losartan-potassium and Brain-Ischemia

Perinatal hypoxic-ischemic encephalopathy (HIE) remains a major cause of morbidity and mortality. Moderate hypothermia (33.5 °C) is currently the sole established standard treatment. However, there are a large number of infants for whom this therapy is ineffective. This inspired global research to find neuroprotectants to potentiate the effect of moderate hypothermia. Here we examine erythropoietin (EPO) as a prominent candidate. Neonatal animal studies show that immediate, as well as delayed, treatment with EPO post-injury, can be neuroprotective and/or neurorestorative. The observed improvements of EPO therapy were generally not to the level of control uninjured animals, however. This suggested that combining EPO treatment with an adjunct therapeutic strategy should be researched. Treatment with EPO plus hypothermia led to less cerebral palsy in a non-human primate model of perinatal asphyxia, leading to clinical trials. A recent Phase II clinical trial on neonatal infants with HIE reported better 12-month motor outcomes for treatment with EPO plus hypothermia compared to hypothermia alone. Hence, the effectiveness of combined treatment with moderate hypothermia and EPO for neonatal HIE currently looks promising. The outcomes of two current clinical trials on neurological outcomes at 18-24 months-of-age, and at older ages, are now required. Further research on the optimal dose, onset, and duration of treatment with EPO, and critical consideration of the effect of injury severity and of gender, are also required.

Topics: Brain Ischemia; Child, Preschool; Erythropoietin; Humans; Hypothermia, Induced; Infant; Infant, Newborn; Infant, Newborn, Diseases; Neuroprotection

Erythropoietin has been researched for its neuroprotective effects in ischemic stroke for over 30 years. Although erythropoietin can cause side effects that need to be controlled, it has been suggested to be effective in enhancing the prognosis of patients who are out of the therapeutic time window and have not received recombinant tissue plasminogen activator therapy. Studies on the mechanism of the function of erythropoietin have shown that it has various protective effects in ischemic brain injury after stroke, including promoting neurogenesis. In this review, we discuss the effects of erythropoietin on neurogenesis after ischemic brain injury and provide references for effective treatments for ischemic stroke, which is one of the leading causes of death worldwide.

Topics: Animals; Brain Ischemia; Erythropoietin; Humans; Ischemic Stroke; Neurogenesis; Neuroprotective Agents

The loss of blood-brain barrier (BBB) integrity leading to vasogenic edema and brain swelling is a common feature of hypoxic/ischemic brain diseases such as stroke, but is also central to the etiology of other CNS disorders. In the past decades, numerous proteins, belonging to the family of angioneurins, have gained increasing attention as potential therapeutic targets for ischemic stroke, but also other CNS diseases attributed to BBB dysfunction. Angioneurins encompass mediators that affect both neuronal and vascular function. Recently, increasing evidence has been accumulated that certain angioneurins critically determine disease progression and outcome in stroke among others through multifaceted effects on the compromised BBB. Here, we will give a concise overview about the family of angioneurins. We further describe the most important cellular and molecular components that contribute to structural integrity and low permeability of the BBB under steady-state conditions. We then discuss BBB alterations in ischemic stroke, and highlight underlying cellular and molecular mechanisms. For the most prominent angioneurin family members including vascular endothelial growth factors, angiopoietins, platelet-derived growth factors and erythropoietin, we will summarize current scientific literature from experimental studies in animal models, and if available from clinical trials, on the following points: (i) spatiotemporal expression of these factors in the healthy and hypoxic/ischemic CNS, (ii) impact of loss- or gain-of-function during cerebral hypoxia/ischemia for BBB integrity and beyond, and (iii) potential underlying molecular mechanisms. Moreover, we will highlight novel therapeutic strategies based on the activation of endogenous angioneurins that might improve BBB dysfuntion during ischemic stroke.

Topics: Angiopoietins; Animals; Blood-Brain Barrier; Brain Ischemia; Erythropoietin; Humans; Stroke; Vascular Endothelial Growth Factor A

Erythropoietin (EPO) for treating acute ischemic stroke (AIS) has been investigated in many studies. However, the evidence was inconsistent. Thus, a systematic review and metaanalysis were performed to elucidate the role of EPO in treating patients with AIS.. Two electronic databases (PubMed and EMBASE) were used. 30-day NIHSS measures primary outcome while all-cause mortality in the follow up and 90-day Barthel Index were regarded as secondary outcome. Results are presented as relative risk (RR), standardized mean difference (SMD) and 95% confidence intervals (CI). We employed Stata software to perform the meta-analysis.. Four randomized controlled trials (RCTs) involving 784 patients were contained in this metaanalysis. The total combined results on 30-day NIHSS were (SMD = -0.52, 95% CI: -1.39, 0.34) with random-effects model and sensitivity analysis showed a significant difference after excluding the Ehrenreich 2009 trial. The total combined secondary measured results were (RR=1.72, 95% CI: 1.10, 2.70) and (SMD = 0.01, 95% CI: -0.14, 0.16) for all-cause mortality and 90-day Barthel Index. In the subgroup analysis by using recombinant tissue plasminogen activator (rtPA) earlier, the rtPA group showed increased all-cause mortality with the result of (RR = 1.92, 95% CI: 1.04, 3.52), but not in non-rtPA group.. To our systematic review and meta-analysis, we didn't recommend EPO administration for patients with AIS, especially with the combination of rtPA. Large RCTs are warranted to examine EPO efficacy in AIS patients in the future.

Topics: Animals; Brain Ischemia; Erythropoietin; Humans; Randomized Controlled Trials as Topic; Recombinant Proteins; Stroke; Tissue Plasminogen Activator

Over recent decades, experimental and clinical stroke studies have identified a number of neurorestorative treatments that stimulate neural plasticity and promote functional recovery. In contrast to the acute stroke treatments thrombolysis and endovascular thrombectomy, neurorestorative treatments are still effective when initiated days after stroke onset, which makes them applicable to virtually all stroke patients. In this article, selected physical, pharmacological and cell-based neurorestorative therapies are discussed, with special emphasis on interventions that have already been transferred from the laboratory to the clinical setting. We explain molecular and structural processes that promote neural plasticity, discuss potential limitations of neurorestorative treatments, and offer a speculative viewpoint on how neurorestorative treatments will evolve.

Topics: Axons; Brain; Brain Ischemia; Cytidine Diphosphate Choline; Erythropoietin; Granulocyte Colony-Stimulating Factor; Humans; Myelin Proteins; Neovascularization, Physiologic; Neurogenesis; Neurological Rehabilitation; Neuronal Plasticity; Nogo Proteins; Nootropic Agents; Phosphodiesterase 5 Inhibitors; Recovery of Function; Regeneration; Selective Serotonin Reuptake Inhibitors; Stem Cell Transplantation; Stroke; Stroke Rehabilitation

Subarachnoid haemorrhage (SAH) caused by a ruptured aneurysm accounts for only 5 % of strokes, but occurs at a fairly young age and carries a poor prognosis. Delayed cerebral ischaemia (DCI) is an important cause of death and dependence after aneurysmal SAH. The current mainstay of preventing DCI is nimodipine and maintenance of normovolemia, but even with this strategy DCI occurs in a considerable proportion of patients.Several drugs have been developed that have the potential to limit cerebral vasospasm and delayed ischaemic neurologic deficit, thus improving outcome for patients. However, although numerous agents can prevent arterial narrowing and/or block the excitatory cascade of events leading to ischaemic neuronal death in experimental conditions, there is still no pharmacologic agent that has been shown conclusively to improve the outcome in clinical practice.Erythropoietin (EPO) is a well-known erythropoietic hormone recently found to exert neuroprotective properties and has been shown to reduce cerebral vasospasm and infarct volume after experimental SAH. In humans, although EPO treatment did not impact the overall incidence of vasospasm, it significantly reduced the incidence of severe vasospasm, the incidence of delayed ischaemic deficits with new cerebral infarcts, and the duration of impaired autoregulation. The current study provides new evidence for the potential benefit and relative safety of EPO for the treatment of SAH in humans. Future clinical trials will hopefully provide definite evidence whether EPO treatment is beneficial in SAH patients.

Topics: Animals; Brain Ischemia; Erythropoietin; Humans; Stroke; Subarachnoid Hemorrhage; Vasospasm, Intracranial

Amounts of researches show that EPO is characterized with neurotrophic and neuroprotective manner, especially in brain stroke, which attracts a large numbers of researchers to study it. With the accumulating researches on its neuroprotection, many related mechanisms were revealed, such as antioxidant, anti-apoptosis, angiogenesis, anti-inflammatory, which suggests a multiple targets role of EPO on brain stroke. However, because of the high risk of thromboembolism in clinical administration of rhEPO and its analogs, the herbs are potential to be a replacer for its less side effects. Many researchers suggested that a larger of herbs were founded having the action of increasing the endogenous EPO in the model of anemia and cerebral ischemia. At the same time, there herbs were also proved that they had the action of against cerebral ischemia while some without considering the role of EPO in the reports. Considering of the action of promoting EPO of these herbs and the neural protection of EPO, this essay mainly summarizes the studies of herbs promoting EPO in the cerebral ischemia and discusses the mechanism of regulating the EPO of these herbs, for the aim of finding the potential drugs against cerebral ischemia.

Topics: Animals; Brain Ischemia; Drugs, Chinese Herbal; Erythropoietin; Humans; Neuroprotection; Plants, Medicinal

Ischemic brain injury inflicted by stroke and cardiac arrest ranks among the leading causes of death and long-term disability in the United States. The brain consumes large amounts of metabolic substrates and oxygen to sustain its energy requirements. Consequently, the brain is exquisitely sensitive to interruptions in its blood supply, and suffers irreversible damage after 10-15 min of severe ischemia. Effective treatments to protect the brain from stroke and cardiac arrest have proven elusive, due to the complexities of the injury cascades ignited by ischemia and reperfusion. Although recombinant tissue plasminogen activator and therapeutic hypothermia have proven efficacious for stroke and cardiac arrest, respectively, these treatments are constrained by narrow therapeutic windows, potentially detrimental side-effects and the limited availability of hypothermia equipment. Mounting evidence demonstrates the cytokine hormone erythropoietin (EPO) to be a powerful neuroprotective agent and a potential adjuvant to established therapies. Classically, EPO originating primarily in the kidneys promotes erythrocyte production by suppressing apoptosis of proerythroid progenitors in bone marrow. However, the brain is capable of producing EPO, and EPO's membrane receptors and signaling components also are expressed in neurons and astrocytes. EPO activates signaling cascades that increase the brain's resistance to ischemia-reperfusion stress by stabilizing mitochondrial membranes, limiting formation of reactive oxygen and nitrogen intermediates, and suppressing pro-inflammatory cytokine production and neutrophil infiltration. Collectively, these mechanisms preserve functional brain tissue and, thus, improve neurocognitive recovery from brain ischemia. This article reviews the mechanisms mediating EPO-induced brain protection, critiques the clinical utility of exogenous EPO to preserve brain threatened by ischemic stroke and cardiac arrest, and discusses the prospects for induction of EPO production within the brain by the intermediary metabolite, pyruvate.

Topics: Brain; Brain Ischemia; Erythropoietin; Humans; Neuroprotective Agents; United States

Ischemic tolerance exists in many organs, among which the cerebral ischemic tolerance and its potential clinical applications are most notable. The discovery of new triggers of cerebral ischemic tolerance has brought more interesting insights into the molecular mechanisms. The remote ischemic preconditioning and pharmacological induction of cerebral ischemic tolerance have shown promising clinical safety and feasibility, and therefore may be important breakthroughs in the clinical application of cerebral ischemic tolerance.

Topics: Brain Ischemia; Erythropoietin; Humans; Ischemic Preconditioning

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

Recombinant erythropoietin (rEPO) failed in a recent clinical study to protect from damages induced by ischemic stroke. The lack of acute treatments in ischemic stroke and the promising outcome in numerous preclinical studies in vivo demands a more critical evaluation of the future use of EPO as an acute treatment.. The current use and administration of rhEPO and its analogs in animal models and the future use of this cytokine in the treatment of ischemic stroke.. In this review the potential reasons for the failure of EPO in the clinical trial are analysed and whether the preclinical trials sufficiently evaluated the true potential of recombinant EPO and its analogs is assessed. Alternative methods for administration of EPO to enhance its potential as a neuroprotective drug in ischemic stroke are discussed.. Failure in clinical trial does not necessarily indicate the lack of therapeutic potential of EPO. This review encourages further investigation of the true potential of EPO as a candidate drug for the treatment of ischemic stroke by improved preclinical experimental design and utilization of alternative administration methods.

Topics: Administration, Intranasal; Animals; Brain Ischemia; Drug Delivery Systems; Erythropoietin; Humans; Injections, Intraventricular; Nanoparticles; Neuroprotective Agents; Recombinant Proteins; Stroke

Since the realization that erythropoietin (EPO) molecules have 'neuroprotective' properties, they have been investigated as treatments for acute ischemic stroke (AIS), but not systematically. The results of the 2009 clinical trial showed that EPO was ineffective as a stroke treatment, and moreover, increased mortality when combined with tissue plasminogen activator. Currently, CEPO, an EPO analog, is entering into a safety, tolerability and pharmacokinetic clinical trial for the treatment of AIS.. This review covers translational and clinical studies carried out over the period 1998 - 2010.. The primary aim of this article is to review the information available regarding the pharmacological and biological characteristics of EPO molecules. Second, based upon the translational research with EPO molecules in preclinical stroke models, a recommendation is made regarding the continued development of EPO molecules as an option to treat AIS.. EPO, CEPO and helix B peptide EPO analogs have significant neuroprotective activity is preclinical stroke models. However, given the detrimental effect of EPO in a recent clinical trial, preclinical safety studies of EPO molecules in embolic stroke models that parallel acute ischemic stroke in humans are warrented.

Topics: Adult; Aged; Aged, 80 and over; Brain Ischemia; Clinical Trials as Topic; Drug Evaluation, Preclinical; Erythropoietin; Female; Humans; Male; Middle Aged; Signal Transduction; Stroke; Translational Research, Biomedical; Young Adult

Erythropoietin (Epo) modulates the survival of developing erythroid cells and the production of new erythrocytes in the bone marrow and is a key molecule in the adaptation to hypoxia and anaemia. Epo receptors have been found to be widely expressed on non-haematopoietic cells, and Epo has been shown to have diverse actions (in particular, preventing ischaemic damage to tissues of the central nervous system). Recently, Epo has been shown to improve the outcome in a murine model of malaria, and high plasma levels of Epo in children with cerebral malaria were associated with a better outcome. Here, we review the biological importance of Epo, its mechanisms of action and the rationale for the proposed use of Epo as an adjunct treatment in cerebral malaria.

Topics: Brain; Brain Ischemia; Erythrocytes; Erythropoietin; Humans; Malaria, Cerebral

Ischemic stroke causes brain damage by multiple pathways. Previous stroke trials have demonstrated that drugs targeting one or only a few of these pathways fail to improve clinical outcome after stroke. Drugs with multimodal actions have been suggested to overcome this challenge. In this review, we describe the mechanisms of action of agents approved for secondary prevention of ischemic stroke, such as antiplatelet, antihypertensive, and lipid-lowering drugs. These drugs exhibit considerable properties beyond their classical mechanisms, including neuroprotective and neuroregenerative properties. In addition, candidate stroke drugs currently studied in clinical phase III trials are described. Among these, albumin, hematopoietic growth factors, and citicoline have been identified as promising agents with multiple mechanisms. These drugs offer hope that additional treatment options for the acute phase after a stroke will become available in the near future.

Topics: Albumins; Animals; Antihypertensive Agents; Aspirin; Brain Ischemia; Clopidogrel; Cytidine Diphosphate Choline; Dipyridamole; Erythropoietin; Granulocyte Colony-Stimulating Factor; Humans; Hypolipidemic Agents; Neuroprotective Agents; Platelet Aggregation Inhibitors; Stroke; Ticlopidine

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

Topics: Acidosis; Apoptosis; Brain Ischemia; Erythropoietin; Fever; Fibrinolytic Agents; Forecasting; Humans; Hyperglycemia; Myocardial Ischemia; Tissue Plasminogen Activator

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

Recombinant human erythropoietin was produced soon after the discovery of the erythropoietin gene in 1985 and since then, it is used in various clinical conditions such as chronic renal failure. Moreover, experimental studies have shown that erythropoietin exerts neuroprotective action as well. Recently, a clinical trial yielded promising results concerning the use of erythropoietin in stroke management. In this review, we summarize the main data which suggest that recombinant human erythropoietin and its analogues may indeed have a role in stroke treatment.

Topics: Acute Disease; Animals; Brain; Brain Ischemia; Clinical Trials as Topic; Erythropoietin; Humans; Neuroprotective Agents; Recombinant Proteins; Stroke; Treatment Outcome

Basic and clinical investigations have been performed, focusing on the mechanism of ischemic brain and spinal cord injuries, and preventive measures against ischemic insults such as drug therapy, hypothermia, maintenace of blood flow to brain and spinal cord, preconditioning, and no use of high dose fentanyl. In this special issue, five experts have provided new relevant information concerning brain and spinal cord protection. Further research in brain and spinal cord protection will contribute to better understanding of ischemic central nervous system injuries and to the establishment of novel therapies for protection of central nervous system.

Topics: Analgesics, Opioid; Anesthetics, Inhalation; Brain Ischemia; Erythropoietin; Heart Arrest, Induced; Humans; Hyperbaric Oxygenation; Hypothermia, Induced; Intraoperative Complications; Ischemic Preconditioning; Perfusion; Spinal Cord Ischemia; Xenon

Cardiovascular surgery is constantly progressing. However, the prevalence of central nervous system injury is greater after operations on the aortic arch than after other types of aortic or cardiac surgery. The central nervous system injury is a frequent cause of death or complications after such operations and is likely to occur as a result of the embolization of particulate matter or severe global ischemia during the interval of circulatory arrest. Several different methods are currently being used to protect them during operations on the aortic arch. This thesis is organized about the protection methods for these procedures.

Topics: Aorta, Thoracic; Brain Ischemia; Cardiopulmonary Bypass; Cardiovascular Surgical Procedures; Cerebrospinal Fluid; Drainage; Erythropoietin; Evoked Potentials, Motor; Heart Arrest, Induced; Humans; Hypothermia, Induced; Intercostal Muscles; Intraoperative Complications; Monitoring, Intraoperative; Perfusion; Perioperative Care; Postoperative Complications; Spinal Cord Ischemia

Recent studies have suggested that the brain preconditioning could induce tolerance to ischemia in humans. It has been believed that newly synthesized proteins are required for the acquisition of delayed tolerance in the brain and spinal cord. However, the mechanism other than the synthesis of neuroprotective proteins may also play a pivotal role. Preconditioning may reprogram the response to ischemic injury as seen during hibernation. Preconditioning with hyperbaric oxygen, volatile anesthetics, and xenon seems to be the focus of the attention from the standpoint of the clinical setting. Strong neuroprotection by the preconditioning with isoflurane and xenon is reported in animal experiments and may change the traditional idea of neuroprotection by anesthetics. The discovery that erythropoietin exerts neuroprotective properties has opened new therapeutic avenues. Erythropoietin is induced in the brain by hypoxic preconditioning and by the pharmacological preconditioning. In addition, the intravenous administration of erythropoietin has been shown to be safe and beneficial for acute stroke in humans. Therefore, erythropoietin is now one of the most promising neuroprotective agents. The research in the brain and spinal cord preconditioning will contribute to the elucidation of the mechanism of ischemic injury and to the establishment of new therapies for neuroprotection.

