allopurinol and Reperfusion-Injury

Ischemia-reperfusion injury is associated with serious clinical manifestations, including myocardial hibernation, acute heart failure, cerebral dysfunction, gastrointestinal dysfunction, systemic inflammatory response syndrome, and multiple organ dysfunction syndrome. Ischemia-reperfusion injury is a critical medical condition that poses an important therapeutic challenge for physicians. In this review article, we present recent advances focusing on the basic pathophysiology of ischemia-reperfusion injury, especially the involvement of reactive oxygen species and cell death pathways. The involvement of the NADPH oxidase system, nitric oxide synthase system, and xanthine oxidase system are also described. When the blood supply is re-established after prolonged ischemia, local inflammation and ROS production increase, leading to secondary injury. Cell damage induced by prolonged ischemia-reperfusion injury may lead to apoptosis, autophagy, necrosis, and necroptosis. We highlight the latest mechanistic insights into reperfusion-injury-induced cell death via these different processes. The interlinked signaling pathways of cell death could offer new targets for therapeutic approaches. Treatment approaches for ischemia-reperfusion injury are also reviewed. We believe that understanding the pathophysiology ischemia-reperfusion injury will enable the development of novel treatment interventions.

Topics: Anti-Inflammatory Agents; Apoptosis; Autophagy; Humans; Mitophagy; NADPH Oxidases; NF-kappa B; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

Sickle cell disease is a class of hemoglobinopathy in humans, which is the most common inherited disease in the world. Although complications of sickle cell disease start from polymerization of red blood cells during its deoxygenating phase, the oxidative stress resulting from the biological processes associated with this disease (ischaemic and hypoxic injuries, hemolysis and inflammation) has been shown to contribute to its pathophysiology. It is widely known that chronic exercise reduces oxidative stress in healthy people, mainly via improvement of antioxidant enzyme efficiency. In addition, recent studies in other diseases, as well as in sickle cell trait carriers and in a mouse model of sickle cell disease, have shown that regular physical activity could decrease oxidative stress. The purpose of this review is to summarize the role of oxidative stress in sickle cell disease and the effects of acute and chronic exercise on the pro-oxidant/antioxidant balance in sickle cell trait and sickle cell disease.

Topics: Anemia, Sickle Cell; Animals; Disease Models, Animal; Exercise; Heme; Hemoglobin, Sickle; Hemolysis; Humans; Hypoxia; Iron; Mice; NADPH Oxidases; Nitric Oxide; Oxidative Stress; Reperfusion Injury; Sickle Cell Trait; Xanthine Oxidase

To update the reader on the recent literature in liver, kidney, pancreas, and intestine static cold preservation, and to identify which solutions are most advantageous for each organ.. The comparison of randomized trials of histidine-tryptophan-ketoglutarate (HTK), Celsior, and University of Wisconsin solutions has shown equivalent risk of delayed graft function after kidney transplantation. Similar outcomes have been observed after pancreas preservation with University of Wisconsin, HTK, and Celsior solution. In live-donor liver transplantation, University of Wisconsin and HTK solution have shown equivalent results, whereas in the recent trials of deceased-donor liver transplantation, University of Wisconsin, HTK, and Celsior solutions have shown equivalence. Contrary to the most clinical trials, national registry data in kidney, pancreas, and liver transplantation demonstrate more detrimental effects and earlier graft loss after preservation with HTK versus University of Wisconsin solution. Early outcomes after intestinal transplantation with University of Wisconsin or HTK solution have shown no significant difference and animal studies indicate intraluminal preservation may be beneficial.. The University of Wisconsin solution is the standard criterion static cold preservation for the procurement of liver, kidney, pancreas, and intestine. University of Wisconsin, HTK, and Celsior solutions all provide similar allograft outcomes in most clinical trials, but subtle differences have become more apparent in the recent studies and registry reports.

Topics: Abdomen; Adenosine; Allografts; Allopurinol; Cryopreservation; Disaccharides; Electrolytes; Glucose; Glutamates; Glutathione; Histidine; Humans; Insulin; Intestines; Kidney; Liver; Mannitol; Organ Preservation Solutions; Organ Transplantation; Pancreas; Potassium Chloride; Procaine; Raffinose; Reperfusion Injury; Tissue Donors

Allopurinol is a xanthine oxidase inhibitor and antioxidant free radical scavenger which facilitates the protection of ischemic organs in part via this mechanism of action. The accumulation of free radicals during ischemia and reperfusion is in great manner overcome by inhibitors of xanthine oxidase and by the development of endogenous antioxidants. The ischemic lesion generates a well-established inflammatory response with the subsequent production of inflammatory molecules characteristically present at the first stages of the injury. Inflammatory cytokines, chemokines, adhesion molecules, and other cellular and molecular compounds are consequently produced as the lesion sets in. Under these conditions, allopurinol diminishes the effect of inflammatory mediators during the ischemic inflammatory response. This study reviews the literature associated with allopurinol and renal ischemia making special emphasis on the best dose and time of administration of allopurinol regarding its protective effect. It also defines the most accepted mechanism of protection on ischemichally damaged kidneys.

Topics: Allopurinol; Animals; Calcium Channels; Cell Adhesion Molecules; Cytokines; Enzyme Inhibitors; Free Radical Scavengers; Humans; Immune System; Ischemia; Kidney; Lipid Peroxidation; Metabolic Networks and Pathways; NF-kappa B; Nitric Oxide; Reactive Oxygen Species; Receptors, Tumor Necrosis Factor; Reperfusion Injury; Tumor Necrosis Factor-alpha; Xanthine Oxidase

Allopurinol was first introduced, in 1963, as a xanthine oxidase inhibitor when it was investigated for concomitant use with cancer chemotherapy drugs. Today it is used in gout and hyperuricemia. Due to its additive benefit in preventing oxidative damage, attention has shifted towards the use of allopurinol in organ ischemia and reperfusion.. Currently, the mechanism by which allopurinol exerts a protective benefit in ischemia reperfusion related events is not fully understood. There are various theories: it may act by inhibiting the irreversible breakdown of purine substrates, and/or by inhibiting the formation of reactive oxygen species, and/or by protecting against damage to the mitochondrial membrane.. This work focuses on liver ischemia and reperfusion injury in an effort to better understand the mechanisms associated with allopurinol and with this pathological entity.. The current research, mainly in animal models, points to allopurinol having a protective benefit, particularly if used pre-ischemically in liver ischemia reperfusion injury. Furthermore, after reviewing allopurinol dosing and administration, it was found that 50 mg/kg is statistically the most effective dose in attenuating liver ischemia reperfusion injury. Owing to the limited number of samples, the time of administration did not show statistical difference, but allopurinol was often beneficial when given around 1 h before ischemia.. In conclusion, allopurinol, through its known xanthine oxidase inhibitory effect, as only one of the potential mechanisms, has demonstrated its potential application in protecting the liver during ischemia and reperfusion.

Topics: Allopurinol; Animals; Enzyme Inhibitors; Humans; Liver Diseases; Reperfusion Injury; Xanthine Oxidase

Several experimental models on adult and newborn animals showed that in cerebral hypoxic-ischemic conditions similar to clinical states the main source of the excessive production of free oxygen radicals is the highly activated xanthine oxidase (XO) enzyme reaction. Long before this data were available, it became known that the main role of allopurinol (AP) is the inhibition of XO. On the basis of these results, many therapeutic trials with AP were performed both in experimental and clinical studies of ischemia and reperfusion. However, it has been shown that only preventive administration of AP has favorable effects. The explanation for the poor results of AP treatment in human fetal brain damage (FBD) cases is that the drug was applied postnatally. The clinical studies performed in healthy laboring mothers whose deliveries were complicated with FBD showed that placental transfer after prenatal administration of AP may be effective in protecting newborns at increased risk of hypoxic-ischemic cerebral damage. Further controlled trials are required to determine if the prophylactic use of the drug might prevent hypoxic-ischemic injuries when the drug is administered immediately prior to impending fetal hypoxia, or even in deliveries at risk of developing hypoxia.

Topics: Allopurinol; Animals; Animals, Newborn; Brain Injuries; Enzyme Inhibitors; Female; Fetal Hypoxia; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Pregnancy; Reperfusion Injury; Xanthine Oxidase

Xanthine oxidoreductase (XOR) is a ubiquitous complex cytosolic molybdoflavoprotein which controls the rate limiting step of purine catabolism by converting xanthine to uric acid. It is known that optimum concentrations of uric acid (UA) and reactive oxygen species (ROS) are necessary for normal functioning of the body. The ability of XOR to perform detoxification reactions, and to synthesize UA and reactive oxygen species (ROS) makes it a versatile intra- and extra-cellular protective "housekeeping enzyme". It is also an important component of the innate immune system. The enzyme is a target of drugs against gout and hyperuricemia and the protein is of major interest as it is associated with ischemia reperfusion (I/R) injury, vascular disorders in diabetes, cardiovascular disorders, adipogenesis, metabolic syndrome, cancer, and many other disease conditions. Xanthine oxidoreductase in conjugation with antibodies has been shown to have an anti-tumor effect due to its ability to produce ROS, which in turn reduces the growth of cancer tissues. Apart from this, XOR in association with nitric oxide synthase also participates in myocardial excitation-contraction coupling. Although XOR was discovered over 100 years ago, its physiological and pathophysiological roles are still not clearly elucidated. In this review, various physiological and pathophysiological functional aspects of XOR and its association with various forms of cancer are discussed in detail.

Topics: Cardiovascular Diseases; Cardiovascular System; Diabetes Mellitus; Excitation Contraction Coupling; Humans; Molecular Conformation; Neoplasms; Purines; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

Allopurinol is an inhibitor of xanthine oxidoreductase (XOR) and inhibits the generation of uric acid (UA) as the final product of purine catabolism, as well as the resulting generation of superoxide (O2(-)), in humans. Elevation of the serum UA (SUA) level, referred to as hyperuricemia (HU), eventually leads to gout and allopurinol has been used for the treatment of HU and gout. Studies have revealed the role of elevated SUA levels and the associated oxidative stress (OS) in a broad spectrum of pathological conditions and it is anticipated that these findings would also expand the use of allopurinol as a therapeutic drug. This article presents a review of reports, mainly of recent studies, on the efficacy of allopurinol in various diseases and explores novel potential uses of the drug. Important novel and potential uses of great interest include metabolic syndrome (MetS) and related disorders, chronic kidney disease (CKD), nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Ischemia-reperfusion injury and mucositis, encountered as adverse effects of cancer treatment, have also been under investigation as potential targets for allopurinol.

Topics: Allopurinol; Enzyme Inhibitors; Fatty Liver; Humans; Liver Diseases; Metabolic Syndrome; Mucositis; Renal Insufficiency, Chronic; Reperfusion Injury; Uric Acid; Xanthine Dehydrogenase

Topics: Animals; Antioxidants; Apoptosis; Free Radicals; Glutathione Peroxidase; Humans; Reactive Oxygen Species; Reperfusion Injury; Selenium Compounds; Xanthine Dehydrogenase; Xanthine Oxidase

Xanthine oxidase (XO) is a cytosolic metalloflavoprotein that has been implicated in the pathogenesis of a wide spectrum of diseases, and is thought to be the most important source of oxygen-free radicals and cell damage during re-oxygenation of hypoxic tissues. Clinical studies have already shown that XO inhibition is safe and effective for the treatment of gout, tumour-lysis syndrome, and to reduce complications such as post-operative arrhythmias, myocardial infarction and mortality in cardiovascular surgery. Here, we review the evidence from two decades of animal studies that have investigated the effects of XO inhibition during intra-abdominal surgery.. A search of the Ovid MEDLINE database from 1950 through January 2007 was carried out using the following search terms: xanthine oxidase, allopurinol, ischemia, reperfusion, intestine, bowel, and general surgery.. The inhibition of XO has been shown to reduce oxidative stress, neutrophil priming, damage to intestinal mucosa due to ischemia reperfusion injuries, intestinal anastomotic dehiscence, bacterial translocation, adhesion formation, distant organ injury and mortality.. Despite this evidence which very strongly suggests a likely clinically beneficial role for XO inhibition in the elective and acute operative setting, it is surprising that such an approach has not been investigated in general surgery. There is now sufficient evidence to justify dedicated studies to determine the clinical benefits, dosing and duration of XO inhibition before and after gastrointestinal surgery.

Topics: Animals; Digestive System Diseases; Laparotomy; Oxidative Stress; Reperfusion Injury; Surgical Wound Dehiscence; Tissue Adhesions; Xanthine Oxidase

The prototypical xanthine oxidase (XO) inhibitor allopurinol, has been the cornerstone of the clinical management of gout and conditions associated with hyperuricemia for several decades. More recent data indicate that XO also plays an important role in various forms of ischemic and other types of tissue and vascular injuries, inflammatory diseases, and chronic heart failure. Allopurinol and its active metabolite oxypurinol showed considerable promise in the treatment of these conditions both in experimental animals and in small-scale human clinical trials. Although some of the beneficial effects of these compounds may be unrelated to the inhibition of the XO, the encouraging findings rekindled significant interest in the development of additional, novel series of XO inhibitors for various therapeutic indications. Here we present a critical overview of the effects of XO inhibitors in various pathophysiological conditions and also review the various emerging therapeutic strategies offered by this approach.

Topics: Allopurinol; Animals; Cardiovascular Diseases; Enzyme Inhibitors; Humans; Inflammatory Bowel Diseases; Reperfusion Injury; Superoxides; Xanthine Oxidase

Uric acid is the final product of purine metabolism in humans. The final two reactions of its production catalyzing the conversion of hypoxanthine to xanthine and the latter to uric acid are catalysed by the enzyme xanthine oxidoreductase, which may attain two inter-convertible forms, namely xanthine dehydrogenase or xanthine oxidase. The latter uses molecular oxygen as electron acceptor and generates superoxide anion and other reactive oxygen products. The role of uric acid in conditions associated with oxidative stress is not entirely clear. Evidence mainly based on epidemiological studies suggests that increased serum levels of uric acid are a risk factor for cardiovascular disease where oxidative stress plays an important pathophysiological role. Also, allopurinol, a xanthine oxidoreductase inhibitor that lowers serum levels of uric acid exerts protective effects in situations associated with oxidative stress (e.g. ischaemia-reperfusion injury, cardiovascular disease). However, there is increasing experimental and clinical evidence showing that uric acid has an important role in vivo as an antioxidant. This review presents the current evidence regarding the antioxidant role of uric acid and suggests that it has an important role as an oxidative stress marker and a potential therapeutic role as an antioxidant. Further well designed clinical studies are needed to clarify the potential use of uric acid (or uric acid precursors) in diseases associated with oxidative stress.

Topics: Animals; Antioxidants; Humans; Laparoscopy; Oxidative Stress; Reperfusion Injury; Uric Acid; Xanthine Oxidase

Ischemia-reperfusion injury of the intestine is a significant problem in abdominal aortic aneurysm surgery, small bowel transplantation, cardiopulmonary bypass, strangulated hernias, and neonatal necrotizing enterocolitis. It can also occur as a consequence of collapse of systemic circulation, as in hypovolemic and septic shock. It is associated with a high morbidity and mortality. This article is a comprehensive review of the current status of the molecular biology and the strategies to prevent ischemia-reperfusion injury of the intestine. Various treatment modalities have successfully been applied to attenuate reperfusion injury in animal models of reperfusion injury of the intestine. Ischemic preconditioning has been found to be the most promising strategy against reperfusion injury during the last few years, appearing to increase the tolerance of the intestine to reperfusion injury. Although ischemic preconditioning has been shown to be beneficial in the human heart and the liver, prospective controlled studies in humans involving ischemic preconditioning of the intestine are lacking. Research focused on the application of novel drugs that can mimic the effects of ischemic preconditioning to manipulate the cellular events during reperfusion injury of the intestine is required.

Topics: Animals; Biomarkers; Endothelins; Heat-Shock Proteins; Humans; Inflammation Mediators; Intestine, Large; Intestine, Small; Ischemia; Ischemic Preconditioning; Nitric Oxide; Prognosis; Rats; Reperfusion Injury; Severity of Illness Index; Survival Analysis; Xanthine Oxidase

Topics: Humans; Ischemic Preconditioning; Liver Transplantation; Oxidative Stress; Reperfusion Injury; Xanthine Oxidase

Xanthine oxidoreductase (XOR) is a complex molybdoflavoenzyme, present in milk and many other tissues, which has been studied for over 100 years. While it is generally recognized as a key enzyme in purine catabolism, its structural complexity and specialized tissue distribution suggest other functions that have never been fully identified. The publication, just over 20 years ago, of a hypothesis implicating XOR in ischemia-reperfusion injury focused research attention on the enzyme and its ability to generate reactive oxygen species (ROS). Since that time a great deal more information has been obtained concerning the tissue distribution, structure, and enzymology of XOR, particularly the human enzyme. XOR is subject to both pre- and post-translational control by a range of mechanisms in response to hormones, cytokines, and oxygen tension. Of special interest has been the finding that XOR can catalyze the reduction of nitrates and nitrites to nitric oxide (NO), acting as a source of both NO and peroxynitrite. The concept of a widely distributed and highly regulated enzyme capable of generating both ROS and NO is intriguing in both physiological and pathological contexts. The details of these recent findings, their pathophysiological implications, and the requirements for future research are addressed in this review.

Topics: Animals; Humans; Models, Biological; Models, Chemical; Models, Molecular; Nitric Oxide; Reactive Oxygen Species; Reperfusion Injury; Structure-Activity Relationship; Xanthine Oxidase

It is well known that biomembranes and subcellular organelles are susceptible to lipid peroxidation. There is a steadily increasing body of evidence indicating that lipid peroxidation is involved in basic deteriorative mechanisms, e.g., membrane damage, enzyme damage, and nucleic acid mutagenicity. The formation of lipid peroxides can be induced by enzymatic or nonenzymatic peroxidation in the presence of oxygen. The mechanisms of formation and removal of reactive oxygen species, lipid peroxides, and free radicals in biological systems are briefly reviewed. In recent years, there has been renewed interest in the role played by lipid peroxidation in many disease states. Xanthine oxidase has been shown to generate reactive oxygen species, superoxide (O2-.), and hydrogen peroxide (H2O2) that are involved in the peroxidative damage to cells that occurs in ischemia-reperfusion injury. During ischemia, this enzyme is induced from xanthine dehydrogenase. We have shown that peroxynitrite (a reactive nitrogen species) has the potential to convert xanthine dehydrogenase to oxidase. The following biological effects of lipid peroxidation were found: a) the lipid peroxidation induced by ascorbic acid and Fe2+ affects the membrane transport in the kidney cortex and the cyclooxygenase activity in the kidney medulla, and b) the hydroperoxy adducts of linoleic acid and eicosapentaenoic acid inhibit the cyclooxygenase activity in platelets. The balance between the formation and removal of lipid peroxides determines the peroxide level in cells. This balance can be disturbed if cellular defenses are decreased or if there is a significant increase in peroxidative reactions. Once lipid peroxidation is initiated, the reactive intermediate formed induces cell damage.

Topics: Animals; Arachidonic Acid; Cell Death; Free Radicals; Humans; Lipid Peroxidation; Lipid Peroxides; Prostaglandin-Endoperoxide Synthases; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

Xanthine oxidoreductase (XOR) is a ubiquitous metalloflavoprotein that appears in two interconvertible yet functionally distinct forms: xanthine dehydrogenase (XD), which is constitutively expressed in vivo; and xanthine oxidase (XO), which is generated by the posttranslational modification of XD, either through the reversible, incremental thiol oxidation of sulfhydryl residues on XD or the irreversible proteolytic cleavage of a segment of XD, which occurs at low oxygen tension and in the presence of several proinflammatory mediators. Functionally, both XD and XO catalyze the oxidation of purines to urate. However, whereas XD requires NAD+ as an electron acceptor for these redox reactions, thereby generating the stable product NADH, XO is unable to use NAD+ as an electron acceptor, requiring instead the reduction of molecular oxygen for this purine oxidation and generating the highly reactive superoxide free radical. Nearly 100 years of study has documented the physiologic role of XD in urate catabolism. However, the rapid, posttranslational conversion of XD to the oxidant-generating form XO provides a possible physiologic mechanism for rapid, posttranslational, oxidant-mediated signaling. XO-generated reactive oxygen species (ROS) have been implicated in various clinicopathologic entities, including ischemia/reperfusion injury and multisystem organ failure. More recently, the concept of physiologic signal transduction mediated by ROS has been proposed, and the possibility of XD to XO conversion, with subsequent ROS generation, serving as the trigger of the microvascular inflammatory response in vivo has been hypothesized. This review presents the evidence and basis for this hypothesis.

Topics: Animals; Endothelium, Vascular; Humans; Reperfusion Injury; Signal Transduction; Uric Acid; Vasculitis; Xanthine Oxidase

Flavonoids are a group of naturally occuring compounds which are widely distributed in nature. Epidemiological evidence suggests an inverse relationship between dietary intake of flavonoids and cardiovascular risk. The biological activities of flavonoids are related to their antioxidative effects. But a number of studies have found both anti and prooxidant effects for many of these compounds. This review article presents the synthetic pathways of flavonoids and discusses the structure-activity relationships between, xanthine oxidase inhibitive activities and their chemical structures, between the antioxidant and prooxidant activities and the chemical structure. Then we will show the antioxidant properties of new flavonoids in a few models. In these compounds one or two di-tert-butylhydroxyphenyl (DBHP) groups replace the catechol moiety at the position 2 of the benzopyrane heterocycle. New structures are compared with quercetin and BHT in an LDL-oxidation system, in protecting cultured bovine aortic endothelial cells against mO-LDL cytotoxicity and on myocardial functional recovery during reperfusion after 30 min global ischemia in isolated rat hearts.

Topics: Animals; Antioxidants; Flavonoids; Free Radicals; Humans; Oxidation-Reduction; Oxidative Stress; Reperfusion Injury; Xanthine Oxidase

During last years considerable interest has been devoted to understand the role of oxygen radicals in the ischemia induced cell injury associated with reperfusion. In the brain and in others tissues, free radicals play a role as modulators of vascular tone as well as a cytotoxic role as part of the ischemia associated pathology. This review discusses methods for free radical detection in brain and in other tissues, mechanisms of radical production in the course of the ischemia reperfusion process, and the efficacy of potential antioxidant agents in post ischemia therapy, especially with respect to allopurinol, an inhibitor of xanthine oxidase, and the role of taurine and its derivatives as antioxidants in different organs including the brain.

Topics: Antioxidants; Enzyme Inhibitors; Free Radicals; Humans; Ischemia; Neutrophils; Reactive Oxygen Species; Reperfusion Injury; Taurine; Xanthine Oxidase

The roles of enzymatic antioxidant defenses in the natural tolerance of environmental stresses that impose changes in oxygen availability and oxygen consumption on animals is discussed with a particular focus on the biochemistry of estivation and metabolic depression in pulmonate land snails. Despite reduced oxygen consumption and PO2 during estivation, which should also mean reduced production of oxyradicals, the activities of antioxidant enzymes, such as superoxide dismutase and catalase, increased in 30 day-estivating snails. This appears to be an adaptation that allows the snails to deal with oxidative stress that takes place during arousal when PO2 and oxygen consumption rise rapidly. Indeed, oxidative stress was indicated by increased levels of lipid peroxidation damage products accumulating in hepatopancreas within minutes after arousal was initiated. The various metabolic sites responsible for free radical generation during arousal are still unknown but it seems unlikely that the enzyme xanthine oxidase plays any substantial role in this despite being implicated in oxidative stress in mammalian models of ischemia/reperfusion. We propose that the activation of antioxidant defenses in the organs of Otala lactea during estivation is a preparative mechanism against oxidative stress during arousal. Increased activities of antioxidant enzymes have also observed under other stress situations in which the actual production of oxyradicals should decrease. For example, antioxidant defenses are enhanced during anoxia exposure in garter snakes Thamnophis sirtalis parietalis (10 h at 5 degrees C) and leopard frogs Rana pipiens (30 h at 5 degrees C) and during freezing exposure (an ischemic condition due to plasma freezing) in T. sirtalis parietalis and wood frogs Rana sylvatica. It seems that enhancement of antioxidant enzymes during either anoxia or freezing is used as a preparatory mechanism to deal with a physiological oxidative stress that occurs rapidly within the early minutes of recovery during reoxygenation or thawing. Thus, a wide range of stress tolerant animals display coordinated changes in antioxidant defenses that allow them to deal with oxidative stress that occurs as part of natural cycles of stress/recovery that alter oxygen levels in tissues. The molecular mechanisms that trigger and regulate changes in antioxidant enzyme activities in these species are still unknown but could prove to have key relevance for the development of new interventio

Topics: Animals; Antioxidants; Estivation; Humans; Lipid Peroxidation; Models, Biological; Oxidative Stress; Oxygen Consumption; Reactive Oxygen Species; Reperfusion Injury; Snails; Vertebrates; Xanthine Oxidase

Topics: Animals; Humans; Liver; Milk; Recombinant Fusion Proteins; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

Topics: Animals; Endothelium, Vascular; Humans; NADPH Oxidases; Neutrophils; Oxidants; Reactive Oxygen Species; Reperfusion Injury; Signal Transduction; Xanthine Oxidase

Reperfusion injury, precipitated by lack of oxygen, is likely to play a major role in many clinical conditions, including shock, coronary artery occlusion disease, and solid organ transplantation. Certain tissues, such as the intestinal mucosa, may be especially susceptible because of the specific microvascular anatomy. Structural changes include not only swelling of the organelles but also the entire cell due to the entry of water and electrolytes. Lysosomal ruptures precede cell death. Other key substances which either participate in or are part of oxygen free radical formation in tissue injury are calcium ions, leukocytes, and bacteria. Leukocyte adhesion has been implicated as a critical step in vascular endothelium injury, leading to increased microvascular permeability and thrombosis. Induction of neutropenia or the administration of antileukocyte adhesion monoclonal antibodies, preventing typical injuries, implies a central role of the white blood cells in reperfusion injury. Specifically, oxygen free radical formation in the intestines may trigger or cause injury in other distant organs, e.g., the heart and lungs, and affect overall vascular function. So-called "bacterial translocation" from the intestines to the lymphatic vessels and the bloodstream is a more recently discovered phenomenon whose role is largely unknown. Ischemic preconditioning is still another concept, mainly tested in the canine heart, that has potential clinical applications. Reperfusion of ischemic tissue occurs with solid organ transplantation, often after considerable cold ischemia time. Protective mechanisms include oxygen free radical scavengers, i.e., allopurinol and superoxide dismutase. Other measures proven to be effective during the implantation are blood volume expansion with colloid solutions and/or electrolyte solutions, and the administration of a calcium antagonist. The mechanisms of these measures are likely related to improved renal microcirculation and relief of vasospasm.

Topics: Animals; Bacterial Translocation; Calcium; Free Radicals; Humans; Ischemic Preconditioning; Leukocytes; Organ Transplantation; Reperfusion Injury; Xanthine Oxidase

It is well known that ischemia causes functional and structural damage to liver cells, and that the status of energy metabolism provides an important means of assessing the functional viability of the ischemic organ. However, the specific sequence leading to ischemic liver cell injury is not yet fully understood; therefore, it is clinically and pathophysiologically important to elucidate the mechanism of cellular injury during hepatic ischemia and subsequent reperfusion. Whereas the conventional view attributes this injury process to the ischemia itself, recent studies have demonstrated that a variable but often substantial proportion of this injury is caused by reactive oxygen metabolites that are generated at the time of reperfusion. This article presents an outline of the mechanism of cellular injury caused during hepatic ischemia and subsequent reperfusion resulting from certain types of surgery, with special reference to the xanthine-xanthine oxidase system and the activation of neutrophils and macrophages.

Topics: Animals; Cell Adhesion Molecules; Free Radicals; Humans; Liver; Liver Transplantation; Macrophage Activation; Neutrophil Activation; Reactive Oxygen Species; Reperfusion Injury; Xanthine; Xanthine Oxidase; Xanthines

Topics: Acute Disease; Adenosine; Allopurinol; Animals; Chronic Disease; Glutathione; Graft Rejection; Humans; Insulin; Intestine, Small; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Transplantation, Homologous

Xanthine oxidase (xanthine dehydrogenase) is composed of two identical subunits of approximately 150,000 daltons. Each subunit contains four oxdation-reduction active cofactors/monomers. In vivo, the enzyme exists mostly as the dehydrogenase type (the NAD-dependent type). The cDNA has been cloned from human liver, and the amino acid sequence has been determined. As xanthine oxidase seems to produce superoxide in postischemic reperfusion, the relation between the superoxide and postischemic tissue injury has been discussed. It has also been reported that inhibition of xanthine oxidase by allopurinol may cause severe 6-mercaptopurine toxicity.

Topics: DNA; Humans; Hypoxanthine; Protein Conformation; Reperfusion Injury; Superoxides; Uric Acid; Xanthine Dehydrogenase; Xanthine Oxidase

Topics: Animals; Antioxidants; Arachidonic Acid; Electron Transport; Free Radicals; Humans; Mitochondria, Heart; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Neutrophils; Oxygen; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

Although mammalian xanthine oxidase exists originally as a dehydrogenase form in freshly prepared samples, it is converted to an oxidase form during purification, either irreversibly by proteolysis or reversibly by sulfhydryl oxidation of the protein molecule. However, avoiding proteolysis the mammalian enzyme can be purified as an interconvertible form and thus can be used to compare directly the properties of xanthine dehydrogenase and the oxidase derived from the same enzyme molecule. The cDNAs encoding the enzyme have been cloned from several sources, and structural information is becoming available. The most significant difference between the two forms is the protein conformation around FAD, which changes the redox potential of the flavin and the reactivity of FAD with the electron acceptors, NAD and molecular oxygen. The flavin semiquinone is thermodynamically stable in xanthine dehydrogenase, but is unstable in xanthine oxidase. Detailed analyses by stopped-flow techniques suggest that the flavin semiquinone reacts with oxygen to form superoxide anion while the fully reduced flavin reacts to form hydrogen peroxide. Although xanthine dehydrogenase can produce greater amounts of superoxide anion than xanthine oxidase during xanthine-oxygen turnover, it seems to be physiologically insignificant because NAD inhibits almost completely the formation of superoxide anion. Although the involvement of this enzyme in reperfusion injury has been proposed, this seems to be more complex than originally envisaged and still remains to be established.

Topics: Animals; Molecular Structure; Reperfusion Injury; Structure-Activity Relationship; Xanthine Dehydrogenase; Xanthine Oxidase

Topics: Animals; Blood Platelets; Capillary Permeability; Cell Adhesion; Endopeptidases; Endothelium, Vascular; Intestinal Diseases; Intestinal Mucosa; Lipid Peroxides; Mast Cells; Microcirculation; Neutrophils; Oxidation-Reduction; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

Topics: Animals; Calcium; Embryo, Mammalian; Humans; Ischemia; Kidney; Liver; Lung; Mitochondria, Heart; Muscles; Myocardial Reperfusion Injury; Myocardium; Phagocytes; Placenta; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

Reperfusion of ischemic organs can result in tissue injury that is manifested as microvascular and parenchymal cell dysfunction. Reactive oxygen metabolites and polymorphonuclear leukocytes (PMN) have been implicated in the pathobiology of reperfusion injury. Reactive oxygen metabolites mediate the lipid peroxidation detected in postischemic tissues and promote the formation of inflammatory agents that recruit and activate PMN. These PMN appear to inflict reperfusion-induced tissue injury. Drugs that scavenge or inhibit the formation of reactive oxygen metabolites and/or prevent the recruitment of PMN may be useful in the treatment of reperfusion injury.

Topics: Animals; Cell Adhesion; Free Radical Scavengers; Humans; Lipid Peroxidation; Neutrophils; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

Primary biochemical role of xanthine oxidoreductase (XOR, EC 1.1.1.204 and EC 1.1.3.22.) is the hydroxylation of hypoxathine and xanthine to form uric acid. This enzyme may produce very reactive oxygen forms which can be pathogenic. Although it is one of the longest known and most continuously studied enzymes, its biological function remains unclear. The review of these aspects is discussed in relation to the authors' experimental results.

Topics: Animals; Humans; Oxidation-Reduction; Reactive Oxygen Species; Reperfusion Injury; Xanthine; Xanthine Dehydrogenase; Xanthine Oxidase; Xanthines

Review the histologic and pathophysiologic alterations seen after intestinal ischemia and reperfusion.. Current literature review.. The most pertinent, current, and representative articles describing results from both animal and human investigations are utilized and discussed.. Postischemic intestinal tissue damage appears to be due to the formation of oxygen radicals and the activation of phospholipase A2. The initial source of oxygen radicals seems to be the hypoxanthine-xanthine oxidase system. Oxygen radicals react directly with poly-unsaturated fatty acids, leading to lipid peroxidation within the cell membranes. Indirectly, the radicals trigger the accumulation of neutrophils within the affected tissue initiating inflammatory processes that lead to severe mucosal lesions. Similarly, phospholipase A2 also initiates postischemic mucosal lesions. Phospholipase A2 is a hydrolytic enzyme capable of increasing formation of cytotoxic lysophospholipids within the tissue. Enhanced activity of phospholipase A2 also stimulates the production of prostaglandins and leukotrienes. Various substances (superoxide dismutase, catalase, dimethyl sulfoxide, allopurinol, and deferoxamine, etc.) are able to detoxify oxygen radicals or inhibit the mechanisms leading to their enhanced generation, thus attenuating the postischemic lesions of the mucosa.. Oxygen radicals and the activation of phospholipase A2 during reperfusion seem to be instrumental for the development of hemorrhagic mucosal lesions after intestinal ischemia. Radical scavengers and phospholipase A2 inhibitors may prevent reperfusion damage of the intestine, even when the treatment starts during ischemia but before reperfusion.

Topics: Animals; Antioxidants; Cats; Disease Models, Animal; Free Radical Scavengers; Free Radicals; Gangrene; Gastrointestinal Hemorrhage; Humans; Hypoxanthines; Inflammation; Intestines; Leukotrienes; Lipid Peroxidation; Neutrophils; Phospholipases A; Phospholipases A2; Prostaglandins; Rats; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

Topics: Cell Death; Free Radicals; Humans; Ischemia; Leg; Microcirculation; Oxygen; Reperfusion; Reperfusion Injury; Vascular Surgical Procedures; Xanthine Oxidase

In conclusion, a large body of evidence demonstrates that reperfusion of ischemic intestine results in significant microvascular and parenchymal cell injury. Reperfusion injury appears to be mediated by both reactive oxygen metabolites and activated polymorphonuclear leukocytes. Xanthine oxidase-derived oxidants initiate the production and release of proinflammatory agents, which in turn lead to polymorphonuclear leukocyte adherence and emigration. The adherent leukocytes mediate microvascular injury by either release of proteases, physical disruption of the endothelial barrier, or both.

Topics: Animals; Humans; Intestines; Ischemia; Neutrophils; Oxygen; Reperfusion Injury; Xanthine Oxidase

Topics: Animals; Kinetics; Liver; Rats; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

Topics: Allopurinol; Animals; Catalase; Free Radical Scavengers; Glutathione; Humans; Lung Diseases; Molecular Weight; Oxygen; Reperfusion Injury; Superoxide Dismutase; Ubiquinone; Vitamin E; Xanthine Oxidase

Topics: Adenosine; Allopurinol; Free Radicals; Glutathione; Humans; Insulin; Liver; Liver Circulation; Organ Preservation Solutions; Oxygen; Preservation, Biological; Preservatives, Pharmaceutical; Raffinose; Reperfusion Injury; Solutions; Specimen Handling; Tissue Preservation

Topics: Adenine Nucleotides; Adolescent; Atrial Natriuretic Factor; Calcium Channel Blockers; Epoprostenol; Female; Free Radicals; Humans; Hypothermia, Induced; Kidney; Kidney Transplantation; Reperfusion Injury; Thyroid Hormones; Tissue Donors; Xanthine Oxidase

Ischemia-reperfusion insults are common clinical problems which involve most notably the heart (myocardial infarction) and brain (stroke). However, these and other organs are susceptible to damage following warming after cold injury, trauma, shock, and/or preparation for transplantation. Although the mechanisms responsible for reperfusion damage following ischemia and reperfusion are unknown, they are the focus of intense interest and investigation. This review briefly addresses our recent research related to the potential contributions of blood cells to the development of ischemia-reperfusion injury.

Topics: Animals; Blood Cells; Catalase; Neutrophils; Oxygen; Reperfusion Injury; Xanthine Oxidase

Experimental evidence available currently indicates that injury to the muscle during reperfusion is most likely mediated by oxygen-derived free radicals (oxyradicals). The major oxyradical generating system in the skeletal muscle has yet to be identified so that an appropriate oxyradical-generating inhibitor and/or scavenger can be selected for the prevention of ischemia-induced reperfusion injury to the skeletal muscle. In addition, apart from generating oxyradicals, the activated leukocytes, platelets, and vascular endothelial cells are also known to synthesize and release vasoconstrictive substances and/or chemoattractants for leukocytes. The role of these locally released chemical substances in the pathogenesis of ischemia-induced reperfusion injury in muscle flaps has yet to be elucidated. The pathophysiology of ischemia-induced reperfusion injury to the skeletal muscle in muscle flaps is reviewed.

Topics: Free Radicals; Leukocytes; Oxidation-Reduction; Oxygen; Reperfusion Injury; Surgical Flaps; Xanthine Dehydrogenase; Xanthine Oxidase

Topics: Allopurinol; Free Radicals; Hot Temperature; Humans; Ischemia; Kidney; Kidney Transplantation; Organ Preservation; Reperfusion Injury; Solutions; Superoxide Dismutase; Superoxides; Time Factors

Topics: Allopurinol; Free Radicals; Humans; Reperfusion Injury; Xanthine Oxidase

Several lines of evidence point to a major role of oxygen free radicals in the pathogenesis of cell death or dysfunction in a variety of disease processes. Recent studies from this as well as other laboratories have demonstrated that oxygen free radicals play a major role in the pathogenesis of post-ischemic reperfusion injury in the heart. We have recently developed methods for direct measurement of radical species and/or specific byproducts of radical injury. Timely administration of oxygen radical scavengers reduced the quantity of free radicals generated following reperfusion and in addition improved recovery of post-ischemic ventricular function and metabolism. In a regionally ischemic model the free radical scavenger recombinant human superoxide dismutase also administered at the time of reflow was shown to limit infarct size. In this article we review the biophysical and molecular mechanisms of oxygen free radical generation that are viewed as contributing to post-ischemic reperfusion injury. We also discuss the mechanisms by which the body defends against free radical attack and the interrelationships of free radical injury to other mechanisms of tissue injury.

Topics: Animals; Antioxidants; Cardiac Surgical Procedures; Catalase; Catecholamines; Chemical Phenomena; Chemistry; Free Radicals; Glutathione Peroxidase; Humans; Leukocytes; Mitochondria, Heart; Myocardial Reperfusion Injury; Neutrophils; Oxidative Phosphorylation; Oxygen; Prostaglandins; Rabbits; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

Ischemia reperfusion injury (IRI) is the main cause of early allograft dysfunction (EAD) and subsequent primary allograft failure (PAF).. The purpose of this study is to compare IRI, EAD, and PAF in liver transplantation in a cohort of patients perfused with histidine-tryptophan-ketoglutarate (HTK) solution and University of Wisconsin (UW) solution versus HTK alone.. A randomized trial was performed to compare outcomes in liver recipients who underwent transplantation surgery in the University Regional Hospital of Malaga, Spain. Forty patients were randomized to two groups. Primary endpoints included IRI, EAD, PAF, re-intervention, acute cellular rejection, retransplantation, arterial complications, and biliary complications at postoperative day 90.. Postoperative glutamic oxaloacetic transaminase (1869.15 ± 1559.75 UI/L vs. 953.15 ± 777.27 UI/L; P = .004) and glutamic pyruvic transaminase (1333.60 ± 1115.49 U/L vs. 721.70 ± 725.02 U/L; P = .023) were significantly higher in patients perfused with HTK alone. A clear tendency was observed in recipients perfused with HTK alone to present moderate to severe IRI (7 patients in the HTK + UW solution group vs. 15 patients in the HTK-alone solution group; P = .06), EAD (0 patients in the HTK + UW solution group vs. 0 patients in the HTK-alone solution group; P = .76), and PAF (3 patients in the HTK + UW solution group vs. 8 patients in the HTK-alone solution group; P = .15).. Initial perfusion with HTK solution followed by UW solution in liver transplantation improves early liver function as compared to perfusion with HTK alone.

Topics: Adenosine; Adult; Alanine Transaminase; Allopurinol; Aspartate Aminotransferases; Cohort Studies; Drug Therapy, Combination; Female; Glucose; Glutathione; Graft Rejection; Humans; Insulin; Liver; Liver Transplantation; Male; Mannitol; Middle Aged; Organ Preservation Solutions; Perfusion; Postoperative Period; Potassium Chloride; Procaine; Raffinose; Reoperation; Reperfusion Injury; Spain; Treatment Outcome

One of the main concerns in liver transplant is the prolonged ischemia time, which may lead to primary graft nonfunction or delayed function. N-acetylcysteine is known as a hepato-protective agent in different studies, which may improve human hepatocyte viability in steatotic donor livers. This study investigated whether N-acetylcysteine can decrease the rate of ischemia-reperfusion syndrome and improve short-term outcome in liver transplant recipients.. This was a double-blind, randomized, control clinical trial of 115 patients. Between April 2012 and January 2013, patients with orthotopic liver transplant were randomly divided into 2 groups; in 49 cases N-acetylcysteine was added to University of Wisconsin solution as the preservative liquid (experimental group), and in 66 cases standard University of Wisconsin solution was used (control group). We compared postreperfusion hypotension, inotrope requirement before and after portal reperfusion, intermittent arterial blood gas analysis and potassium measurement, pathological review of transplanted liver, in-hospital complications, morbidity, and mortality.. There was no significant difference between the groups regarding time to hepatic artery reperfusion, hospital stay, vascular complications, inotrope requirement before and after portal declamping, and blood gas analysis. Hypotension after portal reperfusion was significantly more common in experimental group compared with control group (P = .005). Retransplant and in-hospital mortality were comparable between the groups.. Preservation of the liver inside Univer-sity of Wisconsin solution plus N-acetylcysteine did not change the rate of ischemia reperfusion injury and short-term outcome in liver transplant recipients.

Topics: Acetylcysteine; Adenosine; Allopurinol; Cold Ischemia; Double-Blind Method; Female; Glutathione; Hospital Mortality; Humans; Hypotension; Insulin; Iran; Length of Stay; Liver Transplantation; Male; Middle Aged; Operative Time; Organ Preservation Solutions; Perfusion; Protective Agents; Raffinose; Reperfusion Injury; Risk Factors; Time Factors; Treatment Outcome; Warm Ischemia

To investigate the effect of 6% hydroxyethyl starch (HES) (130/0.4) infusion on ischemia-reperfusion determinants in minor lower extremity operations with tourniquet application.. Prospective, randomized, clinical trial.. University hospital operation room.. American Society of Anesthesiologists I and II 40 patients between 18 and 65 years of age who were scheduled to undergo knee arthroscopy and below-knee minor orthopedic surgery.. Patients were randomized into 2 groups (normal saline [NS] group and HES group). Unilateral spinal anesthesia with 2-mL 0.5% hyperbaric bupivacaine after 10 mL/kg NS intravenous infusion to NS group and 10 mL/kg 6% HES intravenous infusions to HES group.. Blood samples were obtained from antecubital vein for malondialdehyde, xanthine oxidase, and hypoxanthine before tourniquet inflation and after 10 minutes of tourniquet deflation.. There was no significant difference between groups with respect of hemodynamic data. There were no significant differences between 2 groups in terms of malondialdehyde values before tourniquet inflation and after tourniquet deflation. In the HES group, xanthine oxidase levels after tourniquet deflation were significantly lower than xanthine oxidase levels before tourniquet inflation (P < .05). In the HES group, hypoxanthine levels after tourniquet deflation were similar with the basal levels; however, they were significantly higher than levels obtained before tourniquet inflation in the SF group (P < .05).. HES infusion may be beneficial for reduction of tourniquet-associated ischemia-reperfusion injury; however, further large-scale studies are needed to fully elucidate its mechanism.

Topics: Adult; Anesthesia, Spinal; Arthroscopy; Female; Humans; Hydroxyethyl Starch Derivatives; Hypoxanthine; Knee Joint; Lower Extremity; Male; Malondialdehyde; Middle Aged; Orthopedic Procedures; Plasma Substitutes; Prospective Studies; Reperfusion Injury; Tourniquets; Xanthine Oxidase; Young Adult

Free-radical-induced reperfusion injury has been recognised as an important cause of brain tissue damage after birth asphyxia. Allopurinol reduces the formation of free radicals, thereby potentially limiting the amount of hypoxia-reperfusion damage. In this study the long-term outcome of neonatal allopurinol treatment after birth asphyxia was examined.. Follow-up of 4 to 8 years of two earlier performed randomised controlled trials.. Leiden University Medical Center, University Medical Center Groningen and University Medical Center Utrecht, The Netherlands.. Fifty-four term infants were included when suffering from moderate-to-severe birth asphyxia in two previously performed trials.. Infants either received 40 mg/kg allopurinol (with an interval of 12 h) starting within 4 h after birth or served as controls.. Children, who survived, were assessed with the Wechsler Preschool and Primary Scales of Intelligence test or Wechsler Intelligence Scale for Children and underwent a neurological examination. The effect of allopurinol on severe adverse outcome (defined as mortality or severe disability at the age of 4-8 years) was examined in the total group of asphyxiated infants and in a predefined subgroup of moderately asphyxiated infants (based on the amplitude integrated electroencephalogram).. The mean age during follow-up (n=23) was 5 years and 5 months (SD 1 year and 2 months). There were no differences in long-term outcome between the allopurinol-treated infants and controls. However, subgroup analysis of the moderately asphyxiated group showed significantly less severe adverse outcome in the allopurinol-treated infants compared with controls (25% vs 65%; RR 0.40, 95%CI 0.17 to 0.94).. The reported data may suggest a (neuro)protective effect of neonatal allopurinol treatment in moderately asphyxiated infants.

Topics: Allopurinol; Asphyxia Neonatorum; Birth Weight; Developmental Disabilities; Female; Follow-Up Studies; Free Radical Scavengers; Gestational Age; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Intelligence; Male; Neuroprotective Agents; Neuropsychological Tests; Reperfusion Injury; Treatment Outcome

Celsior solution (CS) is a high-sodium, low-potassium, low-viscosity extracellular solution that has been used for liver graft preservation in recent years, although experience with it is still limited. We performed an open-label randomized active-controlled trial comparing CS with the University of Wisconsin solution (UW) for liver transplantation (LT), with a follow-up period of 5 years.. Adult transplant recipients (n=102) were prospectively randomized to receive either CS (n=51) or UW (n=51). The two groups were comparable with respect to donor and recipient characteristics. The primary outcome measure was the incidence of postreperfusion syndrome (PRS). Secondary outcome measures included primary nonfunction (PNF) or primary dysfunction (PDF), liver retransplantation, and graft and patient survival. Other secondary outcome measures were days in the intensive care unit (ICU) and the rates of acute rejection, chronic rejection, infectious complications, postoperative reoperations, and vascular and biliary complications.. In all, 14 posttransplant variables revealed no significant differences between the groups. There were no cases of PNF or PDF. The incidence of PRS was 5.9% in the CS group and 21.6% in the UW group (P=0.041). After reperfusion, CS revealed greater control of serum potassium (P=0.015), magnesium levels (P=0.005), and plasma glucose (P=0.042) than UW. Respective patient survivals at 3, 12, and 60 months were 95.7, 87.2, and 82.0% for the CS group and 95.7, 83.3, and 66.6% for the UW group (P=0.123).. While retaining the same degree of safety and effectiveness as UW for LT, CS may yield postliver graft reperfusion benefits, as shown in this study by a significant reduction in the incidence of PRS and greater metabolic control.

Topics: Adenosine; Adolescent; Adult; Aged; Allopurinol; Disaccharides; Electrolytes; Female; Follow-Up Studies; Glutamates; Glutathione; Histidine; Humans; Insulin; Liver Transplantation; Male; Mannitol; Middle Aged; Organ Preservation; Organ Preservation Solutions; Postoperative Complications; Prospective Studies; Raffinose; Reperfusion Injury; Syndrome; Time Factors; Young Adult

Celsior solution (CS), which has recently become available, that might theoretically offer a new means for improving graft preservation quality. The present prospective, randomized study was designed to evaluate the efficacy of CS compared with University of Wisconsin (UW) for pancreas allografts. Between January 2001 and January 2007, 88 patients underwent pancreatic transplantation, including the last 30 consecutive simultaneous pancreas kidney patients who were randomly assigned to either CS or UW. There was no case of graft thrombosis in either group. There were 2 cases of pancreatitis in the UW group compared with 1 in the CS group. No case of primary nonfunction occurred in either group. There were 2 cases of early duodenal stump fistulae in the CS group that required transplantectomy, whereas this complication was not observed in the UW group. Relaparotomy in the UW group was required in 3 cases due to infection and treated by close drainage that which, progressed to fatal sepsis in 1 patient. In the UW group with 6 months of follow-up, there were 12 patients insulin free. In the CS group, 6 patients underwent relaparotomy, 3 for transplantectomy and the others for intra-abdominal infection, which was fatal in 2 cases. In the CS group with 6 months of follow-up, there were 10 patients insulin free. Two patients died with functioning grafts. These results provided indirect evidence that CS solution is at least as safe as UW to mitigate postreperfusion graft edema and pancreatitis, as well as graft thrombosis.

Topics: Adenosine; Adult; Allopurinol; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disaccharides; Electrolytes; Female; Glutamates; Glutathione; Histidine; Humans; Insulin; Kidney Transplantation; Male; Mannitol; Middle Aged; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose; Reperfusion Injury; Safety; Young Adult

To investigate whether postnatal allopurinol would reduce free radical induced reperfusion/reoxygenation injury of the brain in severely asphyxiated neonates.. In an interim analysis of a randomised, double blind, placebo controlled study, 32 severely asphyxiated infants were given allopurinol or a vehicle within four hours of birth.. The analysis showed an unaltered (high) mortality and morbidity in the infants treated with allopurinol.. Allopurinol treatment started postnatally was too late to reduce the early reperfusion induced free radical surge. Allopurinol administration to the fetus with (imminent) hypoxia via the mother during labour may be more effective in reducing free radical induced post-asphyxial brain damage.

Topics: Allopurinol; Asphyxia Neonatorum; Brain Ischemia; Double-Blind Method; Free Radical Scavengers; Free Radicals; Humans; Infant, Newborn; Reperfusion Injury; Treatment Outcome

We studied the evolution of the liver graft with preservation in Celsior (CS) compared with University of Wisconsin solution (UW).. A randomized prospective clinical study in 80 liver transplants (OLTs) from May 2001 to October 2003, compared CS (group I; n = 40) and UW (group II; n = 40). The characteristics of the donors were homogeneous, with no significant differences in 15 variables. CS was perfused with 4 L through the aorta, 2 L through the portal vein, and 1 L, through the portal vein on the back table; and the UW, as 3 L, 2 L, and 1 L, respectively. All OLTs were performed using the piggyback technique.. Group I experienced reperfusion syndrome (n = 2; 5.9%), primary graft nonfunction (n = 0); vascular complications (n = 0); biliary anastomosis stenosis (n = 8; 22.9%), intensive care unit (ICU) days (n = 4.1 +/- 1), death within 30 days (n = 1; 3.1%). The patient and graft survivals at 1, 3, 6, 12, and 24 months were 93.7%, 93.7%, 90.2%, 85.7%, 85.7%, and 94.3%, 88.5%, 85.2%, 78%, 78%, respectively. For group II; the reperfusion syndrome occured in 6 patients (17.6%); primary graft nonfunction (n = 0); vascular complications (n = 0), biliary anastomosis stenosis (n = 3; 8.6%), ICU days (n = 4.9 +/- 2.4) and death within 30 days (n = 1; 3.1%); The patient and graft survival at 1, 3, 6, 12, and 24 months were 96.9%, 93.5%, 89.8%, 79.8%, 79.8% and 94.3%, 88.3%, 84.9%, 75.5%, 66.1%, respectively.. CS offers the similar safety to UW for preservation of liver grafts within these ischemia times.

Topics: Adenosine; Adult; Aged; Allopurinol; Disaccharides; Electrolytes; Female; Glutamates; Glutathione; Histidine; Humans; Insulin; Liver; Liver Transplantation; Male; Mannitol; Middle Aged; Organ Preservation; Organ Preservation Solutions; Portal Vein; Postoperative Complications; Prospective Studies; Raffinose; Reperfusion; Reperfusion Injury; Treatment Outcome; Vascular Diseases

To explore the protective effect of propofol on liver during hepatic ischemia/reperfusion injury (HIRI) and its mechanisms in patients undergoing liver surgery.. Eighteen patients who were scheduled for selective hepatic surgery were randomly divided into control group (n=9) and propofol treatment group (n=9). Changes of several parameters in plasma and effects of propofol on them were observed before liver ischemia, at end of ischemia and at reperfusion for 25 minutes, parameters of which included superoxide dismutase (SOD), xanthine oxidase (XO), lipid peroxide (LPO) and alanine aminotransferase (ALT) activity, and the ultrastructure changes in liver tissue were observed under electron microscope at 25 minutes after reperfusion.. SOD activity decreased remarkably (P<0.01); XO activity, LPO concentration and ALT value increased significantly (P<0.01) during HIRI, and there were abnormal changes of the hepatic ultrastructure at 25 minutes after reperfusion. Afer treatment with propofol, the variation of all parameters were alleviated markedly (P<0.05 and P<0.01).. Propofol has protective effects on HIRI by reducing oxygen free radical level and inhibiting lipid peroxidation after hepatic ischemia/reperfusion in patients undergoing liver cancer surgery.

Topics: Adult; Carboxypeptidases; Female; Free Radical Scavengers; Humans; Lipid Peroxides; Liver; Liver Neoplasms; Male; Middle Aged; Propofol; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

Renal failure after lower torso ischemia is a serious problem, partly caused by hypotension and indirect reperfusion injury. This injury is partly due to the formation of oxygen free radicals by activated neutrophils. This injury results in albuminuria and renal function impairment. There are indications that free radical damage in indirect reperfusion injury can be diminished by administering extra antioxidants before and during reperfusion.. In this prospective randomised study we have looked at the influence of a multi-antioxidant supplementation on renal function in patients undergoing an elective open infrarenal abdominal aneurysm repair. The patients received either standard treatment (n=22) or standard treatment with additional antioxidants perioperatively (Allopurinol, vitamin E and C, N-acetylcysteine and mannitol). For renal function we have looked at the albumin/creatinine ratio in urine and 24 hr creatinine clearance.. Despite significantly increased serum total antioxidant capacity, the group receiving extra antioxidants showed no decrease in the albumin/creatinine ratio in urine. There was however a significantly higher creatinine clearance in this group at day 2.. The results indicate that the diminished renal function after infrarenal aneurysm repair may be influenced by antioxidant therapy.

Topics: Acetylcysteine; Aged; Albuminuria; Allopurinol; Antioxidants; Aortic Aneurysm, Abdominal; Ascorbic Acid; Female; Humans; Kidney Function Tests; Male; Mannitol; Prospective Studies; Renal Insufficiency; Reperfusion Injury; Vitamin E

Topics: Adenosine; Adult; Allopurinol; Cholagogues and Choleretics; Glutathione; Humans; Insulin; Liver; Liver Transplantation; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Taurochenodeoxycholic Acid; Transplantation, Homologous

The clinical effects of allopurinol on the attenuation of reperfusion injury in thumb replantations after prolonged ischaemic time were studied in a randomized control trial with a 2-year follow-up. There were 60 patients, in the trial group, and 38 patients in the control group. All were young, healthy labourers who had sharp or locally crushed amputations of the thumb at the proximal phalanx with a total ischaemic time of more than 10 hours. The standard management for thumb replantation was used in these patients, except that 300 mg allopurinol was given orally in the trial group on admission and a further 300 mg for another 5 days. After operation, the trial group had a lower infection rate, and less postoperative pain and chronic swelling than the control group. Recovery of sensation was also better in the trial group.

Topics: Adolescent; Adult; Allopurinol; Amputation, Traumatic; Child; Female; Follow-Up Studies; Free Radical Scavengers; Humans; Infant; Male; Middle Aged; Reperfusion Injury; Replantation; Thumb

Thirty horses were randomly assigned to 1 of 5 groups. All horses were anesthetized and subjected to ventral midline celiotomy, then the large colon was exteriorized and instrumented. Colonic arterial blood flow was reduced to 20% of baseline (BL) and was maintained for 3 hours. Colonic blood flow was then restored, and the colon was reperfused for an additional 3 hours. One of 5 drug solutions was administered via the jugular vein 30 minutes prior to colonic reperfusion: group 1, 0.9% NaCl; group 2, dimethyl sulfoxide: 1 g/kg of body weight; group 3, allopurinol: 25 mg/kg; group 4, 21-aminosteroid U-74389G: 10 mg/kg; and group 5, manganese chloride (MnCl2): 10 mg/kg. Hemodynamic variables were monitored and recorded at 30-minutes intervals. Systemic arterial, systemic venous (SV), and colonic venous (CV) blood samples were collected for measurement of blood gas tensions, oximetry, lactate concentration, PCV, and plasma total protein concentration. The eicosanoids, 6-keto prostaglandin F1 alpha, prostaglandin E2, and thromboxane B2, were measured in CV blood, and endotoxin was measured in CV and SV blood. Full-thickness biopsy specimens were harvested from the left ventral colon for histologic evaluation and determination of wet weight-to-dry weight ratios (WW:DW). Data were analyzed, using two-way ANOVA for repeated measures, and statistical significance was set at P < 0.05. Heart rate, mean arterial pressure, and cardiac output increased with MnCl2 infusion; heart rate and cardiac output remained increased throughout the study, but mean arterial pressure returned to BL values within 30 minutes after completion of MnCl2 infusion. Other drug-induced changes were not significant. There were significant increases in mean pulmonary artery and mean right atrial pressures at 2 and 2.5 hours in horses of all groups, but other changes across time or differences among groups were not observed. Mean pulmonary artery pressure remained increased through 6 hours in all groups, but mean right atrial pressure had returned to BL values at 3 hours. Mean colonic arterial pressure was significantly decreased at 30 minutes of ischemia and remained decreased through 6 hours; however, by 3.25 hours it was significantly higher than the value at 3 hours of ischemia. Colonic arterial resistance decreased during ischemia and remained decreased throughout reperfusion in all groups; there were no differences among groups for colonic arterial resistance.(ABSTRACT TRUNCATED AT 400 WO

Topics: Allopurinol; Analysis of Variance; Animals; Antioxidants; Chlorides; Colon; Dimethyl Sulfoxide; Endotoxins; Hemodynamics; Horses; Manganese Compounds; Peroxidase; Pregnatrienes; Reperfusion Injury

Topics: Adenosine; Allopurinol; Biopsy; Glutathione; Graft Rejection; Humans; Hypertonic Solutions; Insulin; Liver Transplantation; Muromonab-CD3; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Retrospective Studies; Solutions; Transplantation, Homologous

Renal ischemia/reperfusion (I/R) is a common cause of acute kidney injury and remote liver damage is an ultimate negative outcome. Current treatments for renal I/R typically involve the use of antioxidants and anti-inflammatory to protect against oxidative stress and inflammation. Xanthine oxidase (XO) and PPAR-γ contribute to renal I/R-induced oxidative stress; however, the crosstalk between the two pathways remains unexplored. In the present study, we report that XO inhibitor, allopurinol (ALP), protects kidney and liver after renal I/R by PPAR-γ activation. Rats with renal I/R showed reduced kidney and liver functions, increased XO, and decreased PPAR-γ. ALP increased PPAR-γ expression and improved liver and kidney functions. ALP also reduced inflammation and nitrosative stress indicated by reduction in TNF-α, iNOS, nitric oxide (NO), and peroxynitrite formation. Interestingly, rats co-treated with PPAR-γ inhibitor, BADGE, and ALP showed diminished beneficial effect on renal and kidney functions, inflammation, and nitrosative stress. This data suggests that downregulation of PPAR-γ contributes to nitrosative stress and inflammation in renal I/R and the use of ALP reverses this effect by increasing PPAR-γ expression. In conclusion, this study highlights the potential therapeutic value of ALP and suggests targeting XO-PPAR-γ pathway as a promising strategy for preventing renal I/R injury.

Topics: Acute Kidney Injury; Allopurinol; Animals; Inflammation; Kidney; PPAR gamma; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Oxidase

To investigate the effects of cerebrolysin on inflammation, oxidative stress, apoptosis, and neurologic recovery in the setting of an experimental rabbit model of spinal cord ischemia/reperfusion injury (SCIRI).. Rabbits were randomly divided into five groups: control, ischemia, vehicle, methylprednisolone (30 mg/kg), and cerebrolysin (5 ml/kg) group. The rabbits in the control group underwent only laparotomy; the other groups underwent spinal cord ischemia and reperfusion injury for 20 minutes. Neurologic examination after 24 hours was based on the Modified Tarlov scale. Myeloperoxidase activities, catalase and malondialdehyde levels, and caspase-3 concentrations were determined in serum and tissue samples. Serum xanthine oxidase levels were studied and histopathological and ultrastructural changes were examined.. After SCIRI, serum and tissue myeloperoxidase activities, malondialdehyde levels, caspase-3 concentrations, and serum xanthine oxidase activities were increased (p < 0.01?0.001). Catalase levels were significantly diminished (p < 0.001). Cerebrolysin treatment correlated with reduced myeloperoxidase and xanthine oxidase activities, malondialdehyde levels and caspase-3 concentrations; and with increased catalase levels (p < 0.001, for all). The cerebrolysin group showed improved histopathological, ultrastructural, and neurological outcomes.. For the first time in the literature, the current study reports anti-inflammatory, antioxidant, antiapoptotic, and neuroprotective effects of cerebrolysin in a SCIRI rabbit model.

Topics: Animals; Antioxidants; Caspase 3; Catalase; Malondialdehyde; Neuroprotective Agents; Peroxidase; Rabbits; Reperfusion Injury; Spinal Cord; Spinal Cord Ischemia; Xanthine Oxidase

The enhancement of inflammation and reactive oxygen species leads to the damage of renal tubular cells in acute kidney injury (AKI), and the upregulation of inflammation increases the risk of AKI being converted into chronic kidney disease (CKD). Hydralazine has shown renoprotective effects in multiple kidney diseases and was shown to be a potent xanthine oxidase (XO) inhibitor. This study aimed to investigate the mechanisms of hydralazine in ischemia-reperfusion (I/R)-stimulated renal proximal tubular epithelial cells in vitro and in AKI animals in vivo.. The effects of hydralazine in AKI-to-CKD transition were also evaluated. Human renal proximal tubular epithelial cells were stimulated by I/R conditions in vitro. To generate a mouse model of AKI, a right nephrectomy was performed, followed by left renal pedicle I/R using a small atraumatic clamp.. In the in vitro part, hydralazine could protect renal proximal tubular epithelial cells against insults from the I/R injury through XO/NADPH oxidase inhibition. In the in vivo part, hydralazine preserved renal function in AKI mice and improved the AKI-to-CKD transition by decreasing renal glomerulosclerosis and fibrosis independently of blood pressure lowering. Furthermore, hydralazine exerted antioxidant, anti-inflammatory, and anti-fibrotic effects both in vitro and in vivo.. Hydralazine, as a XO/NADPH oxidase inhibitor, could protect renal proximal tubular epithelial cells from the insults of I/R and prevent kidney damage in AKI and AKI-to-CKD. The above experimental studies strengthen the possibility of repurposing hydralazine as a potential renoprotective agent through its antioxidative mechanisms.

Topics: Acute Kidney Injury; Animals; Enzyme Inhibitors; Fibrosis; Humans; Hydralazine; Inflammation; Kidney; Mice; NADPH Oxidases; Renal Insufficiency, Chronic; Reperfusion Injury; Xanthine Oxidase

The University of Wisconsin (UW) solution is the gold standard for preserving the liver, kidneys, and pancreas. For renal preservation, the addition of the flavonoid, quercetin (QE), to the preservation solution reduces damage to renal tubular cells, and the addition of sucrose (Suc) is also beneficial for preservation. The aim of this study was to investigate the protective effects of QE and Suc on porcine livers in terms of warm and cold injury and to evaluate whether their use improves ischemia-reperfusion (I/R) injury after simple cold storage (CS).. We tested porcine livers procured after 30 minutes of warm ischemia followed by preservation for 6 hours under the following 2 conditions: group 1, preserved with the CS/UW solution (n = 4); group 2, preserved with the CS/UW solution containing Que 33.1 μM and Suc 0.1 M (n = 6). All livers were evaluated using an ex vivo isolated liver reperfusion model with saline-diluted autologous blood.. Aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase levels in group 2 were significantly lower at 30 minutes of reperfusion than in group 1. Furthermore, histologic evaluation by hematoxylin and eosin staining showed significantly fewer morphologic changes in group 2 than in group 1, as indicated by the total Suzuki score. Group 2 also had significantly better scores for sinusoidal congestion and hepatocyte cytoplasmic vacuolization.. Adding Que and Suc to the UW solution can effectively prevent cold injury in livers donated after circulatory death.

Topics: Allopurinol; Animals; Cold Injury; Glutathione; Humans; Insulin; Liver; Organ Preservation; Organ Preservation Solutions; Quercetin; Raffinose; Reperfusion Injury; Swine

Cold ischemia-reperfusion injury (IRI) is an inevitable event that increases post-transplant complications. We have previously demonstrated that supplementation of University of Wisconsin (UW) solution with non-FDA-approved hydrogen sulfide (H. Thirty Lewis rats underwent bilateral nephrectomy followed by syngeneic orthotopic transplantation of the left kidney after 24-hour preservation in either UW or UW+STS solution at 4 °C. Rats were monitored to post-transplant day 14 and sacrificed to assess renal function (urine output, serum creatinine and blood urea nitrogen). Kidney sections were stained with H&E, TUNEL, CD68, and myeloperoxidase (MPO) to detect acute tubular necrosis (ATN), apoptosis, macrophage infiltration, and neutrophil infiltration.. UW+STS grafts showed significantly improved graft function immediately after transplantation, with improved recipient survival compared to UW grafts (p < 0.05). Histopathological examination revealed significantly reduced ATN, apoptosis, macrophage and neutrophil infiltration and downregulation of pro-inflammatory and pro-apoptotic genes in UW+STS grafts compared to UW grafts (p < 0.05).. We show for the first time that preservation of renal grafts in STS-supplemented UW solution protects against prolonged cold IRI by suppressing apoptotic and inflammatory pathways, and thereby improving graft function and prolonging recipient survival. This could represent a novel clinically applicable therapeutic strategy to minimize the detrimental clinical outcome of prolonged cold IRI in kidney transplantation.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Blood Urea Nitrogen; Cold Ischemia; Creatinine; Glutathione; Insulin; Kidney Function Tests; Kidney Transplantation; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Survival Rate; Thiosulfates

The protective effects of carbon monoxide (CO) against ischemia/reperfusion (IR) injury during organ transplantation have been extensively investigated. Likewise, CO-releasing molecules (CORMs) are known to exert a variety of pharmacological activities via liberation of controlled amounts of CO in organs. Therefore, we hypothesized that intraluminal administration of water-soluble CORM-3 during cold storage of intestinal grafts would provide protective effects against IR injury.. Orthotopic syngeneic intestinal transplantation was performed in Lewis rats following 6 h of cold preservation in Ringer solution or University of Wisconsin solution. Saline containing CORM-3 (100 µmol/L) or its inactive counterpart (iCORM-3) was intraluminally introduced in the intestinal graft before cold preservation.. Histopathological analysis of untreated and iCORM-3-treated grafts revealed a similar erosion and blunting of the intestinal villi. These changes in the mucosa structure were significantly attenuated by intraluminal administration of CORM-3. Intestinal mucosa damage caused by IR injury led to considerable deterioration of gut barrier function 3 h postreperfusion. CORM-3 significantly inhibited upregulation of proinflammatory mRNA levels, ameliorated intestinal morphological changes, and improved graft blood flow and mucosal barrier function. Additionally, CORM-3-treated grafts increased recipient survival rates. Pharmacological blockade of soluble guanylyl cyclase activity significantly reversed the protective effects conferred by CORM-3, indicating that CO partially mediates its therapeutic actions via soluble guanylyl cyclase activation.. Our study demonstrates that luminally delivered CORM-3 provides beneficial effects in cold-stored rat small intestinal grafts and could be an attractive therapeutic application of CO in the clinical setting of organ preservation and transplantation.

Topics: Adenosine; Allopurinol; Animals; Carbon Monoxide; Glutathione; Humans; Insulin; Ischemia; Organ Preservation Solutions; Organometallic Compounds; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Soluble Guanylyl Cyclase; Water

Shock and subsequent resuscitation provoke ischemia-reperfusion injury. Trimetazidine (TMZ), allopurinol (ALO), and histidine-tryptophan-ketoglutarate (HTK) solution, can protect from ischemia-reperfusion injury in chronic coronary syndromes and in transplantation. The objective of the current study is to compare, in a hemorrhagic shock and standard resuscitation animal model, organ damage parameters between placebo and treatment with TMZ, ALO, or HTK. Shock was induced in Wistar rats by controlled arterial bleeding, maintaining mean arterial pressure between 38 and 42 mm Hg for 60 minutes; then, drawn blood was reinfused. Animals were divided into: Sham (n = 4), Control (n = 6), TMZ (n = 7), ALO (n = 9), and HTK (n = 7). At the end of the experiment, animals were sacrificed and tissue harvested. TMZ, ALO and HTK decreased histopathologic damage in heart [Control: 1.72 (1.7-1.77); TMZ: 1.75 (1.72-1.79); ALO: 1.75 (1.74-1.8); HTK: 1.82 (1.78-1.85); all P < 0.05], kidney [Control: 3 (2-3); TMZ: 1 (1-2); ALO: 1 (1-1); HTK: 1(1-1); all P < 0.05] and intestine [Control: 3 (2-3); TMZ: 1 (1-2); ALO: 1 (1-1); HTK: 1 (0-2); all P < 0.05]. Also, treatment with TMZ, ALO, and HTK increased immunohistochemical expression of thioredoxin-1 in heart [Control: 6.6 (5.6-7.4); TMZ: 9.5 (8.1-9.7); ALO: 9.1 (8.4-10.2); HTK: 14.2 (12.6-15); all P < 0.05]; and kidney [Control: 4.6 (4-5.1); TMZ: 9.7 (9.3-9.9); ALO: 9.6 (9-9.9); HTK: 16.7 (16.1-17); all P < 0.05]. In an experimental model of hemorrhagic shock, TMZ, ALO, and HTK solution attenuated cell damage in multiple parenchyma and increased antioxidant defenses.

Topics: Allopurinol; Animals; Cardiovascular Agents; Disease Models, Animal; Glucose; Glutathione; Insulin; Mannitol; Organ Preservation Solutions; Potassium Chloride; Procaine; Rats; Rats, Wistar; Reperfusion Injury; Shock, Hemorrhagic

Dexpanthenol (DXP) reportedly protects tissues against oxidative damage in various inflammation models. This study aimed to evaluate its effects on oxidative stress, inflammation, apoptosis, and neurological recovery in an experimental rabbit spinal cord ischemia/reperfusion injury (SCIRI) model.. Rabbits were randomized into 5 groups of 8 animals each: group 1 (control), group 2 (ischemia), group 3 (vehicle), group 4 (methylprednisolone, 30 mg/kg), and group 5 (DXP, 500 mg/kg). The control group underwent laparotomy only, whereas other groups were subjected to spinal cord ischemia by aortic occlusion (just caudal to the 2 renal arteries) for 20 min. After 24 h, a modified Tarlov scale was employed to record neurological examination results. Malondialdehyde and caspase-3 levels and catalase and myeloperoxidase activities were analyzed in tissue and serum samples. Xanthine oxidase activity was measured in the serum. Histopathological and ultrastructural evaluations were also performed in the spinal cord.. After SCIRI, serum and tissue malondialdehyde and caspase-3 levels and myeloperoxidase and serum xanthine oxidase activities were increased (P < 0.05-0.001). However, serum and tissue catalase activity decreased significantly (P < 0.001). DXP treatment was associated with lower malondialdehyde and caspase-3 levels and reduced myeloperoxidase and xanthine oxidase activities but increased catalase activity (P < 0.05-0.001). Furthermore, DXP was associated with better histopathological, ultrastructural, and neurological outcome scores.. This study was the first to evaluate antioxidant, anti-inflammatory, antiapoptotic, and neuroprotective effects of DXP on SCIRI. Further experimental and clinical investigations are warranted to confirm that DXP can be administered to treat SCIRI.

Topics: Animals; Antioxidants; Caspase 3; Catalase; Disease Models, Animal; Inflammation; Malondialdehyde; Neuroprotective Agents; Peroxidase; Rabbits; Reperfusion Injury; Spinal Cord; Spinal Cord Ischemia; Xanthine Oxidase

Oxidative damage is critical in the pathogenesis of ovarian ischaemia/reperfusion (I/R) injury, and statins have been reported to exert antioxidant activity. However, the role of VCAM-1 and xanthine oxidase (XO)/uric acid (UA) in ovarian I/R injury is not known. Also, whether or not atorvastatin exerts antioxidant activity like other statins is unclear.. This study investigated the involvement of VCAM-1 and XO/UA in ovarian I/R injury and the likely protective role of atorvastatin.. Forty female Wistar rats were randomized into sham-operated, ischaemia, ischaemia/reperfusion (I/R), ischaemia and atorvastatin, and I/R and atorvastatin.. In comparison with the sham-operated group, atorvastatin blunted ischaemia and I/R-induced distortion of ovarian histoarchitecture and follicular degeneration. Also, atorvastatin alleviated ischaemia and I/R-induced rise in XO, UA, and malondialdehyde, which was accompanied by inhibition of ischaemia and I/R-induced reductions in reduced glutathione level, enzymatic antioxidant activities and increase in myeloperoxidase activity and TNF-α and IL-6 levels by atorvastatin treatment. Additionally, atorvastatin blocked ischaemia and I/R-induced increase in VCAM-1 expression, caspase 3 activity, 8-hydroxydeoxyguanosine level and ovarian DNA fragmentation index.. For the first time, this study revealed that atorvastatin-mediated downregulation of VCAM-1 and XO/UA/caspase 3 signaling averts oxidative injury, inflammation, and apoptosis induced by ovarian ischaemia/reperfusion injury.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Apoptosis; Atorvastatin; Caspase 3; Down-Regulation; Female; Glutathione; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Interleukin-6; Ischemia; Malondialdehyde; Oxidative Stress; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Tumor Necrosis Factor-alpha; Uric Acid; Vascular Cell Adhesion Molecule-1; Xanthine Oxidase

Static cold storage (SCS) is currently the most widely used method for organ preservation, but a number of limitations are associated including tissue damage and restricted opportunity for organ repair. Thus, the development of improved hypothermic storage solutions is an urgent need. Herein, using a renal epithelial cell model (LLC-PK1), we tested the benefits of ADD10, a novel clinical grade antioxidant product, in reducing damages associated with ischemia-reperfusion (IR). Cells were stored up to 24h at 4 °C in University of Wisconsin (UW) solution without or in the presence of 1% ADD10 with following reperfusion up to 24h at 37 °C. The presence of ADD10 significantly decreased cells damages, cell death, and the level of reactive oxygen species (ROS) (P < 0.05). Concomitantly, ADD10 supplementation also favored an increased oxygen consumption rate (OCR) and improved bioenergetics of LLC-PK1 cells (P < 0.05). Finally, preliminary in vivo studies suggested a benefit of ADD10 on the renal function post-transplantation. In conclusion, these results demonstrate that the addition of ADD10 to the preservation solution not only efficiently protects renal cells during SCS, but also improves the functionality of cold-stored organs during transplantation.

Topics: Allopurinol; Animals; Cold Injury; Cold Temperature; Energy Metabolism; Glutathione; Humans; Insulin; Kidney; Kidney Transplantation; LLC-PK1 Cells; Organ Preservation Solutions; Reperfusion Injury; Swine

The effect of different drugs on ischemia and reperfusion (I/R; induced oxygen free radical damage) was examined in small bowel tissue because the intestine is extremely sensitive to this pathology. Different drugs (allopurinol and dantrolene) can remove oxygen free radicals or inhibit the mechanisms leading to their generation, thus reducing mucosal lesions. We investigated the protective potential of combination therapy in the intestine against I/R damage.. Forty-eight male Wistar rats were separated into 8 groups: one sham (control), one I/R (ischemia 60 min + reperfusion at 24 h), and 6 groups treated with allopurinol, dantrolene, or combination therapy. The grade of injury in the small bowel was established by the lipid peroxidation (MDA) and antioxidant enzymatic activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in tissue samples. Moreover, the collected samples were subjected to histological study.. Results obtained in the experimental process indicate that the administration of antioxidants protects against intestinal damage by I/R. Overall, combination therapy may protect intestinal tissue from I/R injury.

Topics: Allopurinol; Animals; Dantrolene; Intestine, Small; Male; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase

Topics: Allopurinol; Calcium Channel Blockers; Dantrolene; Humans; Intestine, Small; Reperfusion Injury

The possible reno-protective effect of a controlled brief heat-shock treatment during isolated ex vivo machine perfusion of donor grafts prior to reperfusion should be investigated in a primary in vitro study. Porcine kidneys (n = 14) were retrieved after 20 minutes of cardiac standstill of the donor and subjected to 20 hours of static cold storage in University of Wisconsin solution. Prior to reperfusion, kidneys were subjected to 2 hours of reconditioning machine perfusion with gradual increase in perfusion temperature up to 35°C. In half of the kidneys (n = 7), a brief hyperthermic impulse (10 minutes perfusion at 42°C) was implemented in the machine perfusion period. Functional recovery of the grafts was observed upon normothermic reperfusion in vitro. Hyperthermic treatment resulted in a 50% increase of heat shock protein (HSP) 70 and HSP 27 mRNA and was accompanied by ~ 50% improvement of tubular re-absorption of sodium and glucose upon reperfusion, compared with the controls. Furthermore, renal loss of aspartate aminotransferase was significantly reduced to one-third of the controls as was urinary protein loss, evaluated by the albumin to creatinine ratio. It is concluded that ex vivo heat-shock treatment seems to be an easily implementable and promising option to enhance renal self-defense machinery against reperfusion injury after preservation that merits further investigation in preclinical models.

Topics: Adenosine; Allopurinol; Animals; Female; Glutathione; Insulin; Kidney; Kidney Transplantation; Models, Animal; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Reperfusion Injury; Rewarming; Sus scrofa; Tissue and Organ Harvesting

This study aimed to explore the potential antioxidant, anti-inflammatory, and anti-apoptotic effects of omega 3 fatty acid (Ω-3) in a rat model of testicular torsion/detorsion (T/D). Under ketamine/xylazine anaesthesia, age-matched adult male Wistar rats of comparable weight underwent sham-operation or testicular torsion by fixing the left testis rotated at 720° for two and half hours. After detorsion, animals were treated with either olive oil as vehicle or Ω-3 subcutaneously for three days. On post-operative day 3, rats were culled and the ipsilateral and contralateral testes, as well as obtained blood samples, were analyzed. Our findings revealed that T/D led to significant poor weight gain, distorted gross anatomy, and cytoarchitecture of the testes, low sperm quality, redox imbalance, and inflammation of the ipsilateral and contralateral testes. This was accompanied by reduced circulatory testosterone, a decline in testicular lactate metabolism and transport, upregulation of xanthine oxidase/uric acid signaling, and increased testicular DNA fragmentation. Administration of Ω-3 attenuated T/D-induced damage to the testes and sperm cells with a significant rise in the level of serum testosterone. Enhancement of lactate transport and down-regulation of xanthine oxidase/uric acid signaling by Ω-3 may be beneficial in protecting against T/D-related oxido-inflammatory damage and male infertility.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Fatty Acids, Omega-3; Lactates; Male; Rats; Rats, Wistar; Reperfusion Injury; Signal Transduction; Spermatic Cord Torsion; Spermatozoa; Testis; Testosterone; Uric Acid; Xanthine Oxidase

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cold Temperature; Glutathione; Humans; Insulin; Mice; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury

The eIF5A hypusination inhibitor GC7 (N1-guanyl-1,7-diaminoheptane) was shown to protect from ischemic injuries. We hypothesized that GC7 could be useful for preconditioning kidneys from donors before transplantation. Using a preclinical porcine brain death (BD) donation model, we carried out in vivo evaluation of GC7 pretreatment (3 mg/kg iv, 5 minutes after BD) at the beginning of the 4h-donor management, after which kidneys were collected and cold-stored (18h in University of Wisconsin solution) and 1 was allotransplanted. Groups were defined as following (n = 6 per group): healthy (CTL), untreated BD (Vehicle), and GC7-treated BD (Vehicle + GC7). At the end of 4h-management, GC7 treatment decreased BD-induced markers, as radical oxygen species markers. In addition, GC7 increased expression of mitochondrial protective peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC1α) and antioxidant proteins (superoxyde-dismutase-2, heme oxygenase-1, nuclear factor [erythroid-derived 2]-like 2 [NRF2], and sirtuins). At the end of cold storage, GC7 treatment induced an increase of NRF2 and PGC1α mRNA and a better mitochondrial integrity/homeostasis with a decrease of dynamin- related protein-1 activation and increase of mitofusin-2. Moreover, GC7 treatment significantly improved kidney outcome during 90 days follow-up after transplantation (fewer creatininemia and fibrosis). Overall, GC7 treatment was shown to be protective for kidneys against BD-induced injuries during donor management and subsequently appeared to preserve antioxidant defenses and mitochondria homeostasis; these protective effects being accompanied by a better transplantation outcome.

Topics: Adenosine; Allopurinol; Animals; Brain Death; Glutathione; Insulin; Kidney; Kidney Transplantation; Organ Preservation Solutions; Peptide Initiation Factors; Raffinose; Reperfusion Injury; RNA-Binding Proteins; Swine

Reperfusion surgery following testicular ischemia is a reproductive health threatening status and may result with organ dysfunction in men. The high level of reactive oxygen species (ROS) and cease of blood flow to the testis are the most important reasons of this testicular injury. Until today, numerous experimental studies reported that antioxidants might be efficient to alleviate oxidative stress induced organ dysfunction. For this purpose, in this study, we have investigated the protective effects of xanthine oxidase (XO) inhibitor, allopurinol, and ROS scavenger, trolox, in a comparative perspective in testicular ischemia reperfusion injury subjected rats.. Twenty-eight adult male Sprague Dawley rats were divided into four groups of seven animals in each; control, ischemia/reperfusion (I/R), allopurinol and trolox. The rats in control group did not receive any application. Animals in I/R, allopurinol and trolox groups were subjected to 2 h testicular reperfusion injury following 5 h ischemia. Intraperitoneally (i.p.) 1 ml isotonic, 200 mg/kg allopurinol and 50 mg/kg trolox were administered to the animals in these groups 30 min prior reperfusion. At the end of experiment, all animals were sacrificed and blood serum malondialdehyde (MDA) levels were measured. Histological sections were obtained from the testis and stained with hematoxylin and eosin (H&E), proliferating cell nuclear antigen (PCNA) and cleaved caspase-3. Apoptotic index was evaluated with TUNEL Assay.. Severe morphological degenerations, increased serum MDA, cleaved caspase-3 and TUNEL Assay positivity rate, but reduced PCNA positivity rate was observed in ischemia and reperfusion group. Morphological degenerations, MDA level, apoptotic index and PCNA positive cell rate were slightly alleviated in allopurinol administered animals compared with ischemia and reperfusion group. Protection with trolox was more successful and the results of the analysis were similar to the control group.. Ischemia that leading to testicular torsion is a reproductive health affecting problem and current surgical treatment methods might be insufficient to recover testis. Various types of ROS generating mechanisms in cell are limiting protective potency of allopurinol, and cocktail administration of different ROS inhibitors might be more effective. However, our results indicate that free radical scavenger trolox might be a candidate drug to alleviate degenerative effects of testicular ischemia reperfusion injury.. This is the first study that demonstrates antioxidant trolox was more successful than XO inhibitor allopurinol to protect testis against ischemia and reperfusion injury in rats.

Topics: Allopurinol; Animals; Chromans; Humans; Ischemia; Male; Malondialdehyde; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Spermatic Cord Torsion; Testis

Essential amino acid α-keto acid analogs are used in the treatment of chronic kidney disease to delay the symptoms of uremia. However, it is unknown whether essential amino acid α-keto acid analogs affect the oxidative stress and the inflammation in acute renal injury such as those produced by ischemia-reperfusion.. To evaluate the effect of essential amino acid α-keto acid analogs on renal ischemia-reperfusion injury in Wistar rats.. Rats were divided into 11 groups (n=6/group): Two groups received physiological saline with or without ischemia-reperfusion injury (45 min/24 h), six groups received essential amino acid α-keto acid analogs (400, 800, or 1,200 mg/kg/24 h/7d) with or without ischemia-reperfusion injury (essential amino acid α-keto acid analogs + ischemia-reperfusion), and two groups received allopurinol (50 mg/kg/24 h/7d) with or without ischemia-reperfusion injury. Biochemical markers included creatinine and blood urea nitrogen (BUN), proinflammatory cytokines (IL-1β, IL-6, and TNF-α), renal damage markers (cystatin C, KIM-1, and NGAL), and markers of oxidative stress such as malondialdehyde (MDA) and total antioxidant activity.. The essential amino acid α-keto acid analog- and allopurinol-treated groups had lower levels of creatinine, BUN, renal damage markers, proinflammatory cytokines, and MDA than their corresponding ischemia-reperfusion groups. These changes were related to the essential amino acid α-keto acid analogs dosage. Total antioxidant activity was lower in essential amino acid α-keto acid analog- and allopurinol-treated groups than in the corresponding ischemia-reperfusion groups.. This is a new report on the nephroprotective effects of essential amino acid α-keto acid analogs against ischemia-reperfusion injury. Essential amino acid α-keto acid analogs decreased the levels of biochemical markers, kidney injury markers, proinflammatory cytokines, and MDA while minimizing total antioxidant consumption.. Introducción. Los α-cetoanálogos de aminoácidos esenciales se utilizan en el tratamiento de la enfermedad renal crónica para retrasar los síntomas de la uremia. Sin embargo, se desconoce si los α-cetoanálogos de aminoácidos esenciales afectan el estrés oxidativo y la inflamación en la lesión renal aguda tal como en la producida por la isquemia-reperfusión.\ Objetivo. Evaluar el efecto de las α-cetoanálogos de aminoácidos esenciales sobre la lesión renal por isquemia-reperfusión en ratas Wistar.\ Materiales y métodos. Se emplearon 11 grupos de ratas (n=6): dos grupos recibieron solución salina fisiológica con lesión isquemia-reperfusión o sin ella (45 min/24 h), seis grupos recibieron α-cetoanálogos de aminoácidos esenciales (400, 800 o 1.200 mg/kg/24 h/7d) con lesión isquemia-reperfusión o sin ella (α-cetoanálogos de aminoácidos esenciales + isquemia-reperfusión), y dos grupos recibieron (50 mg/kg/24 h/7d) con lesión isquemia-reperfusión o sin ella. Los marcadores bioquímicos incluyeron creatinina y nitrógeno ureico en sangre (BUN), citocinas proinflamatorias (IL-1β, IL-6 y TNF-α), marcadores de daño renal (cistatina C, KIM-1 y NGAL) y marcadores del estrés oxidativo como el malondialdehído (MDA) y la actividad antioxidante total.\ Resultados. Los grupos tratados con α-cetoanálogos de aminoácidos esenciales y alopurinol tuvieron niveles inferiores de creatinina, BUN, marcadores de daño renal, citocinas proinflamatorias, actividad antioxidante total y MDA que los grupos isquemia-reperfusión correspondientes. Estos cambios se asociaron con la dosis. La actividad antioxidante total fue menor en los grupos tratados con α-cetoanálogos de aminoácidos esenciales que en los grupos isquemia-reperfusión correspondientes.\ Conclusiones. Este es un nuevo informe de los efectos nefroprotectores de las α-cetoanálogos de aminoácidos esenciales contra la lesión isquemia-reperfusión. Los α-cetoanálogos de aminoácidos esenciales disminuyeron los niveles de los marcadores bioquímicos, de los de lesión renal, de las citocinas proinflamatorias y el MDA, a la vez que minimizaron el consumo total de antioxidantes.

Topics: Allopurinol; Amino Acids, Essential; Animals; Antioxidants; Biomarkers; Blood Urea Nitrogen; Creatinine; Cystatin C; Cytokines; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Keto Acids; Kidney; Lipocalin-2; Male; Malondialdehyde; Oxidative Stress; Random Allocation; Rats; Rats, Wistar; Reperfusion Injury

Topics: Allopurinol; Animals; Chromans; Ischemia; Male; Rats; Reperfusion Injury; Testis

Topics: Allopurinol; Animals; Chromans; Ischemia; Male; Rats; Reperfusion Injury; Testis

Although oxidative stress plays central roles in postischemic renal injury, region-specific alterations in energy and redox metabolism caused by short-duration ischemia remain unknown. Imaging mass spectrometry enabled us to reveal spatial heterogeneity of energy and redox metabolites in the postischemic murine kidney. After 10-minute ischemia and 24-hour reperfusion (10mIR), in the cortex and outer stripes of the outer medulla, ATP substantially decreased, but not in the inner stripes of the outer medulla and inner medulla. 10mIR caused renal injury with elevation of fractional excretion of sodium, although histological damage by oxidative stress was limited. Ischemia-induced NADH elevation in the cortex indicated prolonged production of reactive oxygen species by xanthine oxidase (XOD). However, consumption of reduced glutathione after reperfusion suggested the amelioration of oxidative stress. An XOD inhibitor, febuxostat, which blocks the degradation pathway of adenine nucleotides, promoted ATP recovery and exerted renoprotective effects in the postischemic kidney. Because effects of febuxostat were canceled by silencing of the hypoxanthine phosphoribosyl transferase 1 gene in cultured tubular cells, mechanisms for the renoprotective effects appear to involve the purine salvage pathway, which uses hypoxanthine to resynthesize adenine nucleotides, including ATP. These findings suggest a novel therapeutic approach for acute ischemia/reperfusion renal injury with febuxostat through salvaging high-energy adenine nucleotides.

Topics: Acute Kidney Injury; Adenine Nucleotides; Animals; Enzyme Inhibitors; Febuxostat; Kidney; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

To investigate the effect of Picroside II on testicular ischemia and reperfusion (l/R) injury and the underlying mechanism.. Sprague-Dawley rats were randomly divided into 4 groups: sham operated group (Sham), Sham with Picroside II treatment group (Sham+ Pic II), l/R group (l/R) and l/R with Picroside II treatment group (I/R+ Pic II). l/R model was established by rotating the left testis 720° in a clock-wise direction for 4 hours. The histopathologic and spermatogenetic evaluation was performed. The apoptosis changes and the levels of HO-1 (heme oxygenase-1), MPO (myeloperoxidase), NOX (NADPH oxidase), SOD (superoxide dismutase), XO (xanthine oxidase) and NOS (nitric oxide synthase) were measured.. The seminiferous tubules were damaged in l/R rats, but Picroside II alleviated the changes induced by l/R. The increased level of apoptosis was decreased by Picroside II (P=0.01, 9.05±0.35 vs. 4.85±0.25). The activities of HO-1, MPO, NOX, XO and MDA content were increased and the SOD activity was decreased in l/R (P<0.05) and could be reversed by Picroside II (P=0.03, 405.5±7.5 vs. 304±17U/mgprot; P=0.02, 0.99±0.05 vs. 0.52±0.04 mgprot; P=0.01, 260+7 vs. 189±2 mgprot; P=0.04, 10.95+0.55 vs. 8.75+0.35 U/mgprot; P=0.045, 6.8+0.7 vs. 3.75+0.35 mgprot; P=0.04, 44.5+3.5 vs. 57.5+3.5 mgprot). Western blot showed that the expression of iNOS, nNOS and eNOS were increased in l/R (P<0.05); however, they were decreased after Picroside II treatment (P<0.05).. Picroside II attenuated testicular I/R injury in rats mainly through suppressing apoptosis and oxidative stress through reduction of nitric oxide synthesis.

Topics: Animals; Apoptosis; Blotting, Western; Cinnamates; Heme Oxygenase-1; In Situ Nick-End Labeling; Iridoid Glucosides; Male; Malondialdehyde; NADP; Nitric Oxide; Oxidative Stress; Peroxidase; Random Allocation; Rats, Sprague-Dawley; Reperfusion Injury; Reproducibility of Results; Superoxide Dismutase; Testis; Xanthine Oxidase

Ex vivo machine perfusion of the liver after cold storage has found to be most effective if combined with controlled oxygenated rewarming up to (sub)-normothermia. On disconnection of the warm graft from the machine, most surgeons usually perform a cold flush of the organ as protection against the second warm ischemia incurred upon implantation. Experimental evidence, however, is lacking and protective effect of deep hypothermia has been challenged for limited periods of liver ischemia in other models. A first systematic test was carried out on porcine livers, excised 30 min after cardiac arrest, subjected to 18 h of cold storage in UW and then machine perfused for 90 min with Aqix-RSI solution. During machine perfusion, livers were gradually rewarmed up to 20 °C. One group (n = 6) was then reflushed with 4 °C cold Belzer UW solution whereas the second group (n = 6) remained without cold flush. All livers were exposed to 45 min warm ischemia at room temperature to simulate the surgical implantation period. Organ function was evaluated in an established reperfusion model using diluted autologous blood. Cold reflush after disconnection from the machine resulted in a significant increase in bile production upon blood reperfusion, along with a significant reduction in transaminases release alanine aminotransferase and of the intramitochondrial enzyme glutamate dehydrogenase. Interestingly, free radical-mediated lipid peroxidation was also found significantly lower after cold reflush. No differences between the groups could be evidenced concerning histological injury and recovery of hepatic energy metabolism (tissue content of adenosine triphosphate). Post-machine preservation cold reflush seems to be beneficial in this particular setting, even if the organs are warmed up only to 20 °C, without notion of adverse effects, and should therefore be implemented in the protocol.

Topics: Adenosine; Allopurinol; Animals; Bile; Cold Temperature; Female; Glutathione; Insulin; Lipid Peroxidation; Liver; Liver Function Tests; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Reperfusion; Reperfusion Injury; Rewarming; Swine; Time Factors

Liver transplantation (LT) is considered the standard treatment for end-stage liver disease, but ideal donors remain in limited supply, resulting in an unavoidable increase in the need to use grafts from marginal donors. The attenuation of ischemia-reperfusion injury (IRI) in such marginal donors is therefore crucial for reducing the possibility of the primary non-function of grafts and graft loss. Some reports have found that molecular-hydrogen showed antioxidant and anti-inflammatory effects in preventing IRI in some non-hepatic transplant models. Therefore, we investigated whether or not molecular-hydrogen could attenuate IRI in LT model rats.. We used a hydrogen-rich water bath to dissolve hydrogen into solution and graft tissues and performed isogenic and orthotopic LT in Lewis rats with University of Wisconsin (UW) solution. Blood and tissue samples were collected 6 h after the reperfusion. Hepatic enzymes in serum were measured. Pathological findings including the expressions of cytokines and heme oxygenase (HO)-1 in liver tissues were evaluated.. The concentration of hydrogen inside the graft tissues increased depending on the storage time, plateauing after 1 h. Serum liver enzyme levels were significantly lower and the histology score of liver damage markedly attenuated in the group given grafts preserved in hydrogen-rich UW solution than in the control group. The hydrogen-rich UW solution group also showed less oxidative damage and hepatocyte apoptosis than the control group, and the expression of proinflammatory cytokines tended to be lower while the protein levels of HO-1 were significantly increased (n = 3-12 per group, P < 0.05).. Storage of liver grafts in hydrogen-rich UW solution resulted in superior functional and morphologic protection against IRI via the up-regulation of HO-1 expression.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Cold Temperature; Glutathione; Hepatocytes; Hydrogen; Hydrogen-Ion Concentration; Inflammation; Insulin; Kidney; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; RNA, Messenger

Hypothermic machine perfusion (HMP) has become standard care in many center's to preserve kidneys donated after circulatory death (DCD). Despite a significant reduction in metabolism at low temperatures, the remaining cellular activity requires oxygen. Because of the role and safety of oxygen during HMP has not been fully clarified, its supply during HMP is not standard yet. This study investigates the effect of administering oxygen during HMP on renal function in a porcine DCD model.. After 30 minutes of warm ischemia, porcine slaughterhouse kidneys were preserved for 24 hours by means of cold storage (CS), or HMP with Belzer Machine Perfusion Solution supplemented with no oxygen, 21% or 100% oxygen. Next, kidneys were reperfused for 4 hours in a normothermic machine perfusion setup.. HMP resulted in significantly better kidney function during normothermic machine perfusion. Thiobarbituric acid-reactive substances, markers of oxidative stress, were significantly lower in HMP preserved kidneys. HMP preserved kidneys showed significantly lower aspartate aminotransferase and lactate dehydrogenase levels compared with kidneys preserved by CS. No differences were found between the HMP groups subjected to different oxygen concentrations. Adenosine triphosphate levels significantly improved during HMP when active oxygenation was applied.. This study showed that preservation of DCD kidneys with HMP is superior to CS. Although the addition of oxygen to HMP did not result in significantly improved renal function, beneficial effects were found in terms of reduced oxidative stress and energy status. Oxygen addition proofed to be safe and did not show detrimental effects.

Topics: Adenosine; Allografts; Allopurinol; Animals; Biopsy; Disease Models, Animal; Glutathione; Humans; Hypothermia, Induced; Insulin; Kidney; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Oxygen; Perfusion; Raffinose; Reperfusion; Reperfusion Injury; Swine; Tissue and Organ Harvesting; Warm Ischemia

Ischemia‑reperfusion injury (IRI) is a notable cause of tissue damage during surgical procedures and a major risk factor in graft dysfunction in liver transplantation. Livers obtained from donors after circulatory death (DCD) are prone to IRI and toll‑like receptor 4 (TLR4) serves a prominent role in the inflammatory response associated with DCD liver IRI. The present study was designed to investigate whether TAK242, a specific TLR4 inhibitor, improves hepatic IRI following a DCD graft and to investigate its underlying protective mechanisms. Male Sprague‑Dawley rats were randomized into 4 groups: Control, TAK242, DCD and DCD+TAK242 groups. Rats were pretreated with TAK242 or its vehicle for 30 min, then the livers were harvested without warm ischemia (control group and TAK242 group) or with warm ischemia in situ for 30 min. The livers were stored in cold University of Wisconsin solution for 24 h and subsequently perfused for 60 min with an isolated perfused rat liver system. Rat liver injury was evaluated thereafter. When compared with the DCD group, DCD livers with TAK242 pretreatment displayed significantly improved hepatic tissue injury and less tissue necrosis (P<0.05). Compared with DCD livers, mechanistic experiments revealed that TAK242 pretreatment alleviated mitochondrial dysfunction, reduced reactive oxygen species and malondialdehyde levels and inhibited apoptosis. Additionally, TAK242 significantly inhibited the IRI‑associated inflammatory response, indicated by the decreased expression of TLR4, interleukin (IL)‑1β, IL‑6 and cyclooxygenase 2 at the mRNA and protein levels (P<0.05). TAK242 ameliorates DCD liver IRI via suppressing the TLR4 signaling pathway in rats. The results of the present study have revealed that TAK242 pretreatment harbors a potential benefit for liver transplantation.

Topics: Adenosine; Allopurinol; Animals; Anti-Inflammatory Agents; Glutathione; Insulin; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Sulfonamides; Toll-Like Receptor 4; Warm Ischemia

storage is the most prevalent method for graft preservation in kidney transplantation (KTX). The protective effects of various preservation solutions have been studied extensively in both clinical trials and experimental animal models. However, a paucity of studies have examined the effect of different preservation solutions on graft function in mouse KTX; in addition, the tolerance of the transplanted grafts to further insult has not been evaluated, which was the objective of the present study. We performed mouse KTX in three groups, with the donor kidneys preserved in different solutions for 60 min: saline, mouse serum, and University of Wisconsin (UW) solution. The graft functions were assessed by kidney injury markers and glomerular filtration rate (GFR). The grafts that were preserved in UW solution exhibited better functions, reflected by 50 and 70% lower plasma creatinine levels as well as 30 and 55% higher plasma creatinine levels in GFR than serum and saline groups, respectively, during the first week after transplants. To examine the graft function in response to additional insult, we induced ischemia-reperfusion injury (IRI) by clamping the renal pedicle for 18 min at 4 wk after KTX. We found that the grafts preserved in UW solution exhibited ~30 and 20% less injury assessed by kidney injury markers and histology than in other two preservation solutions. Taken together, our results demonstrated that UW solution exhibited a better protective effect in transplanted renal grafts in mice. UW solution is recommended for use in mouse KTX for reducing confounding factors such as IRI during surgery.

Topics: Adenosine; Allopurinol; Animals; Biomarkers; Creatinine; Cytoprotection; Glomerular Filtration Rate; Glutathione; Hepatitis A Virus Cellular Receptor 1; Insulin; Kidney; Kidney Transplantation; Lipocalin-2; Male; Mice, Inbred C57BL; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Saline Solution; Time Factors

University of Wisconsin (UW) solution is not optimal for preservation of marginal organs. Polyethylene glycol (PEG) could improve protection. Similarly formulated solutions containing either 15 or 20 g/L PEG 20 kDa or 5, 15 and 30 g/L PEG 35 kDa were tested in vitro on kidney endothelial cells, ex vivo on preserved kidneys, and in vivo in a pig kidney autograft model. In vitro, all PEGs provided superior preservation than UW in terms of cell survival, adenosine triphosphate (ATP) production, and activation of survival pathways. Ex vivo, tissue injury was lower with PEG 20 kDa compared to UW or PEG 35 kDa. In vivo, function recovery was identical between UW and PEG 35 kDa groups, while PEG 20 kDa displayed swifter recovery. At three months, PEG 35 kDa 15 and 30 g/L animals had worse outcomes than UW, while 5 g/L PEG 35 kDa was similar. PEG 20 kDa was superior to both UW and PEG 35 kDa in terms of function and fibrosis development, with low activation of damage pathways. PEG 20 kDa at 15 g/L was superior to 20 g/L. While in vitro models did not discriminate between PEGs, in large animal models of transplantation we showed that PEG 20 kDa offers a higher level of protection than UW and that longer chains such as PEG 35 kDa must be used at low doses, such as found in Institut George Lopez (IGL1, 1g/L).

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cell Hypoxia; Disease Models, Animal; Endothelial Cells; Glutathione; Insulin; Kidney; Kidney Function Tests; Kidney Transplantation; Male; Molecular Weight; Organ Preservation; Organ Preservation Solutions; Polyethylene Glycols; Primary Cell Culture; Raffinose; Recovery of Function; Reperfusion Injury; Swine; Transplantation, Autologous

Topics: Animals; Apoptosis; Calcium; Endoplasmic Reticulum Stress; Gene Expression Regulation; Humans; Hydrogen Peroxide; Lipopolysaccharides; Mice; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Necrosis; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; Reperfusion Injury; Xanthine Oxidase

Carbon monoxide (CO) inhalation protects organ by reducing inflammation and cell death during transplantation processes in animal model. However, using CO in clinical transplantation is difficult due to its delivery in a controlled manner. A manganese-containing CO releasing molecules (CORM)-401 has recently been synthesized which can efficiently deliver 3 molar equivalents of CO. We report the ability of this anti-inflammatory CORM-401 to reduce ischemia reperfusion injury associated with prolonged cold storage of renal allografts obtained from donation after circulatory death in a porcine model of transplantation.. To stimulate donation after circulatory death condition, kidneys from large male Landrace pig were retrieved after 1 hour warm ischemia in situ by cross-clamping the renal pedicle. Procured kidneys, after a brief flushing with histidine-tryptophan-ketoglutarate solution were subjected to pulsatile perfusion at 4°C with University of Wisconsin solution for 4 hours and both kidneys were treated with either 200 μM CORM-401 or inactive CORM-401, respectively. Kidneys were then reperfused with normothermic isogeneic porcine blood through oxygenated pulsatile perfusion for 10 hours. Urine was collected, vascular flow was assessed during reperfusion and histopathology was assessed after 10 hours of reperfusion.. We have found that CORM-401 administration reduced urinary protein excretion, attenuated kidney damage markers (kidney damage marker-1 and neutrophil gelatinase-associated lipocalin), and reduced ATN and dUTP nick end labeling staining in histopathologic sections. CORM-401 also prevented intrarenal hemorrhage and vascular clotting during reperfusion. Mechanistically, CORM-401 appeared to exert anti-inflammatory actions by suppressing Toll-like receptors 2, 4, and 6.. Carbon monoxide releasing molecules-401 provides renal protection after cold storage of kidneys and provides a novel clinically relevant ex vivo organ preservation strategy.

Topics: Adenosine; Allografts; Allopurinol; Animals; Carbon Monoxide; Cold Ischemia; Glutathione; Insulin; Kidney; Kidney Transplantation; Male; Manganese; Models, Animal; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Sus scrofa

Cold ischemia-reperfusion injury is unavoidable during organ transplantation, and prolonged preservation is associated with poorer function recovery. Cardiotrophin-1 (CT-1) is an IL-6 family cytokine with cytoprotective properties. This preclinical study in rats tested whether CT-1 mitigates cold renal ischemia-reperfusion injury in the context of the transplantation of long-time preserved kidneys.. Kidneys were flushed with cold (4°C) University of Wisconsin solution containing 0.2 μg/mL CT-1 and stored for several periods of time at 4°C in the same solution. In a second approach, kidneys were first cold-preserved for 6 hours and then were perfused with University of Wisconsin solution containing CT-1 (0, 16, 32, or 64 μg/mL) and further cold-preserved. Organ damage markers were measured in the kidneys at the end of the storage period. For renal transplantation, recipient consanguineous Fischer rats underwent bilateral nephrectomy and received a previously cold-preserved (24 hours) kidney as described above. Survival and creatinine clearance were monitored over 30 days.. Cardiotrophin-1 in perfusion and preservation fluids reduced oxidative stress markers (superoxide anion and inducible nitric oxide synthase), inflammation markers (NF-κB and tumor necrosis factor-α), and vascular damage (vascular cell adhesion molecule-1) and activated leukemia inhibitory factor receptor and STAT-3 survival signaling. Transplantation of kidneys cold-preserved with CT-1 increased rat survival and renal function (ie, lower plasma creatinine and higher creatinine clearance) and improved kidney damage markers after transplantation (ie, lower superoxide anion, tumor necrosis factor-α, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 and higher NF-κB).. Cardiotrophin-1 represents a novel therapeutic strategy to reduce ischemia-reperfusion and cold preservation injury to rescue suboptimal kidneys and, consequently, to improve the clinical outcomes of renal transplantation.

Topics: Adenosine; Allografts; Allopurinol; Animals; Cold Ischemia; Cytokines; Disease Models, Animal; Glutathione; Graft Survival; Humans; Insulin; Kidney; Kidney Function Tests; Kidney Transplantation; Male; Nephrectomy; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Inbred F344; Reperfusion Injury; Tissue and Organ Harvesting

The aim of this study was to evaluate the effects of the antioxidant allopurinol and ischemic post-conditioning on the deleterious effects of ischemia followed by reperfusion (I/R) in a standardized model of ischemia involving infra-renal aortic occlusion in rats.. The animals were randomly divided into five groups: (A) animals not subjected to ischemia; (B) animals subjected to 2 h of ischemia and reperfusion only once; (C) animals given an allopurinol dose by gavage, then subjected to 2 h of ischemia and reperfusion only once; (D) animals subjected to 2 h of ischemia and post-conditioning and (E) animals that received allopurinol, then subjected to 2 h of ischemia and post-conditioning. The blood samples and small intestine segments were harvested for analysis after 3 days.. The protective effects of the use of allopurinol and ischemic post-conditioning were observed by measuring aspartate aminotransferase, alanine aminotransferase and lactate levels. The benefits of post-conditioning were evident from the total antioxidant capacity and creatinine levels, but these could not ascertain any positive effects of allopurinol. The histological analysis of mesentery revealed that both methods were effective in minimizing the harmful effects of the ischemia and reperfusion process.. Individual protocols significantly reduced I/R systemic injuries, but no additional protection was observed when the two strategies were combined.

Topics: Allopurinol; Animals; Antioxidants; Aorta, Abdominal; Biomarkers; Disease Models, Animal; Female; Ischemic Postconditioning; Lower Extremity; Rats, Wistar; Regional Blood Flow; Reperfusion Injury

Renal transplantation is the preferred treatment for end-stage renal disease. Currently, there is a large gap between the supply and demand for transplantable kidneys. The use of sub-optimal grafts obtained via donation after cardiac death (DCD) is on the rise. While static cold storage (SCS) in University of Wisconsin (UW) solution on ice (4°C) is the clinical standard of care for renal graft preservation, cold storage has been associated with negative graft outcomes. The alternative, normothermic machine perfusion, involves mechanical perfusion of the organ at physiological or normothermic temperature (37°C) and this technique is expensive, complicated and globally inaccessible. As such, simpler alternatives are of interest. Preliminary results revealed that UW solution is more protective at 21°C than 37°C and subnormothermic preservation is of interest because it may facilitate the use of existing solutions while preventing cold injury. We have previously shown that SCS in UW solution supplemented with mitochondria-targeted H

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Cell Line; Cell Survival; Cytoprotection; Epithelial Cells; Glutathione; Hydrogen Sulfide; Insulin; Kidney; Kidney Transplantation; Organ Preservation; Organ Preservation Solutions; Organophosphorus Compounds; Perfusion; Raffinose; Reperfusion Injury; Swine; Temperature; Thiones

Deceased donor kidneys are exposed to cold ischemic insult which makes them particularly susceptible to the effects of cold ischemic injury during hypothermic preservation resulting in high rates of delayed graft function. Bone morphogenetic protein-7 (BMP-7) is a valuable reagent in the field of tissue regeneration and preservation under ischemic conditions. Following these insights, we investigated the effect of recombinant human BMP-7 (rhBMP-7) on graft preservation during cold ischemia. The study was conducted on an experimental model of kidney cold ischemia in rats. Kidneys were perfused with University of Wisconsin (UW) saline solution, rhBMP-7, or rhBMP-7 + UW, and exposed to cold ischemia for 6, 12, and 24 hours. In tubular epithelial cells of kidneys perfused with rhBMP-7 and rhBMP-7+UW solution, the expression of BMP-7 and E-cadherin was observed after 24 hours of cold ischemia. In kidneys not perfused with rhBMP-7, high expression of transforming growth factor-β and α-smooth muscle actin was found. Also, in kidneys perfused with rhBMP-7 solution, statistically higher levels of Smad1, Smad5, and Smad8 messenger RNA expressions were proven. BMP-7 maintains the morphology of kidney tissue better than UW solution during 24 hours of cold ischemia. BMP-7 prevents epithelial to mesenchymal transformation and consequently maintains epithelial phenotype of tubular cells.

Topics: Adenosine; Allopurinol; Animals; Bone Morphogenetic Protein 7; Cold Ischemia; Delayed Graft Function; Glutathione; Insulin; Kidney Transplantation; Male; Organ Preservation Solutions; Raffinose; Rats; Reperfusion Injury

During liver transplantation, graft ischemia-reperfusion injury leads to a systemic inflammatory response producing postoperative organ dysfunctions. The aim of this observational and prospective study was to compare the impact of Solution de conservation des organes et tissus (SCOT) 15 and University of Wisconsin (UW) preservation solutions on early cytokine release, postreperfusion syndrome and postoperative organ dysfunctions.. Thirty-seven liver transplantations were included: 21 in UW Group and 16 in SCOT 15 group. Five cytokines were measured in systemic blood after anesthetic induction, 30minutes after unclamping portal vein and on postoperative day 1.. Following unclamping portal vein, cytokines were released in systemic circulation. Systemic cytokine concentrations were higher in UW than in SCOT 15 group: Interleukin-10, Interleukine-6. In SCOT 15 group, significant reduction of postreperfusion syndrome incidence and acute kidney injury were observed. Alanine and aspartate aminotransferase peak concentrations were higher in SCOT 15 group than in UW group. However, from postoperative day 1 to day 10, aminotransferase returned to normal values and did not differ between groups.. Compared to UW, SCOT 15 decreases systemic cytokine release resulting from graft ischemia-reperfusion injury and reduces incidence of postreperfusion syndrome and postoperative renal failure.

Topics: Adenosine; Allopurinol; Cytokines; Female; Glutathione; Humans; Insulin; Liver Transplantation; Male; Middle Aged; Multiple Organ Failure; Organ Preservation Solutions; Postoperative Complications; Prospective Studies; Raffinose; Reperfusion Injury; Time Factors

We examined the roles of xanthine oxidoreductase (XOR) in renal ischemia reperfusion (IR) injury.. XOR+/+ and XOR+/- mice were subjected to 24-h reperfusion after a 45-min bilateral renal artery occlusion or sham operation. We evaluated the renal damage based on the concentrations of blood urea nitrogen (BUN) and serum creatinine (Cr), and histological changes were detected by PAS staining. Xanthine dehydrogenase, oxidase (XO) and XOR activities, amounts of blood and urine 8-OHdG, and expressions of TNF-α and MCP-1 mRNA were examined. F4/80 and nitrotyrosine-positive cells were assessed by immunohistochemical staining.. The BUN and Cr concentrations in the XOR+/+IR mice were increased significantly compared to those in XOR+/-IR and allopurinol-treated XOR+/+IR mice. XO and XOR activity, which were increased in IR mice, were reduced in the allopurinol-treated XOR+/+IR and XOR+/-IR mice compared to the XOR+/+IR mice. The concentrations of blood and urine 8-OHdG, and the expressions of MCP-1 and TNF-α mRNA were increased significantly in the XOR+/+IR mice compared to those in the XOR+/-IR mice. The histological analysis revealed that the XOR+/-IR and allopurinol-treated XOR+/+IR mice showed less tubular injury than the XOR+/+IR mice in the cortex regions, with the reduction of inflammation and oxidative stress assessed by the immunohistological staining for F4/80 and nitrotyrosine.. Both the disruption of XOR gene in XOR+/- mice and the reduction of XOR activity in allopurinol-treated XOR+/+IR mice attenuated renal tissue injury in this IR model. Reduced XOR activity during renal IR could be a beneficial treatment target.

Topics: Allopurinol; Animals; Blood Urea Nitrogen; Creatinine; Disease Models, Animal; Enzyme Inhibitors; Inflammation; Kidney Diseases; Male; Mice; Mice, Knockout; Oxidative Stress; Reperfusion Injury; Xanthine Dehydrogenase

Kidney donation after circulatory death (DCD) is a less than ideal option to meet organ shortages. Hypothermic machine perfusion (HMP) with Belzer solution (BS) improves the viability of DCD kidneys, although the graft clinical course remains critical. Mesenchymal stromal cells (MSC) promote tissue repair by releasing extracellular vesicles (EV). We evaluated whether delivering MSC-/MSC-derived EV during HMP protects rat DCD kidneys from ischaemic injury and investigated the underlying pathogenic mechanisms. Warm ischaemic isolated kidneys were cold-perfused (4 hrs) with BS, BS supplemented with MSC or EV. Renal damage was evaluated by histology and renal gene expression by microarray analysis, RT-PCR. Malondialdehyde, lactate, LDH, glucose and pyruvate were measured in the effluent fluid. MSC-/EV-treated kidneys showed significantly less global ischaemic damage. In the MSC/EV groups, there was up-regulation of three genes encoding enzymes known to improve cell energy metabolism and three genes encoding proteins involved in ion membrane transport. In the effluent fluid, lactate, LDH, MDA and glucose were significantly lower and pyruvate higher in MSC/EV kidneys as compared with BS, suggesting the larger use of energy substrates by MSC/EV kidneys. The addition of MSC/EV to BS during HMP protects the kidney from ischaemic injury by preserving the enzymatic machinery essential for cell viability and protects the kidney from reperfusion damage.

Topics: Adenosine; Allopurinol; Animals; Biomarkers; Energy Metabolism; Extracellular Vesicles; Gene Expression; Gene Expression Profiling; Glucose; Glutathione; Insulin; Ion Transport; Kidney; Kidney Transplantation; L-Lactate Dehydrogenase; Lactic Acid; Malondialdehyde; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Organ Preservation; Organ Preservation Solutions; Perfusion; Pyruvic Acid; Raffinose; Rats; Rats, Inbred F344; Rats, Transgenic; Reperfusion Injury

The outcomes of liver transplantation (LT) from donation after cardiac death (DCD) donors remain poor due to severe warm ischemia injury. Perfluorocarbon (PFC) is a novel compound with high oxygen carrying capacity. In the present study, a rat model simulating DCD LT was used, and the impact of improved graft oxygenation provided by PFC addition on liver ischemia/reperfusion injury (IRI) and survival after DCD LT was investigated. Orthotopic liver transplants were performed in male Lewis rats, using DCD liver grafts preserved with cold University of Wisconsin (UW) solution in the control group and preserved with cold oxygenated UW solution with addition of 20% PFC in the PFC group. For experiment I, in a 30-minute donor warm ischemia model, postoperative graft injury was analyzed at 3 and 6 hours after transplantation. For experiment II, in a 50-minute donor warm ischemia model, the postoperative survival was assessed. For experiment I, the levels of serum aspartate aminotransferase, alanine aminotransferase, hyaluronic acid, malondialdehyde, and several inflammatory cytokines were significantly lower in the PFC group. The hepatic expression levels of tumor necrosis factor α and interleukin 6 were significantly lower, and the expression level of heme oxygenase 1 was significantly higher in the PFC group. Histological analysis showed significantly less necrosis and apoptosis in the PFC group. Sinusoidal endothelial cells and microvilli of the bile canaliculi were well preserved in the PFC group. For experiment II, the postoperative survival rate was significantly improved in the PFC group. In conclusion, graft preservation with PFC attenuated liver IRI and improved postoperative survival. This graft preservation protocol might be a new therapeutic option to improve the outcomes of DCD LT. Liver Transplantation 23 1171-1185 2017 AASLD.

Topics: Adenosine; Allografts; Allopurinol; Animals; Disease Models, Animal; Fluorocarbons; Glutathione; Graft Survival; Humans; Insulin; Liver; Liver Function Tests; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Perfusion; Postoperative Period; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Survival Rate; Time Factors; Treatment Outcome; Warm Ischemia

Recent studies have demonstrated the neuroprotective and immunomodulatory effects of 1,25-dihydroxyvitamin D3 (calcitriol), but no previous study has examined these effects on spinal cord ischemia/reperfusion (I/R) injury. Therefore, the present study aimed to evaluate whether calcitriol protects the spinal cord from I/R injury.. Rabbits were randomized into four groups of eight animals: group 1 (laparotomy control), group 2 (ischemia control), group 3 (30mg/kg intraperitoneal methylprednisolone at surgery), and group 4 (0.5μg/kg, intraperitoneal calcitriol for 7 days before I/R injury). The rabbits in the laparotomy control group underwent laparotomy only, whereas all rabbits in the other groups were subject to spinal cord ischemia by aortic occlusion for 20min, just caudal to the renal artery. Malondialdehyde and catalase levels, myeloperoxidase and xanthine oxidase activities, and caspase-3 concentrations were analyzed. Finally, histopathological, ultrastructural, and neurological evaluations were performed.. After I/R injury, increases in malondialdehyde levels, myeloperoxidase and xanthine oxidase activities, and caspase-3 concentrations were found (p<0.001 for all); by contrast, catalase levels decreased (p<0.001). Calcitriol pretreatment was associated with lower malondialdehyde levels (p<0.001), reduced myeloperoxidase (serum, p=0.018; tissue, p<0.001) and xanthine oxidase (p<0.001) activities, and caspase-3 concentrations (p<0.001), but increased catalase levels (p<0.001). Furthermore, calcitriol pretreatment was associated with better histopathological, ultrastructural, and neurological scores.. Calcitriol pretreatment provided significant neuroprotective benefits following spinal cord I/R injury.

Topics: Animals; Caspase 3; Catalase; Disease Models, Animal; Male; Malondialdehyde; Methylprednisolone; Peroxidase; Rabbits; Reperfusion Injury; Spinal Cord Ischemia; Vitamin D; Xanthine Oxidase

To analyze the effects of allopurinol and of post-conditioning on lung injuries induced by lower-limb ischemia and reperfusion.. Thirty rats were used. They were divided in 5 groups: (1) group A: abdominal aortic dissection only, (2) group B: ischemia and reperfusion, (3) group C: administered allopurinol (100mg/Kg) a few hours before procedure, (4) group D: post-conditioned and (5) group E: administered allopurinol and post-conditioned. With the exception of group A, all groups were submitted to infrarenal aortic ischemia for 2 hours, and reperfusion for 72 hours. After euthanasia, lungs were removed for histological analysis. They were graded under two scores: pulmonary injury (neutrophil infiltration, interstitial edema, vascular congestion, and destruction of lung architecture) and lymphocytic score (neutrophil infiltration, lymphoid aggregate and secondary follicle).. On the pulmonary injury score, the degree of injury was smaller than in groups D and E, when compared to group B, p<0.05. Group C did not obtain the same result (p>0,05). On the lymphocytic score, there was no statistic difference among groups, p>0.05.. Both post-conditioning and the combination of allopurinol and post-conditioning were effective in remote lung protection induced by lower-limbs I/R. When used in isolation, allopurinol showed no protective effect.

Topics: Allopurinol; Animals; Antimetabolites; Disease Models, Animal; Female; Ischemic Postconditioning; Lung Injury; Male; Rats; Rats, Wistar; Reperfusion Injury

To compare the effect of University of Wisconsin (UW) solution with or without metformin, an AMP-activated protein kinase (AMPK) activator, for preserving standard and marginal liver grafts of young and aged rats. Eighteen young (4 mo old) and 18 aged (17 mo old) healthy male SD rats were selected and randomly divided into three groups: control group, UW solution perfusion group (UWP), and UW solution with metformin perfusion group (MUWP). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), interleukin-18 (IL-18), and tumor necrosis factor-alpha (TNF-α) in the perfused liquid were tested. The expression levels of AMPK and endothelial nitric oxide synthase (eNOS) in liver sinusoidal endothelial cells were also examined. Additionally, microscopic evaluation of the harvested perfused liver tissue samples was done.. AST, ALT, LDH, IL-18 and TNF-α levels in the young and aged liver-perfused liquid were, respectively, significantly lower in the MUWP group than in the UWP group (. The addition of metformin to the UW preservative solution for

Topics: Adenosine; Alanine Transaminase; Allopurinol; AMP-Activated Protein Kinases; Animals; Aspartate Aminotransferases; Cold Ischemia; Glutathione; Hepatocytes; Humans; Infusion Pumps; Insulin; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Male; Metformin; Microscopy, Electron, Transmission; Models, Animal; Nitric Oxide Synthase Type III; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tissue and Organ Harvesting

Superoxide, nitric oxide (NO), and peroxynitrite are important mediators in the pathogenesis of ischemia-reperfusion (I/R) injury. We tested the renoprotective effects of allopurinol (ALP), a xanthine oxidase inhibitor, N-nitro-L-arginine methyl ester (L-NAME), and 5,10,15,20-tetrakis (N-methyl-4-pyridyl) porphyrinato iron (III) (FeTMPyP) by selective inhibition of superoxide, NO, and peroxynitrite, respectively.. Male Sprague-Dawley rats were randomly assigned to 5 groups (n = 6 per group). Group 1 was a sham-operated group. Group 2 was the renal I/R group (30-minute ischemia followed by 24-hour reperfusion). Rats in groups 3, 4, and 5 received ALP, L-NAME, or FeTMPyP, respectively, at 5 minutes before the reperfusion. Serum creatinine (Cr), blood urea nitrogen (BUN), renal tissue malondialdehyde, superoxide dismutase, histological changes, apoptosis, and monocyte infiltration were evaluated. In addition, the combined treatment with ALP and L-NAME was compared with FeTMPyP in a second independent experiment.. The administration of ALP, L-NAME, and FeTMPyP diminished the increase in Cr (P = .0066 for all) and BUN (P = .0066 for ALP; and P = .013 for L-NAME) induced by I/R injury and decreased the histological damage (P = .0066 for all). In addition, ALP, L-NAME, and FeTMPyP attenuated the oxidative stress response as determined by a decrease in malondialdehyde level (P = .0066 for all), apoptotic renal tubular cells (P = .0066 for all), and monocyte infiltration (P = .0066 for all). The combined treatment of ALP and L-NAME decreased Cr and BUN levels to a greater degree than FeTMPyP (P = .016 for Cr; P = .0079 for BUN).. Superoxide, NO, and peroxynitrite are involved in renal I/R injury. The reduction of peroxynitrite formation, via inhibition of superoxide or NO, or the induction of peroxynitrite decomposition may be beneficial in renal I/R injury.

Topics: Allopurinol; Animals; Antioxidants; Apoptosis; Blood Urea Nitrogen; Creatinine; Cytoprotection; Disease Models, Animal; Enzyme Inhibitors; Kidney; Kidney Diseases; Lipid Peroxidation; Male; Metalloporphyrins; Monocytes; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Peroxynitrous Acid; Rats, Sprague-Dawley; Reperfusion Injury; Superoxides; Xanthine Oxidase

Allopurinol is a well-known antioxidant that protects tissue against ischemia and reperfusion injury, blocking purine catabolism, and possibly reducing TNF-α and other cytokines. It also plays a significant role in reducing the inflammatory processes by inhibiting chemotaxis and other inflammatory mediators. The objective of this study was to define the role of allopurinol regarding kidney ischemic injury particularly as to its effect on inflammatory molecules such as TNF-α, IL-1β, and IL-6 response. One hundred and twenty five rats were subjected to warm renal ischemia. Five more animals were included as sham. Animal survival and plasma levels of lipid peroxidation, myeloperoxidase, lactate dehydrogenase, glutathione, urea, creatinine, and cytokines were determined. Inflammatory parameters (TNF-α, IL-1β, and IL-6) were measured in all groups by quantitative immunosorbent assay. Further, immunohistological and histopathological studies were carried out on animals treated prior to, or following reperfusion with 10 and 50 mg/kg of Allopurinol. The statistical analysis included ANOVA and Fisher test as well as χ

Topics: Acute Kidney Injury; Allopurinol; Animals; Drug Evaluation, Preclinical; Gout Suppressants; Interleukin-18; Interleukin-6; Kidney; Male; Rats, Wistar; Reperfusion Injury; Tumor Necrosis Factor-alpha

To investigate the potential protective effect of allopurinol on reperfusion injury by determining the inflammatory response through the measurement of tumor necrosis factor-alpha (TNF-alpha).. Sixty rats were distributed into two groups: control and allopurinol and each group was divided into three subgroups, ischemia for two hours, ischemia for three hours and ischemia simulation. Allopurinol group rats received 100mg/kg dose of allopurinol, whereas control group rats received an equivalent dose of saline. Clamping of the infrarenal aorta was performed for two or three hours depending on the subgroup. Ischemia simulation subgroups did not suffer ischemia, just aortic dissection, and maintenance for three hours. After 72 hours of reperfusion, blood was collected by cardiac puncture for TNF-alpha measurement.. Allopurinol reduced TNF-alpha significantly (p <0.001) when compared to the matching control subgroups (control X allopurinol in ischemia for two hours and for three hours).. Allopurinol reduced the concentrations of serum TNF-alpha when used at different times of ischemia followed by reperfusion, which might indicate reduction of the inflammation provoked by the reperfusion injury.

