythropoietic agents exacerbated anaemia, even when administered immediately prior to CRT in an attempt to "prime" erythroid cells for the effects of CRT.

Topics: Anemia; Animals; Antineoplastic Agents; Carboplatin; Combined Modality Therapy; Darbepoetin alfa; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Erythropoietin; Female; Hematopoiesis; Hematopoietic Stem Cells; Hemoglobins; Mice; Mice, Inbred Strains; Radiation Injuries; Radiotherapy; Recombinant Proteins

Hypoxic-ischemic encephalopathy seen in survivors of perinatal asphyxia is a frequently encountered and a major clinical problem for which there is currently no effective treatment. Hematopoietic neuroprotective agents, such as erythropoietin (EPO) may rescue neurons from cell death in this setting. EPO is a cytokine hormone that has neuroprotective effect in vitro and in vivo. In this study, we evaluated the effect of posthypoxic EPO administration in an animal model of neonatal hypoxic-ischemic injury. Our results show that a single intracerebroventricular injection of EPO immediately after hypoxic-ischemic insult in neonatal rat model of hypoxic-ischemia reduced the extent of hypoxic-ischemic brain damage. The mean infarct volume assessed 7 days after hypoxia was significantly smaller in EPO-treated group than in the control group. These findings suggest that EPO may provide benefit after hypoxic-ischemic events in the developing brain, a major contributor to static encephalopathy and cerebral palsy.

Topics: Animals; Animals, Newborn; Brain Injuries; Disease Models, Animal; Erythropoietin; Hypoxia-Ischemia, Brain; Injections, Intraventricular; Neuroprotective Agents; Rats; Rats, Wistar

Erythropoietin (Epo) has been shown to have potent anti-apoptotic activity in central nervous system neurons in animal models of ischaemic injury. Recently, Epo and its receptor (EpoR) have been identified in the peripheral nervous system [Campana & Myers (2001), FASEB J., 15, 1804-1806]. Herein, we demonstrate that in painful neuropathy caused by L5 spinal nerve crush (SNC), therapy with recombinant human Epo (rhEpo) reduced dorsal root ganglion (DRG) apoptosis and pain behaviours. Quantification of both DRG neurons and satellite cells revealed that vehicle-treated, crush-injured DRGs had 35.5 +/- 8.3% apoptotic neurons and 23.5 +/- 2.36% satellite cells compared with 7.5 +/- 6.3% apoptotic neurons and 6.4 +/- 3.94% satellite cells in rhEpo-treated, crush-injured DRGs (P < 0.05). While rhEpo-treated animals were not initially protected from mechanical allodynia associated with L5 SNC, rhEpo did significantly improve recovery rates compared to vehicle-treated animals (P < 0.01). Systemic rhEpo therapy increased JAK2 phosphorylation, a key anti-apoptotic signalling molecule for Epo-induced neuroprotection, in DRGs after crush. Dual immunofluorescence demonstrated Epo-induced JAK2-p was associated with both neuronal and glial cells. JAK2-p was associated with NF200-positive large neurons and with smaller neurons. This population of small neurons did not colocalize with IB4, a marker of nonpeptidergic, glial derived growth factor-responsive neurons. The findings link anti-apoptosis activities of Epo/EpoR/JAK2 in DRG neurons capable of inducing protracted pain states with reductions in pain behaviours, and therefore support a role for Epo therapy in the treatment of neuropathic pain.

Topics: Animals; Apoptosis; Behavior, Animal; Blotting, Western; Carbocyanines; Cell Count; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoietin; Female; Fluorescent Antibody Technique; Functional Laterality; Ganglia, Spinal; Glial Fibrillary Acidic Protein; Humans; Hyperalgesia; In Situ Nick-End Labeling; Janus Kinase 2; Microscopy, Confocal; Neurofilament Proteins; Pain; Pain Measurement; Pain Threshold; Peripheral Nervous System Diseases; Plant Lectins; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Ribosome Inactivating Proteins; Satellite Cells, Perineuronal; Time Factors

TNF is considered one of the inflammatory cytokines and contributes mainly to the generation of anemia of chronic disease (ACD). In nude mice TNF has been reported to impair iron metabolism and erythropoiesis, leading to anemia with a low serum iron and preserved iron stores. In this work, we established a murine model for ACD based on sublethal cecal ligation and puncture (CLP) with ensuing protracted peritonitis. Starting on Day 3 after CLP, a severe protracted depression of erythropoiesis in the bone marrow was noted. Two weeks after CLP, we observed a moderate normochromic anemia, low serum iron concentration, and preserved iron stores consistent with transient ACD. To determine whether TNF contributes to the development of ACD in vivo, we neutralized TNF after CLP shortly before and during the phase of most severe bone marrow depression to prevent anemia. Additionally, we studied TNF-deficient mice undergoing CLP. Two weeks after CLP, we determined red blood count, hemoglobin concentration, hematocrit, serum iron concentration, and iron stores in spleens of wild-type mice, TNF-deficient mice, and mice after neutralization of TNF. Neutralization of TNF after CLP could not prevent mice from contracting anemia. Accordingly, TNF-deficient mice developed anemia to the same extent as wild-type mice. Serum iron concentration was lowered and iron stores were overloaded in both TNF-deficient and wild-type mice after CLP. Our results clearly demonstrate that TNF is not a mediator of ACD in our model with transient anemia induced by protracted septic peritonitis.

Topics: Anemia; Animals; Antibodies, Blocking; Bone Marrow; Cecum; Disease Models, Animal; Erythrocyte Count; Erythropoiesis; Erythropoietin; Female; Hemoglobins; Iron; Ligation; Male; Mice; Mice, Inbred Strains; Mice, Knockout; Peritonitis; Sepsis; Tumor Necrosis Factor-alpha

The source of the erythropoietin (EPO) that circulates in the fetus is unknown although it is known that EPO does not cross the placenta and that fetal kidneys, liver, and placenta express the EPO gene. This study tested to what extent in vivo EPO secretion by the fetal kidneys and placenta can be demonstrated under normoxic and hypoxic conditions.. Renal arterial and venous EPO concentrations were determined in eight late-gestation chronically catheterized fetal sheep made progressively anemic by exchange transfusion with saline solution over 5 to 8 days. In a separate additional series of experiments, umbilical arterial and venous EPO concentrations were determined in nine normoxic fetuses and in nine fetuses subjected to 12 hours of hypoxia induced by lowering maternal-inspired oxygen content. Organ secretion rates were calculated as the product of plasma flow rate and the arteriovenous concentration differences.. Renal vein plasma EPO concentration was higher than the arterial concentration in 36 of 40 paired samples (P<.0001) by 16.3%+/-2.7% (mean+/-SE). This difference was concentration independent over a range of 12 to 4100 mU/mL. Renal EPO secretion rates were variable and averaged 155+/-105 mU/min when hematocrit was 31.3%+/-1.6% (n=5) and 1124+/-300 mU/min post-exchange transfusion when hematocrit was 15.6%+/-0.8% (n=12). In contrast, umbilical venous and arterial EPO concentrations (range 9-35 mU/mL), although highly correlated (r=0.94), were not different during normoxia (Po(2)=21.6+/-0.5 mm Hg, n=9). Under hypoxic conditions (Po(2)=15.6+/-0.4 mm Hg, n=9), umbilical vein EPO concentration (range 151-1245 mU/mL) was higher than arterial concentration (range 140-951 mU/mL) in eight of nine paired samples by 13.6%+/-3.3% (P<.01). Under these conditions, estimated umbilical EPO secretion rate was 27,900+/-11,500 mU/min.. Under nonanemic, normoxic basal conditions, the kidneys secreted EPO into the fetal circulation, whereas secretion by the placenta was not demonstrated. In the phlebotomy-induced fetal anemia experiments, the kidney demonstrated marked, progressive increases in the rate of EPO production. Similarly, in the fetal hypoxemia experiments, the placenta demonstrated progressive increases--albeit an order of magnitude greater than the kidneys--in EPO production rate. As an extension of these findings, we speculate that the hypoproliferative neonatal anemia that invariably occurs in the early weeks after birth is in part the result of loss of EPO production by the placenta.

Topics: Anemia; Animals; Disease Models, Animal; Erythropoiesis; Erythropoietin; Female; Fetal Blood; Hypoxia; Kidney; Oxygen; Oxygen Consumption; Placenta; Pregnancy; Pregnancy, Animal; Probability; Reference Values; Sheep, Domestic

Topics: Animals; Disease Models, Animal; Erythropoietin; Glucocorticoids; Humans; Models, Neurological; Neurons; Neuroprotective Agents; Rats; Recovery of Function; Spinal Cord Injuries

Existing animal models of anemia inadequately reflect the hematocrit usually present in chronic renal failure (CRF) patients and do not permit long-term treatment studies. The transgenic mouse strain 134.3LC (Epo-TAg(H)) displays a severe chronic anemia resembling that observed clinically during CRF, while displaying an active, normal life span. This phenotype makes it a particularly interesting mouse model for testing erythropoietin (Epo)-based gene transfer strategies.. Ex vivo gene therapy was employed to administer mouse Epo to homozygous anemic Epo-TAg(H) mice. Encapsulated C(2)C(12) myoblasts genetically engineered to secrete 163 IU mouse Epo/10(6) cells/day were subcutaneously transplanted on the dorsal flank of the mice. Efficacy of delivered Epo was monitored by weekly measurements of animal hematocrit.. Most treated homozygous Epo-TAg(H) mice displayed only a transient rise in hematocrit before eventually decreasing to levels as low as 3%. Administering the immunosuppressor anti-CD4+ monoclonal antibody (mAb) to homozygous Epo-TAg(H) mice, beginning at the time of implantation, permitted a rise in hematocrit that remained stable at elevated levels in cases of continued immunosuppression.. Mice having the T antigen insertion in both Epo alleles appeared to develop an immune response to the natural mouse Epo delivered by encapsulated cells. By preventing this reaction using immunosuppression, we demonstrate that encapsulated myoblasts can deliver therapeutic doses of mouse Epo systemically and restore hemopoiesis in a genetic model of severe anemia.

Topics: Anemia; Animals; Capsules; Cells, Cultured; Disease Models, Animal; Erythropoietin; Genetic Therapy; Hematocrit; Homozygote; Immunosuppression Therapy; Kidney Failure, Chronic; Mice; Mice, Transgenic; Myoblasts; Transplants; Treatment Outcome

In recent work we reported that systemically administered erythropoietin (EPO) crosses the blood-brain barrier and has protective effects in animal models of cerebral ischemia, brain trauma and in a rat model of experimental autoimmune encephalomyelitis (EAE). Here we characterize the effect of systemic EPO on the inflammatory component of actively induced, acute EAE in Lewis rats. Administration of EPO at doses of 500-5000 U/kg bw i.p., daily from day 3 after immunization with myelin basic protein (MBP), delayed the onset of EAE and decreased its clinical score at peak time (days 12-13). Immunohistochemical analysis of the spinal cord using anti-glial fibrillary acidic protein (GFAP) and anti-CD11b antibodies showed that EPO markedly diminished inflammation and glial activation/proliferation. EAE induced significant levels of TNF and IL-6 in the spinal cord, where IL-6 was maximum at the onset of the disease (day 10) and TNF at its peak (day 12). EPO delayed the increase of TNF levels, without altering their peak levels, and markedly reduced those of IL-6 suggesting that the decreased inflammation and clinical score may be in part upon attenuation of IL-6. On the other hand, EPO was without effect in a model of adjuvant-induced arthritis in Lewis rats, suggesting a specificity towards autoimmune demyelinating diseases. These data suggest that EPO might act as a protective cytokine in inflammatory pathologies of the CNS.

