3-nitrotyrosine and Reperfusion-Injury

The formation of peroxynitrite and nitrotyrosine was examined in a variety of in vitro and in vivo animal models and its relation to cell or tissue damage was examined. polymorphonuclear leukocyte (PMN)-induced injury to cardiac myocytes endothelial cells, activated PMN produced peroxynitrite. Peroxynitrite appears to be responsible for the injury but it was not a major mediator of endothelial cell injury. In the experiment of ischemia-reperfusion injury of the rat brain nitrotyrosine was formed in the peri-infarct and core-of infarct regions. The degradation curve of nitrotyrosine revealed that its t(1/2) was about 2.2 hours. In the radiation-induced lung injury of rats, nitrotyrosine was also formed but it was not the sole mechanism for the injury. Levels of nitrotyrosine correlated with the severity of myocardial dysfunction in the canine model of cytokine-induced cardiac injury. Inhibition of NO generation abolished the formation of peroxynitrite and nitrotyrosine in all experiments. In conclusion; although nitrotyrosine is formed in a variety of pathological conditions where the generation of NO is increased, its presence does not always correlate with the severity of injury.

Topics: Animals; Brain Ischemia; Cell Survival; Cells, Cultured; Coculture Techniques; Disease; Dogs; Endothelium, Vascular; Lung Diseases; Male; Myocardium; Neutrophils; Radiation Injuries; Rats; Reperfusion Injury; Tyrosine

It has been reported that hyperglycemia following hypoglycemia produces an ischemia-reperfusion-like effect in type 1 diabetes. In this study the possibility that GLP-1 has a protective effect on this phenomenon has been tested.. 15 type 1 diabetic patients underwent to five experiments: a period of two hours of hypoglycemia followed by two hours of normo-glycemia or hyperglycemia with the concomitant infusion of GLP-1 or vitamin C or both. At baseline, after 2 and 4 hours, glycemia, plasma nitrotyrosine, plasma 8-iso prostaglandin F2alpha, sCAM-1a, IL-6 and flow mediated vasodilation were measured.. After 2 h of hypoglycemia, flow mediated vasodilation significantly decreased, while sICAM-1, 8-iso-PGF2a, nitrotyrosine and IL-6 significantly increased. While recovering with normoglycemia was accompanied by a significant improvement of endothelial dysfunction, oxidative stress and inflammation, a period of hyperglycemia after hypoglycemia worsens all these parameters. These effects were counterbalanced by GLP-1 and better by vitamin C, while the simultaneous infusion of both almost completely abolished the effect of hyperglycemia post hypoglycemia.. This study shows that GLP-1 infusion, during induced hyperglycemia post hypoglycemia, reduces the generation of oxidative stress and inflammation, improving the endothelial dysfunction, in type 1 diabetes. Furthermore, the data support that vitamin C and GLP-1 may have an additive protective effect in such condition.

Topics: Adult; Antioxidants; Ascorbic Acid; Biomarkers; Blood Glucose; Diabetes Mellitus, Type 1; Dinoprost; Female; Glucagon-Like Peptide 1; Humans; Hyperglycemia; Hypoglycemia; Hypoglycemic Agents; Inflammation; Inflammation Mediators; Infusions, Parenteral; Intercellular Adhesion Molecule-1; Interleukin-6; Male; Oxidative Stress; Reperfusion Injury; Time Factors; Treatment Outcome; Tyrosine; Vasodilation; Young Adult

The prophylactic administration of inhaled nitric oxide (NO) during reperfusion after lung transplantation has been shown to reduce neutrophil-induced injury in animal models. There remain questions regarding efficacy in the clinical setting and concerns regarding increased free radical injury. We sought to assess the efficacy of NO in reducing neutrophil infiltration and associated injury if administered from the very onset of reperfusion in clinical lung transplantation.. Twenty bilateral sequential lung transplant recipients were randomized to receive 20-ppm inhaled NO (NO group) or a standard anesthetic gas mixture (control group) from the onset of ventilation. Bronchoalveolar lavage was performed immediately prior to implantation and after 30 minutes of reperfusion and analyzed for inflammatory cytokine levels and free radical surrogates. Primary graft dysfunction (PGD) scoring was performed prospectively for 72 hours post-transplant.. The prophylactic administration of NO during the first 30 minutes of reperfusion had no statistically significant effect on the development of Grade II to III PGD (5 of 10 in NO group and 7 of 10 in control group, p = 0.36) or gas exchange (area under the curve: 429 +/- 296 vs 336 +/- 306; p = 0.64) in the NO and control groups, respectively. Pulmonary neutrophil sequestration, as measured by the transpulmonary arteriovenous neutrophil difference, was not influenced by the administration of NO. Prophylactic NO did not significantly alter the concentration of interleukin-8, myeloperoxidase or nitrotyrosine during transplantation.. This study could not demonstrate a significant effect of inhaled NO during the first 30 minutes of reperfusion in the prevention of neutrophil injury and primary graft dysfunction after lung transplantation.

Topics: Administration, Inhalation; Adult; Bronchoalveolar Lavage Fluid; Female; Free Radical Scavengers; Free Radicals; Graft Rejection; Humans; Interleukin-8; Lung; Lung Transplantation; Male; Middle Aged; Neutrophils; Nitric Oxide; Peroxidase; Reperfusion Injury; Tyrosine

The liver endothelium plays a key role in the progression and resolution of liver diseases in young and adult individuals. However, its role in older people remains unknown. We have herein evaluated the importance of the sinusoidal endothelium in the pathophysiology of acute liver injury, and investigated the applicability of simvastatin, in aged animals. Eighteen-months-old male Wistar rats underwent 60 minutes of partial warm ischemia followed by 2 hours of reperfusion (WIR). A group of aged rats received simvastatin for 3 days before WIR. Endothelial phenotype, parenchymal injury, oxidative and nitrosative stress, and fenestrae dynamics were analyzed. The effects of WIR and simvastatin were investigated in primary LSEC from aged animals. The results of this study demonstrated that WIR significantly damages the liver endothelium and its effects are markedly worse in old animals. WIR-aged livers exhibited reduced vasodilation and sinusoidal capillarization, associated with liver damage and cellular stress. Simvastatin prevented the detrimental effects of WIR in aged livers. In conclusion, the liver sinusoidal endothelium of old animals is highly vulnerable to acute insult, thus targeted protection is especially relevant in preventing liver damage. Simvastatin represents a useful therapeutic strategy in aging.

Topics: Age Factors; Animals; Disease Models, Animal; Endothelial Cells; Liver; Male; Nitric Oxide; Phenotype; Rats; Rats, Wistar; Reperfusion Injury; Simvastatin; Tyrosine

Ischaemia reperfusion (IR) injury occurs during vascular graft harvesting and implantation during vascular/cardiac surgery. Elevated intracellular cyclic guanosine monophosphate (cGMP) levels contribute to an effective endothelial protection in different pathophysiological conditions. The hypothesis that the phosphodiesterase-5 inhibitor vardenafil would protect vascular grafts against IR injury by upregulating the nitric oxide-cGMP pathway in the vessel wall of the bypass graft was investigated.. Lewis rats (n = 6-7/group) were divided into Group 1, control; Group 2, donor rats received intravenous saline; Group 3, received intravenous vardenafil (30 μg/kg) 2 h before explantation. Whereas aortic arches of Group 1 were immediately mounted in an organ bath, aortic segments of Groups 2 and 3 were stored for 2 h in saline and transplanted into the abdominal aorta of the recipient. Two hours after transplantation, the implanted grafts were harvested. Endothelium dependent and independent vasorelaxations were investigated. TUNEL, CD-31, ICAM-1, VCAM-1, α-SMA, nitrotyrosine, dihydroethidium and cGMP immunochemistry were also performed.. The results support the view that impairment of intracellular cGMP signalling plays a role in the pathogenesis of the endothelial dysfunction of an arterial graft after bypass surgery, which can effectively be prevented by vardenafil. Its clinical use as preconditioning drug could be a novel approach in vascular/cardiac surgery.

Topics: Actins; Animals; Aorta, Thoracic; Cold Ischemia; Cyclic GMP; Cytoprotection; DNA Damage; Intercellular Adhesion Molecule-1; Male; Nitrosative Stress; Phosphodiesterase 5 Inhibitors; Platelet Endothelial Cell Adhesion Molecule-1; Rats, Inbred Lew; Reperfusion Injury; Signal Transduction; Tissue and Organ Harvesting; Tyrosine; Vardenafil Dihydrochloride; Vascular Cell Adhesion Molecule-1; Vascular System Injuries; Vasodilator Agents; Warm Ischemia

Topics: Animals; Anthraquinones; Apoptosis; Brain; Brain Ischemia; Caspase 3; Disease Models, Animal; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Nitric Oxide; Nitrosation; Oxidative Stress; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Tyrosine

Our objective was to determine whether controlled reflow on one side and/or the other side after bilateral carotid occlusion release could reduce cell death in focal ischemic P14 rats. Arterial blood flow was measured using ultrasonography. Cell death, inflammation and nitrotyrosine were measured using immunofluorescence. When reflow was first induced in the contralateral side, we observed improved outcome markers compared with those when reflow was first induced in the ipsilateral side and/or simultaneous reflow was induced in both sides. Our data suggest that progressive rerouting of arterial flow through the circle of Willis toward the ischemic site reduced cell death.

Topics: Animals; Brain Ischemia; Cell Death; Cerebral Arteries; Cerebrovascular Circulation; Cyclooxygenase 2; Disease Models, Animal; Prostaglandin-E Synthases; Rats, Wistar; Reperfusion; Reperfusion Injury; Treatment Outcome; Tyrosine; Ultrasonography

Stroke is a major health issue in women. Our previous studies in male rats showed decreased myogenic tone in middle cerebral arteries (MCAs) after ischemia and reperfusion (I/R), while tone in parenchymal arterioles (PAs) was increased. This vascular response may aggravate stroke damage in males by limiting reperfusion; however, the effect in females is not known. The current study investigated the effect of I/R and tissue plasminogen activator (tPA) on myogenic tone and reactivity of MCAs and PAs in female rats. Nitrosative stress by peroxynitrite and recruitment of inflammatory neutrophils to the microvasculature were also studied. Female rats were subjected to 2-h MCA filament occlusion (n = 16) or sham surgery (n = 17) and given tPA (1 mg/kg, i.v) or vehicle followed by 30-min reperfusion. Myogenic tone and reactivity were measured in isolated and pressurized MCAs and PAs from the same animals. Cerebrovascular F-actin, 3-nitrotyrosine (3-NT, peroxynitrite marker), and intravascular neutrophils were quantified. Myogenic tone and constriction to the nitric oxide synthase inhibitor Nω-nitro-L-arginine were decreased in MCAs but unchanged in PAs after I/R with no effect of tPA. F-actin and 3-NT expression were unaffected by I/R or tPA. Our study showed that MCAs from females, similar to what has been seen in males, are dilated after I/R and have decreased myogenic tone while tone in PAs was unchanged. Increased small vessel resistance may contribute to decreased reperfusion and worse outcome after stroke.

Topics: Actins; Amides; Animals; Cerebrovascular Circulation; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Fibrinolytic Agents; Infarction, Middle Cerebral Artery; Middle Cerebral Artery; Muscle, Smooth; Neutrophils; Ovariectomy; Pyridines; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury; Tissue Plasminogen Activator; Tyrosine

Ischemia/reperfusion and the resulting oxidative/nitrative stress impair cerebral myogenic tone via actin depolymerization. While it is known that NADPH oxidase (Nox) family is a major source of vascular oxidative stress; the extent and mechanisms by which Nox activation contributes to actin depolymerization, and equally important, the relative role of Nox isoforms in this response is not clear.. To determine the role of Nox4 in hypoxia-mediated actin depolymerization and myogenic-tone impairment in cerebral vascular smooth muscle.. Control and Nox4 deficient (siRNA knock-down) human brain vascular smooth muscle cells (HBVSMC) were exposed to 30-min hypoxia/45-min reoxygenation. Nox2, Nox4, inducible and neuronal nitric oxide synthase (iNOS and nNOS) and nitrotyrosine levels as well as F:G actin were determined. Myogenic-tone was measured using pressurized arteriography in middle cerebral artery isolated from rats subjected to sham, 30-min ischemia/45-min reperfusion or ex-vivo oxygen glucose deprivation in the presence and absence of Nox inhibitors.. Nox4 and iNOS expression were significantly upregulated following hypoxia or ischemia/reperfusion. Hypoxia augmented nitrotyrosine levels while reducing F actin. These effects were nullified by inhibiting nitration with epicatechin or pharmacological or molecular inhibition of Nox4. Ischemia/reperfusion impaired myogenic-tone, which was restored by the selective inhibition of Nox4.. Nox4 activation in VSMCs contributes to actin depolymerization after hypoxia, which could be the underlying mechanism for myogenic-tone impairment following ischemia/reperfusion.

Topics: Actin Cytoskeleton; Actins; Animals; Cells, Cultured; Humans; Hypoxia; Membrane Glycoproteins; Middle Cerebral Artery; Muscle, Smooth, Vascular; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidases; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Rats; Reperfusion Injury; RNA, Small Interfering; Tyrosine; Up-Regulation

The aim of this study was to investigate the efficacy of PEA+silymarin as a combination treatment in a mouse model of renal I/R and to verify whether PEA+silymarin could exert more potent effects compared to the single substances even if administered at lower doses. Mice were subjected to bilateral renal artery occlusion (30min) and reperfusion (6h) and received intraperitoneally silymarin (100, 30 and 10mg/kg) or PEA (1mg/kg) or PEA (1mg/kg)+silymarin (10mg/kg) 15min before release of clamps. Specific indicators of renal dysfunction, tubular injury, myeloperoxidase activity and malondialdehyde levels were measured. The nuclear factor κB pathway and apoptotic mechanisms were also investigated. The treatment with silymarin reduced kidney dysfunction, histological damage, neutrophil infiltration and oxidative stress in a dose dependent manner. Furthermore, PEA+silymarin showed a significant potentiated effect. Therefore, NF-κB and apoptosis pathways were also significantly inhibited. Our results clearly demonstrate that PEA+silymarin treatment attenuated the degree of renal inflammation.

Topics: Active Transport, Cell Nucleus; Amides; Animals; Apoptosis; Cell Nucleus; Chymases; Cytokines; Disease Models, Animal; Disease Progression; Drug Interactions; Ethanolamines; Gene Expression Regulation; Kidney; Male; Mice; Nitrates; Nitrites; Oxidative Stress; Palmitic Acids; Reperfusion Injury; Risk Factors; Silymarin; Transcription Factor RelA; Tyrosine

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

The cellular prion protein (PrPC), a protein most noted for its link to prion diseases, has been found to play a protective role in ischemic brain injury. To investigate the role of PrPC in the kidney, an organ highly prone to ischemia/reperfusion (IR) injury, we examined wild-type (WT) and PrPC knockout (KO) mice that were subjected to 30-min of renal ischemia followed by 1, 2, or 3 days of reperfusion. Renal dysfunction and structural damage was more severe in KO than in WT mice. While PrP was undetectable in KO kidneys, Western blotting revealed an increase in PrP in IR-injured WT kidneys compared to sham-treated kidneys. Compared to WT, KO kidneys exhibited increases in oxidative stress markers heme oxygenase-1, nitrotyrosine, and Nε-(carboxymethyl)lysine, and decreases in mitochondrial complexes I and III. Notably, phosphorylated extracellular signal-regulated kinase (pERK) staining was predominantly observed in tubular cells from KO mice following 2 days of reperfusion, a time at which significant differences in renal dysfunction, histological changes, oxidative stress, and mitochondrial complexes between WT and KO mice were observed. Our study provides the first evidence that PrPC may play a protective role in renal IR injury, likely through its effects on mitochondria and ERK signaling pathways.

Topics: Animals; Extracellular Signal-Regulated MAP Kinases; Heme Oxygenase-1; Kidney; Kidney Diseases; Mice; Mice, Knockout; Mitochondria; Oxidative Stress; Prions; Reperfusion Injury; Tyrosine

The small G protein RhoA and its downstream effector Rho-kinase play an important role in various physiopathological processes including ischemia/reperfusion (I/R) injury. Reactive oxygen and nitrogen species produced by iNOS and NADPH oxidase are important mediators of inflammation and organ injury following an initial localized I/R event. The aim of this study was to determine whether RhoA/Rho-kinase signaling pathway increases the expression and activity of MEK1, ERK1/2, iNOS, gp91(phox), and p47(phox), and peroxynitrite formation which result in oxidative/nitrosative stress and inflammation leading to hindlimb I/R-induced injury in kidney as a distant organ and gastrocnemius muscle as a target organ. I/R-induced distant and target organ injury was performed by using the rat hindlimb tourniquet model. I/R caused an increase in the expression and/or activity of RhoA, MEK1, ERK1/2, iNOS, gp91(phox), p47(phox), and 3-nitrotyrosine and nitrotyrosine levels in the tissues. Although Rho-kinase activity was increased by I/R in the kidney, its activity was decreased in the muscle. Serum and tissue MDA levels and MPO activity were increased following I/R. I/R also caused an increase in SOD and catalase activities associated with decreased GSH levels in the tissues. Y-27632, a selective Rho-kinase inhibitor, (100µg/kg, i.p.; 1h before reperfusion) prevented the I/R-induced changes except Rho-kinase activity in the muscle. These results suggest that activation of RhoA/Rho-kinase/MEK1/ERK1/2/iNOS pathway associated with oxidative/nitrosative stress and inflammation contributes to hindlimb I/R-induced distant organ injury in rats. It also seems that hindlimb I/R induces target organ injury via upregulation of RhoA/MEK1/ERK1/2/iNOS pathway associated with decreased Rho-kinase activity.

Topics: Amides; Animals; Catalase; Glutathione; Inflammation; Kidney; Male; Malondialdehyde; MAP Kinase Kinase 1; MAP Kinase Signaling System; Muscle, Skeletal; NADPH Oxidases; Nitric Oxide Synthase Type II; Oxidative Stress; Peroxidase; Peroxynitrous Acid; Pyridines; Rats; Rats, Wistar; Reperfusion Injury; rho-Associated Kinases; rhoA GTP-Binding Protein; Superoxide Dismutase; Tyrosine

Topics: Biomarkers; Case-Control Studies; End Stage Liver Disease; Humans; Liver Transplantation; Nitroso Compounds; Oxidative Stress; Reactive Nitrogen Species; Reperfusion Injury; Risk Factors; Serum Albumin; Serum Albumin, Human; Time Factors; Treatment Outcome; Tyrosine

To verify the effects of N-acetylcysteine (NAC) administered before and after ischaemia in an animal model of lung ischaemia-reperfusion (IR) injury.. Twenty-four Wistar rats were subjected to an experimental model of selective left pulmonary hilar clamping for 45 min followed by 2 h of reperfusion. The animals were divided into four groups: control group (SHAM), ischaemia-reperfusion, N-acetylcysteine-preischaemia (NAC-Pre) and NAC-postischaemia (NAC-Post). We recorded the haemodynamic parameters, blood gas analysis and histology. We measured the thiobarbituric acid reactive substances concentration; the expression of superoxide dismutase (SOD), inducible nitric oxide synthase (iNOS), nitrotyrosine, cleaved caspase 3, nuclear factor κB (NF-κB), NF-kappa-B inhibitor alpha (IκB-α), tumour necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β); myeloperoxidase activity (MPO).. No significant differences were observed in the haemodynamic parameters, blood gas analysis and SOD activity among the groups. Lipid peroxidation was significantly higher in the IR and NAC-Pre groups (P < 0.01). The expression of nitrotyrosine, cleaved caspase 3, NF-κB, IκB-α, TNF-α and IL-1β were significantly higher in the IR group when compared with the SHAM and NAC groups (P < 0.01). The NAC-Pre group showed a significantly higher expression of these proteins when compared with the SHAM and NAC-Post groups (P < 0.05). After reperfusion, the expression of iNOS increased almost uniformly in all groups when compared with the SHAM group (P < 0.01). The histological analysis showed fewer inflammatory cells in the NAC groups.. The intravenous administration of NAC demonstrated protective properties against lung IR injury. The use of NAC immediately after reperfusion potentiates its protective effects.

Topics: Acetylcysteine; Administration, Intravenous; Animals; Anti-Inflammatory Agents; Antioxidants; Caspase 3; Cytoprotection; Disease Models, Animal; Hemodynamics; I-kappa B Kinase; Inflammation Mediators; Interleukin-1beta; Lipid Peroxidation; Lung; Lung Injury; NF-kappa B; Nitric Oxide Synthase Type II; Oxidative Stress; Peroxidase; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine

Hericium erinaceus, an edible mushroom, has been demonstrated to potentiate the effects of numerous biological activities. The aim of this study was to investigate whether H. erinaceus mycelium could act as an anti-inflammatory agent to bring about neuroprotection using a model of global ischemic stroke and the mechanisms involved. Rats were treated with H. erinaceus mycelium and its isolated diterpenoid derivative, erinacine A, after ischemia reperfusion brain injuries caused by the occlusion of the two common carotid arteries. The production of inflammatory cytokines in serum and the infracted volume of the brain were measured. The proteins from the stroke animal model (SAM) were evaluated to determine the effect of H. erinaceus mycelium. H. erinaceus mycelium reduced the total infarcted volumes by 22% and 44% at a concentration of 50 and 300 mg/kg, respectively, compared to the SAM group. The levels of acute inflammatory cytokines, including interleukin-1β, interleukin-6 and tumor necrosis factor á, were all reduced by erinacine A. Levels of nitrotyrosine-containing proteins, phosphorylation of p38 MAPK and CCAAT enhancer-binding protein (C/EBP) and homologous protein (CHOP) expression were attenuated by erinacine A. Moreover, the modulation of ischemia injury factors present in the SAM model by erinacine A seemed to result in the suppression of reactive nitrogen species and the downregulation of inducible NO synthase (iNOS), p38 MAPK and CHOP. These findings confirm the nerve-growth properties of Hericium erinaceus mycelium, which include the prevention of ischemic injury to neurons; this protective effect seems to be involved in the in vivo activity of iNOS, p38 MAPK and CHOP.

Topics: Animals; Basidiomycota; Brain Ischemia; CCAAT-Enhancer-Binding Proteins; Cell Death; Cytokines; Diterpenes; Male; Mycelium; Neurons; Neuroprotective Agents; Nitric Oxide Synthase Type II; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Transcription Factor CHOP; Tyrosine

The present study was designed to investigate the potential role of miR-23a-3p in experimental brain ischemia-reperfusion injury. Cerebral ischemia reperfusion was induced by transient middle cerebral artery occlusion (MCAO) for 1h in C57/BL6 mice. And miR-23a-3p angomir was transfected to upregulate the miR-23a-3p level. Our results showed that miR-23a-3p levels were transiently increased at 4h after reperfusion in the peri-infarction area, while markedly increased in the infarction core at reperfusion 4h and 24h. Importantly, in vivo study demonstrated that miR-23a-3p angomir treatment through intracerebroventricular injection markedly decreased cerebral infarction volume after MCAO. Simultaneously, miR-23a-3p reduced peroxidative production nitric oxide (NO) and 3-nitrotyrosine (3-NT), and increased the expression of manganese superoxide dismutase (MnSOD). In vitro study demonstrated that miR-23a-3p decreased hydrogen peroxide (H2O2)-induced lactate dehydrogenase (LDH) leakage dose-dependently, and reduced protein levels of activated caspase-3 in neuro-2a cells. In addition, miR-23a-3p reduced H2O2-induced production of NO and 3-NT dose-dependently, and reversed the decreased activity of total SOD and MnSOD in neuro-2a cells. Our study indicated that miR-23a-3p suppressed oxidative stress and lessened cerebral ischemia-reperfusion injury.

Topics: Animals; Brain; Brain Ischemia; Caspase 3; Cell Line; Disease Models, Animal; Hydrogen Peroxide; Infarction, Middle Cerebral Artery; L-Lactate Dehydrogenase; Male; Mice, Inbred C57BL; MicroRNAs; Nitric Oxide; Oxidative Stress; Reperfusion Injury; Superoxide Dismutase; Transfection; Tyrosine; Up-Regulation

Re-canalization of cerebral vessels in ischemic stroke is pivotal to rescue dysfunctional brain areas that are exposed to moderate hypoxia within the penumbra from irreversible cell death. Goal of the present study was to evaluate the effect of moderate hypoxia followed by reoxygenation (MHR) on the evolution of reactive oxygen species (ROS) and blood-brain barrier (BBB) integrity in brain endothelial cells (BEC). BBB integrity was assessed in BEC in vitro and in microvessels of the guinea pig whole brain in situ preparation. Probes were exposed to MHR (2 hours 67-70 mmHg O2, 3 hours reoxygenation, BEC) or towards occlusion of the arteria cerebri media (MCAO) with or without subsequent reperfusion in the whole brain preparation. In vitro BBB integrity was evaluated using trans-endothelial electrical resistance (TEER) and transwell permeability assays. ROS in BEC were evaluated using 2',7'-dichlorodihydrofluorescein diacetate (DCF), MitoSox and immunostaining for nitrotyrosine. Tight-junction protein (TJ) integrity in BEC, stainings for nitrotyrosine and FITC-albumin extravasation in the guinea pig brain preparation were assessed by confocal microscopy. Diphenyleneiodonium (DPI) was used to investigate NADPH oxidase dependent ROS evolution and its effect on BBB parameters in BEC. MHR impaired TJ proteins zonula occludens 1 (ZO-1) and claudin 5 (Cl5), decreased TEER, and significantly increased cytosolic ROS in BEC. These events were blocked by the NADPH oxidase inhibitor DPI. MCAO with or without subsequent reoxygenation resulted in extravasation of FITC-albumin and ROS generation in the penumbra region of the guinea pig brain preparation and confirmed BBB damage. BEC integrity may be impaired through ROS in MHR on the level of TJ and the BBB is also functionally impaired in moderate hypoxic conditions followed by reperfusion in a complex guinea pig brain preparation. These findings suggest that the BBB is susceptible towards MHR and that ROS play a key role in this process.

Topics: Animals; Blood-Brain Barrier; Cell Membrane; Claudin-5; Endothelial Cells; Guinea Pigs; Hypoxia; Microvessels; Mitochondria; Oxidative Stress; Permeability; Reactive Oxygen Species; Reperfusion Injury; Tight Junction Proteins; Tight Junctions; Tyrosine; Zonula Occludens-1 Protein

Ischemic preconditioning (IPC) strongly protects against ischemia/reperfusion (I/R) injury; however, the molecular mechanism involved in delayed preconditioning-induced endothelial protection in peripheral arteries is unknown. Therefore, we examined using functional, morphologic and molecular biologic studies whether delayed IPC decreases formation of reactive oxygen species and upregulates endothelial nitric oxide synthase (eNOS) that in turn contributes to vascular endothelial protection. Adult male Sprague-Dawley rats were subjected to 30-min ischemia induced by mesenteric artery occlusion followed by 60-min reperfusion 24 h after sham surgery or preconditioning (three cycles of 5-min ischemia/5-min reperfusion). Delayed preconditioning prevented the I/R-induced impairment of endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 91.4±2.2%; I/R 54.0±4.0%; IPC 80.2±6.3%). This protective effect was abolished by NOS inhibitor N(G)-nitro-L-arginine methyl ester and not changed by ascorbic acid. Electron microscopy showed marked endothelial damage after I/R and the ultrastructural changes were prevented by delayed preconditioning. Following I/R, the impairment of eNOS phosphorylation and expression was observed in mesenteric vessels. Furthermore, phosphatidylinositol 3-kinase (PI3K) and Akt phosphorylation were reduced, although total PI3K and Akt remained unchanged. IPC restored I/R-induced impairment of eNOS expression and activity. This was possibly the result of the recovery of PI3K/Akt phosphorylation. Furthermore, I/R increased serum level of malondialdehyde, intravascular superoxide and nitrotyrosine generation, which were abrogated by IPC. These results suggest that delayed preconditioning prevented I/R-induced endothelial injury in peripheral resistance vasculature, both in terms of functional and structural changes. Endothelial protection afforded by delayed IPC is associated with inhibition of oxidative stress and upregulation of PI3K/Akt/eNOS pathway.

Topics: Acetylcholine; Animals; Blotting, Western; DNA Primers; Endothelial Cells; Fluorescent Antibody Technique; Immunohistochemistry; Ischemic Preconditioning; Male; Malondialdehyde; Microscopy, Electron, Transmission; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type III; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinase; Phosphorylation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Tyrosine

Nitric oxide (NO) seems to play an important role during renal ischemia/reperfusion (I/R) injury. We investigated whether rutin inhibits inducible nitric oxide synthase (iNOS) and reduces 3-nitrotyrosine (3-NT) formation in the kidneys of rats during I/R.. Wistar albino rats were nephrectomized unilaterally and, 2 weeks later, subjected to 45 minutes of left renal pedicle occlusion followed by 3 hours of reperfusion. We intraperitoneally administered L-N6-(1-iminoethyl)lysine (L-NIL; 3 mg/kg) for 30 minutes or rutin (1 g/kg) for 60 minutes before I/R. After reperfusion, kidney samples were taken for immunohistochemical analysis of iNOS and 3-NT. We measured plasma nitrite/nitrate and cyclic guanosine monophosphate (cGMP) to evaluate NO levels.. Ischemia/reperfusion caused plasma cGMP to increase significantly. Similarly, plasma nitrite/nitrate was elevated in the I/R group compared with the control group. Histochemical staining was positive for iNOS and 3-NT in the I/R group. Pretreatment with L-NIL or rutin significantly mitigated the elevation of plasma cGMP and nitrite/nitrate. These changes in biochemical parameters were also associated with changes in immunohistochemical appearance. Pretreatment with L-NIL or rutin significantly decreased the incidence and severity of iNOS and 3-NT formation in the kidney tissues.. Our findings suggest that high activity of iNOS causes renal I/R injury, and that rutin exerts protective effects, probably by inhibiting iNOS.

Topics: Animals; Biomarkers; Cyclic GMP; Enzyme Inhibitors; Immunohistochemistry; Infusions, Parenteral; Kidney; Lysine; Male; Nephrectomy; Nitrates; Nitric Oxide Synthase Type II; Nitrites; Protective Agents; Rats; Rats, Wistar; Reperfusion Injury; Rutin; Tyrosine

Excessive generation of superoxide and mitochondrial dysfunction has been described as being important events during ischemia-reperfusion (I/R) injury. Our laboratory has demonstrated that manganese superoxide dismutase (MnSOD), a major mitochondrial antioxidant that eliminates superoxide, is inactivated during renal transplantation and renal I/R and precedes development of renal failure. We hypothesized that MnSOD knockdown in the kidney augments renal damage during renal I/R. Using newly characterized kidney-specific MnSOD knockout (KO) mice the extent of renal damage and oxidant production after I/R was evaluated. These KO mice (without I/R) exhibited low expression and activity of MnSOD in the distal nephrons, had altered renal morphology, increased oxidant production, but surprisingly showed no alteration in renal function. After I/R the MnSOD KO mice showed similar levels of injury to the distal nephrons when compared with wild-type mice. Moreover, renal function, MnSOD activity, and tubular cell death were not significantly altered between the two genotypes after I/R. Interestingly, MnSOD KO alone increased autophagosome formation, mitochondrial biogenesis, and DNA replication/repair within the distal nephrons. These findings suggest that the chronic oxidative stress as a result of MnSOD knockdown induced multiple coordinated cell survival signals including autophagy and mitochondrial biogenesis, which protected the kidney against the acute oxidative stress following I/R.