Topics: Abdominal Cavity; Allopurinol; Animals; Antimetabolites; Inflammation; Ischemia; Models, Animal; Random Allocation; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Time Factors; Tumor Necrosis Factor-alpha

The purpose of this study is to explore whether normothermic machine perfusion (NMP) preservation is superior to cold preservation during reduced-size liver transplantation (RSLT) in pigs. Twenty-four healthy Ba-Ma mini pigs were used (aged >13 months; weight 25-35 kg; regardless of sex). The animals were randomized into 2 groups. In group A (NMP), donor livers were harvested without warm ischemia time and heartbeats and then were connected to the NMP system to reduce the livers' size under the normothermic condition. In group B (University of Wisconsin [UW] solution), donor livers were harvested without warm ischemia time and heartbeats after being perfused by UW solution and were then preserved in 0°C-4°C UW solution to reduce the livers' size under cold conditions. After that, liver transplantation without venovenous bypass was performed. General RSLT information of the pigs from the 2 groups was recorded; the serological indices were measured; and routine pathological examination of liver tissue was observed. A significant difference was observed in the intraoperative bleeding between the 2 groups (P < 0.05), whereas no significant difference was found in the other indices (all P > 0.05). Significant differences of alanine aminotransferase levels, aspartate aminotransferase levels, and lactate dehydrogenase levels between the 2 groups were observed between postoperative days 3 and 5 (P < 0.05). Significant differences of lactic acid levels between the 2 groups were observed between postoperative days 2 and 5 (P < 0.05). Compared with the cold preservation group, the liver tissues of the NMP preservation group only rarely experienced liver cell necrosis and maintained integrities in the hepatic sinusoid spaces and endothelial cells. In conclusion, NMP preservation is superior to cold preservation during RSLT in pigs. Liver Transplantation 22 968-978 2016 AASLD.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Cold Ischemia; Glutathione; Hepatocytes; Humans; Insulin; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Necrosis; Organ Preservation; Organ Preservation Solutions; Perfusion; Postoperative Period; Raffinose; Reperfusion Injury; Swine; Swine, Miniature; Temperature

To investigate the potential effects of pretreatment with allopurinol on renal ischemia/reperfusion injury (IRI) in a rat model.. Twenty four rats were subjected to right kidney uninephrectomy were randomly distributed into the following three groups (n=8): Group A (sham-operated group); Group B (ischemic group) with 30 min of renal ischemia after surgery; and Group C (allopurinol + ischemia group) pretreated with allopurinol at 50 mg/kg for 14 days. At 72 h after renal reperfusion, the kidney was harvested to assess inflammation and apoptosis.. Pretreatment with allopurinol significantly improved renal functional and histological grade scores following I/R injury (p<0.05). Compared with Group B, the expression levels of caspase-3 and Bax were markedly reduced in Group C, meanwhile, whereas expression of bcl-2 was clearly increased (p<0.05). A newly described marker of inflammation, High Mobility Group Box 1(HMGB1), showed reduced expression in Group C (p<0.05).. Pretreatment with allopurinol had a protective effect on kidney ischemia/reperfusion injury, which might be related to the inhibition of HMGB1 expression.

Topics: Allopurinol; Animals; Apoptosis; Blood Urea Nitrogen; Disease Models, Animal; HMGB1 Protein; Inflammation; Ischemic Preconditioning; Kidney; Male; Peroxidase; Protective Agents; Random Allocation; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase

Carbon monoxide poisoning is one of the important emergency situations manifested by primarily acute and chronic anoxic central nervous system (CNS) injuries and other organ damages. Current descriptions and therapeutic approaches have been focused on the anoxic pathophysiology. However, this point of view incompletely explains some of the outcomes and needs to be investigated extensively. Considering this, we propose that reactive oxygen species (ROS) including especially nitric oxide (NO) are likely to be a key concept to understand the emergency related to CO poisoning and to discover new therapeutic modalities in CO toxicity. If we consider the hypothesis that ROS is involved greatly in acute and chronic toxic effects of CO on CNS and some other vital organs such as heart, it follows that the antioxidant and anti-NO therapies might give the clinicians more opportunities to prevent deep CNS injury. In support of this, we review the subject in essence and summarize clinical and experimental studies that support a key role of ROS in the explanation of pathophysiology of CO toxicity as well as new treatment modalities after CO poisoning.

Topics: Animals; Antioxidants; Brain; Carbon Monoxide; Carbon Monoxide Poisoning; Central Nervous System; Humans; Hypoxanthine; Hypoxia; Microdialysis; Models, Theoretical; Nitric Oxide; Oxygen; Rats; Reactive Oxygen Species; Reperfusion Injury; Superoxides; Uric Acid; Xanthine Oxidase

To observe the protective effects of safflor Injection (SI) and extract of Ginkgo biloba (EGB) on lung ischemia-reperfusion injury (LIRI) and investigate its mechanism.. In vivo rabbit model of LIRI was reconstructed. Forty rabbits were randomly and equally divided into four groups: sham-operation group (sham group), ischemia-reperfusion group (model group), ischemia-reperfusion plus SI group (safflor group) and ischemia-reperfusion plus EGB injection group (EGB group). Malondialdehyde (MDA) content, superoxide dismutase (SOD) and xanthine oxidase (XO) activity in serum were measured. The wet/dry weight ratio (W/D) of the lung tissue and activity of myeloperoxidase (MPO) were also tested. Ultrastructure change of the lung tissue was observed by the electron microscope. The expression of intercellular adhesion molecule-1 (ICAM-1) was measured by immunohistochemistry (IHC).. In the model group, MDA and XO increased and SOD decreased in serum compared with the sham group (P<0.01). The values of W/D, MPO and ICAM-1 of the model group were higher than those of the sham group (P<0.01), but those of the safflor group and EGB group were significantly lower than those of the model group (P<0.01). The IHC demonstrated that ICAM-1 expression in lung tissue of the model group was significantly higher than those of the safflor group (P<0.01). Compared with safflor group, in the EGB group MDA, XO, MPO decreased, SOD and ICAM-1 expression increased (P<0.05), but the change of W/D was not statistically significant (P>0.05).. SI and EGB may attenuate LIRI through antioxidation, inhibition of neutrophil aggregation and down-regulation of ICAM-1 expression. But EGB had more effect on the antioxidation, while SI did better on regulating ICAM-1 expression.

Topics: Animals; Female; Ginkgo biloba; Immunohistochemistry; Injections; Intercellular Adhesion Molecule-1; Lung; Male; Malondialdehyde; Plant Extracts; Protective Agents; Rabbits; Reperfusion Injury; Safflower Oil; Superoxide Dismutase; Xanthine Oxidase

Deceased after cardiac death donors represent an important source of organs to reduce organ shortage in transplantation. However, these organs are subjected to more ischaemia-reperfusion injury (IRI). Reducing IRI by targeting coagulation is studied here in an experimental model.. The effect of an anti-Xa compound (fondaparinux) was evaluated using an autotransplanted kidney model in pigs. Kidneys were clamped for 60 min (warm ischaemia) and then preserved for 24 h at 4 °C in University of Wisconsin solution (UW). The anti-Xa compound was injected intravenously before warm ischaemia and used during cold storage, and its effects were compared with those of intravenous injection of unfractionated heparin (UFH) before warm ischaemia and use during cold storage, or use of UW alone during cold storage.. At 3 months after transplantation, anti-Xa treatment improved recovery of renal function and chronic serum creatinine levels compared with UW and UFH (mean(s.e.m.) 89(4), 250(4) and 217(8) µmol/l respectively). The anti-Xa treatment also reduced fibrosis, and decreased tissue expression of markers of the epithelial-mesenchymal transition compared with UW and UFH. Cleaved protease-activated receptor 2 was overexpressed in the UW group compared with the anti-Xa and UFH groups. Leucocyte infiltrates were decreased in the anti-Xa group compared with the UW and UFH groups. Macrophage invasion was also decreased by anticoagulation treatment.. Peritransplant anticoagulation therapy was beneficial to graft outcome, in both the acute and chronic phases. Moreover, specific inhibition of coagulation Xa protease further protected kidney grafts, with better recovery and decreased expression of chronic lesion markers. Surgical relevance The increasing use of marginal donors highlights the importance of organ quality in transplantation. Renal ischaemia-reperfusion injury (IRI), which includes a deleterious activation of coagulation, plays a central role in determining graft quality and outcome. Using an established porcine renal autotransplantation preclinical model with high clinical relevance, the benefits of anticoagulation therapy using an antifactor Xa molecule were evaluated. Peritransplantion anticoagulation treatment, specifically with an anti-Xa compound, protected marginal kidney grafts, improving functional recovery and reducing chronic lesions. This study demonstrates the benefits of anticoagulation therapy at the time of organ collection, particularly for marginal organs, encountered in cases of extended criteria and deceased after circulatory death donors. This anticoagulation strategy could be an important addition to current donor and organ management protocols in order to limit IRI and improve outcome.

Topics: Adenosine; Allopurinol; Animals; Anticoagulants; Constriction; Cytokines; Fondaparinux; Glutathione; Insulin; Kidney; Kidney Transplantation; Leukocytes; Nephritis; Organ Preservation Solutions; Polysaccharides; Raffinose; Reperfusion Injury; Swine; Transplantation, Autologous; Warm Ischemia

The aim of this study is to investigate the potential effects of borax on ischemia/reperfusion injury of the rat spinal cord.. Twenty-one Wistar albino rats were divided into 3 groups: sham (no ischemia/reperfusion), ischemia/reperfusion, and borax (ischemia/reperfusion + borax); each group was consist of 7 animals. Infrarenal aortic cross clamp was applied for 30 minutes to generate spinal cord ischemia. Animals were evaluated functionally with the Basso, Beattie, and Bresnahan scoring system and inclined-plane test. The spinal cord tissue samples were harvested to analyze tissue concentrations of nitric oxide, nitric oxide synthase activity, xanthine oxidase activity, total antioxidant capacity, and total oxidant status and to perform histopathological examination.. At the 72nd hour after ischemia, the borax group had significantly higher Basso, Beattie, and Bresnahan and inclined-plane scores than those of ischemia/reperfusion group. Histopathological examination of spinal cord tissues in borax group showed that treatment with borax significantly reduced the degree of spinal cord edema, inflammation, and tissue injury disclosed by light microscopy. Xanthine oxidase activity and total oxidant status levels of the ischemia/reperfusion group were significantly higher than those of the sham and borax groups (P < .05), and total antioxidant capacity levels of borax group were significantly higher than those of the ischemia/reperfusion group (P < .05). There was not a significantly difference between the sham and borax groups in terms of total antioxidant capacity levels (P > .05). The nitric oxide levels and nitric oxide synthase activity of all groups were similar (P > .05).. Borax treatment seems to protect the spinal cord against injury in a rat ischemia/reperfusion model and improve neurological outcome.

Topics: Animals; Antioxidants; Borates; Locomotion; Male; Nervous System Diseases; Neuroprotective Agents; Nitric Oxide; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Spinal Cord Ischemia; Xanthine Oxidase

By binding to T cell immunoglobulin mucin-3 (TIM-3) on activated Th1 cells, galectin-9 (Gal-9) negatively regulates Th1-type alloimmunity. Although T cells contribute to hepatic ischemia-reperfusion injury (IRI), it is unknown whether negative T cell-dependent TIM-3 co-stimulation may rescue IR-stressed orthotopic liver transplants from innate immunity-driven inflammation.. We used wild type (WT) and TIM-3 transgenic (Tg) mice (C57BL/6) as liver donors and recipients in a clinically-relevant model of hepatic cold storage (20 h at 4°C in UW solution) and syngeneic orthotopic liver transplantation (OLT).. Orthotopic liver transplants in WT or TIM-3Tg→TIM-3Tg groups were resistant against IR-stress, evidenced by preserved hepatocellular function (serum ALT levels) and liver architecture (Suzuki's score). In contrast, orthotopic liver transplants in WT or TIM-3Tg→WT groups were susceptible to IRI. TIM-3 induction in circulating CD4+ T cells of the recipient: (1) depressed T-bet/IFN-γ, while amplifying GATA3 and IL-4/IL-10 expression in orthotopic liver transplants; (2) promoted T cell exhaustion (PD-1, LAG-3) phenotype; and (3) depressed neutrophil and macrophage infiltration/function in orthotopic liver transplants. In parallel studies, we documented for the first time that Gal-9, a natural TIM-3 ligand, was produced primarily by and released from IR-stressed hepatocytes, both in vivo and in vitro. Moreover, exogenous recombinant Gal-9 (rGal-9) potentiated liver resistance against IRI by depressing T cell activation and promoting apoptosis of CD4+ T cells.. Harnessing TIM-3/Gal-9 signalling at the T cell-hepatocyte interface facilitates homeostasis in IR-stressed orthotopic liver transplants. Enhancing anti-oxidant hepatocyte Gal-9 potentiates liver IR-resistance. Negative regulation by recipient TIM-3+CD4+ cells provides evidence for cytoprotective functions of a discrete T cell subset, which should be spared when applying T cell-targeted immunosuppression in transplant recipients.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Cell Differentiation; Disease Models, Animal; Galectins; Glutathione; Hepatitis A Virus Cellular Receptor 2; Hepatocytes; Immunity, Innate; In Vitro Techniques; Insulin; Liver Transplantation; Lymphocyte Activation; Macrophages; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Neutrophils; Organ Preservation; Organ Preservation Solutions; Raffinose; Receptors, Virus; Reperfusion Injury; Signal Transduction; T-Lymphocytes

The aim of this experimental study was to determine whether combination of N-acetylcysteine and allopurinol can reduce the ischemia/reperfusion injury of spinal cord in a rat model.. Twenty-seven Spraque Dawley rats, all male, weighing between 220 to 370 (mean 325) gr were used in the study. 27 rats were divided into three groups: sham group, control group and experimental group. Abdominal aortic occlusion between the renal arteries and iliac bifurcations was carried out for 60 min with proximal and distal clip in control and experimental groups. Hindlimb motor functions were evaluated at 24, and 48 h using the Tarlov Scale. Besides, spinal cord samples were taken for determination of superoxide dismutase, and catalase activities as antioxidant enzymes, and malondialdehyde as an indicator of lipid peroxidation and xanthine oxidase levels as source hydroxyl radical for biochemical studies. Also, histopathological evaluation was made from cord tissue samples.. The experimental group subjects had better neurological functions than control group subjects. In experimental group; superoxide dismutase and catalase levels increased, while malondialdehyde and xantine oxidase levels decreased as compared with control group. Histopathological examination showed that experimental group had less cell degeneration, hemorrhage, edema and inflammation loss than control group.. This study offers that combined use of N-acetylcysteine and allopurinol might help protect the spinal cord against ischemia/reperfusion injury.

Topics: Acetylcysteine; Allopurinol; Animals; Aorta, Thoracic; Disease Models, Animal; Drug Therapy, Combination; Free Radical Scavengers; Ligation; Male; Neuroprotective Agents; Rats; Reperfusion Injury; Spinal Cord Ischemia

We demonstrated that 3-nitrotyrosine and 4-hydroxy-2-nonenal levels in mouse brain were elevated from 1 h until 8 h after global brain ischemia for 14 min induced with the 3-vessel occlusion model; this result indicates that ischemia reperfusion injury generated oxidative stress. Reactive oxygen species production was observed not only in the hippocampal region, but also in the cortical region. We further evaluated the neuroprotective effect of xanthine oxidoreductase inhibitors in the mouse 3-vessel occlusion model by analyzing changes in the expression of genes regulated by the transcription factor nuclear factor-kappa B (including pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), matrix metalloproteinase-9 and intercellular adhesion molecules-1). Administration of allopurinol resulted in a statistically significant decrease in IL-1β and TNF-α mRNA expression, whereas febuxostat had no significant effect on expression of these genes; nevertheless, both inhibitors effectively reduced serum uric acid concentration. It is suggested that the neuroprotective effect of allopurinol is derived not from inhibition of reactive oxygen species production by xanthine oxidoreductase, but rather from a direct free-radical-scavenging effect.

Topics: Aldehydes; Allopurinol; Animals; Biomarkers; Brain; Brain Ischemia; Disease Models, Animal; Interleukin-1beta; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Neuroprotective Agents; NF-kappa B; Oxidative Stress; Reactive Oxygen Species; Reperfusion Injury; RNA, Messenger; Tumor Necrosis Factor-alpha; Tyrosine; Uric Acid; Xanthine Dehydrogenase

BACKGROUND Results in small bowel transplantation are inferior compared to other solid organ transplantations, among other reasons, due to a specific vulnerability to ischemia/reperfusion injury. New strategies are needed to improve organ storage. Here we compare static cold storage in University of Wisconsin solution to storage supplemented with molecular oxygen gas insufflation. MATERIAL AND METHODS Rat small bowel was retrieved and either stored unoxygenated (UW) or oxygenated (UW+O2) for 18 h at 4°C. Biochemical parameters, mucosal function, Toll-like receptor upregulation, and parameters of structural integrity were evaluated following isolated reperfusion in vitro for 30 min at 37°C. RESULTS Oxygenation showed: ATP concentration was 82 times higher; lactate dehydrogenase release was continuously lower over 30 min; malondialdehyde, a final product of lipid peroxidation (UW+O2 vs. UW; 2.7±0.92 nmol/mL vs. 17.22±10.1 nmol/mL; P<0.05) and nitric oxide concentration (0.87±0.27 µmol/L vs. 2.17±0.29 µmol/L; P<0.001) were significantly lower; whereas mucosal functional integrity (galactose uptake) was better preserved (0.47±0.18 mg/dL vs. 0.35±0.05 mg/dL). Amelioration of tissue damage could be demonstrated by reduced apoptosis (3.3±1.2 AU vs. 28.4±10 AU; P>0.05), and preserved subcellular integrity. Toll-like receptors were significantly less upregulated (TLR2 0.32±0.1 vs. 2.1±1.5-fold and TLR4 1.53±1.14 vs. 11.79±5.4-fold; P<0.05). CONCLUSIONS Oxygenated storage is superior to standard storage in University of Wisconsin solution in terms of energetics, tissue damage, and mucosal integrity.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Insufflation; Insulin; Intestine, Small; Male; Organ Preservation; Organ Preservation Solutions; Oxygen; Raffinose; Rats; Rats, Wistar; Reperfusion Injury

Bladder ischemia-reperfusion (I/R) injury results in the generation of reactive oxygen species (ROS) and markedly elevates the risk of lower urinary tract symptoms (LUTS). Allopurinol is an inhibitor of xanthine oxidase (XO) and thus can serve as an antioxidant that reduces oxidative stress. Here, a rat model was used to assess the ability of allopurinol treatment to ameliorate the deleterious effects of urinary bladder I/R injury. I/R injury reduced the in vitro contractile responses of longitudinal bladder strips, elevated XO activity in the plasma and bladder tissue, increased the bladder levels of tumor necrosis factor-α (TNF-α), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase, reduced the bladder levels of extracellular regulated kinase (ERK), and decreased and increased the bladder levels of Bcl-2 and Bax, respectively. I/R injury also elevated lipid peroxidation in the bladder. Allopurinol treatment in the I/R injury was generated significantly ameliorating all I/R-induced changes. Moreover, an in situ fluorohistological approach also showed that allopurinol reduces the generation of intracellular superoxides enlarged by I/R injury. Together, the beneficial effects of allopurinol reducing ROS production may be mediated by normalizing the activity of the ERK, JNK, and Bax/Bcl-2 pathways and by controlling TNF-α expression.

Topics: Allopurinol; Animals; bcl-2-Associated X Protein; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; JNK Mitogen-Activated Protein Kinases; Lipid Peroxidation; Male; Malondialdehyde; p38 Mitogen-Activated Protein Kinases; Protective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Signal Transduction; Tumor Necrosis Factor-alpha; Up-Regulation; Urinary Bladder; Xanthine Oxidase

Decreasing organ quality is prompting research toward new methods to alleviate ischemia reperfusion injury (IRI). Oxidative stress and nuclear factor kappa beta (NF-κB) activation are well-described elements of IRI. We added cyclodextrin-complexed curcumin (CDC), a potent antioxidant and NF-κB inhibitor, to University of Wisconsin (UW) solution (Belzer's Solution, Viaspan), one of the most effective clinically approved preservative solutions. The effects of CDC were evaluated on pig endothelial cells and in an autologous donation after circulatory death (DCD) kidney transplantation model in large white pigs. CDC allowed rapid and lasting uptake of curcumin into cells. In vitro, CDC decreased mitochondrial loss of function, improved viability and lowered endothelial activation. In vivo, CDC improved function recovery, lowered histological injury and doubled animal survival (83.3% vs. 41.7%). At 3 months, immunohistochemical staining for epithelial-to-mesenchymal transition (EMT) and fibrosis markers was intense in UW grafts while it remained limited in the UW + CDC group. Transcriptional analysis showed that CDC treatment protected against up-regulation of several pathophysiological pathways leading to inflammation, EMT and fibrosis. Thus, use of CDC in a preclinical transplantation model with stringent IRI rescued kidney grafts from an unfavorable prognosis. As curcumin has proved well tolerated and nontoxic, this strategy shows promise for translation to the clinic.

Topics: Adenosine; Allopurinol; Animals; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Cells, Cultured; Chemistry, Pharmaceutical; Curcumin; Cyclodextrins; Disease Models, Animal; Fibrosis; Flow Cytometry; Glutathione; Graft Rejection; Humans; Inflammation; Insulin; Kidney Transplantation; Kidney Tubules; Male; Organ Preservation Solutions; Oxidative Stress; Prostate; Raffinose; Real-Time Polymerase Chain Reaction; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Swine

Although liver transplantation from non-heart-beating donors (NHBDs) is an effective way to overcome shortage of donors, primary graft nonfunction is often noted in these grafts. We have previously reported that edaravone, a free radical scavenger, has a cytoprotective effect on warm ischemia-reperfusion injury and improves the function of liver grafts from NHBDs in a rat model of ischemia-reperfusion. The purpose of this study was to investigate the effects of edaravone on liver transplantations from NHBDs.. Pigs were divided into three groups: (1) a heart-beating (HB) group (n = 5), in which liver grafts were retrieved from HB donors; (2) a non-heart-beating (NHB) group (n = 4), in which liver grafts were retrieved under apnea-induced NHB conditions; and (3) an edaravone-treated (ED) group (n = 5), in which liver grafts were retrieved in the same manner as the NHB group and treated with edaravone at the time of perfusion (3 mg/L in University of Wisconsin [UW] solution), cold preservation (1 mg/L in UW solution), and after surgery (1 mg/kg/d). The grafts from all groups were transplanted after 4 hours of cold preservation.. In the ED group, the 7-day survival rate was significantly higher than that in the NHB group (80% versus 0%, P = .0042, Kaplan-Meier log-rank test). Furthermore, on histologic examination, the structure of sinusoids in the ED group was well preserved and similar to that in the HB group.. Edaravone may improve the viability of liver grafts from NHBDs.

Topics: Adenosine; Allopurinol; Animals; Antipyrine; Biomarkers; Cold Ischemia; Cytoprotection; Edaravone; Free Radical Scavengers; Glutathione; Graft Survival; Insulin; Liver; Liver Transplantation; Male; Models, Animal; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Swine; Time Factors; Tissue and Organ Harvesting; Tissue Survival; Warm Ischemia

Use of grafts from donors after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. However, this requires the development of novel preservation methods to recover the organ from changes due to warm ischemia time (WIT).. Porcine livers were perfused with a newly developed machine perfusion (MP) system. The livers were perfused with modified University of Wisconsin solution (UW) - gluconate. All grafts were procured after acute hemorrhagic shock with the ventilator off. For group 1 (n = 6), grafts were procured after WIT of 60 minutes and preserved by hypothermic MP (HMP) for 3 hours. For group 2 (n = 5), grafts were preserved with 2 hours of simple cold storage (SCS) and HMP for 2 hours. For group 3 (n = 6), grafts were preserved with 2 hours of SCS and rewarming up to 25°C by MP for 2 hours (RMP). The preserved liver grafts were transplanted orthotopically.. The alanine aminotransferase level in perfusate in RMP during perfusion preservation was maintained at less than that of HMP. The levels of aspartate aminotransferase and lactate dehydrogenase in the 2 hours after reperfusion were significantly lower in group 3. Histologically, the necrosis of hepatocytes was less severe in group 3. The survival rate in group 3 was 2/4, but 0/4 in the other group.. RMP is expected to facilitate the recovery of the DCD liver grafts.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Biomarkers; Cold Ischemia; Disease Models, Animal; Female; Glutathione; Graft Survival; Heart Arrest; Hepatectomy; Insulin; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Necrosis; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Reperfusion Injury; Rewarming; Sus scrofa; Time Factors; Tissue and Organ Harvesting; Warm Ischemia

The effect of cold ischemia (CI) in vascularized composite allotransplantation is unknown. We herein assess tissue-specific damage, acceptable CI time, and the effect of preservation solutions in a syngenic rat hindlimb transplant model.. Lewis rat limbs were flushed and stored for 2, 10, or 30 hr CI in saline, histidine-tryptophan-ketoglutarate or University of Wisconsin preservation solution before transplantation. Morphologic alterations, inflammation, and damage of the individual tissues were analyzed on day 10 using histomorphology, confocal, light, and transmission-electron microscopy.. Two-hour CI led to mild inflammation of tissues on day 10, whereas 10-hr and 30-hr CI resulted in massive inflammation and tissue damage. Although muscle was mainly affected after prolonged CI (≥10 hr), nerve was affected in all CI groups. A perineural cell infiltrate, hypercellular appearance, pronounced vacuolization, and mucoid degeneration, appearing as Wallerian degeneration, were observed. Staining with propidium iodide and Syto 16 revealed a decrease in viable muscle cell nuclei in the anterior tibial muscle on day 10 in all groups, which was most pronounced in 10-hr and 30-hr CI animals. Transmission-electron microscopy indicated that a large number of mitochondria were degenerated in the 10-hr and 30-hr CI groups. Histidine-tryptophan-ketoglutarate preservation solution slightly decreased inflammation and tissue damage compared to University of Wisconsin-treated and saline-treated animals, especially in skin and muscle when CI times did not exceed 10 hr.. Severe inflammation and tissue damage are observed after prolonged CI in muscle and nerve. Ischemia times in vascularized composite allotransplantation should be kept as short as possible and certainly below 10 hr.

Topics: Adenosine; Allopurinol; Animals; Cold Ischemia; Dose-Response Relationship, Drug; Extremities; Glucose; Glutathione; Inflammation; Insulin; Male; Mannitol; Microscopy, Confocal; Microscopy, Electron, Transmission; Muscle, Skeletal; Organ Preservation; Organ Preservation Solutions; Potassium Chloride; Procaine; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Sciatic Nerve; Time Factors

Based on the fact that the acute phase of ischemic stroke is accompanied by the development of heart damage, manifestations of which are oxidative stress, morphological changes in the myocardium, in the model of brain focal ischemia-reperfusion, we investigated the oxidative stress in rat heart mitochondria and possible mechanisms of cardioprotective effect of ecdysterone. Under the conditions of brain focal ischemia-reperfusion, there is an increase rate of the generation of reactive oxygen species: superoxide (*O2-) and hydroxyl radicals (*OH), pools of stable hydrogen peroxide (H2O2), accumulate products of lipid peroxidation (diene conjugates and malonic dialdehyde), as a result of activation xanthine oxidase (marker uric acid), lipooxygenase (marker leukotriene C4) and cyclooxygenase (marker tromboksane B2) ways of *O2-(generating). In animals that received ecdysterone for 18 days, under conditions of brain focal ischemia-reperfusion, the rate of reactive oxygen species generation and the pools of lipid peroxidation products were decreased, and the survival of animals was increased. The obtained results support the development of oxidative stress in heart mitochondria of rats, powerful antiradical properties ofecdysterone, its cardioprotective effect, in conditions of brain focal ischemia-reperfusion.

Topics: Animals; Brain Ischemia; Cardiotonic Agents; Ecdysterone; Heart; Hydrogen Peroxide; Hydroxyl Radical; Invertebrate Hormones; Lipid Peroxidation; Lipoxygenase; Malondialdehyde; Mitochondria, Heart; Oxidative Stress; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Reperfusion Injury; Superoxides; Survival Analysis; Xanthine Oxidase

To investigate whether allopurinol exerts a protective effect on kidneys by measuring new kidney injury biomarkers (NGALp, NGALu, KIM 1 and IL 18) and analysing the renal function and histology in uninephrectomised rats subjected to ischaemia-reperfusion injury.. Thirty two Wistar rats were randomly allocated to four groups: Sham (S): laparotomy; Control (C): laparotomy and ischaemia-reperfusion in the left kidney; Control Allopurinol (CA): laparotomy and allopurinol at a dose of 100mg·kg 1·d 1; and Allopurinol (A): laparotomy ischaemia-reperfusion in the left kidney and allopurinol at a dose of 100mg·kg 1·d 1. The NGALp, NGALu, KIM 1, IL 18 and creatinine levels and the kidney histology were analysed. The significance level was established as p<0.05.. Creatinine level increased in all the groups, with A ≈ C > S ≈ CA. The NGALp, NGALu and IL 18 levels exhibited similar behaviour in all the groups. KIM 1 was higher in group A than C and showed intermediate values in groups S and CA. Severity of injury in the left kidney was greater in groups C and A compared to S and CA.. Allopurinol did not exert protective or damaging effects on the kidneys of rats subjected to ischaemia-reperfusion injury.

Topics: Acute-Phase Proteins; Allopurinol; Animals; Antimetabolites; Biomarkers; Creatinine; Interleukin-18; Ischemia; Kidney; Lipocalin-2; Lipocalins; Male; Proto-Oncogene Proteins; Random Allocation; Rats, Wistar; Reperfusion Injury

Biolasol solution (Pharmaceutical Research and Production Plant "Biochefa," Sosnowiec, Poland) is a novel extracellular perfusion and ex vivo hypothermic kidney preservation solution. It ensures maintenance of homeostasis, reduces tissue edema, has low viscosity, and allows the graft to preserve structural and functional integrity. It minimizes ischemia-reperfusion damage.. Perfundates from control and transplanted kidneys flushed with Biolasol or ViaSpan solutions (Arkas, Warszawa, Poland) were analyzed. Parameters of serum and urine collected from 12 pigs after auto-transplantation were also analyzed. Renal medulla was investigated for structural alterations by analyzing hematoxylin and eosin-stained slides. The mean survival time of pigs after the auto-transplantation procedure was the measure for the novel Biolasol solution effectiveness.. We observed a statistically significant decrease in marker enzyme levels alanine transaminase, aspartate transaminase, lactic dehydrogenase, and ions (Na and K) in pigs with grafts flushed with Biolasol. Histopathologic examination revealed that the renal cortex structure was not damaged after the use of Biolasol solution.. Biolasol solution protects kidneys against ischemia damage and does not differ significantly from the "golden standard" ViaSpan solution.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Creatinine; Glutathione; Insulin; Kidney; Kidney Transplantation; L-Lactate Dehydrogenase; Organ Preservation Solutions; Poland; Raffinose; Reperfusion Injury; Swine; Transplantation, Autologous

To test whether a new rinse solution containing polyethylene glycol 35 (PEG-35) could prevent ischemia-reperfusion injury (IRI) in liver grafts.. Sprague-Dawley rat livers were stored in University of Wisconsin preservation solution and then washed with different rinse solutions (Ringer's lactate solution and a new rinse solution enriched with PEG-35 at either 1 or 5 g/L) before ex vivo perfusion with Krebs-Heinseleit buffer solution. We assessed the following: liver injury (transaminase levels), mitochondrial damage (glutamate dehydrogenase activity), liver function (bile output and vascular resistance), oxidative stress (malondialdehyde), nitric oxide, liver autophagy (Beclin-1 and LCB3) and cytoskeleton integrity (filament and globular actin fraction); as well as levels of metalloproteinases (MMP2 and MMP9), adenosine monophosphate-activated protein kinase (AMPK), heat shock protein 70 (HSP70) and heme oxygenase 1 (HO-1).. When we used the PEG-35 rinse solution, reduced hepatic injury and improved liver function were noted after reperfusion. The PEG-35 rinse solution prevented oxidative stress, mitochondrial damage, and liver autophagy. Further, it increased the expression of cytoprotective heat shock proteins such as HO-1 and HSP70, activated AMPK, and contributed to the restoration of cytoskeleton integrity after IRI.. Using the rinse solution containing PEG-35 was effective for decreasing liver graft vulnerability to IRI.

Topics: Adenosine; Allopurinol; Animals; Autophagy; Biomarkers; Cold Ischemia; Cytoprotection; Cytoskeleton; Disease Models, Animal; Glutathione; Hepatectomy; Insulin; Liver; Liver Function Tests; Male; Mitochondria, Liver; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Polyethylene Glycols; Raffinose; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Time Factors

S-Nitrosated human serum albumin (SNO-HSA) is useful in preventing liver ischemia/reperfusion injury, and SNO-HSA should thus be able to prevent cell injury during liver transplantation. However, the potential protective effect of SNO-HSA on a combination of cold and warm ischemia, which is obligatory when performing liver transplantation, has not been examined. Therefore, we evaluated the protective effect of SNO-HSA added to University of Wisconsin (UW) solution during cold or/and warm ischemia in situ and in vitro. First, we observed that apoptotic and necrotic cell death were increased during cold and warm ischemia, respectively. SNO-HSA, which possesses anti-apoptosis activity at low NO concentrations, can inhibit cold ischemia injury both in situ and in vitro. In contrast, SNO-HSA had no significant effect on warm liver ischemia injury which, however, can be reduced by UW solution. We also demonstrated that the cellular uptake of NO from SNO-HSA can occur during cold ischemia resulting in induction of heme oxygenase-1 within 3h of cold ischemia. Our results indicate that treatment with SNO-HSA or UW solution alone is not sufficient to inhibit liver injury during a period of both cold and warm ischemia. However, a combination of SNO-HSA and UW solution can be used to prevent the two types of ischemia. SNO-HSA-added UW solution could be very useful in transplantation, because the previously imposed constraints on preservation time can be removed. This is a great advantage in a situation as the present one with increased utilization of scarce donor organs for more recipients.

Topics: Adenosine; Allopurinol; Analysis of Variance; Animals; Apoptosis; Glutathione; Hep G2 Cells; Humans; Insulin; Liver; Liver Diseases; Liver Transplantation; Male; Necrosis; Nitric Oxide Donors; Nitroso Compounds; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Serum Albumin; Serum Albumin, Human

The anti-infammatory and cardioprotective effect of acetylcholine (ACh) has been reported; nevertheless, whether and how ACh exhibits an antioxidant property against ischemia/reperfusion (I/R)-induced oxidative stress remains obscure.. In the present study, H9c2 rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) to mimic I/R injury. We estimated intracellular different sources of reactive oxygen species (ROS) by measuring mitochondrial ROS (mtROS), mitochondrial DNA (mtDNA) copy number, xanthine oxidase (XO) and NADPH oxidase (NOX) activity and expression of rac 1. Cell injury was determined by lactate dehydrogenase (LDH) release and cleaved caspase-3 expression. The siRNA transfection was performed to knockdown of M2 acetylcholine receptor (M2 AChR) expression.. 12-h hypoxia followed by 2-h reoxygenation resulted in an abrupt burst of ROS in H9c2 cells. Administration of ACh reduced the levels of ROS in a concentration-dependent manner. Compared to the H/R group, ACh decreased mtROS, recovered mtDNA copy number, diminished XO and NOX activity, rac 1 expression as well as cell injury. Co- treatment with atropine rather than hexamethonium abolished the antioxidant and cardioprotective effect of ACh. Moreover, knockdown of M2 AChR by siRNA showed the similar trends as atropine co-treatment group.. ACh inhibits mitochondria-, XO- and NOX-derived ROS production thus protecting H9c2 cells against H/R-induced oxidative stress, and these benefcial effects are mainly mediated by M2 AChR. Our findings suggested that increasing ACh release could be a potential therapeutic strategy for treatment and prevention of I/R injury.

Topics: Acetylcholine; Animals; Antioxidants; Caspase 3; Cell Hypoxia; Cell Line; Cytosol; DNA, Mitochondrial; Mitochondria; Myocytes, Cardiac; NADPH Oxidases; Oxidative Stress; Rats; Reactive Oxygen Species; Receptor, Muscarinic M2; Reperfusion Injury; RNA Interference; RNA, Small Interfering; Xanthine Oxidase

To evaluate the therapeutic utility of hyperbaric oxygen (HBO) therapy on testicular ischemia/reperfusion (I/R) injury and elucidate the underlying molecular mechanism, we tested whether HBO therapy provided rescue of the testes after torsion in rats.. Sprague-Dawley rats were randomly divided into 4 groups: control group, control plus HBO therapy, I/R group, and I/R plus HBO therapy. The I/R model was induced by torsion of the right testis.. I/R in the testis resulted in disrupted seminiferous tubules, germ cell-specific apoptosis, followed by a marked reduction in testis weight and daily sperm production. HBO therapy preserved seminiferous tubules, suppressed apoptosis, and prevented testicular atrophy in I/R testes. HBO therapy abated oxidative stress in I/R testes, marked by reduced malondialdehyde formation, enhanced activities of superoxide dismutase and heme oxygenase 1 (HO-1), and decreased activities of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and xanthine oxidase. HBO therapy resulted in a reduction of myeloperoxidase (MPO) activity in I/R testes, a marker of neutrophil recruitment. HBO therapy suppressed inflammation in I/R testes, marked by reduced messenger RNA (mRNA) levels of tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1β), and CD44. Furthermore, HBO therapy suppressed the activation of nuclear factor kappa B (NFκB), p38, and c-JUN-N-terminal kinase (JNK) signaling pathways in I/R testes. In addition, HBO therapy reduced nitric oxide formation in I/R testes through suppression of inducible nitric oxide synthase and dimethylarginine dimethylaminohydrolase.. HBO therapy in rats attenuated I/R-induced testicular injury, possibly through abating oxidative stress, suppressing inflammation, and reducing nitric oxide formation.

Topics: Animals; Apoptosis; Heme Oxygenase-1; Hyaluronan Receptors; Hyperbaric Oxygenation; Inflammation; Interleukin-1beta; Male; Malondialdehyde; MAP Kinase Signaling System; NADPH Oxidases; NF-kappa B; Nitric Oxide; Oxidative Stress; Peroxidase; Rats; Reperfusion Injury; RNA, Messenger; Seminiferous Tubules; Spermatozoa; Superoxide Dismutase; Testicular Diseases; Testis; Torsion Abnormality; Tumor Necrosis Factor-alpha; Xanthine Oxidase

Methotrexate was developed as a cytostatic agent, but at low doses, it has shown potent anti-inflammatory activity. Previous studies have demonstrated that the anti-inflammatory effects of methotrexate are primarily mediated by the release of adenosine. In this study, we hypothesized that low-dose methotrexate has protective effects in spinal cord ischemia-reperfusion injury. Rabbits were randomized into the following four groups of eight animals each: group 1 (control), group 2 (ischemia), group 3 (methylprednisolone) and group 4 (methotrexate). In the control group only a laparotomy was performed. In all the other groups, the spinal cord ischemia model was created by the occlusion of the aorta just caudal to the renal artery. Neurological evaluation was performed with the Tarlov scoring system. Levels of myeloperoxidase, malondialdehyde and catalase were analyzed, as were the activities of xanthine oxidase and caspase-3. Histopathological and ultrastructural evaluations were also performed. After ischemia-reperfusion injury, increases were found in the serum and tissue myeloperoxidase levels, tissue malondialdehyde levels, xanthine oxidase activity and caspase-3 activity. In contrast, both serum and tissue catalase levels were decreased. After the administration of a low-dose of methotrexate, decreases were observed in the serum and tissue myeloperoxidase levels, tissue malondialdehyde levels, xanthine oxidase activity and caspase-3 activity. In contrast, both the serum and tissue catalase levels were increased. Furthermore, low-dose methotrexate treatment showed improved results concerning the histopathological scores, the ultrastructural score and the Tarlov scores. Our results revealed that low-dose methotrexate exhibits meaningful neuroprotective activity following ischemia-reperfusion injury of the spinal cord.

Topics: Animals; Caspase 3; Catalase; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Malondialdehyde; Methotrexate; Methylprednisolone; Peroxidase; Rabbits; Reperfusion Injury; Spinal Cord; Xanthine Oxidase

The small intestine is one of the most sensitive organs to ischemia-reperfusion injury during transplantation. Cytoprotective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) is well known. The aim of our study was to measure changes of PACAP-38-like immunoreactivities and cytokine levels in intestinal grafts stored PACAP-38 containing preservation solution.. Small-bowel autotransplantation was performed on male Wistar rats (n = 56). Grafts were stored in University of Wisconsin (UW) solution at 4 °C for 1 (GI), 3 (GII), and 6 hours (GIII); and in PACAP-38 containing UW solution for 1 (GIV), 3 (GV), and 6 hours (GVI). Reperfusion lasted 3 hours in each group. Intestinal PACAP-38 immunoreactivities were measured by radioimmunoassay. To measure cytokine from tissue homogenates we used rat cytokine array and Luminex Multiplex Immunoassay.. Levels of PACAP-38-like and PACAP-27-like immunoreactivities decreased by preservation time compared to control. This decrease was significant following 6 hours cold storage (p < 0.05). Values remained significantly higher in grafts stored in PACAP-38 containing UW. Expressions of sICAM-1, L-selectin, tissue inhibitor of metalloproteinase-1 were increased in GIII and were decreased in GVI.. PACAP-38 increased tissue levels of PACAP-38 and PACAP-27, and decreased cytokine expression. This indicates that PACAP-38 has anti-inflammatory and cytoprotective effects in intestinal autotransplantation model.. Bevezetés: A vékonybél ischaemia-reperfusióval szembeni fokozott érzékenysége a szerv transzplantációjakor is jelen lévő probléma. Ismert a hypophysis adenilát-cikláz aktiváló polipeptid (PACAP) sejtvédő hatása. Munkánkban azt vizsgáltuk, hogy PACAP-38-at tartalmazó University of Wisconsin (UW) oldatban történő konzerválás hogyan befolyásolja a szöveti PACAP- és citokinszinteket. Anyag és módszer: Wistar-patkányokon (n = 56) vékonybél-autotranszplantációt végeztünk. A graftokat 4 °C-os UW oldatban tároltuk 1 (I. csoport), 3 (II.) és 6 órán (III.), illetve 100 μg PACAP-38-at tartalmazó UW oldatban 1 (IV.), 3 (V.) és 6 órán (VI.) át. A reperfusio 3 óra volt. Bélmintákból a PACAP-38- és PACAP-27-szinteket radioimmunassayjel határoztuk meg, míg a citokinexpressiót kemilumineszcens módszerrel és Luminex Multiplex Immunoassayjel mértük. Eredmények: A szöveti PACAP-38-szint a kontrollhoz (57,32 ± 3,5 fmol/mg) képest a konzerválás idejével csökkent, és 6 óra után szignifikáns volt (III.: 32,6 ± 3,9 fmol/mg, p < 0,05), míg a IV–VI. csoportoknál szignifikánsan nőtt. A PACAP-27 szöveti értéke is hasonló tendenciával változott. Az sICAM-1, L-selectin és a metalloproteáz-1 szöveti inhibitorának emelkedett expressióját mértük a III. csoportban, és jelentős csökkenés volt a VI. csoportban. Következtetés: UW oldathoz adott PACAP-38 növelte a szöveti PACAP-38- és PACAP-27-szinteket, és csökkentette a citokinexpressiót. Mindez a PACAP-38 citoprotektív és anti-inflammatórikus hatását jelzi bél-autotranszplantációs modellben. Támogatta: OTKA (PD77474, 104984, CNK78480), MTA Bolyai-ösztöndíj és Lendület program.

Topics: Adenosine; Allopurinol; Animals; Anti-Inflammatory Agents; Antioxidants; Cytokines; Cytoprotection; Down-Regulation; Glutathione; Graft Survival; Growth Substances; Insulin; Intercellular Adhesion Molecule-1; Intestine, Small; L-Selectin; Male; Organ Preservation; Organ Preservation Solutions; Pituitary Adenylate Cyclase-Activating Polypeptide; Radioimmunoassay; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Time Factors; Tissue Inhibitor of Metalloproteinase-1; Transplantation, Autologous

It is investigated whether preoperative allopurinol administration protects lung injury induced by cardiopulmonary bypass (CPB).. Sixty patients with coronary artery disease who need elective coronary artery bypass grafting operations by using CPB were taken into this study. They were divided into two groups; control and allopurinol. Allopurinol (300 mg/day) was administered to the latter group during the preoperative period of 5 days. Standard CPB procedures were used in all cases. Blood was sampled for TNF-alpha, IL-6, IL-8, IL-10 before anesthesia (T0), after anesthesia and before skin incision (T1), before CPB (T2), after aortic declamping (T3), at the end of CPB (T4), 6 hours after operation (T5), 12 hours after operation (T6), and 24 hours after operation (T7). Pulmonary function test (PFT) was performed before and following the 6th day of operation.. TNF-alpha, IL-6, IL-8 increased in both groups at T3, T4, T5 and T6 compared to control (p<0.05). TNF-alpha, IL-6, and IL-8 levels were lower in group A at T3, T4, T5 and T6 (p<0.05). Creatinin phosphokinase (CK) levels were lower in group A at T6 (p<0.05). CK-MB levels were lower in group A than in group C (p<0.05). Pulmonary function test (PFT) did not yield any differences between the groups.. Preoperative allopurinol administration decreases the inflammation and myocardial injury according to biochemical markers of ischemia reperfusion injury. However, this biochemical success does not rebound to PFT (Tab. 5, Ref. 15).

Topics: Allopurinol; Cardiopulmonary Bypass; Humans; Intraoperative Complications; Lung; Middle Aged; Preoperative Care; Reperfusion Injury

To assess application of cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) as biomarkers for renal ischemic injury. We also evaluated the use of allopurinol as a renoprotective agent. A second goal was to assess cystatin C as a biomarker in patients undergoing partial nephrectomy.. Using 58 Sprague-Dawley rats, we evaluated urinary cystatin C (n=26) and NGAL (n=32) as a biomarker for renal ischemia injury. Half of the rats were pretreated with allopurinol; the other cohort served as a control. The right renal hilum was ligated in all rats, thereby creating a solitary kidney model. After a 30-minute stabilization period, the left hilum was clamped for time periods of 15, 30, and 60 minutes. Urinary levels of cystatin C and NGAL were then measured at the following time points: Preclamp (after the 30-minute stabilization period) and postclamp (30, 45, and 60 minute periods after unclamping). For our clinical subjects, serum cystatin C levels (n=17) were obtained preoperatively, at the induction of anesthesia before robot-assisted partial nephrectomy, immediately postoperatively, and on postoperative days 1 and 2. Three of these patients had their tumors excised off clamp and served as controls. We then estimated glomerular filtration rate by using the Creatinine-Cystatin C Equation.. Urinary levels of cystatin C and NGAL increased after renal clamping. The 30-minute period of ischemia demonstrated the greatest increase of these biomarkers. Allopurinol did appear to serve a renoprotective function in those animals undergoing 30-minute clamp times. In our clinical patients, the serum cystatin C levels did increase at each postoperative time point, but remained nonelevated in the control group.. Cystatin C and NGAL both appear to be useful biomarkers of renal injury. Studies with larger numbers are needed, however. Also, allopurinol does exhibit renoprotective effects against ischemic injury.

Topics: Acute-Phase Proteins; Allopurinol; Animals; Biomarkers; Case-Control Studies; Creatinine; Cystatin C; Disease Models, Animal; Glomerular Filtration Rate; Humans; Kidney; Kidney Diseases; Lipocalin-2; Lipocalins; Male; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors

We aimed to investigate whether caffeic acid phenethyl ester (CAPE) prevents detrimental systemic effects of intestinal ischemia-reperfusion (IR) injury on colonic anastomotic wound healing.. This experimental study was conducted on 48 male Wistar albino rats. The rats were randomly allocated into four groups and a left colonic anastomosis was performed in all rats: (i) sham-operated group (n = 12), laparatomy without intestinal IR injury; (ii) sham + CAPE group (n = 12), identical to Group 1 except for CAPE treatment (10 μmol/kg, intravenously); (iii) intestinal IR group (n = 12), 60 min of superior mesenteric ischemia followed by reperfusion; and (iv) IR + CAPE-treated group (n = 12) (10 μmol/kg, intravenously, 30 min before the construction of colonic anastomosis). On the postoperative day 7, the rats were subjected to relaparotomy for in vivo measurement of the colonic anastomotic bursting pressure. A colonic segment including the anastomotic site was resected for histopathological evaluation and biochemical analyses. The plasma proinflammatory cytokine levels were measured. Body weight changes were examined.. CAPE treatment significantly increased colonic anastomotic bursting pressures, and colonic anastomotic tissue hydroxyproline contents and antioxidant parameters (p < .05), and significantly decreased oxidative stress markers in colonic anastomotic tissues and plasma proinflammatory cytokine levels (p < .05). Histopathological scores were significantly better due to CAPE administration (p < .05).. This study clearly showed that CAPE treatment prevented the delaying effects of remote IR injury on colonic anastomotic wound healing. Further clinical studies are required to determine whether CAPE has a useful role in the enhancement of gastrointestinal anastomotic wound healing during particular surgeries in which IR-induced organ injury occurs.

Topics: Anastomosis, Surgical; Animals; Anti-Inflammatory Agents, Non-Steroidal; Caffeic Acids; Colon; Cytokines; Drug Evaluation, Preclinical; Hydroxyproline; Laparotomy; Male; Malondialdehyde; Mesenteric Artery, Superior; Oxidative Stress; Phenylethyl Alcohol; Random Allocation; Rats; Rats, Wistar; Reperfusion Injury; Surgical Wound Dehiscence; Wound Healing; Xanthine Oxidase

Xanthine oxidase has been implicated in the pathogenesis of a wide spectrum of diseases, and is thought to be the most important source of oxygen-free radicals and cell damage during re-oxygenation of hypoxic tissues.. The present study was undertaken to demonstrate whether febuxostat is superior to allopurinol in prevention of the local and remote harmful effects of small intestinal ischemia/reperfusion injury in rats.. Intestinal ischemia was induced by superior mesenteric artery ligation. The rats were assigned to five groups: the sham control; the intestinal ischemia/reperfusion; the allopurinol; and the febuxostat 5 and 10 mg/kg pretreated ischemia/reperfusion groups. Treatment was administered from 7 days before ischemia induction. After the reperfusion, the serum and tissues were obtained for biochemical, pharmacological, and histological studies.. Intestinal reperfusion led to an elevation in the serum levels of alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor-α, malondialdehyde, and xanthine oxidase as well as intestinal myeloperoxidase, malonadialdehyde, and xanthine oxidase/xanthine dehydrogenase activity. Furthermore, the ischemia/reperfusion induced a reduction in the contractile responsiveness to acetylcholine. These changes were significantly regulated by the pretreatment with febuxostat compared to allopurinol. The degree of pathological impairment in the intestinal mucosa, liver, and lung tissues were lighter in the pretreated groups.. Febuxostat may offer advantages over allopurinol in lessening local intestinal injury as well as remote hepatic and lung injuries induced by small intestinal ischemia/reperfusion.

Topics: Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Dose-Response Relationship, Drug; Febuxostat; Gout Suppressants; Intestinal Mucosa; Lipid Peroxidation; Lung; Male; Malondialdehyde; Muscle Contraction; Muscle, Smooth; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Thiazoles; Tumor Necrosis Factor-alpha; Xanthine Dehydrogenase; Xanthine Oxidase

To investigate the relationship between renal ischemia injury and concentrations of 8-isoprostane in a rat kidney model during renal hilar clamping and their correlation with the administration of allopurinol before clamping.. Reperfusion injury occurs after the reintroduction of blood flow after a prolonged period of ischemia. Thought to be due to oxygen free radicals released by the endothelial, mitochondrial, and parenchymal cells, this process leads to a cascade of events whereby infiltrative leukocytes generate cytokines and reactive oxygen species. The present study was performed in 2 parts. Our primary objective was to first develop a method of quantitating the renal damage using a prostaglandin compound formed in vivo, specifically isoprostane. After the development of this animal model of quantitating renal injury, our second objective was to apply this model and investigate allopurinol's nephroprotective abilities. A microdialysis probe was inserted into the renal parenchyma of rats to allow continuous dialysis and collection of the effluent for isoprostane levels. After clamping of the renal vessels to induce ischemia, the interstitial effluent from the probe was collected and subsequently analyzed for 8-isoprostane levels with and without allopurinol pretreatment.. Clamping of the renal hilum in this rat model significantly increased 8-isoprostane levels. After 60 minutes of clamp time, the largest absolute increase in 8-isoprostane levels resulted, representing a 3.2-fold increase from baseline. However, the rats that had been pretreated with allopurinol demonstrated significantly less isoprostane levels, to baseline levels.. Allopurinol has demonstrated significant benefits by reducing reperfusion injury in rat kidneys, as demonstrated by the use of 8-isoprostane as a tool for the real-time measurement of ischemic injury.

Topics: Allopurinol; Animals; Dinoprost; Disease Models, Animal; Free Radical Scavengers; Kidney; Male; Microdialysis; Rats; Rats, Sprague-Dawley; Renal Artery; Reperfusion Injury; Time Factors

Organ cold storage and subsequent transplantation are associated with significant ischemia-reperfusion injury, leading to cell death, graft inflammation and decreased graft function.. CORM-3s reduce oxidative stress and prevent inflammation in kidneys stored at 4C and subsequently transplanted. Graft survival and function are markedly improved compared to kidneys preserved and stored in University of Wisconsin solution alone. We determined whether CORM-3 has direct antiapoptotic effects on in vitro preparations of human HUVECs exposed to anoxic conditions. We also determined whether direct administration of CORM-3 to renal grafts before and/or after cold storage would prevent renal damage during the transplantation process.. CORM-3 supplementation led to a significantly increased frequency of live cells (mean ± SD 72.3% ± 1.9%, p <0.01), reduced apoptosis (14.9% ± 6.1%, p <0.01) and decreased mitochondrial transmembrane potential (40.2% ± 7.2%, p <0.05) in HUVECs exposed to 20 hours of cold storage compared to controls (11.6% ± 3.5%, 82.2% ± 2.3% and 78.2% ± 3.2%, respectively). In keeping with this antiapoptotic effect CORM-3 supplementation led to a mean 7.4 ± 2.1-fold up-regulation in Bcl-2 gene expression. CORM-3 supplementation in standard preservation solution was most beneficial at initial ischemic injury and before cold storage exposure. However, additional reflushing before vascular reperfusion showed an additive benefit to graft survival and function after transplantation. This was confirmed by decreased glomerular and tubular necrosis, and apoptosis in double flushed grafts.. CORM-3 supplementation in standard University of Wisconsin solution has a significant impact on decreasing cellular and graft injury, and improving survival through its antiapoptotic effects, which are likely mediated through mitochondrial membrane stabilization.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Glutathione; Graft Survival; Inflammation; Insulin; Kidney Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Organometallic Compounds; Oxidative Stress; Raffinose; Rats, Inbred Lew; Reperfusion Injury

The main objective of the study was to determine the neuroprotective effect of allopurinol and nimesulide against the cerebral ischemic reperfusion injury in diabetic and nondiabetic rats.. In this study, Wistar albino rats of either sex weighing 150-250 g were procured from authorized suppliers. Rats were anesthetized by giving thiopentone sodium (45 mg/kg) by i.p. Under anesthesia, midline incision was given. Common carotid arteries were identified and isolated carefully from vago-sympathetic nerve. Rats were made ischemic by occluding bicommon carotid arteries with thread for 30 min, followed by reperfusion for 4 h by removing the occlusion. The drugs allopurinol (15, 30 mg/kg) and nimesulide (20, 40 mg/kg) were administered 10 min before reperfusion. Then after 4 h reperfusion, animals were sacrificed and immediately brain was removed, homogenized, centrifuged and supernatant was collected, various enzyme estimations were done and same procedure was followed in streptozotocin (STZ: 45 mg/kg; i.p.) induced diabetic rats.. Ischemia reperfusion (I/R) group showed significant increase in malondialdehyde (MDA), myeloperoxidase (MPO) and depletion in catalase (CAT) and superoxide dismutase (SOD) levels. Treatment with allopurinol and nimesulide significantly decreased the MDA and MPO levels whereas increased the SOD and CAT levels when compared I/R group in both non-diabetic and diabetic rats.. These findings suggest the cerebral injury due to over production of free radicals was inhibited by allopurinol and nimesulide that exert a neuroprotective effect probably by radical scavenging and antioxidant activities.

Topics: Allopurinol; Animals; Brain Ischemia; Catalase; Diabetes Mellitus, Experimental; Female; Male; Neuroprotective Agents; Oxidative Stress; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Streptozocin; Sulfonamides; Superoxide Dismutase

Cyclosporin-A (CsA) has been reported to protect livers from warm ischemia/reperfusion (I/R) injury. To study if CsA has also a protective effect on cold I/R injury, two models were used: the isolated perfused rat liver (IPRL) and the orthotopic rat liver transplantation (ORLT). (1) IPRL: Livers were preserved for 24 h (5°C) in University of Wisconsin (UW) solution alone (group 1), with CsA (400 nM) dissolved in dimethylsulfoxide (50 μM) (group 2), and with dimethylsulfoxide (DMSO) alone (group 3). Livers were reperfused for 60 min (37°C) (n = 8/group). Cell necrosis was evaluated by trypan blue uptake and apoptosis by laddering and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and by caspase-3 activation. Marked and similar sinusoidal endothelial cell necrosis was found in the three groups while apoptosis was found similarly deceased in groups 2 and 3 compared with group 1. (2) ORLT: Donors received either CsA (5 mg/kg) or corn oil 24 h before transplantation. Recipients were sacrificed after 240 min; cell necrosis and apoptosis were then evaluated. No difference was found between treated and control groups. The current data strongly suggest that CsA has no protective effect on hepatic cold I/R injury. Hepatocyte apoptosis can be reduced by antioxidants, as occurred with DMSO, but introduction of CsA does not provide additional protective effect.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Caspase 3; Cold Ischemia; Cyclosporine; Dimethyl Sulfoxide; Endothelium; Glutathione; Hepatocytes; Insulin; Liver; Liver Transplantation; Male; Models, Animal; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Regional Blood Flow; Reperfusion Injury

Ischemia/reperfusion (I/R) injury remains as a serious deleterious factor in kidney transplantation (KTx). We hypothesized that carbon monoxide (CO), an endogenous potent cytoprotective molecule, inhibits hypothermia-induced apoptosis of kidney grafts. Using the rat KTx model mimicking the conditions of donation after cardiac death (DCD) as well as nontransplantable human kidney grafts, this study examined effects of CO in preservation solution in improving the quality of marginal kidney grafts. After cardiac cessation, rat kidneys underwent 40 min warm ischemia (WI) and 24 h cold storage (CS) in control UW or UW containing CO (CO-UW). At the end of CS, kidney grafts in control UW markedly increased mitochondrial porin release into the cytosol and resulted in increased cleaved caspase-3 and PARP expression. In contrast, grafts in CO-UW had significantly reduced mitochondrial breakdown and caspase pathway activation. After KTx, recipient survival significantly improved with CO-UW with less TUNEL(+) cells and reduced mRNA upregulation for proinflammatory mediators (IL-6, TNF-α, iNOS). Furthermore, when nontransplantable human kidney grafts were stored in CO-UW for 24 h, graft PARP expression, TUNEL(+) cells, and proinflammatory mediators were less than those in control UW. CO in UW inhibited hypothermia-induced apoptosis and significantly improved kidney graft function and outcomes of KTx.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Carbon Monoxide; Cold Temperature; Cytosol; Death; Glutathione; Humans; Inflammation; Insulin; Kidney; Kidney Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; RNA, Messenger; Treatment Outcome

Pathophysiological alterations in the endothelial phenotype result in endothelial dysfunction. Flow cessation, occurring during organ procurement for transplantation, triggers the endothelial dysfunction characteristic of ischemia/reperfusion injury, partly due to a reduction in the expression of the vasoprotective transcription factor Kruppel-like Factor 2 (KLF2). We aimed at (1) characterizing the effects of flow cessation and cold storage on hepatic endothelial phenotype, and (2) ascertaining if the consequences of cold stasis on the hepatic endothelium can be pharmacologically modulated, improving liver graft function. Expression of KLF2 and its vasoprotective programs was determined in (i) hepatic endothelial cells (HEC) incubated under cold storage conditions with or without the KLF2-inducer simvastatin, and (ii) rat livers not cold stored or preserved in cold University of Wisconsin solution (UWS) supplemented with simvastatin or its vehicle. In addition, upon warm reperfusion hepatic vascular resistance, endothelial function, nitric oxide vasodilator pathway, apoptosis, inflammation, and liver injury were evaluated in not cold stored livers or livers preserved in cold UWS supplemented with simvastatin or vehicle. Expression of KLF2 and its vasoprotective programs decrease in HEC incubated under cold storage conditions. Cold-stored rat livers exhibit a time-dependent decrease in KLF2 and its target genes, liver injury, increased hepatic vascular resistance, and endothelial dysfunction. The addition of simvastatin to the storage solution, maintained KLF2-dependent vasoprotective programs, prevented liver damage, inflammation, and oxidative stress and improved endothelial dysfunction.. Our results provide a rationale to evaluate the beneficial effects of a vasoprotective preservation solution on human liver procurement for transplantation.

Topics: Adenosine; Allopurinol; Animals; Cells, Cultured; Cryoprotective Agents; Endothelium, Vascular; Glutathione; Insulin; Kruppel-Like Transcription Factors; Liver; Liver Transplantation; Male; Models, Animal; Organ Preservation; Organ Preservation Solutions; Phenotype; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Simvastatin

Laparoscopic and robotic partial nephrectomy involves temporary clamping of the renal artery, making the kidney susceptible to ischemic damage. Isoprostane represents one potential marker of oxidative injury. The objective was to determine if renal interstitial isoprostane levels can quantitate renal damage secondary to warm ischemia. A second goal is to investigate allopurinol for renoprotective abilities using this model. We chose to investigate potential renoprotection of allopurinol because previous studies have demonstrated transplant kidneys pretreated with allopurinol to have less damage from ischemia.. A microdialysis probe was inserted into the renal parenchyma of rats to allow continuous dialysis and collection of the effluent for isoprostane levels. After clamping of the renal vessels for predefined intervals of ischemia, the interstitial effluent from the probe was collected and subsequently analyzed for isoprostane levels with and without allopurinol pretreatment.. Clamping of the renal artery and vein produced increases in isoprostane levels during the ischemic period and larger increases during reperfusion. There was a trend for increased postclamp isoprostane levels as clamp times increased. When comparing isoprostane levels in rats that did not receive allopurinol, there were significant differences between the clamp and postclamp levels of isoprostane, with allopurinol offering protection to the kidney from ischemic changes caused by clamping the renal hilum.. Our data have demonstrated that isoprostane levels are a potential real-time marker of renal ischemia and reperfusion injury. We also found allopurinol administration demonstrated a trend toward renoprotective abilities in the hilar occluded kidney.

Topics: Allopurinol; Animals; Constriction; Disease Models, Animal; Ischemia; Isoprostanes; Kidney; Male; Microdialysis; Rats; Rats, Sprague-Dawley; Renal Artery; Reperfusion Injury

We evaluated whether IGL-1, a graft preservation solution containing polyethylene glycol, improves the outcome of small bowel grafts in comparison to the University of Wisconsin (UW) solution in a pig allotransplantation model.. Seventeen pigs were randomly allocated to group 1 (n = 10; intestinal allotransplantation with IGL-1) and group 2 (n = 7; allotransplantation with UW). Pigs received no immunosuppression and were sacrificed on postoperative d (POD) 8. Intestinal specimens were obtained from the animal immediately before cold flushing (T0), 2 h after graft reperfusion (T1), and at sacrifice (T2).. Survival rate to POD 8 was 50% in group 1 compared with 16% in group 2 (P < 0.05); 62% of pigs in group 1 did not present any acute cellular rejection (ACR) compared to 16% in group 2 (P < 0.05). Severe ACR rate was 25% in group 1 and 66% in group 2 (P < 0.05). iNOS activity and intestinal caspase 3 levels increased significantly between T0 and T1 in group 1 compared to group 2 (P < 0.05). Cell necrosis increased significantly between TO and T1 in group 2 compared with group 1 (P < 0.05) whereas cell apoptosis was significantly higher at T1 compared with T0 in group 1 in comparison to group 2.. Our results show that IGL-1 improves intestinal graft viability as compared to UW solution, possibly by reducing graft immunogenicity and by favoring intestinal epithelial repair.

Topics: Acute Disease; Adenosine; Allopurinol; Animals; Apoptosis; Caspase 3; Female; Glutathione; Graft Rejection; Graft Survival; Immunosuppression Therapy; Insulin; Intestinal Mucosa; Intestine, Small; Organ Preservation Solutions; Polyethylene Glycols; Raffinose; Reperfusion Injury; Sus scrofa; Transplantation, Homologous

Pneumoperitoneum-associated ischemia-reperfusion (IR) may initiate renal dysfunction. Whether oxidants are responsible for renal structural damage, such as cell apoptosis, has not yet been evaluated. We investigated such eventuality in an isolated rat kidney model.. Thirty-five rat kidneys with their vessels and ureter were harvested and perfused within a closed environment at flow of 15 ml min(-1). After stabilization, kidneys were assigned to one of five groups (n = 7 per group): CO(2)-induced intrachamber pressure of 8, 12, or 0 mmHg (control), and 8 or 12 mmHg pressure applied to kidneys from rats treated pre-experimentally with tungsten for 14 days. Pressurization lasted 60 min.. Organ perfusion pressure raised as intrachamber pressure increased. Urinary output decreased in the two pressurized nonpretreated groups. Intrachamber pressure was directly associated with an increase in postexperimental xanthine oxidase tissue levels. Twofold apoptosis was documented (p < 0.05) in cortex of nonpretreated kidney in the 12 mmHg group compared with the 8 or 0 mmHg groups. Tungsten pretreatment significantly (p < 0.05) attenuated the abnormalities documented in the 12 mmHg group, but less so in the 8 mmHg pressurized nontreated counterparts.. Pneumoperitoneal pressure applied to isolated perfused kidney is associated with renal apoptosis. This rapidly induced structural renal damage is oxidant dependent and can be attenuated by antioxidants. Further studies may shed more light on the role of antioxidants in preventing pneumoperitoneum-induced kidney dysfunction.

Topics: Animals; Antioxidants; Apoptosis; Kidney; Male; Oxidants; Pneumoperitoneum, Artificial; Pressure; Rats; Rats, Wistar; Reperfusion Injury; Tungsten Compounds; Urine; Xanthine Dehydrogenase; Xanthine Oxidase

Matrix metalloproteinases and tissue inhibitor of metalloproteinases play an important role in the regulation of mesangial cell proliferation and may be involved in ischemia-reperfusion injuries. Preservation solutions are thought to diminish the ischemic injury and appropriate choice of the solution should guarantee a better graft function and good prognosis for graft survival. The aim of the study was to examine the effect of preservation solutions UW and EC on the expression of matrix metalloproteinase II and tissue inhibitor of metalloproteinase II genes in rat kidney. The study was carried out on Wistar rat kidneys divided into 3 groups: kidneys perfused with 0.9% NaCl (control group), with UW, and with EC preservation solution. The results show an enhancement of MMP-2 and TIMP-2 gene expression after 12 min of cold ischemia. This increase was more expressed in kidneys preserved with UW solution in comparison with kidneys perfused with EC solution and 0.9% NaCl. After 24 h of cold ischemia the expression of MMP-2 and TIMP-2 genes in kidney perfused with UW solution decreased, while in kidneys perfused with EC it was increased. After warm ischemia the MMP-2 and TIMP-2 gene expression increased, whereas it was significantly lower in kidneys perfused with EC solution.

Topics: Adenosine; Allopurinol; Animals; Gene Expression; Glutathione; Graft Survival; Insulin; Kidney; Kidney Transplantation; Male; Matrix Metalloproteinase 2; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Tissue Inhibitor of Metalloproteinase-2

Injury due to cold ischemia reperfusion (I/R) is a major cause of primary graft non-function following liver transplantation. We postulated that I/R-induced cellular damage during liver transplantation might affect the secretory pathway, particularly at the endoplasmic reticulum (ER). We examined the involvement of ER stress in organ preservation, and compared cold storage in University of Wisconsin (UW) solution and in Institute Georges Lopez-1 (IGL-1) solution. In one group of rats, livers were preserved in UW solution for 8 h at 4 °C, and then orthotopic liver transplantation was performed according to Kamada's cuff technique. In another group, livers were preserved in IGL-1 solution. The effect of each preservation solution on the induction of ER stress, hepatic injury, mitochondrial damage and cell death was evaluated. As expected, we found increased ER stress after liver transplantation. IGL-1 solution significantly attenuated ER damage by reducing the activation of three pathways of unfolded protein response and their effector molecules caspase-12, C/EBP homologous protein-10, X-box-binding protein 1, tumor necrosis factor-associated factor 2 and eukaryotic translation initiation factor 2. This attenuation of ER stress was associated with a reduction in hepatic injury and cell death. Our results show that IGL-1 solution may be a useful means to circumvent excessive ER stress reactions associated with liver transplantation, and may optimize graft quality.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Caspase 12; Cold Ischemia; Cold Temperature; DNA-Binding Proteins; Endoplasmic Reticulum Stress; Gene Expression; Glutathione; Insulin; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Regulatory Factor X Transcription Factors; Reperfusion Injury; Signal Transduction; TNF Receptor-Associated Factor 2; Transcription Factors; Unfolded Protein Response

Renal injury induced by brain death is characterized by ischemia and inflammation, and limiting it is a therapeutic goal that could improve outcomes in kidney transplantation. Brain death resulted in decreased circulating nitrite levels and increased infiltrating inflammatory cell infiltration into the kidney. Since nitrite stimulates nitric oxide signaling in ischemic tissues, we tested whether nitrite therapy was beneficial in a rat model of brain death followed by kidney transplantation. Nitrite, administered over 2 h of brain death, blunted the increased inflammation without affecting brain death-induced alterations in hemodynamics. Kidneys were transplanted after 2 h of brain death and renal function followed over 7 days. Allografts collected from nitrite-treated brain-dead rats showed significant improvement in function over the first 2 to 4 days after transplantation compared with untreated brain-dead animals. Gene microarray analysis after 2 h of brain death without or with nitrite therapy showed that the latter significantly altered the expression of about 400 genes. Ingenuity Pathway Analysis indicated that multiple signaling pathways were affected by nitrite, including those related to hypoxia, transcription, and genes related to humoral immune responses. Thus, nitrite therapy attenuates brain death-induced renal injury by regulating responses to ischemia and inflammation, ultimately leading to better post-transplant kidney function.

Topics: Allopurinol; Animals; Benzoates; Brain Death; Gene Expression; Hemodynamics; Imidazoles; Inflammation; Kidney; Kidney Transplantation; Lipid Peroxidation; Male; Nitrites; Rats; Rats, Inbred Lew; Reperfusion Injury; Signal Transduction; Sodium Nitrite

Hepatic ischemia-reperfusion (I/R) can lead to liver failure in association with remote organ damage, both of which have significant rates of morbidity and mortality. In this study, novel spin trapping and histopathological techniques have been used to investigate in vivo free radical formation in a rat model of warm liver I/R injury. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was administered to rats via intraperitoneal injection at a single dose of 1.5g of pure DMPO/kg body wt 2h before the initiation of liver ischemia. Blood vessels supplying the median and left lateral hepatic lobes were occluded with an arterial clamp for 60min, followed by 60min reperfusion. The effects of DMPO on I/R injury were evaluated by assessing the hepatic ultrastructure via transmission electron microscopy and by histopathological scoring. Immunoelectron microscopy was performed to determine the cellular localization of DMPO nitrone adducts. Levels of nitrone adducts were also measured to determine in situ scavenging of protein and DNA radicals. Total histopathological scoring of cellular damage was significantly decreased in hepatic I/R injury after DMPO treatment. DMPO treatment significantly decreased the hepatic conversion of xanthine oxidase and 4-hydroxynonenal formation in I/R injury compared to the untreated I/R group. The distribution of gold-nanoparticle-labeled DMPO nitrone adducts was observed in mitochondria, cytoplasm, and nucleus of hepatocytes. The formation of protein- and DNA-nitrone adducts was increased in DMPO-treated I/R livers compared to DMPO controls, indicating increased in situ protein and DNA radical formation and scavenging by DMPO. These results suggest that DMPO reduces I/R damage via protection against oxidative injury.

Topics: Alanine Transaminase; Animals; Cyclic N-Oxides; Free Radical Scavengers; Free Radicals; Immunohistochemistry; Ischemia; Lipid Peroxidation; Liver; Male; Malondialdehyde; Microscopy, Electron, Transmission; Oxidation-Reduction; Rats; Rats, Wistar; Reperfusion Injury; Spin Trapping; Tissue Distribution; Xanthine Dehydrogenase; Xanthine Oxidase

Renal ischemia-reperfusion (I/R) injury is unavoidable in kidney transplantation and frequently influences both short- and long-term allograft survival rates. One of the major events in I/R injury is the generation of cytotoxic oxygen radicals. Recently, hydrogen gas has been reported to display antioxidant properties and protective effects against organ dysfunction induced by various I/R injuries. We investigated whether hydrogen-rich University of Wisconsin (HRUW) solution attenuates renal cold I/R injury.. We prepared HRUW solution by a novel method involving immersion of centrifuge tubes containing UW solution into hydrogen-saturated water. Hydrogen readily permeates through the centrifuge tubes, and thus, the hydrogen concentration of the UW solution gradually increases in a time-dependent manner. Syngeneic rat kidney transplantation was performed, and the animals were divided into three groups: recipients with nonpreserved grafts (control group), recipients with grafts preserved in UW solution for 24 to 48 hr (UW group), and recipients with grafts preserved in HRUW solution for 24 to 48 hr (HRUW group).. In the early phases, HRUW solution decreased oxidative stress, tubular apoptosis, and interstitial macrophage infiltration in the kidney grafts. Consequently, HRUW solution improved renal function and prolonged recipient survival rate compared with simple cold storage using UW solution. Histopathologically, HRUW treatment alleviated tubular injury and suppressed development of interstitial fibrosis.. HRUW solution improved graft function and prolonged graft survival compared with simple cold storage using UW solution by protecting tubular epithelial cells from inflammation and apoptosis. Our new method of organ preservation is a groundbreaking, safe, and simple strategy that may be applied in the clinical setting.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Cold Temperature; Glutathione; Graft Survival; Hydrogen; Insulin; Kidney; Kidney Transplantation; Kidney Tubules; Macrophages; Male; Organ Preservation Solutions; Oxidative Stress; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Survival Rate

Prolonged cold preservation frequently causes delayed renal graft function resulting from tubular epithelial injury. Inhibition of signal transduction downstream from protein kinase C (PKC) may reduce renal ischemia-reperfusion injury and confer renal graft protection. We therefore evaluated the effect of sotrastaurin, a small-molecule inhibitor of Ca²⁺-dependent and Ca²⁺-independent PKC isoforms, in comparison with mycophenolic acid (MPA) on rat renal transplants with prolonged cold preservation.. Donor kidneys from male Lewis rats were cold stored in University of Wisconsin solution for 24 hr before syngeneic grafting. Recipients received sotrastaurin (30 mg/kg twice daily), MPA (20 mg/kg/day), or vehicle through gavage starting 1 hr after surgery. Renal function was evaluated by serum creatinine and histology on day 2 (acute injury) and day 7 (repair phase) after transplantation. Postreperfusion inflammation was determined by real-time polymerase chain reaction of proinflammatory genes and histology. Signaling mechanisms were studied by Western blotting and immunohistochemistry.. Sotrastaurin enhanced immediate transplant function, attenuated epithelial injury, and accelerated renal function recovery compared with MPA. Despite the stronger anti-inflammatory capacity of MPA, only sotrastaurin treatment achieved significant cellular protection with persisting reduced apoptosis of tubular epithelial cells. Decreased phosphorylation of extracellular signal-regulated protein kinase and p66Shc adaptor protein, both involved in cellular stress and apoptosis, were likely the responsible mechanism of action.. The PKC inhibitor sotrastaurin effectively ameliorated ischemia-reperfusion organ damage and promoted cytoprotection in a clinically relevant model of extended renal cold preservation followed by transplantation. Pharmacologic targeting of PKC may be beneficial for recipients receiving renal transplants at risk for delayed graft function.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Biomarkers; Blotting, Western; Cell Proliferation; Cold Temperature; Creatinine; Cytokines; Cytoprotection; Delayed Graft Function; Glutathione; Immunohistochemistry; Inflammation Mediators; Insulin; Kidney; Kidney Transplantation; Male; Mycophenolic Acid; Organ Preservation; Organ Preservation Solutions; Protein Kinase C; Protein Kinase Inhibitors; Pyrroles; Quinazolines; Raffinose; Rats; Rats, Inbred Lew; Real-Time Polymerase Chain Reaction; Reperfusion Injury; Signal Transduction; Time Factors

Febuxostat is a novel selective inhibitor of xanthine oxidase (XO), approved for treating hyperuricemia. XO inhibits the generation of uric acid (UA) as well as the resulting generation of superoxide. During renal ischemia-reperfusion (I/R) injury, the burst of reactive oxygen species (ROS) can trigger the inflammation and the tubular cell injury. As XO is a critical source of ROS, inhibition of XO could be a therapeutic target for I/R injury. Therefore, we performed this study to test the therapeutic effect of febuxostat on renal I/R injury. Sprague-Dawley rats, received vehicle or febuxostat, were subjected to right nephrectomy and left renal I/R injury. Febuxostat significantly suppressed XO activity, and thereby reduced oxidative stress, assessed by nitrotyrosine, thiobarbituric acid-reactive substances (TBARS) and urine 8-isoprostane. Furthermore, febuxostat reduced the induction of endoplasmic reticulum (ER) stress, assessed by GRP-78, ATF4, and CHOP. Vehicle-treated I/R injured rats exhibited elevated serum creatinine and UN, which were significantly suppressed in febuxostat-treated I/R-injured rats. Histological analysis revealed that fubuxostat-treated rats showed less tubular injury and interstitial fibrosis with reduction in ED1-positive macrophage infiltration, TUNEL positive apoptotic tubular cells, and interstitial smooth muscle α actin (SMαA) expression, compared to vehicle-treated rats. In conclusion; novel XO inhibitor, febuxostat, can protect kidney from renal I/R injury, and may contribute to preserve kidney function.

Topics: Animals; Apoptosis; Endoplasmic Reticulum Stress; Febuxostat; Hyperuricemia; Kidney; Kidney Tubules; Male; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Thiazoles; Xanthine Dehydrogenase; Xanthine Oxidase

To determine the influence of allopurinol and nimesulide in the protection of the pancreas from acute ischemia-reperfusion (I/R) injury.. A total of 30 rabbits were divided into 3 groups, group A: acute I/R only; group B: allopurinol (30 mg/kg) was administered intravenously 10 minutes before ischemia; group C: nimesulide (50 mg/kg) was given intraperitoneally 20 minutes before ischemia. Neopterin and superoxide dismutase (SOD) levels were examined. Pancreatic biopsies were obtained for electron microscopy study.. The mean neopterin concentrations in group A are 3.56 ± 3.41, 7.74 ± 3.59, and 8.94 ± 2.86 ng/mL, respectively, in the stabilization, ischemia, and reperfusion phases; group B: 3.40 ± 3.03, 7.45 ± 8.89, and 10.64 ± 7.47 ng/mL; and group C: 3.41 ± 2.71, 5.67 ± 2.76, and 4.34 ± 2.87 ng/mL. The mean SOD concentrations in group A are 4.25 ± 1.79, 4.48 ± 1.60, and 5.57 ± 1.15 ng/mL; group B: 4.32 ± 0.81, 5.08 ± 1.10, and 4.45 ± 1.31 ng/mL; and group C: 4.10 ± 0.99, 5.23 ± 1.60, and 3.72 ± 1.30 ng/mL. Histopathology showed the least deterioration in group C.. Nimesulide is more efficient than allopurinol in protecting pancreas from acute I/R injury.

Topics: Allopurinol; Animals; Biomarkers; Biopsy; Cytoprotection; Disease Models, Animal; Infusions, Intravenous; Injections, Intraperitoneal; Neopterin; Pancreas; Protective Agents; Rabbits; Reperfusion Injury; Sulfonamides; Superoxide Dismutase; Time Factors

We aimed to investigate the effects of caffeic acid phenethyl ester (CAPE) on intestinal mucosal injury induced by superior mesenteric occlusion.. This experimental study was conducted on 48 male Wistar-albino rats. The animals were randomly allocated into four groups: (i) Sham-operated group, laparotomy without intestinal ischemia/reperfusion (IR) injury (n = 12); (ii) Sham + CAPE group, identical to group 1 except for CAPE treatment (10 μmol/kg, intravenously) (n = 12); (iii) Intestinal IR group, 60 min of superior mesenteric ischemia followed by 3 hr of reperfusion (n = 12); and (iv) (IR + CAPE)-treated group, 10 μmol/kg injection of CAPE intravenously 30 min before the reperfusion period (n = 12). We evaluated the degree of intestinal mucosal injury on a grading scale, histopathologically, and by measuring oxidative stress markers and antioxidant parameters, biochemically. Intestinal edema was estimated by using wet/dry weight ratios. The plasma proinflammatory cytokine levels were measured. Animal survival was observed up to one week.. Intestinal mucosal injury scores were significantly decreased with CAPE administration (p < .05). CAPE treatment significantly reduced oxidative stress markers in the intestinal tissues (p < .05) and the plasma proinflammatory cytokine levels (p < .05), and significantly increased antioxidant parameters in the intestinal tissues (p < .05). Intestinal edema was significantly alleviated by CAPE treatment (p < .05). The survival rates of CAPE-treated IR animals were significantly higher than IR-subjected rats (p < .05).. This study clearly showed that CAPE treatment significantly alleviated the intestinal mucosal injury caused by superior mesenteric ischemia/reperfusion. Further clinical studies are required to clarify whether CAPE has a useful role in reperfusion injury during particular surgeries in which IR-induced organ injury occurs.

Topics: Animals; Antioxidants; Caffeic Acids; Cytokines; Glutathione Peroxidase; Intestine, Small; Male; Malondialdehyde; Models, Animal; Nitrates; Oxidative Stress; Peroxidase; Phenylethyl Alcohol; Plant Bark; Plant Extracts; Rats; Rats, Wistar; Regional Blood Flow; Reperfusion Injury; Xanthine Oxidase

The initial flush of an organ is important to remove any cellular components from the microcirculation before storage. The aim of this study was to assess graft function after an ex vivo warm flush with a novel non-phosphate buffered preservation solution AQIX RS-I (AQIX) compared with a traditional cold flush.. Porcine kidneys were either warm-flushed with AQIX RS-I at 30°C, or cold-flushed at 4°C with University of Wisconsin solution (UW) or hyperosmolar citrate (HOC) preservation solution at a pressure of 100 cmH2O (n = 6). Renal function was measured ex vivo by perfusing the organs with autologous blood at 37°C on an isolated organ perfusion system.. The AQIX group flushed significantly quicker than the cold stored groups (22 ± 1.8 versus UW 4.9 ± 1.6 versus HOC 10 ± 1.6 mL/min/100g; P = 0.001) and gained less weight than the UW group (19 ± 2.9 versus UW 30 ± 3.4 versus HOC 21% ± 7.7%; P = 0.025). The AQIX group also had superior acid-base homeostasis. Functional results, histologic analysis, and ADP: ATP levels were comparable between the groups.. Flushing kidneys with AQIX at 30°C cleared the renal microcirculation of blood more rapidly without any detrimental effects when compared to traditional cold flushing with UW or HOC at 4°C. Warm initial flushing has potential to be developed as part of normothermic renal preservation techniques.

Topics: Acid-Base Equilibrium; Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Allopurinol; Animals; Citrates; Glutathione; Hypothermia, Induced; Insulin; Kidney; Kidney Transplantation; Microcirculation; Organ Preservation Solutions; Phosphates; Raffinose; Reperfusion Injury; Spouses; Temperature

Reports of ischemia-reperfusion (I/R) injury in vivo describe experiments in which lesions are induced by the physical procedure of clamping, (I/RIC). We compare this procedure with a pharmacologic technique in which I/R injury is induced by drug superfusion (I/RID).. We used rat intestine to determine whether the responses provoked by I/RIC, such as changes in reactive oxygen species (ROS) and nitric oxide levels, are also provoked by I/RID. To this end, rats were treated with allopurinol, SOD, catalase, L-NAME, and L-arginine. In both I/R models ischemia was maintained for 60 min, followed by 30 min of reperfusion.. In both ischemia models, we observed significant differences in Evans blue (vascular permeability) and LDH (tissue injury) concentrations during the reperfusion period compared with the control group. I/RIC always induced greater injury. However, proportionally, the degree of protection was similar in the two models for the different treatments assayed. This indicates that the pathophysiologic mechanisms are the same.. Our I/RID model induces a significant intestinal alteration during the reperfusion period and, also in general terms, this alteration is prevented or worsened in a similar and proportional way to that observed when using the classic I/RIC model. The I/RID model helps to explain the development and evolution of pathologies characterized by the induction of intermittent vasospasms that produce transitory reductions in vascular perfusion, which in turn can generate ROS though an I/R mechanism.

Topics: Allopurinol; Animals; Arginine; Catalase; Constriction; Intestine, Small; Male; Models, Animal; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Time Factors

This study was designed to investigate the possible effect of sitagliptin on renal damage induced by renal ischemia reperfusion (I/R) in diabetic rats. T2DM in rats was induced by the administration of nicotinamide (230 mg/kg, i.p.), 15 min prior to a single dose of streptozotocin (65 mg/kg, i.v.). In vivo renal I/R was performed in both T2DM and normal rats. Each protocol comprised ischemia for 30 min followed by reperfusion for 24h and a treatment period of 14 days before induction of ischemia. Sitagliptin treated diabetic rats that underwent renal I/R demonstrated significant decrease in the serum concentrations of aspartate aminotransferase (p < 0.01), urea nitrogen (p < 0.01) and creatinine (p < 0.001) compared to renal I/R in diabetic rats. Lipid peroxidation, xanthine oxidase activity, myeloperoxidase activity and nitric oxide level in renal tissue were significantly (p < 0.05, p < 0.001, p < 0.01, p < 0.05 respectively) decreased after renal I/R in sitagliptin treated rats compared to diabetic rats. Antioxidant enzymes like glutathione (p < 0.05), glutathione peroxidase (p < 0.001), superoxide dismutase (p < 0.05) and catalase (p < 0.001) were significantly increased after renal I/R in sitagliptin treated diabetic rats compared to non treated diabetic rats. The typical DNA laddering was observed when renal I/R performed in diabetic rats, which indicates cell apoptosis. Sitagliptin treated rats demonstrated a decrease in DNA fragmentation and apoptosis. Furthermore, renal histopathology preserved in sitagliptin treated rats verified protection against renal I/R in diabetes. The results of present investigation established sitagliptin treatment attenuated renal damage induced by renal I/R in diabetic rats.

Topics: Animals; Apoptosis; Catalase; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; DNA Fragmentation; Female; Glutathione; Glutathione Peroxidase; Kidney; Kidney Diseases; Lipid Peroxidation; Male; Oxidative Stress; Peroxidase; Pyrazines; Rats; Rats, Wistar; Reperfusion Injury; Sitagliptin Phosphate; Superoxide Dismutase; Triazoles; Xanthine Oxidase

To investigate the duration of apoptosis caused by ischemia-reperfusion in the intestine in a new double jejunum-segment model, and to analyze the protective effects of allopurinol or ischemic preconditioning (IPC).. In Experiment I for harvesting the double jejunum-segment model after laparotomy a 30-cm-long jejunum part was selected on mongrel dogs (n=24). End-to-end anastomoses were performed at both ends and in the middle of the jejunum part, creating two equal segments. In one segment ischemia was induced by occluding the supplying vessels, the other segment served as control. Tissue samples for detecting apoptosis were taken at 30th minutes, 1st, 2nd, 4th, 6th, 8th, 12th and 24th hours of reperfusion. In Experiment II using the same model the 4-hour reperfusion time period, allopurinol (50 mg/kg) pre-treated and IPC (3 cycles of 5x1) groups (n=5 per each) were also investigated.. In Experiment I the greatest apoptotic activity was detected at the 4th and 6th hour of reperfusion (14.2 ± 1.31 and 16.3 ± 1.05 per visual field at 40x magnification). In Experiment II Using the 4-hour reperfusion time period allopurinol pre-treatment increased the apoptotic activity (10.72 ± 0.47 per 50 intestinal villi) approximately two-fold than the IPC (6.72 ± 0.46 per 50 intestinal villi) did (p<0.05).. Apoptotic activity has a characteristic time curve, reaching the highest values between the 4th and 6th hours after 30-minute intestinal ischemia. Ischemic preconditioning seemed to be protective against the morphological changes caused by intestinal ischemia-reperfusion.

Topics: Allopurinol; Anastomosis, Surgical; Animals; Apoptosis; Disease Models, Animal; Dogs; Female; Immunohistochemistry; Ischemic Preconditioning; Jejunal Diseases; Jejunum; Male; Rats; Reperfusion Injury; Time Factors

Intestinal ischemia/reperfusion (I/R) injury is implicated in many clinical conditions, and it performs a fundamental role in their pathophysiologies. Oral administration of antioxidants and nitric oxide (NO) donors ameliorate intestinal injury. Here, the effects of l-arginine, allopurinol and N(G)-nitro-l-arginine methyl ester (l-NAME) were investigated.. One hundred twenty-eight male Wistar rats were separated into 4 groups and subjected to occlusion of the superior mesenteric artery for 60 min. The Control group did not receive any substance before the surgical operation. However, the 3 other groups received the following: l-arginine (800 mg/kg body weight; l-Arg group), l-NAME (50mg/kg; l-NAME group) or allopurinol (100mg/kg; Allo group). Each substance was given by mouth in 3 equal doses 24, 12 and 1h before the surgical operation. Each group was then divided into 4 subgroups, which underwent different durations of reperfusion (0, 1, 8 or 24h). At the end of each time point, blood and tissue samples were collected, and histological examinations were performed. Serum nitrite and catalase, intestinal tissue myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT) levels were determined.. At each reperfusion time point, the Allo group exhibited the mildest histological lesions in contrast to the l-NAME group, which showed the most severe lesions. MPO was decreased significantly in the Allo and l-Arg groups during reperfusion, and allopurinol administration caused earlier and stronger effect. iNOS and NT levels were higher in the l-Arg group and lower in the Allo group. Serum nitrite and catalase were increased in the l-NAME group after 24h.. Oral administration of allopurinol exerted a strong and protective effect on the intestinal tissue that was subjected to I/R earlier than l-arginine. This finding was also supported with the MPO, iNOS and NT data.

Topics: Administration, Oral; Allopurinol; Animals; Arginine; Catalase; Intestines; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type II; Nitrites; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Time Factors; Tyrosine

The insults sustained by transplanted livers (hepatectomy, hypothermic preservation, and normothermic reperfusion) could compromise hepatic function. Hydrogen sulfide (H₂S) is a physiologic gaseous signaling molecule, like nitric oxide (NO) and carbon monoxide (CO). We examined the effect of diallyl disulfide as a H₂S donor during hypothermic preservation and reperfusion on intrahepatic resistance (IVR), lactate dehydrogenase (LDH) release, bile production, oxygen consumption, bromosulfophthalein (BSP) depuration and histology in an isolated perfused rat liver model (IPRL), after 48 h of hypothermic storage (4 °C) in University of Wisconsin solution (UW, Viaspan). Livers were retrieved from male Wistar rats. Three experimental groups were analyzed: Control group (CON): IPRL was performed after surgery; UW: IPRL was performed in livers preserved (48 h-4 °C) in UW; and UWS: IPRL was performed in livers preserved (48 h-4 °C) in UW in the presence of 3.4 mM diallyl disulfide. Hypothermic preservation injuries were manifested at reperfusion by a slight increment in IHR and LDH release compared with the control group. Also, bile production for the control group (1.32 µL/min/g of liver) seemed to be diminished after preservation by 73% in UW and 69% in UW H₂S group at the end of normothermic reperfusion. Liver samples analyzed by hematoxylin/eosin clearly showed the deleterious effect of cold storage process, partially reversed (dilated sinusoids and vacuolization attenuation) by the addition of a H₂S delivery compound to the preservation solution. Hepatic clearance (HC) of BSP was affected by cold storage of livers, but there were no noticeable differences between livers preserved with or without diallyl disulfide. Meanwhile, livers preserved in the presence of H₂S donor showed an enhanced capacity for BSP uptake (k(A) CON = 0.29 min⁻¹; k(A) UW = 0.29 min⁻¹ ; k(A) UWS = 0.36 min ⁻¹). In summary, our animal model suggests that hepatic hypothermic preservation for transplantation affects liver function and hepatic depuration of BSP, and implies that the inclusion of an H₂S donor during hypothermic preservation could improve standard methods of preparing livers for transplant.

Topics: Adenosine; Allopurinol; Allyl Compounds; Animals; Bile; Cold Ischemia; Disulfides; Gases; Glutathione; Glycogen; Hydrogen Sulfide; Insulin; L-Lactate Dehydrogenase; Liver; Liver Circulation; Liver Function Tests; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Oxygen Consumption; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Sulfobromophthalein; Time Factors; Vascular Resistance

The rapid uniform delivery of University of Wisconsin solution (UW) to the microcirculation may be compromised by its vasoactivity.. In 2 different rodent models, we tested whether UW-mediated vasoconstriction could be reversed with nicardipine.. In the perfused, splanchnic circulation, intravascular control solutions (lactated Ringers [LR], Hextend [HEX], histidine-tryptophan-ketoglutarate [HTK]) or UW (± nicardipine) evoked pressure changes in 3 protocols (series 1; n = 35). In the cremaster muscle, topical control solutions or UW (± nicardipine) evoked vascular responses measured by video microscopy in 4 protocols (series 2; n = 47). In series 1A, 37°C UW increased perfusion pressure, but there was no change caused by LR, HEX, or HTK. In series 1B, 4°C UW caused a similar, albeit transient, increase. In series 1C, nicardipine reversed 37°C UW-mediated vasoconstriction in a dose-related manner. In series 2A, UW caused a 30%-59% constriction that varied with arteriolar branching order. In series 2B, the recovery from UW-induced vasoconstriction varied with duration of exposure, but nicardipine fully reversed residual vasoconstriction. In series 2C, cold and warm UW were equipotent, near maximal, vasoconstrictors. In series 2D, UW potentiated no-reflow.. UW causes a potent temperature-independent vasoconstriction by a calcium-mediated mechanism and this effect can be mitigated with nicardipine.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Insulin; Male; Microcirculation; Nicardipine; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Temperature; Vasodilator Agents

Hepatic ischemia-reperfusion causes histologic injury to the intestinal mucosa. We investigated the effects of diosmin, a phelobotrophic drug with antioxidant and antiinflammatory effects, on intestinal injury in the experimental liver ischemia-reperfusion model.. Fourty rats were divided into four groups: sham group (Group 1), control group (Group 2), perop diosmin group (50 mg/kg) treatment group (Group 3) and preop 10-day diosmin (50 mg/kg) treatment group (Group 4). Ischemia-reperfusion model was carried out by clamping the hepatic pedicle for 60 min and then reperfusing the liver for 90 min. At the end of procedures, blood and ileum tissue samples were obtained for biochemical and histopathological assessments.. According to the results of liver function tests (AST, ALT and LDH) there was a significant difference between the control and other groups (p < 0.001 for all). According to the plasma and ileum oxidative stress parameters (MDA, GSH-Px and XO), there was a significant difference between the control and other groups (p < 0.05 for all). Histopathologically; the specimens in Group 2 showed specific morphological abnormalities (the epithelial lining of the apical surface of villi was degenerated and desquamated to the lumen). Group 3 and 4 showed ileal histomorphology similar to the sham group. Pathological scores were significantly different between Group 2 and other groups.. Diosmin can be administered for protection from destructive effects of hepatic ischemia-reperfusion injury on intestine in both emergent and elective hepatic surgical operations in which the possible ischemic periods are expected (Tab. 4, Fig. 1, Ref. 39).

Topics: Animals; Diosmin; Female; Glutathione Peroxidase; Ileum; Liver; Malondialdehyde; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Oxidase

Our study investigated the effect of ischemic postconditioning (IPO) in intestinal warm ischemia/reperfusion (I/R) and autotransplantation models.. Warm ischemia was performed by occlusion of superior mesenteric artery for 1, 3 and 6 hours in white domestic pigs (n = 15). Prior to 3 hours reperfusion the intestine was postconditioned by 3 cycles of 30-seconds ischemia and 30-seconds reperfusion (IPO protocol). In the cold ischemia group (n = 15) the bowel was preserved in University of Wisconsin solution for 1, 3, and 6 hours. Prior to 3 hours reperfusion IPO protocol was applied, too. Tissue samples were collected after laparotomy (control) and at the end of the reperfusion periods. As far as oxidative stress markers, malondialdehyde and reduced glutathione (GSH) levels and superoxide dismutase (SOD) activity were determined. Tissue damage was evaluated by qualitative (Park-classification) and quantitative (Scion Image) methods.. As regards oxidative stress parameters, lipidperoxidation decreased and the protective effect of endogenous antioxidants (GSH, SOD) retained significantly by IPO procedure at the end of reperfusion. Tissue injury correlated significantly by the duration of warm ischemia and cold preservation. Quantitative analysis demonstrated that IPO ameliorated tissue injury in each group (p < 0.05).. IPO significantly attenuated intestinal oxidative stress and morphological damages in warm and cold I/R models.

Topics: Adenosine; Allopurinol; Animals; Antioxidants; Disease Models, Animal; Glutathione; Insulin; Intestines; Ischemia; Ischemic Postconditioning; Laparotomy; Lipid Peroxidation; Malondialdehyde; Organ Preservation Solutions; Oxidative Stress; Raffinose; Reperfusion Injury; Superoxide Dismutase; Sus scrofa; Time Factors; Transplantation, Autologous; Warm Ischemia

Orthotopic liver transplantation (OLT) has been the standard treatment for end-stage acute and chronic liver disease. Ischemia-reperfusion (I/R) injury is one of the major causes of poor graft function early after OLT, and adversely influencing graft and patient survivals. It is unknown whether I/R injury influences liver fibrogenesis.. Livers from 25 adult male Wistar rats were randomly assigned into 5 experimental groups according to the preservation solution: saline solution (SS); University of Wisconsin (UW) solution; Fructose 1, 6-biphosphate (FBP); S-Nitroso-N-Acetylcysteine (SNAC): or UW+SNAC (SNAC+UW). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) were determined in preservation solution samples at 2, 4, and 6 hours. After 6 hours of cold ischemia, ex situ reperfusion was applied to the liver for 15 minutes. Serum AST, ALT, LDH, and renin levels were determined. Fresh liver slices were processed for histological studies, determination of thiobarbituric acid reactive substances, catalase, and glutathione, and expression of TGF-β1 and angiotensin II AT1 receptor.. AST was significantly lower during cold storage with UW than with the older media (P=.001); ALT was lower in the FBP group (P=.023) and LDH was lower in the FBP and SNAC groups (P=.007). After reperfusion, serum AST, ALT, LDH, and TBARS showed no significant differences among the groups. Catalase was significantly lower in the SS and FBP groups (P=.008 and P=.006, respectively). Compared with UW, glutathione concentrations were significantly higher in SS, FBP, and SNAC 200 (P=.004). Renin levels were significantly lower in the FBP group (P=.022). No histological signs of preservation injury were observed in the hepatic sample. No expressions were detected of TGF-β1 or AT1 receptor.. In this experimental model of early reperfusion injury, preservation changes related to higher levels of renin, which suggest its role in fibrogenesis. FBP was associated with lower renin levels than other solutions including UW.

Topics: Acetylcysteine; Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Biomarkers; Catalase; Disease Models, Animal; Fructosediphosphates; Glutathione; Insulin; L-Lactate Dehydrogenase; Liver; Liver Cirrhosis; Liver Transplantation; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Receptor, Angiotensin, Type 1; Renin; Reperfusion Injury; Thiobarbituric Acid Reactive Substances; Time Factors; Transforming Growth Factor beta1

Ischemia and subsequent reperfusion (IR) may induce local and remote organ reperfusion injury. It may be propagated by xanthine oxidase (XO)-generated oxidant stress. We investigated whether pancreas IR directly and acutely induces renal dysfunction and if this outcome could be prevented by mannitol.. Rat pancreases were isolated and perfused with Krebs-Henseleit solution enriched with 5% bovine albumin. Other rats donated kidneys that were perfused at constant pressure mode. Each pancreas underwent 45 min of either perfusion (control) or ischemia (no flow). Both organ perfusion systems were then combined and the kidneys were perfused with the pancreatic 15-min reperfusate for 2 h. A third group consisted of paired ischemic pancreases and nonischemic kidneys treated with mannitol 250 mg/kg body weight during reperfusion.. The controls demonstrated no abnormal perfusion or metabolite changes. Pancreas and renal perfusion pressures increased by >50% in the ischemia group immediately upon reperfusion; it remained above the values of controls during the 2-h kidney reperfusion. Conversely, perfusion pressure in the treatment group was not significantly different from the control. The reduced glutathione level increased significantly, as did XO, immediately upon starting reperfusion in both organs appertaining to the ischemic group; this misbalance was not documented in the controls and the mannitol-treated groups. Urine output was severely reduced in the IR kidneys.. Ischemia/reperfusion of the rat pancreas evokes immediate renal dysfunction. Kidney oxidant-antioxidant balance is disturbed, but can be prevented with mannitol. These two figures underline the role of oxidative stress in promoting acute renal damage in the presence of pancreas IR.

Topics: Animals; Diuretics, Osmotic; In Vitro Techniques; Kidney; Kidney Diseases; Male; Mannitol; Oxidative Stress; Pancreas; Pancreas Transplantation; Pancreatitis; Perfusion; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Oxidase

Renal ischemia/reperfusion (I/R) which is an important cause of renal dysfunction is inevitable in renal transplantation, surgical revascularization of the renal artery, partial nephrectomy and treatment of suprarenal aortic aneurysms.. The purpose of this study was to investigate the efficacy of α-tocopherol and erdosteine combination in the reduction in injury induced by ROS in a rat model of renal ischemia-reperfusion.. Thirty-six- male Wistar albino rats weighing 200-250 g were utilized for this study. Rats were divided into six groups, and each group was consistent of six rats: (1) sham-operated (control), (2) ischemia group (3) I/R group, (4) I/R/α-tocoferol group (5) I/erdosteine group (6). I/R/α-tocoferol and erdosteine group. Biochemically tissue MDA, XO and SOD activities, light and electron microscopic findings were evaluated.. The erdosteine and α-tocoferol significantly reversed the effect of protein oxidation and lipid peroxidation induced by I/R shown by the decreased levels of MDA and XO activities. Both MDA and XO levels were found to be lower in group 6 compared to single agent treatment groups, and this was significantly different. All treatment groups showed increased SOD activity, which accounts for their oxidative properties. The mean Paller score of the combination treatment group (group 6) was lower than all groups except the sham group (3.67 ± 1.2), and this finding was statistically significant (0.05). Our results showed that the antioxidant pretreatment with α-tocopherol and erdosteine combination reduced lipid peroxidation of renal cellular membranes in a model of normothermic renal ischemia-reperfusion in rats. Combination of erdosteine and α-tocopherol has a synergistic effect of protection against oxidative processes. Long-term use of α-tocopherol seems to have a greater effect on the prevention of IR injury. However, further investigations are needed for the clinical applications of our findings.

Topics: alpha-Tocopherol; Animals; Antioxidants; Kidney; Male; Malondialdehyde; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Thioglycolates; Thiophenes; Xanthine Oxidase

This study investigated the effect of astaxanthin (ASX; 3,3-dihydroxybeta, beta-carotene-4,4-dione), a water-dispersible synthetic carotenoid, on liver ischemia-reperfusion (IR) injury. Astaxanthin (5 mg/kg/day) or olive oil was administered to rats via intragastric intubation for 14 consecutive days before the induction of hepatic IR. On the 15th day, blood vessels supplying the median and left lateral hepatic lobes were occluded with an arterial clamp for 60 min, followed by 60 min reperfusion. At the end of the experimental period, blood samples were obtained from the right ventricule to determine plasma alanine aminotransferase (ALT) and xanthine oxidase (XO) activities and animals were sacrificed to obtain samples of nonischemic and postischemic liver tissue. The effects of ASX on IR injury were evaluated by assessing hepatic ultrastructure via transmission electron microscopy and by histopathological scoring. Hepatic conversion of xanthine dehygrogenase (XDH) to XO, total GSH and protein carbonyl levels were also measured as markers of oxidative stress. Expression of NOS2 was determined by immunohistochemistry and Western blot analysis while nitrate/nitrite levels were measured via spectral analysis. Total histopathological scoring of cellular damage was significantly decreased in hepatic IR injury following ASX treatment. Electron microscopy of postischemic tissue demonstrated parenchymal cell damage, swelling of mitochondria, disarrangement of rough endoplasmatic reticulum which was also partially reduced by ASX treatment. Astaxanthine treatment significantly decreased hepatic conversion of XDH to XO and tissue protein carbonyl levels following IR injury. The current results suggest that the mechanisms of action by which ASX reduces IR damage may include antioxidant protection against oxidative injury.

Topics: Alanine Transaminase; Animals; Antioxidants; Disease Models, Animal; Glutathione; Liver; Male; Nitrates; Nitric Oxide Synthase Type II; Nitrites; Protein Carbonylation; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase; Xanthophylls

To investigate the protective effects of 17beta-estradiol postconditioning against ischemia/reperfusion (I-R)-induced gastric mucosal injury in rats.. The animal model of gastric ischemia/reperfusion was established by clamping of the celiac artery for 30 min and reperfusion for 30 min, 1h, 3h, 6h, 12h or 24h. 17beta-estradiol at doses of 5, 50 or 100 microg/kg (rat) was administered via peripheral veins 2 min before reperfusion. In a subgroup of rats, the estrogen receptor antagonist fulvestrant (Ful, 2mg/kg) was intravenously injected prior to 17beta-estradiol administration. Histological and immunohistochemical methods were employed to assess the gastric mucosal injury index and gastric mucosal cell apoptosis and proliferation. The malondialdehyde (MDA) concentration, superoxide dismutase (SOD) activity, xanthine oxidase (XOD) activity and hydroxyl free radical (-OH) inhibitory ability were determined by colorimetric assays. Subsequently, the expression of Bcl-2 and Bax in rat gastric mucosa was examined by western blotting.. 17beta-estradiol dose-dependently inhibited gastric I-R (GI-R) injury, and 17beta-estradiol (50 microg/kg) markedly attenuated GI-R injury 1h after reperfusion. 17beta-estradiol inhibited gastric mucosal cell apoptosis and promoted gastric mucosal cell proliferation in addition to increasing SOD activity and -OH inhibitory ability and decreasing the MDA content and XOD activity. The Bax protein level increased 1h after GI-R and was markedly reduced by intravenous administration of 17beta-estradiol. In contrast, the level of Bcl-2 protein decreased 1h after GI-R and was restored to normal levels by intravenous administration of 17beta-estradiol. These effects of 17beta-estradiol were inhibited by pretreatment with fulvestrant.. 17beta-estradiol postconditioning should be investigated further as a possible strategy against gastric mucosal injury.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Proliferation; Estradiol; Estrogen Antagonists; Fulvestrant; Gastric Mucosa; Hydroxyl Radical; Male; Malondialdehyde; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

There is increasing support for the use of hypothermic machine perfusion (HMP) in an attempt to reduce preservation injury. However, experimental evidence is needed to further examine the effects of HMP on renal ischemia reperfusion injury.. Porcine kidneys were subjected to 10 min of warm ischemia followed by 18 hr of static cold storage with hyperosomolar citrate (HOC), histidine-tryptophan-ketoglutarate (HTK), or University of Wisconsin (UW) solutions or 18 hr HMP with Kidney Perfusion Solution using the Lifeport perfusion system. Renal function, oxidative damage, and morphology were assessed during 3 hr of reperfusion with autologous blood using an isolated organ perfusion system.. During reperfusion, intrarenal resistance was significantly lower in the HMP group compared with HOC and UW (area under the curve; HMP 3.8+/-1.7, HOC 9.1+/-4.3, UW 7.7+/-2.2, HTK 5.6+/-1.9 mm Hg/min; P=0.006), and creatinine clearance was significantly higher compared with the UW group (area under the curve creatinine clearance; HMP 9.8+/-7.3, HOC 2.2+/-1.7, UW 1.8+/-1.0, HTK 2.1+/-1.8 mL/min/100 g; P=0.004). Tubular function was significantly improved in the HMP group (P<0.05); however, levels of lipid peroxidation were significantly higher (P=0.005).. HMP demonstrated a reduced level of preservation injury compared with the static techniques resulting in improved renal and tubular function and less tubular cell inflammation during reperfusion.

Topics: Acid-Base Equilibrium; Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Biomarkers; Citrates; Cold Ischemia; Creatinine; Disease Models, Animal; Equipment Design; Glucose; Glutathione; Hypothermia, Induced; Insulin; Kidney; Kidney Transplantation; L-Lactate Dehydrogenase; Lipid Peroxidation; Mannitol; Organ Preservation Solutions; Oxidative Stress; Perfusion; Peroxidase; Potassium Chloride; Procaine; Raffinose; Reperfusion Injury; Swine; Time Factors

Here we examine the effect of adding carvedilol (CVD) to University of Wisconsin (UW) solution on the preservation of steatotic and nonsteatotic livers during cold ischemia and after normothermic reperfusion. We used an isolated perfused rat liver model. The following protocols were evaluated. Protocol 1 concerned the effect of CVD after cold ischemia. Steatotic and nonsteatotic livers were preserved for 24 hours in UW solution alone or with CVD. Livers without cold ischemia were used as controls. Transaminases were evaluated in the flushing effluent. Protocol 2 involved the effect of CVD after reperfusion. Both liver types were preserved for 24 hours in UW solution alone or with CVD and then perfused ex vivo for 2 hours at 37 degrees C. Livers flushed and perfused without ischemia were used as controls. Hepatic injury and functionality [transaminases, bile production, and hepatic clearance of sulfobromophthalein (BSP)] were evaluated after reperfusion. In addition, factors potentially involved in hepatic ischemia-reperfusion injury, including oxidative stress (malondialdehyde and superoxide anion levels), mitochondrial damage (glutamate dehydrogenase activity), microcirculatory disorders (flow rate and vascular resistance), and adenosine triphosphate (ATP) depletion, were evaluated after reperfusion. After cold ischemia, steatotic livers preserved in UW solution showed higher transaminase levels than nonsteatotic livers. After reperfusion, steatotic livers preserved in UW solution showed higher transaminase levels and lower bile production and BSP clearance than nonsteatotic livers. Alterations in the perfusion flow rate and vascular resistance, mitochondrial damage, and reduced ATP content were more evident in steatotic livers preserved in UW solution. The addition of CVD to UW solution reduced hepatic injury, obstructed its mechanisms, and improved hepatic functionality in both liver types. We conclude that CVD is a useful additive for UW solution that improves the preservation of steatotic and nonsteatotic livers subjected to prolonged cold ischemia.

Topics: Adenosine; Adenosine Triphosphate; Adenylate Kinase; Adrenergic beta-Antagonists; Allopurinol; Animals; Bile; Carbazoles; Carvedilol; Cold Temperature; Fatty Liver; Glutathione; Insulin; Liver; Liver Function Tests; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Postoperative Complications; Propanolamines; Raffinose; Rats; Rats, Zucker; Reperfusion Injury; Vascular Resistance

Fingolimod (FTY720) is a potent agonist of sphingosine 1 phosphate receptors and thereby interferes with lymphocyte trafficking. We previously showed that FTY720 protects from mild preservation reperfusion injury induced by 4 hr of cold ischemia. The purpose of this study was to explore the role of FTY720 in ischemic injury and regeneration using a clinically relevant rat renal transplant model with 24 hr of cold ischemia.. Donor kidneys were cold stored in the University of Wisconsin solution for 24 hr before transplantation into bilaterally nephrectomized syngeneic recipients (n=6 per group), which received 0.5 mg/kg/d FTY720 or vehicle through oral gavage. Grafts were harvested 2 or 7 days posttransplantation. Renal tissue was examined histologically, stained for apoptosis, proliferation, inflammatory cell infiltrates, and studied for transforming growth factor-beta, and tumor necrosis factor-alpha expression. Rat proximal tubular cells were incubated with 0.1 to 30 micromol/L of phosphorylated FTY720 to test for in vitro cytopathic effects.. FTY720 induced peripheral lymphopenia and significantly reduced intragraft CD3 and ED1 infiltrates. Acute tubular damage scores and graft function were not influenced by FTY720. Tubular apoptosis was significantly reduced, whereas the number of proliferating cell nuclear antigen-positive tubular cells were markedly increased. FTY720 attenuated renal tumor necrosis factor-alpha and transforming growth factor-beta expression. In vitro, pharmacologic concentrations up to 1 micromol/L of phosphorylated FTY720 did not affect tubular cell viability.. FTY720 confers tubular epithelial protection in the presence of severe preservation reperfusion injury. Beneficial effects may in part be due to reduction in cell-mediated immune mechanisms. Furthermore, FTY720 could be helpful in patients with delayed graft function.

Topics: Adenosine; Allopurinol; Animals; Cell Culture Techniques; Cell Division; Cell Survival; Fingolimod Hydrochloride; Flow Cytometry; Glutathione; Immunohistochemistry; Immunosuppressive Agents; Inflammation; Insulin; Kidney Transplantation; Male; Organ Preservation Solutions; Propylene Glycols; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Sphingosine

Orthotopic liver transplantation (OLT) is today the gold standard treatment of the end-stage liver disease. Different solutions are used for graft preservation. Our objective was to compare the results of cadaveric donor OLT, preserved with the University of Wisconsin (UW) or Celsior solutions in the portal vein and Euro-Collins in the aorta.. We evaluated retrospectively 72 OLT recipients, including 36 with UW solution (group UW) and 36 with Celsior (group CS). Donors were perfused in situ with 1000 mL UW or Celsior in the portal vein of and 3000 mL of Euro-Collins in the aortia and on the back table managed with 500 mL UW or Celsior in the portal vein, 250 mL in the hepatic artery, and 250 mL in the biliary duct. We evaluated the following variables: donor characteristics, recipient features, intraoperative details, reperfusion injury, and steatosis via a biopsy after reperfusion. We noted grafts with primary nonfunction (PNF), initial poor function (IPF), rejection episodes, biliary duct complications, hepatic artery complications, re-OLT, and recipient death in the first year after OLT.. The average age was 33.6 years in the UW group versus 41 years in the CS group (P = .048). There was a longer duration of surgery in the UW group (P = .001). The other recipient characteristics, ischemia-reperfusion injury, steatosis, PNF, IPF, rejection, re-OLT, and recipient survival were not different. Stenosis of the biliary duct occured in 3 (8.3%) cases in the UW group and 8 (22.2%) in the CS (P = .19) with hepatic artery thrombosis in 4 (11.1%) CS versus none in the UW group (P = .11).. Cadaveric donor OLT showed similar results with organs preserved with UW or Celsior in the portal vein and Euro-Collins in the aorta.

Topics: Adenosine; Adolescent; Adult; Aged; Allopurinol; Aorta, Abdominal; Cadaver; Child; Child, Preschool; Disaccharides; Electrolytes; Female; Glutamates; Glutathione; Histidine; Humans; Hypertonic Solutions; Immunosuppressive Agents; Insulin; Liver Failure; Liver Transplantation; Male; Mannitol; Middle Aged; Organ Preservation; Organ Preservation Solutions; Portal Vein; Postoperative Complications; Prospective Studies; Raffinose; Reperfusion Injury; Retrospective Studies; Tissue Donors

The two-layer organ preservation method (TLM) based on oxygenated perfluorocarbon overlaid with University of Wisconsin (UW) solution has been successfully used in clinical islet and experimental heart and intestine transplantation. We tested whether this technique would prevent tissue damage and improve kidney function in a model of syngeneic kidney transplantation with prolonged ischemia time.. Kidneys were stored for 24 h either in UW solution (n = 16), with TLM (n = 16) or transplanted immediately (control group, n = 12). In half of the animals, survival was observed and in the other animals grafts were procured for semiquantitative histological scoring and TUNEL apoptosis assessment 24 h after transplantation.. One-month survival rates in the UW, TLM and control groups were 12.5, 62.5 and 100%, respectively (UW vs. TLM, p < 0.01). Median creatinine levels 24 h after transplantation were 381, 299 and 121 microM, respectively (UW vs. TLM, p < 0.02). Histological scoring showed more severe tissue damage in the UW group than in the TLM group (p < 0.05). Apoptosis was more frequent in the UW group than in the TLM group (p < 0.05).. We demonstrated for the first time that conservation with TLM significantly improves the outcome of kidney transplantation in a rat model and should therefore be further studied in larger animals.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Blood Substitutes; Cold Ischemia; Creatinine; Fluorocarbons; Glutathione; Graft Survival; Insulin; Kidney; Kidney Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred BN; Reperfusion Injury; Time Factors; Transplantation, Isogeneic

The detrimental role of superoxide anion (O(2)(-)) has been well documented in the pathogenesis of ischemia-reperfusion (I/R) injury. Our and other studies suggested that one critical source of O(2)(-) generation may be xanthine oxidase (XO). We thus hypothesized that I/R injury could be protected by inhibiting XO activity, which would reduce the amount of O(2)(-) and hence reduce pathogenic consequences. Among various XO inhibitors, we previously found 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP) exhibited potent XO inhibitory activity. Here, we report that the covalent conjugate of AHPP with amphipathic styrene-maleic acid copolymer (SMA-AHPP) showed protective effect against I/R-induced injury in a rat hepatic I/R model. Liver ischemia was induced by occluding both the portal vein and the hepatic artery for 30 min, and followed by reperfusion. SMA-AHPP was administered via the tail vein two hours before ischemia was initiated. A remarkable increase of liver enzymes in plasma (aspartate aminotransferase, AST; alanine aminotransferase, ALT and lactate dehydrogenase, LDH) was detected three hours after reperfusion, whereas prior injection of SMA-AHPP greatly suppressed this increase of AST, ALT and LDH. Moreover, induction of inflammatory cytokines, i.e. tumor necrosis factor-alpha (TNF-alpha), interleukin-12 (IL-12) and monocyte chemotactic protein-1 (MCP-1) by I/R were significantly inhibited by SMA-AHPP treatment. Accordingly, cytotoxic effect or apoptosis in the liver caused by I/R was clearly reduced by SMA-AHPP pretreatment. Furthermore, thiobarbituric acid-reactive substance assay showed a significant decrease of lipid peroxidation in rat liver after the administration of SMA-AHPP, which is parallel with the decreased XO activity after SMA-AHPP treatment, indicating the involvement of reactive oxygen species generated by XO. In addition, SMA-AHPP was found to bind to albumin, thus to exhibit prolonged in vivo (plasma) half-life. These results suggest that SMA-AHPP exerted a potent cytoprotective effect against I/R injury in rat liver, by inhibiting XO activity and the subsequent generation of O(2)(-).

Topics: Albumins; Animals; Apoptosis; Cytokines; Enzyme Inhibitors; Heme Oxygenase-1; Liver; Male; Maleates; Molecular Weight; Oxypurinol; Polystyrenes; Rats; Rats, Wistar; Reperfusion Injury; Superoxides; Thiobarbituric Acid Reactive Substances; Tissue Distribution; Xanthine Oxidase

This study examined the effects of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) supplementation to University of Wisconsin solution (UW) in steatotic and nonsteatotic livers during cold storage. Hepatic injury and function were evaluated in livers preserved for 24 hours at 4 degrees C in UW and in UW with EGF and IGF-I (separately or in combination) and then perfused ex vivo for 2 hours at 37 degrees C. AKT was inhibited pharmacologically. In addition, hepatic injury and survival were evaluated in recipients who underwent transplantation with steatotic and nonsteatotic livers preserved for 6 hours in UW and UW with EGF and IGF-I (separately or in combination). The results, based on isolated perfused liver, indicated that the addition of EGF and IGF-I (separately or in combination) to UW reduced hepatic injury and improved function in both liver types. A combination of EGF and IGF-I resulted in hepatic injury and function parameters in both liver types similar to those obtained by EGF and IGF-I separately. EGF increased IGF-I, and both additives up-regulated AKT in both liver types. This was associated with glycogen synthase kinase-3beta (GSK3(beta)) inhibition in nonsteatotic livers and PPAR gamma overexpression in steatotic livers. When AKT was inhibited, the effects of EGF and IGF-I on GSK3(beta), PPAR gamma, hepatic injury and function disappeared. The benefits of EGF and IGF-I as additives in UW solution were also clearly seen in the liver transplantation model, because the presence of EGF and IGF-I (separately or in combination) in UW solution reduced hepatic injury and improved survival in recipients who underwent transplantation with steatotic and nonsteatotic liver grafts. In conclusion, EGF and IGF-I may constitute new additives to UW solution in steatotic and nonsteatotic liver preservation, whereas a combination of both seems unnecessary.

Topics: Adenosine; Allopurinol; Animals; Cell Survival; Cold Ischemia; Disease Models, Animal; Epidermal Growth Factor; Fatty Liver; Glutathione; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Insulin; Insulin-Like Growth Factor I; Liver; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Perfusion; PPAR gamma; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Raffinose; Rats; Rats, Zucker; Recombinant Proteins; Reperfusion Injury; Time Factors

Xanthine oxidoreductase (XOR) and its active forms, dehydrogenase (XD) and oxidase (XO), act as double-edged swords during ischemia-reperfusion injury. On the one hand, their action generates antioxidants, such as uric acid (UA); however, they may strongly enhance production of free radicals. In this study, we examined the association between post-transplant graft function and perioperative xanthine metabolizing enzymes (XME) activity in kidney transplant recipients divided into early (EGF), slow (SGF), and delayed graft function (DGF) groups.. XME activity and UA levels were measured in blood samples collected directly before and during the first and fifth minutes of reperfusion.. Results demonstrated an increase in XO and XOR activity in all groups; however, these parameters were lower in the EGF than in the DGF group (p < 0.005; p < 0.05). XD activity increased in SGF and DGF patients (p = 0.01); nevertheless, the XD/total XOR coefficient decreased only in DGF individuals (p = 0.0007). XME sensitivity, specificity, and positive and negative predictive values in discriminating SGF/DGF from EGF were 73.3% to 78%, 54% to 62.5%, 76% to 78.6%, and 56.5%, respectively. Moreover, mixed model analysis revealed that recipients classified according to results of XOR(5) and XO(5) significantly differ in 1-year post-transplant allograft function (p = 0.04 and p = 0.02, respectively), but not in the frequency of acute rejection episodes (p = 0.66 and p = 0.90, respectively).. During renal transplantation, significant changes in XME occur that are associated with early post-transplant graft function and have potential value to discern between EGF and SGF/DGF.

Topics: Area Under Curve; Biomarkers; Delayed Graft Function; Female; Graft Survival; Humans; Kidney Transplantation; Male; Middle Aged; Multivariate Analysis; Oxidative Stress; Perioperative Period; Postoperative Period; Reperfusion Injury; Retrospective Studies; Transplantation, Homologous; Uric Acid; Xanthine Dehydrogenase; Xanthine Oxidase

Improved kidney preservation methods are needed to reduce ischemia-reperfusion (IR) injury in kidney allografts. Lifor is an artificial preservation solution comprised of nutrients, growth factors, and a non-protein oxygen and nutrient carrier. The current study compared the effectiveness of Lifor to University of Wisconsin solution (UW) in protecting rat kidneys from warm IR and cold storage injury.. In a warm IR model, rat kidneys were perfused in situ with either saline, UW, or Lifor for 45 min. Renal function and histology were assessed 24 h later. In a cold IR model, kidney slices were cold-stored in saline, UW, or Lifor at 4°C. Kidney injury was assessed by the release of lactate dehydrogenase (LDH) and immunoblot analysis for cleaved caspase-3.. Lifor perfusion significantly mitigated renal dysfunction and tubular injury at 24 h compared with saline or UW. Lifor and UW prevented LDH release in hypoxic kidney slices in vitro, however activation of caspase-3 following hypoxia-reoxygenation was attenuated only with Lifor. Cold storage with Lifor or UW significantly decreased LDH release from kidney slices or normal rat kidney cells in comparison to storage in saline or culture media. After 24 h of cold storage there was a significant decrease in cleaved caspase-3 in Lifor stored slices compared that seen following cold storage in saline or UW solution.. Lifor solution mitigates both warm and cold renal IR and appears to provide greater protection from apoptosis compared with UW solution.