Topics: Animals; Anti-Inflammatory Agents; Arthritis, Experimental; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Erythropoietin; Female; Interleukin-6; Multiple Sclerosis; Rats; Rats, Inbred Lew; Spinal Cord; Tumor Necrosis Factor-alpha

Erythropoietin (EPO), a haemopoietic growth factor and a primary regulator of erythropoiesis, is widely used to treat anaemia in various chronic complications of rheumatoid arthritis (RA). Fibroblast-like cells, found in the pannus tissue of joints, are thought to contribute to the inflammatory pathology of RA. Thus for the current study we investigated the effects of recombinant human EPO (rHuEPO) on NO metabolism, using an interleukin-1beta (IL-1beta)-stimulated Swiss 3T3 fibroblast monolayer as a model for fibroblast activity in RA. The results show that, over 3 days, both alone and in combination with the pro-inflammatory cytokine IL-1beta (10 ng/ml), rHuEPO (25 micro-units/ml) induced significant production of nitrite in cell culture supernatants. This is an indicator of NO production by nitric oxide synthase (NOS), which is a well-documented mediator of metalloproteinase-mediated tissue remodelling in RA. It therefore appears that, through modulation of NOS-dependent NO production, rHuEPO may influence remodelling of connective tissue in RA, independently of its established erythropoietic role.

Topics: 3T3 Cells; Animals; Arthritis, Rheumatoid; Disease Models, Animal; Erythropoietin; Fibroblasts; Humans; Interleukin-1; Mice; Nitric Oxide; Nitrites; Recombinant Proteins; Synovial Membrane; Time Factors

Anemia in premature infants is extremely common. Precise quantitation of iron status and determination of iron incorporation into erythrocytes are important in monitoring therapy for anemia in premature infants, especially when treating with recombinant human erythropoietin (rhEPO). However, the traditional indices of the iron status have limited usefulness in this population. The goal of the current work is to develop an experimental animal model system that addresses the clinical issue relating to quantitation of iron delivery to erythrocytes. We first determined normal hematological values for nontreated, dam-suckled Sprague-Dawley rats by measuring markers of erythropoiesis and iron status during the first 12 postnatal days (PND). The experimental group of rats were administered parenteral rhEpo (430 IU.kg(-1). day(-1)) for 8 days (from PND 4 until PND 12) in the absence (rhEpo(-Fe)) or presence (rhEpo(+Fe)) of oral iron supplementation (6 mg.kg(-1).day(-1)). Rat pups receiving oral iron only (control(+Fe)) and pups that were sham fed with the orogastric tube (control(-Fe)) were included as controls. Hematological parameters were measured in blood and bone marrow. In a pattern similar to that seen in premature infants during the first 2 months of life, the levels of these hematopoietic markers were dynamic and changed during the first 12 PND. After challenging experimental animals with subcutaneous rhEpo, evidence of iron-deficient erythropoiesis was seen in the rhEpo(-Fe) group. Red blood cell levels and absolute reticulocyte counts were higher in both groups receiving rhEpo as compared with the controls. However, the rhEpo(-Fe) group experienced a lower hemoglobin level, a lower mean red cell volume, a greater red cell distribution width, and a higher zinc protoporphyrin/heme (ratio than the rhEpo(+Fe) group. The neonatal rat is an excellent model of iron-deficient erythropoiesis and will be useful in designing future mechanistic studies examining the interplay between iron and erythropoiesis in the anemic, iron-challenged premature neonate.

Topics: Aging; Animals; Animals, Newborn; Disease Models, Animal; Erythrocyte Count; Erythrocyte Indices; Erythrocytes; Erythropoiesis; Erythropoietin; Hemoglobins; Humans; Iron; Iron Deficiencies; Platelet Count; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reticulocyte Count

The study was designed to examine the impact of anemia on the antitumor efficacy of photodynamic therapy (PDT) in a murine colon-26 adenocarcinoma model syngeneic with BALB/c mice.. Acute hemolytic anemia was induced by a single i.p. injection of phenylhydrazine hydrochloride (150 mg/kg). Anemia induced by i.p. administration of carboplatin (100 mg/kg) was corrected by s.c. treatment with recombinant human erythropoietin (1000 units/kg/day). The effectiveness of PDT (10 mg/kg Photofrin, 150 J/cm2 laser dose) was evaluated by measurements of the footpad edema and tumor volume. All of the RBC-related parameters were measured from the tail vein.. Phenylhydrazine hydrochloride injection resulted in a blunted response of normal tissues to Photofrin-mediated PDT-induced edema formation. Similarly, the antitumor response in mice with hemolytic anemia was nearly completely abrogated. The antitumor effectiveness of PDT was also significantly diminished in a more realistic clinical situation when anemia was induced by administration of carboplatin. Importantly, administration of recombinant human erythropoietin completely restored the sensitivity of the tumor to PDT in carboplatin-treated mice.. These results indicate that anemia can negatively influence the therapeutic effectiveness of PDT. For optimal antitumor response anemia should be corrected before PDT procedure.

Topics: Acute Disease; Anemia; Anemia, Hemolytic; Animals; Antineoplastic Agents; Carboplatin; Disease Models, Animal; Erythropoietin; Humans; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms, Experimental; Phenylhydrazines; Photochemotherapy; Recombinant Proteins; Tumor Cells, Cultured

Erythropoietin (EPO) functions as a tissue-protective cytokine in addition to its crucial hormonal role in red cell production. In the brain, for example, EPO and its receptor are locally produced, are modulated by metabolic stressors, and provide neuroprotective and antiinflammatory functions. We have previously shown that recombinant human EPO (rhEPO) administered within the systemic circulation enters the brain and is neuroprotective. At present, it is unknown whether rhEPO can also improve recovery after traumatic injury of the spinal cord. To evaluate whether rhEPO improves functional outcome if administered after cord injury, two rodent models were evaluated. First, a moderate compression of 0.6 N was produced by application of an aneurysm clip at level T3 for 1 min. RhEPO (1,000 units per kg of body weight i.p.) administered immediately after release of compression was associated with partial recovery of motor function within 12 h after injury, which was nearly complete by 28 days. In contrast, saline-treated animals exhibited only poor recovery. In the second model used, rhEPO administration (5,000 units per kg of body weight i.p. given once 1 h after injury) also produced a superior recovery of function compared with saline-treated controls after a contusion of 1 N at level T9. In this model of more severe spinal cord injury, secondary inflammation was also markedly attenuated by rhEPO administration and associated with reduced cavitation within the cord. These observations suggest that rhEPO provides early recovery of function, especially after spinal cord compression, as well as longer-latency neuroprotective, antiinflammatory and antiapoptotic functions.

Topics: Animals; Apoptosis; Disease Models, Animal; Erythropoietin; Female; Humans; Oligodendroglia; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Spinal Cord Injuries; Time Factors

Mutations causing truncations of the cytoplasmic domain of the human erythropoietin receptor (EPOR) result in a dominantly inherited disorder-primary familial congenital polycythemia. This disorder is characterized by increased numbers of erythrocytes (polycythemia) and by in vitro hypersensitivity of erythroid precursors to erythropoietin. The consequences of EPOR truncation in nonerythroid tissues are unknown. We replaced the murine EPOR gene with a wild-type human EPOR gene and a mutant human EPOR gene that we initially identified in a patient with polycythemia. This mutation leads to an EPOR truncated after the first tyrosine residue of the intracellular domain. Mice heterozygous for this mutant allele and a wild-type human EPOR allele mimicked the human disorder. Interestingly, mice that were homozygous for the mutant human allele were severely polycythemic but viable. Our results provide a model for functional studies of EPOR-triggered signaling pathways in erythropoiesis. These animals can now be used to investigate the molecular pathophysiology of this gain-of-function EPOR mutation in erythroid tissue and in those nonerythroid tissues that express EPOR.

Topics: Animals; Disease Models, Animal; Erythroid Precursor Cells; Erythropoietin; Heterozygote; Mice; Mice, Transgenic; Polycythemia; Receptors, Erythropoietin; Recombination, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Sequence Deletion; Stem Cells; Transcription, Genetic

Erythropoietin (EPO) promotes neuronal survival after hypoxia and other metabolic insults by largely unknown mechanisms. Apoptosis and necrosis have been proposed as mechanisms of cellular demise, and either could be the target of actions of EPO. This study evaluates whether antiapoptotic mechanisms can account for the neuroprotective actions of EPO. Systemic administration of EPO (5,000 units/kg of body weight, i.p.) after middle-cerebral artery occlusion in rats dramatically reduces the volume of infarction 24 h later, in concert with an almost complete reduction in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling of neurons within the ischemic penumbra. In both pure and mixed neuronal cultures, EPO (0.1--10 units/ml) also inhibits apoptosis induced by serum deprivation or kainic acid exposure. Protection requires pretreatment, consistent with the induction of a gene expression program, and is sustained for 3 days without the continued presence of EPO. EPO (0.3 units/ml) also protects hippocampal neurons against hypoxia-induced neuronal death through activation of extracellular signal-regulated kinases and protein kinase Akt-1/protein kinase B. The action of EPO is not limited to directly promoting cell survival, as EPO is trophic but not mitogenic in cultured neuronal cells. These data suggest that inhibition of neuronal apoptosis underlies short latency protective effects of EPO after cerebral ischemia and other brain injuries. The neurotrophic actions suggest there may be longer-latency effects as well. Evaluation of EPO, a compound established as clinically safe, as neuroprotective therapy in acute brain injury is further supported.

Topics: Animals; Apoptosis; Brain Ischemia; Cells, Cultured; Disease Models, Animal; Erythropoietin; In Situ Nick-End Labeling; Male; Mice; Motor Neurons; Nerve Growth Factors; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Stress, Physiological

A new intramuscular DNA electrotransfer method for erythropoietin (EPO) expression was evaluated in the natural mouse model of human beta-thalassemia (Hbb-thal1) in terms of its ability to reverse the anemia and improve the thalassemic features of erythrocytes.. Intramuscular injection of small amounts of a plasmid encoding mouse EPO, immediately followed by controlled electric pulses, was used.. This procedure induced very high hematocrit levels in beta-thalassemic mice compared to nonelectrotransferred mice. The hematocrit increase was dose dependent, still increased 4 months after injection of plasmid DNA, and associated with a high transgenic EPO blood level in all mice (up to 2500 mU/mL of plasma). EPO gene electrotransfer not only led to a long-lasting and dose-dependent increase in the hematocrit but also to a 100% increase in the lifespan of erythrocytes of thalassemic mice. This was related to a nearly complete reestablishment of alpha/beta globin chain balance, as demonstrated by a marked decrease in unpaired alpha globin chain. Eight months after the first electrotransfer of pCMV-mEPO plasmid, reinjection of the same construct raised the hematocrit to a level close to that observed following the first electrotransfer.. This is the first description of the use of plasmid DNA to achieve long-term improvement in a mouse model of a human genetic disorder.