Topics: Anesthetics, Inhalation; Animals; Brain Ischemia; Erythropoietin; Humans; Hyperbaric Oxygenation; Ischemic Preconditioning, Myocardial; Neuroprotective Agents; Nitrous Oxide; Perioperative Care; Spinal Cord Ischemia; Xenon

The review describes the capability oferythropoietin to activate and protect the CNS cells and myocardium against ischemia and hypoxia.

Topics: Animals; Brain Ischemia; Central Nervous System; Erythropoietin; Humans; Ischemic Preconditioning; Myocardium

The hemopoietic growth factor erythropoietin (EPO) has been recognized to be a multifunctional cytokine that plays a key role in ischemic preconditioning in the brain and heart. The EPO receptor is expressed widely in the kidney, and we review the important findings from the use of EPO in experimental models of acute renal failure that show that EPO reduces tubular cell death and hence the dysfunction induced by ischemia reperfusion injury, and we explore how these observations may be translated into the clinical arena.

Topics: Acute Kidney Injury; Animals; Apoptosis; Brain Ischemia; Erythropoietin; Humans; Kidney; Kidney Tubules, Proximal; Recombinant Proteins; Reperfusion Injury

Topics: Animals; Apoptosis; Brain Diseases; Brain Ischemia; Cerebral Hemorrhage; Cerebrovascular Disorders; Erythropoietin; Humans; Ischemia; Neurons; Neuroprotective Agents; Stroke

Topics: Animals; Brain Ischemia; Erythropoietin; Humans; Ischemic Preconditioning; Neuroprotective Agents

Perinatal brain injury is a major contributor to perinatal morbidity and mortality, and a considerable number of these children will develop long term neurodevelopmental disabilities. Despite the severe clinical and socio-economic significance and the advances in neonatal care over the past twenty years, no therapy yet exists that effectively prevents or ameliorates detrimental neurodevelopmental effects in cases of perinatal/neonatal brain injury. Our objective is to review recent evidence in relation to the pervading hypothesis for targeting time-dependent molecular and cellular repair mechanisms in the developing brain. In addition we review several potential neuroprotective strategies specific to the developing nervous system, with a focus on erythropoietin (Epo) because of its potential role in protection as well as repair.

Topics: Animals; Antioxidants; Apoptosis; Brain; Brain Injuries; Brain Ischemia; Erythropoietin; Free Radicals; Humans; Models, Biological; Neurons; Oxidants; Receptors, Erythropoietin; Wound Healing

The vascular events that happen during ischemic stroke worsen outcomes in patients by causing edema, hemorrhagic transformation, and general neurologic tissue compromise. In the past 2 decades, clinical trials in patients after ischemic stroke focused on neuroprotection, but these strategies have failed in providing actual benefit. Vascular protection represents a new field to be explored in acute ischemic stroke in order to develop new approaches to therapeutic intervention.. We identified tactics likely to provide vascular protection in patients with ischemic stroke. These tactics are based on knowledge of the molecular processes involved.. The pathologic processes due to vascular injury after an occlusion of a cerebral artery can be separated into acute (those occurring within hrs), subacute (hrs to days), and chronic (days to mo). Targets for intervention can be identified for all three stages. In the acute phase, superoxide is the predominant mediator, followed by inflammatory mediators and proteases in the subacute phase. In the chronic phase, proapoptotic gene products have been implicated. Many already-marketed therapeutic agents (statins, angiotensin modulators, erythropoietin, minocycline, and thiazolidinediones), with proven safety in patients, have been shown to have activity against some of the key targets of vascular protection.. Currently available pharmacologic agents are poised for clinical trials of vascular protection after acute ischemic stroke.

Topics: Acute Disease; Angiotensin-Converting Enzyme Inhibitors; Brain; Brain Edema; Brain Ischemia; Cerebral Hemorrhage; Chronic Disease; Erythropoietin; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Minocycline; Stroke; Thiazolidinediones; Thrombolytic Therapy

This review discusses the potential usefulness of several selected polypeptide growth factors as treatments for stroke. Distinctions between global vs. focal cerebral ischemia, permanent vs. temporary focal ischemia, and acute stroke vs. stroke recovery are first discussed. Potential routes of administration of growth factors are also considered. The growth factors basic fibroblast growth factor (bFGF), osteogenic protein-1 (OP-1), vascular endothelial growth factor (Veg-f), erythropoietin (EPO), and granulocyte colony stimulating factor (G-CSF) all show potential usefulness in animal models of acute stroke and stroke recovery. Two of these factors, bFGF and EPO, have reached human clinical trials for acute stroke, and the data are discussed. Future directions in this field are also discussed.

Topics: Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Brain Ischemia; Drug Administration Routes; Erythropoietin; Fibroblast Growth Factor 2; Granulocyte Colony-Stimulating Factor; Growth Substances; Humans; Mice; Rats; Stroke; Transforming Growth Factor beta; Vascular Endothelial Growth Factors

Erythropoietin, a hematopoietic growth-factor possessing manifold, potent neuroprotective properties, after multiple testing in cell culture and animal studies now gradually finds its way into clinical neuroscience. The first time this took place was in 1998 with a pilot study in stroke patients, the "Göttingen EPO-Stroke-Trial". This study was able to demonstrate that EPO is perfectly well tolerated and safe with this indication. Furthermore, the EPO-treated patients showed a significantly better outcome regarding their clinical progress as well as regarding the infarct size as observed by MRI, when compared to the placebo treated patients. At the moment a multicenter study is being carried out in Germany.

Topics: Anemia; Brain Ischemia; Cerebral Infarction; Clinical Trials as Topic; Erythropoietin; Hematinics; Humans; Hypoxia, Brain; Pilot Projects; Safety; 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

Erythropoietin (EPO) is a cytokine which is commonly associated with its central role in erythropoiesis. The clinical applications of the recombinant hormone are currently restricted to the treatment of anemia in renal failure and cancer. Recent studies, however, have suggested a new role for EPO as an anti-inflammatory and neuroprotective drug. EPO and its receptor are expressed in neurons, glial cells and brain capillary endothelial cells, and the system is upregulated in conditions of cerebral ischaemia and hypoxia. Animal studies have now established that intracerebroventricular administration of recombinant EPO exerts neuroprotection in models of stroke. The mechanisms appear to involve an upregulation of specific anti-apoptotic and anti-inflammatory pathways. In addition, neurotrophic and angiogenetic effects of EPO may contribute in a long latency protection. Interestingly, also systemic administration of recombinant EPO ameliorates neuronal damage after brain ischaemia, and prevents the loss of autoregulation of cerebral blood flow following experimental subarachnoid haemorrhage. Recombinant human EPO is a safe and non-toxic drug, and clinical studies are currently investigating the neuroprotective potential of EPO in humans.

Topics: Animals; Brain Ischemia; Erythropoietin; Humans; Injections, Intraventricular; Neurons; Neuroprotective Agents; Receptors, Erythropoietin; Recombinant Proteins; Stroke

Erythropoietin and its receptor function as primary mediators of the normal physiological response to hypoxia. Erythropoietin is recognized for its central role in erythropoiesis, but studies in which recombinant human erythropoietin (epoetin alfa) is injected directly into ischaemic rodent brain show that erythropoietin also mediates neuroprotection. Abundant expression of the erythropoietin receptor has been observed at brain capillaries, which could provide a route for circulating erythropoietin to enter the brain. In confirmation of this hypothesis, systemic administration of epoetin alfa before or up to 6 h after focal brain ischaemia reduced injury by 50-75%. Epoetin alfa also limited the extent of concussive brain injury, the immune damage in experimental autoimmune encephalomyelitis and excitotoxicity induced by kainate. Thus, systemically administered epoetin alfa in animal models has neuroprotective effects, demonstrating its potential use after brain injury, trauma and multiple sclerosis. It is evident that erythropoietin has biological activities in addition to increasing red cell mass. Given the excellent safety profile of epoetin alfa, clinical trials evaluating systemically administered epoetin alfa as a general neuroprotective treatment are warranted.

Topics: Acute Disease; Animals; Autoimmune Diseases; Brain; Brain Ischemia; Cerebral Cortex; Encephalitis; Epoetin Alfa; Erythropoietin; Humans; Kainic Acid; Neuroprotective Agents; Recombinant Proteins; Stroke; Wounds, Nonpenetrating

Erythropoietin (EPO) is a glycoprotein that has been shown to mediate response to hypoxia, and is most notably recognized for its central role in erythropoiesis. In a series of experiments using rodent models, the ability of systemically administered recombinant human erythropoietin (r-HuEPO, epoetin alfa) to cross the blood-brain barrier and affect the outcome of neuronal injury or cognitive function was evaluated. It was shown that EPO and EPO receptors are expressed at capillaries of the brain-periphery interface, and that systemically administered epoetin alfa crossed the blood-brain barrier. Compared with control animals, epoetin alfa significantly reduced tissue damage in an ischemic stroke model when administered 24 hours before inducing stroke, with significant protection still evident when epoetin alfa was administered 6 hours poststroke. Epoetin alfa reduced injury by blunt trauma when administered 24 hours before trauma, with a significantly smaller volume of tissue necrosis noted when compared with controls. The observation that epoetin alfa may reduce nervous system inflammation was confirmed when an experimental autoimmune encephalomyelitis model in which rats were shown to have significantly delayed onset and reduced severity of experimental autoimmune encephalomyelitis symptoms after treatment with epoetin alfa. Epoetin alfa also was shown to ameliorate the latency and severity of seizures, and significantly increase survival versus controls when exposed to kainate. These findings suggest future potential therapeutic uses for epoetin alfa beyond its current use to increase erythropoiesis.

Topics: Animals; Blood-Brain Barrier; Brain; Brain Ischemia; Central Nervous System; Encephalomyelitis; Epoetin Alfa; Erythropoietin; Humans; Kainic Acid; Mice; Models, Animal; Neuroprotective Agents; Receptors, Erythropoietin; Recombinant Proteins; Seizures

The aim of this study was to establish erythropoietin as a protective factor against brain ischemia during open heart surgery.. A total of 36 consecutive patients scheduled for revascularization heart surgery were included in the study. Of the patients 18 received 3 intravenous doses of recombinant human erythropoietin (rHuEpo, 24,000 IU) and 18 patients received a placebo. Magnetic resonance imaging (MRI) to detect new brain ischemic lesions was performed. Additionally, S100A, S100B, neuron-specific enolase A and B (NSE-A and B) and the concentration of antibodies against N‑methyl-D-aspartate receptors (NMDAR) to identify new neurological complications were determined.. Patients who received rHuEpo showed no postoperative ischemic changes in the brain on MRI images. In the control group 5 (27.8 %) new ischemic lesions were found. The NMDAR antibody concentration, S100A, S100B and NSE showed no significant differences between the groups for new cerebral ischemia. High levels of lactate before and after external aortic compression (p = 0.022 and p = 0.048, respectively) and duration of operation could predict new ischemic lesions (p = 0.009).. The addition of rHuEpo reduced the formation of lesions detectable by MRI in the brain and could be used clinically as neuroprotection in cardiac surgery.

Topics: Aged; Brain Ischemia; Cardiac Surgical Procedures; Coronary Artery Bypass; Double-Blind Method; Erythropoietin; Female; Humans; Magnetic Resonance Imaging; Male; Neuroprotective Agents; Placebo Effect; Premedication; Preoperative Care; Prognosis; Prospective Studies; Thoracotomy; Treatment Outcome

Mortality and disability following ischemic stroke (IS) remains unacceptably high with respect to the conventional therapies. This study tested the effect of erythropoietin (EPO) on long-term neurological outcome in patients after acute IS. This study aimed to evaluate the safety and efficacy of two consecutive doses of EPO (5,000 IU/dose, subcutaneously administered at 48 hours and 72 hours after acute IS) on improving the 90-day combined endpoint of recurrent stroke or death that has been previously reported. A secondary objective was to evaluate the long-term (that is, five years) outcome of patients who received EPO.. This was a prospective, randomized, placebo-controlled trial that was conducted between October 2008 and March 2010 in a tertiary referral center. IS stroke patients who were eligible for EPO therapy were enrolled into the study.. The results showed that long-term recurrent stroke and mortality did not differ between group 1 (placebo-control; n = 71) and group 2 (EPO-treated; n = 71). Long-term Barthel index of <35 (defining a severe neurological deficit) was lower in group 2 than group 1 (P = 0.007). Multiple-stepwise logistic-regression analysis showed that EPO therapy was significantly and independently predictive of freedom from a Barthel index of <35 (P = 0.029). Long-term major adverse neurological event (MANE; defined as: death, recurrent stroke, or long-term Barthel index < 35) was lower in group 2 than group 1 (P = 0.04). Log-Rank test showed that MANE-free rate was higher in group 2 than group 1 (P = 0.031). Multiple-stepwise Cox-regression analysis showed that EPO therapy and higher Barthel Index at day 90 were independently predictive of freedom from long-term MANE (all P <0.04).. EPO therapy significantly improved long-term neurological outcomes in patients after IS.. ISRCTN71371114 . Registered 10 October 2008.

Topics: Aged; Brain Ischemia; Endothelial Progenitor Cells; Erythropoietin; Female; Humans; Male; Middle Aged; Multivariate Analysis; Neuroprotective Agents; Prospective Studies; Stroke; Time Factors; Treatment Outcome

Preclinical studies suggest that growth factors in the early days after stroke improve final outcome. A prior study found three doses of human choriogonadotropin alfa followed by three doses of erythropoietin to be safe after stroke in humans. A proof of concept trial (REGENESIS) was initiated but placed on regulatory hold during review of an erythropoietin neuroprotective trial. Due to financial constraints, the trial was largely moved to India, using lower erythropoietin doses, as the REGENESIS-LED trial.. Entry criteria included National Institutes of Health Stroke Scale 8-20, supratentorial ischemic stroke, and 24-48 h poststroke at start of therapy. Patients were randomized to three QOD doses of subcutaneous human choriogonadotropin alfa followed by three QD doses of intravenous erythropoietin (three escalating dose cohorts, 4000-20,000 IU/dose) vs. placebo. Primary outcomes were safety and neurological recovery.. The study was halted early by the sponsor after 96 enrollees. There was no significant difference across treatment groups in the proportion of patients experiencing death, serious adverse events, or any adverse event. There was no significant difference in National Institutes of Health Stroke Scale score change from baseline to Day 90 between placebo and active treatment, whether active cohorts were analyzed together or separately, and no exploratory secondary measure of neurological recovery showed a significant difference between groups.. Administration of human choriogonadotropin alfa followed by erythropoietin is safe after a new ischemic stroke. At the doses studied, placebo and active groups did not differ significantly in neurological recovery. Study limitations, such as the use of multiple assessors, differences in rehabilitation care, and being underpowered to show efficacy, are discussed.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Brain Ischemia; Chorionic Gonadotropin; Cohort Studies; Double-Blind Method; Epoetin Alfa; Erythropoietin; Female; Hematinics; Humans; International Cooperation; Magnetic Resonance Imaging; Male; Middle Aged; Nervous System Diseases; Outcome Assessment, Health Care; Recombinant Proteins; Severity of Illness Index; Stroke; Time Factors; Tomography Scanners, X-Ray Computed; Treatment Outcome; Young Adult

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

Cardiac surgery and cardiopulmonary bypass (CPB) induce ischemia-reperfusion and subsequent cellular injury with inflammatory reaction. Clinical and experimental studies suggest that recombinant human erythropoietin (EPO) independently of its erythropoietic effect may be used as a cytoprotective agent against ischemic injury. We tested the hypothesis that one large dose of EPO administered shortly before CPB prevents the elevation of cardiac and cerebral ischemic blood markers as well as the systemic inflammatory response induced by cardiac surgery with CBP through this randomized double-blind placebo-controlled pilot trial. Fifty patients scheduled for coronary artery bypass graft (CABG) surgery with CPB were randomly allocated to EPO or control groups. EPO (800 IU/kg intravenously) or placebo (saline) was administered before CPB. The primary end point was to study the effect of EPO administration on several blood markers of myocardial and cerebral ischemia in relation to CABG with CPB. In both groups, surgery increased plasma concentrations of cardiac (troponin T, NT-proBNP, and creatine kinase MB) and cerebral (S100β protein) markers ischemic as well as the pro-inflammatory marker interleukin-6. Compared with the placebo, EPO administration before CPB did not prevent an increase of all these markers following CPB. In conclusion, one large dose of EPO, given shortly before CPB, did not protect against cardiac and cerebral ischemia and inflammatory response occurring during CABG surgery with CPB. Although the long-term clinical implications remain unknown, the findings do not support use of EPO at this dose as a cytoprotective agent in patients undergoing cardiac surgery.

Topics: Aged; Biomarkers; Brain Ischemia; Cardiopulmonary Bypass; Cytokines; Cytoprotection; Data Interpretation, Statistical; Dose-Response Relationship, Drug; Double-Blind Method; Electrocardiography; Erythropoietin; Female; Humans; Male; Middle Aged; Myocardial Ischemia; Myocardial Reperfusion Injury; Nerve Growth Factors; Pilot Projects; Recombinant Proteins; Reperfusion Injury; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Treatment Outcome; Troponin T

Currently, no data on the optimal time point after acute ischemic stroke (IS) at which high-sensitivity C-reactive protein (hs-CRP) level is most predictive of unfavorable outcome. We tested the hypothesis that hs-CRP levels during both acute (48 h after IS) and convalescent (21 days after IS) phases are equally important in predicting 90-day clinical outcome after acute IS. We further evaluated the impact of erythropoietin (EPO), an anti-inflammatory agent, on level of hs-CRP after acute IS.. Totally 160 patients were prospectively randomized to receive either EPO therapy (group 1, n = 80) (5,000 IU each time, subcutaneously) at 48 h and 72 h after acute IS, or placebo (group 2, n = 80). Serum level of hs-CRP was determined using ELISA at 48 h and on day 21 after IS and once in 60 healthy volunteers.. Serum level of hs-CRP was substantially higher in all patients with IS than in healthy controls at 48 h and day 21 after IS (all p < 0.001). Levels of hs-CRP did not differ between group 1 and 2 at 48 h and day 21 after IS (all p > 0.5). Multivariate analysis showed that hs-CRP levels (at 48 h and day 21) were independently predictive of 90-day major adverse neurological event (MANE) (defined as recurrent stroke, NIHSS≥8, or death) (all p < 0.03), whereas EPO therapy was independently predictive of reduced 90-day MANE (all p < 0.02).. EPO therapy which was independently predictive of freedom from 90-day MANE did not alter the crucial role of hs-CRP levels measured at 48 h and 21-day in predicting unfavorable clinical outcome after IS.