Topics: Animals; Autophagy; Cell Proliferation; DNA Repair; Kidney; Mice; Mice, Knockout; Mitochondrial Turnover; Models, Animal; Nephrons; Oxidative Stress; Reperfusion Injury; Superoxide Dismutase; Tyrosine

The role of the tumor necrosis factor (TNF)-α in the pathophysiology of renal ischemia/reperfusion (I/R) injury is unclear. We investigate the effects of TNF-αR1 gene deletion and infliximab administration on the degree of renal injury induced by I/R. TNF-αR1 knockout (TNF-αR1KO) and wild-type (TNF-αWT) mice were subjected to bilateral renal artery occlusion (30min) and reperfusion (24h). Infliximab (10mg/kg subcutaneously, s.c.) was administered 1h before ischemia. At the end of experiments, urea, creatinine, γGT, and AST were measured to assess renal function and reperfusion injury. Markers of oxidative stress, pro-inflammatory mediators, iNOS, COX-2, and NF-κB signaling pathway were measured. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured to study polymorphonuclear cell infiltration and lipid peroxidation. TNF-αR1 gene deletion and infliximab administration prevented the increase of urea, creatinine, γGT, kidney AST levels, iNOS and COX-2 expression, NF-κB translocation, MPO activity and MDA levels. TNF-αR1 gene deletion and infliximab administration lowered the histological evidence of renal damage associated with I/R and caused a reduction of nitrotyrosine suggesting reduced nitrosative stress. Our results demonstrate that TNF-α plays an important role in I/R injury and put forward the hypothesis that modulation of TNF-α expression may represent a novel and possible strategy.

Topics: Acute Kidney Injury; Animals; Antibodies, Monoclonal; Apoptosis; Biomarkers; Cell Adhesion Molecules; Gene Deletion; Gene Expression Regulation; Gene Knockout Techniques; Inflammation; Infliximab; Interleukin-1beta; Kidney; Lipid Peroxidation; Male; Mice; Neutrophil Infiltration; Oxidative Stress; Receptors, Tumor Necrosis Factor, Type I; Reperfusion Injury; Tumor Necrosis Factor-alpha; Tyrosine

Lung ischaemia-reperfusion induces nitric oxide synthesis and reactive nitrogen species, decreasing nitric oxide bioavailability. We hypothesized that in the ventilated lung, this process begins during ischaemia and intensifies with reperfusion, contributing to ischaemia-reperfusion-induced pulmonary vasoconstriction. The aim was to determine whether ischaemia-reperfusion alters inducible and endothelial nitric oxide synthase expression/activity, reactive nitrogen species generation, and nitric oxide bioavailability, potentially affecting pulmonary perfusion.. Ischaemia-reperfusion was induced for various times in anesthetized rabbits with ventilated lungs by reversibly occluding the right pulmonary artery and initiating reperfusion. Nitric oxide synthase activity/expression and phosphorylation, reactive nitrogen species generation and total nitrate/nitrite were determined in lung tissue.. Inducible nitric oxide synthase expression and activity, and reactive nitrogen species formation coincided with increased pulmonary vascular resistance during reperfusion and increased with ischaemia duration, further increasing after 2-h reperfusion. Total nitrate/nitrite also increased with ischaemia but decreased after 2-h reperfusion. Pre-treatment with an inducible nitric oxide synthase inhibitor (1400W; Cayman Chemical Company, Ann Arbor, MI, USA) attenuated inducible nitric oxide synthase activity, reactive nitrogen species generation and pulmonary vascular resistance, but did not affect total nitrate/nitrite. Endothelial nitric oxide synthase expression was unchanged by ischaemia-reperfusion; however, its phosphorylation on serine 1177 and dephosphorylation on threonine 495 was uncoupled, suggesting decreased endothelial nitric oxide synthase activity. 1400W prevented uncoupling of endothelial nitric oxide synthase phosphorylation, maintaining its activity during reperfusion.. Ischaemia-reperfusion up-regulates inducible nitric oxide synthesis and/activity, which coincides with reduced endothelial nitric oxide synthase activity as suggested by its uncoupling and may contribute to ischaemia-reperfusion-induced pulmonary vasoconstriction.

Topics: Animals; Disease Models, Animal; Enzyme Inhibitors; Lung; NADPH Oxidases; Nitrates; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Phosphorylation; Pulmonary Artery; Pulmonary Circulation; Rabbits; Reactive Nitrogen Species; Reperfusion Injury; Respiration, Artificial; Time Factors; Tyrosine; Vascular Resistance; Vasoconstriction

To assess the effects of ischemia and reperfusion on indicators of oxidative stress, activation of eosinophils, and apoptosis in the large colonic mucosa of horses.. 40 horses.. In 1 or two 20-cm-long segments of the pelvic flexure, ischemia was induced for 1 or 2 hours followed by no reperfusion or 30 minutes and 18 hours of reperfusion in anesthetized horses. Mucosal specimens were collected before (controls; n = 20 horses) and after each period of ischemia, and full-thickness tissue samples were collected after each period of reperfusion. Sections of colonic tissues were stained for histomorphometric analysis or assessment of eosinophil accumulation. Nitrotyrosine was identified immunohistochemically, and severity of apoptosis was determined via the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method.. Numbers of mucosal eosinophils were similar before induction of ischemia, after ischemia, and after ischemia-reperfusion. Eosinophil nitrotyrosine production increased significantly during ischemia and continued through 30 minutes of reperfusion; production was decreased at 18 hours of reperfusion but remained greater than that of the controls. In other leukocytes, nitrotyrosine generation peaked at 1 hour of ischemia and again at 18 hours of reperfusion. Compared with control findings, epithelial apoptosis increased gradually at 1 through 2 hours of ischemia with no further progression after reperfusion.. Results suggested that resident eosinophils in the large colon of horses react to mucosal injury from ischemia and reperfusion and may undergo oxidative stress under those conditions. Epithelial apoptosis could contribute to tissue damage.

Topics: Animals; Apoptosis; Colon; Colonic Diseases; Eosinophils; Female; Horse Diseases; Horses; In Situ Nick-End Labeling; Intestinal Mucosa; Ischemia; Male; Microscopy, Electron, Scanning; Oxidative Stress; Random Allocation; Reperfusion Injury; Tyrosine

Retinal ischemia/reperfusion (I/R) results in neuronal death and generation of reactive oxygen species. The aim of this study was to investigate the neuroprotective effect of manganese superoxide dismutase (SOD2) on retinal ganglion cells (RGCs) in an I/R-induced retinal injury model. One eye of each Wistar rat was pretreated with recombinant adeno-associated virus containing the SOD2 gene (AAV-SOD2) or recombinant AAV containing the GFP gene (AAV-GFP) by intravitreal injection 21 days before initiation of I/R injury. Retinal I/R injury was induced by elevating intraocular pressure for 1h, and reperfusion was established immediately afterward. The number of RGCs and the inner plexiform layer (IPL) thickness were measured by Fluorogold retrograde labeling and hematoxylin and eosin staining at 6 h, 24 h, 72 h, and 5 days after injury. Superoxide anion, the number of RGCs, IPL thickness, malondialdehyde (MDA) level, 8-hydroxy-2-deoxyguanosine (8-OHdG) level, MnSOD (manganese superoxide dismutase) activity, and nitrotyrosine level were measured by fluorescence staining, immunohistochemistry, and enzyme-linked immunosorbent analysis at 5 days after I/R injury. Severe RGC loss, reduced IPL thickness, reduced MnSOD activity, and increased superoxide ion, MDA, 8-OHdG, and nitrotyrosine production were observed after I/R injury. Administration of AAV-SOD2 significantly reduced the levels of superoxide ion, MDA, 8-OHdG, and nitrotyrosine and prevented the damage to RGCs and IPL. Delivery of the antioxidant gene inhibited I/R-induced RGC and IPL damage by reducing oxidative stress and nitrative stress, suggesting that MnSOD may be relevant for the neuroprotection of the inner retina from I/R-related diseases.

Topics: Animals; Deoxyadenosines; Dependovirus; DNA Adducts; Female; Gene Transfer Techniques; Genetic Therapy; Ischemia; Malondialdehyde; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Wistar; Recombinant Proteins; Reperfusion Injury; Retina; Retinal Ganglion Cells; Retinal Neurons; Retinal Vessels; Superoxide Dismutase; Superoxides; Tyrosine

NCX 434 is a nitric oxide (NO)-donating triamcinolone acetonide (TA), shown to enhance optic nerve head (ONH) oxygen saturation in non-human primate eyes. Here, the effects of a single intravitreal (IVT) injection of TA were compared with those of NCX 434 on intraocular pressure (IOP), retinal function and retrobulbar haemodymamics in endothelin-1 (ET-1) induced ONH ischaemia/reperfusion in rabbits. Biochemical changes were also assessed in the aqueous humour and in retinal biopsies.. IOP and resistivity index of ophthalmic artery (RI-OA) were recorded using TonoPen and ecocolor Doppler, respectively. Retinal function was assessed using photopic electroretinography. Cytokine expression and oxidative stress markers were evaluated with immunoassay techniques.. At 4 weeks post IVT treatment, TA increased IOP and RI-OA while NCX 434 did not (IOP(Vehicle)=13.6±1.3, IOP(NCX 434)=16.9±2.2, IOP(TA)=20.9±1.9 mm Hg; p<0.05 vs vehicle; RI-OA(Vehicle)=0.44±0.03; RI-OA(NCX 434)=0.47±0.02; RI-OA(TA)=0.60±0.04). Both NCX 434 and TA reversed ET-1 induced decrease in electroretinography amplitude to similar extents. NCX 434 attenuated ET-1 induced oxidative stress markers and nitrotyrosine in retinal tissue, and interleukin-6 and tumour necrosis factor-α in aqueous humour more effectively than TA.. NCX 434 attenuates ET-1 induced ischaemia/reperfusion damage without increasing IOP, probably due to NO release. If data are confirmed in other species and models, this compound could represent an interesting new therapeutic option for retinal and ONH diseases, including diabetic retinopathy.

Topics: Animals; Caspase 3; Cytokines; Electroretinography; Endothelin-1; Glutathione; Hemodynamics; Intraocular Pressure; Intravitreal Injections; Laser-Doppler Flowmetry; Male; Nitrates; Nitric Oxide Donors; Ophthalmic Artery; Optic Disk; Rabbits; Reperfusion Injury; Retina; Retinal Diseases; Superoxide Dismutase; Tonometry, Ocular; Triamcinolone Acetonide; Tyrosine

Gastrointestinal methane generation has been demonstrated in various stress conditions, but it is not known whether nonasphyxiating amounts have any impact on the mammalian pathophysiology. We set out to characterize the effects of exogenous methane administration on the process of inflammatory events arising after reoxygenation in a large animal model of ischemia-reperfusion.. A randomized, controlled in vivo animal study.. A university research laboratory.. Inbred beagle dogs (12.7 6 2 kg).. Sodium pentobarbital-anesthetized animals were randomly assigned to sham-operated or ischemia-reperfusion groups, where superior mesenteric artery occlusion was maintained for 1 hr and the subsequent reperfusion was monitored for 3 hrs. For 5 mins before reperfusion, the animals were mechanically ventilated with normoxic artificial air with or without 2.5% methane. Biological responses to methane-oxygen respirations were defined in pilot rat studies and assay systems were used with xanthine oxidase and activated canine granulocytes to test the in vitro bioactivity potential of different gas concentrations.. The macrohemodynamics and small intestinal pCO(2) gap changes were recorded and peripheral blood samples were taken for plasma nitrite/nitrate and myeloperoxidase analyses. Tissue superoxide and nitrotyrosine levels and myeloperoxidase activity changes were determined in intestinal biopsy samples; structural mucosal damage was measured by hematoxylin and eosin staining. Methane inhalation did not influence the macrohemodynamics but significantly reduced the magnitude of the tissue damage and the intestinal pCO(2) gap changes after reperfusion. Furthermore, the plasma and mucosal myeloperoxidase activity and the intestinal superoxide and nitrotyrosine levels were reduced, whereas the plasma nitrite/nitrate concentrations were increased. Additionally, methane effectively and specifically inhibited leukocyte activation in vitro.. These data demonstrate the anti-inflammatory profile of methane. The study provides evidence that exogenous methane modulates leukocyte activation and affects key events of ischemia-reperfusion-induced oxidative and nitrosative stress and is therefore of potential therapeutic interest in inflammatory pathologies.

Topics: Administration, Inhalation; Animals; Blood Gas Analysis; Dogs; Granulocytes; Inflammation; Intestinal Diseases; Intestinal Mucosa; Intestines; Male; Methane; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Superoxides; Tyrosine

This study was designed to assess a protective effect of palmitoylethanolamide (PEA) in the development of inflammation after ischemia-reperfusion injury of the kidney. Moreover, to suggest a possible mechanism, renal ischemia-reperfusion was performed in mice with targeted disruption of peroxisome proliferator-activated receptor α (PPAR-α) gene (PPAR-αKO) to explain whether the observed PEA effect was dependent on PPAR-α pathway. Peroxisome proliferator-activated receptor-αKO and littermate wild-type controls (PPAR-αWT) were subjected to bilateral renal artery occlusion (30 min) and reperfusion (6 h) and received PEA (10 mg/kg i.p.) 15 min before release of clamps. Serum and urinary indicators of renal dysfunction and tubular and reperfusion injury were measured, specifically serum urea, creatinine, aspartate aminotransferase and γ-glutamyl transferase, and creatinine clearance. In addition, renal sections were used for histological scoring of renal injury and for immunologic evidence of nitrotyrosine formation, poly[adenosine diphosphate-ribose] (PAR), and adhesion molecules expression. The oxidative stress-sensitive nuclear factor κB signaling pathway was also investigated by Western blot analysis. Kidney myeloperoxidase activity and malondialdehyde levels were measured for assessment of polymorphonuclear leukocyte cell infiltration and lipid peroxidation, respectively. Apoptotic mechanisms were also investigated. Moreover, the infiltration and activation of mast cells were explored. In vivo, PEA administration during ischemia significantly reduced the increase in (i) creatinine, γ-glutamyl transferase, aspartate aminotransferase; (ii) nuclear translocation of nuclear factor κB p65; (iii) kidney myeloperoxidase activity and malondialdehyde levels; (iv) nitrotyrosine, PAR, and adhesion molecules expression; (v) the infiltration and activation of mast cells; and (vi) apoptosis. Our results clearly demonstrate that PEA significantly attenuated the degree of renal dysfunction, injury, and inflammation caused by ischemia-reperfusion injury. Moreover, the positive effects of PEA were at least in part dependent on PPAR-α pathway.

Topics: Amides; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspartate Aminotransferases; Cell Adhesion Molecules; Endocannabinoids; Ethanolamines; gamma-Glutamyltransferase; Gene Expression Regulation; Kidney; Leukocytes; Mice; Mice, Knockout; Palmitic Acids; Poly Adenosine Diphosphate Ribose; PPAR alpha; Reperfusion Injury; Tyrosine

Gastrointestinal methane generation has been demonstrated in various conditions, but it is not known whether it has any impact on the mammalian physiology or pathophysiology. Our aim was to characterize the effects of exogenous methane on the process of inflammatory events induced by reoxygenation in a canine model of ischemia-reperfusion.. Sodium pentobarbital-anesthetized inbred beagle dogs (n = 18) were randomly assigned to sham-operated or ischemia-reperfusion (I/R) groups. I/R was induced by occluding the superior mesenteric artery for 1 h, and the subsequent reperfusion was monitored for 3 h. For 5 min before reperfusion, the animals were mechanically ventilated with normoxic artificial air with or without 2.5% methane. The macrohemodynamics and small intestinal pCO2 gap changes were recorded and tissue superoxide and nitrotyrosine levels and myeloperoxidase activity changes were determined in intestinal biopsy samples. Structural mucosal damage was measured via light microscopy and HE staining.. Methane inhalation positively influenced the macrohemodynamic changes, significantly reduced the intestinal pCO2 gap changes and the magnitude of the tissue damage after reperfusion. Further, the intestinal myeloperoxidase activity, the superoxide and nitrotyrosine levels were reduced.. These data demonstrate the anti-inflammatory profile of methane. The study provides evidence that exogenous methane modulates leukocyte activation and affects key events of I/R-induced oxidative and nitrosative stress.

Topics: Administration, Inhalation; Animals; Anti-Inflammatory Agents; Biomarkers; Carbon Dioxide; Disease Models, Animal; Dogs; Hemodynamics; Inflammation; Intestine, Small; Leukocytes; Methane; Peroxidase; Random Allocation; Reperfusion Injury; Superoxides; Tyrosine

Cerebral reperfusion injury may account for complications of thrombolysis and endovascular recanalization. Experimental studies have shown that brain matrix metalloproteinase (MMP) activity increases during reperfusion and is correlated with oxidative/nitrative stress. Increased plasma MMP levels have been reported in stroke, but no information is available for reperfusion-induced plasma MMP and 3-nitrotyrosine (3-NT, a marker of oxidative/nitrative stress) changes immediately after recanalization. We obtained plasma from 29 patients undergoing endovascular recanalization, 12 patients treated with thrombolysis, and six control patients having diagnostic angiogram before and 1,3, and 24 h after treatment to investigate the effect of cerebral reperfusion on plasma MMP gelatinolytic activity and 3-NT level. Hypoperfusion was shown distal to the stenotic artery in endovascular treatment patients. Presence of an occluded artery and recanalization was documented in thrombolysis patients. A significant increase was detected in plasma 3-NT levels 3 and 24 h after stenting/angioplasty. Plasma MMP-9 gelatinolytic activity rose more than 50% of the pre-treatment level in 12 of 29 patients. However, this was not statistically significant and not correlated with any of the clinical or radiological correlates of reperfusion injury (e.g., hyperperfusion and hemorrhage). After thrombolysis, a significant increase in plasma MMP-9 gelatinolytic activity at 3 and 24 h and the cleaved form of MMP-9 were detected. 3-NT levels increased by 44% and 62% at 3 and 24 h, which did not achieve statistical significance, but was highly correlated with admission NIH Stroke Scale (r = 0.930 p < 0.001). No change was detected in MMP-2 in all groups. In conclusion, these data suggest that the increased plasma MMP-9 levels is not a direct measure of MMP-9 activity in the reperfused brain but rather a consequence of tissue plasminogen activator infusion, whereas plasma 3-NT levels appear to originate from the reperfused brain vasculature. The changes in 3-NT levels may therefore be useful to monitor oxygen/nitrogen radical formation during reperfusion with serial measurements.

Topics: Adult; Aged; Brain Ischemia; Cerebral Angiography; Diffusion Magnetic Resonance Imaging; Endovascular Procedures; Female; Humans; Male; Matrix Metalloproteinase 2; Middle Aged; Oxidative Stress; Reperfusion Injury; Thrombolytic Therapy; Time Factors; Tomography, X-Ray Computed; Tyrosine; Ultrasonography, Doppler, Transcranial

NADPH oxidase is a major complex that produces reactive oxygen species (ROSs) during the ischemic period and aggravates brain damage and cell death after ischemic injury. Although many approaches have been tested for preventing production of ROSs by NADPH oxidase in ischemic brain injury, the regulatory mechanisms of NADPH oxidase activity after cerebral ischemia are still unclear. The aim of this study is identifying apocynin as a critical modulator of NADPH oxidase and elucidating its role as a neuroprotectant in an experimental model of brain ischemia in rat. Treatment of apocynin 5min before of reperfusion attenuated cerebral ischemia in rats. Administration of apocynin showed marked reduction in infarct size compared with that of control rats. Medial carotid artery occlusion (MCAo)-induced cerebral ischemia was also associated with an increase in, nitrotyrosine formation, as well as IL-1β expression, IκB degradation and ICAM expression in ischemic regions. These expressions were markedly inhibited by the treatment of apocynin. We also demonstrated that apocynin reduces levels of apoptosis (TUNEL, Bax and Bcl-2 expression) resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. This new understanding of apocynin induced adaptation to ischemic stress and inflammation could suggest novel avenues for clinical intervention during ischemic and inflammatory diseases.

Topics: Acetophenones; Animals; Apoptosis; bcl-2-Associated X Protein; Brain; Cytochromes c; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; I-kappa B Proteins; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Intercellular Adhesion Molecule-1; Interleukin-1beta; Male; NADPH Oxidases; Neurologic Examination; Peptide Fragments; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine

Increased production of reactive oxygen and nitrogen species following cerebral ischemia-reperfusion is a major cause for neuronal injury. In hypercholesterolemic apolipoprotein E knockout (ApoE-KO) mice, 2h of middle cerebral artery (MCA) occlusion followed by 22h of reperfusion led to an enhanced expression of NADPH oxidase subunits (NOX2, NOX4 and p22phox) and isoforms of nitric oxide synthase (neuronal nNOS and inducible iNOS) in the ischemic hemisphere compared with the non-ischemic contralateral hemisphere. This was associated with elevated levels of 3-nitrotyrosine, an indicator of peroxynitrite-mediated oxidative protein modification. Pre-treatment with betulinic acid (50mg/kg/day for 7days via gavage) prior MCA occlusion prevented the ischemia reperfusion-induced upregulation of NOX2, nNOS and iNOS. In parallel, betulinic acid reduced the levels of 3-nitrotyrosine. In addition, treatment with betulinic acid enhanced the expression of endothelial eNOS in the non-ischemic hemispheres. Finally, betulinic acid reduced infarct volume and ameliorated the neurological deficit in this mouse stroke model. In conclusion, betulinic acid protects against cerebral ischemia-reperfusion injury in mice. This is likely to result from a reduction of oxidative stress (by downregulation of NOX2) and nitrosative stress (by reduction of nNOS and iNOS), and an enhancement of blood flow (by upregulation of eNOS).

Topics: Animals; Betulinic Acid; Brain; Brain Ischemia; Male; Mice; Mice, Knockout; NADPH Oxidases; Nitric Oxide Synthase; Oxidative Stress; Pentacyclic Triterpenes; Reactive Nitrogen Species; Reperfusion Injury; RNA, Messenger; Stress, Physiological; Triterpenes; Tyrosine

Changes in intestinal function, notably impaired transit, following ischemia/reperfusion (I/R) injury are likely to derive, at least in part, from damage to the enteric nervous system. Currently, there is a lack of quantitative data and methods on which to base quantitation of changes that occur in enteric neurons. In the present work, we have investigated quantifiable changes in response to ischemia of the mouse small intestine followed by reperfusion from 1 h to 7 days. I/R caused distortion of nitric oxide synthase (NOS)-containing neurons, the appearance of a TUNEL reaction in neurons, protein nitrosylation and translocation of Hu protein. Protein nitrosylation was detected after 1 h and was detectable in 10% of neurons by 6 h in the ischemic region, indicating that reactive peroxynitrites are rapidly produced and can interact with proteins soon after reperfusion. Apoptosis, revealed by TUNEL staining, was apparent at 6 h. The profile sizes of NOS neurons were increased by 60% at 2 days and neurons were still swollen at 7 days, both in the ischemic region and proximal to the ischemia. The distribution of the enteric neuron marker and oligonucleotide binding protein, Hu, was significantly changed in both regions. Hu protein translocation to the nucleus was apparent by 3 h and persisted for up to 7 days. Particulate Hu immunoreactivity was observed in the ganglia 3 h after I/R but was never observed in control. Our observations indicate that effects of I/R injury can be detected after 1 h and that neuronal changes persist to at least 7 days. Involvement of NO and reactive oxygen species in the changes is indicated by the accumulation of nitrosylated protein aggregates and the swelling and distortion of nitrergic neurons. It is concluded that damage to the enteric nervous system, which is likely to contribute to functional deficits following ischemia and re-oxygenation in the intestine, can be quantified by Hu protein translocation, protein nitrosylation, swelling of nitrergic neurons and apoptosis.

Topics: Animals; Apoptosis; Calbindin 2; ELAV Proteins; Enteric Nervous System; Intestine, Small; Male; Mice; Mice, Inbred C57BL; Neurons; Nitric Oxide Synthase; Nitroso Compounds; Reperfusion Injury; S100 Calcium Binding Protein G; Tyrosine

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

Retinal ischemia/reperfusion (I/R) injury causes profound tissue damage, especially retinal ganglion cell (RGC) death. The aims of the study were to investigate whether catalase (CAT) has a neuroprotective effect on RGC after I/R injury in rats, and to determine the possible antioxidant mechanism. Wistar female rats were randonmized into four groups: normal control group (Control group), retinal I/R with vehicle group (I/R with vehicle group), retinal I/R with AAV-CAT group (I/R with AAV-CAT group), and normal retina with AAV-CAT group (normal with AAV-CAT group). One eye of each rat was pretreated with recombinant adeno-associated virus containing catalase gene (I/R with AAV-CAT group or normal with AAV-CAT group) and recombinant adeno-associated virus containing GFP gene (I/R with vehicle group) by intravitreal injection 21 days before initiation of I/R injury. Retinal I/R injury was induced by elevating intraocular pressure to 100mmHg for 1h. The number of RGC and inner plexiform layer (IPL) thickness were measured by fluorogold retrograde labeling and hematoxylin and eosin staining at 6h, 24h, 72 h and 5d after injury. Hydrogen peroxide (H(2)O(2)), the number of RGC, IPL thickness, malondialdehyde(MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), CAT activity and nitrotyrosine were measured by fluorescence staining, immunohistochemistry and enzyme-linked immunosorbent assay analysis at 5 days after injury. Electroretinographic (ERG) evaluation was also used. Pretreatment of AAV-CAT significantly decreased the levels of H(2)O(2), MDA, 8-OHdG and nitrotyrosine, increased the catalase activity, and prevented the reduction of a- and b- waves in the I/R with AAV-CAT group compare with the I/R with vehicle group (p<0.01). Catalase attenuated the I/R-induced damage of RGC and IPL and retinal function. Therefore, catalase can protect the rat retina from I/R-induced injury by enhancing the antioxidative ability and reducing oxidative stress, which suggests that catalase may be relevant for the neuroprotection of inner retina from I/R-related diseases.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Apoptosis; Catalase; Cell Count; Cell Survival; Deoxyguanosine; Dependovirus; Electroretinography; Enzyme-Linked Immunosorbent Assay; Female; Genetic Therapy; Genetic Vectors; Immunohistochemistry; Malondialdehyde; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion Injury; Retina; Retinal Diseases; Retinal Ganglion Cells; Tyrosine

Stachybotrys microspora triprenyl phenol-7 (SMTP-7) is a novel fibrinolytic agent with anti-inflammatory effect. Previous study demonstrated that SMTP-7 further ameliorated infarction volume in a mouse embolic stroke model compared with tissue type plasminogen activator (tPA), but the reason SMTP-7 has more beneficial effect than tPA has not yet been determined. In the present study, we investigated whether SMTP-7 has an intrinsic neuroprotective effect against transient focal cerebral ischemia (tFCI). Sprague-Dawley rats were subjected to tFCI by intraluminal middle cerebral artery occlusion for 2h. Following induction of tFCI, rats were randomized into two groups based on the agent administered: SMTP-7 group and vehicle group. We examined cerebral infarction volume 24h after reperfusion, and evaluated superoxide production, the expressions of nitrotyrosine and matrix metalloproteinase-9 (MMP-9), which play major roles in secondary brain injury and hemorrhagic transformation. The findings showed that SMTP-7 significantly suppressed superoxide production, the expression of nitrotyrosine and MMP-9 after tFCI, and consequently attenuated ischemic neuronal damage. These results suggest that SMTP-7 has an intrinsic neuroprotective effect on ischemia/reperfusion injury through the suppression of oxidative stress and MMP-9 activation.

Topics: Animals; Benzopyrans; Blotting, Western; Brain Injuries; Fibrinolytic Agents; Ischemic Attack, Transient; Male; Matrix Metalloproteinase 9; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Stachybotrys; Superoxides; Tyrosine

Post-ischemic oxidative stress and vasomotor dysfunction in cerebral arteries may increase the likelihood of cognitive impairment and secondary stroke. However, the underlying mechanisms of post-stroke vascular abnormalities, as distinct from those causing primary brain injury, are poorly understood. We tested whether augmented superoxide-dependent dysfunction occurs in the mouse cerebral circulation following ischemia-reperfusion, and evaluated the role of Nox2 oxidase.. Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and Nox2-deficient (Nox2(-/-)) mice by middle cerebral artery occlusion (MCAO; 0.5 h), followed by reperfusion (23.5 h). Superoxide production by MCA was measured by L-012-enhanced chemiluminescence. Nitric oxide (NO) function was assessed in cannulated and pressurized MCA via the vasoconstrictor response to N(ω)-nitro-L-arginine methyl ester (L-NAME; 100 µmol/L). Expression of Nox2, the nitration marker 3-nitrotyrosine, and leukocyte marker CD45 was assessed in cerebral arteries by Western blotting.. Following ischemia-reperfusion, superoxide production was markedly increased in the MCA of WT, but not Nox2(-/-) mice. In WT mice, L-NAME-induced constriction was reduced by ∼50% in ischemic MCA, whereas ischemia-reperfusion had no effect on responses to L-NAME in vessels from Nox2(-/-) mice. In ischemic MCA from WT mice, expression of Nox2 and 3-nitrotyrosine were ∼1.4-fold higher than in the contralateral MCA, or in ischemic or contralateral vessels from Nox2(-/-) mice. Vascular CD45 levels were unchanged by ischemia-reperfusion.. Excessive superoxide production, impaired NO function and nitrosative stress occur in mouse cerebral arteries after ischemia-reperfusion. These abnormalities appear to be exclusively due to increased activity of vascular Nox2 oxidase.

Topics: Animals; Cerebral Arteries; Cerebrovascular Circulation; Crosses, Genetic; Ischemia; Leukocyte Common Antigens; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Middle Cerebral Artery; Models, Biological; NADPH Oxidase 2; NADPH Oxidases; Oxidative Stress; Reperfusion Injury; Stroke; Superoxides; Tyrosine

Experimental evidence has shown that some garlic-derived products have a protective effect against ischemic brain injury. The present study was designed to investigate the effect of aged garlic extract (AGE), establish the therapeutic window, and determine its protective mechanism in a cerebral ischemia model. Animals were subjected to middle cerebral artery occlusion (MCAO) for 2h and treated with 1.2ml/kg body wt.(i.p.) of AGE 30min before, at the beginning of (0R), or 1h after reperfusion. The 0R treatment significantly reduced the size of the infarct area after 2h of reperfusion. Repeated doses subsequent to the 0R treatment (at 1, 2, or 3h after reperfusion) had no effect on the temporal window of protection. The protective 0R treatment with AGE prevented the increase in nitrotyrosine and the decrease in total superoxide dismutase, glutathione peroxidase, and extracellular superoxide dismutase activities induced by MCAO. These data indicate that AGE delays the effects of ischemia/reperfusion-induced neuronal injury. However, this treatment itself was not associated with a noticeable improvement in the neurological outcome, or with an effect on the inflammatory response. We conclude that the neuroprotective effect of AGE in the 0R treatment might be associated with control of the free-radical burst induced by reperfusion, preservation of antioxidant enzyme activity, and the delay of other pathophysiological processes.