Topics: Adenosine; Allopurinol; Animals; Caspase 3; Glutathione; In Situ Nick-End Labeling; Insulin; Kidney; Kidney Transplantation; L-Lactate Dehydrogenase; Male; Nephrectomy; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Renal Circulation; Reperfusion Injury; Transplantation, Homologous

An isolated perfused rat liver model was used to investigate biochemical and histologic changes during 2 hours of reperfusion after 24 hours of cold storage to compare Leeds solution (LS) with University of Wisconsin solution (UW). Compared with livers stored in UW, those perfused with LS showed significantly higher bile flow and lower enzyme production (P < .05 by 1-way analysis of variance). For example, after 120 minutes, alanine aminotransferase results were: LS 38.9 U/L vs UW 66.8 U/L and bile flows were LS 10.3 μg/15 min/g liver vs UW 9.2 μg/15 min/g liver. Histologically the reticulin breakdown was greater and its reformation slower in UW-preserved livers. Liver tissue was viable in both groups, as shown by the increased glycogen content after reperfusion in both groups, but seen at a higher rate among LS, perfused livers. In conclusion, LS compared favorably with UW to prevent ischemic damage and so could offer an alternative perfusion medium to UW.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Analysis of Variance; Animals; Aspartate Aminotransferases; Bile; Cold Ischemia; Glutathione; Glycogen; In Vitro Techniques; Insulin; L-Lactate Dehydrogenase; Liver; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Reticulin; Time Factors; Tissue Survival

Despite the development of immunosuppressive regimens in kidney transplantation, long-term graft survival rates have not increased significantly. One of the causes of long-term graft loss is ischemia-reperfusion insult. Hepatocyte growth factor (HGF) is a regenerative factor produced in response to injury.. Our aim was to assess the effect of HGF and xanthine oxidase (indicators of ischemia/reperfusion insult) on early and late kidney function.. In 17 patients, HGF levels in urine and xanthine oxidase activity in blood were examined 1, 7, 14, 30 days, 3 and 6 months after kidney transplantation. We also measured 24-hour diuresis and serum creatinine levels after transplantation.. Urinary HGF levels were highest 1 day after transplantation. During the following week, it rapidly decreased and was maintained at similar levels in the later period. Creatinine at 1 day showed a positive correlation with urinary HGF levels at 1 day and at 3 months (R = 0.54, P <0.05 and R = 0.82, P <0.01, respectively). Creatinine at 7 days positively correlated with HGF levels at 6 months (R = 0.82, P <0.05). HGF levels at 1 day and at 6 months positively correlated with xanthine oxidase activity at 1 day (R = 0.73, P <0.001 and R = 0.77, P <0.02, respectively). A negative correlation was observed between HGF levels at 6 months and diuresis 1 and 7 days after transplantation (R = -0.99, P <0.00 001 and R = -0.77, P <0.05, respectively).. Urinary HGF is a good marker of perioperative kidney damage and may affect long-term graft function.

Topics: Adult; Aged; Biomarkers; Female; Hepatocyte Growth Factor; Humans; Kidney Transplantation; Male; Middle Aged; Poland; Postoperative Period; Reperfusion Injury; Xanthine Oxidase; Young Adult

To investigate the effects of dextrans of various molecular weights (Mw) during a 12 h cold storage time-course on energetics, histology and mucosal infiltration of fluorescein isothiocyanate (FITC)-dextran.. Rodent intestines were isolated and received a standard University of Wisconsin vascular flush followed by intraluminal administration of a nutrient-rich preservation solution containing dextrans of varying Mw: Group D1, 73 kdal; Group D2, 276 kdal; Group D3, 534 kdal; Group D4, 1185 kdal; Group D5, 2400 kdal.. Using FITC-labeled dextrans, fluorescent micrographs demonstrated varying degrees of mucosal infiltration; lower Mw (groups D1-D3: 73-534 kdal) dextrans penetrated the mucosa as early as 2 h, whereas the largest dextran (D5: 2400 kdal) remained captive within the lumen and exhibited no permeability even after 12 h. After 12 h, median injury grades ranged from 6.5 to 7.5 in groups D1-D4 (73-1185 kdal) representing injury of the regenerative cryptal regions and submucosa; this was in contrast to group D5 (2400 kdal) which exhibited villus denudation (with intact crypts) corresponding to a median injury grade of 4 (P < 0.05). Analysis of tissue energetics reflected a strong positive correlation between Mw and adenosine triphosphate (r(2) = 0.809), total adenylates (r(2) = 0.865) and energy charge (r(2) = 0.667).. Our data indicate that dextrans of Mw > 2400 kdal act as true impermeant agents during 12 h ischemic storage when incorporated into an intraluminal preservation solution.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cold Ischemia; Dextrans; Energy Metabolism; Fluorescein-5-isothiocyanate; Glutathione; Insulin; Intestinal Absorption; Intestinal Mucosa; Intestine, Small; Molecular Weight; Organ Preservation Solutions; Osmosis; Oxidative Stress; Permeability; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors

To investigate the correlation of the ultrasonographic appearance of different degrees of experimentally induced acute unilateral testicular ischemia with the protective effect of allopurinol.. Forty-two male white rabbits were equally randomized into 7 groups: sham-operation control, ischemic A, B and C, and treatment D, E and F. Models of different degrees of unilateral testicular ischemia were established in the ischemic and treatment groups under the dynamic observation by color Doppler ultrasound. The ischemic testes showed slightly decreased homogeneous echoes and flow signals in groups A and D, obviously decreased heterogeneous echoes and flow signals in groups B and E, and radial or fragmental low-echo areas and disappearance of flow signals in groups C and F. The ischemic groups received reperfusion after the appearance of the above ultrasonographic changes, while the treatment groups following the intraperitoneal injection of allopurinol at 200 mg/kg. Contrast-enhanced ultrasonography (CEUS) was performed on the bilateral testes before and 3 days after the reperfusion. After 3 days of breeding, the histological changes and malondialdehyde (MDA) contents of the ischemic testes were observed, and the correlation was analyzed between the protective effect of allopurinol and the ultrasonographic appearance of different degrees of acute unilateral testicular ischemia.. CEUS showed fast wash-in and fast wash-out in the sham-operation control group, slow wash-in and slow wash-out in groups A and B and extensive central filling defect in group C before the reperfusion. Fast wash-in and slow wash-out were observed in all the ischemic groups 3 days after the reperfusion, most obviously in group C. Groups D, E and F exhibited the same CEUS appearance as A, B and C before and 3 days after the reperfusion. Johnsen's scores were significantly increased in groups D (9.10 +/- 0.23) and E (7.03 +/- 0.20) in comparison with A (8.53 +/- 0.22) and B (5.82 +/- 0.33) (P < 0.05), but with no significant differences between C (2.30 +/- 0.53) and F (2.45 +/- 0.33) (P > 0.05). The rates of apoptosis were significantly decreased in groups D ([1.68 +/- 0.43]%) and E ([12.53 +/- 0.59]%) compared with A ([7.12 +/- 0.84]%) and B ([20.87 +/- 1.59]%) (P < 0.05), but with no significant differences between C ([52.93 +/- 2.62 ]%) and F ([51.23 +/- 2.53 ]%) (P > 0.05). Significant decreases of MDA contents in the ischemic testes were observed in groups D ([0.64 +/- 0.05] nmol/mg prot), E ([1.59 +/- 0.06] nmol/mg prot) and F ([3.10 +/- 0.17] nmol/mg prot) in comparison with A ([1.38 +/- 0.07] nmol/mg prot), B ([2.11 +/- 0.08] nmol/mg prot) and C ([3.25 +/- 0.14] nmol/mg prot) (P < 0.05).. Allopurinol contributes to the recovery of spermatogenesis when testicular ischemia is sonographically shown to be mild or moderate, but produces no significant effect when it is shown to be severe. Ultrasonography helps to choose the right therapy of testicular torsion and predict spermatogenesis of ischemic testes after reperfusion.

Topics: Allopurinol; Animals; Disease Models, Animal; Ischemia; Male; Rabbits; Reperfusion Injury; Testicular Diseases; Testis; Ultrasonography

Intestinal ischemia-reperfusion (II/R) induced acute lung injury is mediated by activated neutrophils and formation of free radicals. Several antioxidants have been shown to attenuate such remote organ injury. We studied the effects of zinc aspartate on lung injury induced by II/R in rats.. Twenty-four Sprague-Dawley rats were randomized into three groups. Group I was the control. Animals in Groups II and III (II/R + zinc aspartate [ZA]) underwent 60 min of ischemia and 60 min of reperfusion, respectively. Rats in Group III also received 50 mg/kg zinc aspartate before 15 min of reperfusion. Lung tissue samples and bronchoalveolar lavage fluid were obtained to assess lung tissue myeloperoxidase (MPO), adenosine deaminase (ADA), xanthine oxidase (XO), glutathione peroxidase (GPx) activities, and nitric oxide (NO), malondialdehyde (MDA) levels. Also, the levels of MDA, NO, and MPO activity were determined in bronchoalveolar lavage fluid.. Compared with the control, lung tissue MDA, NO levels, and MPO, ADA, XO activities were markedly increased (P < 0.05), whereas GPx activity significantly decreased in the II/R group (P < 0.05). However, administration of ZA significantly reversed these effects by reducing the levels of MDA, NO, and decreasing MPO, ADA, XO activities (P < 0.05). In addition, ZA significantly increased GPx activity (P < 0.05). The activity of MPO and the levels of NO and MDA were found to be higher in bronchoalveolar lavage fluid in II/R group than the control (P < 0.05). Zinc aspartate significantly diminished MPO activity and the levels of NO and MDA compared with that of control rats (P < 0.05).. Our results indicate that zinc aspartate alleviates lung injury induced by II/R attributable to its antioxidant and antiinflammatory effects.

Topics: Adenosine Deaminase; Animals; Antioxidants; Aspartic Acid; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Free Radicals; Glutathione Peroxidase; Intestines; Lung; Lung Injury; Male; Neutrophils; Nitric Oxide; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Xanthine Oxidase; Zinc

In this work, we evaluated the effects of allopurinol (ALO), an inhibitor of xanthine oxidase (XO), on hepatic lesions caused by ischemia/reperfusion (I/R) in the rabbit liver. Rabbits were pretreated with ALO (10 mg/kg IV) or saline solution 0.9% before the hepatic I/R procedure. The effects of ALO on hepatic injury were evaluated before and after I/R. A standard, warm hepatic I/R procedure caused profound acute liver injury, as indicated by elevated serum aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase levels, as well as a high apoptotic cell count. All of these changes were reversed by the administration of ALO before the hepatic I/R procedure. In conclusion, ALO exerted protective effects on hepatic I/R lesions. This protective effect of ALO was probably associated with blocking the generation of superoxide anions during the hepatic I/R procedure by inhibiting XO activity.

Topics: Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Enzyme Inhibitors; L-Lactate Dehydrogenase; Liver Diseases; Male; Rabbits; Reperfusion Injury; Xanthine Oxidase

An emerging theme in the study of the pathophysiology of persistent pain is the role of reactive oxygen species (ROS). In the present study, we examined the hypothesis that the exogenous supply of antioxidant drugs during peri-reperfusion would attenuate pain induced by ischemia/reperfusion (IR) injury. We investigated the analgesic effects of three antioxidants administered during peri-reperfusion using an animal model of complex regional pain syndrome-type I consisting of chronic post-ischemia pain (CPIP) of the hind paw.. Application of a tight-fitting tourniquet for a period of 3 h produced CPIP in male Sprague-Dawley rats. Low-dose allopurinol (4 mg/kg), high-dose allopurinol (40 mg/kg), superoxide dismutase (SOD, 4000 U/kg), N-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), or SOD (4000 U/kg)+L-NAME (10 mg/kg) was administered intraperitoneally just after tourniquet application and at 1 and 2 days after reperfusion for 3 days. The effects of antioxidants in rats were investigated using mechanical and cold stimuli. Each group consisted of seven rats.. Allopurinol caused significant alleviation in mechanical and cold allodynia for a period of 4 weeks in rats with CPIP. Both SOD and L-NAME, which were used to investigate the roles of superoxide (O2(-)) and nitric oxide (NO) in pain, also attenuated neuropathic-like pain symptoms in rats for 4 weeks.. Our findings suggest that O2(-) and NO mediate IR injury-induced chronic pain, and that ROS scavengers administered during the peri-reperfusion period have long-term analgesic effects.

Topics: Allopurinol; Animals; Chronic Disease; Cold Temperature; Enzyme Inhibitors; Free Radical Scavengers; Ischemia; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Pain; Physical Stimulation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

Oxidants and their generator, xanthine oxidase (XO), play a major role in the damaging of the structural and functional integrity of the lung. Such damage has been recently demonstrated in the presence of pancreas ischemia-reperfusion (IR). We investigated whether mannitol, a clinically used agent and antioxidant, prevented lung damage after pancreas IR. Rats (n = 48) were anesthetized, after which each pancreas was isolated and perfused (controls), or made ischemic (IR) for 40 min, or made ischemic and treated upon reperfusion with four different doses of mannitol administered in the perfusate (8 replicates/group). Ischemia was followed by in-series 15-min pancreas plus normal isolated lung reperfusion. Isolated lungs were subsequently perfused for 45 min with the 15-min accumulated effluents. Pancreas injury occurred in all IR organs as demonstrated by abnormal reperfusion pressure, the wet-to-dry ratio, amylase and lipase leakage into the circulation, and XO activity and reduced glutathione (GSH) pool in the tissues. Pulmonary plateau pressure increased by 80%, and final PO(2)/FiO(2) decreased by 28% in the IR-untreated paired lungs. Bronchoalveolar lavage volume increased by 50% and 2- to 8-fold increase in their contained XO and GSH were recorded as well. The above indices of injury in lungs perfused with 0.77 mM mannitol were the least detected, compared with negligible efficacy of other (0.55 < 0.22 < 1.1 mM) dosages. Amylase and lipase did not contribute to lung injury. Ex vivo acute pancreatitis induces acute lung injury via oxidants/antioxidants imbalance, which is preventable by mannitol.

Topics: Acute Lung Injury; Amylases; Animals; Antioxidants; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glutathione; Lipase; Lung; Male; Mannitol; Oxidative Stress; Pancreas; Pancreatic Diseases; Rats; Rats, Wistar; Reperfusion Injury; Time Factors; Xanthine Oxidase

Diabetes mellitus is characterized by a chronic hyperglycemia and might cause skin pathologies resulting from an ischemic insult. A variety of mechanisms have been suggested for the damage provided by ischemia-reperfusion injury (IRI) or for hyperglycemic conditions. Yet, the association between IRI and hyperglycemia together in skin has been poorly investigated even thought they are both present in diabetic patients.. To examine the effect of a dual stress combining IRI and hyperglycemia on human keratinocytes-its ability to cause oxidative damage and inflammatory response via the enzymes xanthine oxidase (XO) and inducible nitric oxide synthase (iNOS).. HaCaT cells were used as a model to induce IRI and hyperglycemia. In order to assess the oxidative damage, total antioxidant scavenging capacity (TSC) and GSH/GSSG ratio were evaluated. iNOS expression was evaluated and its metabolite nitric oxide was estimated by measuring nitrite levels. XO activity was assessed by uric acid quantification and by superoxide radical formation. Inflammatory response was determined through interleukin-6 secretion.. Our observations demonstrate different responses of the cells exposed to single stress (IRI) compared to dual stress combining also hyperglycemia. However, cells response exhibited similarity during reperfusion, by enhancing iNOS expression as well as superoxide levels. While ischemia led to changes in TSC and redox state, reperfusion restored them to basal levels. IRI also caused the enhancement of secreted IL-6 and uric acid levels.. iNOS and XO play a major role in IRI and hyperglycemia. Inhibition of one of these enzymes may be beneficial to skin cells under these conditions.

Topics: Biomarkers; Cell Hypoxia; Cell Line; Cell Survival; Glucose; Glutathione; Glutathione Disulfide; Humans; Hyperglycemia; Inflammation Mediators; Interleukin-6; Ischemia; Keratinocytes; Nitric Oxide; Nitric Oxide Synthase Type II; Nitrites; Oxidative Stress; Reperfusion Injury; Superoxides; Uric Acid; Xanthine Oxidase

Oxidants (and their generator, xanthine oxidase [XO]) play a role in inducing acute lung injury (ALI) expressed both structurally and functionally. Such damage has recently been demonstrated in the presence of pancreas ischemia-reperfusion (IR). We now investigated whether methylene blue (MB), a clinically used coloring agent and antioxidant in itself, protected the lung exposed to pancreas IR.. Isolated pancreata (eight replicates/group) were (1) continuously perfused (controls), (2) made ischemic (IR-0) for 40 min and reperfused without treatment, (3) organs procured from allopurinol-treated rats made ischemic and reperfused with allopurinol, and (4) made ischemic and treated upon reperfusion with three different doses of MB contained in the perfusate. All perfusate solutions were directed into the isolated lungs' circulation whereby they were perfused for 60 min.. Pancreas injury was documented in all IR organs by abnormally high reperfusion pressure, wet-to-dry ratio, amylase and lipase concentrations, and abnormal XO activity and reduced glutathione in the circulation. Lungs paired with IR-0 pancreata developed approximately 60% increase in ventilatory plateau pressure and final PO(2)/FiO(2) decrease by 35%. Their weight during reperfusion and bronchoalveolar lavage (BAL) volume and contents increased 1.5-2.5 times the normal values; XO and reduced glutathione values were abnormal both in the BAL and in the lung tissues. Lungs exposed to IR effluents containing allopurinol or 68 microM MB were minimally damaged, whereas perfusion solutions containing 42 or 128 microM MB were ineffective in preventing lung injury.. Ex vivo pancreas IR-induced ALI is preventable by MB, although at a narrow dose range.

Topics: Acute Lung Injury; Allopurinol; Amylases; Analysis of Variance; Animals; Antioxidants; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Dose-Response Relationship, Drug; Glutathione; Lipase; Male; Methylene Blue; Pancreas; Probability; Pulmonary Circulation; Random Allocation; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Oxidase

We compared the susceptibility of liver grafts from lean and obese Zucker rats to preservation injury, using two organ-preservation techniques: conventional static preservation (SP) and machine perfusion (MP) preservation. SP: livers preserved by UW solution at 4, 8 or 20 degrees C for 6-h. MP: livers perfused for 6-h with an improved oxygenated Krebs-Henseleit solution (KH) at 4, 8 or 20 degrees C. Reperfusion with KH (2-h) was performed either with the SP or MP preserved livers. Fatty livers tolerate SP poorly at 4, 8 and 20 degrees C as compared with MP at the same temperatures. SP induced a decrease in the ATP/ADP ratio both at 8 and 20 degrees C in obese rats while an increase in energy status was found with MP at 8 and 20 degrees C. Nitrate/nitrite (NOx) concentration was higher and bile flow lower in livers preserved with SP than MP. In lean rats, no differences were observed between MP and SP as regards enzyme release, bile production and NOx levels except for SP at 20 degrees C in which high enzyme release and low bile flow were observed. In lean rats ATP/ADP was higher and NOx was lower with MP at 20 degrees C than with SP at 20 degrees C. To optimize steatotic liver preservation SP should be avoided because it is particularly detrimental as compared with MP.

Topics: Adenosine; Allopurinol; Animals; Cryopreservation; Fatty Liver; Glucose; Glutathione; Insulin; Liver Transplantation; Male; Obesity; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Zucker; Reperfusion Injury; Tromethamine

We recently reported that excessive superoxide anion radical (O(2)(-)) was generated in the jugular vein during reperfusion in rats with forebrain ischemia/reperfusion using a novel electrochemical sensor and excessive O(2)(-) generation was associated with oxidative stress, early inflammation, and endothelial injury. However, the source of O(2)(-) was still unclear. Therefore, we used allopurinol, a potent inhibitor of xanthine oxidase (XO), to clarify the source of O(2)(-) generated in rats with forebrain ischemia/reperfusion. The increased O(2)(-) current and the quantified partial value of electricity (Q), which was calculated by the integration of the current, were significantly attenuated after reperfusion by pretreatment with allopurinol. Malondialdehyde (MDA) in the brain and plasma, high-mobility group box 1 (HMGB1) in plasma, and intercellular adhesion molecule-1 (ICAM-1) in the brain and plasma were significantly attenuated in rats pretreated with allopurinol with dose-dependency in comparison to those in control rats. There were significant correlations between total Q and MDA, HMGB, or ICAM-1 in the brain and plasma. Allopurinol pretreatment suppressed O(2)(-) generation in the brain-perfused blood in the jugular vein, and oxidative stress, early inflammation, and endothelial injury in the acute phase of forebrain ischemia/reperfusion. Thus, XO is one of the major sources of O(2)(-)- in blood after reperfusion in rats with forebrain ischemia/reperfusion.

Topics: Allopurinol; Analysis of Variance; Animals; Dose-Response Relationship, Drug; Enzyme Inhibitors; HMGB1 Protein; Immunoassay; Intercellular Adhesion Molecule-1; Male; Malondialdehyde; Oxidative Stress; Prosencephalon; Rats; Rats, Wistar; Reperfusion Injury; Superoxides; Xanthine Oxidase

Hepatic ischemia/reperfusion (I/R) injury significantly influences short-term and long-term outcomes after liver transplantation (LTx). The critical step initiating the injury is known to include sinusoidal endothelial cell (SEC) alteration during the cold preservation period. As carbon monoxide (CO) has potent cytoprotective functions on vascular endothelial cells, this study examined if CO treatment of excised liver grafts during cold storage could protect SECs and ameliorate hepatic I/R injury. Rat liver grafts were preserved in University of Wisconsin (UW) solution containing 5% CO (CO-UW solution) for 18 to 24 hours and were transplanted into syngeneic Lewis rats. After 18 hours of cold preservation, SEC damage was evident with propidium iodide (PI) nuclear staining on SECs, and the frequency of PI(+) SECs was significantly lower in grafts stored in CO-UW solution versus those stored in control UW solution. SEC protection with CO was associated with decreased intercellular cell adhesion molecule translocation and less matrix metalloproteinase release during cold preservation. After LTx with 18 hours of cold preservation, serum alanine aminotransferase levels and hepatic necrosis were significantly less in the CO-UW group than in the control UW group. With 24 hours of cold storage, 35% (7/20) survived with control UW solution, whereas the survival with CO-UW solution improved to 80% (8/10). These beneficial effects of CO-UW solution were associated with a significant reduction of neutrophil extravasation, down-regulation of hepatic messenger RNA for tumor necrosis factor alpha and intercellular cell adhesion molecule 1, and less hepatic extracellular signal-regulated kinase activation. Liver grafts from Kupffer cell-depleted donors or pseudogerm-free donors showed less SEC death during cold preservation, and CO-UW solution further reduced SEC death. In conclusion, CO delivery to excised liver grafts during cold preservation efficiently ameliorates SEC damage and hepatic I/R injury.

Topics: Adenosine; Allopurinol; Animals; Carbon Monoxide; Cryopreservation; Cryoprotective Agents; Endothelial Cells; Glutathione; Graft Survival; Hepatitis; Insulin; Kupffer Cells; Liver Transplantation; Male; MAP Kinase Signaling System; Matrix Metalloproteinases; Neutrophils; Organ Preservation; Organ Preservation Solutions; Primary Graft Dysfunction; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

This study was planned to investigate the neuroprotective potentials of three commercially available prostaglandin analogues (PGA), in the ischemia and reperfusion model (I/R). Thirty New Zealand rabbits were divided into 5 groups and except for the control group (non-ischemic, non-treated), 0.9% NaCl, bimatoprost, latanoprost, or travoprost were applied to both eyes of animals of the respective groups for 1 week. At the end of treatment, ischemia was induced in both eyes of the 4 treatment groups by anterior chamber irrigation of the animals for 60 min. Following 24 h reperfusion, the animals were sacrificed. Enucleated eyes and retinal tissues were investigated by light microscopy, electron microscopy, immunohistochemicstry for retinal histopathology, intracellular and apoptotic cells and by retinal morphometry. Vitreous samples were biochemically investigated for probable role of reactive oxygen species, by measuring xanthine oxidase (XO) activity. Analysis of morphometric measurements and vitreous XO activity revealed significant differences between the PGA-treated groups and the NaCl-treated group (P<0.05). Similarly, apoptotic cell counts in different retinal layers showed that PGA-treated groups had fewer apoptotic cells in all retinal layers than the NaCl-treated ischemic group (P<0.05). PGA may have high protective potential for different retinal layers and cells. Biochemical analysis of vitreous showed that all PGAs decreased vitreous XO activity significantly compared to the NaCl-treated group (P<0.05). However we could not find any statistically significant differences among the analogues. PGAs may reduce the injury induced by I/R, through the inhibition of XO activity, and it seems that their effects are elicited through numerous pathways.

Topics: Amides; Animals; Antihypertensive Agents; Apoptosis; Bimatoprost; Cloprostenol; Disease Models, Animal; Latanoprost; Male; Neuroprotective Agents; Prostaglandins F, Synthetic; Prostaglandins, Synthetic; Rabbits; Reactive Oxygen Species; Reperfusion Injury; Retina; Retinal Diseases; Travoprost; Vitreous Body; Xanthine Oxidase

Several studies have been carried out investigating different preservation methods and preservation solutions for the pancreata of various species. Attention has to be drawn to the extreme vulnerability of porcine pancreata (PP) to oxidative stress due to the lack of endogenous antioxidants. This study sought to evaluate the influence of cannulation and infusion of different volumes of University of Wisconsin (UW) solution immediately after organ retrieval on PP organ quality.. PP from 24 slaughterhouse pigs were harvested with immediate cannulation of the pancreatic duct for infusion of 10 mL, 20 mL, 50 mL, or 100 mL UW solution. The organs were stored in cold UW solution. Control organs were only stored in UW. After 6 hours of cold ischemia, tissue and supernate samples were analyzed for markers of oxidative cell damage, adenosine triphosphate (ATP) levels, and occurrence of apoptosis.. The fewest apoptotic cells were detected in the PP infused with 50 mL UW via the pancreatic duct (PP 50) as compared with all other groups. Oxidative cell damage was lowest and ATP levels were highest in the PP 50 group.. Because PP 50 showed significantly better results when compared with all other groups, we suggest that infusion of 50 mL UW via the pancreatic duct immediately after organ retrieval may be useful to minimize oxidative cell damage and cell death in PP.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Insulin; Lipase; Models, Animal; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Pancreas; Pancreas Transplantation; Raffinose; Reperfusion Injury; Swine; Tissue and Organ Harvesting

Cold flush preservation prolongs tissue viability during ischemia. However, there is little understanding of the effects of various preservation fluids on events during this period. A study of cold ischemia in rat livers was undertaken to compare biochemical and histological changes over time, using three preservation solutions: University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), and Leeds solution (LS) under development at our institution. Leeds solution is a phosphate-based sucrose solution that like UW contains the impermeant lactobionate and the metabolite allopurinol (1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one) which acts as a competitive inhibitor of xanthine oxidase, stopping the breakdown of hypoxanthine to xanthine by oxidizing it to alloxanthine, inhibiting both the conversion of hypoxanthine to xanthine and the conversion of xanthine to uric acid.. At various time points, samples were analyzed for adenosine triphospate (ATP) and metabolites by high-performance liquid chromatography as well as for histological changes.. In all livers, ATP, ADP, and AMP degraded over 4 hours. In UW and LS groups, degradation beyond hypoxanthine was halted, and it continued in the HTK group. This blockade led to a significant reduction in the accumulation of xanthine and uric acid. Histological analysis showed protected architecture and maintenance of reticulin scaffolds in the UW and LS groups, whereas tissue breakdown was seen from earlier time points in the HTK group. Additionally, throughout ischemia, signs of pathological injury were more pronounced with UW- than with LS-preserved tissue.. These results implied that cold ischemia in the liver is characterized by dynamic biochemical changes coincident with pathological injury which are initiated from the time of organ perfusion and influenced by the choice of the perfusion fluid. Allopurinol in UW and LS appears to be critical. We hypothesized that it may also affect the degree of subsequent reperfusion injury. The data supported the assertion that LS offerred improved preservation over UW, adding to the impetus to shorten ischemic times in clinical transplantation.

Topics: Adenosine; Adenosine Monophosphate; Adenosine Triphosphate; Allopurinol; Animals; Energy Metabolism; Glutathione; Hypoxanthine; Insulin; Ischemia; Kinetics; Liver; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Reticulin; Uric Acid; Uridine; Xanthine

To evaluate the effect of curcumin, a potent antioxidant, on testicular ischemia-reperfusion injury caused by overgeneration of reactive oxygen species (ROS) after testicular torsion-detorsion.. Controlled experimental study using rats.. Research laboratory.. Sixty adult male Sprague-Dawley rats.. Rats in the control group underwent a sham operation of the left testis. In the torsion-detorsion group, the left testis was rotated 720 degrees for 2 hours. Rats in treatment group received the same surgical procedure as the torsion-detorsion group, but curcumin was administered IV at repair of testicular torsion.. Testicular activity of xanthine oxidase, which catalyzes production of ROS; malondialdehyde level (an indicator of ROS content); protein expression level of heme oxygenase-1, which catalyzes antioxidant generation; and spermatogenesis.. Unilateral testicular torsion-detorsion caused significant increases in xanthine oxidase activity, malondialdehyde level, and heme oxygenase-1 protein expression level and caused a significant decrease in testicular spermatogenesis in ipsilateral testes. The rats treated with curcumin had significant decreases in xanthine oxidase activity and malondialdehyde level and had significant increases in heme oxygenase-1 protein expression level and testicular spermatogenesis in ipsilateral testes, compared with the torsion-detorsion group.. The curcumin exerts a protective effect on testicular ischemia-reperfusion injury.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Curcumin; Disease Models, Animal; Male; Malondialdehyde; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Spermatic Cord Torsion; Testis; Xanthine Oxidase

Inorganic nitrite (NO(2)(-)) is emerging as a regulator of physiological functions and tissue responses to ischemia, whereas the more stable nitrate anion (NO(3)(-)) is generally considered to be biologically inert. Bacteria express nitrate reductases that produce nitrite, but mammals lack these specific enzymes. Here we report on nitrate reductase activity in rodent and human tissues that results in formation of nitrite and nitric oxide (NO) and is attenuated by the xanthine oxidoreductase inhibitor allopurinol. Nitrate administration to normoxic rats resulted in elevated levels of circulating nitrite that were again attenuated by allopurinol. Similar effects of nitrate were seen in endothelial NO synthase-deficient and germ-free mice, thereby excluding vascular NO synthase activation and bacteria as the source of nitrite. Nitrate pretreatment attenuated the increase in systemic blood pressure caused by NO synthase inhibition and enhanced blood flow during post-ischemic reperfusion. Our findings suggest a role for mammalian nitrate reduction in regulation of nitrite and NO homeostasis.

Topics: Adult; Allopurinol; Animals; Blood Pressure; Disease Models, Animal; Female; Homeostasis; Humans; Liver; Male; Mice; Middle Aged; Nitrate Reductase; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Organ Specificity; Oxidation-Reduction; Rats; Reperfusion Injury; Xanthine Dehydrogenase

Activated protein C (APC) is a serine protease with anticoagulant and ant-inflammatory activities. APC has been shown to attenuate deleterious effects of ischemia/reperfusion (I/R) injury in many organs. In this study, we aimed to investigate the effects of APC on intestinal mucosal injury induced by superior mesenteric occlusion.. Male Wistar-albino rats were allocated into four groups: (1) sham-operated group, laparotomy without I/R injury (n = 12); (2) sham + APC group, identical to Group 1 except for APC treatment (n = 12); (3) I/R group, 60 min of ischemia followed by 3-h of reperfusion (n = 12); and (4) I/R + APC-treated group, 100 mug/kg injection of APC intravenously, 15 min before reperfusion (n = 12). We evaluated the degree of intestinal mucosal injury on a grading scale from 0 to 5, histopathologically, and by measuring activities of oxidative and antioxidative enzymes as well as nitrate/nitrite levels, biochemically. Intestinal edema was estimated by using wet/dry weight ratios. The plasma levels of proinflammatory cytokines and D-dimer were measured. Animal survival was observed up to 1 wk.. Intestinal mucosal injury scores were significantly decreased with APC administration (P < 0.05). APC treatment significantly reduced activities of oxidative enzymes and nitrate/nitrite levels in the intestinal tissues, and plasma levels of proinflammatory cytokines and D-dimer, and also significantly increased activities of antioxidative enzymes in the intestinal tissues (P < 0.05). Intestinal edema was significantly alleviated with APC treatment (P < 0.05). The survival rate of rats in the APC-treated group were significantly higher than that of the I/R-treated group (P < 0.05).. This study clearly showed that APC treatment significantly attenuated intestinal mucosal injury caused by superior mesenteric ischemia/reperfusion. Further clinical studies are required to clarify whether APC has a useful role in reperfusion injury during particular surgeries in which I/R-induced organ injury occurs.

Topics: Animals; Anticoagulants; Edema; Fibrin Fibrinogen Degradation Products; Glutathione; Glutathione Reductase; Interleukin-6; Intestinal Mucosa; Male; Malondialdehyde; Mesenteric Artery, Superior; Mesenteric Vascular Occlusion; Nitrates; Nitrites; Peroxidases; Protein C; Rats; Rats, Wistar; Reperfusion Injury; Tumor Necrosis Factor-alpha; Warm Ischemia; Xanthine Oxidase

To study the effect of oral administration of a nitric oxide (NO) donor L-arginine (L-Arg), a NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and an inhibitor of xanthine oxidase, allopurinol (Allo), on serum NO concentration and catalase activity after intestinal ischemia/reperfusion (I/R) in rats.. Male Wistar rats receivedper os L-Arg (800 mg/kg) or L-NAME (50 mg/kg) or Allo (100 mg/kg) 24 hrs, 12 hrs and 1 hr before underwent 1 hr occlusion of superior mesenteric artery followed by 1 hr of reperfusion (L-Arg(IR1), L-NAME(IR1) and Allo(IR1) respectively) or 1 hr occlusion followed by 8 hrs of reperfusion (L-Arg(IR8), L-NAME(IR8) and Allo(IR8) respectively). There was one group underwent 1 hr occlusion (I), a group underwent 1 hr occlusion followed by 1 hr reperfusion (IR1), a group subjected to 1 hr occlusion followed by 8 hrs of reperfusion (IR8) and a last group that served as control (C). Serum NO concentration and catalase activity were measured.. After 1 hr of reperfusion serum NO concentration was elevated in IR1 and L-Arg(IR1) groups compared with group C but not in L-NAME(IR1) and Allo(IR1) group. Catalase activity was enhanced in L-NAME(IR1) group. Interestingly, serum NO concentration was increased after 8 hrs of reperfusion in all groups (IR8, L-Arg(IR8), L-NAME(IR8) and Allo(IR8)) compared with control while catalase activity did not show significant difference in any group.. The results of the present study show that NO concentration is elevated in serum after intestinal I/R and the elevation sustained after administration of L-Arg but not after administration of L-NAME or Allo after 1 hr reperfusion. However, after 8 hrs of reperfusion NO concentration was increased in all groups studied, focusing attention on its possible important role in a complicated situation such as intestinal I/R that involves intestine and other organs. Serum catalase activity does not seem to be affected by per os supplementation of L-Arg or Allo in intestinal I/R.

Topics: Administration, Oral; Allopurinol; Animals; Arginine; Catalase; Disease Models, Animal; Enzyme Inhibitors; Intestinal Mucosa; Intestines; Male; Mesenteric Artery, Superior; Mesenteric Vascular Occlusion; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Rats; Rats, Wistar; Reperfusion Injury; Time Factors; Xanthine Oxidase

To study the effect of prophylactic agents allopurinol, verapamil, dexamethasone and trifluoperazine on ischemic intestinal damage.. Sixty albino Wistar rats weighing 120-150 g were used. Group 1 animals underwent sham laparotomy without ischemia-reperfusion injury. Group 2 animals underwent laparotomy and occlusion of superior mesenteric arteries for 30 minutes followed by 20 minutes of reperfusion without pretreatment. The allopurinol group received 50 mg/kg/day allopurinol intraperitoneally 2 days before ischemia-reperfusion injury. The verapamil group was given 0.3 mg/kg verapamil intravenously 5 minutes before laparotomy. The dexamethasone group was given 1.5 mg/kg dexamethasone intraperitoneally before laparotomy. The trifluoperazine group was given 10 mg/kg trifluoperazine intraperitoneally 6 hours before laparotomy.. In the allopurinol group, generally the mucosa and villar structure were protected. In the verapamil group, shedding of cells at the tip of the villi is apparent. In the dexamethasone group, desquamation of the villar apex was seen. In the trifluoperazine group, edema and necrosis were seen in the mucosa. In statistical analysis of the MDA level, data were obtained after a respective measurement in all groups.. In this study histopathologic results with agents that are effective on preventing the neutrophyle functions are mostly achieved.

Topics: Allopurinol; Animals; Dexamethasone; Intestine, Small; Male; Rats; Rats, Wistar; Reperfusion Injury; Trifluoperazine; Verapamil

Oxidative stress may have a role in liver damage after acute renal injury due to various reasons such as ischemia reperfusion (IR). Diabetes mellitus (DM) is an important disease for kidneys and may cause nephropathy as a long term complication. The aim of this study was to investigate protective effect of melatonin, a potent antioxidant, against distant organ injury on liver induced by renal IR in rats with or without DM. The rats were divided into six groups: control (n=7), DM (n=5), IR (n=7), DM+IR (n=7), melatonin+IR (Mel+IR) (melatonin, 4 mg/ kg during 15 days) (n=7), and Mel+DM+IR groups (n=7). Diabetes developed 3 days after single i.p. dose of 45 mg/kg streptozotocin. After 15 day, the left renal artery was occluded for 30 min followed 24 h of reperfusion in IR performed groups. DM did not alter oxidative parameters alone in liver tissue. The levels of malondialdehyde, protein carbonyl and nitric oxide with activities of xanthine oxidase and myeloperoxidase were increased in liver tissues of diabetic and non-diabetic IR groups. Nitric oxide level in DM was higher than control. The activities of catalase and superoxide dismutase were increased in IR groups in comparison with control and DM. ALT and AST levels were higher in IR and DM+IR groups than control and DM. Melatonin treatment reversed all these oxidant and antioxidant parameters to control values as well as serum liver enzymes. We concluded that renal IR may affect distant organs such as liver and oxidative stress may play role on this injury, but DM has not an effect on kidney induced distant organ injury via oxidant stress. Also, it was concluded that melatonin treatment may prevent liver oxidant stress induced by distant injury of kidney IR.

Topics: Alanine Transaminase; Animals; Antioxidants; Aspartate Aminotransferases; Catalase; Diabetes Mellitus, Experimental; Kidney; Liver; Liver Diseases; Male; Malondialdehyde; Melatonin; Nitric Oxide; Oxidative Stress; Peroxidase; Protein Carbonylation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

The activation of p38 mitogen-activated protein kinases (MAPK) is implicated in cold ischemia-reperfusion injury of donor organs. The islet isolation process, from pancreas procurement through islet collection, may activate p38MAPK leading to cytokine release and islet damage. This damage may be prevented by treating pancreata with a p38MAPK inhibitor (p38IH) before cold preservation.. Pancreata removed from Beagle dogs were infused with University of Wisconsin solution containing the p38IH, SB203580, and Pefabloc (n=6) or vehicle (dimethyl sulfoxide and Pefabloc) alone (n=7), through the pancreatic duct and preserved using the two-layer method. After 20 to 22 hr, islets were isolated and 3000 IEQ/kg were autotransplanted into the corresponding dog to monitor glucose metabolism.. p38IH-treated pancreata yielded significantly more islets than control pancreata (IEQ/g: 2134+/-297 vs. 1477+/-145 IEQ/g or 65,012+/-9385 vs. 45,700+/-5103 IEQ/pancreas; P<0.05). Apoptotic beta-cell percentages assessed by laser scanning cytometry were lower in p38IH-treated than the controls (44%+/-9.4% vs. 61.6%+/-4.8%, P<0.05). Tumor necrosis factor-alpha expression assessed by real-time reverse transcription polymerase chain reaction was significantly lower in the p38IH-treated group than controls. All dogs (3000 IEQ/kg) transplanted with p38IH-treated islets (n=5) became euglycemic versus four of five dogs that received untreated islets. Plasma C-peptide levels after glucagon challenge were higher in animals receiving p38IH-treated islets (n=5) versus untreated islets (n=4) (0.40+/-0.78 vs. 0.21+/-0.05 ng/mL, P<0.05).. Infusion of pancreata with University of Wisconsin solution containing p38IH through the duct before preservation suppresses cytokine release, prevents beta-cell apoptosis, and improves islet yield significantly with no adverse effect on islet function after transplantation. p38IH treatment of human pancreata may improve islet yield for use in clinical transplantation.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Cytokines; Dogs; Enzyme Activation; Enzyme Inhibitors; Glutathione; Insulin; Insulin-Secreting Cells; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Organ Preservation; Organ Preservation Solutions; p38 Mitogen-Activated Protein Kinases; Raffinose; Reperfusion Injury

Hypoxic pulmonary vasoconstriction (HPV) is a well-known phenomenon to temporarily offset a ventilation/perfusion mismatch. Sustained HPV may lead to pulmonary hypertension. In this protocol, we studied the relationships between the HPV response and oxygen radical release after hypoxia/reoxygenation (H/R) challenge in an isolated perfused lung model.. We used an in situ isolated rat lung preparation. Two hypoxic challenges (5% CO2-95% N2) were administered for 10 minutes each with administration of antioxidants of superoxide dismutase (SOD; 2 mg/kg), catalase (20,000 IU/kg), dimethylthiourea (DMTU; 100 mg/kg), dimethylsulfoxide (DMSO; 1 mL/kg), or allopurinol (30 mg/kg) between 2 challenges. We measured pulmonary arterial pressure changes before, during, and after H/R challenge. We measured blood concentration changes in hydroxyl radicals and nitric oxide (NO) before and after H/R. mRNA expressions of SOD and catalase in lung tissue were measured after the experiments.. Hypoxia induced pulmonary vasoconstriction by increasing pulmonary arterial pressure and consecutive hypoxic challenges did not show tachyphylaxis. Blood concentrations of hydroxyl radicals and NO increased significantly after H/R challenges. mRNA expressions of SOD and catalase increased significantly, however, neither SOD nor catalase showed attenuated effects on HPV responses. Small molecules of DMTU, DMSO, and allopurinol attenuated the HPV responses.. H/R induced increases in the expressions of SOD and catalase in lung tissues. DMTU, DMSO, and allopurinol antioxidants attenuated the HPV responses by reducing the oxygen radical release.

Topics: Allopurinol; Animals; Antioxidants; Catalase; Dimethyl Sulfoxide; Free Radical Scavengers; Gene Expression Regulation, Enzymologic; Hypoxia; Lung; Polymerase Chain Reaction; Pulmonary Artery; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Superoxide Dismutase; Thiourea; Vasoconstriction

Ischemia-reperfusion (IR) injury is a major insult to postcapillary venules. We hypothesized that IR increases postcapillary venular hydraulic conductivity and that IR-mediated changes in hydraulic conductivity result from temporally and mechanistically separate processes. A microcannulation technique was used to determine hydraulic conductivity (Lp) in rat mesenteric postcapillary venules serially throughout ischemia (45 min) and reperfusion (5 h) induced by superior mesenteric artery occlusion and release. Mesenteric IR resulted in a biphasic increase in Lp. White blood cell (WBC) adhesion slowly increased with maximal adhesion corresponding to the second peak (P < 0.005). After IR, tissue was harvested for RT-PCR analysis of ICAM-1, E-selectin, and P-selectin mRNA. Intercellular adhesion molecule-1 (ICAM-1) mRNA in the gut showed the most significant upregulation. Quantitative real-time PCR revealed that ICAM-1 mRNA was upregulated 60-fold in the gut. An ICAM-1 antibody was therefore used to determine the effect of WBC adhesion on Lp during IR. ICAM-1 inhibition attenuated Lp during the first peak and completely blocked the second peak (P < 0.005). When rats were fed a tungsten diet to inhibit xanthine oxidase and then underwent IR, Lp was dramatically attenuated during the first peak and mildly decreased the second peak (P < 0.005). Inhibition of xanthine oxidase by oxypurinol decreased Lp during IR by over 60% (P < 0.002). Tempol, a superoxide dismutase mimetic, decreased Lp during IR by over 30% (P < 0.01). We conclude that IR induces a biphasic increase in postcapillary hydraulic conductivity. Reactive oxygen species impact both the first transient peak and the sustained second peak. However, the second peak is also dependent on WBC-endothelial cell adhesion. These serial measurements of postcapillary hydraulic conductivity may lead the way for optimal timing of pharmaceutical therapies in IR injury.

Topics: Animals; Antibodies; Cell Adhesion; Cricetinae; Cyclic N-Oxides; Disease Models, Animal; E-Selectin; Endothelium, Vascular; Enzyme Inhibitors; Female; Intercellular Adhesion Molecule-1; Leukocytes; Male; Mesentery; Mesocricetus; Oxypurinol; P-Selectin; Permeability; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; RNA, Messenger; Spin Labels; Time Factors; Tungsten Compounds; Venules; Xanthine Oxidase

Tumour necrosis factor-alpha (TNF-alpha) plays a key role in causing ischaemia/reperfusion (I/R) injury. I/R also causes activation of xanthine oxidase and dehydrogenase (XDH + XO) system that, via generated free radicals, causes organ damage. We investigated the effect of ischaemia, reperfusion and non-ischaemic prolonged perfusion (NIP) on TNF-alpha and XDH + XO production in an isolated perfused rat liver model.. Rat livers underwent 150 min NIP (control group) or two hours of ischaemia followed by reperfusion (I/R group). TNF-alpha (TNF-alpha mRNA and protein level), XDH + XO production and bile secretion were determined in tissue and effluent at baseline, at 120 min of ischaemia, after 30 min of reperfusion (I/R group) and after 120 and 150 min of prolonged perfusion (control).. Unexpectedly, neither ischaemia nor reperfusion had any effect on TNF-alpha production. TNF-alpha in effluent was 11 +/- 4.8 pg mL(-1) at baseline, 7 +/- 3.2 pg mL(-1) at the end of ischaemia, and 13 +/- 5.3 pg mL(-1) after 30 min of reperfusion. NIP, however, caused a significant increase of TNF-alpha synthesis and release. TNF-alpha effluent level after 120 and 150 min of perfusion was 392 +/- 78.7 pg mL(-1) and 408 +/- 64.3 pg mL(-1), respectively. TNF-alpha mRNA in tissue was also significantly elevated compared to baseline levels (1.31 +/- 0.2 P < 0.001 and 1.38 P < 0.002, respectively). Decrease of liver function (expressed by bile secretion) during I/R and NIP was accompanied by significant XDH + XO elevation.. This is the first evidence that NIP, and not I/R, is the decisive trigger for TNF-alpha production. This study leads to a better understanding of pathogenesis of liver I/R and perfusion damage.

Topics: Animals; Liver; Male; Rats; Rats, Wistar; Reperfusion Injury; Tumor Necrosis Factor-alpha; Xanthine Dehydrogenase; Xanthine Oxidase

To determine if blockade of P-selectin in the isolated blood-perfused cold ex vivo rat liver model protects the liver from ischemia-reperfusion injury.. The effect of P-selectin blockade was assessed by employing an isolated blood-perfused cold ex vivo rat liver with or without P-selectin antibody treatment before and after 6 h of cold storage in University of Wisconsin solution.. In our isolated blood-perfused rat liver model, pre-treatment with P-selectin antibody failed to protect the liver from ischemia-reperfusion injury, as judged by the elevated aspartate aminotransferase activity. In addition, P-selectin antibody treatment did not significantly reduced hepatic polymorphonuclear leukocyte accumulation after 120 min of perfusion. Histological evaluation of liver sections obtained at 120 min of perfusion showed significant oncotic necrosis in liver sections of both ischemic control and P-selectin antibody-treated groups. However, total bile production after 120 min of perfusion was significantly greater in P-selectin antibody-treated livers, compared to control livers. No significant difference in P-selectin and ICAM-1 mRNAs and proteins, GSH, GSSG, and nuclear NF-kappaB was found between control and P-selectin antibody-treated livers.. In conclusion, we have shown that blockade of P-selectin alone failed to reduced polymorphonuclear leukocyte accumulation in the liver and protect hepatocytes from ischemia-reperfusion injury in the isolated blood-perfused cold-ex vivo rat liver model.

Topics: Adenosine; Allopurinol; Animals; Antibodies; Aspartate Aminotransferases; Bile; Cold Ischemia; Glutathione; Glutathione Disulfide; In Vitro Techniques; Insulin; Intercellular Adhesion Molecule-1; Liver; Male; Necrosis; Neutrophils; NF-kappa B; Organ Preservation Solutions; P-Selectin; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Time Factors

University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) solutions are the 2 most commonly used liver preservation solutions. The aim of this study was to compare cardiovascular stability, acid-base status, and potassium concentrations between patients who received grafts preserved in either UW or HTK solution in orthotopic liver transplantation (OLT).. In this retrospective study, 87 patients who underwent living donor OLT were divided into 2 groups: UW (n = 28) and HTK (n = 59). Group HTK was subdivided into group NF-HTK (n = 31; nonflushed before reperfusion) and group F-HTK (n = 28; flushed before reperfusion). We determined mean arterial pressure (MAP) and heart rate every minute for 5 minutes after reperfusion and the maximum change in these values and incidence of postreperfusion syndrome (PRS). Body temperature, cardiovascular and acid-base parameters, as well as potassium concentrations were compared at 5 minutes before and 5 and 30 minutes after reperfusion.. The maximum decreases in MAP within 5 minutes after reperfusion were significantly greater in both the NF-HTK and the F-HTK groups. The rate of PRS was significantly greater in the NF-HTK compared with the UW group. Flushing with HTK solution decreased the rate of PRS; there was no significant difference between the F-HTK and UW groups. All serial changes in body temperature, cardiovascular and acid-base parameters, as well as potassium concentrations were similar among the 3 groups.. The incidence of PRS was greater using HTK compared with UW solution during the reperfusion period. Therefore, careful hemodynamic management is advised when using HTK solution.

Topics: Acid-Base Equilibrium; Adenosine; Adult; Allopurinol; Blood Pressure; Carcinoma, Hepatocellular; Female; Glucose; Glutathione; Hemodynamics; Humans; Insulin; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Living Donors; Male; Mannitol; Middle Aged; Organ Preservation Solutions; Portal Vein; Potassium; Potassium Chloride; Procaine; Raffinose; Reperfusion Injury; Retrospective Studies

To observe protective effects of safflower injection (SI) on lung ischemia/reperfusion injury (LIRI) and investigate its potential mechanism.. Rabbit lung model of ischemia/reperfusion injury was constituted in vivo. The rabbits were randomly divided into three groups: sham-operation group (S group), ischemia/reperfusion group (I/R group) and ischemia/reperfusion plus safflower injection group (SI group). Malondialdehyde (MDA) content, superoxide dismutase (SOD) and xanthine oxidase (XO) activities in serum were measured. The lung tissue sampled at the end of the experiment was assayed for wet/dry weight ratio (W/D), injured alveoli rate (IAR) and ultrastructural changes were observed under electron microscope. The expression of COX-1 and COX-2 were measured by immunohistochemistry (IHC). The expressions of COX-1mRNA and COX-2mRNA were observed by in situ hybridization (ISH).. In I/R group, XO and MDA increased and SOD decreased in serum, while the same changes happened in SI group but less severely(P<0.01). The value of W/D and IAR was much higher in I/R group than S group, but decreased in SI group. Electron microscope showed obvious ultrastructural injury brought by LIRI in I/R group, which was greatly attenuated in SI group. The IHC and ISH demonstrated that COX-2 and COX-2mRNA in pulmonary tissue of I/R group were significantly higher than those of SI group (P < 0.01). The difference of COX-1 and COX-1mRNA in pulmonary tissue among the three groups was not significant.. The ischemia/reperfusion lung injury insults induced the regulation of COX-2 in lung. Safflower injection may attenuate lung ischemia/reperfusion injury through inhibiting cyclooxygenase-2 expression.

Topics: Animals; Carthamus tinctorius; Cyclooxygenase 1; Cyclooxygenase 2; Lung; Malondialdehyde; Rabbits; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

[corrected] The goal of this experimental study was to investigate whether erdosteine has a protective effect against lung injury as a remote organ after hind-limb ischemia-reperfusion (I/R).. The rats were divided into three groups: control, I/R, and I/R + erdosteine. After the experimental procedure, nitric oxide (NO) levels, myeloperoxidase (MPO), adenosine deaminase (ADA), and the activities of xanthine oxidase (XO) were determined on the lung tissue. The levels of NO and activities of MPO were also measured on the bronchial alveolar lavage (BAL). In addition, the lung tissue was examined by histopathology.. The lung tissue ADA and XO activities were increased in the I/R group compared with the control group (P < 0.05). In the I/R group, the levels of NO were higher than the control group (P < 0.05), whereas the erdosteine treatment did not alter the NO levels (P < 0.05). The MPO activities increased after I/R in the I/R group compared to both control and I/R + erdosteine group (P < 0.05). The activity of MPO increased in the IR group in comparison with the control group in BAL (P < 0.05). The activity of MPO in the I/R + erdosteine group was significantly lower than the I/R group in BAL (P < 0.05). NO levels increased in all I/R groups compared to control group in BAL (P < 0.05). However, treatment of erdosteine significantly decreased NO levels compared to I/R group (P < 0.05). The animals of the I/R group had total destruction of normal alveolar structure with the intense presence of infiltrating neutrophils and mononuclear phagocytes in histopathological examination. The rat lung exhibited mild degrees of destruction in the erdosteine group.. As a result, erdosteine may be a protective effect for lung injury, decreasing oxidative stress and neutrophil accumulation after hind-limb I/R in rats.

Topics: Adenosine Deaminase; Animals; Antioxidants; Bronchoalveolar Lavage Fluid; Hindlimb; Male; Neutrophils; Nitric Oxide; Oxidative Stress; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Respiratory Distress Syndrome; Thioglycolates; Thiophenes; Xanthine Oxidase

Recent evidence suggests that apoptosis is involved in germ cell loss following testicular ischemia-reperfusion (IR) injury. Allopurinol (Allo) is as a free radical scavenger which prevents tissue damage caused by reperfusion and oxygenation after ischemia; however, its effect on apoptosis in this type of injury has not been studied. To examine the effect of allopurinol on germ cell apoptosis following testicular IR in a rat. Forty rats were divided randomly into 4 experimental groups of 10 rats each: group A (Sham)-Sham operated animals; group B (Sham-Allo)-Sham operated rats treated with allopurinol given PO (by gavage) at a dose of 200 mg/kg, once daily, immediately before and 24 h following operation; group C (IR)-rats underwent 90 min of unilateral testicular ischemia and 48 h of reperfusion; group D (IR-Allo)-rats underwent IR and were treated with allopurinol similar to group B. The ipsilateral and contralateral testes were harvested 48 h following operation. Johnsen's criteria and the number of germinal cell layers were used to categorize spermatogenesis. TUNEL assay was used to determine germ cell apoptosis. Statistical analysis was performed using one-way ANOVA test, with P < 0.05 considered statistically significant. Testicular ischemia in rats led to histological damage in the ipsilateral testis. In the contralateral testis minimal damage was observed. Treatment with allopurinol increased significantly Johnsen's score in both the ischemic (7.3 +/- 0.5 vs 5.6 +/- 0.5, P < 0.05) and contralateral (8.9 +/- 0.1 vs 8.3 +/- 0.2, P < 0.05) testis, compared to IR-animals. Germ cell apoptosis in both the ischemic and the contralateral testis increased significantly after IR. Treatment with allopurinol resulted in a significant decrease in germ cell apoptosis in the ipsilateral testis, expressed as the number of positive tubules per 100 tubules (AI-1, (apoptotic index) threefold decrease, P < 0.005) and the number of apoptotic cells per 100 tubules (AI-2, fivefold decrease, P < 0.005) as well as a significant decrease in germ cell apoptosis in the contralateral testis (AI-1, 3.5-fold decrease, P < 0.05, AI-2- sixfold decrease, P < 0.005) compared to IR animals. In a rat model of testicular IR, treatment with allopurinol decreases germ cell apoptosis in both ischemic and contralateral testes and improves spermatogenesis.

Topics: Allopurinol; Animals; Apoptosis; Disease Models, Animal; Free Radical Scavengers; In Situ Nick-End Labeling; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Spermatogenesis; Spermatozoa; Testis; Treatment Outcome

Oxidative stress mediates cell injury during ischaemia/reperfusion. On the other hand, experimental findings suggest that ROS (reactive oxygen species) induce processes leading to ischaemic preconditioning. The extent and source of oxidative stress and its effect on antioxidant status in the human liver during intermittent ischaemia and reperfusion remains ill-defined. Therefore the aim of the present study was to investigate the occurrence of oxidative stress in humans undergoing liver resection. Liver biopsies, and arterial and hepatic venous blood samples were taken from ten patients undergoing hepatectomy with an intermittent Pringle manoeuvre. Plasma MDA (malondialdehyde) and hepatic GSSG levels were measured as markers of oxidative stress and plasma uric acid as a marker of xanthine oxidase activity. In addition, changes in hepatosplanchnic consumption of plasma antioxidants and hepatic levels of carotenoids and glutathione (GSH) were measured. After ischaemia, hepatosplanchnic release of MDA and increased hepatic GSSG levels were found. This was accompanied by the release of uric acid, reflecting xanthine oxidase activity. During reperfusion, ongoing oxidative stress was observed by further increases in hepatic GSSG content and hepatosplanchnic MDA release. Uric acid release was minimal during reperfusion. A gradual decrease in plasma antioxidant capacity and net hepatosplanchnic antioxidant uptake was observed upon prolonged cumulative ischaemia. Oxidative stress occurs during hepatic ischaemia in man mainly due to xanthine oxidase activity. Interestingly, the gradual decline in plasma antioxidant capacity and net hepatosplanchnic antioxidant uptake during prolonged cumulative ischaemia, preserved both hydrophilic and lipophilic hepatic antioxidant levels. Decreasing plasma levels and net hepatosplanchnic uptake of plasma antioxidants may warrant antioxidant supplementation, although it should be clarified to what extent limitation of oxidative stress compromises ROS-dependent pathways of ischaemic preconditioning.

Topics: Aged; Antioxidants; Female; Hepatectomy; Humans; Intraoperative Period; Liver; Liver Neoplasms; Male; Malondialdehyde; Middle Aged; Oxidative Stress; Reactive Oxygen Species; Reperfusion Injury; Splanchnic Circulation; Uric Acid; Xanthine Oxidase

1. Testicular ischaemia-reperfusion injury is commonly seen in childhood. Infertility occurs in 25% of patients after unilateral testicular ischaemia. It is has been reported that methylene blue has a positive effect in the reparation of ischaemia-reperfusion injury in different tissues. Therefore, we hypothesized that methylene blue may prevent the hazardous effects of ischaemia-reperfusion injury in testicular tissue after unilateral testicular torsion. 2. Thirty-two prepubertal Wistar-albino rats were divided into four groups. Testicular torsion was created by rotating the right testis 720 degrees in a clockwise direction for 5 h in all groups except for Group C, which was the sham control group. In Group T, bilateral orchiectomy was performed following the torsion period. In Group TD, both testes were removed 5 days after the torsion period. In Group MB, methylene blue (1 mg/kg, i.p.) was administered 40 min before detorsion and once daily over 5 days; then, both testes were harvested. Tissue levels of malondialdehyde (MDA), serum levels of creatine kinase (CK), mean testicular biopsy score (MTBS) and mean seminifer tubule diameter (MSTD) were determined. 3. There was a significant difference in MTBS between Groups T and TD (P < 0.05) in both ipsilateral and contralateral testes. In the contralateral testis, treatment with methylene blue decreased MTBS and MSTD (P < 0.05) and increased MDA levels (P < 0.05). In Group T, mean serum CK concentrations were higher than in any of the other groups (P < 0.05). 4. After 5 h of unilateral testicular torsion and a 5 day reperfusion period, serious tissue damage occurred on both the ipsilateral and contralateral sides. Serum CK concentrations may be an indicator for ischaemia, but not for ischaemia-reperfusion injury. Contrary to our hypothesis, methylene blue increased contralateral testicular damage after unilateral testicular torsion and exacerbated oxidative events.

Topics: Animals; Creatine Kinase; Disease Models, Animal; Enzyme Inhibitors; Male; Malondialdehyde; Methylene Blue; Nitric Oxide Synthase; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; Spermatic Cord Torsion; Testicular Diseases; Testis; Time Factors; Xanthine Oxidase

Reactive oxygen species (ROS) participate in tissue injury after ischemia-reperfusion. Their implication in leukocyte adherence and increase in permeability at the venular side of the microcirculation have been reported, but very little is known about ROS production in arterioles. The objective of this work was to evaluate, in the arteriole wall in vivo, the temporal changes in superoxide anion production during ischemia and reperfusion and to identify the source of this production. Mouse cremaster muscle was exposed to 1 h of ischemia followed by 30 min of reperfusion, and superoxide anion production was assessed by a fluorescent probe, i.e., intracellular dihydroethidium oxidation. During ischemia, we found a significant increase in dihydroethidium oxidation; however, we observed no additional increase in fluorescence during the subsequent reperfusion. This phenomenon was significantly inhibited by pretreatment with superoxide dismutase. Allopurinol (xanthine oxidase inhibitor) or stigmatellin [Q(o)-site (oriented toward the intermembrane space) inhibitor of mitochondrial complex III] or simultaneous administration of these two inhibitors significantly reduced superoxide production during ischemia to 80%, 88%, and 72%, respectively, of that measured in the untreated ischemia-reperfusion group. By contrast, no significant inhibition was found when NADPH oxidase was inhibited by apocynin or when mitochondrial complex I or complex II was inhibited by rotenone or thenoyltrifluoroacetone. A significant increase in ROS was found with antimycin A [Q(i)-site (located in the inner membrane and facing the mitochondrial matrix) inhibitor of mitochondrial complex III]. We conclude that a significant increase in ROS production occurs during ischemia in the arteriolar wall. This increased production involves both a cytoplasmic source (i.e., xanthine oxidase) and the mitochondrial complex III at the Q(o) site.

Topics: Animals; Arterioles; Blood Pressure; Ethidium; Fluorescent Dyes; Ischemia; Mice; Mice, Inbred BALB C; Microcirculation; Mitochondria, Muscle; Muscle, Skeletal; Reactive Oxygen Species; Regional Blood Flow; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

Cold ischemia time and preservation of organs are limited by I/R injury leading to primary nonfunction of the graft. In a rat heart transplant model, we compared cardioplegic St Thomas (ST) to histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin preservation solutions in terms of contractile function, and mitochondrial respiratory and enzymatic defects after prolonged cold ischemia and reperfusion. Contractile function was scored after transplantation and 24 h of reperfusion. Mitochondrial function was investigated by high-resolution respirometry of permeabilized myocardial fibers. Graft performance in terms of contractile function declined with the duration of cold storage. Recovery was significantly improved after 10 h of cold storage in HTK compared with ST (cardiac scores, 3.3+/-0.5 and 1.8+/-0.8, respectively). Tissue lactate dehydrogenase was better preserved in HTK than ST. Increase of tissue water content (edema) was less pronounced in HTK than ST (3.33+/-0.14 and 3.73+/-0.21 mg/mg dry weight, respectively). Similar cardiac scores (2.6+/-0.9 and 2.9+/-1.2, respectively) and mitochondrial respiratory parameters were obtained after preservation in HTK and University of Wisconsin. Decline in contractile function of individual grafts correlated well with loss of mitochondrial respiratory capacity, whereas citrate synthase activity remained largely preserved, indicating specific damage of respiratory complexes. Our data provide evidence for the superiority of preservation solutions versus a cardioplegic solution for prolonged cold storage of the heart. The correlation of graft performance and mitochondrial function indicates the potential of high-resolution respirometry for quantitative assessment of myocardial injury upon cold I/R, providing a basis for diagnostic approaches and evaluation of improved preservation solutions for heart transplantation.

Topics: Adenosine; Allopurinol; Animals; Cardioplegic Solutions; Citrate (si)-Synthase; Glutathione; Heart Transplantation; Insulin; Ischemia; Male; Mitochondria; Myocardial Contraction; Myocardium; Organ Preservation Solutions; Permeability; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

This study investigated the role of a novel nutrient-rich preservation solution in alleviating intestinal ischemia-reperfusion (IR) injury in a large animal model.. Porcine intestines were treated in vivo with the following intraluminal flush solutions: group 1, none; group 2, University of Wisconsin solution; group 3, an amino acid-based solution, previously shown to be effective in reducing IR injury in rodent models. Intestinal ischemia was induced in vivo for 60 min, followed by 180 min reperfusion. Key metabolic aspects were assessed in relation to two fundamental kinase mechanisms that govern cell fate, AMP kinase, and Jun kinase.. After 180 min reperfusion, groups 1 and 2 exhibited clefting, denudation, and mucosal hemorrhage, whereas injury was markedly reduced in group 3 (median grades 4.5 and 5 vs. 0; P<0.05). In contrast to groups 1 and 2, group 3 tissues exhibited a full recovery of adenylates (ATP, total adenylates) and an effective control of oxidative stress throughout reperfusion. Neutrophil-mediated inflammation was abrogated in group 3. An up-regulation of two key enzymes (glutaminase and alanine aminotransferase) provided a mechanism for the superior recovery of energetics and the preservation of mucosal integrity in group 3. A strong activation of AMP-activated protein kinase resulting in the up-regulation of a primary proapoptotic kinase mechanism, Jun kinase, was evident in groups 1 and 2.. A strategy of intraluminal administration of a nutrient-rich solution represents a potential therapy for alleviating intestinal IR injury; these findings suggest a more effective method for the ischemic storage of intestine.

Topics: Adenosine; Adenylate Kinase; Alanine Transaminase; Allopurinol; Animals; Glutaminase; Glutathione; Hemorrhage; Insulin; Intestinal Mucosa; Intestine, Small; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Swine

Activated protein C (APC) is a serine protease with anticoagulant and antiinflammatory activities. The delaying effects of remote reperfusion injury on the wound-healing process in colonic anastomoses have been previously shown. In this study, we aimed to investigate whether APC protects against deleterious systemic effects of intestinal ischemia/reperfusion (I/R) injury on colonic anastomotic wound healing process.. Male Wistar-albino rats were randomly allocated into 4 groups, and a left colonic anastomosis was performed in all animals: (1) sham-operated group, simultaneously with left colonic anastomosis, the superior mesenteric artery and collateral branches were divided from the celiac axis, and the inferior mesenteric artery were isolated but not occluded (group 1, n = 12), (2) sham + APC group, identical to group 1 except for APC treatment (100 microg/kg, intravenously, 15 minutes before construction of the colonic anastomosis), (group 2, n = 12), (3) intestinal I/R group, 60 minutes of superior mesenteric ischemia followed by reperfusion (group 3, n = 12), and (4) APC-treated group, (100 microg/kg, intravenously, 15 minutes before reperfusion) (group 4, n = 12). All animals were sacrificed, and colonic anastomotic bursting pressures were measured in vivo on day 7. Tissue samples were obtained for analysis of hydroxyproline contents, nitrate/nitrite levels, and activities of oxidative and antioxidative enzymes. The plasma levels of proinflammatory cytokines and D-dimer were also measured.. Intestinal I/R led to significant decreases in colonic anastomotic bursting pressures, tissue hydroxyproline contents, and activities of antioxidative enzymes, along with increases in tissue nitrate/nitrite levels, activities of oxidative enzymes, and plasma levels of proinflammatory cytokines and D-dimer (P < .05). However, APC treatment led to significant increases in colonic anastomotic bursting pressures, tissue hydroxyproline contents, and activities of antioxidative enzymes, along with decreases in tissue nitrate/nitrite levels, activities of oxidative enzymes, and plasma levels of proinflammatory cytokines and D-dimer (P < .05).. This study clearly showed that APC treatment prevented the delaying effects of remote I/R injury on colonic anastomotic wound healing process. Further clinical studies are required to determine whether APC has a useful role in the enhancement of colonic anastomotic wound healing after particular operations in which I/R injury occurs.

Topics: Analysis of Variance; Anastomosis, Surgical; Animals; Chi-Square Distribution; Glutathione; Glutathione Reductase; Interleukin-6; Intestines; Ischemia; Male; Malondialdehyde; Mesenteric Arteries; Mice; Nitrates; Nitrites; Peroxidase; Protein C; Random Allocation; Reperfusion Injury; Tumor Necrosis Factor-alpha; Wound Healing; Xanthine Oxidase

ET-Kyoto solution (ET-K) is an extracellular-type organ preservation solution containing the cytoprotective disaccharide, trehalose. A previous study reported the supplement of dibutyryl cyclic adenosine monophosphate (db-cAMP) in conventional ET-K to attenuate lung ischemia-reperfusion injury. In this study, the efficacy of this modified ET-K for liver preservation was investigated by comparison with University of Wisconsin solution (UW). ET-K was supplemented with db-cAMP (2 mmol/L). Lewis rats were randomly assigned to two groups, and liver grafts were flushed and stored at 40C for 24 h with ET-K or UW before syngeneic liver transplantation. The graft function and histological changes at 4 h posttransplant as well as 7-day survival were evaluated. Recipient rat survival rate was significantly higher in the ET-K group than in the UW group. Preservation in ET-K resulted in a significant reduction in serum parenchymal transaminase level and promotion of bile production in comparison with UW. The serum hyaluronic acid level, an indicator of sinusoidal endothelial cell injury, was significantly lower after ET-K preservation than that in UW. Histologically, at 4 h after transplantation, the liver grafts preserved in UW solution demonstrated a greater degree of injury than those in ET-K, which appeared to be apoptosis, rather than necrosis. The continuity of the sinusoidal lining was better preserved in ET-K than in UW. In conclusion, ET-K supplemented with db-cAMP is superior to UW in rat liver preservation. This modified ET-K might therefore be a novel candidate for the procurement and preservation of multiple organs.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Aspartate Aminotransferases; Bucladesine; Gluconates; Glutathione; Graft Survival; Hydroxyethyl Starch Derivatives; Insulin; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Phosphates; Raffinose; Rats; Reperfusion Injury; Trehalose

To determine the effects of allopurinol, an inhibitor of xanthine oxidase, and apocynin, an inhibitor of NADPH oxidase, on oxidant stress and liver injury caused by hepatic ischemia/reperfusion (I/R) procedure in mice.. Mice were pretreated with a xanthine oxidase inhibitor, allopurinol, or NADPH oxidase (NOX) inhibitor, apocynin before the hepatic I/R procedure. Then treated or untreated mice underwent the hepatic I/R procedure. The effects on hepatic injury and superoxide anions were determined after starting reperfusion.. A standard warm hepatic I/R procedure led to a marked increase in superoxide anion production as indicated by a superoxide anion tracer, MCLA. At the same time, the procedure caused profound acute liver injury, as indicated by elevated serum alanine aminotransferase and tumor necrosis factor-alpha levels, reduced liver glutathione levels and elevated malondialdehyde contents, as well as a high apoptotic cell count. All these changes were reversed by the use of apocynin or allopurinol prior to the hepatic I/R procedure.. Allopurinol and apocynin exerted protective effects on hepatic ischemia/reperfusion injury. The protection is associated with blocking the generation of superoxide anions during the hepatic I/R procedure by inhibiting xanthine oxidase and NADPH oxidase activity.

Topics: Acetophenones; Allopurinol; Animals; Apoptosis; Enzyme Inhibitors; Glutathione; Liver; Malondialdehyde; Mice; Mice, Inbred ICR; NADPH Oxidases; Reperfusion Injury; Superoxides; Tumor Necrosis Factor-alpha; Warm Ischemia; Xanthine Oxidase

Experimental study.. To determine the neuroprotective effects of zinc and melatonin on spinal cord ischemia-reperfusion (I/R) injuries of rabbits.. The Experimental Research Centre of Selçuk University, Konya, Turkey.. Twenty-four male rabbits underwent spinal cord ischemia by clamping the thoraco-abdominal aorta for 20 min. Twenty minutes before the aortic clamping, animals received zinc, melatonin or a combination of both. Neurological examination of the animals was performed three times during reperfusion period. The animals were killed 24 h after reperfusion. Spinal cord samples were taken for biochemical and histopathological evaluation.. Pre-treated animals with zinc, melatonin or combination displayed better neurological outcomes than the I/R group (P<0.05). Zinc, melatonin and combined treatment prevented spinal cord injury by reducing apoptosis rate (P<0.05) and preserving intact ganglion cell numbers (P<0.05). Zinc pre-treatment protected spinal cord by preventing malondialdehyde (MDA) formation (P=0.002), increasing glutathione peroxidase (GPx) activity (P=0.002) and decreasing xanthine oxidase enzyme activity (P=0.026) at molecular level. Melatonin treatment also resulted with MDA formation (P=0.002), increased GPx activity (P=0.002) and decreased xanthine oxidase activity (P=0.026).. The results of the study showed that prophylactic zinc and melatonin use in spinal cord I/R not only suppressed lipid peroxidation by activating antioxidant systems but also had significant neuroprotective effects by specifically improving the neurological and histopathological situation.

Topics: Analysis of Variance; Animals; Antioxidants; Disease Models, Animal; Glutathione Peroxidase; Male; Malondialdehyde; Melatonin; Neurologic Examination; Rabbits; Reperfusion Injury; Spinal Cord Ischemia; Time Factors; Trace Elements; Xanthine Oxidase; Zinc

This study investigates how the addition of trimetazidine (TMZ) and aminoimidazole-4-carboxamide ribonucleoside (AICAR) to University of Wisconsin (UW) solution protects steatotic livers. Steatotic and nonsteatotic livers were preserved for 24 hours at 4 degrees C in UW and UW with TMZ and AICAR (separately or in combination) and then perfused ex vivo for 2 hours at 37 degrees C. Adenosine monophosphate-activated protein kinase (AMPK) or nitric oxide (NO) synthesis inhibition in livers preserved in UW with TMZ was also investigated. Hepatic injury and function (transaminases, bile production, and sulfobromophthalein clearance) and factors potentially involved in the susceptibility of steatotic livers to ischemia-reperfusion (I/R), including vascular resistance, mitochondrial damage, adenosine triphosphate depletion, and oxidative stress were evaluated. AMPK, NO synthase (NOS), nitrate, and nitrite levels were also determined. The addition of TMZ and AICAR (separately or in combination) to UW reduced hepatic injury, improved functionality, and protected against the mechanisms responsible for the vulnerability of steatotic livers to I/R. Like AICAR, TMZ increased AMPK, constitutive NOS, and nitrates and nitrites, and conversely, AMPK or NO synthesis inhibition abolished the benefits of TMZ. In conclusion, TMZ, by means of AMPK, increased NO, thus protecting steatotic livers against their vulnerability to I/R injury. TMZ and AICAR may constitute new additives to UW solution in steatotic liver preservation, whereas a combination of both seems unnecessary.

Topics: Adenine Nucleotides; Adenosine; Allopurinol; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Animals; Enzyme Activators; Fatty Liver; Glutathione; Insulin; Liver; Mitochondria, Liver; Multienzyme Complexes; Nitric Oxide; Nitric Oxide Synthase; Organ Preservation Solutions; Oxidative Stress; Protein Serine-Threonine Kinases; Raffinose; Rats; Rats, Zucker; Reperfusion Injury; Ribonucleotides; Trimetazidine; Vascular Resistance

It has now been firmly established that, not only ischemia/reperfusion, but also cold itself causes damage during kidney transplantation. Iron chelators or anti-oxidants applied during the cold plus rewarming phase are able to prevent this damage. At present, it is unknown if these measures act only during the cold, or whether application during the rewarming phase also prevents damage. We aimed to study this after cold normoxic and hypoxic conditions. LLC-PK1 cells were incubated at 4 degrees C in Krebs-Henseleit buffer for 6 or 24h, followed by 18 or 6h rewarming, respectively. Cold preservation was performed under both normoxic (95% air/5% CO2) and hypoxic (95% N2/5% CO2) conditions. The iron chelator 2,2'-DPD (100 microM), anti-oxidants BHT (20 microM) or sibilinin (200 microM), and xanthine oxidase inhibitor allopurinol (100 microM) were added during either cold preservation plus rewarming, or rewarming alone. Cell damage was assessed by LDH release (n=3-9). Addition of 2,2'-DPD and BHT during cold hypoxia plus rewarming did, but during rewarming alone did not prevent cell damage. When added during rewarming after 6h cold normoxic incubation, BHT and 2,2'-DPD inhibited rewarming injury compared to control (p<0.05). Allopurinol did not prevent cell damage in any experimental set-up. Our data show that application of iron chelators or anti-oxidants during the rewarming phase protects cells after normoxic but not hypoxic incubation. Allopurinol had no effect. Since kidneys are hypoxic during transplantation, measures aimed at preventing cold-induced and rewarming injury should be taken during the cold.

Topics: 2,2'-Dipyridyl; Allopurinol; Animals; Antioxidants; Butylated Hydroxytoluene; Ethanol; Hypoxia; Iron Chelating Agents; L-Lactate Dehydrogenase; LLC-PK1 Cells; Organ Preservation; Organ Preservation Solutions; Reperfusion Injury; Rewarming; Silybin; Silymarin; Sodium Hydroxide; Swine

Topics: Adenosine; Allopurinol; Animals; Cold Ischemia; Glutathione; Graft Survival; Humans; Insulin; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Polyethylene Glycols; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Changes in hemorheological parameters were studied in dogs following unilateral renal artery clamping (45-minute ischemia then reperfusion), with and without preoperative administration of allopurinol. Sham-operated animals were also evaluated. Blood samples were collected preoperatively, at beginning and at 30, 60 and 120 minutes of reperfusion, then on the 1st, 3rd, 5th and 7th days. Filtration properties of erythrocytes (relative cell transit time, RCTT), whole blood and plasma viscosity (WBV, PV), fibrinogen level and hematology parameter were determined. RCTT significantly increased for both ischemic groups at 30 minutes of reperfusion, and remained elevated on the 1st and 2nd postoperative days; these changes were abolished by allopurinol pretreatment. WBV and hematocrit increased on the 1st day, and PV and fibrinogen level showed elevation on 1st-5th postoperative days. We thus conclude that decreases of RBC deformability (i.e., higher RCTT) were characteristic and specific on early postoperative days after renal ischemia-reperfusion and that these alterations were prevented by pre-ischemia administration of allopurinol.

Topics: Allopurinol; Animals; Blood Viscosity; Disease Models, Animal; Dogs; Erythrocyte Deformability; Erythrocyte Indices; Free Radical Scavengers; Renal Circulation; Reperfusion Injury

We investigated the effect of alpha-lipoic acid (LA) on reperfusion injury in a rat ovarian torsion-detorsion model. The changes in tissue and plasma levels of malondialdehyde (MDA), end-product of lipid peroxidation, superoxide dismutase (SOD), xanthine oxidase (XO) and nitric oxide (NO), were determined. Ovarian histopathological findings were scored and compared among groups.. Thirty-two female Sprague-Dawley rats were divided into four groups. Sham operation was performed in group I; in group II only ovarian torsion was performed. Group III received intraperitoneal injections of saline, and group IV received LA via intraperitoneal injections (LA group: aqueous solution at 36 mg/kg of body weight per day, saline group: equal volume of saline) 21, nine, and one hour before torsion of the ovary. Rats in the torsion group were killed after 360 degrees clockwise adnexial torsion for three hours, and ovaries were harvested. After three hours of adnexial detorsion, the rats in saline group and LA group were killed and adnexa were surgically removed.. Ovarian tissue damage scores were significantly different among groups and were seen to correlate with tissue MDA levels. Ovarian tissue and serum MDA, NO and serum XO levels in the group III were significantly higher than those of the groups I and IV (P<0.05). The serum levels of SOD in the group III were significantly lower than those of the groups I and IV (P<0.05).. These results suggest that LA pretreatment has beneficial effects in the prevention of ischaemia-reperfusion injury of the ovaries.

Topics: Animals; Disease Models, Animal; Female; Malondialdehyde; Nitric Oxide; Ovary; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Thioctic Acid; Xanthine Oxidase

Oxidative stress occurs in remote liver injury, but the origin of the oxidant generation has yet to be thoroughly delineated. Some reports suggest that the source of the distant oxidative stress originates from the site of initial insult [i.e., xanthine oxidase (XO)]; however, it could also be derived from sources such as phagocytic and/or vascular NAD(P)H oxidase (Nox) enzymes. With a murine model of bilateral hindlimb ischemia-reperfusion, we describe here a mechanism for Nox-dependent oxidant production that contributes, at least in part, to remote hepatic parenchymal injury and sinusoidal endothelial cell (SEC) dysfunction. To determine whether Nox enzymes were the source of oxidants, mice were treated immediately after the onset of hindlimb ischemia with specific inhibitors to XO (50 mg/kg ip allopurinol) or Nox (10 mg/kg ip gp91ds-tat and 3 mg/kg ip apocynin). After 1 h of ischemia, hindlimbs were reperfused for either 3 or 6 h. Inhibition of XO failed to provide any improvement in parenchymal injury, SEC dysfunction, neutrophil accumulation, or microvascular dysfunction. In contrast, the inhibition of Nox enzymes prevented the progression (6 h) of parenchymal injury, significantly protected against SEC dysfunction, and completely prevented signs of neutrophil-derived oxidant stress. At the same time, however, inhibition of Nox failed to protect against the early parenchymal injury and microvascular dysfunction at 3 h of reperfusion. These data confirm that microvascular perfusion deficits are not essential for the pathogenesis of remote hepatic parenchymal injury. The data also suggest that Nox enzymes, not XO, are involved in the progression of compromised hepatic parenchymal and endothelial integrity during a systemic inflammatory response.

Topics: Animals; Disease Progression; Endothelium; Liver; Liver Diseases; Male; Mice; Mice, Inbred C57BL; Microcirculation; Models, Biological; NADPH Oxidases; Neutrophils; Oxidative Stress; Peroxidase; Reperfusion Injury; Tyrosine; Xanthine Oxidase

In kidney transplantation, cold storage is the dominant modality used to prolong organ viability ex vivo, but is inevitably followed by a period of warm ischemia. Preservation fluids limit tissue damage during the ischemic period, but there is little information on the influence of preservation fluids on the physiologic consequences of warm ischemia alone, or on the comparative ability of such preservation fluids to limit warm ischemic injury. In this study, warm ischemia was induced in rat kidneys by crossclamping the left renal pedicle for 45 min with contralateral nephrectomy. The ischemic kidneys were flushed with Euro-Collins (EC), hyper osmolar citrate (HOC), University of Wisconsin (UW), or phosphate buffered sucrose (PBS)140 solution. Over a period of 2 h after reperfusion, urine and blood samples were collected and physiological parameters related to the function of the postischemic kidneys were assessed. The data show that postischemic renal function can be influenced by the choice of preservation fluid. Essentially, the continued use of EC as a renal preservation solution finds little support in these data, and, while HOC and UW solutions were better able to limit the decline in renal function after warm ischemia than EC, the solution most able to limit functional impairment after warm ischemia was PBS140. This analysis compares the efficacies of the commonly used preservation solutions and could form the basis for future solid-organ transplant studies that may ultimately allow us to propose best-practice guidelines and an optimum platform for improved preservation solutions.

Topics: Adenosine; Allopurinol; Animals; Citric Acid; Glutathione; Hypertonic Solutions; In Vitro Techniques; Insulin; Ischemia; Kidney; Kidney Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Osmolar Concentration; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Sugar Phosphates; Temperature

Cold ischemia and reperfusion during renal transplantation result in release of reactive oxygen species. The aim of this study is to examine whether cold storage induced cell injury can be ameliorated by adding flavonoids directly to preservation solutions.. Cultured renal tubular epithelial cells (LLC-PK1) were stored in University of Wisconsin (UW) or Euro-Collins (EC) solution at 4 degrees C for 20 hours. Preservation solutions were supplemented with various flavonoids. After rewarming, structural and metabolic cell integrity was measured by lactate dehydrogenase (LDH) release and MTT-test, and lipid peroxidation was assessed from generation of thiobarbituric acid-reactive substances (TBARS).. Twenty hours of cold storage resulted in a substantial loss of cell viability in both preservation solutions (in EC: LDH release 92.4+/-2.7%; MTT-test 0.5+/-0.7%). Addition of luteolin, quercetin, kempferol, fisetin, myricetin, morin, catechin, and silibinin significantly reduced cell injury (for luteolin in EC: LDH release 2.4+/-1.6%; MTT-test 110.3+/-10.4%, P<0.01; TBARS-production (related to cold stored control cells) 8.9+/-2.6%). No cytoprotection was found for apigenin, naringenin, and rutin. Protective potency of flavonoids depends on number of hydroxyl-substituents and lipophilicity of the diphenylpyran compounds.. Cold storage induced injury of renal tubular cells was substantially ameliorated by adding selected flavonoids directly to preservation solutions.

Topics: Adenosine; Allopurinol; Animals; Cell Survival; Cold Temperature; Cryoprotective Agents; Flavonoids; Glutathione; Hypertonic Solutions; In Vitro Techniques; Insulin; Kidney Transplantation; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Lipid Peroxidation; LLC-PK1 Cells; Luteolin; Molecular Structure; Organ Preservation; Organ Preservation Solutions; Quercetin; Raffinose; Reperfusion Injury; Swine

To investigate the impact of garlic extract (GE), which is known for its antioxidant activity, on a testicular torsion/detorsion model in animals and to help understand how to prevent both ischemic and reperfusion injuries after testicular torsion and detorsion.. Six groups of rats (n=7 in each group) were used. The animals in the control group (Group I) did not receive any treatment. The animals in the sham group (Group II) underwent scrotal incision and testicular fixation only. The animals in Groups III-VI underwent 720 degrees of left testicular torsion for 2 h; subsequent detorsion was performed for 2h in Groups IV and VI only. Animals in Groups V and VI were treated exactly the same as those in Groups III and IV, respectively except that they were pretreated with oral GE for 5 days at a dosage of 5 ml/kg. Both testicles in all rats were removed and tissue malondialdehyde (MDA) levels and enzymatic activities of xanthine oxidase (XO) were studied, in addition to a histological evaluation after hematoxylin-eosin staining.. Testicular MDA levels and XO activities were higher in Group III compared to Group II (p<0.05). Pretreatment with GE prevented these increases. Detorsion caused more damage and resulted in a further increase in MDA levels but MDA levels were not increased in animals pretreated with GE. Histologically, torsion caused some separation between germinative cells in the seminiferous tubules, which became much more prominent in Group IV and was attenuated by GE pretreatment. There were no significant changes in any of the above-mentioned enzymatic activities or histopathologic changes in the contralateral testicle in any of the groups.. We believe that both testicular torsion and detorsion result in testicular tissue damage by means of lipid peroxidation, which is evident by an increase in the tissue levels of MDA. Dietary supplementation with GE seems to attenuate the generation of toxic free radicals, as evidenced indirectly by low tissue MDA levels.

Topics: Animals; Antioxidants; Disease Models, Animal; Free Radical Scavengers; Immunohistochemistry; Ischemia; Male; Malondialdehyde; Probability; Protective Agents; Random Allocation; Rats; Rats, Inbred BN; Reference Values; Reperfusion Injury; Risk Factors; Sensitivity and Specificity; Spermatic Cord Torsion; Statistics, Nonparametric; Testis; Tissue Culture Techniques; Xanthine Oxidase

Preconditioning treatments hold significant potential for improving outcomes in solid organ transplantation. Protective phenotypes can be induced using certain drugs. Curcumin is a biologically active component of turmeric and has been reported to induce stress proteins in certain cell lines, leading to cell protection. This study investigates in detail the effect of curcumin on the stress-response in human hepatocytes, in particular its effect on heme oxygenase 1 (HO-1) and its cytoprotective effect. Pretreatment with curcumin protected hepatocytes in a model of oxidative injury and this protection was mediated through HO-1. In a model of cold preservation injury, curcumin pretreatment resulted in elevation of HO-1 throughout the cold storage and rewarming period, and was cytoprotective against oxidative injury. This is the first study to demonstrate that curcumin induces HO-1 in human hepatocytes, and that the protective effects of curcumin pretreatment may have clinical potential in hepatic transplantation.

Topics: Adenosine; Allopurinol; Curcumin; Gene Expression Regulation, Enzymologic; Glutathione; Heme Oxygenase-1; Hepatocytes; HSP70 Heat-Shock Proteins; Humans; Insulin; Liver Circulation; Liver Transplantation; Organ Preservation Solutions; Polymerase Chain Reaction; Raffinose; Reperfusion Injury; Tissue Preservation; Transplantation Conditioning

During ischemia-reperfusion, free oxygen radicals which directly affect renal cells may cause delayed graft function. We investigated whether there was a difference regarding antioxidant enzyme content between use of Ringer's lactate (RL) versus University of Wisconsin (UW) perfusion solutions in kidney transplantation. Ischemia was achieved by clamping the renal pedicle for 20 minutes followed by perfusion with either solution for 20 minutes and reperfusion for another 20 minutes. A parenchymal biopsy was taken before and after the ischemia, perfusion, and reperfusion (IPR) process. The levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) were investigated in the biopsy specimens. We used paired t tests within groups and t tests for comparisons between groups. The results were expressed as mean values +/- SEM with P < .05 accepted as statistically significant. After IPR, SOD, GPx, and MDA were decreased in all groups: only GPx (P = .001) and MDA (P = .04) for the RL group and SOD (P = .001) and MDA (P = .05) for the UW group were statistically significant. In the control group, we did not observe any difference (P > .05). Comparisons between groups did not reveal differences (P > .05). In our study, no difference was observed between RL and UW regarding their effects on antioxidant enzymes following renal I/R injury in pigs. More investigations are needed to evaluate graft function in this setting.

Topics: Adenosine; Allopurinol; Animals; Disease Models, Animal; Glutathione; Glutathione Peroxidase; Insulin; Isotonic Solutions; Kidney Transplantation; Male; Malondialdehyde; Organ Preservation Solutions; Perfusion; Raffinose; Renal Circulation; Reperfusion Injury; Ringer's Lactate; Solutions; Swine; Thiobarbituric Acid Reactive Substances

In pancreas transplantation (PTx), ischemia/reperfusion-induced deterioration of graft-microcirculation is accompanied by alterations of intermittent capillary perfusion (IP; alternating cessation and resumption of capillary blood flow) is known to counteract malperfusion. Incidence and effectiveness of IP following short- versus long-term preservation of pancreas grafts with University of Wisconsin (UW) solution has not been examined so far. PTx was performed in Lewis rats following 2-h or 18-h preservation in UW solution. Using intravital fluorescence microscopy, functional capillary density (FCD), red blood cell (RBC) velocity, IP-incidence and -frequency were analyzed. Laser Doppler flowmetry allowed for the determination of erythrocyte flux and velocity. Measurements were performed at 30, 60 and 120 min after reperfusion. Nontransplanted animals served as controls. FCD, RBC-velocity and -flux remained unchanged in the 2-h group. IP was encountered in 87% of all observation areas at 120 min. After 18-h ischemia, FCD was significantly reduced, which was paralleled by a 50% incidence of IP at 120 min. Tissue edema and leukocyte infiltration in pancreas grafts following 18-h preservation were significantly enhanced. Therefore, IP is an important mechanism aimed at improving microcirculation and UW solution is suitable to preserve vasomotion-activities enabling long-term preservation in a pancreas graft.

Topics: Adenosine; Allopurinol; Animals; Blood Flow Velocity; Capillaries; Glutathione; In Vitro Techniques; Insulin; Male; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Perfusion; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Time Factors

There is a great evidence that reactive oxygen species (ROS) play an important role in the pathophysiology of ischemia-reperfusion (I/R) injury in skeletal muscle. Caffeic acid phenethyl ester (CAPE) is a component of honeybee propolis. It has antioxidant, anti-inflammatory and free radical scavenger properties. The aim of this study is to determine the protective effects of CAPE against I/R injury in respect of protein oxidation, neutrophil in filtration, and the activities of xanthine oxidase (XO) and adenosine deaminase (AD) on an in vivo model of skeletal muscle I/R injury. Rats were divided into three equal groups each consisting of six rats: Sham operation, I/R, and I/R plus CAPE (I/R+CAPE) groups. CAPE was administered intraperitoneally 60 min before the beginning of the reperfusion. At the end of experimental procedure, blood and gastrocnemius muscle tissues were used for biochemical analyses. Tissue protein carbonyl (PC) levels and the activities of XO, myeloperoxidase (MPO) and AD in I/R group were significantly higher than that of control (p < 0.01, p < 0.05, p < 0.01, p < 0.005, respectively). Administration of CAPE significantly decreased tissue PC levels, MPO and XO activities in skeletal muscle compared to I/R group (p < 0.01, p < 0.05, p < 0.05, respectively). In addition, plasma creatine phosphokinase (CPK), XO and AD activities were decreased in I/R+CAPE group compared to I/R group (p < 0.05, p < 0.05, p < 0.001). The results of this study revealed that free radical attacks may play an important role in the pathogenesis of skeletal muscle I/R injury. Also, the potent free radical scavenger compound, CAPE, may have protective potential in this process. Therefore, it can be speculated that CAPE or other antioxidant agents may be useful in the treatment of I/R injury as well as diffused traumatic injury of skeletal muscle.

Topics: Adenosine Deaminase; Animals; Caffeic Acids; Creatine Kinase; Male; Muscle, Skeletal; Peroxidase; Phenylethyl Alcohol; Protein Carbonylation; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Oxidase

Carbon monoxide (CO), a byproduct of heme catalysis, was shown to have potent cytoprotective and anti-inflammatory effects. In vivo recipient CO inhalation at low concentrations prevented ischemia/reperfusion (I/R) injury associated with small intestinal transplantation (SITx). This study examined whether ex vivo delivery of CO in University of Wisconsin (UW) solution could ameliorate intestinal I/R injury. Orthotopic syngenic SITx was performed in Lewis rats after 6 h cold preservation in control UW or UW that was bubbled with CO gas (0.1-5%) (CO-UW). Recipient survival with intestinal grafts preserved in 5%, but not 0.1%, CO-UW improved to 86.7% (13/15) from 53% (9/17) with control UW. At 3 h after SITx, grafts stored in 5% CO-UW showed improved intestinal barrier function, less mucosal denudation and reduced inflammatory mediator upregulation compared to those in control UW. Preservation in CO-UW associated with reduced vascular resistance (end preservation), increased graft cyclic guanosine monophosphate levels (1 h), and improved graft blood flow (1 h). Protective effects of CO-UW were reversed by ODQ, an inhibitor of soluble guanylyl cyclase. In vitro culture experiment also showed better preservation of vascular endothelial cells with CO-UW. The study suggests that ex vivo CO delivery into UW solution would be a simple and innovative therapeutic strategy to prevent transplant-induced I/R injury.

Topics: Adenosine; Allopurinol; Animals; Antimetabolites; Carbon Monoxide; Disease Models, Animal; Glutathione; Graft Survival; Insulin; Intestinal Mucosa; Intestine, Small; Male; Microscopy, Electron, Transmission; Organ Preservation; Organ Preservation Solutions; Organ Transplantation; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Treatment Outcome

To investigate whether tanshinone IIA could improve the effect of UW solution for skeletal muscle preservation and to determine the dose range of tanshinone IIA providing optimal protection during ischemia and reperfusion.. Ischemic rat limbs were perfused with UW solution or UW plus tanshinone IIA (UW+T, 0.05, 0.1, or 0.2 mg/mL) for 0.5 h before reperfusion; controls (I/R) received no perfusion. Serum creatine phosphokinase (CPK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were measured pre-ischemia and after reperfusion (2-h, 4-h, and 6-h). Muscle water content, superoxide dismutase (SOD), malondialdehyde (MDA), adenosine triphosphatase (ATPase) were assessed pre-reperfusion and after 6-h reperfusion. Intercellular adhesion molecule-1 (ICAM-1) and apoptosis were detected after 6-h reperfusion. Reperfusion blood flow was monitored during reperfusion period.. UW and UW+T prevented luxury perfusion during reperfusion and inhibited ICAM-1 expression and apoptosis after 6-h reperfusion. Serum CPK, AST, and LDH levels in UW rats were significantly less than those in controls after 2-h reperfusion (no difference, 4-h or 6-h reperfusion). After 4-h ischemia, there were significant differences in water content, MDA, SOD, and ATPase between UW and controls, but no difference after 6-h reperfusion. All tests with UW+T rats were significantly different from control results at corresponding durations. Higher tanshinone doses improved results.. UW plus tanshinone IIA increased protection against I/R injury, suggesting that tanshinone IIA has clinical value.

Topics: Abietanes; Adenosine; Adenosine Triphosphatases; Allopurinol; Animals; Apoptosis; Aspartate Aminotransferases; Creatine Kinase; Dose-Response Relationship, Drug; Glutathione; Hindlimb; Insulin; Intercellular Adhesion Molecule-1; L-Lactate Dehydrogenase; Male; Malondialdehyde; Muscle, Skeletal; Organ Preservation Solutions; Phenanthrenes; Raffinose; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase

Ischemia-reperfusion-induced acute renal failure (ARF) is associated with a high mortality in patients with hypertension and with an unfavorable outcome of kidney transplants from marginal donors.. The influence of allopurinol and enalapril on urinary nitrate/nitrite (UNOx), glomerular filtration rate, plasma and urinary sodium, and hemodynamic parameters was examined in spontaneously hypertensive rats (SHR) with ARF.. ARF was induced by right-kidney removal and clamping the left renal artery for 40 min in 50 male 26-week-old SHR weighing 300 +/- 23 g. The rats were randomly allocated to five groups: (1) sham operated; (2) ARF; (3) ARF after pretreatment with 40 mg/kg allopurinol; (4) ARF after pretreatment with 40 mg/kg enalapril, and (5) ARF after pretreatment with 40 mg/kg allopurinol and 40 mg/kg enalapril. Creatinine clearance, UNOx (Griess reaction), cardiac output (dye dilution technique), mean arterial blood pressure, and renal blood flow were measured 24 h after reperfusion. Total vascular resistance and renal vascular resistance were calculated and compared between the groups.. A nonsignificant decrease was found in both daily UNOx excretion and creatinine clearance when pretreated ARF groups and the ARF group without pretreatment were compared (p > 0.05). Significantly lower plasma sodium values (139.5 +/- 4.86 mmol/l) in the allopurinol-pretreated ARF group were found than in the ARF group without pretreatment, in the ARF group pretreated with enalapril, and in the sham SHR group (p = 0.029). The urinary sodium loss was greater in the enalapril-pretreated than in the allopurinol-pretreated ARF group (p = 0.047). Allopurinol and/or enalapril pretreatment decreased total vascular resistance (p = 0.003) in comparison with the sham SHR group.. Neither allopurinol nor enalapril nor both were protective against ischemia-reperfusion injury in SHR, nor altered glomerular filtration rate and UNOx in a favorable direction.

Topics: Acute Kidney Injury; Allopurinol; Animals; Antihypertensive Agents; Antimetabolites; Drug Combinations; Enalapril; Male; Nitrates; Nitrites; Rats; Rats, Inbred SHR; Reperfusion Injury; Sodium; Treatment Outcome

Cold ischemia--warm reperfusion (CI/WR) injury of liver transplantation involves hepatocyte cell death, the nature and underlying mechanisms of which remain unclear. Isolated hepatocytes and isolated perfused livers were used to determine the prevalence of necrosis and apoptosis as well as mitochondrial dysfunction. In isolated cells, propidium iodide and Hoechst 33342 staining showed a cold-storage, time-dependent increase in necrosis, whereas apoptosis was minimal even after 48 h of hypothermia. Nonetheless, a progressive loss of mitochondrial membrane potential was observed. Translocation of mitochondrial cytochrome c toward microsomes occurred within 24 h of CI/WR, with cytochrome c reaching the cytosol later. Mitochondria isolated from whole livers subjected to CI/WR also display reduced metabolic parameters and increased susceptibility to swelling. These events are associated with increased activity of major initiator (caspase 9) and effector (caspase 3) caspases. The results demonstrate that CI/WR induces mitochondrial dysfunction in isolated cells and in the whole organ; only in the latter is that sufficient to trigger the classical mitochondrial pathway of apoptosis. Our study also provides evidence for the involvement of endoplasmic reticulum stress in CI/WR hepatocyte injury. Combined protection of mitochondria and endoplasmic reticulum may thus represent an innovative therapeutic avenue to enhance liver graft viability and functional integrity.

Topics: Adenosine; Allopurinol; Animals; Caspase 3; Caspase 9; Caspases; Cell Death; Cold Temperature; Cytochromes c; Glutathione; Hepatocytes; In Vitro Techniques; Insulin; Liver; Liver Transplantation; Male; Membrane Potentials; Mitochondria; Mitochondrial Swelling; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury

This study examined the effects of ischemic preconditioning (IPC), allopurinol (Allo) or a combination of both on the extent of mitochondrial injury caused by hepatic ischemia/reperfusion (I/R). I/R increased the serum aminotransferase activity and the level of mitochondrial lipid peroxidation, whereas it decreased the mitochondrial glutathione level. Either IPC or Allo alone attenuated these changes with Allo+IPC having a synergistic effect. Allo increased the serum nitrite and nitrate level after brief ischemia. The significant peroxide production observed after 10 min of reperfusion after sustained ischemia was markedly attenuated by Allo+IPC. The mitochondria isolated after I/R were swollen, which was reduced by Allo+IPC. At the end of ischemia, the hepatic ATP level was lower and there was significant xanthine accumulation, which was attenuated by Allo+IPC. These results suggest that IPC and Allo act synergistically to protect cells against mitochondrial injury and preserve the hepatic energy metabolism during hepatic I/R.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Ischemic Preconditioning; Liver; Male; Malondialdehyde; Mitochondria; Mitochondrial Swelling; Nitrates; Nitric Oxide; Peroxides; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Transaminases; Xanthine

Acute pancreatitis can lead to increased pulmonary vascular permeability and respiratory failure. Oxidants (and their generator, xanthine oxidase (XO)) play an important role in injuring the structural integrity of the pulmonary epithelium and endothelium, but their importance in the induction of acute lung injury following pancreas ischaemia-reperfusion (IR) has not been defined.. Rats (n = 48) received a regular or a tungsten (oxidoreductase inhibitor)-enriched diet for 14 days. Their isolated pancreases were then either perfused (controls) or made ischaemic (IR) for 40 min (12 replicates/group). This was followed by in-series pancreas plus normal isolated lung reperfusion for 15 min. Lungs only were subsequently perfused with the 15-min accumulated pancreas effluents for 45 min.. Injury was induced in all IR pancreases as expressed by reperfusion pressure, wet-to-dry ratio and amylase and lipase concentrations. Tissue XO activity was high and reduced glutathione pool was low in the tungsten-free IR pancreases. Pulmonary plateau pressure increased by 46% and final PO(2)/FiO(2) decreased by 24%. Capillary pressure and weight rose two- to fourfold in lungs paired with IR non-treated pancreases. Twofold increases in bronchoalveolar lavage volume and contents, including XO, were also recorded in this group of lungs. Lungs exposed to tungsten-treated ischaemic pancreas effluents were minimally damaged and tissue XO content was low compared to controls.. Ex-vivo acute pancreatitis induces acute lung injury via oxidants/antioxidants misbalance, which may be prevented by attenuating pancreas oxidative stress.

Topics: Acute Disease; Animals; Lung Diseases; Male; Oxidative Stress; Pancreas; Pancreatitis; Rats; Reperfusion Injury; Xanthine Oxidase

The aim of this study was to investigate whether the protective effect of endothelin-A (ET(A)) receptor antagonist BQ-123 against renal ischemia reperfusion (I/R) injury is related to nitric oxide (NO) production. Sprague-Dawley rats were divided into six groups: control, I/R, N sup omega nitro-L-arginine methyl ester (L-NAME), BQ, BQ+L-NAME, BQ+L-NAME+L-Arg groups. After urethane anesthesia, 30min renal ischemia and 2h reperfusion were performed in all groups except control group. Mean arterial pressures (MAP) during reperfusion in all L-NAME-treated groups were higher than during pre-ischemia and ischemia, however, MAP at 60th and 120th minute of reperfusion in control and BQ groups were lower than during ischemia. MAP of L-NAME-treated groups were significantly higher than the other groups during reperfusion period. The I/R caused lipid peroxidation and protein oxidation, however, BQ-123 treatment prevented oxidant injury. The inhibition of NO production prevented effect of BQ-123 treatment. Also, BQ-123 treatment caused an increase in superoxide dismutase and catalase activities. Both BQ-123 and L-NAME treatments prevented high xanthine oxidase activity. BQ-123 prevented risen myeloperoxidase activity and L-NAME reversed this effect of BQ-123 just like the addition of L-arginine to the treatment altered the effect of L-NAME. The plasma BUN was affected as increasing manner from L-NAME treatments; on the other hand, plasma Cr and Na concentrations were affected as decreasing manner from BQ-123 treatments. ET(A) receptor antagonist BQ-123 may be revealed a protective agent against renal I/R injury with a possible secondary pathway via its antioxidant effects. We suggest that BQ-123 may mediate the protective effect via a NO-dependent mechanism.

Topics: Animals; Blood Pressure; Blood Urea Nitrogen; Catalase; Endothelin A Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Glutathione Peroxidase; Kidney; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Organ Size; Peptides, Cyclic; Peroxidase; Rats; Rats, Sprague-Dawley; Renal Circulation; Reperfusion Injury; Sodium; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Xanthine Oxidase

The transplanted lung suffers ischemia-reperfusion injury and many efforts have been made to improve preservation. The aim of this study was to compare the effectiveness of intracellular high-potassium Belzer solution versus extracellular Celsior in 24-hour lung preservation.. Sixteen pigs weighing 25.1-28.1 kg underwent single, left-lung allotransplantation. Sixteen other pigs were donors of the left lung. Heart and lungs were retrieved from the donors using single-flush perfusion by a randomly selected solution that was also used for cold storage (4 degrees C) for 24 hrs. Biopsies were taken from the right lung before storage, after the preservation, and finally three hours following transplantation. The observation period lasted three hours following transplantation.. Pulmonary artery pressure and pulmonary vascular resistance were significantly higher in the Belzer group (p<0.05), while the pO2/FiO2 ratio was much higher in the Celsior group (p<0.05). Compliance was reduced approximately equally in both groups. Histology showed less edema, atelectasis, and hemorrhagic infiltration in the Celsior group than in the Belzer group, which developed more interstitial thickening and presented more leukocyte infiltration and desquamation of alveoli cells.. According to hemodynamic, respiratory, and histological data, the extra-cellular solution Celsior offers improved prolonged preservation compared with Belzer.

Topics: Adenosine; Allopurinol; Anesthesia; Animals; Blood Pressure; Disaccharides; Electrolytes; Glutamates; Glutathione; Graft Survival; Histidine; Insulin; Lung; Lung Transplantation; Mannitol; Organ Preservation Solutions; Potassium; Pulmonary Artery; Raffinose; Reperfusion Injury; Swine; Temperature; Time Factors

Production of free radical species in cells and body tissues is known to cause many pathological disorders. Therefore, free radical scavengers play an important role in the prevention of various human diseases. Bamboo grass, Sasa senanensis, is a native Japanese plant. Sasa has been used for medicine in Japan for many centuries. In this study, we investigated the antioxidative activity of Absolutely Hemicellulose Senanensis (AHSS), a novel extract from Sasa. In the first part of this study, we found that AHSS has antioxidant activities by the assay using superoxide anion-2-methyl-6-methoxyphenylethynylimidazopyrazynone (MPEC) reaction kit. We then confirmed its antioxidative activity using a rat ischemia and subsequent reperfusion (I/R) injury model. Breakdown of the intestinal wall caused by intestinal I/R was attenuated by pretreatment with AHSS. Moreover, AHSS inhibited the production of lipid peroxide by intestinal I/R. AHSS could be an important source of ingredients for use in functional foods and other applications.

Topics: Allopurinol; Animals; Antioxidants; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Intestine, Small; Lipid Peroxides; Luminescence; Male; Mesenteric Vascular Occlusion; Phytotherapy; Plant Extracts; Rats; Rats, Wistar; Reperfusion Injury; Sasa; Xanthine Oxidase

There is increasing evidence to suggest that toxic oxygen radicals play an essential role in the pathogenesis of ischemia/reperfusion (I/R) injury in the kidney. This study was designed to investigate the effects of leflunomide, an isoxazole derivative and a unique immunomodulatory agent, in I/R-induced renal injury in rats.. Forty female Sprague-Dawley rats were divided equally into four groups: (I) control (only leflunomide 10 mg/kg, intragastrically treated); (II) sham operated (only unilateral nephrectomy); (III) I/R; and (IV) leflunomide (10 mg/kg for two doses prior to experiment) plus I/R groups. In groups III and IV, after unilateral nephrectomy, the rats were subjected to 60 min of left renal pedicle occlusion, followed by 6 h of reperfusion. At the end of the reperfusion period, rats were killed and kidneys and blood were removed. Catalase, myeloperoxidase and xanthine oxidase activities, and malondialdehyde, nitric oxide and protein carbonyl levels were determined in renal tissue. Serum creatinine, blood urea nitrogen and aspartate aminotransferase were measured for the evaluation of renal function. In histopathological examination, renal damage was scored 0-3.. Group III animals demonstrated severe deterioration of renal function, renal morphology and a significant renal oxidative stress. Pretreatment of animals with leflunomide markedly attenuated renal dysfunction, morphological alterations, reduced elevated oxidative stress products levels and restored the depleted renal antioxidant enzyme.. The findings imply that oxygen radicals play a causal role in I/R-induced renal injury, and leflunomide exerts renoprotective effects probably by the radical scavenging and antioxidant activities with immunomodulatory effect.

Topics: Animals; Antioxidants; Aspartate Aminotransferases; Blood Urea Nitrogen; Catalase; Creatinine; Female; Immunosuppressive Agents; Isoxazoles; Kidney; Leflunomide; Lipid Peroxidation; Nitric Oxide; Oxidative Stress; Peroxidase; Protein Carbonylation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Xanthine Oxidase

Graft pancreatitis (GP) is one the main technical problems associated with pancreas transplant (PT). It occurs in 20% of patients representing a risk factor for thrombosis and cause of graft loss. GP is related to oxidative effects from oxygen-derived free radicals (OFR) in ischemia-reperfusion injury. We evaluated lipid peroxidation by the OFR in the PT of pig organs preserved with either Celsior or Wisconsin solutions.. In Landrace pigs we performed 24 pancreas allografts, which were preserved 18 or 24 hours: 12 with Celsior solution (CS) and 12 with Wisconsin solution (UW). No immunosuppression was administered. The oxidative effects were determined by quantification of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) and of the carbonyl groups of proteins in our pancreatic tissue samples and measured at different times: (A) baseline in the donor, (B) after perfusion of the graft, (C) after the ischemia period, and (D) 30 minutes after ischemia-reperfusion of the graft.. The MDA and 4-HDA values were similar in conditions A, B, and C, but showed an extraordinary increase after ischemia-reperfusion in D, among both the 18- or 24-hour preserved grafts and in the same proportion with CS and UW. The carbonyl groups of the proteins rose in conditions B and C (cold ischemia), but less so in state D (reperfusion).. The oxidative injury of a pancreatic graft preserved for 18 or 24 hours occurs during reperfusion, with an extraordinary intensity, but similarly with CS and UW, an observation that may help to explain graft pancreatitis.

Topics: Adenosine; Allopurinol; Animals; Disaccharides; Electrolytes; Glutamates; Glutathione; Histidine; Insulin; Mannitol; Models, Animal; Organ Preservation Solutions; Oxidative Stress; Pancreas; Pancreas Transplantation; Postoperative Complications; Raffinose; Reperfusion Injury; Swine

To investigate the role of oxygen free radicals (OFR) in hepatic ischemia and reperfusion injury (HI/RI) and effect of propofol on them.. The rabbits were randomly divided into three groups (n=10), sham operated group (Control), HIR group(I/R) and HIR + propofol group (PRO). Changes of several parameters which included malondialdehyde (MDA), superoxide dismutase (SOD), xanthine oxidase (XO) and alanine aminotransferase(ALT) were measured before ischemia, 45 minutes after ischemia and 45 minutes after reperfusion in plasma. Meanwhile MDA concentration, SOD, XO activities and ALT value in liver tissue were measured, and the ultrastructure changes in liver tissue were observed under electron microscope at 45 minutes after reperfusion.. As compared with group control, XO, MDA and ALT increased and SOD decreased during HI/RI (P < 0.05 and P < 0.01) in plasma, and XO, MDA increased as well as SOD decreased at 45 minutes after reperfusion (P < 0.05 and P < 0.01) in liver tissue and there were abnormal changes of the hepatic ultrastructure at 45 minutes after reperfusion. Propofol reversed the results of mentioned indices as above markedly (P < 0.05 and P < 0.01).. OFR is an important factor during HI/RI, propofol may attenuate hepatic ischemia-reperfusion injury by dropping OFR level (raising SOD activity and dropping XO activity) and antagonizing lipid pe-reoxidation (reducing MDA content).

Topics: Alanine Transaminase; Animals; Free Radical Scavengers; Liver; Malondialdehyde; Oxidation-Reduction; Propofol; Rabbits; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

This study aimed to explore the hypothesis that activated complement components contribute significantly to I/R (ischaemia/reperfusion) injury in skeletal muscle. After 50, 70 and 90 min of tourniquet ischaemia and 24 h of reperfusion, viability of the medial gastrocnemius muscle in CBA-C57BL/6 wild-type mice, assessed histochemically by reduction of NBT (Nitro Blue Tetrazolium) dye, was 60, 21 and 8% respectively. Skeletal muscle viability after 70 min of ischaemia and 24 h of reperfusion in transgenic mice expressing a combination of human CD46, CD55 and CD59, all inhibitors of complement activation, was 45% compared with 24% in ischaemic reperfused wild-type mice (P=0.008; n=6 per group). Muscle from sham-treated transgenic mice and wild-type littermates had no significant loss of viability relative to normal contralateral gastrocnemius muscle. A significant reduction in myeloperoxidase activity (a measure of neutrophil infiltration), xanthine oxidase activity (a source of free radicals) and water content (a measure of oedema) was observed in ischaemic reperfused muscle from transgenic mice compared with ischaemic reperfused wild-type muscle (P<0.05). Haematoxylin and eosin-stained histological sections also showed less damage and less apparent leucocyte infiltration in muscles from ischaemic reperfused transgenic mice than those from wild-type animals given the same degree of injury. Muscles from sham-treated transgenic and wild-type controls were almost identical with normal muscle. It is concluded that complement activation contributes to the pathogenesis of I/R injury in murine skeletal muscle, resulting in increased neutrophil infiltration into the injured muscle, increased free radical production and vascular permeability during reperfusion, and a net detrimental effect on muscle viability.

Topics: Animals; Antigens, CD; CD55 Antigens; CD59 Antigens; Complement Inactivator Proteins; Edema; Hindlimb; Humans; Membrane Cofactor Protein; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Models, Animal; Muscle, Skeletal; Neutrophil Infiltration; Peroxidase; Reperfusion Injury; Xanthine Oxidase

To assess the effect of five antioxidants on exocrine function of rabbit testes retained in situ for 24 h and 3 months after experimental torsion.. The left testes of peripubertal rabbits were clamped for 60 min, after which the clamps were removed and the testes allowed to reperfuse. The right testes served as internal controls. There were eight rabbits in each of the following experimental groups: (a) sham; (b) 60-min ischaemia followed by reperfusion; (c) 60-min ischaemia followed by left orchidectomy. In five further groups, rabbits were exposed to 60-min ischaemia followed by reperfusion, but received one of the following antioxidants before reperfusion: acetyl salicylic acid, ascorbic acid, allopurinol, quercetin or superoxide dismutase. Both testes were excised at 24 h or 3 months. The degree of lipid peroxidation, a measure of free radical damage, was assessed in testicular tissue homogenates by measuring the tissue levels of malondialdehyde (MDA). The Johnsen score was used to assess the morphological damage at 24 h and 3 months for each group.. At 3 months twisted viable testes allowed to reperfuse had higher MDA levels than controls; the left testes of rabbits treated with allopurinol had significantly lower MDA levels than untreated rabbits and rabbits given other antioxidants. Rabbits given quercetin, ascorbic acid or superoxide dismutase had lower (but not significantly) left testicular MDA levels than untreated rabbits, while rabbits given acetyl salicylic acid had even higher levels. Allopurinol-treated rabbits had a Johnsen score of > 7.6 and those given other antioxidants had scores of < 7.6 at 3 months.. The twisted viable testis treated by orchidopexy contains high free radical levels at 3 months. Of the antioxidants studied, only allopurinol had a beneficial long-term effect, by significantly reducing testicular MDA levels at 3 months.

Topics: Allopurinol; Animals; Antioxidants; Drug Evaluation, Preclinical; Free Radical Scavengers; Male; Orchiectomy; Rabbits; Reperfusion Injury; Spermatic Cord Torsion; Testis; Time Factors

Cold ischemia is the best known method to preserve kidneys for transplant. However, it produces several detrimental effects. First, cellular necrosis. Secondarily, during the hypothermic period a mitochondrial injury process develops which makes the cell entering a pre-apoptotic state. This apoptosis occurs definitively in the reperfusion. Preservation solutions currently available are not perfect and are not able to avoid cold-related cell injuries. The addition of certain substances to UW solution (desferrioxamine) has shown experimentally a reduction in mitochondrial cold-related lesions. Isolated hypothermic kidney perfusion reduces initial graft dysfunction about 20% in comparison to hypothermic storage. This fact relates to important either economical as functional consequences.

Topics: Adenosine; Allopurinol; Cold Temperature; Glutathione; Graft Survival; Humans; Insulin; Ischemia; Kidney; Kidney Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury

We analysed the effect of ischemia-reperfusion injury to renal parenchyma after unilateral renal artery clamping using urinary N-acetyl-beta-D-glucosaminidase (NAG) that is a sensitive parameter of early renal tubular injury. In the study 60 mongrel dogs were divided into 3 groups: in the 1st group the left renal artery was clamped for 45 minutes, in the 2nd group Allopurinol was administered before the clamping, the 3rd was the control group, where only laparotomy and closure of the abdomen was performed. Urinary NAG activity referring to urinary creatinine (NAG index) was determined before the operation, at the beginning of the reperfusion, in the 60th and 120th minute of the reperfusion then 1, 2, 3 and 5 days after the operation. The highest NAG indices relating to injury of the proximal tubuli were found at the beginning of the reperfusion, in the 60th and 120th minutes of the reperfusion, then NAG returned to preoperative level in each group. Significantly higher NAG indices were found in the ischemia-reperfusion group compared to the group with Allopurinol pretreatment. Renal ischemia-reperfusion injury and the protective effect of Allopurinol could be detected by lysosomal NAG enzyme. The injury of the tubular function was reversible so it could be a change in tubular function.

Topics: Acetylglucosaminidase; Allopurinol; Animals; Creatinine; Dogs; Enzyme Inhibitors; Kidney Tubules; Reperfusion Injury; Time Factors

It was demonstrated that xanthine oxidoreductase (XOR), during ischemia, catalyzes the formation of nitric oxide (NO) from nitrite (NO2-) and this NO2- -derived NO protects the isolated perfused rat heart against the damaging effects of ischemia-reperfusion (I/R) when conventional nitric oxide synthase (NOS)-dependent NO production is impaired. Liver is one of the organs with the highest XOR concentration. This study was designed to determine whether NO2- -derived NO by XOR protects liver against I/R injury in vivo. For its minute amounts and active reactivity, NO can not be detected directly in real time in vivo by this time. We have to prove the above hypothesis indirectly.. Wistar rats were pretreated with saline, NOS inhibitor L-NAME (10 mg/kg intravenously), XOR inhibitor allopurinol (1.5 mg/kg orally), L-NAME +allopurinol and NO scavenger carboxy-PTIO (0.6 mg/kg intravenously) respectively (12 animals per group). And then, they were subjected to total liver ischemia for 40 minutes followed by reperfusion. Blood samples and liver tissues were obtained for analysis after 3 hours of reperfusion. Survival was also investigated.. Allopurinol-treated animals exhibited further increased serum alanine aminotransferase(ALT) levels and liver myeloperoxidase(MPO) activities, but further decreased liver adenosine triphosphate(ATP) stores after I/R compared to saline-treated counterparts (830.5+/-108.3 U/L, 56.5+/-11.0 U/mg protein and 1.93+/-0.47 mumol/g vs. 505.8+/-184.2 U/L, 41.5+/-10.2 U/mg protein and 3.05+/-0.55 micromol/g respectively, P < 0.01, P < 0.05 and P < 0.01 respectively). The hepatocyte injury was further exacerbated and the overall survival rate was significantly decreased after I/R in animals given by allopurinol compared to those pretreated by saline (P < 0.05). L-NAME and allopurinol co-treated animals exhibited more severe liver injury (P < 0.05 and P<0.01)and a further decreased overall survival rate (P < 0.05)compared to L-NAME or allopurinol alone-treated counterparts, but they were not different from carboxy-PTIO treated animals (P > 0.05).. NO2- -derived NO by XOR in the hypoxic and acidic environment induced by hepatic I/R protects the liver against I/R injury in vivo.

Topics: Adenosine Triphosphate; Alanine Transaminase; Allopurinol; Animals; Enzyme Inhibitors; Female; Liver; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Survival Analysis; Xanthine Dehydrogenase

Topics: Allopurinol; Animals; Free Radical Scavengers; Male; Models, Animal; Rabbits; Reperfusion Injury; Spermatic Cord Torsion

Allopurinol is a xanthine oxidase inhibitor that prevents the generation of free radicals and may play a role in the protection of the cells during cerebral ischemia.. We evaluated the protective and therapeutic effect of allopurinol on reversible focal cerebral ischemia-reperfusion model in rats. Cerebral blood flow to the left hemisphere of adult Sprague-Dawley rats (n = 40) was temporarily interrupted by middle cerebral artery (MCA) and bilateral common carotid artery (CCA) occlusion for 3 hours in 5 groups of 8 rats each. Allopurinol (50 mg/kg) was given intraperitoneally 2 hours and immediately before ischemia and immediately and 2 hours after reperfusion in 4 different groups of rats, respectively. Animals were kept alive 24 hours after reperfusion. After sacrifice, infarction volumes and ratios of the brain slices were calculated, and the results were compared with those of the control group.. The difference between the allopurinol-administered group and the control group 2 hours before for both infarction volumes and infarction ratios achieved statistical significance. Regarding the allopurinol-administered group immediately before ischemia, infarction volumes and infarction ratios were diminished, but there was no statistically significant difference. The difference between allopurinol-administered and control group immediately after and 2 hours after reperfusion for both infarction volumes and infarction ratios achieved no statistical significance.. This study showed that allopurinol has a protective effect, but not a therapeutic effect, on cerebral ischemia.

Topics: Allopurinol; Animals; Brain Ischemia; Disease Models, Animal; Drug Administration Schedule; Enzyme Inhibitors; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Xanthine Oxidase

The aims of the present study were to determine the effects and mechanisms of angiotensin II (Ang II) on leukocyte-endothelium interactions and the role of Ang II in a novel model of ischemia/reperfusion (I/R) in the mouse colon. Ang II dose-dependently increased leukocyte rolling and adhesion in colonic venules. Importantly, Ang II-induced leukocyte rolling was completely inhibited by immunoneutralization of P-selectin, and leukocyte adhesion was abolished in lymphocyte function antigen-1 (LFA-1)-deficient mice. The P-selectin-dependent rolling was found to be a precondition for the subsequent LFA-1-dependent leukocyte adhesion. Moreover, Ang II-induced leukocyte responses involved generation of reactive oxygen species and up-regulation of CXC chemokines. Notably, CXC chemokines, but not Ang II, stimulated leukocyte chemotaxis in vitro. I/R increased gene expression of angiotensin converting enzyme (ACE) in the colon and plasma concentrations of Ang II. Inhibition of ACE and the type 1 angiotensin (AT1) receptor significantly decreased the I/R-induced leukocyte adhesion. Taken together, these novel findings demonstrate that Ang II exerts potent pro-inflammatory effects in the colonic microcirculation and that inhibition of Ang II expression or function protects against I/R-induced leukocyte responses in the colon. Thus, it is suggested that Ang II is a major target to control pathological inflammation in the colon.

Topics: Allopurinol; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Capillaries; Captopril; Cell Adhesion; Chemokines; Chemotaxis, Leukocyte; Colon; Constriction; Disease Models, Animal; Endothelium, Vascular; Enzyme Induction; Gene Expression Regulation; Inflammation; Ischemia; Leukocyte Count; Leukocytes; Losartan; Male; Mesenteric Artery, Superior; Mice; Mice, Inbred C57BL; Mice, Knockout; P-Selectin; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase

The influence of hyperbaric oxygen (HBO) treatment on the activities of superoxide dismutase (SOD) and Na(+),K(+)-ATPase was determined during different time periods of reperfusion in rats exposed to global cerebral ischemia. Ischemic animals were either sacrificed or exposed to the first HBO treatment 2, 24, 48 or 168 h after ischemic insult (for SOD activities measurement) or immediately, 0.5, 1, 2, 6, 24, 48, 72 or 168 h after ischemic procedure (for Na(+),K(+)-ATPase activities measurement). Hyperbaric oxygenation procedure was repeated for seven consecutive days. The results of presented experiments demonstrated the statistically significant increase in the hippocampal SOD activity 24 and 48 h after global cerebral ischemia followed by a decrease in the enzymatic activity 168 h after ischemic insult. In the ischemic rats treated with HBO the level of hippocampal SOD activity was significantly higher after 168 h of reperfusion in comparison to the ischemic, non HBO-treated animals. In addition, it was found that global cerebral ischemia induced a statistically significant decrease of the hippocampal Na(+),K(+)-ATPase activity starting from 1 to 168 h of reperfusion. Maximal enzymatic inhibition was obtained 24 h after the ischemic damage. Decline in Na(+),K(+)-ATPase activity was prevented in the animals exposed to HBO treatment within the first 24 h of reperfusion. Our results suggest that global cerebral ischemia induces significant alterations in the hippocampal SOD and Na(+),K(+)-ATPase activities during different periods of reperfusion. Enhanced SOD activity and preserved Na(+),K(+)-ATPase activity within particular periods of reperfusion, could be indicators of a possible beneficial role of HBO treatment in severe brain ischemia.