Topics: Aged; Brain Ischemia; C-Reactive Protein; Case-Control Studies; Convalescence; Erythropoietin; Female; Humans; Logistic Models; Male; Middle Aged; Multivariate Analysis; ROC Curve; Statistics, Nonparametric; Stroke; Treatment Outcome

The German Multicenter EPO Stroke Trial, which investigated safety and efficacy of erythropoietin (EPO) treatment in ischemic stroke, was formally declared a negative study. Exploratory subgroup analysis, however, revealed that patients not receiving thrombolysis most likely benefited from EPO during clinical recovery, a result demonstrated in the findings of the Göttingen EPO Stroke Study. The present work investigated whether the positive signal on clinical outcome in this patient subgroup was mirrored by respective poststroke biomarker profiles. All patients of the German Multicenter EPO Stroke Trial nonqualifying for thrombolysis were included if they (a) were treated per protocol and (b) had at least two of the five follow-up blood samples for circulating damage markers drawn (n = 163). The glial markers S100B and glial fibrillary acid protein (GFAP) and the neuronal marker ubiquitin C-terminal hydrolase (UCH-L1) were measured by enzyme-linked immunosorbent assay in serum on d 1, 2, 3, 4 and 7 poststroke. All biomarkers increased poststroke. Overall, EPO-treated patients had significantly lower concentrations (area under the curve) over 7 d of observation, as reflected by the composite score of all three markers (Cronbach α = 0.811) and by UCH-L1. S100B and GFAP showed a similar tendency. To conclude, serum biomarker profiles, as an outcome measure of brain damage, corroborate an advantageous effect of EPO in ischemic stroke. In particular, reduction in the neuronal damage marker UCH-L1 may reflect neuroprotection by EPO.

Topics: Aged; Area Under Curve; Biomarkers; Brain Ischemia; Epoetin Alfa; Erythropoietin; Female; Humans; Male; Neuroprotective Agents; Recombinant Proteins; Stroke; Treatment Outcome

Animal data suggest the use of beta-human chorionic gonadotropin followed by erythropoietin to promote brain repair after stroke. The current study directly translated these results by evaluating safety of this sequential growth factor therapy through a 3-center, single-dose, open-label, noncontrolled, Phase IIa trial.. Patients with ischemic stroke 24 to 48 hours old and National Institutes of Health Stroke Scale score of 6 to 24 started a 9-day course of beta-human chorionic gonadotropin (once daily on Days 1, 3, and 5 of study participation) followed by erythropoietin (once daily on Days 7, 8, and 9 of study participation). This study also evaluated performance of serially measured domain-specific end points.. A total of 15 patients were enrolled. Two deaths occurred, neither related to study medications. No safety concerns were noted among clinical or laboratory measures, including screening for deep vein thrombosis and serial measures of serum hemoglobin. In several instances, domain-specific end points provided greater insight into impairments as compared with global outcome measures.. Results support the safety of this sequential, 2-growth factor therapy initiated 24 to 48 hours after stroke onset.

Topics: Adult; Aged; Aged, 80 and over; Brain Ischemia; Chorionic Gonadotropin, beta Subunit, Human; Drug Therapy, Combination; Erythropoietin; Female; Hemoglobins; Humans; Male; Middle Aged; Stroke; Venous Thrombosis; Young Adult

Delayed ischemic deficits (DIDs), a major source of disability following aneurysmal subarachnoid hemorrhage (aSAH), are usually associated with severe cerebral vasospasm and impaired autoregulation. Systemic erythropoietin (EPO) therapy has been demonstrated to have neuroprotective properties acting via EPO receptors on cerebrovascular endothelia and ischemic neurons. In this trial, the authors explored the potential neuroprotective effects of acute EPO therapy following aSAH.. Within 72 hours of aSAH, 80 patients (age range 24-82 years) were randomized to receive intravenous EPO (30,000 U) or placebo every 48 hours for a total of 90,000 U. Primary end points were the incidence, duration, and severity of vasospasm and impaired autoregulation on transcranial Doppler ultrasonography. Secondary end points were incidence of DIDs and outcome at discharge and at 6 months.. Randomization characteristics were balanced except for age, with the EPO group being older (mean age 59.6 vs 53.3 years, p=0.034). No differences were demonstrated in the incidence of vasospasm and adverse events; however, patients receiving EPO had a decreased incidence of severe vasospasm from 27.5 to 7.5% (p=0.037), reduced DIDs with new cerebral infarcts from 40.0 to 7.5% (p=0.001), a shortened duration of impaired autoregulation (ipsilateral side, p<0.001), and more favorable outcome at discharge (favorable Glasgow Outcome Scale score, p=0.039). Among the 71 survivors, the EPO group had fewer deficits measured with National Institutes of Health Stroke Scale (median Score 2 vs 6, p=0.008).. This preliminary study showed that EPO seemed to reduce delayed cerebral ischemia following aSAH via decreasing severity of vasospasm and shortening impaired autoregulation.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Blood Pressure; Blood Transfusion; Brain Ischemia; Double-Blind Method; Erythropoietin; Female; Follow-Up Studies; Homeostasis; Humans; Male; Middle Aged; Neuroprotective Agents; Placebos; Subarachnoid Hemorrhage; Treatment Outcome; Ultrasonography, Doppler, Transcranial; Vasospasm, Intracranial; Young Adult

Numerous preclinical findings and a clinical pilot study suggest that recombinant human erythropoietin (EPO) provides neuroprotection that may be beneficial for the treatment of patients with ischemic stroke. Although EPO has been considered to be a safe and well-tolerated drug over 2 decades, recent studies have identified increased thromboembolic complications and/or mortality risks on EPO administration to patients with cancer or chronic kidney disease. Accordingly, the double-blind, placebo-controlled, randomized German Multicenter EPO Stroke Trial (Phase II/III; ClinicalTrials.gov Identifier: NCT00604630) was designed to evaluate efficacy and safety of EPO in stroke.. This clinical trial enrolled 522 patients with acute ischemic stroke in the middle cerebral artery territory (intent-to-treat population) with 460 patients treated as planned (per-protocol population). Within 6 hours of symptom onset, at 24 and 48 hours, EPO was infused intravenously (40,000 IU each). Systemic thrombolysis with recombinant tissue plasminogen activator was allowed and stratified for.. Unexpectedly, a very high number of patients received recombinant tissue plasminogen activator (63.4%). On analysis of total intent-to-treat and per-protocol populations, neither primary outcome Barthel Index on Day 90 (P=0.45) nor any of the other outcome parameters showed favorable effects of EPO. There was an overall death rate of 16.4% (n=42 of 256) in the EPO and 9.0% (n=24 of 266) in the placebo group (OR, 1.98; 95% CI, 1.16 to 3.38; P=0.01) without any particular mechanism of death unexpected after stroke.. Based on analysis of total intent-to-treat and per-protocol populations only, this is a negative trial that also raises safety concerns, particularly in patients receiving systemic thrombolysis.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Brain Ischemia; Double-Blind Method; Drug Administration Schedule; Drug Interactions; Drug-Related Side Effects and Adverse Reactions; Erythropoietin; Female; Humans; Infarction, Middle Cerebral Artery; Injections, Intravenous; Male; Middle Aged; Mortality; Neuroprotective Agents; Patient Selection; Placebo Effect; Recombinant Proteins; Stroke; Tissue Plasminogen Activator; Treatment Outcome; Young Adult

Topics: Adult; Antioxidants; Brain Chemistry; Brain Ischemia; Catalysis; Chelating Agents; Erythropoietin; Ferritins; Ferrous Compounds; Hematocrit; Hemoglobins; Humans; Iron; Male; Neuroprotective Agents; Oxidation-Reduction; Oxidative Stress; Recombinant Proteins; Transferrin

Within the hippocampus, the CA1 and dentate gyrus (DG) regions are considered the most and the least susceptible to damage by cerebral ischemia, respectively. In addition, it has been tested that rHuEPO exhibits neuroprotective properties. This work investigates the effect of different intranasal doses of rHuEPO, applied in different ischemic post-damage times in the DG, and the effect of the rHuEPO on astroglial reactivity after cerebral ischemia. Additionally, an effective dose for neuroprotection and an administration time was used to evaluate gene and protein expression changes of EPO and EPOR in the DG region. We observed a considerable loss of cells on the granular layer and an increased number of GFAP immunoreactive cells in this region only 72 h after the onset of ischemia/damage. When rHuEPO was administered, the number of morphologically abnormal cells and immunoreactivity decreased. In the analysis of protein and gene expression, there is no correlation between expression level of these molecules, although the rHuEPO amplifies the response to ischemia of EPO and EPOR gene for each evaluated time; in the case of the protein only at 2 h this effect was observed. We demonstrated the susceptibility of the DG to ischemia; so granular cells damage was observed, moreover of the astrocytic response, which is accompanied by molecular changes in signaling mediated by rHuEPO intranasal administration.

Topics: Administration, Intranasal; Brain Ischemia; Cerebral Infarction; Dentate Gyrus; Erythropoietin; Gliosis; Humans

Previous studies have indicated that EPO maintains the M2 microglia phenotype that contributes to white matter repair after ischemic stroke in young mice (2 months old). However, the underlying mechanisms that regulate microglial polarization are poorly defined. This study investigated the neuroprotective effects of nonerythropoietic mutant EPO (MEPO) on white matter and the underlying mechanism in middle-aged (9-month-old) male mice following cerebral ischemia. Middle-aged male C57 BL/6 mice were treated with MEPO (5000 IU/kg) or vehicle after middle cerebral artery occlusion (MCAO) and reperfusion. The specific inhibitor AG490 was used to block the JAK2/STAT3 pathway. Neurological function was assessed by beam walking and adhesive removal tests. Immunofluorescence staining and western blotting were used to assess the severity of white matter injury, phenotypic changes in the microglia and the expression of the signaling molecules. MEPO significantly improved neurobehavioral outcomes, alleviated brain tissue loss, and ameliorated white matter injury after MCAO compared with the vehicle group. Moreover, MEPO promoted oligodendrogenesis by shifting microglia toward M2 polarization by promoting JAK2/STAT3 activation and inhibiting the expression of C/EBPβ at 14 days after cerebral ischemia-reperfusion. However, the MEPO's effect on microglial M2 polarization and oligodendrogenesis was largely suppressed by AG490 treatment. Collectively, these data indicate that MEPO treatment improves white matter integrity after cerebral ischemia, which may be partly explained by MEPO facilitating microglia toward the beneficial M2 phenotype to promote oligodendrogenesis via JAK2/STAT3 and the C/EBPβ signaling pathway. This study provides novel insight into MEPO treatment for ischemic stroke.

Topics: Animals; Behavior, Animal; Brain Ischemia; CCAAT-Enhancer-Binding Protein-beta; Cell Polarity; Erythropoietin; Janus Kinase 2; Male; Mice; Mice, Inbred C57BL; Microglia; Mutation; Oligodendroglia; Recovery of Function; Reperfusion Injury; Signal Transduction; STAT3 Transcription Factor; Steroid Isomerases; Tyrphostins; White Matter

The level of erythropoietin (EPO) and vascular endothelial growth factor (VEGF-A) was investigated in blood serum and brain of Wistar rats by the enzyme immunoassay with specific rat antibodies. These growth factors are actively studied as biomarkers of ischemia or cytoprotection, as well as targets for agents initiating preconditioning (PreC). Pharmacological (amtizol administration), hypoxic (hypobaric hypoxia), and combined PreC (amtizol+hypobaric hypoxia) were used as neuroprotective approaches in this experimental work. In normoxia groups blood and brain tissue were collected 1 h (early period) or 48 h (delayed period) after the PreC. In addition we studied groups of animals with cerebral ischemia (induced by bilateral ligation of the common carotid arteries) 1 h and 48 h after the combined PreC: the levels of EPO and VEGF-A in the blood serum and the brain supernatant were determined in one day after the ligation. Experiments have shown that amtizol (3,5-diamino-1,2,4-thiadiazole) in normoxia increased the EPO level in the brain, and did not change EPO in blood serum and VEGF-A levels in both serum and the brain. A three-day (60 min exposure with 48 h intervals) hypobaric hypoxia (410 mm Hg) increased EPO and VEGF-A in the blood serum and brain tissues, but in most experimental groups differences did not reach the level of statistical significance versus intact control. The combined PreC was accompanied by a significant increase of EPO and VEGF-A in normoxia conditions both in early and delayed period of PreC. In cerebral ischemia the EPO level in the blood serum and brain tissues was higher than in intact control. The serum level of VEGF-A of the ischemia control group tended to increase while the brain level of VEGF-A remained basically unchanged versus the intact control group. In combined PreC before ischemia, the EPO level was lower in serum as compared with the ischemia control in the delayed PreC period, but did not differ significantly from the ischemia control in serum in early period and in brain tissues in both PreC periods. The VEGF-A level in the groups of combined PreC was significantly lower in serum as compared with the ischemia control in both the early and delayed PreC; in brain tissues it did not differ from the level of both the intact and ishemia control in early PreC period and was higher than in both control groups in the delayed PreC period.. Soderzhanie éritropoétina (EPO) i faktora rosta éndoteliia sosudov (VEGF-A) v syvorotke krovi i golovnom mozge (GM) krys linii Vistar izuchali s pomoshch'iu immunofermentnogo analiza s ispol'zovaniem spetsificheskikh krysinykh antitel. Dannye rostovye faktory aktivno issleduiutsia v kachestve biomarkerov kletochnogo povrezhdeniia ili, naprotiv, tsitoprotektsii, a takzhe misheneĭ dlia sredstv, sposobnykh initsiirovat' prekonditsionirovanie (PreK). V kachestve izuchaemykh neĭroprotektivnykh podkhodov ispol'zovali farmakologicheskoe (vvedenie amtizola), gipoksicheskoe (gipobaricheskaia gipoksiia) i kombinirovannoe (amtizol+gipobaricheskaia gipoksiia) PreK. Cherez 1 ch (ranniĭ period) i 48 ch (pozdniĭ period) posle okonchaniia PreK proizvodili zabor materiala (seriia opytov v usloviiakh normoksii). V drugoĭ serii opytov zhivotnym cherez 1 ch i 48 ch posle okonchaniia PreK modelirovali ishemiiu GM putem pereviazki obshchikh sonnykh arteriĭ i cherez sutki posle operatsii opredeliali soderzhanie EPO i VEGF-A. Pri sravnenii éffektivnosti farmakologicheskogo i gipoksicheskogo PreK v vozmozhnoĭ induktsii EPO i VEGF-A pri normoksii vyiavleno, chto amtizol (3,5-diamino-1,2,4-tiadiazol) povyshaet uroven' EPO v GM, ne izmeniaia soderzhanie EPO v syvorotke krovi i VEGF-A v oboikh substratakh. Trekhkratnaia (60 min ékspozitsii s intervalom 48 ch) gipobaricheskaia gipoksiia (410 mm rt.st.) povyshaet soderzhanie EPO i VEGF-A v syvorotke krovi i GM, no v bol'shinstve opytnykh grupp razlichiia s intaktnym kontrolem okazyvaiutsia statisticheski neznachimymi. Kombinirovannoe PreK privodit k znachimomu povysheniiu soderzhaniia EPO i VEGF-A pri normoksii kak v rannem, tak i pozdnem periode PreK. Pri ishemii GM v kontrol'noĭ gruppe soderzhanie EPO kak v syvorotke krovi, tak i v tkaniakh GM bylo vyshe znacheniĭ intaktnogo kontrolia. Pri otsenke soderzhaniia VEGF-A v kontrol'noĭ gruppe s ishemieĭ obnaruzhena tendentsiia k povysheniiu ego v syvorotke krovi i otsutstvie znachimykh razlichiĭ v tkaniakh GM. Pri ispol'zovanii do operatsii ishemii kombinirovannogo PreK soderzhanie EPO bylo nizhe v syvorotke krovi v sravnenii s kontrolem s ishemieĭ v pozdniĭ period PreK, ne otlichalos' znachimo ot znacheniĭ kontrolia s ishemiĭ v syvorotke krovi v ranniĭ period i v tkaniakh GM v oba perioda PreK. Soderzhanie VEGF-A pri ispol'zovanii kombinirovannogo PreK bylo znachimo nizhe v syvorotke krovi v sravnenii s kontrolem s ishemieĭ kak v ranniĭ, tak i v pozdniĭ periody PreK, v tkaniakh GM krys n

Topics: Animals; Brain Ischemia; Erythropoietin; Hypoxia; Rats; Rats, Wistar; Vascular Endothelial Growth Factor A

In this study, the molecular mechanism by which EPO regulates the angiogenesis after cerebral ischemia through AMPK-KLF2 signaling pathway was investigated.. Sixty healthy, male, C57BL/6 mice were randomly divided into three groups of 20 mice: a sham group, the middle cerebral artery occlusion (MCAO) group, and a MCAO+EPO treatment group. The MCAO model was established using a modified ZeaLonga method. Mice in the EPO treatment group were injected with EPO immediately after reperfusion (5000 IU/kg), and EPO was injected the following day. The number of mouse deaths and neurologic function scores were recorded during the experiment. On day 7 after cerebral ischemia, brain tissue proteins were extracted. The following proteins expressions were detected by western blot assay: EPO, vascular endothelial growth factor (VEGE), vascular endothelial growth factor receptor (KDR), adenosine activated protein kinase (AMPK), and alpha HIF-1α alpha (HIF-1α), KLF2 and nitric oxide synthase (eNOS).. Compared with the MCAO group, the survival rate of mice in the EPO group was significantly improved and neurological function was significantly improved (P < 0.01). Western blot results showed that the content of EPO in brain tissue in MCAO group significantly increased compared with sham group. The content of EPO in the brain tissue of mice in the MCAO+EPO treatment group was significantly higher than in that of the MCAO group, which indicates that EPO increased the content of EPO in mouse brain tissue. Compared with the sham group, the protein expression of vascular endothelial growth factor (VEGE) and its receptor (KDR) in brain tissue of the MCAO group significantly decreased. However, the protein expression of VEGE and its receptor KDR in brain tissue of rats treated with MCAO+EPO was significantly higher than in that of the MCAO group. Thus, in this study, EPO was associated with vascular endothelial differentiation after cerebral ischemia in mice. The results of AMPK and KLF2 showed that the expression levels of AMPK and KLF2 in brain tissues of MCAO group mice significantly decreased compared with the sham group. However, the expression levels of AMPK and KLF2 in brain tissues of mice treated with MCAO+EPO were significantly higher than those in the MCAO group. Thus, EPO can activate AMPK and upregulate the expression of the transcription factor KLF2. The protein expression of HIF-1α in the brain tissue of mice in the MCAO group significantly increased compared with the sham group. However, the expression of HIF-1α in mice brain tissues in the MCAO+EPO treatment group was significantly lower than in that of the MCAO group, indicating that EPO was involved in regulating HIF-1α expression. The eNOS results showed that, compared with Sham group, the protein expression of eNOS in brain tissue of MCAO group mice significantly decreased. In the MCAO+EPO treatment group, the protein expression of eNOS was significantly higher in the brain tissue of the mice than in that of the MCAO group, indicating that EPO was involved in the synthesis of NO and promoted the angiogenesis.. EPO promotes VEGE and its receptor (KDR) expression and participates in the regulation of HIF-1α and eNOS protein expression through the activation of AMPK-KLF2 signaling pathways to promote new vascular development after cerebral ischemia.