Topics: Animals; Antioxidants; Brain; Brain Ischemia; Cerebral Infarction; Disease Models, Animal; Garlic; Glutathione Peroxidase; Infarction, Middle Cerebral Artery; Male; Neuroprotective Agents; Oxidative Stress; Phytotherapy; Plant Extracts; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Tyrosine

In the present study, we tested the efficacy of treatment with the selective adenosine A2A receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-50-ethylcarboxamidoadenosine (CGS 21680) on ischemia and reperfusion injury of the multivisceral organs. Ischemia and reperfusion injury was induced in mice by clamping both the superior mesenteric artery and the celiac artery for 30 min, followed thereafter by reperfusion. Sixty minutes after reperfusion, animals were killed for histological examination and biochemical studies. Injured vehicle-treated mice developed a significant increase of ileum TNF-alpha levels, myeloperoxidase activity, and marked histological injury and apoptosis. Ischemia and reperfusion injury of the multivisceral organs was also associated with significant mortality. Reperfused ileum sections from injured vehicle-treated mice showed positive staining for P-selectin and intercellular adhesion molecule 1. The intensity and degree of P-selectin and intercellular adhesion molecule 1 were markedly reduced in tissue sections from injured CGS 21680-treated mice. Ischemia and reperfusion-injured mice that have been treated with CGS 21680 showed also a significant reduction of neutrophil infiltration into the intestine, a reduction of apoptosis, and improved histological status of the intestine and survival. Taken together, our results clearly demonstrate that selective activation of adenosine A2A receptors plays an important role in the regulation of ischemia and reperfusion injury and results put forward the hypothesis that selective activation of adenosine A2A receptors may represent a novel and possible strategy.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Fas Ligand Protein; Intercellular Adhesion Molecule-1; Intestine, Small; Male; Mice; Mitogen-Activated Protein Kinases; NF-kappa B; P-Selectin; Peroxidase; Phenethylamines; Poly Adenosine Diphosphate Ribose; Proto-Oncogene Proteins c-bcl-2; Receptor, Adenosine A2A; Reperfusion Injury; Signal Transduction; Tumor Necrosis Factor-alpha; Tyrosine

It is well known that insulin possesses a cardioprotective effect and that insulin resistance is closely related to cardiovascular diseases. Peroxynitrite (ONOO(-)) formation may trigger oxidative/nitrative stress and represent a major cytotoxic effect in heart diseases. This study was designed to investigate whether insulin attenuates ONOO(-) generation and oxidative/nitrative stress in acute myocardial ischemia/reperfusion (MI/R). Adult male rats were subjected to 30 min of myocardial ischemia and 3 h of reperfusion. Rats randomly received vehicle, insulin, or insulin plus wortmannin. Arterial blood pressure and left ventricular pressure were monitored throughout the experiment. Insulin significantly improved cardiac functions and reduced myocardial infarction, apoptotic cell death, and blood creatine kinase/lactate dehydrogenase levels following MI/R. Myocardial ONOO(-) formation was significantly attenuated after insulin treatment. Moreover, insulin resulted in a significant increase in Akt and endothelial nitric oxide (NO) synthase (eNOS) phosphorylation, NO production, and antioxidant capacity in ischemic/reperfused myocardial tissue. On the other hand, insulin markedly reduced MI/R-induced inducible NOS (iNOS) and gp91(phox) expression in cardiac tissue. Inhibition of insulin signaling with wortmannin not only blocked the cardioprotection of insulin but also markedly attenuated insulin-induced antioxidative/antinitrative effect. Furthermore, the suppression on ONOO(-) formation by either insulin or an ONOO(-) scavenger uric acid reduced myocardial infarct size in rats subjected to MI/R. We concluded that insulin exerts a cardioprotective effect against MI/R injury by blocking ONOO(-) formation. Increased physiological NO production (via eNOS phosphorylation) and superoxide anion reduction contribute to the antioxidative/antinitrative effect of insulin, which can be reversed by inhibiting phosphatidylinositol 3'-kinase. These results provide important novel information on the mechanisms of cardiovascular actions of insulin.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Creatine Kinase; Electrocardiography; Heart Function Tests; Hypoglycemic Agents; In Situ Nick-End Labeling; Insulin; L-Lactate Dehydrogenase; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Necrosis; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Nitrogen Species; Reperfusion Injury; Tyrosine

To investigate the beneficial effects of inchinkoto (ICKT) in the liver after 70% hepatectomy following ischemia reperfusion.. Wistar rats were divided into 3 groups: simple laparotomy and 70% hepatectomy (Hx), 70% hepatectomy following ischemia reperfusion (IR) with vehicle (IRHxV), 70% hepatectomy following IR with ICKT (1 or 2 g/kg of body weight; IRHxK). Vehicle or ICKT was administered for 3 days preoperatively. The hepatoduodenal ligament was clamped for 15 minutes before hepatectomy in the IRHx groups. Rats were killed 1 hours after hepatectomy. In other experiments, the hepatoduodenal ligament was clamped for 30 minutes, with or without ICKT treatment, to evaluate the effect of ICKT on IR injury-induced mortality. Serum transaminase levels and the gene expression of inflammatory cytokines and inducible nitric oxide synthase in the remnant liver were determined. Furthermore, the expression of antioxidant genes was evaluated by PCR array.. The elevation of serum transaminase levels, the upregulation of genes for inflammatory cytokines and inducible nitric oxide synthase, and the increased formation of nitrotyrosine observed in the remnant livers of the IRHxV group were all significantly attenuated by preoperative administration of ICKT in the IRHxK group. The expression of antioxidant genes was also higher in the IRHxK group compared with that of the IRHxV group. Moreover, administration of ICKT significantly reduced the mortality induced by IRHx after 30-minute ischemia.. Preoperative administration of ICKT provides beneficial effects through attenuating inflammatory responses and oxidative stress in the liver following IR and subsequent hepatectomy.

Topics: Animals; Antioxidants; Cytokines; Drugs, Chinese Herbal; Glutathione; Hepatectomy; Inflammation Mediators; Iridoid Glycosides; Iridoids; Liver; Male; Nitric Oxide Synthase; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Polymerase Chain Reaction; Rats; Rats, Wistar; Reactive Nitrogen Species; Reactive Oxygen Species; Reperfusion Injury; Survival Rate; Transaminases; Tyrosine

Neuroglobin (Ngb) is a recently discovered globin that affords protection against hypoxic/ischemic-induced cell injury in brain. Hypoxic/ischemic injury is associated with accumulation of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). In previous studies, we found that Ngb has antioxidative properties, and protects PC-12 cells against hypoxia- and β-amyloid-induced cell death. To further delineate the potential role of Ngb in protection against cerebral ischemia-reperfusion injury in vivo, we developed a transgenic mouse line that overexpresses Ngb. Hippocampal ischemia-reperfusion injury was induced by a 10-minute bilateral occlusion of the common carotid arteries, and the animal brains were assessed 3 days later. CA1 neural injury was determined by cresyl violet staining. Lipid peroxidation was assessed using a malonyldialdehyde assay kit, whereas ROS/RNS accumulation was determined by Het staining in the CA1 hippocampal region. Hippocampal Ngb mRNA and protein expressions were assessed by reverse transcriptase-PCR and western blotting, respectively. Neuroglobin was successfully overexpressed in the hippocampus of Ngb transgenic mice. After ischemia-reperfusion, CA1 ROS/RNS production and lipid peroxidation were markedly decreased in Ngb transgenic mice compared with wild-type mice. Furthermore, CA1 neuronal injury was also markedly reduced. Thus, Ngb may confer protection against ischemia-reperfusion injury in the brain through its intrinsic antioxidant properties.

Topics: Animals; Brain Ischemia; Catalase; Globins; Glutathione Peroxidase; Hippocampus; Humans; Lipid Peroxidation; Mice; Mice, Transgenic; Nerve Tissue Proteins; Neuroglobin; Neurons; Neuroprotective Agents; Oxidative Stress; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Tyrosine; Up-Regulation

Intestinal ischemia/reperfusion causes tissue hypoxia and damage, leading to the pathophysiology of inflammation. The aim of this study was to investigate the effects of glutamine on the tissue injury caused by ischemia/reperfusion of the gut. Ischemia/reperfusion injury of the intestine was caused by clamping both the superior mesenteric artery and the celiac trunk for 30 min followed by the release of the clamp allowing reperfusion for 1h. This procedure results in splanchnic artery occlusion-injury. Based on our findings we propose that the amino acid glutamine, administered 15 min before reperfusion at the dose of 1.5mg/kg, i.v. may be useful in the treatment of various ischemia and reperfusion diseases. The present study was performed in order to determine the pharmacological effects of glutamine ischemia/reperfusion-induced intestinal injury in rats. In particular, to gain a better insight into the mechanism(s) of action of glutamine, we evaluated the following endpoints of the inflammatory response: (1) histological damage; (2) neutrophil infiltration of the reperfused intestine (MPO activity); (3) NF-kappaB activation and cytokines production; (4) expression of ICAM-1 and P-selectin during reperfusion; (5) nitrotyrosine and poly-ADP-ribose formation; (6) pro-inflammatory cytokine production; (7) inducible nitric oxide synthase expression; (8) apoptosis as shown by TUNEL staining and (9) Bax/Bcl-2 expression.

Topics: Animals; Apoptosis; Cell Adhesion Molecules; Glutamine; I-kappa B Kinase; Ileal Diseases; Ileum; Male; Neutrophil Infiltration; NF-kappa B; Nitric Oxide Synthase Type II; Poly Adenosine Diphosphate Ribose; Protein Transport; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Splanchnic Circulation; Time Factors; Tyrosine

Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) provides neuroprotection against apoptosis in a transient middle cerebral artery occlusion (MCAo) model. This study was to further investigate the anti-apoptotic effect of FA during reperfusion after cerebral ischemia.. Rats were subjected to 90 min of cerebral ischemia followed by 3 or 24 h of reperfusion after which they were sacrificed.. Intravenous FA (100 mg/kg) administered immediately after middle cerebral artery occlusion (MCAo) or 2 h after reperfusion effectively abrogated the elevation of postsynaptic density-95 (PSD-95), neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), nitrotyrosine, and cleaved caspase-3 levels as well as apoptosis in the ischemic cortex at 24 h of reperfusion. FA further inhibited Bax translocation, cytochrome c release, and p38 mitogen-activated protein (MAP) kinase phosphorylation. Moreover, FA enhanced the expression of gamma-aminobutyric acid type B receptor subunit 1 (GABA(B1)) in the ischemic cortex at 3 and 24 h of reperfusion. In addition, nitrotyrosine-positive cells colocalized with cleaved caspase-3-positive cells, and phospho-p38 MAP kinase-positive cells colocalized with nitrotyrosine- and Bax-positive cells, indicating a positive relationship among the expression of nitrotyrosine, phospho-p38 MAP kinase, Bax, and cleaved caspase-3. The mutually exclusive expression of GABA(B1) and nitrotyrosine revealed that there is a negative correlation between GABA(B1) and nitrotyrosine expression profiles. Additionally, pretreatment with saclofen, a GABA(B) receptor antagonist, abolished the neuroprotection of FA against nitric oxide (NO)-induced apoptosis.. FA significantly enhances GABA(B1) receptor expression at early reperfusion and thereby provides neuroprotection against p38 MAP kinase-mediated NO-induced apoptosis at 24 h of reperfusion.

Topics: Animals; Apoptosis; Brain Ischemia; Coumaric Acids; Disease Models, Animal; Free Radical Scavengers; Gene Expression Regulation; Male; Neuroprotective Agents; Nitric Oxide; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Sprague-Dawley; Receptors, GABA-B; Reperfusion Injury; Tyrosine

Females suffer a less severe ischemic acute renal failure than males, apparently because of higher nitric oxide (NO) bioavailability and/or lower levels of oxidative stress. Because the renal ischemic injury is associated with outer medullary (OM) endothelial dysfunction, the present study evaluated sex differences in OM changes of NO and peroxynitrite levels (by differential pulse voltammetry and amperometry, respectively) during 45 min of ischemia and 60 min of reperfusion in anesthetized Sprague-Dawley rats. Endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) protein expression and their phosphorylated forms [peNOS(Ser1177) and pnNOS(Ser1417)], 3-nitrotyrosine, reduced sulfhydryl groups (-SH), and glomerular filtration rate (GFR) were also determined. No sex differences were observed in monomeric eNOS and nNOS expression, NO, or 3-nitrotyrosine levels in nonischemic kidneys, but renal -SH content was higher in females. Ischemia increased dimeric/monomeric eNOS and nNOS ratio more in females, but the dimeric phosphorylated peNOS(Ser1177) and pnNOS(Ser1417) forms rose similarly in both sexes, indicating no sex differences in nitric oxide synthase activation. However, NO levels increased more in females than in males (6,406.0 ± 742.5 and 4,058.2 ± 272.35 nmol/l respectively, P < 0.05), together with a lower increase in peroxynitrite current (5.5 ± 0.7 vs. 12.7 ± 1.5 nA, P < 0.05) and 3-nitrotyrosine concentration, (28.7 ± 3.7 vs. 48.7 ± 3.7 nmol/mg protein, P < 0.05) in females than in males and a better preserved GFR after ischemia in females than in males (689.7 ± 135.0 and 221.4 ± 52.5 μl·min(-1)·g kidney wt(-1), P < 0.01). Pretreatment with the antioxidants N-acetyl-L-cysteine or ebselen abolished sex differences in peroxynitrite, nitrotyrosine, and GFR, suggesting that a greater oxidative and nitrosative stress worsens renal damage in males.

Topics: Acute Kidney Injury; Animals; Antioxidants; Disease Models, Animal; Enzyme Activation; Female; Glomerular Filtration Rate; Kidney Medulla; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Oxidative Stress; Peroxynitrous Acid; Phosphorylation; Protein Multimerization; Rats; Rats, Sprague-Dawley; Reactive Nitrogen Species; Reperfusion Injury; Sex Factors; Sulfhydryl Compounds; Time Factors; Tyrosine

To date, the role that NO derived from endothelial NO synthase (eNOS) plays in the development of the injuries occurring under hypoxia/reoxygenation (H/R) in the lung remains unknown and thus constitutes the subject of the present work. A follow-up study was conducted in Wistar rats submitted to H/R (hypoxia for 30 min; reoxygenation of 0 h, 48 h and 5 days), with or without prior treatment using the eNOS inhibitor L-NIO (20 mg/kg). Lipid peroxidation, apoptosis, protein nitration and NO production (NOx) were analysed. The results showed that L-NIO administration lowered NOx levels in all the experimental groups. Contrarily, the lipid peroxidation level and the percentage of apoptotic cells rose, implying that eNOS-derived NO may have a protective effect against the injuries occurring during H/R in the lung. These findings could open the possibility of future studies to design new therapies for this type of hypoxia based on NO-pharmacology.

Topics: Animals; Cytoprotection; Enzyme Inhibitors; Hypoxia; In Situ Nick-End Labeling; Lung; Male; Nitric Oxide; Nitric Oxide Synthase Type III; Ornithine; Oxygen; Rats; Rats, Wistar; Reperfusion Injury; Thiobarbituric Acid Reactive Substances; Tyrosine

Depletion of the nitric oxide synthase cofactor tetrahydrobiopterin (H4B) during ischemia and reperfusion is associated with severe graft pancreatitis. Since clinically feasible approaches to prevent ischemia reperfusion injury (IRI) by H4B-substitution are missing we investigated its therapeutic potential in a murine pancreas transplantation model using different treatment regimens. Grafts were subjected to 16 h cold ischemia time (CIT) and different treatment regimens: no treatment, 160 μM H4B to perfusion solution, H4B 50 mg/kg prior to reperfusion and H4B 50 mg/kg before recovery of organs. Nontransplanted animals served as controls. Recipient survival and endocrine graft function were assessed. Graft microcirculation was analyzed 2 h after reperfusion by intravital fluorescence microscopy. Parenchymal damage was assessed by histology and nitrotyrosine immunohistochemistry, H4B tissue levels by high pressure liquid chromatography (HPLC). Compared to nontransplanted controls prolonged CIT resulted in significant microcirculatory deterioration. Different efficacy according to route and timing of administration could be observed. Only donor pretreatment with H4B resulted in almost completely abrogated IRI-related damage showing graft microcirculation comparable to nontransplanted controls and restored intragraft H4B levels, resulting in significant reduction of parenchymal damage (p < 0.002) and improved survival and endocrine function (p = 0.0002 each). H4B donor pretreatment abrogates ischemia-induced parenchymal damage and represents a promising strategy to prevent IRI following pancreas transplantation.

Topics: Animals; Biopterins; Cold Ischemia; Male; Mice; Mice, Inbred C57BL; Microcirculation; Models, Animal; Pancreas; Pancreas Transplantation; Peroxynitrous Acid; Reperfusion Injury; Tissue Donors; Transplantation, Isogeneic; Tyrosine

Retinal ischemia/reperfusion (I/R) occurs in many ocular diseases and leads to neuronal death. Lutein, a potent antioxidant, is used to prevent severe visual loss in patients with early age-related macular degeneration (AMD), but its effect on I/R insult is unclear. The objective of the present study is to investigate the neuroprotective effect of lutein on retinal neurons after acute I/R injury.. Unilateral retinal I/R was induced by the blockade of internal carotid artery using intraluminal method in mice. Ischemia was maintained for 2 hours followed by 22 hours of reperfusion, during which either lutein or vehicle was administered. The number of viable retinal ganglion cells (RGC) was quantified. Apoptosis was investigated using TUNEL assay. Oxidative stress was elucidated using markers such as nitrotyrosine (NT) and poly(ADP-ribose) (PAR).. In vehicle-treated I/R retina, severe cell loss in ganglion cell layer, increased apoptosis as well as increased NT and nuclear PAR immunoreactivity were observed. In lutein-treated I/R retina, significantly less cell loss, decreased number of apoptotic cells, and decreased NT and nuclear PAR immunoreactivity were seen.. The neuroprotective effect of lutein was associated with reduced oxidative stress. Lutein has been hitherto used principally for protection of outer retinal elements in AMD. Our study suggests that it may also be relevant for the protection of inner retina from acute ischemic damage.

Topics: Acute Disease; Animals; Antioxidants; Apoptosis; Calbindin 2; Disease Models, Animal; In Situ Nick-End Labeling; Lutein; Male; Mice; Mice, Inbred C57BL; Neurons; Neuroprotective Agents; Oxidative Stress; Poly Adenosine Diphosphate Ribose; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells; S100 Calcium Binding Protein G; Tyrosine

Oxidative stress and apoptosis are important factors in the etiology of renal ischemia-reperfusion (I/R) injury. The present study tested the hypothesis that the cell-permeant SOD mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) protects the kidney from I/R-mediated oxidative stress and apoptosis in vivo. Male Sprague-Dawley rats (175-220 g) underwent renal I/R by bilateral clamping of the renal arteries for 45 min followed by reperfusion for 24 h. To examine the role of reactive oxygen species (ROS) in renal I/R injury, a subset of animals were treated with either saline vehicle (I/R Veh) or MnTMPyP (I/R Mn) (5 mg/kg ip) 30 min before and 6 h after surgery. MnTMPyP significantly attenuated the I/R-mediated increase in serum creatinine levels and decreased tubular epithelial cell damage following I/R. MnTMPyP also decreased TNF-alpha levels, gp(91phox), and lipid peroxidation after I/R. Furthermore, MnTMPyP inhibited the I/R-mediated increase in apoptosis and caspase-3 activation. Interestingly, although MnTMPyP did not increase expression of the antiapoptotic protein Bcl-2, it decreased the expression of the proapoptotic genes Bax and FasL. These results suggest that MnTMPyP is effective in reducing apoptosis associated with renal I/R injury and that multiple signaling mechanisms are involved in ROS-mediated cell death following renal I/R injury.

Topics: Animals; Antioxidants; Apoptosis; Caspase 3; Creatinine; Gene Expression; Kidney; Kidney Diseases; Kidney Function Tests; Lipid Peroxidation; Male; Metalloporphyrins; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase; Superoxides; Tumor Necrosis Factor-alpha; Tyrosine

Acute intestinal ischemia reperfusion (I/R) injury affects not only the intestines but also remote organs due to pro-inflammatory and tissue injurious factors. Thus, we aimed to investigate the roles of melatonin (a powerful antioxidant) and 1400W (a strong inhibitor of inducible nitric oxide) in a rat intestinal I/R injury model, since oxidative and nitrosative injury are believed to be the major causes.. A total of 56 Wistar albino rats were used, with seven rats in each group. After I/R induction in the intestines by clamping/unclamping the superior mesenteric artery, we measured malondialdehyde, superoxide dismutase, glutathione peroxidase, nitric oxide, and 3-nitrotyrosine levels in lung, kidney, and liver tissues (to evaluate remote organ injury) as well as in the intestines. Study groups received melatonin, 1400W or both to examine the roles of these molecules in the pathogenesis of injury following I/R.. Melatonin and 1400W had an ameliorating effect on both oxidative and nitrosative stress in the intestine and the lung against mesenteric I/R injury in rats. Moreover, each of these two agents had an inhibitory effect on oxidative injury and histopathological changes in the intestine and the lung. Furthermore, the combination of both agents (melatonin and 1400W) was more effective than either of the agents alone (P < 0.05).. Melatonin and 1400W, either alone or in combination, were efficient in ameliorating experimental I/R injury of the intestines.

Topics: Amidines; Animals; Antioxidants; Benzylamines; Enzyme Inhibitors; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Intestinal Mucosa; Intestines; Kidney; Liver; Lung; Male; Malondialdehyde; Melatonin; Mesenteric Vascular Occlusion; Nitric Oxide Synthase Type II; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Survival Rate; Tyrosine

Recently, kidney fibrosis following transplantation has become recognized as a main contributor of chronic allograft nephropathy. In transplantation, transient ischemia is an inescapable event. Reactive oxygen species (ROS) play a critical role in ischemia and reperfusion (I/R)-induced acute kidney injury, as well as progression of fibrosis in various diseases such as hypertension, diabetes, and ureteral obstruction. However, a role of ROS/oxidative stress in chronic kidney fibrosis following I/R injury remains to be defined. In this study, we investigated the involvement of ROS/oxidative stress in kidney fibrosis following kidney I/R in mice. Mice were subjected to 30 min of bilateral kidney ischemia followed by reperfusion on day 0 and then administered with either manganese (III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP, 5 mg/kg body wt ip), a cell permeable superoxide dismutase (SOD) mimetic, or 0.9% saline (vehicle) beginning at 48 h after I/R for 14 days. I/R significantly increased interstitial extension, collagen deposition, apoptosis of tubular epithelial cells, nitrotyrosine expression, hydrogen peroxide production, and lipid peroxidation and decreased copper-zinc SOD, manganese SOD, and glucose 6-phosphate dehydrogenase activities in the kidneys 16 days after the procedure. MnTMPyP administration minimized these postischemic changes. In addition, MnTMPyP administration significantly attenuated the increases of alpha-smooth muscle actin, PCNA, S100A4, CD68, and heat shock protein 47 expression following I/R. We concluded that kidney fibrosis develops chronically following I/R injury, and this process is associated with the increase of ROS/oxidative stress.

Topics: Actins; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antioxidants; Apoptosis; Collagen; Disease Models, Animal; Disease Progression; Fibrosis; Glucosephosphate Dehydrogenase; HSP47 Heat-Shock Proteins; Hydrogen Peroxide; Ischemia; Kidney; Lipid Peroxidation; Male; Metalloporphyrins; Mice; Mice, Inbred BALB C; Oxidation-Reduction; Oxidative Stress; Proliferating Cell Nuclear Antigen; Reactive Oxygen Species; Reperfusion Injury; S100 Calcium-Binding Protein A4; S100 Proteins; Superoxide Dismutase; Time Factors; Tyrosine; Vitamin E

Many studies have indicated leukocytes are a major contributor to brain injuries caused by intracerebral hemorrhage (ICH). Leukocyte-expressed CD18 is important for neutrophil-endothelial interactions in the vasculature, and CD18 deficiency protects against ischemia-reperfusion injury. We investigated whether CD18 deficiency provides protection against ICH-induced brain injury. Male wild-type (WT) CD18(+/+) mice and CD18(-/-) -knockout mice were used in this study. ICH was induced by a collagenase injection. Mortality, neurological function, brain edema, and myeloperoxidase (MPO) activity as well as tissue expression of nitrotyrosine and MPO were evaluated 24 hr after ICH. We discovered significantly reduced brain edema and diminished mortality with a concomitant decrease in MPO and nitrotyrosine immunoreactivity in brains of CD18-knockout mice.

Topics: Animals; Brain Edema; CD18 Antigens; Cerebral Hemorrhage; Immunohistochemistry; Male; Mice; Mice, Knockout; Peroxidase; Reperfusion Injury; Tyrosine

Although dapsone (4,4'-diaminodiphenylsulfone) has been described as a neuroprotective agent in occlusive focal ischemia in rats, its mechanism of action is still unknown. To explore this mechanism, oxidative, inflammatory and apoptotic processes were evaluated in the striatum of adult rats using a model of ischemia-reperfusion (I/R), either with or without dapsone treatment. Male Wistar rats were submitted to transient middle cerebral artery occlusion for 2 hr, followed by reperfusion. Rats were dosed either with dapsone (12.5 mg/kg i.p.) or vehicle 30 min before or 30 min after the ischemia onset. Lipid peroxidation (LP) and nitrotyrosine contents were measured 22 hr after reperfusion, and myeloperoxidase activity was evaluated 46 hr after I/R. Different markers for apoptosis and necrosis were also evaluated both at 24 and 72 hr after I/R experimental procedure. LP increased by 37% in ischemic animals vs controls, and this effect was reversed by dapsone treatments. A similar effect was observed regarding nitrotyrosine striatal contents. Myeloperoxidase activity, a marker of inflammatory response, increased 3.7-fold in ischemic animals vs. control rats, and dapsone treatment antagonized that effect. Although apoptosis was increased by the effect of ischemia at both evaluation times, dapsone antagonized that effect only at 72 hr after surgery. Dapsone antagonized all of the I/R end points measured, showing a remarkable ability to decrease markers of damage through antioxidant, antiinflammatory, and anti-apoptotic effects.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Brain Ischemia; Caspase 3; Caspase 9; Chromatography, High Pressure Liquid; Dapsone; Immunoblotting; In Situ Nick-End Labeling; Lipid Peroxidation; Male; Neuroprotective Agents; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine

Ovariectomy resulted in decreased blood flow and hypoxia to the bladder mucosa and smooth muscle. Nitric oxide (NO) played an important role in regulating bladder function during bladder ischemia and reperfusion. This study was designed to evaluate the role of NO on bladder function in the first few days after ovariectomy. Female rabbits were separated into three groups, one which received no medication, premedicated with N(omega)-nitro-L-arginine methyl ester (L-NAME) and the third treated with L-arginine. Non-ovariectomized controls and at 1 and 3 days post-ovariectomy, animals from each group were euthanized. Cystometry and in vitro isometric contractile responses were recorded and the oxidative stress markers, nitrotyrosine and protein carbonylation were determined. L-NAME treatment did not significantly alter bladder function after ovariectomy. L-Arginine fed, ovariectomized rabbits had lower intravesical pressure and better contractile responses to all forms of stimulation than the ovariectomized rabbits with or without L-NAME. Furthermore, the ovariectomized ones with or without L-NAME had higher oxidative stress markers than L-arginine fed rabbits. This study clearly demonstrates that feeding rabbits with L-arginine can protect the bladder from oxidative free radical damage following short-term ovariectomy.

Topics: Analysis of Variance; Animals; Arginine; Blotting, Western; Carbachol; Female; Free Radicals; Isometric Contraction; NG-Nitroarginine Methyl Ester; Nitric Oxide; Ovariectomy; Oxidative Stress; Potassium Chloride; Protein Carbonylation; Rabbits; Reperfusion Injury; Tyrosine; Urinary Bladder; Urination

Ischemia-reperfusion (I/R) is a major mechanism of liver injury following hepatic surgery or transplantation. Despite numerous reports on the role of oxidative/nitrosative stress and mitochondrial dysfunction in hepatic I/R injury, the proteins that are oxidatively modified during I/R damage are poorly characterized. This study was aimed at investigating the oxidatively modified proteins underlying the mechanism for mitochondrial dysfunction in hepatic I/R injury. We also studied the effects of a superoxide dismutase mimetic/peroxynitrite scavenger metalloporphyrin (MnTMPyP) on oxidatively modified proteins and their functions.. The oxidized and/or S-nitrosylated mitochondrial proteins from I/R-injured mouse livers with or without MnTMPyP pretreatment were labeled with biotin-N-maleimide, purified with streptavidin-agarose, and resolved by 2-dimensional gel electrophoresis. The identities of the oxidatively modified proteins were determined using mass spectrometric analysis. Liver histopathology, serum transaminase levels, nitrosative stress markers, and activities of oxidatively modified mitochondrial proteins were measured.. Comparative 2-dimensional gel analysis revealed markedly increased numbers of oxidized and S-nitrosylated mitochondrial proteins following hepatic I/R injury. Many key mitochondrial enzymes involved in cellular defense, fat metabolism, energy supply, and chaperones were identified as being oxidatively modified proteins. Pretreatment with MnTMPyP attenuated the I/R-induced increased serum transaminase levels, histologic damage, increased inducible nitric oxide synthase expression, and S-nitrosylation and/or nitration of various key mitochondrial proteins. MnTMPyP pretreatment also restored I/R-induced suppressed activities of mitochondrial aldehyde dehydrogenase, 3-ketoacyl-CoA thiolases, and adenosine triphosphate synthase.. These results suggest that increased nitrosative stress is critically important in promoting S-nitrosylation and nitration of various mitochondrial proteins, leading to mitochondrial dysfunction with decreased energy supply and increased hepatic injury.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Energy Metabolism; Enzymes; Liver; Male; Metalloporphyrins; Mice; Mice, Inbred C57BL; Mitochondrial Proteins; Nitric Oxide Synthase Type II; Nitrites; Nitrogen; Oxidation-Reduction; Oxidative Stress; Reperfusion Injury; Sequence Analysis, Protein; Superoxide Dismutase; Tandem Mass Spectrometry; Tyrosine

Reactive oxygen species (ROS) is massively produced in the brain after cerebral ischemia and reperfusion. It reacts strongly with cellular components, which has detrimental effects and leads to neuronal cell death. DJ-1, which was found to be the causative gene of familial Parkinson's disease PARK7, is a multifunction protein, which plays a key role in transcriptional regulation, and a molecular chaperone. In this study, we investigated the neuroprotective effect of DJ-1 against neurodegeneration caused by ischemia/reperfusion injury. Cerebral ischemia was induced in rats by 120 mins of middle cerebral artery occlusion (MCAO) using an intraluminal introduction method. The intrastriatal injection of recombinant glutathione S-transferase-tagged human DJ-1 (GST-DJ-1) markedly reduced infarct size in 2,3,5-triphenyltetrazolium chloride staining at 3 days after MCAO. In addition, we performed a noninvasive evaluation of ischemic size using magnetic resonance imaging and found a significant reduction of infarct size with the administration of GST-DJ-1. In GST-DJ-1-treated rats, behavioral dysfunction and nitrotyrosine formation were significantly inhibited. Furthermore, GST-DJ-1 markedly inhibited H(2)O(2)-mediated ROS production in SH-SY5Y cells. These results indicate that GST-DJ-1 exerts a neuroprotective effect by reducing ROS-mediated neuronal injury, suggesting that DJ-1 may be a useful therapeutic target for ischemic neurodegeneration.