Topics: Adenosine Triphosphate; Animals; Brain Ischemia; Hippocampus; Hyperbaric Oxygenation; Rats; Rats, Wistar; Reperfusion Injury; Sodium-Potassium-Exchanging ATPase; Superoxide Dismutase; Xanthine; Xanthine Oxidase

In organ transplantation, ischemia-reperfusion injury (IRI) has been implicated in delayed graft function (DGF) as well as in short- and long-term complications. Using an autotransplant pig kidney model, changes in renal function and morphology were determined after different periods of cold ischemia in kidneys preserved in the University of Wisconsin solution (UW), high-Na(+) version of UW (HEH) or Celsior (CEL) a newly developed high-Na(+) solution, with or without trimetazidine (TMZ). Kidney function was better preserved in CEL, UW and particularly HEH in combination with TMZ, particularly after 48 and 72 h. Mitochondria integrity was improved in TMZ-preserved groups. This study indicates that TMZ is efficiently protective against IRI even after prolonged preservation and in different preservation solutions.

Topics: Adenosine; Allopurinol; Animals; Biomarkers; Cryopreservation; Disaccharides; Disease Models, Animal; Drug Evaluation, Preclinical; Electrolytes; Glutamates; Glutathione; Histidine; Insulin; Kidney Function Tests; Kidney Transplantation; Mannitol; Mitochondria; Organ Preservation; Organ Preservation Solutions; Raffinose; Receptors, GABA-A; Reperfusion Injury; Swine; Time Factors; Transplantation, Autologous; Trimetazidine

We assessed the effect of adding exogenous fructose-1,6-biphosphate (F16BP) to the preservation solution (University of Wisconsin storage solution) used during an experimental procedure of small bowel transplantation in rats.. We studied levels of the nucleotides hypoxanthine/xanthine and adenosine in tissue after cold ischemia, as well as histologic changes and associated deleterious processes such as bacterial translocation produced by the reperfusion associated with the transplantation.. The groups of rats treated with F16BP showed the lowest levels of hypoxanthine/xanthine and uric acid, the highest levels of adenosine, and the lowest levels of histologic damage and lactate dehydrogenase release to the bloodstream. Consumption of intestinal hypoxanthine during reperfusion was lowest in the groups treated with F16BP, as was the incidence of bacterial translocation.. This study shows a protective effect of exogenous F16BP added to University of Wisconsin solution during experimental intestinal transplantation in rats. This protective effect, reflected by decreased intestinal damage and bacterial translocation, was related to a decrease in adenosine triphosphate depletion during cold ischemia before intestinal transplantation, and to the reduced availability of xanthine oxidase substrates for free radical generation during reperfusion.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Bacterial Translocation; Cryopreservation; Cytoprotection; Fructosediphosphates; Glutathione; Hypoxanthine; Insulin; Intestine, Small; Intestines; L-Lactate Dehydrogenase; Male; Organ Preservation Solutions; Oxidative Stress; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Uric Acid; Xanthine

This study aimed to evaluate the relation between apoptosis of enterocytes and oxygen-free radical injury in scalded rats with delayed resuscitation as well as the role of antioxidants in the prevention of enterocyte apoptosis.. For this study, 150 male Wistar rats were divided randomly into four groups representing early resuscitation (ER), delayed resuscitation (DR), N-acetylcysteine (NAC) treatment, and allopurinol (Allo) treatment. The animals were subjected to a 30% total body surface area, full-thickness scald. Fluid therapy was started 6 hours after the injury in the DR and treatment groups. Apoptosis of enterocytes was identified by DNA fragmentation (ap%), DNA agarose gel electrophoresis, and terminal deoxynucleotidyl transferace (TdT)-mediated dUPT-biotin nick end labeling (TUNEL). The contents of malondialdehyde (MDA), total sulfhydryl (TSH), and nonprotein sulfhydryl (NPSH) and the activity of xanthine oxidase in intestinal mucosa were determined after the burn in the four groups.. Apoptosis of enterocytes increased significantly in all the groups. The animals in the DR group showed an earlier and greater increase in ap% than the animals in the ER group. Similar results were seen for electrophoresis, TUNEL assay, and levels of MDA, xanthine oxidase (XO), TSH, and NPSH. Treatment with NAC was associated with a decrease in ap% and MDA, but not XO, as compared with the levels in the DR group, whereas treatment with Allo was associated with a decrease in MDA and XO, but not ap%. Delayed resuscitation was associated with significant decreases in TSH and NPSH, as compared with the levels in the ER group, whereas both the NAC and Allo groups had significantly higher levels of TSH and NPSH than the DR group.. Significant apoptosis of enterocytes was induced by oxidative stress in the intestinal mucosa after a burn in rats. The findings show that NAC blunted intestinal apoptosis induced by oxygen-free radical, which was generated in the process of ischemia-reperfusion injury after a burn because of delayed resuscitation.

Topics: Acetylcysteine; Allopurinol; Animals; Apoptosis; Burns; DNA Fragmentation; Enterocytes; Glutathione; Ilium; In Situ Nick-End Labeling; Intestinal Mucosa; Male; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Resuscitation; Shock; Xanthine Oxidase

The mechanisms underlying liver graft dysfunction are not completely defined, although much of the injury derives from oxidative stress in organ reperfusion. The antioxidant glutathione in its reduced form (GSH) is an important agent to detoxify oxygen species after reperfusion. However, this effect might be limited by low concentrations at the end of cold storage. The objective of this study was to evaluate GSH and glutathione oxidized (GSSG) hepatic levels pre- and postreperfusion and correlate with hepatocellular injury and liver function in the 5 subsequent days after transplantation.. Liver biopsies were taken immediately before implant and 2 hours after venous reperfusion in 34 grafts, determining GSH, GSSG levels, and GSSG/GSH ratio. Aminotransferases (ALT, AST) and PT were measured for 5 days.. There was a strong decrease in GSH concentration (P <.0001), increase of GSSG levels (P <.01), and increase of the GSSG/GSH ratio (P <.0001). No correlations were found between GSH, GSSG, or GSH/GSSH levels and AST, ALT, and PT.. Glutathione levels showed significant changes after 2 hours of reperfusion, due to intense oxidative stress. Therapies to replenish GSH should be considered as a protective measure to avoid liver graft dysfunction after transplantation.

Topics: Adenosine; Adult; Allopurinol; Cause of Death; Female; Glutathione; Glutathione Disulfide; Hepatocytes; Humans; Insulin; Liver; Liver Function Tests; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Raffinose; Reperfusion Injury; Retrospective Studies

Topics: Allopurinol; Animals; Blood Flow Velocity; Intercellular Adhesion Molecule-1; Intestines; Ischemia; Male; Rats; Reperfusion Injury

This study investigated the effects of pyrrolidine dithiocarbamate (PDTC), a novel NF-kappaB inhibitor, on the expression of multiple inflammatory mediators and on neutrophilic inflammation of the graft in rats following liver transplantation. Orthotopic liver transplantation (OLT) was performed after 24 hr of cold storage using University of Wisconsin (UW) solution that contained various concentrations of PDTC. We determined the time course of NF-kappaB activation and of the expression of multiple inflammatory signals: tumor necrosis factor-alpha (TNF-alpha), cytokine-inducible neutrophil chemoattractant (CINC), and intercellular adhesion molecule-1 (ICAM)-1. Serum alanine aminotransferase (ALT), intrahepatic myeloperoxidase (MPO/WBC ratio, a measure of neutrophil accumulation), and Mac-1 expression (CD11b/CD18, a measure of circulating neutrophil activity) were also evaluated. The results showed that PDTC decreased OLT-induced NF-kappaB activation in a dose-dependent manner (from 20 mmol/L to 60 mmol/L), diminished TNF-alpha, CINC, and ICAM-1 protein levels in the graft, and reduced the OLT-induced increase of serum TNF-alpha level. Pretreatment with PDTC significantly suppressed OLT-induced neutrophilic inflammation of the graft. The PDTC-exposed livers (PDTC, 40 mmol/L), in comparison with the control livers, had a significant reduction of MPO/WBC ratio (7.04+/-0.97 vs 14.07+/-1.31) and Mac-1 expression (181+/-11.3% vs 281+/-13.2%) at 6 hr after reperfusion. Furthermore, PDTC inhibited the increase of serum ALT levels after liver transplantation. In conclusion, PDTC inhibited NF-kappaB activation and the expression of the inflammatory mediators. These effects were associated with improved graft viability through inhibited intrahepatic neutrophilic inflammation. A therapeutic strategy directed at inhibition of NF-kappaB activation within the transplanted liver might be effective in reducing intrahepatic neutrophilic inflammation, and be beneficial to prolonged graft storage.

Topics: Adenosine; Allopurinol; Animals; Antioxidants; Chemokines, CXC; Glutathione; Insulin; Intercellular Signaling Peptides and Proteins; Liver Transplantation; Neutrophils; NF-kappa B; Organ Preservation Solutions; Pyrrolidines; Raffinose; Rats; Reperfusion Injury; Thiocarbamates; Time Factors; Tumor Necrosis Factor-alpha

D-Allose, a rare sugar, is one of the potent inhibitors of ischemia/reperfusion injury of the rat liver. To investigate the potency of this powerful agent we examined its effect against ischemia/reperfusion injury and compared it to that of allopurinol and superoxide dismutase.. Male Lewis rats were given water ad libitum preoperatively for 12 h and anesthetized by isoflurane inhalation anesthesia. Drugs were administered through a polyethylene catheter inserted into the portal vein for 2 h (D-allose), 10 min (allopurinol), or 5 min (superoxide dismutase) before ischemia, and the livers were then subjected to 70% ischemia, induced by crossclamping the vessels to the lateral and median lobes of the liver for 90 min. Rats were divided into four groups: group 1, pretreated with vehicle (normal saline); group 2, treated with D-allose; group 3, treated with allopurinol; and group 4, treated with superoxide dismutase. The effects of the drugs were evaluated by liver hemodynamics, neutrophil count, myeloperoxidase, liver enzymes, and histological studies.. D-Allose improved liver hemodynamics (P < 0.001) and postischemic animal survival (P < 0.05) significantly compared with the control group and nonsignificantly compared with the allopurinol and superoxide dismutase groups. Myeloperoxidase activity in the postischemic liver tissue was decreased significantly (P < 0.05) by D-allose compared with all other treatment and control groups. Neutrophil count was also significantly (P < 0.05) decreased in the D-allose group compared with than that in the control group, as well as the superoxide dismutase group. Only D-allose produced a statistically significant decrease in the level of liver enzymes, compared with levels in the control group.. The moderately protective effect of D-allose, which caused no clinical side effects, is encouraging. D-Allose had the best protective effect against neutrophil-related postischemic injury of the liver tissue, followed by allopurinol and superoxide dismutase. However, a more extensive study is needed to ensure the effects as well as the mechanisms of the effect of this rare sugar.

Topics: Allopurinol; Animals; Enzyme Inhibitors; Free Radical Scavengers; Glucose; Leukocyte Count; Male; Neutrophils; Rats; Rats, Inbred Lew; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

The guanidine compound ME10092 (1-(3,4-dimethoxy-2-chlorobenzylideneamino)-guanidine), which possesses a strong cardioprotective effect to ischemia-reperfusion, was assessed for different pharmacological actions that may underlie its cardioprotective effect. In the living rat ME10092 decreased the blood pressure and heart rate in a dose-dependent manner. We found ME10092 to bind to alpha 1- and alpha 2-adrenoreceptors with moderate affinity (Ki values 1-4 microM), and to block adrenaline-elicited contractile responses in isolated guinea pig aortas. Our results indicate that ME10092 possesses a certain anti-oxidant profile. Thus, in a competitive manner and with low affinity it inhibited the bovine milk xanthine oxidase enzyme, as well as NAD(P)H oxidase driven oxyradical formation in membrane fractions isolated from the rat brain. By using electron paramagnetic resonance we here show that, after its systemic administration, ME10092 modulates the nitric oxide (NO) content in several tissues of the rat in a time-dependent manner. However, in vitro ME10092 inhibited the activities of nitric oxide synthases nNOS and eNOS, but not that of iNOS. Our data give evidence that the cardioprotective effect of ME10092 could be mediated through pharmacological mechanisms that include some modulation of NO production, as well as possible inhibition of radical formation during ischemia-reperfusion.

Topics: Animals; Aorta; Blood Pressure; Brain; Cardiotonic Agents; Chlorocebus aethiops; COS Cells; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Guanidines; Guinea Pigs; Heart Rate; Humans; Ileum; Injections, Intravenous; Liver; Male; Muscle Contraction; Muscle, Smooth; Myocardial Ischemia; NAD; NADPH Oxidases; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Rats; Rats, Wistar; Receptors, Adrenergic, alpha; Reperfusion Injury; Xanthine Oxidase

Ischemic preconditioning has shown to reduce apoptosis in the intestinal mucosa during ischemia/reperfusion. This study evaluated if the decrease of apoptotic events found during preconditioning could be related with a reduction of the substrate (i.e., xanthine/hypoxanthine) available for xanthine oxidase (XO). Animals were randomly assigned to the following study groups: C, control; I/R, ischemia/reperfusion; P+I/R, ischemic preconditioning; P+I/R+H/X, ischemic preconditioning plus hypoxanthine/xanthine, and P+I/R+H/X+Allo, ischemic preconditioning plus hypoxanthine/xanthine plus allopurinol. Caspase-3 activity, DNA fragmentation and TUNEL staining increased in the I/R group compared to control. Ischemic preconditioning (P+I/R group) was able to reverse these apoptotic variables to a level similar to that of control rats. The addition of hypoxanthine/xanthine to rats subjected to ischemic preconditioning (P+I/R+H/X group) showed the highest apoptotic activity; however, further addition of allopurinol (P+I/R+H/X+Allo group) decreased significantly apoptotic activity and events. In conclusion, intestinal ischemic preconditioning is able to reduce apoptosis during the following sustained ischemia/reperfusion event because of a reduced accumulation of xanthine/hypoxanthine nucleotide.

Topics: Allopurinol; Animals; Apoptosis; Caspases; Enzyme Inhibitors; Hypoxanthines; Intestinal Mucosa; Intestine, Small; Ischemic Preconditioning; Male; Malondialdehyde; Rats; Rats, Wistar; Reperfusion Injury; Xanthine; Xanthine Oxidase

This experimental study was designed to determine the effects of melatonin on the levels of malondialdehyde (MDA), reduced glutathione (GSH), xanthine oxidase (XO) after adnexial torsion/detorsion (ischemia/reperfusion, I/R) of the ovaries of in rats. Forty adult albino rats were divided into five groups: sham operation, torsion, I/R plus saline, I/R plus melatonin and torsion plus melatonin. Rats in the sham-operated group underwent a surgical procedure similar to the other groups but the adnexa was not occluded. Rats in the torsion group were killed after adnexal torsion for 3 hr. Melatonin and saline were injected intraperitoneally (10 mg/kg) 30 min before detorsion to the I/R plus melatonin group and I/R plus saline group respectively. After 3 hr of ovarian detorsion, the rats were killed and ovaries were removed. Melatonin was injected intraperitoneally (10 mg/kg) 30 min before torsion to the torsion plus melatonin group. After 3 hr of ovarian torsion, the rats were killed and ovaries were harvested. The tissue levels of MDA, GSH and XO were measured. MDA and XO levels in the I/R plus saline group increased significantly when compared with torsion and sham-operated groups (P < 0.001). MDA and XO levels in the I/R plus melatonin group were lower than I/R plus saline and differences between the two groups were statistically significant (P < 0.001). GSH levels in the I/R plus saline group decreased significantly when compared with ischemia and sham-operated groups (P < 0.001). GSH levels in the I/R plus melatonin treated rats were significantly higher than I/R plus saline and ischemia groups (P < 0.001). The tissue levels of XO, MDA and GSH were similar between ischemia and ischemia plus melatonin groups. Morphologically, polymorphonuclear neutrophil infiltration and vascular dilatation were obvious in the I/R-damaged ovaries, and the changes also partially reversed by melatonin. This study demonstrates that melatonin protects the ovaries against oxidative damage associated with reperfusion following an ischemic insult.

Topics: Animals; Antioxidants; Female; Glutathione; Malondialdehyde; Melatonin; Ovary; Rats; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

Myocardial ischemia--reperfusion (MI/R) represents a clinically relevant problem associated with thrombolysis, angioplasty, and coronary bypass surgery. MI/R injury is known to occur on restoration of coronary flow after a period of myocardial ischemia. Injury of myocardium caused by I/R includes cardiac contractile dysfunction, arrhythmias, as well as irreversible myocyte damage. Prevention of myocardial death in acute coronary syndromes is the immediate goal of therapy. The main factor concerned with the experimental generation of reperfusion damage is oxygen-derived free radicals. This MI/R injury has been shown to be salvaged by supplementing antioxidants to diseased hearts. Caffeic acid phenethyl ester (CAPE), an active component of propolis extract, has antioxidant and anti-inflammatory properties, and may function in cardiac protection against I/R-induced damage. To test this hypothesis, we randomly assigned 14 male Wistar rats for necrosis experiments. To produce myocardial necrosis, the left main coronary artery was occluded for 30 min, followed by 120 min of reperfusion in anesthetized rats. CAPE (50 microM kg-1) was given intravenously 10 min before occlusion and continued during ischemia by infusion pump. The volume of infarct and the risk zone was determined by planimentry of each tracing and multiplying by the slice thickness. Infarct was normalized by expressing it as a percentage of the area at risk. Compared to control group, CAPE administration statistically reduced the myocardial infarct size/area of risk zone (50 +/- 4% and 32 +/- 6%, respectively) and the myocardial infarct size (23 +/- 3% and 9 +/- 4%, respectively) in rat model of ischemia-reperfusion. In conclusion, this result shows that CAPE is important in reducing I/R-induced myocardial damage.

Topics: Animals; Antioxidants; Caffeic Acids; Free Radicals; Lipoxygenase; Male; Myocardial Infarction; Myocardium; Neutrophils; Oxidative Stress; Oxygen; Phenylethyl Alcohol; Rats; Rats, Wistar; Reperfusion Injury; Time Factors; Xanthine Oxidase

In August rats, local myocardial ischemia caused by 30-min occlusion of the coronary artery induced a slight depression of the contractile function of the heart; the latter was restored after 15-min reperfusion more rapidly than in Wistar rats. In August rats, the activities of antioxidant protection enzymes were lower than in Wistar rats. In comparison with Wistar rats, these enzyme activities were decreased in a lesser degree under ischemia and were restored in a greater degree under reperfusion. It may thus be concluded that the higher stability of antiradical protection parameters in August rats is one of the mechanisms responsible for the enhanced resistance of the heart to ischemia- and reperfusion-induced injuries.

Topics: Animals; Blood Pressure; Catalase; Disease Models, Animal; Heart Rate; Male; Myocardial Contraction; Myocardium; Rats; Rats, Inbred Strains; Rats, Wistar; Reperfusion Injury; Species Specificity; Superoxide Dismutase; Xanthine Oxidase

The aim of this study was to assess whether depression of cardiac Na+,K(+)-ATPase activity during ischemia/reperfusion (I/R) is associated with alterations in Na+,K(+)-ATPase isoforms, and if oxidative stress participates in these I/R-induced changes. Na+,K(+)-ATPase alpha1, alpha2, alpha3, beta1, beta2, and beta3 isoform contents were measured in isolated rat hearts subjected to I/R (30 min of global ischemia followed by 60 min of reperfusion) in the presence or absence of superoxide dismutase plus catalase (SOD+CAT). Effects of oxidative stress on Na+,K(+)-ATPase isoforms were also examined by perfusing the hearts for 20 min with 300 microM hydrogen peroxide or 2 mM xanthine plus 0.03 U/ml xanthine oxidase (XXO). I/R significantly reduced the protein levels of all alpha and beta isoforms. Treatment of I/R hearts with SOD+CAT preserved the levels of alpha2, alpha3, beta1, beta2, and beta3 isoforms, but not that of the alpha1 isoform. Perfusion of hearts with hydrogen peroxide and XXO depressed all Na+,K(+)-ATPase alpha and beta isoforms, except for alpha1. These results indicate that the I/R-induced decrease in Na+,K(+)-ATPase may be due to changes in Na+,K(+)-ATPase isoform expression and that oxidative stress plays a role in this alteration. Antioxidant treatment attenuated the I/R-induced changes in expression of all isoforms except alpha1, which appears to be more resistant to oxidative stress.

Topics: Animals; Antioxidants; Catalase; Hydrogen Peroxide; In Vitro Techniques; Isoenzymes; Male; Myocardium; Oxidants; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sarcolemma; Sodium-Potassium-Exchanging ATPase; Superoxide Dismutase; Xanthine; Xanthine Oxidase

Ischemia / reperfusion (I / R) injury is related to tissue graft energy status. Insulin, which is currently used in the University of Wisconsin (UW) preservation solution with insulin (UWI), is an anabolic hormone and was shown to exacerbate the hepatic I / R injury in our previous study. In this study, the energy status and regulation of metabolism genes by insulin were investigated in liver grafts preserved by UW solution. Insulin could significantly decrease adenosine triphosphate (ATP) level after 3 hours of preservation, as well as total adenine nucleotides (TANs) and energy charge (EC) levels. Energy regeneration deteriorated in the grafts preserved by insulin in terms of ATP and EC levels at 24 hours after transplantation. The insulin signal was transduced through the insulin receptor substrate-2 (IRS-2) pathway and the activity of IRS-2 was decreased gradually at the messenger ribonucleic acid (mRNA) level during cold preservation. Downstream targeting genes such as sterol regulatory element-binding protein-1c (SREBP-1c), glucokinase (GKC), and fatty acid synthase (FAS) genes, as well as phospho-glycogen synthase kinase-3beta (GSK-3beta) were activated and they showed the similar expression profiles during cold preservation. Lipoprotein metabolism was accelerated by insulin through upregulation of the activity of apolipoprotein C-III (Apo C-III) during cold preservation. The insulin-like growth factor-binding protein-1 pathway was inhibited during cold preservation. In conclusion, insulin in UW solution exacerbates hepatic I / R injury by energy depletion as the graft maintains its anabolic activity. The key enzyme activities of the energy-consuming process of glycogen and fatty acid synthesis as well as lipoprotein metabolism were accelerated by insulin through the IRS-2 / SREBP-1c pathway.

Topics: Adenosine; Allopurinol; Animals; Apolipoprotein C-III; Apolipoproteins C; Blotting, Western; CCAAT-Enhancer-Binding Proteins; DNA-Binding Proteins; Energy Metabolism; Glutathione; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hypoglycemic Agents; Insulin; Insulin Receptor Substrate Proteins; Intracellular Signaling Peptides and Proteins; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Phosphoproteins; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Signal Transduction; Sterol Regulatory Element Binding Protein 1; Transcription Factors; Up-Regulation

Oxygen-derived free radicals have been suggested as important in degeneration after spinal cord ischemia. The aim of this study was to investigate whether erdosteine has a protective effect against spinal cord ischemia during aortic cross clamping.. New Zealand White rabbits (n=21) were divided into three groups. In the ischemia/reperfusion group (I/R) (n=8), the infrarenal aorta of rabbits was cross clamped for 21 min and then reperfused. In erdosteine group, the administration of erdosteine solution (50 mg/kg) was started two days before aortic cross-clamping and rabbits (n=8) were subjected to ischemia and reperfusion. Animals in control group (n=5) underwent a surgical procedure similar to the other groups but the aorta was not clamped. The animals were sacrificed at 72 h and histopathological, and biochemical analyses were carried out on the lumbar spinal cords.. Erdosteine treatment was associated with improved neurological function in the postoperative period. Histopathological examination of spinal cord tissues in erdosteine group revealed changes consistent with mild ischemic injury, but rabbits in I/R group with paraplegia had total destruction of the motor neurons. Biochemical analyses of spinal cord tissues, in the I/R group, revealed a significant increase in the superoxide dismutase, xanthine oxidase, adenosine deaminase and myeloperoxidase activities, and a significant depletion in glutathione peroxidase activity when compared to that of control rabbits. Erdosteine treatment prevented the increase of all these enzymes except adenosine deaminase. Ischemia/reperfusion produced a significant increase in the tissue malondialdehyde levels. Ischemia/reperfusion-induced increments in malondialdehyde content of the spinal cord were significantly prevented by erdosteine treatment.. The present study demonstrated that erdosteine treatment before aortic cross clamping ameliorates neurological outcome, neuronal injury and oxidative stress in the rabbit spinal cord.

Topics: Adenosine Deaminase; Animals; Aorta, Abdominal; Biomarkers; Disease Models, Animal; Expectorants; Glutathione Peroxidase; Malondialdehyde; Models, Cardiovascular; Motor Neurons; Neuroprotective Agents; Nitric Oxide; Oxidative Stress; Psychomotor Performance; Rabbits; Reperfusion Injury; Spinal Cord; Spinal Cord Ischemia; Superoxide Dismutase; Thioglycolates; Thiophenes; Treatment Outcome; Xanthine Oxidase

This study examined the potential therapeutic effects of a combination therapy consisting of 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR) and N-acetyl cysteine (NAC) to attenuate ischemia-reperfusion (I/R) injury in a canine model of autologous renal transplantation.. Male mongrel dogs (15-20 kg) underwent left nephrectomy followed by flushing and static preservation of the kidney in University of Wisconsin (UW) solution for 48 hr. The treatment group received AICAR (50 mg/kg) plus NAC (100 mg/kg) intravenously before the left nephrectomy. The compounds were added to the UW solution as well. All dogs underwent right nephrectomy 48 hr later followed by autotransplantation of the preserved left kidney. Treated dogs received a second dose of AICAR and NAC before implantation of the renal autograft.. The treated dogs had excellent urine output posttransplant, with peak serum creatinine of 7.26 mg/dL on postoperative day (POD) 3 that normalized after 14 days. The control group were anuric and developed clinical symptoms of uremia on POD 1. Morphologic evaluation supported the protective effects of combination therapy. Immunohistochemical analysis revealed decrease of tumor necrosis factor-alpha, interferon-gamma, and inducible nitric oxide synthase; and TUNEL assay showed decreased apoptosis in the treated group.. Combination therapy with AICAR and NAC attenuates renal I/R injury and improves the outcome of the transplanted kidney after prolonged cold preservation.

Topics: Acetylcysteine; Adenosine; Allopurinol; Aminoimidazole Carboxamide; Animals; Apoptosis; Creatinine; Disease Models, Animal; Dogs; Glutathione; Graft Survival; Immunohistochemistry; Insulin; Kidney; Kidney Transplantation; Male; Nephrectomy; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Ribonucleotides; Transplantation, Autologous; Tumor Necrosis Factor-alpha

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a nuclear hormone receptor super family that has recently been implicated in atherosclerosis, inflammation, cancer, infertility, and demyelination. Oxidative stress, neutrophil infiltration, proinflammatory cytokines, and the exhibition of luminal acid play a role in the pathogenesis of gastric injury induced by ischemia-reperfusion. Rosiglitazone, a specific PPAR-gamma ligand, has been shown to have antiinflammatory activity, but its effects on experimental ischemia-reperfusion gastric injury remain unknown. We have investigated the effects of the rosiglitazone on gastric injury caused by ischemia following reperfusion in rats. Tumour necrosis factor-alpha (TNF-alpha) levels and changes in enzymatic activities of myeloperoxidase, as a marker of neutrophils infiltration, xanthine oxidase, superoxide dismutase, and glutathione peroxidase, were determined. Histological analysis of the lesions was also carried out. Pretreatment with 1 or 4 mg/kg of rosiglitazone ameliorated the gastric damage induced by clamping the celiac artery for 30 min followed by 60 min of reperfusion. It significantly (P<0.05) reduced the index of neutrophil infiltration and the levels of the cytokine. Rosiglitazone did not revert the reduced glutathione peroxidase activity but enhanced significantly (P<0.01) the decreased xanthine oxidase and superoxide dismutase activities in gastric mucosa of ischemic rats. In conclusion, rosiglitazone reduces the damage in ischemia-reperfusion gastric injury and alleviates the inflammatory response and the oxidative events.

Topics: Animals; Cyclooxygenase 1; Cyclooxygenase 2; Gastric Mucosa; Glutathione Peroxidase; Immunohistochemistry; Male; Membrane Proteins; Necrosis; Neutrophils; Oxidative Stress; Peroxidase; PPAR gamma; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Rosiglitazone; Stomach; Superoxide Dismutase; Thiazolidinediones; Tumor Necrosis Factor-alpha; Up-Regulation; Xanthine Oxidase

The aim of this experimental study was to investigate whether nebivolol has protective effects against neuronal damage induced by spinal cord ischemia/reperfusion (I/R). Twenty-one rabbits were divided into three groups: group I (control, no I/R), group II (only I/R) and group III (I/R+nebivolol). Spinal cord ischemia was induced by clamping the aorta both below the left renal artery and above the aortic bifurcation. Seventy-two hours postoperatively, the motor function of the lower limbs was evaluated in each animal. The animals were sacrificed at 72 h, and histopathological and biochemical analyses were carried out in the lumbar spinal cords. The motor deficit scores in nebivolol group were different from I/R group at 72 h (3.25+/-0.70 vs. 1.75+/-1.28, p=0.01). I/R produced a significant increase in the superoxide dismutase (SOD), xanthine oxidase (XO), adenosine deaminase (ADA) and myeloperoxidase (MPO) activities in spinal cord tissue when compared with control group. Nebivolol treatment prevented the increase of all those enzymes activities produced by I/R. A significant decrease in spinal cord glutathione peroxidase (GSH-Px) level was seen in I/R group and nebivolol treatment prevented the decrement in the spinal cord tissue GSH-Px contents. On the other hand, I/R produced a significant increase in the spinal cord tissue malondialdehyde (MDA) and nitric oxide (NO) contents, this was prevented by nebivolol treatment. In conclusion, this study demonstrates a considerable neuroprotective effect of nebivolol on neurological, biochemical and histopathological status during periods of spinal cord I/R in rabbits.

Topics: Adenosine Deaminase; Adrenergic beta-Antagonists; Animals; Benzopyrans; Ethanolamines; Free Radicals; Glutathione Peroxidase; Malondialdehyde; Nebivolol; Nerve Tissue Proteins; Neuroprotective Agents; Nitric Oxide; Oxidative Stress; Peroxidase; Rabbits; Reperfusion Injury; Spinal Cord; Spinal Cord Injuries; Superoxide Dismutase; Xanthine Oxidase

Cold ischemic injury plays an important role in short- and long-term function of kidneys after transplant. Antimicrobial peptides have not previously been studied for their impact on cold ischemia in transplanted kidneys.. Bactenecin (L- and D-forms) was added to University of Wisconsin (UW) preservation solution for 3-day cold storage of dog kidneys. Effects on membrane permeability were studied in synthetic liposomes and in kidney cortex tissue slices. The role of bactenecin as a tissue mitogen and direct cytoskeletal stabilizer were studied with cultured cells and in vitro.. Bactenecin (both L- and D- forms) resulted in significant decreases in postoperative serum creatinine and time required for return of creatinine to the normal range showing the effect was independent of chirality. Bactenecin permeabilized synthetic liposomes and altered kidney cortex tissue slice membrane permeability characteristics, irrespective of chirality. Neither did bactenecin act as a mitogen for either primary renal tubule or Madin-Darby canine kidney (MDCK) cells stored in UW solution, nor did it appear to directly affect cytoskeletal dynamics.. These results show that the antimicrobial peptide bactenecin can improve the quality of static cold storage of kidneys. The mechanism of its action is independent of receptor binding and does not appear to involve either an effect on the cytoskeleton or via activity as a mitogen. Current evidence best supports the hypothesis that bactenecin protects against cold ischemic injury by a controlled permeabilization of the membranes of the kidney during cold storage.

Topics: Actins; Adenosine; Allopurinol; Animals; Antimicrobial Cationic Peptides; Cell Line; Cold Temperature; Cryopreservation; Culture Media, Serum-Free; Cytoskeleton; Dogs; Dose-Response Relationship, Drug; Female; Fluoresceins; Glutathione; Insulin; Ischemia; Kidney; Kidney Tubules; Liposomes; Membranes; Microtubules; Mitogens; Organ Preservation; Organ Preservation Solutions; Paclitaxel; Peptides, Cyclic; Permeability; Protein Binding; Raffinose; Reperfusion Injury; Stereoisomerism; Temperature; Time Factors

Increases in intracellular calcium ion (Ca(2+)) levels of sinusoidal endothelial cell (SEC) may have a crucial role in mediating the expression of adhesion molecules and thus contribute to the microcirculatory disturbances observed in primary graft dysfunction. The effect of changes in the composition and/or temperature of the reperfusion solution on cytosolic Ca(2+) was studied in isolated rat SECs. Cells were preserved in cold University of Wisconsin (UW) solution for 0, 12, or 24 hours and loaded with Fura-2AM dye (Cedarlane, Eugene, OR) at 20 degrees C in N-2-hydroxyethylpiperazine-propanesulfonic acid (HEPES)-buffered physiological solution (HEPES 20 degrees C) or UW solution (UW 20 degrees C). SEC Ca(2+) levels were measured by cytofluorimetry. Basal steady-state Ca(2+) levels were much lower when SECs were loaded in UW 20 degrees C (37 +/- 2 nmol/L) than in HEPES 20 degrees C (114 +/- 32 nmol/L). In unstored controls (0 hour), going from UW 20 degrees C to HEPES 37 degrees C induced a large transient increase (185 +/- 31 nmol/L) in SEC Ca(2+) levels, which was greatly inhibited (43 +/- 13 nmol/L) in Ca(2+)-free HEPES 37 degrees C. A similar large transient increase was observed going from UW 20 degrees C to HEPES 20 degrees C (163 +/- 22 nmol/L). Changing temperature only (20 degrees C to 37 degrees C) in UW or HEPES solution had a much smaller effect on SEC Ca(2+) levels (14 +/- 2 and 60 +/- 18 nmol/L, respectively). These changes were similar in cold-preserved cells. In unstored controls, solution changes greatly attenuated the intensity of subsequent Ca(2+) responses to the purinergic agonist adenosine triphosphate (ATP). Cold preservation (CP) greatly attenuated both the frequency of appearance and intensity of ATP-induced Ca(2+) responses. Hence, changing reperfusion solution composition has a greater impact on SEC steady-state Ca(2+) levels than changing temperature. Cold preservation does not significantly affect changes in SEC steady-state Ca(2+) levels, but greatly impairs the capacity of SECs to subsequently respond to Ca(2+)-mobilizing agonists.

Topics: Adenosine; Allopurinol; Animals; Calcium; Cells, Cultured; Cryopreservation; Cytosol; Endothelium, Vascular; Glutathione; HEPES; Homeostasis; Hot Temperature; Insulin; Liver Circulation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury; Temperature

Using in situ fluorescence microscopy with Sprague Dawley rats, we studied the hypothesis of compromised microvascular kidney perfusion on organ harvest in non-heart-beating donors (NHBDs), and we evaluated the potential benefit of an additional preflush with saline solution containing streptokinase. Aortal flush of NHBD kidneys solely with University of Wisconsin solution resulted in a significantly (P <0.05) reduced functional capillary density (FCD) with increased perfusion heterogeneity compared with kidneys of heart-beating controls. This was associated with an increased lactate dehydrogenase (LDH) release on 24 hr postpreservation rinse of the grafts (76.7+/-18.9 U/L). Warm preflush with low-viscosity Ringer's lactate (RL) solution alone did not influence the decreased renal FCD and the postpreservation LDH release (76.2+/-29.1 U/L). In contrast, the addition of streptokinase to the RL preflush solution resulted in a significant (P <0.05) improvement of FCD with values not statistically different from those of heart-beating controls. This was associated with an attenuation of perfusion heterogeneity and a significantly lowered postpreservation LDH release (17.0+/-2.5 U/L). Furthermore, in transplanted and reperfused NHBD kidney grafts, the use of streptokinase-supplemented RL for preflush during organ harvest significantly (P <0.05) reduced early manifestation of tubular necrosis (29%+/-8%) when compared with kidneys preflushed exclusively with University of Wisconsin solution (56%+/-4%). Thus, we conclude that kidney harvest from NHBDs is prone to severe microvascular perfusion deficits, which are likely to preclude successful preservation of organ integrity during cold storage. Temporary fibrinolytic preflush with streptokinase may represent a feasible tool to improve microvascular graft equilibration, which effectively protects the renal integrity during both cold storage and posttransplant reperfusion.

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Fibrinolytic Agents; Glutathione; Graft Survival; Heart Arrest; Insulin; Kidney; Kidney Transplantation; Male; Microcirculation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sodium Chloride; Streptokinase

Prompted by the observation of ischaemia development during the treatment of tumours by photodynamic therapy (PDT) that is typically followed by a restoration of tumour blood flow and by the indications of secondary superoxide generation after PDT, we aimed in this study to obtain evidence of the induction of ischaemia-reperfusion (I/R) injury in PDT-treated tumours. Using subcutaneous mouse FsaR fibrosarcoma model and Photofrin-based PDT treatment, we have examined the activity of xanthine oxidase (XO, a key enzyme in the I/R injury development) in tumours before and after the therapy. Compared to the levels in nontreated tumours, there was a five-fold increase in the activity of this enzyme in tumours excised immediately after PDT. This burst of elevated XO activity declined rapidly, returning to the pretreatment levels within the next 30 min. Visible reflectance spectroscopy confirmed the occurrence of a PDT-induced strong but temporary reduction in tumour oxygenation. The administration of XO inhibitor oxypurinol prevented this PDT-induced rise in XO activity. The oxypurinol treatment also decreased the extent of neutrophil accumulation in PDT-treated tumours and reduced the level of PDT-mediated cures. These results demonstrate the induction of I/R injury in PDT-treated tumours, and show that it can contribute to the therapy outcome. Since I/R injury is a well-recognised proinflammatory insult, we suggest that its induction in PDT-treated tumours promotes the development of inflammatory response that has become established as a key element of the antitumour effect of PDT.

Topics: Animals; Flow Cytometry; Mice; Mice, Inbred C3H; Neoplasms, Experimental; Photochemotherapy; Reperfusion Injury; Xanthine Oxidase

Cold ischemia (CI)-warm reperfusion (WR) liver injury remains a problem in liver transplantation. CI-WR initially causes sinusoidal endothelial cell (SEC) apoptosis through a caspase-dependent mechanism. We previously showed that the caspase inhibitor IDN-1965 prevents CI-WR-induced SEC apoptosis. However, this agent required to be administered to the donor, preservation solution, and recipient for efficacy. Here, we show that a second-generation caspase inhibitor, IDN-6556, effectively prevents CI-WR-induced SEC injury when added only to University of Wisconsin (UW) cold storage media. Rat livers were stored in UW solution for 24 hours at 4 degrees C and reperfused for 1 hour at 37 degrees C. Apoptosis was quantitated using terminal deoxynucleotide transferasemediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and caspase 3 activation determined by biochemical measurement and immunohistochemical analysis. Pan-caspase inhibitors (IDN-8066, IDN-7503, IDN-7436, IDN-1965, and IDN-6556) were applied at preischemic, cold preservation, or reperfusion periods. TUNEL-positive SEC and caspase 3-like activity in the liver was increased by CI-WR. Three caspase inhibitors (IDN-8066, IDN-1965, and IDN-6556) effectively attenuated SEC apoptosis and caspase 3 activation. The most potent inhibitor, IDN-6556, reduced SEC apoptosis and caspase 3 activity by 55% and 94%, respectively. Prevention of SEC apoptosis by IDN-6556 was not reduced when this agent was administered only during the cold preservation period. When added to the preservation solution, the caspase inhibitor IDN-6556 appears to be a feasible therapeutic agent against ischemia-reperfusion injury in liver transplantation.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Endothelium; Enzyme Inhibitors; Glutathione; Insulin; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Ischemic-type biliary lesions (ITBLs) lead to considerable morbidity after orthotopic liver transplantation (OLT). The exact pathogenesis is unknown. We tested the hypothesis that insufficient perfusion of biliary arterial vessels might be responsible for ITBLs. This could be prevented by improved perfusion techniques. Since February 2000, we performed a controlled study using arterial back-table pressure perfusion (AP) to achieve reliable perfusion of the biliary-tract capillary system, which may be impaired by the high viscosity of University of Wisconsin solution. We retrospectively analyzed 190 OLTs performed between September 1997 and July 2002 with regard to ITBLs. One hundred thirty-one grafts were preserved by in situ standard perfusion (SP), including portal perfusion, whereas in 59 cases, additional AP was performed. Donor-related factors, recipient age, indication for OLT, OLT technique, immunosuppression, and ischemia time were similar in both groups. In the SP group, 21 of 131 patients (16%) developed ITBLs. Only 1 of 59 patients with grafts receiving AP developed ITBLs. This difference was highly significant (P =.004). Peak aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels within the first 3 days were significantly lower in the AP group (AST, P =.016; ALT, P =.007). Multivariate analysis showed a significant influence of AP (P =.010) and donor age (P =.003) on the development of ITBLs. AP is an easy and reliable method to prevent ITBLs in OLT. It therefore should be used as the standard technique in liver procurement.

Topics: Adenosine; Adult; Aged; Allopurinol; Bile Ducts; Glutathione; Graft Rejection; Graft Survival; Humans; Immunosuppressive Agents; Insulin; Liver Diseases; Liver Transplantation; Middle Aged; Organ Preservation Solutions; Perfusion; Postoperative Complications; Pressure; Raffinose; Reperfusion Injury; Retrospective Studies; Survival Rate; Viscosity

Xanthine oxidoreductase (XOR) catalyzes the final reactions of purine catabolism and may account for cell damage by producing reactive oxygen metabolites in cells reoxygenated after hypoxia. We found a three- to eightfold higher XOR activity in cultured human bronchial epithelial cells exposed to hypoxia (0.5-3% O2) compared with cells grown in normoxia (21% O2) but no difference in XOR protein or mRNA. XOR promoter constructs failed to respond to hypoxia. The cellular XOR activity at 3% O2 returned to basal levels when the cells were returned to 21% O2, and hyperoxia (95% O2) abolished enzyme activity with no change in XOR protein. Our data suggest reversible enzyme inactivation by oxygen or its metabolites. NADH was normally oxidized by the oxygen-inactivated enzyme, which rules out damage to the flavin adenine dinucleotide cofactor. Hydrogen peroxide partially inactivated the molybdenum center of XOR, as shown by a parallel decrease in XOR-catalyzed xanthine oxidation and dichlorophenolindophenol reduction. We conclude that the transcription or translation of XOR is not influenced by hypoxia or hyperoxia. Instead, the molybdenum center of XOR is posttranslationally inactivated by oxygen metabolites in "normal" (21% O2) cell culture atmosphere. This inactivation is reversed in hypoxia and accounts for the apparent induction.

Topics: Cell Hypoxia; Cell Line, Transformed; Cell Survival; Cobalt; Enzyme Activation; Epithelial Cells; Gene Expression Regulation, Enzymologic; Humans; Hyperoxia; Oxygen; Promoter Regions, Genetic; Protein Processing, Post-Translational; Reperfusion Injury; Respiratory Mucosa; RNA, Messenger; Substrate Specificity; Transcription, Genetic; Xanthine Oxidase

Moderate consumption of red wine has been shown to exert cardioprotection against ischemia/reperfusion. Because oxidant-dependent leukocyte infiltration plays a critical role in ischemia/reperfusion-induced tissue injury, we hypothesized that resveratrol, a red wine constituent polyphenol would attenuate postischemic leukocyte recruitment and subsequent endothelial dysfunction. Intravital microscopic approaches were used to quantify leukocyte/endothelial cell interactions and venular protein leakage in rat mesenteries exposed to either 20 min ischemia and 60 min reperfusion (I/R), oxidants generated by the reaction of hypoxanthine and xanthine oxidase (HX/XO), platelet-activating factor (PAF), or leukotriene B4 (LTB4). I/R or HX/HX produced marked increases in the number of adherent (LA) and emigrated (LE) leukocytes, which were associated with significant increases in venular albumin leakage (VAL). Intravenous administration of resveratrol or superoxide dismutase (SOD) attenuated these increases in LA, LE, and VAL. Superfusion of the mesentery with PAF or LTB4 also markedly increased LA, LE, and VAL. While resveratrol attenuated the proinflammatory effects of PAF, LTB4-induced changes were not affected by resveratrol. Resveratrol prevents leukocyte recruitment and endothelial barrier disruption induced by a number of superoxide-dependent proinflammatory stimuli, including I/R, HX/XO, or PAF. These salutary effects appear to be related to the antioxidant properties of resveratrol and contribute to the cardioprotective actions associated with consumption of red wine.

Topics: Animals; Antioxidants; Cell Adhesion; Flavonoids; Free Radicals; Hypoxanthine; Inflammation; Leukocytes; Leukotriene B4; Male; Oxidants; Phenols; Platelet Activating Factor; Polyphenols; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Resveratrol; Stilbenes; Superoxide Dismutase; Superoxides; Time Factors; Xanthine Oxidase

The effect of University of Wisconsin (UW) solution perfusion for extremity preservation is still unknown although it is widely used. The purpose of this study is to examine the effect of UW solution perfusion on skeletal muscle preservation in a rat model.. Rat hindlimbs were amputated and either preserved with UW solution perfusion (UW perfusion group) or given no perfusion (no-perfusion group) for 5 h at 25 degrees C. They were then transplanted to other isogeneic rats. ATP in the muscle and serum creatine phosphokinase were measured after 24 h of reperfusion. The vascular endothelial function of the femoral artery rings was measured before and after 24 h of reperfusion in the presence or absence of indomethacin (cyclooxygenase inhibitor) and L-NMMA (nitric oxide synthase inhibitor). TEA (calcium-activated potassium channel inhibitor) was also used to verify the vasodilator function. Reperfusion blood flow was monitored during the first 2 h of reperfusion.. ATP in the UW perfusion group was significantly decreased after 24 h of reperfusion, while that in the no-perfusion group recovered. Reperfusion blood flow in the UW solution perfusion group was significantly lower than that in the no-perfusion group. Acetylcholine-induced relaxation in the UW perfusion group was significantly reduced before and after 24 h of reperfusion compared to that in the no-perfusion group and was mostly diminished by indomethacin and L-NMMA administration.. Skeletal muscle injury is augmented by UW solution perfusion, probably due to deterioration of the vascular endothelial function resulting in blood supply diminution.

Topics: Adenosine; Allopurinol; Animals; Endothelium, Vascular; Exocrine Glands; Glutathione; Hindlimb; In Vitro Techniques; Insulin; Male; Muscle, Skeletal; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

Celsior, a new preservation solution in thoracic organ transplantation was evaluated for efficacy in cold preservation of human hepatocytes and compared with University of Wisconsin solution (UW) and histidine-tryptophan-ketoglutarate solution (HTK, Custodiol). Human hepatocyte cultures were preserved at 4 degrees C in Celsior, UW and HTK for 2, 6, 12, 24 and 48 h with 6 h of reperfusion. Levels of lactate dehydrogenase (LDH; cell necrosis), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; mitochondrial function), and adenosine 5'-triphosphate (ATP; loss of intracellular energy) were measured. Cell necrosis, mitochondrial dysfunction, and loss of ATP were significantly ( P<0.001, P<0.001, P<0.002, respectively) lower in Celsior than in HTK. The amount of cell necrosis and mitochondrial dysfunction in Celsior solution (CS) and UW was equal ( P=n.s.) up to 24 h and significantly lower in UW after 48 h ( P<0.001). Additionally, the intracellular level of ATP was significantly higher after ischemia ( P<0.001) and reperfusion from long-term ischemia (24, 48 h) ( P<0.002). We can conclude that Celsior was superior to HTK and equal to UW in the protection of human hepatocytes against cold preservation injury from ischemia and reperfusion. Furthermore, Celsior was effective in long-term preservation of human hepatocytes.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Cells, Cultured; Cryopreservation; Disaccharides; Electrolytes; Glucose; Glutamates; Glutathione; Hepatocytes; Histidine; Humans; Insulin; L-Lactate Dehydrogenase; Mannitol; Organ Preservation Solutions; Oxidation-Reduction; Potassium Chloride; Procaine; Raffinose; Reperfusion Injury; Tetrazolium Salts; Thiazoles

Heme oxygenase (HO) catalyzes the rate-limiting enzymatic step of heme degradation and regulates the cellular heme content. Gene expression of the inducible isoform of HO, HO-1, is upregulated in response to various oxidative stress stimuli. To investigate the regulatory role of anoxia and reoxygenation (A/R) on hepatic HO-1 gene expression, primary cultures of rat hepatocytes were exposed after an anoxia of 4 hr to normal oxygen tension for various lengths of time. For comparison, gene expression of the noninducible HO isoform, HO-2, and that of the heat-shock protein 70 (HSP70) were determined. During reoxygenation, a marked increase of HO-1 and HSP70 steady-state mRNA levels was observed, whereas no alteration of HO-2 mRNA levels occurred. Corresponding to HO-1 mRNA, an increase of HO-1 protein expression was determined by Western blot analysis. The anoxia-dependent induction of HO-1 was prevented by pretreatment with the transcription inhibitor, actinomycin D, but not by the protein synthesis inhibitor, cycloheximide, suggesting a transcriptional regulatory mechanism. After exposure of hepatocytes to anoxia, the relative levels of oxidized glutathione increased within the first 40 min of reoxygenation. Pretreament of cell cultures with the antioxidant agents, beta-carotene and allopurinol, before exposure to A/R led to a marked decrease of HO-1 and HSP70 mRNA expression during reoxygenation. An even more pronounced reduction of mRNA expression was observed after exposure to desferrioxamine. Taken together, the data demonstrate that HO-1 gene expression in rat hepatocyte cultures after A/R is upregulated by a transcriptional mechanism that may be, in part, mediated via the generation of ROS and the glutathione system.

Topics: Allopurinol; Animals; Antioxidants; beta Carotene; Cell Hypoxia; Cells, Cultured; Cycloheximide; Dactinomycin; Deferoxamine; Free Radical Scavengers; Gene Expression Regulation, Enzymologic; Glutathione; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hepatocytes; HSP70 Heat-Shock Proteins; Iron Chelating Agents; Male; Oxygen; Protein Synthesis Inhibitors; Rats; Rats, Wistar; Reperfusion Injury

To investigate the possible protection provided by oral quercetin pretreatment against hepatic ischemia-reperfusion injury in rats.. The quercetin (0.13 mmol/kg) was orally administrated in 50 min prior to hepatic ischemia-reperfusion injury. Ascorbic acid was also similarly administered. The hepatic content of quercetin was assayed by high performance liquid chromatography (HPLC). Plasma glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) activities and malondialdehyde (MDA) concentration were measured as markers of hepatic ischemia-reperfusion injury. Meanwhile, hepatic content of glutathione (GSH), activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and xanthine oxidase (XO), total antioxidant capacity (TAOC), contents of reactive oxygen species (ROS) and MDA, DNA fragmentation were also determined.. Hepatic content of quercetin after intragastric administration of quercetin was increased significantly. The increases in plasma GPT, GOT activities and MDA concentration after hepatic ischemia-reperfusion injury were reduced significantly by pretreatment with quercetin. Hepatic content of GSH and activities of SOD, GSH-Px and TAOC were restored remarkably while the ROS and MDA contents were significantly diminished by quercetin pretreatment after ischemia-reperfusion injury. However, quercetin pretreatment did not reduce significantly hepatic XO activity and DNA fragmentation. Ascorbic acid pretreatment had also protective effects against hepatic ischemia-reperfusion injury by restoring hepatic content of GSH, TAOC and diminishing ROS and MDA formation and DNA fragmentation.. It is indicated that quercetin can protect the liver against ischemia-reperfusion injury after oral pretreatment and the underlying mechanism is associated with improved hepatic antioxidant capacity.

Topics: Administration, Oral; Animals; Antioxidants; Ascorbic Acid; Biological Availability; Biomarkers; DNA Fragmentation; Glutathione Peroxidase; Liver; Male; Malondialdehyde; Quercetin; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Transaminases; Xanthine Oxidase

Acute renal failure commonly follows reduced renal perfusion or ischemia. Reperfusion is essential for recovery but can itself cause functional and structural injury to the kidney. The separate contributions of ischemia and of reperfusion were examined in the isolated perfused rat kidney. Three groups were studied: brief (5 min) ischemia, 20 min ischemia, and repetitive brief ischemia (4 periods of 5 min) with repetitive intervening reperfusion of 5 min. A control group had no intervention, the three ischemia groups were given a baseline perfusion of 30 min before intervention and all groups were perfused for a total of 80 min. In addition, the effects of exogenous *NO from sodium nitroprusside and xanthine oxidase inhibition by allopurinol were assessed in the repetitive brief ischemia-reperfusion model. Brief ischemia produced minimal morphological injury with near normal functional recovery. Repetitive brief ischemia-reperfusion caused less functional and morphological injury than an equivalent single period of ischemia (20 min) suggesting that intermittent reperfusion is less injurious than ischemia alone over the time course of study. Pretreatment with allopurinol improved renal function after repetitive brief ischemia-reperfusion compared with the allopurinol-untreated repetitive brief ischemia-reperfusion group. Similarly, sodium nitroprusside reduced renal vascular resistance but did not improve the glomerular filtration rate or sodium reabsorption in the repetitive brief ischemia-reperfusion model. Thus, these studies show that the duration of uninterrupted ischemia is more critical than reperfusion in determining the extent of renal ischemia-reperfusion injury and that allopurinol, in particular, counteracts the oxidative stress of reperfusion.

Topics: Allopurinol; Animals; Disease Models, Animal; Free Radical Scavengers; Glomerular Filtration Rate; Kidney Diseases; Kidney Medulla; Kidney Tubules, Proximal; Male; Natriuresis; Necrosis; Nitroprusside; Rats; Rats, Sprague-Dawley; Reperfusion; Reperfusion Injury; Severity of Illness Index; Vascular Resistance; Vasodilator Agents

University of Wisconsin (UW) solution (Viaspan) is currently used to preserve organs from nonheartbeating donors. Histidine-tryptophan-ketoglutarate (HTK) solution (Custodiol) is of proven efficacy in experimental pancreas preservation, but its efficacy in combined warm ischemia (WI) and cold ischemia (CI) is unknown. The viability of HTK-preserved porcine pancreatic grafts was assessed after various periods of WI and compared with grafts flushed and preserved with UW solution.. A total of 14 pigs were used: G1 (n=4, UW) and G2 (n=4, HTK) with 15-min WI and 16-hr cold storage; G3 (n=3, UW) and G4 (n=3, HTK) with 30-min WI and 16-hr cold storage.. All animals in G1 and G2 were normoglycemic, whereas only 66% of pancreases were functioning in G3 and G4. HTK perfusion was associated with increased wet weight. Transient hyperinsulinemia was noted in all the groups on postoperative day 1 (mean range: 8.9-12.4 microU/L). Postoperative serum amylase and lipase were more pronounced in G3 and G4. However, HTK-stored grafts exhibited less evidence of biochemical pancreatitis as compared with UW-stored grafts on the first postoperative day in the group with 15-min WI. Mean K values of intravenous glucose tolerance tests on postoperative day 14 were similar in both groups. Vascular congestion was uniformly observed and was considered a typical feature of WI.. Porcine pancreatic grafts are viable after 16-hr CI following 15-min WI in this experimental nonheartbeating donor model. HTK solution seems to provide reliable graft function in this setting and to be equivalent to UW.

Topics: Adenosine; Allopurinol; Amylases; Animals; Cold Temperature; Edema; Glutathione; Graft Survival; Hot Temperature; Hyperinsulinism; Hypoglycemia; Insulin; Lipase; Models, Animal; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose; Reperfusion Injury; Swine

The hypothesis was tested that treatment with allopurinol, a xanthine oxidase inhibitor, or deferoxamine, a chelator of nonprotein-bound iron, preserved cerebral energy metabolism, attenuated development of edema, and improved histologic outcome in the newborn piglet at 24 h after hypoxia-ischemia. Thirty-two newborn piglets were subjected to 1 h of hypoxia-ischemia by occluding both carotid arteries and reducing the fraction of inspired oxygen; five newborn piglets served as sham-operated controls. The depth of hypoxia-ischemia was controlled by phosphorous magnetic resonance spectroscopy. Upon reperfusion and reoxygenation, piglets received vehicle (n= 12), allopurinol (30 mg/kg/d, n = 10), or deferoxamine (12.5 mg/kg/d, n = 10). The cerebral energy status was determined with phosphorous magnetic resonance spectroscopy. The presence of vasogenic edema was assessed by T2-weighted magnetic resonance imaging. Brain cell injury was assessed with caspase-3 activity, histology, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end (TUNEL)-labeling. At 24 h after hypoxia-ischemia, the phosphocreatine/inorganic phosphate ratios were significantly decreased in vehicle-treated, but not in allopurinol- or deferoxamine-treated piglets. Water T2 values were significantly increased at 24 h after hypoxia-ischemia in cerebral cortex, thalamus, and striatum of vehicle-treated piglets, but not in allopurinol- and deferoxamine-treated piglets. No differences in caspase-3 activity, histologic outcome, or TUNEL-labeling were demonstrated between the three treatment groups. We suggest that allopurinol and deferoxamine may have an additional value in the treatment of perinatal hypoxia-ischemia with other neuroprotective agents or in combination with hypothermia.

Topics: Allopurinol; Animals; Animals, Newborn; Brain; Caspase 3; Caspases; Deferoxamine; Enzyme Inhibitors; Hypoxia-Ischemia, Brain; In Situ Nick-End Labeling; Iron; Iron Chelating Agents; Magnetic Resonance Imaging; Reperfusion Injury; Swine

Ischemia-reperfusion injury has been associated with both early and late effects on allografts in the form of delayed graft function and decreased graft survival. Recent studies demonstrated that functional parameters were influenced by cold storage conditions and particularly the ratio of Na+:K+ of the preservation solution.. We have extended this study to examine whether the high-Na+ low-K+ formulation of Belzer's solution (HEH) was efficient in an autotransplanted pig kidney model when compared with the classical low-Na+ high-K+ University of Wisconsin solution and the new high-Na+ low-K+ Celsior solution. Kidneys were harvested, cold flushed, and preserved for 24, 48, or 72 hr with HEH, Celsior solution, or University of Wisconsin solution and autotransplanted. Renal function was determined on days 1, 3, 7, and 14, and at 4 to 16 weeks after autotransplantation. Histologic changes and cell infiltration were assessed on kidney biopsy specimens taken after reperfusion (30-40 min), at days 5 and 14, and at 4 to 5 and 10 to 12 weeks after surgery. Peripheral benzodiazepine receptor (PBR), a structural mitochondrial protein, was also studied.. Cold storage in HEH resulted in reduction of delayed graft function and renal damage, with a decrease in interstitial inflammation. HEH reduced interstitial fibrosis, tubular atrophy, and improved PBR expression.. This study suggests that cold preservation in HEH has a beneficial action in in vivo renal preservation and reduces tubular necrosis, interstitial inflammation, and fibrosis in these groups. In addition, PBR detection was correlated to the level of preservation integrity.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Graft Survival; Insulin; Kidney; Kidney Transplantation; Male; Models, Animal; Organ Preservation; Organ Preservation Solutions; Potassium; Raffinose; Reperfusion Injury; Sodium; Swine; Time Factors; Transplantation, Autologous

Insulin keeps the liver in a metabolically vigorous state. However, organ preservation aims to decrease the metabolic rate. The objective of this study was to clarify the effect of insulin used in University of Wisconsin (UW) preservation solution on the liver graft.. The liver grafts were preserved by UW solution with or without insulin for 7, 9, and 24 hr, respectively. The influence of insulin was studied by 7-day survival rate, liver function, morphology, and intragraft gene expression 24 hr after transplantation. Morphology was studied on the preserved grafts.. The morphology of the graft in the insulin group showed more severe ischemia-reperfusion injury. The 7-day graft survival rates of the 7-hr subgroups with and without insulin were 55% and 93%, respectively (P=0.02). In the 9-hr subgroups, the survival rates were 0% and 78%, respectively (P=0.002). The serum levels of aspartate aminotransferase (AST) (P=0.008) and alanine aminotransferase (ALT) (P=0.032) were higher in the 7-hr subgroup with insulin. The same trend was found in the 9-hr subgroups (AST, P=0.016; ALT, P=0.016). The expression level of 215 genes were much lower at 24 hr after transplantation in the grafts preserved with insulin than in those preserved without insulin, and most of the genes were related to metabolic activities.. Insulin in UW solution may exacerbate graft ischemic injury and decrease the graft survival rate in rat liver transplantation. Insulin, in the absence of glucose in UW solution, may exhaust the metabolic activity of the liver graft. It is harmful rather than helpful for isolated rat liver grafts preserved in UW solution.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Gene Expression Regulation; Glutathione; Graft Survival; Insulin; Liver; Liver Circulation; Liver Function Tests; Liver Transplantation; Oligonucleotide Array Sequence Analysis; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Time Factors; Transplantation, Isogeneic

Although classic ischemia-reperfusion injury is mediated by reactive oxygen intermediaries, increasing evidence implicates a role for immune-mediated apoptosis during ischemic injury in transplantation. Herein, we report the effects of polyclonal rabbit anti-thymocyte globulin (rATG) on mediators of hepatic apoptosis during cold storage.. Three-month-old male Lewis rats were placed under halothane anesthesia and the portal vein cannulated. University of Wisconsin (UW) solution (35 ml) with (n = 5) and without (n = 5) 20 mg/kg anti-rat rATG was infused before hepatectomy. The liver was stored in UW solution +/- rATG (143 ng/ml) at 4 degrees C for various times up to 24 h. Specimens were terminal deoxyuridine nick end labeling-stained for apoptosis. Tissue lysates were analyzed by Western blotting and densitometry.. Compared to UW alone, significantly fewer apoptotic cells were present in UW + rATG perfused and stored livers. There were early and sustained significant increases in Bcl-X(L) and decreases in Bcl-X(S) with rATG. There was an initial, but not sustained, significant decrease in Bax with rATG. Moreover, there was a significant one-third decrease in caspase-9 production with rATG at 0, 6, 12, and 18 h.. Decreased proapoptotic Bcl-X(S) and increased antiapoptotic Bcl-X(L), as well as decreased downstream proapoptotic caspase-9 expression, during liver ischemia after treatment with rATG, all favor cell survival. Because apoptotic ischemic injury results in allograft dysfunction, preservation strategies that ameliorate such immunological effects may improve organ function.

Topics: Adenosine; Adjuvants, Immunologic; Allopurinol; Animals; Apoptosis; bcl-X Protein; Caspase 9; Caspases; Cold Temperature; Glutathione; Immunoglobulins; Insulin; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Proto-Oncogene Proteins c-bcl-2; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

Heme oxygenase (HO)-1 preconditioning through genetic or pharmacologic interventions was shown experimentally to improve posttransplant outcome of liver grafts. However, its clinical application requires careful consideration because of the complexity and economic costs of the procedures. This study aimed to examine if graft preconditioning with HO-1 could be substituted by a simple treatment with heme-degrading products such as bilirubin. Rats were pretreated with or without hemin, an HO-1 inducer for preconditioning. Their livers were harvested as grafts in University of Wisconsin (UW) solution for 16 hours at 4 degrees C and followed by reperfusion ex vivo or by transplantation in vivo. The control grafts were also treated with a rinse buffer containing varied concentrations of unconjugated bilirubin with different time intervals. The HO-1-preconditioned grafts ex vivo exhibited a marked improvement of bile output and cell injury that was cancelled by blocking HO with zinc protoporphyrin-IX. The aggravation of the graft viability by the inhibitor was repressed by supplementation of bilirubin but not by that of carbon monoxide. Furthermore, a short-term rinse treatment with micromolar levels of bilirubin attenuated biliary dysfunction and cell injury of the grafts both ex vivo and in vivo even without HO-1 preconditioning. The protective effects of HO-1 preconditioning or bilirubin rinse appeared to involve its inhibitory effects on lipid peroxidation in hepatocytes. In conclusion, these results suggest that bilirubin rinse serves as a simple strategy to ameliorate hyperacute oxidative stress and hepatobiliary dysfunction of the transplanted grafts, mimicking effects of HO-1-mediated preconditioning.

Topics: Adenosine; Allopurinol; Animals; Bile; Bilirubin; Carbon Monoxide; Cholestasis, Intrahepatic; Glutathione; Graft Survival; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hemin; Insulin; Ischemic Preconditioning; Liver Transplantation; Male; Organ Preservation Solutions; Oxidative Stress; Raffinose; Rats; Rats, Wistar; Reperfusion Injury

Ischemic biliary lesions are a threatening complication following orthotopic liver transplantation. Their exact pathophysiological mechanism is unknown so far, but insufficient perfusion of biliary arterial vessels might be responsible for the development of these lesions. This might be changed by improved perfusion techniques. We performed a controlled study of cases since February 2000.. We used arterial back table pressure perfusion to achieve reliable perfusion of the capillary system of the biliary tract, which may be impaired by the high viscosity of University of Wisconsin solution. In this study, 190 orthotopic liver transplantations performed between September 1997 and July 2002 were investigated with regard to ischemic biliary lesions.. One hundred thirty-one grafts were preserved by in situ standard perfusion including portal perfusion,whereas additional arterial back table pressure perfusion was performed in 59 cases. Donor-related factors, recipient age, indications for transplantation, transplantation techniques, and ischemia times were comparable between groups. Twenty-one (16%) of the patients in the standard perfusion group and only one of the those receiving arterial back table pressure perfusion developed ischemic biliary lesions. This difference was highly significant (P=0.004). Maximal aspartate aminotransferase and alanine aminotransferase levels in the first 3 days were significantly lower in the arterial back table pressure perfusion group (P>0.05).. Arterial back table pressure perfusion is an easy and reliable method for preventing ischemic biliary lesions in orthotopic liver transplantation. It should, therefore, be the standard technique in liver procurement.

Topics: Adenosine; Adult; Allopurinol; Angiography; Biliary Tract; Capillaries; Cholestasis; Female; Follow-Up Studies; Glutathione; Hepatic Artery; Humans; Hydrostatic Pressure; Insulin; Intraoperative Complications; Ischemia; Liver Function Tests; Liver Transplantation; Male; Middle Aged; Organ Preservation Solutions; Perfusion; Postoperative Complications; Raffinose; Reperfusion Injury

Oxygen-derived free radicals have been implicated in the pathogenesis of cerebral injury after ischaemia-reperfusion. Caffeic acid phenethyl ester (CAPE), an active component of propolis extract, exhibits antioxidant properties. The purpose of the present study was to investigate the effects of ischaemia and subsequent reperfusion on rat brain and to investigate the effects of two free radical scavengers, CAPE and alpha-tocopherol, on this in vivo model of cerebral injury. Ischaemia was induced by bilateral occlusion of the carotid arteries for 20 min and reperfusion was achieved by releasing the occlusion to restore the circulation for 20 min. Control rats underwent a sham operation. CAPE at 10 micromol kg(-1) or alpha-tocopherol at 25 micromol kg(-1) was administered intraperitoneally before reperfusion. Reperfusion led to significant increase in the activity of xanthine oxidase and higher malondialdehyde levels in the brain. Acute administration of both CAPE and alpha-tocopherol suppressed ischaemia-reperfusion-induced cerebral lipid peroxidation and injury, but CAPE seems to offer a better therapeutic advantage over alpha-tocopherol.

Topics: Adenosine Deaminase; alpha-Tocopherol; Animals; Brain; Brain Chemistry; Caffeic Acids; Catalase; Data Interpretation, Statistical; Glutathione Peroxidase; Heart; Male; Malondialdehyde; Myocardium; Nitric Oxide; Phenylethyl Alcohol; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Statistics, Nonparametric; Superoxide Dismutase; Xanthine Oxidase

Reactive nitrogen and oxygen species are implicated in the damage of ischemic tissue that is reperfused. One important pathway may involve xanthine oxidase. Xanthine oxidase uses xanthine, a product of ATP degradation in ischemic tissue, to produce superoxide and hydrogen peroxide. Superoxide reacts rapidly with nitric oxide to form peroxynitrite, a powerful oxidant. Another potential source of reactive nitrogen species is the myeloperoxidase-hydrogen peroxide-nitrite system of activated phagocytes. We demonstrate that peroxynitrite and myeloperoxidase nitrate xanthine in vitro. Through 13C NMR spectroscopy, UV/visible spectroscopy, and mass spectrometry, the major product was identified as 8-nitroxanthine. Xanthine nitration by peroxynitrite was optimal at neutral pH and was markedly stimulated by physiological concentrations of bicarbonate. Xanthine nitration by myeloperoxidase required hydrogen peroxide and nitrite. However, it was independent of chloride ion and little affected by scavengers of hypochlorous acid, suggesting that the reactive agent is a nitrogen dioxide-like species. 8-Nitroxanthine was generated by a low, steady flux of peroxynitrite, and also by the myeloperoxidase-hydrogen peroxide-nitrite system of activated human neutrophils, suggesting that the reactions may be physiologically relevant. 8-Nitroxanthine may exert biological effects because it markedly increased the production of superoxide by the xanthine oxidase-xanthine system. Our observations suggest a mechanism for the enhanced formation of superoxide in reperfused tissue, which might increase the production of peroxynitrite and 8-nitroxanthine. Generation of 8-nitroxanthine by peroxynitrite and myeloperoxidase could represent a positive feedback mechanism that enhances further the production of both reactive oxygen and nitrogen species in ischemic tissue that is reperfused.

Topics: Humans; Neutrophil Activation; Neutrophils; Peroxidase; Peroxynitrous Acid; Reperfusion Injury; Superoxides; Xanthine Oxidase; Xanthines

Activation and accumulation of leukocytes constitute a rate-limiting step in ischemia/reperfusion (I/R)-induced tissue injury. The signalling mechanisms, however, that regulate leukocyte rolling and adhesion in the colonic microcirculation are not known. The objective of the study was to define the role of CXC chemokines (MIP-2 and KC) in I/R-induced leukocyte-endothelial cell interactions in the mouse colon. In C57/B16 mice, colonic ischemia was induced by clamping the superior mesenteric artery for 30 min and leukocyte rolling and stationary adhesion were examined in venules after 120 and 240 min of reperfusion. I/R provoked a clear-cut increase in leukocyte rolling and adhesion in colonic venules. Both MIP-2 and KC were upregulated at the gene and protein level in the reperfused colon. Immunoneutralization of MIP-2 and KC by monoclonal antibodies reduced reperfusion-induced firm adhesion of leukocytes by 73% and 75%, respectively. Interestingly, combined inhibition of MIP-2 and KC additionally decreased leukocyte rolling by 79%, but did not further reduce the number of firmly adherent leukocytes. To study the role of oxygen free radicals (OFRs) in the regulation of CXC chemokine expression, additional animals were pretreated with the xanthine-oxidase inhibitor allopurinol. In fact, allopurinol treatment reduced the colonic levels of MIP-2 and KC by 62% and 64%, respectively. This study elucidates important interactions between OFRs and chemokines in the I/R-induced leukocyte response in the mouse colon. Moreover, our data demonstrate that CXC chemokines play a fundamental role in colonic I/R and that functional interference with CXC chemokines may protect against pathological inflammation in the colon.

Topics: Allopurinol; Animals; Cell Adhesion; Chemokine CXCL1; Chemokine CXCL2; Chemokines; Chemokines, CXC; Colon; Cytokines; Endothelial Cells; Gene Expression Regulation; Leukocyte Rolling; Leukocytes; Male; Mice; Mice, Inbred C57BL; Monokines; Reactive Oxygen Species; Reperfusion Injury; RNA, Messenger

The aim of this study was to evaluate the effect of allopurinol, methylene blue, and a monoclonal antibody to the adhesion molecule ICAM-1 in intestinal ischemia and reperfusion injury.. The rats were divided into 5 groups. CG (n = 8) was untreated controls, SISG (n = 11) received sterile isotonic saline solution, ICAMG (n = 12) received a monoclonal antibody to rat ICAM-1, ALLOG (n = 12) received allopurinol, and MBG (n = 14) received methylene blue. Intestinal ischemia was performed for 60 minutes followed by 60 minutes of reperfusion. The agents were injected 10 minutes before the reperfusion to animals. After 60 minutes of reperfusion, the plasma samples for myeloperoxidase (MPO) activity, tumor necrosis factor alpha (TNF-alpha) and uric acid levels, and the intestinal biopsies of ileum and jejunum for histopathologic examination were taken.. The mucosal damage was attenuated, and TNF-alpha level significantly decreased in ALLOG and ICAMG compared with SISG. The MPO activity was the lowest in ICAMG, and uric acid level was significantly decreased in ALLOG compared with the other groups. Methylene blue decreased TNF-alpha response to reperfusion injury but significantly increased the grade of the mucosal damage and the MPO activity.. This study shows that prereperfusion application of allopurinol and monoclonal antibody to the adhesion molecule ICAM-1 may attenuate the damage caused by intestinal ischemia and reperfusion, but the different time-points for application, the effects observed in the different ischemia and reperfusion durations, and the long-term results also should be investigated in the same experimental model before the final conclusion. Methylene blue was not effective to prevent or attenuate the intestinal tissue injury, but because this was the first study examining the effect of methylene blue on intestinal reperfusion injury, further studies with the different doses, ischemic duration, and application times will be needed.

Topics: Allopurinol; Animals; Antibodies, Monoclonal; Drug Evaluation, Preclinical; Ileum; Intercellular Adhesion Molecule-1; Intestines; Ischemia; Jejunum; Male; Methylene Blue; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tumor Necrosis Factor-alpha; Uric Acid

To investigate the effects of scutellarin (Scu) on liver function after brain ischemia/reperfusion in Wistar rats.. Rats were pretreated with Scu for 7 d and then subjected to a brain ischemia/reperfusion injury induced by a middle cerebral artery occlusion. The levels of nitric oxide (NO), xanthine oxidase (XOD), alanine transaminase (ALT), and aspartate aminotransferase (AST) in serum or liver tissues and the activities of antioxidant enzymes and cytochrome P450-dependent monooxygenases (CYPs) in liver tissues after brain ischemia/reperfusion were determined.. In vehicle-treated rats, XOD, ALT, and AST activities (P<0.01) in serum and the MDA level (P<0.05) in liver tissues were all elevated but were significantly reduced (P<0.05) by Scu pretreatment. The NO levels in serum and liver tissues were decreased (P<0.01) dramatically in vehicle-treated rats and returned to the levels in the sham-operated animals when pretreated with Scu. SOD (P<0.05) and GSH-PX (P<0.01) activities in cytosol fraction were increased significantly by Scu pretreatment. Furthermore, a loss of CYP3A activity (P<0.01), but no changes of CYP1A1, CYP1A2, and CYP2E1 activities in liver were observed after brain ischemia/reperfusion in rats. Scu had no effect on them.. These results demonstrated that pretreatment with Scu could attenuate hepatocellular damage elicited by brain ischemia/reperfusion in rats and this protection is in major part by its antioxidant activity.

Topics: Alanine Transaminase; Animals; Antioxidants; Aryl Hydrocarbon Hydroxylases; Aspartate Aminotransferases; Brain Ischemia; Cytochrome P-450 CYP3A; Glutathione Peroxidase; Liver; Male; Malondialdehyde; Nitric Oxide; Oxidoreductases, N-Demethylating; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

Sirolimus (SRL) seems to impair renal recovery from ischemic injury in animal models and delayed graft function after clinical renal transplantation. This study determined the impact of SRL on renal recovery after ischemia-reperfusion injury in a rat kidney transplant model.. Syngeneic kidneys were preserved in University of Wisconsin solution before transplantation into bilaterally nephrectomized rats. Recipients received vehicle or SRL targeting for whole-blood trough levels of 10 to 20 ng/mL as measured by high-performance liquid chromatography. Renal function was assessed by animal survival or daily serum creatinine. Tissue samples were collected for histologic examination.. Median SRL whole-blood concentrations were 16.6 +/- 1.6 ng/mL on postoperative day (POD) 1 and 12.0 +/- 0.9 ng/mL on POD 3. Transplantation of kidneys after 39 hr of cold storage resulted in 30% survival in the SRL-treated group compared with 100% survival in the control group (P=0.002). Transplantation of kidneys after 24 hr of cold storage resulted in no survival differences, but there were significant differences in renal function. Daily serum creatinine (PODs 1-4) was higher in the SRL-treated group compared with the control group (P<0.05 at all time points). Grafts from SRL-treated animals showed more severe tubular necrosis compared with control animals.. When given at therapeutic immunosuppressive doses, SRL compromises renal function after ischemia-reperfusion injury in a rat kidney transplant model. The antiproliferative effect of SRL may translate into impairment of tubular repair and regeneration necessary for recovery after such injury.

Topics: Adenosine; Allopurinol; Animals; Creatinine; Glutathione; Immunosuppressive Agents; Insulin; Kidney; Kidney Transplantation; Organ Preservation Solutions; Raffinose; Rats; Reperfusion Injury; Sirolimus; Time Factors; Transplantation, Isogeneic; Treatment Outcome

To explore the role of ligustrazin on dynamic changes of lipid peroxidation in hepatic ischemia/reperfusion injury (HIRI) and its mechanism.. The HIRI model was used. Twenty rabbits were randomly divided into control group (n = 10) and ligustrazin group (n = 10). The xanthine oxidase (XO) activity, superoxide dismutase (SOD) activity,malondialdehyde (MDA) content and glutamic pyruvic transaminase (GPT) activity in plasma were observed before ischemia and at ischemia 25 min, reperfusion 25 min, reperfusion 60 min and reperfusion 120 min.. The XO activity, SOD activity, MDA content and GPT activity of ligustrazin group, as compared with control group, showed significant differences (P < 0.05 or P < 0.01) at total time points of reperfusion.. Ligustrazin has notable anti-lipid peroxidation effect on HIRI, which is due to its inhibiting the generation of oxygen free radicals and its strengthening scavenging of oxygen free radicals.

Topics: Alanine Transaminase; Animals; Female; Lipid Peroxidation; Liver; Male; Malondialdehyde; Pyrazines; Rabbits; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

The objectives of this study were to compare the protective influence of exogenous nitric oxide on renal ischemia reperfusion (I/R) injury with that of the antioxidant vitamins C and E. Sprague-Dawley rats were divided into three groups ( n=12 per group). Normal saline solution was given in group 1, a vitamin C (200 mg/kg/d) plus vitamin E (100 mg/kg/d) combination in group 2 for 3 days before operating and Na-nitroprusside (5 mg/kg/d) in group 3 before reperfusion. The left kidneys were exposed to warm ischemia for 40 min followed by reperfusion for 90 min. The right kidneys were used as internal controls. After both kidneys were removed, histopathological examinations were performed, and oxidative and antioxidative parameters were measured. In the postischemic reperfused rat kidneys, the renal lipid peroxidation level was significantly lower, and the renal GSH level higher in the group given Na-nitroprusside compared with groups 1 and 2. Renal specific xanthine oxidase activity was also significantly lower in the group treated with Na-nitroprusside than in the groups given vitamins or saline. There was a significant, negative correlation between lipid peroxidation and reduced glutathione levels. Our results suggest that the exogenous nitric oxide (Na-nitroprusside) inhibits xanthine oxidase, and has more apparent preventive features for renal I/R injury than the antioxidant vitamins C+E.

Topics: Animals; Antioxidants; Ascorbic Acid; Glutathione; Ischemia; Kidney; Lipid Peroxides; Male; Nitric Oxide; Rats; Rats, Sprague-Dawley; Reference Values; Renal Circulation; Reperfusion Injury; Vitamin E; Xanthine Oxidase

The peroxidation of membranous phospholipids induced by ischemia reperfusion was inhibited in Cu/Zn superoxide dismutase (SOD) overexpressing mice, suggesting a detrimental role for intracellular reactive oxygen species (ROS) in reoxygenated cell injury. To ascertain the in-vitro relevance of this hypothesis, the present study examined the participation of intracellular ROS in reoxygenation injury.. This examination was done in two experimental models: Cu/Zn-SOD transgenic (Tg) mice that underwent hypoxia-reoxygenation in vitro and normal mice pretreated with a specific inhibitor of xanthine oxidase, BOF-4272, followed by in vitro hypoxia-reoxygenation.. The release of aspartate aminotransferase (AST) and the peroxidation of phospholipids were both ameliorated in hepatocytes from the Tg mice compared with findings in hepatocytes from normal mice. Similar findings were seen in the BOF-4272-pretreated cells, in which there was a decrease in AST and phospholipid peroxides.. These results support the pivotal role of intracellular ROS generated by xanthine oxidase in reoxygenated cell injury, and suggest the viability of using an intracellular antioxidative therapy for reperfusion injury of the liver.

Topics: Adenosine Triphosphate; Animals; Aspartate Aminotransferase, Cytoplasmic; Enzyme Inhibitors; Free Radical Scavengers; Hepatocytes; In Vitro Techniques; Lipid Peroxidation; Liver; Mice; Mice, Transgenic; Models, Biological; Phosphatidylcholines; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Triazines; Xanthine Oxidase

Oxidative stress is believed to be implicated in the pathogenesis of postischaemic cerebral injury. Many antioxidants were shown to be neuroprotective in experimental models of cerebral ischaemia/reperfusion (I/R). The present study was designed to investigate the potential protective effects of curcumin (CUR) against I/R insult in rat forebrain. The model adopted was that of surgically-induced forebrain ischaemia, performed by means of bilateral common carotid artery occlusion (BCCAO) for 1 h, followed by reperfusion for another 1h. The effects of a single i.p. dose of CUR (50, 100 or 200 mg kg(-1)), administered 0.5 h after the onset of ischaemia, were investigated by assessing oxidative stress-related biochemical parameters in rat forebrain. CUR, at the highest dose level (200 mg kg(-1)), decreased the I/R-induced elevated xanthine oxidase (XO) activity, superoxide anion (O(2)*(-)) production, malondialdehyde (MDA) level and glutathione peroxidase (GPx), superoxide dismutase (SOD), and lactate dehydrogenase (LDH) activities. On the other hand, CUR did not affect the declined reduced glutathione (GSH) content due to I/R insult. Worth mentioning is that the activity of catalase (CAT) did not change in response to either I/R insult or drug treatment. In conclusion, CUR was found to protect rat forebrain against I/R insult. These protective effects may be attributed to its antioxidant properties and/or its inhibitory effects on xanthine dehydrogenase/xanthine oxidase (XD/XO) conversion and resultant O(2)*(-) production.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antioxidants; Brain Ischemia; Catalase; Curcumin; Glutathione Peroxidase; L-Lactate Dehydrogenase; Male; Malondialdehyde; Neuroprotective Agents; Prosencephalon; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase; Superoxides; Xanthine Oxidase

Ischemic preconditioning (IPC) is a phenomenon of protection in various tissues from normothermic ischemic injury by previous exposure to short cycles of ischemia-reperfusion. The ability of IPC to protect hepatocytes from a model of hypothermic transplant preservation injury was tested in this study. Rat hepatocytes were subjected to 30min of warm ischemia (37 degrees C) followed by 24 or 48h of hypothermic (4 degrees C) storage in UW solution and subsequent re-oxygenation at normothermia for 1h. Studies were performed with untreated control cells and cells treated with IPC (10min anoxia followed by 10min re-oxygenation, 1 cycle). Hepatocytes exposed to IPC prior to warm ischemia released significantly less LDH and had higher ATP concentrations, relative to untreated ischemic hepatocytes. IPC significantly reduced LDH release after 24h of cold storage before reperfusion and after 48h of cold storage and after 60min of warm re-oxygenation, relative to the corresponding untreated hepatocytes. ATP levels were also significantly higher when IPC was used prior to the warm and cold ischemia-re-oxygenation protocols. In parallel studies, IPC increased new protein synthesis and lactate after cold storage and reperfusion compared to untreated cells but no differences in the patterns of protein banding were detected on electrophoresis between the groups. In conclusion, IPC significantly improves hepatocyte viability and energy metabolism in a model of hypothermic preservation injury preceded by normothermic ischemia. These protective effects on viability may be related to enhanced protein and ATP synthesis at reperfusion.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Glutathione; Hepatocytes; In Vitro Techniques; Insulin; Ischemic Preconditioning; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Oxygen Consumption; Protein Biosynthesis; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Temperature

Topics: Adenosine; Adult; Alanine Transaminase; Allopurinol; Aspartate Aminotransferases; Brain Death; Glutathione; Glycine; Hepatectomy; Humans; Infusions, Intravenous; Insulin; Liver; Liver Function Tests; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Tissue and Organ Harvesting; Tissue Donors

Although bilirubin, which crosses the blood-brain barrier, can cause irreversible brain damage, it also possesses antioxidant properties that may be protective against oxidative stress. Intestinal ischemia-reperfusion (IR) injury results in cell destruction, mediated via the generation of reactive oxygen species. Although increased serum bilirubin is correlated with increased antioxidant potential in the face of hyperoxia, evidence of bilirubin-associated protective effect against IR injury remains nonspecific. We therefore sought to investigate whether hyperbilirubinemia would be protective against IR injury to the intestine.. Young adult rats were randomly assigned to one of three groups: 1) IR/control (n = 12); 2) IR/hyperbilirubinemia (n = 10), in which IR was generated while the rats were treated with a continuous infusion of bilirubin; and 3) hyperbilirubinemia controls (n = 10). Blood and intestinal tissue samples were obtained to determine serial thiobarbituric acid reducing substances (index of lipid peroxidation) and for xanthine oxidase/xanthine dehydrogenase and glutathione/glutathione disulfide ratios. Intestinal histopathology was graded from 1 (normal) to 4 (severe necrotic lesions).. Histopathologic scoring and circulating and tissue thiobarbituric acid reducing substances were highest in the IR/control animals compared with either the IR/hyperbilirubinemics or the controls. All of these are consistent with the most severe injury in this group. Xanthine oxidase/xanthine dehydrogenase ratios were not significantly different among the groups.. Hyperbilirubinemia ameliorates the extent of intestinal IR injury in our model and appears to act as an antioxidant. This study supports the concept that bilirubin possesses some beneficial properties in vivo, although no direct clinical conclusions can be drawn from these data.

Topics: Animals; Bilirubin; Disease Models, Animal; Enterocolitis, Necrotizing; Glutathione Disulfide; Intestines; Rats; Reperfusion Injury; Thiobarbituric Acid Reactive Substances; Time Factors; Xanthine Dehydrogenase; Xanthine Oxidase

Ischaemia/reperfusion injury of the liver is the major cause of liver dysfunction and cellular death in transplantation and in liver resection with hepatic pedicle clamping. Many agents are used to prevent this phenomenon, which occurs following interaction of different mediators during both ischaemia and reperfusion. In this study, we aimed to assess the effects of allopurinol, a xanthine oxidase inhibitor, and pentoxifilline, on liver ischaemia/reperfusion injury when used together and to compare these with the effects of using these agents singly.. Thirty-two rats were divided into four groups consisting of eight rats: Group C, control; Group P, pentoxifilline; Group A, allopurinol; and Group PA, pentoxifilline + allopurinol. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) levels were measured before hepatic pedicle clamping, on the 45th minute of ischaemia and 15 and 45 minutes after reperfusion. Group P rats were injected with 50 mg/kg pentoxifilline, Group A rats 50 mg/kg allopurinol and Group PA rats were injected with both agents 15 minutes before hepatic pedicle clamping.. Ischaemia/reperfusion injury was produced by hepatic pedicle clamping, as demonstrated by AST, ALT and LDH increase. Injury prevention occurred in Groups P, A and PA. No significantly different (better) prevention was provided by giving allopurinol plus pentoxifilline to the rats. Furthermore, no difference was observed between the allopurinol and pentoxifilline injected groups in terms of preventing ischaemia/reperfusion injury.. Pretreatment with allopurinol or pentoxifilline resulted in significantly lower hepatic enzyme elevation than that in controls in the rat liver ischaemia/reperfusion model. Using both agents does not provide better protection than using either agent alone.

Topics: Allopurinol; Animals; Disease Models, Animal; Drug Therapy, Combination; Enzyme Inhibitors; Ischemia; Liver Circulation; Pentoxifylline; Rats; Reperfusion Injury

Hepatic transplantation may result in coagulopathy caused by the release of mast-cell-derived heparin, and xanthine oxidase (XO) inhibition stabilizes mast cells. Thus, XO inactivation could decrease coagulopathy after hepatoenteric ischemia-reperfusion. Rabbits were fed a standard or XO-inactivating diet before hepatoenteric ischemia for 35 min and before 30 min of reperfusion. Hemostasis was assessed by thrombelastography. Heparin activity was quantified by anti-IIa. XO inactivation resulted in clot formation after reperfusion in all animals, whereas only 37.5% of animals with XO activity clotted (P<0.05). Anti-IIa activity was less in animals at baseline and after reperfusion with XO inactivation (45+/-5 and 65+/-5 mU/mL, respectively) compared to animals with XO activity (51+/-4 and 71+/-5 mU/mL, respectively) (P<0.05). Clot strength, which was mediated by coagulation proteins, was significantly greater at baseline and after reperfusion in animals with XO inactivation. XO inactivation enhances hemostasis by decreasing circulating heparin activity and increasing coagulation protein function before ischemia-reperfusion.

Topics: Animals; Blood Coagulation Disorders; Blood Coagulation Factors; Blood Platelets; Hemostasis; Heparin; Liver; Liver Transplantation; Male; Mast Cells; Rabbits; Reperfusion Injury; Xanthine Oxidase

Leucocyte recruitment is a key feature in ischaemia-reperfusion (I/R)-triggered tissue injury. However, the mechanisms underlying leucocyte-endothelium interactions in the large bowel remain elusive because of a previous lack of models to examine the colonic microcirculation. The aim of this study was to develop and validate a novel method for studying reperfusion-induced leucocyte-endothelium interactions in the colon.. The superior mesenteric artery was occluded for 30 min in male C57/Bl6 mice and leucocyte responses were analysed in colonic venules after 30-240 min of reperfusion. Analysis of leucocyte rolling and adhesion in colonic venules was made possible by an inverted approach using intravital fluorescence microscopy.. Thirty minutes of ischaemia and 120 min of reperfusion induced the strongest and most reproducible increase in leucocyte rolling and adhesion. This was associated with a significant increase in colonic levels of malondialdehyde (MDA). Administration of allopurinol and superoxide dismutase reduced I/R-induced leucocyte responses in a dose-dependent manner. Pretreatment with allopurinol attenuated the tissue content MDA in the colon by more than 60 per cent.. A new method for examining I/R-induced leucocyte responses in the colonic microcirculation is described. Oxygen free radicals play an important role in triggering leucocyte rolling and adhesion in colonic venules.

Topics: Allopurinol; Animals; Colitis, Ischemic; Colon; Endothelium, Vascular; Free Radical Scavengers; Free Radicals; Leukocytes; Ligation; Male; Mesenteric Artery, Superior; Mice; Mice, Inbred C57BL; Reperfusion Injury; Superoxide Dismutase

The effect of Wen-Pi-Tang extract on renal injury induced by peroxynitrite (ONOO-) production was investigated using rats subjected to intravenous lipopolysaccharide (LPS) injection and then renal ischemia followed by reperfusion. The plasma level of 3-nitrotyrosine, a marker of cytotoxic ONOO formation in vivo, was enhanced markedly in control rats subjected to LPS plus ischemia-reperfusion, but was significantly reduced by the oral administration of Wen-Pi-Tang extract, at doses of 62.5 and 125 mg/kg body weight/day, for 30 days prior to LPS plus ischemia-reperfusion. The activities of inducible nitric oxide synthase (iNOS) and xanthine oxidase (XOD) in renal tissue of control and Wen-Pi-Tang extract-treated rats did not change significantly, while those of the antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase, were significantly increased by the administration of Wen-Pi-Tang extract, indicating that Wen-Pi-Tang improved the defense system by scavenging free radicals, not by directly inhibiting nitric oxide and superoxide production by iNOS and XOD. In addition, the levels of the hydroxylated products, m- and p-tyrosine, declined, whereas that of phenylalanine increased, after oral administration of Wen-Pi-Tang extract. Furthermore, the elevated plasma urea nitrogen and creatinine levels resulting from LPS plus ischemia-reperfusion process were significantly reduced by Wen-Pi-Tang extract, implying amelioration of renal impairment. The present study indicates that Wen-Pi-Tang extract contributes to the regulation of ONOO- formation and plays a beneficial role against ONOO(-) -induced oxidative injury and renal dysfunction in vivo.

Topics: Animals; Chromatography, High Pressure Liquid; Creatinine; Drugs, Chinese Herbal; Kidney; Lipid Peroxides; Lipopolysaccharides; Models, Chemical; Nitric Oxide Synthase; Nitrogen; Oxygen; Peroxynitrous Acid; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine; Urea; Xanthine Oxidase

The effects of the BuOH fraction from mustard leaf in rats subjected to renal ischemia-reperfusion were examined. The elevated serum superoxide anion (O2-) level and renal xanthine oxidase (XOD) activity in rats subjected to 6-h reperfusion following 1-h ischemia significantly and dose-dependently declined after oral administration of the BuOH fraction at doses of 50 and 200 mg/kg body weight/d for 10 d prior to ischemia-reperfusion. These findings indicate that this fraction might scavenge O2- or inhibit the generation of O2- through XOD activated by the ischemia-reperfusion process. In addition, the thiobarbituric acid-reactive substance level of the renal mitochondrial fraction of rats given the BuOH fraction orally was significantly lower than that of control rats given physiological saline (vehicle), implying that this fraction exerted protective action against lipid peroxidation caused by ischemia-reperfusion. Furthermore, oral administration of the BuOH fraction reduced the serum urea nitrogen and creatinine levels, indicators of renal function. These results suggest that the BuOH fraction has protective effects against ischemia-reperfusion injury, acting as an antioxidant by scavenging O2-, inhibiting O2- generation through XOD, protecting against lipid peroxidation and ameliorating renal functional impairment.

Topics: Animals; Blood Urea Nitrogen; Butanols; Creatinine; Disease Models, Animal; Kidney; Male; Mustard Plant; Phytotherapy; Plant Leaves; Rats; Rats, Wistar; Reperfusion Injury; Superoxides; Thiobarbituric Acid Reactive Substances; Xanthine Oxidase

Reperfusion injury can occur when blood flow is restored after a transient period of ischaemia. The resulting cascade of reactive oxygen species damages tissue. This mechanism may contribute to the tissue damage produced by 5-aminolaevulinic acid-induced photodynamic therapy, if this treatment temporarily depletes oxygen in an area that is subsequently reoxygenated. This was investigated in the normal colon of female Wistar rats. All animals received 200 mg kg(-1) 5-aminolaevulinic acid intravenously 2 h prior to 25 J (100 mW) of 628 nm light, which was delivered continuously or fractionated (5 J/150 second dark interval/20 J). Animals were recovered following surgery, killed 3 days later and the photodynamic therapy lesion measured macroscopically. The effects of reperfusion injury were removed from the experiments either through the administration of free radical scavengers (superoxide dismutase (10 mg kg(-1)) and catalase (7.5 mg kg(-1)) in combination) or allopurinol (an inhibitor of xanthine oxidase (50 mg kg(-1))). Prior administration of the free radical scavengers and allopurinol abolished the macroscopic damage produced by 5-aminolaevulinic acid photodynamic therapy in this model, regardless of the light regime employed. As the specific inhibitor of xanthine oxidase (allopurinol) protected against photodynamic therapy damage, it is concluded that reperfusion injury is involved in the mechanism of photodynamic therapy in the rat colon.

Topics: Allopurinol; Aminolevulinic Acid; Animals; Catalase; Colon; Female; Necrosis; Photochemotherapy; Rats; Rats, Wistar; Reperfusion Injury

The term "ischemic reperfusion injury" encompasses all toxic events in a cell that occur during ischemia and subsequent reoxygenation. These reactions have a significant effect, for example, on the rate of organ survival in kidney transplantation. Reactive oxygen intermediates (ROI) play an important role in the process of postischemic reperfusion. The basic mechanisms of generation and detoxification of ROI as well as the possibilities for their registration and quantification under conditions of ischemic reperfusion injury in the rat kidney are demonstrated in this report. A prerequisite to developing cytoprotective strategies is understanding the precise course of these mechanisms to minimize damage caused by ischemia and the subsequent reperfusion, thus retaining the organ's function to the greatest extent.

Topics: Animals; Humans; Kidney; Kidney Transplantation; Rats; Reactive Oxygen Species; Reperfusion Injury; Tissue Survival; Xanthine Oxidase

Intestinal ischemia-reperfusion affects hemodynamics. We studied intratracheal vs. intraperitoneal methylene blue (MB) attenuation of hemodynamic and metabolic deterioration following superior mesenteric artery (SMA) clamping/unclamping. Murine SMAs (5/group) were clamped for 1 h. MB (2, 6, 20, or 60 mg/kg [MB-2, MB-6, MB-20, and MB-60]) was administered intraperitoneally or intratracheally 10 min before unclamping. Observation continued for another 3 h. Circulating xanthine oxidase and base deficit levels doubled among ischemia non-treated and ischemia MB-2- and MB-60-treated groups, blood pressure decreased by 50%, and heart rate increased by 35%, compared to controls (non-clamped/unclamped and non-MB-treated rats, P < 0.01). These three ischemia groups needed 3-fold the amount of fluid to maintain systolic pressure > or =60 mmHg than controls (P < 0.01). Only the MB-6 and MB-20 intraperitoneal and intratracheal regimens similarly afforded hemodynamic stability in ischemic animals; base deficit and resuscitation volumes normalized as well. No drug regimen affected heart rate. We concluded that intraperitoneal and intratracheal MB at specific doses prevented systemic derangement following SMA clamping/unclamping.

Topics: Animals; Blood Pressure; Dose-Response Relationship, Drug; Heart Rate; Hemodynamics; Injections, Intraperitoneal; Male; Mesenteric Artery, Superior; Methylene Blue; Rats; Rats, Wistar; Reperfusion Injury; Respiration; Xanthine Oxidase

Many studies indicate that oxygen free-radical formation after reoxygenation of liver may initiate the cascade of hepatocellular injury. It has been demonstrated that controlled ozone administration may promote an oxidative preconditioning or adaptation to oxidative stress, preventing the damage induced by reactive oxygen species (ROS) and protecting against liver ischaemia-reperfusion (I/R) injury. On the basis of those results we postulated that ozone treatment in our experimental conditions has biochemical parameters similar to the ischaemic preconditioning (IscheP) mechanism. Four groups of rats were classified as follows: (1) sham-operated animals subjected to anaesthesia and laparotomy, plus surgical manipulation; (2) I/R animals were subjected to 90 min of right-lobe hepatic ischaemia, followed by 90 min of reperfusion; (3) IscheP, previous to the I/R period (as in group 2): animals were subjected to 10 min of ischaemia and 10 min of reperfusion; (4) ozone oxidative preconditioning (OzoneOP), previous to the I/R period (as in group 2): animals were treated with ozone by rectal insufflation 1 mg kg (-1). The rats received 15 ozone treatments, one per day, of 5-5.5 ml at the ozone concentration of 50 microg ml (-1). The following parameters were measured: serum transaminases (AST, ALT) and 5'-nucleotidase (5 '-NT), with morphological determinations, as indicators or hepatocellular injury; total sulfhydryl groups, calcium levels and calpain activity as mediators which take part in xanthine deshydrogenase (XDH) conversion to xanthine oxidase (XO) (reversible and irreversible forms, respectively); XO activities and malondialdehyde + 4-hydroyalkenals as indicators of increased oxidative stress. AST, ALT levels were attenuated in the IscheP (130 +/- 11.4 and 75 +/- 5.7 U l (-1)) with regard to the I/R group (200 +/- 22 and 117 +/- 21.7 U l (-1)) while the OzoneOP maintained both of the enzyme activities ( 89.5 +/- 12.6 and 43.7 +/- 10 U l (-1)) without statistical differences (P< 0.05) in comparison with the sham-operated ( 63.95 +/- 11 and 19.48 +/- 3.2 U l (-1)). Protective effects of both the preconditioning settings on the preservation of total sylfhydryl groups (IscheP: 6.28 +/- 0.07, OzoneOP: 6.34 +/- 0.07 micromol mg prot (-1)), calcium concentrations (IscheP: 0.18 +/- 0.09, OzoneOP: 0.20 +/- 0.06 micromol mg prot (-1)), and calpain activity (IscheP: 1.04 +/- 0.58, OzoneOP: 1.41 +/- 0.79 U mg prot (-1)) were observed. Both of the preconditionings a

Topics: Animals; Calcium; Calpain; Ischemic Preconditioning; Lipid Peroxidation; Liver; Liver Circulation; Liver Function Tests; Male; Oxidants, Photochemical; Ozone; Proteins; Rats; Rats, Wistar; Reperfusion Injury; Sulfhydryl Compounds; Xanthine Dehydrogenase; Xanthine Oxidase

Topics: Adenosine; Allopurinol; Animals; Endoplasmic Reticulum; Glutathione; Insulin; Liver; Mitochondria, Liver; Organ Preservation; Organ Preservation Solutions; Polyethylene Glycols; Raffinose; Rats; Reperfusion Injury

While the effects of transient intestinal ischemia on mucosa have been well investigated, less is known about its effect on motor function. An experimental study was designed to investigate the effects of ischemia-reperfusion (I/R) on intestinal motility and intestinal muscular microcirculation. Wistar albino rats were divided into four groups: (1) baseline, (2) sham operation, (3) I/R, and (4) I/R with allopurinol pretreatment. Ischemia was induced by clamping the superior mesenteric artery (SMA) for 10 min. Gastroanal transit time (GATT) was measured with serial x-rays after instillation of barium sulfate to the stomach. Intestinal muscular microcirculation was evaluated by determining the number of carbon-perfused intestinal muscular microvessels (CPIMM). I/R prolonged GATT and decreased CPIMM significantly (P < 0.01). Pretreatment with allopurinol prevented prolongation of GATT and returned the number of CPIMM to the level of sham treatment (P < 0.01). In conclusion, reperfusion after 10 min of SMA ischemia alters intestinal motility. The no-reflow phenomenon plays an important role in this alteration of motility. Administration of allopurinol before reperfusion preserves intestinal motility by preventing the occurrence of no-reflow phenomenon.

Topics: Allopurinol; Animals; Free Radical Scavengers; Gastrointestinal Motility; Microcirculation; Rats; Rats, Wistar; Reperfusion Injury

The formation of reactive oxygen species (ROS) may be important in the pathogenesis of microvascular dysfunction and injury in ischemic retinopathies. The authors hypothesized that retinal endothelial cells can generate injurious levels of superoxide radical in response to ischemia/reperfusion, that endothelial xanthine oxidase and cyclooxygenase are important enzymatic sources of superoxide radical under these conditions, and that superoxide scavengers and inhibitors of these enzymes can protect endothelium from ischemic injury. The authors used confluent cultures of mouse retinal endothelial cells (MREC) subjected to exogenously generated superoxide or simulated ischemia-reperfusion to test these hypotheses. Cell injury was assessed biochemically by lactate dehydrogenase release into the culture medium. MREC were injured in a duration-dependent fashion by exposure to the superoxide-generating mix of hypoxanthine and xanthine oxidase. Increasing periods of oxygen and glucose deprivation (OGD) for 5-9 hr followed by replenishment of substrates for 2 hr led to progressive increases in endothelial cell injury; a significant proportion of the injury occurred during the period of substrate replenishment. Significant MREC protection was achieved by the superoxide scavengers SOD (1000 U ml(-1)) and a carboxylic acid derivative of carboxyfullerene (10 microM), the xanthine oxidase inhibitors oxypurinol (100 microM) and diphenyleneiodonium (DPI) (100 n M), and the cyclooxygenase inhibitors indomethacin (300 microM) and ibuprofen (300 microM). It is concluded that MREC are vulnerable to auto-oxidative injury by superoxide radical generated following a period of OGD. Both xanthine oxidase- and cyclooxygenase-dependent pathways are important enzymatic sources of superoxide formation in this setting. These enzymes and the ROS produced from their activity may be viable therapeutic targets to reduce microvascular dysfunction and injury in ischemic retinopathies.

Topics: Animals; Cell Hypoxia; Cells, Cultured; Endothelium, Vascular; Female; Hypoxanthine; L-Lactate Dehydrogenase; Mice; Prostaglandin-Endoperoxide Synthases; Reperfusion Injury; Retinal Diseases; Retinal Vessels; Superoxide Dismutase; Superoxides; Xanthine Oxidase

Three newly synthesised lipid peroxidation inhibitors (7, 11, 14) were evaluated for their effects on myocardial functional recovery during reperfusion after 30 min global ischemia in isolated rat hearts. The flavonoid compounds (7, 11, 14, rutin) reduce ischemia/reperfusion-induced cardiac dysfunction.

Topics: Allopurinol; Animals; Catalase; Chalcone; Flavones; Flavonoids; Glutathione Peroxidase; Heart; In Vitro Techniques; Lipid Peroxidation; Male; Molecular Structure; Myocardium; Perfusion; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Rutin; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Ventricular Pressure

The relationship between zonal oxidative stress and cell death after ischemia-reperfusion injury in rat liver was investigated. Oxidative stress was detected in situ by perfusion with nitroblue tetrazolium, which is converted to insoluble blue formazan by reducting agents. Cell death was detected in situ by perfusion with trypan blue. When isolated liver was perfused after 30 or 60 min of warm ischemia, oxidative stress was detected in periportal parenchymal cells after 30 min of reperfusion. It spread in the shape of a doughnut to midzonal cells after 60 min of reperfusion. On the other hand, cell death was observed in parenchymal cells that were within the doughnut-like area in which oxidative stress was detected. The extent of oxidative stress and cell death was higher after 60 min of ischemia than after 30 min of ischemia. In nonparenchymal cells, oxidative stress was observed in midzonal and pericentral regions after only 12 min of reperfusion, but minor cell death was observed only in periportal and midzonal regions after 30 min of reperfusion. Administration of allopurinol, an inhibitor of xanthine oxidase, suppressed oxidative stress and cell death in periportal parenchymal cells. These findings indicate that periportal and midzonal parenchymal cell death can be caused by zone-specific and xanthine-oxidase-mediated oxidative stress in parenchymal cells.

Topics: Allopurinol; Animals; Cell Death; Free Radical Scavengers; Ischemia; Liver; Liver Circulation; Male; Microcirculation; Oxidative Stress; Portal Vein; Rats; Rats, Wistar; Reperfusion Injury; Time Factors

Reactive oxygen species generated by xanthine oxidase during reperfusion of ischemic liver might in part be responsible for ischemic organ injury. In normothermic ischemia/reperfusion rat model, we investigated whether allopurinol pretreatment improved ischemia-induced mitochondrial dysfunction. Rats were subjected to 60 min of hepatic ischemia and to 1 h and 5 h of reperfusion thereafter. At 18 h and 1 h before ischemia, the animals received 0.25 mL of either saline or allopurinol (50 mg/kg) i.p. In saline-treated ischemic rats, serum aspartate aminotransferase levels increased significantly at 5 h (4685 +/- 310 IU/L) and were significantly reduced with allopurinol pretreatment. Similarly, mitochondrial lipid peroxidation was elevated in the saline-treated ischemic group, but this elevation was prevented by allopurinol. In contrast, mitochondrial glutamate dehydrogenase activity and ketone body ratio decreased in the saline-treated group, but this decrease was also inhibited by allopurinol. Hepatic ATP levels in the saline-treated rats were 42% lower 5 h after reperfusion. However, treatment with allopurinol resulted in significantly higher ATP levels. Allopurinol treatment preserved the concentration of AMP in ischemic liver but inhibited the accumulation of xanthine in reperfused liver. Our findings suggest allopurinol protects against mitochondrial injury, which prevents a mitochondrial oxidant stress and lipid peroxidation and preserves the hepatic energy metabolism.

Topics: Allopurinol; Animals; Aspartate Aminotransferases; Energy Metabolism; Enzyme Inhibitors; Glutamate Dehydrogenase; Ischemia; Ketones; Lipid Peroxidation; Liver; Male; Mitochondria, Liver; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Xanthine Oxidase

We investigated the effect of a hydroxyl radical scavenger, 1,3-dimethyl-2-thiourea (dimethylthiourea), and two xanthine oxidase inhibitors, oxypurinol and allopurinol, on the threshold shift of the compound action potential (CAP) after transient ischemia of the cochlea. Transient ischemia of 30 min duration was induced in albino guinea pigs via a skull base approach. The animals were treated with perilymphatic perfusion of dimethylthiourea, oxypurinol or allopurinol from 10 min before the onset of ischemia to 4 h after the termination of ischemia. Dimethylthiourea ameliorated the CAP threshold shifts at 4 h after the onset of reperfusion in a dose-dependent manner. However, oxypurinol and allopurinol did not affect the post-ischemic cochlear dysfunction. These results imply that the hydroxyl radical plays an important role in generation of cochlear dysfunction induced by ischemia-reperfusion and that xanthine oxidase may not be the primary source of this radical.

Topics: Action Potentials; Allopurinol; Animals; Cochlea; Enzyme Inhibitors; Free Radical Scavengers; Guinea Pigs; Hydroxyl Radical; Ischemia; Oxypurinol; Reperfusion Injury; Thiourea; Xanthine Oxidase

Allopurinol has been widely used to reduce the severity of the reperfusion injury. However, conflicting data have been reported regarding the dosage, the duration of the timing, and the administrative regimen of the drug. The aim of this study was, therefore, to evaluate the effects of short versus long periods of allopurinol pretreatment on the anastomotic healing of intestines, directly after being subjected to ischemia-reperfusion (IR) stress. Furthermore, the effects of an allopurinol pretreatment on the survival rate following IR stress, was also assessed. One hundred thirty-seven male Wistar rats with a median weight of 235 (range, 180-275) g used in the study. In group I (control group, N = 20) superior mesenteric artery (SMA) and collateral vessels were isolated but not occluded. In group II, the profound IR group (PIR, N = 42), the SMA was occluded immediately distal to the aorta with collateral interruption using an atraumatic arterial clip for 30 min. In group III [two days of allopurinol (ALL) pretreatment group, 2ALL, N = 38], allopurinol (100 mg/kg body wt) was given intraperitoneally on a daily basis for two days prior to the experiment. In group IV (seven days of allopurinol pretreatment group, 7ALL, N = 37), the same pretreatment and the allopurinol schedule was performed for seven days before surgery. All animals underwent 3 cm of ileal resection and primary anastomosis, 10 cm proximal to ileocecal valve. Within each group, animals were anesthetized either on the third or seventh postoperative days. Abdominal wound healing, intraabdominal adhesions, anastomotic complications, anastomotic bursting pressure measurements, and bursting site were recorded as were the histopathologic evaluation. No rats in group I, 20 rats in group II, 18 rats in group III, and 7 rats in group IV died (P = 0.0003). Anastomotic dehiscence was found in one of 20 group I, in 11 of 22 in group II, in 9 of 20 in group III, and in 3 of 30 in group IV (P = 0.0003). On the third and seventh days, the median bursting pressures of the anastomosis were determined: 42 and 235 mm Hg in group I, 17 and 105 mm in Hg in group II, 22 and 183 mm Hg in group III, and 36 and 214 mm Hg in group IV (P < 0.0001). The burst occurred at the anastomoses in all animals tested on the third postoperative day, one in group I, six in group II, four in group III and one in group IV on the seventh postoperative day (P < 0.01). All deleterious effects of reperfusion injury on intestinal anasto

Topics: Allopurinol; Anastomosis, Surgical; Animals; Disease Models, Animal; Drug Evaluation, Preclinical; Free Radical Scavengers; Ileum; Injections, Intraperitoneal; Male; Rats; Rats, Wistar; Reperfusion Injury; Tensile Strength; Time Factors; Wound Healing; Xanthine Oxidase

The complex series of pathophysiologic alterations associated with obstruction nephropathy includes renal ischemia. Free-radical production follows relief of obstruction. We sought to determine whether free radicals caused additional reduction in blood flow after relief of obstruction.. In White-Landrace pigs, the left ureter was divided 6 cm distal to the ureteropelvic junction, and a nephrostomy tube was passed into the renal pelvis and exteriorized. A catheter was placed in the renal vein and exteriorized. Mean renal blood flow was measured before manipulation and during and after 6 hours of obstruction in animals receiving (N = 7) or not receiving (N = 7) allopurinol. The control groups were not subjected to ureteral obstruction and received (N = 7) or did not receive (N = 7) allopurinol. Free radicals in venous blood were measured by the Fox-1 assay for lipid peroxidation.. After obstruction, renal blood flow declined significantly by the sixth hour (-28.73% +/- 1.81). The increase after relief of obstruction was only temporary, and by the third hour, the blood flow was again reduced (-20.14% +/- 2.67). Free radical production was significantly increased, with a peak of +24.63% being found 60 minutes after relief of obstruction. Allopurinol prevented free radical production after relief of obstruction and was associated with a return of blood flow to baseline values.. Free radicals contribute to renal blood flow reduction after relief of ureteral obstruction. Functional impairment may be preventable by free radical blockade, but further studies are required to confirm this hypothesis.

Topics: Allopurinol; Animals; Female; Free Radical Scavengers; Free Radicals; Ischemia; Renal Circulation; Renal Veins; Reperfusion Injury; Swine; Ureteral Obstruction

The generation of oxygen-derived free radicals has been suggested to be significantly responsible for ischemia-reperfusion injury in gastrointestinal tissues. Biochemical mechanisms include the xanthine-oxidase-derived oxidants mainly the superoxide anion. Both in vitro and in vivo studies have demonstrated that the pineal hormone melatonin possesses free radical scavenging and antioxidant properties. The indolamine has been effective in reducing the induced-oxidative damage in several tissues and biological systems. The aim of this study was to elucidate additional antioxidant mechanisms responsible for the gastroprotection afforded by the indolamine in ischemia-reperfusion gastric injury. Therefore, changes of related enzymes such as xanthine-oxidase, superoxide dismutase, glutathione reductase and total glutathione were investigated. Our results showed that treatment with 5, 10 or 20 mg kg(-1) of melatonin, administered i.p., clearly diminished the percentage of damage to 49.56 +/- 17.20, 37.54 +/- 11.40 and 26.70 +/- 8.12 respectively. Histologically there was a reduction of exfoliation of superficial cells and blood cell infiltration. These protective effects were related to a significant reduction of xanthine-oxidase activity (2.23 +/- 0.38 U/mg prot x 10(-4) with the highest tested dose of melatonin) and significant increases in superoxide dismutase reaching a value of 6.20 +/- 0.56 U/mg prot with 25 mg/Kg of melatonin and glutation reductase activities (417.44 +/- 29.72 and 649.43 +/- 81.11 nmol/min/mg prot with 10 and 20 mg/Kg of melatonin). We conclude that the free radical scavenger properties of melatonin mainly of the superoxide anion, probably derived via the xanthine-oxidase pathway, and the increase of antioxidative enzymes significantly contributes to mediating the protection by the hormone against ischemia-reperfusion gastric injury.

Topics: Animals; Antioxidants; Female; Free Radical Scavengers; Gastric Mucosa; Glutathione; Glutathione Reductase; Male; Melatonin; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

Human lung allografts can only be preserved for 6 hours. Experimental interventions that reduce ischemia-reperfusion (I/R) lung injury can be used to improve the properties of the preservation solution. The best solution for lung preservation is still a matter of controversy. The purpose of this study was to compare the protective effects of various solutions on I/R lung injury in Sprague-Dawley rats.. The following solutions were compared: a physiological salt solution; an intracellular preservation solution (the University of Wisconsin Solution, UW); an extracellular preservation solution (EP3); and the extracellular preservation solution with the addition of various protective agents--EP3 plus dexamethasone (Dex) (EP3-a), plus glutathione (GLU) and allopurinol (ALL) (EP3-b), and EP3 plus GLU, ALL, lactobionate (LACT), and raffinose (RAF) (EP3-c). I/R lung injury was induced by ischemia for either 45 or 60 minutes, followed by reperfusion for 60 minutes. Hemodynamic changes, lung weight gain (LWG), and capillary filtration coefficients were measured.. Both EP3 and UW preservation solutions had partial attenuation effects on I/R lung injury, but UW produced a better attenuation effect than EP3. Use of modified EP3 solutions containing either protective agents (GLU, ALL, or Dex) or impermeants (LACT and RAF) improved the ability of EP3 to reduce I/R lung injury. The LWG using the modified EP3-c solution was the lowest among all groups. UW induced pulmonary hypertension. After I/R challenge, pulmonary arterial pressure with EP3-c was lower than with UW. Based on a lower LWG and the changes in hemodynamics, EP3-c is a better lung preservation solution than UW and EP3.. Based on the attenuation of I/R injury, we conclude that there is no significant difference between intracellular UW and extracellular (EP3-a, EP3-b) preservation solutions in this rat model, but the addition of protective agents and impermeants to the solution are important. The findings suggest that EP3-c might be a better lung preservation solution than UW.

Topics: Adenosine; Allopurinol; Animals; Dextrans; Glucose; Glutathione; Hemodynamics; Heparin; Insulin; Ischemia; Lung; Lung Transplantation; Male; Organ Preservation Solutions; Organ Size; Phosphates; Prednisolone; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury

University of Wisconsin (UW) solution has been proven able to prevent liver injury during cold ischemia. During rewarming ischemia, however, the efficacy of this solution in preserving hepatocyte function is unclear. The aim of the present study was to investigate to what extent UW solution protects rat liver during rewarming ischemia.. Livers were washed out with cool physiologic saline or with UW solution and subjected to rewarming ischemia for periods of 20 min or 45 min followed by reperfusion using a blood-free perfusion model.. In comparison with controls, ischemia for 20 min in saline-treated livers led to mild depression of hepatocyte function, while UW solution afforded complete protection of the liver. In UW-treated livers, compared with saline-treated livers exposed to ischemia for 45 min, portal flow was slightly but significantly higher, bile production was increased by 62%, and lactate dehydrogenase leakage into the perfusate was reduced by 61%. In an attempt to explain mechanisms of liver protection by UW solution, we found that UW solution inhibited conversion of hypoxanthine into uric acid, but this effect was not associated with decreased degradation of adenine nucleotides in the liver during ischemia. Following 30 min reperfusion, UW solution increased tissue levels of adenosine triphosphate (not significantly) and adenosine diphosphate (significantly). Further, UW solution markedly reduced tumor necrosis factor-alpha release by the liver both after ischemia and after reperfusion.. These results create the hypothesis that UW solution may protect liver tissue during ischemia in liver surgery as well as during the implantation stage of liver transplantation.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Hot Temperature; Insulin; Liver; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury

Our objective was to evaluate graft equilibration with high viscosity (University of Wisconsin solution [UW]) or low viscosity (Bretschneider's histidine-tryptophan-ketoglutarate [HTK]) during liver procurement from non-heart beating donors (NHBD) and the potential impact of a preceding fibrinolysis with streptokinase on postpreservation viability.. After 60 min of cardiac arrest, rat livers were perfused by gravity (60 cm H2O) via the portal vein with either 60 ml of HTK, 20 ml of UW, or 20 ml of Ringer's solution (22 degrees C including 7500U of streptokinase) and, subsequently, 20 ml of UW. After 24 h of storage at 4 degrees C, viability of the livers was assessed upon isolated reperfusion in vitro.. Magnetic resonance imaging revealed severe perfusion deficits, which were mildly attenuated with HTK, upon flush-out with UW. After preflush with streptokinase, a mostly homogenous distribution of the preservation solution was observed throughout the liver tissue. The choice of the flush-out solution (UW or HTK) had no influence on parenchymal enzyme leakage, hepatic bile production, or tissue levels of ATP after reperfusion of the livers. Fibrinolytic preflush, however, resulted in a relevant and significant improvement of structural integrity as well as functional and metabolic recovery.. Compromised vascular tissue perfusion upon organ harvest in NHBD triggers graft dysfunction after cold storage and can easily be circumvented by temporary fibrinolysis before graft retrieval.

Topics: Adenosine; Allopurinol; Animals; Energy Metabolism; Fibrinolytic Agents; Glucose; Glutathione; Heart Arrest; Insulin; Liver; Magnetic Resonance Imaging; Male; Mannitol; Organ Preservation Solutions; Potassium Chloride; Preoperative Care; Procaine; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Streptokinase; Tissue and Organ Harvesting; Tissue Donors; Tissue Survival

Activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in the development of ischemia/reperfusion injury in nonhepatic organs, such as the heart. However, the role of p38 MAPK activation in the liver is unclear. We examined the effects of FR167653, a novel p38 MAPK inhibitor, as an additive to University of Wisconsin (UW) solution in rat liver transplantation.. Rat orthotopic liver transplantation was performed after 30 hr of cold storage using UW solution with or without FR167653. Ten-day survival rates, serum alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels, liver tissue blood flow, histological findings, and activities of p38 MAPK and p46/p54 c-Jun N-terminal kinase (JNK) in liver grafts were evaluated.. The addition of FR167653 significantly increased animal survival rates. FR167653 significantly suppressed serum ALT and LDH levels and improved liver tissue blood flow after transplantation. FR167653 also ameliorated histological damage to the liver graft. Neither p38 MAPK nor p46/p54 JNKs was activated during cold storage, whereas both were markedly activated within 30 min of reperfusion and remained activated until 60 min after reperfusion. FR167653 inhibited the activation of p38 MAPK both 30 and 60 min after reperfusion, but it did not affect the activation of p46/p54 JNKs.. The addition of FR167653 to UW solution improved liver graft viability and animal survival rates associated with the inhibition of p38 MAPK activation. These results suggest that inhibiting the activation of p38 MAPK may attenuate ischemia/reperfusion injury in liver transplantation.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Cryopreservation; Enzyme Inhibitors; Glutathione; Insulin; JNK Mitogen-Activated Protein Kinases; L-Lactate Dehydrogenase; Liver; Liver Circulation; Liver Transplantation; Male; Mitogen-Activated Protein Kinases; Organ Preservation Solutions; p38 Mitogen-Activated Protein Kinases; Protein Isoforms; Pyrazoles; Pyridines; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Survival Analysis

Oxygen-derived free radicals have been implicated in the pathogenesis of renal injury after ischemia-reperfusion. Caffeic acid phenethyl ester (CAPE), an active component of propolis extract, exhibits antioxidant and anti-inflammatory properties. To determine whether CAPE offers any advantage over alpha-tocopherol, we compared their effects on an in vivo model of renal ischemia-reperfusion injury in rats. CAPE at 10 micromol/kg or alpha-tocopherol at 10 mg/kg was administered intraperitoneally before reperfusion. Acute administration of CAPE suppressed ischemia-reperfusion induced renal lipid peroxidation and tissue injury more than alpha-tocopherol. CAPE may therefore offer a therapeutic advantage in acute injury settings.

Topics: alpha-Tocopherol; Animals; Antioxidants; Caffeic Acids; Female; Kidney; Phenylethyl Alcohol; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Oxidase

Functional irregularities due to damage after ischaemia-reperfusion vary depending upon the organs affected. High energy phosphates such as ATP and ADP are destroyed after ischaemia-reperfusion damage. Subsequently, protons and inorganic phosphates accumulate within the cells and the proton pumps such as adenosine triphosphatase (ATPase), which maintain intracellular ion balance are damaged. In the present study, malondialdehyde (MDA), a product of lipid peroxidation, was measured as an indicator of tissue damage. Additionally, we measured sodium-potassium-ATPase levels and determined the interactions between MDA and Na+-K+ ATPase levels. A total of 31 female guinea pigs were divided into four groups: sham operated guinea pigs (group 1), ischaemia-reperfusion (group 2), ischaemia-reperfusion + superoxide dismutase (SOD) (group 3), ischaemia-reperfusion + allopurinol (group 4). Following reperfusion, the livers of guinea pigs in each group were removed for histopathological examination and the levels of MDA and Na+-K+ ATPase were determined in homogenized tissue samples. There was a statistically significant (p < 0.05) reduction in tissue MDA levels in group 2 when compared with group 1. The level of tissue MDA in groups 3 and 4 was significantly lower than tissue MDA levels of group 2. However, there was a statistically significant (p < 0.05) reduction in tissue Na+-K+ ATPase levels of group 2 when compared with group 1. Similarly, the level of tissue Na+-K+ ATPase in groups 3 and 4 was significantly higher than the tissue Na+-K+ ATPase levels of group 2. The results of the histopathologic examination also revealed the beneficial effects of the use of SOD and allopurinol in preventing liver damage in cases of ischaemia-reperfusion. Although the levels of MDA and Na+-K+ ATP ase in group 2 were not equal to the level in group 1, antioxidant therapy significantly improved the tendency to reverse the effects of ischaemia-reperfusion and to protect the liver from damage due to ischaemia-reperfusion.

Topics: Adenosine Triphosphate; Allopurinol; Animals; Antimetabolites; Antioxidants; Disease Models, Animal; Female; Free Radical Scavengers; Guinea Pigs; Ischemia; Lipid Peroxidation; Liver; Malondialdehyde; Reactive Oxygen Species; Reperfusion Injury; Sodium-Potassium-Exchanging ATPase; Superoxide Dismutase

Topics: Administration, Oral; Allopurinol; Analysis of Variance; Animals; Antioxidants; Free Radical Scavengers; Injections, Intraperitoneal; Intestinal Mucosa; Intestine, Small; Leukocyte Count; Lipid Peroxidation; Malondialdehyde; Rats; Rats, Wistar; Regression Analysis; Reperfusion Injury; Thiobarbituric Acid Reactive Substances; Uric Acid

Ischaemia/reperfusion (I/R) lung injury using University of Wisconsin solution (UW) as perfusate has not been well studied. Isolated rat lungs were challenged with various periods of ischaemia and/or reperfusion. Haemodynamics, lung weight gain (LWG), capillary filtration coefficient (K(fc)), tissue pathology, the concentrations of cytokines in the perfusate, and mRNAs for the various cytokines in the lung tissues were measured. I/R induced a permeability type of pulmonary oedema, as reflected by increases in LWG and K(fc). LWG and K(fc) in the I(45)R(60)(UW) group (45 min of ischaemia followed by 60 min of reperfusion with UW) were only 2% and 5% respectively of those in the I(45)R(60)(NS) group (where NS is normal saline). LWG and K(fc) in the UW group had both increased by 180 min, to values similar to those in the I(45)R(60)(NS) group. However, these findings show that UW was remarkably effective at preventing LWG after 60 min of reperfusion, and was more than 3-fold more effective than NS in delaying LWG. For longer ischaemic times only, or the same period of ischaemia followed by longer reperfusion periods, greater lung injury occurred. I/R lung injury also induced increased concentrations of tumour necrosis factor-alpha (TNF-alpha), interleukin 1 and interleukin 6 in the perfusate, and increased the mRNAs for these cytokines in lung tissue. A significant correlation was obtained between TNF-alpha concentration and LWG. TNF-alpha production in the I(45)R(60)(UW) group was only 7% of that in the I(45)R(60)(NS) group. However, TNF-alpha mRNA expression in the I(45)R(60)(UW) group was 80% of that in the I(45)R(60)(NS) group. This indicates that transcription/translation do not correlate well with cytokine production, and also suggests that one reason for the effectiveness of UW in delaying LWG may be because it delays TNF-alpha production. In summary, ischaemia or I/R caused a permeability-type pulmonary oedema that was associated with leucocyte infiltration and the up-regulation of various cytokines, regardless of the perfusion fluid. Except for pulmonary hypertension, less severe I/R lung injury and delayed cytokine production in lungs perfused with UW, the pattern of injury associated with I/R challenge was similar to that in lungs perfused with NS. We propose that more or long-acting protective agents are required as additives in order to modify UW to produce an optimal preservation solution.