Topics: AMP-Activated Protein Kinases; Angiogenesis Modulating Agents; Animals; Brain; Brain Ischemia; Erythropoietin; Gene Expression Regulation; Kruppel-Like Transcription Factors; Male; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Random Allocation; Signal Transduction

Previously study has proved the non-erythropoietic mutant erythropoietin (MEPO) exerted neuroprotective effects against ischemic cerebral injury, with an efficacy similar to that of wild-type EPO. This study investigates its effects on neurogenesis, angiogenesis, and gliogenesis in cerebral ischemic mice. Male C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO) and reperfusion. EPO (5000 U/kg), MEPO (5000 U/kg) or equal volume of normal saline was injected intraperitoneally. Neurological function was evaluated by Rota-rod test, Neurological severity scores (NSS) and Adhesive removal test. After ischemia and reperfusion (I/R), the survival rate, brain tissue loss, neurogenesis, angiogenesis and gliogenesis were detected by Nissl staining, Immunofluorescence and Western blot, respectively. The results shown that MEPO significantly increased survival rate, reduced brain tissue loss, and improved neurological function after MCAO (P < 0.05). Furthermore, MEPO obviously enhanced the proliferation of neuronal precursors (DCX) and promoted its differentiation into mature neurons (NeuN) (P < 0.05). In addition, compared to normal saline treatment mice, MEPO increased the number of BrdU-positive cells in the cerebral vasculature (P < 0.05). Whereas, MEPO treatment also reduced the numbers of newly generated astrocytes (GFAP) and microglia (Iba1) (P < 0.05). Among all the tests in this study, there was no significant difference between EPO group and MEPO group. Taken together, MEPO promoted the regeneration of neurons and blood vessels in peripheral area of infarction, and suppressed the gliogenesis, thus promoting neurogenesis, improving neurological function and survival rate. Our findings suggest that the MEPO may be a therapeutic drug for ischemic stroke intervention.

Topics: Animals; Brain Ischemia; Cell Proliferation; Doublecortin Protein; Erythropoietin; Male; Mice; Mice, Inbred C57BL; Mutation; Neovascularization, Physiologic; Neurogenesis; Neuroglia; Neuroprotection; Stroke

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

Erythropoietin, a multitarget molecule exhibited neuroprotective properties, especially against cerebral ischemia. However, little effort has been made to determinate both the administration pathway and doses that diminishes neuronal damage. In this study, we investigate the effect on CA1 region of different intranasal doses of rHuEPO (500, 1000 and 2500 IU/kg) applied in distinct post-damage times (1, 6, and 24 h) against ischemic cellular damage. Furthermore, most effective dose and time were used to evaluate gen and protein expression changes in 3 key molecules (EPO, EPOR, and βcR). We established that CA1-region present histopathological damage in this ischemia model and that rHuEPO protects cells against damage, particularly at 1000 IU dose. Molecular data shows that EPO and EPOR gene expression are upregulated in a short term after damage treatment with rHuEPO (1 h); oppositely, BcR is upregulated in ischemic and Isc + EPO. Protein expression data displays no changes on EPO expression in evaluated times after treatment, but a tendency to increase 24 h after damage; in the opposite way, EPOR is upregulated significantly 6 h after treatment and this effect last until 24 h. So, our data suggest that a single intranasal dose of rHuEPO (1 h post-injury) provides histological neurorestoration in CA1 hippocampal region, even if we did not observe a dose-dependent dose effect, the medium dose evaluated (1000 UI/kg of b.w.) was more effective and sufficient for induces molecular changes that provides a platform for neuroprotection.

Topics: Administration, Intranasal; Animals; Brain Ischemia; CA1 Region, Hippocampal; Erythropoietin; Humans; Male; Neuroprotective Agents; Rats; Rats, Wistar

Among the properties of lactoferrin (LF) are bactericidal, antianemic, immunomodulatory, antitumour, antiphlogistic effects. Previously we demonstrated its capacity to stabilize in vivo HIF-1-alpha and HIF-2-alpha, which are redox-sensitive multiaimed transcription factors. Various tissues of animals receiving recombinant human LF (rhLF) responded by expressing the HIF-1-alpha target genes, hence such proteins as erythropoietin (EPO), ceruloplasmin, etc. were synthesized in noticeable amounts. Among organs in which EPO synthesis occurred were brain, heart, spleen, liver, kidneys and lungs. Other researchers showed that EPO can act as a protectant against severe brain injury and status epilepticus in rats. Therefore, we tried rhLF as a protector against the severe neurologic disorders developed in rats, such as the rotenone-induced model of Parkinson's disease and experimental autoimmune encephalomyelitis as a model of multiple sclerosis, and observed its capacity to mitigate the grave symptoms. Moreover, an intraperitoneal injection of rhLF into mice 1 h after occlusion of the medial cerebral artery significantly diminished the necrosis area measured on the third day in the ischaemic brain. During this period EPO was synthesized in various murine tissues. It was known that EPO induces nuclear translocation of Nrf2, which, like HIF-1-alpha, is a transcription factor. In view that under conditions of hypoxia both factors demonstrate a synergistic protective effect, we suggested that LF activates the Keap1/Nrf2 signaling pathway, an important link in proliferation and differentiation of normal and malignant cells. J774 macrophages were cultured for 3 days without or in the presence of ferric and ferrous ions (RPMI-1640 and DMEM/F12, respectively). Then cells were incubated with rhLF or Deferiprone. Confocal microscopy revealed nuclear translocation of Nrf2 (the key event in Keap1/Nrf2 signaling) induced by apo-rhLF (iron-free, RPMI-1640). The reference compound Deferiprone (iron chelator) had the similar effect. Upon iron binding (in DMEM/F12) rhLF did not activate the Keap1/Nrf2 pathway. Added to J774, apo-rhLF enhanced transcription of Nrf2-dependent genes coding for glutathione S-transferase P and heme oxygenase-1. Western blotting revealed presence of Nrf2 in mice brain after 6 days of oral administration of apo-rhLF, but not Fe-rhLF or equivalent amount of PBS. Hence, apo-LF, but not holo-LF, induces the translocation of Nrf2 from cytoplasm to the nucle

Topics: Animals; Brain Ischemia; Encephalomyelitis, Autoimmune, Experimental; Erythropoietin; Female; Humans; Lactoferrin; Male; Mice; Mice, Inbred BALB C; Multiple Sclerosis; Neuroprotection; Neuroprotective Agents; NF-E2-Related Factor 2; Parkinson Disease; Rats; Rats, Wistar; Recombinant Proteins

Salidroside is being investigated for its therapeutic potential in stroke because it is neuroprotective over an extended therapeutic window of time. In the present study, we investigated the mechanisms underlying the anti-inflammatory effects of salidroside (50 mg/kg intraperitoneally) in rats, given 1 h after reperfusion of a middle cerebral artery that had been occluded for 2 h. After 24 h, we found that salidroside increased the neuronal nuclear protein NeuN and reduced the marker of microglia and macrophages CD11b in the peri-infarct area of the brain. Salidroside also decreased IL-6, IL-1β, TNF-α, CD14, CD44, and iNOs mRNAs. At the same time, salidroside increased the ratio of phosphorylated protein kinase B (p-Akt) to total Akt. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 prevented this increase in p-Akt and reversed the inhibitory effects of salidroside on CD11b and inflammatory mediators. Salidroside also elevated the protein levels of hypoxia-inducible factor (HIF) subunits HIF1α, HIF2α, HIF3α, and of erythropoietin (EPO). The stimulatory effects of salidroside on HIFα subunits were blocked by LY294002. Moreover, YC-1, a HIF inhibitor, abolished salidroside-mediated increase of HIF1α and prevented the inhibitory effects of salidroside on CD11b and inflammatory mediators. Taken together, our results provide evidence for the first time that all three HIFα subunits and EPO can be regulated by PI3K/Akt in cerebral tissue, and that salidroside entrains this signaling pathway to induce production of HIFα subunits and EPO, one or more of which mediate the anti-inflammatory effects of salidroside after cerebral IRI.

Topics: Animals; Anti-Inflammatory Agents; Brain Ischemia; Erythropoietin; Glucosides; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation; Phenols; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Reperfusion Injury; Signal Transduction

The human brain requires uninterrupted delivery of blood-borne oxygen and nutrients to sustain its function. Focal ischemia, particularly, ischemic stroke, and global ischemia imposed by cardiac arrest disrupt the brain's fuel supply. The resultant ATP depletion initiates a complex injury cascade encompassing intracellular Ca

Topics: Animals; Brain; Brain Ischemia; Erythropoietin; Humans; Neuroprotective Agents; Oxidative Stress; Reperfusion Injury

Erythropoietin (EPO) has been well known as a hematopoietic cytokine over the past decades. However, recent reports have demonstrated that EPO plays a neuroprotective role in the central nervous system, and EPO has been considered as a therapeutic target in neurodegenerative diseases such as ischemic stroke. Despite the neuroprotective effect of EPO, clinical trials have shown its unexpected side effects, including undesirable proliferative effects such as erythropoiesis and tumor growth. Therefore, the development of EPO analogs that would confer neuroprotection without adverse effects has been attempted. In this study, we examined the potential of a novel EPO-based short peptide, MK-X, as a novel drug for stroke treatment in comparison with EPO. We found that MK-X administration with reperfusion dramatically reduced brain injury in an in vivo mouse model of ischemic stroke induced by middle cerebral artery occlusion, whereas EPO had little effect. Similar to EPO, MK-X efficiently ameliorated mitochondrial dysfunction followed by neuronal death caused by glutamate-induced oxidative stress in cultured neurons. Consistent with this effect, MK-X significantly decreased caspase-3 cleavage and nuclear translocation of apoptosis-inducing factor induced by glutamate. MK-X completely mimicked the effect of EPO on multiple activation of JAK2 and its downstream PI3K/AKT and ERK1/2 signaling pathways, and this signaling process was involved in the neuroprotective effect of MK-X. Furthermore, MK-X and EPO induced similar changes in the gene expression patterns under glutamate-induced excitotoxicity. Interestingly, the most significant difference between MK-X and EPO was that MK-X better penetrated into the brain across the brain-blood barrier than did EPO. In conclusion, we suggest that MK-X might be used as a novel drug for protection from brain injury caused by ischemic stroke, which penetrates into the brain faster in comparison with EPO, even though MK-X and EPO have similar protective effects against excitotoxicity.

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Caspase 3; Cell Death; Cerebral Cortex; Cerebrovascular Disorders; Embryo, Mammalian; Erythropoietin; Gene Expression Regulation; Glutamic Acid; Janus Kinase 2; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Middle Cerebral Artery; Mitochondria; Neurons; Neuroprotective Agents; Peptides; Permeability; Phosphatidylinositol 3-Kinases; Primary Cell Culture; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Stroke

Erythropoietin (EPO), which is inversely associated with blood haemoglobin (Hb), exerts neuroprotective effects in experimental ischaemic stroke (IS). However, clinical treatment trials have so far been negative. Here, in patients with IS, we analysed whether serum EPO is associated with (1) initial stroke severity, (2) recovery and (3) functional outcome.. Prospective. Controls available at baseline.. A Swedish hospital-initiated study with outpatient follow-up after 3 months.. Patients (n=600; 64% males, mean age 56 years, controls n=600) were included from the Sahlgrenska Academy Study on IS (SAHLSIS).. In addition to EPO and Hb, initial stroke severity was assessed by the Scandinavian Stroke Scale (SSS) and compared with SSS after 3 months (follow-up) as a measure of recovery. Functional outcome was evaluated using the modified Rankin Scale (mRS) at follow-up. Serum EPO and SSS were divided into quintiles in the multivariate regression analyses.. Serum EPO was 21% and 31% higher than in controls at the acute phase of IS and follow-up, respectively. In patients, acute serum EPO was 19.5% higher in severe versus mild IS. The highest acute EPO quintile adjusted for sex, age and Hb was associated with worse stroke severity quintile (OR 1.70, 95% CI 1.00 to 2.87), better stroke recovery quintile (OR 1.93, CI 1.09 to 3.41) and unfavourable mRS 3-6 (OR 2.59, CI 1.15 to 5.80). However, the fourth quintile of EPO increase (from acute to follow-up) was associated with favourable mRS 0-2 (OR 3.42, CI 1.46 to 8.03). Only the last association withstood full adjustment.. The crude associations between EPO and worse stroke severity and outcome lost significance after multivariate modelling. However, in patients in whom EPO increased, the association with favourable outcome remained after adjustment for multiple covariates.

Topics: Brain Ischemia; Erythropoietin; Female; Hemoglobins; Humans; Male; Middle Aged; Prognosis; Prospective Studies; Severity of Illness Index

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

This article seeks to clarify if gender-based differences occur in the pharmacokinetics of metoprolol in the elderly patients. There are a series of physiologic changes that occur in the elderly ranging from decreased hepatic blood flow to increased adiposity causing higher plasma concentrations at therapeutic doses as compared to the healthy young population.. Population pharmacokinetic modeling were performed using MONOLIX and Monte-Carlo simulations were conducted using MATLAB. The data was based from a previously published dataset where elderly patients, having multiple comorbidities, were administered a 50mg dose of metoprolol.. Gender stratified doses resulting in an equivalent systemic metoprolol exposure in geriatric patients have been identified. Metoprolol doses resulting a similar AUC in a healthy young male administered 50mg tablet were 15mg for geriatric women and 25mg for geriatric men. Further, Metoprolol doses of 25mg for geriatric women and 50mg for geriatric men resulted in an equivalent AUC to a healthy young males dosed with a 100mg tablet. A 15mg Metoprolol tablet may need to be compounded to account for the gender differences in Metoprolol pharmacokinetics.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antigens, Ly; Apoptosis; Astrocytes; Biomarkers; Biomarkers, Tumor; Blotting, Western; Bone Marrow Cells; Brain Ischemia; Bromodeoxyuridine; Carcinoma, Renal Cell; Case-Control Studies; Cell Hypoxia; Cell Movement; Cell Proliferation; Cells, Cultured; Chemokines; Diabetes, Gestational; Down-Regulation; Erythropoietin; Female; Gestational Age; Glucose; Heme Oxygenase-1; Heterozygote; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoenzyme Techniques; Insulin-Like Growth Factor Binding Protein 1; Insulin-Like Growth Factor Binding Protein 2; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Kidney Neoplasms; Longitudinal Studies; Male; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Mitogen-Activated Protein Kinases; Myeloid Cells; Neoplasm Grading; Neoplasm Staging; Neuroprotection; Odds Ratio; Oxidation-Reduction; Oxygen; Pregnancy; Prognosis; Rats; Real-Time Polymerase Chain Reaction; Receptors, CCR2; Receptors, Erythropoietin; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; RNA, Messenger; Signal Transduction; Stearoyl-CoA Desaturase; Survival Rate; TRPV Cation Channels; Tumor Cells, Cultured; Uterus; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Young Adult

Endothelial colony-forming cells (ECFCs) are promising candidates for cell therapy of ischemic diseases, as less than 10% of patients with an ischemic stroke are eligible for thrombolysis. We previously reported that erythropoietin priming of ECFCs increased their in vitro and in vivo angiogenic properties in mice with hindlimb ischemia. The present study used SPECT/CT to evaluate whether priming of ECFCs with erythropoietin could enhance their homing to the ischemic site after transient middle cerebral artery occlusion (MCAO) followed by reperfusion in rats and potentiate their protective or regenerative effect on blood-brain barrier (BBB) disruption, cerebral apoptosis, and cerebral blood flow (CBF).. Erythropoietin priming increased homing of ECFCs to the ischemic hemisphere (ECFC. Priming with erythropoietin before cell transplantation is an efficient strategy to amplify the migratory and engraftment capacities of ECFCs and their beneficial impact on BBB disruption, apoptosis, and CBF.

Topics: Animals; Brain Ischemia; Cells, Cultured; Endothelial Cells; Erythropoietin; Male; Premedication; Rats; Rats, Sprague-Dawley; Single Photon Emission Computed Tomography Computed Tomography; Stem Cell Transplantation; Treatment Outcome

Intra-artery infusion of recombinant human erythropoietin (rhEPO) has recently been reported to confer neuroprotection against cerebral ischemia-reperfusion injury in animal models; however, the molecular mechanisms are still under investigation. The present study focused on the specific mechanism involved in blood-brain barrier (BBB) disruption.. Thirty-six male and nine female Sprague Dawley rats were subjected to middle cerebral artery (MCA) occlusion to induce focal cerebral ischemia, and administrated rhEPO at a dose of 800 U/kg through MCA infusion at the beginning of reperfusion. Neurobehavioral deficits, brain edema, and infarct volume were evaluated after 2 h of ischemia and 24 h of reperfusion. BBB permeability was assessed by quantifying the extravasation of Evans blue (EB) dye. The expression of tight junction proteins and matrix metalloproteinases (MMPs) (Claudin-5, Occludin, MMP-2, and MMP-9) in microvessels were detected by immunofluorescence and western blot. The activities of MMPs in the cerebral microvessels were determined by gelatin zymography.. Treatment with rhEPO through the MCA strongly alleviated infarct volume, brain edema, and improved neurobehavioral outcomes in male and female rats. In addition, rhEPO remarkably suppressed the EB extravasation induced by brain ischemia. Furthermore, rhEPO prevented degradation of Claudin-5 and Occludin, and reduced the expression and activity of MMP-2 and MMP-9 in isolated brain microvessels.. Treatment with rhEPO through MCA infusion prevented brain edema formation and infarction through inhibition of MMP-mediated BBB disruption in acute ischemic stroke.

Topics: Animals; Blood-Brain Barrier; Brain Edema; Brain Ischemia; Cerebral Infarction; Erythropoietin; Female; Humans; Infarction, Middle Cerebral Artery; Infusions, Intra-Arterial; Male; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury

Local infusion of low dose erythropoietin (EPO) alleviates cerebral ischemia and reperfusion (I/R) injury in rats; however, the underlying molecular mechanisms are still unclear. The present study investigated the effect of low dose EPO treatment on I/R-induced endoplasmic reticulum (ER) stress in brain tissue and isolated microvessels in rodents. Sprague-Dawley rats were subjected to 2 h ischemia/24 h reperfusion by middle cerebral artery (MCA) occlusion, then administered fluorescein isothiocyanate-labeled EPO via MCA infusion (MCAI) or subcutaneous injection (SI) to compare the efficiency of two modes of delivery. Neurobehavioral deficits and infarct volume, and the expression of ER stress-associated proteins and apoptosis in brain tissue or isolated microvessels, as well as the transcriptional activity of 16 factors involved in ER stress and the unfolded protein response in brain tissue was asscessed. A higher EPO level in cerebrospinal fluid and brain tissue was observed in rats treated with EPO by MCAI (800 IU/kg) than by SI (5000 IU/kg). Moreover, neurobehavioral deficits and infarct volume were reduced in rats treated with EPO by MCAI and salubrinal. EPO suppressed the expression of ER stress signals glucose-regulated protein 78, activating transcription factor (ATF) 6α, and CCAAT enhancer-binding protein homologous protein (CHOP), as well as that of the pro-apoptotic protein caspase-3 in brain microvessels, and decreased the number of CHOP-positive, apoptotic neurons. EPO treatment also reduced the transcriptional activities of CHOP, forkhead box protein O1, and ATF4. These results provide evidence that low dose EPO treatment via MCAI provides neuroprotection following acute ischemic stroke by inhibiting the ER stress response.