Topics: Animals; beta-Thalassemia; Cytomegalovirus; Disease Models, Animal; DNA, Recombinant; Electroporation; Erythropoietin; Female; Gene Deletion; Genetic Therapy; Genetic Vectors; Globins; Hematocrit; Injections, Intramuscular; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Promoter Regions, Genetic; Recombinant Fusion Proteins; Transgenes

Mechanisms governing the induction of effective erythropoiesis in response to erythropoietin (Epo) oversecretion have been investigated in beta thalassemic C57Bl/6(Hbbth) mice. Naked DNA encoding an expression vector for mouse Epo was introduced into skeletal muscles by electrotransfer. A transient increase of serum Epo concentrations with a proportional augmentation of hematocrit values was observed. Various parameters relevant to beta thalassemia were surveyed in blood samples taken before treatment, at the peak of Epo secretion, and when the phenotype reverted to anemia. We measured globin messenger RNA (mRNA) levels in reticulocytes by real-time quantitative polymerase chain reaction, globin chain synthesis levels, and several indicators of erythrocyte membrane quality, including bound alpha chains, bound immunoglobulins, main protein components, and iron compartmentalization. Data indicated that high serum Epo levels primarily affect betaminor-globin mRNA accumulation in reticulocytes. Other changes subsequent to intense Epo stimulation, like increased betaminor/alpha-globin chain synthesis ratio, reduced levels of alpha chains and immunoglobulins bound to membranes, improved spectrin/band 3 ratio, increased red blood cell survival, and improved erythropoiesis appeared as consequences of increased betaminor-globin mRNA levels. This conclusion is consistent with models postulating that intense Epo stimulation induces the expansion and differentiation of erythroid progenitors committed to fetal erythropoiesis. Although phenotypic correction was partial in mice, and comparable achievements will probably be more difficult to obtain in humans, naked DNA electrotransfer may provide a safe and low-cost method for reassessing the potentials of Epo as an inducer of fetal erythropoiesis reactivation in patients with beta thalassemia.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; beta-Thalassemia; Cell Compartmentation; Cell Differentiation; Disease Models, Animal; DNA, Complementary; Electroporation; Erythrocytes; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Genetic Complementation Test; Globins; Injections, Intramuscular; Iron; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Phenotype; Polymerase Chain Reaction; Recombinant Fusion Proteins; Reticulocytes; RNA, Messenger; Spectrin; Transfection

Recombinant human erythropoietin (rHuEpo) has been used successfully in the treatment of cancer-related anemia. Clinical observations with several patients with multiple-myeloma treated with rHuEpo has shown, in addition to the improved quality of life, a longer survival than expected, considering the poor prognostic features of these patients. Based on these observations, we evaluated the potential biological effects of rHuEpo on the course of tumor progression by using murine myeloma models (MOPC-315-IgAlambda(2) and 5T33 MM-IgG(2b)). Here we report that daily treatment of MOPC-315 tumor-bearing mice with rHuEpo for several weeks induced complete tumor regression in 30-60% of mice. All regressors that were rechallenged with tumor cells rejected tumor growth, and this resistance was tumor specific. The Epo-triggered therapeutic effect was shown to be attributed to a T cell-mediated mechanism. Serum Ig analysis indicated a reduction in MOPC-315 lambda light chain in regressor mice. Intradermal inoculation of 5T33 MM tumor cells followed by Epo treatment induced tumor regression in 60% of mice. The common clinical manifestation of myeloma bone disease in patients with multiple-myeloma was established in these myeloma models. Epo administration to these tumor-bearing mice markedly prolonged their survival and reduced mortality. Therefore, erythropoietin seems to act as an antitumor therapeutic agent in addition to its red blood cell-stimulating activity.

Topics: Animals; Blotting, Western; Disease Models, Animal; Disease Progression; Erythropoietin; Female; Hemoglobins; Humans; Immunoglobulin lambda-Chains; Immunophenotyping; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred Strains; Multiple Myeloma; Remission Induction; Survival Rate; T-Lymphocytes

Acute cerebral vasoconstriction and subsequent brain ischemia, often occurring in the early phase of subarachnoid hemorrhage (SAH), are critical problems in the management of patients affected by ruptured intracranial aneurysms. It is known that nitric oxide (NO) decreases during SAH with impairment of cerebrovascular relaxation, and glutamate is mainly involved in the consequent brain ischemic damage. Recently, erythropoietin (EPO) has shown to exert a neuroprotective effect during cerebral ischemia by enhancing the NO system activity. In the present study the effect of systemic administration of recombinant human erythropoietin (rHuEPO) has been investigated in a rabbit model of SAH.. Thirty-two rabbits were assigned to four groups: 1) Control; 2) SAH; 3) SAH plus placebo; 4) SAH plus rHuEPO. Experimental SAH was induced by injecting autologous blood into the cisterna magna. rHuEPO, at a dose of 1000 IU/kg, and placebo were given 5 minutes after SAH. Administration was repeated three times during 24 hours. The animals were killed 24 hours after SAH by a perfusion-fixation method. Luminal cross-sections of the basilar artery were measured by computer-assisted morphometric analysis. Ischemic injury was histologically evaluated by analysis of the frequency of ischemia-induced damaged cortical neurons.. Administration of rHuEPO significantly reversed the vasoconstriction of the basilar artery in Group 4 compared with the other groups (p<0.05). Histological examination showed a significant reduction in total damaged neurons count in Group 4 compared with the other groups (p<0.01).. These results suggest that rHuEPO is effective in attenuating acute cerebral vasoconstriction and ischemic brain injury following experimental SAH.

Topics: Animals; Brain Ischemia; Cerebrovascular Circulation; Disease Models, Animal; Erythropoietin; Glutamic Acid; Humans; Male; Neuroprotective Agents; Nitric Oxide; Rabbits; Recombinant Proteins; Subarachnoid Hemorrhage; Vasoconstriction

In the present study we examined the effect of recombinant human erythropoietin (rhEPO) on intestinal malondialdehyde (MDA) as an index of lipid peroxidation, related to iron-catalysed free radical reaction and platelet-activating factor (PAF) synthesis in the experimental model of necrotizing enterocolitis (NEC). Three groups, each consisting of eight 1-day-old Wistar albino rat pups, were studied; Group 1, hypoxia-reoxygenation; Group 2, hypoxia-reoxygenation and rhEPO pretreatment; Group 3, control. rhEPO was given 750 U/kg/week by intraperitoneal injection three times a week for 2 weeks. On day 15th of life, hypoxia was induced by placing rat pups in a 100% CO2 chamber for 5 min. After hypoxia, the rat pups were reoxygenated for 10 min with 100% oxygen and returned to their mothers. All pups were killed at 4h following hypoxia-reoxygenation. The abdomen was opened and representative samples of injured areas were taken for histopathologic examination. MDA and PAF levels were determined in the intestine. Significantly increased intestinal MDA content was found in Group 1 rat pups compared to Group 2 and Group 3 pups (p < 0.001 and p < 0.001, respectively). However, PAF concentrations were highly elevated in the intestine of Group 1 and Group 2 pups (p>0.05) when compared to the intestine of Group 3 pups (p < 0.001 and p < 0.001, respectively). Histopathologic findings did not differ between Groups 1 and 2. The present study demonstrates that oxygen-derived free radicals and PAF are involved in the pathophysiological mechanism of the development of NEC. This study also shows that administration of rhEPO significantly decreases lipid peroxidation; however, PAF generation was not inhibited in hypoxia-induced bowel necrosis.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Enterocolitis, Necrotizing; Erythropoietin; Female; Injections, Intraperitoneal; Lipid Peroxidation; Male; Malondialdehyde; Platelet Activating Factor; Probability; Rats; Rats, Wistar; Recombinant Proteins; Reference Values; Sensitivity and Specificity; Statistics, Nonparametric

Long-term loss of fetal blood can occur with fetomaternal hemorrhage, vasoprevia, or placental previa. Our objective was to determine the effects of progressive fetal blood loss over 10 days on fetal plasma erythropoietin (EPO) concentration and its relationship to arterial PO(2), hematocrit, and the volume of blood loss. Late-gestation fetal sheep (n = 8) were hemorrhaged daily at a rate of 1 ml/min over 10 days. The extent of hemorrhage differed in each fetus and ranged from 30 to 80 ml/day, with the cumulative volume removed ranging from 78 to 236 ml/kg estimated fetal weight. Four fetuses served as time controls. EPO concentration measurements were by radioimmunoassay. Statistical analyses included regression, correlation, and analysis of variance. We found that EPO and arterial PO(2) were unchanged until the cumulative hemorrhage volume exceeded 20-40 ml/kg. Once this threshold was exceeded, plasma EPO concentration increased progressively throughout the study and averaged 14.3 +/- 3.2 times basal values on day 10. EPO concentration, arterial PO(2), and hematocrit changes were related curvilinearly to cumulative hemorrhage volume (P < 0.01), whereas the relationship between plasma EPO and arterial PO(2) was log linear (P < 0.001). We conclude that 1) fetal plasma EPO concentration and arterial PO(2) are insensitive to a slow, mild-to-moderate blood loss over several days; 2) unlike the rapid return of EPO to normal within 48 h after acute hemorrhage, fetal EPO concentration undergoes a progressive increase with moderate-to-severe blood loss over several days; 3) the long-term hemorrhage-induced changes in EPO are best correlated with arterial PO(2); and 4) the fetal EPO response to hemorrhage does not appear to be limited by the fetus's ability to produce EPO.

Topics: Animals; Arteries; Blood Gas Analysis; Blood Volume; Chronic Disease; Disease Models, Animal; Erythropoietin; Female; Fetomaternal Transfusion; Fetus; Hematocrit; Iron; Multivariate Analysis; Oxygen; Pregnancy; Sheep

We developed a rodent model of noninfectious systemic inflammation to examine the pathogenesis of the associated anemia of chronic disorders (ACD), to evaluate the similarity of this ACD model to human ACD, and to evaluate the potential efficacy of novel erythropoiesis stimulating protein (darbepoetin alfa) as an ACD therapy.. Lewis rats were immunized with peptidoglycan-polysaccharide polymers (PG-APS), the chronic inflammation and associated ACD were characterized, and the effects of darbepoetin alfa treatment on complete blood counts (CBC), red blood cell (RBC) indices, and iron metabolism were analyzed weekly.. Acutely inflamed rats had reduced peripheral blood (PB) RBC counts and hemoglobin (Hb) concentrations and increased reticulocyte counts. PB RBC numbers normalized during chronic inflammation, but RBC remained hypochromic and microcytic. Consequently, the rats remained chronically anemic. Anemic rats had fluctuating serum erythropoietin (EPO) concentrations, but mean EPO concentrations never varied significantly from baseline control levels. Histology of anemic rat spleen sections revealed reticuloendothelial siderosis. Total serum iron concentrations were chronically low. Peritoneal exudate cells (PEC) isolated from anemic rats and stimulated with PG-APS in vitro produced more interleukin (IL)-1alpha and interferon (IFN)-gamma, and significantly more tumor necrosis factor (TNF)-alpha and IL-10 than control cultures. Darbepoetin alfa restored Hb concentrations to baseline levels within 2 to 7 weeks, depending on dosage. A refined treatment strategy restored Hb to baseline and maintained those levels with reduced dosing.. ACD in this rodent model closely replicates human ACD. Darbepoetin alfa treatment reversed ACD in this model by increasing RBC production and RBC hemoglobinization while reducing siderosis and hypoferremia.

Topics: Anemia; Animals; Ascitic Fluid; Blood Cell Count; Chronic Disease; Disease Models, Animal; Erythrocyte Count; Erythropoietin; Female; Hemoglobins; Inflammation; Mononuclear Phagocyte System; Peptidoglycan; Polysaccharides; Rats; Rats, Inbred Lew; Reticulocyte Count; Siderosis

There is limited information available on which to base decisions regarding red blood cell (RBC) transfusion treatment in anemic newborn infants. Using a conscious newborn lamb model of progressive anemia, we sought to identify accessible metabolic and cardiovascular measures of hypoxia that might provide guidance in the management of anemic infants. We hypothesized that severe phlebotomy-induced isovolemic anemia and its reversal after RBC transfusion result in a defined pattern of adaptive responses. Anemia was produced over 2 days by serial phlebotomy (with plasma replacement) to Hb levels of 30-40 g/l. During the ensuing 2 days, Hb was restored to pretransfusion baseline levels by repeated RBC transfusion. Area-under-the-curve methodology was utilized for defining the Hb level at which individual study variables demonstrated significant change. Significant reciprocal changes (P < 0.05) of equivalent magnitude were observed during the phlebotomy and transfusion phases for cardiac output, plasma erythropoietin (Epo) concentration, oxygen extraction ratio, oxygen delivery, venous oxygen saturation, and blood lactate concentration. No significant change was observed in resting oxygen consumption. Cardiac output and plasma Epo concentration increased at Hb levels <75 g/l, oxygen delivery and oxygen extraction ratio decreased at Hb levels <60 g/l, and venous oxygen saturation decreased and blood lactate concentration increased at Hb levels <55 g/l. We speculate that plasma Epo and blood lactate concentrations may be useful measures of clinically significant anemia in infants and may indicate when an infant might benefit from a RBC transfusion.