Topics: Animals; Behavior, Animal; Cerebral Infarction; Humans; Intracellular Signaling Peptides and Proteins; Magnetic Resonance Imaging; Nerve Degeneration; Oncogene Proteins; Protein Deglycase DJ-1; Rats; Reactive Oxygen Species; Reperfusion Injury; Tyrosine

The aim of this experimental study was to investigate whether dimethylsulfoxide (DMSO) has protective effects on spinal cord ischemia-reperfusion (I/R) injury. New Zealand rabbits were enrolled in the study. In addition to the control group, the study group received 0.1 mL/kg DMSO prior to ischemia. Blood samples were taken to obtain nitrite-nitrate levels during the surgical procedure. After neurological evaluation at 24 hr of reperfusion, lumbar spinal cords were removed for electron microscopic evaluation and malondialdehyde and myeloperoxidase measurements. The mean Tarlov score of the DMSO group was higher than that of the control group. Electron microscopic examination was carried out with tissue samples at 24 hr of reperfusion. The DMSO group had better preservation with the electron microscopic scoring compared to the control group. Malondialdehyde and myeloperoxidase levels were decreased in the DMSO group compared to the control group. Nitrite-nitrate levels were also lower in the DMSO group compared to control at 5 and 30 min of reperfusion. This study demonstrates a considerable neuroprotective effect of DMSO on neurological, biochemical, and histopathological analyses during periods of spinal cord I/R injury in rabbits. Although there was a difference between the DMSO and control groups in all measured parameters in our study, this was not statistically significant. DMSO deserves further investigation related with spinal cord ischemia and reperfusion. We should also consider the effect of DMSO when we use it as a solvent or vehicle during experimental I/R models.

Topics: Animals; Aorta, Abdominal; Dimethyl Sulfoxide; Disease Models, Animal; Free Radical Scavengers; Ligation; Malondialdehyde; Neuroprotective Agents; Nitrates; Nitrites; Peroxidase; Rabbits; Reperfusion Injury; Solvents; Spinal Cord; Spinal Cord Ischemia; Time Factors; Tyrosine

Ischemia-reperfusion injury (IRI) is characterized by ATP depletion in the ischemic phase, followed by a rapid increase in reactive oxygen species, including peroxynitrite in the reperfusion phase. In this study, we examined the role of peroxynitrite on cytotoxicity and apoptosis in an in vitro model of ATP depletion-recovery. Porcine proximal tubular epithelial (LLC-PK(1)) cells were ATP depleted for either 2 h (2/2) or 4 h (4/2) followed by recovery in serum free medium for 2 h. A subset of cells was treated with 100 microM of the peroxynitrite scavenger, iron (III) tetrakis (N-methyl-4'pyridyl) porphyrin pentachloride (FeTMPyP) 30 min prior to and during treatment/recovery. Treatment with FeTMPyP reduced cytotoxicity and superoxide levels at both the 2/2 and 4/2 time points, however FeTMPyP decreased nitric oxide only at the 2/2 time point. FeTMPyP also partially blocked caspase-3 and caspase-8 activation at both 2/2 and 4/2 time points. At the 4/2 time point, FeTMPyP also partially inhibited the ATP depletion mediated increase in tumor necrosis factor alpha (TNF-alpha) and decreased Bax and FasL gene expression. These data show that peroxynitrite induces apoptosis by activation of multiple pathways depending on length and severity of insult following ATP depletion-recovery.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Enzyme Activation; L-Lactate Dehydrogenase; LLC-PK1 Cells; Metalloporphyrins; Mitochondria; Models, Animal; Peroxynitrous Acid; Reperfusion Injury; Swine; Tyrosine

The role of iron in the pathogenesis of cardio-vascular disorders is still controversial. We studied the effects of iron perturbations on myocardial injury upon temporary ischemia/reperfusion. C57BL/6J male mice were injected with iron dextran for 2 weeks while controls received saline. Mice were then subjected to 30 min of myocardial ischemia and subsequent reperfusion for 6-24 h. Tissue damage was quantified histologically and by troponin T determination. The expressions of tumor necrosis factor-alpha (TNF-alpha), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) were investigated in non-ischemic and ischemic regions of both groups. After myocardial ischemia and reperfusion, troponin T levels, as a marker of myocardial damage, were significantly reduced in iron-treated mice as compared to control mice (P < 0.05). Under the same conditions the infarction area and damage score were significantly lower in iron-treated animals. In parallel, TNF-alpha and SOD expressions were increased in infarcted regions of iron-treated mice as compared to controls, whereas myocardial iNOS expression was significantly lower in iron-treated mice. Although, iron challenge increased radical formation and TNF-alpha expression in vivo, this did not result in myocardial damage which may be linked to the parallel induction of SOD. Importantly, iron treatment inhibited iNOS expression. Since, an increased nitric oxide (NO) formation has been linked to cardiac damage after acute myocardial infarction, iron may exert short time cardio-protective effects after induction of ischemia/reperfusion via decreasing iNOS formation.

Topics: Animals; Iron; Male; Mice; Mice, Inbred C57BL; Myocardial Ischemia; Nitric Oxide Synthase Type II; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Tyrosine

2,2,5,7,8-Pentamethyl-6-hydroxychromane (PMC) is the most potent analogue of alpha-tocopherol for anti-oxidation. It is more hydrophilic than other alpha-tocopherol derivatives and has potent free radical-scavenging activity. In the present study, PMC significantly attenuated middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Administration of PMC at 20mg/kg, showed marked reductions in infarct size compared with that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in HIF-1alpha, active caspase-3, iNOS, and nitrotyrosine expressions in ischemic regions. These expressions were markedly inhibited by treatment with PMC (20mg/kg). In addition, PMC (4-12 microM) inhibited respiratory bursts in human neutrophils stimulated by fMLP (800 nM) and PMA (320 nM). Furthermore, PMC (6, 12, and 60 microM) also significantly inhibited neutrophil migration stimulated by leukotriene B(4) (160 nM). An electron spin resonance (ESR) method was conducted on the scavenging activity of PMC on the free radicals formed. PMC (12 microM) greatly reduced the ESR signal intensities of superoxide anion, hydroxyl radical, and methyl radical formation. In conclusion, we demonstrate a potent neuroprotective effect of PMC on MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by inhibition of free radical formation, followed by inhibition of HIF-1alpha activation, apoptosis formation (active caspase-3), neutrophil activation, and inflammatory responses (i.e., iNOS and nitrotyrosine expressions), resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, PMC treatment may represent a novel approach to lowering the risk or improving function in ischemia-reperfusion brain injury-related disorders.

Topics: alpha-Tocopherol; Animals; Antioxidants; Caspase 3; Chromans; Gene Expression Regulation; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Ischemia, Brain; Male; Molecular Structure; Neuroprotective Agents; Neutrophil Activation; Neutrophils; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine

In normal conditions, nitric oxide (NO) is oxidized to the anion nitrite, but in hypoxia, this nitrite may be reduced back to NO by the nitrite reductase action of deoxygenated hemoglobin, acidic disproportionation, or xanthine oxidoreductase (XOR). Herein, is investigated the effects of topical sodium nitrite administration in a rat model of renal ischemia/reperfusion (I/R) injury. Rats were subjected to 60 min of bilateral renal ischemia and 6 h of reperfusion in the absence or presence of sodium nitrite (30 nmol) administered topically 1 min before reperfusion. Serum creatinine, serum aspartate aminotransferase, creatinine clearance, fractional excretion of Na(+), and plasma nitrite/nitrate concentrations were measured. The nitrite-derived NO-generating capacity of renal tissue was determined under acidic and hypoxic conditions by ozone chemiluminescence in homogenates of kidneys that were subjected to sham, ischemia-only, and I/R conditions. Nitrite significantly attenuated renal dysfunction and injury, an effect that was abolished by previous treatment of rats with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (2.5 mumol intravenously 5 min before ischemia and 50 nmol topically 6 min before reperfusion). Renal tissue homogenates produced significant amounts of NO from nitrite, an effect that was attenuated significantly by the xanthine oxidoreductase inhibitor allopurinol. Taken together, these findings demonstrate that topically administered sodium nitrite protects the rat kidney against I/R injury and dysfunction in vivo via the generation, in part, of xanthine oxidoreductase-catalyzed NO production. These observations suggest that nitrite therapy might prove beneficial in protecting kidney function and integrity during periods of I/R such as those encountered in renal transplantation.

Topics: Animals; Disease Models, Animal; Kidney; Male; Nitrates; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Rats; Rats, Wistar; Renal Circulation; Reperfusion Injury; Tyrosine; Xanthine Dehydrogenase

Damage of the placenta resulting from ischemia-reperfusion is important to the pathophysiology of preeclampsia. Here we investigated whether low concentrations of glyceryl trinitrate (GTN), a nitric oxide mimetic with anti-apoptotic properties, inhibit hypoxia/reoxygenation-induced apoptosis in the syncytiotrophoblast of chorionic villous explants from human placentas. Compared with villi analyzed immediately after delivery or maintained under normoxic conditions, villi exposed to a 6-hour cycle of hypoxia/reoxygenation exhibited greater numbers of syncytiotrophoblasts with terminal dUTP nick-end labeling (TUNEL)-positive nuclei in the syncytiotrophoblast. This increased number of TUNEL-positive nuclei was paralleled by higher levels of 4-hydroxynonenal (marker of lipid peroxidation), nitrotyrosine residues, and active caspase-3 and polyADP-ribose polymerase expression. Morphological analysis of explants exposed to hypoxia/reoxygenation revealed apoptotic and aponecrotic features similar to those of chorionic villi from preeclamptic pregnancies. Treatment with GTN during the hy-poxia/reoxygenation cycle blocked the increases in the number of TUNEL-positive nuclei and in the levels of 4-hydroxynonenal, nitrotyrosine, and active caspase-3. Incubation with GTN also attenuated the hypoxia/reoxygenation-induced polyADP-ribose polymerase expression and the apoptotic and aponecrotic morphological alterations. These results suggest that small concentrations of nitric oxide protect chorionic villi from hypoxia/reoxygenation-induced damage and provide a rationale for the use of low doses of nitric oxide mimetics in the treatment and/or prevention of preeclampsia.

Topics: Aldehydes; Apoptosis; Blotting, Western; Caspase 3; Collagen Type XI; Female; Humans; Hypoxia; Immunohistochemistry; In Situ Nick-End Labeling; Microscopy, Electron, Transmission; Nitroglycerin; Organ Culture Techniques; Pre-Eclampsia; Pregnancy; Reperfusion Injury; Tocolytic Agents; Trophoblasts; Tyrosine

During renal ischemia-reperfusion (I/R) injury, apoptosis has been reported as a very important contributor to final kidney damage. The determinant role of cytoskeleton derangement in the development of apoptosis has been previously reported, but a clear description of the different mechanisms involved in this process has not been yet provided. The aim of our study was to know the role of peroxynitrite as an inductor of cytoskeleton derangement and apoptosis during renal I/R. Based on a rat kidney I/R model, using experiments in which both the actin cytoskeleton and peroxynitrite generation were pharmacologically manipulated, results indicate that the peroxynitrite produced during the I/R-derived oxidative stress state is able to provoke cytoskeleton derangement and apoptosis development. Thus control of peroxynitrite generation during I/R could be an effective tool for the improvement of cytoskeleton damage and reduction of apoptosis incidence in renal I/R injury.

Topics: Animals; Apoptosis; Blood Pressure; Blood Urea Nitrogen; Blotting, Western; Caspase 3; Cytoskeleton; In Situ Nick-End Labeling; Kidney Diseases; Kidney Function Tests; Male; Microscopy, Confocal; Peroxynitrous Acid; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine

Reactive oxygen and nitrogen species have been implicated in ischemia-reperfusion (I/R) injury. Metalloporphyrins (MP) are stable catalytic antioxidants that can scavenge superoxide, hydrogen peroxide, peroxynitrite and lipid peroxyl radicals. Studies were conducted with three manganese-porphyrin (MnP) complexes with varying superoxide dimutase (SOD) and catalase catalytic activity to determine if the MnP attenuates I/R injury in isolated perfused mouse livers. The release of the hepatocellular enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) was maximal at 1 min reperfusion, decreased rapidly and increased gradually by 90 min. Manganese tetrakis-(N-ethyl-2 pyridyl) porphyrin (MnTE-2-PyP) decreased ALT, AST, LDH at 1-90 min reperfusion, while manganese tetrakis-(N-methyl-2 pyridyl) porphyrin (MnTM-2-PyP) and manganese tetrakis-(ethoxycarbonyl) porphyrin (MnTECP) decreased ALT and LDH from 5 to 90 min reperfusion. The release of thiobarbituric acid-reacting substances (TBARS) was diminished by MnTE-2-PyP and MnTM-2-PyP at 90 min. The extent of protein nitration (nitrotyrosine, NT) was decreased in all three MnPs treated livers. These results demonstrate that MnP complexes can attenuate hepatic I/R injury and may have therapeutic implications in disease states involving oxidants.

Topics: Alanine Transaminase; Animals; Antioxidants; Aspartate Aminotransferases; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Ischemia; L-Lactate Dehydrogenase; Lipid Peroxidation; Liver; Liver Diseases; Male; Metalloporphyrins; Mice; Mice, Inbred C57BL; Nitrosation; Reactive Oxygen Species; Reperfusion Injury; Thiobarbituric Acid Reactive Substances; Tyrosine

To determine whether age influences the nitric oxide system response to ischemia in the cerebellum, we have analyzed the levels of nitrogen oxides (NOx) and the expression of the different nitric oxide synthase isoforms (NOS) in mature adult (4-5 months old) and aged rats (24-27 months old) subjected to a transient global ischemia/reperfusion (I/R) model. We also analyzed the nitrated proteins and the glial fibrillary acidic protein (GFAP) expression. NOx concentration in adult rats, which more than doubled the values found in the aged rats, decreased after the ischemia and reperfusion. However, in the aged animals, these NOx levels did not significantly change after I/R. Constitutive isoforms were first down-regulated in the ischemic period, in both adult and aged animals. However, after 6 h of reperfusion, these isoforms were up-regulated, but only in aged rats. After I/R, iNOS was up-regulated in adults but down-regulated in the aged rats. Hence, after an episode of transient global ischemia and reperfusion, the aged cerebellum maintains a balanced NO production, silencing the iNOS isoform and inducing a weak expression of nNOS and eNOS; this allows NO physiological functions while avoiding possible undesirable effects such as the nitrative damage or astrocyte activation.

Topics: Aging; Animals; Astrocytes; Brain Ischemia; Cerebellar Diseases; Cerebellum; Down-Regulation; Glial Fibrillary Acidic Protein; Gliosis; Isoenzymes; Male; Nerve Tissue Proteins; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Rats; Reperfusion Injury; Time Factors; Tyrosine; Up-Regulation

Nuclear factor-kappaB is a key transcriptional factor in the regulation of inflammatory factors that are involved in tissue reperfusion injury, but conflicting data have been presented in the literature. The proteasome regulates proteins that control cell-cycle progression and apoptosis, and inhibition of the proteasome has been shown to reduce nuclear factor-kappaB activation and reperfusion injury. Although bortezomib is a potent proteasome inhibitor, its role in skeletal muscle reperfusion injury has not been documented, and its effects on the regulation of inflammatory factors in reperfused tissue are unclear. In this study, the authors investigated the role of nuclear factor-kappaB in skeletal muscle reperfusion injury and the effect of bortezomib (a proteasome inhibitor) on reperfusion injury.. Pedicled cremaster muscle flaps from bortezomib-treated and phosphate-buffered saline-treated control mice were subjected to 4.5 hours of ischemia and 90 minutes of reperfusion.. During reperfusion, arterial diameters and blood flow recovered earlier and more completely in bortezomib-treated muscle than in controls. Compared with controls, Western blot analysis demonstrated a significant reduction in degradation of nuclear factor-kappaB inhibitory protein and expression of inducible nitric oxide synthase protein in bortezomib-treated muscle at the end of reperfusion. Immunohistochemistry showed decreased nuclear factor-kappaB p65-binding activity and down-regulated protein expression of intercellular adhesion molecule-1 and nitrotyrosine, accompanied by less muscle edema and inflammation as proven by histologic examination.. Bortezomib effectively blocks nuclear factor-kappaB activation in attenuating muscle reperfusion injury through inhibiting nuclear factor-kappaB inhibitory protein degradation. Therefore, inhibition of proteasome activity may provide a novel therapeutic strategy for the treatment of skeletal muscle reperfusion injury.

Topics: Animals; Blood Vessels; Boronic Acids; Bortezomib; Drug Evaluation, Preclinical; Intercellular Adhesion Molecule-1; Intracellular Signaling Peptides and Proteins; Laser-Doppler Flowmetry; Male; Mice; Microscopy, Video; Muscle, Skeletal; NF-kappa B p50 Subunit; Nitric Oxide Synthase Type II; Protease Inhibitors; Proteasome Inhibitors; Proteins; Pyrazines; Random Allocation; Reperfusion Injury; Surgical Flaps; Tyrosine

Partial bladder outlet obstruction (PBOO) results in cellular damage due to ischemia and reperfusion injury. Our study seeks to establish how early this damage can occur and the role that nitric oxide may play in its pathophysiology. Surgical PBOO (1, 3, and 7 days) were performed on male New Zealand White rabbits. Half of the animals were premedicated for 3 days with N(G)-nitro-l-arginine methyl ester(l-NAME), an inhibitor of nitric oxide synthase before obstruction. Bladder weight increased with duration of PBOO but was significantly lower at 3 and 7 days in animals treated with l-NAME compared with their untreated counterparts. Contractile function decreased progressively with PBOO duration. At 1 day postobstruction, bladder contractility was significantly lower in the l-NAME rabbits than in the untreated rabbits. At 3 and 7 days, contractility of the l-NAME bladders was equal or higher than the untreated bladders. The level of hypoxia at 1 day after obstruction was significantly higher in the l-NAME-treated animals than in the untreated controls but equal at 3 and 7 days obstruction. Increased nitrotyrosine was seen by Western blot in all obstructed animals. However, the amount was significantly less in the l-NAME-treated animals at 3 and especially at 7 days. Nerve density decreased progressively after obstruction; however, it decreased to a significantly lesser degree in the l-NAME-treated bladders than in the untreated groups. These results suggest that l-NAME pretreatment enhanced ischemic damage at 1 day after obstruction but protected the bladder from nitric oxide-generated free radical damage at the later time periods by inhibiting the generation of nitrotyrosine.

Topics: Animals; In Vitro Techniques; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Organ Size; Prostatic Hyperplasia; Rabbits; Reperfusion Injury; Tyrosine; Urinary Bladder; Urinary Bladder Neck Obstruction

We have demonstrated previously that preischemic treatment with FK409 [(+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide], a spontaneous nitric oxide (NO) donor, markedly improves ischemia/reperfusion-induced renal injury. However, there is conflicting information (renoprotective or cytotoxic) as to the contribution of NO to ischemic acute renal failure (ARF). In the present study, we investigated the effect of postischemic treatment with FK409 (1, 3, and 10 mg/kg i.v.) at 6 h after reperfusion on ischemic ARF, in comparison with the preischemic treatment effect. Ischemic ARF was induced by clamping of the left renal artery and vein for 45 min, followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function in ARF rats markedly decreased at 24 h after reperfusion. Histopathological examination of the kidney of ARF rats revealed severe renal damage. In contrast to the renoprotective effect by preischemic treatment, postischemic treatment with FK409 aggravated the ischemia/reperfusion-induced renal dysfunction and histological damage. Immunohistochemical analysis of renal sections obtained from ARF rats revealed positive staining for nitrotyrosine, a biomarker of peroxynitrite formation, in injured tubular cells, and more intense staining was observed in renal tissues from the animals that received postischemic treatment with FK409. On the other hand, the formation of nitrotyrosine, neutrophil infiltration into renal tissues, and renal superoxide production, all of which were enhanced in ARF rats, were efficiently attenuated by the preischemic treatment with FK409. These results demonstrate that, although preischemic treatment with an NO donor is renoprotective, postischemic treatment with the same agent aggravates the ischemia/reperfusion-induced renal injury, probably through peroxynitrite overproduction.

Topics: Acute Kidney Injury; Animals; Blood Urea Nitrogen; Ischemia; Kidney; Male; Neutrophil Infiltration; Nitric Oxide Donors; Nitric Oxide Synthase Type II; Nitro Compounds; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Superoxides; Time Factors; Tyrosine

This study investigated the effect of peroxynitrite (ONOO-) on pressure-induced myogenic activity and vascular smooth muscle (VSM) actin of isolated posterior cerebral arteries (PCAs).. Histochemical staining of nitrotyrosine (NT) was used to demonstrate the presence of ONOO- in the cerebrovasculature after 1 hour of middle cerebral artery occlusion with 30 minutes of reperfusion. To determine the effect of ONOO- on pressure-induced myogenic activity, third-order PCAs from nonischemic animals were isolated and mounted in an arteriograph chamber. Diameter in response to changes in pressure was determined in the absence and presence of ONOO- (10(-8) to 10(-4) mol/L). Filamentous actin (F-actin) and globular actin (G-actin) were quantified using confocal microscopy in PCAs with and without exposure to ONOO-.. NT staining of vascular cells was greater in ischemic brain versus sham animals (56+/-3% versus 35+/-3%; P<0.01). Addition of low concentrations of ONOO- (< or =10(-6) mol/L) to isolated PCAs caused constriction from 129+/-16 microm to 115+/-15 microm (P<0.01), whereas concentrations >10(-6) mol/L caused dilation of spontaneous tone and loss of myogenic activity in the physiological range of 50 to 125 mm Hg, increasing diameter from 130+/-6 to 201+/-5 microm at 75 mm Hg (P<0.01). In addition, the diminished myogenic activity was associated with a 4.5-fold decrease in F-actin content of VSM and a 27% increase in G-actin content (P<0.01).. This study demonstrates that ONOO- affects the myogenic activity of cerebral arteries and causes F-actin depolymerization in VSM, a consequence that could promote vascular damage during reperfusion injury and further brain injury.

Topics: Actins; Animals; Blood Pressure; Brain; Cerebral Arteries; Humans; Infarction, Middle Cerebral Artery; Ischemia; Male; Microscopy, Confocal; Muscle, Smooth, Vascular; Peroxynitrous Acid; Posterior Cerebral Artery; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Time Factors; Tyrosine

In this study, we evaluated the effect of tyrphostin AG126, a tyrosine kinase inhibitor, in the splanchnic artery occlusion (SAO) shock mediated injury. SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min. After 1 h of reperfusion, SAO shocked rats developed a significant fall in mean arterial blood pressure. Ileum analysis revealed that SAO shock is characterized by a significant (P<0.01) induction in TNF-alpha and IL-1 ileum levels, while immunohistochemistry examination of necrotic ileum demonstrated a marked increase in the immunoreactivity in intracellular adhesion molecule (ICAM-1) and nitrotyrosine formation. A significant increase in myeloperoxidase activity (P<0.01) was also observed in rats subjected to ischemia-reperfusion injury. Tyrphostin AG126, given intraperitoneally 30 min before ischemia at the dose of 5 mg/kg, significantly improved mean arterial blood pressure, markedly reduced TNF-alpha and IL-1beta levels and the positive staining of ICAM-1 into the reperfused ileum. Tyrphostin AG126 significantly improved the histological status of the reperfused tissue. In conclusion, this study demonstrates that tyrphostin AG126 exerts multiple protective effects in splanchnic artery occlusion/reperfusion shock and suggests that this tyrosine kinase inhibitor may be a candidate for consideration as a therapeutic intervention for ischemia-reperfusion injury.

Topics: Animals; Blood Pressure; Disease Models, Animal; Immunohistochemistry; Intercellular Adhesion Molecule-1; Interleukin-1beta; Intestines; Lipid Peroxidation; Male; Mesenteric Artery, Superior; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine; Tyrphostins

Previous studies indicate that endothelial nitric oxide synthase (eNOS) function is impaired in diabetes as a result of increased vascular generation of reactive oxygen species. We hypothesized that eNOS gene therapy would augment NO. bioavailability and protect against hepatic ischemia-reperfusion (I-R) injury in type 2 diabetes mellitus. We developed a transgenic (Tg) diabetic mouse in which eNOS is systemically overexpressed. We also examined the effects of hepatic eNOS adenovirus therapy in diabetic mice. Diabetic (db/db) and nondiabetic mice were subjected to hepatic I-R injury. In nondiabetic mice, genetic overexpression of eNOS (both eNOS-Tg and eNOS adenovirus) resulted in hepatoprotection. In contrast, hepatic I-R injury was significantly increased in the db/db eNOS-Tg mouse, as serum alanine aminotransaminase (ALT) levels were increased by 3.3-fold compared with diabetic controls. Similarly, eNOS adenovirus treatment resulted in a 3.2-fold increase in serum ALT levels as compared with diabetic controls. We determined that hepatic eNOS was dysfunctional in the db/db mouse and increased genetic expression of eNOS resulted in greater production of peroxynitrite. Treatment with the eNOS cofactor tetrahydrobiopterin (BH4) or the BH4 precursor sepiapterin resulted in a significant decrease in serum ALT levels following I-R injury. We present clear examples of the protective and injurious nature of NO. therapy in I-R. Our data indicate that eNOS exists in an "uncoupled" state in the setting of diabetes and that "recoupling" of the eNOS enzyme with cofactor therapy is beneficial.

Topics: Animals; Biological Availability; Biopterins; Diabetes Mellitus, Type 2; Drug Synergism; Genetic Therapy; Liver; Metalloporphyrins; Mice; Mice, Inbred Strains; Mice, Transgenic; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Nitrites; Phenotype; Phosphorylation; Pterins; Reperfusion Injury; Severity of Illness Index; Tyrosine

In this study we investigated the effect of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases, on ischemia-reperfusion injury (IRI) following murine pancreas transplantation. Pancreatic grafts were exposed to prolonged cold ischemia times (CIT) and different treatment regimens: normal saline (S), S + 16 h CIT, BH4 50 mg/kg + 16 h CIT. Nontransplanted animals served as controls. Graft microcirculation was analyzed by means of functional capillary density (FCD) and capillary diameters (CD) after 2 h reperfusion using intravital microscopy. Quantification of inflammatory responses (mononuclear infiltration) and endothelial disintegration (edema formation) was done by histology (hematoxylin and eosin), and peroxynitrite formation assessed by nitrotyrosine immunostaining. FCD was significantly reduced after prolonged CIT, paralleled by increased peroxynitrite formation as compared with controls (all p < 0.05). Microcirculatory changes correlated significantly with intragraft peroxynitrite generation (Spearman: r = -0.56; p < 0.01). Pancreatic grafts treated with BH4 displayed markedly higher FCD values (p < 0.01) and abrogated nitrotyrosine staining (p = 0.03). CD were not significantly different in any group. Histology showed increased inflammation, interstitial edema, hemorrhage, acinar vacuolization and focal areas of necrosis after 16 h CIT, which was diminished by BH4 administration (p < 0.01). BH4 treatment significantly reduces post-ischemic deterioration of microcirculation as well as histologic damage and might be a promising novel strategy in attenuating IRI following pancreas transplantation.

Topics: Animals; Biopterins; Male; Mice; Mice, Inbred C57BL; Microcirculation; Pancreas Transplantation; Peroxynitrous Acid; Reperfusion Injury; Transplantation, Homologous; Tyrosine

There is increasing evidence to suggest that reactive oxygen and nitrogen species play a role in the pathogenesis of renal ischemia-reperfusion (I/R) injury. This study was designed to determine the possible protective effects of trapidil treatment against oxidative and nitrosative tissue injury of kidney induced by I/R. A renal I/R injury was induced by a left renal pedicle occlusion by ischemia for 45 minutes, followed by 1 hour of reperfusion with contralateral nephrectomy in I/R and I/R + trapidil groups. Trapidil (8 mg/kg intravenously) was administrated immediately before reperfusion phase. At the end of the reperfusion period, rats were killed. Then, renal tissue samples were taken for biochemical analysis and histopathological evaluation, and blood samples were obtained to determinate serum urea, aspartate aminotransferase (AST), and tumor necrosis factor-alpha (TNF-alpha) levels. Ischemia-reperfusion injury caused significant increases in myeloperoxidase activity and malondialdehyde and 3-nitrotyrosine levels in renal tissue and elevated serum urea, AST, and TNF-alpha levels. In addition, severe deterioration of renal morphology was seen in the I/R group. Trapidil treatment significantly reduced in biochemical parameters, as well as serum urea, AST, and TNF-alpha levels. Furthermore, renal tissue injury was markedly attenuated with trapidil treatment. These data suggest that reactive oxygen species and reactive nitrogen species play a causal role in I/R-induced renal tissue, and trapidil has a renoprotective effect against oxidative and nitrosative kidney damage.

Topics: Animals; Kidney; Malondialdehyde; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Trapidil; Tumor Necrosis Factor-alpha; Tyrosine; Vasodilator Agents

Here, we investigate the effects of renal ischemia/reperfusion (I/R) on the degree of renal injury, dysfunction, and inflammation in interleukin (IL)-6 knockout (IL-6(-/-)) mice and mice administered a monoclonal antibody against IL-6. IL-6(-/-) mice were subjected to bilateral renal artery occlusion (30 min) and reperfusion (24 h). At the end of experiments, indicators and markers of renal dysfunction, injury, and inflammation were measured. Kidneys were used for histological evaluation of renal injury. Renal expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and P-selectin, as well as nitration of proteins in the kidney, were determined using immunohistochemistry. In addition, wild-type mice were pretreated (24 and 1 h before ischemia) with an IL-6 antibody to mimic the effects that would be seen in IL-6(-/-) mice. IL-6(-/-) mice and wild-type mice administered the IL-6 antibody demonstrated significantly reduced plasma urea and creatinine levels, indicating reduction of renal dysfunction caused by I/R. Neutrophil infiltration was also significantly reduced in IL-6(-/-) mice and wild-type mice administered the IL-6 antibody subjected to renal I/R. Proinflammatory cytokines (tumor necrosis factor-alpha and IL-1beta) in renal tissues were significantly attenuated in IL-6(-/-) mice to levels seen in wild-type mice. IL-6(-/-) mice demonstrated reduced histological evidence of tubular injury and markedly reduced immunohistochemical evidence of ICAM-1, P-selectin, and nitrotyrosine when subjected to renal I/R. We propose that endogenous IL-6 enhances the degree of renal injury, dysfunction, and inflammation caused by I/R of the kidney by promoting the expression of adhesion molecules and subsequent oxidative and nitrosative stress.