Topics: Animals; Apoptosis Regulatory Proteins; Brain Ischemia; Capillaries; Cerebrovascular Circulation; Endoplasmic Reticulum Stress; Epoetin Alfa; Erythropoietin; Heat-Shock Proteins; Infarction, Middle Cerebral Artery; Infusions, Intra-Arterial; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury

Preconditioning (PC) using a preceding sublethal ischemic insult is an attractive strategy for protecting neurons by inducing ischemic tolerance in the brain. Although the underlying molecular mechanisms have been extensively studied, almost all studies have focused on neurons. Here, using a middle cerebral artery occlusion model in mice, we show that astrocytes play an essential role in the induction of brain ischemic tolerance. PC caused activation of glial cells without producing any noticeable brain damage. The spatiotemporal pattern of astrocytic, but not microglial, activation correlated well with that of ischemic tolerance. Interestingly, such activation in astrocytes lasted at least 8 weeks. Importantly, inhibiting astrocytes with fluorocitrate abolished the induction of ischemic tolerance. To investigate the underlying mechanisms, we focused on the P2X7 receptor as a key molecule in astrocyte-mediated ischemic tolerance. P2X7 receptors were dramatically upregulated in activated astrocytes. PC-induced ischemic tolerance was abolished in P2X7 receptor knock-out mice. Moreover, our results suggest that hypoxia-inducible factor-1α, a well known mediator of ischemic tolerance, is involved in P2X7 receptor-mediated ischemic tolerance. Unlike previous reports focusing on neuron-based mechanisms, our results show that astrocytes play indispensable roles in inducing ischemic tolerance, and that upregulation of P2X7 receptors in astrocytes is essential.

Topics: Animals; Astrocytes; Brain Ischemia; Erythropoietin; Hypoxia-Inducible Factor 1, alpha Subunit; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Ischemic Preconditioning; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Receptors, Purinergic P2X7

rhEPO has previously been shown to exert neuroprotective action in focal cerebral ischemia. However, its mechanism is not clear. We established the model of permanent focal cerebral ischemia. rhEPO was administered (5000 IU/kg i.p.) 2 h later after the successful ischemia model in rhEPO group and increased translation of Nrf2 and HO-1 and decreased the H2O2 concentration in the brain confirming activation of the Keap1-Nrf2/ARE pathway. The results show that rhEPO activate Keap1-Nrf2/ARE pathway after ischemia to protect the brain tissue.

Topics: Animals; Body Water; Brain Chemistry; Brain Ischemia; Cerebral Infarction; Erythropoietin; Heme Oxygenase-1; Hydrogen Peroxide; Intracellular Signaling Peptides and Proteins; Kelch-Like ECH-Associated Protein 1; Male; NF-E2-Related Factor 2; Rats; Rats, Sprague-Dawley; Recombinant Proteins

Topics: Brain Ischemia; Erythropoietin; Female; Humans; Male; Neuroprotective Agents; Subarachnoid Hemorrhage

To perform a feasibility and safety study with recombinant human erythropoietin (rhEPO) in neonates with perinatal arterial ischemic stroke.. Neonates with a magnetic resonance imaging-confirmed perinatal arterial ischemic stroke (n = 21) were treated with 1000 IU/kg rhEPO immediately after diagnosis and at 24 and 48 hours after the first dose. Repeat magnetic resonance imaging was performed when the patients were 3 months of age. Coagulation and hematologic variables (red blood cells, white blood cells, platelet counts) were performed in the first week after initiation of treatment. We also compared 10 patients who were treated with rhEPO with 10 historic infants with perinatal arterial ischemic stroke matched for the involved arterial branch to investigate whether rhEPO reduces the residual size of the infarction and subsequent brain growth between first and second scan.. Seizures were a first symptom in 20 of 21 neonates. Heart rate, blood pressure, and coagulation function were in the normal range, as were red blood cells, white blood cells, and platelet counts. In a subgroup of 10 rhEPO-treated neonates, no differences were detected in residual infarction volumes or neurodevelopmental outcome compared with their historical nontreated counterparts.. rhEPO in neonates with perinatal arterial ischemic stroke had no adverse effects on red blood cells, white blood cells, platelets counts, or coagulation. rhEPO, 3000 IU/kg in total, given during a 3-day period, appears to be a safe therapy. The beneficial effects remains to be demonstrated in a larger, randomized, double-blind, placebo-controlled trial.

Topics: Blood Cell Count; Brain Ischemia; Cerebral Palsy; Erythropoietin; Feasibility Studies; Female; Hematocrit; Hemoglobins; Humans; Infant, Newborn; Magnetic Resonance Imaging; Male; Matched-Pair Analysis; Neuroprotective Agents; Recombinant Proteins; Seizures; Stroke

Using proteomics, we identified nucleoside diphosphate kinase A (NDPKA; also known as NME/NM23 nucleoside diphosphate kinase 1: NME1) to be up-regulated in primary cortical neuronal cultures by erythropoietin (EPO) preconditioning. To investigate a neuroprotective role of NDPKA in neurons, we used a RNAi construct to knock-down and an adenoviral vector to overexpress the protein in cortical neuronal cultures prior to exposure to three ischemia-related injury models; excitotoxicity (L-glutamic acid), oxidative stress (hydrogen peroxide), and in vitro ischemia (oxygen-glucose deprivation). NDPKA down-regulation had no effect on neuronal viability following injury. By contrast, NDPKA up-regulation increased neuronal survival in all three-injury models. Similarly, treatment with NDPKA recombinant protein increased neuronal survival, but only against in vitro ischemia and excitotoxicity. These findings indicate that the NDPKA protein may confer a neuroprotective advantage following injury. Furthermore, as exogenous NDPKA protein was neuroprotective, it suggests that a cell surface receptor may be activated by NDPKA leading to a protective cell-signaling response. Taken together both NDPKAs intracellular and extracellular neuroprotective actions suggest that the protein is a legitimate therapeutic target for the design of drugs to limit neuronal death following stroke and other forms of brain injury.

Topics: Animals; Brain Ischemia; Cell Survival; Cells, Cultured; Cerebral Cortex; Erythropoietin; Gene Expression Regulation; HEK293 Cells; Humans; Neurons; Neuroprotective Agents; Nucleoside-Diphosphate Kinase; Rats; Rats, Sprague-Dawley; Up-Regulation

Erythropoietin (EPO) is a neuroprotective agent against cerebral ischemia/reperfusion (I/R)-induced brain injury. However, its crossing of blood-brain barrier is limited. Focused ultrasound (FUS) sonication with microbubbles (MBs) can effectively open blood-brain barrier to boost the vascular permeability. In this study, we investigated the effects of MBs/FUS on extending the therapeutic time window of EPO and its neuroprotective effects in both acute and chronic phases. Male Wistar rats were firstly subjected to two common carotid arteries and right middle cerebral artery occlusion (three vessels occlusion, 3VO) for 50 min, and then the rats were treated with hEPO (human recombinant EPO, 5000 IU/kg) with or without MBs/FUS at 5 h after occlusion/reperfusion. Acute phase investigation (I/R, I/R+MBs/FUS, I/R+hEPO, and I/R+hEPO+MBs/FUS) was performed 24 h after I/R; chronic tests including cylinder test and gait analysis were performed one month after I/R. The experimental results showed that MBs/FUS significantly increased the cerebral content of EPO by bettering vascular permeability. In acute phase, both significant improvement of neurological score and reduction of infarct volume were found in the I/R+hEPO+MBs/FUS group, as compared with I/R and I/R+hEPO groups. In chronic phase, long-term behavioral recovery and neuronal loss in brain cortex after I/R injury was significantly improved in the I/R+hEPO+MBs/FUS group. This study indicates that hEPO administration with MBs/FUS sonication even at 5 h after occlusion/reperfusion can produce a significant neuroprotection.

Topics: Animals; Behavior, Animal; Blood-Brain Barrier; Brain Ischemia; Drug Delivery Systems; Erythropoietin; Gait; Infarction, Middle Cerebral Artery; Male; Microbubbles; Neurons; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion Injury; Sound

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

We tested the influence of erythropoietin (EPO), a basic cytokine in erythropoiesis regulation, on the process of motor function and cognition after focal brain ischemia induced by a local application of endothelin. Endothelin-1 (ET-1) induced short lasting strong vasoconstriction, with described impact on the structure and on the function of neuronal cells. Neurological description of motor function and Morris water maze test (the swimming test is one of most widely used methods for studying cognitive functions in rodents) were used to study the process of learning and memory in three-month-old male albino Wistar rats (n=52). Both tests were performed one week before, and three weeks after ischemia induction (endothelin application on the cortex in the area of a. cerebri media dx.). Experimental group received i.p. injection of EPO (5,000 IU/kg body weight, 10 min before endothelin application). Control group of animals received one i.p. injection of saline at the dose of 1 ml/kg body weight at the same time. Only sham surgery was performed in the third group of animals. Rats with EPO pretreatment before the experimental lesion exhibited significantly better motor and cognitive function then those with saline injection. No significant changes in the motor and cognitive function were found in the third group of rats (sham operated controls).

Topics: Animals; Brain Ischemia; Cognition; Endothelin-1; Erythropoietin; Male; Motor Activity; Rats; Rats, Wistar

Erythropoietin in the nervous system is a potential neuroprotective factor for cerebral ischemic damage due to specific-binding to the erythropoietin receptor, which is associated with survival mechanisms. However, the role of its receptor is unclear. Thus, this work assessed whether a low dose (500UI/Kg) of intranasal recombinant human erythropoietin administered 3h after ischemia induced changes in the activation of its receptor at the Tyr456-phosphorylated site in ischemic hippocampi in rats. The results showed that recombinant human erythropoietin after injury maintained cell survival and was associated with an increase in receptor phosphorylation at the Tyr456 site as an initial signaling step, which correlated with a neuroprotective effect.

Topics: Administration, Intranasal; Animals; Brain Ischemia; Cell Survival; Cytoprotection; Erythropoietin; Hippocampus; Humans; Male; Neuroprotective Agents; Phosphorylation; Rats, Wistar; Receptors, Erythropoietin; Recombinant Proteins; Tyrosine

Animal models constitute an indispensable tool to investigate human pathology. Here we describe the procedure to induce permanent and transient cerebral ischemia in the mouse and the rat. The model of transient occlusion of the middle cerebral artery (MCA) is performed by the insertion of an occlusive filament until the origin of the MCA while the permanent occlusion described in the mice is performed by a distal electrocoagulation of the MCA. Those models allow evaluating the efficiency of therapeutic strategy of ischemia from tissular aspect to behavioral and cognitive impairment assessment. They were widely used in the literature to evaluate the efficiency of different drugs including the cytokines and especially erythropoietin (EPO) or its derivatives.

Topics: Animals; Brain Ischemia; Erythropoietin; Infarction, Middle Cerebral Artery; Mice; Middle Cerebral Artery; Rats

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

SMND-309 is a novel derivative of salvianolic acid B, and has shown protective effects against rat cortical neuron damage in vitro and in vivo. However the molecular mechanisms through which SMND-309 affords this protection are unclear. The present study aimed to investigate the mechanisms associated with the protective activities of SMND-309 in a cerebral ischemia and reperfusion injury rat model. In this study, we used AG490, a specific inhibitor of the signaling pathway involving the Janus Kinase 2 (JAK2)/Signal Transducers and Activators of Transcription 3 (STAT3) signaling molecules and suramin, a potent inhibitor of vascular endothelial growth factor (VEGF), to investigate the mechanisms of SMND-309. The cerebral ischemia and reperfusion injury model was induced by performing middle cerebral artery occlusion (MCAO) in the rats. SMND-309 mitigated the effects of ischemia and reperfusion injury on brain by decreasing the infract volume, improving neurological function, increasing the survival of neurons and promoting angiogenesis by increasing the levels of erythropoietin (EPO), erythropoietin receptor (EPOR), phosphorylated JAK2 (P-JAK2), phosphorylated STAT3 (P-STAT3), VEGF and VEGF receptor 2 (Flk-1) in the brain. Our results suggest that SMND-309 provides significant neuroprotective effects against cerebral ischemia and reperfusion injury. The mechanisms of this protection may be attributed to the increased VEGF expression occurring from the JAK2/STAT3 pathway, activated by the increased EPO/EPOR expression in the brain.

Topics: Animals; Axons; Benzofurans; Brain; Brain Ischemia; Caffeic Acids; Cerebral Infarction; Dendrites; Erythropoietin; Gene Expression Regulation; Infarction, Middle Cerebral Artery; Janus Kinase 2; Male; Neovascularization, Physiologic; Neuroprotective Agents; Phosphoproteins; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Recovery of Function; Reperfusion Injury; Signal Transduction; STAT3 Transcription Factor; Survival Analysis; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Water

Understanding the endogenous survival pathways induced by ischemic tolerance may yield targets for neuroprotection from stroke. One well-studied pathway reported to be evoked by preconditioning stimuli is the transcription factor HIF (hypoxia-inducible factor). However, whether HIF induction by ischemic insults is neuroprotective or toxic is still unclear. We examined the ability of three prolyl-hydroxylase inhibitors, which induce HIF, to protect hippocampal cultures from oxygen-glucose deprivation. Hippocampal cultures were exposed to ischemic preconditioning or various concentrations of cobalt chloride, deferoxamine (DFO) or dimethyloxylalyglycine (DMOG), prior to lethal oxygen-glucose deprivation (OGD). Cell survival of neurons and astrocytes was determined with dual-label immunocytochemistry. The induction of HIF targets was assessed in mixed as well as astrocyte-enriched cultures. Ischemic preconditioning, as well as low concentrations of cobalt and DFO, enhanced the survival of neurons following OGD. However, DMOG exacerbates OGD-induced neuronal death. At low concentrations, all three prolyl-hydroxylase (PHD) inhibitors increased the survival of astrocytes. Neuroprotective concentrations of cobalt induced the transcription of the cytokine erythropoietin (EPO) in astrocyte cultures. In addition, pretreatment with recombinant human erythropoietin (rH-EPO) also protected neurons from OGD. Our data suggest that HIF-induced EPO, released from astrocytes, protects neurons from OGD. However, the three PHD inhibitors each exhibited different neuroprotective profiles at low concentrations, suggesting that not all PHD inhibitors are created equal. The protective effects at low doses is reminiscent of HIF involvement in ischemic tolerance, in which sub-lethal insults induce HIF pathways resulting in neuroprotection, whereas the high-dose toxicity suggests that over-activation of HIF is not always protective. Therefore, the choice of inhibitor and dose may determine the clinical utility of these compounds. Deferoxamine exhibited little toxicity even at higher doses, and therefore appears a promising candidate for clinical use.

Topics: Amino Acids, Dicarboxylic; Animals; Astrocytes; Brain Ischemia; Cell Survival; Cells, Cultured; Cobalt; Deferoxamine; Erythropoietin; Hippocampus; Hypoxia-Inducible Factor 1; Immunohistochemistry; Ischemic Preconditioning; Mice; Mice, Inbred C57BL; Neurons; Neuroprotective Agents; Polymerase Chain Reaction; Prolyl-Hydroxylase Inhibitors

Erythropoietin (EPO) has been demonstrated to possess significant neuroprotective effects in stroke. We determined if the nano-drug form of human recombinant EPO (PLGA-EPO nanoparticles [PLGA-EPO-NP]) can enhance neuroprotection at lower dosages versus human recombinant EPO (r-EPO).. Established neonatal rat model of unilateral ischemic stroke was used to compare r-EPO, PLGA-EPO-NP and phosphate-buffered saline, given by daily intraperitoneal injections, followed by infarction volume and Rotarod Performance Test assessment.. PLGA-EPO-NP significantly reduced infarction volumes 72 hours after injury compared with the same concentrations of r-EPO. Functional deficits were significantly reduced by 300 U/kg PLGA-EPO-NP versus controls, with deficit attenuation apparent at significantly lower dosages of PLGA-EPO-NP versus r-EPO.. PLGA-EPO-NP is neuroprotective and beneficial against deficits after brain ischemia, at significantly reduced dosages versus r-EPO.

Topics: Animals; Animals, Newborn; Behavior, Animal; Brain Ischemia; Cerebral Infarction; Erythropoietin; Excipients; Female; Hypoxia, Brain; Injections, Intraperitoneal; Lactic Acid; Male; Nanoparticles; Nervous System Diseases; Neuroprotective Agents; Organ Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Postural Balance; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Rotarod Performance Test

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

To study whether recombinant human erythropoietin (rhEPO) reduces neuronal apoptosis through inhibiting over-expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in nucleus induced by brain ischemia/reperfusion in rats, 48 adult Sprague-Dawley rats were randomly divided into 3 groups: sham, saline and EPO groups. Animal models of brain ischemia/reperfusion were established by middle cerebral artery occlusion in rats. The effects of EPO on the sizes of ischemia tissue were observed by TTC staining. The over-expression of GAPDH in nucleus was detected by Hoechst-33258 and anti-GAPDH antibody double staining. The neuronal apoptosis in penumbral was detected by Nissl's staining and Hoechst-33258 immunofluorescence, respectively. The results showed that rhEPO treatment (3 000 U/kg, three times daily, i.p.) apparently reduced the sizes of infarct brain tissue in ischemia/reperfusion rats. rhEPO inhibited over-expression of GAPDH in nucleus of apoptotic neurons. In the meantime rhEPO decreased the number of apoptotic neurons in ischemia/reperfusion rats. These results suggest that rhEPO may induced reduction of neuronal apoptosis in penumbra may be through inhibiting over-expression of GAPDH in nucleus of apoptotic neurons induced by ischemia/reperfusion. Reduction of GAPDH over-expression in nucleus may play a pivotal role in EPO inhibiting neuronal apoptosis in cerebral ischemia/reperfusion rats, providing experimental evidence for EPO neuro-protecting effects against ischemia/reperfusion.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Erythropoietin; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Humans; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury

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

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

The promotion of post-ischaemic motor recovery remains a major challenge in clinical neurology. Recently, plasticity-promoting effects have been described for the growth factor erythropoietin in animal models of neurodegenerative diseases. To elucidate erythropoietin's effects in the post-acute ischaemic brain, we examined how this growth factor influences functional neurological recovery, perilesional tissue remodelling and axonal sprouting of the corticorubral and corticobulbar tracts, when administered intra-cerebroventricularly starting 3 days after 30 min of middle cerebral artery occlusion. Erythropoietin administered at 10 IU/day (but not at 1 IU/day), increased grip strength of the contralesional paretic forelimb and improved motor coordination without influencing spontaneous locomotor activity and exploration behaviour. Neurological recovery by erythropoietin was associated with structural remodelling of ischaemic brain tissue, reflected by enhanced neuronal survival, increased angiogenesis and decreased reactive astrogliosis that resulted in reduced scar formation. Enhanced axonal sprouting from the ipsilesional pyramidal tract into the brainstem was observed in vehicle-treated ischaemic compared with non-ischaemic animals, as shown by injection of dextran amines into both motor cortices. Despite successful remodelling of the perilesional tissue, erythropoietin enhanced axonal sprouting of the contralesional, but not ipsilesional pyramidal tract at the level of the red and facial nuclei. Moreover, molecular biological and histochemical studies revealed broad anti-inflammatory effects of erythropoietin in both hemispheres together with expression changes of plasticity-related molecules that facilitated contralesional axonal growth. Our study establishes a plasticity-promoting effect of erythropoietin after stroke, indicating that erythropoietin acts via recruitment of contralesional rather than of ipsilesional pyramidal tract projections.