Topics: Acclimatization; Anemia; Animals; Animals, Newborn; Disease Models, Animal; Erythrocyte Transfusion; Erythropoietin; Heart Rate; Hemodynamics; Humans; Infant, Newborn; Lactates; Oxygen; Partial Pressure; Phlebotomy; Sheep; Stroke Volume; Time Factors; Vascular Resistance

In addition to its better-known hemopoietic action, erythropoietin (Epo) has neurotrophic properties and neuroprotective effects in some models of hypoxic-ischemic injury. To define further the cellular mechanisms underlying neuroprotection by Epo, we studied the effects of Epo on hypoxia with glucose deprivation in cultured rat cortical neurons and astroglia and on exposure to excitotoxins in cultured rat cortical neurons. Epo (30 pM) reduced neuronal, but not astroglial, cell death from hypoxia with glucose deprivation, and also attenuated the neurotoxic effect of (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), but not other excitotoxins. Epo appears to protect against cerebral ischemia through a direct effect on neurons that may be mediated in part by AMPA receptors.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Astrocytes; Cell Culture Techniques; Cell Survival; Cerebral Cortex; Disease Models, Animal; Drug Interactions; Embryo, Mammalian; Erythropoietin; Glucose; Hypoxia, Brain; Neurons; Neuroprotective Agents; Neurotoxins; Rats; Rats, Sprague-Dawley

We have investigated the efficacy of a gene transfer strategy based on plasmid DNA electroinjection for the correction of anemia associated with renal failure. An expression plasmid encoding the rat erythropoietin (EPO) cDNA under the control of the CMV promoter as constructed and utilized for this work. Electroinjection of pCMV/rEPO in different rat muscles yielded sustained and long-term EPO production and secretion. The muscle-produced EPO corrected the anemia in five of six nephrectomized rats, used as a model of renal failure. The efficiency of muscle transduction was comparable in rats and mice injected with equivalent amounts of DNA per kilogram of body weight. These results demonstrate that gene electrotransfer can be applied to produce therapeutically significant levels of erythropoietin in chronic renal failure.

Topics: Anemia; Animals; Cytomegalovirus; Disease Models, Animal; Erythropoietin; Gene Transfer Techniques; Genetic Therapy; Hematocrit; Injections; Kidney Failure, Chronic; Mice; Mice, Inbred BALB C; Muscle, Skeletal; Nephrectomy; Plasmids; Promoter Regions, Genetic; Rabbits; Rats; Rats, Sprague-Dawley; Transduction, Genetic

The involvement of proximal kidney tubules in erythropoietin production and/or in its control has been a subject of controversy among researchers. The present study addresses the problem whether acute tubular impairment affects erythropoietin production and thus alters the basic hematological parameters. For this purpose rats with hypobaric hypoxia-stimulated (42.55 kPa) erythropoesis were studied and the rat model of gentamicin-induced (50 mg/kg daily for 15 days) tubular damage was employed. According to the study protocol 4 different groups of rats were used: control rats, rats exposed to hypobaric hypoxia, rats treated with gentamicin prior to hypoxic exposure and finally rats given a dose of 50 U human recombinant erythropoietin for 2 days following treatment with gentamicin. The evaluated hematological parameters included: hemoglobin and hematocrit levels, reticulocyte count and plasma creatinine concentration. Histological analysis of kidney sections was also used. INCSTAR (USA) immunoassay was employed to determine plasma erythropoietin level. Evidence (histological and laboratory-elevated plasma creatinine) of distinct tubular lesions was found in rats injected with gentamicin. Hemoglobin and hematocrit levels were decreased in rats treated with gentamicin but a rise in reticulocyte count was seen on day 3 after hypoxic exposure--39@1000 +/- 10 vs. 5@1000 +/- 1.3 on day 0 (p < 0.001). Reduced reticulocyte counts on day 0 were not seen only in rats injected with human recombinant erythropoietin--36.33@1000 (p < 0.01). Hypoxia-induced increase in plasma erythropoietin was inhibited in animals treated with gentamicin--15.3 mU/ml (on day 0). Both hypoxic exposure and erythropoietin administration were associated with a rise in plasma erythropoietin on day 0--40.5 +/- 4.4 mU/ml and 42.7 +/- 4.1 mU/ml respectively, which normalised on day 3. The results of the study support the hypothesis that the erythropoietin response in rats after stimulation is related to the functional condition of the proximal kidney tubules.

Topics: Anemia; Animals; Disease Models, Animal; Erythropoiesis; Erythropoietin; Gentamicins; Hematologic Tests; Hypoxia; Kidney Tubular Necrosis, Acute; Kidney Tubules, Proximal; Male; Rats; Rats, Wistar; Recombinant Proteins

To investigate the effects of erythropoietin (EPO) gene transfer into skeleton muscle mediated by electroporation on renal anemia.. Renal failure models were created by adenine-excessive diet (150 mg per day). Plasmid vectors encoding EPO were transferred by electroporation after 80 days when mean blood urea nitrogen level (BUN) had increased from 3.4 mmol/L +/- 1.3 mmol/L to 18.1 mmol/L +/- 4.1 mmol/L and the hematocrit had decreased from 45.6% +/- 2.1% to 25.4% +/- 3.7%. During the process of treatment, adenine-excessive diet was given. Hb, HCT, BUN and Cre in blood were tested by automatic analyzer; EPO level in the serum was tested by EPO ELISA kit, EPO gene expression was proved by RT/PCR. The survival rate was calculated.. Hematocrit increased to 34.4% +/- 7.5% only 7 days after the treatment and reached 91.4% of normal level (46% +/- 2%) after 5 weeks. The survival rate of test models after 9 weeks was 77.8%, which was remarkably higher than that of controls (16.7%). mRNA level of EPO gene expression was indicated by RT/PCR.. Electroporation can increase the efficiency of EPO gene transfer and thus greatly improve hematocrit in mice and prolong the life-span of chronic renal anemia models. This method can provide a new way for treatment of EPO-responsive anemias.

Topics: Adenine; Anemia; Animals; Disease Models, Animal; Electroporation; Erythropoietin; Gene Expression; Gene Transfer Techniques; Hematocrit; Kidney; Muscle, Skeletal; Rats; Rats, Wistar; Renal Insufficiency

Topics: Animals; Aorta; Aortic Diseases; Arteriosclerosis; Cholesterol Esters; Disease Models, Animal; Disease Progression; Erythropoietin; Genetic Predisposition to Disease; Humans; Hyperlipidemias; Lipids; Male; Rabbits; Recombinant Proteins

The in-vivo survival of 51Cr-labelled murine red blood cells (RBCs) loaded with recombinant human erythropoietin (rhEpo-RBCs) was slightly lower than that of normal RBCs. Intravenous administration to normal mice of the encapsulated rhEpo shows the pharmacokinetic bicompartmental profile typical of the free rhEpo. Distribution and elimination half-life values for the RBC-entrapped rhEpo were no longer than those for the free protein. The area under the curve value was significantly increased for rhEpo-RBCs. Hypertransfused polycythaemic mice were evaluated as an adequate animal model to study the in vivo biological activity of encapsulated rhEpo. rhEpo-RBCs stimulate the erythropoiesis of polycythaemic mice in a linear dose-radio-iron incorporation response relationship. These results suggest that rhEpo-RBCs may behave as an alternative to the administration of free rhEpo in the clinical field.

Topics: Animals; Cell Division; Disease Models, Animal; Drug Carriers; Drug Delivery Systems; Erythrocyte Aging; Erythrocytes; Erythropoietin; Humans; Mice; Mice, Inbred Strains; Polycythemia; Recombinant Proteins

beta thalassemia (beta thal) in DBA/2J mice is a consequence of the spontaneous and complete deletion of the beta major globin gene. Homozygous beta thal mice have clinical and biological features similar to those observed in human beta thal intermedia. Erythrocytes in human beta thal are characterized by a relative cell dehydration and reduced K+ content. The role of this erythrocyte dehydration in the reduced erythrocyte survival, which typifies the disease, has not previously been evaluated. We examined for 1 month the effects on the anemia and the erythrocyte characteristics of beta thal mice of daily treatment with either clotrimazole (CLT), an inhibitor of red blood cell (RBC) dehydration via the Gardos channel, or human recombinant erythropoietin (r-HuEPO), or hydroxyurea (HU). The use of either r-HuEPO or HU induced a significant increase in hemoglobin (Hb), hematocrit (Hct), erythrocyte K+ and a decrease in percent reticulocytes, suggesting improved erythrocyte survival. CLT alone decreased only mean corpuscular hemoglobin concentration (MCHC) and cell density and increased cell K+. Thus, though the Gardos channel plays a major role in cell dehydration of murine beta thal erythrocyte survival. Combination therapy with r-HuEPO plus HU produced no incremental benefit beyond those of single drug therapy. However, addition of CLT to r-HuEPO, to HU, or to combined r-HuEPO plus HU led to statistically significant increase in Hb, Hct, and erythrocyte K+ compared with any of the regimens without CLT. These results suggest that CLT not only inhibits erythrocyte dehydration, but also potentiates the erythropoietic and cellular survival responses to r-HuEPO and HU.

Topics: Animals; beta-Thalassemia; Body Water; Calcimycin; Calcium; Calcium Channel Blockers; Chlorides; Clotrimazole; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Erythrocyte Aging; Erythrocyte Count; Erythrocyte Deformability; Erythrocytes, Abnormal; Erythropoietin; Female; Gene Deletion; Globins; Hematocrit; Humans; Hydroxyurea; Intracellular Fluid; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Mutant Strains; Potassium; Potassium Channels; Recombinant Proteins; Reticulocytes; Rubidium

Regular administration of recombinant erythropoietin (EPO) is frequently complicated by a rise in arterial blood pressure. We therefore asked if prolonged stimulation of endogenous EPO production has the same effect. To this end, male Sprague-Dawley rats were placed in a hypobaric chamber (390 mm Hg) for 24 days. The control (NL) group was placed in the chamber at normobaric condition. The animals were then removed from the chamber and monitored through day 108. Plasma EPO peaked within 24 hours and returned to baseline by day 7 and remained so thereafter. Hematocrit rose steadily during the hypoxic phase and declined steadily during the normobaric phase, reaching the baseline on day 45. This was accompanied by parallel changes in erythrocyte mass and blood volume. The rise in hematocrit during hypoxia was accompanied by a parallel rise in blood pressure which peaked on day 24. Despite the restoration of normal hematocrit, erythrocyte mass and blood volume following resumption of normoxia, blood pressure remained elevated throughout the observation period. To dissect the role of hypoxia from that of the associated rise in hematocrit, the experiments were repeated using a group of rats whose hematocrits were kept constant by repeated phlebotomies. These animals exhibited a sustained rise in blood pressure identical to that found in the original group. Thus, prolonged hypobaric hypoxia leads to a severe hematocrit-independent systemic hypertension (HTN) that persists long after the restoration of normoxia. Given the transient nature of the rise in its plasma concentration, endogenous EPO does not appear to play a role in the genesis of the observed systemic HTN. The authors believe that this animal model can be used for future studies of the mechanism, consequences and treatment of acquired HTN.