Topics: Adenosine; Allopurinol; Animals; Cytokines; Glutathione; Hemodynamics; Insulin; Lung; Male; Organ Preservation Solutions; Organ Size; Polymerase Chain Reaction; Pulmonary Edema; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Up-Regulation

Inhibition of protein synthesis and polyribosome disaggregation are the early events in cell injury provoked by various pathogenic mechanisms, including energy depletion. Polyribosome disaggregation might be expected to occur during ischaemia-reperfusion injury due to ischaemic energy depletion, but also due to detrimental effects of reactive oxygen species on various macromolecules and cellular structures.. Mouse kidney ischaemia-reperfusion injury was provoked by temporary clamping of the renal artery. The polyribosome sedimentation pattern was analyzed by sucrose density centrifugation of kidney postmitochondrial supernatant.. Ischaemia for 5 min in the mouse kidney provoked polyribosome disaggregation and an increase of monomer ribosome fraction which was augmented during 10-360 min of reperfusion. Recovery of polyribosome aggregates appeared between 6 and 24 h of reperfusion. Cycloheximide pretreatment prevented only polyribosome disaggregation caused by ischaemia and not that caused by reperfusion. This indicates different mechanisms of polyribosome disaggregation during ischaemia and reperfusion. It probably occurs in the former due to inhibition of initiation of translation, resulting in accumulation of unprogrammed monomer ribosomes, and in the latter due to the splitting of mRNA and breakdown of polyribosomes. Reperfusion did not increase ribonuclease activity in kidney cytosol, but increased the tissue concentration of malonaldehyde, indicating an augmentation in oxygen free radical generation. Possibly these may have caused a non-enzymatic breakdown of polyribosomes. However, pretreatment with allopurinol did not prevent polyribosome breakdown during ischaemia-reperfusion injury.

Topics: Allopurinol; Animals; Free Radical Scavengers; Free Radicals; Kidney Diseases; Male; Mice; Polyribosomes; Reperfusion Injury

A recent clinical study demonstrated that in renal allografts preserved in the cold apoptosis occurred soon after reperfusion. The mode of cell death during cold storage is generally considered necrotic. Whether apoptosis occurs as a part of cold storage is uncertain. The objective was to determine in human renal tubular cells whether apoptosis is specific for rewarming or it also occurs during cold storage and whether it could be modified.. Cold storage (4 degrees C) of primary human renal proximal tubular epithelial (RPTE) in University of Wisconsin (UW) solution up to 48 hr caused a time-dependent increase in cell death measured by lactic dehydrogenase (LDH) release and vital dye exclusion methods. Transmission electron microscopy (TEM) demonstrated that cell death in the cold was necrotic, involving considerable mitochondrial disruption, and was not apoptotic. The TUNEL assay that provides a specific, quantitative measure for apoptosis showed no increase in TUNEL-positivity during flow cytometry of cells stored in cold: 37 degrees C, 0.23+/-0.14%; 24 hr cold, 0.23+/-0.1%; 48 hr cold, 1.79+/-0.58%. Annexin-V staining, a sensitive method for detecting early apoptosis, similarly showed no increase in positively stained cells during cold storage. Addition of antioxidants 2-methyl aminochroman and deferoxamine to UW solution inhibited necrotic cell death and preserved mitochondrial structure. In contrast to cold storage alone, rewarming (37 degrees C for 24 hr) of cold stored cells, however, resulted in significant apoptosis (TUNEL positive: 48 hr cold: 2+/-0.6%, 48 hr cold and 24 hr rewarming: 54+/-17%), which was confirmed by the TEM based on typical apoptotic features. Addition of 2-MAC and DFO significantly inhibited rewarming-induced apoptotic cell death (plus 2-MAC: 3+/-1%, plus DFO: 3+/-2%).. Our study in human tubular cells provides evidence that cold storage per se does not result in apoptosis, but is primarily necrotic. However, rewarming is associated with significant apoptosis in the presence of ongoing necrosis, speculatively due to the activation of the apoptotic enzymic process of sublethally injured cells. Inclusion of antioxidants in the storage solution confers protection against both cold storage and rewarming-induced necrosis and apoptosis.

Topics: Adenosine; Allopurinol; Annexin A5; Apoptosis; Cells, Cultured; Cold Temperature; Epithelial Cells; Glutathione; Hot Temperature; Humans; In Situ Nick-End Labeling; In Vitro Techniques; Insulin; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Microscopy, Electron; Necrosis; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Tissue Preservation; Trypan Blue

Oxygen-derived free radicals have been implicated in the pathogenesis of renal injury after ischaemia-reperfusion. Caffeic acid phenethyl ester (CAPE), an active component of propolis extract, exhibits antioxidant properties. To investigate whether treatment with either CAPE or alpha-tocopherol modifies the levels of the endogenous indices of oxidant stress, we examined their effects on an in vivo model of renal ischaemia-reperfusion injury in rats. CAPE at 10 micromol kg(-1) or alpha-tocopherol at 10 mg kg(-1) was administered intraperitoneally before reperfusion. Acute administration of both CAPE and alpha-tocopherol altered the indices of oxidative stress differently in renal ischaemia-reperfusion injury.

Topics: Adenosine Deaminase; alpha-Tocopherol; Animals; Antioxidants; Caffeic Acids; Female; Kidney; Malondialdehyde; Oxidative Stress; Phenylethyl Alcohol; Rats; Reperfusion Injury; Thiobarbituric Acid Reactive Substances; Xanthine Oxidase

Hypoventilation, associated with hypercapnic acidosis (HCA), may improve outcome in acute lung injury (ALI). We have recently reported that HCA per se protects against ALI. The current study explored whether the mechanisms of protection with HCA were related to acidosis versus hypercapnia. Because CO(2) equilibrates rapidly across cell membranes, we hypothesized that (1) HCA would afford greater protection than metabolic acidosis. We further hypothesized that (2) buffering HCA would attenuate its protection. Forty isolated perfused rabbit lung preparations were randomized to: control (normal pH, PCO(2)); HCA; metabolic acidosis; or buffered hypercapnia. After ischemia-reperfusion (IR) injury wet:dry ratio was greatest with control and buffered hypercapnia, and rank order of capillary filtration coefficient was: control approximately buffered hypercapnia > metabolic acidosis > HCA. Isogravimetric pressure reduction was greatest with buffered hypercapnia. Despite comparable injury, pulmonary artery pressure elevation was less with buffered hypercapnia versus control. In vitro xanthine oxidase (XO) activity depended on pH, not PCO(2). We conclude that: (1) HCA and metabolic acidosis are protective, but HCA is the most protective; (2) buffering HCA attenuates its protection; (3) buffering HCA causes pulmonary vasodilation; (4) because metabolic acidosis and HCA similarly inhibit in vitro XO activity, the differential effects cannot be explained solely on the basis of extracellular XO activity.

Topics: Acidosis, Respiratory; Animals; Blood Gas Analysis; Capillary Permeability; Disease Models, Animal; Hydrogen-Ion Concentration; Hypercapnia; Hyperventilation; In Vitro Techniques; Lung; Male; Rabbits; Reperfusion Injury; Respiratory Distress Syndrome; Vasodilation; Xanthine Oxidase

The relative contribution of xanthine oxidase (XO) and leukocytes to tissue injury after short-term ischemia is unknown. In this study, we subjected three groups of rat spinotrapezius muscles to 30-min ischemia and 1-h reperfusion: 1) ischemia-reperfusion (I/R) + 0.9% saline, 2) I/R + superoxide dismutase, and 3) I/R + oxypurinol. A fourth group served as nonischemic control. We quantified the increase in resistance (%DeltaR) caused by leukocyte-capillary plugging concurrently with myocyte uptake of propidium iodide (PI) [expressed as no. of PI spots per total volume of perfused tissue (N(PI)/V)] and performed assays to quantify XO activity, thiobarbituric acid-reactive substances (TBARS), and myeloperoxidase (MPO). Groups 2 and 3 exhibited significant decreases in N(PI)/V relative to group 1. MPO levels and TBARS were similar among all groups, and mean %DeltaR was significantly reduced in groups 2 and 3 relative to group 1. However, elevated XO was observed in groups 1 and 2 relative to group 3 and nonischemic controls. These data are consistent with the hypothesis that XO, rather than toxic species produced by plugging or venule-adherent leukocytes, is responsible for postischemic damage in this model.

Topics: Animals; Capillaries; Cell Adhesion; Cell Movement; Female; Ischemia; Leukocytes; Lipid Peroxides; Muscle, Skeletal; Oxidants; Oxypurinol; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sodium Chloride; Superoxide Dismutase; Time Factors; Vascular Resistance; Xanthine Oxidase

In clinical transplantation, exposure of donors to gut-derived endotoxin occurs frequently and may adversely affect liver transplantation therapy. The aim of this study was to investigate: 1) whether brief exposure of rats to endotoxin before liver procurement aggravates the early phase of reperfusion injury of hepatic explants; and if so 2) whether Kupffer cell activation is a contributing factor to liver injury; and 3) whether heparin and pentoxifylline could minimize this effect.. Male Wistar rats were injected with 0.2-4.0 mg/kg of Escherichia coli lipopolysaccharide 2 h prior to liver harvest. After preservation in University of Wisconsin cold-storage solution, the livers were reperfused using a blood-free perfusion model. To inactivate Kupffer cells, some rats were pretreated with gadolinium chloride or liposome-encapsulated dichloromethylene-diphosphonate before lipopolysaccharide administration. The other rats received lipopolysaccharide with heparin or pentoxifylline.. In a dose-independent fashion, lipopolysaccharide impaired portal flow during graft reperfusion. In a dose-dependent way, lipopolysaccharide increased lactate dehydrogenase release into the perfusate and decreased bile flow and bromosulfophthalein excretion. Gadolinium chloride, liposomal dichloromethylene-diphosphonate, heparin, and pentoxifylline reduced lactate dehydrogenase release by 34%, 43%, 59%, and 64%, respectively, and improved functional parameters of the liver. A 52-fold increased neutrophil infiltration in the liver sinusoids after lipopolysaccharide exposure was not affected significantly by the drugs studied; however, heparin reduced markedly neutrophil activation.. The results of this investigation provide direct evidence that aggravation of preservation-reperfusion injury of rat liver by endotoxin is mediated by Kupffer cell-dependent mechanism(s) and it can be minimized by heparin and pentoxifylline.

Topics: Adenosine; Allopurinol; Animals; Bile; Clodronic Acid; Cryopreservation; Dose-Response Relationship, Drug; Escherichia coli; Glutathione; Heparin; Insulin; Kupffer Cells; L-Lactate Dehydrogenase; Lipopolysaccharides; Liver; Male; Neutrophils; Organ Preservation; Organ Preservation Solutions; Pentoxifylline; Perfusion; Raffinose; Rats; Rats, Wistar; Reperfusion Injury

To evaluate the putative relation of platelet activating factor (PAF), xanthine oxidase, reactive oxidants, and leukocytes in the pathogenesis of hepatic injury after shock/resuscitation (S/R) in vivo.. Reactive oxygen metabolites generated by xanthine oxidase at reperfusion have been found to trigger postischemic injury in many organs, including the liver. However, the precise linear sequence of the mechanism of consequent hepatic injury after S/R remains to be characterized.. Unheparinized male rats were bled to a mean blood pressure of 45 +/- 3 mmHg. After 2 hours of shock, they were resuscitated by reinfusion of shed blood (anticoagulated with citrate-phosphate-dextrose) and crystalloid and observed for the next 6 or 24 hours.. S/R caused the oxidation of hepatic glutathione and generated centrolobular leukocyte accumulation at 6 hours, followed by predominantly centrolobular hepatocellular injury at 24 hours. Each of these components was attenuated by PAF inhibition with WEB 2170, xanthine oxidase inhibition with allopurinol, antioxidant treatment with N-acetylcysteine, or severe leukopenia induced by vinblastine. In each case, the degree of leukocyte accumulation at 6 hours correlated with the hepatocellular injury seen at 24 hours. However, xanthine oxidase inhibition with allopurinol failed to attenuate further the small level of residual hepatocellular injury seen in leukopenic rats.. These findings suggest that reactive oxidants generated by xanthine oxidase at reperfusion, stimulated by PAF, mediate hepatocellular injury by triggering leukocyte accumulation, primarily within the centrolobular sinusoids.

Topics: Animals; Disease Models, Animal; Drug Interactions; Leukocytes; Liver; Male; Oxidative Stress; Platelet Activating Factor; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion; Reperfusion Injury; Resuscitation; Shock, Hemorrhagic; Time Factors; Xanthine Oxidase

Acute lung reperfusion injury (ALI) frequently follows an ischemic event in another organ, such as organ transplantation. We recently demonstrated that lung priming with N-acetyl-L-cysteine (NAC) prevented liver ischemia-reperfusion (IR)-induced ALI pending on reduced glutathione (GSH) amount of replenishment. We now assessed the therapeutic effect of NAC-in preventing ALI caused by liver IR-if administered to the lung during liver reperfusion.. Rat isolated livers were stabilized (30 min) and then perfused with modified Krebs-Henseleit solution (control, n=20) or made globally ischemic (IR, n=20) for 2 hr. Rat lungs were isolated separately, ventilated, and stabilized (30 min) with Krebs plus 5% bovine albumin. Pairs of liver and lung were then reperfused together for 15 min, followed by only lung recirculation with the liver effluent for another 45 min. Three more controls (n=20 each) and three ischemic groups (n=20 each) included lungs which were treated with 100, 150 or 225 mg x kg(-1) NAC (0.5, 0.74, or 1.1 mmol, respectively) during the 15-min liver and lung reperfusion period.. Pulmonary artery and ventilatory pressures and vascular resistance increased by 60-80% of baseline, compliance decreased, and bronchoalveolar lavage volume and content were abnormally high in the IR-nontreated and the IR-100 lungs. Most parameters in IR-150 and IR-225 lungs remained almost similar to controls. Postinsult GSH content in IR-100, -150, and -225 lungs was at 20%, 110%, and 90% above the IR-nontreated lungs, respectively.. Lung treatment with NAC during its reperfusion with IR liver effluent prevented ALI. Lung GSH replenishment accounted for lung protection, but its content did not correlate directly with grade of protection; NAC itself seemingly afforded lung protection as well.

Topics: Acetylcysteine; Animals; Dose-Response Relationship, Drug; Free Radical Scavengers; Glutathione; Ischemia; Liver; Liver Circulation; Lung; Lung Diseases; Male; Perfusion; Pulmonary Circulation; Rats; Rats, Wistar; Reperfusion Injury; Respiration; Xanthine Oxidase

Hepatocyte membrane potential differences (PDs) may be altered by the preservation solutions used in liver transplantation. Such alterations could impact on the survival of the donor liver, extent of biochemical injury, and flux of important ionic compounds. The purpose of the present study was to document these outcomes in the presence of four different preservation solutions.. Livers of adult male Sprague-Dawley rats (N = 3 to 4 per group) were impaled with intracellular microelectrodes prior to and at various time periods for 6 hours following complete hepatic resection. Just prior to resection, each liver was perfused with preservation solutions associated with high (normal saline [NS]), moderate (Euro-Collins [EC]), and low (University of Wisconsin solution [UW]) risks of reperfusion injury.. Baseline (in situ) PDs were similar in all groups (-37 +/- 4 mV, mean +/- SD). Ten minutes postresection, hepatic PDs were as follows: NS, -23.8 +/- 3.5 mV; EC, -11.4 +/- 0.4 mV; and UW, -8.7 +/- 0.3 mV (P <0.01 for all groups). Maximum depolarization occurred at 6 hours postresection (NS, -8.1 +/- 1.1 mV; EC, -7.7 +/- 1.3 mV; and UW, -8.6 +/- 1.0 mV). To determine whether these changes are of pathophysiologic importance, the NS solution was modified (addition of 0.1% ethanol) to achieve similar PD changes as those observed with UW. Liver transplants were then performed where the donor livers had been perfused and preserved for 6 hours with either NS or the modified NS (MNS) solution. Posttransplant (10 day) survival was 1 of 6 (17%) in the NS group and 4 of 6 (67%) in the MNS group (P <0.05). Regarding the effects of PD changes on ionic flux, intracellular calcium levels were documented for up to 4 hours by fluorescence video microscopy using Fura-2 in isolated hepatocytes exposed to NS, UW, and MNS solutions. Intracellular calcium levels were similar in all solutions at each time point studied.. The results of this study indicate that hepatocytes undergo prompt and marked depolarization following hepatic resection, and the extent of the depolarization correlates with survival following transplantation.

Topics: Adenosine; Allopurinol; Animals; Calcium; Calcium Signaling; Ethanol; Fluorescent Dyes; Fura-2; Glutathione; Graft Survival; Hepatectomy; Hypertonic Solutions; Insulin; Liver; Liver Transplantation; Male; Membrane Potentials; Microscopy, Fluorescence; Microscopy, Video; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sodium Chloride; Statistics, Nonparametric; Treatment Outcome

We here show that the novel N-hydroxyguanidine derivative PR5 (1-(3, 4-dimethoxy-2-chlorobenzylideneamino)-3-hydroxyguanidine) is acting as an alternative electron acceptor in xanthine oxidase catalyzed oxidation of xanthine. The reduction product is the corresponding guanidine derivative 1-(3, 4-dimethoxy-2-chlorobenzylideneamino)guanidine (PR9). The reaction occurs under both anaerobic and aerobic conditions. Moreover, EPR measurements show that the action of PR5 is associated with the inhibition of superoxide radical formation seen under aerobic conditions. PR5 also supports xanthine oxidase catalyzed anaerobic oxidation of NADH. Kinetic studies indicate that increasing xanthine concentration significantly increases the apparent K(m) of PR5, but it remains unaltered by changing NADH concentration. Moreover, the molybdenum center inhibitor allopurinol inhibits the PR5-sustained oxidation of xanthine and NADH equally well, whereas the flavin adenine dinucleotide site inhibitor diphenyliodonium (DPI) markedly inhibits only the PR5-sustained oxidation of NADH. We suggest that PR5 binds and becomes reduced at the molybdenum center of the xanthine oxidase. We also found that both PR5 and its reduction product PR9 can inhibit the oxygen-sustained xanthine oxidase reaction. The properties of PR5 suggest that it is a member of a novel class of compounds which we have termed xanthine oxidase electron acceptor-inhibitor drugs. The potential use of xanthine oxidase electron acceptor-inhibitors in the prevention of free radical mediated tissue damage in organ ischemia-reperfusion diseases is discussed.

Topics: Allopurinol; Animals; Binding Sites; Catalysis; Cattle; Chromatography, High Pressure Liquid; Electron Spin Resonance Spectroscopy; Guanidines; Hydroxylamines; Inhibitory Concentration 50; Kinetics; Milk; Models, Chemical; Molybdenum; NAD; Onium Compounds; Oxidation-Reduction; Oxygen; Reperfusion Injury; Superoxides; Uric Acid; Xanthine; Xanthine Oxidase

We describe the synthesis and biological applications of a novel nitrogen-15-labeled nitrone spin trap, 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide ([(15)N]EMPO) for detecting superoxide anion. Superoxide anion generated in xanthine/xanthine oxidase (100 nM min(-1)) and NADPH/calcium-calmodulin/nitric oxide synthase systems was readily detected using EMPO, a nitrone analog of 5,5'-dimethyl-1-pyrroline N-oxide (DMPO). Unlike DMPO-superoxide adduct (DMPO-OOH), the superoxide adduct of EMPO (EMPO-OOH) does not spontaneously decay to the corresponding hydroxyl adduct, making spectral interpretation less confounding. Although the superoxide adduct of 5-(diethoxyphosphoryl)-5-methyl-pyrroline N-oxide is more persistent than EMPO-OOH, the electron spin resonance spectra of [(14)N]EMPO-OOH and [(15)N]EMPO-OOH are less complex and easier to interpret. Potential uses of [(15)N]EMPO in elucidating the mechanism of superoxide formation from nitric oxide synthases, and in ischemia/reperfusion injury are discussed.

Topics: Calmodulin; Citrulline; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Half-Life; Hydroxyl Radical; Kinetics; NADP; Nitric Oxide Synthase; Nitrogen Isotopes; Nitrogen Oxides; Reperfusion Injury; Sensitivity and Specificity; Spin Labels; Spin Trapping; Superoxides; Xanthine; Xanthine Oxidase

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Chemokine CCL2; Glutathione; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; HSP70 Heat-Shock Proteins; Insulin; Kidney; Organ Preservation Solutions; Raffinose; Rats; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction

Intestinal ischemia/reperfusion provokes a local inflammatory response leading to a systemic inflammatory state. In this study we aimed to assess the effects of intestinal ischemia/reperfusion injury on anastomotic healing in the left colon with an intact vascular supply. A total of 94 Wistar albino rats were divided into three groups: sham-operated control (group I, n = 25), 30 minutes of intestinal ischemia/reperfusion (group II, n = 40), and 7-day allopurinol pretreatment and intestinal ischemia/reperfusion (group III, n = 29). After the reperfusion experiment, a segmental left colon resection and anastomosis were done. On postoperative days 3 and 7 anastomotic bursting pressure, anastomotic and operative complications, and intraabdominal adhesions were assessed. Mortality rates were 1/25, 16/40, and 4/29 for groups I, II, and III, respectively (p = 0.001). There was no difference among the groups for wound and anastomotic healing parameters evaluated by macroscopic criteria. On postoperative day 7 the mean bursting pressures were 220.3 +/- 18.5, 162.0 +/- 21.0, and 213.9 +/- 24.7 for groups I, II, and II, respectively (p = 0.000). Significantly dense adhesions were found in group II (p = 0.000). Allopurinol pretreatment prevented the effects of ischemia/reperfusion on anastomotic healing of the left colon. Intestinal/ischemia reperfusion causes impairment of anastomotic healing of the left colon. In addition to remote organ effects, reperfusion injury may affect anastomotic healing in the viscera with an intact vascular supply.

Topics: Allopurinol; Anastomosis, Surgical; Animals; Colon; Colonic Diseases; Female; Free Radical Scavengers; Postoperative Complications; Rats; Rats, Wistar; Reperfusion Injury; Tissue Adhesions; Wound Healing

Ischemia followed by reperfusion (I/R) induces gastric lesions, probably due to excessive formation of free radicals, but the role of the scavenger of these radicals, proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), in the healing of these lesions has not been extensively studied. It is also unknown whether expression of intercellular adhesion molecule-1 (ICAM-1), which mediates neutrophil-induced injury and neutrophil infiltration, is involved in the recovery from I/R lesions.. I/R lesions were induced in Wistar rats by applying a small clamp to the celiac artery for 30 min (ischemia phase), followed by the removal of the clamp for 60 min (reperfusion phase). The influence of I/R on gastric secretion was also tested in rats equipped with a gastric fistula (GF) without or with the exposure to a standard period of I/R. Two series of rats (A and B) were used to determine the effects of exogenous and endogenous superoxide dismutase SOD (series A) and allopurinol, a xanthine oxidase inhibitor (series B), on the mucosal recovery from the gastric lesions induced by I/R. The animals were killed immediately after the exposure to I/R (0 h) and at 3 h, 24 h, or 3, 5, or 10 days after this I/R, the area of gastric lesions being measured by planimetry, and the gastric blood flow (GBF) determined by the H2 gas clearance method. Blood was withdrawn for measurement of plasma IL-1beta and TNF-alpha levels with enzyme-linked immunosorbent assay, and plasma gastrin with radioimmunoassay. Biopsy samples of oxyntic mucosa were taken for the assessment of SOD, IL-1beta, TNF-alpha, and ICAM-1 mRNAs by reverse-transcription polymerase chain reaction and Southern blot.. Exposure to I/R resulted in acute gastric erosions, with the maximal increase of the area of these lesions observed 3 h after the end of I/R. This effect was accompanied by a decrease in the GBF, a significant increase in blood free radicals and plasma gastrin increments, and almost complete suppression of gastric secretion. Starting 24 h after I/R, the gastric superficial lesions progressed into deeper ulcers that healed progressively within 10 days, and this was accompanied by gradual restoration of the gastric secretion and the GBF. Treatment with SOD and allopurinol accelerated significantly the healing of I/R erosions, and this effect was accompanied by a significant increase in the GBF and the attenuation of blood free radicals. At 0, 3, and 12 h after I/R a significant decrease in SOD mRNA was observed, whereas expression of TNF-alpha, IL-1beta, and ICAM-1 showed a progressive increase starting immediately after I/R, reaching a maximum on day 3. The plasma level of TNF-alpha and IL-1beta started to increase on day 3 and peaked on day 5 after I/R, being still significantly higher at day 10 than that measured in the vehicle-treated control gastric mucosa. On day 10 the gastric ulcers were almost completely healed, and a decrease in the expression for TNF-alpha, IL-1beta, and ICAM-1 mRNA and an increase in the expression of SOD mRNA were observed.. 1) exposure to I/R produces gastric lesions mediated by the excessive formation of free radicals, resulting in suppression of both gastric microcirculation and secretory activity of the stomach; 2) SOD and allopurinol accelerate the healing of I/R lesions, probably due to suppression of oxygen free radicals and improvement of gastric microcirculation; and 3) the upregulation of SOD mRNA, with subsequent increase in the SOD production and local release of IL-1beta and TNF-alpha, may activate ICAM-1 expression and neutrophil infiltration, which appear to play an important role in the progression of I/R-induced acute gastric erosions into chronic ulcers.

Topics: Allopurinol; Animals; Cytokines; Free Radical Scavengers; Free Radicals; Gastric Juice; Gastric Mucosa; Gene Expression Regulation; Intercellular Adhesion Molecule-1; Interleukin-1; Male; Rats; Rats, Wistar; Reperfusion Injury; Stomach Ulcer; Superoxide Dismutase; Tumor Necrosis Factor-alpha

Although postischemic cardiac or pulmonary dysfunction can relate to the impact of remotely generated oxygen stress mediators on the heart, their direct effect on the vascular bed remains unresolved. Thus, we tested these remote effects in an ex-vivo double organ model.. After stabilization With Krebs-Henseleit solution, isolated rat livers were either perfused or made ischemic for 2 hours. Aortic rings were stabilized, immersed in postischemic liver perfusates and their functions were tested. Some organs originated from donors fed with tungstate, whereas others had mannitol (0.25 g/kg) in the buffer.. Incubation of aortic rings with postischemic hepatic effluent resulted in protracted contraction. Spasm was slightly lesser when the livers were pretreated with tungstate or exposed to mannitol, but worse in pretreated rings. The return to basal tone was abrupt in all ischemia-reperfusion aortae. The response of the rings to phenylephrine under the influence of the ischemia-reperfusion hepatic effluent was deficient. Mannitol prevented most abnormal responses.. Aortal tone impairment can occur by direct influence of the ischemia-reperfusion liver. It cannot be attributed entirely to xanthine oxidase, but also to other hepatic-released factors.

Topics: Animals; Aorta; In Vitro Techniques; Liver; Male; Mannitol; Muscle Tonus; Phenylephrine; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Tungsten Compounds; Xanthine Oxidase

Reperfusion of tissues after interruption of their vascular supply causes free-radical generation that leads to tissue damage, a scenario referred to as "reperfusion injury." Because sickle disease involves repeated transient ischemic episodes, we sought evidence for excessive free-radical generation in sickle transgenic mice. Compared with normal mice, sickle mice at ambient air had a higher ethane excretion (marker of lipid peroxidation) and greater conversion of salicylic acid to 2,3-dihydroxybenzoic acid (marker of hydroxyl radical generation). During hypoxia (11% O(2)), only sickle mice converted tissue xanthine dehydrogenase to oxidase. Only the sickle mice exhibited a further increase in ethane excretion during restitution of normal oxygen tension after 2 hours of hypoxia. Only the sickle mice showed abnormal activation of nuclear factor-kappaB after exposure to hypoxia-reoxygenation. Allopurinol, a potential therapeutic agent, decreased ethane excretion in the sickle mice. Thus, sickle transgenic mice exhibit biochemical footprints consistent with excessive free-radical generation even at ambient air and following a transient induction of enhanced sickling. We suggest that reperfusion injury physiology may contribute to the evolution of the chronic organ damage characteristic of sickle cell disease. If so, novel therapeutic approaches might be of value.

Topics: Allopurinol; Anemia, Sickle Cell; Animals; Biomarkers; Enzyme Inhibitors; Ethane; Hemoglobin, Sickle; Humans; Hydroxybenzoates; Hydroxyl Radical; Kidney; Lipid Peroxidation; Liver; Mice; Mice, Inbred C57BL; Mice, Transgenic; NF-kappa B; Reference Values; Reperfusion Injury; Salicylic Acid; Xanthine Dehydrogenase; Xanthine Oxidase

A brief period of liver ischemia decreases sinusoidal endothelial cell killing after cold liver storage and improves graft survival after liver transplantation, a phenomenon called ischemic preconditioning. In this study, we investigated the mechanism of sinusoidal endothelial cell protection after ischemic preconditioning. Livers were preconditioned by 5 minutes of ischemia and 5 minutes of reperfusion. Subsequently, livers were stored for 30 hours in cold University of Wisconsin (UW) solution and reperfused briefly with physiological buffer containing Trypan blue. Ischemic preconditioning decreased sinusoidal endothelial cell killing after storage/reperfusion, as assessed by Trypan blue staining of nonparenchymal cells. Adenosine A(2) receptor blockade prevented the protective effect of ischemic preconditioning. By contrast, adenosine A(1) receptor blockade did not prevent protective ischemic preconditioning. Other rat livers were treated with adenosine A(1) and A(2) receptor agonists or dibutyryl-cyclic adenosine monophosphate (DB-cAMP) before storage. The adenosine A(2) receptor agonist, CGS-21680, and DB-cAMP decreased sinusoidal endothelial cell killing to the same extent as ischemic preconditioning, but the adenosine A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA), had no effect. The adenosine A(2) agonist and prostaglandin E(2), another agent that preconditions sinusoidal endothelial cells against storage/reperfusion injury, but not the adenosine A(1) agonist, increased cAMP levels in cultured sinusoidal endothelial cells. In conclusion, an adenosine A(2) receptor pathway coupled to increased cAMP mediates sinusoidal endothelial cell protection by ischemic preconditioning.

Topics: Adenosine; Allopurinol; Animals; Cyclic AMP; Endothelium, Vascular; Glutathione; Insulin; Ischemia; Ischemic Preconditioning; Liver; Male; Organ Preservation Solutions; Purinergic P1 Receptor Agonists; Raffinose; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Reperfusion Injury

Delayed graft function (DGF) has remained an important complication after renal transplantation. The exact causes of DGF remain to be clarified, particularly the impact of retrieval conditions and preservation factors. In the present investigation, (1)HNMR spectroscopy of urine was assessed in order to detect the influence of retrieval condition on renal medulla damage.. The isolated perfused pig kidney (IPK) was used to assess initial renal function from multiorgan donors (MOD) or single organ donors (SOD) after in situ cold flush and 24-h cold storage (CS) preservation with two standard preservation solutions: Euro-Collins (EC) and University of Wisconsin (UW) solutions. Kidneys flushed with cold heparinized saline and immediately perfused were used as the control group. Kidneys were perfused for 90 min at 37.5 degrees C for functional evaluation. During reperfusion, renal perfusion flow rate (PF) was measured. Glomerular filtration rate (GFR), tubular reabsorption of Na(+), and lactate dehydrogenase (LDH) and N-acetyl-beta-d-glucosaminidase (NAG) excretions were determined. Ischemia-reperfusion impairment was also determined by histological techniques and (1)HNMR spectroscopy.. PF, GFR, and tubular reabsorption of Na(+) were significantly decreased in experimental groups when compared to the control group but there was no significant difference between experimental SOD groups. GFR was significantly greater in UW-MOD than in EC-MOD and tubular reabsorption of Na(+) was significantly greater in UW-MOD than in EC-MOD after 45 min of reperfusion. The release of LDH in the effluent and the urinary excretion of NAG were not significantly different after 24-h CS in the various experimental groups. The most relevant resonances determined by (1)HNMR spectroscopy were citrate, trimethylamine-N-oxide, lactate, acetate, and amino acids. Excretion of these markers was significantly different when compared to biochemical markers. A resonance (P) detected particularly in EC-MOD after 24-h CS was identified and well correlated to renal dysfunction. Histological study showed that ultrastructural damage and mitochondrial injury were more pronounced in the EC-MOD group.. These results show that retrieval condition influences renal medullary damage. NMR spectroscopy, which is a noninvasive and nondestructive technique, is more efficient in assessing renal damage than conventional histology and biochemical analysis.

Topics: Acetylglucosaminidase; Adenosine; Allopurinol; Animals; Glutathione; Insulin; Kidney Medulla; Kidney Transplantation; L-Lactate Dehydrogenase; Magnetic Resonance Spectroscopy; Male; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Reperfusion Injury; Swine; Tissue Donors

Ischemia-reperfusion injury involves free radical production, polymorphonuclear neutrophil chemotaxis/degranulation, and production of proteolytic enzymes, complement components, coagulation factors, and cytokines. Activated polymorphonuclear neutrophils, endothelial cells, and macrophages produce platelet activating factor, which further promotes these inflammatory reactions. The recently cloned plasma form of platelet activating factor-acetylhydrolase (PAF-AH) demonstrates antiinflammatory effects by degrading platelet activating factor. We evaluated the effects of PAF-AH in an isolated perfused rat lung model by adding it to the flush solutions or to the reperfusion blood.. Rat lungs were isolated, flushed with EuroCollins (EC) or University of Wisconsin (UW) solution, stored at 4 degrees C for 6 or 12 hours, and reperfused using a cross-circulating syngeneic support rat. During reperfusion, oxygenation, compliance, and capillary filtration coefficient were calculated. There were four groups in the study; group I (control) had no PAF-AH added, group II had PAF-AH added to the flush solution, group III had PAF-AH added to reperfusion blood, and group IV had PAF-AH added to both flush solution and reperfusion blood.. After 6 hours of storage, oxygenation, compliance, and capillary filtration coefficient significantly improved for EC in group IV. For UW, oxygenation improved in group IV whereas compliance improved in groups II, III, and IV. After 12 hours of storage, compliance improved for EC in group IV and capillary filtration coefficient improved in groups III and IV. For UW, oxygenation and compliance improved in groups II and IV, whereas capillary filtration coefficient improved in group IV.. Addition of PAF-AH to intracellular organ preservation solutions and to the blood reperfusate significantly improves postreperfusion oxygenation and compliance, and reduces lung capillary permeability.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Adenosine; Allopurinol; Animals; Glutathione; Hypertonic Solutions; In Vitro Techniques; Insulin; Lung; Male; Organ Preservation Solutions; Oxidative Stress; Phospholipases A; Platelet Activating Factor; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

Topics: Adenosine; Allopurinol; Blood Pressure; Glutathione; Hemodynamics; Humans; Insulin; Intestines; Lactates; Mannitol; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Short Bowel Syndrome; Transplantation, Homologous

Some studies have shown that postischemic hepatic dysfunction is mainly due to oxygen free radicals that are generated by xanthine oxidase. The present study was undertaken to determine the effect of allopurinol, an inhibitor of xanthine oxidase, on oxidative stress, liver injury and histologic alterations induced by hepatic ischemia-reperfusion in rats.. One hundred and sixty Wistar rats were used and divided into three groups. Group 1: sham operation; group 2: 50 min of ischemia followed by 1 h of reperfusion, and group 3: pretreatment with allopurinol and 50 min of ischemia followed by 1 h of reperfusion. The effect of allopurinol was evaluated by plasma levels of alanine aminotransferase and aspartate aminotransferase, histopathologic studies, and lipid peroxidation measured by the thiobarbituric acid reactive substances method and chemiluminescence initiated by tert-butyl hydroperoxide technique.. Ischemia followed by reperfusion promoted an increase in lipid peroxidation of the hepatic cells when compared to the sham-operated group (p<0.05). This increase was attenuated in the group treated with allopurinol (p< 0.05). Allopurinol also showed a protective effect on hepatocellular necrosis (p<0.05), and the plasma levels of liver enzymes returned earlier to the normal range in rats pretreated with allopurinol in comparison to those that did not receive the drug (p<0.05).. Allopurinol exerted a protective effect on hepatic ischemia and reperfusion in rats. The administration of this drug prior to liver operations should be considered to be submitted to trials in humans.

Topics: Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Enzyme Inhibitors; Ischemia; Lipid Peroxides; Liver; Liver Circulation; Male; Rats; Rats, Wistar; Reperfusion Injury; Time Factors

Reperfusion injury with pulmonary edema continues to be a major complication after lung transplantation. Alveolar fluid homeostasis is regulated by Na+/K+-ATPase activity on the basolateral surface of alveolar epithelial cells. Intact Na+/K+-ATPase is essential to the resolution of pulmonary edema. We characterized the effects of cold ischemia and reperfusion on expression of Na+/K+-ATPase mRNA and protein.. Baseline values for Na+/K+-ATPase mRNA and protein were determined from freshly harvested lungs with no cold storage time or reperfusion (group I). Group II lungs were analyzed after cold storage times of 12 or 24 hr without subsequent reperfusion. Group III lungs were analyzed after cold storage times of 12 or 24 hr with subsequent reperfusion. Lungs were flushed with either Euro-Collins (EC) or University of Wisconsin (UW) solution in each group. All samples were quantified for Na+/K+-ATPase mRNA and Na+/K+-ATPase protein. Physiological parameters including oxygenation and compliance were also measured.. There were no significant differences in the level of mRNA and protein for samples that were cold stored without reperfusion (group II). With reperfusion (group III) there was a significant increase in the level of the Na+/K+-ATPase mRNA after 12 hr of storage for both EC and UW. After 24 hr of storage and subsequent reperfusion, lungs flushed with EC had significantly decreased Na+/K+-ATPase protein and mRNA, although lungs preserved with UW maintained their increased levels of Na+/K+-ATPase protein and mRNA.. Our data suggest that ischemia-reperfusion injury results in an initial up-regulation of Na+/K+-ATPase mRNA. With prolonged injury in lungs preserved with EC, the level of the mRNA decreased with a corresponding decrease in the Na+/K+-ATPase protein. The different response seen in EC versus UW may be explained by better preservation of pump function with UW than EC and correlates with improved physiological function in lungs preserved with UW solution.

Topics: Adenosine; Allopurinol; Animals; Blotting, Western; Cryopreservation; Gene Expression; Glutathione; Insulin; Lung; Lung Compliance; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Ribonucleases; RNA, Messenger; Sodium-Potassium-Exchanging ATPase; Tissue Preservation

Hyperoxic reperfusion from global ischemia worsens functional outcome because of oxygen radical-mediated injury. This study tested the hypothesis that hyperoxic reperfusion would exacerbate postischemic renal dysfunction.. Twenty-nine healthy, uninephrectomized, male mongrel rabbits (Oryctolagus cuniculus) in 3 groups were subjected to 30 minutes of complete normothermic renal ischemia followed by reperfusion under hyperoxic or normoxic conditions. The groups were: hyperoxically reperfused (n = 8), normoxically reperfused (n = 8), hyperoxic sham (no ischemia, n = 5), and allopurinol-pretreated (50 mg/kg, intravenously), hyperoxically reperfused animals (n = 8). Plasma concentrations of creatinine, urea nitrogen and electrolytes were measured at 0, 24, 48, and 72 hours after ischemia and served as functional outcome markers. Histopathologic analysis of kidneys for injury was performed by an expert who was blinded to the procedures.. Plasma creatinine in hyperoxically reperfused rabbits was significantly elevated above normoxic (P =.02) and sham (P =.003) animals by 48 hours and remained elevated to 72 hours. Plasma urea nitrogen in hyperoxically reperfused rabbits was significantly elevated above the normoxic group (P = .01), the sham group (P = .02), and the allopurinol group (P = .04) by 72 hours. These coincided with a significantly elevated histopathologic injury score in hyperoxically reperfused rabbits compared with sham (P = .019), normoxic (P = .035), and allopurinol-pretreated hyperoxically reperfused animals (P = .037).. Hyperoxic reperfusion exacerbates renal dysfunction after 30 minutes of complete normothermic ischemia. This dysfunction may be mediated by oxygen radical-related injury.

Topics: Allopurinol; Animals; Antioxidants; Blood Urea Nitrogen; Creatinine; Hyperoxia; Ischemia; Kidney; Male; Rabbits; Reference Values; Renal Circulation; Reperfusion Injury

The bioactivity of 3-methyl-1-phenyl-pyrazolin-5-one (MCI-186) was examined based on histochemical changes in drastic global ischemic rat brains. Rats with mean arterial blood pressure reduction were subjected to 60 min cerebral ischemia/80 min reperfusion. Infusion of MCI-186 at 3.0 mg/Kg reduced brain infarction from 21 +/- 4% (saline control, n= 15) to 11 +/- 3% (n=16, p<0.05). By comparison, infusion of up to 20 mg/Kg propyl galalate (PG)--a well documented antioxidant--produced an infarct percentage of 14 +/- 5% (n=8), close to the saline control. Biochemically, the neuroprotective effect of MCI-186 was demonstrated by diminishing the release of creatine kinase (CK) in serum from 3363 +/- 608 U/L (n=14) in saline control to 1989 +/- 293 U/L (n= 15) in MCI group (p<0.05), while PG did not lower the activity of CK significantly. MCI-186 behaves as a free radical scavenger by suppressing the formation of superoxide anion in xanthine oxidase (XO)-hypoxanthine (HP) system (p<0.05). Our data supported our contention that MCI-186 has potent anti-stroke effect with antioxidant activities.

Topics: Animals; Antipyrine; Brain; Brain Infarction; Creatine Kinase; Cytochrome c Group; Edaravone; Free Radical Scavengers; Free Radicals; Hypoxanthine; Male; Neuroprotective Agents; Propyl Gallate; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Xanthine Oxidase

Questions as to the critical stress factor and primary targets of cold ischemia/reperfusion (CIR) injury were addressed by comparing mitochondrial defects caused by (1) CIR injury and (2) intracellular Ca2+ overload. CIR was simulated in transformed human umbilical vein endothelial cell cultures (tEC) by 8 h cold anoxia in University of Wisconsin solution and reoxygenation at 37 degrees C. Intracellular Ca2+ concentrations were changed by permeabilization of suspended cells with digitonin in culture medium (RPMI, 0.4 mM Ca2+). Binding of free Ca2+ by ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid in RPMI or mitochondrial incubation medium served as controls. Extracellular Ca2+ protected the cell membrane against permeabilization. Mitochondrial functions were determined before and after permeabilization of the cell membrane. After CIR, mitochondrial respiratory capacity declined, but oxygen consumption remained coupled to adenosine triphosphate (ATP) production. In contrast, Ca2+ overload caused uncoupling of mitochondrial respiration. High intracellular Ca2+ overload, therefore, does not reproduce cold ischemia/reperfusion injury in endothelial cells.

Topics: Adenosine; Allopurinol; Calcium; Cell Hypoxia; Cell Line, Transformed; Cell Membrane Permeability; Cells, Cultured; Cold Temperature; Egtazic Acid; Endothelium, Vascular; Glutathione; Humans; Insulin; Ischemia; Mitochondria; Organ Preservation Solutions; Oxygen Consumption; Raffinose; Reperfusion Injury; Umbilical Veins

Topics: Adenosine; Allopurinol; Animals; Cell Line; Glutathione; Hot Temperature; HSP70 Heat-Shock Proteins; Insulin; Intestinal Mucosa; Organ Preservation Solutions; Raffinose; Rats; Reperfusion Injury; Tissue Preservation

Topics: Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Glutathione; In Vitro Techniques; Insulin; Isotonic Solutions; Liver; Organ Preservation Solutions; Oxygen; Partial Pressure; Raffinose; Rats; Reperfusion Injury; Ringer's Lactate

Topics: Adenosine; Allopurinol; Animals; Glutathione; Heart Arrest; Hepatic Veins; Insulin; Ischemia; Liver; Liver Circulation; Male; Organ Preservation; Organ Preservation Solutions; Oxygen; Raffinose; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury; Superoxide Dismutase; Tissue Donors

The therapeutic benefits of allopurinol pretreatment in renal ischaemia-reperfusion injury were investigated by monitoring renal malondialdehyde (MDA) and ATP levels together with calculated MDA/ATP ratio in ischaemic (45 min) and reperfused (15 min) rat kidneys. MDA levels remained unchanged during ischaemia, but increased after the subsequent reperfusion. ATP content of the ischaemic kidney was decreased significantly and the recovery of ATP was incomplete after the reperfusion, whereas the MDA/ATP ratio increased at both periods. Allopurinol pretreatment (40 mg kg(-1) iv) maintained higher ATP levels during the ischaemia and inhibited the MDA formation during the reperfusion and decreased the MDA/ATP ratio at both periods. Our findings demonstrate that allopurinol exerts a biphasic protective action by preserving tissue ATP and by inhibiting lipid peroxidation during ischaemia and the reperfusion period, respectively. These findings suggest the selective involvement of two protective mechanisms in the different periods of renal ischaemia-reperfusion injury. The MDA/ATP ratio could be a useful parameter for monitoring these protective actions of allopurinol simultaneously.

Topics: Allopurinol; Animals; Kidney Diseases; Male; Malondialdehyde; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Injury during the transplant process affects the alloantigen-dependent factors and the alloantigen-independent processes of "chronic" rejection. Consequently, the determination of reliable parameters for the assessment of ischemic damage is essential for the prediction of renal changes after ischemia/reperfusion injury. The aim of this study was to assess the ability of (1)H NMR spectroscopy to predict the early graft dysfunction in an ischemia/reperfusion model after preservation in two standard preservation solutions, Euro-Collins (EC) and University of Wisconsin (UW). The second aim was to specify the role of the UW solution in preventing renal medullary injury. Urine and plasma samples from three experimental groups were examined during 2 weeks: control group (n = 5), EC group (cold flushed and 48-h cold storage of kidney in EC and autotransplantation, n = 12), and UW group (cold flushed and 48-h cold storage of kidney in UW and autotransplantation; n = 12). We also examined these kidneys 30-40 min after implantation and on the sacrifice day. Creatinine clearance was significantly reduced in the EC group during the second week. Fractional excretion of sodium and urine N-acetyl-beta-d-glucosaminidase activity were improved but not significantly different in the preserved groups. Urinary concentrations of the alpha-class glutathione S-transferase were significantly greater in the EC group during the first week after transplantation. The most relevant resonances for evaluating renal function after transplantation determined by (1)H NMR spectroscopy were those arising from citrate, dimethylamine (DMA), lactate, and acetate in urine and trimethylamine-N-oxide (TMAO) in urine and plasma. These findings suggest that graft dysfunction is associated with damage to the renal medulla determined by TMAO release in urine and plasma associated with DMA and acetate excretion. Citrate is also a urinary marker that can discriminate kidneys with a favorable evolution. Our results suggest that (1)H NMR spectroscopy is an efficient technique for detecting ischemic damage when accurate and precise data on graft injury is required. In addition, this study outlines the specific impact of the UW solution against injury to the renal medulla.

Topics: Acetic Acid; Acetylglucosaminidase; Acute Kidney Injury; Adenosine; Allopurinol; Animals; Biomarkers; Citric Acid; Cold Temperature; Creatinine; Dimethylamines; Glutathione; Glutathione Transferase; Hypertonic Solutions; Insulin; Isoenzymes; Kidney; Kidney Medulla; Kidney Transplantation; Lactic Acid; Magnetic Resonance Spectroscopy; Male; Methylamines; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Swine; Transplantation, Autologous

Experimental interventions that reduce ischaemia/reperfusion (I/R) lung injury can be used to improve the properties of preservation solutions. We attempted to increase the attenuation of I/R injury by University of Wisconsin solution (UW) by adding an antibody against tumour necrosis factor-alpha (TNF-alpha), to neutralize TNF-alpha, and/or by adding 3-deaza-adenosine (c3-Ado), to inhibit leucocyte adhesion and the biosynthesis of ICAM-1 (intercellular cell-adhesion molecule 1). We examined I/R injury using an isolated rat lung model. Six different solutions were perfused individually, followed by evaluation of I/R injury: (1) 0.9% NaCl (normal saline; NS), (2) NS+anti-TNF-alpha antibody, (3) UW alone, (4) UW+anti-TNF-alpha, (5) UW+c3-Ado and (6) UW+anti-TNF-alpha+c3-Ado. Haemodynamic changes, lung weight gain, capillary filtration coefficient, TNF-alpha levels and lung pathology were analysed in order to evaluate I/R injury. Compared with lungs perfused with NS, lungs treated with NS+anti-TNF-alpha showed less I/R injury. The addition of anti-TNF-alpha and/or c3-Ado to UW reduced I/R injury compared with unmodified UW. Among the six solutions tested, UW containing anti-TNF-alpha antibody reduced I/R injury to the greatest extent. We conclude that addition of anti-TNF-alpha antibody or c3-Ado protects against I/R lung injury when using UW. Further investigation of the improved properties of modified UWs would be beneficial with regard to lung transplantation research.

Topics: Adenosine; Allopurinol; Animals; Antibodies, Monoclonal; Glutathione; Hemodynamics; Insulin; Lung; Lung Transplantation; Male; Organ Culture Techniques; Organ Preservation; Organ Preservation Solutions; Organ Size; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tubercidin; Tumor Necrosis Factor-alpha

The lung is particularly susceptible to reperfusion injury, both experimentally and clinically after transplantation. The extracellular-type preservation solution Celsior, which has been predominantly studied in cardiac preservation, has components designed to prevent cell swelling, free radical injury, energy depletion, and calcium overload. Using an isolated blood-perfused rat lung model, we investigated whether Celsior would decrease preservation injury and improve lung function after cold ischemic storage and reperfusion compared to Euro-Collins (EC) and University of Wisconsin (UW) solutions.. Lewis rat lungs were isolated, flushed with the respective cold preservation solution, and then stored at 4 degrees C for 6 or 12 hr. After ischemic storage, the lung block was suspended from a force transducer, ventilated with 100% O2, and reperfused for 90 min with fresh blood via a cannula in the pulmonary artery. Lung compliance, alveolar-arterial oxygen difference, and outflow oxygen tension were all measured. The capillary filtration coefficient (Kf), a sensitive measure of changes in microvascular permeability, was determined.. For 6 hr of cold storage, lungs stored in Celsior had lower Kf values than those stored in EC, indicating decreased microvascular permeability. No other significant differences were noted between Celsior and EC or UW. For 12 hr of cold storage, Celsior provided increased oxygenation, decreased alveolar-arterial O2 differences, increased compliance, and decreased Kf values as compared to both EC and UW.. Celsior provides better lung preservation than EC or UW as demonstrated by increased oxygenation, decreased capillary permeability, and improved lung compliance, particularly at 12-hr storage times. These results are highly relevant, inasmuch as EC and UW are the most common clinically used lung preservation solutions. Further studies of Celsior in experimental and clinical lung transplantation, as well as in other solid organs, are indicated.

Topics: Adenosine; Allopurinol; Animals; Arteries; Capillary Permeability; Disaccharides; Electrolytes; Glutamates; Glutathione; Histidine; Hypertonic Solutions; Insulin; Lung; Lung Compliance; Male; Mannitol; Organ Preservation Solutions; Oxygen; Partial Pressure; Pulmonary Alveoli; Pulmonary Circulation; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

Sinusoidal endothelial cells are the primary target of ischemia-reperfusion injury following liver preservation. The present study was undertaken to examine the susceptibility of sinusoidal endothelial cells to hypoxia-reoxygenation and the potential role of oxygen free radicals in the induction of cell injury.. Sinusoidal endothelial cells were isolated from rat liver. After 2 3 days of primary culture, the cells were exposed to hypoxia (N2/CO2 95/5) for 120 min and reoxygenation (O2/CO2 95/5) for 90 min. Control cells were exposed to hypoxia alone, to 95% O2 alone or were maintained under normoxic conditions. Human umbilical vein endothelial cells were used as a model of vascular endothelial cells and submitted to the same protocol. Cell viability and lipid peroxidation were assessed by LDH leakage and malondialdehyde production, respectively. In order to test the potential role of xanthine oxidase and mitochondrial dysfunction in cell injury, the cells were treated with allopurinol and potassium cyanide (KCN) respectively.. The different gaseous treatments did not affect LDH leakage in human umbilical vein endothelial cells. In sinusoidal endothelial cells, the sequential hypoxia-reoxygenation caused a significant increase in LDH release, malondialdehyde production and xanthine oxidase activity while hypoxia alone had no effect except on xanthine oxidase activity. Allopurinol inhibited xanthine oxidase without preventing cell injury or lipid peroxidation in this latter cell type.. The results suggest that sinusoidal endothelial cells, as opposed to vascular endothelial cells, are susceptible to a direct cytotoxic effect of hypoxia-reoxygenation. This effect occurs in combination with an increase in xanthine oxidase activity and lipid peroxidation, although cell injury is mediated at least in part by mechanisms independent of xanthine oxidase such as mitochondrial dysfunction.

Topics: Animals; Cell Hypoxia; Cell Survival; Cells, Cultured; Endothelium, Vascular; L-Lactate Dehydrogenase; Lipid Peroxidation; Liver; Male; Malondialdehyde; Oxygen; Potassium Cyanide; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

The objective of this study was to determine if allopurinol (AL) and/or trifluoperazine (TFP) added to the Belzer machine preservation solution (MPS) improves the function of non-heart-beating donor (NHBD) canine kidneys.. Anesthetized canines underwent bilateral dissection of the renal vessels, obtaining baseline flow. After removing one kidney (heart-beating donor [HBD]), the dog was exsanguinated. After remaining in situ for 120 min (30-min warm ischemia time, 90-min cold ischemia time), the second kidney was removed (NHBD), flushed, biopsied, and weighed. The kidneys were machine-perfused separately for 20 hr, and pressure, flow, and resistance were measured serially. The kidneys were randomly assigned to a perfusate group (G): G1=MPS, G2=MPS+TFP, G3=MPS+AL, and G4=MPS+TFP+AL. Kidneys were implanted separately into a single recipient dog. Flow, resistance, and urine output were measured serially for 4 hr. Blood and urine samples and kidney biopsies were then obtained. All measurements were standardized to 100 g of kidney weight.. HBD kidneys functioned better than NHBD kidneys in all groups, as expected. Although perfusate G1 was the most effective solution for HBD kidneys, the TFP additive (perfusate G2) more effectively reversed the vasospastic effects of ischemia/reperfusion for NHBD than the MPS solution (G1) with or without other additives. In HBD kidneys, the addition of AL resulted in the best creatinine clearance; however, AL was less effective than MPS alone in NHBD kidneys. TFP+AL together were completely ineffective in preserving renal function, regardless of whether the kidneys were from HBD or NHBD.. MPS+TFP more effectively protected renal function against reperfusion injury in the NHBD than MPS alone, AL, or AL+TFP. AL exerts a salutary effect on creatinine clearance in HBD but not in the NHBD. The TFP and AL combination should not be used together with the MPS in machine preservation of kidneys.

Topics: Adenosine; Allopurinol; Animals; Diuresis; Dogs; Female; Glutathione; Heart Arrest; Insulin; Kidney; Kidney Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Renal Circulation; Reperfusion Injury; Trifluoperazine

In situ manipulation by touching, retracting, and moving liver lobes gently during harvest dramatically reduces survival after transplantation (P. Schemmer, R. Schoonhoven, J. A. Swenberg, H. Bunzendahl, and R. G. Thurman. Transplantation 65: 1015-1020, 1998). The development of harvest-dependent graft injury upon reperfusion can be prevented with GdCl3, a rare earth metal and Kupffer cell toxicant, but it cannot be used in clinical liver transplantation because of its potential toxicity. Thus the effect of glycine, which prevents activation of Kupffer cells, was assessed here. Minimal dissection of the liver for 12 min plus 13 min without manipulation had no effect on survival (100%). However, gentle manipulation decreased survival to 46% in the control group. Furthermore, serum transaminases and liver necrosis were elevated 4- to 12-fold 8 h after transplantation. After organ harvest, the rate of entry and exit of fluorescein dextran, a dye confined to the vascular space, was decreased about twofold, indicating disturbances in the hepatic microcirculation. Pimonidazole binding, which detects hypoxia, increased about twofold after organ manipulation, and Kupffer cells isolated from manipulated livers produced threefold more tumor necrosis factor-alpha after lipopolysaccharide than controls. Glycine given intravenously to the donor increased the serum glycine concentration about sevenfold and largely prevented the effect of gentle organ manipulation on all parameters studied. These data indicate for the first time that pretreatment of donors with intravenous glycine minimizes reperfusion injury due to organ manipulation during harvest and after liver transplantation.

Topics: Adenosine; Allopurinol; Animals; Female; Gadolinium; Glutathione; Glycine; Graft Survival; Hepatectomy; Infusions, Intravenous; Insulin; Kupffer Cells; Liver; Liver Transplantation; Microcirculation; Necrosis; Nitrites; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Tumor Necrosis Factor-alpha; Valine

This study was designed to determine whether oxygen-derived free radicals play a role in the pathogenesis of gastric mucosal lesions produced by haemorrhagic shock and reperfusion experimental model in the rat. Ranitidine (H2-receptor blocker) in different doses, allopurinol, an inhibitor of xanthine oxidase and SOD (superoxide dysmutase) pre-treatment were used against haemorrhagic shock and reperfusion induced gastric mucosal lesions. Altogether 67 rats were divided into seven different groups. The area of gastric mucosal lesions was measured, the activity of endogenous peroxidase was examined histochemically and histological grading was made. Evans blue was used to demonstrate the improved permeability of gastric mucosal membranes. Ranitidine, in high dose, allopurinol and superoxide dysmutase significantly protected against haemorrhagic shock-induced gastric mucosal lesions, against improved membrane permeability and peroxidation.

Topics: Allopurinol; Animals; Free Radical Scavengers; Free Radicals; Gastric Mucosa; Histamine H2 Antagonists; Histocytochemistry; Male; Peroxidase; Ranitidine; Rats; Rats, Wistar; Reperfusion Injury; Shock; Shock, Hemorrhagic; Superoxide Dismutase; Xanthine Oxidase

A complication in liver transplantation is increased clotting times due to inhibition of protein synthesis resulting from prolonged hypothermic preservation. Protein synthesis is also blocked in cold preserved hepatocytes. In this study, the mechanism of inhibition of protein synthesis in cold preserved hepatocytes was investigated.. Hepatocytes prepared from rat liver were cold preserved in University of Wisconsin solution for 4, 24, and 48 hr. Protein synthesis was measured as incorporation of radiolabeled leucine into acid precipitable proteins. Hepatocytes were treated with antioxidants (dithiothreitol, trolox or deferoxamine, nitric oxide synthase inhibitor (N(G)-monomethyl-L-arginine monoacetate), steroids (dexamethasone or methylprednisolone), methods to keep adenosine triphosphate high (aerobic storage), and cytoskeletal disrupting agents (cytochalasin D or colchicine).. There was a 26% decrease in protein synthesis after only 4 hr of cold storage and a further 25% decrease at 24 hr. Antioxidants, elevated adenosine triphosphate, and N(G)-monomethyl-L-arginine monoacetate did not affect the rate of loss of protein synthesis. Protein synthesis was not due to inhibition of amino acid transport or lack of amino acids in the storage medium. Steroid pretreatment of hepatocytes had no effect on the loss of protein synthesis occurring in the first 4 hr of storage but did suppress the loss occurring during the next 44 hr of storage. Cytoskeletal disrupting agents, added to freshly isolated cells, inhibited protein synthesis.. The mechanism of loss of protein synthesis in cold preserved liver cells is not mediated by: (1) oxygen free radical generation or improved by antioxidant therapy, (2) nitric oxide generation in hepatocytes, (3) an adenosine triphosphate-sensitive destruction of cell viability, and (4) decreased permeability of amino acids or loss of amino acids from the cells. Loss of protein synthesis due to hypothermic storage appears biphasic. The first phase, occurring within 4 hr of storage, may be the result of the effects of hypothermia on the cell cytoskeletal system and may be untreatable. The second phase, which occurs during the next 24 to 48 hr is sensitive to steroid pretreatment. This phase may be amenable to improved preservation methodology. Improved preservation of the liver may require the use of steroids to conserve protein synthetic capabilities.

Topics: Adenosine; Allopurinol; Animals; Antioxidants; Chromans; Cryopreservation; Cytosol; Deferoxamine; Dithiothreitol; Glutathione; Insulin; Leucine; Liver; Organ Preservation Solutions; Protein Biosynthesis; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tritium; Vitamin E

Intestinal ischemia-reperfusion (IR) injury results in cell destruction, which may be mediated by the generation of reactive oxygen species, potentially toxic metabolites of xanthine oxidase. Pentoxifylline (PTX) possesses a variety of biochemical and antioxidant properties that can improve capillary flow and tissue oxygenation. Because of these combined effects, it has been hypothesized that pentoxifylline would protect against intestinal IR.. Young adult rats were randomly assigned to one of four experimental groups: IR/Placebo (n = 12) in which superior and inferior mesenteric arteries were clamped for 45 minutes and then reopened; IR/PTX (n = 11) in which IR was induced as in the Placebo group, but with 25 mg/kg PTX at 0, 30, and 60 minutes; No IR/Placebo (n = 12); and No IR/PTX (n = 6) in which placebo and PTX were applied with no IR. Blood and intestinal samples were taken for serial thiobarbituric acid-reducing substances (TBARS; index of lipid peroxidation), for xanthine oxidase-xanthine dehydrogenase ratios, glutathione, myeloperoxidase, and histopathology.. Animals in the IR/PTX group had lower TBARS and the least severe histopathologic injury. Xanthine oxidasexanthine dehydrogenase ratios were elevated only in IR/ Placebo (0.67+/-0.22 vs. 0.45+/-0.14 in IR/PTX; 0.42+/-0.22 in No IR/Placebo; and 0.40+/-0.11 in No IR/PTX; p = 0.0009). Reduced glutathione was diminished in IR/PTX animals (38.9 +/-1.35 vs. 46.1+/-7.0 in IR/Placebo; 41.1+/-2.5 in No IR/ Placebo; 43.6+/-1.0 in No IR/PTX; p = 0.048). No differences were recorded in myeloperoxidase levels among groups.. Pentoxifylline ameliorates histopathologic signs of injury and decreases lipid peroxidation (TBARS). Normal xanthine oxidase-xanthine dehydrogenase ratios in the treated compared with IR-only animals imply that the protective effect of PTX is at least partially mediated through inhibition of xanthine oxidase.

Topics: Animals; Evaluation Studies as Topic; Free Radical Scavengers; Intestines; Lipid Peroxidation; Pentoxifylline; Random Allocation; Rats; Reperfusion Injury; Xanthine Oxidase

Free radical scavengers have been utilized to prevent the consequences of ischemia, however, results do not seem conclusive. In our study we analyzed the blood flow, function, and histology of rat liver tissue after warm liver ischemia, in order to assess the effect of free radicals in liver reperfusion injury. N-acetyl cysteine (NAC), tocopherol, allopurinol, and superoxide dismutase (SOD), pharmacological agents expected to protect from injury mediated by free radicals, were investigated. Laser Doppler flowmetry and photometry were utilized to measure post-ischemic microcirculatory changes as an expression of ischemia-reperfusion injury in a model of segmental liver ischemia in the rat, with an ischemic time of 45 min. Galactose elimination capacity, ALT and histology were used to assess the functional and morphological consequences of ischemia after 24 h of reperfusion. The overall mean blood flow over 1 hour after reperfusion was of 33.9% (SD 11.2) of the normal, non-ischemic control. NAC (31.2% SD 10.9) did not show any protective effect and in some cases the effect seemed to be negative. Tocopherol (41.7% SD 5.1) marginally improved post ischemic liver tissue blood flow. Treatment with allopurinol did not show any beneficial effects (37.5% SD 14.2). Only animals treated with SOD showed an improvement of the post ischemic liver microcirculation (57.9% SD 14.4)(P < 0.001) and function. Only SOD produced statistically significant differences in galactose elimination capacity, compared with those of the ischemic control group. This moderately protective effect of SOD is encouraging, however, the relevance of all these compounds in a broader pathophysiological setting remains unproven.

Topics: Acetylcysteine; Allopurinol; Animals; Free Radical Scavengers; Galactose; Ischemia; Liver; Liver Function Tests; Male; Microcirculation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase; Vitamin E

Renal ischemia injures the renal tubular cell by disrupting the vital cellular metabolic machinery. Further cell damage is caused when the blood flow is restored by oxygen free radicals that are generated from xanthine oxidase. Oxygen radicals cause lipid peroxidation of cell and organelle membranes, disrupting the structural integrity and capacity for cell transport and energy metabolism. In the present study, the possible therapeutic usefulness of the adenosine deaminase inhibitor, 2'-deoxycoformycin (DCF), during renal ischemia and reperfusion injury was investigated. The effects of DCF on renal malondialdehyde (MDA) and ATP levels were studied after 45 min ischemia and 15 min subsequent reperfusion in rat kidneys. MDA levels remained unchanged during ischemia, but increased after the subsequent reperfusion. DCF pretreatment (2.0 mg/kg i.m.) decreased MDA and increased ATP levels during the ischemia-reperfusion period. DCF exerts a dual protective action by facilitating purine salvage for ATP synthesis and inhibiting oxygen radical-induced lipid peroxidation. These results suggest that DCF therapy could be beneficial in the treatment of ischemia-reperfusion renal injuries.

Topics: Adenosine; Adenosine Deaminase Inhibitors; Adenosine Triphosphate; Animals; Antioxidants; Enzyme Inhibitors; Ischemia; Kidney; Lipid Peroxidation; Male; Malondialdehyde; Oxidative Stress; Pentostatin; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Thiobarbituric Acid Reactive Substances; Xanthine Oxidase

Initial ischemia-reperfusion injury is associated with organ retrieval, storage, and transplantation adversely affects early graft function and influences the development of chronic graft dysfunction. We have recently shown that the protective agent trimetazidine (TMZ) added to preservation solutions: Euro-collins (EC) and University of Wisconsin (UW) was efficient to protect kidneys from ischemia-reperfusion injury in an isolated perfused kidney model. We extended these observations to investigate the role of this drug in the development and progression of organ dysfunction in the autotransplant pig kidney model.. Five experimental groups were studied. After 48-hr cold preservation, autotransplantation and immediate controlateral nephrectomy was then performed in group EC (EC+placebo (n=8), EC+TMZ (n=8), UW+placebo (n=7), and (UW+TMZ) (n=7) and compared with control group (uninephrectomized, n=4) during 14 days. Blood and urine samples were collected for the measurement of creatinine and blood urea nitrogen on postoperative days 1, 3, 5, 7, 11, and 14. Histological analysis was performed after reperfusion and at day 14.. Survivals were 100% in group B and D versus 42% in group A and 57% in group C. Urine production occurred earlier after autotransplantation from TMZ preserved kidneys than in placebo preserved groups. Peak creat and blood urea nitrogen was significantly greater in groups B and D than in groups A and C. TMZ was also efficient both to reduce ischemia-reperfusion injury and to decrease cellular infiltration.. These results support the beneficial effect of TMZ against ischemia-reperfusion injury and its early effects on grafts in the form of delayed graft function and decreased graft survival. In addition, TMZ reduces inflammatory cellular infiltration in the renal parenchyma.

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Cryoprotective Agents; Glutathione; Graft Survival; Hypertonic Solutions; In Vitro Techniques; Insulin; Kidney; Kidney Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Swine; Trimetazidine

Reperfusion injury following lung preservation has been associated with free radical formation and subsequent endothelial cell damage. Trolox is a water-soluble analogue of the free radical scavenger alpha-tocopherol. We hypothesized that addition of this form of vitamin E to University of Wisconsin (UW) solution would decrease reperfusion injury and improve lung function after cold ischemic preservation.. Bovine aortic endothelial cells were cultured and stored at 4 degrees C for 12, 24, and 48 h in UW or UW + Trolox (UWT). Endothelial cell viability after storage was assessed by dimethylthiazole tetrazolium cytotoxicity assay. An isolated rat perfused lung (IPL) model was used and lungs were flushed with the respective solutions with cold storage times of 6 and 12 h. Following storage, the lungs were reperfused with fresh blood and lung function was assessed by blood gas analysis, alveolar-arterial gradient, and compliance.. There was no difference in endothelial cell viability between UW and UWT after 12 or 24 h; however, UWT had higher endothelial cell viability than UW with 48 h of cold ischemic storage. Using the IPL model, the pO2 was higher with UWT than UW after 6 and 12 h of cold ischemia. The alveolar-arterial oxygen difference was significantly lower for UWT versus UW at 6 h. UWT provided increased compliance at 6 and 12 h of ischemia.. The addition of a water-soluble vitamin E analogue to UW solution resulted in increased endothelial cell viability after prolonged storage and improved whole lung preservation in the postreperfusion period as evidenced by higher oxygenation and increased compliance. These results are clinically relevant as the lung is extremely sensitive to reperfusion injury and UW solution is being increasingly used in lung transplantation and remains the predominant solution in abdominal organ transplantation.

Topics: Adenosine; Allopurinol; Animals; Cattle; Cells, Cultured; Child; Chromans; Endothelium, Vascular; Glutathione; Humans; In Vitro Techniques; Insulin; Ischemia; Lung; Organ Preservation Solutions; Pulmonary Circulation; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solubility; Vitamin E

The extent of lipid peroxidation after ischemia-reperfusion (I-R) injury in rat kidney has been controversial. After I, xanthine oxidase (XO) is thought to be the main oxygen radical-generating system and malondialdehyde (MDA) is considered to be a marker of lipid peroxidation (LPO). In young rats (10 weeks old) a unilateral warm I of 40 and 60 min duration with subsequent R up to 1 h was conducted. Beside the "footprints" of oxidative stress, the cytosolic antioxidative capacity, expressed as superoxide anion (SOA) scavenging capacity, and the renal catalase were also investigated. There was only a moderate and transient increase of renal MDA 5 and 10 min after the onset of reoxygenation (133.57/70. 67 and 97.84/91.57 vs. 49.47 nmol/g ww in preischemic controls). ATP breakdown (to 83/65 from 2947 nmol/g ww) with consecutive accumulation of hypoxanthine (up to 1105 nmol/g ww) at the end of ischemic period and the subsequent rapid decline of hypoxanthine by XO during reperfusion were used for an assessment of the SOA-generating capacity of these kidneys. Superoxide dismutase (SOD) activity, glutathione (GSH) and the high activity of catalase (18000 U/g ww) remained nearly unchanged during R. Only 1/25-1/50 of the kidney cytosol was able to scavenge the whole amount of SOA generated by the total XO activity of rat kidney. Thus, it could be analytically and stoichiometrically shown that after IR there is only a moderate oxidative stress in kidneys of young rats; this is due to their high SOA-scavenging capacity compared with their SOA-generating ability.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Catalase; Glutathione; Hypoxanthine; Kidney; Lipid Peroxidation; Male; Malondialdehyde; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Superoxides; Time Factors; Xanthine Oxidase

1. This study examined the impact of allopurinol on the renal functional responses to a 30 min period of ischaemia in anaesthetized rats. 2. Immediately on reperfusion, blood pressure rose transiently, while renal blood flow remained stable throughout at control values. Glomerular filtration rate was decreased by some 90% over the first and 80% over the sixth hour (P<0.001). 3. Allopurinol, 50 or 100 mg kg-1, had no effect on the blood pressure or renal blood flow responses over the 6 h reperfusion period but glomerular filtration decreased by 60% initially, and to less than 30% of basal at 6 h. 4. Urine flow and absolute sodium excretion increased 2 - 3 fold in the first 2 h but decreased thereafter. Fractional sodium excretion was 30 times higher for the first 2 h but decreased reaching some 10 fold higher at 6 h. In the presence of allopurinol, urine flow and absolute sodium excretion increased by 5 - 6 fold in the first 2 h, and fell by half by 6 h which was greater than in the vehicle group (P<0.01). Fractional sodium excretion increased 20 fold in the allopurinol animals in the first 2 h period, but fell at a faster rate (P<0.01) than in untreated rats. 5. Potassium excretion decreased (P<0.05) by one half for the 6 h reperfusion period but in the allopurinol animals it was minimally altered. 6. Allopurinol largely ameliorated the decrease in kidney haemodynamic and excretory function following an ischeamic period for the initial few hours of reperfusion.

Topics: Acute Disease; Allopurinol; Anesthesia; Animals; Blood Pressure; Disease Models, Animal; Glomerular Filtration Rate; Ischemia; Kidney; Male; Potassium; Rats; Rats, Wistar; Renal Artery; Renal Circulation; Reperfusion Injury; Sodium; Time Factors; Xanthine Oxidase

Topics: Acetylcysteine; Allopurinol; Animals; Free Radical Scavengers; Ischemia; Liver; Liver Circulation; Male; Microcirculation; Organ Preservation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase; Temperature; Time Factors; Vitamin E

Topics: Acetylcysteine; Adenosine Triphosphate; Allopurinol; Animals; Glutathione; Heart; Intestine, Small; Ischemia; Liver; Lung; Malondialdehyde; Mesenteric Arteries; Multiple Organ Failure; Myocardium; Oxidative Stress; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Somatostatin

Current methods of preserving lung tissue for transplantation are inadequate. In this study, we tested whether the combination of hypothermia plus prostaglandin E(1) (PGE(1)) treatment would have synergistic attenuation on ischemia-reperfusion (I/R) lung injury. Isolated rat lung experiments with ischemia for 1 h then reperfusion for 1 h, were conducted using six different perfusates: (1) University of Wisconsin solution (UW) at 30 degrees C (n = 5), (2) UW at 22 degrees C (n = 5), (3) UW at 10 degrees C (n = 4), (4) UW+PGE(1) at 30 degrees C (n = 4), (5) UW+PGE(1) at 22 degrees C (n = 4), and (6) UW+PGE(1) at 10 degrees C (n = 4). Hemodynamic changes, lung weight gain, capillary filtration coefficients, and lung pathology were analyzed to evaluate the I/R injury. Compared with 30 degrees C UW, animals treated with 22 degrees C UW and 10 degrees C UW had less I/R lung injury, with the groups receiving 22 degrees C UW showing superior results to group receiving 10 degrees C UW. The addition of PGE(1) to UW solution produced more attenuation of I/R injury than did UW alone. Among the six groups, 10 degrees C UW+PGE(1) produced the most reduction of I/R injury. This study has shown that hypothermia can attenuate I/R injury with the optimal flushing temperature being near 22 degrees C. PGE(1) also has a protective effect on I/R. Furthermore, hypothermia and PGE(1) have synergistic attenuation of I/R lung injury. We propose that pulmonary artery flushed with cooling UW+PGE(1) might improve lung preservation and improve results in lung transplantation.

Topics: Adenosine; Allopurinol; Alprostadil; Animals; Capillary Permeability; Glutathione; Hypothermia, Induced; Insulin; Lung; Male; Organ Preservation; Organ Preservation Solutions; Organ Size; Pulmonary Circulation; Pulmonary Wedge Pressure; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Vascular Resistance; Vasodilator Agents

The inclusion of the colon in the intestinal graft resulted in worsening patient and graft outcome and increased the incidence of infection and rejection. In this study, we examine the role of ischemia on the barrier function of the epithelium during cold ischemia. Samples were collected from 15 harvested and transplanted human donor grafts (colon, 10; ileum, 6), which were immersed in University of Wisconsin (UW) solution. Ischemia (6, 12, 24, and 48 h) and reoxygenation were performed to evaluate the mucosal electrical status using the Ussing chamber technique. The functions of enterocytes and crypt cells were tested by glucose and theophylline challenge. Modified Park's classification was applied to evaluate the severity of mucosal damage under light microscopy. The colon had higher levels of baseline potential difference, short-circuit current, and resistance than the ileum during 6 48 h of ischemia. Colonic epithelial cells responded well to theophylline stimulation at 24 h of ischemia, while there was no ileal response. The colonic mucosa was histopathologically well preserved in UW solution for 48 h, and mucosal damage induced by reoxygenation was less than in the ileum. In conclusion, electrophysiologically and histopathologically, the colon is less susceptible to cold preservation damage than the ileum during storage with UW solution.

Topics: Adenosine; Allopurinol; Cold Temperature; Colon; Electrophysiology; Glutathione; Humans; Ileum; In Vitro Techniques; Insulin; Intestinal Mucosa; Organ Preservation Solutions; Raffinose; Reperfusion Injury

Pyruvate dehydrogenase has been thought to be involved in the improved recovery of livers, from fasted donors, reperfused with alanine after cold preservation. The aim of this work was to investigate the effect on perfused mouse liver of dichloroacetate, an activator of this enzyme. Livers from fed and fasted animals were perfused with oxygenated Krebs-Henseleit buffer for 30 min, then stored at 4 degrees C in University of Wisconsin solution for 48 h. Then reperfusion at 37 degrees C was performed with Krebs-Henseleit buffer containing 2 mM dichloroacetate for 1 h. (3-(13)C)Alanine (8 mM) was then added and perfusion was continued for a second hour. (31)P-NMR was used to measure nucleoside triphosphate recovery of the livers. At the end of reperfusion, (13)C-NMR spectra of perfusates were recorded. Dichloroacetate (DCA) was found to activate pyruvate dehydrogenase in all cases. However, it decreased the functional recovery of livers from both fed and fasted mice. In order to study the effect of alanine on this DCA deleterious effect, we reperfused the livers according to a modified protocol. The first hour of perfusion without alanine was omitted and the organs were reperfused directly for 1 h in the presence of 2 mM dichloroacetate and 8 mM (3-(13)C)alanine. In this protocol, the deleterious effect of DCA was completely suppressed for livers from fasted mice. These results led to the conclusion that the specific beneficial effect of alanine on livers from fasted livers persists in the presence of DCA and thus cannot be explained solely by the induction of a greater pyruvate dehydrogenase reaction rate.

Topics: Adenosine; Alanine; Allopurinol; Animals; Carbon Isotopes; Cold Temperature; Cryopreservation; Dichloroacetic Acid; Fasting; Glutamine; Glutathione; Insulin; Ischemia; Liver; Male; Mice; Nuclear Magnetic Resonance, Biomolecular; Organ Preservation; Organ Preservation Solutions; Phosphorus; Raffinose; Reperfusion Injury; Tissue Donors

Cardiovascular, respiratory, and vascular dysfunction can follow trauma-induced no-flow-reflow states: hemorrhage, blunt trauma, or neurogenic shock. Liver ischemia-reperfusion (IR) induces remote lung damage by means of xanthine oxidase (XO) pro-oxidant activity. This damage was not proven in the heart, neither was the independent role of radical oxygen species (ROS) established in such cases. We investigated whether multiple organ dysfunction after a trauma-like IR is XO and ROS related and whether clinically used ROS scavengers could be beneficial.. A controlled, randomized trial in which isolated rat livers, hearts, lungs, and aortic rings were perfused with Krebs-Henseleit solutions. After stabilization, livers were either perfused or made ischemic (2 hours). Then, pairs of liver plus heart, lung, or ring were reperfused in series (15 minutes), and then the second organ circulated alone for 45 minutes. Remote organ protection against the pro-oxidant hepatic-induced toxicity was evaluated by using allopurinol (1 mmol/L, heart), mannitol (0.25 g/kg, lung), or methylene blue (40 mg/kg, ring).. IR liver effluents typically contained high lactate dehydrogenase, XO, and uric acid concentrations compared with control organs. IR was associated with doubled lung peak inspiratory pressure and reduced static compliance. Myocardial velocity of contraction and relaxation decreased by one third of baseline, and rings contracted abnormally and responded inadequately to phenylephrine. Wet-weight to dry-weight ratios in the remote organs increased as well. Most remote reperfusion injuries were attenuated by the drugs.. Liver no-flow-reflow directly induces myocardial, pulmonary, and vascular dysfunction. These are likely mediated by XO and ROS. The tested drugs protected against these pro-oxidants, even in the presence of circulating XO.

Topics: Allopurinol; Animals; Aorta; Coronary Vessels; Disease Models, Animal; Drug Evaluation, Preclinical; Free Radical Scavengers; Heart Arrest; In Vitro Techniques; Liver; Lung; Male; Mannitol; Methylene Blue; Multiple Organ Failure; Multiple Trauma; Myocardium; Random Allocation; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

1. The potential for the N-hydroxyguanidine compound PR5 (N-(3, 4-dimethoxy-2-chlorobenzylideneamino)-N'-hydroxyguanidine) as a cardioprotective agent in heart ischaemia and reperfusion injury was investigated using rat models. 2. Administration of 1-10 mg kg-1 of PR5 5 min before 10 min of left coronary artery occlusion, followed by 20 min reperfusion, strongly inhibited reperfusion burst of arrhythmias and markedly improved the survival of the animals (e.g. ventricular fibrillation incidence 93 vs 43% (P<0.05); mortality 47 vs 0% (P<0.05), for controls and for 3 mg kg-1 of PR5, respectively). 3. Administration of 3 mg kg-1 of PR5 1 min before reperfusion to rats subjected to 10 min occlusion, 20 min reperfusion was most effective in reducing arrhythmias and decreasing mortality (43 vs 0%, P<0.05), but effects were also seen when PR5 was administered 0, 1 and 5 min after start of reperfusion. 4. Coronary occlusion/reperfusion (10 - 20 min) increased malondialdehyde (MDA) of rat hearts (0.88+/-0.13 for sham vs 1.45+/-0.12 nmol mg-1 protein for ischaemic; P<0.05). In rats where 3 mg kg-1 PR5 were administered 1 min before reperfusion the increase was attenuated (MDA being 1.04+/-0.12; P<0.05 vs ischaemic). 5. PR5 caused a substantial reduction of the infarction size in rats subjected to 180 min left coronary artery occlusion, followed by 120 min of reperfusion; the necrotic zone being 326+/-32 mg for controls vs 137+/-21 mg for animals treated with 3x3 mg kg-1 of PR5 (P<0.01). 6. PR5 reduced the elevation of the ST-segment of the ECGs, as well as caused pronounced attenuation of the rapid blood pressure drop seen at the start of reperfusion following coronary artery occlusion. 7 We conclude that the N-hydroxyguanidine PR5 provides remarkable protection against ischaemia and reperfusion induced myocardial necrosis and life-threatening arrhythmias. These effects of PR5 are discussed in relation to a recently discovered ability of N-hydroxyguanidines to function as electron acceptors at the xanthine oxidase enzyme.

Topics: Animals; Antihypertensive Agents; Arrhythmias, Cardiac; Cardiovascular Agents; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanabenz; Guanidines; Hydroxylamines; Male; Malondialdehyde; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Oxidation-Reduction; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Oxidase

To establish the antioxidant status of rat intestinal tissues after ischemia-reperfusion and to determine if pretreatment with an allopurinol and antioxidant vitamin combination gives any protection against mucosal injury.. Twenty rats were divided into 4 groups of 5 animals each.. Group 1 (control) rats were not subjected to ischemia-reperfusion and received no allopurinol plus vitamin combination; group 2 rats received vitamins C (200 mg/kg) and E (100 mg/kg) and allopurinol (50 mg/kg) combination daily for 3 days preoperatively; group 3 rats were subjected to ischemia-reperfusion only; and group 4 rats were subjected to ischemia-reperfusion and received the vitamin and allopurinol combination.. Activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) enzymes, the level of thiobarbituric acid-reagent substances (TBARS) and histologic grading of tissue samples.. SOD and GSH-Px activities were decreased, but the CAT activity and TBARS level increased. Pretreatment of the rats with the allopurinol-vitamin C-vitamin E combination did not have any significant effect on the enzyme activities. However, it resulted in important reductions in the TBARS tissue levels. Histologic investigation revealed significant mucosal injury in group 3 rats compared with group 4 rats (mean [and standard deviation] for grading, 4.6 [0.5] versus 1.8 [0.4]).. The enzymatic antioxidant defence system was significantly changed after ischemia-reperfusion and intestinal tissue was exposed to increased oxidant stress, the results of which were peroxidation of some cellular structures and increased concentrations of oxidative products. Although antioxidant treatment did not drastically affect the enzyme activities or afford complete protection of cellular structures against deformation, it apparently could eliminate oxygen radicals and prevent peroxidative reactions.

Topics: Allopurinol; Animals; Antioxidants; Ascorbic Acid; Catalase; Drug Therapy, Combination; Free Radicals; Glutathione Peroxidase; Intestinal Mucosa; Lipid Peroxidation; Oxidative Stress; Premedication; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Vitamin E

Topics: Adenosine; Allopurinol; Anastomosis, Surgical; Animals; Cell Survival; Gadolinium; Glutathione; Hepatic Artery; Insulin; Kupffer Cells; Liver; Liver Circulation; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Portal Vein; Raffinose; Regional Blood Flow; Reperfusion Injury; Swine; Vena Cava, Inferior

Orthotopic rat liver transplantation (ORLT) following extended cold preservation in University of Wisconsin (UW) solution has been shown to induce alterations of the hepatic microcirculation, mainly characterized by areas of no-reflow. The present study was performed to determine whether these alterations were related to the portal vein clamping time (PVCT), shown to be the main determinant of survival after ORLT. The hepatic microcirculation was evaluated using the multiple-indicator dilution curve (MIDC) technique after ORLT following 24-hour cold ischemia in UW solution. Two groups of rats were studied: one with PVCTs of less than 14 min (survival conditions) and one with PVCTs of more than 18 min (nonsurvival conditions). Four hours after ORLT, only long PVCTs were associated with small, but significant, nonperfused areas, about 10% of the liver not being perfused by water; however, in both survival and nonsurvival conditions, the sinusoidal sieving function was well-maintained in perfused areas. In addition, liver viability parameters and hepatocyte function were similarly and minimally altered. The hepatic microcirculation is minimally altered 4 h after ORLT following extended cold preservation in UW solution, whatever the survival condition. Although only found after long PVCTs, the low magnitude of areas of no-reflow should not be associated with lethal injury of the transplanted liver, a finding further supporting the concept that survival after ORLT following 24-hour cold preservation in UW solution is mainly influenced by extrahepatic factors.

Topics: Adenosine; Allopurinol; Animals; Constriction; Cryopreservation; Glutathione; Graft Survival; Insulin; Ischemia; Liver; Liver Circulation; Liver Transplantation; Male; Microcirculation; Organ Preservation; Organ Preservation Solutions; Portal Vein; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Time Factors; Tissue and Organ Harvesting

Xanthine dehydrogenase and oxidase activities increased by 87% in rat brain slices after 30 min in vitro ischaemia. A further 41% increase was induced by 30 min simulated reperfusion of ischaemic slices. No conversion from the dehydrogenase to the oxidase activity was observed. The increment of enzyme activity was not due to neosynthesis of the enzyme, since it was not affected by the addition of cycloheximide during the ischaemic incubation. The increased oxygen-dependent form of the enzyme could aggravate the ischaemic brain injury by free radicals production, in particular after reperfusion.

Topics: Animals; Brain; Cycloheximide; In Vitro Techniques; Ischemic Attack, Transient; Male; Models, Biological; Oxygen; Protein Synthesis Inhibitors; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

Pretransplant rinse solutions have been shown to reduce reperfusion injury in cold-stored liver grafts, especially at the nonparenchymal level in sinusoidal endothelial cells (SEC). In this study, different rinse temperatures were tested in a rat liver preservation model. Livers were washed out in situ via the portal vein with cold (4 degrees C) University of Wisconsin (UW) solution, and after hepatectomy (t0), were stored for 8, 16, or 24 h of cold ischemia time (CIT). After storage, livers were flushed with UW solution at either 4 degrees C, 20 degrees C, or 37 degrees C and reperfused for 90 min (37 degrees C). Control livers were reperfused at t0 without preflush. Levels of hyaluronic acid (HA), purine nucleoside phosphorylase (PNP), AST, and LDH were measured in the reperfusion medium. Bile production was monitored during reperfusion. At the end of reperfusion, liver biopsies were taken for enzyme hystochemistry (5'-nucleotidase and LDH). After 8-h CIT and a flush at 4 degrees C, a release of endogenous HA (-7%) was observed, whereas uptake of exogenous HA occurred after the 20 degrees C flush (2%, P = NS) and after the 37 degrees C flush (24%, p < 0.001). HA release occurred at all three preflush temperatures after the 16-h CIT but was significantly lower when flushed at 37 degrees C (-10%) that at 4 degrees C and 20 degrees C (-64% and -17%, respectively, p = 0.05). After the 24-h CIT, the release of endogenous HA increased in the 4 degrees C and 20 degrees C preflush groups, but not in the 37 degrees C group. Levels of PNP and AST increased until the 24-h CIT in all groups but were significantly lower after preflush at 37 degrees C. Release of LDH did not increase with increasing periods of cold storage in any of the flush series. Compared to control livers, mean bile production during reperfusion was significantly decreased following preflush at 4 degrees C or 37 degrees C after all periods of CIT. No differences in mean bile production could be demonstrated in the three preflush groups after any period of CIT. LDH activity in liver tissue was best preserved after the 8 and 16-h CIT in combination with the 37 degrees C preflush, indicating less hepatocellular damage. In conclusion, in cold stored rat livers flushed at 37 degrees C before reperfusion, SEC and hepatocellular damage is attenuated.

Topics: Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Bile; Cold Temperature; Female; Glutathione; Hot Temperature; Hyaluronic Acid; Insulin; L-Lactate Dehydrogenase; Liver; Organ Preservation Solutions; Purine-Nucleoside Phosphorylase; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Tissue Preservation

Oxygen free radicals, generated by cerebral ischemia, have been widely implicated in the damage of vascular endothelium. Endothelial cells have been proposed as a significant source of oxygen free radicals. In the present study, we developed an anoxia-reoxygenation (AX/RO) model using pure cultures of cerebral endothelial cells (CECs) isolated from piglet cortex to measure CEC oxygen free radical production and determine its role in AX/RO-induced CEC injury. CEC injury, as measured by lactate dehydrogenase efflux into the culture medium, increased progressively with the duration of anoxic exposure, becoming significant after 10 h. Reoxygenation significantly increased CEC anoxic injury in a time-dependent manner. A 55% increase in oxygen free radical production, determined by fluorescence detection of dihydroethidium oxidation, was measured at the end of 4-h reoxygenation in CECs subjected to AX/RO conditions that killed 40% of the cells. Blockade of oxygen free radical production with superoxide dismutase (SOD; 250 and 1000 U/ml) or oxypurinol (50 and 200 microM), a potent xanthine oxidase inhibitor, reduced this injury by 32-36% and 30-39%, respectively. Results from our in vitro model indicate that CECs produce significant amounts of oxygen free radicals following ischemia, primarily from the xanthine oxidase pathway. These radicals ultimately have a cytotoxic effect on the very cells that produced them. Thus, reductions in oxygen free radical-mediated vascular injury may contribute to improvements in neurophysiologic outcome following treatment with oxygen free radical inhibitors and scavengers.

Topics: Animals; Brain Ischemia; Cells, Cultured; Cerebrovascular Circulation; Endothelium, Vascular; Enzyme Inhibitors; Microcirculation; Oxypurinol; Reperfusion Injury; Superoxide Dismutase; Superoxides; Swine; Xanthine Oxidase

50 Sprague-Dawley rats were used to study the effect of allopurinol and prostaglandin E1 (PGE1) on renal ischemia-reperfusion injury. They underwent left renal ischemia for 1 h and reperfusion. A right nephrectomy was performed, and 5 groups were made. Group AP received allopurinol 50 mg/kg and PGE1 20 micrograms/kg; group A, allopurinol; group P, PGE1; group C, control, and group S, sham group. Five animals from each group were used to study renal functions and 5 for renal histology. The serum creatinine values were lower in the treatment groups compared to the controls on days 1-3 and 7 (p < 0.05). The blood urea nitrogen values showed a similar trend. Maximum histological damage was seen in group C, followed by groups A, P and AP, in this order. We conclude that allopurinol and PGE1 attenuate renal ischemia-reperfusion injury in rats.

Topics: Allopurinol; Alprostadil; Animals; Blood Urea Nitrogen; Creatinine; Drug Therapy, Combination; Ischemia; Kidney; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Lung injury often occurs after hepatoenteric ischemia, with xanthine oxidase (XO, an oxidant-generating enzyme), released from reperfusing liver and intestines, mediating a significant component of this injury. Since pentastarch administration decreases intestinal reperfusion injury, we determined whether resuscitation with PentaLyte (a pentastarch-containing solution) would decrease hepatoenteric reperfusion injury, xanthine oxidase release, and concomitant lung injury after aortic occlusion- reperfusion. Aortic occlusion was established in rabbits for 40 min, and was followed by 3 h of reperfusion, during which either PentaLyte or lactated Ringer's solution-based resuscitation was administered. Sham-operated animals served as controls. Hepatoenteric reperfusion injury, as manifested by release of the enzyme aspartate aminotransferase and decreased gastric intramucosal pH, was significantly (p < 0.0167) attenuated by PentaLyte administration after aortic occlusion-reperfusion, as compared with its occurrence in animals given lactated Ringer's solution. The release of XO after aortic occlusion-reperfusion was 4-fold smaller after PentaLyte administration than after resuscitation with lactated Ringer's solution (p < 0.05). Pulmonary injury, as defined by an increase in bronchoalveolar lavage fluid (BALF) protein content and lactate dehydrogenase (LDH) activity, was 4-fold less after PentaLyte administration following aortic occlusion-reperfusion than after administration of lactated Ringer's solution (p < 0.05). We conclude that remote pulmonary injury is significantly decreased by concomitant PentaLyte-mediated reduction of hepatoenteric reperfusion injury and XO release.

Topics: Animals; Aorta, Thoracic; Aspartate Aminotransferases; Blood Proteins; Bronchoalveolar Lavage Fluid; Constriction; Electrolytes; Gastric Mucosa; Glucose; Hydrogen-Ion Concentration; Hydroxyethyl Starch Derivatives; Infusions, Intravenous; Intestines; Isotonic Solutions; L-Lactate Dehydrogenase; Liver; Male; Phenylephrine; Plasma Substitutes; Rabbits; Reperfusion Injury; Respiratory Distress Syndrome; Resuscitation; Ringer's Lactate; Sodium Bicarbonate; Xanthine Oxidase

We hypothesized that the extent of injury and release of xanthine oxidase, an oxidant generator, into the circulation would be less in normal-flow hypoxia-reoxygenation than in equal duration no-flow ischemia-reperfusion.. Randomized study.. University-based animal research facility.. Male Sprague-Dawley rats.. The livers were isolated, perfused, and then randomly subjected to 2 hrs of hypoxia (normal flow, low oxygen) or ischemia (no flow, no oxygen), and 2 hrs of reperfusion. Hepatocytes were also isolated, and were subjected to either: a) hypoxia (0, 2, 4, and 6 hrs); or b) hypoxia (2 and 4 hrs) with reoxygenation (2 hrs).. The extent of liver injury (as assessed by release of hepatocellular enzymes) and the release of xanthine oxidase were measured from isolated-perfused rat livers and cultured hepatocytes. The pattern of release of xanthine oxidase in isolated-perfused liver effluent was different in hypoxia-reoxygenation compared with ischemia-reperfusion. During hypoxia, xanthine oxidase gradually increased in the effluent; then, the xanthine oxidase decreased to low concentrations during reoxygenation. After ischemia, there was a sharp spike in xanthine oxidase at 1 min of reperfusion, with a rapid decrease to low concentrations. The total release of xanthine oxidase during hypoxia-reoxygenation was similar to that during ischemia-reperfusion. Lactate dehydrogenase and other markers of liver injury showed a pattern of release that was similar to that of xanthine oxidase, but the total release of markers was not different between the two groups. In hepatocytes, most of the release of enzymes occurred in hypoxia, and the rate of release was not different between hypoxia and hypoxia-reoxygenation.. Hypoxia-reoxygenation results in as much damage to the liver as ischemia-reperfusion, and results in the release of a similar amount of oxidant-producing xanthine oxidase into the circulation.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Hypoxia; Liver; Male; Oxygen; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Xanthine Oxidase

Pulmonary injury occurs after vascular surgery, with xanthine oxidase (an oxidant generator) released from reperfusing liver and intestines mediating a significant component of this injury. Because halogenated anesthetics have been observed to enhance oxidant-mediated injury in vitro, the authors hypothesized that desflurane would increase alveolar-capillary membrane permeability mediated by circulating xanthine oxidase after thoracic occlusion and reperfusion.. Rabbits were assigned to one of five groups: aorta occlusion groups administered desflurane (n=14), desflurane and tungstate (xanthine oxidase inactivator, n=12), fentanyl plus droperidol (n=13), and two sham-operated groups (desflurane, n=7 and fentanyl plus droperidol, n=7). Aortic occlusion was maintained for 45 min with a balloon catheter, followed by 3 h of reperfusion. Alveolar-capillary membrane permeability was assessed by measurement of bronchoalveolar lavage fluid protein. Xanthine oxidase activity was determined in plasma and lung tissue. Ascorbic acid content (an antioxidant) was determined in lung tissue.. Desflurane was associated with significantly increased alveolar-capillary membrane permeability after aortic occlusion-reperfusion when compared with the fentanyl plus droperidol anesthesia or sham-operated groups (P < 0.05). Inactivation of xanthine oxidase abrogated the alveolar-capillary membrane compromise associated with desflurane. Although significantly greater than for sham-operated animals, plasma xanthine oxidase activities released after aortic occlusion-reperfusion were not different between the two anesthetics. There were no anesthetic-associated differences in lung tissue xanthine oxidase activity. However, desflurane anesthesia resulted in a significant reduction in lung ascorbic acid after aortic occlusion-reperfusion compared with the sham-operated animals.. Desflurane anesthesia increased xanthine oxidase-dependent alveolar-capillary membrane compromise after aortic occlusion-reperfusion in concert with depletion of a key tissue antioxidant.

Topics: Anesthetics, Inhalation; Anesthetics, Intravenous; Animals; Capillary Permeability; Desflurane; Droperidol; Fentanyl; Isoflurane; Male; Pulmonary Alveoli; Rabbits; Reperfusion Injury; Xanthine Oxidase

The isolated perfused rat liver model was used to assess graft viability after 24 h of cold preservation. Two solutions were compared for liver preservation: Belzer's original UW solution (high-K+ UW) and a solution containing the same components but with inverted concentrations of sodium and potassium (high-Na+ UW). During the 120 min of normothermic reperfusion, livers preserved in the high-Na+ UW solution released lower levels of creatine kinase-BB isoenzyme, transaminases (ALT and AST), and potassium than those preserved in the high-K+ UW solution. Bile flow and biliary excretion of indocyanine green increased when livers were preserved in the high-Na+ UW solution. We found no statistical differences for oxygen consumption and tissue ATP concentration. The results of this study support the concept that a high-Na+ UW solution is a more effective means of preserving rat livers, at least after 24 h of cold-storage and 120 min of reperfusion in the isolated perfused model, than the original high-K+ UW solution. Liver preservation in the high-Na+ UW solution reduces damage to sinusoidal endothelial and hepatocellular cells. The use of an extracellular-like Belzer cold storage solution eliminates potassium-related problems in cold preservation and subsequent normothermic reperfusion while keeping all the qualities of the original UW solution.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Creatine Kinase; Cryopreservation; Glutathione; Indocyanine Green; Insulin; Isoenzymes; Liver; Liver Circulation; Liver Transplantation; Male; Microcirculation; Organ Preservation Solutions; Potassium; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Sodium

Xanthine oxidase (XO) is conventionally known as a generator of reactive oxygen species (ROS) which contribute to hypoxic-reperfusion injury in tissues. However, this role for human XO is disputed due to its distinctive lack of activity towards xanthine, and the failure of allopurinol to suppress reperfusion injury. In this paper, we have employed native gel electrophoresis together with activity staining to investigate the role human xanthine dehydrogenase (XD) and XO in hypoxic reperfusion injury. This approach has provided information which cannot be obtained by conventional spectrophotometric assays. We found that both XD and XO of human umbilical vein endothelial cells (HUVECs) and lymphoblastic leukaemic cells (CEMs) catalysed ROS generation by oxidising NADH, but not hypoxanthine. The conversion of XD to XO was observed in both HUVECs and CEMs in response to hypoxia, although the level of conversion varied. Purified human milk XD generated ROS more efficiently in the presence of NADH than in the presence of hypoxanthine. This NADH oxidising activity was blocked by the FAD site inhibitor, diphenyleneiodonium (DPI), but was not suppressible by the molybdenum site inhibitor, allopurinol. However, in the presence of both DPI and allopurinol the activities of XD/XO were completely blocked with either NADH or hypoxanthine as substrates. We conclude that both human XD and XO can oxidise NADH to generate ROS. Therefore, the conversion of XD to XO is not necessary for post-ischaemic ROS generation. The hypoxic-reperfusion injury hypothesis should be reappraised to take into account the important role played by XD and XO in oxidising NADH to yield ROS.

Topics: Allopurinol; Cell Hypoxia; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Endothelium, Vascular; Enzyme Inhibitors; Humans; Hypoxia; Ischemia; Lymphocytes; Milk, Human; NAD; Onium Compounds; Oxidation-Reduction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Reactive Oxygen Species; Reperfusion Injury; Spectrophotometry; Substrate Specificity; Tumor Cells, Cultured; Umbilical Veins; Xanthine Dehydrogenase; Xanthine Oxidase

Organ preservation injury is associated with endothelial cell damage, destabilization of mitochondrial and cell membranes, and the release of proteolytic enzymes. In addition to its well-known clinical effect of reducing perioperative blood loss, aprotinin has antiproteolytic and membrane-stabilizing properties. We hypothesized that adding aprotinin to Euro-Collins (EC) and University of Wisconsin (UW) solutions would decrease preservation injury in cultured endothelial cells and a whole organ rat lung model.. Bovine aortic endothelial cells were cultured and stored in the respective solution at 4 degrees C for 12 or 48 hours. Endothelial cell viability after storage was assessed by dimethylthiazole tetrazolium cytotoxicity assay. In the whole organ model, rat lungs were isolated, flushed with the respective solution, and stored at 4 degrees C for 6 or 12 hours. The lungs were ventilated with 100% O2 and reperfused with fresh blood. Alveolar-arterial O2 difference, O2 tension, capillary filtration coefficient, and compliance were determined.. Endothelial cell viability was optimized with the addition of aprotinin to EC and UW at a dose of 150 KIU/mL (0.02 mg/mL). In the isolated perfused lung model, after 6 hours of ischemic storage, aprotinin-enhanced (100 KIU/mL [0.014 mg/mL]) EC and UW decreased alveolar-arterial O2 difference, increased O2 tension, and decreased capillary filtration coefficient compared with EC and UW alone. After 12 hours of ischemic storage, aprotinin-enhanced EC and UW decreased alveolar-arterial O2 difference, increased O2 tension, decreased capillary filtration coefficient, and increased compliance compared with EC and UW alone.. The addition of aprotinin to EC and UW solutions increases endothelial cell viability in hypoxic cold storage conditions. In terms of whole organ function, aprotinin improves lung preservation as demonstrated by increased oxygenation and compliance, and decreased capillary permeability. This study is clinically applicable as there is already extensive experience with the use of aprotinin in heart and lung transplant recipients, in addition to its routine use in conventional cardiac operations.

Topics: Adenosine; Allopurinol; Animals; Aprotinin; Capillaries; Cattle; Cell Membrane; Cell Survival; Cells, Cultured; Cryopreservation; Endopeptidases; Endothelium, Vascular; Glutathione; Hemostatics; Hypertonic Solutions; Insulin; Lung Compliance; Lung Diseases; Lung Transplantation; Male; Mitochondria; Organ Preservation Solutions; Oxygen; Oxygen Consumption; Protease Inhibitors; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Ventilation-Perfusion Ratio

To investigate the effect of methylene blue, an inhibitor of oxygen radicals, on lung injury caused by reperfusion of ischemic tissue.. Intestinal ischemia-reperfusion injury was induced in rats by clamping the superior mesenteric artery for 1 hour. Thereafter, the experimental group was administered 1% methylene blue intraperitoneally and the control group received saline. After 4 hours, pulmonary histopathologic features were assessed, and lung wet-weight to dry-weight ratios and tissue xanthine oxidase were determined.. The control group suffered from severe pulmonary parenchymal damage, compared with slight damage in the experimental group. The number of sequestered neutrophils was significantly higher in the control group (319 +/- 60 polymorphonuclear cells per 10 high-power fields) than in the methylene blue-treated group (91 +/- 8 polymorphonuclear cells per 10 high-power fields; p < 0.001). The wet-weight to dry-weight ratio was significantly increased in the saline-treated rats compared with the methylene blue-treated group (6.19 +/- 0.28 vs. 5.07 +/- 0.21; p < 0.001). Xanthine oxidase activity was similar in both groups.. Methylene blue attenuated lung injury after intestinal ischemia-reperfusion. Inhibition of oxygen free radicals may be the protective mechanism.

Topics: Animals; Antioxidants; Disease Models, Animal; Drug Evaluation, Preclinical; Intestines; Leukocyte Count; Male; Methylene Blue; Neutrophils; Organ Size; Random Allocation; Rats; Rats, Wistar; Reperfusion Injury; Respiratory Distress Syndrome; Xanthine Oxidase

1. An intervention to reduce ischaemia-reperfusion lung injury will be an important advance in transplant medicine. Although the mechanisms associated with producing ischaemia-reperfusion endothelial injury have not been completely elucidated, many of the injury mediators have been studied in detail. While no single pharmacological therapy is likely to be totally effective in eliminating this complex injury, we have developed a mixture of agents that are known to block pathways involved in producing ischaemia-reperfusion-associated lung vascular injury.2. The present study modified University of Wisconsin solution (UW) by adding one of the protective agents prostaglandin E1 (PGE1), dexamethasone (Dex) or dibutyryl cAMP (Bt2-cAMP), or a combination of these, to the perfusate of rat lungs exposed to 4 h of cold ischaemia followed by 1 h of reperfusion. Nine modified UW solutions were studied: (1) UW+Dex, (2) UW+PGE1, (3) UW+Bt2-cAMP, (4) UW+Dexx3, (5) UW+PGE1x3, (6) UW+Bt2-cAMPx3, (7) UW+Dex+PGE1, (8) UW+Dex+Bt2-cAMP, (9) UW+PGE1+Bt2-cAMP. These solutions were utilized in individual experiments to assess haemodynamic changes, lung weight gain, the capillary filtration coefficient (Kfc) and pathology in all lungs.3. The results indicate that lung weight gain and Kfc values were significantly lower than with UW alone in groups 1, 2 and 3, which contained only one additional protective agent. In groups 4, 5 and 6, which contain three times the concentration of each protective agent, both Kfc and lung weight gain were similar to those measured in groups 1, 2 and 3, i.e. lungs were protected but the protection was not dose dependent. In groups 7, 8 and 9, which contained two protective agents, lung weight gain and Kfc were greatly reduced compared with UW alone. Histopathological studies showed similar decreases in the injury profiles of lungs.4. Although UW contains several antioxidant protective agents such as allopurinol and glutathione, it did not provide effective protection in our ischaemia-reperfusion lung injury model. UW modified with an additive of PGE1, Dex or Bt2-cAMP attenuated ischaemia-reperfusion injury. Furthermore, UW containing two of these protective agents augmented the protection. Among the modified solutions, it appears that UW+PGE1+Bt2-cAMP protects the lungs to a greater extent than all other solutions used in our study. We suggest that preservation solutions containing PGE1-Bt2-cAMP will provide additional protective effects to organs s

Topics: Adenosine; Allopurinol; Analysis of Variance; Animals; Anti-Inflammatory Agents; Bucladesine; Dexamethasone; Glutathione; Insulin; Lung; Lung Transplantation; Male; Organ Preservation Solutions; Organ Size; Prostaglandins E; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tissue Preservation; Vascular Resistance

Topics: Adenosine; Alkanesulfonates; Allopurinol; Animals; Enzyme Inhibitors; Glutathione; Insulin; Intestinal Mucosa; Intestines; Microvilli; Organ Preservation; Organ Preservation Solutions; Phospholipases A; Phospholipases A2; Raffinose; Rats; Reperfusion Injury

Topics: Adenosine; Allopurinol; Animals; Glutathione; Insulin; Intestine, Small; Ischemia; Male; Organ Preservation; Organ Preservation Solutions; Phospholipases A; Proteins; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Time Factors

Topics: Adenosine; Allopurinol; Animals; Female; Glutathione; Graft Rejection; Insulin; Intestinal Mucosa; Intestine, Small; Muscle, Smooth; Myenteric Plexus; Neurons; Organ Preservation; Organ Preservation Solutions; Phosphopyruvate Hydratase; Raffinose; Reperfusion Injury; S100 Proteins; Swine; Transplantation, Homologous

Topics: Acetylcysteine; Allopurinol; Animals; Glutathione; Intestine, Small; Ischemia; Lung; Malondialdehyde; Myocardium; Necrosis; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury

Nonspecific injury in cadaveric renal transplants adversely affects early graft function and influences long-term graft survival after organ transplantation. Trimetazidine (TMZ) has been reported to exert a protective action against normothermic ischemia and reperfusion injury in several experimental and clinical studies. In an isolated perfused pig kidney model, we investigated the effects of TMZ added to University of Wisconsin solution (UW) during 48 or 72 h of cold storage (CS) and the consequence during reperfusion. Under all conditions tested renal perfusate flow rate (PFR), renal functions, and tubular injury markers were determined during a 120-min perfusion period. Lipid peroxidation and histological examination (optical and electron microscopy) were also determined after CS and reperfusion. The addition of TMZ (10(-6) M) to the UW solution improved dramatically the quality of preserved kidneys and consequently the functional recovery during reperfusion. TMZ + UW also significantly had a protecting role against reperfusion injury and lipid peroxidation when compared to UW alone. These results were correlated with both a better preservation of the proximal brush border membrane and reduced cellular and mitochondrial swelling. These results also suggested that the TMZ-induced renoprotection correlated well with the observed decrease membrane lipid peroxidation. Therefore, trimetazidine may be useful for clinical kidney graft preservation.

Topics: Adenosine; Allopurinol; Animals; Citric Acid; Cold Temperature; Glomerular Filtration Rate; Glutathione; Humans; Insulin; Kidney; Kidney Transplantation; Kidney Tubules; Lactic Acid; Lipid Peroxidation; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Reperfusion Injury; Swine; Trimetazidine

Cyclic nucleotides mediate intracellular signal transduction of several vasodilators. In addition to its vascular relaxant effects, cAMP is known to protect endothelial cells and to suppress Kupffer cell activation. On the other hand, cGMP potently ameliorates adhesion of leukocytes and platelets. We tested the effects of two analogs of cyclic nucleotides (8bromo cyclic adenosine monophosphate [8br-cAMP] and 8bromo cyclic guanosine monophosphate [8br-cGMP]) in rat liver preservation.. In experiment 1, either analog (0.1-1.0 mM) alone was added to University of Wisconsin (UW) solution in a survival study. In experiment 2, donors and recipients were also treated with 8br-cAMP or 8br-cGMP, with the following three groups tested: group 1=control; group 2=administration of 8br-cAMP to donors, UW solution, and recipients; group 3=administration of 8br-cGMP to donors, UW solution, and recipients. Experiment 3 tested combined treatments: group 4=administration of 8br-cGMP to donors and UW solution, and cAMP to recipients; group 5=administration of 8br-cAMP to donors and UW solution, and 8br-cGMP to recipients. To elucidate the roles of each nucleotide, two further groups were tested: group 6=administration of 8br-cAMP to donors and UW solution; group 7=administration of 8br-cGMP to recipients. In experiment 4, rats in groups 1, 5, 6, and 7 were killed at several time points after reperfusion, and percent graft blood flow (%BF), number of accumulated neutrophils, plasma levels of tumor necrosis factor-alpha and interleukin-1, and serum alanine aminotransferase levels were examined.. In experiments 1 and 2, no significant effect was observed on animal survival. In experiment 3, a significant increase in animal survival was observed only in group 5 (100%, 7/7, P=0.0004 vs. group 1: 16.7%, 2/12). In group 5, no improvement of %BF was observed during the early phase of reperfusion (15 and 30 min) compared with that in group 1. On the other hand, the %BF of group 5 was significantly higher in the later phase (6 hr), consistent with the decrease in accumulation of neutrophils observed then. Production of tumor necrosis factor-alpha and serum alanine aminotransferase levels were also reduced with this treatment. Histologically, the bleeding and segmental necrosis, observed in group 1, were completely prevented in group 5.. We conclude that restoration of grafts with cAMP and administration of cGMP to recipients led to successful transplantation, and that the two analogs acted synergistically in opposing preservation and reperfusion injury without improvement of graft blood flow during the early phase of reperfusion. The effect was due to their regulation of neutrophil activation and sequestration.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Allopurinol; Animals; Cyclic GMP; Drug Combinations; Drug Synergism; Glutathione; Insulin; Interleukin-1; Liver; Liver Circulation; Male; Organ Preservation; Organ Preservation Solutions; Postoperative Period; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Tissue Donors; Tumor Necrosis Factor-alpha

Relative hypoventilation, involving passively-or "permissively"-generated hypercapnic acidosis (HCA), may improve outcome by reducing ventilator-induced lung injury. However, the effects of HCA per se on pulmonary microvascular permeability (Kf,c) in noninjured or injured lungs are unknown. We investigated the effects of HCA in the isolated buffer-perfused rabbit lung, under conditions of: (1) no injury; (2) injury induced by warm ischemia-reperfusion; and (3) injury induced by addition of purine and xanthine oxidase. HCA (fraction of inspired carbon dioxide [FICO2] 12%, 25% versus 5%) had no adverse microvascular effects in uninjured lungs, and prevented (FICO2 25% versus 5%) the increase in Kf,c following warm ischemia-reperfusion. HCA (FICO2 25% versus 5%) reduced the elevation in Kf,c, capillary (Pcap), and pulmonary artery (Ppa) pressures in lung injury induced by exogenous purine/xanthine oxidase; inhibition of endogenous NO synthase in the presence of 25% FICO2 had no effect on Kf,c, but attenuated the reduction of Pcap and Ppa. HCA inhibited the in vitro generation of uric acid from addition of xanthine oxidase to purine. We conclude that in the current models, HCA is not harmful in uninjured lungs, and attenuates injury in free-radical-mediated lung injury, possibly via inhibition of endogenous xanthine oxidase.

Topics: Acidosis; Analysis of Variance; Animals; Blood Pressure; Capillaries; Capillary Permeability; Carbon Dioxide; Free Radicals; Hydrostatic Pressure; Hypercapnia; Lung; Male; Microcirculation; Nitric Oxide Synthase; Pulmonary Artery; Purines; Rabbits; Reperfusion Injury; Respiration, Artificial; Respiratory Distress Syndrome; Uric Acid; Vascular Resistance; Xanthine Oxidase

We investigated the effects of the xanthine oxidase inhibitor, BOF-4272, on the production of cytokine-induced neutrophil chemoattractant (CINC) following reperfusion injury in rat liver. Ischemia was induced for 30 minutes by portal vein occlusion. Animals were pretreated with intravenous injection of BOF-4272 (1 mg/kg) or heparin (50 U/kg) 5 minutes before vascular clamp. Both BOF-4272 and heparin limited increases in the chemoattractant compared with nonpretreated rats. Pretreatment with BOF-4272 plus heparin resulted in an additive effect. Most cells immunostained for chemoattractant were macrophages in sinusoids. In vitro chemoattractant production by Kupffer cells isolated from animals pretreated with heparin or BOF-4272 was significantly lower than by Kupffer cells from nonpretreated animals. Expression of transcripts in liver for chemoattractant peaked 3 hours after reperfusion in nonpretreated animals, while pretreatment with heparin or BOF-4272 significantly decreased chemoattractant mRNA levels. In vitro chemoattractant transcription and production could be induced in naive Kupffer cells by hypoxanthine and xanthine oxidase, but BOF-4272 prevented these increases. We conclude that Kupffer cells release chemoattractant in response to oxygen radicals reducible by xanthine oxidase inhibition.

Topics: Animals; Chemokines, CXC; Chemotactic Factors; Enzyme Inhibitors; Growth Substances; Heparin; Intercellular Signaling Peptides and Proteins; Ischemia; Kupffer Cells; Liver; Liver Circulation; Male; Neutrophils; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Triazines; Xanthine Oxidase

Topics: Adenosine; Adolescent; Adult; Aged; Allopurinol; Aspartate Aminotransferases; Cold Temperature; Glutathione; Humans; Insulin; Ischemia; Liver; Liver Circulation; Liver Transplantation; Microcirculation; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose; Regression Analysis; Reperfusion Injury; Time Factors

Topics: Adenosine; Allopurinol; Animals; Antioxidants; Cold Temperature; Creatinine; Diuresis; Dogs; Glutathione; Hypertonic Solutions; In Vitro Techniques; Insulin; Kidney; Kidney Transplantation; Male; Niacinamide; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Transplantation, Autologous

Topics: Acetylcysteine; Adenosine; Allopurinol; Animals; Antibodies, Monoclonal; Choline Deficiency; Fatty Liver; Glutathione; Insulin; Intercellular Adhesion Molecule-1; Ischemia; Liver; Male; Methionine; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury

The effect of ad libitum oral-administration of (-)catechin solution on ischemia-reperfusion-induced cell death of hippocampal CA1 in the gerbil was histologically examined. When (-)catechin solution instead of drinking water was orally administered ad libitum for 2 weeks, dose-dependent protection against neuronal death following by transient ischemia and reperfusion was observed. To evaluate the involvement of reduction of reactive-oxygen-species (ROIs) by the antioxidant activity of (-)catechin in this protection, the superoxide scavenging activity of the brain in catechin-treated gerbils was measured by ESR and spin-trapping using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The superoxide scavenging activities of the brains obtained from catechin-treated gerbils were significantly higher than those of catechin-untreated animals. From these results, it was suggested that orally administered (-)catechin was absorbed, passed through the blood-brain barrier and that delayed neuronal death of hippocampal CA1 after ischemia-reperfusion was prevented due to its antioxidant activities.

Topics: Administration, Oral; Animals; Antioxidants; Blood-Brain Barrier; Catechin; Cell Death; Cyclic N-Oxides; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Gerbillinae; Hippocampus; Ischemic Attack, Transient; Neurons; Prosencephalon; Reactive Oxygen Species; Reperfusion Injury; Spin Labels; Superoxides; Xanthine Oxidase

Ventricular arrhythmias were studied in rat isolated hearts subjected to coronary artery occlusion and reperfusion. Free radicals in the perfusate were detected by continuous flow luminol-enhanced chemiluminescence. Administration of purine (2.3 mM) and xanthine oxidase (0.12 U ml-1 min-1) did not significantly modify the severity of reperfusion-induced arrhythmias but did generate free radicals. No free radical generation was detected during the period of coronary artery occlusion or reperfusion. Superoxide dismutase (SOD) 20-80 U ml-1 did not alter the severity of reperfusion arrhythmias but, in the presence of 80 U ml-1 SOD, occlusion-induced arrhythmias were augmented. SOD did not produce any effect on haemodynamics at the concentrations tested. Ventricular arrhythmias and cardiac haemodynamics were also not significantly changed by the combination of scavengers, SOD (10 U ml-1), catalase (100 U ml-1) and mannitol (20 mM). These data suggest that the superoxide free radical is unlikely to be the primary cause of reperfusion induced arrhythmias in rat isolated hearts subjected to regional ischaemia.

Topics: Animals; Arrhythmias, Cardiac; Catalase; Coronary Disease; Free Radical Scavengers; Free Radicals; In Vitro Techniques; Luminescent Measurements; Male; Mannitol; Oxygen Compounds; Purines; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

Triple therapy with S-adenosylmethionine (SAM) (given to the donor animal, included in University of Wisconsin solution [UW], and added to the reperfusing medium) has been shown to reduce the sequential cold and warm ischemia/reperfusion injuries characteristic of the liver transplantation procedure. To clarify the actions of SAM during different stages of ischemia/ reperfusion, we have compared its benefit in five dosage regimens, using perfused rat livers after sequential periods of 24 hr cold and 20 min rewarming ischemia. When added only to UW, the presence of SAM throughout ischemia improved hepatic blood flow by 26% after 15 min of reperfusion versus no treatment (2.32+/-0.18 vs. 1.84+/-0.11 ml/min/g liver, P<0.05). SAM also improved blood flow by 23% during the 3-hr perfusion overall (P<0.05). Oxygen consumption and the release of purine nucleoside phosphorylase (PNP) were decreased (both P<0.05). When added to both UW and the perfusate, SAM additionally increased bile production at 15 min (7.14+/-1.21 vs. 2.31+/-0.74 mg/h/g liver, P<0.01). By pretreating the liver donor with SAM in vivo, and including it in the preservation and reperfusing media, it was possible to prolong and amplify the benefits on blood flow (P<0.001) and bile production (P<0.05) and to sustain glucose uptake (P<0.01). An acute exposure to SAM, when used in saline to flush UW from the graft before reperfusion, increased blood flow at 15 min (by 68%) and over a 3-hr period (both P<0.001), but no indices of metabolic activity were improved. Oxygen consumption and PNP release were both decreased (P<0.05). When added to the perfusate (present throughout reperfusion), SAM increased blood flow at 15 min (58%) and over a 3-hr period (P<0.01 in both cases). Net glucose uptake was increased (P<0.05), whereas oxygen consumption (P<0.001) and PNP release fell (P<0.05). Actions of SAM achieved acutely and over the intermediate- and long-term all seem to underlie its benefits in reducing ischemia/reperfusion injuries.

Topics: Adenosine; Allopurinol; Animals; Bile; Glutathione; Insulin; Ischemia; Liver; Liver Transplantation; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; S-Adenosylmethionine

The effects of free radical generating systems on basal and ischemia/reperfusion-evoked release of amino acids into cortical superfusates was examined in the rat using the cortical cup technique. Xanthine oxidase plus xanthine significantly enhanced GABA levels 358 fold over controls during 20 min of four vessel occlusion. Glutamate and phosphoethanolamine release following reperfusion were also elevated. Prostaglandin synthase plus arachidonic acid significantly enhanced the ischemia-evoked release of all amino acids (aspartate 360 fold; glutamate 433 fold; glycine 6 fold; GABA 689 fold; phosphoethanolamine 69 fold) and increased the pre-ischemic levels of glutamate, glycine and phosphoethanolamine. Administration of H2O2 plus ferrous sulfate significantly elevated both pre-ischemic amino acid release and ischemia-evoked release. A role for free radical generating systems in the development of ischemic injury is supported by the ability of superoxide dismutase plus catalase to reduce ischemia-evoked amino acid efflux into cortical superfusates. Thus, the species of free radical produced, as well as the amount generated, may after the pattern of amino acid release under both ischemic and non-ischemic conditions.

Topics: Animals; Basal Metabolism; Cerebral Cortex; Free Radicals; Ischemic Attack, Transient; Male; Neurotransmitter Agents; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Xanthine Oxidase

Allopurinol (AP) protects skeletal muscle function against ischaemia-induced injury, but the mechanism is not yet clear. As AP acts as a competitive xanthine oxidase inhibitor, both a reduction of oxygen-derived free radicals and an enhancement of purine resynthesis (salvage pathway) might be involved. We investigated the in vivo kinetics of high-energy phosphates in skeletal muscle after AP pretreatment using 31P-magnetic resonance spectroscopy during 2 h of ischaemia and 3 h of reperfusion in rat hindlimbs. Three animals (group A) were pretreated with a total of 160 mg/kg AP i.p., 3 control animals (group B) received the same amount of 0.9% saline solution. ATP decreased to 18.6 +/- 1.3% of the pre-ischaemic value in group A and to 17.3 +/- 2.8% in group B after 2 h of ischaemia, and rose to only 47.7 +/- 1.5 and 50.5 +/- 1.8%, respectively, after 3 h of reperfusion. Phosphocreatine fell to 7.2 +/- 2.9 and 7.6 +/- 2.2% of pre-ischaemic values after 2 h of ischaemia and rose again to 36.5 +/- 12.9 and 45.4 +/- 20.4% after 3 h of reperfusion. Inorganic phosphate (Pi) increased 5-fold after 2 h of ischaemia, irrespective of the treatment. After 3 h of reperfusion, Pi was still 4 times the pre-ischaemic value. The kinetics of ATP, PCr, and Pi levels were not statistically different between the two groups. These results indicate that the ATP salvage pathway does not play an important role in AP-induced attenuation of ischaemia/reperfusion-induced muscle damage.

Topics: Adenosine Triphosphate; Allopurinol; Animals; Ischemia; Magnetic Resonance Spectroscopy; Male; Muscle, Skeletal; Phosphocreatine; Rats; Rats, Wistar; Reperfusion Injury

Oxygen free radical generation by xanthine oxidase (XO) is a possible mechanism in the injury following reperfusion of transplanted organs. This study was undertaken to investigate XO in human lung, and to investigate whether XO is released into the blood stream during the immediate postoperative period after lung transplantation. XO activity was measured in healthy human lung tissue, and XO protein and the adenine nucleotide catabolic products hypoxanthine, xanthine and uric acid were analysed in the plasma samples collected during human heart-lung transplantation (n=4), double lung transplantation (n=2), and single lung transplantation (n=1). Neutrophil degranulation was assessed by plasma lactoferrin measurements. The results indicated that XO activity (detection limit 5 pmol x min(-1) x mg(-1) protein) and protein (detection limit 5 ng x mg-1 protein) were undetectable in the lungs of five healthy individuals. Similarly, no XO protein could be found in the plasma samples from the right ventricle or left atrium during and after the transplantation in any of the cases. Plasma xanthine and hypoxanthine concentrations were elevated 2-10 fold immediately after the reperfusion of the transplant, indicating washout of high-energy phosphate degradation products from the ischaemic lung. Plasma uric acid decreased rather than increased immediately after the surgery and during the following 24 h. Lactoferrin was elevated during the surgery. In conclusion, these results show that XO activity in human lung is low, it is not released into the blood stream during human heart-lung transplantation, and it is unlikely to contribute to postoperative complications in these patients.