Topics: Analysis of Variance; Animals; Blotting, Western; Brain; Brain Ischemia; Erythropoietin; Exploratory Behavior; Hand Strength; Immunohistochemistry; Male; Mice; Motor Activity; Neuronal Plasticity; Neurons; Recovery of Function; Reverse Transcriptase Polymerase Chain Reaction; Rotarod Performance Test; Stroke

The purpose of this study was to evaluate the neuroprotective effects of intranasally delivered recombinant human neuronal erythropoietin (Neuro-EPO) on brain injury induced by unilateral permanent ischemia in the Mongolian gerbil. Expression of EPO receptor (EPOR) and neuroglobin (Ngb) over 5 weeks after intranasal treatment with Neuro-EPO was determined using immunohistochemistry. Mortality of Neuro-EPO-treated gerbils decreased after surgery, and the sensory and motor function was significantly improved. Histopathological mapping showed that Neuro-EPO significantly reduced delayed neuronal death in the brain. Expression of Ngb was upregulated in the cerebral cortex at most time points (expect for 10 min and 48 hr) and in the hippocampus at 10 min and from 48 hr to 5 weeks, whereas EPOR was almost downregulated or unchanged in the brain (expect for 48 hr). The 10 min and 48 hr seemed to be two time points for the brain to switch the expression of both Ngb and EPOR to early and late recovery phase, respectively. In addition, there were two phases, 10 min to 1 hr and 24 hr to 72 hr, respectively, closing to the "golden hour" of about 60 min and the "silver day" of 1 to 3 days, for the brain to recover from stroke onset with intranasal Neuro-EPO treatment. Therefore, the results suggest that the intranasal administration of Neuro-EPO is effective in the treatment of acute brain ischemia. The different expression patterns of Ngb and EPOR is probably due to ischemic tolerance in the cerebral cortex and ischemic sensitivity in the hippocampus.

Topics: Administration, Intranasal; Animals; Brain; Brain Ischemia; Cerebral Cortex; Erythropoietin; Gene Expression Regulation; Gerbillinae; Globins; Hippocampus; Male; Nerve Tissue Proteins; Neuroglobin; Neuroprotective Agents; Organ Specificity; Receptors, Erythropoietin; Recombinant Proteins; Time Factors

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

It has been demonstrated that conditioned medium from astrocytes challenged by in vitro ischemia (oxygen-glucose deprivation, OGD) improved neuronal survival. In addition, preconditioning stimuli can be cross-tolerant, safeguarding against other types of injury. We therefore hypothesized that hyperthermia-conditioned astrocyte-cultured medium (ACM) might also have protective effect on neurons against ischemic injury. The cultured-media, named 38ACM and 40ACM respectively, were collected after astrocytes had been incubated at 38 °C or 40 °C for 6h, followed by incubation at 37 °C for 24h. It was found that ischemia for 6h induced a significant reduction in the number of neuronal cells and cell-viability, and an increase in lactate dehydrogenase (LDH) release and the percentage of apoptotic nuclei in neurons. Pre-treatment with 38ACM or 40ACM for 24h significantly diminished ischemia injury, enhanced cell viability, reduced LDH release and reversed apoptosis. Western blot analysis showed that treatment with 38ACM or 40ACM for 24h led to a significant increase in hypoxia-inducible factor-1 (HIF-1) alpha expression. The EMSA demonstrated that the ACM increased the binding activity of HIF-1 in ischemic neurons. The data implied that hyperthermia-conditioned ACM protects neurons from ischemic injury by up-regulating HIF-1 alpha, and the increased binding activity of HIF-1 and anti-apoptotic ability.

Topics: Animals; Apoptosis; Astrocytes; Brain Ischemia; Cell Hypoxia; Cell Survival; Culture Media, Conditioned; Erythropoietin; Hyperthermia, Induced; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemic Preconditioning; L-Lactate Dehydrogenase; Mice; Neurons; Response Elements; Up-Regulation

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

Topics: Amino Acids; Animals; Brain Ischemia; Cell Hypoxia; Chemokine CXCL12; Endothelial Cells; Erythropoietin; Microglia; Neovascularization, Physiologic; Nerve Regeneration; Neurogenesis; Rats; Receptors, CXCR4; Stem Cell Niche; Stem Cell Transplantation; Swine

1. Asialoerythropoietin (aEPO), a derivative of cytokine erythropoietin, has been shown to have neuroprotective effects without haematological complications when administered in single or repeated doses. The present study examines our hypothesis that aEPO may provide neuroprotection against programmed apoptotic cell death when administered in a continuous low dose. 2. Focal cerebral ischaemia was introduced by occlusion of the middle cerebral artery using a surgically placed intraluminal filament in young male Sprague Dawley rats (9 weeks old). After 90 min ischaemia, reperfusion was established by filament removal. Both study and control groups had implanted osmotic minipumps through which they received either aEPO (1 microL/h; 20 microg/kg per 24 h) or normal saline (1 microL/h) for 4 days. On Day 4, infarct volume, the number of apoptotic cells and concentrations of activated caspase 3 and 9 were evaluated in the penumbra region. 3. Asialoerythropoietin was detected in the cerebrospinal fluid of the study group, whereas none was detected in the control group. Although there were no significant changes in haematocrit levels or behaviour scores (on Days 1 and 4) between the study and control groups, aEPO administration significantly reduced infarct volume in the study group compared with the control group (168 +/- 19 vs 249 +/- 28 mm(3), respectively; P < 0.05). 4. The number of terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL)-positive cells and the concentration of activated caspase 3 and 9 in the penumbra region were significantly lower in the study group compared with the control group. 5. In conclusion, our data suggest that aEPO provides a short-term, possibly histological, protection in young adult male rats when administered immediately after reperfusion.

Topics: Animals; Apoptosis; Asialoglycoproteins; Brain Ischemia; Caspase 3; Caspase 9; Cerebral Infarction; Erythropoietin; Infarction, Middle Cerebral Artery; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley

Topics: Brain Ischemia; Endothelium, Vascular; Erythropoietin; Humans; Recombinant Proteins; Stroke

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

After 1 clinical study in which recombinant erythropoietin (EPO) protected against ischemic stroke and improved clinical outcome, the German multicenter EPO trial recently reported increased mortality in stroke patients receiving EPO after tissue-plasminogen activator (t-PA)-induced thrombolysis. The reasons for the adverse effects of EPO in t-PA-treated patients are unknown.. Mice were submitted to 90 minutes of middle cerebral artery occlusion. Immediately after reperfusion, animals were treated with normal saline or t-PA (10 mg/kg). Animals subsequently received injections of normal saline or EPO that were administered after reperfusion and 12 hours later (2500 IU/kg each). Ischemic injury and brain edema were analyzed at 24 hours after reperfusion by cresyl violet staining and terminal transferase biotinylated-dUTP nick end labeling. Blood-brain barrier integrity was assessed by histochemistry for extravasated serum IgG. Matrix metalloproteinase activity was evaluated by gelatinase zymography.. EPO did not influence ischemic infarct size but reduced brain swelling. This effect was abolished by t-PA, which exacerbated serum IgG extravasation in ischemic tissue. Gelatinase zymographies revealed that EPO promoted matrix metalloproteinase-9 activity that was markedly elevated by t-PA. Add-on treatment with t-PA increased the density of DNA-fragmented cells in ischemic tissue of EPO-treated, but not vehicle-treated, mice.. Our data demonstrate a hitherto unknown interaction of t-PA with EPO at the blood-brain interface, ie, promotion of vascular permeability and extracellular matrix breakdown, which may account for the unfavorable actions of EPO in t-PA-treated patients. After t-PA-induced thrombolysis, EPO may not be suitable as stroke treatment.

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Capillary Permeability; DNA Fragmentation; Drug Therapy, Combination; Erythropoietin; Extracellular Matrix; Male; Mice; Mice, Inbred C57BL; Tissue Plasminogen Activator

Topics: Brain Ischemia; Drug Evaluation, Preclinical; Erythropoietin; Humans; Meta-Analysis as Topic; Stroke

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

Topics: Animals; Brain Edema; Brain Ischemia; Erythropoietin; Female; Hippocampus; Male; Nitric Oxide; Protective Agents; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase

Topics: Brain Ischemia; Erythropoietin; Humans; Neurosurgical Procedures; Recombinant Proteins; Subarachnoid Hemorrhage; Vasospasm, Intracranial

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

Topics: Brain; Brain Ischemia; Erythropoietin; Fluorescein Angiography; Humans; Malaria, Cerebral; Neuroprotective Agents; Treatment Outcome

The hematopoietic growth factor erythropoietin (EPO) and its neuroprotective, but not hematopoietic, variants asialoEPO, carbamylated EPO (CEPO), and low sialic acid EPO (Neuro-EPO) are attractive candidates for stroke treatment. Due to their large molecular weight, these proteins enter the brain only to a minor extent when intravenously administered, which has raised the question for alternative delivery strategies, among which intranasal delivery may certainly be an attractive choice, as the review by Garcia Rodriguez and Sosa Teste in this journal points out. Before this strategy may be considered clinically applicable, however, more and, in particular, quantitative information is needed about (a) the temporospatial accumulation of EPO and its variants in the brain tissue both in animals and nonhuman primates, and (b) the accumulation of EPO and its variants in the human cerebrospinal fluid.

Topics: Administration, Intranasal; Asialoglycoproteins; Blood-Brain Barrier; Brain Ischemia; Erythropoietin; Humans; Recombinant Proteins; Stroke

Facilitation of endogenous neuroprotective pathways, such as the erythropoietin (Epo) pathway, has been proposed as adjuvant treatment strategies in cerebral malaria. Whether different endogenous protein expression levels of Epo or differences in the abundance of its receptor components could account for the extent of structural neuropathological changes or neurological complications in adults with severe malaria was investigated.. High sensitivity immunohistochemistry was used to assess the frequency, distribution and concordance of Epo and components of its homodimeric and heteromeric receptors, Epo receptor and CD131, within the brainstem of adults who died of severe malaria. The following relationships with Epo and its receptor components were also defined: (i) sequestration and indicators of hypoxia; (ii) vascular damage in the form of plasma protein leakage and haemorrhage; (iii) clinical complications and neuropathological features of severe malaria disease. Brainstems of patients dying in the UK from unrelated non-infectious causes were examined for comparison.. The incidence of endogenous Epo in parenchymal brain cells did not greatly differ between severe malaria and non-neurological UK controls at the time of death. However, EpoR and CD131 labelling of neurons was greater in severe malaria compared with non-neurological controls (P = .009). EpoR labelling of vessels was positively correlated with admission peripheral parasite count (P = .01) and cerebral sequestration (P < .0001). There was a strong negative correlation between arterial oxygen saturation and EpoR labelling of glia (P = .001). There were no significant correlations with indicators of vascular damage, neuronal chromatolysis, axonal swelling or vital organ failure.. Cells within the brainstem of severe malaria patients showed protein expression of Epo and its receptor components. However, the incidence of endogeneous expression did not reflect protection from vascular or neuronal injury, and/or clinical manifestations, such as coma. These findings do not provide support for Epo as an adjuvant neuroprotective agent in adults with severe malaria.

Topics: Adult; Aged; Aged, 80 and over; Brain Ischemia; Brain Stem; Case-Control Studies; Cytokine Receptor Common beta Subunit; Erythropoietin; Female; Humans; Immunohistochemistry; Malaria, Falciparum; Male; Middle Aged; Plasmodium falciparum; Receptors, Erythropoietin; United Kingdom; Vietnam; Young Adult

Carbamylated erythropoietin (CEPO) is a novel neuroprotective agent that does not bind to the classical erythropoietin receptor or affect hematocrit. Since CEPO has not been systematically studied in a fully blinded and randomized manner in an embolic stroke model, we determined if CEPO would be useful to attenuate clinical deficits associated with multiple infarct ischemia using the rabbit small clot embolic stroke model (RSCEM). Rabbits were embolized and treated with vehicle or CEPO within 6 h of embolization and behavioral analysis was conducted 48 h after embolization. Using quantal analysis, we determined the quantity of blood clot (mg) in brain that produce neurologic dysfunction in 50% of the rabbits (P(50)), with intervention considered beneficial if it increased the P(50) compared to controls. CEPO administered between 5 min and 3 h after embolization significantly (p<0.05) improved behavioral function and increased the P(50) value by 55-216%. However, CEPO administration did not improve behavior when administered 6 h following embolization. In conclusion, in the RSCEM, CEPO had a therapeutic window of at least 3 h, where it effectively improved clinical rating scores and motor function. Our results suggest that CEPO may be useful to treat acute ischemic stroke and supports the study of CEPO in stroke patients.

Topics: Animals; Behavior, Animal; Brain Infarction; Brain Ischemia; Erythropoietin; Intracranial Embolism; Male; Neuroprotective Agents; Rabbits; Recovery of Function; Time

Topics: Animals; Brain; Brain Ischemia; Erythropoietin; Heart Arrest; Injections, Intravenous; Rats; Recovery of Function; Treatment Outcome

Uncontrolled expression of vascular endothelial growth factor (VEGF) in vivo may cause unexpected side effects, such as brain hemangioma or tumor growth. Because hypoxia-inducible factor-1 (HIF-1) is upregulated during cerebral ischemia and regulates gene expression by binding to a cis-acting hypoxia-responsive element (HRE), we therefore used a novel HRE, originating in the 3'-end of the erythropoietin (Epo) gene, to control gene expression in the ischemic brain. A concatemer of nine copies (H9) of the consensus sequence of HRE was used to mediate hypoxia induction. Three groups of adult CD-1 mice received AAVH9-VEGF, AAVH9-lacZ or saline injection, and then underwent 45 min of transient middle cerebral artery occlusion (tMCAO). Results show that HIF-1 was persistently expressed in the ischemic brain. VEGF was overexpressed in the ischemic perifocal region in AAVH9-VEGF-transduced mice. Double-labeled immunostaining showed that VEGF expressed in neurons and astrocytes but not endothelial cells, suggesting that adeno-associated virus (AAV) vectors transduced neurons and astrocytes predominantly. The total number of microvessels/enlarged microvessels was greatly increased in the AAVH9-VEGF-transduced mice (180+/-29/27+/-4) compared to the AAVH9-lacZ (118+/-19/14+/-3) or saline-treated (119+/-20/14+/-2) mice after tMCAO (P<0.05). Cell proliferation examination demonstrated that these microvessels were newly formed. Regional cerebral blood flow recovery in the AAVH9-VEGF-transduced mice was also better than in AAVH9-lacZ or saline-treated mice (P<0.05). Our data indicated that HRE is a novel trigger for the control of VEGF expression in the ischemic brain. VEGF overexpression through AAVH9-VEGF gene transfer showed stable focal angiogenic effects in post-ischemic repair process, providing an opportunity to rebuild injured brain tissue.

Topics: 3' Untranslated Regions; Animals; Astrocytes; Brain Ischemia; Cerebrovascular Circulation; Dependovirus; Erythropoietin; Gene Expression; Genetic Therapy; Genetic Vectors; Hypoxia; Hypoxia-Inducible Factor 1; Immunohistochemistry; Mice; Neovascularization, Physiologic; Neurons; Transduction, Genetic; Vascular Endothelial Growth Factor A

We studied the effect of hyperbaric oxygen (HBO) preconditioning on the molecular mechanisms of neuroprotection in a rat focal cerebral ischemic model. Seventy-two male Sprague-Dawley rats were pretreated with HBO (100% O(2), 2 atmospheres absolute, 1 h once every other day for 5 sessions) or with room air. In experiment 1, HBO-preconditioned rats and matched room air controls were subjected to focal cerebral ischemia or sham surgery. Postinjury motor parameters and infarction volumes of HBO-preconditioned rats were compared with those of controls. In experiment 2, HBO-preconditioned rats and matched room air controls were killed at different time points. Brain levels of hypoxia-inducible factor-1alpha (HIF-1alpha) and its downstream target gene erythropoietin (EPO) analyzed by Western blotting and RT-PCR as well as HIF-1alpha DNA-binding and transcriptional activities were determined in the ipsilateral hemisphere. HBO induced a marked increase in the protein expressions of HIF-1alpha and EPO and the activity of HIF-1alpha, as well as the expression of EPO mRNA. HBO preconditioning dramatically improved the neurobehavioral outcome at all time points (3.0 +/- 2.1 vs. 5.6 +/- 1.5 at 4 h, 5.0 +/- 1.8 vs. 8.8 +/- 1.4 at 8 h, 6.4 +/- 1.8 vs. 9.7 +/- 1.3 at 24 h; P < 0.01, respectively) and reduced infarction volumes (20.7 +/- 4.5 vs. 12.5 +/- 3.6%, 2,3,5-Triphenyltetrazolium chloride staining) after cerebral ischemia. This observation indicates that the neuroprotection induced by HBO preconditioning may be mediated by an upregulation of HIF-1alpha and its target gene EPO.

Topics: Animals; Behavior, Animal; Blotting, Western; Brain Ischemia; Cerebral Infarction; Cerebrovascular Circulation; DNA; Erythropoietin; Forelimb; Hyperbaric Oxygenation; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemic Preconditioning; Male; Neuroprotective Agents; Oxygen; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazolium Salts; Up-Regulation

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

CNS neurons use robust cytoprotective mechanisms to ensure survival and functioning under conditions of injury. These involve pathways induced by endogenous neuroprotective cytokines such as erythropoietin (EPO). Recently, in contrast to its well known deleterious roles, TNF has also been shown to exhibit neuroprotective properties. In the present study, we investigated the molecular mechanisms by which TNF receptor (TNFR)I mediates neuroprotection by comparing the gene expression profiles of lesioned cortex from WT and TNFRI KO mice after permanent middle cerebral artery occlusion. Several known neuroprotective molecules were identified as TNFRI targets, notably members of the Bcl-2 family, DNA repair machinery and cell cycle, developmental, and differentiation factors, neurotransmitters and growth factors, as well as their receptors, including EPO receptor (EPOR), VEGF, colony-stimulating factor receptor 1, insulin-like growth factor (IGF), and nerve growth factor (NGF). Further analysis showed that induction of EPOR and VEGF expression in primary cortical neurons after glucose deprivation (GD) largely depended on TNFRI and was further up-regulated by TNF. Also, EPO- and VEGF-induced neuroprotection against GD, oxygen-glucose deprivation, and NMDA excitotoxicity depended significantly on TNFRI presence. Finally, EPO prevented neuronal damage induced by kainic acid in WT but not TNFRI KO mice. Our results identify cross-talk between tissue protective cytokines, specifically that TNFRI is necessary for constitutive and GD-induced expression of EPOR and VEGF and for EPO-mediated neuroprotection.

Topics: Animals; Brain Ischemia; Cell Death; Cytoprotection; Erythropoietin; Excitatory Amino Acid Agonists; Gene Expression Profiling; Kainic Acid; Male; Mice; Mice, Knockout; N-Methylaspartate; Neurons; Oligonucleotide Array Sequence Analysis; Receptors, Erythropoietin; Receptors, Tumor Necrosis Factor, Type I; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

The most effective way to augment neural progenitor proliferation after ischemia is still unknown. We administered various agents into the rat cerebral ventricle after transient global ischemia and compared the neural progenitor response in the anterior subventricular zone (aSVZ), dentate gyrus subgranular zone, posterior periventricle, and hypothalamus. We demonstrated that cocktail administration of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) remarkably increased the numbers of neural progenitors in all four regions examined. The addition of Notch ligand DLL4 to the cocktail elicited the largest progenitor response in the aSVZ and hypothalamus. Our results suggest that EGF and FGF-2, combined with DLL4, represent the universally applicable regimen for the expansion of the neural progenitor pool following ischemia.