Topics: Air Pressure; Animals; Arginine Vasopressin; Atrial Natriuretic Factor; Blood Pressure; Blood Volume; Disease Models, Animal; Erythropoietin; Hematocrit; Hypertension; Hypoxia; Male; Rats; Rats, Sprague-Dawley; Time Factors

Iron is an important catalyst for free oxygen radicals and lipid peroxidation reactions which may play a role in the pathogenesis of several diseases in premature infants. During the early neonatal period, extracellular iron is available in excessive amounts. We hypothesized that administration of erythropoietin (EPO) mobilizes iron from plasma and inhibits iron-catalyzed reactions. To evaluate this hypothesis, recombinant human EPO (rhEPO) was administered s.c. to premature rabbits delivered at 29-d gestation: one group was kept in room air (RA) and the other in a 100% oxygen environment. Within each group, the animals were randomized to receive placebo or rhEPO at 400 or at 800 U/kg on d 0 and 2 of life. On d 3 or 4, plasma iron and iron saturation of transferrin were assessed. Lipid peroxidation was analyzed in plasma and bronchoalveolar lavage fluid (BAL). Nonsedimentable protein (NSP) and phospholipid content were measured in BAL. Erythropoiesis was evaluated in liver and bone marrow. Treatment with rhEPO decreased plasma iron, decreased iron saturation of transferrin, increased reticulocytes, and increased erythropoiesis in liver and bone marrow in both RA and hyperoxia group. Oxygen exposure increased NSP in BAL and decreased the ability of BAL to inhibit lipid peroxidation as measured by malondialdehyde (MDA) generation compared with RA exposure. In O2-exposed animals, EPO treatment increased the ability of both plasma (EPO 800) and BAL (EPO 400 and 800) to inhibit lipid peroxidation and decreased NSP in BAL (EPO 400). In addition, rhEPO treatment decreased alveolar thickening and proteinaceous exudate in the hyperoxia group. We propose that by stimulating erythropoiesis, rhEPO mobilizes non-heme iron and decreases oxidant injury that depends on the availability of transient metal.

Topics: Animals; Animals, Newborn; Antioxidants; Catalysis; Disease Models, Animal; Drug Evaluation, Preclinical; Erythropoiesis; Erythropoietin; Free Radicals; Gestational Age; Humans; Hyperoxia; Infant, Newborn; Infant, Premature, Diseases; Iron; Lung Diseases; Oxidative Stress; Rabbits; Recombinant Proteins

Chronic renal failure (CRF) is associated with a hyporegenerative anemia, which is caused primarily by inadequate production of erythropoietin (EPO) by the diseased kidneys and is responsive to exogenous EPO administration. Little is known about compensatory mechanisms that might supervene in anemia with low levels of EPO. Multiple investigations in vitro suggest an important role for insulin-like growth factor-1 (IGF-1) as well as EPO in erythropoiesis. Recently, both EPO and IGF-1 in vitro have been found to stimulate erythroid colony forming units in the mouse. However, no studies have examined the effect of IGF-1, singly and in combination with EPO, in CRF in vivo. This study examined mice with surgically-induced renal failure of six weeks duration that were treated for three weeks with the combination of subtherapeutic doses of both EPO and IGF-1. The single administration of each cytokine caused no significant change in hemoglobin in all CRF mice. In marked contrast the combined administration of the two cytokines produced a striking rise in hemoglobin, resulting in anemia correction in the majority of animals. The response to the combination therapy was comparable to the maximal response obtained with a single EPO dose (10 U) in a dose-finding study. Although the data are limited to utilizing one dose of each cytokine and one preparation of IGF-1, the large increase in hemoglobin observed with the combination therapy indicates that these two cytokines work in concert to stimulate erythroid precursors in CRF. In addition, untreated CRF mice showed markedly increased serum levels of low molecular weight binding proteins for IGF-1, potentially reducing the bioavailability of IGF-1. These findings taken together suggest that the anemia of CRF may represent both an EPO and a functional IGF-1 deficient state.

Topics: Anemia; Animals; Blotting, Western; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Therapy, Combination; Erythropoietin; Female; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Kidney Failure, Chronic; Mice; Mice, Inbred C57BL

The anemia of chronic renal failure (CRF) is largely due to decreased production of erythropoietin (EPO) by the kidney. A small amount of EPO also originates from extra-renal sources, and this would be expected to assume a more important role in maintaining erythropoiesis when renal production is impaired. In this study, we examined the production of EPO mRNA by RT-PCR in kidney, liver, and bone marrow tissues isolated from normal mice, mice rendered acutely anemic by phlebotomy, and from mice with surgically induced CRF. The induction of acute anemia results in an expected increase in the expression of EPO mRNA in renal and hepatic tissue. In contrast, while the expression of EPO mRNA was expectedly reduced in the kidney from CRF mice, it was completely absent in the liver of these same animals. EPO mRNA expression was also absent in the bone marrow in both states of acute anemia and CRF. These results show that CRF can directly or indirectly can suppress the extrarenal production of EPO by the liver and that this effect may further aggravate the anemia of CRF.

Topics: Anemia; Animals; Bone Marrow; Disease Models, Animal; Erythropoietin; Female; Kidney; Kidney Failure, Chronic; Liver; Mice; Mice, Inbred C57BL; RNA, Messenger

An elevation of blood pressure is observed in approximately 30% of dialysis patients treated with recombinant human erythropoietin (rHuEPO). Various studies have been performed in order to elucidate possible underlying mechanisms, but it is not yet well understood whether there is one major mechanism involved. In this present study, samples were obtained from male normotensive Wistar-Kyoto rats (WKY) and genetically hypertensive rats (SHR) at the age of 5 and 20 weeks. Thoracic aorta rings, with or without endothelium, isolated from WKY and SHR were used to evaluate the direct effect of rHuEPO on vascular smooth muscle by measuring the tension of vascular smooth muscle induced by various concentrations (1-100 IU/ml) of rHuEPO. Also, rHuEPO (10, 100, 1000 and 10,000 IU/kg) was intravenously administrated and the changes in mean blood pressure were recorded for 5-10 min. rHuEPO produced no significant contraction in the rat aortae in any of the preparation studies, in the presence or the absence of endothelium. The intravenous administration of rHuEPO had no immediate effect on mean blood pressure in 5- and 20-week-old WKY and SHR. These results suggest that the elevation of blood pressure observed in the clinical setting following the administration of rHuEPO is not due to a direct pressor effect on vascular smooth muscle.

Topics: Animals; Aorta; Blood Pressure; Disease Models, Animal; Erythropoietin; Hypertension; Infusions, Intravenous; Male; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Vasoconstriction

In a subset of dialysis patients, erythropoietin (rHuEpo) treatment exacerbates hypertension. The mechanism of this pressor effect is unknown; however, it has been suggested that decreased endogenous nitric oxide (NO) activity may play a role. To explore this hypothesis, Sprague-Dawley rats were given rHuEpo (150 U/kg s.c. three times per week) or corresponding vehicle. Blood pressure, haematocrit, and urinary excretion of the stable NO metabolites, nitrite (NO2) and nitrate (NO3), were determined at baseline and 3 weeks. After 3 weeks of rHuEpo treatment there was a significant increase in blood pressure and haematocrit, while in vehicle-treated rats blood pressure and haematocrit remained at basal levels. Urinary excretion of NO2+NO3 increased compared to basal in rHuEpo, but not vehicle rats. Thus in normal rats rHuEpo does have a significant pressor effect, but this is not associated with decreased activity of the endogenous NO system. Thus decreased endogenous NO activity is not responsible for rHuEpo-associated hypertension. These data further suggest that endogenous NO activity is increased in rHuEpo-treated rats, perhaps as a counter-regulatory mechanism that limits the pressor effect. Whether this mechanism is active in the setting of rHuEpo-treated chronic renal failure in humans is unknown.

Topics: Animals; Blood Pressure; Disease Models, Animal; Erythropoietin; Hematocrit; Hydrogen-Ion Concentration; Hypertension, Renal; Male; Nitrates; Nitric Oxide; Nitrites; Random Allocation; Rats; Rats, Sprague-Dawley; Recombinant Proteins

Anemia is an invariable consequence of end-stage renal failure (ESRF) and recombinant erythropoietin has dramatically improved the quality of life of patients with ESRF. As an alternative approach, we developed a myoblast gene transfer system for the systemic delivery of human erythropoietin (EPO). We recently reported that transplantation of 4 x 10(7) cells of a C2 myoblast cell clone that stably secretes high level of functional human EPO, increased hematocrit from 44.6 +/- 3.0 to 71.2 +/- 7.9(%) in 2 wk, and the increase was sustained for at least 12 wk in nude mice. A renal failure model was created by a two-step nephrectomy in nude mice, and myoblasts were transplanted 3 wk after the second nephrectomy, when mean blood urea nitrogen level had increased from 26.3 +/- 6.1 to 85.4 +/- 24.0 (mg/dl) and the hematocrit had decreased from 45.2 +/- 2.7 to 33.9 +/- 3.7(%). After transplantation, the hematocrit markedly increased to 68.6 +/- 4.2(%) 2 wk, and to 68.5 +/- 4.0(%) 7 wk after the transplantation. Serum human EPO concentration determined by ELISA indicated a persistent steady EPO production from the transplanted muscle cells 8 wk after the transplantation. The fate of transplanted myoblasts in uremic mice was monitored by transplanting the EPO-secreting clone which had also been transduced with BAG retrovirus bearing the beta-galactosidase gene. 8 wk later, X-gal positive myofibers were detected in the entire transplanted area. The results demonstrate that myoblasts can be transplanted in uremic mice, and that myoblast gene transfer can achieve sufficient and sustained delivery of functionally active EPO to correct anemia associated with renal failure in mice.

Topics: Anemia; Animals; Cell Transplantation; Disease Models, Animal; Erythropoietin; Genetic Therapy; Hematocrit; Humans; Kidney Failure, Chronic; Mice; Mice, Nude; Muscles; Nephrectomy; Time Factors

1. There is no experimental proof that renal insufficiency is a necessary condition for hypertension during erythropoietin treatment. 2. The present study compares the effect of 3 weeks treatment with r-hu EPO (50 i.u./kg) on systolic blood pressure (SBP), haematocrit and plasma cGMP in an animal model of chronic renal failure (remnant kidney model excision) and sham-operated rats. 3. Sub-total nephrectomy induced a significant fall in haematocrit and a significant increase in plasma creatinine levels. Treatment with r-hu EPO resulted in a significant haematocrit increase in uraemic as well as in non-uraemic rats. Despite this effect, r-hu EPO treatment had no effect on SBP in sham-operated rats. On the contrary, this treatment caused significant SBP elevation in uraemic rats; in these rats, SBP increase did not correlate with haematocrit increase. 4. Plasma cGMP concentrations were significantly higher in uraemic compared to sham-operated rats and were not modified by r-hu EPO treatment. 5. This study provides evidence that renal insufficiency in rats is a prerequisite for the development of hypertension during erythropoietin treatment.