Topics: Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Free Radicals; Heart-Lung Transplantation; Humans; Hypoxanthine; Lactoferrin; Lung; Lung Transplantation; Middle Aged; Postoperative Complications; Reperfusion Injury; Uric Acid; Xanthine; Xanthine Oxidase; Xanthines

We have revealed that acute gastric mucosal injury induced by a single ischemia-reperfusion (I-R) treatment develops into an ulcerative lesion within a few days. In the present studies, we examined the effects of oral administration of sucralfate on gastric damage induced by I-R. Sucralfate (1-100 mg/kg, 15 min before I-R) significantly reduced the total erosion area observed immediately after I-R. A high dose of sucralfate (30-100 mg/kg) inhibited the increase in the thiobarbituric acid-reactive substances, an index of lipid peroxidation, induced by I-R, although a low dose of it failed. When sucralfate (30 mg/kg, once a day) was orally administered after I-R, it prevented mucosal damage from developing into gastric ulcers: the total area of the ulcers was significantly reduced compared to that without sucralfate administration 72 h after I-R. High concentrations of sucralfate (3-10 mg/ml) reduced the superoxide radicals generated by leukocytes or xanthine-xanthine oxidase, and protected erythrocyte membrane ghosts against lipid peroxidation induced by hydrogen peroxide and Fe2+ in vitro. These results indicate that sucralfate may prohibit both the generation of acute gastric mucosal injury and its progression to ulcer induced by I-R, probably due to a cytoprotective action on the mucosal surface. However, the protective mechanism may involve an inhibitory action on superoxide and hydroxyl radicals at high doses.

Topics: Animals; Anti-Ulcer Agents; Erythrocyte Membrane; Gastric Mucosa; Hydroxyl Radical; Leukocytes; Lipid Peroxides; Male; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Stomach Ulcer; Sucralfate; Superoxides; Thiobarbituric Acid Reactive Substances; Xanthine Oxidase

Intestinal ischemic injury is exacerbated by reperfusion in rodent and feline models because of xanthine oxidase-initiated reactive oxygen metabolite formation and neutrophil infiltration. Studies were conducted to determine the relevance of reperfusion injury in the juvenile pig, whose low levels of xanthine oxidase are similar to those of the human being.. Ischemia was induced by means of complete mesenteric arterial occlusion, volvulus, or hemorrhagic shock. Injury was assessed by means of histologic examination and measurement of lipid peroxidation. In addition, myeloperoxidase, as a marker of neutrophil infiltration, and xanthine oxidase-xanthine dehydrogenase were measured.. Significant ischemic injury was evident after 0.5 to 3 hours of complete mesenteric occlusion or 2 hours of shock or volvulus. In none of these models was the ischemic injury worsened by reperfusion. To maximize superoxide production, pigs were ventilated on 100% O2, but only limited reperfusion injury (1.2-fold increase in histologic grade) was noted. Xanthine oxidase-xanthine dehydrogenase levels were negligible (0.4 +/- 0.4 mU/gm).. Reperfusion injury may not play an important role in intestinal injury under conditions of complete mesenteric ischemia and low-flow states in the pig. This may result from low xanthine oxidase-xanthine dehydrogenase levels, which are similar to those found in the human being.

Topics: Animals; Female; Intestinal Mucosa; Intestines; Ischemia; Male; Peroxidases; Reperfusion Injury; Swine; Xanthine Dehydrogenase; Xanthine Oxidase

This study investigated metabolic and biochemical consequences of colonic ischemia/reperfusion (I/R) in the rat and evaluated whether antioxidants prevent I/R-induced functional damage in the rat colon. The surgical preparation involved a 10 cm segment of the colon and occlusion of the superior mesenteric artery (SMA) to induce I/R. Arterial blood from the aorta and venous blood from the superior mesenteric vein (SMV) was collected to measure blood gases, lactic acid (LA) and arachidonic acid (AA) metabolites. Tissue xanthine oxidase (XO) and thiobarbituric acid (TBA) derivatives were measured before and after reperfusion. In addition, vascular and mucosal permeability, and the effect of MDL 73404 (a water soluble vitamin E analog) and 5-aminosalicylic acid on LA, AA, XO and TBA was measured. After ischemia, the colon displayed a metabolic shift from aerobic to anaerobic course by increasing lactic acid production in the colon (183% increase in SMV lactate level compared 87% in the SMA; p < 0.03). After 10 minutes of reperfusion, circulating 6-keto-prostaglandin F1 alpha increased by 3.85 fold (p < 0.001) and thromboxane B2 increased by 2 to 3 fold. An Ischemia time longer than 60 minutes was required to cause changes in tissue XO levels. Tissue TBA levels showed a good dose response corresponding with I/R time. I/R (60 minutes) caused a three and 16 fold increase (p < 0.01) in vascular and mucosal permeability, respectively. MDL 73404 and 5-aminosalicylic acid significantly inhibited the vascular permeability and decreased LA, AA, XO and TBA. These observations provide the first direct experimental evidence for I/R-induced damage in the colon and some of its effects can be reversed by conventional and novel antioxidants.

Topics: 6-Ketoprostaglandin F1 alpha; Aerobiosis; Aminosalicylic Acids; Anaerobiosis; Animals; Antioxidants; Arachidonic Acid; Colon; Disease Models, Animal; Glutathione; Lactic Acid; Male; Mesalamine; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Thiobarbituric Acid Reactive Substances; Thromboxane B2; Vitamin E; Xanthine Oxidase

The relationship between the changes of active oxygen metabolism and blood flow and the formation, progression, and recovery of lesions was examined in the gastric mucosa of rats treated once with compound 48/80, a mast cell degranulator. Gastric mucosal lesions appeared 0.5 hr after compound 48/80 treatment, became worst at 3 hr, and recovered fairly well at 12 hr. Increases in gastric mucosal lipid peroxide content and xanthine oxidase and myeloperoxidase activities and decreases in gastric mucosal vitamin E and hexosamine contents and Se-dependent glutathione peroxidase activity occurred with the formation and progression of gastric mucosal lesions. These changes were attenuated with the recovery of the lesion. Gastric mucosal nonprotein SH content decreased with the formation of gastric mucosal lesions, and this decreased SH content returned to near the original level with lesion progression. No changes in gastric mucosal superoxide dismutase and catalase activities occurred with the formation, progression, and recovery of gastric mucosal lesions. Gastric mucosal blood flow decreased with the formation of gastric mucosal lesions, and this decreased blood flow recovered with lesion progression. Serum serotonin concentration, an index of mast cell degranulation, increased with the formation of gastric mucosal lesions, and this increased serotonin level was attenuated with lesion progression and recovery. Pretreatment with ketotifen, a connective tissue mast cell stabilizer, prevented the formation of gastric mucosal lesions, the increases of gastric mucosal lipid peroxide content, xanthine oxidase and myeloperoxidase activities, and serum serotonin level; and the decreases of gastric mucosal nonprotein SH content, glutathione peroxidase activity, and blood flow found at 0.5 hr after compound 48/80 treatment. These results indicate that the changes of gastric mucosal active oxygen metabolism and blood flow are closely related to the formation, progression, and recovery of gastric mucosal lesions in rats with a single compound 48/80 treatment. The present results also suggest that this compound 48/80-induced gastric mucosal injury could be a kind of ischemia-reperfusion-induced injury occurring through degranulation of connective tissue mast cells.

Topics: Animals; Cell Degranulation; Gastric Mucosa; Glutathione Peroxidase; Hexosamines; Ketotifen; Lipid Peroxides; Male; Mast Cells; Oxygen Consumption; p-Methoxy-N-methylphenethylamine; Peroxidase; Pyrimidines; Rats; Rats, Wistar; Regional Blood Flow; Reperfusion Injury; Sulfhydryl Compounds; Vitamin E; Xanthine Oxidase

The effects of hypothermic injury to the liver were investigated on an isolated perfusion circuit by comparing porcine livers with varying degrees of preservation injury.. A group of unstored livers (n = 5) were compared to livers stored in University of Wisconsin (UW) solution for 18 h (n = 5), and a group of livers stored in Hartmann's solution for 18 h (n = 5).. We observed that the degree of platelet sequestration was directly related to the severity of the preservation injury. After 2 h of isolated liver perfusion, the perfusate platelet count fell from 148 +/- 14 x 10(9)/L to 84 +/- 13 x 10(9)/L for control livers. In comparison for livers stored in UW solution, the platelet count fell from 173 +/- 43 x 10(9)/L to 61 +/- 14 x 10(9)/L representing a 64.8% fall, while for those stored in Hartmann's solution, an even more profound fall from 152 +/- 36 x 10(9)/L to 19 +/- 9 x 10(9)/L (87.5% fall) was observed. The difference between the UW-stored and Hartmann's-stored livers was significant (P < 0.05). However, using this model, the degree of leukocyte sequestration did not differentiate the groups. Both histological and ultrastructural examination of liver biopsies taken immediately following revascularization demonstrated that for mild degrees of preservation injury following hypothermic storage, changes occur to the sinusoidal lining cells well before changes to the parenchymal elements.. These findings substantiate the hypothesis that the primary injury associated with hypothermia involves the sinusoidal lining cells (non-parenchymal elements), that it is predominantly a reperfusion phenomenon and that efforts at improving preservation should therefore be targeted primarily at these cells and not the hepatocytes.

Topics: Adenosine; Allopurinol; Animals; Biopsy; Cryopreservation; Disease Models, Animal; Glutathione; Insulin; Isotonic Solutions; Liver; Liver Function Tests; Organ Preservation; Organ Preservation Solutions; Platelet Count; Raffinose; Reperfusion Injury; Ringer's Lactate; Swine

Deterioration of energy metabolism and oxidative stress represent fundamental mechanisms in ischemia and reperfusion injury. In a normothermic ischemia/reperfusion rat model, we investigated whether allopurinol (ALL) may improve the scavenging ability of the liver after ischemia. ALL was given prior to ischemia and reperfusion (concentration 100 or 50 mg/kg) and controls were given a placebo. After a basal period of 30 min, 1 h normothermic ischemia was induced in the median and left liver lobes followed by 24 h observation. The overall liver function was assessed by bile secretion, and free oxygen production was assessed by glutathione efflux into bile during the first 60 min of reperfusion and at 24 h. Allopurinol (concentration 100 mg/kg) protected hepatocyte function as bile flow improved significantly in this group after 1 and 24 h of reperfusion compared with that of controls. Oxidative stress was also significantly attenuated in this group, as efflux of glutathione into bile was significantly higher in the ALL group (100 mg/kg) after 24 h but not after 1 h of reperfusion compared with that of controls. All given in a concentration 50 mg/kg had some, but a non-significant, effect. We conclude that biliary glutathione is an important marker of oxidative stress and may reflect the scavenging ability of the liver after ischemic injury. Significant correlation of bile flow with biliary glutathione during reperfusion indicates that oxidative stress is an important mechanism attenuating liver function after ischemia/reperfusion injury.

Topics: Allopurinol; Animals; Bile; Disease Models, Animal; Enzyme Inhibitors; Glutathione; Liver; Male; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

Proteases as well as alterations in intracellular calcium have important roles in hepatic preservation-reperfusion injury, and increased calpain activity recently has been demonstrated in liver allografts. Experiments were designed to evaluate (i) hepatic cytosolic calpain activity during different periods of cold ischemia (CI), rewarming, or reperfusion, and (ii) effects of inhibition of calpain on liver graft function using the isolated perfused rat liver and arterialized orthotopic liver transplantation models. Calpain activity was assayed using the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin (AMC) and expressed as mean +/- SD pmol AMC released/min per mg of cytosolic protein. Calpain activity rose significantly after 24 hr of CI in University of Wisconsin solution and further increased with longer preservation. Activity also increased within 30 min of rewarming, peaking at 120 min. Increased durations of CI preceding rewarming resulted in significantly higher activity (P < 0.01). Calpain activity increased rapidly upon reperfusion and was significantly enhanced by previous CI (P < 0.01). Calpain inhibition with Cbz-Val-Phe methyl ester significantly decreased aspartate aminotransferase released in the isolated perfused rat liver perfusate (P < 0.05). Duration of survival after orthotopic liver transplantation using livers cold-preserved for 40 hr was also significantly increased (P < 0.05) with calpain inhibitor. In conclusion, calpain proteases are activated during each phase of transplantation and are likely to play an important role in the mechanisms of preservation-reperfusion injury.

Topics: Adenosine; Allopurinol; Animals; Calpain; Female; Glutathione; Insulin; Liver; Liver Transplantation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Rats, Wistar; Reperfusion Injury; Tissue Preservation

Recent evidence suggests that oxygen-derived free radicals are involved in mediating gastric microvascular and parenchymal cell injuries induced by ischaemia and reperfusion. Therefore, the effect of the locally acting anti-ulcer drug, sucralfate, was studied on ischaemia and reperfusion (e.g. induced gastric lesions, intraluminal bleeding, changes in vascular permeability and non-protein sulfhydryl levels in the rat stomach). Allopurinol was used as a known standard antioxidant drug. Rats were subjected to 30 min of gastric ischaemia in the presence of 100 mmol/L hydrochloric acid and reperfusion periods of 15, 30 or 60 min duration. The gastric lesions were assessed microscopically under an inverted microscope. The vascular permeability was quantified by measuring the extravasated Evans blue in the stomach. There were significantly greater numbers of gastric lesions, intraluminal bleeding and leakage of Evans blue during all reperfusion periods as compared with those of ischaemia, with maximum effects occurring at 60 min following reperfusion. Pretreatment with sucralfate (31.25-250 mg/kg, p.o.) or allopurinol (12.5-50 mg/kg, i.p.) 30 min before the procedure, dose-dependently reduced the gastric lesions, intraluminal bleeding, and decreased the vascular permeability induced by ischaemia and reperfusion. Furthermore, sucralfate dose-dependently reverses the ischaemia and reperfusion-induced depletion of mucosal non-protein sulfhydryl levels and inhibited the superoxide radical production in both cell-free xanthine-xanthine oxidase and in the stimulated polymorphonuclear cellular systems. These results suggest that the protection produced by sucralfate against gastric injury may be due to its antioxidant effects.

Topics: Allopurinol; Animals; Capillary Permeability; Coloring Agents; Dose-Response Relationship, Drug; Evans Blue; Gastric Mucosa; Ischemia; Male; Neutrophils; Rats; Rats, Wistar; Reperfusion Injury; Sucralfate; Sulfhydryl Compounds; Superoxide Dismutase; Tetradecanoylphorbol Acetate; Time Factors; Xanthine Oxidase; Xanthines

This study examines the hypothesis that intestinal ischemia-reperfusion (I/R) injury contributes to renal dysfunction by altered renal eicosanoid release. Anesthetized Sprague-Dawley rats underwent 60 min of sham or superior mesenteric artery (SMA) occlusion with 60 min of reperfusion. The I/R groups received either allopurinol, pentoxifylline, 1-benzylimidazole, or carrier before SMA occlusion. In vivo renal artery blood flow was measured by Transonic flow probes, the kidneys were then perfused in vitro for 30 min, and the effluent was analyzed for eicosanoid release and renal function. Intestinal I/R caused a twofold increase in the ratio of renal release of thromboxane B2 to prostaglandin E2 and to 6-ketoprostaglandin F1alpha compared with the sham level, with a corresponding 25% decrease in renal sodium and inulin clearance and renal blood flow. Pentoxifylline or allopurinol pretreatment restored renal eicosanoid release and renal sodium and inulin clearance to the sham level but did not alter renal blood flow. Pretreatment with 1-benzylimidazole restored renal function, eicosanoid release, and renal blood flow to sham levels. These data suggest that severe intestinal I/R contributes to the downregulation of renal function. The decrease in renal function is due in part to toxic oxygen metabolites, which occur in the milieu of altered renal eicosanoid release, reflecting a decrease in vasodilator and an increase in vasoconstrictor eicosanoids.

Topics: Allopurinol; Animals; Blood Pressure; Eicosanoids; Imidazoles; In Vitro Techniques; Inulin; Ischemia; Kidney; Male; Pentoxifylline; Rats; Rats, Sprague-Dawley; Renal Circulation; Reperfusion Injury; Sodium; Splanchnic Circulation

A method to reduce ischemia-reperfusion (I/R) injury can be an important criterion to improve the preservation solution. Although University of Wisconsin solution (UW) works as a lung preservation solution, its attenuation effect on I/R injury has not been investigated. We attempted to determine whether, by adding various protective agents, modified UW solutions will enhance the I/R attenuation by UW. We examined the I/R injury in an isolated rat lung model. Various solutions, e.g., physiological salt solution (PSS), UW, and modified UW solutions containing various protective agents such as prostaglandin E1, dexamethasone, U-74389G, or dibutyryl adenosine 3',5'-cyclic monophosphate were perfused individually to evaluate the I/R injury. Isolated rat lung experiments, with ischemia for 45 min, then reperfusion for 60 min, were conducted in a closed circulating system. Hemodynamic changes, lung weight gain (LWG), capillary filtration coefficient (Kfc), protein content of lavage fluid, concentration of cytokines, and lung histopathology were analyzed. Results showed that the acute I/R lung injury with immediate permeability pulmonary edema was associated with an increase in tumor necrosis factor-alpha (TNF-alpha) production. A significant correlation existed between TNF-alpha and Kfc (r = 0.8, P < 0.0001) and TNF-alpha and LWG (r = 0. 9, P < 0.0001), indicating that TNF-alpha is an important cytokine modulating early I/R injury. Significantly lower levels of Kfc, LWG, TNF-alpha, and protein concentration of lung lavage (P < 0.05) were found in the UW-perfused group than in the control group perfused with PSS. Modified UW promoted the protective effect of UW to further decrease Kfc, LWG, and TNF-alpha (P < 0.05). Histopathological observations also substantiated this evidence. In the UW+U-74389G group, bronchial alveolar lavage fluid contained lowest protein concentration. We conclude that the UW solution attenuates I/R injury of rat lung and that the modified UW solutions further enhance the effect of UW in reducing I/R injury. Among modified solutions, UW+U-74389G is the best. Further investigation of the improved effects of the modified UW solutions would be beneficial in lung transplantation.

Topics: Adenosine; Allopurinol; Alprostadil; Animals; Bucladesine; Dexamethasone; Drug Synergism; Glutathione; Hemodynamics; In Vitro Techniques; Insulin; Interleukin-1; Ischemia; Lung; Male; Organ Preservation Solutions; Pregnatrienes; Pulmonary Circulation; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tumor Necrosis Factor-alpha

We hypothesized that multiple organ injury and concentrations of xanthine oxidase (an oxidant-generating enzyme released after hepatoenteric ischemia) would be decreased by the administration of a bolus of a colloid solution at reperfusion.. Randomized, masked, controlled animal study.. University-based animal research facility.. Fifty-four New Zealand white male rabbits, weighing 2 to 3 kg.. Anesthetized rabbits were assigned to either the hepatoenteric ischemia-reperfusion group (n = 27) or the sham-operated group (n = 27). Hepatoenteric ischemia was maintained for 40 mins with a balloon catheter in the thoracic aorta, followed by 3 hrs of reperfusion. Each group was randomly administered a bolus of one of three fluids at the beginning of reperfusion: Hextend (hetastarch solution); 5% human albumin; or lactated Ringer's solution. The investigators were masked as to the identity of the fluid administered.. Multiple organ injury was assessed by the release of lactate dehydrogenase activity into the plasma and by indices of gastric and pulmonary injury. Circulating lactate dehydrogenase activity was significantly greater (p < .001) in animals receiving lactated Ringer's solution than in rabbits receiving either colloid solution. Gastric injury (tissue edema, Histologic injury Score) was significantly decreased (p < .01) by administration of both colloid solutions. Lung injury (bronchoalveolar lavage lactate dehydrogenase activity) was significantly decreased (p < .05) by the hetastarch solution administration. The hetastarch solution administration resulted in 50% less xanthine oxidase activity release during reperfusion compared with albumin or lactated Ringer's solution administration (p < .001).. We conclude that multiple organ injury and xanthine oxidase release after hepatoenteric ischemia-reperfusion are decreased by colloid administration.

Topics: Animals; Disease Models, Animal; Drug Evaluation, Preclinical; Hydroxyethyl Starch Derivatives; Intestines; L-Lactate Dehydrogenase; Liver; Male; Multiple Organ Failure; Plasma Substitutes; Rabbits; Random Allocation; Reperfusion Injury; Xanthine Oxidase

Plaunatol, an anti-ulcer drug, increases prostaglandin content in gastric tissue but its effect on radical-mediated gastric damage or activity against reactive oxygen species is unknown. We examined the effects of oral administration of plaunotol (Kelnac) on the acute gastric mucosal lesion and its progression to ulcer lesion induced by ischaemia-reperfusion in rats. Plaunotol (30 and 100 mg kg-1, 15 min before ischaemia) significantly reduced the total erosion area observed immediately after ischaemia-reperfusion. When plaunotol (30 and 100 mg kg-1, once a day) was administrated orally 60 min after reperfusion, it prevented the progression from erosion to ulcer. At 72 h after ischaemia-reperfusion, the total area of ulcers lesions was significantly reduced compared with that without plaunotol administration. Furthermore, treatment with plaunotol (100 mg kg-1) significantly increased prostaglandin E2 content in gastric tissues of both acute gastric mucosal lesion and gastric ulcer lesion. In in-vitro experiments, plaunotol (1-3 mg mL-1) reduced the superoxide radicals generated by leucocytes, but not by xanthine oxidase. These results indicate that plaunotol has protective effects on both the onset of acute gastric mucosal injury and its progression to ulcer lesion induced by ischaemia-reperfusion. Both effects of plaunotol on increase in prostaglandin content in gastric tissues and inhibition of superoxide radical from leucocytes may play important roles on the protection against gastric mucosal injury.

Topics: Animals; Anti-Ulcer Agents; Dinoprostone; Diterpenes; Fatty Alcohols; Gastric Mucosa; Humans; In Vitro Techniques; Leukocytes; Male; Rats; Rats, Wistar; Reperfusion Injury; Stomach Ulcer; Superoxides; Xanthine Oxidase

Animal models of liver ischaemia and reperfusion are frequently used to study the consequences on liver cells of transient oxygen deprivation. In 3 different rat models we studied ischaemia/reperfusion effects on liver cell membrane integrity, cytoplasmic enzyme proteins and enzyme activities by in situ histochemical techniques. In vivo ischaemia, as well as no-flow hypoxia, or N2-induced hypoxia in isolated perfused livers, reduced the activity of 5'-nucleotidase, a sensitive marker for plasma membrane damage in hepatocytes. As little as 2 minutes of reoxygenation in each model resulted in leakage of soluble enzymes from parenchymal and non-parenchymal liver cells, as shown by decreased protein level and activity of cytoplasmic enzymes. Whereas a multifocal decrease was observed after in vivo reperfusion, a decrease was found in all periportal and midzonal cells after blood-free reoxygenation. As judged by alkaline phosphatase activity and immunohistochemistry, an influx of inflammatory cells was not found in the in vivo model. Our findings indicate that reoxygenation itself, rather than restoration of flow, accounts for the loss of soluble enzymes from liver cells after a period of hypoxia. In situ detection of enzyme protein and activity proved useful for the examination of very early ischaemia/reperfusion effects on rat liver cells.

Topics: 5'-Nucleotidase; Animals; Disease Models, Animal; Glucosephosphate Dehydrogenase; Immunoenzyme Techniques; L-Lactate Dehydrogenase; Liver; Male; Organ Culture Techniques; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

To investigate the role of allopurinol in the attenuation of ischaemia- and reperfusion-induced corporeal injury in a rat model of veno-occlusive priapism.. Placebo or allopurinol were given to eight groups of rats before priapism (ischaemia) was induced using a vacuum-constriction device for a duration of 6 or 12 h. Half of the groups of rats undergoing the same duration of priapism had 1 h of detumescence after the constriction band was removed (reperfusion). A ninth group was not treated and received no drug, serving as controls. Corporeal homogenates were then examined for malondialdehyde (MDA) accumulation, derived from lipid peroxidation, using a thiobarbituric acid assay.. The accumulation of MDA was significantly higher in the groups treated with placebo and undergoing ischaemia/reperfusion compared with the control group (P < 0.001), but was not significantly different between the placebo-treated ischaemic and reperfused groups (P > 0.05). Rats undergoing 6 and 12 h of ischaemia and reperfusion, and receiving allopurinol had significantly less accumulation of MDA compared with those receiving placebo (P < 0.005).. Lipid peroxidation, an indicator of injury induced by reactive oxygen metabolites, occurred in corporeal tissue during and after veno-occlusive priapism in this rat model; when assessed by lipid peroxidation, allopurinol appeared to protect rat corporeal tissue against this injury.

Topics: Allopurinol; Animals; Antimetabolites; Ischemia; Lipid Peroxidation; Male; Malondialdehyde; Penis; Priapism; Rats; Rats, Wistar; Reperfusion Injury

Little is known about the possible contribution of apoptosis to ischemia-reperfusion injury in human liver transplantation. Therefore, we studied postreperfusion surgical biopsy specimens of 16 human liver allografts using the TUNEL assay for in situ demonstration of apoptotic cells. In all patients, a variable proportion of hepatocytes and sinusoidal endothelial cells presented labeled nuclei. The mean +/- standard deviation percentages of positive hepatocytes were 18.7% +/- 12.2% in the whole section, 30.4% +/- 18.7% in the subcapsular region, 14.5% +/- 13.5% in the centrilobular zones, and 10.3% +/- 9.5% in the periportal zones. The percentage of positive hepatocytes were not correlated with the duration of cold ischemia but was higher in grafts harvested from donors with elevated preoperative aspartate aminotransferase (AST) levels. The percentage of positive hepatocytes was correlated with postoperative serum levels of AST (P = .015) and inversely correlated with postoperative serum levels of factor V (P = .019). Apoptotic biliary epithelial cells were detected in only 3 cases. In conclusion, apoptosis is a frequent event in postreperfusion biopsy specimens of liver allografts and probably contributes to preservation injury of hepatocytes.

Topics: Adenosine; Allopurinol; Apoptosis; Biopsy; Cryopreservation; Deoxyuracil Nucleotides; DNA Nucleotidyltransferases; Female; Glutathione; Humans; Immunoenzyme Techniques; Insulin; Liver; Liver Diseases; Liver Function Tests; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Random Allocation; Reperfusion Injury; Transplantation, Homologous

To investigate whether liver ischemia and reperfusion (IR) directly affect functions of remote organs.. Cardiovascular and respiratory dysfunction follows hemorrhage, spinal shock, or trauma as a result of no-flow-reflow phenomena. Hepatic IR induces remote organ damage probably by xanthine oxidase and oxygen species.. Isolated rat livers, lungs, and hearts were perfused with Krebs-Henseleit solutions. After stabilization, livers were either perfused or made ischemic. Then, livers and hearts or livers and lungs were reperfused in series, and the liver was disconnected and the second organ continued to perfuse with the accumulated effluents.. Ischemic and reperfused liver effluent contained high lactate dehydrogenase and uric acid concentrations compared with controls; xanthine oxidase increased 60 to 100 times. Ischemic and reperfused lung peak inspiratory pressure almost doubled; airway static compliance halved; myocardial contractility decreased to 70% of baseline; wet weight-to-dry weight ratios of lungs and livers increased.. Ischemic and reperfused liver can directly induce myocardial and pulmonary dysfunction, presumably by oxidant-induced injury.

Topics: Acute Disease; Animals; Heart Diseases; In Vitro Techniques; L-Lactate Dehydrogenase; Liver; Lung Diseases; Male; Myocardial Contraction; Rats; Rats, Wistar; Reperfusion Injury; Uric Acid; Xanthine Oxidase

Topics: Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Energy Metabolism; Glutathione; Hypoxanthine; Insulin; Ischemia; Jejunum; Mesenteric Artery, Superior; Organ Preservation; Organ Preservation Solutions; Oxygen; Phosphocreatine; Portal Vein; Raffinose; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury

Ischemia caused by cold storage (CS) and reperfusion of the kidney is often responsible for delayed graft function after transplantation. Significant attention has been focused on the cascade of events involved in ischemia-reperfusion injury, with the objective of identifying drugs to ameliorate the functional damage that occurs.. The purpose of this study was to evaluate the renal function of isolated perfused pig kidneys after 48 hr of CS with Euro-Collins (EC) solution plus trimetazidine (EC+TMZ), standard EC solution, or University of Wisconsin (UW) solution. Normothermic isolated perfused pig kidneys were randomized into five experimental groups: (A) control group (cold flush with cold heparinized saline and immediately reperfused; n=6); (B) cold flush with cold heparinized saline with TMZ (10(-6) M), n=6; (C) 48 hr of CS with EC and reperfusion (n=8); (D) 48 hr of CS with EC+TMZ alone and reperfusion (n=8); (E) 48 hr of CS with UW and reperfusion (n=8). Proton nuclear magnetic resonance spectroscopy and biochemical studies were performed for the functional evaluation during reperfusion. Lipid peroxidation was also determined. Histological examination (optical and electron microscopy) was performed after CS and reperfusion.. Using TMZ, the renal perfusate flow rate as well as the glomerular filtration rate and proximal tubular function were significantly improved. This improvement of renal function during reperfusion was correlated with a less significant cellular and interstitial edema. In addition, tubular injury markers were significantly lower in the group preserved with EC+TMZ, and TMZ reduced lipid peroxidation dramatically during reperfusion.. The addition of TMZ to the EC solution increased the preservation quality and renal tubular function, and gave protection from reperfusion injury better than EC alone or UW. These results strongly suggest that TMZ has a cytoprotective effect and may therefore be useful for kidney preservation.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Hypertonic Solutions; In Vitro Techniques; Insulin; Ischemia; Kidney; Lipid Peroxidation; Magnetic Resonance Spectroscopy; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Renal Circulation; Reperfusion Injury; Swine; Trimetazidine

Topics: Alanine; Amino Acid Substitution; Animals; Cloning, Molecular; Liver; Mutagenesis, Site-Directed; Peptide Fragments; Rats; Recombinant Proteins; Reperfusion Injury; Threonine; Trypsin; Xanthine Dehydrogenase; Xanthine Oxidase

Topics: Animals; Cats; Colic; Horse Diseases; Horses; Intestinal Diseases; Intestinal Mucosa; Intestine, Small; Laparotomy; Necrosis; Peroxidase; Rats; Reperfusion Injury; Swine; Xanthine Dehydrogenase; Xanthine Oxidase

Exposure to high-concentration oxygen (O2) increases lipid peroxidation of the cellular membrane, leading to tissue injury which may involve hepatic ischemia-reperfusion (I/R) injury. We examined the effects of inhaling high-concentration O2 on hepatic I/R injury with allopurinol, which is a xanthine oxidase inhibitor. Partial hepatic ischemia was performed in rats with or without allopurinol under 21 or 100% O2 inhalation. Levels of lipid peroxide, serum liver enzymes, and hepatocellular oxidative stress in the 100% O2 group were significantly higher than in the 21% O2. Administration of allopurinol significantly inhibited those changes in the 100% O2 group. Severe degeneration of mitochondria were noted in the 100% O2 group, but appeared to be reduced by allopurinol. Results suggest that inhalation of high-concentration O2 during liver surgery may increase lipid peroxidation and exacerbate hepatic I/R injury, but those changes may be prevented by allopurinol.

Topics: Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Enzyme Inhibitors; Glutathione; Glutathione Disulfide; Lipid Peroxidation; Liver; Male; Microscopy, Electron; Oxidative Stress; Oxygen; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Oxidase

Topics: Adenosine; Allopurinol; Animals; Bile; Enalapril; Epoprostenol; Glutathione; Insulin; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Transplantation, Isogeneic

Topics: Adenosine; Allopurinol; Cell Membrane; Cell Membrane Permeability; Cells, Cultured; Digitonin; Glucose; Glutathione; Humans; Insulin; Lung Neoplasms; Mannitol; Mitochondria; Organ Preservation Solutions; Oxygen Consumption; Potassium Chloride; Procaine; Raffinose; Reperfusion Injury; Rotenone; Tumor Cells, Cultured; Umbilical Veins

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Calcium; Glutathione; In Vitro Techniques; Insulin; Kidney Cortex; Kinetics; Male; Mitochondria; Organ Preservation Solutions; Permeability; Quinacrine; Rabbits; Raffinose; Reperfusion Injury; Temperature

Resuscitated hemorrhagic shock causes global ischemia reperfusion with generation of toxic oxygen metabolites. We hypothesized that the immunosuppression that follows hemorrhagic shock may be linked to this process.. Forty-five male Sprague-Dawley rats (weight, 250-300 g) were bled to a mean arterial pressure of 30 mm Hg for 60 minutes, then were resuscitated with three times the maximum blood loss of lactated Ringer's solution. Immune response was assessed by splenocyte proliferation and interleukin-2 (IL-2) production 72 hours after hemorrhage. Allopurinol (50 mg/kg) was given after hemorrhage and immediately before resuscitation.. Hemorrhagic shock caused significant decreases in splenocyte proliferation (cpm: (157,880 +/- 22,068 (mean +/- SD) vs. 37,787 +/- 15,849) and IL-2 production (1/2 max U/ml: 79.6 +/- 7.9 vs. 48.0 +/- 7.7) (both p < 0.05). Hepatic xanthine oxidase was significantly increased with hemorrhage and resuscitation. Hepatic xanthine oxidase activity after hemorrhage and resuscitation was significantly decreased after treatment with allopurinol (74.2 +/- 41.7 vs. 9.2 +/- 9.40). Allopurinol did not affect splenocyte proliferation (cpm: 21,875 +/- 9,316) or IL-2 production (1/2 max U/ml: 45.0 +/- 7.1).. These results demonstrate that inhibition of xanthine oxidase by allopurinol after hemorrhagic shock did not affect splenocyte proliferation or IL-2 production. We conclude that the immunosuppression after hemorrhagic shock is not dependent on xanthine oxidase-induced production of toxic oxygen metabolites.

Topics: Allopurinol; Animals; Drug Evaluation, Preclinical; Enzyme Inhibitors; Immune Tolerance; Interleukin-2; Male; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Resuscitation; Shock, Hemorrhagic; Spleen; Xanthine Oxidase

Intestinal ischaemia-reperfusion (IR) injury has largely been attributed to cellular necrosis. Apoptosis, a distinct form of cell death has been observed following IR to the brain, heart, adrenals and the kidneys. In order to characterize the role of apoptosis in intestinal IR, small bowel grafts were stored in saline (n = 6) or modified University of Wisconsin solution (n = 6) at 4 degrees C for 12 h and reperfused for 6 h in syngeneic rats. Samples of normal, stored and reperfused intestines at 1, 3 and 6 h were analysed by light and electron microscopy. Following reperfusion, there was crypt and villous epithelial apoptosis, loss of crypt and villous structures, and an increase in mucosal inflammatory cell infiltration. Ongoing apoptosis was maximum at 1 h, its degree decreasing with increasing reperfusion intervals. Large numbers of apoptotic bodies dominated the picture from 3 h of reperfusion. This study has demonstrated the induction of apoptosis by intestinal IR injury, which begins within an hour of reperfusion and is probably responsible for the observed crypt and villous loss. This has potential therapeutic implications as, opposed to necrosis, apoptosis is an active process with genetic regulators and biochemical effectors, which can be specifically targeted to prevent or alleviate IR injury.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Glutathione; Insulin; Intestinal Mucosa; Intestine, Small; Male; Microscopy, Electron; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Sodium Chloride; Tissue Preservation

This study focuses upon two discrete components of posttransplant hepatic reticuloendothelial system (RES) function-phagocytosis and killing of bacteria-under various conditions of ischemic preservation. We had previously reported that, following intravenous injection of rats with 51Cr and 125I double-labeled Escherichia coli, hepatic 51Cr levels can be used to reliably quantify hepatic phagocytic clearance of the bacteria from the blood (HPC), while the subsequent release of 125I from the liver accurately parallels hepatic bacterial killing. Here, Wistar rats were transplanted with syngeneic livers perfused with either normal saline (NS) or University of Wisconsin solution (UW) and stored at 4 degrees C for 1, 2, or 3 hr prior to implantation. Control rats underwent laparotomy and hepatic artery ligation. Using the double-labeled E coli assay, HPC was decreased in all transplanted animals when compared with controls, reaching a nadir on the third postoperative day (P < 0.05). In rats transplanted with livers preserved in NS, the fraction of phagocytosed organisms that were subsequently killed (hepatic killing efficiency=HKE) was increased to 142%, 129%, or 112% of normal following 1, 2, or 3 hr of cold ischemia, respectively; P < 0.05). Conversely, preservation of donor allografts in UW was associated with marked depression of HKE. Moreover, rats receiving NS- or UW-preserved livers tolerated an intravenous challenge with Streptococcus pneumoniae poorly (50% mortality) compared with hepatic artery ligated controls (12% mortality) at 7 days. Ischemic preservation of rat livers in NS resulted in a dose (of ischemia)-dependent reduction of hepatic phagocytosis coupled with a potentiation of HKE. Preservation in UW, however, produced a striking suppression of both components of hepatic RES function. Following a septic challenge survival was reduced in both groups of transplanted rats.

Topics: Adenosine; Allopurinol; Animals; Escherichia coli; Escherichia coli Infections; Glutathione; Insulin; Kupffer Cells; Liver; Liver Transplantation; Male; Organ Preservation Solutions; Phagocytosis; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Tissue Preservation

In the presence of its substrates hypoxanthine and xanthine, xanthine oxidase generates oxygen free radicals that cause postischemic injury. Recently, it has been demonstrated that the burst of xanthine oxidase-mediated free radical generation in the reperfused heart is triggered by a large increase in substrate formation, which occurs secondary to the degradation of adenine nucleotides during ischemia. It is not known, however, whether blocking this substrate formation is sufficient to prevent radical generation and functional injury. Therefore, studies were performed in isolated rat hearts in which xanthine oxidase substrate formation was blocked with the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), and measurements of contractile function and free radical generation were performed. Chromatographic measurements of the intracellular adenine nucleotide pool showed that preischemic administration of EHNA blocked postischemic hypoxanthine, xanthine, and inosine formation. Electron paramagnetic resonance spin trapping measurements of free radical generation showed that inhibition of adenosine deaminase with EHNA blocked free radical generation and that it also increased the recovery of contractile function by more than 2-fold. Exogenous infusion of hypoxanthine and xanthine totally reversed the protective effects of EHNA. These results demonstrate that blockade of xanthine oxidase substrate formation by adenosine deaminase inhibition can prevent free radical generation and contractile dysfunction in the postischemic heart.

Topics: Adenine; Adenine Nucleotides; Adenosine Deaminase Inhibitors; Animals; Coronary Circulation; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Female; Free Radicals; Hydroxyl Radical; Myocardial Ischemia; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxides; Xanthine Oxidase

Topics: Adenosine; Allopurinol; Animals; Cells, Cultured; Free Radical Scavengers; Glutathione; Glutathione Peroxidase; Insulin; Lipid Peroxidation; Liver; Liver Transplantation; Male; Mitochondria, Liver; Organ Preservation; Organ Preservation Solutions; Phosphatidylcholines; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Swine; Time Factors

Topics: Adenosine; Allopurinol; Analysis of Variance; Animals; Anticoagulants; Bile; Cold Temperature; Gabexate; Glutathione; Insulin; Ischemia; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Male; Microcirculation; Organ Preservation; Organ Preservation Solutions; Portal System; Raffinose; Rats; Rats, Inbred Lew; Regional Blood Flow; Reperfusion; Reperfusion Injury; Time Factors

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Anti-Ulcer Agents; Biomarkers; Glutathione; Insulin; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Pentoxifylline; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Time Factors; Tumor Necrosis Factor-alpha; Vasodilator Agents; Xanthines

Topics: Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Biomarkers; Cold Temperature; Glutathione; Hyaluronic Acid; Insulin; Kinetics; L-Lactate Dehydrogenase; Liver; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Reperfusion Injury; Time Factors

This study determined the role that oxygen-derived free radicals played in the production of gastric injury in rats challenged orally with concentrated ethanol or subjected to vascular compromise. In the ethanol study, rats were pretreated with a variety of free radical scavengers or enzyme inhibitors prior to exposing the stomach to 100% ethanol. At sacrifice, the degree of macroscopic damage to the glandular gastric mucosa was quantified. In separate studies, the effects of ethanol on gastric mucosal levels of enaldehydes (malondialdehyde and 4-hydroxynonenal) were examined as an index of lipid peroxidation. Superoxide dismutase and catalase pretreatment were without benefit in reducing injury in our ethanol model, excluding potential contributory roles for the superoxide anion or hydrogen peroxide, respectively. Dimethyl sulfoxide and desferoxamine were likewise without protective capabilities, eliminating a role for the hydroxyl radical. Allopurinol, a xanthine oxidase inhibitor, provided no protection under acute conditions, even though partial protection was noted when administered chronically. Further, enaldehyde levels were not increased over control levels in alcohol-exposed mucosa, indicating no enhanced lipid peroxide formation. In contrast, in animals in which ischemia to the stomach was induced followed by reperfusion, marked gastric injury was observed in combination with enhanced enaldehyde levels. Prevention of enaldehyde formation by a 21-aminosteroid concomitantly prevented injury induced by ischemia-reperfusion. These findings support the conclusion that ischemia-reperfusion injury to the stomach is an oxygen-derived free radical process whereas ethanol-induced injury clearly involved some other process. Although allopurinal was partially protective against ethanol damage when administered chronically, observations in other models of injury suggest that this action is independent of its inhibitory effect on xanthine oxidase.

Topics: Allopurinol; Animals; Antioxidants; Catalase; Deferoxamine; Dimethyl Sulfoxide; Ethanol; Free Radicals; Gastric Mucosa; Lipid Peroxidation; Pregnatrienes; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase

Topics: Adenosine; Allopurinol; Animals; Blood; Blood Pressure; Glutathione; Hypertonic Solutions; Insulin; Ischemia; Lung; Lung Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Perfusion; Pneumonectomy; Pulmonary Artery; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Trehalose

Topics: Adenosine; Allopurinol; Animals; Benzamidines; Blood Glucose; Glutathione; Graft Survival; Guanidines; Insulin; Male; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Protease Inhibitors; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Time Factors; Transplantation, Isogeneic

Topics: Adenosine; Allopurinol; Analysis of Variance; Animals; Arginine; Cyclic GMP; Enzyme Inhibitors; Glutathione; Insulin; Kidney; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Temperature

Extracellular yeast glycoproteins (YG) produced by Rhodosporidium toruloides have been shown to increase the survival rate of different yeast species after storage in liquid nitrogen. The purpose of this study was to investigate the effect of YG on cold-stored rat livers. Water-soluble YG produced either by Phaffia rhodozyma (G3) or by Leucosporidium antarcticum (G4) were added to a modified University of Wisconsin solution (mUW) and used for cold storage (1 degree C) of isolated livers. The functional status of each liver was then assessed under conditions of 90-min normothermic reperfusion. The 46-h cold storage in mUW without G3 and G4 resulted in serious preservation-reperfusion injury of the liver. The addition of G3 to mUW for 46-h preservation of the liver resulted in significantly higher bile flow (4.32 +/- 0.35 vs 2.35 +/- 0.49 microliters/min/10 g at 75-90 min), higher portal blood flow (10.99 +/- 0.2 vs 4.78 +/- 1.07 ml/min/g at 90 min), lower liver weight after reperfusion (102.4 +/- 1.5 vs 116.7 +/- 6.6% of weight before preservation), and lower total tissue water after reperfusion (2.49 +/- 0.05 vs 2.92 +/- 0.13 g water/g dry weight). However, the activity of ALT, AST, and LDH in perfusate was not changed. The beneficial effect of G4 was less pronounced. The 24-h storage in mUW resulted in a significant increase of AST and LDH activity in perfusate; the addition of G3 to mUW for 24-h preservation did not affect these parameters. In conclusion, the addition of 0.05% G3 or G4 to mUW was only partially beneficial in improving rat liver preservation.

Topics: Adenosine; Allopurinol; Animals; Basidiomycota; Cold Temperature; Evaluation Studies as Topic; Female; Fungal Proteins; Glutathione; Glycoproteins; In Vitro Techniques; Insulin; Liver; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Time Factors

Many studies show that allopurinol can reduce skeletal muscle I-R (ischemia-reperfusion) injury, but the mechanism of the effect is still unclear and some studies suspect the effect. In this study, we determined whether allopurinol really reaches to reperfused muscle and reduces tissue injury by inhibiting xanthine oxidase or not.. In this study, microdialysis method combined with HPLC (high performance liquid chromatography) were employed and purines, MDA (malondialdehyde), allopurinol in gracilis muscle were measured continuously in I-R injury model of canine gracilis muscle. The effect was compared between Group N (no treatment: n=8), Group P (pre-ischemic treatment: n=8) and Group R (pre-reperfusion treatment: n=8).. Allopurinol reduced the increase of xanthine, uric acid, MDA in the muscle and CPK in blood effluent from gracilis muscle after reperfusion. Tissue protecting effect of allopurinol was more effective in group R than in group P.. By continuous measurement of purines, allopurinol and MDA in canine gracilis muscle during 5 hr ischemia and 2 hr reperfusion, it was proved that allopurinol was delivered to reperfused skeletal muscle and reduced I-R injury by inhibiting xanthine oxidase.

Topics: Allopurinol; Animals; Chromatography, High Pressure Liquid; Creatine Kinase; Dogs; Enzyme Inhibitors; Extracellular Space; Female; Male; Malondialdehyde; Microdialysis; Muscle, Skeletal; Purines; Reperfusion Injury; Time Factors; Xanthine Oxidase

To determine if gastric intramucosal pH is affected by hepatoneteric ischemia-reperfusion. We additionally proposed to determine if changes in gastric mucosal hydrogen ion concentration are associated with liver and lung injury following hepatoenteric ischemia-reperfusion. Finally, we hypothesized that gastric intramucosal pH is influenced by xanthine oxidase, an oxidant-generating enzyme released after hepatoenteric ischemia-reperfusion.. Randomized, controlled, animal study.. University-based animal research facility.. Thirty-six New Zealand white male rabbits (2 to 3 kg).. Anesthetized rabbits were randomly assigned to one of four groups (n = 9 per group): a) sham-operated group; b) sham-operated group pretreated with sodium tungstate (xanthine oxidase inactivator); c) aorta occlusion group; and d) aorta occlusion group pretreated with sodium tungstate. Descending thoracic aorta occlusion was maintained for 40 mins with a 4-Fr Fogarty embolectomy catheter, followed by 2 hrs of reperfusion.. Gastric tonometry was performed after completion of the surgical preparation (30-min equilibration) and at 30, 60, 90, and 120 mins of reperfusion. Plasma alanine aminotransferase activity was determined at 120 mins of reperfusion to assess hepatic injury. Bronchoalveolar lavage of the right lung was performed after 120 mins of reperfusion, and the protein content was determined as a measure of pulmonary alveolar-capillary membrane compromise. Descending thoracic aorta occlusion resulted in a significant decrease in gastric intramucosal pH as compared with sham-operated rabbits (p < .001). The change in gastric mucosal hydrogen ion concentration was significantly associated with plasma alanine aminotransferase activity (r2 = .48, p < .01) and bronchoalveolar protein content (r2 = .51, p < .01). Xanthine oxidase inactivation significantly improved gastric intramucosal pH after aortic occlusion and reperfusion (p < .001), with a concomitant attenuation of the release of plasma alanine aminotransferase (p < .05) and accumulation of bronchoalveolar protein (p < .05) during reperfusion.. Gastric intramucosal pH was significantly decreased after hepatoenteric ischemia-reperfusion. Furthermore, an increase in gastric intramucosal hydrogen ion concentration was associated with a concomitant increase in tissue injury, a presumed harbinger of multiple organ failure. Gastric intramucosal pH values improved during reperfusion after xanthine oxidase inactivation, concomitant with attenuation of hepatic and pulmonary injury. Gastric tonometry is an important clinical tool that can provide critical insight into the pathogenesis of multiple organ injury after hepatoenteric ischemia-reperfusion. Gastric tonometry may aid in the rapid assessment of pharmacologic interventions designed to attenuate multiple organ injury in similar clinical settings (e.g., trauma, shock, major vascular surgery).

Topics: Animals; Gastric Mucosa; Hydrogen-Ion Concentration; Ischemia; Liver; Lung; Male; Manometry; Phenylephrine; Rabbits; Random Allocation; Reperfusion Injury; Sodium Bicarbonate; Xanthine Oxidase

Rat livers were flushed with different preservation solutions and stored at 4 degrees C for 24 h before being reperfused with a synthetic air-equilibrated, water-based solution. Four solutions were tested using this isolated rat liver model: Marshall's hypertonic citrate (HC); modified University of Wisconsin solution (Mod UW); a histidine-based solution (HIS); and a histidine-lactobionate-raffinose-based solution (HLR). After storage, livers were perfused at 4 degrees C for a period of 2 h and biopsy samples taken, at different time points, to investigate energy metabolism. Livers stored in HLR and HIS had higher 24-h storage levels of ATP (0.41 and 0.24 mumol/g respectively; P < 0.05) than those stored in Mod UW and HC. On reperfusion, all groups regenerated ATP by 1-2 h. However, significantly greater levels of ATP regeneration occurred in livers stored in the HLR (1.6 +/- 0.08 mumol/g) and Mod UW (1.3 +/- 0.18) than HC (0.58 +/- 0.19) and HIS (0.96 +/- 0.12); P < 0.05. Energy charge (1) (EC) recovered in all groups but was significantly higher in HLR and Mod UW (0.79 and 0.68, respectively; P < 0.05) than HC and HIS. These represent 95% (HLR) and 80% (Mod UW) of values observed in FIL. During the reperfusion period, total adenine nucleotide levels (TA) did not vary significantly within each storage group, except in the HIS solution. However, TAs were greater with livers stored for 24 h in HLR (2.5 +/- 0.25) and Mod UW (2.7 +/- 0.20) than those the other two storage groups (P < 0.05 in each case). This study has demonstrated that it was possible to resuscitate liver energetics after prolonged hypothermic ischemia by a period of cold reperfusion, and the method can differentiate between preservation solutions. The livers stored in each solution showed varying degrees of success in regeneration of ATP and EC, demonstrating that oxygen was not a limiting factor when using an air-equilibrated perfusate. The solutions providing the better preservation conditions gave the greater resuscitation of liver energetics (Mod UW and HLR). Overall, livers stored in HLR had the greatest resuscitation of energy metabolism, which correlates with survival data from other studies (29-31, 33).

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Disaccharides; Energy Metabolism; Evaluation Studies as Topic; Glutathione; Histidine; Hypertonic Solutions; In Vitro Techniques; Insulin; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Solutions

Simple models are needed that effectively test the variables that may be important in liver preservation. Two such models are isolated hepatocytes and tissue slices. In this study the effects of hypothermic preservation on the viability of hepatocytes (HC) and tissue slices (TS) from rat livers were measured by LDH leakage after cold storage and rewarming. We compared how glycine, calcium, and fasting, shown previously to affect preservation injury in hepatocytes, affected both HC and TS viability. Hepatocytes were cold-stored in University of Wisconsin organ preservation solution for up to 48 h and rewarmed in Krebs-Henseleit Bicarbonate (KHB) for 120 min. Tissue slices were studied in two ways. Either livers were cold-stored intact and then tissue slices (TS-A) prepared and rewarmed in KHB, or tissue slices were prepared from the fresh liver, cold-stored, and then rewarmed (TS-B). The latter method may be similar to cold storage of HC. Freshly prepared samples (HC, TS-A, or TS-B) showed < 15% LDH leakage during the rewarming phase. Cold storage for 24 h resulted in < 30% LDH leakage in all preparations. After 48 h cold storage there was a significant increase in LDH leakage (HC, 65.1 +/- 5.1%; TS-A, 52.9 +/- 0.8%, TS-B, 53.6 +/- 2.6%). Glycine (3 mM) or calcium (1.5 mM) included in the KHB significantly reduced LDH leakage from 48 h cold-stored HC to 20.7 +/- 1.8 and 26.3 +/- 2.4%, respectively. These agents caused a smaller decrease in LDH release from tissue slices (around 40%). Hepatocytes appear more susceptible to preservation/reperfusion damage than the more structurally intact tissue slices as suggested by the greater release of LDH. Another difference was that the agents which improved preservation quality of HC were not as effective in TS. Hepatocytes may be more vulnerable to preservation/reperfusion damage because of the harsh methods used in their preparation. The damage induced during preparation appears amenable to suppression by glycine or calcium. Tissue slices, which are intact pieces of liver tissue, may be more suitable for studies related to development of better methods for liver preservation. The intact cells in TS have not been exposed to harsh conditions and maintain a more natural cell-cell relationship.

Topics: Adenosine; Allopurinol; Animals; Calcium; Cell Separation; Cold Temperature; Fasting; Glutathione; Glycine; In Vitro Techniques; Insulin; L-Lactate Dehydrogenase; Liver; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tissue Preservation

We examined the potential protective effect of pretreatment with corticosteroids or antioxidants (ascorbic acid or allopurinol) in rabbits with reperfusion-induced damage to skeletal muscle after ischemia. 4 hours of limb ischemia induced by a pneumatic tourniquet, followed by reperfusion for 1 hour, caused a considerable amount of ultrastructural damage to the anterior tibialis muscles accompanied by a rise in circulating creatine kinase activity. Pretreatment of animals with depomedrone by a single 8 mg bolus injection led to a preservation of the anterior tibialis structure on both light and electron microscopy. High-dose continuous intravenous infusion with ascorbic acid (80 mg/hr) throughout the period of ischemia and reperfusion also preserved skeletal muscle structure, although allopurinol in various doses had no protective effect. These data are fully compatible with a mechanism of ischemia/reperfusion-induced injury to skeletal muscle, involving generation of oxygen radicals and neutrophil sequestration and activation. They also indicate that damage to human skeletal muscle caused by prolonged use of a tourniquet is likely to be reduced by simple pharmacological interventions.

Topics: Allopurinol; Animals; Anti-Inflammatory Agents; Antimetabolites; Antioxidants; Ascorbic Acid; Ischemia; Methylprednisolone; Methylprednisolone Acetate; Muscles; Rabbits; Reperfusion Injury

We have shown that 24-hour preservation by a two-layer [University of Wisconsin solution (UW)/perfluorochemical (PFC)] cold storage method at 4 degrees C allowed tissue ATP synthesis and resuscitated canine pancreases subjected to 90 minutes of warm ischemia. The purpose of this study was to examine whether the two-layer (UW/PFC) mild hypothermic storage method at 20 degrees C could shorten a preservation period for recovery of ischemically damaged pancreas and clarify changes of tissue adenine nucleotide metabolism and tissue perfusions. After 90 minutes of warm ischemia, canine pancreas grafts were preserved by the two-layer method and then autotransplanted. Tissue adenine nucleotide levels at the end of preservation and tissue perfusions after reperfusion were measured. Pancreas grafts subjected to 90 minutes of warm ischemia did not survive (0 of 5), without preservation. During a 5-hour preservation by the two-layer cold storage method the grafts did not synthesize enough ATP to repair damaged cell, although tissue perfusions were maintained after reperfusion. Consequently, ischemically damaged pancreases were not resuscitated (0 of 3). However, during 5-hour preservation by the two-layer mild hypothermic storage method, the grafts supplied enough ATP for processes that repair damaged cells, and tissue perfusions were maintained after reperfusion. As a result, ischemically damaged grafts were resuscitated (5 of 5). We conclude that 5-hour preservation by the two-layer mild hypothermic storage method accelerates ATP synthesis, which is essential for repairing damaged cells and protects the vascular microcirculation. This method can resuscitate ischemically damaged pancreas faster and holds promise for pancreas-kidney transplantation from cardiac arrest donors.

Topics: Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Cryopreservation; Dogs; Female; Fluorocarbons; Glutathione; Graft Survival; Insulin; Male; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose; Reperfusion Injury

Altered cellular calcium (Ca) homeostasis may be important in mediating hypothermic injury in preserved kidneys. In this study the effect of hypothermic (5 degrees C) storage on ionized intracellular Ca concentration ([Ca]i) in rabbit tubules was examined using Indo-1. Tubules were stored up to 250 min in UW-gluconate solution containing either 0.0, 0.5, 1.5, or 5.0 mM Ca (yielding about 3.6, 62, 371, and 1,010 microM ionized solution Ca (Ca2+) at 5 degrees C, respectively). [Ca]i increased to about 1,600 nM within 1 min after suspension in UW solution followed by a decrease in [Ca]i during the subsequent 60 min in all groups, suggesting mitochondrial Ca sequestration. Thereafter, [Ca]i either 1) increased in tubules incubated with 1.5 and 5.0 mM Ca to levels greater than 2,500 nM; 2) decreased to about 800 nM in tubules incubated with 0.5 mM Ca and then remained stable; or 3) continued to decrease in tubules incubated with 0.0 mM added Ca to reach an apparent steady-state concentration of about 175 nM after 180 min of incubation. The early spike in [Ca]i was unaffected by adding EGTA (solution Ca2+ = 50 nM). Ryanodine eliminated the [Ca]i spike, indicating that cooling in UW-gluconate solution caused release of endoplasmic reticulum Ca. This study shows that [Ca]i initially increases after exposure to UW-gluconate solution and appears to be transiently buffered through intracellular, probably mitochondrial, sequestration. Saturation of cellular buffer mechanisms resulted in a sustained dependence of [Ca]i on extracellular Ca2+. These results support the hypothesis that the effect of Ca on kidney viability is related to solution-induced alterations in [Ca]i.

Topics: Adenosine; Allopurinol; Animals; Calcium; Cattle; Cold Temperature; Gluconates; Glutathione; Homeostasis; Hypothermia; In Vitro Techniques; Insulin; Intracellular Fluid; Kidney Tubules; Kinetics; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Tissue Preservation

Although reactive oxygen metabolites may play a pivotal role in mediating microvascular reperfusion injury, the source of these radicals is still a matter of controversy. With the use of spectrophotometry and intravital microscopy we studied the role of xanthine oxidase and superoxide radicals in portal triad crossclamping-induced microvascular injury in rats. After 20 min of global hepatic ischemia and splanchnic vascular congestion, followed by 40 min of reperfusion (n = 8), xanthine oxidase activities in hepatic venous (26.9 +/- 4.7 nmol/ml x min) and systemic arterial blood (16.3 +/- 2.5 nmol/ml x min) were found significantly (p < .01) increased when compared with sham-operated controls (6.8 +/- 0.9 and 6.0 +/- 0.8 nmol/ml x min, n = 8). The increase of xanthine oxidase activity was accompanied by oxygen radical-mediated intravascular hemolysis. Intravital microscopy (n = 6) revealed accumulation of leukocytes within the postischemic hepatic microvasculature with stasis in sinusoids (75.9 +/- 8.9 per liver lobule) and adherence to the endothelial lining of postsinusoidal venules (534.7 +/- 125.3 per mm2 endothelial surface). Concomitantly, compromised microvascular reperfusion was characterized by perfusion deficits of individual sinusoids (25.6 +/- 4.0% nonperfused sinusoids). The xanthine oxidase inhibitor allopurinol (50 mg/kg b.wt., orally, n = 6) and the radical scavenger superoxide dismutase (60000 IU/kg b.wt., IV, n = 6) effectively (p < .01) inhibited both sinusoidal leukostasis (16.1 +/- 2.6 and 32.1 +/- 3.1 cells/lobule) and venular leukocyte adherence (247.6 +/- 7.9 and 205.0 +/- 38.0 cells/mm2), and, hence, reduced microcirculatory deteriorations, indicated by the attenuation of sinusoidal perfusion failure (2.8 +/- 0.8 and 9.0 +/- 3.1%). Our results support the hypothesis that portal triad crossclamping-induced microvascular reperfusion injury is triggered by superoxide radicals derived from the xanthine oxidase system.

Topics: Allopurinol; Animals; Constriction; Enzyme Inhibitors; Free Radicals; Hemolysis; Hepatic Artery; Liver; Microcirculation; Portal Vein; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase; Superoxides; Xanthine Oxidase

The possibility that verapamil (CAS 52-53-9) may intensify the efficacy of vitamin E in preventing the ischemia-reperfusion-caused biochemical dearrangement in rat cerebral cortex was investigated. A daily injection of vitamin E at i.m. dose of 175 mg/kg b.wt. for 7 days prior to subjecting the rats to 1 h bilateral occlusion of the common carotid arteries followed by reperfusion for another 1 h, moderately diminished the ischemia-reperfusion-induced increase in the activity of lactate dehydrogenase and in formation of conjugated dienes as well as in the conversion of xanthine dehydrogenase-->xanthine oxidase in cerebral cortex of rats. However, concomitant injection of verapamil at i.m. dose of 0.68 mg/kg b.wt. 15 min prior to ischemia-reperfusion together with vitamin E pretreatment afforded an elegant combined therapy that effectively abolished the dearrangement caused by ischemia-reperfusion in the above parameters. These results indicated that the protective efficacy of vitamin E against ischemia/reperfusion-induced biochemical dearrangement in cerebral cortex was intensified by concomitant use of verapamil.

Topics: Animals; Calcium Channel Blockers; Cerebral Cortex; L-Lactate Dehydrogenase; Lipid Peroxidation; Male; Rats; Rats, Wistar; Reperfusion Injury; Verapamil; Vitamin E; Xanthine Dehydrogenase; Xanthine Oxidase

Despite suggestions of a connection between endothelial damage and permeability alterations after ischemia and reperfusion in pulmonary tissue undergoing transplantation, no direct correlation between vascular endothelial discontinuity and parenchymal edema has yet been shown.. Forty-two rat lungs were harvested and stored for 48 or 72 hours under hypothermic and ischemic conditions. Stored pulmonary tissue was studied before transplantation and 5 minutes or 24 hours after transplantation by light microscopy and scanning electron microscopy of arterial vascular endothelium.. Stored lungs not subjected to revascularization showed moderate perivascular edema, with small intercellular gaps in endothelial monolayers. Five minutes after transplantation, pulmonary tissue appeared congested, with perivascular and alveolar edema. Examination of vascular endothelium by scanning electron microscopy showed detachment of endothelial cells. Twenty-four hours after transplantation, edema, hemorrhage, and vascular congestion were found in all specimens. Arterial vascular endothelium showed weak intercellular connections, numerous intercellular gaps, and widespread cell detachment. Bronchial epithelial cells appeared damaged after storage, with loss of cilia, blebbing of apical cytoplasm, and cellular rounding. These changes were maintained 5 minutes after transplantation but appeared totally reversed after 24 hours in specimens stored 48 hours, whereas bronchial denudation was observed in 72-hour stored lungs. Statistically significant positive correlations (Kendall p < 0.001) between revascularization time and alveolar edema and hemorrhage were found for both storage periods.. The results from this study demonstrate correlation between loss of endothelial monolayer continuity and histologic evidence of vascular permeability increases in pulmonary tissue before and after lung transplantation.

Topics: Adenosine; Allopurinol; Animals; Bronchi; Capillary Permeability; Cold Temperature; Endothelium; Endothelium, Vascular; Female; Glutathione; Insulin; Lung; Lung Transplantation; Microscopy, Electron, Scanning; Organ Preservation; Organ Preservation Solutions; Pulmonary Edema; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

The uptake of hyaluronic acid (HA) was used to assess preservation damage to sinusoidal endothelial cells (SEC) during cold storage and subsequent normothermic reperfusion of rat livers. After 8, 16, 24, and 48 h storage in University of Wisconsin (UW) solution, livers were gravity-flushed via the portal vein with a standard volume of cold UW solution containing 50 micrograms/l HA. The effluent was collected for analysis of HA, aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). The mean uptake of HA at 0 h was 59.1% +/- 4.6% (mean +/- SEM). After 8 h of storage, HA uptake was similar (55.5% +/- 7.3%), whereas after 16 h of storage it was reduced to 34.7% +/- 5.8%. At 24 and 48 h of storage, no uptake of HA was found. In a second series of experiments, livers were stored in UW solution and subsequently reperfused for 90 min with a Krebs-Henseleit solution (37 degrees C) in a recirculating system containing 150 micrograms/l HA. Following 8 h of storage, 34.6% +/- 8.0% of the initial HA concentration was taken up from the perfusate. After 16 and 24 h of storage, no uptake of HA was found. The results of this study indicate that damage to SEC occurs progressively during storage, leading to zero uptake of HA by the rat livers at 24 h of cold ischemia time. Additional reperfusion injury to the SEC was demonstrated by the reduced ability of the SEC to take up HA following normothermic reperfusion. The uptake of exogenous HA in preserved livers, used as a tool to assess SEC injury, enables the detection of early preservation damage.

Topics: Adenosine; Allopurinol; Animals; Biomarkers; Cold Temperature; Endothelium; Female; Glucose; Glutathione; Hyaluronic Acid; Insulin; Ischemia; Liver; Organ Preservation; Organ Preservation Solutions; Oxygen; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Temperature; Tromethamine

We investigated the role of adhesion molecules in the early phase of reperfusion following cold ischemia. Livers of male Lewis rats were preserved for 0 h (group A) or 24 h in University of Wisconsin (UW) solution without additives (group B) or in UW solution with anti-ICAM-1 antibody (group C) or anti-E-selectin-1, SLe(x) and SLe(a) antibodies (group D). The livers were then reperfused with diluted rat whole blood (DWB; groups A and B). DWB containing anti-ICAM-1 and LFA-1 antibodies (group C) or DWB containing anti-L-selectin, SLe(x) and SLe(a) antibodies (group D). The reperfusion was performed at 37 degrees C for 1 h at 5 cm H2O of perfusion pressure. During reperfusion, hepatic microcirculation was assessed by monitoring portal and peripheral tissue blood flow. Bile production was significantly reduced in group B livers compared with those in group A. Anti-ICAM-1 and LFA-1 antibodies failed to improve hepatic microcirculation, whereas anti-LECAM-1, SLe(x) and SLe(a) antibodies significantly improved the microcirculation. Bile production in group C and D livers was comparable to that in group B livers. Preservation for 24 h significantly increased the release of TNF-alpha from 0.207 to 43.7 pg/g per hour during reperfusion. Monoclonal antibodies to the adhesion molecules did not suppress the release of TNF-alpha in groups C and D. Histological examination demonstrated a lack of leukocyte infiltration or thrombus in hetapic microvessels. The extent of hepatocyte necrosis did not differ among groups B, C, and D. We conclude that the microcirculatory disturbance in the early phase of reperfusion occurs as a result of the tethering of leukocytes through the interaction of the selectin family and their ligands, and that the ICAM-1-LFA-1 pathway is not involved in this step. The lack of improvement in bile production with antibodies to the selectin family and their ligands strongly suggests that other mechanisms participate in the deterioration of hepatic function.

Topics: Adenosine; Allopurinol; Animals; Antibodies, Monoclonal; Antibody Specificity; Bile; CA-19-9 Antigen; Cell Adhesion; Cold Temperature; E-Selectin; Glutathione; Insulin; Intercellular Adhesion Molecule-1; Ischemia; L-Lactate Dehydrogenase; L-Selectin; Leukocytes; Liver; Lymphocyte Function-Associated Antigen-1; Male; Microcirculation; Necrosis; Oligosaccharides; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion; Reperfusion Injury; Sialyl Lewis X Antigen; Tumor Necrosis Factor-alpha

The time course of the energy metabolism after reperfusion, the relationship between the conversion of xanthine dehydrogenase to xanthine oxidase (D-to-O conversion) during ischemia, and the changes of the energy metabolism after reperfusion were studied using an ischemia-reperfusion model in the small intestine of the rat. The rat jejunum underwent an occlusion of the superior mesenteric artery and vein for either 30 minutes (group 1, n = 6) or 90 minutes (group 2, n = 6) with collateral interruption, and then it was reperfused. The contents of the adenine nucleotides in the small intestine of the rat were measured by high-performance liquid chromatography (HPLC) before ischemia, and 30, 60, and 90 minutes of ischemia, as well as 30, 60, 120, and 180 minutes after reperfusion. The recovery level of adenosine triphosphate (ATP) in group 1 (6.05 +/- 0.80 mumol/g dry weight) 30 minutes after reperfusion was significantly higher than that in group 2 (2.28 +/- 1.12 mumol/g dry weight) (P < .001). In addition, the ATP content after reperfusion in group 2 did not change from 30 to 180 minutes after reperfusion. The D-to-O conversion during ischemia in group 1 was not significantly greater than that before ischemia; however, that of group 2 did increase significantly during ischemia (P < .005). These results suggest that the tissue damage from ischemia-reperfusion injury after reperfusion under 90 minutes' ischemia is accomplished within the first 30 minutes after reperfusion. Therefore, the ATP level at 30 minutes after reperfusion may be useful for the evaluation of intestinal viability. Thus, the conversion of the xanthine oxidase enzyme system might play an important role in the expression of ischemia-reperfusion injury.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Animals; Chromatography, High Pressure Liquid; Energy Metabolism; Intestine, Small; Male; Rats; Rats, Inbred Strains; Reperfusion Injury; Time Factors; Tissue Survival; Xanthine Oxidase

To test the possible inhibitory effect of allopurinol on reperfusion injury, caused by oxygen-derived free radicals, of sheep large intestine.. An ultrastructural study on caecal tissues from control and treated groups.. Fifty sheep in four ischaemic and reperfused (treatment) groups and one control group. Three of the treatment groups were subdivided for half to be injected with allopurinol and the other half with its solvent, potassium hydroxide (KOH).. Ischaemia of the caecum was induced in the four treatment groups for 60 minutes by clamping the apex. Allopurinol and its KOH solvent were injected intravenously in three treatment groups prior to ischaemia. Samples were collected before and 1 hour after induction of ischaemia and 1 min, 1 h and 8 h after reperfusion. Tissues were processed and examined with an electron microscope.. Untreated and solvent injected sheep showed minor ultrastructural changes following ischaemia. With reperfusion, there was severe mitochondrial, goblet cell and basement membrane damage. Tissues from allopurinol-treated sheep were preserved and appeared similar to tissues from the control group.. Pre-treatment with allopurinol prevented damage to tissues whereas untreated or allopurinol solvent-treated showed severe damage following reperfusion. It is believed that allopurinol, an analogue of hypoxanthine and xanthine, prevents reperfusion injury by competitively binding with xanthine oxidase. This reduces or inhibits the xanthine oxidase mediated conversion of hypoxanthine to xanthine thereby preventing the formation of oxygen-derived free radicals.

Topics: Allopurinol; Animals; Cecum; Cell Membrane; Enzyme Inhibitors; Hydroxides; Injections, Intravenous; Male; Microscopy, Electron; Mitochondria; Potassium Compounds; Reperfusion Injury; Sheep; Sheep Diseases

The University of Wisconsin solution is widely used for organ preservation. We show that this fluid is often contaminated by traces of iron catalytic for free radical reactions, as demonstrated by the bleomycin assay and the ability of the iron to stimulate lipid peroxidation. Reduced glutathione (GSH) added to University of Wisconsin solution during its commercial preparation had oxidized to the disulfide in all samples examined. Addition of GSH to UW solution showed its half-life to be about one day. The metal ion chelators desferrioxamine and phenanthroline did not substantially delay the loss of GSH, although desferrioxamine was able to suppress iron-mediated lipid peroxidation induced by the contaminant iron in the preservation solution. Additional iron was released from rat liver after a period of cold storage/rewarming.

Topics: Adenosine; Allopurinol; Animals; Catalysis; Copper; Drug Stability; Free Radicals; Glutathione; Insulin; Ions; Iron; Lipid Peroxidation; Organ Preservation Solutions; Raffinose; Rats; Reperfusion Injury; Tissue Preservation

Gut ischemia has been implicated in the pathogenesis of necrotizing enterocolitis. Cyclosporine A and rapamycin, both potent novel immunosuppressants which act on signal transduction pathways in CD4+ T-cells, could potentially modulate immune/inflammatory cellular reactions involved in tissue ischemia/reperfusion injury. We hypothesized that cyclosporine A and rapamycin would preserve mucosal cell function and attenuate inflammatory T-cell-mediated cellular changes associated with small bowel ischemic injury. Forty Sprague-Dawley rats underwent 60 min of gut ischemia by vascular occlusion of the superior mesenteric vessels. Animals were randomized to four groups (n = 10): cyclosporine A (CSA, 5 mg/kg/day SQ), rapamycin (RAP, 2 mg/kg/day SQ), cyclosporine A and rapamycin (C&R), and vehicle given to controls (CON). Following 1 hr of reperfusion, small bowel was harvested for xanthine oxidase (XO, units/mg protein) and maltase (MALT, mM substrate degraded/min/g protein) assays. Blood was obtained from the portal vein for tumor necrosis factor-alpha (TNF-alpha, pg/ml) assay. The results of the study are presented below (mean +/- SEM, *, P < 0.05 versus controls). (Table in text) The results indicate that cyclosporine and rapamycin each play a significant role in attenuating ischemia/reperfusion injury in the gut. These data suggest that there are cytoprotective and anti-inflammatory mechanisms of these drugs independent of T-cell signal transduction that provide some protective effect in small bowel ischemia. Furthermore, T-cell-mediated immune mechanisms may not be associated with the adverse effects of small bowel ischemia/reperfusion injury. Additional investigation will be necessary in order to define the role of T-cell-mediated immune injury in the gut and how this relates to the beneficial effect of immunosuppression in small bowel mucosal ischemic injury.

Topics: alpha-Glucosidases; Animals; Cyclosporine; Immunosuppressive Agents; Intestinal Mucosa; Intestine, Small; Ischemia; Male; Polyenes; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Tumor Necrosis Factor-alpha; Xanthine Oxidase

One of the most important mechanisms in the production of ischemic damage after replantation surgery is the rise of oxygen free radicals during revascularization of ischemic tissues. Free radicals produce damage in the cell membranes (lipoperoxydation). This occurs not only in muscle tissue, but also in endothelial cells, with a consequent increase of local edema and the risk of compartment syndrome. This study attempted to interrupt the ischemic-reperfusion injury process in ischemic rat hindlimbs. Complete ischemia was induced for different numbers of hours (3, 6, 9, 12 hr) in four groups of rats (24 animals in each group). Allopurinol, an oxygen free radical scavenger, was tested in solution, 12.5 mg/kg b.w., in half the studied animals (n = 12). Collected data showed an increase (mean value: 0.60 nM/mg 3 hri 0.90 nM/mg at 6 hr; 0.80 nM/mg at 9 hr; 0.89 nM/mg at 12 hr; mean value in nonischemic muscle = 0.526 nM/mg) in lipoperoxides (NS between treated/untreated groups, p > 0.05) and high tissue pressure values in the posterior compartment of the ischemic rat hindlimbs. Allopurinol reduced the pressure values (p < 0.05 in Groups 1-3; p < 0.1 in Group 4), but was not effective in reducing lipoperoxides in skeletal muscle.

Topics: Allopurinol; Animals; Hindlimb; Muscle, Skeletal; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury

Topics: Adenosine; Allopurinol; Animals; Biomarkers; Glutathione; Insulin; Intestinal Mucosa; Jejunum; L-Lactate Dehydrogenase; Organ Preservation; Organ Preservation Solutions; Phospholipases A; Phospholipases A2; Raffinose; Rats; Rats, Inbred Lew; Reperfusion; Reperfusion Injury; Time Factors

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Glutaminase; Glutamine; Glutathione; Insulin; Intestinal Mucosa; Intestine, Small; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Time Factors; Transplantation, Isogeneic

Topics: Adenosine; Allopurinol; Glutathione; Humans; Insulin; Intestinal Mucosa; Intestine, Small; Jejunum; Microvilli; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Tissue Donors

Topics: Adenosine Triphosphate; Animals; Energy Metabolism; Intestinal Mucosa; Ischemia; Jejunum; Organ Preservation; Rats; Rats, Inbred Strains; Reperfusion; Reperfusion Injury; Xanthine Oxidase

Topics: Adenine Nucleotides; Adenosine; Allopurinol; alpha-Glucosidases; Animals; Dogs; Glutathione; Hypertonic Solutions; Insulin; Intestinal Mucosa; Intestine, Small; Isotonic Solutions; Organ Preservation; Organ Preservation Solutions; Peroxidase; Raffinose; Reperfusion; Reperfusion Injury; Ringer's Lactate

Topics: Adenine Nucleotides; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Allopurinol; Animals; Glutathione; Hypertonic Solutions; Insulin; Intestinal Mucosa; Intestine, Small; Ischemia; Isotonic Solutions; Male; Mesenteric Artery, Superior; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion; Reperfusion Injury; Ringer's Lactate; Time Factors

Topics: Adenosine; Allopurinol; Biomarkers; Colon; Glutathione; Humans; Ileum; Insulin; Intestinal Mucosa; Membrane Potentials; Muscle, Smooth; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Sodium-Potassium-Exchanging ATPase; Theophylline; Transplantation, Homologous

Topics: Administration, Oral; Allopurinol; Animals; Dogs; Intestinal Mucosa; Ischemia; Jejunum; Reperfusion Injury; Time Factors

Oxidant stress plays a major role in the pathophysiologic processes associated with ischemia-reperfusion injury. Xanthine oxidase (XO) is often implicated as a significant source of oxidants and increases in the circulation after hepatoenteric ischemia-reperfusion. We hypothesized that pulmonary injury is associated with hepatic ischemia-reperfusion resulting from descending thoracic aorta occlusion-reperfusion (AoOR). We also proposed that this remote pulmonary injury is attenuated through inactivation of circulating and tissue XO by tungstate, implicating an XO-dependent mechanism. Aortic occlusion was established in rabbits (standard or tungstate diet) for 40 min by 2 h reperfusion. Sham operated rabbits (standard or tungstate diet) served as controls. Hepatic reperfusion injury, as manifested by release of the hepatocellular enzyme alanine aminotransferase (ALT), was markedly increased after AoOR. Suprarenal-infrahepatic occlusion failed to increase ALT release. Tungstate pretreatment significantly (p < 0.05) reduced XO activity and ameliorated liver and intestinal injury (p < 0.05). Lung injury, manifested by increased bronchoalveolar lavage (BAL) protein concentration, BAL lactate dehydrogenase (LDH) activity and increased lung edema was significantly associated with liver injury (p < 0.05) and circulating XO activity (p < 0.001). XO inactivation significantly decreased BAL protein concentration, BAL LDH activity, and lung edema (p < 0.05). We conclude that remote pulmonary injury is significantly influenced by the extent of liver injury and circulating XO activity.

Topics: Administration, Oral; Alanine Transaminase; Animals; Bronchoalveolar Lavage Fluid; Liver; Lung; Male; Rabbits; Reperfusion Injury; Tungsten Compounds; Xanthine Oxidase

Livers of male Wistar rats (250-300 g) were isolated and flushed with 10 ml of Ringer's solution and 10 ml of UW preservation solution. Then the organs were stored for 24 h at 4 degrees C in UW solution. Livers of Group 1 were rinsed with 10 ml of Ringer's solution and reperfused after hypothermic storage with oxygenated Krebs-Henseleit solution (95% O2; 5% CO2) in a nonrecirculating system at constant pressure (10 mmHg) and 37 degrees C. Livers of Group 2 were incubated for 30 min at 37 degrees C prior to reperfusion, in order to simulate rewarming of the organ upon surgical implantation. Livers of Group 3 were treated like Group 2, but taurine was admixed to the UW solution (1 mM). Livers of Group 1 showed little signs of a preservation/reperfusion injury, with low enzyme activities of the parenchymal ALT and endothelial purine nucleoside phosphorylase (PNP) in the postischemic rinse solution (ALT: 19.9 +/- 12.4; PNP: 3.3 +/- 0.4 U/liter), adequate portal flow values about 3 ml/g/min and high O2 uptake at the end of the experiment (VO2: 3.2 +/- 0.4 ml/100g/min). Livers of Group 2 exhibited nearly tenfold higher enzyme activities in the rinse solution (ALT: 247.0 +/- 94.7*; PNP: 29.5 +/- 17.0* U/l) and disturbed tissue perfusion with significantly reduced flow values of about 2 ml/g/min during the first 10 min of reperfusion. As a result, the recovery of O2 uptake was only 2.2 +/- 0.3 ml/100 g/min*. Addition of taurine (Group 3) resulted in a significant reduction of the enzyme loss (ALT: 96.2 +/- 50.0#; PNP:12.4 +/- 7.0# U/liter) and improved portal flow values and O2 uptake at the end of reperfusion (2.7 +/- 0.3 ml/100 g/min#). The results give evidence for the importance of the rewarming period after hypothermic storage, which is inevitable during implantation of the organ in vivo. Taurine seems to exert a protective effect, affecting both the vascular endothelium and parenchymal tissue (*p < 0.05 vs Group 1; # p < 0.05 vs Group 2).

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Biomarkers; Cold Temperature; Glutamate Dehydrogenase; Glutathione; In Vitro Techniques; Insulin; Liver; Male; Organ Preservation; Organ Preservation Solutions; Purine-Nucleoside Phosphorylase; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Taurine

Gut ischemia has been implicated in the pathogenesis of necrotizing enterocolitis. Cyclosporine A (CSA), a potent immunosuppressant, attenuates immune/inflammatory cellular reactions. CSA also might be useful for inhibiting cellular immune responses involved in tissue ischemia/reperfusion injury. The authors hypothesized that CSA would attenuate inflammatory cellular changes associated with gut ischemic injury and that these effects could be quantified by computerized morphometry.. Twenty Sprague-Dawley rats underwent 60 minutes of gut ischemia by vascular occlusion of the superior mesenteric vessels. After 1 hour of reperfusion, the ischemic small bowel was harvested for histopathological examination and computerized morphometry, as well as xanthine oxidase (XO, U/mg protein) and maltase (MALT, mmol/L substrate degraded/min/mg protein) assays. CSA (5 mg/kg/d subcutaneously) was given to experimental animals (CSA, n = 10) for 5 days before ischemia, and vehicle was given to controls (CON, n = 10). The computer morphometric parameters studied were: surface index (SI, mucosal surface length per linear unit of intestine), average villous thickness (AVT), and average villous height (AVH).. Results are provided in Table 1.. The results of this study show that CSA may play a role in attenuating ischemia/reperfusion injury in the gut. Enzymatic analysis showed a beneficial role in the preservation of mucosal cell function after gut ischemia/reperfusion injury, as demonstrated by an elevated maltose level. Computerized morphometry demonstrated significant differences in all parameters in the experimental group, showing that CSA does confer gut mucosal protection during ischemia.

Topics: alpha-Glucosidases; Animals; Cyclosporine; Disease Models, Animal; Enterocolitis, Pseudomembranous; Image Interpretation, Computer-Assisted; Immunosuppressive Agents; Intestinal Mucosa; Intestine, Small; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Xanthine Oxidase

Hepatic vascular exclusion allows the performance of major hepatic resections with minimal intraoperative blood loss. We have previously shown that normothermic ischemia can be tolerated by a healthy liver for up to 90 minutes, and this period is increased to 4 hours if the liver is cooled to 4 degrees C using University of Wisconsin solution.. This study assessed whether these techniques could be successfully applied for patients requiring resection of a diseased liver, which is more sensitive to ischemic damage. Between July 1990 and May 1994, 12 patients (6 men, 6 women; mean age, 57.8 years) in whom the planned hepatic resection was believed to require hepatic vascular exclusion for more than 1 hour were treated with perfusion with the University of Wisconsin solution. The surgical procedures were right hepatectomy (one patient), extended right hepatectomy (seven patients), and extended left hepatectomy (four patients). The underlying hepatic disease was cirrhosis or severe fibrosis with hepatocellular carcinoma (four patients), cholestasis (due to cholangiocarcinoma and biliary stricture, one patient each), and more than 30 percent steatosis after treatment of hepatic metastases with chemotherapy (six patients). The University of Wisconsin solution that had been cooled to 4 degrees C was perfused through a cannula placed in the portal vein or the hepatic arterial branch of the segment to be resected, but with flow directed toward the liver that should be retained and effluent fluid drained through a cavotomy. Before reperfusion, the liver was rinsed with Ringer's lactate solution, which was also 4 degrees C.. The mean duration of hepatic ischemia was 121 minutes (range, 65 to 250 minutes), and venovenous bypass was used in three cases. The mean amount of blood transfused intraoperatively was 4.3 +/- 4 U; four cases required no transfusion. One patient died on postoperative day seven of portal vein thrombosis. The median hospital stay was 21 days (range, 12 to 56 days). Postoperative complications consisted of pneumonia (one patient), liver insufficiency (one patient, who recovered spontaneously), and subphrenic abscess (one patient). The postoperative tests of hepatic function were altered to the same degree as that seen after hepatic vascular exclusion of less than 1-hour duration in healthy livers. All patients who left the hospital were alive at 1 year.. Cooling of the hepatic parenchyma allowed us to perform major hepatic resection in patients with diseased livers using hepatic vascular exclusion for longer than 1 hour without increased morbidity or mortality. However, because of particular difficulties due to the size or location of the lesions, the application of these new techniques should only be considered for the largest and most complex hepatic resections for which hepatic vascular exclusions longer than 1 hour are foreseen.

Topics: Adenosine; Adult; Aged; Allopurinol; Blood Loss, Surgical; Cryopreservation; Female; Follow-Up Studies; Glutathione; Hepatectomy; Hepatic Artery; Humans; Hypothermia, Induced; Insulin; Liver; Liver Circulation; Liver Diseases; Male; Middle Aged; Organ Preservation Solutions; Portal Vein; Raffinose; Reperfusion Injury; Tissue Preservation

Preservation with University of Wisconsin (UW) solution can maintain liver graft function and produces survival rates of recipients higher than that with Euro Collins (EC) solution. To explore the underlying mechanisms, we transplanted rat livers following cold preservation with EC or UW solution for 18 hr, and measured hepatic adenine nucleotide levels, the percentage of water content, lactate levels, and endogenous antioxidant levels (alpha-tocopherol [alpha-Toc], reduced coenzyme Q9 [CoQ9H2], reduced coenzyme Q10, [CoQ1OH2] and reduced glutathione [GSH] during preservation and after transplantation. The adenosine triphosphate levels of the liver grafts preserved with UW solution recovered after reperfusion more rapidly and reached a higher level than those preserved with EC solution. UW solution caused a reduction in hepatic water content during preservation. Conversely, EC solution induced remarkable tissue edema. In addition, UW solution reduced the rate of hepatic lactate production both during preservation and after reperfusion. The concentrations of hepatic GSH, alpha-Toc, CoQ9H2, and CoQ1OH2 immediately after the graftectomy, and after the 18 hr of preservation with both EC and UW solutions, did not differ from those in the normal liver, and decreased only after transplantation. However, UW solution suppressed significantly the reduction in hepatic GSH, alpha-Toc, and CoQ9H2 after reperfusion, compared with EC solution. These results suggest that long-term cold storage induces tissue edema, reflecting a disturbance of the microcirculation during preservation, followed by parenchymal cell damage mediated by free radicals after reperfusion. The protective effects of UW solution could be attributable to the inhibition of free radical production after reperfusion.

Topics: Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Antioxidants; Cold Temperature; Glutathione; Insulin; Lactates; Liver; Liver Transplantation; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Survival Rate; Tissue Preservation; Ubiquinone; Vitamin E; Water

In this study, the changes of arachidonic acid metabolites after an ischemia-reperfusion (I/R) period are investigated. The cyclooxygenase and lipoxygenase metabolites were found to be significantly increased after a 45 min period of ischemia followed by 5 min of reperfusion. Prostaglandin E2 (PGE2)- and leukotriene C4 (LTC4)-like activities did not change in the ischemic period, but they both increased after reperfusion. A cyclooxygenase inhibitor indomethacin and lipoxygenase inhibitor nordehydroguaretic acid (NDGA) decreased PGE2- and LTC4-like activities, respectively, while allopurinol and superoxide dismutase (SOD) decreased both activities. According to our results, it can be assumed that free oxygen radicals are responsible for the elevation of PGE2- and LTC4-like activities and both of these arachidonic acid metabolites and free oxygen radicals are the main necrotizing agents in ischemia-reperfusion induced damage.

Topics: Alkaline Phosphatase; Allopurinol; Animals; Antioxidants; Creatine Kinase; Cyclooxygenase Inhibitors; Dinoprostone; Enzyme Inhibitors; Female; Guinea Pigs; Indomethacin; Ischemia; Leukotriene C4; Male; Masoprocol; Mesentery; Rats; Reperfusion Injury; Superoxide Dismutase; Tocolytic Agents

The aim of this study was to determine whether the administration of free radical antagonists, immediately before and during the early minutes of reperfusion, improves muscle survival 24 hr after a period of ischemia. Rabbit rectus femoris muscles were isolated, made ischemic for 3 1/2 hr and treated with either desferrioxamine (DFX), an Fe3+ chelator, superoxide dismutase and catalase (SOD & CAT), which quench superoxide and hydrogen peroxide, or allopurinol, an inhibitor of xanthine oxidase (XO). After 24 hr reperfusion, muscle viability (+/-s.e.m.), measured by the nitro blue tetrazolium (NBT) vital staining technique, was 41.6 +/- 11.3% for saline-treated ischemic controls, 30.6 +/- 7.6% for DFX-treated, 46.7 +/- 10.3% for SOD & CAT-treated, and 43.3 +/- 9.5% for allopurinol-treated muscles. None of the treated groups differed significantly from the ischemic control group. Tissue myeloperoxidase, ATP and reduced glutathione levels, and plasma lactate dehydrogenase (LDH) and aspartate transaminase (AST) levels were increased by ischemia and reperfusion in all groups, but the changes did not differ between the treatment groups. Levels of XO in the rabbit muscle were determined and found to be very low in both normal and postischemic muscle. As XO is the target enzyme of allopurinol, its absence provides a basis for the lack of effect of this agent. However, it is not clear why DFX and SOD & CAT had no protective effect.

Topics: Allopurinol; Animals; Catalase; Deferoxamine; Enzyme Inhibitors; Female; Free Radical Scavengers; Male; Muscle, Skeletal; Rabbits; Reperfusion Injury; Superoxide Dismutase; Time Factors; Xanthine Oxidase

Topics: Allopurinol; Animals; Dinoprostone; Endothelins; Female; Ischemia; Leukotriene C4; Malondialdehyde; Mesenteric Artery, Superior; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Topics: Adenine Nucleotides; Allopurinol; Animals; Body Water; Catalase; Energy Metabolism; Femoral Artery; Hindlimb; Mannitol; Muscle, Skeletal; Necrosis; Neutrophils; Peroxidase; Rabbits; Reperfusion; Reperfusion Injury; Superoxide Dismutase

Topics: Adenosine Triphosphate; Allopurinol; Animals; Energy Metabolism; Ischemia; Male; Muscle Fatigue; Muscle, Skeletal; Phosphocreatine; Rats; Rats, Wistar; Reference Values; Reperfusion Injury

Previous studies have demonstrated that reactive oxygen species are involved in ischemic injury. The present work was undertaken to determine in vivo the role of xanthine oxidase in the oxygen free radical production during rat liver ischemia and to examine the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) during the same period. Our results indicate a 4-fold increase in xanthine oxidase activity between 2 and 3 hours of normothermic ischemia, in parallel with a decrease in cell viability. Moderate hypothermia delays both events. Under the same conditions, the activity of oxygen radical scavenging enzymes remains unchanged. Moreover, we have compared in vitro the susceptibility of isolated liver cells to an oxidative stress induced by O2.-, H2O2 and .OH. Our results reveal that endothelial cells are much more susceptible to reactive oxygen species than hepatocytes, probably because they lack H2O2-detoxifying enzymes. These findings suggest that xanthine oxidase might play a major role in the ischemic injury mainly at the level of the sinusoidal space where most endothelial cells are located.

Topics: Animals; Catalase; Cell Separation; Endothelium, Vascular; Glutathione Peroxidase; In Vitro Techniques; Liver; Male; Oxidative Stress; Rats; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

Topics: Adenosine; Allopurinol; Animals; Cell Adhesion; Cold Temperature; Endothelium, Vascular; Glutathione; Graft Survival; Insulin; Ischemia; Leukocytes; Liver Circulation; Liver Transplantation; Microcirculation; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred ACI; Reperfusion Injury; Tacrolimus; Transplantation, Isogeneic

We examined the cytology of the exudate in preserved intestinal grafts on reperfusion and compared it with the histological findings in rat small intestinal transplantation. The jejunal graft was harvested from the Lewis rat and was preserved in University of Wisconsin solution for 6, 12, 24 and 48 h at 4 degrees C (n = 6, in each group) and was then syngeneically transplanted. On reperfusion, the exudate was collected and studied cytologically. Full thickness biopsies were performed at the end of the preservation and at 30 min after reperfusion for histological examination. Histological examination after reperfusion showed that the crypt layer was preserved until 24 h. However, it was destroyed by 48 h preservation. The cytological findings correlated with the depth of tissue injury shown histologically. The degeneration of villus epithelial cells, the decrease in the content of mucin in both the goblet cells as well as villus cells, and the appearance of crypt cells are all considered to be signs of poor graft viability. Cytological examination is therefore recommended as an effective, non-invasive and real-time method for evaluating graft viability just after reperfusion in small intestinal transplantation.

Topics: Adenosine; Allopurinol; Animals; Cell Death; Glutathione; Graft Survival; Insulin; Intestine, Small; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Tissue Preservation

The implication of different eicosanoids and oxygen free radicals in the development of pancreatic injury after an ischemia-reperfusion process has been evaluated. For this purpose we have compared the effect of allopurinol and indomethacin administration on the pancreatic levels of eicosanoids in a rat model of pancreatic ischemia-reperfusion. After 60 min of pancreatic ischemia and 2 h of reperfusion, significant increases in 6-keto-PGF1 alpha, PGE2, and LTB4 in pancreas tissue were detected. Allopurinol before the ischemic period reduced 6-keto-PGF1 alpha, PGE2, and LTB4 levels to the range of basal values, while prior indomethacin treatment significantly reduced 6-keto-PGF1 alpha and PGE2 levels, with LTB4 remaining unmodified. Increased postischemic plasma lipases were also significantly reduced by allopurinol to the range of sham-operated animals whereas indomethacin did not modify these levels. The data suggest a role for lipoxygenase metabolites in the development of pancreatic injury and the importance of the enzyme xanthine oxidase as an inductor of eicosanoid biosynthesis.

Topics: 6-Ketoprostaglandin F1 alpha; Allopurinol; Animals; Biomarkers; Cyclooxygenase Inhibitors; Dinoprostone; Free Radicals; Indomethacin; Ischemia; Leukotriene B4; Lipase; Lipoxygenase; Male; Oxidative Stress; Pancreas; Pancreatitis; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

"Declamping shock" is observed after aortic crossclamping, with hypovolemia, hypotension, and metabolic acidemia invariably present. We hypothesized that oxidants derived from xanthine oxidase influence the resuscitative interventions required to maintain baseline hemodynamic and acid-base status after aortic occlusion and reperfusion in rabbits. We also hypothesized that inactivation of xanthine oxidase with sodium tungstate could reduce systemic injury as assessed by the release of lactate dehydrogenase and alkaline phosphatase. To test these hypotheses, we established aortic occlusion in rabbits (n = 10, standard diet; n = 8, tungstate diet) for 40 minutes by inflation of a 4F Fogarty catheter in the descending thoracic aorta followed by 2 hours of reperfusion. Sham-operated rabbits (n = 10, standard diet; n = 9, tungstate diet) served as controls. Tungstate-pretreated rabbits required significantly less Ringer's solution (28%), phenylephrine (68%), and sodium bicarbonate (30%) during reperfusion (p < 0.005). Lactate dehydrogenase and alkaline phosphatase release during reperfusion was significantly attenuated by tungstate pretreatment (p < 0.05). Tungstate pretreatment resulted in plasma xanthine oxidase activities significantly lower than those in the sham group administered a standard diet (p = 0.007). Resuscitation requirements and systemic injury were reduced by inactivation of xanthine oxidase in a rabbit model that simulates the situation of human thoracic aorta operations.

Topics: Acid-Base Equilibrium; Alkaline Phosphatase; Analysis of Variance; Animals; Aorta, Thoracic; Constriction; Enzyme Activation; Hemodynamics; Isotonic Solutions; L-Lactate Dehydrogenase; Male; Phenylephrine; Rabbits; Reperfusion Injury; Resuscitation; Ringer's Solution; Shock, Surgical; Sodium Bicarbonate; Tungsten Compounds; Xanthine Oxidase

Rat livers were orthotopically transplanted after 90-min cold ischemia (group 1) or after 20-min warm and 70-min cold ischemia without (group 2) or with (group 3) allopurinol treatment (AT) (50 mg/kg i.v. 10 min prior to warm ischemia into the donor, flush perfusates with 1 mmol/l). Recovery processes were followed up for 60 min of reperfusion. Liver tissue levels of ATP and total adenine nucleotides were restored in group 1 to almost preischemic ranges within 15-30 min, remained significantly reduced by 30 and 20%, respectively, in group 2, and recovered with AT within 60 min in group 3 to almost the same extent as in group 1. A massive increase in the tissue malondialdehyde concentration, indicative of lipid peroxidation, occurred in the beginning of reperfusion of warm-ischemically damaged donor livers, which in group 3 with AT tended to be less pronounced than in group 2 without AT. The GSSG/GSH ratio reflecting intracellular oxidant stress averaged 3.3 x 10(-3) in group 1 between 15 and 60 min reperfusion. In group 3 AT resulted in comparably low values averaging 3.8 x 10(-3), while in warm-ischemically damaged livers without AT of group 2 this ratio was significantly and continuously elevated averaging 5.8 x 10(-3).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenine Nucleotides; Adenosine Triphosphate; Allopurinol; Animals; Antimetabolites; Energy Metabolism; Free Radicals; Glutathione; Lipid Peroxidation; Liver; Liver Transplantation; Male; Rats; Rats, Wistar; Reperfusion Injury; Temperature

A study was designed to investigate the possibility of reducing peritoneal adhesion formation in mice by pretreatment with allopurinol. Allopurinol, at a dose of 35 mg/kg of body weight/d significantly reduced the severity of peritoneal adhesions (P < .001), and also the neutrophil response to ischemia (P < .05). Tissue myeloperoxidase activity at the site of ischemic injury was significantly lower in the allopurinol-treated mice at the end of 2 weeks (P < .001). However, xanthine oxidase was undetectable in both control and allopurinol-treated mice. These observations suggest that allopurinol reduces the severity of peritoneal adhesion formation in mice, possibly by reducing the neutrophil response to ischemia.

Topics: Allopurinol; Animals; Laparotomy; Male; Malondialdehyde; Mice; Neutrophils; Peritoneal Diseases; Peritoneum; Peroxidase; Pilot Projects; Postoperative Complications; Premedication; Reperfusion Injury; Tissue Adhesions; Xanthine Oxidase

To ascertain whether surgery causes ischaemia-reperfusion (I-R) related injury, if this injury is augmented by preoperative shock, and reduced with low dose allopurinol.. Randomised blind placebo controlled trial.. Surgical laboratory.. 22 pigs were randomly allocated to four groups; OP = operation/placebo, OA = operation/ allopurinol, SOP = shock + operation/placebo, SOA = shock + operation/allopurinol. An aortic tube prosthesis was inserted in all. In groups SOP and SOA preoperative shock was induced by exsanguination. Allopurinol was administered in group OA on the preoperative day and peroperatively, in group SOA during shock and peroperatively.. Perioperative blood concentrations of thiobarbituric acid reactive species (TBARS), ascorbic acid (AA), albumin, 99mTc-albumin and creatine phosphokinase (CPK) as indicators of oxidative membrane damage, antioxidant activity, microvascular permeability changes and muscular cell damage respectively.. In the OP and OA groups TBARS gradually increased, while AA, 99mTc-albumin and CPK remained unchanged and albumin decreased. No effect of allopurinol was observed in these groups. In the SOP group TBARS and AA were not significantly different from groups OP and OA. Yet, albumin, 99mTc-albumin and CPK decreased significantly more in the SOP group. Compared with the SOP group, allopurinol treatment (SOA) produced lower TBARS and higher AA levels, and reduced the effect of shock on albumin, 99mTc-albumin and CPK concentrations.. Aortic surgery causes no I-R related damage. Pre-operative shock produces I-R related damage, which is reduced by allopurinol.

Topics: Allopurinol; Animals; Antioxidants; Aorta, Abdominal; Ascorbic Acid; Blood Vessel Prosthesis; Capillary Permeability; Creatine Kinase; Female; Granulocytes; Ischemia; Muscle, Skeletal; Oxidation-Reduction; Placebos; Random Allocation; Reperfusion Injury; Serum Albumin; Shock; Single-Blind Method; Swine; Technetium; Thiobarbituric Acid Reactive Substances

In this study, the kinetics of copper-zinc superoxide dismutase (CuZn-SOD) in experimentally induced ischemia-reperfusion injury of the canine jejunum were examined using immunohistochemical procedures, and evaluated as an index for the viability of transplants. A pedicled jejunal graft was subjected to arterial reperfusion after clamping the supplying blood vessels for 30 min. Under nonischemic conditions, some of the goblets in the goblet cells and the mucin covering the surface of the villi were stained positively with luxol fast blue, von Kossa, and immunohistochemistry for CuZn-SOD. Between 5 and 30 min after reperfusion, the appearance of goblets with positive immunoreaction for CuZn-SOD in the intestinal glands and the disappearance of these goblets in the villi were observed in the grafts of animals that received arterial reperfusion after 30 min of clamping of the arteries and veins at room temperature. Thereafter, the former disappeared gradually and the latter returned toward the nonischemic condition. The administration of allopurinol led to a decrease in tissue damage and a significantly higher number of goblets with positive immunoreaction for CuZn-SOD than in untreated animals. Furthermore, the goblets in the intestinal glands showed a negative reaction for CuZn-SOD 5 to 30 min after reperfusion. Preservation at 4 degrees C during ischemia revealed similar results to those observed in the animals given allopurinol. Thus, the distribution and intensity of CuZn-SOD positive goblets seems to be a useful indicator for the evaluation of tissue damage induced by free radicals mediating ischemia-reperfusion injury.

Topics: Allopurinol; Animals; Disease Models, Animal; Dogs; Immunohistochemistry; Jejunum; Kinetics; Reperfusion Injury; Superoxide Dismutase

Rat abdominal skin flaps were subjected to total venous occlusion for 8 h. Five minutes before release of the vascular occlusion, mannitol, mannitol plus anisodamin, anisodamin or placebo (0.9% normal saline) was administered intravenously. Drug treated flaps showed a statistically significant increase in the proportion of area surviving (P < 0.001). The combination of mannitol and anisodamin was not more effective than either agent alone in increasing the proportion of area surviving. The results of biochemical analyses indicated that neither mannitol nor anisodamin affected xanthine oxidase activity (p > 0.05) but that both agents significantly reduced the increase of malondialdehyde (MDA) concentration caused by ischaemia-reperfusion (p < 0.01). Treatment with mannitol or anisodamin also prevented the increase of lactate and water content and the decrease in glucose content in the island skin flap tissue which occurred on reperfusion. The data indicate that mannitol and/or anisodamin have the potential to salvage anticipated flap necrosis. It is possible that the mechanism of action is inhibition of damage caused by toxic oxygen species and improvement in capillary reperfusion.

Topics: Animals; Atropine Derivatives; Double-Blind Method; Drug Evaluation, Preclinical; Drug Therapy, Combination; Glucose; Graft Survival; Lactates; Male; Malondialdehyde; Mannitol; Rats; Rats, Wistar; Reperfusion Injury; Surgical Flaps; Xanthine Oxidase

A growing body of experimental data indicates that the "no-reflow" phenomenon is a type of reperfusion injury in skeletal muscle which may, in part, be mediated by oxygen free radicals, and thus may be attenuated by using agents that scavenge or inhibit formation of these reactive oxygen metabolites. This study was undertaken to assess the efficacy of recombinant human manganese superoxide dismutase (rhMnSOD) in reducing reperfusion injury in skeletal muscle. The specific advantage of this agent over other SOD types is a much longer plasma half-life (5 to 7 hr), allowing better equilibration between extra- and intracellular compartments. The rat cremaster model was used to study "no-reflow" in skeletal muscle. Reperfusion injury in the muscle was assessed by fluorescein dye perfusion, myocyte creatine phosphokinase (CPK) release, and contractile function in response to electrical field stimulation. Compared with untreated saline control animals, those treated with rhMnSOD after 5 hr of cremasteric ischemia, had a significantly higher percentage area of blood reflow (78 percent +/- 6 percent of normal), a greater percentage tetanic (66 percent +/- 9 percent of normal) and twitch (56 percent +/- 9 percent of normal) contractile strength, and less CPK release (21.5 percent higher than pre-reperfusion baseline CPK levels) (p < 0.05). Untreated saline control CPK release (21.5 percent higher than the prereperfusion level. Animals treated with allopurinol also had a significantly higher percentage twitch contraction (47 percent +/- 14 percent of normal) and a lower CPK release (11.1 percent of the prereperfusion value) 45 min after reperfusion than untreated saline controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Abdominal Muscles; Allopurinol; Animals; Creatine Kinase; Electric Stimulation; Fluorescein; Fluoresceins; Humans; In Vitro Techniques; Male; Muscle Contraction; Muscle, Skeletal; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Regional Blood Flow; Reperfusion Injury; Superoxide Dismutase

Topics: Adenosine; Allopurinol; Animals; Bacterial Physiological Phenomena; Glutathione; Insulin; Intestine, Small; Ischemia; Isotonic Solutions; Lipid Peroxidation; Male; Malondialdehyde; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Ringer's Lactate; Transplantation, Isogeneic

Topics: Allopurinol; Antimutagenic Agents; Antioxidants; Cardiac Surgical Procedures; Chromosome Aberrations; Heart Arrest, Induced; Humans; Mutagens; Reperfusion Injury

Acute inflammatory lung injury often complicates hemorrhagic shock, a systemic ischemia-reperfusion syndrome. Because oxygen radicals are generated during ischemia-reperfusion, and oxygen radicals can activate nuclear regulatory factors that affect transcription of proinflammatory cytokines, we examined the premise that oxygen radicals increase interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) expression in lung mononuclear cells after hemorrhage. Intraparenchymal pulmonary mononuclear cells isolated 1 h after hemorrhage from control mice had increased levels of mRNA for IL-1 beta (P < 0.001) and TNF-alpha (P < 0.05) compared with cells from sham-hemorrhaged mice. Hemorrhaged mice treated with the oxygen radical scavenger dimethylthiourea (DMTU) had decreased levels of mRNA for IL-1 beta in pulmonary mononuclear cells, compared with hemorrhaged controls (P < 0.05). In hemorrhaged mice depleted of xanthine oxidase (XO) by a tungsten-enriched diet, pulmonary mononuclear cell mRNA levels for IL-1 beta and TNF-alpha were significantly decreased (P < 0.01 and 0.05, respectively), compared with cells from hemorrhaged control mice fed a normal diet. Similarly, mRNA transcripts for IL-1 beta and TNF-alpha among pulmonary mononuclear cells from hemorrhaged mice treated with allopurinol, an inhibitor of XO, were also significantly reduced (P < 0.05 and 0.001, respectively), compared with hemorrhaged control mice not treated with allopurinol. Our results indicate that XO-derived oxygen radicals contribute to the increased expression of mRNA for IL-1 beta and TNF-alpha, which occurs among pulmonary mononuclear cell populations immediately after hemorrhage.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Allopurinol; Animals; Cytokines; Gene Expression; Lung; Male; Mice; Mice, Inbred BALB C; Reactive Oxygen Species; Reperfusion Injury; Shock, Hemorrhagic; Thiourea; Tungsten; Xanthine Oxidase

Free radicals are important mediators of reperfusion injury; however, the mechanism(s) of oxyradical production after liver reimplantation are not well understood. A model of cold storage and reperfusion using low-level chemiluminescence to directly measure oxyradical production during reperfusion was developed.. Rat livers were harvested and stored at 4 degrees C in University of Wisconsin cold-storage solution or Euro-Collins solution for 0-48 hours and then flushed and reperfused with warm oxygenated (37 degrees C) Krebs-Henseleit buffer. Liver chemiluminescence was measured using a sensitive photomultiplier tube, and hepatocellular injury was assessed by measuring aspartate aminotransferase release into the perfusate.. Chemiluminescence reached a maximum within 5 minutes of reperfusion and then decreased to a baseline within 30 minutes. There was a marked increase in chemiluminescence after only a short period of storage in University of Wisconsin cold-storage solution. Chemiluminescence decreased with longer periods of storage but steadily increased again after 16 hours of storage. Chemiluminescence after 22 hours of storage, but not after 3 hours of storage, was decreased by pretreatment with the Kupffer cell inactivator gadolinium chloride.. The data suggest two mechanisms of oxyradical production during cold storage and reperfusion of the rat liver. The later phase seems to be Kupffer cell dependent.

Topics: Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Cold Temperature; Free Radicals; Glutathione; Hypertonic Solutions; Insulin; Kupffer Cells; Least-Squares Analysis; Liver; Luminescent Measurements; Male; Organ Preservation; Organ Preservation Solutions; Oxygen; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion; Reperfusion Injury

To apply an electron spin resonance (ESR) spectroscopic technique as a means of determining the oxidising capacity of reactive oxygen species produced during hypoxia and reoxygenation of diseased human synovial tissue.. Twenty four specimens of fresh synovial tissue were obtained from patients undergoing primary total knee joint replacement and graded according to the degree of inflammation present. Tissue samples were subjected to an ex vivo hypoxia-reoxygenation cycle in the presence of the nitroso based spin trap, 3,5-dibromo-4-nitrosobenzene sulphonate. The degree of oxidation of the spin trap to a stable free radical was determined and followed with time. Control samples were subjected to hypoxia only.. The results indicate that the oxidising capacity of reactive oxygen species produced by human synovial tissue varies with the degree of inflammation present. Only the more inflamed specimens, from both rheumatoid arthritis and osteoarthritis patients, demonstrated increased production of reactive oxygen species when subjected to a hypoxia-reoxygenation cycle. This change was reduced by both competitive and non-competitive inhibitors of the endothelial based enzyme xanthine oxidase. The relative concentration of reactive oxygen species generated by the synovial tissue samples correlated with the mean capillary density of the specimens.. This study supports the hypothesis of movement induced hypoxicreperfusion injury of the chronically inflamed joint by demonstrating the generation of reactive oxygen species within inflamed human synovium following an ex vivo hypoxia-reoxygenation cycle. Evidence is presented that the microvascular endothelial based enzyme xanthine oxidase is the predominant source of ESR detectable oxidising species in inflamed synovial specimens exposed to hypoxia-reoxygenation.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Culture Techniques; Electron Spin Resonance Spectroscopy; Humans; Knee Joint; Middle Aged; Osteoarthritis; Oxypurinol; Reactive Oxygen Species; Reperfusion Injury; Synovial Membrane; Thiazoles; Xanthine Oxidase

Reactive oxygen species (ROS) generated from xanthine oxidase (XO) play an important role in ischemia-induced injury. We hypothesize that XO and xanthine dehydrogenase (XDH) are released into the circulation with ischemia reperfusion to the human liver and intestine. Blood was drawn from a patient, before and at intervals after an aortic cross-clamp procedure. Plasma was incubated in the presence of xanthine, with NAD+ (for XD +XO) and without NAD+ (for XO). The amount of urate formed was quantified using a high-performance liquid chromatograph (HPLC). The calculated XDH+XO and XO activity increased from 1.88 and 1.66 microU/mg protein, respectively, before the cross clamp to 3.77 and 3.11 microU/mg, respectively, 7 minutes after reperfusion to the superior mesenteric, celiac, and right renal artery beds. The release of a significant biological source of ROS may explain the damage to lung or heart observed after ischemia to the human liver and intestine.

Topics: Aged; Anastomosis, Surgical; Aorta; Aortic Aneurysm; Chromatography, High Pressure Liquid; Constriction; Humans; Intestines; Ischemia; Liver; Male; Reactive Oxygen Species; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

The present study investigated the effect of the administration of oxypurinol (40 mg/kg), an inhibitor of xanthine oxidase, on adenosine and adenine nucleotide levels in the rat brain during ischemia and reperfusion. The brains of the animals were microwaved before, at the end of a 20-min period of cerebral ischemia, and after 5, 10, 45, and 90 min of reperfusion. Cerebral ischemia was elicited by four-vessel occlusion with arterial hypotension to 45-50 mm Hg. Adenosine and adenine nucleotide levels in the oxypurinol-pretreated (administered intravenously 20 min before ischemia) rats were compared with those in nontreated animals exposed to the same periods of ischemia and reperfusion. Oxypurinol administration resulted in significantly elevated ATP levels at the end of ischemia and 5 min after ischemia, but not at 10 min after ischemia. ADP levels were also elevated, in comparison with those in the control rats, at the end of the ischemic period. Conversely, AMP levels were significantly reduced at the end of ischemia and during the initial (5 min) period of reperfusion. Adenosine levels were lower in oxypurinol-treated rats, during ischemia, and in the initial reperfusion phase. Oxypurinol administration resulted in a significant increase in the energy charge both during ischemia and after 5 min of reperfusion. Physiological indices, namely, time to recovery of mean arterial blood pressure and time to onset of respiration, were also shortened in the oxypurinol-treated animals. These beneficial effects of oxypurinol may have been a result of its purine-sparing (salvage) effects and of its ability to inhibit free radical formation by the enzyme xanthine oxidase. Preservation of high-energy phosphates during ischemia likely contributes to the cerebroprotective potency of oxypurinol.

Topics: Adenine Nucleotides; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Blood Pressure; Electrochemistry; Hydrogen-Ion Concentration; Ischemic Attack, Transient; Kinetics; Male; Oxypurinol; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Respiration; Xanthine Oxidase

It has been proposed that xanthine oxidase-derived superoxide mediates reperfusion injury in the liver; however, there is a little direct evidence to support this hypothesis. In this paper we describe a model system to directly and noninvasively measure oxyradical formation and hepatic injury in isolated perfused rat liver. Using this sensitive chemiluminescent technique, we clearly demonstrate the theorized burst in oxygen radical production upon reperfusion of previously ischemic liver, without perturbing the system with chemical luminescence enhancers. This increase in chemiluminescence (CL) upon reperfusion was diminished by the free radical scavengers trolox and ascorbate, as well as N-2-mercaptoproprionyl-glycine (MPG), thereby confirming the oxyradical nature of this signal. Additionally, superoxide dismutase and the xanthine oxidase inhibitor allopurinol, but not catalase, attenuated the reperfusion effect, providing the most direct evidence so far that XOD derived superoxide anion is formed during liver reperfusion. Hepatic injury (AST release) did not appear to relate to increased CL, supporting the notion that the oxyradical flux may serve as a signal for other events leading to tissue injury. Further studies using this sensitive chemiluminescent technique should aid in delineating the detailed mechanism(s) of reperfusion injury.

Topics: Animals; Aspartate Aminotransferases; Free Radical Scavengers; Liver; Luminescent Measurements; Male; Perfusion; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxides; Time Factors; Xanthine Oxidase

1. The role of allopurinol in the protection of kidney function following ischaemia-reperfusion injury has been investigated using the novel technique of near-infrared spectroscopy. 2. An in vivo model of rat kidney ischaemia was used, with the expected falls in blood and tissue oxygenation seen and confirmed by near-infrared spectroscopy. 3. Allopurinol infusion increased the rate of reperfusion of oxygenated blood seen in control rats (P < 0.05). 4. Allopurinol enhanced the rate of tissue oxygenation during early reperfusion (P < 0.01). 5. This study provides further evidence for the proposed benefits of allopurinol in ischaemia-reperfusion injury. Furthermore, the potential of near-infrared spectroscopy as a technique of value in interventional studies of this nature is confirmed.

Topics: Allopurinol; Animals; Hemoglobins; Ischemia; Kidney; Male; Oxyhemoglobins; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury

We investigated the effects of allopurinol on renal damage following renal ischemia. Male Wistar rats weighing 250-300 g were classified into enflurane and allopurinol groups and anesthetized for 5 minutes using 1.7 MAC of enflurane in 30% oxygen. Then the left renal artery was dissected and clamped. Arterial occlusion was performed under 1.3 MAC enflurane for 30 minutes. Anesthesia was maintained for an additional 90 minutes after releasing the clip. In the allopurinol group, the rats were administered with allopurinol 3 mg.kg-1 intravenously prior to renal ischemia. At the end of anesthesia and 24 hours after the discontinuation of anesthesia, the necrotic areas, kidney weight/body weight ratios, gamma-GTP and NAG activities of the kidney which had been clamped were examined. Urinary gamma-GTP and NAG activities and serum inorganic fluoride concentrations were also measured. The necrotic area was significantly smaller in the allopurinol group than in the enflurane group. The activity of gamma-GTP in the kidney was higher in the allopurinol group than in the enflurane group. The kidney weight/body weight ratio was lower in the allopurinol group than in the enflurane group. There was no difference in serum inorganic fluoride concentration between the allopurinol and enflurane groups. These results suggest that allopurinol decreases renal damage following renal ischemia under enflurane anesthesia.

Topics: Allopurinol; Anesthesia, Inhalation; Animals; Constriction; Enflurane; Ischemia; Kidney; Male; Premedication; Rats; Rats, Wistar; Reperfusion Injury

Oxygen radicals have been proposed to be involved in the induction of liver cell damage during reperfusion after ischemia. The role of xanthine oxidase in this process and the potential of the antioxidant system have been studied in a model of in vivo ischemia of rat liver followed by 1 h reperfusion by the use of enzyme histochemistry. Based on decreased lactate dehydrogenase activity in certain areas of liver parenchyma, cell damage could already be detected at 1 h reperfusion after ischemia. Incubations performed on serial sections showed that the same areas contained decreased activities of xanthine oxidoreductase, xanthine oxidase, catalase and glucose-6-phosphate dehydrogenase. Some individual cells in the undamaged liver parenchyma expressed a very high glucose-6-phosphate dehydrogenase, which suggests that these cells have a good defence against oxidative stress. It is concluded that oxygen radicals derived from xanthine oxidase do not play a decisive role in the induction of cell damage immediately at reperfusion after ischemia. However, it cannot be excluded that xanthine oxidase present in the blood stream can give rise to the development of additional damage later on.

Topics: Animals; Disease Models, Animal; Glucosephosphate Dehydrogenase; Histocytochemistry; Kupffer Cells; L-Lactate Dehydrogenase; Liver; Male; Monocytes; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

In the model of the perfused rat liver, we investigated the alterations of sinusoidal cells in the pathogenesis of liver injury caused by hypoxia and reperfusion. In sinusoidal endothelial cells, the activity of xanthine oxidase (XOX), a cytoplasmic marker enzyme, was located cytochemically and determined biochemically. Kupffer cells, identified by their endogenous peroxidase staining, were studied with regard to changes in their ultrastructure. In our experiments, parenchymal cells were shown to be severely damaged in contrast to sinusoidal lining cells, which showed minor signs of injury. In comparison with the control group, XOX activity increased significantly in the sinusoidal endothelial cells after low-flow hypoxia; however, after reoxygenation of only 5 minutes, that activity was lower after hypoxia but higher after control perfusion. In Kupffer cells, hypoxia resulted in a strong suppression of phagocytic and endocytotic activity and in a disappearance of the lamellopodia. Kupffer cells were flattened, resembling sinusoidal endothelial cells. After reoxygenation phagocytic vesicles, lamellopodia, and cell volume of Kupffer cells increased markedly in comparison with the control group. In the hypoxia/reperfusion injury model, our observations revealed significant alterations of sinusoidal lining cells. It appears that sinusoidal endothelial cells respond to the hypoxic phase by producing oxygen-derived free radicals and that Kupffer cells respond to the subsequent reperfusion phase by activation followed by the release of toxic mediators.

Topics: Animals; Cell Nucleus; Endothelium; Female; Glutamate Dehydrogenase; Hypoxia; Kupffer Cells; L-Lactate Dehydrogenase; Liver; Microbodies; Oxygen; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury; Xanthine Oxidase

Topics: Animals; Fasting; Ischemia; Liver; Rats; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

Kupffer cell activation is hypothesized to play an etiopathogenic role in storage-related graft failure after liver transplantation. The aim of this study was to verify whether the elimination of Kupffer cells modifies the magnitude of cold ischemia/reperfusion injury of the liver.. Rat Kupffer cells were eliminated by an intravenous injection of liposome-encapsulated dichloromethylene diphosphonate. Livers from control and treated rats were isolated and perfused before and after 24-hour cold ischemia in the University of Wisconsin solution (4 degrees C). Hepatocyte and sinusoidal endothelial cell functions were evaluated by taurocholate and hyaluronic acid elimination, respectively. Liver transplantation was also performed using control and treated donor livers stored under identical conditions.. Compared with baseline values, similar alterations were found in both groups after cold ischemia for hepatocyte function (intrahepatic resistance, bile secretion, lactate dehydrogenase release, oxygen consumption, and taurocholate intrinsic clearance) and for sinusoidal endothelial cell function (hyaluronic acid intrinsic clearance). The 10-day survival rate of animals undergoing transplantation was not different between the groups (6 of 15 vs. 4 of 15, control vs. treated donor livers, respectively).. The presence or absence of Kupffer cells does not modify the effect of 24-hour cold ischemia/reperfusion on the rat liver.

Topics: Adenosine; Allopurinol; Analysis of Variance; Animals; Bile; Glutathione; Hyaluronic Acid; Hypothermia, Induced; In Vitro Techniques; Insulin; Ischemia; Kupffer Cells; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Macrophage Activation; Male; Metabolic Clearance Rate; Organ Preservation; Organ Preservation Solutions; Oxygen Consumption; Raffinose; Rats; Reperfusion Injury; Taurocholic Acid; Tissue Survival

We investigated the effects of D-neopterin (NP) and its reduced form, 5,6,7,8-tetrahydro-D-neopterin (NPH4), in two models of ischemia-reperfusion injury, i.e., ischemic paw edema in mice and gastric ischemia in rats. In ischemic paw edema, iv administration of either NP or NPH4 more potently inhibited the increase of paw thickness after release from ischemia than did administration of superoxide dismutase plus catalase or allopurinol. In gastric ischemia, NP and NPH4 also significantly suppressed the formation of gastric mucosal erosions. Lipid peroxidation in the stomach was increased by ischemia-reperfusion treatment, and the increase was inhibited by the administration of NP or NPH4. The minimum dose of NPH4 required to suppress the gastric ischemic injury in this experiment was 0.3 mg/kg of body weight. These results suggest that neopterin may be effective as a protective agent against ischemia-reperfusion injury, in which active oxygen species are believed to play a major role.

Topics: Allopurinol; Animals; Biopterins; Catalase; Disease Models, Animal; Dose-Response Relationship, Drug; Edema; Gastric Mucosa; Lipid Peroxidation; Male; Mice; Neopterin; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase

To investigate the role of platelet-activating factor (PAF) in the preservation/reperfusion injury of the liver graft, the effect of treatment with a potent PAF antagonist (E5880) was evaluated in a pig orthotopic liver transplantation model. The graft liver was flushed out and preserved for 8 hr at 4 degrees C using a simplified University of Wisconsin solution. The PAF antagonist was administered into the University of Wisconsin solution (1 mg/L), into the rinsing solution (1 mg/L), and to a recipient pig (0.3 mg/kg d.i.v.) in group 1. The PAF antagonist was not given in the control group (group 2). Postoperative survival of more than 12 hr was 100% (9/9) in group 1 and 56% (5/9) in group 2 (P < 0.05). At 12 hr after reperfusion of the graft (RPF), the arterial ketone body ratio (acetoacetate to 3-hydroxybutyrate) increased to 1.54 +/- 0.15 (mean +/- SEM) in group 1, compared with 0.95 +/- 0.09 (P < 0.05) in group 2. In group 2, blood leukocyte count decreased to 8.3 +/- 0.9 (x 10(3)/microliters) at 2 hr after RPF, in contrast to a slight increase in group 1 (14.3 +/- 1.8 x 10(3)/microliter, P < 0.01). At 4 hr after RPF, glutamic oxaloacetic transaminase (461 +/- 59 vs. 712 +/- 97 U/L, P < 0.05), glutamic pyruvic transaminase (65 +/- 4 vs. 82 +/- 5 U/L, P < 0.05), and the lactate level (6.2 +/- 1.1 vs. 9.4 +/- 1.0 mmol/L, P < 0.05) in arterial blood were significantly lower in group 1 than in group 2. Light and electron microscopic study at 1 hr after RPF showed neutrophil sludging in the sinusoids and sinusoidal endothelial cell damage in group 2, while these findings were attenuated in group 1. It is suggested that PAF plays a key role in microcirculatory disturbance of the liver graft manifested on reperfusion, and that the treatment with E5880 has a protective effect against preservation/reperfusion injury of the graft in liver transplantation.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Graft Rejection; Insulin; Leukocyte Count; Liver; Liver Transplantation; Microscopy, Electron; Mitochondria, Liver; Organ Preservation Solutions; Piperidines; Platelet Activating Factor; Pyridinium Compounds; Raffinose; Reperfusion Injury; Swine; Tissue Preservation; Transaminases

The role of eicosanoid metabolism and its relationship with nitric oxide production in the ischemia-reperfusion associated with pancreas transplantation in the rat is explored in this study. Twenty-six male Sprague-Dawley rats were randomized into 3 groups, as follows: group 1, control animals not surgically manipulated; group 2, pancreas transplantation, after 12 hr of organ preservation in University of Wisconsin solution; group 3, same as group 2 but with administration of NG-nitro-L-arginine methyl ester (a nitric oxide synthase inhibitor) (10 mg/kg) before organ revascularization. The results show posttransplantation increases in edema and in 6-keto-prostaglandin F1 alpha (x1.9), thromboxane B2 (x4), and prostaglandin E2 (x5) levels in pancreatic tissue. Nitric oxide synthase inhibition reversed the increases in edema and eicosanoid production, which suggests that eicosanoid generation in the recipient rat would be mediated, in part, through a nitric oxide-dependent mechanism.