Topics: Animals; Creatinine; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-6; Kidney; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; Nephritis; P-Selectin; Reperfusion Injury; Tumor Necrosis Factor-alpha; Tyrosine

In hepatic ischaemia/reperfusion injury, activated liver macrophages (Kupffer cells) are dominantly regulated by a transcription factor, nuclear factor kappaB (NFkappaB), with respect to expression of inflammatory cytokines, acute phase response proteins, and cell adhesion molecules.. We assessed whether inactivation of NFkappaB in the liver could attenuate total hepatic warm ischaemia/reperfusion injury.. We studied rats with hepatic overexpression of inhibitor kappaBalpha super-repressor (IkappaBalpha SR) caused by a transgene introduced using an adenoviral vector. Hepatic ischaemia/reperfusion injury was induced under warm conditions by total occlusion of hepatoduodenal ligament structures for 20 minutes, followed by reperfusion. Controls included uninfected and control virus (AdLacZ) infected rats.. IkappaBalpha SR was overexpressed in Kupffer cells as well as in hepatocytes, blocking nuclear translocation of NFkappaB (p65) into the nucleus after reperfusion. Gene transfection with IkappaBalpha SR, but not with LacZ, markedly attenuated ischaemia/reperfusion injury, suppressing inducible nitric oxide synthase and nitrotyrosine expression in the liver. Moreover, no remarkable hepatocyte apoptosis was detected under IkappaBalpha SR overexpression.. Adenoviral transfer of the IkappaBalpha SR gene in the liver ameliorates short term warm ischaemia/reperfusion injury, possibly through attenuation of hepatic macrophage activation.

Topics: Adenoviridae; Aldehydes; Animals; Blotting, Western; Gene Transfer Techniques; Hepatocytes; I-kappa B Proteins; Kupffer Cells; Liver; Male; NF-kappa B; NF-KappaB Inhibitor alpha; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tumor Necrosis Factor-alpha; Tyrosine

Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants, and/or a depletion of antioxidants. A considerable body of recent evidence suggests that oxidative stress and exaggerated production of reactive oxygen species play a major role in several aspects ischemia and reperfusion. Hypericum perforatum is a medicinal plant species containing many polyphenolic compounds, namely flavonoids and phenolic acids. Because polyphenolic compounds have high antioxidant potential, in this study we evaluated the effect of H. perforatum extract on splanchnic artery occlusion (SAO) shock-mediated injury. SAO shock was induced in rats by clamping the superior mesenteric artery and the celiac trunk for 45 min. After 1 h of reperfusion, SAO-shocked rats developed a significant fall in mean arterial blood pressure. Treatment of rats with H. perforatum extract (applied at 25 mg/kg 15 min before reperfusion) significantly reduced a significant fall in mean arterial blood pressure and the migration of polymorphonuclear cells caused by SAO-shock. H. perforatum extract also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase and malondialdehyde caused by SAO shock in the ileum. Immunohistochemical analysis for nitrotyrosine and for poly ADP-ribosylated proteins revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine and poly ADP-ribosylated proteins was markedly reduced in tissue sections obtained from SAO-shocked rats that had received H. perforatum extract. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin and for intercellular adhesion molecule-1 in the vascular endothelial cells. H. perforatum extract treatment markedly reduced the intensity and degree of P-selectin and intercellular adhesion molecule-1 in tissue section from SAO-shocked rats. H. perforatum extract treatment significantly improved survival. In conclusion, this study demonstrates that H. perforatum extract exerts multiple protective effects in splanchnic artery occlusion-reperfusion shock and suggests that H. perforatum extract may be a candidate for consideration as a therapeutic intervention for ischemia-reperfusion injury.

Topics: Animals; Antioxidants; Blood Pressure; Cytokines; Densitometry; Flavonoids; Hydroxybenzoates; Hypericum; Immunohistochemistry; Intercellular Adhesion Molecule-1; Lipid Peroxidation; Male; Malondialdehyde; Mesenteric Artery, Superior; Neutrophils; P-Selectin; Peroxidase; Phenols; Phytotherapy; Plant Extracts; Poly(ADP-ribose) Polymerases; Polyphenols; Random Allocation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Shock; Time Factors; Treatment Outcome; Tyrosine

The role of nitric oxide (NO) production because of inducible nitric oxide synthase (iNOS) in the pathogenesis of renal ischemia/reperfusion (I/R) injury is unclear. In this study the roles of both iNOS and NO were characterized in a rat model of renal I/R injury. In addition, the effect of iNOS inhibition on renal function was evaluated.. Sprague-Dawley rats underwent 45 min of left renal ischemia and contralateral nephrectomy followed by various periods of reperfusion and renal function analysis [plasma creatinine, fractional excretion of sodium (FENa), creatinine clearance (CrCl), and measurement of plasma and urine NO levels]. In addition, the effect of treatment with 1400W, a highly selective iNOS inhibitor, was evaluated.. Renal dysfunction peaked at 48 h after reperfusion and immunohistochemistry studies revealed iNOS expression in the vasculature (3 h) and renal tubules (48 h) after reperfusion. Renal function improved significantly in treated animals compared to controls [creatinine of 1.1 v. 1.9 mg/dl (P < 0.05) and CrCl of 0.54 v. 0.31 ml/min (P < 0.05), respectively]. In addition, FENa was decreased by 50%, plasma NO levels were significantly lower (32.7 v. 45.7 micromol/L, P < 0.01), and deposition of nitrotyosine in the tubules of treated rats was less than in control animals.. These data support the hypothesis that iNOS and NO are involved in the pathogenesis of renal I/R injury and suggests that use of iNOS inhibitors may be a valuable therapeutic strategy clinical situations where renal I/R may be prevalent.

Topics: Amidines; Animals; Benzylamines; Creatinine; Disease Models, Animal; Endothelins; Enzyme Inhibitors; Kidney Tubules; Male; Nitric Oxide; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tyrosine

Oxidative stress contributes to ischemia/reperfusion neuronal damage in a consecutive 2-phase pattern: an immediate direct cytotoxic effect and subsequent redox-mediated inflammatory insult. The present study was designed to assess the neuroprotective mechanisms of edaravone, a novel free radical scavenger, through antioxidative and anti-inflammatory pathways, from the early period to up to 7 days after ischemia/reperfusion in mice.. Mice were subjected to 60-minute ischemia followed by reperfusion. They were divided into the edaravone group (n=72; with different schedules for first administration) and the vehicle (control) group (n=36). Infarct volume and neurological deficit scores were evaluated at several time points after ischemia. Immunohistochemical analysis for 4-hydroxy-2-nonenal (HNE), 8-hydroxy-deoxyguanosine (8-OHdG), ionized calcium-binding adapter molecule 1 (Iba-1), inducible NO synthase (iNOS), and nitrotyrosine were performed at 24 hours, 72 hours, or 7 days after reperfusion.. Edaravone, even when administrated 6 hours after onset of ischemia/reperfusion, significantly reduced the infarct volume (68.10+/-6.24%; P<0.05) and improved the neurological deficit scores (P<0.05) at 24 hours after reperfusion. Edaravone markedly suppressed the accumulation of HNE-modified protein and 8-OHdG at the penumbra area during the early period after reperfusion (P<0.05) and reduced microglial activation, iNOS expression, and nitrotyrosine formation at the late period.. Our results indicated that edaravone exerts an early neuroprotective effect through the early free radicals scavenging pathway and a late anti-inflammatory effect and suggested that edaravone is important for expansion of the therapeutic time window in stroke patients.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Anti-Inflammatory Agents; Antioxidants; Antipyrine; Brain; Brain Ischemia; Deoxyguanosine; DNA Damage; Edaravone; Electrophoresis, Polyacrylamide Gel; Free Radical Scavengers; Immunoblotting; Immunohistochemistry; Inflammation; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Oxidative Stress; Reperfusion Injury; Time Factors; Tyrosine

It has been established that hypothyroidism protects rats against renal ischemia and reperfusion (IR) oxidative damage. However, it is not clear if hypothyroidism is able to prevent protein tyrosine nitration, an index of nitrosative stress, induced by IR or if antioxidant enzymes have involved in this protective effect. In this work it was explored if hypothyroidism is able to prevent the increase in nitrosative and oxidative stress induced by IR. In addition the activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase was studied. Control and thyroidectomized (HTX) rats were studied 24 h of reperfusion after 60 min ischemia.. Male Wistar rats weighing 380 +/- 22 g were subjected to surgical thyroidectomy. Rats were studied 15 days after surgery. Euthyroid sham-operated rats were used as controls (CT). Both groups of rats underwent a right kidney nephrectomy and suffered a 60 min left renal ischemia with 24 h of reperfusion. Rats were divided in four groups: CT, HTX, IR and HTX+IR. Rats were sacrificed and samples of plasma and kidney were obtained. Blood urea nitrogen (BUN) and creatinine were measured in blood plasma. Kidney damage was evaluated by histological analysis. Oxidative stress was measured by immunohistochemical localization of protein carbonyls and 4-hydroxy-2-nonenal modified proteins. The protein carbonyl content was measured using antibodies against dinitrophenol (DNP)-modified proteins. Nitrosative stress was measured by immunohistochemical analysis of 3-nitrotyrosine modified proteins. The activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase was measured by spectrophotometric methods. Multiple comparisons were performed with ANOVA followed by Bonferroni t test.. The histological damage and the rise in plasma creatinine and BUN induced by IR were significantly lower in HTX+IR group. The increase in protein carbonyls and in 3-nitrotyrosine and 4-hydroxy-2-nonenal modified proteins was prevented in HTX+IR group. IR-induced decrease in renal antioxidant enzymes was essentially not prevented by HTX in HTX+IR group.. Hypothyroidism was able to prevent not only oxidative but also nitrosative stress induced by IR. In addition, the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase seem not to play a protective role in this experimental model.

Topics: Animals; Catalase; Glutathione Peroxidase; Hypothyroidism; Immunohistochemistry; Kidney; Male; Oxidative Stress; Oxidoreductases; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Tyrosine

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are closely involved in the mechanism of skeletal muscle ischemia/reperfusion (I/R) injury. This study was designed to determine the effects of inducible nitric oxide synthase (iNOS) inhibitor 1400 W on the reperfused cremaster muscle in extracellular super-oxide dismutase knockout (EC-SOD(-/-)) mice. The muscle was exposed to 4.5 h of ischemia, followed by 90 min of reperfusion. Mice received either 3 mg/kg of 1400 W or the same amount of phosphate-buffered saline (PBS, as a control) subcutaneously at 10 min before the start of reperfusion. 1400 W treatment markedly improved the recovery speed of vessel diameter and blood flow in the reperfused cremaster muscle of EC-SOD(-/-) mice compared to controls. Histological examination showed reduced edema in the interstitial space and muscle fiber, and reduced density of nitrotyrosine (a marker of total peroxi-nitrate (ONOO(-)) level) in 1400 W-treated muscles compared to controls. Our results suggest that iNOS and ONOO(-) products are involved in skeletal muscle I/R injury. Reduced I/R injury by using selective inhibition of iNOS perhaps works by limiting cytotoxic ONOO(-) generation, a reaction product of nitric oxide (NO) and super-oxide anion (O(2) (-)). Thus, inhibition of iNOS appears to be a treatment strategy for reducing clinical I/R injury.

Topics: Amidines; Animals; Benzylamines; Enzyme Inhibitors; Immunohistochemistry; Male; Mice; Mice, Knockout; Microcirculation; Muscle, Skeletal; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Reperfusion Injury; Superoxide Dismutase; Tyrosine

In this study we evaluated the effect of calpain inhibitor I on splanchnic artery occlusion (SAO) shock-mediated injury. SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min. After 1 h of reperfusion, SAO-shocked rats developed a significant fall in mean arterial blood pressure. Western blot analysis of ileum revealed a marked decrease in of IkappaB-alpha expression, and immunohistochemical examination of necrotic ileum demonstrated a marked increase in the immunoreactivity to P-selectin, intracellular adhesion molecule (ICAM-1), nitrotyrosine formation, and nuclear enzyme poly[adenosine diphosphate (ADP)-ribose] synthase (PARS) activation. An increase in myeloperoxidase activity (143 +/- 22 4.5 U/100 mg wet tissue vs. 4.5 +/- 2.5 U/100 mg wet tissue of sham-operated rats) and in malondialdehyde levels (13.12 +/- 1.2 micromol/100 mg wet tissue vs. 3.9 +/- 1.1 micromol/100 mg wet tissue of sham-operated rats) was also observed in rats subjected to ischemia-reperfusion injury. Calpain inhibitor I, given intraperitoneally 30 min before ischemia at a dose of 15 mg/kg, significantly improved mean arterial blood pressure, markedly reduced IkappaB-alpha degradation and the intensity of P-selectin and ICAM-1 in the reperfused ileum. Calpain inhibitor I also significantly prevented neutrophil infiltration (32.95 +/- 9.82 U/100 mg wet tissue), reduced malondialdehyde levels (6.76 +/- 0.98 micromol/100 mg wet tissue) and markedly improved the histological status of the reperfused tissue. In conclusion, this study demonstrates that calpain inhibitor I exerts multiple protective effects in splanchnic artery occlusion-reperfusion shock and suggests that calpain inhibitor I may be a candidate for consideration as a therapeutic intervention for ischemia-reperfusion injury.

Topics: Animals; Blood Pressure; Blotting, Western; Cysteine Proteinase Inhibitors; Glycoproteins; I-kappa B Proteins; Ileum; Immunohistochemistry; Intercellular Adhesion Molecule-1; Lipid Peroxidation; Male; Malondialdehyde; Neutrophils; NF-KappaB Inhibitor alpha; P-Selectin; Peroxidase; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Shock; Splanchnic Circulation; Time Factors; Tyrosine

This study was carried out to elucidate whether the protective activity of (-)-epicatechin 3-O-gallate (ECg) against excessive peroxynitrite (ONOO(-)) production, is distinct from the activity of several well-known free radical inhibitors, the ONOO(-) inhibitors ebselen and uric acid, the superoxide anion (O(2)(-)) scavenger copper zinc superoxide dismutase (CuZnSOD) and the selective inducible nitric oxide synthase inhibitor L-N(6)-(1-iminoethyl)lysine hydrochloride (L-NIL). To generate ONOO(-), male Wistar rats (n = 6/group) were subjected to ischaemia-reperfusion process together with lipopolysaccharide (LPS) injection. Although ECg did not scavenge the ONOO(-) precursors nitric oxide (NO) and O(2)(-), it reduced the 3-nitrotyrosine level, a property similar to that of uric acid, but distinct from L-NIL. In addition, the elevation in myeloperoxidase activity was reversed by the administration of ECg, uric acid and SOD, but not by that of L-NIL. Furthermore, ECg was the more potent scavenger of the ONOO(-) decomposition product, the hydroxyl radical (*OH), than any other free radical inhibitor tested. The LPS plus ischaemia-reperfusion process resulted in renal dysfunction, estimated by measuring the parameters of renal function--serum urea nitrogen and creatinine levels. However, administration of ECg ameliorated renal dysfunction more than that of the other free radical inhibitors. Moreover, ECg reduced the excessive uric acid level, while the others did not, suggesting a property of ECg distinct from the others. Furthermore, proteinuria, which was demonstrated by the low- and high-molecular weight (LMW and HMW) protein bands of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern, caused by LPS plus ischaemia-reperfusion, was attenuated by administration of ECg and L-NIL, after which the HMW band intensities decreased and LMW protein bands were absent. This study indicates that, in an in-vivo model of ONOO(-) generation, ECg, L-NIL and uric acid exert stronger protective activity against ONOO(-)-induced oxidative damage than SOD and ebselen, and that the mechanism whereby ECg protects against ONOO(-) is distinct from that of L-NIL or uric acid.

Topics: Animals; Azoles; Blood Urea Nitrogen; Catechin; Copper; Creatinine; Disease Models, Animal; Dose-Response Relationship, Drug; Free Radical Scavengers; Isoindoles; Japan; Kidney; Lipopolysaccharides; Lysine; Male; Nitric Oxide; Organoselenium Compounds; Peroxidase; Phytotherapy; Plant Extracts; Plant Roots; Proteinuria; Rats; Rats, Wistar; Reperfusion Injury; Rheum; Superoxide Dismutase; Tyrosine; Uric Acid; Zinc

Use of thrombolysis in stroke is limited by a short therapeutic window because delayed reperfusion may cause brain hemorrhage and edema. Available evidence suggests a role for superoxide, NO, and peroxynitrite in reperfusion-induced injury. However, depending on their cellular origin and interactions between them, these molecules may exert protective or deleterious actions, neither of which is characterized in the intact brain.. Using fluorescent probes, we determined superoxide and peroxynitrite formation within neurons, astrocytes, and endothelium, and the association between oxidative/nitrative stress and vascular injury in mice brains subjected to 2-hour middle cerebral artery occlusion and 3 or 5 hours of reperfusion.. Both signals were colocalized, suggesting that the main source of peroxynitrite in the reperfused brain was a reaction between superoxide and NO. Superoxide and peroxynitrite formation was particularly intense in microvessels and astrocytic end-feet surrounding them, and overlapped with dense mitochondrial labeling. Sites of oxidative/nitrative stress on microvessels were colocalized with markers of vascular injury such as Evans blue (EB) leakage and matrix metalloproteinase-9 (MMP-9) expression, suggesting an association between peroxynitrite and microvascular injury. Supporting this idea, partial inhibition of endothelial NO synthesis at reperfusion with a low dose of L-nitroarginine (1 mg/kg IP) reduced 3-nitrotyrosine formation in microvessels and EB extravasation.. During reperfusion, intense superoxide, NO, and peroxynitrite formation on microvessels and surrounding end-feet may lead to cerebral hemorrhage and edema by disrupting microvascular integrity. Combination of thrombolysis with agents diminishing oxidative/nitrative stress may reduce reperfusion-induced injury and extend the therapeutic window for thrombolysis.

Topics: Animals; Blood-Brain Barrier; Brain; Brain Ischemia; Coloring Agents; Enzyme Inhibitors; Evans Blue; Matrix Metalloproteinase 9; Mice; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Peroxynitrous Acid; Reperfusion Injury; Superoxides; Tyrosine

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the tissue injury associated with ischemia-reperfusion and inflammation. Splanchnic artery occlusion and reperfusion causes an enhanced formation of reactive oxygen species which contribute to the pathophysiology of shock. The aim of the present study was to investigate the effects of 5-aminoisoquinolinone (5-AIQ), a potent water-soluble inhibitor of poly(ADP-ribose) polymerase (PARP), in the pathogenesis of splanchnic artery occlusion shock. Splanchnic artery occlusion shock was induced in rats by clamping both the superior mesenteric artery and the celiac artery for 45 min, followed thereafter by release of the clamp (reperfusion). At 60 min after reperfusion, all animals were sacrificed for histological examination and biochemical studies. Treatment of rats with 5-AIQ (3 mg/kg i.v.), attenuated the fall of mean arterial blood pressure caused by splanchnic artery occlusion shock. 5-AIQ also attenuated the ileum injury as well as the increase in the tissue levels of myeloperoxidase and malondialdehyde caused by splanchnic artery occlusion shock in the ileum. The immunohistochemical examination also demonstrated a marked increase in the immunoreactivity to PAR, nitrotyrosine, and intercellular adhesion molecule (ICAM-1) in the necrotic ileum from splanchnic artery occlusion-shocked rats. 5-AIQ treatment significantly reduced the increase of positive staining for PAR, nitrotyrosine and ICAM-I. In conclusion, these results show that 5-AIQ, a new water-soluble potent inhibitor of poly(ADP-ribose) polymerase, exerts multiple protective effects in splanchnic artery occlusion/reperfusion shock.

Topics: Animals; Celiac Artery; Disease Models, Animal; Intercellular Adhesion Molecule-1; Intestine, Small; Ischemia; Isoquinolines; Lipid Peroxidation; Male; Malondialdehyde; Mesenteric Artery, Superior; Mesenteric Vascular Occlusion; Peroxidase; Poly(ADP-ribose) Polymerase Inhibitors; Rats; Rats, Sprague-Dawley; Reperfusion; Reperfusion Injury; Solubility; Tyrosine; Water

High-density lipoproteins (HDLs) have been shown to reduce the organ injury and mortality in animal models of shock by reducing the expression of adhesion molecules and proinflammatory enzymes. However, there is limited evidence that HDL treatment reduces inflammation. As inflammation plays an important role in the development of colitis as well as ischemia/reperfusion (I/R) injury of the intestine, we have investigated the effects of HDL in animal models of associated with gut injury and inflammation (splanchnic artery occlusion [SAO] shock and dinitrobenzene sulfonic acid [DNBS]-induced colitis). We report here for the first time that the administration of reconstituted HDLs (recHDLs; 80 mg/kg i.v. bolus 30 min prior to ischemia in the SAO-shock model or 40 mg/kg i.v. every 24 h in the colitis model) exerts potent anti-inflammatory effects (e.g., reduced inflammatory cell infiltration and histological injury, and delayed the development of the clinical signs) in vivo. Furthermore, recHDL reduced the staining for nitrotyrosine and poly(ADP-ribose) (immunohistochemistry) and the expression of intercellular adhesion molecule-1 in the ileum of SAO-shocked rats and in the colon from DNBS-treated rats. Thus, recHDL reduces the inflammation caused by intestinal I/R and colitis. HDLs may represent a novel therapeutic approach for the therapy of inflammation of the gut.

Topics: Animals; Colitis; Dinitrofluorobenzene; Immunohistochemistry; Inflammation; Intercellular Adhesion Molecule-1; Intestines; Lipoproteins, HDL; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tyrosine

Acute gastric mucosal lesions (AGMLs) are an important cause of gastrointestinal bleeding. Herein, we demonstrate that peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of a nuclear receptor family, functions as an endogenous anti-inflammatory pathway in a murine model of AGML induced by ischemia-reperfusion (I/R). Treatment with specific PPARgamma ligands such as BRL-49653, pioglitazone, or troglitazone was examined in a model of AGML induced by I/R. PPARgamma-deficient and wild-type mice were also examined for their response to I/R in stomach. Specific PPARgamma ligands exhibited dramatic and rapid protection against AGML formation associated with I/R in mice in a dose-dependent manner. In contrast, the AGML induced by I/R in PPARgamma-deficient mice was more severe than that observed in wild-type mice. Administration of the PPARgamma ligand significantly inhibited the upregulation of TNF-alpha, ICAM-1, inducible nitric oxide synthase, apoptosis, and nitrotyrosine formation induced by I/R in the stomach. These data indicate that an endogenous pathway associated with PPARgamma plays an important role in the pathogenesis of I/R-associated injury in the stomach.

Topics: Animals; Gastric Mucosa; Intercellular Adhesion Molecule-1; Ligands; Mice; Mice, Inbred BALB C; Mice, Knockout; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Receptors, Cytoplasmic and Nuclear; Reperfusion Injury; RNA, Messenger; Stomach; Transcription Factors; Tumor Necrosis Factor-alpha; Tyrosine

Altered nitric oxide (NO) metabolism has been shown to contribute to ischemia-reperfusion (IR) injury in animal models. However, similar studies have not been performed in human liver transplantation (LT). In this study, we examined nitrate, nitrite, and nitrosothiols (NOx), NO synthases (endothelial [constitutive] nitric oxide synthase [eNOS] and inducible nitric oxide synthase [iNOS]), and nitrotyrosine in early IR injury after human LT.. Paired biopsies were obtained from nine donor livers before cold ischemia (retrieval biopsy) and after reimplantation (reperfusion biopsy). Sections were graded for reperfusion injury using the Suzuki score. NO was detected by chemiluminescence after reduction of NOx. Expression of eNOS and iNOS was by Western blot and reverse transcriptase polymerase chain reaction and peroxynitrite by immunodetection of 3-nitrotyrosine.. Reperfusion biopsies showed histologic evidence of injury (median Suzuki score: retrieval 2, reperfusion 6, P=0.008) and neutrophil infiltration. NOx was reduced after reperfusion from 5.41 microM/100 mg (median, range 2.17-13.39 microM) to 3.51 microM (1.45-5.66 microM, P=0.05). eNOS protein was reduced after reperfusion from 0.6 units (median, range 0.45-1 unit) in retrieval biopsies to 0.39 units in reperfusion biopsies (range 0.2-0.79 units, P=0.007). There was no change in eNOS or iNOS mRNA expression or iNOS protein. Western blotting showed increased nitrotyrosine formation after reperfusion, median 0.42 (range 0.16-0.87) units in retrieval biopsies and 0.68 (0.29-1.06) units in reperfusion samples (P=0.007) and localized to periportal regions.. iNOS protein may not contribute to early reperfusion injury during human LT. However, reduced NO bioavailability caused by reduced eNOS may contribute significantly to damage at this time point.

Topics: Adolescent; Adult; Aged; Cadaver; Female; Humans; Immunohistochemistry; Liver Transplantation; Male; Middle Aged; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Reoperation; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Donors; Tyrosine

Ischemia-reperfusion (IR) is characterized by microvascular disfunction and this involves both direct effected organ and remote organ by systemic inflammatory response. These remote effects of IR are most frequently observed in the lung and cardiovascular system. In this study we aim to determine lung damage which induced IR, and endothelial and microvascular disfunction using nitrosative markers. Previous studies suggest that caffeic acid phenethyl ester (CAPE) has some antioxidant effects. Therefore, we also investigated whether it has a role associated with nitric oxide during IR condition. Twenty-two adult male Wistar rats were divided into three groups: control (n= 7), IR (n= 7), and CAPE + IR (n= 8). 8 h IR period was performed on right hindlimb in the IR and the CAPE with IR group. In the CAPE with IR group, animals received CAPE 10 microM 1 h before the reperfusion. At the end of the reperfusion period, blood, bronchoalveolar lavage (BAL) and lung tissue were obtained, and were used for biochemical and histopathological examination. There was a significantly elevation in serum nitrate, BAL MPO, and leukocyte infiltration in the lung in the IR group compared to the CAPE + IR group. But, serum nitrite and lung 3-NT levels were not different between these groups. While nitrate (p< 0.0001), MPO (p< 0.0001) and leukocyte infiltration (chi2= 27.163, p= 0.0001), reduce by using CAPE before reperfusion, tissue 3-NT levels did not change. In conclusion, peripheral IR leads to systemic inflammatory responses and endothelial disfunction-induced NO production, and these harmful effects may reduced by CAPE.

Topics: Animals; Bronchoalveolar Lavage Fluid; Caffeic Acids; Capillary Permeability; Cytokines; Hindlimb; Lung; Male; Malondialdehyde; Neutrophil Infiltration; NF-kappa B; Phenylethyl Alcohol; Rats; Rats, Wistar; Reperfusion Injury; Respiratory Distress Syndrome; Tourniquets; Tyrosine

Ischemia-reperfusion (I/R) of remote organs is a common cause of lung injury. We observed that lung injury after partial hepatic I/R in mice coincides with the appearance of 3-nitrotyrosine (NT) in the lung tissue, a marker of peroxynitrite involvement and oxidant stress. Peroxynitrite can cause mitochondrial dysfunction by inactivation of manganese superoxide dismutase (MnSOD), the major antioxidant enzyme in mitochondria. Our aims were to examine whether pulmonary MnSOD is a target of nitration following hepatic I/R and whether nitrated MnSOD (N-MnSOD) correlates with acute lung injury.. Five 20-25-g male C57BL/6 mice underwent laparotomy, and atraumatic occlusion of the portal and arterial blood supply to the upper three lobes of the liver for 90 min. This warm ischemic period was followed by 4 h of reperfusion, and the animals were then euthanized. Lung injury was assessed by LDH and protein levels in bronchoalveolar lavage (BAL) fluid. Pulmonary MnSOD activity in pulmonary homogenates was measured by the cytochrome c reduction method. The presence of N-MnSOD was determined by immunoprecipitation (IP) and Western Blot analysis. Controls (N = 5) underwent sham operation.. Elevated plasma transaminases confirmed hepatic injury. Lung injury was demonstrated by elevation in BAL protein and LDH levels (495.7 (48.4) versus 644.9 (37.3) [p < 0.05] and 56.5 (11.8) versus 345.2 (80) [p < 0.01], respectively). Immunoprecipitation and Western blot demonstrated N-MnSOD in the lung tissue of I/R animals but not controls. MnSOD activity decreased following I/R (8.1 (0.7) versus 10.8 (0.3) [p < 0.05]).. Pulmonary MnSOD is both nitrated and inactivated following hepatic I/R and is associated with acute lung injury. These findings suggest that MnSOD incapacitance may contribute to I/R-induced lung injury and provide a therapeutic target in attenuating multisystem injury following hepatic I/R.

Topics: Animals; Biomarkers; Biopsy, Needle; Disease Models, Animal; Immunohistochemistry; Ischemia; Liver; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Reperfusion; Reperfusion Injury; Respiratory Distress Syndrome; Sensitivity and Specificity; Severity of Illness Index; Superoxide Dismutase; Tyrosine

The peroxisome proliferator-activated receptor-alpha (PPAR-alpha) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors related to retinoid, steroid, and thyroid hormone receptors. WY 14643 is a potent PPAR-alpha ligand that modulates the transcription of target genes. The aim of this study was to investigate the effect of WY 14643 on the tissue injury caused by ischemia-reperfusion (I/R) of the gut. I/R injury of the intestine was caused by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamp, allowing reperfusion for 2 h or 4 h. This procedure results in splanchnic artery occlusion (SAO) shock. Rats subjected to SAO developed a significant fall in mean arterial blood pressure, and only 20% of the animals survived for the entire 4-h reperfusion period. Surviving animals were sacrificed for histological examination and biochemical studies. Rats subjected to SAO displayed a significant increase in tissue myeloperoxidase (MPO) activity, significant increases in plasma tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta levels, and marked injury to the distal ileum. Increased immunoreactivity to nitrotyrosine and polyadenosine diphosphate [ADP]-ribose (PAR) was observed in the ileum of rats subjected to SAO. Staining of sections of the ileum obtained from SAO rats with anti-intercellular adhesion molecule (ICAM-1) antibody or with anti-P-selectin antibody resulted in diffuse staining. Administration of WY 14643 (1 mg/kg i.v.) 30 min before the onset of gut ischemia significantly reduced the (a) fall in mean arterial blood pressure, (b) mortality rate, (c) infiltration of the reperfused intestine with polymorphonuclear neutrophils (MPO activity), (d) production of proinflammatory cytokines (TNF-alpha and IL-1beta), and (e) histological evidence of gut injury. Administration of WY 14643 also markedly reduced the nitrotyrosine formation, poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) activation, up-regulation of ICAM-1, and expression of P-selectin during reperfusion. These results demonstrate that the PPAR-alpha agonist WY 14643 significantly reduces I/R injury of the intestine.