Topics: Animals; Blood Pressure; Creatinine; Cyclic GMP; Disease Models, Animal; Erythropoietin; Hematocrit; Hypertension; Kidney Failure, Chronic; Male; Nephrectomy; Rats; Rats, Wistar; Uremia

The Han:SPRD rat model for inherited polycystic kidney disease (PKD) was characterized (clinical parameters, morphology, immunohistochemistry and in situ hybridization). Homozygous animals died of uremia after three to four weeks with severe cystic transformation of virtually all nephrons and collecting ducts (serum urea: 616 +/- 195 mg/dl; kidney-to-body weight ratio: > 20%). In heterozygotes, slow progression of the disease led to death between the 12th and 21st month (median: 17 months; serum urea levels above 200 mg/dl). Kidney enlargement was moderate, and cysts were restricted to the cortex and outer medulla. Immunohistochemical markers showed that approximately 75% of the cysts were derived from the proximal tubule. Cystic transformation started in the proximal tubule with a sharp onset of basement membrane alteration and a loss of epithelial differentiation restricted to small focal areas. In these areas, alpha 1(IV) collagen and laminin B1 mRNA were enhanced as revealed by isotopic and non-isotopic in situ hybridization. Fibroblasts underlying the affected tubular portions were involved in matrix overexpression resulting in subepithelial accumulation of immunoreactive collagen IV and laminin. In later stages of cystic transformation distal nephron segments were affected as well. A reversal in epithelial polarity as judged from Na,K-ATPase-immunoreactivity was not observed. Renal immunoreactive renin-status was significantly decreased. Hematocrit was lowered in heterozygotes (40.4 +/- 5.8 vol% compared to 46.7 +/- 1.99 vol% in controls; P < 0.05) and total renal EPO mRNA was reduced to 36 +/- 14% of the mean value of control animals, whereas serum EPO levels were not significantly altered. We conclude that the Han:SPRD rat is a useful model for the study of human ADPKD since both diseases are similar in several aspects. The model is particularly suitable for the study of epithelial-mesenchymal interactions at the beginning of tubular cystic transformation.

Topics: Animals; Disease Models, Animal; Erythropoietin; Extracellular Matrix Proteins; Female; Hypertrophy; Kidney; Male; Mucoproteins; Polycystic Kidney, Autosomal Dominant; Rats; Rats, Mutant Strains; Rats, Sprague-Dawley; Renin; RNA, Messenger; Sodium-Potassium-Exchanging ATPase; Uromodulin

The risks inherent in the use of homologous blood products have increased efforts toward identifying alternatives to transfusion. We have previously shown that the administration of recombinant human erythropoietin (rhEpo) enhances the erythropoietic response to acute blood loss. Recombinant human interleukin-3 (rh-IL-3) is a hematopoietic growth factor that has been shown to act synergistically with rhEpo in accelerating erythropoiesis in vitro. The purpose of this study in a primate model was to determine if the administration of rhIL-3 in combination with rhEpo could augment the erythropoietic response to acute blood loss more than rhEpo therapy alone. Twenty-four adult male baboons were randomized into four groups. The induction of acute normovolemic anemia to a hematocrit of 20% was accomplished via exchange-transfusion with 6% hetastarch. The groups were then treated for 7 consecutive days with the following growth factors: group I (n = 7), no growth factors; group II (n = 5), rhIL-3 alone (100 micrograms/kg/d); group III (n = 6), rhEpo alone (1000 U/kg/d); group IV (n = 6), rhEpo (1000 U/kg/d) plus rhIL-3 (100 micrograms/kg/d). All animals received folate, vitamin B12, and intravenous iron-dextran immediately following the exchange-transfusion. Response to therapy was monitored for 35 days. There were no adverse reactions following growth factor administration. The analysis of erythropoietic rates between study days 1 through 11, as determined via linear regression analysis, revealed that hematocrits increased significantly faster in the groups receiving rhEpo compared to controls. The administration of rhIL-3, however, did not increase the rate of erythropoiesis when compared to controls, nor did it augment response when added to the rhEpo regimen. The results of this study demonstrate that the administration of rhIL-3 alone had no significant effect on erythropoiesis in this setting of acute blood loss. Further, despite promising in vitro data, rhIL-3 provided no additional stimulation of erythropoiesis in animals receiving rhEpo. Nevertheless, the study confirms that the pharmacologic acceleration of erythropoiesis by rhEpo alone remains an attractive alternative to homologous transfusion.

Topics: Acute Disease; Analysis of Variance; Anemia; Animals; Blood Cell Count; Disease Models, Animal; Drug Therapy, Combination; Erythropoiesis; Erythropoietin; Interleukin-3; Male; Papio; Recombinant Proteins

Hydroxyurea, a cell-cycle-specific cytotoxic agent, has been shown to increase fetal hemoglobin (HbF) production. This property makes it an attractive drug for treatment of sickle cell disease and severe beta thalassemia. Its potential efficacy is limited because of a variable and often suboptimal response. Combinations of hydroxyurea and other drugs may induce more clinically significant increases in HbF. We have utilized chronically phlebotomized rhesus monkeys, treated with oral hydroxyurea, to investigate the capacity of several other agents to further augment HbF synthesis. Recombinant human erythropoietin, in super-pharmacologic doses, increased F-reticulocyte production when given on a weekly sequential schedule (3 of 7 days) with hydroxyurea (4 of 7 days), but it was less effective on an alternate day schedule when hydroxyurea was given daily. Neither recombinant human interleukin 3 (IL-3) nor recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), when infused individually, increased F-reticulocytes in animals receiving daily hydroxyurea. Sequential, overlapping infusions of IL-3 and GM-CSF produced a small but statistically significant increase in F-reticulocytes in one of two hydroxyurea-treated animals. Infusions of sodium butyrate produced a substantial augmentation in F-reticulocyte production in animals chronically treated with hydroxyurea. Thus, our studies have identified several agents that may prove useful in combination with hydroxyurea to achieve clinically beneficial levels of HbF.

Topics: Administration, Oral; Anemia; Animals; Bloodletting; Butyrates; Butyric Acid; Cell Division; Chromatography, High Pressure Liquid; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Therapy, Combination; Erythrocytes; Erythropoietin; Female; Fetal Hemoglobin; Granulocyte-Macrophage Colony-Stimulating Factor; Hematocrit; Hematopoietic Cell Growth Factors; Hydroxyurea; Injections, Intravenous; Interleukin-3; Macaca mulatta; Reticulocytes; Time Factors

In mice, SNB-5001 released reticulocytes dose-dependently. In rats, rabbits and dogs given the same doses of SNB-5001, each dose-response curve of hemopoiesis almost showed parallelism. SNB-5001 induced nearly the same extent of hemopoiesis in these animals. After the hemopoiesis caused by SNB-5001, reticulocytes decreased within a week in both rats and dogs, but numbers of red blood cells (RBC) were higher than each control group for over 2 weeks in rats and for over 3 weeks in dogs. In polycythemic rats given excessive doses (200-5000 U/kg) of SNB-5001, blood volume increased, but blood pressure did not change. In the renal anemic rats produced by partial nephrectomy, dose-related and cumulative hemopoiesis were observed in both groups given SNB-5001 with different administration schedules (once a day for a week or once a week for 3 weeks). In the phrebotomized rats, SNB-5001 accelerated the recovery from the anemia induced by phrebotomy when given in a large quantity (6 ml/rat) and prevented the progressive anemia induced by intermittent phlebotomies when given in a small quantity (1 ml/rat x 3). SNB-5001 also improved the anemia caused by chronic inflammation in rats. However, increases of hemoglobin and hematocrit were smaller than that of RBC. Those results were caused by impaired release of iron from the reticuloendothelial system.

Topics: Anemia; Animals; Disease Models, Animal; Dogs; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Hemodynamics; Mice; Rabbits; Rats; Rats, Inbred Strains; Recombinant Proteins

Hilltop (H) and Madison (M) strains of Sprague-Dawley rats exhibit strikingly different susceptibilities to the effects of chronic altitude exposure. The H rats develop greater polycythemia, hypoxemia, and pulmonary hypertension. We studied ventilation, pulmonary gas exchange, tissue oxygenation, and hematologic adaptations in the two rat strains during a 50-day exposure to a simulated altitude (HA) of 5,500 m (18,000 ft). There were no strain differences among the variables we studied under sea level (SL) conditions. Within the first 14 days of hypoxic exposure, the only significant strain differences were that erythropoietin (EPO) rose much higher and erythroid activity was greater in the H rats, even though arterial Po2 and PCo2 (Pao2 and PaCo2, respectively), renal venous PO2 (Prvo2), and ventilation (VE) were equivalent in the two strains during this time. By day 14 at HA, the H rats had significantly higher erythroid activity, hematocrit (Hct), and EPO levels, significantly lower PaO2 and PrvO2, but equivalent VE and PaCO2. These changes persisted for the remainder of the exposure, except that the Hct continued to rise and the increase was greater in H rats. Despite the greater O2-carrying capacity of H rats in the later stages of hypoxic exposure, PaO2 and PrvO2 were significantly lower in H rats. There were no strain differences at either SL or HA in ventilatory responses to hypercapnia or hypoxia, in blood O2 affinity or 2,3-diphosphoglycerate, in extrarenal production of EPO, or in EPO clearance. We conclude that early in the hypoxic exposure the H rats produce more EPO at apparently equivalent levels of hypoxia, and this is the first step in the pathogenesis of the maladaptation to HA manifest by H rats. We find no consistent evidence that differences in VE contribute to the variable susceptibility to hypoxia in the two rat strains.

Topics: Altitude Sickness; Animals; Disease Models, Animal; Erythropoietin; Hematopoiesis; Hypoxia; Kidney; Male; Oxygen; Rats; Rats, Inbred Strains; Respiration; Species Specificity

Azidothymidine (AZT) induces severe anemia in patients with acquired immune deficiency syndrome (AIDS). To evaluate the mechanism of anemia in immune-suppressed animals, a murine model of AIDS (MAIDS), caused by infection with LP-BM5 murine leukemia virus (LP-BM5 MuLV) was used at early and late stages of the disease. AZT-induced anemia was dose- and time-dependent. An increased percentage of erythroblasts in bone marrow was observed, with an increased ratio of early to late erythroblasts in both disease stages. Increases in splenic erythroid burst-forming units (BFUe) were observed in early-stage AZT-treated mice. Mean plasma erythropoietin (EPO) levels were increased by AZT in both groups in a dose-dependent manner and were inversely proportional to hematocrit values. These data suggest that the anemia induced by AZT stimulated a response by immature erythroid elements, but that the maturation or survival of early erythroblasts may be impaired.

Topics: Acquired Immunodeficiency Syndrome; Anemia; Animals; Bone Marrow; Disease Models, Animal; Dose-Response Relationship, Drug; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Female; Hematocrit; Mice; Mice, Inbred C57BL; Pregnancy; Reticulocytes; Time Factors; Zidovudine

Mechanisms for the development of anemia and the effects of recombinant human erythropoietin (r-HuEPO) on hematological parameters were studied in new congenital adult type polycystic kidney (DBA/2FG-pcy) mice. The majority of DBA/2FG-pcy mice showed progressive anemia and an elevation of blood urea nitrogen, while a minority showed progressive anemia following polycythemia. Kidneys with numerous cysts in the cortex and medulla occupied virtually the entire abdominal cavity, and the combined kidney weight taken as a percentage of body weight reached 13.5% in the DBA/2FG-pcy mouse. The osmotic fragility of DBA/2FG-pcy mice erythrocytes was significantly increased compared with that of normal control mice. In addition, two-fold increases in serum EPO levels, determined by radioimmunoassay, and a decreased number of colony forming unit-erythroid (CFU-E) were observed in the DBA/2FG-pcy mice. The administration of r-HuEPO during anemia significantly increased the red blood cell count, hemoglobin concentration, hematocrit and reticulocyte percentage in a dose-dependent manner. These findings indicate that anemia in the DBA/2FG-pcy mouse is due to increased fragility of erythrocytes, a deficiency in EPO for the degree of anemia and a decreased number or a decreased response of erythroid progenitor cells. We suggest that the DBA/2FG-pcy mouse is a useful spontaneous model of chronic progressive renal failure.