Topics: Adenosine; Allopurinol; Animals; Arginine; Dinoprostone; Edema; Glutathione; Insulin; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Organ Preservation; Organ Preservation Solutions; Pancreas Transplantation; Prostaglandins F; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Thromboxane B2

Topics: Adenosine; Allopurinol; Analysis of Variance; Animals; Glutathione; Insulin; Kupffer Cells; Liver; Liver Circulation; Liver Transplantation; Microcirculation; Organ Preservation Solutions; Phagocytosis; Raffinose; Rats; Rats, Inbred Lew; Reperfusion; Reperfusion Injury; Tissue Preservation; Transplantation, Isogeneic

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Glucose; Glutathione; Glycine; Graft Survival; Insulin; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Reperfusion Injury; Solutions; Time Factors; Transplantation, Homologous; Tromethamine

Topics: Adenosine; Allopurinol; Animals; Benzamidines; Glutathione; Graft Survival; Guanidines; Heart; Heart Transplantation; Insulin; Organ Preservation; Organ Preservation Solutions; Pancreas; Pancreas Transplantation; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solutions; Transplantation, Isogeneic

The gastric mucosal microcirculation and purine nucleotide metabolism were studied in rats after hemorrhagic shock and retransfusion. The mucosal surface density of perfused vessels (SDPV) and the mucosal levels of ATP, ADP, AMP, IMP, hypoxanthine and uric acid were measured following 15 min of hemorrhagic shock and 10 and 30 min after retransfusion, and the effects of pretreatment with allopurinol or ascorbate were studied. During shock there was a dephosphorylation of nucleotides and a decline in the SDPV. Retransfusion led to an additional reduction in the SDPV, but a complete restoration of preshock nucleotide levels 30 min after retransfusion. Allopurinol accelerated early rephosphorylation of nucleotides without effects upon SDPV while ascorbate completely preserved the mucosal level of energy-rich nucleotides 15 min after hemorrhagic shock and increased SDPV during early reperfusion. The results showed that there was a renewal of energy stores in gastric mucosa after hemorrhagic shock and retransfusion although parts of the vascular bed were not reperfused. The mucosal energy depletion after 15 min of hemorrhagic shock and part of the mucosal vessel injury after retransfusion were prevented by pretreatment with ascorbate.

Topics: Allopurinol; Animals; Ascorbic Acid; Blood Transfusion, Autologous; Gastric Mucosa; Male; Microcirculation; Purine Nucleotides; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Shock, Hemorrhagic

The relationship between lipid peroxidation and alterations in hepatic secretory function and microsomal function during hepatic ischemia/reperfusion was studied. Rats pretreated with free radical scavengers were subjected to 60 min of hepatic ischemia and to 1 and 5 h of reperfusion thereafter. Serum aminotransferase level and microsomal lipid peroxidation were markedly increased by ischemia/reperfusion. These increases were significantly attenuated by rebamipide, alpha-tocopherol or allopurinol. Bile flow and cholate output were markedly decreased by ischemia/reperfusion and free radical scavengers, especially rebamipide, restored their secretion. NADPH-cytochrome P450 reductase activity and cytochrome P450 content were decreased by ischemia/reperfusion. Rebamipide prevented the decrease of the NADPH-cytochrome P450 reductase activity but had little effect on the cytochrome P450 content. Aminopyrine N-demethylase activity was decreased and aniline p-hydroxylase was increased by ischemia/reperfusion, which were prevented by alpha-tocopherol and allopurinol, but not by rebamipide. Our findings suggest that ischemia/reperfusion diminishes hepatic secretory function and microsomal function by increasing lipid peroxidation, and rebamipide significantly ameliorates these changes through its free radical scavenging activity.

Topics: Alanine; Alanine Transaminase; Allopurinol; Animals; Bile; Enzyme Inhibitors; Free Radical Scavengers; Lipid Peroxidation; Liver Diseases; Male; Microsomes, Liver; Mixed Function Oxygenases; NADPH-Ferrihemoprotein Reductase; Organ Size; Quinolones; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Vitamin E; Xanthine Oxidase

Topics: Animals; Brain Ischemia; Cats; Female; Free Radicals; Humans; Male; Middle Aged; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

We have investigated the effect of two doses of allopurinol (ALL) (100 and 50 mg/kg) administered i.v. on liver function after 1 h of normothermic ischemia. ALL given in a concentration of 100 mg/kg significantly improved bile output after 1 and 24 h of reperfusion. Hepatocyte injury reflected by alanine aminotransferase (ALT) and lactic dehydrogenase (LDH) in plasma was also significantly reduced at 24 h, but not at 1 h of reperfusion compared with controls. ALL administered at a concentration of 50 mg/kg had some protective effect. Significant correlation between circulating liver enzymes and bile output at 24 h after reperfusion indicates an important pathophysiologic link between hepatocyte function and injury in this time window.

Topics: Alanine Transaminase; Allopurinol; Animals; Bile; Body Temperature; L-Lactate Dehydrogenase; Liver; Male; Random Allocation; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Reperfusion Injury

This study was conducted to determine the beneficial effects of treating digestive disorders of (6E,12E)-tetradecadiene-8,10-diyne-1,3-diol diacetate (TDEYA) detected in the plasma in hydrolyzed form: (6E,12E)-tetradecadiene-8,10-diyne-1,3-diol (TDEY), following the oral administration of a decoction of Atractylodes rhizome to rats. Assessment was also made of the efficacy of TDEYA in experimental gastric disorder models. Oral administration of TDEYA at doses of 300 to 500 mg/kg suppressed the formation of gastric lesions induced by indometacin in a dose-dependent manner. TDEYA at a dose of 200 mg/kg suppressed gastric lesions induced by an ischemia-reperfusion injury model. TDEYA at doses of 100 to 300 mg/kg did not show suppressive effects on water immersion stress-induced gastric lesions. TDEYA showed no active oxygen species scavenging action, nor did it have any effect on superoxide dismutase activity in the stomach tissue. TDEYA at doses of 200 to 500 mg/kg significantly suppressed xanthine oxidase (XO) activity in the stomach tissue following its oral administration. The suppressive effects of TDEYA on lesion formation induced by indometacin and ischemia-reperfusion injury models would thus appear to be due in part to the inhibition of XO activity in the stomach tissue.

Topics: Acetylene; Administration, Oral; Alkynes; Animals; Anti-Ulcer Agents; Disease Models, Animal; Diynes; Dose-Response Relationship, Drug; Fatty Alcohols; Free Radical Scavengers; Gastric Mucosa; Indomethacin; Male; Plant Extracts; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Stomach Ulcer; Superoxide Dismutase; Xanthine Oxidase

In the present study the authors investigated the effect of pretreatment with exogenous antioxidants such as deferoxamine (iron chelating drug), allopurinol (competitive inhibitor of xanthine oxidase) and vitamin E (scavenger of oxygen free radicals) on lipid peroxidation in rat cerebral cortex after post-ischaemic reperfusion, and on the survival rate. The effect of pretreatment with two calcium-antagonist drugs (diltiazem and verapamil) was also evaluated under the same experimental conditions. Pretreatment with exogenous antioxidants and with calcium-antagonist drugs significantly decreased cerebral conjugated diene levels (index of lipoperoxidation) with a concomitant increase in survival with respect to untreated ischaemic rats.

Topics: Allopurinol; Animals; Antioxidants; Brain Ischemia; Deferoxamine; Diltiazem; Lipid Peroxidation; Male; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury; Verapamil; Vitamin E

Xanthine oxidase (XO) activity and hydroxyl radical (.OH) formation are widely proposed mediators of renal reperfusion injury, potentially altering the severity of, and recovery from, postischemic acute renal failure. The goal of this study was to ascertain whether combination XO inhibitor (oxypurinol) and .OH scavenger (Na benzoate) therapy, given at the time of renal ischemia, alters the extent of: (1) tubular necrosis and filtration failure; (2) DNA fragmentation/apoptosis (assessed in situ by terminal deoxynucleotidyl transferase reactivity); (3) early tubular regenerative responses (proliferating cell nuclear antigen expression; (3H)thymidine incorporation); and (4) the rate and/or degree of functional and morphologic repair. The effects of XO inhibition, .OH scavengers, and "catalytic" iron (FeSO4) on human proximal tubular cell proliferation in vitro were also assessed with a newly established cell line (HK-2). Male Sprague-Dawley rats were subjected to 35 min of bilateral renal arterial occlusion with or without oxypurinol/benzoate therapy. These agents did not alter the extent of tubular necrosis or filtration failure, proliferating cell nuclear antigen expression or thymidine incorporation, or the rate/extent of renal functional/morphologic repair. DNA fragmentation did not precede tubular necrosis, and it was unaffected by antioxidant therapy. By 5 days postischemia, both treatment groups demonstrated regenerating epithelial fronds that protruded into the lumina. These structures contained terminal deoxynucleotidyl transferase-reactive, but morphologically intact, cells, suggesting the presence of apoptosis. Oxypurinol and .OH scavengers (benzoate; dimethylthiourea) suppressed in vitro tubular cell proliferation; conversely, catalytic Fe had a growth-stimulatory effect. These results suggest that: (1) XO inhibition/.OH scavenger therapy has no discernible net effect on postischemic acute renal failure; (2) DNA fragmentation does not precede tubular necrosis, suggesting that it is not a primary mediator of ischemic cell death; and (3) antioxidants can be antiproliferative for human tubular cells, possibly mitigating their potential beneficial effects.

Topics: Acute Kidney Injury; Animals; Antioxidants; Apoptosis; Benzoates; Benzoic Acid; Cell Division; Cells, Cultured; DNA Damage; DNA Nucleotidylexotransferase; Free Radical Scavengers; Humans; Ischemia; Kidney; Kidney Tubular Necrosis, Acute; Male; Nuclear Proteins; Oxypurinol; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley; Regeneration; Reperfusion Injury; Xanthine Oxidase

Using isolated rat lungs, we compared prevention of ischemia-reperfusion injury provided by flushing the lungs with modified Euro-Collins solution (EC), University of Wisconsin solution (UW), low-potassium-dextran solution (LPD), or Wallwork solution (WA). After 4 hours' and 6 hours' cold ischemia, reperfusion injury was assessed on the basis of changes in filtration coefficients (Kfc) and pressure-flow curves, characterized by the slope of the curves (incremental resistance) and the extrapolation of this slope to zero flow (pulmonary pressure intercept [Ppi]). After 4 hours, Kfc and Ppi were higher with EC than with UW, LPD, and WA, and the incremental resistance was higher with EC and UW. After 6 hours, Kfc and incremental resistance Ppi were higher with LPD than with WA. Because ischemia-reperfusion injury is associated with decreased endothelial synthesis of prostacyclin and nitric oxide, we tested whether the addition of prostacyclin or the nitric oxide precursor L-arginine to WA would improve preservation. The Kfc and Ppi were lower with both treatments. In conclusion, ischemia-reperfusion injury was best prevented by using WA. The favorable effect of prostacyclin or L-arginine emphasizes the role played by endothelial dysfunction in ischemia-reperfusion injury.

Topics: Adenosine; Albumins; Allopurinol; Animals; Blood Pressure; Cardioplegic Solutions; Chlorides; Epoprostenol; Glutathione; Hypertonic Solutions; Hypothermia, Induced; In Vitro Techniques; Insulin; Lung; Lung Compliance; Male; Mannitol; Organ Preservation; Organ Preservation Solutions; Organ Size; Potassium Compounds; Propionates; Pulmonary Artery; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors; Vascular Resistance

Topics: Animals; Antioxidants; Free Radicals; Humans; Hypoxanthine; Hypoxanthines; Hypoxia; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

To assess right colic artery blood flow and relevance of xanthine dehydrogenase/xanthine oxidase after experimentally induced strangulation obstruction and reperfusion of the colon, 5 ponies were subjected to 2.5 hours of complete ischemia of the left dorsal and ventral colons, allowed to recover from surgery, and monitored during a 48-hour reperfusion period. Five ponies were subjected to sham surgery and served as controls. All ponies had a Doppler ultrasound blood flow monitor implanted on the right colic artery near the pelvic flexure 10 to 14 days prior to the ischemic period. Colic artery blood flow was monitored prior to, during, and for 4 hours after surgery. Blood samples from the right colic artery and vein distal to the obstruction site were collected during surgery (prior to ischemia, after 1 and 2 hours of ischemia, and after 10 and 60 minutes of reperfusion) for determination of arterial and venous blood gas tensions and electrolytes. Prior to surgery, blood selenium and plasma vitamin E (alpha-tocopherol) concentrations and blood glutathione peroxidase (GPX) activity were determined to assess the status of endogenous antioxidants. Combined xanthine dehydrogenase (XDH) plus xanthine oxidase (XO) activity, and XO activity alone (nanomoles per minute per gram of tissue) were determined, using a dual-spectrophotometric technique. Xanthine dehydrogenase and oxidase activities were determined prior to ischemia, after 1 and 2 hours of ischemia, and at 1 and 48 hours after reperfusion. Median blood flow in the experimental and control groups (156 ml/min and 110 ml/min, respectively) was not statistically different before surgery, and was significantly (P < 0.02) lower in the experimental (4 ml/min) vs the control group (72.5 ml/min) during the ischemic period. Experimental ponies had significantly (P < 0.03) lower right colic artery blood flow during the 4 hours immediately after recovery from anesthesia. Significant difference was not observed in right colonic venous bicarbonate concentration between groups at any time. Median right colonic venous PCO2, pH, and standard base excess were different (P < 0.001) between groups during the ischemic period only. Median venous oxygen saturation and median venous PO2 were significantly (P < 0.001) lower in the experimental ponies at the end of 2 hours of ischemia, but were significantly (P < 0.05) increased during the reperfusion phase. Median venous potassium concentration was significantly (P < 0.01) hig

Topics: Animals; Antioxidants; Blood Flow Velocity; Colic; Colon; Disease Models, Animal; Electrolytes; Free Radicals; Horse Diseases; Horses; Hydrogen-Ion Concentration; Intestinal Obstruction; Oxygen; Reperfusion Injury; Vascular Resistance; Xanthine Oxidase

Iron chelators have been reported to protect tissues against reperfusion injury. This implies that iron is being released into the plasma or is made accessible in tissues for oxidation-reduction reactions. It has been postulated that ferritin is a likely source for this iron. This report demonstrates that adrenergic agents with the catechol structure, which includes the endogenous catecholamines norepinephrine and epinephrine, are capable of releasing iron from ferritin. It is shown that the net release of iron from ferritin by epinephrine is significantly enhanced under anaerobic conditions. The findings suggest that catecholamines can mediate iron release from ferritin under conditions that can occur during ischemia/reperfusion. Catecholamines are also shown to interact with the released iron and xanthine oxidase to produce highly reactive hydroxyl radicals. The implications of this interaction for ischemia/reperfusion are discussed.

Topics: Aerobiosis; Anaerobiosis; Catecholamines; Epinephrine; Ferritins; Free Radicals; Hydroxides; In Vitro Techniques; Iron; Reperfusion Injury; Structure-Activity Relationship; Xanthine Oxidase

It has been widely postulated that the central mechanism of hepatic reperfusion injury involves the conversion, during ischemia, of the enzyme xanthine dehydrogenase (XDH) to its free radical-producing form, xanthine oxidase (XOD). However, this theory has been questioned because (a) XDH to XOD conversion in whole liver occurs very slowly; (b) the cellular distribution of XDH/XOD is unclear; and (c) the direct demonstration of XDH to XOD conversion in viable cells is lacking. In this paper, we address all three issues by measuring XDH to XOD conversion and cell viability in purified populations of hepatic endothelial cells (EC), Kupffer cells (KC), and hepatocytes (HEP). Although XDH/XOD activity on a cellular basis was greater in hepatocytes (0.92 +/- 0.12 mU/10(6) cells) than ECs (0.03 +/- 0.01) or KCs (0.12 +/- 0.04), XDH + XOD specific activity was similar in all three cell types (HEP 1.85 +/- 0.10 U/g protein; EC 1.69 +/- 0.54; KC 2.30 +/- 0.22). Over 150 min of warm (37 degrees C) or 24 h of cold (4 degrees C) hypoxia, percent XOD activity increased slowly in ECs, from 21 +/- 2% (basal) to 39 +/- 3% (warm) and 49 +/- 5% (cold) and in HEPs (29 +/- 2% to 38 +/- 3% and 49 +/- 2%), but converted significantly faster in KCs (28 +/- 3% to 91 +/- 7% and 94 +/- 4%). The dramatic changes in Kupffer cell XOD during cold hypoxia occurred despite only minor changes in cell viability. When hypoxic KCs were reoxygenated after 16 h of cold hypoxia, there was a marked increase in cell death that was significantly blocked by allopurinol. These data suggest that significant conversion to the free radical-producing state occurs within viable KCs, and that Kupffer cell XOD may play an important role in mediating reperfusion injury in the liver.

Topics: Animals; Cell Separation; Cell Survival; Cold Temperature; Endothelium; Hot Temperature; Hypoxia; Kupffer Cells; Liver; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors; Tissue Distribution; Xanthine Dehydrogenase; Xanthine Oxidase

To determine the contribution of ischemia-reperfusion injury (IRI) to the blood flow deficit and hepatocellular dysfunction seen after resuscitation from hemorrhagic shock, the xanthine oxidase inhibitor allopurinol was given to rats as a 50 mg/kg bolus after shock but before resuscitation and continued as a 25 mg/kg/h infusion. Resuscitation with shed blood and lactated Ringer's restored cardiac output and blood pressure in both groups. Control animals demonstrated a reduction in total hepatic and effective hepatic blood flow to 59% and 43% of baseline values, respectively. Allopurinol resulted in a return to baseline values of both variables. Allopurinol treatment resulted in a 350% increase in xanthine, a 630% increase in hypoxanthine, and a 70% reduction in uric acid concentrations. These data suggest that IRI contributes to the organ dysfunction and blood flow deficits seen after resuscitated hemorrhagic shock the effect of which can be attenuated by the addition of the xanthine oxidase inhibitor allopurinol to standard resuscitation.

Topics: Adenosine Triphosphate; Allopurinol; Animals; Liver; Liver Circulation; Purines; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Shock, Hemorrhagic; Xanthine Oxidase

Cultured human umbilical vein endothelial cells (EC) were exposed to hypoxia for various time periods followed by reoxygenation, and production of both O2- and H2O2 by EC was measured using the cytochrome c and scopoletin methods, respectively. Production of significant amounts of O2- was observed as early as 1 min after reoxygenation, reached maximum at 3 min and seemed to decline thereafter. Production of H2O2 was observed slightly later than that of O2-. Endothelial cell injury during hypoxia/reoxygenation was determined by both the trypan blue dye exclusion and chromium release methods. Significant increase of EC injury was detected after reoxygenation by both methods. This injury was significantly inhibited by allopurinol but not by superoxide dismutase or catalase. These results indicate that the production of reactive oxygen metabolites by EC and EC injury occur very rapidly after reoxygenation, which has not been reported, and these should be taken in consideration for the understanding and treatment of the clinical reperfusion injury.

Topics: Allopurinol; Cells, Cultured; Endothelium, Vascular; Humans; Hypoxia; Oxides; Peroxides; Reperfusion Injury; Superoxides; Time Factors

This study examines the hypothesis that pentoxifylline protects splanchnic PGI2 synthesis during severe mesenteric ischemia/reperfusion injury. Anesthetized Sprague-Dawley rats (300 grams) were subjected to sham or superior mesenteric artery occlusion for 20 minutes followed by 30 minutes of reperfusion. The ischemia/reperfusion groups received either enteral allopurinol (10 mg/kg) daily for 5 days prior to ischemia, PTX (50 mg/kg) 10 minutes prior to ischemia or carrier. The superior mesenteric artery was cannulated and removed with its intact intestine (SV + SI). The SV + SI was perfused in vitro with oxygenated Krebs buffer. The venous effluent was collected and assayed for release of 6-keto-PGF1 alpha, PGE2 and thromboxane B2 by enzyme immunoassay. Severe mesenteric ischemia/reperfusion decreased SV + SI 6-keto-PGF1 alpha release by 40% compared to the sham group but did not alter release of PGE2 or thromboxane B2. Pretreatment of the animals with PTX and not allopurinol preserved SV + SI 6-keto-PGF1 alpha release at all times of perfusion to a level similar to the sham group. These data showed that severe mesenteric ischemia/reperfusion injury abolished release of endogenous splanchnic PGI2. PTX exerted a protective effect against severe mesenteric ischemia/reperfusion injury by maintaining release of splanchnic PGI2, a potent endogenous splanchnic vasodilator.

Topics: Allopurinol; Animals; Drug Carriers; Epoprostenol; Hemodynamics; Male; Mesenteric Arteries; Pentoxifylline; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Splanchnic Circulation

Topics: Adenosine; Allopurinol; Animals; Antioxidants; Cold Temperature; Edetic Acid; Glutathione; Graft Survival; Hypertonic Solutions; Insulin; Intestine, Small; Ischemia; Lipid Peroxidation; Male; Organ Preservation Solutions; Pregnatrienes; Raffinose; Rats; Rats, Inbred WF; Reperfusion Injury; Temperature; Tissue Preservation

Topics: Adenosine; Adenosine Diphosphate; Adenosine Triphosphate; Allopurinol; Animals; Biomarkers; Cats; Cold Temperature; Energy Metabolism; Enzymes; Female; Glutathione; Insulin; Intestinal Mucosa; Intestine, Small; Male; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Tissue Preservation; Transplantation, Autologous

Topics: Animals; Free Radicals; Glutathione; Ischemia; Liver; Liver Diseases; Mitochondria, Liver; Rats; Reperfusion Injury; Ubiquinone; Vitamin E; Xanthine Dehydrogenase; Xanthine Oxidase

Glycine has been shown to decrease membrane injury in isolated cells due to hypoxia or cold ischemia. The mechanisms of action of glycine are not known, but glycine may be useful in organ preservation solutions or in treating recipients of liver transplantation. In this study the isolated, perfused rabbit liver was used to measure how glycine affected liver performance after 48-h preservation in University of Wisconsin (UW) solution without added glutathione. UW solution is less effective for 48-h liver preservation when glutathione is omitted. Rabbit livers stored for 48 h without glutathione show a large increase in enzyme release (LDH and AST) from the liver and a reduction in bile production. The addition of 15 mM glycine to UW solution, in place of glutathione, did not improve bile production or reduce enzyme release. However, infusion of 10 mM glycine into the reperfused liver lowered LDH release significantly (from 2383 +/- 562 units/100 g to 1426 +/- 286 units/100 g) during the initial reperfusion of the 48-h preserved liver. Hepatamine, a parenteral nutrition solution containing glycine, as well as other amino acids, was also effective in lowering LDH release from the preserved liver. Although glycine reduced LDH release, it did not decrease the amount of AST released from the liver, nor did it improve bile production. Thus, we conclude that glycine, either in UW solution or given to the liver upon reperfusion, has no significantly beneficial effect as tested in this model. Further testing of glycine, however, should be conducted in an orthotopic transplant model in the rat or dog.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Glycine; Insulin; Liver; Liver Function Tests; Organ Preservation; Organ Preservation Solutions; Perfusion; Rabbits; Raffinose; Reperfusion Injury

Liver endothelial cells appear to be particularly vulnerable to cold ischemia reperfusion. However, their function has not yet been evaluated, except using electron microscopic changes and trypan blue exclusion (an index of cell death). Hyaluronic acid is a polysaccharide highly extracted by normal liver endothelial cells. We thus evaluated liver endothelial cell function by measuring hyaluronic acid elimination in a model of ischemia-reperfusion injury using isolated perfused Wistar rat livers. We compared the effects of two preservation solutions during cold ischemia (4 degrees C): normal saline with 2 mM CaCl2 (4 h and 8 h ischemia) and the University of Wisconsin solution (8 h and 24 h ischemia). Eliminations were measured during two 40-min periods before and after ischemia; during each period, hyaluronic acid (150 ng/ml) and also, to evaluate hepatocyte function, propranolol (100 ng/ml) were infused into the reservoir. We show that, whatever the preservation solution or time used, liver endothelial cell function is altered to a larger extent than hepatocyte function. University of Wisconsin solution does not appear to protect liver endothelial cells during preservation, particularly after 24 h of cold ischemia. Hyaluronic acid elimination can be a useful tool in the investigation of an ideal preservation solution to protect liver endothelial cells from ischemia-reperfusion damage.

Topics: Adenosine; Allopurinol; Animals; Endothelium; Glutathione; Hyaluronic Acid; In Vitro Techniques; Insulin; Liver; Liver Circulation; Male; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Sodium Chloride; Tissue Preservation; Trypan Blue

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Female; Glutathione; Hypertonic Solutions; In Vitro Techniques; Insulin; Ischemia; Kidney; Kidney Transplantation; Microscopy, Electron; Mitochondria; Models, Biological; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury

In spite of the interest in free radicals as mediators of ischemic damage, most information on these species in biological systems is derived from indirect measurements. Our aim was to obtain more direct information concerning sources of free radical production during ischemia and reperfusion.. We have performed simultaneous measurement of radical generation, purine metabolites, reduced glutathione, neutrophil infiltration and morphological appearance in the cat small intestine in vivo during 60 minutes of ischemia followed by 60 minutes of reperfusion.. Radical formation increased abruptly on reperfusion and remained elevated in untreated animals. Inhibition by a monoclonal antibody (IB4) against the neutrophil and by allopurinol treatment was paralleled by improvement of biochemical and morphological parameters. The radicals detected during reperfusion could be divided into one component arising directly from the neutrophils, one due to the xanthine oxidase reaction, and one unknown source.. Neutrophils are a major source of radical production during reperfusion after ischemia. Radicals formed in the xanthine oxidase reaction seem to function as a primer for the neutrophils. The nonsignificant linear correlation between radical formation and morphological appearance suggests that factors other than free radicals are important for the development of intestinal damage after a period of ischemia.

Topics: Allopurinol; Animals; Antibodies, Monoclonal; Biopsy; Cats; Female; Hypoxanthine; Hypoxanthines; Intestinal Mucosa; Intestine, Small; Ischemia; Male; Neutrophils; Peroxidase; Reactive Oxygen Species; Reperfusion Injury; Xanthine Oxidase

We evaluated the damage to the gastric epithelium produced by local ischemia-reperfusion (IR) with or without luminal perfusion with 0.1 N HCl. Local gastric ischemia was induced by clamping the left gastric artery. Use of radioactive microsphere technique revealed a significant reduction in blood flow induced only in the corpus (67% reduction). Because no measurable gross lesion was observed in this model, the blood-to-lumen clearance of 51Cr-labeled EDTA (51Cr-EDTA) served as an index of epithelial damage. In the absence of exogenous acid, the histological damage was minimum and could not be quantified. However, a significant increase in 51Cr-EDTA clearance was observed shortly after reperfusion in a manner that depended on the duration of ischemia. This increase in clearance reached a maximum approximately 10 min after reperfusion and returned rapidly toward control levels within 40-50 min after reperfusion. In the presence of exogenous acid, EDTA clearance increased significantly during ischemia, increased further during reperfusion, and did not recover for at least 60 min after reperfusion. The acid infused after reperfusion (no acid before reperfusion) did not significantly aggravate the mucosal damage that followed reperfusion. However, the acid infused before reperfusion (no acid after reperfusion) showed an effect on EDTA clearance similar to that induced by continuous acid perfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Allopurinol; Animals; Chromium Radioisotopes; Dimethyl Sulfoxide; Edetic Acid; Gastric Mucosa; Hydrochloric Acid; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase

The present study in Lewis rats was designed to assess the predictive value of the mucosal enzyme activities of glutaminase, maltase, and xanthine oxidase and of histology as parameters to delineate the degree of small bowel preservation injury. Small bowel grafts were flushed with saline or a modified phosphate-buffered sucrose (PBS) solution, stored at 8 degrees C for 1, 6, or 12 hr, and transplanted heterotopically. Tissue samples for determination of mucosal enzyme activities were taken after the cold storage period, 20 min after reperfusion and 2 and 7 days postoperatively. Biopsies for light microscopic evaluations were obtained at the same time points, but not after cold storage. Glutaminase activity was well maintained after cold storage, regardless of the duration of preservation. Enzyme activities measured 20 min after reperfusion decreased with increasing duration of preservation (saline: R2 = 32.8%; P < 0.01; PBS: R2 = 52.3%; P < or = 0.001) and with increasing histologic preservation injury. Glutaminase activities were predictive for survival of grafts preserved with the PBS solution (R2 = 49.6%; P < or = 0.001; sensitivity 92%; specificity 100%), while the activities of maltase and of xanthine oxidase failed to do so. The degree of histologic preservation injury seen in graft specimens obtained 20 min after reperfusion was a good predictor of graft survival with a sensitivity of 90% for saline-preserved grafts and 92% for PBS-preserved grafts and a specificity of 88 and 67%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: alpha-Glucosidases; Animals; Glutaminase; Graft Survival; Intestinal Mucosa; Intestine, Small; Male; Organ Preservation; Rats; Rats, Inbred Lew; Reperfusion Injury; Xanthine Oxidase

The purpose of this study was to clarify the role of adenosine in preservation of ischemically damaged pancreas by the two-layer (Euro-Collins solution [EC]/perfluorochemical [PFC]) method using a canine model. Twenty-four-hour preservation of the pancreas graft subjected to 60-min warm ischemia was successful by the two-layer (EC with adenosine/PFC) method (4/5, 80%), but neither simple cold storage in EC (0/5, 0%), nor EC with adenosine (1/5, 20%), nor the two-layer (EC/PFC) method (0/3, 0%) was successful. Tissue ATP concentrations at the end of preservation by the two-layer (EC with adenosine/PFC) method were significantly higher compared with the two-layer (EC/PFC) method (7.23 +/- 2.17 vs. 1.56 +/- 0.40 mumol/g dry weight, P < 0.01). Studies with [2-3H]adenosine demonstrated that only part of adenosine was converted to inosine, hypoxanthine, and adenine, whereas the remainder was incorporated into adenine nucleotides in the pancreas graft. In addition, hypoxanthine, inosine, and adenine did not substitute for adenosine. We conclude that provision of adenosine to ischemically damaged pancreas during preservation by the two-layer (EC/PFC) method allows ATP synthesis within the graft via direct phosphorylation of adenosine. Metabolic processes vital to repair damaged cells and maintain cellular integrity can be maintained, which makes it possible to preserve ischemically damaged pancreas.

Topics: Adenosine; Adenosine Triphosphate; Allopurinol; Animals; Dogs; Female; Fluorocarbons; Glutathione; Graft Survival; Hot Temperature; Hypertonic Solutions; Insulin; Male; Nucleosides; Nucleotides; Organ Preservation; Organ Preservation Solutions; Pancreas; Raffinose; Reperfusion Injury

Fourteen domestic swine were divided into two groups. Group A (n = 7) was the control group, in which no pharmacologic intervention was applied. In group B (n = 7), the ischemic-reperfused spinal cord was treated with the combination of allopurinol (50 mg/kg/day for 3 days before the day of operation) and deferoxamine (Desferal, 50 mg/kg administered intravenously over 3 to 4 hours). The administration of deferoxamine was completed 1 hour before crossclamping. The crossclamp was placed on the descending aorta just distal to the left subclavian artery for 30 minutes. Proximal hypertension was controlled with sodium nitroprusside and volume depletion. Methods of assessment included an evaluation of the neurologic status of the animals by quantitative Tarlov criteria, blood flow by radiolabeled microspheres, and histologic examination of the spinal cord. All animals in the control group, group A, were completely paraplegic with 0% recovery by Tarlov criteria at 24 hours after the removal of the crossclamp. In contrast, all animals in group B, in which the combination of allopurinol and deferoxamine was used, completely recovered (100% recovery by Tarlov criteria), and at 24 hours after the ischemic episode they were able to walk with no difficulty and had intact sensation. Functional parameters of these animals fully correlated with the morphologic findings. Widespread acute neuronal injury and vacuolation of neuropil were observed in the control group of animals. In contrast, animals in group B showed much less pronounced morphologic changes after the same period of ischemia. In summary, the combined use of these agents significantly (p < 0.001) reduced the incidence of paraplegia induced by aortic crossclamping with 82% additivity.

Topics: Allopurinol; Animals; Aorta, Thoracic; Constriction; Deferoxamine; Drug Therapy, Combination; Female; Paraplegia; Reperfusion Injury; Spinal Cord; Swine; Time Factors

Cardiopulmonary and other organ dysfunction often occurs after operation on the descending thoracic aorta. Though there are multiple causes of organ dysfunction in this setting, free radical injury may play a prominent role. Xanthine oxidoreductase, an enzyme that generates oxidants after exposure to ischemia, could be released from ischemic liver and intestine during reperfusion. To test this hypothesis, we created aortic occlusion in eight rabbits for 40 minutes by inflation of a 4F Fogarty balloon catheter in the descending thoracic aorta. Eight sham-operated rabbits served as a control group. Two hours of reperfusion followed removal of the balloon catheter. Hemodynamic and acid-base status were maintained near baseline values during reperfusion. Plasma samples were obtained for determination of the activity of the hepatocellular enzymes xanthine oxidoreductase, aspartate aminotransferase, alanine transferase, and lactate dehydrogenase. Plasma xanthine oxidoreductase activity increased significantly (p < 0.001) during reperfusion (729 +/- 140 microU/ml, mean +/- standard error of the mean) compared with baseline (132 +/- 18 microM/mL). The other enzymes followed a similar pattern of release. We report the release of xanthine oxidoreductase in an animal model that simulates the situation of human thoracic aorta operations. The oxidants produced by the circulating xanthine oxidoreductase observed during reperfusion would likely be toxic to vascular endothelium, potentially contributing to multiple organ dysfunction.

Topics: Alanine Transaminase; Animals; Aorta, Thoracic; Aspartate Aminotransferases; Catheterization; Constriction; Free Radicals; L-Lactate Dehydrogenase; Liver; Male; Rabbits; Reperfusion Injury; Time Factors; Xanthine Dehydrogenase; Xanthine Oxidase

Biopsies taken from 13 human liver grafts at different stages of the transplantation process were used for study of the morphology of sinusoidal cells prior to harvesting (5 biopsies), after preservation in UW solution (10 biopsies), and after complete revascularization (13 biopsies). The mean cold ischemic period was 12 h 30. Immediate follow up was uneventful and the mean peak of post-operative transaminases below 1300 IU/l. Biopsies were perfusion-fixed by the transparenchymal route to ensure satisfactory ultrastructural results. There were no loose sinusoidal endothelial cells in the lumen and no signs of cellular death. Some endothelial cells presented signs of activation at the end of the preservation period, and even more after revascularization, with numerous lucent vacuoles resembling endosomes in the cytoplasm. Kupffer cells also presented signs of activation, particularly after reperfusion. The retraction of endothelial cell processes which formed large gaps during cold ischemia proved to be partly reversible after reperfusion. Signs of endothelial cell damage with gaps and partial rupture of the plasmic membrane were also observed, particularly after revascularization, in areas which contained numerous inflammatory cells adhering to the wall. The Disse space was not generally enlarged and contained no inflammatory cells. The sinusoidal pole of hepatocytes was occasionally damaged with the formation of blebs. These results strongly suggest that any drug or preservation solution that will inhibit endothelial and Kupffer cell activation could be beneficial in the prevention of preservation and reperfusion injury.

Topics: Adenosine; Adult; Allopurinol; Biopsy; Endothelium; Female; Glutathione; Humans; Insulin; Kupffer Cells; Liver; Liver Transplantation; Male; Microscopy, Electron; Middle Aged; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury

The aim of this study was to test whether conversion of xanthine dehydrogenase into xanthine oxidase as induced by fasting, ischemia of the liver or both is an in vivo process or only occurs in vitro in homogenates. For this purpose, the conversion rate of xanthine dehydrogenase into xanthine oxidase was studied in liver homogenates obtained from rats after normal feeding or 24 hr of fasting followed or not by 2 hr of ischemia of the liver. In fed rats, the conversion rate of xanthine dehydrogenase into xanthine oxidase was studied as well in liver homogenates after different periods of reperfusion after 2 hr of ischemia. Homogenization was carried out under strictly controlled conditions, after which the supernatants were incubated at 37 degrees C in buffer for 0 to 5 hr. Enzyme activities were assayed spectrophotometrically by measuring urate production at 295 nm. Conversion started only after 2 to 3 hr of incubation of supernatants of control fed livers, whereas conversion started immediately after 24 hr of fasting. The percentage oxidase activity of total xanthine oxidoreductase activity in ischemic livers from fed animals was slightly higher (26.7% +/- 1.7%; p < 0.05) than in control livers (19.3% +/- 1.4%), whereas the percent oxidase activity in ischemic livers from fasted animals (16.7% +/- 1.0%) was not different from that in control animals (16.8% +/- 1.1%). Ischemia for 2 hr caused in vitro a substantial increase in the conversion rate in supernatants of livers of fed and fasted rats as compared with their controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Histocytochemistry; Liver; Male; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

The resumption of blood flow to organs following ischemia may cause a further increase in tissue damage through an increase in peroxidation of lipids in cell membranes. An experimental study was conducted to investigate the prevention of reperfusion injury after testicular torsion through changes in the lipid peroxide content of the testis. Adult male albino rats were divided into 11 groups, each containing 10 rats. One group served to determine base values of the lipid peroxide content of the testis and kidney; 3 groups were subjected to unilateral testicular torsion lasting 1, 3 and 5 hours; 3 groups were subjected to detorsion following torsion lasting 1, 3 and 5 hours; 3 groups were treated with allopurinol before detorsion following torsion lasting 1, 3 and 5 hours; and 1 group underwent sham operation as a control. Thiobarbituric acid reactive products of lipid peroxidation (TBAR) were assessed in testicular and renal tissues. Testicular torsion caused a significant increase in TBAR in the testis (p < 0.01), but not in the kidneys. Detorsion caused a further significant increase in testicular TBAR (p < 0.01). Pretreatment with allopurinol prevented this further increase (p < 0.01). It is concluded that, biochemically, reperfusion injury occurs in the testis following detorsion after testicular torsion of 720 degrees lasting as long as 5 hours. Pretreatment with allopurinol before detorsion prevents such reperfusion injury.

Topics: Allopurinol; Animals; Kidney; Male; Rats; Rats, Wistar; Reperfusion Injury; Spermatic Cord Torsion; Testis; Thiobarbituric Acid Reactive Substances

Recently, it has been demonstrated that the use of both cold Carolina rinse (CR, 4 degrees C) as well as warm Ringer's lactate (RL, 37 degrees C) attenuates microvascular perfusion failure and leukocyte (WBC) accumulation in liver grafts. The aim of this study was to analyse in vivo whether warming of CR can also lead to a reduction in microvascular reperfusion injury in rat liver transplantation. Syngeneic orthotopic liver transplantation, including arterial reconstruction, was performed in male Lewis rats (180-300 g). Livers were stored in University of Wisconsin (UW) solution for 24 h and rinsed with 15 ml CR which was either cold 4 degrees C (n = 7) or warm 37 degrees C (n = 8) prior to reperfusion. Hepatic microcirculation and WBC accumulation were assessed by intravital fluorescence microscopy, and graft function was determined by analysis of bile flow during the 90-min reperfusion period. Warm CR yielded significantly (P < 0.01) improved sinusoidal perfusion when compared with cold CR; however, the extent of WBC adherence in both sinusoids and postsinusoidal venules did not vary between the groups. In addition, bile flow was slightly increased after warm CR. We conclude that after 24 h of cold storage in UW solution, warming of CR may offer additional benefit in the prevention of microcirculatory reperfusion injury without affecting WBC accumulation.

Topics: Adenosine; Allopurinol; Animals; Bile; Glutathione; Hemodynamics; Insulin; Isotonic Solutions; Liver; Liver Transplantation; Male; Microcirculation; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Ringer's Lactate; Solutions; Transplantation, Isogeneic

Recently, we reported that SPC-100270, a sphingosine derivative and inhibitor of protein kinase C (50-90 microM) in mixed micelle assays, reduced reperfusion injury resulting from hypoxia in a low-flow, reflow model of liver perfusion. Here we report that SPC-100270 has similar beneficial effects following liver transplantation in vivo. Rat liver transplantation was performed using nonarterial and rearterial techniques. Livers from syngenic rats were harvested surgically, prepared with vascular cuffs and a splint, and stored for 24 or 48 h in University of Wisconsin (UW) cold storage solution. Just prior to completion of vascular reconstruction, the organ was rinsed with 3 or 10 ml of Ringer's solution, vehicle, or a solution containing SPC-100270 (up to 500 microM). Following implantation surgery, low doses of SPC-100270 were ineffective at reducing both parenchymal and nonparenchymal cell death, yet significant (P < 0.05) reductions were observed with 500 microM. Further, nonparechnymal cell viability was improved nearly four fold by the drug. SPC-100270 (500 microM) tended to increase survival following 48 h cold storage in UW solution, but the improvement was not statistically significant. SPC-100270 also did not diminish carbon-centered free radical formation in transplanted livers from alcohol-treated rats. Collectively, these data support the hypothesis that pretreatment of donor livers with an inhibitor of protein kinase C is effective in vivo at reducing reperfusion injury, particularly to nonparenchymal cells, following orthotopic liver transplantation in the rat.

Topics: Adenosine; Allopurinol; Animals; Cell Survival; Enzyme Inhibitors; Fatty Liver; Female; Glutathione; Graft Survival; Insulin; Liver Transplantation; Organ Preservation Solutions; Protein Kinase C; Raffinose; Rats; Reperfusion Injury; Sphingosine; Transplantation, Isogeneic

The effect of Rhizoma Ligustici Chuanxiong and allopurinol on ischemia-reperfusion damage of rabbit ear flap was studied. The results showed that MDA level was higher and SOD activity lower distinctly at 0.5 hour after reperfusion than that at 16 hours of postischemia and preischemia, Rhizoma Ligustici Chuanxiong and allopurinol changed the result markedly with a decrease of necrosis rate. The effect of Rhizoma Ligustici Chuanxiong was more significant than that of allopurinol in promoting microcirculation, preventing clot formation, reducing exudation and hemorrhage.

Topics: Allopurinol; Animals; Drugs, Chinese Herbal; Female; Free Radical Scavengers; Male; Malondialdehyde; Microcirculation; Rabbits; Reperfusion Injury; Skin; Superoxide Dismutase; Surgical Flaps

The aim of this study was to clarify the role of oxygen-derived free radicals and the effect of free radical scavengers on skeletal muscle ischemia/reperfusion injury. Male Wistar rats were divided into a complete ischemia group (C-group) and an incomplete ischemia group (IC-group) and each animal was subjected to 2 h of ischemia and 1 h of reperfusion. In an attempt to decrease reperfusion injury, the rats were given free radical scavengers either as allopurinol 50 mg/kg for 2 days or as superoxide dismutase 60,000 units/kg plus catalase 500,000 units/kg. Tissue malondialdehyde, a product of lipid peroxidation, was measured as an indicator of free radicals, with higher levels indicating higher concentrations of free radicals. The malondialdehyde level in the gastrocnemius muscle after 1 h of reperfusion increased significantly in both groups when compared to the levels before and 2 h after ischemia, although there was no significant difference between the two groups. The water content of the gastrocnemius muscle and serum creatinine phosphokinase MM isoenzyme (CPK-MM) in both groups, and GOT in the C-group, increased significantly after 1 h of reperfusion when compared the values before and 2 h after ischemia. In the C-group, these values were significantly higher than in the IC-group. The administration of free radical scavengers suppressed the increase in malondialdehyde in the gastrocnemius muscle after reperfusion in both groups. The increase in water content and CPK-MM after reperfusion was also suppressed by free radical scavengers in the IC-group, but not in the C-group.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Allopurinol; Animals; Aspartate Aminotransferases; Blood Flow Velocity; Blood Pressure; Body Water; Catalase; Creatine Kinase; Free Radical Scavengers; Free Radicals; Fructose-Bisphosphate Aldolase; Ischemia; Isoenzymes; Lipid Peroxidation; Male; Malondialdehyde; Muscles; Oxygen; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase

Xanthine oxidase, a key source of reactive oxygen species, and purine substrates are detected in the circulation after ischemia-reperfusion. High levels of uric acid, produced by a xanthine oxidase-catalyzed reaction, are found in human plasma. We studied whether uric acid could alter xanthine oxidase activity in plasma obtained from eight adults and eight neonates. Known amounts of uric acid were added to xanthine and xanthine oxidase-supplemented buffer and plasma, and the production of uric acid and superoxide was determined. Uric acid, 150 and 300 microM, decreased the oxidation of xanthine to uric acid in adult plasma by 37.5 +/- 5.6 and 48.9 +/- 6.1% and formation of superoxide by 23.2 +/- 1.9 and 32.0 +/- 2.3%, respectively, compared with plasma without uric acid. In newborn plasma, a similar pattern and extent of inhibition was observed. Superoxide formation, however, was inhibited to a greater extent than in adult plasma. Endogenous xanthine oxidase was detected in newborn plasma in nine additional neonates using HPLC. These results indicate that uric acid is an effective inhibitor of the formation of superoxide and hydrogen peroxide by xanthine oxidase at the levels found in human plasma. Plasma uric acid may play an important role in attenuating the oxidant-mediated tissue damage caused by xanthine oxidase released into the circulation during ischemia-reperfusion.

Topics: Adult; Age Factors; Free Radical Scavengers; Humans; Infant, Newborn; Oxidation-Reduction; Reactive Oxygen Species; Reperfusion Injury; Superoxides; Uric Acid; Xanthine; Xanthine Oxidase; Xanthines

This study investigated whether prostaglandin E1 (PGE1) could reduce hepatic injury to the liver graft caused by harvesting and 24-h preservation in University of Wisconsin (UW) solution in a canine model. The PGE1-treated group was intravenously administered 0.5 microgram/kg per minute of PGE1 for 30 min before harvesting, as well as a concentration of 1 mg/l PGE1 in the washout and UW solutions. In both the PGE1-treated and the control group, all recipients survived for 1 week or more after transplantation. Arterial ketone body ratio (AKBR) remained over 1.0 in the early postoperative period. The PGE1 group showed significant reductions in guanase, GOT, and LDH during the early postoperative period compared to the untreated control group. Histological examination disclosed partial mitochondrial swelling, hepatocyte vacuolation, and necrosis in the control group, while such abnormalities were rarely seen in the PGE1 group. These results suggest that PGE1 can effectively reduce hepatic injury to liver grafts preserved in UW solution prior to transplantation.

Topics: Adenosine; Allopurinol; Alprostadil; Animals; Aspartate Aminotransferases; Dogs; Glutathione; Guanine Deaminase; Insulin; Ketone Bodies; L-Lactate Dehydrogenase; Liver; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury; Time Factors

The effect of adding a 21-aminosteroid, U74500A, and a Ca2+ antagonist, lidoflazine, alone and together to UW solution was assessed in a rat liver preservation model. Following preservation, the livers were reperfused using a closed circuit, and the release of hepatocellular enzymes (ASAT, ALAT, and LDH) into the perfusate was determined with increasing time. Both drugs reduced the amount of enzymes lost from the liver. The combination of the two drugs was better than either drug alone. These data suggest that both agents may be of value in organ preservation for clinical liver transplantation.

Topics: Adenosine; Alanine Transaminase; Allopurinol; Animals; Aspartate Aminotransferases; Glutathione; Insulin; L-Lactate Dehydrogenase; Lidoflazine; Lipid Peroxides; Liver; Male; Organ Preservation; Organ Preservation Solutions; Pregnatrienes; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury

To assess the effects of oxygen free radicals on liver regeneration in rats after 80% hepatectomy.. Open laboratory study.. Institute of Biomedical Sciences, University of Milan, Italy.. Female Sprague-Dawley rats.. After laparotomy 122 rats had ischaemia induced by clamping of the portal vein and hepatic artery, and 93 rats had manipulation of the vessels only. They were then allowed to recover before 80% hepatectomy was done. Absolute controls (n = 16) did not have a laparotomy. Mortality was recorded, and surviving rats were killed one, three, five, and seven days after operation. A further 10 rats had 80% hepatectomies and then underwent a 10 minute period of ischaemia 24 hours later. These animals were killed after three days. In yet more experiments 38 rats underwent 80% hepatectomies and were then divided into four groups: 8 were given allopurinol 50 mg/kg/day starting three days before operation; 8 were given superoxide dismutase 4.16 mg/kg intraperitoneally 30 minutes before induction of ischaemia; 12 were given verapamil 0.1 mg/kg 30 minutes before induction of ischaemia; and 10 were given saline (controls).. Incorporation of tritiated thymidine into DNA, differences in liver weights, and lipid peroxide concentrations.. 43 rats died after ischaemia/reperfusion and 19 after hepatectomy alone. Ischaemia/reperfusion caused a significant reduction in the incorporation of tritiated thymidine into DNA 24 hours after hepatectomy (p < 0.01), and significant inhibition of recovery of liver weight three (p < 0.01) and five (p < 0.05) days after hepatectomy. These effects were associated with high lipid peroxide concentrations at three days. Allopurinol (p < 0.01, p < 0.05), superoxide dismutase (p < 0.01, p < 0.01) and verapamil (p < 0.01, N.S.) reduced the effects of ischaemia/reperfusion on liver weights or lipid peroxide concentrations three days after hepatectomy.. A 10 minute period of ischaemia followed by reperfusion temporarily reduces liver regeneration after 80% hepatectomy in rats.

Topics: Allopurinol; Animals; Female; Free Radicals; Hepatectomy; Ischemia; Lipid Peroxidation; Lipid Peroxides; Liver; Liver Regeneration; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion; Reperfusion Injury; Superoxide Dismutase; Verapamil

Isolated rat hepatocytes were used for the evaluation of nucleotide depletion and oxidative stress as two causal components of postischemic injury following oxygen deficiency. The ATP and GTP loss during anoxia was accompanied by temporary increases of nucleotide degradation products. The critical duration of anoxia for a complete ATP restoration during reoxygenation was between 30 and 60 min. The oxidative stress during reoxygenation was demonstrated by decrease of GSH concentration and increase of TBA-RS level. The tremendous GSH loss could not be balanced by the slight GSSG increase during reoxygenation. Prevention of GSH decrease and TBA-RS increase in parallel to prevention of viability loss in presence of oxipurinol in contrast to lacking improvement of ATP and GTP restoration by this drug speak in favor for the oxidative stress as major causal component for postischemic injury of hepatocytes in comparison with depletion of energy-rich purine nucleotides. The inhibition of formation of reactive oxygen species via xanthine oxidase reactions was found to be the dominant protective effect of oxipurinol against postischemic injury of hepatocytes in comparison with lacking influence on nucleotide salvage and ATP/GTP regeneration and with radical scavenging.

Topics: Adenosine Triphosphate; Allopurinol; Animals; Cell Hypoxia; Cell Survival; Cells, Cultured; Energy Metabolism; Glutathione; Guanosine Triphosphate; Liver; Male; Nucleotides; Oxidation-Reduction; Oxygen; Oxygen Consumption; Oxypurinol; Rats; Rats, Wistar; Reperfusion Injury; Stress, Physiological; Thiobarbituric Acid Reactive Substances

Reactive oxygen metabolites generated from xanthine oxidase play an important role in the pathogenesis of ischemia-induced tissue injury. In a hemorrhagic shock model of ischemia-reperfusion, the intracellular enzyme xanthine oxidase was released into the vasculature. This intravascular source of superoxide (O2.-) and hydrogen peroxide (H2O2) interacted reversibly with glycosaminoglycans of vascular endothelium and markedly concentrated xanthine oxidase at cell surfaces, enhancing its ability to produce extensive damage to remote tissues. Rats were made hypotensive by hemorrhage, maintained for 2h, and reinfused with shed blood. Blood samples were obtained prior to hemorrhage and 15, 30, 60, and 90 min after reperfusion for determination of xanthine oxidase (XO), lactate dehydrogenase (LDH), and alanine transaminase (AST). These enzymes were not significantly elevated in control animals. Reperfusion after hemorrhage-induced ischemia resulted in significantly elevated AST and LDH in both low heparin (100 U/h) and high heparin (1000 U/h) groups. Xanthine oxidase was detected in the circulation only after 90 min reperfusion in the low heparin group and was elevated during the entire reperfusion period in the high heparin group. Studies with cultured vascular endothelium showed significant heparin-reversible binding of XO to cellular glycosaminoglycans. These results suggest that XO can gain access to the circulation following ischemia, where it then binds to the vascular endothelial cells to produce site-specific oxidant injury to organs remote from the site of XO release.

Topics: Alanine Transaminase; Animals; Endothelium, Vascular; Free Radicals; Glycosaminoglycans; Heparin; Hydrogen Peroxide; Ischemia; L-Lactate Dehydrogenase; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Shock, Hemorrhagic; Superoxides; Xanthine Oxidase

The effects of perfusate calcium reduction, allopurinol and dimethylthiourea on reperfusion-induced arrhythmias and purine wash-out in isolated rabbit and rat hearts were compared. The overall incidence of reperfusion-induced ventricular tachycardia (VT) was 88% and 94% and that of ventricular fibrillation (VF) was 44% and 88% in the control rabbit and rat hearts, respectively. VF was reduced to 10% and 0% in rat and rabbit hearts subjected to perfusate calcium reduction (0.4 mM for 1 min before ischemia and for 1 min before and throughout reperfusion), respectively. In allopurinol, 1 mM, perfused rat hearts the overall incidence of VF was not changed and only the incidence of a sustained VF (that lasting for at least 10 min) was reduced. VT and VF were prevented in allopurinol-perfused rabbit hearts. Dimethylthiourea, 10 mM, reduced the incidence of VF in rat hearts to 16% and did not significantly affect VT and VF in rabbit hearts. In untreated rat hearts, the major purine compounds washed out upon reperfusion were inosine, hypoxanthine, xanthine and urate. Allopurinol augmented the wash-out of adenosine and abolished that of xanthine and urate. In untreated rabbit hearts, the major purine washed out were inosine, adenosine and hypoxanthine. Allopurinol did not cause further increase in adenosine wash-out in rabbit hearts. We speculate that: (1) calcium mediated arrhythmogenic mechanism is operating both in reperfused rat and rabbit heart; (2) free radical mediated mechanism is of an importance only in rat heart; (3) neither a decreased free radical production secondary to xanthine oxidase inhibition nor the augmentation of adenosine wash-out is a likely explanation for the antiarrhythmic effect of allopurinol in reperfused hearts; and (4) high level of myocardial adenosine accumulation during ischemia, probably secondary to low xanthine oxidase activity, may play a role of a natural defence mechanism in ischemic/reperfused rabbit heart.

Topics: Adenosine; Allopurinol; Animals; Arrhythmias, Cardiac; Calcium; Female; Free Radicals; Heart; Incidence; Male; Myocardium; Oxidation-Reduction; Purines; Rabbits; Rats; Rats, Wistar; Receptors, Purinergic P1; Reperfusion Injury; Tachycardia, Ventricular; Thiourea; Xanthine Oxidase

This study looks at the role of xanthine oxidase (XO) in ischemia/reperfusion (I/R) induced intestinal mucosal damage using normal and xanthine oxidase deficient rats. Tungstate feeding for 3 days depleted the intestinal mucosal XO by 80%. A ligated loop of the rat small intestine (both normal and XO-deficient) was subjected to 1 h of total ischemia followed by 5 min revascularisation. The ensuing mucosal damage was assessed by biochemical and histological studies. Ischemia or I/R increased the XO levels in normal rats without any change in XO-deficient rats. Myeloperoxidase (a neutrophil marker) level was increased in both group of rats but it was comparatively higher in the XO-deficient rats. Accumulation of peroxidation products such as malondialdehyde, conjugated diene and increased production of hydroxyl radicals by microsomes were seen after ischemia and I/R and were similar in normal and XO-deficient rats. Studies on other parameters of peroxidation showed a decrease in polyunsaturated fatty acids and alpha-tocopherol, an increase in cysteine and cystine levels after I/R and were similar in both normal and XO-deficient rats. Histological results indicated gross morphological changes in the intestinal mucosa due to ischemia and I/R, and the damage was more severe in XO-deficient rats. These observations suggest that oxygen-derived free radicals are involved in the intestinal mucosal damage during I/R and infiltrated neutrophils rather than XO may be the primary source of free radicals under these conditions.

Topics: Animals; Free Radicals; Intestinal Mucosa; Male; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reference Values; Reperfusion Injury; Xanthine Oxidase

This quantitative in vivo fluorescence microscopy study investigated the relative impact of an optimized rinse solution (warm Carolina rinse) and that of an established storage solution (University of Wisconsin solution) on various pathomechanisms of hepatic reperfusion injury after cold storage. Syngeneic orthotopic, arterialized liver transplantation was performed in male Lewis rats after 24 hr of cold ischemia (n = 24). The four experimental groups differed according to the type of preservation/rinse solution used: University of Wisconsin solution/albumin rinse (group 1), autologous blood (just external cooling)/albumin rinse (group 2), blood/Carolina rinse (group 3) and University of Wisconsin solution/Carolina rinse (group 4). Hepatic microvascular perfusion, leukocyte accumulation and phagocytic activity of Kupffer cells were assessed by means of intravital fluorescence microscopy 30 to 90 min after reperfusion. Disturbances of microvascular perfusion were most pronounced in group 2, markedly reduced by University of Wisconsin solution (group 1) and Carolina rinse (group 3) and minimized by combined use of University of Wisconsin solution and Carolina rinse in group 4. Intrahepatic leukocyte-endothelium interaction in sinusoids and postsinusoidal venules was found to depend on the application of Carolina rinse before reperfusion rather than the use of University of Wisconsin solution during cold storage. Activation of phagocytosis by Kupffer cells was most pronounced in group 1, intermediate in groups 2 and 3 and not noticeable in group 4. Hepatocellular excretory function as assessed on the basis of total bile flow and excretion of bile acids during the first 90 min after reperfusion was found to be improved by application of Carolina rinse, both after storage in blood or in University of Wisconsin solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Allopurinol; Animals; Bile; Bile Acids and Salts; Cell Adhesion; Cryopreservation; Glutathione; Insulin; Kupffer Cells; Leukocytes; Liver; Liver Circulation; Liver Transplantation; Male; Microcirculation; Organ Preservation; Organ Preservation Solutions; Phagocytosis; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solutions

The role of leukocyte sticking and permeability changes in the variation of perfused capillaries induced by ischemia reperfusion was studied in the hamster cheek pouch microcirculation. The drugs utilized were the antiperoxidative agents allopurinol, the calcium antagonists verapamil and diltiazem, and phenidone and adenosine, which inhibit leukotriene formation as well as leukocyte adhesion to the endothelium. The microvasculature was visualized by a fluorescence technique. Ischemia was induced by clamping the cheek pouch for 30 minutes followed by 30 minutes of reperfusion. The increase in permeability, the perfused capillary length and the number of adhering leukocytes to venular vessel wall were measured. Ischemia and reperfusion were associated with increased permeability, increased number of leukocytes sticking to the venular wall, and decreased number of functional capillaries. Microvascular injury evidenced by increased permeability was apparent in the first 5 min of ischemia. All the drugs decreased the number of leukocytes sticking to the venular wall; allopurinol, verapamil and adenosine reduced the increase in permeability, whereas phenidone and diltiazem were effective only during ischemia. In addition verapamil and adenosine preserved capillary blood flow during reperfusion. In conclusion, leukocyte sticking is correlated with increased microvascular permeability, but not with decreased perfusion of the capillary bed. These data suggest that leukocytes did not play a prominent role in the reduction of functional capillaries at the end of reperfusion.

Topics: Adenosine; Allopurinol; Animals; Capillary Permeability; Cell Adhesion; Cheek; Cricetinae; Diltiazem; Leukocytes; Male; Mesocricetus; Perfusion; Pyrazoles; Reperfusion Injury; Verapamil

Using isolated perfusion rat lung model, we studied the effect of continuous ventilation without perfusion and allopurinol on the development of ischaemia-reperfusion lung injury. Ischaemia was induced by stopping the perfusion. Normothermic ischaemia for 90 min without ventilation caused significant lung oedema. Continuous ventilation during ischaemia with 21% O2 decreased lung oedema significantly after 60 min of reperfusion. The same protection could be achieved by 100% N2 ventilation during 90 min of ischaemia, suggesting that xanthine oxidase (XO) is unlikely to cause the ischaemia-reperfusion lung injury. On the other hand allopurinol, XO inhibitor, equally inhibited lung oedema after 90 min of ischaemia and 60 min of reperfusion. These results indicate that mechanical movement of alveoli provides successful preservation of ischaemic lung, and allopurinol has some protective effect other than XO inhibition.

Topics: Allopurinol; Animals; Body Water; Ischemia; Lung; Male; Oxygen; Pulmonary Edema; Rats; Rats, Wistar; Reperfusion Injury; Respiration, Artificial; Xanthine Oxidase

Topics: Allopurinol; Animals; Cold Temperature; Dogs; Kidney; Kidney Transplantation; Kinetics; Mannitol; Mitochondria; Organ Preservation; Oxygen Consumption; Reperfusion Injury; Time Factors; Transplantation, Autologous; Verapamil

The effects of ischemia and reperfusion with and without oxygen radical scavengers and xanthine oxidase inhibitors on Ca(2+)-ATPase activity were examined in the rat liver of 5 min ischemia followed by 5 and 10 min reperfusion. Ischemia was produced by the ligation of right hepatic artery and right portal vein. Superoxide dismutase, catalase and allopurinol were administered by subcutaneous injection of 60,000U/kg, 90,000U/kg and 200mg/kg, respectively before ligation. Reaction products of Ca(2+)-ATPase were morphometrically analyzed by RUZEX IIIU. Histochemically, Ca(2+)-ATPase activities were demonstrated on plasma membrane of liver cells, bile canaliculi and Kupffer cells involving mitochondria in liver cells of control rats. Ca(2+)-ATPase activities were depressed in the central lobes of liver after 5 min ischemia followed by 5 and 10min reperfusion. However, the activities of Ca(2+)-ATPase were not depressed by addition of oxygen radical scavengers and xanthine oxidase inhibitor before ischemia. These results suggest that oxygen free radicals may influence Ca(2+)-ATPase activity and contribute to liver cell damage due to ischemia-reperfusion.

Topics: Animals; Calcium-Transporting ATPases; Catalase; Free Radicals; Ischemia; Liver; Male; Oxygen; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

Reactive oxygen metabolites have an important role in ischemia-reperfusion injury. One of the sources of reactive oxygen metabolites is xanthine oxidase, which is present in several tissues but is also released into the circulation after ischemia. We studied the effect of several potentially protective compounds on adenine nucleotide depletion induced by extracellular xanthine oxidase and hypoxanthine, in concentrations relevant to human pathophysiology. In umbilical vein endothelial cells prelabeled with 14C-adenine, cellular adenine nucleotides retained 64 +/- 9% of the initial radioactivity over a 4-h incubation with culture medium (controls), whereas in the presence of xanthine oxidase (80 mU/mL) and hypoxanthine (100 microM), only 3 +/- 4% of radioactivity remained in cellular nucleotides, the rest appearing in catabolic products in the medium. Glutathione and 3-aminobenzamide, an inhibitor of poly-ADP-ribose polymerase, partly prevented the nucleotide depletion (adenine nucleotide radioactivity 15 +/- 6% to 33 +/- 13% of total), but scavengers of the hydroxyl radical, dimethylthiourea and DMSO, as well as vitamins E and C, were without effect. Superoxide dismutase prevented the leakage of nucleotides into the culture medium but not intracellular nucleotide catabolism, whereas the latter process was decreased by catalase, consistent with predominant effects of superoxide and hydrogen peroxide at the cell membrane and interior, respectively.

Topics: Adenine Nucleotides; Antioxidants; Benzamides; Catalase; Cells, Cultured; Endothelium, Vascular; Glutathione; Humans; Hypoxanthine; Hypoxanthines; Poly(ADP-ribose) Polymerase Inhibitors; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Vitamin E; Xanthine Oxidase

Hemorrhagic shock-reinfusion injury produces critical changes in various organs with the generation of oxidant-free radicals. Some papers have reported that shock-reinfusion injury to the intestine is effectively reduced by the scavengers of free radicals; however, few reports mention the central organ damage caused by systemic hemorrhagic shock-reinfusion injury. Using a rat systemic hemorrhagic shock model, injury to the central organs, being the brain, heart, lungs, liver, and kidneys was assessed by measuring malondialdehyde (MDA). The MDA levels in the lungs, kidneys, and liver were elevated significantly after reinfusion, although there was no elevation of MDA in the brain or heart. These data show that the lungs, liver, and kidneys are easily damaged by shock-reinfusion, but that the brain and heart are relatively resistant. The efficacy of the free radical scavengers, superoxide dismutase plus catalase and allopurinol, were evaluated 30 min after reinfusion. Pathological examination showed that superoxide dismutase plus catalase and allopurinol reduced reinfusion injury in the lungs, liver, and kidneys. Moreover, superoxide dismutase plus catalase reduced MDA levels in both the liver and kidneys, whereas allopurinol reduced MDA levels only in the kidneys after reinfusion. However, these free radical scavengers could not suppress the elevation of MDA in the lungs after reinfusion.

Topics: Allopurinol; Animals; Catalase; Disease Models, Animal; Free Radical Scavengers; Male; Malondialdehyde; Rats; Rats, Wistar; Reperfusion Injury; Shock, Hemorrhagic; Superoxide Dismutase

The present study was undertaken to evaluate if allopurinol administration protects mice from bowel necrosis caused by temporary intestinal ischemia followed by indomethacin (INDO). We have previously reported that ischemia (15-minute occlusion of superior mesenteric vessels) followed by intravenous (i.v.) INDO caused significant bowel necrosis in CD-1 mice. Ischemia or INDO alone did not cause necrosis. To investigate protective measures against necrosis, we used CD-1 mice, 25 to 30 g. Forty-four animals were gavage fed 1 mL of water for 7 days and 32 animals were gavage fed 10 mg/kg allopurinol for 7 days. On the seventh day all animals were anesthetized and the superior mesenteric vessels occluded for 15 to 20 minutes, followed by i.v. INDO (0.5 mg/kg) once daily for 3 days. Animals who died were examined for bowel necrosis and all animals were killed 7 days after surgery and necropsied. Of the 44 saline-fed animals, 12 developed bowel necrosis (27%). Of the 32 allopurinol-fed animals, 1 developed necrosis (3%). The result of Fisher's exact two-tailed test was P = .006. Pretreatment with oral allopurinol significantly protects the mice from developing bowel necrosis when the mesenteric vessels are temporarily occluded and INDO is administered. Allopurinol may prevent reperfusion injury by inhibiting formation of xanthine oxidase generated, oxygen-derived free radicals and may be valuable in pretreating premature infants with patent ductus arteriosus who have had an ischemic episode in whom INDO use is contemplated.

Topics: Allopurinol; Animals; Ductus Arteriosus, Patent; Enterocolitis, Pseudomembranous; Gastric Lavage; Indomethacin; Intestines; Mice; Premedication; Reperfusion Injury

Topics: Adenosine; Allopurinol; Cold Temperature; Endothelins; Glutathione; Humans; Insulin; Ischemia; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Raffinose; Reperfusion Injury

Topics: Adenosine; Allopurinol; Animals; Aspartate Aminotransferases; Bile; Glutathione; Hepatic Artery; Insulin; Ischemia; Liver Function Tests; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Organ Size; Pressure; Raffinose; Reperfusion Injury; Swine

In this series of experiments, we surveyed xanthine oxidase activity after microvascular transfer in the venous effluent after reperfusion of human rectus abdominis muscle (n = 8) and jejunum (n = 4). Enzyme activity was correlated with duration of ischemia and biochemical markers of cellular injury. Xanthine oxidase (XO) activity was measured spectrofluorometrically using a pterin assay, whereas cellular injury was measured with commercial creatinine phosphokinase activity assay and lipid peroxidation products using a spectrophotometer. The data demonstrated that XO activity was statistically significantly increased in muscle flaps kept at room temperature during ischemia compared with muscle flaps that were cooled (p < 0.05). Creatinine phosphokinase activity was also increased after 15 minutes of reperfusion in muscle flaps that were not cooled (p < 0.05). Two of the jejunal free flaps had ischemia times of > 1 hour and had elevated XO activity after reperfusion despite cooling (p < 0.05). Two other jejunal flaps had ischemia times of < 1 hour, but in one case, the XO activity was increased before harvest. The other case had no increase in XO activity.

Topics: Abdominal Muscles; Creatine Kinase; Esophageal Neoplasms; Female; Graft Survival; Humans; Jejunum; Leg Injuries; Lipid Peroxidation; Male; Mammaplasty; Pharyngeal Neoplasms; Postoperative Complications; Reperfusion Injury; Surgical Flaps; Xanthine Oxidase

We have investigated the influence of BN 50727, a PAF antagonist, and allopurinol, a free radical scavenger, on the damaging effects of ischaemia-reperfusion and endotoxin in the small intestinal mucosa. Using a rat experimental model, we determined the alterations in intestinal permeability and mucosal levels of PAF and lysoPAF following ischaemia or intravenous administration of endotoxin. Both of these treatments increased intestinal permeability and enhanced PAF levels in the mucosa. Preventive oral or intraduodenal administration of BN 50727 reduced both of these effects, by decreasing mucosal PAF formation, probably as a result of neutrophil infiltration and activation reduction. Pretreatment of the rats with allopurinol also resulted in similar protection except that the free radical scavenger was unable to inhibit the increase in PAF levels after ischaemia, suggesting that oxidative reagents are implicated in this pathology to a much greater extent than PAF.

Topics: Allopurinol; Animals; Azepines; Endotoxins; Free Radical Scavengers; Intestinal Mucosa; Intestines; Male; Permeability; Platelet Activating Factor; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Thienopyridines; Triazoles; Xanthine Oxidase

The effect of Allopurinol on energy metabolism (re-utilisation of hypoxanthine) was studied in a in vivo skeletal muscle ischemia rat model by 31-P-MR spectroscopy. Allopurinol-treatment showed no benefit to the kinetics of PCr/(Pi + PCr) and ATP/(Pi + PCr). The role of re-utilisation of hypoxanthine has to be further investigated.

Topics: Adenosine Triphosphate; Allopurinol; Animals; Energy Metabolism; Hindlimb; Magnetic Resonance Spectroscopy; Male; Muscles; Phosphates; Phosphocreatine; Rats; Rats, Wistar; Reperfusion Injury

Topics: Allopurinol; Animals; Dogs; Electron Spin Resonance Spectroscopy; Endothelium, Vascular; Free Radicals; Granulocytes; Heart-Lung Transplantation; Hydroxides; Hydroxyl Radical; Luminescent Measurements; Oxygen; Reperfusion Injury

Polymorphonuclear leukocyte (PMN) sequestration within the pulmonary microvasculature is known to occur in association with ischemia/reoxygenation (I/R). This sequestration is dependent on eicosanoids and reactive oxygen species. PMN sequestration within the lungs suggests that pulmonary microvascular endothelial cells (MECs) may in part regulate the I/R response. Simulating I/R, we examined the effect of hypoxia/reoxygenation (H/R) on pulmonary MECs in vitro, with and without PMNs. Significant cellular injury, assessed by 51Cr release, occurred upon reoxygenation of MECs (P < .01). Addition of PMNs to the H/R-injured monolayers did not increase MEC injury. Reoxygenation of MECs also resulted in increased thromboxane (Tx) B2 production compared to controls (P < .01). Inhibition of Tx secretion by aspirin reduced H/R-induced PMN adhesion to MECs (P < .01). Furthermore, H/R-induced increases in PMN-MEC adhesion were prevented by allopurinol and superoxide dismutase (P < .01). These data suggest that the pulmonary response to H/R is mediated by MEC generation of reactive oxygen radical species and Tx, which promotes increased PMN adhesion.

Topics: Actins; Allopurinol; Animals; Cattle; Cell Adhesion; Cells, Cultured; Chromium Radioisotopes; Cytoskeleton; Dexamethasone; Eicosanoids; Endothelium, Vascular; Hypoxia; Leukotriene B4; Lung; Masoprocol; Microcirculation; Neutrophils; Radioimmunoassay; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Thromboxane B2; Xanthine Oxidase

Aortic aneurysm repair produces inflammatory mediators, neutrophil activation, and remote organ injury. Reperfusion plasma from these patients produces microvascular injury in an ex vivo chemotactic model. This study investigates the mechanism of this injury. Vena caval blood was obtained before and 15 minutes after aortic clamp removal (n = 16) or at laparotomy (n = 10). Plasma or saline solution was introduced into unit dose chambers fixed atop dermabrasions on the back of depilated anesthetized rabbits. Animals were treated with intravenous saline solution (n = 4); made neutropenic with nitrogen mustard (n = 4); pretreated with the xanthine oxidase inhibitor allopurinol (n = 4); or cotreated intravenously with the free radical scavengers superoxide dismutase (SOD) and catalase (n = 4). Three hours later neutrophil counts (polymorphonuclear cells [PMN]/mm3) and activity (free radical production by flow cytometry), protein leakage, and inflammatory mediators (thromboxane [TX] and leukotriene B4 [LTB4]) were measured. In contrast to control plasma in untreated rabbits, reperfusion plasma produced TX and LTB4 generation (1090 +/- 105 and 794 +/- 91 pg/ml, respectively, p < 0.01), PMN accumulation (1636 +/- 210/mm3, p < 0.01) and activation (276 +/- 31 mean fluorescent units), and microvascular permeability (554 +/- 90 micrograms/ml, p < 0.01). Neutropenia (3 +/- 1 PMN/mm3) and cotreatment with SOD and catalase abolished these responses, whereas pretreatment with allopurinol did not. Human reperfusion plasma contains a soluble factor that stimulates free radical generation by rabbit neutrophils to produce a microvascular injury characterized by de novo TX production, neutrophil accumulation and activation, and increased microvascular permeability to protein.

Topics: Allopurinol; Animals; Aortic Aneurysm, Abdominal; Catalase; Cell Movement; Chemotaxis, Leukocyte; Dermabrasion; Humans; Inflammation; Leukocyte Count; Leukotriene B4; Male; Neutrophils; Proteins; Rabbits; Reperfusion Injury; Skin; Superoxide Dismutase; Thromboxane B2

Neutrophils (PMNs) are believed to play a key role in the pathogenesis of postinjury adult respiratory distress syndrome. We have previously shown that gut ischemia/reperfusion (I/R) produces lung injury by a process that requires PMNs. More recently, we have shown that xanthine oxidase (XO) plays a role. The purpose of this study was to characterize the mechanistic sequencing of XO activity versus the PMN in this model of gut I/R-induced lung injury. Normal and XO-inactivated (tungsten enriched, molybdenum depleted diet) rats underwent 45 min of superior mesenteric artery occlusion. After 6 hr reperfusion, blood was sampled and gut and lungs harvested. Myeloperoxidase (MPO) was used to quantitate PMN presence in the gut and lungs, while circulating PMN priming was measured as the difference in superoxide production with and without the activating stimulus, fMLP. 125I-labeled albumin leak was used as a marker for lung endothelial permeability. We observed that the gut I/R increased gut MPO levels, primed circulating PMNs, increased lung MPO levels, and provoked distant lung leak. XO inactivation abolished gut MPO activity, attenuated circulating PMN priming, and blocked lung leak. In conclusion, XO plays a proximal role in the pathogenesis of remote organ injury following splanchnic hypoperfusion.

Topics: Animals; Intestines; Ischemia; Lung; Lung Diseases; Male; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxides; Xanthine Oxidase

The peroxidation of lipids and changes in the activities of related enzymes, such as xanthine-xanthine oxidase (XOD), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) in the gastric mucosa were studied in rat model of ischemia-reperfusion with pylorus ligation. Myeloperoxidase (MPO), a marker enzyme of leucocytes, was also studied. Thiobarbituric acid reactive substances (TBA RS) in gastric mucosa were significantly increased by clamping the celiac artery for 30 min and reperfusion for 60 min after 3 h of pylorus ligation. XOD activity in gastric mucosa increased with the development of gastric mucosal injury. Allopurinol significantly suppressed XOD activity but did not inhibit mucosal injury or the increase in TBA RS. MPO activity in the gastric mucosa was significantly increased by gastric mucosal injury. Famotidine significantly inhibited the increase in MPO activity in gastric mucosa, while allopurinol did not. SOD and GSH-px activities in the gastric mucosa were decreased significantly by gastric mucosal injury. SOD activity was normal following treatment with famotidine and allopurinol. Moreover, GSH-px activity recovered to the normal level with famotidine and allopurinol treatment. These findings suggest that oxygen radicals and lipid peroxidation can cause gastric mucosal injury by ischemia-reperfusion in the pylorus-ligated rat. The generation of oxygen free radicals may be derived mainly from activated polymorphonuclear leukocytes (PMN), and the decrease in SOD and GSH-px activity in gastric mucosa seems to aggravate mucosal injury by free radicals and lipid peroxidation.

Topics: Allopurinol; Animals; Famotidine; Free Radicals; Gastric Mucosa; Glutathione Peroxidase; Ligation; Lipid Peroxidation; Male; Peroxidase; Pylorus; Rats; Reperfusion Injury; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Xanthine Oxidase

We investigated whether xanthine oxidase (XO) is a major source of oxygen-derived free radicals (oxy-radicals) in the pig and human skeletal muscles. It was observed that xanthine dehydrogenase and XO activities in nonischemic pig latissimus dorsi (LD) and gracilis muscles and human LD and rectus abdominis (RA) muscles were < 0.5 mU/g wet wt. The pig LD muscle hypoxanthine content increased significantly from 0.33 +/- 0.02 to 2.33 +/- 0.44 mumol/g dry wt after 5 h of warm ischemia, but the muscle uric acid content remained unchanged up to 2 h of reperfusion. Similarly, the hypoxanthine content in the human LD and RA muscles increased from 0.33 +/- 0.03 to 0.84 +/- 0.23 mumol/g dry wt after 2.0-3.5 h of warm ischemia, and the muscle uric acid content remained unchanged at the end of 15-90 min of reperfusion. Furthermore, 5 days of allopurinol treatment (25 mg/kg iv twice daily) starting 2 days before ischemia or 3 days of oxypurinol treatment (25 mg/kg iv twice daily) starting 15 min before reperfusion did not attenuate the extent of skeletal muscle necrosis in pig LD muscles subjected to 5 h of ischemia and 48 h of reperfusion. However, deferoxamine treatment (250 mg/kg iv twice daily) starting before or after ischemia, as described above, significantly reduced the extent of pig LD muscle necrosis. Finally, at 2 and 48 h of reperfusion significantly higher muscle neutrophil contents were seen in ischemic than in nonischemic control pig LD muscles. Neutrophil depletion with mechlorethamine (0.75 mg/kg iv) significantly reduced the extent of necrosis in pig LD muscles. These observations indicate that XO is not a major source of oxy-radicals in ischemia/reperfusion injury in the pig gracilis and LD muscles and human RA and LD muscles.

Topics: Adult; Allopurinol; Animals; Blood Cell Count; Chromatography, High Pressure Liquid; Female; Humans; Hypoxanthines; Male; Mechlorethamine; Muscles; Necrosis; Neutrophils; Orchiectomy; Oxypurinol; Peroxidase; Reactive Oxygen Species; Reperfusion Injury; Swine; Uric Acid; Xanthine Dehydrogenase; Xanthine Oxidase; Xanthines

This study sought to examine the acute effects of ocular ischemia and reperfusion on retinal function and determine the extent to which recovery during reperfusion is improved by allopurinol (AP), a blocker of xanthine oxidase (XO). The latter is presumed to be a major factor in the formation of free radicals associated with reperfusion of ischemic tissue.. Electroretinographic (ERG) responses were recorded simultaneously from the two isolated, arterially perfused eyes obtained from the same cat. One eye served as the control and received only the standard perfusate; the other eye was infused with AP before and after a 3 hr period of total ischemia.. After the prolonged period of nonperfusion, recovery of the electroretinographic components was incomplete to varying degrees. Maximum b-wave amplitudes recovered only to 17 +/- 5% (mean +/- SEM) of baseline, whereas the a-wave grew to 60 +/- 10% of its baseline value. For both measures, the recovery of electroretinographic amplitude was significantly greater in AP-treated eyes than in the control eyes.. Generation of free radicals by XO contributes to the retinal damage and loss of function that occurs after a period of ischemia and subsequent reperfusion.

Topics: Allopurinol; Animals; Cats; Dark Adaptation; Electroretinography; Female; Free Radicals; Ischemia; Light; Perfusion; Reperfusion Injury; Retina; Retinal Artery; Xanthine Oxidase

In a recent study, reperfusion mucosal injury was demonstrated in a rat model of total ischemia if venous congestion was avoided. The aims were to examine the possibility of reperfusion damage in a canine model involving 2 hours of complete segmental ischemia and to investigate the effects of antioxidant therapy or pretreatment with nonspecific phospholipase A2 inhibitors on postocclusive mucosal changes. Tissue samples were evaluated histologically in a blind manner, according to a 0 to V grade scale. The degree of mucosal damage was statistically significantly increased during the 30-minute reperfusion period. Similarly, 2 hours of total ischemia followed by 30 minutes of reperfusion produced significantly more tissue lesions than did 2 1/2 hours of ischemia without reperfusion. Oral allopurinol pretreatment supplemented by an intravenous dose, or oral allopurinol in combination with a superoxide radical scavenger, resulted in a significant amelioration of postischemic histologic changes. Pretreatment with a nonspecific phospholipase A2 inhibitor (methylprednisolone, dexamethasone, or quinacrine) was ineffective in diminishing the reperfusion injury in either case. The results suggest that reperfusion injury may develop even after complete intestinal ischemia, and this damage can be attenuated by inhibiting the capacity of xanthine oxidase to generate reactive oxygen intermediates.

Topics: Allopurinol; Animals; Antioxidants; Dexamethasone; Dogs; Female; Ileum; Intestinal Mucosa; Ischemia; Male; Methylprednisolone; Phospholipases A; Phospholipases A2; Quinacrine; Quinolines; Reperfusion Injury

The time course of oxidative stress and tissue damage in zonal liver ischemia-reperfusion in rat liver in vivo was evaluated. After 180 min of ischemia, surface chemiluminescence decreased to zero, state 3 mitochondrial respiration decreased by 70-80%, and xanthine oxidase activity increased by 26% without change in the water content and in the activities of superoxide dismutase, catalase, and glutathione peroxidase. After reperfusion, marked increases in oxyradical production and tissue damage were detected. Mitochondrial oxygen uptake in state 3 and respiratory control as well as the activities of superoxide dismutase, catalase, and glutathione peroxidase and the level of nonenzymatic antioxidants (evaluated by the hydroperoxide-initiated chemiluminescence) were decreased. The severity of the post-reperfusion changes correlated with the time of ischemia. Morphologically, hepatocytes appeared swollen with zonal cord disarrangement which ranged from mild to severe for the tissue reperfused after 60-180 min of ischemia. Neutrophil infiltration was observed after 180 min of ischemia and 30 min of reperfusion. Mitochondria appear as the major source of hydrogen peroxide in control and in reperfused liver, as indicated by the almost complete inhibition of hydrogen peroxide production exerted by the uncoupler carbonylcyanide p-(trifluoromethoxy) phenylhydrazone. Additionally, inhibition of mitochondrial electron transfer by antimycin in liver slices reproduced the inhibition of state 3 mitochondrial respiration and the increase in hydrogen peroxide steady-state concentration found in reperfused liver. Increased rates of oxyradical production by inhibited mitochondria appear as the initial cause of oxidative stress and liver damage during early reperfusion in rat liver.

Topics: Animals; Free Radicals; Hydrogen Peroxide; Ischemia; Liver; Luminescent Measurements; Male; Mitochondria, Liver; Oxygen Consumption; Phenobarbital; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

Lactobionic acid, [4-beta-(galactosido)-D-gluconic acid] = LBA, is the major component of the Wisconsin organ transplantation preservant fluid and may suppress oxygen radical-induced tissue damage upon reperfusion by the control of FeII autoxidation. FeII and FeIII complexes of LBA and the related gluconic acid (GLC) have been studied herein by titrimetric, infrared, and electrochemical methods (CV; DPP). FeII(GLC) forms quickly at pH 7, but FeII(LBA) reacts in two steps, the second requiring 4 hr. The initial complex lacks coordination of the LBA carboxylate (C-1) and is bound by the "2,3,5" hydroxyl groups. The slow rearrangement forms a "1,2,3,6" chelate which FeII(LBA) shares in common with the donor set of the FeIII(LBA) complex. Titration data shows the removal of three protons from LBA through pH 5 and an additional proton from pH 6 to 9 which is indicative of the [FeIII(LBA)(OH)(H2O)]- formulation with LBA donating at the "1,2,3,6" positions. The more stable, second form of FeII(LBA) has been investigated in its oxidation mechanisms with H2O2 and O2 using selected trapping agents for HO. and ferryl intermediates. Eighty-six percent of the oxidation events of FeII(LBA)/H2O2 occurs in steps involving formation and reduction of freely diffusible HO.. These pathways are altered by the known HO. traps t-butanol, dmso, ethanol, and methanol in the manner predictable for beta-oxidizing radicals (from t-butanol or dmso) and alpha-reducing radicals (from ethanol and methanol). Fourteen percent of the FeII(LBA)/H2O2 reaction occurs via FeIVO intermediates not trapped by t-butanol or dmso, but intercepted by primary and secondary alcohols. The HO. generating pathways are responsible for a competitive LBA ligand oxidation at the C-2 position via HO., formed from FeII(LBA) and H2O2 within the original reaction cage. Competitive ligand oxidation at C-2 is absent for the FeII(LBA)/O2 autoxidation, indicative of a different redox mechanism. The FeII(LBA)/O2 reaction rate is first-order in each component and is insensitive to the presence of t-butanol as an HO. trap. These observations support a ferryl intermediate in the autoxidation pathway and the absence of HO. or free H2O2 during autoxidation. Although chelation of FeII by hard ligand donors such as edta4-, Cl-, or HPO4(2-) accelerate the rate of autoxidation of FeII, chelation of carboxylate, alkoxy, and hydroxyl donors of LBA does not accelerate autoxidation. The implications of these findings, and the abse

Topics: Adenosine; Allopurinol; Disaccharides; Electrochemistry; Free Radical Scavengers; Gluconates; Glutathione; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Insulin; Iron; Organ Preservation; Organ Preservation Solutions; Oxidation-Reduction; Raffinose; Reactive Oxygen Species; Reperfusion Injury; Solutions; Spectrophotometry, Infrared

Topics: Adenosine; Allopurinol; Animals; Dogs; Glutathione; Insulin; Lipid Peroxides; Lung; Lung Compliance; Organ Preservation; Organ Preservation Solutions; Pulmonary Circulation; Pulmonary Edema; Raffinose; Reperfusion Injury; Solutions; Time Factors; Vascular Resistance

Topics: Adenosine; Allopurinol; Animals; Free Radicals; Glucose; Glutathione; Graft Survival; Hypertonic Solutions; Insulin; Intestinal Absorption; Intestinal Mucosa; Intestine, Small; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solutions; Superoxide Dismutase; Transplantation, Isogeneic

Oxymyoglobin at 'physiological' concentrations of 20-100 micromolar protected cultured endothelial cells from damage by xanthine/xanthine oxidase or by hydrogen peroxide. Metmyoglobin also provided a degree of protection, but apomyoglobin was ineffective. Protection was enhanced in the presence of ascorbate (0.01-1 mM). Myoglobin may have a physiological role in the protection of muscular tissue from ischaemia/reperfusion-induced damage.

Topics: Adenine; Animals; Aorta; Ascorbic Acid; Cattle; Cells, Cultured; Endothelium, Vascular; Hydrogen Peroxide; Metmyoglobin; Myoglobin; Reperfusion Injury; Xanthine; Xanthine Oxidase; Xanthines

The critical injury to liver during cold preservation is believed to occur to the sinusoidal endothelium. In this study the viability of cultured sinusoidal endothelial cells from rat liver was assessed during storage in University of Wisconsin solution at 4 degrees C. The vast majority of cells (83 +/- 12%) died within 24 hr of storage. Addition of KCN (1 mM) to the solution to simulate hypoxia markedly increased survival: only 3 +/- 2% of cells had lost viability after 24 hr in the presence of cyanide. Further experiments showed that other inhibitors of mitochondrial ATP formation (antimycin A 1 microM, rotenone 1 microM, oligomycin 10 microM, carbonyl cyanide m-chlorophenylhydrazone 1 microM) were protective as well, whereas glucose (10 mM) greatly diminished the protective effect of cyanide (loss of viability 38 +/- 7% after 24 hr). ATP measurements confirmed the correlation between the energy state of the cells and cell death: ATP levels after 6 hr of incubation were 19.9 +/- 4.0 nmol/10(6) cells in UW solution, 13.7 +/- 2.9 nmol/10(6) cells in UW + glucose, 6.9 +/- 1.9 nmol/10(6) cells in UW + KCN + glucose and 1.9 +/- 1.5 nmol/10(6) cells in UW + KCN. In contrast to the protective effect observed in UW solution, addition of KCN to Krebs-Henseleit buffer led to increased endothelial cell damage upon cold storage. We therefore conclude that in UW solution damage to the sinusoidal endothelium is energy-dependent.

Topics: Adenosine; Adenosine Triphosphate; Aerobiosis; Allopurinol; Animals; Antimycin A; Cell Survival; Cryopreservation; Cyanides; Endothelium; Glucose; Glutathione; Hypoxia; Insulin; Liver; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Wistar; Reperfusion Injury; Rotenone; Solutions; Time Factors; Tissue Preservation

Acute ischemic renal failure is of great clinical importance because of its frequent occurrence and the high mortality it causes. Recent observations indicate that reperfusion has its own dangers because of oxygen-derived free radicals. To study this problem, ischemia was evoked in dogs in one kidney, by clamping the left renal artery for 45 min. This was followed by a 90-min period of reperfusion when diuresis, GFR, PAH clearance and sodium and potassium excretion were studied. Besides a control group (n = 6), the following treatment groups were investigated. Allopurinol (n = 7): 50 mg/kg for two days p.o. and 50 mg/kg in physiological saline infusion during the experiment; a small dose of SOD (n = 6): 0.5 mg/kg in infusion, started 1 min before reperfusion and given continuously for 10 min; and a high dose of SOD (n = 7): 5 mg/kg as above. In the first 15 min following reperfusion, the renal functions significantly worsened in all groups. Later on, the renal functions gradually improved and in the last period after reperfusion, GFR in the ischemic kidney was 64%, cPAH 59%, diuresis 60% and sodium and potassium excretion were 65% and 76%, respectively, of the basal values in the control group. Treatment with free radical scavengers did not cause any considerable changes in the renal functions. In some respects, the worst results were observed with low-level SOD treatment (cPAH, diuresis, as well as sodium and potassium excretion). At the end of reperfusion, there was a significant drop in sodium excretion by the right (intact circulation) kidney of the treated animals.

Topics: Allopurinol; Animals; Antioxidants; Dogs; Female; Kidney; Kidney Function Tests; Male; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase

Effects of 60 and 120 minutes of in-vitro ischaemia on the localization of xanthine oxidase activity were studied in rat intestine and liver. A histochemical method was applied on unfixed cryostat sections using a semipermeable membrane. The incubation medium contained hypoxanthine as substrate, cerium ions which capture the enzyme product, hydrogen peroxide, and sodium azide to inhibit catalase and peroxidase activities. In a second step reaction diaminobenzidine was polymerized in the presence of cobalt ions and hydrogen peroxide by decomposition of cerium perhydroxide. Large amounts of final reaction product were found in the cytoplasm of enterocytes and goblet cells of control small intestine. When the incubation was performed in the absence of substrate or in the presence of substrate and allopurinol, a specific inhibitor of xanthine oxidase activity, no reaction product was found. After 60 and 120 minutes of storage of tissue blocks at 37 degrees C enzyme activity was significantly reduced in the apical region of epithelial cells, whereas a high activity was present in the basal region of these cells. A very low xanthine oxidase activity was found in rat liver. Highest activity was present in endothelial cells, whereas in liver parenchymal cells, a more pronounced activity was found in pericentral than in periportal hepatocytes. Ischaemia up to 120 minutes did not affect the enzyme activity in livers. It was concluded that increased xanthine oxidase activity during ischaemia may not be responsible for cell damage during reperfusion in contrast with assumptions in the literature.

Topics: Animals; Culture Techniques; Intestine, Small; Ischemia; Liver; Male; Rats; Rats, Wistar; Reperfusion Injury; Xanthine Oxidase

The conversion from xanthine dehydrogenase (XD) to xanthine oxidase (XO) and the effect of trifluoperazine (TFP), a calmodulin inhibitor, on the conversion were examined during the normothermic ischemia of the rat small intestine. Rat jejunums were stored in lactated Ringer's solution (LR) at 37 degrees C for various hours after intravascular flushing with LR. The extents of the conversion from XD to XO (%XO) constituted 21.1% +/- 3.0%, 36.2% +/- 7.0%, 63.2% +/- 8.1%, and 88.2% +/- 8.6% after 0, 2, 4, and 6 hours of the preservation, respectively (control group). The preservation without the intravascular flushing showed significant increase in the %XO (99.5% +/- 6.0%) only after 6 hours compared with those in the control group (P < .05). When the intestines were stored in LR containing 50 mg/L of TFP at 37 degrees C, or stored in LR at 37 degrees C after the intraperitoneal pretreatment with 10 mg/kg of TFP 1 hour before laparotomy showed significant decrease in the extents of the conversion after 4 hours (P < .005) and 6 hours (P < .025) of the preservation, compared with those in the control group. When the dose of TFP for the pretreatment was increased to 50 mg/kg, the suppressive effect on the conversion was found even after 2 hours (P < .025) as well as after 4 hours (P < .005) and 6 hours (P < .025) of the preservation. These results suggest that TFP could be effective on reducing the XO-mediated postischemic reperfusion injury by means of inhibiting the conversion during ischemia of the rat small intestine.

Topics: Animals; In Vitro Techniques; Ischemia; Jejunum; Male; Rats; Reperfusion Injury; Trifluoperazine; Xanthine Dehydrogenase; Xanthine Oxidase

Flushing hepatic grafts immediately before revascularization with a specially designed rinse solution such as "Carolina rinse" has been reported to improve survival after liver transplantation in the rat. This study investigated the influence of Carolina rinse and adenosine rinse on early graft function, microcirculation, and leukocyte (WBC)-endothelial cell interaction of arterialized syngeneic orthotopic liver transplants in Lewis rats. Livers were preserved for 24 hr in University of Wisconsin solution and flushed immediately before reperfusion with either Ringer's lactate (group A: n = 7), Ringer's lactate + 0.2 mmol/liter adenosine (group B: n = 6), or Carolina rinse (group C: n = 7). Microvascular perfusion and WBC accumulation were assessed by intravital fluorescence microscopy. In group C, acinar perfusion was significantly improved, accompanied by a lower percentage of nonperfused sinusoids 1 hr after reperfusion (mean +/- SEM: 26 +/- 2% [group A], 21 +/- 2% [B], 11 +/- 1% [C], P < 0.01 for C vs. A or B). In addition, Carolina rinse and, to a lesser extent, adenosine rinse reduced the number of WBC sticking in sinusoids and postsinusoidal venules. Better graft function in group C was indicated by increased bile flow during the observation period of 90 min after reperfusion (0.5 +/- 0.3 ml/100 g liver [group A], 1.5 +/- 0.7 [B], 3.7 +/- 0.6 [C], P < 0.01 for C vs. A or B). We conclude that Carolina rinse is able to improve early excretory hepatocellular function, microvascular perfusion, and intrahepatic WBC accumulation after prolonged cold ischemia and reperfusion, but adenosine is unlikely to be the key component of this rinse solution.

Topics: Adenosine; Allopurinol; Animals; Bile; Cell Adhesion; Cell Communication; Endothelium, Vascular; Glutathione; Hemodynamics; Insulin; Leukocytes; Liver; Liver Transplantation; Male; Microcirculation; Microscopy, Fluorescence; Organ Preservation; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solutions

Pharmacologic manipulation of free flaps to enhance tolerance to ischemia has become a subject of great interest in the research literature. In an effort to improve survival, perfusion washout of experimental free flaps was performed following an episode of primary ischemia. The perfusates utilized were lactated Ringer's solution (LR), University of Wisconsin solution (UW), a high-molecular-weight medium used in organ preservation, and urokinase, a thrombolytic agent. Seventy-five rats were used in this study and divided into groups of 5 each. A 3 x 6-cm abdominal free flap based on the superficial inferior epigastric vessels was raised in each rat. The free flaps were subjected to either 12 or 18 hr of primary ischemia. Following the period of ischemia, perfusion washout was performed with either LR, UW solution, or urokinase at increasing concentrations alone or in combination with UW solution. Urokinase was first evaluated as a perfusate alone at increasing concentrations. In the 12-hr ischemia group, free-flap survival was shown to increase from 0 percent in the LR-perfused flaps to 20 percent, 60 percent, and 80 percent in flaps perfused with 12,500, 25,000, and 100,000 U of urokinase, respectively (p < 0.05). A similar increase in survival was demonstrated in the 18-hr ischemia group, where 0 percent, 20 percent, and 40 percent of flaps survived following perfusion with 12,500, 25,000, and 100,000 U of urokinase, respectively (p < 0.05). Urokinase was then perfused along with UW solution to evaluate the combined effect on flap survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Allopurinol; Animals; Drug Combinations; Glutathione; Graft Survival; Insulin; Isotonic Solutions; Organ Preservation Solutions; Perfusion; Raffinose; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Ringer's Solution; Solutions; Surgical Flaps; Time Factors; Tissue Preservation; Urokinase-Type Plasminogen Activator

Both storage injury and reperfusion injury have been reported in association with liver transplantation; however, which predominates is not clear. Therefore, these studies were designed to evaluate whether Carolina Rinse, which minimizes reperfusion injury following orthotopic liver transplantation in the rat, would be effective after long-term (48 h) storage of grafts in University of Wisconsin (UW) cold storage solution where sufficient time for development of storage injury exists. Livers were rinsed with either Ringer's solution or Carolina Rinse solution immediately prior to completion of implantation surgery. In the Ringer's group, 30-day survival was high following 24 h of cold storage (4/5) but was very low after 48 h (1/16). Importantly, survival was increased significantly (5/14) when grafts were rinsed with Carolina Rinse following 48 h of cold storage. In both groups, parenchymal cells appeared normal by scanning electron microscopy, excluded trypan blue, and released SGOT at values only slightly above the normal range immediately (i.e., less than 5 min) after 48 h of cold storage. However, SGOT values rose steadily during the 1st hour postoperatively following reperfusion in the Ringer's rinse group and reached levels around 1,000 U/l. In addition, nonparenchymal cells were not labelled with trypan blue following storage, but significant labelling occurred within 1 h. Both SGOT release and nonparenchymal cell injury were reduced significantly when grafts were rinsed with Carolina Rinse prior to completion of surgery. Liver injury assessed histologically 24 h postoperatively was also reduced about 50% by Carolina Rinse. Oxidative stress appeared to be involved, since radical adducts, most likely of lipid origin, were trapped during the first 5 min after reperfusion with the spin trapping technique and detected by electron paramagnetic resonance spectroscopy. Lipid radical formation was reduced nearly completely on reperfusion by Carolina Rinse. Since Carolina Rinse improved survival of liver grafts following long periods of cold storage and reduced lipid radical formation and hepatocellular injury, we concluded that a reperfusion injury rather than a storage injury predominates following orthotopic transplantation of livers stored for long periods of time in cold UW solution.

Topics: Adenosine; Allopurinol; Animals; Glutathione; Graft Survival; Insulin; Liver; Liver Transplantation; Organ Preservation Solutions; Raffinose; Rats; Reperfusion; Reperfusion Injury; Solutions

Ischemia-reperfusion (I/R)-induced microvascular injury is characterized by capillary "no-reflow" and reflow-associated events, termed "reflow paradox," including leukocyte-endothelium interaction and increase in microvascular permeability. The major objectives of this study were 1) to elucidate the significance of reflow paradox after 4 h of tourniquet-induced ischemia in striated muscle and 2) to determine the role of reactive oxygen metabolites in the pathogenesis of reflow paradox-dependent microcirculatory alterations. By use of in vivo fluorescence microscopy in a striated muscle preparation of hamsters, leukocyte-endothelium interaction in postcapillary venules and macromolecular extravasation from capillaries and venules were quantified before ischemia and after 30 min, 2 h, and 24 h of reperfusion. I/R elicited marked enhancement (P < 0.01) of leukocyte rolling during initial reperfusion and a 20-fold increase of leukocyte adherence (P < 0.01) lasting for the entire postischemic reperfusion period (n = 7). These phenomena were accompanied by significant leakage (P < 0.01) of macromolecules from capillaries and in particular from postcapillary venules (n = 9). Both superoxide dismutase (SOD, 20 mg/kg body wt, n = 7) and allopurinol (50 mg/kg body wt, n = 7) were effective in attenuating I/R-induced leukocyte rolling and adherence. In addition, microvascular leakage was significantly reduced by allopurinol (n = 9) and completely abolished by SOD (n = 9) (P < 0.01). These results support the concept that reactive oxygen metabolites contribute to I/R-induced reflow paradox, resulting in leukocyte accumulation, adherence, and increase in microvascular permeability.

Topics: Allopurinol; Animals; Capillary Permeability; Cricetinae; Dextrans; Endothelium, Vascular; Fluorescein-5-isothiocyanate; Ischemia; Leukocytes; Mesocricetus; Microcirculation; Muscles; Regional Blood Flow; Reperfusion Injury; Superoxide Dismutase

Topics: Animals; Free Radical Scavengers; Free Radicals; Gastric Mucosa; Hypoxanthine; Hypoxanthines; Lipid Peroxidation; Male; Neutrophils; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Thiobarbituric Acid Reactive Substances; Xanthine Oxidase

Ischemia-reperfusion is observed in various diseases such as myocardium infarct. Different theories have been proposed to explain the reperfusion injury, among them that the free radical generation plays a crucial role. To study the mechanisms of the reperfusion injury, a hypoxia (H)-reoxygenation (R) model upon human umbilical vein endothelial cells in culture was developed in order to mimic the in vivo situation. Different parameters were quantified and compared under H or H/R, and we found that oxygen readmission led to damage amplification after a short hypoxia period. To estimate the importance of various causes of toxicity, the effects of various protective molecules were compared. Different antioxidant molecules, iron-chelating agent, xanthine oxidase inhibitors, and energy-supplying molecules were very efficient protectors. Synergy could also be observed between the antioxidants and the energy-supplying molecules or the xanthine oxidase inhibitors. The toxic effect of O2.(-) could be lowered by the presence of SOD or glutathione peroxidase in the culture medium, whereas glutathione peroxidase was the most efficient enzyme when injected into the cells. The production of O2.(-) and of H2O2 by endothelial cells was directly estimated to be, respectively, of 0.17 and 0.035 mumol/min/mg prot during the R period. O2.(-) production was completely inhibited when allopurinol was added during H and R. In addition, a xanthine oxidase activity of 21.5 10(-6) U/mg prot could be observed by a direct assay in cells after H but not in control cells, thus confirming the previous conclusions of xanthine oxidase as a potent source of free radicals in these conditions. Thanks to the use of cultured human endothelial cells, a clear picture was obtained of the overall process leading to cell degenerescence during the reoxygenation process. We particularly could stress the importance of the low energetic state of these cells, which is a critical factor acting synergistically with the oxidant molecules to injure the cells. These results also open new possibilities for the development of new therapeutics for ischemia.

Topics: Cell Hypoxia; Cells, Cultured; Endothelium, Vascular; Free Radicals; Humans; Hydrogen Peroxide; Reperfusion Injury; Superoxides; Umbilical Veins; Xanthine Oxidase

Injury to nonpulmonary organ systems often initiates systemic processes that cause recruitment of neutrophils to the lung. We found that rats subjected to intestinal ischemia-reperfusion (I/R) had increased transvascular leak of 125I-labeled albumin into lungs and decreased lung ATP levels (P less than 0.05). In addition, rats subjected to intestinal I/R had increased plasma xanthine oxidase (XO) activity, plasma leukotactic activity for neutrophils, and lung neutrophil retention (assessed by morphometry and myeloperoxidase activity) compared with sham-treated rats (P less than 0.05). By comparison, after intestinal I/R, rats fed an allopurinol- or tungsten-enriched diet had decreased plasma and intestinal XO activities, decreased plasma leukotacic and lung myeloperoxidase (MPO) activities, decreased lung leak, and increased lung ATP levels compared with rats fed control diets (P less than 0.05). Further studies suggested a more specific role for circulating rather than tissue XO in mediating lung neutrophil accumulation but not lung leak. Plasma XO, plasma leukotactic, and lung MPO activities, but not lung leak, increased in rats administered purified XO intravenously. In addition, plasma XO, plasma leukotactic, and lung MPO activities, but not lung leak, decreased in rats administered antisera against XO and then subjected to intestinal I/R. We conclude that circulating XO increases acutely and may contribute to pulmonary retention of neutrophils after an ischemic intestinal insult.

Topics: Allopurinol; Animals; Cell Movement; Endotoxins; Injections, Intravenous; Intestines; Ischemia; Lung; Male; Neutrophils; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tungsten; Xanthine Oxidase

The effects of ulinastatin (ULN), a human urinary protease inhibitor, on liver injury caused by ischemia-reperfusion were studied in rats. In the liver ischemia-reperfusion model, ULN suppressed the elevation of serum transaminase levels and tissue lipid peroxide levels in the liver. ULN did not exhibit a radical-trapping action on the superoxide and hydroxyl radicals as measured by electron spin resonance (ESR). ULN suppressed formylmethionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA)-induced superoxide production from polymorphonuclear leukocytes (PMNs) as measured by the cytochrome c assay. ULN did not inhibit either xanthine oxidase (XO) activity or the conversion of xanthine dehydrogenase (XDH) to XO during the ischemic period. ULN also strongly protected against the hypotonic hemolysis of rat erythrocytes. These results suggest that ULN's membrane stabilizing action and suppressive effect against PMNs superoxide production might be attributed to its suppressive effect on the liver's lipid peroxidation caused by ischemia-reperfusion.

Topics: Animals; Enzymes; Erythrocyte Membrane; Free Radicals; Glycoproteins; Ischemia; Lipid Peroxides; Liver; Liver Circulation; Male; Neutrophils; Protease Inhibitors; Rats; Rats, Wistar; Reperfusion Injury; Superoxides; Trypsin Inhibitors; Xanthine Oxidase

Recent studies have demonstrated a connection between xanthine oxidase-generated reactive oxygen intermediates and histamine release during ischemia-reperfusion. In the present work, the effect of modulation of the endogenous histamine level on the xanthine oxidase activity was examined during the reperfusion of a canine ileal segment following a 2 hr of complete ischemia. The xanthine oxidase activity and the plasma histamine level peaked simultaneously at the beginning of reperfusion, reaching mean values of 14.9 nmol/ml/min and 12.1 nmol/l, respectively. Pretreatment with aminoguanidine, a blocker of diamine oxidase (histaminase), resulted in significantly higher levels of histamine during reperfusion, but this elevation was not accompanied by a further increase in xanthine oxidase activity. Pretreatment with the mast cell stabilizer cromolyn significantly diminished the rise in plasma histamine level, with an unchanging activity of xanthine oxidase. No significant alteration could be observed in the postocclusive activity of xanthine oxidase following the intra-arterial administration of 0.5, 1, or 5 nmol of histamine during the last 10 min of the ischemic period. These data suggest that the amount of histamine liberated during reperfusion does not result in a further increase in the xanthine oxidase activity. The release of histamine is not a cause, but rather an effect of the elevated activity of intestinal xanthine oxidase.

Topics: Animals; Cromolyn Sodium; Disease Models, Animal; Dogs; Female; Guanidines; Histamine; Histamine Release; Intestines; Ischemia; Male; Mast Cells; Reperfusion Injury; Xanthine Oxidase

The role of sucralphate in prevention of acute gastric injuries and its comparison with free radical blockers such as allopurinol, soybean trypsin inhibitor and superoxidase dismutase in the ischemia-reperfusion model by total occlusion of the coeliac artery in Wistar rats, was studied. The gross gastric mucosal necrotic area was 80%. In contrast with the antioxidant drugs the necrotic area attained was between 7 to 15%, while with sucralphate, an antioxidant-cytoprotective drug that enhances the gastric defensive barrier, the prevention of the secondary aggression induced by free radicals was more important.

Topics: Acute Disease; Allopurinol; Animals; Female; Gastric Mucosa; Rats; Rats, Wistar; Reperfusion Injury; Stomach; Stomach Ulcer; Sucralfate; Superoxide Dismutase; Trypsin Inhibitor, Kunitz Soybean

Under normal conditions the intestinal mucosa is impermeable to potentially harmful materials from the intestinal lumen. Mucosal disruption promotes bacterial translocation, which is postulated to be a fuel source for sepsis and multiorgan failure. We have previously demonstrated that mesenteric ischemia-reperfusion (I/R) injury increases intestinal permeability (IP); however, the mechanism remains unclear. This study was designed to examine the hypothesis that changes in IP, after I/R injury, are mediated by xanthine oxidase-generated, oxygen-derived free radicals. Thirty-three Sprague-Dawley rats (weighing 300 to 400 g) were included in this study. Group 1 (n = 10) received enteral allopurinol, a xanthine oxidase inhibitor, 10 mg/kg daily for 1 week prior to mesenteric ischemia. Group 2 consisted of 11 untreated, ischemic animals. Groups 1 and 2 were subjected to superior mesenteric artery occlusion with interruption of collateral flow for 20 minutes to produce ischemic injury to the intestine. An additional 12 rats (group 3), served as nonischemic controls (sham). A loop of distal ileum was isolated and cannulated proximally and distally to allow luminal perfusion with warmed Ringer's lactate at 1 mL/min. IP was determined in all groups by quantitatively measuring the plasma-to-luminal clearance of chromium (51Cr)-labeled ethylenediaminetetraacetate (EDTA) at baseline, during ischemia and 20, 40, and 60 minutes after reperfusion. Complete ischemia produced significant increases in IP over baseline values in the untreated rats (group 2, baseline: 0.49 +/- 0.006, ischemia: 0.149 +/- 0.039) compared with sham rats (baseline: 0.41 +/- 0.006; ischemia: 0.047 +/- 0.009) or allopurinol-treated rats (baseline: 0.098 +/- 0.020, ischemia: 0.073 +/- 0.012, P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Allopurinol; Animals; Hemodynamics; Intestinal Mucosa; Ischemia; Male; Permeability; Rats; Rats, Wistar; Reperfusion Injury

Ischemia and reperfusion of the small intestine and colon in rats were produced by reversible occlusion (for 30 min and 1 or 3 h) of the superior mesenteric artery and the aorta above the inferior mesenteric artery. Despite a greater reduction of mucosal perfusion in the colon than in the small intestine with 30 min of ischemia, the depth of mucosal damage was significantly smaller in the former than in the latter. Thirty minutes of ischemia followed by 1 h of reperfusion induced an increase in polymorphonuclear leukocyte infiltration in both locations. Exacerbation of mucosal injury occurred only in the small intestine, suggesting that reperfusion injury is independent of polymorphonuclear leukocyte infiltration. Reperfusion after 1 or 3 h of ischemia did not exacerbate mucosal damage in either location. Allopurinol significantly diminished the exacerbation of injury after reperfusion in the small intestine. The protective effect of allopurinol, however, was neither associated with an improvement in perfusion nor a reduction in polymorphonuclear leukocyte infiltration. These data indicate that there is a window (30 min) of reperfusion injury in the small intestine, but there is no evidence of reperfusion injury in the colon.

Topics: Allopurinol; Animals; Intestinal Mucosa; Intestines; Ischemia; Male; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Reperfusion; Reperfusion Injury

The role of platelet-activating factor as a potential mediator of hepatic inflammatory injury associated with liver ischemia/reperfusion was investigated using a partial no-flow model in rats in vivo. Platelet-activating factor levels of livers from sham-operated rats and from animals experiencing hepatic reperfusion for less than 6 hr were very low. They were observed to increase significantly after 12 hr of reperfusion and reached peak levels after a 24-hr reperfusion period, a time when maximal hepatic injury and inflammation occurred. Treatment of experimental rats with WEB2170, a platelet-activating factor receptor antagonist, attenuated the hepatic injury and inflammation, as evidenced by decreases in plasma ALT and in hepatocyte necrosis and neutrophil infiltration. Both inactivation of Kupffer cells with gadolinium chloride and inhibition of the formation of reactive oxygen species with allopurinol reduced platelet-activating factor production in the liver, whereas induction of neutropenia had no effect, suggesting that interaction of Kupffer cells with oxygen-derived free radicals may be a plausible mechanism for hepatic platelet-activating factor accumulation. It is concluded that platelet-activating factor contributes to the inflammatory consequences of ischemia/reperfusion underlying late-phase hepatic injury.

Topics: Alanine Transaminase; Allopurinol; Animals; Azepines; Gadolinium; Hepatitis; Ischemia; Leukocytes; Liver; Male; Necrosis; Platelet Activating Factor; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Triazoles

Lazaroids are a class of novel 21 aminosteroids. They have been reported to be potent inhibitors of lipid peroxidation, which is a major contributing factor to ischemia-reperfusion injury in the lung. A Lewis rat orthotopic left lung isotransplant model was used to investigate the effects of the lazaroid U74500A on pulmonary preservation. The heart-lung blocks of donor rats were flushed with and then stored in either standard University of Wisconsin solution or University of Wisconsin solution with 30 mumol/L of U74500A substituted for the dexamethasone. After 6 or 12 hours of cold storage at 0 degrees C, the left lungs were transplanted into recipient rats and reperfused for 1 hour. Pulmonary function was assessed by measuring oxygen and carbon dioxide tensions in arterial blood after removal of the right lung. Lipid peroxide concentrations were measured as a thiobarbituric acid-reactive substance. Although arterial oxygen and carbon dioxide pressures and water content after 6 hours of preservation followed by reperfusion were similar in both the lazaroid and dexamethasone groups, lipid peroxide concentration was significantly higher in the dexamethasone group (0.88 +/- 0.07 mumol/gm) than in the lazaroid group (0.54 +/- 0.07 mumol/gm) (p < 0.01). After 12 hours of preservation, there were significant differences between the lazaroid and dexamethasone groups in arterial oxygen pressure (339 +/- 70 vs 27 +/- 3 mm Hg, p < 0.01), arterial carbon dioxide pressure (24.3 +/- 2.7 vs 47.7 +/- 7.0 mm Hg, p < 0.001), and lipid peroxide concentrations (0.69 +/- 0.07 vs 1.30 +/- 0.09 mumol/gm, p < 0.001). We conclude that addition of U74500A to the flush and storage solution enhances the preservation of the pulmonary graft in this transplant model.

Topics: Adenosine; Allopurinol; Animals; Body Water; Carbon Dioxide; Cardioplegic Solutions; Cold Temperature; Dexamethasone; Glutathione; Insulin; Ischemia; Lipid Peroxides; Lung; Male; Organ Preservation; Organ Preservation Solutions; Oxygen; Pregnatrienes; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solutions

Oxygen-derived free radicals produced during reperfusion may be responsible for the disturbed pathology which follows prolonged ischaemia. Measurement of hepatic chemiluminescence (low level light emission resulting from the energy released during chemical reactions of free radicals) allowed determination of whether allopurinol could prevent formation of oxygen-derived free radicals during reperfusion of the ischaemic liver. While control animals demonstrated a burst of light emission shortly after reperfusion, the rats pretreated with allupurinol showed no evidence of chemiluminescence during either ischaemia or reperfusion. It is concluded that allopurinol may modify reperfusion-induced free radical formation and possibly ameliorate the organ damage which can follow ischaemia.

Topics: Administration, Oral; Allopurinol; Animals; Free Radicals; Ischemia; Lipid Peroxidation; Liver; Liver Circulation; Luminescent Measurements; Oxygen; Rats; Rats, Wistar; Reperfusion Injury

The enzyme xanthine oxidase has been implicated in the tissue oxidative injury after ischemia-reperfusion. This enzyme, which is a source of oxygen free radicals, is formed from a dehydrogenase form during ischemia. The ratio dehydrogenase/oxidase of rat kidney homogenates decreases during the ischemia and the reperfusion. Two flavonoids, quercetin and silybin, characterized as free radical scavengers, exert a protective effect preventing the decrease in the dehydrogenase/oxidase ratio observed during ischemia-reperfusion. The mechanism of this effect and the role of flavonoids in the ischemia-reperfusion tissue damage is discussed.

Topics: Animals; Free Radical Scavengers; Ischemia; Kidney; Male; Quercetin; Rats; Rats, Wistar; Reperfusion Injury; Silymarin; Uric Acid; Xanthine Dehydrogenase; Xanthine Oxidase

"No reflow" has been implicated as prominent phenomenon in microvascular injury associated with ischemia-reperfusion (I/R). The objectives of this study were 1) to elucidate the significance of no reflow in microvascular I/R injury of striated muscle and 2) to determine whether reactive oxygen metabolites play a role in the development of postischemic no reflow. By use of the hamster dorsal skinfold preparation and intravital microscopy, microvascular perfusion of capillaries and postcapillary venules of striated muscle was quantitatively assessed before and 30 min, 2 h, and 24 h after 4 h of tourniquet-induced ischemia. I/R was characterized by a significant reduction (P < 0.01) in functional capillary density to 35% of baseline values during initial reperfusion, with incomplete recovery after 24 h (n = 9). In addition, capillary perfusion was found to be extremely heterogeneous, and wall shear rate in postcapillary venules was significantly decreased (P < 0.01). Treatment with either superoxide dismutase (SOD; n = 9) or allopurinol (n = 9) resulted in maintenance of capillary density of 60% of baseline (P < 0.05). Furthermore, I/R-induced capillary perfusion inhomogeneities and decrease of wall shear rate in venules were attenuated significantly (P < 0.01) by SOD and allopurinol. Thus part of capillary perfusion disturbances during I/R in striated muscle may be caused by increased postcapillary vascular resistance, probably mediated by reactive oxygen metabolites. However, the fact that in SOD- and allopurinol-treated animals 40% of the capillaries were still found to be nonperfused indicates that mechanisms other than oxygen radicals play an important role in the development of postischemic no reflow.

Topics: Allopurinol; Animals; Capillaries; Cricetinae; Guinea Pigs; Ischemia; Mesocricetus; Microcirculation; Muscles; Regional Blood Flow; Reperfusion Injury; Skin; Superoxide Dismutase; Venules

The efficacy of pharmacologic agents for prevention and control of oxygen-derived free radical damage in ischemia-reperfusion injury of the spinal cord was assessed in a swine model of thoracic and thoracoabdominal aortic crossclamping. Animals were exposed to 30 minutes of ischemia that induced lethal, irreversible injury and paraplegia. The experimental groups were as follows: group A (n = 7), control group, receiving no pharmacologic intervention; group B (n = 7), deferoxamine 50 mg/kg/day administered intravenously over 3 to 4 hours before ischemia; group C (n = 7), allopurinol pretreatment 50 mg/kg/day for 3 days; and group D (n = 7), superoxide dismutase 60,000 units administered with 50,000 units before removal of the aortic crossclamp and 10,000 units over 10 minutes of reperfusion. Proximal hypertension was controlled with sodium nitroprusside and volume depletion. The methods of assessment were neurologic by a modified Tarlov criteria and blood flow by radiolabeled microspheres. Results of blood flow assessment confirmed a true ischemic episode of 30 minutes for all animals in all groups. The blood flow fell significantly during ischemia (p less than 0.01) and a hyperemic response was evident in the early reperfusion period. All animals in control group A were paraplegic. The group B (deferoxamine) results were superior; 85% had grade III function on a modified Tarlov scale, with animals in the group standing and even walking with difficulty. Only one animal in this group had good movements of hind limbs but was unable to stand or walk. Neurologic recovery was limited in the allopurinol group (group C), with 85% showing slight neurologic recovery with limited movement of the hind limbs. The animals in the superoxide dismutase group (group D) all had good recovery, with strong motor response of hind limbs, but were not able to stand. In summary, the results of this experimental protocol confirmed the possible role of oxygen-derived free radicals in the pathophysiology of spinal cord injury, induced by aortic crossclamping. Moreover, it proved that ischemia-reperfusion injury could be altered by pharmacologic interventions.

Topics: Allopurinol; Animals; Aorta; Aortic Aneurysm; Constriction; Deferoxamine; Free Radicals; Paraplegia; Postoperative Complications; Reperfusion Injury; Spinal Cord; Superoxide Dismutase; Swine

The purpose of this study was to investigate the possibility of improving the organ preservation properties of the University of Wisconsin (UW) solution by adding the calcium entry blocker lidoflazine. We also investigated the possibility of decreasing the cold ischemia and reperfusion damage by pretreatment with lidoflazine of the donor and/or recipient. The protective effects of lidoflazine treatment were estimated by measuring the amount of trapped erythrocytes in the rat renal medulla after 48 h of cold storage, subsequent transplantation, and 20 min of reperfusion. Lidoflazine (20 mg/liter) added to the UW solution decreased the amount of erythrocyte trapping from 14.8 +/- 3.1% in controls to 8.6 +/- 1.7% (P less than 0.01). The flow rate of the flush-out solution during the harvesting procedure was also significantly (P less than 0.01) increased when lidoflazine was included in the UW solution (1.10 +/- 0.21 ml/min vs 0.75 +/- 0.22 ml/min). Administration of lidoflazine (0.28 mg/kg body wt) to the donor and/or the recipient did not further reduce the postischemia/reperfusion damage as estimated by the degree of erythrocyte trapping. In conclusion, the results indicate that the preservation properties of the UW solution can be significantly improved by adding lidoflazine to the solution.

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Erythrocytes; Glutathione; Insulin; Kidney; Kidney Transplantation; Lidoflazine; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Strains; Reperfusion Injury; Solutions

The mechanisms of gastric mucosal injury following a period of ischemia remain unclear. The aim of this study was to determine the relative contributions of ischemia, reperfusion, and reactive oxygen metabolites to mucosal injury induced by temporary occlusion of the celiac artery. Rats were subjected to 30 min of gastric ischemia in the presence of 100 mM HCl. Reperfusion periods ranged from 1 min to 24 hr. Drug treatments included allopurinol (100 mg/kg) or a combination of superoxide dismutase (15,000 units/kg), catalase (90,000 units/kg), and desferrioxamine (50 mg/kg). Mucosal injury was assessed by quantitative histology and the extent of macroscopic hemorrhage. Approximately one third of the total injury to the volume of the mucosa (11.8 +/- 9.1%) was due to ischemia alone. Another third was blocked by allopurinol or superoxide dismutase, catalase, and desferrioxamine (22.1 +/- 6.9%, P less than 0.001; and 25.9 +/- 4.6%, P less than 0.01), respectively, compared with control (32.5 +/- 5.1%). In contrast, extensive surface mucosal injury (62.2 +/- 27.6%) occurred primarily during ischemia and was not affected by antioxidants. Macroscopic hemorrhage was halved by treatment with allopurinol (17.5 +/- 12.6%, P less than 0.01) or superoxide dismutase, catalase, and desferrioxamine (15.9 +/- 14.5%, P less than 0.01). We conclude that temporary celiac occlusion results in gastric mucosal damage that consists of both ischemic and reperfusion components. The majority of surface mucosal injury occurred during ischemia, whereas injury to the volume of the mucosa and the vasculature occurred equally during reperfusion and was associated with reactive oxygen metabolites.

Topics: Allopurinol; Animals; Catalase; Deferoxamine; Free Radicals; Gastric Mucosa; Ischemia; Male; Oxygen; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase

We studied the effect of inhibition of oxyradical formation and of endogenous glutathione (GSH) depletion on lesion formation in the gastrointestinal tract in a modified rat hemorrhagic shock model (1 h hypotension and 1 h reperfusion). Allopurinol, an inhibitor of xanthine oxidase, did not protect against lesion formation. This suggests that oxygen radicals generated from xanthine oxidase may not be the major cause of injury under these conditions of prolonged 'ischemia'-reperfusion. Phorone (diisopropylideneacetone), a GSH depletor, decreased mucosal GSH levels in the corpus, duodenum and small intestine, and also significantly reduced lesion formation histologically in the corpus, antrum, duodenum and small intestine. However, there was no significant differences in mucosal blood flow (as estimated by changes in mucosal hemoglobin concentrations and oxygen saturation of mucosal hemoglobin) in the corpus, antrum, duodenum and small intestine between phorone-pretreated and control rats. We conclude that phorone decreased mucosal GSH concentrations and exerted a protective effect against hemorrhagic shock-induced gastrointestinal mucosal lesions. The protective effect appears to be independent of mucosal blood flow.

Topics: Allopurinol; Animals; Duodenum; Free Radicals; Gastric Mucosa; Glutathione; Intestinal Mucosa; Intestine, Small; Ketones; Male; Oxygen Consumption; Rats; Rats, Inbred Strains; Regional Blood Flow; Reperfusion Injury; Shock, Hemorrhagic

A proposal that injury in ischaemic/reperfused rat heart is critically dependent on the availability of free iron rather than on the efficiency of O2-. and H2O2 production was examined.. Isolated working rat hearts from 152 male Wistar rats (200-250 g weight), subjected to 20-40 min of global ischaemia and reperfused for 30 min, were perfused with 10 mumol.litre-1 Fe[III] or Fe[II] and/or 0.6 mmol.litre-1 desferrioxamine, 10 mmol.litre-1 dimethylthiourea, and 1 mmol.litre-1 allopurinol. Curves relating the recoveries of haemodynamic functions and the reperfusion lactate dehydrogenase release to the duration of the preceding ischaemic period were constructed. Morphological examination was also performed.. In the untreated hearts, the duration of ischaemia resulting in 50% loss of cardiac output was 29 min. This time was decreased to 24 min and 20 min by Fe[III] and Fe[II], respectively, and was increased to 36 min and 37 min by desferrioxamine and dimethylthiourea, respectively. Desferrioxamine prevented the effect of Fe[III] but not that of Fe[II], whereas dimethylthiourea prevented the effect of Fe[II]. Neither the effect of Fe[III] nor that of Fe[II] was prevented by allopurinol which, however, proved to be beneficial in the untreated hearts.. The beneficial effect of desferrioxamine and dimethylthiourea suggest that it is intensification of the Fenton reaction by iron which accounts for iron induced aggravation of the reperfusion injury. Thus we speculate that the availability of free iron, rather than O2-. and H2O2, is a limiting factor in the development of injury in an ischaemic/reperfused rat heart. What remains unclear is why allopurinol is unable to prevent iron induced changes.

Topics: Allopurinol; Animals; Biological Availability; Deferoxamine; Ferric Compounds; Ferrous Compounds; Free Radicals; Heart; L-Lactate Dehydrogenase; Male; Microscopy, Electron; Myocardium; Rats; Rats, Inbred Strains; Reperfusion Injury; Thiourea; Time Factors

In this study rat epigastric island flaps were used as a model to investigate selected tissue biochemical changes occurring during secondary ischemia. It was hypothesized that free radical damage, depletion of free radical scavengers, depletion of ATP, and increased edema might explain differences in flap survival between partial (venous obstruction) and total (arteriovenous obstruction) ischemia and decreased flap survival with increasing ischemia time. Flaps were given 2 hr or primary ischemia, 8 hr of normal perfusion, then secondary ischemia of 0, 2, 4, 8, or 12 hr with either arteriovenous obstruction or venous obstruction. Biochemical analysis of the skin was performed after 0, 24, or 96 hr reperfusion. Only minor differences were found between arteriovenous and venous ischemia for any of five biochemical parameters, despite a previous finding that venous ischemic flaps are more susceptible to necrosis. Levels of xanthine oxidase and malonyldialdehyde (both indices of free radical generation) increased with ischemia time. Levels of superoxide dismutase (a free radical scavenger) correspondingly decreased. Tissue levels of ATP decreased after ischemia and recovered to normal for shorter but not for longer ischemia times after 96 hr of reperfusion in parallel with flap survival. Edema increased immediately after the ischemic insult but decreased once the tissue became necrotic. These results imply roles for free radicals, ATP, and edema in secondary ischemia, but do not distinguish between arteriovenous and venous secondary ischemia.

Topics: Adenosine Triphosphate; Animals; Constriction; Edema; Ischemia; Male; Malondialdehyde; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase; Surgical Flaps; Xanthine Oxidase

Activated neutrophils cause conversion of xanthine dehydrogenase to its oxidase form (xanthine oxidase) in endothelial cells, the mechanism of which may be related to the cytotoxic effect of activated neutrophils. The elastase inhibitors, elastatinal, alpha 1-antitrypsin, and MeO-Suc-(Ala)2-Pro-Val-CH2Cl, significantly inhibited xanthine dehydrogenase to oxidase conversion by phorbol myristate acetate-stimulated neutrophils without inhibition of neutrophil adherence to the endothelial cell monolayer. The role of elastase in this enzyme conversion process was confirmed by the ability of purified elastase to cause conversion of xanthine dehydrogenase to xanthine oxidase in intact endothelial cells (or cell extracts) without causing cytotoxicity. In contrast, cathepsin G failed to cause conversion. The kinetics of conversion induced by elastase was relatively rapid, being essentially completed by 30 min. Upon removal of elastase, the effect was slowly (greater than 12 h) reversible and could be inhibited by cycloheximide treatment. Exposure of endothelial cells to hypoxia failed to enhance the elastase-induced conversion. Treatment of endothelial cells with Ca2+ ionophores failed to cause conversion of xanthine dehydrogenase to oxidase, suggesting that intracellular Ca(2+)-activated proteases are not sufficient to induce this process. Neutrophil-induced xanthine dehydrogenase to oxidase conversion was inhibited by concomitant treatment with antibodies to CD11b. The results suggest that activated neutrophils induce conversion of xanthine dehydrogenase to oxidase by secretion of elastase in close proximity to the endothelial cells and that this intimate contact between the two cell types enables high local concentrations of elastase to be attained, which are sufficient to cause xanthine dehydrogenase to xanthine oxidase conversion.

Topics: Animals; Calcimycin; Cell Cycle; Cycloheximide; Endothelium, Vascular; In Vitro Techniques; Ionomycin; Neutrophils; Oxygen; Pancreas; Pancreatic Elastase; Rats; Reperfusion Injury; Tetradecanoylphorbol Acetate; Xanthine Dehydrogenase; Xanthine Oxidase

Acute renal failure following hemorrhagic shock was studied in awake rats. The animals were bled to maintain the mean arterial blood pressure between 40 and 60 mm Hg during 180 min. After this period, the blood was reinfused and the rats were studied 24 h later. Hemorrhagic shock caused a less intensive renal injury than 60-min bilateral renal artery clamping. Renal function in the latter model was worse (p less than 0.05) as shown by serum creatinine (SCr) (0.75 +/- 0.10 vs 1.2 +/- 0.2 mg/dL), blood urea nitrogen (BUN) (26.0 +/- 2.8 vs 53.0 +/- 8.5 mg/dL), fractional excretion of sodium (FENa, %) (0.3 +/- 0.1 vs 1.8 +/- 1.0) and potassium (FEK, %) (41.4 +/- 5.7 vs 76.3 +/- 14.2) and urine/plasma creatine (U/PCr (86.4 +/- 15.7 vs 38.8 +/- 15.5). The rats which received verapamil (10 micrograms/kg/min) prior and during the HS did not show increase in SCr (0.5 +/- 0.06 vs 0.75 +/- 0.1 mg/dL, p less than 0.05). This effect was also observed in the rats which received intravenous allopurinol (40 mg/kg) before HS, SCr did not increase (0.5 +/- 0.04 vs 0.75 +/- 0.1 mg/dL, p = 0.05), suggesting a protective effect of those substances in HS.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acute Kidney Injury; Allopurinol; Animals; Calcium; Dehydration; Free Radicals; Gentamicins; Kidney; Kidney Function Tests; Male; Rats; Rats, Inbred Strains; Reperfusion Injury; Shock, Hemorrhagic; Verapamil

Allopurinol has been reported to improve cell survival in a variety of conditions, including the ischemia-reperfusion injury occurring in skin flaps. It has been suggested that the beneficial effect of allopurinol on rat skin flaps is through blockage of xanthine oxidase-generated oxygen-derived free radicals. We have previously reported on the lack of xanthine oxidase activity in the skin of humans and pigs as compared with that of rats. This current study attempts to improve skin and myocutaneous flap survival in pigs in two separate experiments using allopurinol. In the first experiment, a suspension of 50 mg/kg (N = 12) allopurinol resulted in no significant difference in the survival of control and treated flaps. Because of the negative results in the first experiment, a second experiment was designed making several changes. The length of the global ischemic insult was reduced from 8 to 6 hours, and allopurinol was administered as a solution of 300 mg/kg (N = 14). This higher dose is expected to produce complete inhibition of xanthine oxidase in this animal model. These changes resulted in three operative deaths, no improvement in skin-flap survival, and a decrease in myocutaneous flap survival. Allopurinol's therapeutic effectiveness and its mechanism of action in an ischemia-reperfusion injury model lacking xanthine oxidase activity are discussed.

Topics: Allopurinol; Animals; Female; Graft Survival; Necrosis; Random Allocation; Reperfusion Injury; Surgical Flaps; Swine; Xanthine Oxidase

We investigated whether reoxygenation following anoxia increased biliary permeability and whether or not allopurinol had a protective effect. Isolated rat livers were perfused for 30 min in a one-pass system with buffer equilibrated with 100% nitrogen after stabilization, and then for 60 min with the oxygenated buffer. Hepatic tight junction permeability was assessed by quantifying the early appearance in the bile of horseradish peroxidase (HRP) injected with the perfusate. This early peak represents paracellular passage of HRP, whereas a later second peak results from transcellular passage. In the control livers, 7% of the total HRP passage (93 +/- 50 pg/g liver) was paracellular and 93% was transcellular. After 30 min of reoxygenation following anoxia, however, 516 +/- 20 pg/g liver of HRP passed paracellularly. Addition of allopurinol (5 micrograms/ml) to the perfusate from the start of perfusion reduced paracellular passage of HRP to 219 +/- 49 pg/g liver after anoxia and reperfusion (P less than 0.01). Allopurinol also reduced the cumulative lactate dehydrogenase (LDH) release during the first 30 min of reoxygenation from 2.1 +/- 0.3 x 10(4) to 1.4 +/- 0.4 x 10(4) units/g liver (P less than 0.01). Reduction of the anoxic period from 30 min to 25 min significantly reduced the change in tight junction permeability and the extent of cellular injury: Paracellular passage of HRP was 336 +/- 20 pg/g and LDH release was 0.7 +/- 0.1 x 10(4) units/g liver, both significantly lower than those at 30 min (P less than 0.01). No significant difference in hepatic ATP levels after 60 min of reoxygenation was noted among the experimental groups, but all had lower levels than the control group. The protective effect of allopurinol suggests that the mechanism of biliary reoxygenation injury involves free radical generation. Susceptibility of tight junctions suggests a pattern of injury similar to that involved in anoxic damage of the vascular endothelium.

Topics: Adenosine Triphosphate; Allopurinol; Animals; Cell Membrane Permeability; Horseradish Peroxidase; L-Lactate Dehydrogenase; Liver; Male; Oxygen Consumption; Rats; Rats, Inbred Strains; Reperfusion Injury

We have previously shown that gut ischemia/reperfusion (I/R) causes simultaneous liver and lung dysfunction and that neutrophils play a critical role in this process. The purpose of this study was to ascertain whether xanthine oxidase (XO) was likewise operational. Normal and XO-inactivated rats (given a tungsten-enriched, molybdenum-depleted diet for 3 weeks) underwent 45 minutes of occlusion of the superior mesenteric artery, and control rats were subjected to a sham laparotomy. After zero and six hours of reperfusion, blood was sampled and livers and lungs harvested. Iodine-125-labeled albumin leak was used as a marker for pulmonary and liver capillary permeability barrier function, and serum acetoacetate/3-hydroxybutyrate (AcAc/3-OHB) levels as an index of hepatic mitochondrial redox state. Gut ischemia/six hours of reperfusion (I/R) increased the 125I albumin lung/blood ratio and the 125I albumin liver/blood ratio; AcAc/3-OHB levels decreased significantly. Xanthine oxidase activation eliminated the observed lung and liver capillary leak as well as the hepatic metabolic derangement induced by gut I/R. In conclusion, the simultaneous lung and liver dysfunction produced by gut I/R is mediated by XO.

Topics: Animals; Disease Models, Animal; Evaluation Studies as Topic; Ischemia; Liver; Lung; Male; Mesentery; Multiple Organ Failure; Neutrophils; Rats; Rats, Inbred Strains; Reperfusion Injury; Respiratory Distress Syndrome; Serum Albumin, Radio-Iodinated; Xanthine Oxidase

Optimal techniques for lung preservation are yet to be defined. Platelet activating factor is a phospholipid released by a variety of cells and produces pulmonary abnormalities similar to posttransplantation pulmonary dysfunction. We investigated the strength of the effect of the platelet activating factor antagonist BN 52021 as compared with that of deferoxamine, an iron chelator previously shown to improve lung preservation. Differential lung function and pulmonary hemodynamics were used to assess preservation after a 6-hour period of cold ischemic storage in a modified canine model of left lung allotransplantation. Thirty size- and weight-matched mongrel male dogs were used for 15 transplant procedures randomized to one of three preservation techniques. The University of Wisconsin solution was used as the basic flush solution for all experimental animals. BN 52021 was added to the flush solution in one group (10 mg/kg, n = 5) and deferoxime in another group (10 mg/kg, n = 5). No additives were used for the control animals (n = 5). BN 52021 and deferoxime were administered to respective donor animals 30 minutes before organ harvesting (10 mg/kg) and to recipient animals 30 minutes before reperfusion (10 mg/kg). The pulmonary artery flush solution was administered (40 ml/kg) over 4 minutes. Recipient animals received double-lumen endotracheal tubes and were monitored with balloon-tipped, flow-directed catheters in both pulmonary arteries and dual-angle ultrasonic flow probes around each pulmonary artery. Solid-state high-fidelity micromanometers were used to measure pressures in the pulmonary artery, the left atrium, and the left ventricle. Systemic arterial, right and left pulmonary venous, and mixed venous blood samples were analyzed at 1, 2, 4, and 6 hours after transplantation. Pulmonary venous oxygen tension of the transplanted lung for the control group was 202 +/- 81 mm Hg versus 282 +/- 53 mm Hg for the BN 52021 group 6 hours after transplantation (p less than 0.05). Pulmonary vascular resistance of the transplanted lung for the control group was 319 +/- 54 dynes.sec.cm-5 versus 149 +/- 71 dynes.sec.cm-5 for the BN 52021 group (p less than 0.05). Proton magnetic resonance spectroscopy was performed on segments of upper and lower lobes of the native and transplanted lung from recipient animals to determine total lung water content. The BN 52021 group had a total lung water content of 57.3% as compared with 51.9% for the deferoxime group (p = not s

Topics: Adenosine; Allopurinol; Animals; Deferoxamine; Diterpenes; Dogs; Ginkgolides; Glutathione; Insulin; Lactones; Lung; Lung Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Platelet Activating Factor; Raffinose; Reperfusion Injury; Solutions; Tissue Preservation

Xanthine oxidase was purified from human milk in yields comparable with those obtained from bovine milk. The freshly purified enzyme appeared homogeneous in gel permeation FPLC and SDS-PAGE, consistent with its being a homodimer with total M(r) 290,000 +/- 6000. The ultraviolet/visible absorption spectrum differed only slightly from that of bovine milk enzyme and showed an A280/A450 ratio of 5.13 +/- 0.29, indicating a high degree of purity. Xanthine oxidase activities of purified enzyme varied with batches of milk, ranging between 3 and 46 mU/mg protein; values that are some two to three orders of magnitude smaller than those shown by the most highly purified samples of bovine milk enzyme. Direct comparison with commercially-available bovine milk enzyme showed that activities involving xanthine as reducing substrate were 1-6% that of the bovine enzyme, whereas those involving NADH, in contrast, were of the same order for the two enzymes. Anaerobic bleaching experiments indicated that less than 2% of the human enzyme was present as a form active with xanthine. These findings, together with the activity data, are consistent with a very high content, possibly greater than 98%, of demolybdo- and/or desulpho-forms of human enzyme, both of which occur, to a lesser extent, in bovine xanthine oxidase. Molybdenum assay indicated that demolybdo-enzyme could only account for some 26% of this inactive component, suggesting that desulpho-enzyme may account for the remainder.

Topics: Animals; Antibodies; Humans; Milk; Milk, Human; Molecular Weight; Reperfusion Injury; Spectrophotometry; Xanthine Oxidase; Xanthopterin

In this study, we proposed that oxygen free radicals participate in the acute pulmonary injury that follows limb ischemia/reperfusion. Using an established model of hind limb ischemia, reproducible lung injury occurred after reperfusion. Lung microvascular permeability was measured with 125I-BSA and increased two-fold after 30 minutes of reperfusion. Pulmonary injury was blocked with DMSO, DMTU, allopurinol, indomethacin, and SOD plus catalase. The degree of pulmonary neutrophil sequestration as assessed by tissue myeloperoxidase activity was significantly diminished in animals pretreated with antioxidants. Pretreatment with indomethacin did not attenuate the neutrophil sequestration within the pulmonary parenchyma. These data suggest that increased lung microvascular permeability and neutrophil accumulation occur following hind limb ischemia/reperfusion. Therapeutic interventions with oxygen radical inhibitors blocked this process, while the prostaglandin inhibitor, indomethacin, only reduced lung permeability.

Topics: Allopurinol; Animals; Capillary Permeability; Catalase; Dimethyl Sulfoxide; Free Radicals; Hindlimb; Indomethacin; Lung; Lung Diseases; Neutrophils; Peroxidase; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase; Thiourea

Tissue oxidases, especially xanthine oxidase, have been proposed as primary sources of toxic oxygen radicals in many experimental models of disease states. Among these, ischemia-reperfusion injury may be of the greatest clinical interest. In this paper we propose the use of methylene blue as a means of suppressing the production of superoxide radicals O2- by acting as an alternative electron acceptor for xanthine oxidase. Previous work has indicated that methylene blue accepts electrons from xanthine oxidase at the iron-sulfur center. Initial experiments in our laboratory demonstrated that (1) pairs of electrons from each enzymatic oxidation are transferred to methylene blue, (2) the reduction of methylene blue can be achieved by model iron-sulfur centers, similar to the iron-sulfur center of xanthine oxidase, (3) reduced methylene blue auto-oxidizes to produce H2O2 directly, rather than O2-, and (4) methylene blue is effective at non-toxic levels (2-5 mg/kg) in preventing free radical damage to liver and kidney tissues in an in vitro model of ischemia and reoxygenation. Accordingly, we propose that methylene blue may represent a new class of antioxidant drugs that competitively inhibit reduction of molecular oxygen to superoxide by acting as alternative electron acceptors for tissue oxidases. We have termed these agents "parasitic" electron acceptors.

Topics: Animals; Antioxidants; Hypoxanthine; Hypoxanthines; In Vitro Techniques; Kidney; Lipid Peroxidation; Liver; Methylene Blue; Models, Biological; Oxidation-Reduction; Rats; Reperfusion Injury; Succimer; Superoxides; Xanthine; Xanthine Oxidase; Xanthines

The generation of free oxygen radicals is presumed as a substantial pathogenetic principle in reperfusion injury. Although demonstrated in gut, muscle and kidneys its role in liver reperfusion injury is still under investigation. In an experimental rat model of warm liver ischemia of 60 min and 8 h reperfusion electron resonance spectroscopy assessed the increased generation of free radicals in early reperfusion period, leading to a decrease of polyunsaturated free fatty acids in liver tissue within 15 min of reperfusion. Histologically, single cell death, local and patchy necrosis of hepatic lobuli could be observed after 8 h reperfusion (n = 6). These histologic signs of liver injury could be attenuated by administration of superoxide-dismutase in combination with catalase but not by allopurinol. Best results could be obtained by deferoxamine. This indicates that increased generation of free oxygen radicals in reperfusion is not caused by the known conversion of xanthine-dehydrogenase to -oxidase but is mediated by an increased generation of hydroxyl-radicals, which can be scavenged by deferoxamine.

Topics: Allopurinol; Animals; Deferoxamine; Electron Spin Resonance Spectroscopy; Fatty Acids, Nonesterified; Free Radicals; Ischemia; Liver; Male; Oxygen; Rats; Rats, Inbred Strains; Reperfusion Injury

The aim of this study was to determine the gastric microcirculatory alterations occurring during reperfusion after a period of ischemia and the possible role of oxyradicals in the microcirculatory disturbance. An in vivo microscopy technique was used to observe the superficial mucosal blood flow during reperfusion. After reperfusion, mucosal blood flow resumed quickly and then slowed with eventual cessation of flow. Thirty minutes of ischemia followed by reperfusion resulted in cessation of flow in 50 +/- 4% and 81 +/- 8%, of the capillaries in the microscopic field at 15 and 30 min, respectively, after reperfusion. During this mucosal microcirculatory change, numerous white thrombi were observed flowing in the mucosal microvessels. In rats pretreated with allopurinol to inhibit oxyradical formation, blood flow was maintained to a significant and markedly greater extent. Study of the submucosal microvasculature after reperfusion revealed a marked delay in transit of a fluorescein-albumin bolus from terminal submucosal arterioles through the mucosal microvasculature and back to submucosal collecting venules. Submucosal vascular diameter change could not explain the altered mucosal blood flow. These findings indicate that there is marked slowing and cessation (in many microvessels) of gastric mucosal blood flow during reperfusion after a period of ischemia, and that the obstruction to flow occurs in the mucosal microvessels. The results of the study with allopurinol suggest that oxygen-derived free radicals generated by xanthine oxidase may play a major role in the genesis of this gastric mucosal microcirculatory disturbance.

Topics: Allopurinol; Animals; Free Radicals; Gastric Mucosa; Gastrointestinal Transit; Male; Microcirculation; Oxygen; Rats; Rats, Inbred Strains; Reperfusion Injury; Videotape Recording

Acute tubular necrosis (ATN) after renal transplantation is related to the duration of warm and cold ischemia and leads to temporary or permanent impairment of graft function. An increased incidence of ATN has been reported since the introduction of cyclosporin A. Kidney damage resulting from hypothermic storage is generated in part during reperfusion rather than during ischemia itself. Potential mediators of the reperfusion injury are oxygen-derived free radicals. Therefore, the influence of two oxygen radical antagonists, allopurinol and superoxide dismutase, was evaluated in syngeneic rat kidney transplantation with and without concurrent administration of cyclosporin A. At 15 h cold ischemia, 28-day survival increased from 8% (no treatment) to 22% (superoxide dismutase), 33% (superoxide dismutase and allopurinol), and 73% (allopurinol). Cyclosporin A cotreatment (10 mg/kg over 14 days) resulted in survival rates of 0%, 25%, 17%, and 50% for the respective treatment groups. The results of serum creatinine values and morphological evaluation of biopsies paralleled the survival rates. Cyclosporin A nephrotoxicity was evidenced by significant serum creatinine elevations throughout the 28-day period of observation. In conclusion, allopurinol significantly protects syngeneic rat kidney transplants against a critical duration of cold ischemia. Under the conditions of this experiment, allopurinol was clearly superior to superoxide dismutase treatment. Cyclosporin A nephrotoxicity was, however, not ablated by the oxygen radical antagonists employed.

Topics: Allopurinol; Animals; Cyclosporine; Free Radical Scavengers; Immunosuppression Therapy; Kidney Transplantation; Kidney Tubular Necrosis, Acute; Male; Rats; Rats, Inbred WF; Reperfusion Injury; Superoxide Dismutase

Conflicting data have been reported on the relationship between reactive oxygen intermediates and the formation of oxygenase-derived eicosanoids. Plasma levels of prostacyclin (PGI2, measured as the stable metabolite 6-keto-PGF1 alpha) and thromboxane A2 (TxA2, measured as TxB2) in the effluent blood of a canine ileal segment were determined following 1 or 2 h of ischemia. The synthesis of both eicosanoids was significantly stimulated during reperfusion, but extension of the ischemic interval from 60 to 120 min was not followed by a further increase. The role of oxidants potentially involved in the process was investigated by using materials that inactivate the xanthine-oxidase-generated intermediates. Previous studies on the same in vivo animal model had demonstrated the effectiveness of antioxidant therapy in reducing the postischemic histamine release. There was no significant alteration in the amount of eicosanoids synthesized following oral allopurinol, catalase, dimethylsulfoxide, mannitol or desferrioxamine treatment. Intravenously administered allopurinol, however, significantly elevated the postischemic 6-keto-PGF1 alpha/TxB2 ratio. The results suggest that these antioxidants at doses inhibitory to histamine liberation are not effective in influencing the postischemic eicosanoid release. Intravenously administered allopurinol could exert a potentially beneficial effect through a mechanism other than the blockade of xanthine oxidase.

Topics: Allopurinol; Animals; Antioxidants; Dogs; Epoprostenol; Female; Intestines; Ischemia; Male; Prostaglandin-Endoperoxide Synthases; Reperfusion Injury; Thromboxane A2

We perfused isolated rat livers with Krebs-Ringer buffer, with no recirculation. Bile flow virtually stopped during 30 min of anoxia and resumed following reoxygenation to reach a plateau of 44% of the control level. When taurodehydrocholic acid (TDHC, 50 nmol/min/g liver) was administered during reoxygenation, bile flow increased three-fold (16.1 +/- 1.3 to 45.3 +/- 6.3 microliters/g liver). The increase in bile output with TDHC was 27.8 microliters/g liver, which was 89% of the control output. Bile acid output during this period was 1.4 mumol/g liver, which was 93% of the control level. Addition of allopurinol (50 nmol/min/g liver) without TDHC increased bile flow significantly (16.1 +/- 1.3 to 21.3 +/- 1.2 microliters/g liver), but the change was not significant when allopurinol and TDHC were given. The addition of allopurinol also reduced the cumulative release of lactate dehydrogenase from the liver during the reoxygenation period, but had no effect on hepatic adenosine triphosphate levels. Our data suggest that the bile acid-independent bile flow is sensitive to reoxygenation injury following anoxia whereas bile acid output and bile acid-dependent bile flow are resistant.

Topics: Adenosine Triphosphate; Allopurinol; Animals; Bile; Bile Acids and Salts; Hypoxia; L-Lactate Dehydrogenase; Male; Perfusion; Rats; Rats, Inbred Strains; Reperfusion Injury; Taurocholic Acid

We hypothesized that xanthine oxidase plays a role in the postischemic reperfusion injury in the equine small intestine. Under anesthesia, four horses and two ponies underwent ischemic strangulating obstructions of segments of the proximal jejunum, mid-jejunum and ileum. Prior to vascular occlusion, and at 1 h and 2 h of ischemia, full-thickness intestinal biopsies were collected for histopathological evaluation and for determination of combined xanthine dehydrogenase (XDH) plus xanthine oxidase (XO) activity, and XO activity alone. The level of XO activity was expressed in percentage according to the ratio of XO/(XDH + XO). We found a nearly threefold increase in the combined level of XDH plus XO activity from ileum to duodenum (p less than 0.04). However, the preischemic level of % XO activity did not vary significantly (p = 0.61) between segments of jejuno-ileum. Likewise, no significant difference was noted between intestinal segments after ischemia. Therefore, the data from all intestinal segments were pooled for each time and analyzed using Wilcoxon's signed rank test (one-tailed). Compared to the pre-ischemic level of % XO activity (median 27%), the % XO activity increased after 1 h of ischemia (median 37.0%), reaching statistical significance (p = 0.016). There were no statistical differences between the preischemic % XO activity and the % XO activity in non-ischemic bowel at the end of the anesthetic period. During ischemia, % XO activity increased, which lends credence to the importance of xanthine oxidase in previously-documented reperfusion injury in the equine small intestine.

Topics: Animals; Biopsy; Horse Diseases; Horses; Intestine, Small; Ischemia; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

In the current study we evaluated effluent blood from extremities of human patients undergoing reconstructive surgical treatment which is routinely accompanied by upper extremity exsanguination and application of a tourniquet. Following tourniquet release (reperfusion), there were immediate increases in the plasma levels of xanthine oxidase activity, uric acid, and histamine. Xanthine dehydrogenase activity was not detectable. Plasma also contained products consistent with the formation of oxygen-derived free radicals, namely hemoglobin and fluorescent compounds. Our data indicate in humans that ischemia-reperfusion events are associated with the appearance of xanthine oxidase activity and its products in the plasma effluent.

Topics: Adult; Arm; Free Radicals; Histamine; Humans; Oxygen; Reperfusion Injury; Tourniquets; Uric Acid; Xanthine Dehydrogenase; Xanthine Oxidase

The intestinal reperfusion injury has been studied in Sprague-Dawley rats weighing 200 g. Intestinal ischemia has been induced by clamping the Superior Mesenteric Artery (SMA) for 120 minutes. In order to parallel clinical situations, free radical scavengers (allopurinol [ALLO]) and superoxide-dismutase [SOD] were inoculated at a low perfusion rate through the femoral vein during the last 20 minutes of the ischemic period. Both drugs have decreased the mortality rate (from 70% in control group, to 40%) and the mean percentage of damaged intestine (30.89% vs. 23.84% and 24.70%). Histologically, ALLO was less effective than SOD (12.54 control; 8.40 SOD; 11.54 ALLO). The modification of glucose, SGOT, SGPT and LDH found in all the ischemic animals shows the hepatocellular injury induced by intestinal reperfusion.

Topics: Allopurinol; Animals; Antioxidants; Female; Free Radical Scavengers; Liver Function Tests; Mesenteric Arteries; Mesenteric Vascular Occlusion; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase

The role of Sucralfate in prevention of acute gastric injuries and its comparison with free radicals blockers as Allopurinol, Soybean Trypsin Inhibitor and Superoxide Dismutase was studied in the ischemia-reperfusion model by total occlusion of the celiac axis in Wistar rats. In control rats, the gross gastric mucosal necrotic area was of 80%; in contrast, the antioxidant drugs resulted in a necrotic area of 7%-15% and Sucralfate resulted in a necrotic area of only a 4%. It was concluded that Sucralfate, as antioxidant-cytoprotective drug, by enhancing the gastric defensive barrier was more important than the secondary aggression induced by free radicals.

Topics: Allopurinol; Animals; Female; Gastric Mucosa; Necrosis; Random Allocation; Rats; Rats, Inbred Strains; Reperfusion Injury; Sucralfate; Superoxide Dismutase; Trypsin Inhibitor, Kunitz Soybean

The effects of pretreatment with cyclosporine, allopurinol, or methylprednisolone on ischemia-reperfusion injury of the liver were investigated. A total of 32 adult mongrel dogs that received one of the pretreatments were divided into four groups and were subjected to 90 min liver ischemia. Serum activities of aspartate aminotransferase (s-AST) and lactate dehydrogenase, (s-LDH) as well as animal survivals were used as indicators of liver injury. The elevation of both s-AST and s-LDH was significantly suppressed by pretreatment with cyclosporine as much as by allopurinol. However a significant improvement in animal survival was obtained only in the cyclosporine-pretreated group. Pretreatment with methylprednisolone did not affect either the activities of s-AST and s-LDH or animal survivals when compared with the control group. These data suggest that cyclosporine is a potent protector against ischemic liver injury--as effective as allopurinol or methylprednisolone. Although the precise mechanism of the effect of cyclosporine on liver ischemia still remains unknown, these observations may be of use in liver transplantation.

Topics: Allopurinol; Animals; Aspartate Aminotransferases; Cyclosporins; Dogs; Female; L-Lactate Dehydrogenase; Liver; Male; Methylprednisolone; Reperfusion Injury

Allopurinol (ALL) improves energy metabolism in organs subjected to ischemia-reperfusion injury. The importance of different administration schedules of ALL has been studied in a rat liver model exposed to 60 min of normothermic ischemia followed by reperfusion. ALL (100 mg/kg) that administered in two doses, one prior to ischemia and one prior to reperfusion, improved production of adenosine triphosphate in the liver as well as bile flow during reperfusion. ALL administered in a single dose, either prior to ischemia or prior to reperfusion, was less effective. The concentration of hypoxanthine during ischemia increased in the groups given ALL prior to induction of ischemia. Based on the present findings, we argue that the beneficial effect of ALL administration can be potentiated by different drug-administration schedules. Our data also suggest that the prime mechanism of action for ALL is not only related to inhibition of free-oxygen-radicals production but that preservation of hypoxanthine, which can be used for ATP resynthesis and the scavenging properties of ALL itself, may be equally important.

Topics: Adenosine Triphosphate; Allopurinol; Animals; Bile; Drug Administration Schedule; Energy Metabolism; Hypoxanthine; Hypoxanthines; Liver; Male; Rats; Rats, Inbred Strains; Reperfusion Injury; Xanthine; Xanthines

The role of xanthine oxidase and oxygen free radicals in postischemic reperfusion injury in the rat kidney remains controversial. Proximal tubules, the focal segment affected by ischemic renal injury, were isolated in bulk, assayed for xanthine oxidase activity, and subjected to 60 min of anoxia or hypoxia and 60 min of reoxygenation to evaluate the participation of xanthine oxidase and oxygen radicals in proximal tubule reoxygenation injury. The total xanthine oxidase in isolated rat proximal tubules was 1.1 mU/mg of protein, approximately 30% to 40% of the activity found in rat intestine and liver. Lactate dehydrogenase release, an indicator of irreversible cell damage, increased substantially during anoxia (39.8 +/- 2.3 versus 9.8 +/- 1.8% in controls) with an additional 8 to 12% release during reoxygenation. Addition of 0.2 mM allopurinol, a potent xanthine oxidase inhibitor, and dimethylthiourea, a hydroxyl radical scavenger, failed to protect against the reoxygenation lactate dehydrogenase release. Analysis of xanthine oxidase substrate levels after anoxia and flux rates during reoxygenation indicates that hypoxanthine and xanthine concentrations are in a 15-fold excess over the enzyme Km and 0.3 mU/mg of protein of xanthine oxidase activity exists during reoxygenation. Hypoxic tubule suspensions had a minimal lactate dehydrogenase release during hypoxia and failed to demonstrate accelerated injury upon reoxygenation. In conclusion, although xanthine oxidase is present and active during reoxygenation in isolated rat proximal tubules, oxygen radicals did not mediate reoxygenation injury.

Topics: Adenine Nucleotides; Animals; Free Radicals; Hypoxanthine; Hypoxanthines; Hypoxia; In Vitro Techniques; Kidney Tubules; L-Lactate Dehydrogenase; Male; Oxygen; Rats; Rats, Inbred Strains; Reperfusion Injury; Xanthine; Xanthine Oxidase; Xanthines

Endothelial cells may be damaged by oxygen reactive species produced by granulocytes, by transition metal ions or by xanthine oxidase, an enzyme present in great quantity in these cells. Since it has been observed that propionyl carnitine protects the heart from peroxidation, we have investigated the effect of this compound on the formation of thiobarbituric acid reactive oxidation products (TBAR) in endothelial membranes. The peroxidation systems used were a mixture of Fe3+ and Fe2+, hydrogen peroxide and Fe2+, or xanthine oxidase-- xanthine. Propionyl carnitine at millimolar concentrations decreases TBAR formation. The protection is concentration-dependent and is almost absent in the presence of propionate and carnitine. From these results it appears that propionyl carnitine may protect not only myocardium but also vessels from peroxidative damage that occurs during ischaemia and reperfusion.

Topics: Animals; Carnitine; Cattle; Cell Membrane; Cell Separation; Dose-Response Relationship, Drug; Endothelium, Vascular; Hydrogen Peroxide; Iron; Reperfusion Injury; Thiobarbiturates; Xanthine Oxidase

We reported previously that a transient occlusion followed by reperfusion of the portal vein and the hepatic artery of the rat significantly decreased the transhepatic transport of a cholephilic compound, and that this decrease was prevented by pretreating animals with poly(styrene co-maleic acid butyl ester)-conjugated superoxide dismutase (SM-SOD). To elucidate the mechanism for oxidative injury of the liver and the site for the generation of superoxide radicals, the effect of a portosystemic bypass on the liver function was examined in the rat whose hepatic vessels were temporarily occluded. A portosystemic bypass inhibited the reperfusion-induced decrease in hepatic transport of bromosulfophthalein as effectively as did SM-SOD. Kinetic analysis using 125I-labeled albumin revealed that the permeability of the small intestine markedly increased after a transient occlusion. The increase in intestinal permeability was also inhibited either by SM-SOD or by the portosystemic bypass. Xanthine oxidase activity in portal plasma markedly increased during occlusion and reperfusion, while it remained within normal ranges in the bypassed group. Thus, superoxide radical, and/or its metabolite(s), might play a critical role in increasing the intestinal permeability and in the pathogenesis of reperfusion-induced liver injury.

Topics: Animals; Azides; Blood Pressure; Catalase; Enterohepatic Circulation; Intestinal Mucosa; Liver; Liver Circulation; Luminescent Measurements; Male; Portasystemic Shunt, Surgical; Rats; Rats, Inbred Strains; Reperfusion Injury; Serum Albumin; Sodium Azide; Sulfobromophthalein; Superoxide Dismutase; Xanthine Oxidase; Zymosan

Recently, we described a new solution, Carolina rinse, that prevents nonparenchymal cell injury in vitro after reperfusion of livers stored in University of Wisconsin cold solution (Currin RT, Toole JG, Thurman RG, Lemasters JJ. Transplantation 1990; 50: 1076). The present study was designed to examine the effect of Carolina rinse on graft survival in vivo. Unlike UW cold storage solution, which is high in potassium, Carolina rinse contains extracellular inorganic ions at levels similar to blood, a calcium channel blocker and a radical scavenger. Carolina rinse also contains fructose and mildly acidotic pH to reduce hypoxic cell death. Livers from Lewis rats were explanted, stored in UW cold storage solution under nonsurvival conditions, and rinsed with either 15 ml of Ringer's, UW solution, Carolina rinse, or Carolina rinse saturated with nitrogen prior to completion of implantation surgery. In the Ringer's rinse group, only 4% of recipients survived 30 days postoperatively. In this group, SGOT levels reached maximal values of about 5000 U/L. Survival was also poor (25%) when grafts were rinsed with UW solution. In the Carolina rinse group, however, 9 of 16 rats (56%) survived indefinitely, and maximal postoperative SGOT levels were reduced 3-fold. Liver injury indexed histologically was also decreased about 3-fold by Carolina rinse compared with the control group rinsed with Ringer's solution. Carolina rinse diminished postoperative sinusoidal endothelial cell damage assessed by electron microscopy and reduced carbon particle phagocytosis due to Kupffer cells significantly. Moreover, Carolina rinse diminished graft swelling and improved postoperative hepatic microcirculation compared with the Ringer's rinse group. Taken together, these results indicate that Carolina rinse is a superior alternative to Ringer's solution in vivo to protect liver grafts from reperfusion injury when removing high-potassium-containing cold storage solutions clinically prior to implantation.

Topics: Adenosine; Allopurinol; Animals; Cold Temperature; Endothelium; Female; Glutathione; Graft Survival; Insulin; Liver; Liver Circulation; Liver Transplantation; Organ Preservation Solutions; Postoperative Complications; Raffinose; Rats; Rats, Inbred Lew; Reperfusion Injury; Solutions; Tissue Preservation

An in vitro canine tibia model was used to assess the effects of 48 h of hypothermic (4 degrees C) ischemia on bone vascular resistance and on responsiveness of intraosseous blood vessels to circulating norepinephrine. Three groups of bones were studied: Group I (n = 11), 48 h hypothermic ischemia; Group II (n = 11), 48 h hypothermic ischemia with pretreatment with allopurinol and oxypurinol; and Group III (n = 10), no ischemia. Resting vascular resistance in both ischemic groups (79 and 74 mmHg/ml/min) was significantly higher (p less than 0.0001) than in the nonischemic group (22 mmHg/ml/min). Effects of norepinephrine on vascular resistance were significantly greater in both ischemic groups (p less than 0.004). In all three groups, acetylcholine infusion attenuated the increases in perfusion pressure caused by norepinephrine. This demonstrates secretion of endothelial-mediated relaxing factors (EDRF) and prostaglandin for up to 48 h of hypothermic ischemia. As no significant differences were detected between the two ischemic groups, this study failed to demonstrate any protective effect of xanthine oxidase inhibitors.

Topics: Acetylcholine; Allopurinol; Animals; Cold Temperature; Disease Models, Animal; Dogs; Drug Hypersensitivity; Ischemia; Nitric Oxide; Norepinephrine; Oxypurinol; Regional Blood Flow; Reperfusion Injury; Tibia; Vascular Resistance; Xanthine Oxidase

Ischemia-reperfusion injury has been implicated as playing a major role in the development of necrotizing enterocolitis, a major cause of morbidity and mortality in the newborn. A tungsten-supplemented molybdenum-free diet can reduce xanthine oxidase (XO) enzyme activity in the intestine, which in turn reduces the generation of oxygen radicals after an ischemia-reperfusion insult. This study evaluated the ability of this diet to be effective by indirect means, ie, transplacental and breast-feeding routes. XO activity of the intestine was measured in three groups of CD-1 white rats: I, weanlings fed the tungsten diet or standard chow for 1 week; II, 1-day-old rat pups whose mothers were maintained on the tungsten or standard chow for 7 to 10 days prior to term; and III, rat pups at 1 and 3 weeks after birth whose lactating mothers were maintained on the tungsten or standard chow. Some animals from group III also underwent either a 30- or 60-minute episode of occlusion of the superior mesenteric artery (SMA) to evaluate the protective effects of the diet. XO activity was significantly reduced in all groups receiving the tungsten diet (P less than .0001). Blinded histopathologic studies of the entire small bowel showed significantly less villar necrosis (P less than .05) and fibrosis (P less than .0001) in the tungsten-treated group than in the controls. In the 60-minute occlusion study all tungsten-group animals survived, whereas 7 of 12 in the control group died of intestinal infarction within 24 hours (P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Breast Feeding; Enterocolitis, Pseudomembranous; Female; Free Radicals; Intestine, Small; Placenta; Pregnancy; Rats; Reperfusion Injury; Tungsten; Xanthine Oxidase

Topics: Adenosine; Allopurinol; Amino Acids; Animals; Dogs; Glutathione; Glycine; Insulin; Liver; Liver Function Tests; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Rabbits; Raffinose; Reperfusion Injury; Solutions

The purpose of this study was to assess the protective effects of allopurinol, a blocker of free oxygen radical (FOR) formation, and superoxide dismutase-polyethylene glycol (SOD-PEG), a scavenger of FORs, on ischemic and reperfusion-induced cochlear damage. Fifteen Wistar Kyoto rats (WKY) were randomly assigned to three groups: (1) a control group (5 animals) that was exposed to 15 minutes of cochlear ischemia by clamping the anterior inferior cerebellar artery (AICA), followed by 15 minutes of reperfusion as documented by laser Doppler flowmetry; (2) a drug-treated group (5 animals) that received allopurinol before ischemia/reperfusion; and (3) a drug-treated group (5 animals) that received SOD-PEG before ischemia/reperfusion. In the control group, the tone burst-evoked compound action potential (CAP) recorded from the round window (RW) of the cochlea was abolished, and the cochlear microphonic (CM) was reduced after ischemia. In contrast, both allopurinol and SOD-PEG-treated animals showed post-reperfusion sensitivity in CAP and CM measures. We interpret these results to indicate that damage to the cochlear from ischemia and subsequent reperfusion can be attenuated by pretreatment with allopurinol or SOD-PEG. This provides indirect evidence that FORs may be partially responsible for cochlear damage resulting from ischemic conditions.

Topics: Action Potentials; Allopurinol; Animals; Auditory Threshold; Cochlea; Ischemia; Polyethylene Glycols; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase

Topics: Allopurinol; Animals; Intestinal Diseases; Male; Mesenteric Arteries; Peristalsis; Rabbits; Reperfusion Injury; Xanthine Oxidase

Reperfusion injury characterized by loss of endothelial cell viability occurs after cold ischemic storage of livers for transplantation surgery. Here, ultrastructural changes in stored rat livers were examined by scanning and transmission electron microscopy. With increasing times of storage in Euro-Collins solution (4 to 24 hr) followed by 15 min of reperfusion at 37 degrees C, a sequence of structural alterations was observed involving endothelial and Kupffer cells. Widening of endothelial fenestrations occurred after 4 hr and progressed over 8 to 24 hr to retraction of cellular processes, ball-like rounding, sinusoidal denudation and ultrastructural derangements consistent with loss of cell viability. Kupffer cells exhibited progressive rounding, ruffling of the cell surface, polarization, appearance of wormlike densities, vacuolization and degranulation over a similar time course. By contrast, the structures of parenchymal and fat-storing cells were relatively undisturbed by cold storage and reperfusion. Alterations to endothelial and Kupffer cells were also studied as a function of time of reperfusion. After 24 hr of storage, endothelial cells showed retraction of cytoplasm before reperfusion that progressed quickly to loss of viability and denudation during reperfusion. Kupffer cell activation (ruffling, degranulation) during reperfusion was slower and occurred after deterioration of endothelial cells. Livers stored in Euro-Collins solution were also compared with livers stored in University of Wisconsin cold storage solution, an improved preservation medium for transplantation. University of Wisconsin solution provided better preservation of endothelial structure and markedly reduced parenchymal cell blebbing and swelling before reperfusion. University of Wisconsin solution also reduced Kupffer cell activation and release of lysosomal enzymes. In conclusion, endothelial cell deterioration followed by Kupffer cell activation occurred after increasing times of cold ischemic storage and reperfusion of rat livers. Both changes may contribute to the pathophysiology of graft failure caused by reperfusion-mediated storage injury.

Topics: Acid Phosphatase; Adenosine; Allopurinol; Animals; Endothelium; Glutathione; Hypertonic Solutions; Insulin; Kupffer Cells; Liver; Liver Transplantation; Male; Organ Preservation; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Strains; Reperfusion Injury; Solutions

Topics: Adenosine; Allopurinol; Animals; Glutathione; Insulin; Lipid Peroxides; Organ Preservation; Organ Preservation Solutions; Organ Transplantation; Raffinose; Rats; Rats, Inbred Strains; Reperfusion Injury; Solutions; Time Factors

The effectiveness of superoxide dismutase and allopurinol was evaluated in preventing reperfusion injury in Sprague-Dawley rats utilizing a limb replantation model after 6 hours of warm ischemia. Immediately prior to reperfusion of the replanted limb the animals received (intravenously) either a single bolus of superoxide dismutase (16,000 units/kg), a single bolus of allopurinol (45 mg/kg), both agents, or only control solutions. In the rats that received only control solutions, 30% of the limbs survived the ischemic insult. The group that received both superoxide dismutase and allopurinol had a 75% limb survival (P = 0.005). The animals that had only superoxide dismutase or allopurinol had 58% and 60% of the replanted limbs survive, respectively. This study suggests that the administration of superoxide dismutase and allopurinol may be helpful in the prevention of reperfusion injury in skeletal muscle in an acute injury setting (without pretreatment) and may thereby improve limb salvage after a significant ischemic period.

Topics: Allopurinol; Animals; Hindlimb; Injections, Intravenous; Ischemia; Male; Muscles; Rats; Rats, Inbred Strains; Reperfusion Injury; Replantation; Superoxide Dismutase; Tissue Survival

It has been widely proposed that conversion of xanthine dehydrogenase (XDH) to its free radical-producing form, xanthine oxidase (XOD), underlies ischemic/reperfusion injury, although the relationship of this conversion to hypoxia and its physiologic control have not been defined. This study details the time course and control of this enzymatic interconversion. In a functionally intact, isolated perfused rat liver model, mean % XOD activity increased as a function of both the duration (25 to 45% in 3 h) and degree (r = 0.97) of hypoxia. This process was markedly accelerated in ischemic liver by an overnight fast (45 vs. 30% at 2 h), and by imposing a short period of in vivo ischemia (cardiopulmonary arrest 72%). Moreover, only under these conditions was there a significant rise in the XOD activity due to the conformationally altered XDH molecule (XODc, 18%), as well as concomitant morphologic injury. Neither circulating white blood cells nor thrombosis appeared to contribute to the effects of in vivo ischemia on enzyme conversion. Thus, it is apparent that conversion to the free radical-producing state, with high levels of XOD activity and concurrent cellular injury, can be achieved during a relatively short period of hypoxia under certain well-defined physiologic conditions, in a time course consistent with its purported role in modulating reperfusion injury. These data also suggest that the premorbid condition of organ donors (e.g., nutritional status and relative state of hypoxia) is important in achieving optimal organ preservation.

Topics: Animals; Free Radicals; In Vitro Techniques; Liver; Neutropenia; Oxygen Consumption; Rats; Rats, Inbred Strains; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

Trolox, a hydrophilic analog of vitamin E, was reported to scavenge peroxyl radicals from artificial systems better than its parent compound. Here we examined the possible cytoprotective effect of Trolox in cultured hepatocytes and in the rat liver. In cultured rat hepatocytes, 0.5 to 16 mmol/L Trolox (with optimum between 1 to 2 mmol/L) was observed to prolong the survival of cells exposed to oxyradicals generated with xanthine oxidase-hypoxanthine. The protection by 1 mmol/L Trolox surpassed that provided by either ascorbate, mannitol, superoxide dismutase and/or catalase--each at a level giving its maximal protection in the same system. In both a global and partial model of hepatic ischemia-reperfusion in rats, infusion of Trolox (7.5 to 10 mumol/kg body weight) just before reflow reduced by greater than 80% the liver necrosis sustained in untreated (no Trolox) control rats. Such organ salvage was apparently accompanied by approximately 50% reduction in the amount of hepatic conjugated dienes, which were quantified by a highly specific radiochemical assay. Since conjugated dienes are presumed to be good "markers" of oxyradical damage, our data may have provided a semiquantitative link between free radical-induced necrosis and its chemical imprint in vivo. The data also indicated a relatively rapid and potent antioxidant-like action by Trolox on rat hepatocytes and on the postischemic reperfused rat liver.

Topics: Animals; Cells, Cultured; Chromans; Free Radicals; Hypoxanthine; Hypoxanthines; Ischemia; Liver; Liver Diseases; Male; Rats; Rats, Inbred Strains; Reperfusion Injury; Xanthine Oxidase

Oxygen-derived free radicals have been implicated in a variety of diseases and pathologic processes, including ischemia reperfusion injury (IRI). Based on experimental work with rat skin-flap models, the enzyme xanthine oxidase (XO) has been proposed as a major source of free radicals responsible for tissue injury and flap necrosis. The presence of this enzyme is variable within different tissues of a specific species and between species. Xanthine oxidase levels in pig and human skin have not previously been reported. The activity of xanthine oxidase in the skin of rats (N = 16), pigs (N = 7), and humans (N = 8) was measured after varying intervals of ischemia and in the rat also following reperfusion. Control pig and human skin were found to contain minimal enzyme activity, almost 40 times less than that of the rat. In the rat, xanthine oxidase activity was stable throughout a prolonged period of ischemia, and a significant decrease in activity was found after 12 hours of reperfusion (p less than 0.05). In humans, xanthine oxidase activity was unaffected by ischemia time, and in the pig, it did not increase until 24 hours of ischemia (p less than 0.05). The potential sources of free radicals and the mechanism of action of xanthine oxidase and its inhibitor allopurinol in improving flap survival in different species are reviewed.

Topics: Analysis of Variance; Animals; Female; Free Radicals; Humans; Ischemia; Male; Rats; Rats, Inbred Strains; Reperfusion Injury; Skin; Surgical Flaps; Swine; Time Factors; Xanthine Oxidase

Reperfusion of ischaemic skeletal muscle may lead to increased vascular permeability, oedema and ultimately muscle necrosis. Oxygen-derived free radicals have been suggested as aetiological factors in reperfusion injury. Amputated rabbit hindlimbs were subjected to 4 h of ischaemia followed by 2 h or reperfusion with Krebs' buffer. One limb from each animal was reperfused with oxygen-saturated buffer (reoxygenated limb) while the other limb was reperfused with nitrogen-saturated buffer (non-reoxygenated limb). Six animals received allopurinol orally 2 days prior to the experiment and ten animals received no treatment. The energy charge dropped from 0.90 to 0.54 during ischaemia and increased to 0.82 after reperfusion with oxygenated perfusate. Oedema was determined by limb weight and water content in muscle biopsies and muscle injury was assessed by uptake of [Tc99]methylenediphosphonate ([Tc99]MDP). The results were expressed in ratios, between the reoxygenated and nonreoxygenated limb. Without allopurinol treatment, the increase in water content and limb weight in reoxygenated limbs exceeded (p less than 0.05) non-reoxygenated limbs (ratios = 1.73 and 1.89, respectively). Allopurinol treatment significantly reduced (p less than 0.05 and p less than 0.02, respectively) the increase in water content and limb weight (ratios = 0.54 and 1.01, respectively). Without treatment, [Tc99]MDP-uptake was greater (p less than 0.05) in reoxygenated limbs than in non-reoxygenated limbs (ratio = 1.60). Allopurinol treatment significantly reduced (p less than 0.002) [Tc99]MDP-uptake in reoxygenated limbs (ratio = 0.80). These results demonstrate that additional injury to ischaemic skeletal muscle occurs during reperfusion with oxygen.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Allopurinol; Animals; Female; Free Radical Scavengers; Free Radicals; Hindlimb; Male; Muscles; Oxygen; Rabbits; Reperfusion Injury; Xanthine Oxidase

The reperfusion of previously ischemic tissue may lead to the formation of highly reactive free radicals that promote tissue injury. Xanthine oxidase has been implicated as one source of these free radicals. We examined the role of xanthine oxidase in brain injury using a cerebrospinal fluid compression model of global cerebral ischemia with 15 minutes of ischemia and 4 hours of reperfusion. Seven dogs were pretreated with the xanthine oxidase inhibitor allopurinol (50 mg/kg for 5 days). Neurophysiological recovery was monitored with cortical somatosensory evoked potentials. As an attempt to correlate brain recovery with the mechanism of protection, free brain malondialdehyde was measured at the end of reperfusion by high-performance liquid chromatography. Brain water content was measured by wet-dry weights. Compared with seven untreated control dogs, allopurinol pretreatment significantly improved recovery of somatosensory evoked potentials after 4 hours of reperfusion. However, the amount of free malondialdehyde in the allopurinol-treated dogs was 32% greater than that in the controls. Brain water content was similar in the two groups. These results suggest that xanthine oxidase contributes to brain injury after ischemia and reperfusion. However, tissue damage caused by xanthine oxidase may be mediated through mechanisms other than free radical production.

Topics: Allopurinol; Animals; Brain Ischemia; Cerebrovascular Circulation; Dogs; Evoked Potentials, Somatosensory; Reperfusion Injury; Xanthine Oxidase

Frostbite is characterized by acute tissue injury induced by freezing and thawing. Initial complete ischemia is followed by reperfusion and later, tissue necrosis. These vascular events support the hypothesis that free radical-mediated reperfusion injury at thawing might contribute to tissue necrosis after frostbite in a manner similar to that seen after normothermic ischemia. To test this hypothesis, rabbit ears were frozen at -21 degrees C for 30, 60, 90, or 120 s and rewarmed at room temperature (22 degrees C). Rabbits were treated "blindly" with saline alone, highly purified, pharmaceutical grade superoxide dismutase (SOD), allopurinol, or deferoxamine. The area of ear necrosis was determined 3 weeks after frostbite by "blinded" morphometry. The administration of SOD at the time of thawing significantly improved viability in ears frozen for 60 and 90 s, but not in those frozen for 30 or 120 s. Deferoxamine also improved viability in ears frozen for 60 s. Allopurinol did not significantly affect ear survival. Electron micrographs showed the appearance of severe endothelial cell injury beginning during freezing and extending through early reperfusion. Later, neutrophil adhesion, erythrocyte aggregation, and microvascular stasis were seen. These findings suggest that free radical-mediated reperfusion injury has a role in frostbite, and quantitate the proportion of the injury that is due to this mechanism.

Topics: Allopurinol; Animals; Deferoxamine; Disease Models, Animal; Ear; Free Radicals; Freezing; Frostbite; Male; Necrosis; Rabbits; Reperfusion Injury; Superoxide Dismutase

The influence of diltiazem and/or allopurinol on kidney microcirculation was studied in anaesthetized rats, which were subjected to 60 min unilateral renal ischemia followed by 60 min reflow. In histological sections capillary plasma flow patterns were determined based on the distribution of two different fluorochrome-labelled globulins administered i.v.. In the outer medulla (OM) of untreated postischemic kidneys labelling of the capillary network was greatly diminished. Tissue areas occupied by red blood cells increased 4-6 fold. During reperfusion massive penetration of red cells in the urine was demonstrated by the occurrence of hemoglobin in the urine. Maintenance of the rats on allopurinol-saturated drinking water for six days prior to the experiment (daily intake approximately 50 mg allopurinol/kg body wt) combined with the i.v. administration of diltiazem during the pre- and postischemic period (16 mg/kg body wt) resulted in an almost complete normalization of capillary plasma flow patterns in the OM. In this region tissue areas occupied by red blood cells were much lesser in extent than in the untreated controls. Furthermore, urine hemoglobin content after the combined drug regimen was largely decreased when compared to the untreated ischemic group. Effects of the treatment with either of the drugs alone were qualitatively similar, but significantly less pronounced. In conclusion, a synergistic effect of diltiazem and allopurinol in improving postischemic renal microcirculation is clearly evident, whereas no improvement in kidney function was demonstrable. This supports the hypothesis that disturbed microcirculation is not a prerequisite for the generation of the renal functional deterioration in the clamp-induced ischemia model in the rat.

Topics: Allopurinol; Animals; Capillaries; Capillary Permeability; Diltiazem; Drug Interactions; Erythrocyte Aggregation; Globulins; Kidney; Male; Microcirculation; Rats; Rats, Inbred Strains; Regional Blood Flow; Reperfusion Injury; Time Factors

Xanthine oxidase (XO)-derived oxygen radicals are thought to play an important role in the intestinal injury resulting from ischemia and reperfusion. In vitro data shows enhanced XO activity in the presence of histamine. Histamine is known to be released during intestinal ischemia and reperfusion. The purpose of this study was to evaluate the relationship between histamine and XO in vivo in intestinal ischemia/reperfusion injury. Using an established model of gut ischemia and reperfusion, portal venous plasma was obtained and assayed for histamine levels, XO activity, and xanthine dehydrogenase (XD) activity following injury. Intestinal ischemia for 120 minutes resulted in a 200% increase in plasma histamine levels (263.4 +/- 36.9 nmol/mL control, v 548.7 +/- 35.1 nmol/mL experimental, P less than .05). Reperfusion for 15 minutes resulted in a further increase in plasma histamine (to 658.3 +/- 33.9 nmol/mL), compared with 120 minutes of ischemia alone. No significant change in plasma XO activity resulted after simple ischemia for 120 minutes. However, XO activity doubled within 15 minutes of reperfusion of the ischemic intestine (6.37 +/- 0.53 nmol O2- per milliliter per minute v 3.12 +/- 0.25 nmol O2- per milliliter per minute, P less than .05). Reperfusion for 60 minutes resulted in the maximal observed increase in plasma XO activity (9.49 +/- 0.67 nmol O2- per milliliter per minute). Analysis of XD activity demonstrated no significant decrease compared with controls until 120 minutes of ischemia and 60 minutes of reperfusion (1.62 +/- 0.49 nmol uric acid per milliliter per minute at 60 minutes of reperfusion, versus 5.02 +/- 0.52 nmol uric acid per milliliter per minute control, P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Germ-Free Life; Histamine; Histamine Release; Intestines; Ischemia; Male; Rats; Rats, Inbred Strains; Reperfusion Injury; Time Factors; Xanthine Dehydrogenase; Xanthine Oxidase

This study evaluated the effect of ischemia-reperfusion (I-R) on pulmonary capillary permeability in isolated rabbit lungs and the roles of xanthine oxidase (XO), aldehyde oxidase (AO), and neutrophils (PMN) in producing this lung injury. Effects of XO and AO were studied by inactivation with a tungsten-enriched diet (0.7 g/kg) and inhibition of XO by allopurinol (100 microM) or AO by menadione (3.5 microM). PMN effects were studied by preventing endothelial adhesion with the monoclonal antibody IB4 (10 microM). Vascular permeability was evaluated by determining the capillary filtration coefficient (Kf,c) measured before and after I-R in all experimental conditions. Reperfusion after 2 h of ischemia significantly increased pulmonary capillary permeability (Kf,c changed from 0.096 +/- 0.014 to 0.213 +/- 0.025 ml.min-1. cmH2O-1.100 g-1), and this increase was blocked by the addition of catalase (50,000 U) at reperfusion (baseline Kf,c was 0.125 +/- 0.023 and 0.116 +/- 0.014 ml.min-1.cmH2O-1.100 g-1). XO inactivation with the tungsten-supplemented diet and XO inhibition with allopurinol prevented the Kf,c increase observed after I-R (0.183 +/- 0.030 to 0.185 +/- 0.033 and 0.126 +/- 0.018 to 0.103 +/- 0.005 ml.min-1.cmH2O-1.100 g-1). Inhibition of AO had no effect on I-R injury (Kf,c 0.108 +/- 0.011 to 0.167 +/- 0.014 ml.min-1.cmH2O-1.100 g-1). Preventing PMN adhesion resulted in significant attenuation of the change in Kf,c associated with I-R (0.112 +/- 0.032 to 0.090 +/- 0.065 ml.min-1.cmH2O-1.100 g-1). We conclude that XO and PMN adherence, but not AO, are involved in the increased capillary permeability associated with I-R.

Topics: Aldehyde Oxidase; Aldehyde Oxidoreductases; Allopurinol; Animals; Blood Pressure; Capillaries; Lung; Neutrophils; Pulmonary Circulation; Rabbits; Reperfusion Injury; Vascular Resistance; Vitamin K; Xanthine Oxidase

The effect of organ flushing with the calcium entry blocker verapamil on the conversion of innocent enzyme xanthine dehydrogenase (XDH) to superoxide generating enzyme xanthine oxidase (XOD) in ischemic rat livers was studied. This enzyme conversion progressed over time in warm or cold ischemia. In non-flushed livers, the activities of XOD as percentages of XDH plus XOD after 6 h at 37 degrees C and 6 days at 4 degrees C were 80.3 +/- 5.2 and 31.6 +/- 2.1, respectively. In the livers flushed with Euro-Collins solution, the conversion was inhibited to 37.0 +/- 3.9% (P less than 0.001) after 6 h of warm ischemia, while this inhibitory effect was not found in cold ischemia. Verapamil given through the portal vein on flushing further suppressed the conversion in both warm and cold ischemia (with 5.0 microM of verapamil, 21.2 +/- 5.8% (P less than 0.001) after 6 h of warm ischemia and 25.2 +/- 3.3% (P less than 0.01) after 6 days of cold ischemia). A similar effect was also obtained with the addition of 10 or 30 mM of EGTA instead of verapamil. In contrast, no inhibitory effect on conversion was obtained in livers flushed and homogenized with 10.0 microM of verapamil followed by incubation for 6 h at 37 degrees C. In the livers that were flushed and stored at a warm temperature for 6 h, verapamil reduced the increase of tissue lipid peroxidation product (P less than 0.02) after 15 min of reperfusion. Although the precise mechanisms of these inhibitory effects of verapamil on the enzyme conversion are still uncertain, it is thought that organ flushing with verapamil might reduce the XOD-mediated postischemic reperfusion injury in livers subjected to prolonged ischemia.

Topics: Animals; Calcium; Free Radicals; In Vitro Techniques; Ischemia; Liver; Male; Oxygen; Rats; Rats, Inbred Strains; Reperfusion Injury; Verapamil; Xanthine Dehydrogenase; Xanthine Oxidase

The possible relationship between increased blood levels of thromboxane (TXA2) and tissue levels of free radicals during ischaemia was investigated. Rabbit epigastric skin flaps were subjected to 4 h of body temperature ischaemia, then infused with either the TXA2 synthetase inhibitor UK-38,485, the free radical scavenger superoxide dismutase (SOD), or both immediately prior to reperfusion. After 30 min of reperfusion, increases in the tissue levels of xanthine oxidase (XO) and malonyldialdehyde (MDA), both of which are indices of free radical generation and decreases in the tissue levels of SOD were found. SOD treatment completely restored XO, MDA and SOD levels to normal, whereas UK-38,485 only partially improved all three parameters. None of these changes was statistically significant. Effluent blood thromboxane B2 (TXB2) levels from the flap increased significantly (P less than 0.01) after ischaemia and were reduced significantly by both UK-38,485 and SOD (P less than 0.05). Combined UK-38,485 and SOD treatment was no better than treatment with either agent alone. ATP levels and oedema, which decreased and increased respectively due to ischaemia, were not significantly altered by drug infusion. These results suggest that free radical damage may be related to TXA2-generated thrombosis in ischaemia/reperfusion injury.

Topics: Adenosine Triphosphate; Animals; Body Water; Free Radicals; Imidazoles; Ischemia; Malondialdehyde; Rabbits; Reperfusion Injury; Skin; Superoxide Dismutase; Surgical Flaps; Thromboxane-A Synthase; Thromboxanes; Xanthine Oxidase

The influence of different drugs on ischemia induced oxygen free radical damage was examined in intestinal tissue of rats by determination of thiobarbituric acid reactive substances (TBARS). Some methodical aspects of this method were considered. Experiments were done with and without the use of polymerized stromafree hemoglobin (PHb) as an additional oxygen carrier. Reversible total occlusion of the superior mesenteric artery was performed for 90 min, reperfusion time was 2.5 hours. Despite higher O2 availability PHb did not increase the TBARS level any further. Superoxide dismutase with catalase; allopurinol; ciprofloxacin; and deferoxamine produced a highly significant reduction of TBARS, even if used together with PHb.

Topics: Allopurinol; Animals; Catalase; Deferoxamine; Free Radicals; Hemoglobins; Intestinal Mucosa; Intestines; Ischemia; Lipid Peroxides; Male; Oxygen; Prohibitins; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase

We hypothesized that xanthine oxidase (XO)-derived hydrogen peroxide (H2O2) contributes to ischemic skeletal muscle injury during reperfusion. We found that after ischemia (3 h) and then reperfusion (4 h) rat gastrocnemius muscles had decreased contractile function following direct stimulation. Three lines of investigation suggested that XO-derived H2O2 contributes to reperfusion injury of ischemic skeletal muscle. First, treatment with dimethylthiurea (DMTU), a highly permeant O2 metabolite scavenger, but not urea, just before reperfusion improved muscle function in legs subjected to ischemia and then reperfusion. Second, gastrocnemius muscles from rats fed tungsten or allopurinol had negligible XO activities and increased muscle function after ischemia and reperfusion. Third, as assessed by measurement of skeletal muscle catalase activity in the presence of aminotriazole, H2O2 was measured during reperfusion of ischemic muscles from untreated or urea-treated rats but not during reperfusion of muscles from rats treated with DMTU, tungsten, or allopurinol.

Topics: Allopurinol; Amitrole; Animals; Catalase; Hydrogen Peroxide; Ischemia; Male; Muscle Contraction; Muscles; Rats; Rats, Inbred Strains; Reperfusion Injury; Thiourea; Tungsten; Urea; Xanthine Oxidase

Using a left lung orthotopic isograft model in AS strain rats, we have investigated ultrastructural changes in lungs preserved for 48 h at 0 degrees C after a simple flush technique. Lungs were examined after storage alone and after storage followed by up to 1 h reperfusion with blood in vivo. Grafts were flushed with either isotonic saline (NaCl) or hypertonic citrate solution (HCA) alone, or with HCA containing either verapamil (a Ca2+ channel blocker), desferrioxamine (a Fe2+ antagonist), prostacyclin PGI2, nifedipine (a Ca2+ channel blocker) or allopurinol (a xanthine oxidase inhibitor). These agents were also given intravenously to both donor and recipient. Storage in NaCl produced gross cytoplasmic swelling and disruption with widespread nuclear injury. Reperfusion for 1 h resolved cell swelling but some endothelial loss and alveolar capillary wall rupture were seen. HCA with or without additional agents protected against cell swelling but endothelial blebbing and widening of the basement membrane occurred. Reperfusion for 1 h led to recovery of the basement membrane thickness but widespread endothelial loss was observed which was reduced by the addition of verapamil, desferrioxamine, nifedipine or allopurinol to the flush, but not by prostacyclin. Examination of lungs reperfused for shorter periods (5 and 15 min) identified three main types of damage to the vascular endothelium: (1) gross cell swelling, (2) detachment of intact endothelial cells from the underlying basal lamina, and (3) attenuation of cytoplasm due to blebbing. The results suggest that endothelial injury occurring on reperfusion is partly Fe2+ and Ca2(+)-mediated and that reactions catalysed by xanthine oxidase (which include oxygen free radical production) may also be important.

Topics: Allopurinol; Animals; Basement Membrane; Cryopreservation; Deferoxamine; Endothelium, Vascular; Epoprostenol; Lung; Lung Transplantation; Microscopy, Electron; Nifedipine; Organ Preservation; Rats; Rats, Inbred Strains; Reperfusion Injury; Time Factors; Verapamil

Prolonged ischemia decreases the chances for a successful pedicled or free tissue transfer as well as for major limb replantation. Skeletal muscle is especially sensitive to periods of prolonged ischemia and reperfusion. It is now hypothesized that tissue injury occurs during reperfusion due to the formation of toxic oxygen-free radicals. A replantation model, using the rabbit hind limb tibialis anterior muscle, was developed, to assess muscle function and histological appearance following ischemic intervals of five and eight hours. Muscle strength five weeks after injury was used as a functional measurement of tissue damage. The effects of the superoxide free radical scavenger superoxide dismutase (SOD), the hydroxide radical scavenger dimethylsulfoxide (DMSO) and the xanthine-oxidase inhibitor allopurinol (ALLO), administered systemically just before reperfusion, were studied.

Topics: Allopurinol; Animals; Dimethyl Sulfoxide; Free Radicals; Hindlimb; Microscopy, Electron; Muscle Contraction; Muscles; Pilot Projects; Premedication; Rabbits; Reperfusion Injury; Replantation; Superoxide Dismutase; Superoxides

The role of oxygen-derived free radicals and lipid peroxidation in the pathogenesis of acute gastric mucosal erosion was investigated in rat models produced by burn shock stress, by treatment with regional hyperthermia, platelet activating factor, and compound 48/80, and by ischemia-reperfusion. In all experimental models, the increase in the gastric erosions and in TBA reactants in the gastric mucosa were significantly inhibited by the treatment with superoxide dismutase (SOD) and/or catalase. Pretreatment with allopurinol, a competitive inhibitor of xanthine oxidase, prevented considerably the gastric injury (a) induced by burn shock, (b) produced by treatment with compound 48/80, and (c) caused by ischemia-reperfusion. By the treatment with anti-rat neutrophil antibody, the gastric mucosal injuries induced by regional hyperthermia, platelet activating factor, and compound 48/80 were significantly inhibited; however, burn shock and ischemia-reperfusion injuries were not inhibited. These results suggest that oxygen-free radical and lipid peroxidation contribute to the formation of gastric mucosal lesions, and that the sources of oxygen radicals seem to be different among these experimental models.

Topics: Allopurinol; Animals; Burns; Female; Free Radical Scavengers; Free Radicals; Gastric Mucosa; Hyperthermia, Induced; Lipid Peroxidation; Male; Oxygen; p-Methoxy-N-methylphenethylamine; Platelet Activating Factor; Rats; Reperfusion Injury; Shock, Traumatic

Topics: Allopurinol; Animals; Clinical Trials as Topic; Double-Blind Method; Free Radicals; Humans; Ischemia; Kidney Transplantation; Random Allocation; Renal Circulation; Reperfusion Injury; Superoxide Dismutase; Swine

Topics: Allopurinol; Animals; Dimethyl Sulfoxide; Diterpenes; Female; Free Radicals; Ginkgolides; Intestinal Diseases; Intestinal Mucosa; Ischemia; Lactones; Platelet Activating Factor; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase

Blood granulocyte-mediated reactions involving generation of oxygen-derived free radicals have recently been shown to be capable of causing injury to the lungs. These findings suggest a similar mechanism also to be involved in the development of pulmonary ischemia/reperfusion injury. In the present study, therefore, the effects of three oxygen-derived free radical scavengers, superoxide dismutase (SOD; 1 mg/kg), catalase (20,000 IU/kg) and allopurinol (45 mg/kg), were evaluated during reperfusion in a rabbit model after 2 h normothermic ischemia of the lung. During reperfusion, ischemic lungs were found to have an elevated pulmonary vascular resistance, increased total and extravascular lung water content, and decreased arterial oxygen tension (PaO2) compared to control animals. SOD and catalase, but not allopurinol, were able to reduce pulmonary injury by lowering the pulmonary vascular resistance, but could not prevent pulmonary damage as shown by total lung water (TLW) or PaO2. It is concluded that oxygen-derived free radicals such as hydrogen peroxide and the superoxide anion may play an important role in precipitating pulmonary injury after ischemia. The failure of xanthine oxidase inhibition (allopurinol) to exert protective effects may suggest that oxygen-derived free radical generation following pulmonary ischemia occurs predominantly via leukocyte-mediated reactions.

Topics: Allopurinol; Animals; Cardiac Output; Catalase; Extravascular Lung Water; Free Radical Scavengers; Free Radicals; Humans; Lung; Oxygen; Pulmonary Circulation; Rabbits; Reperfusion Injury; Superoxide Dismutase; Vascular Resistance

The role of allopurinol in the prevention of ischemia-reperfusion injury was assessed in a model of heart-lung transplantation. Fourteen swine were divided into two groups (seven donors and seven recipients). All heart and lung blocks were placed in hypothermic storage after perfusion with cold iso-osmolar cardioplegic solution and modified Collins solution, respectively (t = 8-10 degrees C for heart and t = 16-18 degrees C for lungs). The total ischemic time including the orthotopic transplantation was 6 h. Animals (donors and recipients) were pretreated with allopurinol given orally at a dosage of 50 mg/kg for 4 days. Animals were assessed by monitoring heart and lung function, including extravascular lung water at three time intervals, which included pretransplantation (donor), and 30 min and 2 h posttransplantation (recipient). Erythrocyte peroxidation susceptibility was assessed for 3 days, and surgery was performed on day 4. The malondialdehyde levels determined from erythrocyte exposure to in vitro peroxidative challenge classified three paired donor and recipient animals as responders and four paired donor and recipient animals as nonresponders to the allopurinol pretreatment. A persistent deterioration of lung function was observed over time in nonresponders (p less than .05) (increase of lung water, decrease of partial pressure of oxygen, increase in alveolar-arterial gradient, and decrease in arterial-alveolar tension ratio). Responders showed no significant alterations in lung function. This study in swine, a species devoid of myocardial xanthine oxidase activity, indicates that allopurinol may have a mechanism of action other than xanthine oxidase inhibition in the prevention of ischemia-reperfusion injury. The parallelism between protection of lung function and of red blood cells suggests the involvement of a generalized increase in tissue antioxidant capacity.

Topics: Allopurinol; Animals; Erythrocytes; Heart; Heart-Lung Transplantation; Lipid Peroxidation; Lung; Peroxides; Premedication; Reperfusion Injury; Swine; tert-Butylhydroperoxide; Vascular Resistance

Canine gastric dilatation-volvulus (GDV) is a naturally acquired condition of large-breed dogs primarily and is associated with high mortality. The clinical course suggests that reperfusion injury may be important in the pathogenesis of GDV. To evaluate the role of xanthine oxidase and iron-dependent lipid peroxidation (which are purported mechanisms of reperfusion injury) in the pathogenesis of GDV-related mortality, we created experimental GDV in 21 dogs. These dogs were then treated with either allopurinol (a xanthine oxidase inhibitor), U74006F (an experimental lipid peroxidation inhibitor), or saline solution (NaCl, 0.85%). Three of 8 dogs died in the allopurinol-treated group, none of 5 died in the U74006F-treated group, and 4 of 8 died in the saline solution-treated group. Tissue malondialdehyde concentration, a nonspecific indicator of lipid peroxidation, was significantly (P less than 0.05) greater in the duodenum, jejunum, colon, liver, and pancreas of the saline-solution treated and allopurinol-treated dogs than in the same tissues of the U74006F-treated dogs after surgical correction of the GDV (ie, during reperfusion), compared with malondialdehyde concentrations determined before inducing GDV. The results of this study support the concept that lipid peroxidation associated with reperfusion injury is important in the pathogenesis and high mortality of canine GDV. Furthermore, this lipid peroxidation and mortality may be preventable by appropriate and timely treatment.

Topics: Allopurinol; Animals; Dog Diseases; Dogs; Lipid Peroxides; Pregnatrienes; Reperfusion Injury; Stomach Volvulus

Since hydrogen peroxide (H2O2) can react with ferrous iron (FE++) to form the more toxic hydroxyl radical (OH) in vitro, and since H2O2 is generated brain xanthine oxidase (XO) during ischemia/reperfusion (I/R), we hypothesized that gerbils depleted of iron by dietary restriction or treated with iron chelators would be less susceptible to I/R injury. We found that gerbils fed a low iron diet for 8 weeks had decreased brain and serum iron levels, less neurologic deficits, and decreased brain edema after temporary unilateral carotid ligation (ischemia) and then reperfusion than gerbils fed a control standard iron diet. In addition, brains from gerbils treated with iron-free deferoxamine (an iron chelator), but not iron-loaded deferoxamine, had decreased (P less than .05) brain edema following ischemia and reperfusion. The results indicate that iron may contribute to cerebral ischemia/reperfusion damage.

Topics: Animals; Brain; Brain Chemistry; Brain Edema; Brain Ischemia; Deferoxamine; Female; Gerbillinae; Hematocrit; Iron; Iron Deficiencies; Male; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

The conversion rates of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) were compared with the time course of in vivo lipid peroxidation (LPO) in an ischemia-reperfusion model of acute renal failure in the rat. LPO, measured as the renal release of malondialdehyde (MDA), was found to be markedly elevated only during the first 5 min of blood reflow following a 45-min interval of ischemia (arteriovenous MDA difference -277.3 +/- 53.5 vs. 3.7 +/- 5.7 nmol/l in controls, n = 14). After 30 min of reperfusion, arteriovenous MDA differences nearly reached control values (9.7 +/- 31.8 nmol/l, n = 7). In contrast to enhanced LPO, no significant conversion of XDH to XO was found (XO activity in controls: 23 +/- 1% of XO plus XDH activity vs. 26 +/- 3% after 45 min of ischemia, n = 7). Therefore XO-derived superoxide anion radicals cannot be considered causative for LPO in the reperfusion interval of experimental ischemic acute renal failure.

Topics: Animals; Ischemia; Kidney; Lipid Peroxides; Male; Rats; Rats, Inbred Strains; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

We hypothesized that the pulmonary damage induced by smoke inhalation is the result of ischemic reperfusion injury. We determined the effect of allopurinol (xanthine oxidase inhibitor) on the pulmonary microvascular fluid flux in an ovine model after inhalation of cotton smoke (n = 13) and compared these data with those from untreated similarly smoke-injured (n = 7), as well as sham- (air, n = 9) smoked, animals and sheep given an equivalent dose of CO (n = 7). Smoke injury resulted in an increased lung lymph flow, lymph-to-plasma protein ratio, lung content of polymorphonuclear cells, and extravascular lung water (gravametric), in addition to histological evidence of tissue (pulmonary) edema and destruction. No significant difference was found in these variables between the sheep that were injured with smoke whether or not they were pretreated with allopurinol. The sham-smoked and CO-insufflated animals showed no significant changes in cardiopulmonary function or morphology. We conclude that there are few data to support a role of ischemic reperfusion injury in the pulmonary damage seen after smoke inhalation.

Topics: Allopurinol; Animals; Disease Models, Animal; Female; Lung; Lymph; Reperfusion Injury; Sheep; Sheep Diseases; Smoke Inhalation Injury; Xanthine Oxidase

We evaluated effluent blood from extremities of human patients undergoing reconstructive surgical treatment, which is routinely accompanied by upper-extremity exsanguination and application of a tourniquet, resulting in total interruption of arterial blood flow to one upper extremity. After tourniquet release (reperfusion), there were immediate increases in the plasma levels of xanthine oxidase activity, uric acid, and histamine in the ipsilateral limb and much smaller increases, if any, in levels of the same materials in plasma obtained from the contralateral extremity. There was no detectable xanthine dehydrogenase activity in plasma from either limb. Plasma also contained evidence of products consistent with the formation of oxygen-derived free radicals, namely, the appearance predominantly in the reperfused limb of hemoglobin and fluorescent compounds. These data indicate for the first time in humans that ischemia-reperfusion events are associated with the appearance of xanthine oxidase activity and its products in the plasma effluent.

Topics: Adult; Arm; Fluorescence; Free Radicals; Hemoglobins; Humans; Ischemia; Middle Aged; Reperfusion; Reperfusion Injury; Xanthine Oxidase

Topics: Adenosine Triphosphate; Allopurinol; Animals; Blood Pressure; Dogs; Heart Rate; Hypertonic Solutions; Hypotension; Kidney Transplantation; Organ Preservation; Renal Circulation; Reperfusion Injury; Time Factors; Transplantation, Autologous

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Disease Models, Animal; Histamine; Ischemia; L-Lactate Dehydrogenase; Liver Circulation; Male; Radioimmunoassay; Rats; Reperfusion Injury; Time Factors; Xanthine Oxidase

Skeletal muscle edema secondary to an increase in capillary permeability after reflow is an important cause of the compartment syndrome after acute arterial revascularization. The purpose of this study was to investigate the possible role of oxygen free radicals, generated at reperfusion, in the pathogenesis of the compartment syndrome secondary to acute arterial ischemia/reperfusion. A reproducible model of this syndrome was produced in anesthetized rabbits by femoral artery occlusion after surgical devascularization of collateral branches from the aorta to the popliteal artery. Increasing periods of ischemia from 6 to 12 hours, followed by 2 hours of reperfusion, were associated with corresponding increases in the anterior muscle compartment hydrostatic pressure and inversely proportional decreases in tibialis anterior muscle blood flow within that compartment as assessed by xenon 133 washout (n = 46) (r = -0.62, p less than 0.001). Anterior compartment pressure increased from 5 +/- 1 to 48 +/- 5 mm Hg (n = 46) (p less than 0.001) after 7 hours of total arterial ischemia and 2 hours of reperfusion. Ablation of free radicals generated from xanthine oxidase with either allopurinol (n = 8) or oxypurinol (n = 8), by scavenging the superoxide radical at reperfusion with superoxide dismutase (n = 8), or by blocking secondary hydroxyl radical formation with deferoxamine (n = 8) significantly ameliorated the rise in compartment pressure (p less than 0.05) in each case; it also significantly improved muscle perfusion in the superoxide dismutase-, allopurinol-, and deferoxamine-treated animals (p less than 0.05). These findings indicate that development of the compartment syndrome after acute arterial revascularization may be due, at least in part, to microvascular injury mediated by oxygen-derived free radicals generated from xanthine oxidase at reperfusion.

Topics: Allopurinol; Animals; Blood Pressure; Compartment Syndromes; Deferoxamine; Free Radicals; Ischemia; Oxygen; Oxypurinol; Rabbits; Regional Blood Flow; Reperfusion Injury; Superoxide Dismutase

The present studies show clearly that both dexamethasone and insulin can be omitted without altering the efficacy of UW. Adenosine and glutathione are both helpful additives, as is allopurinol. These findings suggest an important role of reperfusion injury after preservation, and confirm the benefits of adding pharmacological agents likely to reduce this injury. Cold ischemic damage was significantly ameliorated by UW solution in this stringent model of rat kidney preservation for 48 hr. A substantially simplified modification of UW solution has been shown to give equally effective kidney preservation, after removal of hydroxyethyl starch, dexamethasone, and insulin. Adenosine, glutathione, and allopurinol have been confirmed as helpful pharmacological additives. These findings have defined some of the mechanisms of effectiveness of UW solution and suggest avenues of further exploration to improve simple hypothermic storage and to prevent reperfusion injury.

Topics: Adenosine; Allopurinol; Animals; Female; Glutathione; Insulin; Kidney Diseases; Kidney Transplantation; Male; Organ Preservation Solutions; Raffinose; Rats; Rats, Inbred Strains; Reperfusion Injury; Solutions; Tissue Preservation

The major objective of this study was to develop an in vitro model of ischemia/reperfusion (I/R)-induced microvascular injury. Cultured venular endothelial cells were grown to confluency, labeled with 51Cr, and exposed to different durations of anoxia (0.5, 1, 2, 3, and 4 h). 51Cr release and cell detachment (indexes of cell injury) were determined at different times after reoxygenation (1, 2, 4, 6, 8, and 18 h). Because in vivo studies have implicated neutrophils in I/R injury, in some experiments human neutrophils were added to the endothelial cells upon reoxygenation. Periods of anoxia greater than or equal to 2 h resulted in 70-80% 51Cr release and 80-95% cell detachment upon reoxygenation. Under these conditions (near maximal injury), the addition of neutrophils produced negligible effects. Periods of anoxia less than or equal to 1 h resulted in 30-40% 51Cr release and 50-60% cell detachment. Under these conditions (moderate cell injury), addition of neutrophils enhanced endothelial cell injury. Using a 30-min period of anoxia, we also assessed the effects of superoxide dismutase (SOD; 300 U/ml) and allopurinol (20 microM) on anoxia/reoxygenation (A/R)-induced injury in the presence or absence of neutrophils. In the absence of neutrophils, SOD or allopurinol did not protect against A/R-induced injury. However, in the presence of neutrophils, both SOD and allopurinol attenuated the increases in 51Cr release. The results derived using this in vitro model of I/R injury are largely consistent with published in vivo studies. Thus this in vitro model may provide further insights regarding the mechanisms involved in I/R injury.

Topics: Allopurinol; Animals; Cattle; Coronary Vessels; Endothelium, Vascular; Humans; Hypoxia; In Vitro Techniques; Ischemia; Microcirculation; Neutrophils; Pancreatic Elastase; Reperfusion Injury; Superoxide Dismutase; Venules

The purpose of this study was to determine whether gamma-hydroxybutyrate provides protection against intestinal ischemia/reperfusion injury and to compare its effect with that of allopurinol and vitamin E. Thirty minutes of total regional ischemia, followed by 3 hours of reperfusion, produced intestinal damage that was completely prevented by gamma-hydroxybutyrate pretreatment. Naloxone partially blocked this protective effect. Allopurinol provided only partial protection against this injury, whereas vitamin E provided none. Treatment with gamma-hydroxybutyrate after ischemia but before reperfusion also provided significant protection. This study clearly demonstrates that gamma-hydroxybutyrate provides significant protection against intestinal ischemic injury and that it may do so via an opiate receptor-mediated mechanism.

Topics: Allopurinol; Animals; Cricetinae; Hydroxybutyrates; Intestines; Ischemia; Male; Mesocricetus; Naloxone; Reperfusion Injury; Sodium Oxybate; Vitamin E

Recently, oxygen free radicals have appeared to play a major role in injury after ischemia, especially that followed by normoxic reperfusion. To clarify the mechanisms of reperfusion injury, the activities of both oxygen radical producing enzymes and radical scavenging enzymes were measured in the ischemic rat kidney followed by reperfusion. All defensive enzymes activities significantly decreased; superoxide dismutase 2.15 +/- 0.14----1.71 +/- 0.11, catalase 186.6 +/- 12.7----116.5 +/- 7.1, glutathione peroxidase 30.0 +/- 2.6----19.1 +/- 2.9, glutathione reductase 118 +/- 5.1----39.9 +/- 6.8 (U/mg protein). Conversion from xanthine dehydrogenase to xanthine oxidase was only 12% of total activity, and all of them were reversible type oxidase. However, it was suggested by the electron spin resonance method that the tissue xanthine oxidase freed of superoxide dismutase could produce oxygen free radicals. In conclusion, reperfusion injury is caused not only by the increase of oxygen free radicals but by the destruction of scavenging systems.

Topics: Animals; Catalase; Free Radicals; Glutathione Peroxidase; Ischemia; Kidney; Oxygen; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

We have previously described a chronic, in vivo biochemical and histologic model of ischemia and reperfusion injury and wished to test the ability of superoxide dismutase, catalase and allopurinol to protect against the hepatocellular injury demonstrated by this model. Xanthine oxidase inhibitors given preoperatively produced a significant hepatocellular protective effect when compared with a comparable insult delivered to a cohort of control rats. The protection given seemed greater than that produced by pretreatment with free radical scavengers. Possible mechanisms for this observation are discussed. Investigations combining free radical scavengers with xanthine oxidase inhibitors may further define protection for warm hepatic ischemia and reperfusion injury.

Topics: Allopurinol; Animals; Catalase; Free Radicals; Ischemia; Liver; Male; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase

Topics: Allopurinol; Animals; Aspartate Aminotransferases; Coenzymes; Dose-Response Relationship, Drug; Free Radicals; Glutathione; Liver; Necrosis; Oxygen; Rats; Rats, Inbred Lew; Reperfusion Injury; Superoxide Dismutase; Temperature; Ubiquinone

The purposes of this study were to determine whether exercise training induces increases in skeletal muscle antioxidant enzymes and to further characterize the relationship between oxidative capacity and antioxidant enzyme levels in skeletal muscle. Male Sprague-Dawley rats were exercise trained (ET) on a treadmill 2 h/day at 32 m/min (8% incline) 5 days/wk or were cage confined (sedentary control, S) for 12 wk. In both S and ET rats, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were directly correlated with the percentages of oxidative fibers in the six skeletal muscle samples studied. Muscles of ET rats had increased oxidative capacity and increased GPX activity compared with the same muscles of S rats. However, SOD activities were not different between ET and S rats, but CAT activities were lower in skeletal muscles of ET rats than in S rats. Exposure to 60 min of ischemia and 60 min of reperfusion (I/R) resulted in decreased GPX and increased CAT activities but had little or no effect on SOD activities in muscles from both S and ET rats. The I/R-induced increase in CAT activity was greater in muscles of ET than in muscles of S rats. Xanthine oxidase (XO), xanthine dehydrogenase (XD), and XO + XD activities after I/R were not related to muscle oxidative capacity and were similar in muscles of ET and S rats. It is concluded that although antioxidant enzyme activities are related to skeletal muscle oxidative capacity, the effects of exercise training on antioxidant enzymes in skeletal muscle cannot be predicted by measured changes in oxidative capacity.

Topics: Animals; Catalase; Free Radicals; Glutathione Peroxidase; Male; Muscles; Oxygen; Physical Conditioning, Animal; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase; Xanthine Oxidase

Hyperthermia (39.5 degrees C) worsens experimental ischemic acute renal failure (ARF). We assessed whether it does so by affecting the ischemic and/or reperfusion injury phase and if its influence is mediated through changes in kidney ATP content and xanthine oxidase-mediated oxidant stress. Rats were subjected to 25 minutes of renal pedicle occlusion and hyperthermia was imposed during ischemia alone, reflow alone (0 to 30, 30 to 60, and 60 to 90 minutes), ischemia + reflow, or without ischemia. Hyperthermia's effects on ischemic/reperfusion adenylate pools lipid peroxidation (malondialdehyde), and the severity of ARF were assessed in comparison with normothermic ischemic controls. Hyperthermia confined to ischemia profoundly worsened ARF whereas during immediate reflow (0 to 30 minutes) hyperthermia had only a mild ARF-potentiating effect. During late reflow (greater than 30 minutes) or in the absence of ischemia, hyperthermia caused no damage. Hyperthermia had only a brief negative impact on ischemic ATP content, just slightly lowering it during the first 5 minutes of ischemia. Nevertheless, much greater ischemic damage resulted, reflected by increased proximal tubular brush border membrane sloughing at the end of vascular occlusion. Hyperthermia imposed only during reflow did not affect ATP concentrations. Hyperthermia increased end-ischemic purine base concentrations by 10% due to increased ATP degradation. However, reperfusion lipid peroxidation did not result and xanthine oxidase inhibition (by oxypurinol) conferred no protection.. (a) Hyperthermia worsens ARF predominantly by affecting ischemic, not reperfusion, injury; (b) xanthine oxidase is not an important mediator of hyperthermic-ischemic ARF; and (c) hyperthermia has a quantitatively trivial impact on ischemic ATP levels. This suggests that hyperthermia principally worsens ARF by magnifying the consequences of energy depletion (e.g., membrane damage) more than by worsening energy depletion, per se.

Topics: Acute Kidney Injury; Adenosine Triphosphate; Animals; Female; Hyperthermia, Induced; Malondialdehyde; Rats; Rats, Inbred Strains; Reperfusion Injury; Time Factors; Uremia; Xanthine Oxidase

Isolated Langendorff-perfused rat hearts after 10 minutes preperfusion, were subjected to a substrate-free anoxic perfusion (20 minutes) followed by 20 minutes reperfusion with a glucose-containing oxygen-balanced medium. Under the same perfusion conditions, the effect of exogenous 5mM fructose-1,6-bisphosphate has been investigated. The xanthine dehydrogenase to xanthine oxidase ratio, concentrations of high-energy phosphates and of TBA-reactive material (TBARS) were determined at the end of each perfusion period in both control and fructose-1,6-bisphosphate-treated hearts. Results indicate that anoxia induces the irreversible transformation of xanthine dehydrogenase into oxidase as a consequence of the sharp decrease of the myocardial energy metabolism. This finding is supported by the protective effect exerted by exogenous fructose-1,6-bisphosphate which is able to maintain the correct xanthine dehydrogenase/oxidase ratio by preventing the depletion of phosphorylated compounds during anoxia. Moreover, in control hearts, the release of lactate dehydrogenase during reperfusion, is paralleled by a 50% increase in the concentration of tissue TBARS. On the contrary, in fructose-1,6-bisphosphate-treated hearts this concentration does not significantly change after reoxygenation, while a slight but significant increase of lactate dehydrogenase activity in the perfusates is observed. On the whole these data indicate a direct contribution of oxygen-derived free radicals to the worsening of post-anoxic hearts. A hypothesis on the mechanism of action of fructose-1,6-bisphosphate in anoxic and reperfused rat heart and its possible application in the clinical therapy of myocardial infarction are presented.

Topics: Animals; Energy Metabolism; Free Radicals; Fructosediphosphates; Hexosediphosphates; Hypoxia; In Vitro Techniques; Lipid Peroxidation; Male; Oxygen; Rats; Rats, Inbred Strains; Reperfusion Injury; Xanthine Dehydrogenase; Xanthine Oxidase

Previous studies demonstrating protective effects of allopurinol in intestinal ischemia have evaluated intravenous allopurinol (presently unavailable for human use) or enteral allopurinol at supranormal doses and, therefore, have questionable clinical relevance. To address this problem, we evaluated the protective effects of clinically used doses of enteral allopurinol in rats with intestinal ischemia. Forty male Sprague-Dawley rats (weighing 300 to 400 g) received enteral allopurinol (10 mg/kg) or water daily for 1 week. Rats were then subjected to superior mesenteric artery occlusion with interruption of collateral flow for 20 minutes to produce ischemic injury to the intestine. Segmental small bowel resections were performed in 10 control rats and 10 allopurinol-treated rats before and after reperfusion to identify histopathologic evidence of reperfusion injury. Mucosal injury was quantitated using a grading scale of 0 to 5 (5 being most severe). The remaining 20 rats (10 in each group) were observed for mortality (death within 7 days) after reperfusion. Mucosal injury after reperfusion was graded at 4.4 +/- 0.20 in controls versus 2.3 +/- 0.23 in the treated group (P less than .001). In addition, there was a significant increase in mucosal damage in the control group when postreperfusion specimens were compared with specimens taken before reperfusion (2.8 +/- 0.19 before and 4.4 +/- 0.20 after reperfusion, P less than .001). Injury score for the allopurinol-treated group did not significantly increase after reperfusion. Survival was 50% in the water-fed control group compared with 100% survival in allopurinol-treated rats (P = .016). We conclude that enteral allopurinol in the presently available form and dose is effective in reducing mesenteric reperfusion injury.

Topics: Administration, Oral; Allopurinol; Animals; Intestinal Mucosa; Ischemia; Male; Mesentery; Rats; Rats, Inbred Strains; Reperfusion Injury; Splanchnic Circulation; Survival Rate; Time Factors

We have developed a model of reperfusion injury in Krebs buffer-perfused rabbit lungs, characterized by pulmonary vasoconstriction, microvascular injury, and marked lung edema formation. During reperfusion there was a threefold increase in lung superoxide anion (O2-) production, as measured by in vivo reduction of nitroblue tetrazolium, and a twofold increase in the release of O2- into lung perfusate, as measured by reduction of succinylated ferricytochrome c. Injury could be prevented by the xanthine oxidase inhibitor allopurinol, the O2- scavenger SOD, the hydrogen peroxide scavenger catalase, the iron chelator deferoxamine, or the thiols dimethylthiourea or N-acetylcysteine. The protective effect of SOD could be abolished by the anion channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, indicating that SOD consumes O2- in the extracellular medium, thereby creating a concentration gradient favorable for rapid diffusion of O2- out of cells. Our results extend information about the mechanisms of reperfusion lung injury that have been assembled by studies in other organs, and offer potential strategies for improved organ preservation, for treatment of reperfusion injury after pulmonary thromboembolectomy, and for explanation and therapy of many complications of pulmonary embolism.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Antioxidants; Cytochrome c Group; Disease Models, Animal; Free Radicals; Ion Channels; Lung Diseases; Male; Nitroblue Tetrazolium; Rabbits; Reperfusion Injury; Superoxide Dismutase; Superoxides; Xanthine Oxidase

Acute mesenteric ischemia is highly lethal and therefore a serious problem for surgery and intensive care medicine; accordingly its pathophysiology warrants further study. Oxygen free radicals (OFR) play a role in the intestinal mucosal damage that develops during reperfusion after ischemia. Histamine (H) is generally released in various types of tissue ischemia. The link between H release and OFR has only been studied in in vitro systems. We tested the hypothesis that OFR may be involved in H release following reperfusion of the ischemic gut. The artery supplying a segment of the ileum was occluded for 1 or 2 h in anesthetized dogs. On reperfusion, a release of H into the venous effluent of the segment was demonstrated. Pretreatment of the animals with allopurinol (an inhibitor of xanthine oxidase), or with MTDQ-DA [6,6'-methylene-bis(2,2-dimethyl-4-methanesulfonic acid sodium-1,2-dihydroquinoline)], a superoxide anion scavenger, or with a combination of allopurinol and MTDQ-DA resulted in an inhibition of H release. We conclude that OFR may play a role in the local H release following intestinal ischemia.

Topics: Allopurinol; Animals; Antioxidants; Dogs; Female; Free Radicals; Histamine; Histamine Release; Ileum; Ischemia; Male; Mesenteric Vascular Occlusion; Oxygen; Quinolines; Reperfusion Injury

Pulmonary hypoperfusion/ischemia-reperfusion (I/R) may initiate ARDS (nonhydrostatic pulmonary edema). Endothelial damage via xanthine oxidase (XO)-derived oxygen radicals (O2*) may mediate I/R injury. We previously documented Factor VIII antigen (F8) as a marker for endothelial injury. The purpose of this study was to (1) document I/R-induced nonhydrostatic pulmonary edema, (2) identify whether XO or O2* mediates nonhydrostatic edema, and (3) identify the site of injury (? endothelium). Rat lungs were isolated, ventilated, and perfused (100 min, control, or 40 min at 37 degrees C, I (static vent.), + 60 min, R). Effluent was analyzed for F8 release (ELISA: data relative to control). Tungsten-fed rats had negligible lung XO vs rats fed standard diet (3.6 vs 34.5 mU/g, (P less than 0.05). Catalase (CAT) 50 micrograms/ml) was added to perfusate prior to R. Sectioned lungs were fluorescein anti-F8 photographed (IF) and qualitatively assessed. (Table: see text). We conclude that (1) pulmonary hypoperfusion (I/R) leads to nonhydrostatic pulmonary edema, and (2) the edema results in part from XO-generated O2* directed at the capillary endothelium.

Topics: Animals; Antigens; Capillary Permeability; Catalase; Endothelium, Vascular; Factor VIII; Free Radicals; Lung; Male; Organ Size; Oxygen; Pulmonary Edema; Rats; Rats, Inbred Strains; Reperfusion Injury; Tungsten; von Willebrand Factor; Xanthine Oxidase

In this study the role of free radicals, lipid peroxidation, and neutrophil infiltration as mediators of ischemia and reperfusion-induced intestinal mucosal damage were investigated. We used a rat experimental model in which a ligated loop of the distal ileum was subjected to ischemia and reperfusion and the ensuing mucosal damage was assessed by means of lysosomal enzyme release and intestinal permeability measurements. We also determined the mucosal content of malondialdehyde, a lipid peroxidation product, and the mucosal activity of myeloperoxidase, a neutrophil granulocyte marker. Ischemia and revascularization alone caused increased mucosal permeability to sodium fluorescein, increased N-acetyl-beta-glucosaminidase release from the mucosa into the lumen, increased malondialdehyde content in the mucosa, and increased myeloperoxidase activity in the mucosa. Intravenous injection of enzymatic antioxidant, superoxide dismutase, together with xanthine oxidase inhibitor, allopurinol, prevented the malondialdehyde accumulation and caused attenuation of all the other effects of ischemia. Intravenous pretreatment of hydrocortisone sodium succinate (Solu-Cortef), a steroid and also a nonenzymatic antioxidant, prevented not only malondialdehyde accumulation but also neutrophil infiltration and mucosal damage. These data support a concept that neutrophil infiltration is an important element in ischemic mucosal damage. In addition, the blocking of this phenomenon may have clinical significance in attempts to modulate the potential damaging effects of the increased neutrophil infiltration associated with small-intestinal ischemia.

Topics: Acetylglucosaminidase; Allopurinol; Animals; Disease Models, Animal; Fluoresceins; Free Radicals; Hydrocortisone; Intestinal Absorption; Intestinal Mucosa; Ischemia; Lipid Peroxidation; Malondialdehyde; Neutrophils; Oxygen; Peroxidase; Rats; Reperfusion Injury; Superoxide Dismutase

Topics: Allopurinol; Animals; Dogs; Free Radicals; Intestinal Mucosa; Intestine, Small; Microvilli; Reperfusion Injury; Sodium-Potassium-Exchanging ATPase

The purpose of this study was to evaluate the possible involvement of xanthine and xanthine oxidase in reperfusion injury in a low-flow, reflow model of liver perfusion. Livers were perfused at flow rates around 25% of normal for 90 min and then at normal flow rates (4 ml/g/min) for 30 min. When flow was restored to normal, malondialdehyde and lactic dehydrogenase (LDH) were released into the effluent perfusate. Malondialdehyde production rapidly reached values of 300 nmol/g/hr whereas LDH increased from basal levels of 100 to 600 U/l upon reperfusion. Trypan blue was taken up exclusively in cells in pericentral regions of the liver lobule under these conditions. Xanthine and hypoxanthine in the effluent perfusate increased steadily during the low-flow period reaching values around 5 and 10 microM, respectively, and decreased rapidly after the flow was restored to normal. Perfusion with nitrogen-saturated buffer for 3 min before restoration of normal flow rates or infusion of the radical scavenger catechin (400 microM) reduced cell damage by about 50%. Infusion of allopurinol (2-6 mM), an inhibitor of xanthine oxidase, prevented reperfusion injury in a dose-dependent manner. Taken together, these data indicate that a reperfusion injury occurs in liver upon reintroduction of oxygen which is initiated by oxidation of xanthine and hypoxanthine via xanthine oxidase and ultimately leads to production of lipid peroxides. Surprisingly, infusion of xanthine (4 mM), substrate for xanthine oxidase, reduced hepatocellular injury on reperfusion. LDH release was decreased from values around 700 to less than 200 U/l and trypan blue uptake in pericentral region was prevented totally by xanthine.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Female; Hypoxanthine; Hypoxanthines; L-Lactate Dehydrogenase; Liver Circulation; Malondialdehyde; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxides; Uric Acid; Xanthine; Xanthine Oxidase; Xanthines

Free radicals in the small intestine were quantified by using an electron spin resonance spectrometer, and the amounts of TBA (thiobarbituric acid) reactants in arterial plasma, portal venous plasma and intestinal tissue were determined at the several stages. The effects of allopurinol, alpha-tocopherol, the simultaneous occlusion of superior mesenteric artery or the porto-jugular venous bypass, with the temporary occlusion of the portal vein, were also investigated. 1) Free radical concentration (mostly, semiquinones of CoQ and/or flavin in mitochondria) decreased with portal vein occlusion but showed a temporary increase at 10 sec after reperfusion. Allopurinol suppressed such temporary increase. 2) TBA reactants increased with the temporary occlusion of the portal vein. TBA reactants decreased during the portal vein occlusion with alpha-tocopherol and during reperfusion with allopurinol. Lipid peroxidation in the small intestine was also diminished by using the methods of simultaneous occlusion of the superior mesenteric artery or the porto-jugular venous bypass. In conclusion, there may be three sources for the generation of superoxide: the xanthine oxidase system, semiquinone radicals and paramagnetic metal irons. They may induce lipid a peroxidation, which accelerates the injury on the small intestine, in acute portal vein occlusion and the following restoration of portal vein flow in rats.

Topics: Allopurinol; Animals; Constriction; Electron Spin Resonance Spectroscopy; Free Radicals; Intestine, Small; Lipid Peroxidation; Male; Portal Vein; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxides; Thiobarbiturates; Vitamin E

During the last few years, the scientific field has focused its attention on the pathogenic role of free radicals in the process of ischemia-revascularization. It is a well-known fact that xanthine oxidase is an important source of tissular free radicals. Bearing this in mind, we designed an experimental protocol to analyse the effect of allopurinol (a xanthine oxidase inhibitor) in the survival of rats after the occlusion of the superior mesenteric artery during a period of 90 minutes and its action on the superoxide radical liberation. The concentration of oxipurinol and allopurinol in the ischemic area (intestine), liver and blood were measured. We concluded that the administration of allopurinol increased the survival rate, which is correlated to higher concentrations of allopurinol and oxipurinol in the inner part of the intestinal cells. A correlation between the survival rate and superoxide radicals was not found.

Topics: Acute Disease; Allopurinol; Animals; Female; Free Radicals; Intestines; Ischemia; Male; Oxypurinol; Prognosis; Purines; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxides; Xanthine Oxidase

Oxygen-derived free radicals have been implicated as possible mediators in the development of tissue injury induced by ischemia and reperfusion. Clamping of the celiac artery in rats reduced the gastric mucosal blood flow to 10% of that measured before the clamping. The area of gastric erosions and thiobarbituric acid (TBA) reactants in gastric mucosa were significantly increased 60 and 90 min after clamping. These changes were inhibited by treatment with SOD and catalase. Thirty and 60 min after reoxygenation. produced by removal of the clamps following 30 min of ischemia, gastric mucosal injury and the increase in TBA reactants were markedly aggravated compared with those induced by ischemia alone. SOD and catalase significantly inhibited these changes. The serum alpha-tocopherol/cholesterol ratio, an index of in vivo lipid peroxidation, was significantly decreased after long periods of ischemia (60 and 90 min), or after 30 and 60 min of reperfusion following 30 min of ischemia. These results indicated that active oxygen species and lipid peroxidation may play a role in the pathogenesis of gastric mucosal injury induced by both ischemia alone and ischemia-reperfusion. Although, allopurinol inhibited the formation of gastric mucosal injury and the increase in TBA reactants in gastric mucosa, the depletion of polymorphonuclear leukocytes (PMN) counts induced by an injection of anti-rat PMN antibody did not inhibit these changes. As compared with the hypoxanthine-xanthine oxidase system. PMN seem to play a relatively small part in the formation of gastric mucosal injury induced by ischemia-reperfusion.

Topics: Allopurinol; Animals; Catalase; Cholesterol; Free Radicals; Gastric Mucosa; Ischemia; Lipid Peroxidation; Male; Neutrophils; Oxygen; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase; Vitamin E; Xanthine Oxidase

In the feline intestine studies have implicated superoxide (O.-) and other oxygen derived free radicals as initiators of injury as measured by increased capillary permeability during the reperfusion period. Biochemical mechanisms of this free radical generation include: xanthine oxidase dependent O.- production, hydrogen peroxide (H2O2) formation by superoxide dismutase (SOD), hydroxyl radical (OH-) production via the Haber-Weiss reaction, and lipid radical formation from membrane peroxidation. Pathological consequences of these events include inflammatory neutrophil infiltration, damage to the collagen and mucosal basement membrane, increased capillary permeability, edema, cell degeneration and necrosis. Animal models of neonatal necrotizing enterocolitis (NNEC) indicate that intestinal injury occurs after the etiologic factors (hypothermia, hypoxia) are removed. In order to determine the role of active oxygen species in the pathogenesis of NNEC, weanling hamsters and neonatal piglets were cold stressed and activities of pro/antioxidant enzymes were determined, and histopathologic and ultrastructural studies were performed. Cold stressed weanling hamsters showed a 55.7% (P less than 0.05) decrease in xanthine dehydrogenase/xanthine oxidase activity ratio. Light microscopy revealed scattered colonic mucosal erosions and submucosal edema in 50% of cold stressed animals. Transmission electron microscopy demonstrated degeneration of colonic mucosal epithelial cells, enlarged intracellular spaces, cytoplasmic vacuolization, and nuclear membrane swelling. The colonic serosa was also edematous and infiltrated with bacteria. Large intestinal tissue from cold stressed neonatal piglets showed a significant increase (P less than 0.05) in Mn and Cu, Zn, SOD, CAT, GSH-Red, total GSH, and Glc6-PD at 0 and 12 hrs. post stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Animals, Newborn; Cricetinae; Enterocolitis, Pseudomembranous; Free Radicals; Intestines; Oxygen; Reperfusion Injury; Xanthine Oxidase

A model of ischaemia and reperfusion was established which consistently achieved a 50% reduction in viable length of random pattern pedicle skin flaps in pigs. Flaps were subjected to two periods of ischaemia and reperfusion. Superoxide dismutase (SOD) in a dose of 3000 mu/kg given intravenously and allopurinol 70 mg/kg/day orally had no effect on viable skin flap length, skin blood flow or histological characteristics of flaps. SOD polymers, which have been used in other species to delay SOD clearance, were found to cause anaphylactoid reactions in pigs.

Topics: Allopurinol; Animals; Disease Models, Animal; Male; Polymers; Radioisotopes; Reperfusion Injury; Skin; Superoxide Dismutase; Surgical Flaps; Swine; Time Factors

The protective actions of allopurinol in ischemic/reperfusion injury seem critically determined by the drug pretreatment regimen and may involve generalized alterations in tissue antioxidant status. In the present study, 12 male swine to be used as donors and recipients in a heart-lung transplantation study were treated with allopurinol given orally at a dose of 50 mg/kg for 4 days prior to surgery. Red cells from allopurinol-treated animals showed a progressive decrease in susceptibility to in vitro peroxidative challenge. Although the degree and time-course of protection showed some degree of interanimal variation, maximal effects were obtained in most animals after 2-3 days. The extent of red cell protection in both donor and recipient animals correlated significantly with the functional viability of the transplanted lung, as assessed by tissue water content. It is suggested that the susceptibility of red cells to in vitro oxidative damage may provide a useful functional assessment of generalized alterations in antioxidant status produced by pharmacological interventions.

Topics: Allopurinol; Animals; Erythrocytes; Heart Transplantation; Lipid Peroxidation; Lung Transplantation; Male; Malondialdehyde; Reperfusion Injury; Swine

Preserved organs are damaged not only by the ischemic injury due to lack of oxygen. The reperfusion injury mediated by oxygen free radicals is an important factor in the postischemic organ failure. The prevention of free radical-induced reperfusion injury with allopurinol (AP) and superoxide dismutase (SOD) is shown in a warm ischemia kidney model. Rats were treated with allopurinol (40 mg/kg i.v.) one hour, or with SOD (20,000 IU/kg i.v.) one minute before reperfusion after a period of 35 minutes of warm ischemia. Allopurinol and SOD reduced significantly the postischemic kidney failure with a less important increase of creatinine. Creatinine levels on day three in the control group: 517 +/- 87 mumol/ml, in the SOD-group: 206 +/- 105 mumol/ml, and in the AP-group: 163 +/- 81 mumol/ml (anal. of variance: p = 0.0001). AP has a wide therapeutic range. We feel, that it is important to confirm the prevention of reperfusion injury by allopurinol prophylaxis clinically.

Topics: Allopurinol; Animals; Creatinine; Kidney Transplantation; Male; Organ Preservation; Premedication; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase

Postischemic renal dysfunction (PIRD) is characterized by a reduction in glomerular filtration and tubular reabsorption of solute. The relative contribution of oxygen free radicals (OFRs) generated during reperfusion remains unclear. This study characterized the renal response to OFRs--independent of an ischemic insult. Isolated rat kidneys were perfused at 37 degrees C and 90-100 mm Hg with a modified Krebs' buffer. Hypoxanthine (25 mumole) and xanthine oxidase (1 unit) were combined and infused proximal to the kidney. There was a 50% increase in vascular resistance. This was accompanied by a 30% reduction in perfusate flow rate and a 70% reduction in glomerular filtration rate. There was also a significant reduction in urine flow rate and oxygen consumption. The percentage reabsorption of filtered water and sodium by the renal tubules was not diminished, however. This pattern was not observed when the xanthine oxidase was inactivated or when the perfusate was pretreated with superoxide dismutase (250 units/ml) and catalase (500 units/ml). The generation of OFRs, independent of an ischemic insult, causes a decrease in glomerular filtration out of proportion to the decrease in renal flow similar to that observed with PIRD. OFRs may contribute to the hemodynamic and glomerular alterations seen with PIRD. Factors other than OFRs, probably associated with ischemia, must be responsible for the tubular dysfunction.

Topics: Animals; Free Radicals; Glomerular Filtration Rate; Hypoxanthine; Hypoxanthines; Kidney; Male; Rats; Rats, Inbred Strains; Renal Circulation; Reperfusion Injury; Superoxide Dismutase; Urination; Vascular Resistance; Xanthine Oxidase

The effectiveness of superoxide dismutase (SOD), catalase (CAT), dimethyl sulphoxide (DMSO) and allopurinol in prevention of peritoneal adhesion formation induced by complete vascular obstruction and reperfusion of an ileal segment was investigated in rats. The ischaemic period was 30 min. Group A (n = 20) were controls, group B (n = 15) received SOD 15,000 U/kg i.v. and group C (n = 17) the same dose of CAT immediately before induction of ischaemia. In group D (n = 20) DMSO 20 mg/kg was given i.v. 5 min before ischaemia, and group E (n = 20) received allopurinol orally 50 mg/kg daily for 2 days and also 2 hours before ischaemia. Ten days later adhesions had developed in 80% of group A, 40% of group B, 47% of group C and 45% of groups D and E (p less than 0.05). The severity of the adhesions was significantly less in the pretreated groups than in the controls. Oxygen-derived free radicals may be pathogenetically important for such adhesion formation. Xanthine oxidase is the principal source of oxygen radicals after a 30-min period of complete regional intestinal ischaemia.

Topics: Allopurinol; Animals; Catalase; Dimethyl Sulfoxide; Drug Evaluation; Female; Free Radicals; Ileum; Peritoneal Diseases; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase; Tissue Adhesions

The study describes regional changes of xanthine oxidase and succinate dehydrogenase activities as shown by the ischemic and reperfused small intestine of the rat. The results are obtained with enzyme histochemical methods, including densitometrical verifications, and are substantiated with biochemical enzyme determinations. The decrease of xanthine oxidase activity was best visible in the anoxic duodenum and jejunum, where the findings of histochemical enzyme determinations agreed with those achieved biochemically. The activities of succinate dehydrogenase as measured densitometrically may serve as a further control, considering also the typical intracellular distribution of the reaction products.

Topics: Animals; Densitometry; Histocytochemistry; Intestinal Mucosa; Intestine, Small; Ischemia; Male; Rats; Rats, Inbred Strains; Reperfusion Injury; Succinate Dehydrogenase; Xanthine Oxidase

Rabbit kidneys were subjected to 120 min of warm ischaemia or to 120 min of warm ischaemia followed by 60 min reperfusion with blood in vivo before being removed, homogenised and incubated at 37 degrees C for 90 min. Lipid extracts were obtained and monitored for Schiff base (fluorescence emission 400-450 nm, excited at 360 nm), thiobarbituric acid (TBA)-reactive material (emission 553 nm, excited at 515 nm) and diene conjugates (absorbance at 237 nm). Samples removed before incubation were assayed for reduced glutathione (GSH) and oxidised glutathione (GSSG) to provide an index of glutathione redox activity (GSH:GSSG). Allopurinol injected systemically i.v. (a) 15 mins before kidneys were clamped, (b) 15 mins before they were reperfused or (c) as two injections (before clamping and before reperfusion) significantly inhibited these biochemical markers of lipid peroxidation. Administration before reperfusion had a markedly more pronounced effect than when allopurinol was given before warm ischaemia only. It is concluded that allopurinol is probably effective because of its ability to inhibit xanthine oxidase and consequently lipid peroxidation during reperfusion rather than by preventing loss of purine nucleotides from hypoxic cells during ischaemia.

Topics: Allopurinol; Animals; Body Temperature; Free Radicals; Glutathione; Hot Temperature; Kidney; Lipid Peroxidation; Oxidation-Reduction; Rabbits; Reperfusion Injury

We developed a quantitative histologic method for assessing injury in the rat retina due to transient ischemia. We used this technique to test the effectiveness of local hypothermia and allopurinol, an inhibitor of oxygen-free radical formation, in reducing ischemia/reperfusion injury in the rat retina. Retinal ischemia and reperfusion was produced by transient ligation of the optic nerve. Histologic evaluation by a masked observer was based on the average count of nonpyknotic nuclei in the inner nuclear layer of the retina from eight high power fields (X100) in one 5 microns thick sagital section at or near the optic nerve. A sharp increase in tissue damage occurs between 90 and 120 min of ischemia. Ischemia for periods of 60 and 90 min produced mild damage while periods of 120 and 240 min produced severe damage. Hypothermia protected the retina significantly from 120 min of ischemic injury (P less than 0.001 student t-test, compared to 120 min control), while allopurinol had no protective effect.

Topics: Allopurinol; Animals; Hypothermia, Induced; Rats; Rats, Inbred Strains; Reperfusion Injury; Retinal Diseases; Retinal Vessels; Time Factors

This study was performed to clarify the relationship between hemodynamics, congestive damage, lipid peroxidation and intraluminal hemorrhage of the small intestine. Using 51Cr-red blood cells, with a temporary occlusion of the portal vein for 30 min. in rats, the hemodynamic, biochemical and histological changes were investigated. By occluding the portal vein, its pressure increased to a level eight times higher than normal, and destruction of the intestinal mucosa and an increase of TBA reactants were observed. The intraluminal hemorrhage of the intestine increased to a quantity 7.5 times higher than usual during portal vein occlusion, and it decreased gradually after reperfusion. At 120 min. after reperfusion, this amount remained high, but the administration of Allopurinol diminished its level. A technique of temporary simultaneous occlusion of the superior mesenteric artery or the bypass between the portal and jugular veins was effective in reducing the congestive damage on the intestine. During the occlusion of the portal vein, sudden and high pressure of the portal vein primarily causes congestive damage, and superoxide generated by a xanthine oxidase system during reperfusion may cause lipid peroxidation which induces reperfusion injury. Thus, the lipid peroxidation may accelerate the injury on the small intestine.

Topics: Allopurinol; Animals; Gastrointestinal Hemorrhage; Hemodynamics; Intestine, Small; Lipid Peroxidation; Male; Portal Vein; Rats; Rats, Inbred Strains; Reperfusion Injury

We examined the basis of reperfusion-induced pulmonary edema produced by pulmonary artery occlusion and subsequent reperfusion. After a 24-h period of occlusion of a rabbit pulmonary artery followed by a 2-h period of reperfusion, the lungs were removed from the animal and perfused with a 0.5 g% Ringer's-albumin solution. An increase in lung weight was observed within 60 min compared with control lungs (i.e., lungs subjected to pulmonary arterial occlusion but not reperfusion) (p less than 0.05). Shorter periods of occlusion (6 or 12 h) did not result in edema, which suggests that a period of ischemia was required for the reperfusion-induced pulmonary edema. The extravascular lung water content also increased in the contralateral lung (i.e., the lung not subjected to pulmonary arterial occlusion and reperfusion). The capillary filtration coefficient increased in reperfused lungs compared with controls (p less than 0.05), indicating an increase in lung vascular permeability following reperfusion. Infusion of allopurinol (a xanthine oxidase inhibitor) and superoxide dismutase during the reperfusion period prevented the increases in lung weight and vascular permeability; infusion of catalase was ineffective. We conclude that pulmonary reperfusion following pulmonary artery occlusion increases pulmonary vascular permeability, which is mediated by the generation of oxidants.

Topics: Animals; Blood Pressure; Catheterization; Endothelium, Vascular; Female; Lung; Male; Organ Size; Pulmonary Artery; Pulmonary Edema; Pulmonary Wedge Pressure; Rabbits; Reperfusion Injury; Superoxide Dismutase; Xanthine Dehydrogenase; Xanthine Oxidase

Oxygen-free radicals have been implicated as mediators of ischemic damage in a number of tissues, including heart, kidney, small intestine, and skin. Superoxide dismutase, a free radical scavenger, and allopurinol, an inhibitor of xanthine oxidase (a catalyst in the formation of superoxide) have been shown separately to decrease ischemic damage to tissue. Sixty-two male Sprague-Dawley rats (220 to 280 g) were divided into five groups: control, superoxide dismutase only, allopurinol only, high-dose combined allopurinol and superoxide dismutase, and low-dose combined allopurinol and superoxide dismutase. An 18-cm2 ventral island skin flap, based on a single inferior epigastric vessel, was raised and replaced. Blood flow was assessed with a perfusion fluorometer immediately after the flap was replaced, and on postoperative days 1 and 3. Gross necrosis was assessed on postoperative day 7. Gross necrosis was reliably reduced in all groups as compared with the control; however, necrosis in any one experimental group was not significantly different from any other experimental group. Necrosis (expressed as a percentage of the area of the random [distal] half of the flap) was as follows for each group: control, 74%; superoxide dismutase, 43%; allopurinol, 43%; high-dose combined, 48%; low-dose combined, 33%. Blood flow, as represented by the dermofluorescence index, was not changed by any of the treatments. Blood flow was also related to the eventual survival or necrosis for any one portion of tissue. All experimental groups survived with significantly less blood flow than the control.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Allopurinol; Animals; Male; Necrosis; Rats; Rats, Inbred Strains; Regional Blood Flow; Reperfusion Injury; Skin; Superoxide Dismutase; Surgical Flaps

Methylene blue interacts with xanthine oxidase at the iron-sulfide site in the electron pathway (Scheme I) that is known to serve as an electron-sink connecting the reductive and oxidative sites in both the oxidase and dehydrogenase forms. Thus, shunting of electrons to methylene blue at this site effectively diverts their flow away from the FAD site where molecular oxygen is converted to superoxide radicals. Since the electron affinity constants of xanthine oxidase for electron acceptors are FAD greater than iron/sulfide greater than molybdenum, methylene blue falls between the FAD and iron-sulfide site. Thus, methylene blue effectively inhibits superoxide and hydroxyl radical production while accelerating the conversion of xanthine to uric acid. As methylene blue is already approved for medicinal use in humans and is relatively nontoxic, the drug may have a role in reducing tissue injury associated with reperfusion. We are currently investigating this possibility in animal models.

Topics: Animals; Cattle; Cytochrome c Group; Female; Free Radicals; Hydroxides; Hydroxyl Radical; Kinetics; Liver; Methylene Blue; Milk; Rats; Reperfusion Injury; Superoxides; Xanthine Oxidase

Total injury in ischemic skeletal muscle is a function of ischemic damage and reperfusion injury. In an attempt to decrease reperfusion injury, we gave the oxygen-derived free radical scavengers allopurinol, superoxide dismutase, or mannitol during reperfusion of canine gracilis muscle made ischemic for 4 hours. We measured muscle O2 consumption (MVO2), and tissue calcium, water, and adenosine triphosphatase (ATP) before ischemia, after ischemia, and at 5 minutes and 60 minutes of reperfusion. The results at 60 minutes showed no improvement in MVO2 or ATP. In fact, ATP was significantly depressed with allopurinol and superoxide dismutase treatment, and tissue edema did not decrease in any of the groups. We conclude that the simple addition of oxygen-derived free radical scavengers during the initial reperfusion of totally ischemic skeletal muscle does not attenuate reperfusion injury.

Topics: Adenosine Triphosphate; Allopurinol; Animals; Body Water; Calcium; Dogs; Female; Free Radicals; Ischemia; Male; Mannitol; Muscles; Oxygen Consumption; Reperfusion Injury; Superoxide Dismutase

In an isolated, normothermic rat heart model (Langendorff, 37 degrees C), dimethylthiourea (DMTU) infusion only during reperfusion reduced both injury and measurable hydrogen peroxide (H2O2) concentrations after global ischemia. Cardiac function was assessed by measurement of ventricular developed pressure (DP). H2O2 was assessed using H2O2 dependent aminotriazole inactivation of myocardial catalase. Depletion of xanthine oxidase by two methods (tungsten or allopurinol inhibition) also improved recovery of function and H2O2 production. The results indicate that XO derived H2O2 contributes to myocardial reperfusion injury.

Topics: Amitrole; Animals; Catalase; Coronary Circulation; Heart; Hydrogen Peroxide; In Vitro Techniques; Myocardium; Rats; Reperfusion Injury; Thiourea; Xanthine Oxidase

Topics: Acute Disease; Allopurinol; Animals; Deferoxamine; Drug Combinations; Free Radicals; Intestinal Diseases; Rats; Rats, Inbred Strains; Reperfusion Injury; Trifluoperazine

Topics: Allopurinol; Animals; Infusions, Intra-Arterial; Kidney; Kidney Tubular Necrosis, Acute; Male; Rats; Rats, Inbred Strains; Reperfusion Injury; Superoxide Dismutase

Reactive oxygen species generated by xanthine oxidase during reperfusion of ischemic liver might in part be responsible for ischemic organ injury. Therefore, the effect of allopurinol, an inhibitor of xanthine oxidase, on the oxidant stress associated with reperfusion and on hepatic function 24 h after ischemia was assessed in a model of partial hepatic ischemia in rats. The increase in circulating glutathione disulfide (GSSG) was used as an index of oxidant stress. Hepatic function was assessed using a breath test to quantitative the demethylation of aminopyrine in vivo. In control animals the plasma concentration of GSSG 1 h after onset of reperfusion increased from 0.9 mumol/l in sham-operated controls to 4.2, 5.5, and 8.0 mumol/l following 45, 90 and 120 min of ischemia, respectively. The percent of the administered dose of (dimethylamine-14C)-aminopyrine appearing in breath as 14CO2 was not significantly different from sham-operated controls (40.2%) 24 h after 45 min of ischemia (34.1%), but decreased progressively to 26.0% (p less than 0.05) and 20.6% (p less than 0.05) after 90 and 120 min of ischemia, respectively. Allopurinol, administered at a dose of 50 mg/kg 18 h and 1 h prior to ischemia, did not prevent the rise in plasma GSSG, did not alleviate the release of transaminases, and did not improve the demethylation of aminopyrine 24 h after ischemia, suggesting that reactive oxygen species generated by xanthine oxidase during reperfusion of ischemic liver do not contribute significantly to ischemic injury.

Topics: Allopurinol; Animals; Free Radicals; Glutathione; Glutathione Disulfide; Liver; Male; Rats; Rats, Inbred Strains; Reperfusion Injury; Xanthine Oxidase

Topics: Allopurinol; Animals; Creatine Kinase; Deferoxamine; Disease Models, Animal; Heart Arrest; Ischemia; Rats; Reperfusion Injury; Resuscitation

Based on work from our laboratory and studies by others, we propose the following hypothesis to explain the interaction among xanthine oxidase, PMNs, and tissue injury in the postischemic small intestine (Figure 2). During the ischemic period, ATP is catabolized to yield hypoxanthine. The hypoxic stress also triggers the conversion of NAD-reducing xanthine dehydrogenase to the oxygen radical-producing xanthine oxidase via a protease. When the intestine is reperfused, molecular oxygen is reintroduced into the tissue where it reacts with hypoxanthine and xanthine oxidase to produce a burst of superoxide anion and hydrogen peroxide. In the presence of ferric iron, superoxide anion and hydrogen peroxide react via the Haber-Weiss reaction to form hydroxyl radicals. This highly reactive and cytoxic free radical then initiates lipid peroxidation of cell membrane components and the subsequent release of substances that activate, attract, and promote the adherence of PMN to microvascular endothelium. The adherent PMN then causes further endothelial cell injury via the release of superoxide and various proteases.

Topics: Animals; Chemotaxis, Leukocyte; Free Radicals; Intestine, Small; Ischemia; Microcirculation; Neutrophils; Reperfusion Injury; Xanthine Oxidase

Topics: Allopurinol; Animals; Female; Free Radicals; Gastric Mucosa; Ischemia; Necrosis; Platelet Activating Factor; Rats; Rats, Inbred Strains; Reference Values; Reperfusion Injury; Stomach; Superoxide Dismutase