Topics: Animals; Brain Ischemia; Brain-Derived Neurotrophic Factor; Cell Division; Cerebral Ventricles; Dentate Gyrus; Epidermal Growth Factor; Erythropoietin; Fibroblast Growth Factor 2; Hypothalamus; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Nerve Regeneration; Rats; Rats, Wistar; Stem Cells

Carbamylerythropoietin (CEPO) does not bind to the classical erythropoietin (EPO) receptor. Nevertheless, similarly to EPO, CEPO promotes neuroprotection on the histologic level in short-term stroke models. In the present study, we investigated whether CEPO and other nonerythropoietic EPO analogs could enhance functional recovery and promote long-term histologic protection after experimental focal cerebral ischemia. Rats were treated with the compounds after focal cerebral ischemia. Animals survived 1, 7, or 60 days and underwent behavioral testing (sensorimotor and foot-fault tests). Brain sections were stained and analyzed for Iba-1, myeloperoxidase, Tau-1, CD68 (ED1), glial fibrillary acidic protein (GFAP), Fluoro-Jade B staining, and overall infarct volumes. Treatment with CEPO reduced perifocal microglial activation (P<0.05), polymorphomonuclear cell infiltration (P<0.05), and white matter damage (P<0.01) at 1 day after occlusion. Carbamylerythropoietin-treated rats showed better functional recovery relative to vehicle-treated animals as assessed 1, 7, 14, 28, and 50 days after stroke. Both GFAP and CD68 were decreased within the ipsilateral thalamus of CEPO-treated animals 60 days postoperatively (P<0.01 and P<0.05, respectively). Furthermore, behavioral analysis showed efficacy of CEPO treatment even if administered 24 h after the stroke. Other nonerythropoietic derivatives such as carbamylated darbepoetin alfa and the mutant EPO-S100E were also found to protect against ischemic damage and to improve postischemic neurologic function. In conclusion, these results show that postischemic intravenous treatment with nonerythropoietic EPO derivatives leads to improved functional recovery, which may be linked to their long-term effects against neuroinflammation and secondary tissue damage.

Topics: Animals; Brain; Brain Ischemia; Erythropoietin; Humans; Immunohistochemistry; Inflammation; Male; Neuroprotective Agents; Rats; Recovery of Function

Restoration of local blood supply in the post-ischemic brain plays a critical role in tissue repair and functional recovery. The present investigation explored beneficial effects of recombinant human erythropoietin (rhEPO) on vascular endothelial cell survival, angiogenesis, and restoration of local cerebral blood flow (LCBF) after permanent focal cerebral ischemia in adult mice. Saline or rhEPO (5,000 U/kg, intraperitoneal) was administered 30 mins before ischemia and once daily after ischemic stroke. Immunohistochemistry showed an enhancing effect of rhEPO on expression of EPO receptor (EPOR) of endothelial cells in the penumbra region 3 to 21 days after the ischemic insult. The treatment with rhEPO decreased ischemia-induced cell death and infarct volume 3 days after stroke. Specifically, rhEPO reduced the number of terminal deoxynucleotidyl transferase biotin-dUPT nick end labeling- and caspase-3-positive endothelial cells in the penumbra region. Colocalization of the vessel marker glucose transporter-1 (Glut-1) and cell proliferation marker 5-bromo-2'-deoxyuridine indicated enhanced angiogenic activity in rhEPO-treated mice 7 to 21 days after stroke. Western blot showed upregulation of the expression of angiogenic factors Tie-2, Angiopoietin-2, and vascular endothelial growth factor in rhEPO-treated animals. Local cerebral blood flow was measured by laser scanning imaging 3 to 21 days after stroke. At 14 days, LCBF in the penumbra was recovered to preischemia levels in rhEPO-treated mice but not in control mice. Our data suggest that rhEPO treatment upregulates the EPOR level in vascular endothelial cells, confers neurovascular protection, and enhances angiogenesis. We further show a promoting effect of rhEPO on LCBF recovery in the ischemic brain. These rhEPO-induced effects may contribute to therapeutic benefits in the treatment of ischemic stroke.

Topics: Animals; Antimetabolites; Blood Pressure; Blotting, Western; Brain Ischemia; Bromodeoxyuridine; Cerebral Infarction; Cerebrovascular Circulation; Erythropoietin; Fluorescent Antibody Technique; Heart Rate; Humans; In Situ Nick-End Labeling; Laser-Doppler Flowmetry; Male; Mice; Mice, Inbred C57BL; Neovascularization, Physiologic; Recombinant Proteins

In addition to its well-known hematopoietic effects, erythropoietin (EPO) also has neuroprotective properties. However, hematopoietic side effects are unwanted for neuroprotection, underlining the need for EPO-like compounds with selective neuroprotective actions. One such compound, devoid of hematopoietic bioactivity, is the chemically modified, EPO-derivative carbamylerythropoietin (CEPO). For comparison of the neuroprotective effects of CEPO and EPO, we subjected organotypic hippocampal slice cultures to oxygen-glucose deprivation (OGD) or N-methyl-d-aspartate (NMDA) excitotoxicity. Hippocampal slice cultures were pretreated for 24 h with 100 IU/ml EPO (=26 nM) or 26 nM CEPO before OGD or NMDA lesioning. Exposure to EPO and CEPO continued during OGD and for the next 24 h until histology, as well as during the 24 h exposure to NMDA. Neuronal cell death was quantified by cellular uptake of propidium iodide (PI), recorded before the start of OGD and NMDA exposure and 24 h after. In cultures exposed to OGD or NMDA, CEPO reduced PI uptake by 49+/-3 or 35+/-8%, respectively, compared to lesion-only controls. EPO reduced PI uptake by 33+/-5 and 15+/-8%, respectively, in the OGD and NMDA exposed cultures. To elucidate a possible mechanism involved in EPO and CEPO neuroprotection against OGD, the integrity of alpha-II-spectrin cytoskeletal protein was studied. Both EPO and CEPO significantly reduced formation of spectrin cleavage products in the OGD model. We conclude that CEPO is at least as efficient neuroprotectant as EPO when excitotoxicity is modeled in mouse hippocampal slice cultures.

Topics: Animals; Animals, Newborn; Brain Ischemia; Calpain; Coloring Agents; Erythropoietin; Excitatory Amino Acid Agonists; Glucose; Hippocampus; Hypoxia, Brain; In Vitro Techniques; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; N-Methylaspartate; Neuroprotective Agents; Propidium; Tolonium Chloride

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

After transient global cerebral ischaemia, selectively vulnerable areas of the brain show delayed neuronal degeneration. Recent data have demonstrated potent neuroprotective effects of the application of growth hormones like erythropoietin (EPO) after focal cerebral ischaemia. In order to assess possible effects of the application of EPO on cerebral recovery after cardiac arrest in rats, the vulnerable hippocampal CA-1 sector was investigated.. Thirty male Wistar rats were randomised into two groups (EPO versus placebo; n=15 per group). Cardiac arrest was induced by ventricular fibrillation during general anaesthesia. After 6 min of global cerebral ischaemia, animals were resuscitated by external chest compressions combined with defibrillation. Investigator blinded bolus application of EPO (5000 IE/kg bodyweight) and placebo was performed at three different time points, respectively: 5 min before cardiac arrest (i.v.; intravenously), 24h (i.p.; intraperitoneally) and 72 h (i.p.) after ischaemia. At 24h, 72 h and 7 days, animals were tested according to a neurological deficit score. After of reperfusion, coronal brain sections were analysed by TUNEL- and Nissl-staining. A caspase activity assay was done to determine antiapoptotic properties of EPO. Statistical analyses were done using ANOVA.. Neurological deficit scoring did not show differences between the groups. However, in all groups typical delayed neuronal degeneration could be found in the CA-1 sector. There was no difference in neuronal survival between the groups (viable neurons EPO (median [interquartile range]): 15.5 [10.1-21.3]; placebo: 16.8 [7.7-26.3]). Results from TUNEL-staining revealed no differences in the amount of apoptotic cell death between the groups (EPO: 71.2 [58.1-81.8]; placebo: 73.4 [67.8-78.2]). Caspase activity assays demonstrated a strong expression of general caspase activity as well as caspase-3 activity in the CA-1 sector and the nucleus reticularis thalami, without any differences between both groups.. Despite the well known neuroprotective properties of EPO in ischaemia induced neuronal degeneration, this study could not reveal any beneficial effects of EPO after global cerebral ischaemia due to cardiac arrest in rats.

Topics: Analysis of Variance; Animals; Brain Ischemia; Caspases; Erythropoietin; Heart Arrest; In Situ Nick-End Labeling; Injections, Intravenous; Male; Random Allocation; Rats; Rats, Wistar; Statistics, Nonparametric

Majority of severe disabilities in adults are caused by stroke. The aim of our study is to learn the effects of erythropoietin (EPO), on infarct size in cerebral ischemia and to determine neurological behavioral scores and histopathological evaluation.. In this study 30 adult Sprague-Dawney rats were used. Cerebral ischemia was constituted by intraluminal filament method with a 4-0-nylon suture. Reperfusion was started after two hours of middle cerebral artery occlusion. The rats were randomly divided into two groups as follow: control and EPO groups. Saline 0.9% (0.5 ml/kg) and EPO (5 000 U/kg) was administered intraperitoneally in the groups. Three coronal slices in two millimeters thickness were obtained from cerebrum, cerebellum and brain stem, and were stained with a 2% solution of triphenyltetrazolium chloride. Transparent sheets were placed over each section and the areas of the brain and infarct were measured. The neurological scores were determined at 24th, 48th and 72nd hours after reperfusion.. Percent of ischemic area (%) in cerebrum, cerebellum and brain stem level in EPO groups were less than those of control group (p<0.0001). In addition, we determined that EPO group was better than controls of neurologic score and histopathologically after cerebral ischemia.. We concluded that EPO may decrease ischemic area in experimental cerebral ischemia in rats and it seems that EPO may be beneficial.

Topics: Animals; Brain Ischemia; Drug Evaluation, Preclinical; Erythropoietin; Infarction, Middle Cerebral Artery; Injections, Intraperitoneal; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Treatment Outcome

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

The administration of CD34-positive cells after stroke has been shown to have a beneficial effect on functional recovery by accelerating angiogenesis and neurogenesis in rodent models. Granulocyte colony-stimulating factor (G-CSF) is known to mobilize CD34-positive cells from bone marrow and has displayed neuroprotective properties after transient ischemic stress. This led us to investigate the effects of G-CSF administration after stroke in mouse. We utilized permanent ligation of the M1 distal portion of the left middle cerebral artery to develop a reproducible focal cerebral ischemia model in CB-17 mice. Animals treated with G-CSF displayed cortical atrophy and impaired behavioral function compared with controls. The negative effect of G-CSF on outcome was associated with G-CSF induction of an exaggerated inflammatory response, based on infiltration of the peri-infarction area with CD11b-positive and F4/80-positive cells. Although clinical trials with G-CSF have been started for the treatment of myocardial and limb ischemia, our results indicate that caution should be exercised in applying these results to cerebral ischemia.

Topics: Animals; Atrophy; Brain Ischemia; Data Interpretation, Statistical; Erythropoietin; Granulocyte Colony-Stimulating Factor; Immunohistochemistry; Mice; Middle Cerebral Artery; Necrosis; Neovascularization, Pathologic; Recombinant Proteins; Stroke; Treatment Outcome

Metallothioneins (MTs) are small cysteine-rich proteins found widely throughout the mammalian body, including the CNS. MT-1 and -2 protect against reactive oxygen species and free radicals. We investigated the role of MT-1 and -2 using MT-1,-2 knockout (KO) mice. MT-1,-2 KO mice exhibited greater neuronal damage after permanent middle cerebral artery occlusion (MCAO) than wild-type mice. MT-2 mRNA was significantly increased at 6, 12, and 24 h after MCAO in the wild-type mouse brain [as detected by real-time reverse-transcription polymerase chain reaction (RT-PCR)], while MT-1 and MT-3 were decreased at 12 and 24 h. In an immunohistochemical study, MT expression displayed colocalization with glial fibrillary acidic protein (GFAP)-positive cells (astrocytes) in the penumbra area in wild-type mice. Since erythropoietin (EPO) has been reported to induce MT-1 and -2 gene expression in vitro, we examined its effect after permanent MCAO, and explored the possible underlying mechanism by examining MT-1 and -2 induction in vivo. In wild-type mice, EPO significantly reduced both infarct area and volume at 24 h after the ischemic insult. However, in MT-1,-2 KO mice EPO-treatment did not alter infarct volume (vs. vehicle-treatment). In wild-type mice at 6 h after EPO administration, real-time RT-PCR revealed increased MT-1 and -2 mRNA expression in the cerebral cortex (without MCAO). Further, MT-1 and -2 immunoreactivity was increased in the cortex of EPO-treated mice. These findings indicate that MTs are induced, and may be neuroprotective against neuronal damage, after MCAO. Furthermore, EPO is neuroprotective in vivo during permanent MCAO, and this may be at least partly mediated by MTs.

Topics: Animals; Astrocytes; Brain; Brain Infarction; Brain Ischemia; Cytoprotection; Erythropoietin; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Infarction, Middle Cerebral Artery; Male; Metallothionein; Metallothionein 3; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Degeneration; Neuroprotective Agents; RNA, Messenger; Up-Regulation

Animal experiments have suggested that apoptotic programmed cell death is responsible for an important portion of the delayed ischaemic brain damage. Antiapoptotic signalling through erythropoietin (EPO) binding to its receptor (EPOR) is triggered by systemic or local hypoxia and may exist in the post-ischaemic brain, and a neuroprotective effect by EPO was described recently and proposed for clinical stroke treatment. The objective of the study was to determine whether apoptosis occurs in human ischaemic stroke and to describe its topographical distribution. An autopsy cohort consisting of 13 cases of fatal ischaemic stroke (symptom duration from 15 h to 18 days) treated at the Department of Neurology, Helsinki University Central Hospital and 3 controls were studied. DNA damage was investigated by immunofluorescent TUNEL-labelling in combination with apoptotic cell morphology and by visualization of a major signalling system of apoptosis, Fas-FasL (Fas-ligand), by the immunoperoxidase technique. The relationship of EPO and EPOR in the face of TUNEL-labelled and necrotic cell death was co-registered in human cerebral neurons undergoing different stages of ischaemic change. TUNEL-labelled cells with apoptotic morphology were disproportionately more frequent, 148% (30) [mean (SE)] in the periinfarct versus 97% (22) in the core, as percentage of the cells in the contralateral hemisphere (P = 0.027). The apoptotic cell percentage reached up to 26% (2) of all cells in periinfarct area. A linear correlation was found for Fas and its counterpart FasL expression (r(S) = 0.774, P < 0.001). Ischaemia induced widespread neuronal expression of EPOR, which was inversely related to the severity of ischaemic neuronal necrosis (P < 0.05). To conclude, these data verify the predominance of apoptosis in the periphery of human ischaemic infarctions. Fas and FasL were linearly overexpressed supporting that this 'death-receptor' complex may promote the completion of cell death. Increased EPO signalling may be a cellular response for survival in less severely damaged areas. These results support antiapoptotic therapies against delayed neuronal cell death in human ischaemic stroke.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Autopsy; Brain; Brain Chemistry; Brain Ischemia; DNA Fragmentation; Erythropoietin; Fas Ligand Protein; fas Receptor; Female; Fluorescent Dyes; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Indoles; Male; Membrane Glycoproteins; Microscopy, Fluorescence; Middle Aged; Receptors, Erythropoietin; Tumor Necrosis Factors

Cell death induced by the combined insult of hypoxia-ischemia in neonatal rodents has been extensively investigated. Ischemia-only-induced cell death, however, has been much less characterized. Based on the notion that 1) ischemic stroke is a relatively common disorder in human neonates, and 2) developing cells are more susceptible to apoptosis, the present study examined whether typical apoptosis was induced by cerebral ischemia in a new neonatal rat model. Erythropoietin (EPO; Epoetin) was tested for its protective effect against ischemia-induced cell death. Postnatal day 7 rats were subjected to permanent occlusion of the middle cerebral artery branch supplying the right whisker-barrel cortex. Terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeled-positive cells in the ischemic region were detectable 4 h after ischemia and reached a peak level 16 h later. The cell death was preceded by caspase activation and cytochrome c release. Cell body shrinkage was evident among damaged cells. Agarose gel electrophoresis showed DNA damage with a smear pattern as well as DNA laddering. Electron microscopy demonstrated apoptotic features such as cell shrinkage, chromatin condensation, and fragmentation; meanwhile, necrotic alterations coexisted in the cytoplasm. EPO treatment increased signal transducers and activators of transcription-5 and Bcl-2 levels, markedly attenuated apoptotic cell death, and reduced ischemic infarct in the cortex. It is suggested that focal ischemia in the developing brain causes cell death with prominent apoptotic features coexisting with some characteristics of necrosis. This is consistent with the concept of hybrid death described previously in cultures and adult or developing brain. EPO may be explored as a potential therapy for neonatal ischemic stroke.

Topics: Animals; Animals, Newborn; Apoptosis; Brain Ischemia; Cerebral Cortex; Erythropoietin; Female; In Situ Nick-End Labeling; Male; Microscopy, Electron, Transmission; Neuroprotective Agents; Rats; Rats, Wistar; Recombinant Proteins

This study was designed to investigate the neuroprotective effect of intrinsic and extrinsic erythropoietin (EPO) against hypoxia/ischemia, and determine the optimal time-window with respect to the EPO-induced neuroprotection. Experiments were conducted using primary mixed neuronal/astrocytic cultures and neuron-rich cultures. Hypoxia (2%) induces hypoxia-inducible factor-1alpha (HIF-1alpha) activity followed by strong EPO expression in mixed cultures and weak expression in neuron-rich cultures as documented by both western blot and RT-PCR. Immunoreactive EPO was strongly detected in astrocytes, whereas EPOR was only detected in neurons. Neurons were significantly damaged in neuron-rich cultures but were distinctly rescued in mixed cultures. Application of recombinant human EPO (rhEPO) (0.1 U/mL) within 6 h before or after hypoxia significantly increased neuronal survival compared with no rhEPO treatment. Application of rhEPO after onset of reoxygenation achieved the maximal neuronal protection against ischemia/reperfusion injury (6 h hypoxia followed 24 h reoxygenation). Our results indicate that HIF-1alpha induces EPO gene released by astrocytes and acts as an essential mediator of neuroprotection, prove the protective role of intrinsic astrocytic-neuronal signaling pathway in hypoxic/ischemic injury and demonstrate an optimal therapeutic time-window of extrinsic rhEPO in ischemia/reperfusion injury in vitro. The results point to the potential beneficial effects of HIF-1alpha and EPO for the possible treatment of stroke.