Topics: Animals; Blood Pressure; Ileum; Immunohistochemistry; Intercellular Adhesion Molecule-1; Interleukin-1; Male; Neutrophils; P-Selectin; Peroxidase; Peroxisome Proliferators; Poly(ADP-ribose) Polymerases; Pyrimidines; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Shock; Splanchnic Circulation; Tumor Necrosis Factor-alpha; Tyrosine; Up-Regulation

Caspase activation has been implicated in the development of ischemia-reperfusion injury. Here, we investigate the effects of different caspase inhibitors on the renal dysfunction and injury caused by ischemia-reperfusion of the rat kidney. Bilateral clamping of renal pedicles (45 min) followed by reperfusion (6 h) caused significant renal dysfunction and marked renal injury. Caspase-1 inhibitor II (N-acetyl-L-tyrosyl-L-valyl-N-[(1S)-1-(carboxymethyl)-3-chloro-2-oxo-propyl]-L-alaninamide, Ac-YVAD-CMK, 3 mg/kg, administered i.p.) significantly reduced biochemical and histological evidence of renal dysfunction and injury. However, although caspase-3 inhibitor I (N-acetyl-L-aspartyl-L-glutamyl-N-(2-carboxyl-1-formylethyl]-L-valinamide, Ac-DEVD-CHO, 3 mg/kg, administered i.p.) produced a significant improvement of renal (glomerular) dysfunction (reduction of serum creatinine levels), it was not able to reduce tubular dysfunction and injury. Furthermore, the pan-caspase inhibitor caspase inhibitor III (N-tert-butoxycarbonyl-aspartyl(OMe)-fluoromethylketone, Boc-D-FMK, 3 mg/kg, administered i.p.) did not reduce renal dysfunction and injury. Both caspase-1 and -3 inhibitors markedly reduced the evidence of oxidative and nitrosative stress in rat kidneys subjected to ischemia-reperfusion. Overall, these results demonstrate that inhibition of caspase-1 reduces renal ischemia-reperfusion injury to a greater extent than caspase-3 inhibition, supporting the notion that the mode of acute cell death in our model of renal ischemia-reperfusion is primarily via necrosis. Furthermore, our finding that a pan-caspase inhibitor did not reduce the renal dysfunction and injury suggests that activation of some caspases during ischemia-reperfusion could provide protection against acute ischemic renal injury. Overall, these results demonstrate that inhibition of caspase-1 activity reduces renal ischemia-reperfusion injury and that this therapeutic strategy may be of benefit against ischemic acute renal failure.

Topics: Animals; Benzyl Compounds; Biomarkers; Caspase 3; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Hydrocarbons, Fluorinated; Immunohistochemistry; In Vitro Techniques; Intercellular Adhesion Molecule-1; Kidney; Kidney Function Tests; Male; Malondialdehyde; Myocardium; Nitric Oxide; Oligopeptides; Organ Size; Oxidative Stress; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Serpins; Tyrosine; Viral Proteins

The aim of this study was to investigate the role of nitric oxide (NO) in hepatic ischemia-reperfusion (I/R) injury in rats. Immunohistochemistry was used to examine the protein expression of endothelial and inducible nitric oxide synthases (eNOS, iNOS) and nitrotyrosine after I/R challenges to the liver, and blood levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactic dehydrogenase (LDH), hydroxyl radical and NO were measured before ischemia and after reperfusion. Ischemia was induced by occlusion of the common hepatic artery and portal vein for 40 min, followed by reperfusion for 90 min. Reperfusion of the liver induced a significant increase in the blood concentrations of AST, ALT, LDH (n = 8; P < 0.001), hydroxyl radical (n = 8; P < 0.001) and NO (n = 8; P < 0.01). The eNOS, iNOS, nitrotyrosine, SOD1 and SOD2 protein expression was also found to increase significantly after reperfusion (n = 3). Administration of the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (n = 8) had a protective effect on the I/R-related injury, but the NO donor L-arginine (L-Arg) (n = 8) potentiated the damage caused by I/R. These results suggest that reperfusion of the liver induces expression of NOS, which is related to the elevation of blood NO. The increase in hydroxyl radical concentration was accompanied by an increase in antioxidant enzyme expression (SOD1 and SOD2), and an increase in nitrotyrosine expression was also observed, reflecting the increased production of NO and oxygen radicals. We concluded from the protective effect of L-NAME and the potentiation by L-Arg that NOS expression and increases in NO and hydroxyl radical production have deleterious effects on the response to I/R in the liver.

Topics: Alanine Transaminase; Animals; Arginine; Aspartate Aminotransferases; Enzyme Inhibitors; Immunohistochemistry; L-Lactate Dehydrogenase; Liver; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Staining and Labeling; Tyrosine

Acute inflammatory reactions cause neuronal damage in cerebral ischemia-reperfusion. Urinary trypsin inhibitor (UTI), a serine protease inhibitor, is cytoprotective against ischemia-reperfusion injury in the liver, intestine, kidney, heart, and lung through its antiinflammatory activity. Neuroprotective action of UTI on transient global cerebral ischemia has been documented. This is the first study to determine whether UTI is neuroprotective against transient focal cerebral ischemia.. Adult male Wistar rats were randomly assigned to the following treatment groups: 0.9% saline (control, n = 9); 100,000 U/kg UTI (n = 9); and 300,000 U/kg UTI (n = 9). Treatments were performed intravenously 10 min before right middle cerebral artery occlusion for 2 h and subsequent reperfusion. Ninety-six hours after the onset of reperfusion, the motor neurologic deficit and the cerebral infarct size were evaluated. Furthermore, immunohistochemical staining for myeloperoxidase and nitrotyrosine to count infiltrating neutrophils and nitrated cells, respectively, was performed on the brain sections.. Infarct volume in the 300,000 U/kg UTI group was smaller than in the 100,000 U/kg UTI and saline control groups (P < 0.05). Treatment with 300,000 U/kg UTI showed a trend to improve neurologic outcome but did not reach statistical significance (P = 0.0693). The significant decrease in neutrophil infiltration was observed in the ischemic hemisphere treated with 300,000 U/kg UTI compared with saline control (P < 0.05). Nitrotyrosine deposition in the ischemic hemisphere was significantly reduced in the 300,000 U/kg UTI group compared with saline control and 100,000 U/kg UTI groups (P < 0.05).. Intravenous pretreatment with 300,000 U/kg UTI reduces focal ischemia-reperfusion injury in the rat brain, potentially opening a novel therapeutic avenue for the treatment of cerebral ischemia.

Topics: Animals; Behavior, Animal; Brain; Cell Count; Cerebrovascular Circulation; Glycoproteins; Hypoxia-Ischemia, Brain; Immunohistochemistry; Infarction, Middle Cerebral Artery; Laser-Doppler Flowmetry; Male; Nervous System Diseases; Neuroprotective Agents; Neutrophils; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Trypsin Inhibitors; Tyrosine

Generation of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of GW274150, a novel, highly selective, potent and long-acting inhibitor of iNOS activity in rat and mouse models of renal I/R.. Rats were administered GW274150 (5 mg/kg intravenous bolus administered 30 minutes prior to I/R) and subjected to bilateral renal ischemia (45 minutes) followed by reperfusion (6 hours). Serum and urinary indicators of renal dysfunction, tubular and reperfusion injury were measured, specifically, serum urea, creatinine, aspartate aminotransferase (AST) and N-acetyl-beta-d-glucosaminidase (NAG) enzymuria. In addition, renal sections were used for histologic scoring of renal injury and for immunologic evidence of nitrotyrosine formation and poly [adenosine diphosphate (ADP)-ribose] (PAR). Nitrate levels were measured in rat plasma using the Griess assay. Mice (wild-type, administered 5 mg/kg GW274150, and iNOS-/-) were subjected to bilateral renal ischemia (30 minutes) followed by reperfusion (24 hours) after which renal dysfunction (serum urea, creatinine), renal myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured.. GW274150, administered prior to I/R, significantly reduced serum urea, serum creatinine, AST, and NAG indicating reduction of renal dysfunction and injury caused by I/R. GW274150 reduced histologic evidence of tubular injury and markedly reduced immunohistochemical evidence of nitrotyrosine and PAR formation, indicating reduced peroxynitrite formation and poly (ADP-ribose) polymerase (PARP) activation, respectively. GW274150 abolished the rise in the plasma levels of nitrate (indicating reduced NO production). GW274150 also reduced the renal dysfunction in wild-type mice to levels similar to that observed in iNOS-/- mice subjected to I/R. Renal MPO activity and MDA levels were significantly reduced in wild-type mice administered GW274150 and iNOS-/- mice subjected to renal I/R, indicating reduced polymorphonuclear leukocyte (PMN) infiltration and lipid peroxidation.. These results suggest that (1). an enhanced formation of NO by iNOS contributes to the pathophysiology of renal I/R injury and (2). GW274150 reduces I/R injury of the kidney. We propose that selective inhibitors of iNOS activity may be useful against renal dysfunction and injury associated with I/R of the kidney.

Topics: Animals; Creatinine; Enzyme Inhibitors; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peroxidase; Poly Adenosine Diphosphate Ribose; Rats; Rats, Wistar; Reperfusion Injury; Sulfides; Tyrosine; Urea

It is still controversial whether a major mode of cell death during hepatic ischemia-reperfusion (I/R) injuries is apoptosis or necrosis. Moreover, the correlation between these cell deaths and the effects of a novel inducible nitric oxide synthase inhibitor (ONO-1714) has not been studied before.. Pigs were subjected to 180 min of hepatic warm I/R under extracorporeal circulation. The control group was not administered ONO-1714. In the ONO-1714 group, ONO-1714 was administered 5 min before ischemia at a dose of 0.05 mg/kg through a portal vein catheter. The apoptotic and necrotic changes after reperfusion were examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and hematoxylin-eosin staining. Nitrotyrosine, active caspase-3, and cytochrome c were examined by immunohistochemistry. The plasma NO + NO, aspartate aminotransferase, and lactate dehydrogenase levels were also examined.. In the control group, the frequency of apoptotic cells was only 2.6%; nevertheless, that of necrotic cells was 37% at 24 hr after reperfusion. ONO-1714 significantly attenuated apoptosis and necrosis, the expression of nitrotyrosine, and the increases of the plasma aspartate aminotransferase, lactate dehydrogenase, and NO(2)- + NO(3)- levels in the reperfusion phase.. A major mode of cell death during hepatic warm I/R injury was necrosis, and apoptosis was not dominant. These necrotic changes were caused by the excess production of peroxynitrite, and ONO-1714 greatly attenuated I/R injuries as the result of inhibition of the peroxynitrite production.

Topics: Amidines; Animals; Apoptosis; Aspartate Aminotransferases; DNA; Enzyme Inhibitors; Female; Hepatocytes; Heterocyclic Compounds, 2-Ring; Hot Temperature; In Situ Nick-End Labeling; Ischemia; L-Lactate Dehydrogenase; Liver; Liver Circulation; Microscopy, Confocal; Microscopy, Electron; Necrosis; Nitrates; Nitric Oxide Synthase; Nitrites; Reperfusion Injury; Swine; Time Factors; Tyrosine

Liver ischemia-reperfusion injury is a serious problem during liver resection and transplantation. Nitric oxide (NO) has been suggested to have a cytoprotective effect for microcirculation, while the interaction of active oxygen species and NO produces peroxynitrite anion. The present study attempts to clarify the role of NO in liver ischemia-reperfusion injury.. Wistar male rats were subjected to 30 min of hepatic ischemia followed by reperfusion. The model rats were divided into the three following groups: a control group that was not administered NO synthase inhibitors, and two experimental groups that were administered either N(omega)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine. In each group, we examined active oxygen species and nitric oxide production, and investigated liver function by measuring serum transaminase levels. In addition, we conducted histopathologic examinations and microcirculation examinations using intravital videomicroscopy.. In the control group, NO concentrations in the plasma increased with time after reperfusion. A decrease in NO production was detected in the groups administered NO synthase inhibitors. Elevated serum transaminase levels became more prominent after L-NAME administration, while aminoguanidine administration reduced its level. The degree of microcirculation failure was found to be more prominent in the L-NAME-administered group over both the control group and the aminoguanidine-administered group. A significantly lower survival rate was observed at 6 h after reperfusion in the L-NAME-administered group over that of the other groups.. A reduction of the ischemia-reperfusion injury is important in inhibiting the production of high-output NO and peroxynitrite, and in maintaining NO levels necessary for maintenance of microcirculation.

Topics: Animals; Enzyme Inhibitors; Guanidines; Infusions, Parenteral; Liver; Male; Microcirculation; Models, Animal; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Time Factors; Transaminases; Tyrosine

Endogenous tyrosine nitration and inactivation of manganese superoxide dismutase (MnSOD) has previously been shown to occur in both human and rat chronic renal allograft rejection. To elucidate the time course of MnSOD inactivation and mitochondrial dysfunction at earlier times during renal transplantation, we developed a rodent model of renal ischemia/reperfusion (I/R). Renal function was significantly impaired at 16 h reperfusion following 30 min of warm ischemia. Tyrosine nitration of specific mitochondrial proteins, MnSOD and cytochrome c, occurred at the earliest time point examined, an event that preceded significant renal injury. Interestingly, a small percentage of both mitochondrial proteins were also located in the cytosol. This leakage and decreased adenosine 5(')-triphosphate levels indicate loss of mitochondrial membrane integrity during renal I/R. Inactivation of MnSOD occurred rapidly in this model of renal I/R, suggesting that loss of MnSOD activity leads to further renal injury and nitration of other mitochondrial targets.

Topics: Adenosine Triphosphate; Animals; Blotting, Western; Creatinine; Cytochrome c Group; Cytosol; Humans; In Situ Nick-End Labeling; Ischemia; Kidney; Male; Mitochondria; Oxidants; Oxidative Stress; Precipitin Tests; Rats; Rats, Inbred F344; Reperfusion Injury; Superoxide Dismutase; Time Factors; Tyrosine

Hemorrhagic shock and resuscitation cause hepatocellular damage by mechanisms involving oxidative stress. However, the sources of free radicals mediating hepatocellular injury remain controversial. Thus, this study tested the hypothesis that NADPH oxidase plays a role in producing hepatocellular injury after hemorrhagic shock and resuscitation. Both wild-type and NADPH oxidase-deficient mice (p47(phox) knockout mice) were subjected to hemorrhagic shock (3 h at 30 mmHg). The mice were resuscitated over 30 min with the shed blood and additional lactated Ringer's solution (50% of the shed blood volume). Serum alanine aminotransferase (ALT) levels increased at 1 and 6 h postresuscitation in wild-type animals to 4735 +/- 1017 IU/L and 1450 +/- 275 IU/L (mean +/- SE), respectively, whereas in knockout mice, this ALT increase was blunted at both time points (732 +/- 241 IU/L and 328 +/- 69 IU/L, P < 0.05). Liver necrosis assessed histologically 6 h after the end of reperfusion was also attenuated in the knockout mice (3.5% +/- 0.95% of area vs. 0.9% +/- 0.26%, P < 0.05). In hemorrhaged wild-type mice, infiltrating neutrophils were twice as numerous compared with hemorrhaged NADPH oxidase-deficient animals 6 h after reperfusion. In knockout animals, hepatic 4-hydroxynonenal content, indicative of lipid peroxidation from reactive oxygen species, was blunted (6.7% +/- 0.6% vs. 26.4% +/- 2.3% of stained area, P < 0.05), as shown by immunohistochemistry. Immunohistochemical staining for 3-nitrotyrosine, indicative of reactive nitrogen species formation, was also blunted in the livers of knockout mice (11.6% +/- 2.8% vs. 37.4% +/- 3.4, P < 0.05). In conclusion, hemorrhagic shock and resuscitation cause hepatocellular damage via NADPH oxidase-mediated oxidative stress. The absence of NADPH oxidase substantially attenuates hepatocellular injury after hemorrhagic shock and resuscitation, blunts neutrophil infiltration, and decreases formation of reactive oxygen and reactive nitrogen species.

Topics: Alanine Transaminase; Animals; Chemotaxis, Leukocyte; Ischemia; Lipid Peroxidation; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; NADPH Oxidases; Necrosis; Neutrophils; Oxidative Stress; Peroxynitrous Acid; Phosphoproteins; Reperfusion Injury; Resuscitation; Shock, Hemorrhagic; Superoxides; Tyrosine

In recent years the important role of nitric oxide in hepatic ischemia-reperfusion injury has been increasingly recognised. The prevailing consensus is that reperfusion injury may be partly the result of decreased production of nitric oxide from endothelial nitric oxide synthase and excessive production of nitric oxide from the inducible isoform. We therefore undertook this study to characterize the expression of different nitric oxide synthase isoforms during hepatic reperfusion.. Male Wistar rats (n = 6) were subjected to 45 minutes of partial hepatic ischemia (left lateral and median lobes) followed by 6 hours of reperfusion. Control animals (n = 6) were subjected to sham laparotomy. The expression of endothelial and inducible nitric oxide synthase was examined using immunohistochemistry and Western blotting. Liver sections were also stained with nitrotyrosine antibody, a specific marker of protein damage induced by peroxynitrite (a highly reactive free radical formed from nitric oxide).. Liver sections from all the control animals showed normal expression of the endothelial isoform and no expression of inducible nitric oxide synthase. Livers from all the animals subjected to hepatic ischemia showed decreased expression of endothelial nitric oxide synthase, and all but one animal from this group showed expression of the inducible isoform both in inflammatory cells and in hepatocytes. Western blotting confirmed these findings. Staining with the antinitrotyrosine antibody was also confined to five liver sections from animals subjected to hepatic ischemia.. During the reperfusion period after hepatic ischemia, endothelial nitric oxide synthase is downregulated while inducible nitric oxide synthase is expressed in both hepatocytes and inflammatory cells. The presence of nitrotyrosine in livers subjected to hepatic ischemia-reperfusion suggests that the expression of inducible nitric oxide synthase plays an important role in mediating reperfusion injury in this model.

Topics: Animals; Blotting, Western; Disease Models, Animal; Fluorescent Antibody Technique; Ischemia; Liver; Liver Diseases; Liver Function Tests; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine

The protective effect of (-)-epicatechin 3-O-galate (ECg) against peroxynitrite (ONOO-)-mediated damage was examined using an animal model and a cell culture system. In rats subjected to lipopolysaccharide (LPS) administration plus ischemia-reperfusion, the plasma 3-nitrotyrosine level an indicator of ONOO- production in vivo, was elevated, whereas it declined significantly and dose-dependently after the oral administration of ECg at doses of 10 and 20 micromoles/kg body weight/day for 20 days prior to the process. Moreover, oral administration of ECg significantly enhanced the activities of the antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase, and the antioxidant glutathione, showing enhancement of the biological defense system against the damage induced by ONOO-. In addition, the significant increase in the renal mitochondrial thiobarbituric acid-reactive substance level of LPS and ischemic-reperfused control rats was attenuated in rats given ECg. Furthermore, the elevations in the plasma urea nitrogen and creatinine (Cr) levels and the urinary methylguanidine/Cr ratio induced by the procedure were attenuated markedly after oral administration of ECg, implying amelioration of renal impairment. The addition of ECg (25 or 125 microM) prior to 3-morpholinosydnonimine (SIN-1, 800 microM) exposure reduced ONOO- formation and increased the viability of cultured renal epithelial (LLC-PK1) cells in a dose-dependent manner. In particular, ECg inhibited ONOO(-)-mediated apoptotic cell death, which was confirmed by decreases in the DNA fragmentation rate and the presence of apoptotic morphological changes, i.e. small nuclei and nuclear fragmentation. Furthermore, adding ECg before SIN-1 treatment regulated the cell cycle by enhancing G2/M phase arrest. This study provides evidence that ECg has protective activity against the renal damage induced by excessive ONOO- in cellular and in vivo systems.

Topics: Animals; Antioxidants; Caspases; Catechin; Cell Cycle; Cell Line; Cell Survival; DNA Fragmentation; Dose-Response Relationship, Drug; Flow Cytometry; Glutathione; Glutathione Peroxidase; Kidney; Lipid Peroxides; Lipopolysaccharides; LLC-PK1 Cells; Male; Molsidomine; Peroxynitrous Acid; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Rhodamines; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Time Factors; Tyrosine

To evaluate effects of nitric oxide (NO) and peroxynitrite anion (ONOO(-)) on lung injury following intestinal ischemia-reperfusion (IR) in rats.. A rat model of intestinal ischemia was made by clamping superior mesenteric artery and lung injury was resulted from reperfusion. The animals were randomly divided into 3 groups: sham operation (Sham), 2 h ischemia followed by 2 h reperfusion (IR) and IR pretreated with aminoguanidine (AG) - an inhibitor of inducible NO synthase (iNOS) 15 minutes before reperfusion (IR+AG). The lung malondialdehyde (MDA) and nitrate/nitrite (NO(2)(-)/NO(3)(-)) contents and morphological changes were examined. Western blot was used to detect the iNOS protein expression. Immunohistochemical staining was used to determine the change of nitrotyrosine (NT)- a specific "footprint" of ONOO(-).. The morphology revealed evidence for lung edema, hemorrhage and polymorphonuclear sequestration after intestinal IR. Compared with sham group, lung contents of MDA and NO(2)(-)/NO(3)(-) in IR group were significantly increased (12.00+/-2.18 vs 23.44+/-1.25 and 76.39+/-6.08 vs 140.40+/-4.34, P<0.01) and the positive signals of iNOS and NT were also increased in the lung. Compared with IR group, the contents of MDA and NO(2)(-)/NO(3)(-) in IR+AG group were significantly decreased (23.44+/-1.25 vs 14.66+/-1.66 and 140.40+/-4.34 vs 80.00+/-8.56, P<0.01) and NT staining was also decreased.. Intestinal IR increases NO and ONOO(-) production in the lung, which may be involved in intestinal IR-mediated lung injury.

Topics: Animals; Anions; Lung; Male; Malondialdehyde; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Peroxynitrous Acid; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tyrosine

Peroxynitrite is a potent cytotoxic free radical produced by the reaction of nitric oxide with the superoxide ion produced in conditions of oxidative stress. The purpose of the study was to examine the role of this reactive nitrogen species in lung ischemia-reperfusion injury.. Left lungs of male Long-Evans rats were rendered ischemic for 90 minutes and reperfused for up to 4 hours. Treated animals received FP-15 (a water-soluble iron containing metalloporphyrin that acts as a peroxynitrite decomposition catalyst). Injury was quantitated in terms of tissue neutrophil accumulation (myeloperoxidase content) and vascular permeability ((125)I bovine serum albumin [BSA] extravasation) and bronchoalveolar lavage cytokine, transcriptional factor and leukocyte content. Separate tissue samples were processed for immunohistology and nuclear protein analysis.. Lung vascular permeability was reduced in treated animals by 61% compared with control animals (p < 0.005). The protective effects of enhanced peroxynitrite decomposition correlated with a 72% reduction in tissue myeloperoxidase content (p < 0.001) and marked reductions in brochoalveolar lavage leukocyte accumulation. This correlated positively with the diminished expression of pro-inflammatory chemokines and nuclear transcription factors.. The deleterious effects of lung ischemia-reperfusion injury are in part mediated by the formation of peroxynitrite, as enhanced decomposition of this species is protective in this model. The development of potent water-soluble decomposition catalysts represents a potentially useful therapeutic tool in the prevention of lung ischemia-reperfusion injury after lung transplantation.

Topics: Animals; Bronchoalveolar Lavage Fluid; Capillary Permeability; Chemokines; Disease Models, Animal; Immunohistochemistry; Inflammation Mediators; Macrophages, Alveolar; Male; Models, Cardiovascular; NF-kappa B; Peroxidase; Peroxynitrous Acid; Rats; Rats, Long-Evans; Reactive Nitrogen Species; Reperfusion Injury; Respiratory Distress Syndrome; Severity of Illness Index; Statistics as Topic; Transcription Factor AP-1; Transcriptional Activation; Tyrosine

1. The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors related to retinoid, steroid and thyroid hormone receptors. The thiazolidinedione rosiglitazone and the endogenous cyclopentenone prostaglandin (PG)D2 metabolite, 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2), are two PPAR-gamma ligands, which modulate the transcription of target genes. 2. The aim of this study was to investigate the effect of rosiglitazone and 15d-PGJ2 on the tissue injury caused by ischaemia/reperfusion (I/R) of the gut. 3. I/R injury of the intestine was caused by clamping both the superior mesenteric artery and the coeliac trunk for 45 min, followed by release of the clamp allowing reperfusion for 2 or 4 h. This procedure results in splanchnic artery occlusion (SAO) shock. 4. Rats subjected to SAO developed a significant fall in mean arterial blood pressure, and only 10% of the animals survived for the entire 4 h reperfusion period. Surviving animals were killed for histological examination and biochemical studies. Rats subjected to SAO displayed a significant increase in tissue myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels, significant increases in plasma tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta levels and marked injury to the distal ileum. 5. Increased immunoreactivity to nitrotyrosine was observed in the ileum of rats subjected to SAO. Staining of sections of the ileum obtained from SAO rats with anti-intercellular adhesion molecule (ICAM-1) antibody resulted in diffuse staining. 6. Administration at 30 min prior to the onset of gut ischaemia of the two PPAR-gamma agonists (rosiglitazone (0.3 mg kg-1 i.v.) and 15d-PGJ2 (0.3 mg kg-1 i.v.)) significantly reduced the (i) fall in mean arterial blood pressure, (ii) mortality rate, (iii) infiltration of the reperfused intestine with polymorphonuclear neutrophils (MPO activity), (iv) lipid peroxidation (MDA levels), (v) production of proinflammatory cytokines (TNF-alpha and IL-1beta) and (vi) histological evidence of gut injury. Administration of rosiglitazone and 15d-PGJ2 also markedly reduced the nitrotyrosine formation and the upregulation of ICAM-1 during reperfusion. 7. In order to elucidate whether the protective effects of rosiglitazone and 15d-PGJ2 are related to the activation of the PPAR-gamma receptor, we also investigated the effect of a PPAR-gamma antagonist, bisphenol A diglycidyl ether (BA

Topics: Animals; Benzhydryl Compounds; Blood Pressure; Epoxy Compounds; Immunologic Factors; Intercellular Adhesion Molecule-1; Interleukin-1; Intestinal Mucosa; Intestines; Ligands; Male; Malondialdehyde; Peroxidase; Prostaglandin D2; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Reperfusion Injury; Rosiglitazone; Thiazolidinediones; Transcription Factors; Tumor Necrosis Factor-alpha; Tyrosine; Vasodilator Agents

We investigate the effects of tyrphostin AG126, an inhibitor of tyrosine kinase activity, on the renal dysfunction and injury caused by ischemia/reperfusion (I/R) of the kidney.. Tyrphostin AG126 (5 mg/kg intraperitoneally) was administered to male Wistar rats 30 minutes prior to bilateral renal ischemia for 45 minutes followed by reperfusion for up to 48 hours. Biochemical markers of renal dysfunction and injury were measured and renal sections assessed for renal injury. Expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and formation of nitrotyrosine and poly (ADP) ribose (PAR) were assessed using immunohistochemistry. Rat proximal tubular cells (PTCs) were incubated with interferon-gamma (100 IU/mL), bacterial lipopolysaccharide (10 microg/mL), and with increasing concentrations of tyrphostin AG126 (0.0001-1 mmol/L) for 24 hours. Nitric oxide production was measured in both plasma from rats subjected to I/R and in incubation medium from PTCs.. After 6 hours of reperfusion, tyrphostin AG126 significantly reduced the increase in serum and urinary indicators of renal dysfunction and injury caused by I/R and reduced histologic evidence of renal injury. Tyrphostin AG126 also improved renal function (after 24 and 48 hours of reperfusion) and reduced the histologic signs of renal injury (after 48 hours of reperfusion). Tyrphostin AG126 reduced the expression of iNOS and nitric oxide levels in both rat plasma and in PTC cultures, as well as expression of COX-2. Tyrphostin AG126 also reduced nitrotyrosine and PAR formation, suggesting reduction of nitrosative stress and poly (ADP-ribose) polymerase (PARP) activation, respectively.. Taken together, these results show that tyrphostin AG126 significantly reduces the renal dysfunction and injury caused by I/R of the kidney. We propose that inhibition of tyrosine kinase activity may be useful against renal I/R injury.

Topics: Acute Kidney Injury; Animals; Cells, Cultured; Cyclooxygenase 2; Enzyme Inhibitors; Isoenzymes; Kidney Tubules; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Prostaglandin-Endoperoxide Synthases; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine; Tyrphostins

NO is a putative neurotransmitter and neuromodulator in the brain. NO is not functioning as a direct neurotoxin. NO with the superoxide radical product peroxynitrite (ONOO-) is much more cytotoxic under tissue impairment conditions. Caspase-3, a potent effector of apoptosis that is triggered via several different signaling pathways, may play a very important role in neuronal cell death caused by various brain injuries. The relationship between mouse caspase-3 and peroxynitrite remains unclear. In the present study, we examined the in vivo expression of 3-nitrotyrosine (a metabolite of peroxinitrite) and caspase-3 after cerebral ischemia produced in a global ischemia model using mice (i.e., a cardiac arrest model). 3-nitrotyrosine immunoreactivity was detected in neuronal cells in the hippocampal dentate nucleus, and cortical regions starting at 12 hrs after ischemia. In particular, numerous neuronal cells were highly immunoreactive for 3-nitrotyrosine in the cortical regions. In hippocampal CA1 pyramidal neurons, 3-nitrotyrosine immunoreactivity was detected from 24 hrs. Caspase-3 immunopositive cells were observed in approximately the same area in which the positive reaction to the anti-nitrotyrosine antibody was observed. These results provide direct evidence for the induction of 3-nitrotyrosine and caspase-3 expression in vivo in an ischemia model using mice. The present findings suggest that peroxynitrite generated by cerebral ischemia/ reperfusion was strongly cytotoxic and induced neuronal cell death (apoptosis) mediated by caspase-3.

Topics: Animals; Brain Ischemia; Caspase 3; Caspases; Heart Arrest, Induced; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Peroxynitrous Acid; Reperfusion Injury; Time Factors; Tyrosine

Ischemia/reperfusion injury, and thus graft pancreatitis, remains a major problem in pancreas transplantation. Contradictory results about the role of nitric oxide (NO) in pancreatic ischemia/reperfusion have been reported; however, in none of the reports has a detailed comparison between inhibition of NO synthase and NO supplementation been carried out.. Vascular isolation of the pancreatic tail was performed in landrace pigs. After splenectomy catheters placed in the distal part of the splenic vessels allowed collection of the venous effluent and perfusion of the pancreatic tail. Three hours of complete warm ischemia was followed by 6 h of reperfusion. The effect of the NO donor sodium nitroprusside (SNP) and L-arginine was compared to a control group and NO synthase inhibition with L-NAME.. Lipase in the venous effluent of the pancreas was significantly decreased in the SNP and the L-arginine groups. Vascular resistance was markedly elevated in the L-NAME group and reduced in the NO donor groups. Tissue pO2 after reperfusion was only significantly elevated in the SNP group. Granulocyte infiltration and also overall histological tissue injury were most severe in the control group followed by the L-NAME group, the SNP group, and the L-ARG group.. The data show that supplementation of nitric oxide is clearly protective in pancreatic ischemia/reperfusion. However, inhibition of NO synthesis does not lead to an equally clear aggravation of tissue injury.