Topics: Anemia; Animals; Blood Urea Nitrogen; Bone Marrow; Colony-Forming Units Assay; Disease Models, Animal; Erythrocyte Count; Erythropoietin; Hematocrit; Kidney; Mice; Osmotic Fragility; Polycystic Kidney Diseases; Recombinant Proteins

Anaemia is a major complication of chronic renal failure. It is mainly due to a decrease in the production of erythropoietin and at present it can be corrected by recombinant human erythropoietin (rHuEpo). The question has arisen whether aluminium overload, which is frequently observed in uraemic patients, could exert a resistance to the effect of rHuEpo. To answer this question, we submitted two series of rats with two groups in each to an experimental aluminium intoxication. Group II rats received repeated i.p. injections of aluminium, whereas group I (control) rats were given vehicle solution alone. Subsequently, all rats were treated with identical s.c. doses of rHuEpo (100 IU/kg body-weight twice weekly). In the first series, rats were fed ad libitum whereas in the second, rats were pair-fed and received iron supplementation. In the first series, group I rats had an increase of mean haemoglobin in response to rHuEpo: 15.6 +/- 0.3 vs 19.8 +/- 0.3 g/dl, P less than 0.001. In contrast, group II rats had a decrease: 15.1 +/- 0.2 vs 10.1 +/- 0.8 g/dl, P less than 0.001. However, compared to group I rats, group II rats did not gain body-weight and their plasma iron concentration was less. In the second series, mean haemoglobin concentration of group I rats increased from 15.1 +/- 0.2 to 18.9 +/- 0.3 g/dl (P less than 0.001) in response to rHuEpo.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aluminum; Anemia; Animals; Disease Models, Animal; Drug Resistance; Erythropoiesis; Erythropoietin; Iron; Male; Rats; Recombinant Proteins

The metabolism of recombinant human erythropoietin (rHuEpo) labelled with 125I has been investigated in five normal and nine 5/6-nephrectomised rabbits. The plasma erythropoietin half-life was significantly prolonged at 5.1 +/- 1.2 h (mean +/- SD) in the 5/6-nephrectomised rabbits, compared to 3.0 +/- 0.4 h in sham-operated controls (P less than 0.001). The disappearance of 125I-labelled rHuEpo is biphasic. Examination of serum by fast protein liquid chromatography (FPLC) following administration of 125I-labelled rHuEpo by FPLC showed a single peak of radioactivity in all rabbits except two of the nephrectomised group. In serum from both of these animals a second labelled peak was found, corresponding to material of MW 200,000-250,000 D. We conclude that the 5/6-nephrectomised rabbit provides a stable model for the study of hormonal metabolism in chronic renal failure.

Topics: Animals; Disease Models, Animal; Erythropoietin; Female; Half-Life; In Vitro Techniques; Male; Nephrectomy; Rabbits; Uremia

We administered recombinant human interleukin-1 beta (IL-1 beta), the common mediator of inflammation process, to C57B1/6 male mice (0.5 microgram, every 12 hours over five times) intraperitoneally and consequently induced a remarkable thrombocytosis. Day 1 was designated as the following day of the last injection in the morning. A significant thrombocytosis was observed on days 1 through 5 with a peak on day 2 (162 +/- 9 x 10(4)/mm3) compared with the control mice injected with heated IL-1 beta (101 +/- 11 x 10(4)/mm3). A striking increase in mean size of marrow megakaryocytes was noted on days 1 and 2. The incorporation of 75Se-selenomethionine into circulating platelets as a measure of platelet production was about 2.3 times higher in IL-1 beta-treated mice than in control mice. To determine which factor(s) is responsible for elicited thrombocytosis, the in vitro studies and bioassays for several hematopoietic factors were performed. IL-1 beta by itself did not stimulate megakaryocytopoiesis in vitro, suggesting that the thrombocytosis is attributed to other factor(s) via IL-1 beta stimulation. Serum colony-stimulating factor (CSF) activity after a single IL-1 beta (0.5 microgram) injection, monitored by colony assay with 10% tested serum, peaked at 3 hours. Formed colonies were mostly granulocyte (G) and granulocyte-macrophage (GM)-types, and studies using rabbit anti-mouse GM-CSF serum or using human marrow as target cells showed that the CSF activity of the tested serum consisted of, at least, GM-CSF and G-CSF. Addition of IL-3 concomitantly with the tested serum gave rise to a greater number of megakaryocytic colonies. Serum IL-3, monitored by IL-3-dependent cell line 32D clone 5, and erythropoietin activities were not detected at serum level in IL-1 beta-treated mice. Serum IL-6 assay by IL-6-dependent mouse hybridoma cell line MH-60.BSF2 showed high levels of the tested serum with a peak at 2.5 hours with no detection at 10 hours after the injection. Heated IL-1 beta caused an increase of neither IL-6 nor CSF activities. Our data suggest that the thrombocytosis induced by IL-1 beta is mediated by IL-6 or a combination of IL-6 and other cytokine(s), and that IL-6 may play a regulatory role in platelet production in vivo.

Topics: Animals; Blood Platelets; Bone Marrow; Cerebrospinal Fluid; Disease Models, Animal; Erythropoietin; Hematopoiesis; Hematopoietic Stem Cells; Injections, Intraperitoneal; Interleukin-1; Interleukin-3; Interleukin-6; Megakaryocytes; Mice; Platelet Count; Selenium Radioisotopes; Selenomethionine; Spleen; Thrombocytosis

Highly purified, recombinant, human erythropoietin (rh EPO) has been compared to natural urinary derived erythropoietin (nEPO). Both EPO preparations have been characterized biologically: The proliferation of murine spleen cells in vitro after pretreatment with phenylhydrazine and the 59Fe incorporation into the heme of polycythemic mice have been determined. Further, the effect of rh EPO on the erythropoiesis of normal mice was studied. Depending on the applied rh EPO dosage an increase in hematocrit was observed. Treatment with rh EPO in rats with anemia due to subtotal nephrectomy also showed a dose-dependent rise of hematocrit and hemoglobin, and therefore a reversal of the anemic status. The comparison of rh EPO and natural EPO showed full biological activity of the recombinant protein and its equivalence to the natural hormone.

Topics: Anemia; Animals; Disease Models, Animal; DNA Replication; Erythropoietin; Female; Humans; Kidney Failure, Chronic; Mice; Mice, Inbred Strains; Polycythemia; Rats; Rats, Inbred Strains; Recombinant Proteins

Topics: Anemia; Animals; Disease Models, Animal; Erythropoietin; Hematocrit; Hemoglobins; Male; Nephrectomy; Rats; Rats, Inbred Strains; Uremia

Nonhuman primate models of gestational diabetes have produced fetopathies most similar to those of the human infant of the mother with gestational diabetes (IGDM). Fetal hyperglycemia, hyperinsulinemia, macrosomia, selective organomegaly, intrauterine death, and placental hyperplasia are hallmarks of the fetopathy of the IGDM. The chronic infusion of insulin into the fetus of a normal pregnant rhesus monkey results in fetal hyperinsulinemia with normal to low plasma metabolic substrate concentrations. Under these conditions, fetal hyperinsulinemia is sufficient to cause fetal growth and hormone changes observed in the human IGDM. Our studies provide evidence that the soft tissue hyperplasia in the fetal macrosomia syndromes in humans and nonhuman primates in which fetal hyperinsulinemia is observed is the direct result of that chronic in utero hyperinsulinemia.

Topics: Adipose Tissue; Amino Acids; Animals; Birth Weight; Blood Glucose; C-Peptide; Disease Models, Animal; Erythropoietin; Female; Fetal Blood; Fetal Hypoxia; Fetus; Glucagon; Hydrocortisone; Insulin; Macaca mulatta; Pregnancy; Pregnancy in Diabetics

Chronic fetal hyperinsulinemia, similar to that found in human infants of diabetic mothers, was produced in fetal rhesus monkeys during the latter third of gestation. Fetal plasma glucose and amino acid concentrations were found to be inversely logarithmically correlated with plasma insulin concentration. Fetal plasma glucagon concentrations were suppressed by hyperinsulinemia. Fetal plasma erythropoietin concentrations were increased by hyperinsulinemia in a dose/response manner. The activity of the hepatic gluconeogenic enzymes glucose-6-phosphatase and total phosphoenolpyruvate carboxykinase were reduced by hyperinsulinemia. Fatty acid synthase complex activity was, in contrast, increased by hyperinsulinemia while citrate cleavage enzyme and glucose-6-phosphate dehydrogenase were only increased when supraphysiologic hyperinsulinemia was produced. This model provides an opportunity to study the metabolic effects of hyperinsulinemia separate from those of hyperglycemia on the primate fetus, making it a useful model for the study of fetal pathologic conditions in diabetic pregnancies.

Topics: Amino Acids; Animals; Blood Glucose; Disease Models, Animal; Erythropoietin; Female; Fetus; Glucagon; Insulin; Liver; Macaca mulatta; Maternal-Fetal Exchange; Pregnancy; Pregnancy in Diabetics

The presence of inhibitors which accumulate during uraemia has been postulated as a significant factor in the development of anaemia in chronic renal failure (CRF). To determine whether factors in uraemic serum depress erythropoiesis, samples were obtained from sheep prior to and after surgical induction of CRF. The sera were tested in vitro for their effect on erythroid colony growth. The sheep sera were substituted for fetal calf serum (30% concentration) in cultures of serotype-matched or autologous sheep marrow cells at optimal doses of erythropoietin (Ep). Forty-two paired sera from five animals were tested against normal (22) and uraemic marrow (20). In 7/42 random pairs, erythroid colony growth was decreased by 20% in the presence of uraemic serum when compared to a normal sample from the same animal. In the remainder of the cultures, uraemic sera stimulated or supported erythroid colony growth as well as normal sera. When the results were analysed individually, serum from only one of five animals showed minimal (10%) in vitro inhibition of erythroid colony growth. This study, performed in a prospective manner utilizing compatible target cells, disputes the hypothesis that uraemic toxins significantly inhibit in vitro erythropoiesis. These data correlate with the in vivo response to Ep in this sheep model, and suggest Ep would be effective in treating the anaemia of CRF.

Topics: Anemia; Animals; Bone Marrow; Cells, Cultured; Colony-Forming Units Assay; Disease Models, Animal; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Kidney Failure, Chronic; Prospective Studies; Sheep; Uremia

Topics: Anemia; Animals; Bone Marrow; Disease Models, Animal; Erythropoietin; Kidney Failure, Chronic; Mice; Mice, Mutant Strains; Mice, Nude; Spleen

The hypoproliferative anemia in chronic renal failure has been assumed to be the result of decreased erythropoietin (Ep) production by the damaged kidney and of the shortening of erythrocyte survival. However, many in vitro studies suggest that erythropoietic inhibitors in uremic plasma may contribute to the anemia. To determine the in vivo relevance of uremic inhibitors, increasing amounts of Ep as Ep-rich plasma were infused into six uremic sheep, and their erythropoietic responses were compared with those of nine normal sheep receiving similar amounts of Ep-rich plasma. Three sheep were studied in both normal and uremic states. Ep-rich plasma was obtained from phenylhydrazine- and phlebotomy-induced anemic sheep. Stable uremia was created by subtotal nephrectomy. Erythropoiesis was quantitated by reticulocyte response, ferrokinetics (plasma iron turnover and marrow transit time), and by hemoglobin C synthesis. Ep-rich plasma stimulated erythropoiesis similarly in uremic and normal sheep, regardless of the degree of uremia. Nondialyzed uremic sheep responded as well as dialyzed animals. The anemia was corrected in the uremic dialyzed animals. The anemia was corrected in the uremic sheep after 15-40 daily infusions of Ep-rich plasma, the total dosage depending on the severity of the anemia. Polycythemia was induced when the infusions were continued. Reticulocytes, plasma iron turnover, and erythrocyte mass changes increased as the amount of Ep-rich plasma was increased. These dose-response effects, coupled with the identical erythropoietic response in normal and uremic sheep given the same amount of Ep-rich plasma, imply that there are no physiologically significant erythropoietic inhibitors in uremia.