Topics: Animals; Brain Ischemia; Cells, Cultured; Cerebral Cortex; Erythropoietin; Humans; Neurons; Neuroprotective Agents; Rats; Rats, Wistar; Receptors, Erythropoietin; Recombinant Proteins; Reperfusion Injury

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

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

Erythropoietin (Epo) plays a central role in erythropoiesis but also has neuroprotective properties. Recently, Epo-related neuroprotective studies used a hypoxic-ischemic neonatal model, which is different from focal stroke, a frequent cause of neonatal brain injury. We report on the effects of Epo treatment given after focal stroke and its potential neuroprotective mechanisms in postnatal day 7 rats with focal cerebral ischemia (FCI) achieved by occlusion of the middle cerebral artery. The experimental groups included sham operation, FCI plus vehicle, and FCI plus Epo. In the Epo-treated group, pups received a single intraperitoneal injection of 1000 U/kg 15 min after FCI or three injections of 100, 1000, or 5000 U/kg, starting at 15 min and repeated at 1 and 2 d after FCI. Epo treatment produced significant reductions in the mean infarct area and volume at 1 and 3 d after FCI, demonstrated by 2,3,5-triphenyltetrazolium chloride staining. Terminal deoxynucleotidyltransferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining showed a markedly reduced number of TUNEL-positive cells in the Epo-treated group when compared with the vehicle control 3 d after FCI (p<0.01). The most effective dose after FCI was 1000 U/kg for 3 d. Immunoanalyses showed that Epo induced a significant increase in phosphorylated Janus kinase 2 and signal transducer and activator of transcription-5 expressions at 1 and 3 d and up-regulated Bcl-xL expression by 24 h after FCI but did not affect Epo receptor or NF-kappaB expression. In conclusion, Epo given after FCI in neonatal rats provides significant neuroprotection, mediated possibly by activation of the Janus kinase-signal transducer and activator of transcription-Bcl-xL signaling pathways.

Topics: Animals; Animals, Newborn; bcl-X Protein; Brain Ischemia; Cerebral Cortex; DNA-Binding Proteins; Erythropoietin; Female; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Janus Kinase 2; Milk Proteins; NF-kappa B; Pregnancy; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Signal Transduction; STAT5 Transcription Factor; Trans-Activators

Darbepoetin alfa is a novel erythropoiesis-stimulating protein developed for treating anemia. In animal models, exogenous recombinant human erythropoietin has been reported to be beneficial in treating experimental cerebral ischemia. In this study, we determined whether darbepoetin alfa would protect in a rat model of transient focal cerebral ischemia.. Rats received 2-hour middle cerebral artery suture-occlusion. The drug (darbepoetin alfa, 10 microg/kg) or vehicle was administered intraperitoneally 2 hours after onset of middle cerebral artery occlusion. Animals were allowed to survive for 3 or 14 days. Behavioral tests were performed sequentially. Infarct volumes and brain swelling were determined.. Darbepoetin alfa-treated rats showed improved neuroscores relative to vehicle-treated animals beginning within 1 hour of treatment and persisting throughout the 14-day survival period. Darbepoetin alfa significantly reduced corrected total (cortical + subcortical) infarct volume (56.3+/-20.6 and 110.8+/-6.8 mm3, respectively) and total infarct areas at multiple levels compared with vehicle in the 14-day survival group. Brain swelling was not affected by treatment.. Darbepoetin alfa confers behavioral and histological neuroprotection after focal ischemia in rats.

Topics: Animals; Behavior, Animal; Brain Edema; Brain Ischemia; Darbepoetin alfa; Erythropoietin; Infarction, Middle Cerebral Artery; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Recombinant Proteins

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

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

Apart from its hematopoietic function, erythropoietin (Epo) exerts neuroprotective functions in brain hypoxia and ischemia. To examine the mechanisms mediating Epo's neuroprotective activity in vivo, we made use of our transgenic mouse line tg21 that constitutively expresses human Epo in brain without inducing excessive erythrocytosis. We show that human Epo is expressed in tg21 brains and that cortical and striatal neurons carry the Epo receptor. After middle cerebral artery occlusion, human Epo potently protected brains of tg21 mice against ischemic injury, both when severe (90 min) and mild (30 min) ischemia was imposed. Histochemical studies revealed that Epo induced an activation of JAK-2, ERK-1/-2, and Akt pathways in the ischemic brain. This activation was associated with elevated Bcl-XL and decreased NO synthase-1 and -2 levels in neurons. Intracerebroventricular injections of selective inhibitors of ERK-1/-2 (PD98059) or Akt (wortmannin) pathways revealed that both ERK-1/-2 and Akt were required for Epo's neuroprotective function, antagonization of either pathway completely abolishing tissue protection. On the other hand, ERK-1/-2 and Akt blockade did not reverse the neuronal NO synthase-1/-2 inhibition, indicating that Epo down-regulates these NO synthases in an ERK-1/-2 and Akt independent manner. On the basis of our data, the dual activation of ERK-1/-2 and Akt is crucial for Epo's neuroprotective activity.

Topics: Androstadienes; Animals; bcl-X Protein; Brain; Brain Ischemia; Enzyme Activation; Enzyme Inhibitors; Erythropoietin; Flavonoids; Humans; Hypoxia; Immunohistochemistry; Ischemia; Janus Kinase 2; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Biological; Neurons; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Erythropoietin; Signal Transduction; Time Factors; Wortmannin

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

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

Erythropoietin (Epo) prevents ischemia and hypoxia-induced neuronal death in vitro. Recent studies have shown that this cytokine also has in vivo neuroprotective effects in cerebral and spinal ischemia in adult rodents. In this study, we aimed to investigate the effect of systemically administered recombinant human Epo on infarct volume and apoptotic neuronal death in a newborn rat hypoxic-ischemic brain injury model. Our results showed that a single dose of intraperitoneal Epo treatment (1,000 U/kg) significantly decreased the mean infarct volume as compared to the control group. In contrast to the Epo-treated group, histopathological examination by positive terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of the affected brain in control animals revealed widespread neuronal injury associated with numerous apoptotic cells. Morphometric analysis to determine the extent of damage quantitatively ascertained that the mean infarct volume was significantly lower in the Epo-treated group (p < 0.003). These results suggest the beneficial neuroprotective effect of Epo in this model of neonatal hypoxic-ischemic brain injury. To our knowledge, this is the first study that demonstrates a protective effect of Epo against hypoxia-ischemia in the developing brain.

Topics: Animals; Animals, Newborn; Brain; Brain Ischemia; Erythropoietin; Hypoxia, Brain; Neuroprotective Agents; Rats; Rats, Wistar

Erythropoietin (EPO) is a tissue-protective cytokine preventing vascular spasm, apoptosis, and inflammatory responses. Although best known for its role in hematopoietic lineages, EPO also affects other tissues, including those of the nervous system. Enthusiasm for recombinant human erythropoietin (rhEPO) as a potential neuroprotective therapeutic must be tempered, however, by the knowledge it also enlarges circulating red cell mass and increases platelet aggregability. Here we examined whether erythropoietic and tissue-protective activities of rhEPO might be dissociated by a variation of the molecule. We demonstrate that asialoerythropoietin (asialoEPO), generated by total enzymatic desialylation of rhEPO, possesses a very short plasma half-life and is fully neuroprotective. In marked contrast with rhEPO, this molecule at doses and frequencies at which rhEPO exhibited erythropoiesis, did not increase the hematocrit of mice or rats. AsialoEPO appeared promptly within the cerebrospinal fluid after i.v. administration; intravenously administered radioiodine-labeled asialoEPO bound to neurons within the hippocampus and cortex in a pattern corresponding to the distribution of the EPO receptor. Most importantly, asialoEPO exhibits a broad spectrum of neuroprotective activities, as demonstrated in models of cerebral ischemia, spinal cord compression, and sciatic nerve crush. These data suggest that nonerythropoietic variants of rhEPO can cross the blood-brain barrier and provide neuroprotection.

Topics: Animals; Brain Ischemia; Erythropoietin; Hemoglobins; Male; Mice; Neuroprotective Agents; Rats; Rats, Sprague-Dawley

Topics: Brain Ischemia; Clinical Trials, Phase III as Topic; Erythropoiesis; Erythropoietin; Humans; Neuroprotective Agents

Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)-expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R-expressing inflammatory cells.

Topics: Animals; Apoptosis; Brain Ischemia; Cells, Cultured; Coculture Techniques; Cytokines; Erythropoietin; Humans; Infarction, Middle Cerebral Artery; Inflammation; Lipopolysaccharides; Male; Neuroglia; Neurons; Neuroprotective Agents; Rats; Receptors, Erythropoietin; Recombinant Proteins; Tumor Necrosis Factor-alpha

Erythropoietin has recently been studied for its role in the central nervous system (CNS). It has been shown to exert neuroprotective effects in different models of brain injury. We studied whether neuroprotective effects assessed from the reduction of neuronal loss after transient brain ischemia are associated to the preservation of learning ability. Recombinant human erythropoietin (0.5-25 U) was injected in the lateral cerebral ventricle of gerbils that are subjected to temporary (3 min) bilateral carotid occlusion. Post-ischemic histological evaluation of CA1 area neuronal loss and passive avoidance test were performed. Treatment with recombinant human erythropoietin significantly reduced delayed neuronal death in the CA1 area of the hippocampus and prevented cognition impairment in the passive avoidance test. These data indicate that recombinant human erythropoietin neuroprotective effects in brain ischemia are associated with the preservation of learning function.

Topics: Animals; Avoidance Learning; Behavior, Animal; Brain Ischemia; Cognition Disorders; Dose-Response Relationship, Drug; Erythropoietin; Gerbillinae; Humans; Male; Neurons; Recombinant Proteins

Erythropoietin (EPO) promotes neuronal survival after cerebral ischemia in vivo and after hypoxia in vitro. However, the mechanisms underlying the protective effects of EPO on ischemic/hypoxic neurons are not fully understood. The present in vitro experiments showed that EPO attenuated neuronal damage caused by chemical hypoxia at lower extracellular concentrations (10(- 4)-10(-2) U/ml) than were previously considered. Moreover, EPO at a concentration of 10(-3) U/ml up-regulated Bcl-xL mRNA and protein expressions in cultured neurons. Subsequent in vivo study focused on whether EPO rescued hippocampal CA1 neurons from lethal ischemic damage and up-regulated the expressions of Bcl-xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils. EPO was infused into the cerebroventricles of gerbils immediately after 3 min of ischemia for 28 days. Infusion of EPO at a dose of 5 U/day prevented the occurrence of ischemia-induced learning disability. Subsequent light microscopic examinations showed that pyramidal neurons in the hippocampal CA1 field were significantly more numerous in ischemic gerbils infused with EPO (5 U/day) than in those receiving vehicle infusion. The same dose of EPO infusion caused significantly more intense expressions of Bcl-xL mRNA and protein in the hippocampal CA1 field of ischemic gerbils than did vehicle infusion. These findings suggest that EPO prevents delayed neuronal death in the hippocampal CA1 field, possibly through up-regulation of Bcl-xL, which is known to facilitate neuron survival.

Topics: Animals; Avoidance Learning; bcl-X Protein; Brain Ischemia; Cell Count; Cell Hypoxia; Cells, Cultured; Cerebral Cortex; Erythropoietin; Gene Expression; Gerbillinae; Male; Neurons; Neuroprotective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Reaction Time; RNA, Messenger

Tolerance to cerebral ischemia is achieved by preconditioning sublethal stresses, such as ischemia or hypoxia, paradigms in which the decrease of O2 availability may constitute an early signal inducing tolerance. In accordance with this concept, this study shows that hypoxia induces tolerance against focal permanent ischemia in adult mice. Normobaric hypoxia (8% O2 of 1-hour, 3-hour, or 6-hour duration), performed 24 hours before ischemia, reduces infarct volume by approximately 30% when compared with controls. To elucidate the mechanisms underlying this neuroprotection, the authors investigated the effects of preconditioning on cerebral expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and its target genes, erythropoietin and vascular endothelial growth factor (VEGF). Hypoxia, whatever its duration (1 hour, 3 hours, 6 hours), rapidly increases the nuclear content of HIF-1alpha as well as the mRNA levels of erythropoietin and VEGF. Furthermore, erythropoietin and VEGF are upregulated at the protein level 24 hours after 6 hours of hypoxia. The authors' findings show that (1) hypoxia elicits a delayed, short-lasting (<72 hours) tolerance to focal permanent ischemia in the adult mouse brain; (2) HIF-1 target genes could contribute to the establishment of tolerance; and (3) this model might be a useful paradigm to further study the mechanisms of ischemic tolerance, to identify new therapeutic targets for stroke.

Topics: Animals; Brain; Brain Ischemia; DNA-Binding Proteins; Endothelial Growth Factors; Erythropoietin; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Ischemic Preconditioning; Lymphokines; Male; Mice; Nuclear Proteins; Oxygen; Time Factors; Transcription Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

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

There is increasing evidence that erythropoietin (Epo) has a protective function in cerebral ischemia. When used for treatment, high Epo plasma levels associated with increases in blood viscosity, however, may counteract beneficial effects of Epo in brain ischemia. The authors generated two transgenic mouse lines that overexpress human Epo preferentially, but not exclusively, in neuronal cells. In mouse line tg21, a fourfold increase of Epo protein level was found in brain only, whereas line tg6 showed a dramatic increase of cerebral and systemic transgene expression resulting in hematocrit levels of 80%. Cerebral blood flow (CBF), as determined by bolus tracking magnetic resonance imaging, was not altered in the tg6 line. The time-to-peak interval for the tracer, however, increased approximately threefold in polyglobulic tg6 mice. Immunohistochemical analysis revealed an increase in dilated vessels in tg6 mice, providing an explanation for unaltered CBF in polyglobulic animals. Permanent occlusion of the middle cerebral artery (pMCAO) led to similar perfusion deficits in wild-type, tg6, and tg21 mice. Compared with wild-type controls, infarct volumes were not significantly smaller (22%) in tg21 animals 24 hours after pMCAO, but were 49% enlarged (P < 0.05) in polyglobulic tg6 mice. In the latter animals, elevated numbers of Mac-1 immunoreactive cells in infarcted tissue suggested that leukocyte infiltration contributed to enlarged infarct volume. The current results indicate that moderately increased brain levels of Epo in tg21 transgenic mice were not sufficient to provide significant tissue protection after pMCAO. The results with tg6 mice indicate that systemic chronic treatment with Epo associated with elevated hematocrit might deteriorate outcome after stroke either because of the elevated hematocrit or other chronic effects.

Topics: Animals; Blood Flow Velocity; Blood Viscosity; Brain; Brain Ischemia; CD11 Antigens; Cerebral Infarction; Endothelium, Vascular; Erythropoietin; Gene Expression; Hematocrit; Humans; Laminin; Macrophages; Magnetic Resonance Imaging; Male; Mice; Mice, Transgenic; Middle Cerebral Artery; Monocytes; Neurons; Platelet Endothelial Cell Adhesion Molecule-1; Vascular Cell Adhesion Molecule-1

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

Erythropoietin prevents in vitro glutamate-induced neuronal death and could play a role in the central nervous system. We investigated the in vivo effects of recombinant human erythropoietin after intraperitoneal (i.p.; 25-100 U) or intracerebroventricular (i.c.v.; 0.25-25 U) administration on survival, brain malonildialdehyde (MDA) levels, brain edema, hippocampal neuronal death and brain nitric oxide (NO) synthesis after bilateral carotid occlusion (5 min), followed by reperfusion in the Mongolian gerbil. Peripheral posttreatment with recombinant human erythropoietin reduced postischemic MDA levels, brain edema and increased survival. Either peripheral or i.c.v. posttreatment with recombinant human erythropoietin significantly reduced hippocampal CA1 neuronal loss, observed 7 days after the ischemic event. Increase of nitrite and nitrate (as an index of NO formation) in the hippocampus, as observed after ischemia, was reduced in animals treated with recombinant human erythropoietin. These data suggest that in vivo recombinant human erythropoietin effects on brain ischemic injury could be due to inhibition of NO overproduction.

Topics: Aldehydes; Animals; Brain; Brain Edema; Brain Ischemia; Erythropoietin; Gerbillinae; Humans; Injections, Intraperitoneal; Injections, Intraventricular; Male; Neurons; Nitrates; Nitric Oxide; Nitrites; Recombinant Proteins; Survival Rate

Erythropoietin exerts a neuroprotective effect during cerebral ischemia. We investigated the effect of systemic administration of recombinant human erythropoietin in a rabbit model of subarachnoid hemorrhage-induced acute cerebral ischemia. The animals were divided into three groups: group 1, subarachnoid hemorrhage; group 2, subarachnoid hemorrhage plus placebo; group 3, subarachnoid hemorrhage plus recombinant human erythropoietin (each group, n=8). Experimental subarachnoid hemorrhage was produced by injecting autologous blood into the cisterna magna. Treatment with recombinant human erythropoietin and placebo was started 5 min after subarachnoid hemorrhage and was continued every 8 h for 24 h. Before the animals were killed, erythropoietin concentration was measured in the cerebrospinal fluid. The rabbits were killed 24 h after subarachnoid hemorrhage and ischemic brain injury was histologically evaluated. In group 3, the concentration of erythropoietin in the cerebrospinal fluid was significantly increased and a significant reduction in cortical necrotic neuron count was also observed. These findings may encourage the use of erythropoietin in the treatment of cerebral ischemia that often occurs in the early stage of subarachnoid hemorrhage.

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Calcium; Erythropoietin; Male; Rabbits; Recombinant Proteins; Subarachnoid Hemorrhage

The present study describes, for the first time, a temporal and spatial cellular expression of erythropoietin (Epo) and Epo receptor (Epo-R) with the evolution of a cerebral infarct after focal permanent ischemia in mice. In addition to a basal expression of Epo in neurons and astrocytes, a postischemic Epo expression has been localized specifically to endothelial cells (1 day), microglia/macrophage-like cells (3 days), and reactive astrocytes (7 days after occlusion). Under these conditions, the Epo-R expression always precedes that of Epo for each cell type. These results support the hypothesis that there is a continuous formation of Epo, with its corresponding receptor, during the active evolution of a focal cerebral infarct and that the Epo/Epo-R system might be implicated in the processes of neuroprotection and restructuring (such as angiogenesis and gliosis) after ischemia. To support this hypothesis, a significant reduction in infarct volume (47%; P < 0.0002) was found in mice treated with recombinant Epo 24 hours before induction of cerebral ischemia. Based on the above, we propose that the Epo/Epo-R system is an endogenous mechanism that protects the brain against damages consequent to a reduction in blood flow, a mechanism that can be amplified by the intracerebroventricular application of exogenous recombinant Epo.

Topics: Animals; Astrocytes; Blotting, Western; Brain; Brain Chemistry; Brain Ischemia; Cell Death; Cells, Cultured; Cerebral Infarction; Endothelium, Vascular; Erythropoietin; Immunohistochemistry; In Situ Hybridization; Injections, Intraventricular; Mice; Neurons; Receptors, Erythropoietin

Human recombinant erythropoietin (r-huEPO) is very effective in the treatment of anemia of hemodialyzed patients. We describe 4 patients who developed symptoms of central nervous dysfunction during r-huEPO therapy. Three exhibited typical hypertensive encephalopathy, whereas signs of cerebral ischemia were found in the fourth. The increase in blood viscosity with r-huEPO treatment, leading to a rise in peripheral vascular resistance and blood pressure especially in previously hypertensive patients, may be of importance in the pathogenesis of these cerebrovascular incidents; preexistent arteriosclerosis is an possible additional risk factor.

Topics: Adult; Aged; Anemia, Hemolytic; Blood Viscosity; Brain Edema; Brain Ischemia; Cerebrovascular Disorders; Erythropoietin; Female; Humans; Male; Middle Aged; Recombinant Proteins; Renal Dialysis