Topics: Acute Disease; Adenosine Triphosphate; Animals; Arginine; Blood Pressure; Enzyme Inhibitors; Female; Glutathione; Lipase; Microcirculation; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Oxidation-Reduction; Oximetry; Oxygen Consumption; Pancreas Transplantation; Pancreatitis; Regional Blood Flow; Reperfusion Injury; Swine; Tyrosine; Vascular Resistance

Peroxynitrite is assumed to play a crucial role in brain damage associated with the overproduction of nitric oxide (NO). The purpose of this study is to examine time-dependent changes of nitrite and nitrate (NOx) concentration in the circulation, and peroxynitrite formation as well as the expression of inducible nitric oxide synthase (iNOS) in the penumbra of rat brains during transient middle cerebral artery occlusion (MCAO) of Wistar rat for 2 h and reperfusion for 4-70 h. NOx concentration in the circulation was continuously monitored at the right jugular vein by microdialysis. The expression of iNOS was detected at 22-70 h after reperfusion in vascular walls and the cortex. Nitrotyrosine, a marker of peroxynitrite, appeared 4 h after reperfusion in the cortex, increasing substantially at 22-46 h in vascular walls. NOx level in dialysate increased immediately after MCAO. After a gradual decrease, the level increased again 4 h after reperfusion, reaching a maximum at 46 h. Brain myeloperoxidase activity, a marker of neutrophil infiltration, was not detected 4 h after reperfusion, but greatly increased at 22 h and then decreased. These results suggest that a marked increase of NOx level in the circulation might reflect the expression of iNOS, while neuronal NOS may contribute to peroxynitrite formation in the cortex observed at an earlier phase of reperfusion. This study indicates that monitoring NOx level in the circulation serves to assess the progress of stroke, and to determine appropriate therapeutic measures.

Topics: Animals; Cerebral Cortex; Cerebral Infarction; Cerebrovascular Circulation; Chemotaxis, Leukocyte; Disease Models, Animal; Hypoxia-Ischemia, Brain; Infarction, Middle Cerebral Artery; Male; Neutrophils; Nitric Oxide; Nitric Oxide Synthase; Peroxidase; Peroxynitrous Acid; Rats; Rats, Wistar; Reaction Time; Reperfusion Injury; Tyrosine

The aim of this study was to investigate whether in vivo administration of a low, sub-lethal dose of lipoteichoic acid (LTA), a bacterial wall-fragment derived from the Gram-positive bacterium Staphylococcus aureus, protects the kidney against the renal dysfunction and injury caused by ischemia/reperfusion (I/R).. Male Wistar rats were administered LTA from S. aureus (1 mg/kg, IP). After 24 hours, rats were subjected to bilateral renal ischemia (45 min) followed by reperfusion (6 h). Serum and urinary markers were measured for the assessment of renal function, tubular and reperfusion-injury. Renal sections were used for histological grading of renal injury and for immunohistochemical localization of P-selectin, inducible nitric oxide synthase (iNOS) and nitrotyrosine (indicative of peroxynitrite formation). Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of polymorphonuclear (PMN) cell infiltration and lipid peroxidation, respectively. Nitric oxide (NO) production was determined by measurement of plasma nitrite/nitrate levels.. LTA pretreatment significantly reduced renal dysfunction, tubular and reperfusion-injury caused by I/R of the kidney as well as histological evidence of renal injury. LTA also reduced the expression of P-selectin and kidney MPO activity associated with renal I/R. MDA levels were significantly reduced by LTA pretreatment suggesting a reduction in the lipid peroxidation and formation of reactive oxygen species (ROS). LTA pretreatment also markedly reduced both the expression of iNOS and the formation of nitrotyrosine associated with renal I/R. Although LTA significantly reduced plasma nitrite/nitrate levels associated with I/R, nitrite/nitrate levels remained at levels significantly higher than that measured from the plasma obtained from Sham-operated animals.. These data suggest, to our knowledge for the first time, that LTA pretreatment for 24 hours significantly reduces renal I/R injury. We propose that the mechanism of the protective effect involves reduction of the production of NO, ROS and peroxynitrite subsequent to reduced P-selectin and iNOS expression and PMN recruitment. However, although LTA pretreatment resulted in a reduction of iNOS expression and NO production, we hypothesize that the remaining significant levels of NO contribute to the beneficial actions provided by LTA.

Topics: Animals; Antioxidants; Cyclic N-Oxides; Kidney Diseases; Kidney Tubules; Lipopolysaccharides; Male; Malondialdehyde; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; P-Selectin; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Spin Labels; Staphylococcus aureus; Teichoic Acids; Tyrosine

Here we investigate the effects of the stable, water-soluble nitroxyl radical, TEMPONE, on renal dysfunction and injury caused by ischemia/reperfusion (I/R) of the rat kidney in vivo. TEMPONE significantly improved both glomerular and tubular function (serum urea, creatinine, creatinine clearance, and fractional excretion of Na(+)) in a dose-dependent manner and significantly attenuated the reperfusion-injury associated with I/R (urinary N-acetyl-beta-D-glucosaminidase, aspartate aminotransferase, assessment of renal histology). TEMPONE also markedly reduced the immunohistochemical evidence of the formation of nitrotyrosine and poly(ADP-ribose), indicating reduction of nitrosative and oxidative stress, respectively. The latter was reflected in vitro, where TEMPONE significantly reduced cellular injury of primary cultures of rat renal proximal tubular (PT) cells caused by hydrogen peroxide in a dose-dependent manner. Importantly, in contrast to its in vivo metabolite TEMPOL (which also provided protective effects against renal I/R and oxidative stress of PT cells), TEMPONE reduced renal dysfunction and injury without causing a significant reduction in blood pressure upon administration. These results suggest, for the first time, that TEMPONE can reduce the renal dysfunction and injury caused by I/R and the injury caused to PT cells by oxidative stress without producing the adverse cardiovascular effects observed when using other nitroxyl radicals.

Topics: Animals; Coloring Agents; Dose-Response Relationship, Drug; Hydrogen Peroxide; Immunohistochemistry; Kidney; Kidney Diseases; Male; Mice; Nitrogen; Oxidative Stress; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Tetrazolium Salts; Thiazoles; Triacetoneamine-N-Oxyl; Tyrosine; Urine

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

Thioredoxin (TRX) is a 13 kDa protein with antioxidant effect and redox regulating functions. Peroxynitrite is a strong oxidizing and nitrating agent which can react with all classes of biomolecules. In the present study, we focused on the association between TRX and nitrotyrosine, which served as a marker of peroxynitrite formation, in the neonatal hypoxia-ischemia (HI) rat brain. At 4-16 h after HI, the immunoreactivity for TRX was diminished in the injured region in the cortex and striatum, whereas nitrotyrosine immunoreactivity was enhanced. In contrast, around the injured region, TRX immunoreactivity was enhanced in survival neurons at 4-24 h after HI, while the immunoreactivity for nitrotyrosine was mostly not detected. Northern blot analysis showed increased TRX mRNA induction in the cerebral hemisphere ipsilateral to the carotid ligation from 4-24 h after HI but not in the contralateral hypoxic hemisphere. These findings suggest that production of peroxynitrite is involved in HI brain injury, and that induced TRX plays a neuroprotective role against oxidative stress resulting from HI.

Topics: Animals; Animals, Newborn; Blotting, Northern; Brain; Hypoxia; Models, Anatomic; Oxidants; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger; Thioredoxins; Time Factors; Tyrosine

Changes in the nitric oxide (NO) system of the rat cerebral cortex were investigated by immunohistochemistry, immunoblotting, NO synthase (NOS) activity assay, and magnetic resonance imaging (MRI) in an experimental model of global cerebral ischemia and reperfusion. Brains were perfused transcardially with an oxygenated plasma substitute and subjected to 30 minutes of oxygen and glucose deprivation, followed by reperfusion for up to 12 hours with oxygenated medium containing glucose. A sham group was perfused without oxygen or glucose deprivation, and a further group was treated with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) before and during perfusion. Global ischemia led to cerebrocortical injury as shown by diffusion MRI. This was accompanied by increasing morphologic changes in the large type I interneurons expressing neuronal NOS (nNOS) and the appearance of nNOS immunoreactivity in small type II neurons. The nNOS-immunoreactive band and calcium-dependent NOS activity showed an initial increase, followed by a fall after 6 hours of reperfusion. Inducible NOS immunoreactivity appeared in neurons, especially pyramidal cells of layers IV-V, after 4 hours of reperfusion, with corresponding changes on immunoblotting and in calcium-independent NOS activity. Immunoreactive protein nitrotyrosine, present in the nuclear area of neurons in nonperfused controls and sham-perfused animals, showed changes in intensity and distribution, appearing in the neuronal processes during the reperfusion period. Prior and concurrent L-NAME administration blocked the changes on diffusion MRI and attenuated the morphologic changes, suggesting that NO and consequent peroxynitrite formation during ischemia-reperfusion contributes to cerebral injury.

Topics: Animals; Blotting, Western; Brain Ischemia; Calcium; Cerebral Cortex; Disease Models, Animal; Enzyme Inhibitors; Gene Expression; Glucose; Hypoxia; Immunohistochemistry; Magnetic Resonance Imaging; Male; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Peroxynitrous Acid; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine

Peroxynitrite is responsible for nitration in vivo, whereas myeloperoxidase can also catalyze protein nitration in the presence of high NO2(-) levels. Recent reports of myeloperoxidase-mediated enzyme inactivation or lipid peroxidation have suggested a role of myeloperoxidase in various pathological conditions. To clarify the role of myeloperoxidase in ischemic brain injury, the authors measured nitrotyrosine formation and infarct volume in myeloperoxidase-deficient or wild-type mice subjected to 2-hour focal cerebral ischemia-reperfusion. Twenty-four hours after reperfusion, infarct volume was significantly larger in myeloperoxidase-deficient mice than in wild-type mice (81 +/- 20 mm(3) vs. 52 +/- 13 mm(3), P < 0.01), and nitrotyrosine levels in the infarct region were higher in myeloperoxidase-deficient mice than in wild-type mice (13.4 +/- 6.1 microg/mg vs. 9.8 +/- 4.4 microg/mg, P = 0.13). Fourteen hours after reperfusion, the nitrotyrosine level was significantly higher in myeloperoxidase-deficient mice than in wild-type mice (3.3 +/- 2.9 microg/mg vs. 1.4 +/- 0.4 microg/mg, P < 0.05). The authors conclude that the absence of myeloperoxidase increases ischemic neuronal damage in vivo, and that the myeloperoxidase-mediated pathway is not responsible for the nitration reaction in cerebral ischemia-reperfusion.

Topics: Animals; Brain; Brain Infarction; Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Peroxidase; Peroxynitrous Acid; Reperfusion Injury; Time Factors; Tyrosine

Nitric oxide (NO), produced via inducible nitric oxide synthase (iNOS), is implicated in the pathophysiology of renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of the iNOS inhibitors L-N6-(1-iminoethyl)lysine (L-NIL) and aminoethyl-isothiourea (AE-ITU) on (a) renal dysfunction and injury mediated by bilateral I/R of rat kidneys in vivo and (b) cytokine-stimulated NO production by primary cultures of rat proximal tubule (PT) cells.. Male Wistar rats subjected to bilateral renal ischemia (45 min) followed by reperfusion (6 h). Rats were administered either L-NIL (3 mg/kg IV bolus 15 min prior to I/R followed by 1 mg/kg/h throughout I/R) or AE-ITU (1 mg/kg IV bolus 15 min prior to I/R followed by 1 mg/kg/h throughout I/R). Serum and urinary biochemical indicators of renal dysfunction and injury were measured; serum creatinine (SCr, glomerular dysfunction), fractional excretion of Na+ (FENa, tubular dysfunction), serum aspartate aminotransferase (sAST, I/R injury) and urinary N-acetyl-beta-d-glucosaminidase (uNAG, tubular injury). Additionally, renal sections were used for histological grading of renal injury and for immunological evidence of nitrotyrosine formation. Nitrate/nitrate levels in plasma were measured using the Griess assay and used as an indicator of NO production. Primary cultures of rat PT cells were incubated with interferon-gamma(IFN-gamma, 100 IU/mL) and lipopolysaccharide (LPS, 10 microg/mL) for 24 h, either in the absence or presence of increasing concentrations of L-NIL or AE-ITU (0.001 to 1 mmol/L) after which nitrite/nitrate levels were measured using the Griess assay.. L-NIL and AE-ITU significantly reduced the I/R-mediated increases in SCr, FENa, sAST and uNAG, indicating attenuation of I/R-mediated renal dysfunction and injury. Specifically, L-NIL and AE-ITU reduced the I/R-mediated glomerular and tubular dysfunction and biochemical and histological evidence of tubular injury. Both L-NIL and AE-ITU attenuated the plasma levels of nitrate (indicating reduced NO production) and the immunohistochemical evidence of the formation of nitrotyrosine. In vitro, L-NIL and AE-ITU both significantly reduced cytokine-stimulated NO production by primary cultures of rat PT cells in a dose-dependent manner.. These results suggest that L-NIL and AE-ITU reduce the renal dysfunction and injury associated with I/R of the kidney, via inhibition of iNOS activity and subsequent reduction of NO (and peroxynitrite) generation. We propose that selective and specific inhibitors of iNOS activity may be useful against the NO-mediated renal dysfunction and injury associated with I/R of the kidney.

Topics: Animals; Cells, Cultured; Cytokines; Enzyme Inhibitors; Ischemia; Kidney Glomerulus; Kidney Tubules; Kidney Tubules, Proximal; Lysine; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Renal Circulation; Reperfusion Injury; Thiourea; Tyrosine

The present experiment determined the effects of glutathione and ascorbic acid, the two most important hydrophilic antioxidants, on myocardial ischemia-reperfusion injury and evaluated their relative therapeutic values. Isolated rat hearts were subjected to ischemia (30 min) and reperfusion (120 min) and treated with ascorbic acid, glutathione monoethyl ester (GSHme), or their combination at the onset of reperfusion. Administration of 1 mM GSHme alone, but not 1 mM ascorbic acid alone, significantly attenuated postischemic injury (P < 0.05 versus vehicle). Most interestingly, coadministration of ascorbic acid with GSHme markedly enhanced the protective effects of GSHme (P < 0.01 versus vehicle). The protection exerted by the combination of GSHme and ascorbic acid at 1 mM each was significantly greater than that observed with 1 mM GSHme alone (P < 0.05). Moreover, treatment with GSHme alone or GSHme plus ascorbic acid markedly reduced myocardial nitrotyrosine levels, suggesting that these treatments attenuated myocardial peroxynitrite formation. These results demonstrated that 1) GSHme, but not ascorbic acid, exerted protective effects against ischemia-reperfusion injury; and 2) the protective effects of GSHme were further enhanced by coadministration with ascorbic acid, suggesting a synergistic effect between GSHme and ascorbic acid.

Topics: Animals; Antioxidants; Ascorbic Acid; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Glutathione; Heart; Heart Rate; Incidence; Lipid Peroxidation; Myocardial Reperfusion Injury; Myocardium; Protective Agents; Rats; Reperfusion Injury; Tachycardia; Tyrosine; Ventricular Fibrillation

Recently, a novel inhibitor of inducible nitric oxide synthase, ONO-1714, was developed. We evaluated the effect of ONO-1714 on a critical warm I/R model of the pig liver.. Pigs were subjected to 180 min of hepatic warm I/R under the extracorporeal circulation. We investigated the time course of changes in the serum NO2- + NO3- (NOx), the cellular distribution of endothelial and inducible nitric oxide synthase, thrombocyte-thrombi, and nitrotyrosine by immunohistochemistry. The hepatic tissue blood flow (HTBF) was measured continuously using a laser-Doppler blood flowmeter.. ONO-1714 at 0.05 mg/kg improved the survival rate from 54 (control group) to 100%. The serum NOx levels in the ONO-1714 group were significantly lower than those in the control group at 1, 1.5, 2, 3, and 6 hr after reperfusion. The serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels of the ONO-1714 group were significantly lower than the control group, and the HTBF of the ONO-1714 group was significantly higher than the control group. The formation of thrombocyte-thrombi and nitrotyrosine after reperfusion was significantly lower in the ONO-1714 group.. These results indicated that ONO-1714 improved the survival rates and attenuated I/R injury in a critical hepatic warm I/R model of the pig. ONO-1714 will be beneficial for hepatectomy or liver transplantation in the clinical field.

Topics: Amidines; Animals; Aspartate Aminotransferases; Enzyme Inhibitors; Female; Heterocyclic Compounds, 2-Ring; Hot Temperature; Ischemia; L-Lactate Dehydrogenase; Liver; Liver Circulation; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Reperfusion Injury; Survival Analysis; Swine; Thrombosis; Tissue Distribution; Tyrosine

1. Splanchnic artery occlusion shock (SAO) causes an enhanced formation of reactive oxygen species (ROS), which contribute to the pathophysiology of shock. Here we have investigated the effects of M40401, a new S:,S:-dimethyl substituted biscyclohexylpyridine Mn-based superoxide dismutase mimetic (SODm, k(cat)=1.2x10(+9) M(-1) s(-1) at pH=7.4), in rats subjected to SAO shock. 2. Treatment of rats with M40401 (applied at 0.25, 2.5 or 25 microg kg(-1), 15 min prior to reperfusion), attenuated the mean arterial blood and the migration of polymorphonuclear cells (PMNs) caused by SAO-shock. M40401 also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase (MPO) and malondialdehyde (MDA) caused by SAO shock in the ileum. 3. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine was markedly reduced in tissue sections obtained from SAO-shocked rats which had received M40401. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin and for anti-intercellular adhesion molecule (ICAM-1) in the vascular endothelial cells. M40401 treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue sections from SAO-shocked rats. M40401 treatment significantly improved survival. 4. Additionally, the very high catalytic activity of this new mimetic (comparable to the native human Cu/Zn SOD enzyme and exceeding the activity of the human Mn SOD enzyme) translates into a very low dose ( approximately microg kg(-1)) required to afford protection in this SAO model of ischemia reperfusion injury. 5. Taken together, our results clearly demonstrate that M40401 treatment exerts a protective effect, and part of this effect may be due to inhibition of the expression of adhesion molecules and peroxynitrite-related pathways with subsequent reduction of neutrophil-mediated cellular injury.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arterial Occlusive Diseases; Catalysis; Cytokines; Fluorescent Antibody Technique; Ileum; Leukocyte Count; Male; Malondialdehyde; Manganese; Nitrates; Nitrites; Organometallic Compounds; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Splanchnic Circulation; Superoxide Dismutase; Superoxides; Tyrosine

The aim of this study is to determine experimentally whether N-methyl-D-aspartate (NMDA) receptor is involved in nitrotyrosine formation in rat brain subjected to focal ischemia-reperfusion, by using the NMDA receptor antagonist MK-801. Halothane-anesthetized and artificially ventilated rats were given MK-801 (5 mg/kg, i.p.) or vehicle prior to 2 h of focal cerebral ischemia followed by 0.5 h of reperfusion. The brain was then removed and divided into four sections, cortical ischemic core, peri-ischemic cortex, lateral caudate-putamen and non-ischemic cortex. Tissue nitrotyrosine was measured by means of hydrolysis/HPLC. MK-801 significantly attenuated nitrotyrosine formation in the lateral caudate-putamen. We conclude that nitrotyrosine formation required activation of NMDA receptors, at least in part.

Topics: Animals; Brain Ischemia; Calcium Channels; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Male; Neostriatum; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Tyrosine

Reactive nitrogen species (RNS) cause nitration of protein-bound tyrosine that is used as biomarker for detection. We hypothesized that RNS are formed in fetal rabbit brain following acute placental insufficiency. Near-term pregnant rabbits were randomized to either repetitive uterine ischemia or no ischemia, and fetal brains obtained. Only one electrochemical HPLC method (of three tested) was successful in detecting brain nitrotyrosine. Protein nitrotyrosine was significantly increased following cumulative 40 min ischemia and 20 min reperfusion compared to controls. Repetitive hypoxia-ischemia results in the increased formation of RNS in near-term fetal brains.

Topics: Animals; Brain Injuries; Brain Ischemia; Female; Fetal Hypoxia; Fetus; Free Radicals; Hypoxia, Brain; Placental Insufficiency; Pregnancy; Rabbits; Reperfusion Injury; Tyrosine

A perfusion model of global cerebral ischemia was used for the immunohistochemical study of changes in the glutamate-nitric oxide (NO) system in the rat cerebellum and cerebellar nuclei during a 0-14 h reperfusion period after 30 min of oxygen and glucose deprivation, with and without administration of 1.5 mM N(omega)-nitro-L-arginine methyl ester (L-NAME). While immunostaining for N-methyl-D-aspartate receptor subunit 1 (NMDAR1) showed no marked changes during the reperfusion period, neuronal NO synthase (nNOS) immunostaining increased in stellate and basket cells, granule cells and neurons of the cerebellar nuclei. However, global cerebellar nNOS concentrations determined by Western blotting remained largely unchanged in comparison with actin expression. Inducible NOS (iNOS) immunostaining appeared in Purkinje cells and neurons of the cerebellar nuclei after 2-4 h of reperfusion and intensified during the 6-14 h period. This was reflected by an increase in global cerebellar iNOS expression determined by Western blotting. Immunostaining for protein nitrotyrosine was seen in Purkinje cells, stellate and basket cells, neurons of the cerebellar nuclei and glial cells in controls, and showed a progressive translocation in Purkinje cells and neurons of the cerebellar nuclei from an initial perinuclear or nuclear location towards the periphery. At the end of the reperfusion period the Purkinje cell apical dendrites were notably retracted and tortuous. Prior and concurrent L-NAME administration eliminated nitrotyrosine immunostaining in controls and blocked or reduced most of the postischemic changes observed. The results suggest that while nNOS expression may be modified in certain cells, iNOS is induced after a 2-4 h period, and that changes in protein nitration may be associated with changes in cell morphology.

Topics: Animals; Blotting, Western; Cerebellum; Disease Models, Animal; Enzyme Inhibitors; Glucose; Hypoxia-Ischemia, Brain; Immunohistochemistry; Male; Neurons; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Purkinje Cells; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Tyrosine

Previous evidence suggests that both oxygen radicals and nitric oxide (NO) are important mediators of injury during renal ischemia-reperfusion (I-R) injury. However, the generation of reactive nitrogen species (RNS) has not been evaluated in this model at early time points. The purpose of these studies was to examine the development of oxidant stress and the formation of RNS during I-R injury. Male Sprague-Dawley rats were anesthetized and subjected to 40 min of bilateral renal ischemia followed by 0, 3, or 6 h of reperfusion. Control animals received a sham operation. Plasma urea nitrogen and creatinine levels were monitored as markers of renal injury. Glutathione (GSH) oxidation and 4-hydroxynonenal (4-HNE)-protein adducts were used as markers of oxidant stress. 3-Nitrotyrosine (3-NT) was used as a biomarker of RNS formation. Significant increases in plasma creatinine concentrations and urea nitrogen levels were found following both 3 and 6 h of reperfusion. Increases in GSH oxidation, 4-HNE-protein adduct levels, and 3-NT levels were observed following 40 min of ischemia with no reperfusion. Since these results suggested RNS generation during the 40 min of ischemia, a time course of RNS generation following 0, 5, 10, 20, and 40 min of ischemia was evaluated. Significant increases in 3-NT generation was detected as early as 10 min of ischemia and rose to values nearly 10-fold higher than Control at 40 min of ischemia. No additional increase was observed following reperfusion. The data clearly demonstrate that oxidative stress and RNS generation occur in the kidney during ischemia.

Topics: Aldehydes; Animals; Blood Urea Nitrogen; Creatinine; Free Radicals; Glutathione; Kidney; Male; Nitrates; Oxidation-Reduction; Oxidative Stress; Proteins; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Time Factors; Tyrosine

Oxidative stress is a prominent feature of the placenta in many complications of pregnancy, such as preeclampsia. The cause is primarily unknown, although ischemia-reperfusion injury is one possible mechanism. Our aim was to test this hypothesis by examining the oxidative status of human placental tissues during periods of hypoxia and reoxygenation in vitro. Rapid generation of reactive oxygen species was detected using the fluorogenic probe, 2',7'-dichlorofluorescein diacetate, when hypoxic tissues were reoxygenated. The principal sites were the villous endothelium, and to a lesser extent the syncytiotrophoblast and stromal cells. Increased concentrations of heat shock protein 72, nitrotyrosine residues, and 4-hydroxy-2-nonenal were also observed in the villous endothelial and underlying smooth muscle cells, and in the syncytiotrophoblast. Furthermore, preloading placental tissues with the reactive oxygen species scavengers desferrioxamine and alpha-phenyl-N-tert-butylnitrone reduced levels of oxidative stress after reoxygenation. These changes are consistent with an ischemia-reperfusion injury, and mirror those seen in preeclampsia. Consequently, in vitro hypoxia/reoxygenation may represent a suitable model system for investigating the generation of placental oxidative stress in preeclampsia and other complications of pregnancy.

Topics: Aldehydes; Cyclic N-Oxides; Deferoxamine; Female; Fluorescent Antibody Technique; Heat-Shock Proteins; HSP72 Heat-Shock Proteins; Humans; Hypoxia; Immunohistochemistry; Ischemia; Nitrogen Oxides; Oxidative Stress; Oxygen; Placenta; Pregnancy; Pregnancy Complications; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Tissue Distribution; Tyrosine

Generation of reactive oxygen species and nitric oxide in hypoxia-reperfusion injury may form a cytotoxic metabolite, peroxynitrite, which is capable of causing lipid peroxidation and DNA damage. This study was designed to examine the contribution of oxidative and nitrosative stress to the renal damage in ischemic acute renal failure (iARF). iARF was initiated in rats by 45-min renal artery clamping. This resulted in lipid peroxidation, DNA damage, and nitrotyrosine modification confirmed both by Western and immunohistochemical analyses. Three groups of animals were randomly treated with an inhibitor of inducible nitric oxide synthase (NOS), L-N(6)-(1-iminoethyl)lysine (L-Nil), cell-permeable lecithinized superoxide dismutase (SOD), or both. Each treatment resulted in amelioration of renal dysfunction, as well as reduced nitrotyrosine formation, lipid peroxidation, and DNA damage, thus suggesting that peroxynitrite rather than superoxide anion is responsible for lipid peroxidation and DNA damage. Therefore, in a separate series of experiments, a scavenger of peroxynitrite, ebselen, was administered before the reperfusion period. This treatment resulted in a comparable degree of amelioration of iARF. In conclusion, the present study provides the first attempt to elucidate the role of peroxynitrite in initiation of the cascade of lipid peroxidation and DNA damage to ischemic kidneys. The results demonstrate that L-Nil, lecithinized SOD, and ebselen treatments improve renal function due to their suppression of peroxynitrite production or its scavenging, consequently preventing lipid peroxidation and oxidative DNA damage.

Topics: Animals; Azoles; Blotting, Western; Cell Line; Cyclic N-Oxides; DNA Damage; Enzyme Inhibitors; Free Radical Scavengers; Immunohistochemistry; Ischemia; Isoindoles; Kidney; Lipid Peroxidation; Lysine; Macrophages; Male; Mice; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Organoselenium Compounds; Oxidative Stress; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Spin Labels; Superoxide Dismutase; Tyrosine

The importance of reactive oxygen species released through interaction of leukocyte/endothelial cell in ischemia-reperfusion injury of the lung is not yet fully understood. A novel selectin blocker, OJ-R9188, which inhibits the interaction, may alleviate oxidative stress and pulmonary dysfunction after warm ischemia-reperfusion.. Rat lungs were reperfused at 37 degrees C for 60 min with an ex vivo model and were divided into three groups (n = 10). In the fresh group, lungs were reperfused immediately after harvest. In the OJ-R (-) and OJ-R (+) groups, lungs were reperfused after warm ischemia at 37 degrees C for 90 min. In the OJ-R (+) group, rats received 100 microg per body of OJ-R9188 intravenously 10 min before the harvest. The electron spin resonance method was used to assess the direct scavenging activity of OJ-R9188.. Both shunt fractions and wet/dry weight ratios of the lung tissue after reperfusion in the OJ-R (+) group were significantly lower than those in the OJ-R (-) group. Oxidative DNA damage in the alveolar wall of the OJ-R (+) group, assessed by immunohistochemical quantitation of 8-hydroxy-2'-deoxyguanosine, was significantly lower than that of the OJ-R (-) group. Immunostaining of 3-nitro-l-tyrosine, which represents nitric oxide-mediated oxidative damage, was also more intense in the OJ-R (-) group than in the OJ-R (+) group. Direct scavenging activity of OJ-R9188 was not observed, and the number of leukocytes infiltrated to the lung tissue as seen by myeloperoxidase activity was not different between the OJ-R (-) and OJ-R (+) groups.. A novel selectin blocker, OJ-R9188, improves pulmonary function after warm ischemia-reperfusion and alleviates reperfusion-induced oxidative alveolar damage.

Topics: Animals; Deoxyguanosine; Free Radical Scavengers; Ischemia; Lung; Male; Neutrophil Infiltration; Oxidative Stress; Peroxynitrous Acid; Pulmonary Circulation; Rats; Rats, Inbred Lew; Reactive Oxygen Species; Reperfusion Injury; Selectins; Tyrosine

The aim of the present study was to investigate the protective effect of the pineal secretary product melatonin in a model of splanchnic artery occlusion shock (SAO). SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed thereafter by release of the clamp (reperfusion). At 60 min after reperfusion, animals were sacrificed for tissue histological examination and biochemical studies. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO-shocked rats after reperfusion, but not during ischemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, an index of nitrogen species such as peroxynitrite, in the necrotic ileum in shocked rats. SAO-shocked rats developed a significant increase of tissue myeloperoxidase and malondialdehyde activity, and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival at 2 hr after reperfusion). Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin, which was mainly localized in the vascular endothelial cells. Ileum tissue sections obtained from SAO-shocked rats with anti-intercellular adhesion molecule (ICAM-1) antibody showed a diffuse staining. Melatonin (applied at 3 mg/kg, 5 min prior to reperfusion, followed by an infusion of 3 mg/kg per hr), significantly reduced ischemia reperfusion injury in the bowel as evaluated by histological examination. This prevented the infiltration of neutrophils into the reperfused intestine, is evidenced by reduced myeloperoxidase activity and reduced lipid peroxidation. This was evaluated by malondialdehyde activity which reduced the production of peroxynitrite during reperfusion, markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue section from SAO-shocked rats and improved their survival. Taken together, our results clearly demonstrate that melatonin treatment exerts a protective effect and part of this effect may be due to inhibition of the expression of adhesion molecule and peroxynitrite-related pathways and subsequent reduction of neutrophil-mediated cellular injury.