Topics: Anemia; Animals; Disease Models, Animal; Erythropoietin; Female; Iron; Kidney Failure, Chronic; Renal Dialysis; Reticulocytes; Sheep

The four existing animal models of cyclic hematopoiesis are briefly described. The unusual erythropoietin (Ep) responses of the W/Wv mouse, the Sl/Sld mouse, and cyclic hematopoietic dog are reviewed. The facts reviewed indicate that the bone marrow itself is capable of influencing regulatory events of hematopoiesis.

Topics: Anemia; Animals; Bone Marrow; Bone Marrow Cells; Disease Models, Animal; Dog Diseases; Dogs; Erythropoiesis; Erythropoietin; Hematopoiesis; Humans; Hypoxia; Lithium; Mice; Mice, Inbred Strains; Mink; Neutropenia; Periodicity; Prednisolone; Strontium Radioisotopes

The pathogenesis of the increased erythrocytosis and extramedullary erythropoiesis observed in infants of diabetic mothers (IDM) has been obscure. In the present studies, IDM were found to have elevated umbilical plasma erythropoietin (Ep) concentrations by radioimmunoassay. 22 of 61 IDM (36%) had levels above the range of 28 nonasphyxiated, appropriately grown normal infants. In 16 controls and 20 IDM, plasma Ep correlated directly with plasma insulin (P less than 0.001, r = 0.73). To investigate this relationship further, a chronic rhesus model was studied with continuous fetal hyperinsulinemia for 21 d in utero in the last third of pregnancy. In five experimental fetuses, plasma insulin levels averaged 4,210 microU/ml at delivery, whereas plasma Ep was above the range of six controls. In addition, the experimental fetuses had elevated reticulocyte counts in umbilical cord blood. The mechanism for the increased plasma Ep associated with hyperinsulinemia in the fetus is unexplained but may be mediated by fetal hypoxia.

Topics: Animals; Blood Glucose; Carbohydrate Metabolism; Diabetes Mellitus; Disease Models, Animal; Erythropoiesis; Erythropoietin; Female; Fetal Blood; Hemoglobin A; Humans; Hyperinsulinism; Insulin; Macaca mulatta; Pregnancy; Pregnancy in Diabetics

Previous studies in mice deprived of water have suggested that these animals, like men in space, show hemoconcentration due to plasma volume reductions, a weight loss greater than that due to fluid loss alone, and suppression of red blood cell production. To more fully understand the mechanisms responsible for the suppressed erythropoiesis in dehydrated mice, a mathematical model for erythropoietic regulation has been adapted to this rodent. Computer simulations suggested several new experimental studies to more fully understand the erythroid response to dehydration. The investigations were directed to determining whether dehydration was accompanied by: a) a shortened red blood cell survival, b) altered sensitivity of the erythropoietin (Ep)-producing mechanism, c) a shortened red blood cell transit time, d) changes in the Ep serum half-life, e) changes in hemoglobin P50, and f) reduced renal blood flow. All parameters except changes in renal blood flow were investigated in vivo and incorporated into, or omitted from, the mathematical simulations as directed by experimental findings. The mathematical model is able to realistically simulate the in vivo erythroid response to dehydration making only one, experimentally-untested, assumption. Computer simulations confirm conclusions drawn from the animal studies that the primary cause of the suppressed erythropoiesis in dehydrated mice is the reduced food intake, with hemoconcentration playing a relatively minor role. The interaction between computer simulations and animal experiments is shown to be a powerful approach for formulating and testing hypotheses, designing new experiments, and achieving a clearer understanding of the factors controlling erythropoiesis.

Topics: Anemia; Animals; Computers; Disease Models, Animal; Erythrocytes; Erythropoiesis; Erythropoietin; Half-Life; Male; Mice; Mice, Inbred C3H; Models, Biological; Space Flight

During chronic hypoxia, the number of splenic erythroid progenitor cells in mice, particularly CFU-E, increased dramatically but transiently. Since all three classes of erythroid progenitors in the femoral bone marrow were suppressed, a large part of this increase might be attributed to migration of CFU-E and/or their progenitors from the medullary cavity. The changes in CFU-E were preceded 48-72 hours earlier by an increase in serum erythropoietin (Ep) titers which, in turn, had been preceded by a rapid and marked "shift-to-the-right" in the hemoglobin oxygen dissociation curve. During hypoxia, the mice lost a considerable fraction of their body weight. Computer simulations, using a mathematical model of erythropoietic regulation, suggest that this weight loss, either indirectly by reducing the need for red cells in a smaller-than-control animal or by directly altering the sensitivity of the Ep-producing mechanism, is the major cause of the falling Ep titers despite continuation of the hypoxic stress. Because of high endogenous 59Fe incorporation levels, it was not possible to confirm the thesis that animals with an expanded Erythropoietin Responsive Cell (ERC) compartment would be more sensitive to exogenous erythropoietin than are mice with a normal or reduced ERC population.

Topics: Animals; Bone Marrow Cells; Chronic Disease; Colony-Forming Units Assay; Computers; Disease Models, Animal; Erythrocyte Aging; Erythropoiesis; Erythropoietin; Female; Hematopoietic Stem Cells; Hypoxia; Mice; Mice, Inbred C3H; Spleen

ESF deficiency is probably not a major contributing factor in the early stages of the anemia of renal insufficiency. Serum ESF titers are lower in advanced renal failure when compared to that of nonuremic anemic subjects suffering from equivalent anemia. With increasing renal insufficiency a relative ESF deficiency gains increasing importance as a pathogenic factor in reduced erythropoiesis. Kidneys without excretory function may still be erythropoietically effective, since a further increase in the anemia occurs after bilateral nephrectomy. However, a basal erythropoiesis is still maintained by extrarenal ESF production, which is also enhanced by hypoxia. ESF deficiency is compensated after successful renal transplantation. A decreased response of the bone marrow to ESF may be another factor contributing to the hypoproliferative state of erythropoiesis in uremia. As demonstrated in a chronic uremic rabbit model there may be a blockade of further differentiation of the erythroid precursors. The relationship of this blockade in differentiation to the inhibitor of heme synthesis is not clear.

Topics: Anemia; Animals; Disease Models, Animal; Erythropoietin; Hematocrit; Kidney; Kidney Failure, Chronic; Nephrectomy; Rabbits; Reticulocytes

Erythropoietic activity produced by exogenous erythropoietin (Ep) is markedly reduced in Swiss mice rendered uremic by bilateral nephrectomy or bilateral ureteral ligation. Exogenous Ep causes a greater increase in erythropoiesis in germfree Swiss mice as compared with non-germfree mice, but the germfree mice show a more drastic reduction of erythropoiesis in uremia. Heme production in blood, spleen, and femoral marrow retains its typical pattern in uremia; there is no shift of erythropoiesis among the three sites. Production of endogenous Ep in uremic mice is less by a factor of 2-3 than that in intact mice whereas the reduction is by a factor of almost 100 in anephric mice. The data suggest that the anemia associated with uremia is the result of two phenomena: (1) a decreased production of Ep and (2) a diminished erythropoietic response to Ep.

Topics: Anemia; Animals; Disease Models, Animal; Erythropoiesis; Erythropoietin; Female; Germ-Free Life; Kidney; Male; Mice; Nephrectomy; Uremia

Two experimental models for pure red cell aplasia (PRCA) were established. In the first one, administration of PRCA serum IgG in normal mice induced a sustained inhibitory effect on erythropoiesis, a progressive decline of the hematocrit values and an inverse rise of erythropoietin (Ep) levels in serum. Thus, the physiopathological pattern of PRCA type I (or A) was established, In the second model a rabbit producing anti-Ep crossreacting with endogenous Ep was subjected to a booster injection of Ep. The rise of the immune response was associated with decrease of Gct values and disappearance of erythroid precursors from marrow smears, and its subsequent decline with reticulocytosis and regression of the anemia, thus reproducing the physiopathological pattern of PRCA type II (or B).

Topics: Anemia, Aplastic; Animals; Autoantibodies; Autoimmune Diseases; Chronic Disease; Disease Models, Animal; Erythropoietin; Immune Sera; Immunoglobulin G; Mice; Rabbits

Topics: Anemia; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Cell Division; Disease Models, Animal; Dog Diseases; Dogs; Erythrocytes, Abnormal; Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Hemoglobins; Mice; Mutation; Parabiosis; Pigmentation Disorders; Rats; Rodent Diseases; Sex Chromosome Aberrations; Spleen; Transplantation, Homologous

Topics: Adenocarcinoma; Animals; Antibody Formation; Cell Division; Cell-Free System; Diethylstilbestrol; Disease Models, Animal; Erythropoietin; Female; Hematocrit; Kidney Neoplasms; Male; Medroxyprogesterone; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms, Experimental; Sex Factors; Species Specificity; Testosterone; Transplantation, Homologous

Topics: Adenocarcinoma; Animals; Antigens, Neoplasm; Disease Models, Animal; Erythropoietin; Hematocrit; Hormones; Inclusion Bodies, Viral; Kidney Neoplasms; Kinetics; Mice; Microscopy, Electron; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms, Experimental

Topics: Adenocarcinoma; Animals; Disease Models, Animal; Erythropoietin; Hematocrit; Kidney; Kidney Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms, Experimental; Nephrectomy; Organ Size; Splenic Neoplasms; Time Factors

Topics: Animals; Disease Models, Animal; Erythropoiesis; Erythropoietin; Hematocrit; Hypoxia; Iron Isotopes; Male; Mice; Mice, Inbred Strains; Polycythemia; Rats

Topics: Acute Disease; Animals; Disease Models, Animal; Erythropoietin; Hypoxia; Male; Pituitary Gland; Rats

Topics: Animals; Disease Models, Animal; Erythropoietin; Methods; Methylcholanthrene; Mice; Neoplasms, Experimental; Papilloma; Skin Neoplasms

Topics: Abortion, Spontaneous; Animals; Blood; Blood Proteins; Bone Marrow; Bone Marrow Cells; Disease Models, Animal; Dogs; Erythropoietin; Female; Humans; In Vitro Techniques; Mice; Placenta; Pregnancy; Uterine Hemorrhage

Topics: Animals; Culture Techniques; Disease Models, Animal; Erythropoietin; Methods; Mice; Polycythemia; Proteins

Topics: Animals; Culture Techniques; Disease Models, Animal; Erythropoiesis; Erythropoietin; Hematopoiesis; Hematopoietic System; Liver; Liver Transplantation; Methods; Mice; Organ Culture Techniques; Spleen; Transplantation, Homologous

Topics: Anemia; Animals; Bone Marrow Examination; Castration; Disease Models, Animal; Erythrocyte Aging; Erythrocyte Count; Erythropoietin; Estrogens; Female; Hematocrit; Hematologic Diseases; Hemoglobinometry; Homozygote; Leukocyte Count; Leukopenia; Male; Rats; Rodent Diseases; Sex Factors; Spleen; Splenectomy; Splenomegaly