Topics: Animals; Chemotaxis, Leukocyte; Endothelium, Vascular; Free Radical Scavengers; Ileal Diseases; Immunoenzyme Techniques; Intercellular Adhesion Molecule-1; Male; Malondialdehyde; Melatonin; Mesenteric Artery, Superior; Neutrophils; Oxidative Stress; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Splanchnic Circulation; Tyrosine

Nitrotyrosine produced by NO-mediated reaction is a possible marker for cytotoxicity in brain ischemia. In this study, we aimed to determine whether iNOS is responsible for the nitrotyrosine formation and which type of cell is predominantly nitrated. Fifty-eight wild-type and 28 iNOS knockout male mice were used. Under halothane anesthesia the left middle cerebral artery was occluded for 2 h and reperfused for 0.5 or 15 h. The ratio of nitrotyrosine to total tyrosine (%NO2-Tyr) was measured by means of a hydrolysis/HPLC. After 0.5-h reperfusion, %NO2-Tyr in the ischemic cortex of wild-type and knockout mice amounted to 0.037 +/- 0.040% (n = 8) and 0.064 +/- 0.035% (n = 6), respectively, being significantly higher than that in the sham operation group (n = 7) (P < 0.05). After 15-h reperfusion, nitrotyrosine was detected only in wild-type mice (0.039 +/- 0.025%, n = 7), not in knockout or sham-operated mice (P < 0.05). Immunohistochemical reaction for nitrotyrosine was seen predominantly in the vascular wall in the peri-infarct region of the cerebral cortex in wild-type mice after 15-h reperfusion, but not in corresponding knockout mice. Our data suggest that iNOS is responsible for nitrotyrosine formation in the later phase of reperfusion, and that vascular endothelium is the major site of this reaction, at least in the case of 15-h reperfusion.

Topics: Acidosis; Animals; Blood Glucose; Blood Pressure; Carbon Dioxide; Cerebral Cortex; Cerebrovascular Circulation; Chromatography, High Pressure Liquid; Endothelium, Vascular; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxygen; Reperfusion Injury; Tyrosine

Nitric oxide contributes to tissue necrosis after ischemia-reperfusion (IR). A biochemical and immunohistochemical study was made of the amounts and localization of both Ca++-independent nitric oxide synthase (NOS) II and Ca++-dependent (NOS I and NOS III) in rat skeletal muscle after ischemia and 0.5, 2, 8, 16, and 24 hours reperfusion. NOS II was not detectable in control muscle or during ischemia, was first detected after 2 hours reperfusion, increased further by 8 hours, and remained elevated at 24 hours. Both NOS II and nitrotyrosine, a marker of peroxynitrite formation, were localized exclusively to mast cells except after 24 hours reperfusion when some macrophages and neutrophils also showed positive immunoreactivity. Mast cells underwent extensive degranulation during reperfusion. NOS I was not detected in injured or control muscle. The level of NOS III, which was localized to the endothelium of blood vessels of all sizes in control muscle, decreased progressively during ischemia and reperfusion to reach undetectable levels after 16 hours reperfusion. These findings indicate that most of the nitric oxide formed during IR injury is generated by NOS II located almost exclusively in mast cells.

Topics: Animals; Isoenzymes; Mast Cells; Muscle, Skeletal; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Reperfusion Injury; Tyrosine

Poly(ADP-ribose) synthase (PARS), an abundant nuclear protein, has been described as an important candidate for mediation of neurotoxicity by nitric oxide. However, in cerebral ischemia, excessive PARS activation may lead to energy depletion and exacerbation of neuronal damage. We examined the effect of inhibiting PARS on the (a) degree of cerebral injury, (b) process of inflammatory responses, and (c) functional outcomes in a neonatal rat model of focal ischemia. We demonstrate that administration of 3-aminobenzamide, a PARS inhibitor, leads to a significant reduction of infarct volume: 63 +/- 2 (untreated) versus 28 +/- 4 mm(3) (treated). The neuroprotective effects currently observed 48 h postischemia hold up at 7 and 17 days of survival time and attenuate neurological dysfunction. Inhibition of PARS activity, demonstrated by a reduction in poly(ADP-ribose) polymer formation, also reduces neutrophil recruitment and levels of nitrotyrosine, an indicator of peroxynitrite generation. Taken together, our results demonstrate that PARS inhibition reduces ischemic damage and local inflammation associated with reperfusion and may be of interest for the treatment of neonatal stroke.

Topics: Animals; Animals, Newborn; Benzamides; Brain Ischemia; Cell Death; Cerebral Infarction; Encephalitis; Female; Male; Motor Activity; Neurologic Examination; Neuroprotective Agents; Neutrophils; Nitrates; Poly(ADP-ribose) Polymerase Inhibitors; Polymers; Rats; Rats, Wistar; Reperfusion Injury; Signal Transduction; Stroke; Time Factors; Tyrosine

The aim of the present study was to investigate the protective effect of the peroxynitrite decomposition catalyst 5,10,15, 20-tetrakis(2,4,6-trimethyl-3,5-disulfonatophenyl)-porphyrinato iron (III) (FeTMPS) in a model of splanchnic artery occlusion shock (SAO). SAO shock was induced in rats by clamping both the superior mesenteric artery and the celiac trunk for 45 min, followed by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO-shocked rats after reperfusion, but not during ischemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, an index of nitrogen species such as peroxynitrite, in the necrotic ileum in shocked rats. SAO-shocked rats developed a significant increase of tissue myeloperoxidase and malonaldehyde activity, and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival at 2 h after reperfusion). Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin localized mainly in the vascular endothelial cells. Ileum tissue sections obtained from SAO-shocked rats and stained with antibody to ICAM-1 showed a diffuse staining. Administration of FeTMPS significantly reduced ischemia/reperfusion injury in the bowel, and reduced lipid and the production of peroxynitrite during reperfusion. Treatment with PN catalyst also markedly reduced the intensity and degree of P-selectin and ICAM-1 staining in tissue sections from SAO-shocked rats and improved survival. Our results clearly demonstrate that peroxynitrite decomposition catalysts exert a protective effect in SAO and that this effect may be due to inhibition of the expression of adhesion molecules and the tissue damage associated with peroxynitrite-related pathways.

Topics: Animals; Blood Pressure; Catalysis; Celiac Artery; Disease Models, Animal; Endothelium, Vascular; Ferric Compounds; Ileum; Intercellular Adhesion Molecule-1; Leukocyte Count; Lipid Peroxidation; Male; Malondialdehyde; Metalloporphyrins; Nitrates; Nitric Oxide; Nitrites; Oxidative Stress; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Rhodamines; Splanchnic Circulation; Tyrosine

1. Nitric oxide (NO), peroxynitrite, formed from NO and superoxide anion, poly (ADP-ribole) synthetase have been implicated as mediators of neuronal damage following focal ischaemia. Here we have investigated the effects of n-acetylcysteine (NAC) treatment in Mongolian gerbils subjected to cerebral ischaemia. 2. Treatment of gerbils with NAC (20 mg kg(-1) 30 min before reperfusion and 1, 2 and 6 h after reperfusion) reduced the formation of post-ischaemic brain oedema, evaluated by water content. 3. NAC also attenuated the increase in the brain levels of malondialdehyde (MDA) and the increase in the hippocampus of myeloperoxidase (MPO) caused by cerebral ischaemia. 4. Positive staining for nitrotyrosine was found in the hippocampus in Mongolian gerbils subjected to cerebral ischaemia. Hippocampus tissue sections from Mongolian gerbils subjected to cerebral ischaemia also showed positive staining for poly (ADP-ribose) synthetase (PARS). The degree of staining for nitrotyrosine and for PARS were markedly reduced in tissue sections obtained from animals that received NAC. 5. NAC treatment increased survival and reduced hyperactivity linked to neurodegeneration induced by cerebral ischaemia and reperfusion. 6. Histological observations of the pyramidal layer of CA1 showed a reduction of neuronal loss in animals that received NAC. 7. These results show that NAC improves brain injury induced by transient cerebral ischaemia.

Topics: Acetylcysteine; Animals; Brain; Brain Edema; Brain Ischemia; Free Radical Scavengers; Gerbillinae; Male; Malondialdehyde; Motor Activity; Nitric Oxide; Peroxidase; Poly(ADP-ribose) Polymerases; Reperfusion Injury; Time Factors; Tyrosine

The generation of reactive oxygen species (ROS) contributes to the pathogenesis of renal ischemia-reperfusion injury. The aim of this study was to investigate the effects of tempol in (1) an in vivo rat model of renal ischemia/reperfusion injury and on (2) cellular injury and death of rat renal proximal tubular (PT) cells exposed to oxidant stress in the form of hydrogen peroxide (H2O2).. Male Wistar rats underwent bilateral renal pedicle clamping for 45 minutes followed by reperfusion for six hours. Tempol (30 mg/kg/h), desferrioxamine (DEF; 40 mg/kg/h), or a combination of tempol (30 mg/kg/h) and DEF (40 mg/kg/h) were administered prior to and throughout reperfusion. Plasma concentrations of urea, creatinine, Na+, gamma-glutamyl transferase (gammaGT), aspartate aminotransferase (AST), and urinary Na+ and N-acetyl-beta-D-glucosaminidase (NAG) were measured for the assessment of renal function and reperfusion injury. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of polymorphonuclear (PMN) cell infiltration and lipid peroxidation, respectively. Renal sections were used for histologic grading of renal injury and for immunohistochemical localization of nitrotyrosine and poly(ADP-ribose) synthetase (PARS). Primary cultures of rat PT cells were incubated with H2O2 (1 mmol/L for 4 h) either in the absence or presence of increasing concentrations of tempol (0.03 to 10 mmol/L), DEF (0.03 to 10 mmol/L), or a combination of tempol (3 mmol/L) or DEF (3 mmol/L). PT cell injury and death were determined by evaluating mitochondrial respiration and lactate dehydrogenase (LDH) release, respectively.. In vivo, tempol significantly reduced the increase in urea, creatinine, gammaGT, AST, NAG, and FENa produced by renal ischemia/reperfusion, suggesting an improvement in both renal function and injury. Tempol also significantly reduced kidney MPO activity and MDA levels, indicating a reduction in PMN infiltration and lipid peroxidation, respectively. Tempol reduced the histologic evidence of renal damage associated with ischemia/reperfusion and caused a substantial reduction in the staining for nitrotyrosine and PARS, suggesting reduced nitrosative and oxidative stress. In vitro, tempol significantly attenuated H2O2-mediated decrease in mitochondrial respiration and increase in LDH release from rat PT cells, indicating a reduction in cell injury and death. Both in vivo and in vitro, the beneficial actions of tempol were similar to those obtained using the Fe2+ chelator DEF. However, coadministration of DEF and tempol did not produce any additional beneficial actions against renal ischemia/reperfusion injury or against oxidative stress-mediated PT cell injury/death.. Our results suggest that the membrane-permeable radical scavenger, tempol, reduces the renal dysfunction and injury associated with ischemia/reperfusion of the kidney.

Topics: Acute Kidney Injury; Animals; Cell Membrane Permeability; Cell Separation; Cells, Cultured; Chelating Agents; Cyclic N-Oxides; Deferoxamine; Disease Models, Animal; Free Radical Scavengers; Hydrogen Peroxide; Kidney Glomerulus; Kidney Tubules, Proximal; Male; Malondialdehyde; Necrosis; Oxidants; Oxidative Stress; Peroxidase; Poly(ADP-ribose) Polymerases; Rats; Rats, Wistar; Reperfusion Injury; Spin Labels; Tyrosine

Reperfusion injury is one of the factors that unfavorably affects stroke outcome and shortens the window of opportunity for thrombolysis. Surges of nitric oxide (NO) and superoxide generation on reperfusion have been demonstrated. Concomitant generation of these radicals can lead to formation of the strong oxidant peroxynitrite during reperfusion.. We have examined the role of NO generation and peroxynitrite formation on reperfusion injury in a mouse model of middle cerebral artery occlusion (2 hours) and reperfusion (22 hours). The infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride staining; blood-brain barrier permeability was evaluated by Evans blue extravasation. Nitrotyrosine formation and matrix metalloproteinase-9 expression were detected by immunohistochemistry.. Infarct volume was significantly decreased (47%) in animals treated with the nonselective nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NA) at reperfusion. The specific inhibitor of neuronal NOS, 7-nitroindazole (7-NI), given at reperfusion, showed no protection, although preischemic treatment with 7-NI decreased infarct volume by 40%. Interestingly, prereperfusion administration of both NOS inhibitors decreased tyrosine nitration (a marker of peroxynitrite toxicity) in the ischemic area. L-NA treatment also significantly reduced vascular damage, as indicated by decreased Evans blue extravasation and matrix metalloproteinase-9 expression.. These data support the hypothesis that in addition to the detrimental action of NO formed by neuronal NOS during ischemia, NO generation at reperfusion plays a significant role in reperfusion injury, possibly through peroxynitrite formation. Contrary to L-NA, failure of 7-NI to protect against reperfusion injury suggests that the source of NO is the cerebrovascular compartment.

Topics: Animals; Biomarkers; Blood-Brain Barrier; Coloring Agents; Endothelium, Vascular; Enzyme Inhibitors; Evans Blue; Indazoles; Infarction, Middle Cerebral Artery; Matrix Metalloproteinase 9; Mice; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxidants; Permeability; Reperfusion Injury; Tyrosine

Topics: Alprostadil; Animals; Arginine; Burns; Enzyme Inhibitors; Humans; Ischemia; Luminescent Measurements; Models, Biological; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Oxidative Stress; Reperfusion Injury; Sepsis; Shock; Superoxides; Tyrosine

Splanchnic artery occlusion shock (SAO) causes an enhanced formation of reactive oxygen species (ROS), which contribute to the pathophysiology of shock. Here we have investigated the effects of n-acetylcysteine (NAC), a free radical scavenger, in rats subjected to SAO shock.. Treatment of rats with NAC (applied at 20 mg/kg, 5 min prior to reperfusion, followed by an infusion of 20 mg/kg/h) attenuated the mean arterial blood and the migration of polymorphonuclear cells (PMNs) caused by SAO-shock. NAC also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase (MPO) and malondialdehyde (MDA) caused by SAO shock in the ileum. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine in the plasma of the SAO-shocked rats after reperfusion. Immunohistochemical analysis for nitrotyrosine and for poly(ADP-ribose) synthetase (PARS) revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine and PARS were markedly reduced in tissue sections obtained from SAO-shocked rats which had received NAC. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin, which was mainly localised in the vascular endothelial cells. Ileum tissue section obtained from SAO-shocked rats with anti-intercellular adhesion molecule (ICAM-1) antibody showed a diffuse staining. NAC treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue section from SAO-shocked rats. In addition, in ex vivo studies in aortic rings from shocked rats, we found reduced contractions to noradrenaline and reduced responsiveness to a relaxant effect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively). NAC treatment improved contractile responsiveness to noradrenaline, enhanced the endothelium-dependent relaxations and significantly improved survival.. Taken together, our results clearly demonstrate that NAC treatment exert a protective effect and part of this effect may be due to inhibition of the expression of adhesion molecule and peroxynitrite-related pathways and subsequent reduction of neutrophil-mediated cellular injury.

Topics: Acetylcholine; Acetylcysteine; Analysis of Variance; Animals; Aorta; Fluorescent Antibody Technique, Indirect; Free Radical Scavengers; Ileum; In Vitro Techniques; Intercellular Adhesion Molecule-1; Leukocyte Count; Lipid Peroxidation; Male; Malondialdehyde; Nitrates; Nitrites; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Splenic Artery; Tyrosine; Vasodilation

There is evidence that the excessive generation of reactive-oxygen radicals contributes to the brain injury associated with transient, cerebral ischemia. This study investigates the effects of tempol, a small, water-soluble molecule, that crosses biological membranes, on the brain injury caused by bilateral occlusion and reperfusion of both common carotid arteries in the gerbil (BCO). Treatment of gerbils with tempol (30 mg/kg i.p. at 30 min prior to reperfusion and at 1 and 6 h after the onset of reperfusion) reduced the formation of post-ischemic brain oedema. Tempol also attenuated the increase in the cerebral levels of malondialdehyde (MDA) and the hippocampal levels of myeloperoxidase (MPO) caused by cerebral ischemia and reperfusion. The immunohistochemical analysis of the hippocampal region of brains subjected to ischemia-reperfusion exhibited positive staining for nitrotyrosine (an indicator of the generation of peroxynitrite) and poly(ADP-ribose) synthetase (PARS) (an indicator of the activation of this nuclear enzyme secondary to single strand breaks in DNA). In gerbils subjected to BCO, which were treated with tempol, the degree of staining for nitrotyrosine and PARS was markedly reduced. Tempol increased survival and reduced the hyperactivity (secondary to the ischemia-induced neurodegeneration) caused by cerebral ischemia and reperfusion. The loss of neurons from the pyramidal layer of the CA1 region caused by ischemia and reperfusion was also attenuated by treatment of gerbils with tempol. This is the first evidence that the membrane-permeable, radical scavenger tempol reduces the cerebral injury caused by transient, cerebral ischemia in vivo.

Topics: Animals; Arterial Occlusive Diseases; Brain; Brain Edema; Cerebral Arteries; Cyclic N-Oxides; Free Radical Scavengers; Gerbillinae; Hippocampus; Lipid Peroxides; Male; Malondialdehyde; Motor Activity; Neutrophil Infiltration; Nitrates; Nitrites; Peroxidase; Poly(ADP-ribose) Polymerases; Reperfusion Injury; Spin Labels; Survival Analysis; Tyrosine

Recent studies have demonstrated that melatonin is a scavenger of oxyradicals and peroxynitrite and an inhibitor of nitric oxide (NO) production. NO, peroxynitrite (formed from NO and superoxide anion), and poly (ADP-Ribose) synthetase (PARS) have been implicated as mediators of neuronal damage following focal ischemia. In the present study, we have investigated the effects of melatonin treatment in Mongolian gerbils subjected to cerebral ischemia. Treatment of gerbils with melatonin (10 mg kg(-1), 30 min before reperfusion and 1, 2, and 6 hr after reperfusion) reduced the formation of post-ischemic brain edema, evaluated by water content. Melatonin also attenuated the increase in the brain levels malondialdehyde (MDA) and the increase in the hippocampus of myeloperoxidase (MPO) caused by cerebral ischemia. Positive staining for nitrotyrosine was found in the hippocampus of Mongolian gerbils subjected to cerebral ischemia. Hippocampus tissue sections, from Mongolian gerbils subjected to cerebral ischemia, also showed positive staining for PARS. The degrees of staining for nitrotyrosine and for PARS were markedly reduced in tissue sections obtained from animals that received melatonin. Melatonin treatment increased survival and reduced hyperactivity linked to neurodegeneration induced by cerebral ischemia and reperfusion. Histological observations of the pyramidal layer of CA-1 showed a reduction of neuronal loss in animals that received melatonin. These results show that melatonin improves brain injury induced by transient cerebral ischemia.

Topics: Animals; Brain; Brain Edema; Brain Ischemia; Enzyme-Linked Immunosorbent Assay; Free Radical Scavengers; Gerbillinae; Immunoenzyme Techniques; Male; Malondialdehyde; Melatonin; Motor Activity; Nitrates; Nitrites; Oxidative Stress; Peroxidase; Poly(ADP-ribose) Polymerases; Reperfusion Injury; Tyrosine

4-Hydroxy-2-nonenal (HNE)-modified proteins and 3-nitro-L-tyrosine were evaluated as a specific marker of reactive oxygen species (ROS)- and nitric oxide (NO)-mediated peroxynitrite-induced tissue injuries in ischemic and reperfused skin flap by Western blot analysis. Specimens were taken from island skin flaps of rats during the following three conditions: ischemia only, 5 hours of ischemia and reperfusion, and 10 hours of ischemia and reperfusion. HNE-modified proteins and 3-nitro-L-tyrosine increased with ischemic time (3, 6, and 10 hours postischemia). In the reperfused skin flap after both 5 and 10 hours of ischemia, HNE-modified proteins and 3-nitro-L-tyrosine were increased 3 hours postreperfusion, and they reached a maximum 6 hours after reperfusion. HNE-modified proteins and 3-nitro-L-tyrosine 1 hour postreperfusion were higher with 10 hours ischemia-reperfusion than with 5 hours ischemia-reperfusion. These results indicate (1) that ROS- and NO-induced peroxynitrite-mediated cytotoxicity in ischemic flaps is dependent on the ischemic period and (2) that ROS- and NO-induced peroxynitrite-mediated cytotoxicity occurs during an early stage of reperfusion if the ischemic period is long.

Topics: Aldehydes; Animals; Blotting, Western; Free Radicals; Ischemia; Male; Proteins; Rats; Rats, Wistar; Reperfusion Injury; Skin; Specific Pathogen-Free Organisms; Surgical Flaps; Tyrosine

The purpose of this study was to establish the dynamics of nitrotyrosine (NO2-Tyr) formation and decay during the rise of NO2-Tyr in rat brain subjected to 2-hour focal ischemia-reperfusion, and to evaluate the role of inducible nitric oxide synthase in the rise. The authors first determined the half life of NO2-Tyr in rat brain at 24 hours after the start of reperfusion by blocking NO2-Tyr formation with N(G)-monomethyl-L-arginine and after the decay of NO2-Tyr by means of a hydrolysis/HPLC procedure. The values obtained were approximately 2 hours in both peri-infarct and core-of-infarct regions. Using the same hydrolysis/HPLC procedure, the ratio of nitrotyrosine to tyrosine from the 2-hour occlusion to as much as 72 hours after the start of reperfusion was measured in the presence and absence of aminoguanidine (100 mg/kg intraperitoneally twice a day). In the absence of aminoguanidine, the ratio of NO2-Tyr in the peri-infarct and core-of-infarct regions reached 0.95% +/- 0.34% and 0.52% +/- 0.34%, respectively, at 1 hour after the start of reperfusion. The elevated levels persisted until 48 hours, then declined. The peri-infarct region showed the highest percent NO2-Tyr level, followed by the core of infarct, then the caudoputamen. Aminoguanidine significantly reduced NO2-Tyr formation (up to 90% inhibition) during 24 to 48 hours. The authors conclude that inducible nitric oxide synthase is predominantly responsible for NO2-Tyr formation, at least in the late phase of reperfusion. These results have important implications for the therapeutic time window and choice of nitric oxide synthase inhibitors in patients with cerebral infarction.

Topics: Animals; Brain Chemistry; Brain Ischemia; Cerebrovascular Circulation; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Guanidines; Half-Life; Hydrolysis; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; omega-N-Methylarginine; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tyrosine

The purpose of this study was to evaluate the role of neuronal nitric oxide synthase (nNOS) in nitrotyrosine (NO2-Tyr) formation in the early phase of ischemia-reperfusion in mouse brain. Using a hydrolysis/high pressure liquid chromatography (HPLC) procedure (0.6 microM detection limit), we measured %NO2-Tyr (ratio of NO2-Tyr to total tyrosine) in 23 male C57Black/6J mice subjected to 2-h middle cerebral artery occlusion followed by 0.5-h reperfusion, in the presence (25 or 50 mg/kg) and absence of 7-nitroindazole (7-NI), a relatively specific nNOS inhibitor. At 25 mg/kg, 7-NI reduced NO2-Tyr formation to about a half of that in the vehicle-treated group (0.10 +/- 0.07 vs. 0.18 +/- 0.05%), while 50 mg/kg suppressed NO2-Tyr formation to below the limit of detection, indicating that nNOS is responsible for most of the NO2-Tyr formation in the early phase after reperfusion.

Topics: Animals; Brain Ischemia; Chromatography, High Pressure Liquid; Indazoles; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Reperfusion Injury; Tyrosine

We used IL-6 knock-out (KO) mice to evaluate a possible role for IL-6 in the pathogenesis of splanchnic artery occlusion shock (SAO). SAO shock was induced by clamping both the superior mesenteric artery and the celiac trunk, followed by release of the clamp. There was a marked increase in the peroxynitrite formation in the plasma of the SAO-shocked IL-6 wild-type (WT) mice after reperfusion. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine in the necrotic ileum in shocked IL-6 WT mice. SAO-shocked WT mice developed a significant increase of tissue myeloperoxidase (MPO) and malondialdehyde (MDA) activity and marked histological injury to the distal ileum. SAO shock was also associated with a significant mortality (0% survival). Reperfused ileum tissue sections from SAO-shocked WT mice showed positive staining for P-selectin. Little specific staining was observed in sham-WT mice. Staining of ileum tissue obtained from sham-operated WT mice with anti-ICAM-1 antibody showed weak but diffuse staining, demonstrating that ICAM-1 is constitutively expressed. However, after SAO shock the staining intensity increased substantially in the ileum section from WT mice. Intensity and degree of P-selectin and ICAM-1 were markedly reduced in tissue section from SAO-shocked IL-6 KO mice. SAO-shocked IL-6 KO mice also show significant reduction of neutrophil infiltration into the reperfused intestine, as evidenced by reduced MPO activity, improved histological status of the reperfused tissues, reduced peroxynitrite formation, reduced MDA levels, and improved survival. In vivo treatment with anti-IL-6 significantly prevents the inflammatory process. Our results clearly demonstrate that IL-6 plays an important role in ischemia and reperfusion injury and allows the hypothesis that inhibition of IL-6 may represent a novel and possible strategy. Part of this effect may be due to inhibition of the expression of adhesion molecules and subsequent reduction of neutrophil-mediated cellular injury.

Topics: Animals; Antibodies, Monoclonal; Arterial Occlusive Diseases; Celiac Artery; Constriction; Cytokines; Ileum; Immunity, Innate; Intercellular Adhesion Molecule-1; Interleukin-6; Ischemia; Leukocyte Count; Lipid Peroxidation; Malondialdehyde; Mesenteric Artery, Superior; Mice; Mice, Knockout; Neutrophils; Nitrates; Nitric Oxide; P-Selectin; Peroxidase; Reperfusion Injury; Shock; Splanchnic Circulation; Tyrosine

Although inhibition of endothelial nitric oxide synthase (eNOS) has been reported to aggravate hepatic ischemia-reperfusion (I/R) injury, the role of inducible nitric oxide synthase (iNOS) has been still unknown. We investigated the role of NO produced by iNOS, and evaluated the effect of an iNOS inhibitor on prolonged warm I/R injury in the pig liver.. Pigs were subjected to 120 min of hepatic warm I/R under the extracorporeal circulation. We investigated the time course of changes in serum and hepatic microdialysate NO2- + NO3- (NOx) and the cellular distribution of eNOS and iNOS by immunohistochemistry, including a double-immunofluorescence technique in combination with confocal laser scanning microscopy. The effect of iNOS inhibitor was also investigated.. Hepatic I/R induced new nitric oxide production in serum and hepatic microdialysate NOx after reperfusion and severe hepatic damage in the centrilobular region where nitrotyrosine was strongly expressed. Diffuse eNOS expression in sinusoidal endothelium did not differ before and after reperfusion. In contrast, strong iNOS expression in Kupffer cells and neutrophils appeared strongly in the centrilobular region after reperfusion. Pigs with intraportal administration of N(G)-nitro-L-arginine (10 mg/kg) died during the period of ischemia or early in the period of reperfusion with a high mortality rate (80.0%). Intraportal administration of aminoguanidine hemisulfate (10 mg/kg) significantly suppressed nitric oxide production and serum aspartate aminotransferase after reperfusion, inhibited nitrotyrosine expression, and attenuated hepatic damage.. These results indicate that hepatic I/R injury is triggered by centrilobular iNOS expression; and attenuated by inhibition of iNOS.

Topics: Animals; Arteries; Aspartate Aminotransferases; Blood Pressure; Female; Fluorescent Antibody Technique; Liver; Microscopy, Confocal; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Reperfusion Injury; Survival Rate; Swine; Tyrosine

We have investigated the role of oxidative damage in peripheral nerve ischaemia-reperfusion injury using a rat sciatic nerve model. After 5 h ischaemia blood flow to the sciatic nerve was restarted and markers of oxidative damage measured after various times of reperfusion. As a marker of protein oxidative damage, protein carbonyl formation was measured using a sensitive enzyme-linked immunosorbent assay. Protein carbonyl content was unaffected by ischaemia alone, but increased by 55% after 12-18 h reperfusion, correlating with the onset of nerve pathology. Pretreatment with the xanthine oxidase inhibitor allopurinol prevented these abnormalities, suggesting that xanthine oxidase activity is proximal to oxidative damage during reperfusion injury. To determine whether formation of the potent oxidant peroxynitrite from nitric oxide and superoxide contributed to ischaemia-reperfusion injury, we measured the accumulation of 3-nitrotyrosine residues in proteins. Only one protein of 49,000 mol. wt contained significant amounts of 3-nitrotyrosine residues which was shown to be glial fibrillary acidic protein, an abundant cytoskeletal protein in Schwann cells. However glial fibrillary acidic protein contained 3-nitrotyrosine residues prior to ischaemia-reperfusion, and the amount of nitrated tyrosine residues in total glial fibrillary acidic protein did not increase significantly during reperfusion, therefore it was not possible to draw conclusions about the role of peroxynitrite in nerve reperfusion injury.

Topics: Animals; Biomarkers; Enzyme-Linked Immunosorbent Assay; Glial Fibrillary Acidic Protein; Ischemia; Male; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sciatic Nerve; Sensitivity and Specificity; Time Factors; Tyrosine

Oxygen free radicals and nitric oxide (NO.) have been proposed to be involved in acute CNS injury produced by cerebral ischemia; however, controversy remains regarding how they cause injury. Because superoxide generation is triggered during reperfusion, the cytotoxic oxidant peroxynitrite could be formed, but it is not known if this occurs. Dot blot and immunohistochemistry studies were performed on the magnitude and time course of tyrosine nitration and inducible NO synthase (NOS2) in the postischemic rat pup brain. Neonatal ischemia was induced by permanent left middle cerebral artery occlusion in association with 1-h occlusion of the left common carotid artery in 7-day-old Wistar pups. Nitrotyrosine (NT) immunoreactivity was evident in the blood vessels close to the cortical infarct at 48-72 h of recovery, and T lymphocytes were involved with this production. NOS2 immunoreactivity was seen in neutrophils in the same vessels and in the parenchyma at 72 h of recirculation. Whereas NT staining decreased with time, NOS2-positive neutrophils could be still detected in arachnoid vessels at 14 days of recirculation. We conclude that perivascular reactions mediated by peroxynitrite are important in the cascade of events that lead to brain oxidative stress in neonatal ischemia. Moreover, NO-related species may serve as a signaling function instead of directly mediating toxicity.

Topics: Animals; Animals, Newborn; Brain; Cell Death; Female; Immunoenzyme Techniques; Ischemic Attack, Transient; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidants; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine