Studies on hepatic injury and antioxidant enzyme activities in rat subcellular organelles following in vivo ischemia and reperfusion

The activities of rat hepatic subcellular antioxidant enzymes were studied during hepatic ischemia/reperfusion. Ischemia was induced for 30 min (reversible ischemia) or 60 min (irreversible ischemia). Ischemia was followed by 2 or 24 h of reperfusion. Hepatocyte peroxisomal catalase enzyme activity decreased during 60 min of ischemia and declined further during reperfusion. Peroxisomes of normal density (d = 1.225 gram/ml) were observed in control tissues. However, 60 min of ischemia also produced a second peak of catalase specific activity in subcellular fractions corresponding to newly formed low density immature peroxisomes (d = 1.12 gram/ml). The second peak was also detectable after 30 min of ischemia followed by reperfusion for 2 or 24 h. Mitochondrial and microsomal fractions responded differently. MnSOD activity in mitochondria and microsomal fractions increased significantly (p < 0.05) after 30 min of ischemia, but decreased below control values following 60 min of ischemia and remained lower during reperfusion at 2 and 24 h in both organelle fractions. Conversely, mitochondrial and microsomal glutathione peroxidase (GPx) activity increased significantly (p < 0.001) after 60 min of ischemia and was sustained during 24 h of reperfusion. In the cytosolic fraction, a significant increase in CuZnSOD activity was noted following reperfusion in animals subjected to 30 min of ischemia, but 60 min of ischemia and 24 h of reperfusion resulted in decreased CuZnSOD activity. These studies suggest that the antioxidant enzymes of various subcellular compartments respond to ischemia/reperfusion in an organelle or compartment specific manner and that the regulation of antioxidant enzyme activity in peroxisomes may differ from that in mitochondria and microsomes. The compartmentalized changes in hepatic antioxidant enzyme activity may be crucial determinant of cell survival and function during ischemia/reperfusion. Finally, a progressive decline in the level of hepatic reduced glutathione (GSH) and concomitant increase in serum glutamate pyruvate transaminase (SGPT) activity also suggest that greater tissue damage and impairment of intracellular antioxidant activity occur with longer ischemia periods, and during reperfusion.     

Electron spin resonance and spin trapping technique provide direct evidence that edaravone prevents acute ischemia–reperfusion injury of the liver by limiting free radical-mediated tissue damage

A novel free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), is used for the treatment of acute ischemic stroke and is protective in several animal models of organ injury. We tested whether edaravone is protective against acute liver warm ischemia/reperfusion injury in the rat by acting as a radical scavenger. When edaravone was administered prior to ischemia and at the time of initiation of the reperfusion, liver injury was markedly reduced. Production of oxidants in the liver in this model was assessed in vivo by spin-trapping/electron spin resonance (ESR) spectroscopy. Ischemia/reperfusion caused an increase in free radical adducts rapidly, an effect markedly blocked by edaravone. Furthermore, edaravone treatment blunted ischemia/reperfusion-induced elevation in pro-inflammatory cytokines, infiltration of leukocytes and lipid peroxidation in the liver. These results demonstrate that edaravone is an effective blocker of free radicals in vivo in the liver after ischemia/reperfusion, leading to prevention of organ injury by limiting the deleterious effects of free radicals.     

Fetuin-A exerts a protective effect against experimentally induced intestinal ischemia/reperfusion by suppressing autophagic cell death

Intestinal tissue is highly susceptible to ischemia/reperfusion injury in many hazardous health conditions. The anti-inflammatory and antioxidant glycoprotein fetuin-A showed efficacy in cerebral ischemic injury; however, its protective role against intestinal ischemia/reperfusion remains elusive. Therefore, this study investigated the protective role of fetuin-A supplementation against intestinal structural changes and dysfunction in a rat model of intestinal ischemia/reperfusion. We equally divided 72 male rats into control, sham, ischemia/reperfusion, and fetuin-A-pretreated ischemia/reperfusion (100 mg/kg/day fetuin-A intraperitoneally for three days prior to surgery and a third dose 1 h prior to the experiment) groups. After 2 h of reperfusion, the jejunum was dissected and examined for spontaneous contractility. A jejunal homogenate was used to assess inflammatory and oxidative stress enzymes. Staining of histological sections was carried out with hematoxylin, eosin and Masson’s trichrome stain for evaluation. Immunohistochemistry was performed to detect autophagy proteins beclin-1, LC3, and p62. This study found that fetuin-A significantly improved ischemia/reperfusion-induced mucosal injury by reducing the percentage of areas of collagen deposition, increasing the amplitude of spontaneous contraction, decreasing inflammation and oxidative stress, and upregulating p62 expression, which was accompanied by beclin-1 and LC3 downregulation. Our findings suggest that fetuin-A treatment can prevent ischemia/reperfusion-induced jejunal structural and functional changes by increasing antioxidant activity and regulating autophagy disturbances observed in the ischemia/reperfusion rat model. Furthermore, fetuin-A may provide a protective influence against intestinal ischemia/reperfusion complications.     

Heat shock preconditioning reduces the formation of 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal modified proteins in ischemia-reperfused liver of rats

Heat shock preconditioning (HSPC) is a promising strategy for providing ischemic tolerance. The objective of this study is to investigate the effectiveness of HSPC in preventing oxidative damage of cellular proteins and DNA during ischemia-reperfusion of the liver. Male Wistar rats were divided into a heat shock group (group HS) and control (group C). Forty-eight hours prior to ischemia, rats in group HS received HSPC at 42 degrees C for 15 min. All rats received hepatic warm ischemia for 30min and subsequent reperfusion. The formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE) modified proteins in liver tissue, survival rate of the animals, and changes in biochemical and histological parameters were compared between groups. Heat shock protein 72 was produced only in group HS. The 7-day survival of rats was significantly better in group HS (10/10) than in group C (5/10) (p < 0.01). The serum release of alanine aminotransferase (n = 10, p < 0.01) and the concentration of adenosine triphosphate in liver tissue (n = 10, p < 0.01) 40min after reperfusion was significantly better in group HS than in group C. The formation of 8-OHdG in liver tissue measured by high-performance liquid chromatography was suppressed in group HS (p < 0.01). The production of HNE-modified proteins as determined by Western-blot analysis was also decreased in group HS. These results were also confirmed by immunohistochemical analysis. As determined by levels of 8-OHdG and HNE-modified proteins produced during ischemia-reperfusion of the liver, HSPC reduced the oxidative injury of cellular proteins and DNA in the liver tissue.     

Attenuation of mitochondrial injury by L-arginine preconditioning of the liver

The present study was aimed to evaluate the efficacy of L-arginine on mitochondrial function in ischemic and reperfusion (I/R) induced hepatic injury. Adult Wistar rat were subjected to 1 h of partial liver ischemia followed by 3 hour reperfusion. Eighteen wistar rats were divided into three groups viz. sham-operated control group (I) (n = 6), ischemia and reperfusion (I/R) group (II) (n = 6), L-arginine treated group (100 mg/kg body weight/daily by oral route for 7 days before induced ischemia reperfusion maneuver) (III) (n = 6). Mitochondrial injury was assessed in terms of decreased (P < 0.05) activities of mitochondrial antioxidant enzymes (GSH, SOD, CAT), respiratory marker enzymes (NADH dehydrogenase, cytochrome c oxidases) and hepatocytes nitric oxide production. Pre-treatment with L-arginine (10 mg/kg/p.o. for 7 days) significantly counteracted the alternations of hepatic enzymes and mitochondrial respiratory and antioxidant enzymes. In addition, electron microscopy and histopathology study showed the restoration of cellular normalcy and accredits the cytoprotective role of L-arginine against I/R induced hepatocellular injury. On the basis of these findings it may be concluded that L-arginine protects mitochondrial function in hepatic ischemic and reperfused liver.     

Local hypothermia during early reperfusion protects skeletal muscle from ischemia-reperfusion injury

Amputated tissue maintained in a hypothermic environment can endure prolonged ischemia and improve replantation success. The authors hypothesized that local tissue hypothermia during the early reperfusion period may provide a protective effect against ischemia-reperfusion injury similar to that seen when hypothermia is provided during the ischemic period. A rat gracilis muscle flap model was used to assess the protective effects of exposing skeletal muscle to local hypothermia during ischemia only (p = 18), reperfusion only (p = 18), and both ischemia and reperfusion (p = 18). Gracilis muscles were isolated and exposed to hypothermia of 10 degrees C during 4 hours of ischemia, the initial 3 hours of reperfusion, or both periods. Ischemia-reperfusion outcome measures used to evaluate muscle flap injury included muscle viability (percent nitroblue tetrazolium staining), local edema (wet-to-dry weight ratio), neutrophil infiltration (intramuscular neutrophil density per high-power field), neutrophil integrin expression (CD11b mean fluorescence intensity), and neutrophil oxidative potential (dihydro-rhodamine oxidation mean fluorescence intensity) after 24 hours of reperfusion. Nitroblue tetrazolium staining demonstrated improved muscle viability in the experimental groups (ischemia-only: 78.8 +/- 3.5 percent, p < 0.001; reperfusion-only: 80.2 +/- 5.2 percent, p < 0.001; and ischemia-reperfusion: 79.6 +/- 7.6 percent, p < 0.001) when compared with the nonhypothermic control group (50.7 +/- 9.3 percent). The experimental groups demonstrated decreased local muscle edema (4.09 +/- 0.30, 4.10 +/- 0.19, and 4.04 +/- 0.31 wet-to-dry weight ratios, respectively) when compared with the nonhypothermic control group (5.24 +/- 0.31 wet-to-dry weight ratio; p < 0.001, p < 0.001, and p < 0.001, respectively). CD11b expression was significantly decreased in the reperfusion-only (32.65 +/- 8.75 mean fluorescence intensity, p < 0.001) and ischemia-reperfusion groups (25.26 +/- 5.32, p < 0.001) compared with the nonhypothermic control group (62.69 +/- 16.93). There was not a significant decrease in neutrophil CD11b expression in the ischemia-only group (50.72 +/- 11.7 mean fluorescence intensity, p = 0.281). Neutrophil infiltration was significantly decreased in the reperfusion-only (20 +/- 11 counts per high-power field, p = 0.025) and ischemia-reperfusion groups (23 +/- 3 counts, p = 0.041) compared with the nonhypothermic control group (51 +/- 28 counts). No decrease in neutrophil density was observed in the ischemia-only group (40 +/- 15 counts per high-power field, p = 0.672) when compared with the nonhypothermic control group (51 +/- 28 counts). Finally, dihydrorhodamine oxidation was significantly decreased in the reperfusion-only group (45.83 +/- 11.89 mean fluorescence intensity, p = 0.021) and ischemia-reperfusion group (44.30 +/- 11.80, p = 0.018) when compared with the nonhypothermic control group (71.74 +/- 20.83), whereas no decrease in dihydrorhodamine oxidation was observed in the ischemia-only group (65.93 +/- 10.3, p = 0.982). The findings suggest a protective effect of local hypothermia during early reperfusion to skeletal muscle after an ischemic insult. Inhibition of CD11b expression and subsequent neutrophil infiltration and depression of neutrophil oxidative potential may represent independent protective mechanisms isolated to local tissue hypothermia during the early reperfusion period (reperfusion-only and ischemia-reperfusion groups). This study provides evidence for the potential clinical utility of administering local hypothermia to ischemic muscle tissue during the early reperfusion period.     

Nitric oxide mediated oxidative stress injury in rat skeletal muscle subjected to ischemia/reperfusion as evaluated by chemiluminescence.

The involvement of nitric oxide (*NO) in oxidative stress in the rat gastrocnemius muscle subjected to ischemia/reperfusion injury was investigated using a specific and sensitive chemiluminescence (CL) method for measurement of both membrane lipid peroxide and total tissue antioxidant capacity (TRAP). In addition, inhibitors of nitric oxide synthase enzymes were used. The CL time-course curve increased dramatically after 1, 2, and 4 h of reperfusion, reaching values about 12 times higher than those of both control and ischemic rats. Initial velocity (V0) increased from 13.6 cpm mg protein(-1) min(-1) in the ischemic group, to 7341-8524 cpm mg protein(-1) min(-1) following reperfusion. The administration of L-NAME prior to reperfusion significantly reduced (p<0.007) the time-course of the CL curve, decreasing the V(0) value by 51% and preventing antioxidant consumption for 1h following reperfusion. No significant change in CL time-course curve and TRAP values were observed with aminoguanidine treatment. On contrary, after 4h following reperfusion, pre treatment with aminoguanidine led to a significant decrease (p < 0.0001) in the time-course of the CL curve, where V0 decreased by 75% and TRAP returned to control levels. No significant change in CL time-course curve and TRAP values were observed with L-NAME treatment. When RT-PCR was carried out with an iNOS-specific primer, a single band was detected in RNA extracted from muscle tissue of only the 4 h ischemia/4 h reperfusion group. No bands were found in either the control, 4 h ischemia or 4 h ischemia/1 h reperfusion groups. Based on these results, we conclude that *NO plays an important role in oxidative stress injury, possibly via -ONOO, in skeletal muscle subjected to ischemia/reperfusion. Our results also show that cNOS isoenzymes are preferentially involved in *NO generation at the beginning of reperfusion and that iNOS isoenzyme plays an important role in reperfusion injury producing *NO later in the process.     

Warm ischemia-induced alterations in oxidative and inflammatory proteins in hepatic Kupffer cells in rats

The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells. Lean Zucker rats (n = 6 each group) were randomized to 75 min of warm ischemia or sham operation. After reperfusion for 8 h, Kupffer cells were isolated by enzymatic perfusion and density gradient centrifugation. Proteins were tryptically digested into peptides and differentially labeled with iTRAQ (isobaric tags for relative and absolute quantitation) reagent. After fractionation by cation exchange chromatography, peptides were identified by mass spectrometry (ESI-LC-MS/MS). Spectra were interrogated against the Swiss-Prot database and quantified using ProteinProspector. The results for heat shock protein 70 and myeloperoxidase were validated by ELISA. Quantitative information for more than 1559 proteins was obtained. In the ischemia group proteins involved in inflammation were significantly up-regulated. The ratio for calgranulin B in the ischemia/sham group was 1.81 +/- 0.97 (p < 0.0001), for complement C3 the ratio was 1.81 +/- 0.49 (p < 0.0001), and for myeloperoxidase the ratio was 1.30 +/- 0.32. Myeloperoxidase was only recently documented in Kupffer cells. The antioxidative proteins Cu,Zn-superoxide dismutase (1.34 +/- 0.19; p < 0.001) and catalase (1.23 +/- 0.43; p < 0.001) were also elevated. In conclusion, ischemia/reperfusion injury induces alterations in the Kupffer cell proteome. Isotope ratio mass spectrometry is a powerful tool to investigate these reactions. The ability to simultaneously monitor several pathways involved in reperfusion stress may result in important mechanistic insight and possibly new treatment options.     

Antioxidant vitamin levels and glutathione peroxidase activity during ischemia/reperfusion in myocardial infarction

The consequences of increased oxidative stress, measured as the level of malondialdehyde (MDA) during ischemia/reperfusion, were studied in 48 patients in the acute phase of myocardial infarction (AMI) and a control group (21 blood donors). The serum levels of alpha-tocopherol and beta-carotene were followed. Immediately after the treatment onset the level of alpha-tocopherol started to decrease, reaching a plateau after 24 h. The consumption of beta-carotene was delayed by 90 min. Steady decline was detected during the whole time interval studied (48 h). Glutathione peroxidase (GPx) activity, as a representative of antioxidant enzymes, was estimated in whole blood. The influx of oxygenated blood was accompanied by a stimulation of GPx activity, which reached its maximum at the time of completed reperfusion. When comparing the AMI patients with the control group, the levels of MDA were found significantly increased, which indicates that oxidative stress is already increased during ischemia. Lower antioxidant levels found in the patients might either already be the result of vitamin consumption during ischemia or be a manifestation of their susceptibility to AMI. Monitored consumption of alpha-tocopherol and beta-carotene during reperfusion indicated that in the case of patients, whose level of antioxidant vitamins is below the threshold limit, a further substantial decrease of antioxidant vitamins during reperfusion could enhance the oxidative damage of the myocardium.     

Selective antioxidant enzymes during ischemia/reperfusion in myocardial infarction

The study of ischemia/reperfusion injury included 25 patients in the acute phase of myocardial infarction (19 perfused, 6 remained non-reperfused as evaluated according to the time course of creatine kinase and CK-MB isoenzyme activity) and a control group (21 blood donors). Plasma level of malondialdehyde was followed as a marker of oxidative stress. Shortly after reperfusion (within 90 min), a transient increase of malondialdehyde concentration was detected. The return to the baseline level was achieved 6 h after the onset of therapy. The activity of a free radical scavenger enzyme, plasma glutathione peroxidase (GPx), reached its maximum 90 min after the onset of treatment and returned to the initial value after 18 h. The specificity of the GPx response was confirmed by comparing with both non-reperfused patients and the control group, where no significant increase was detected. The erythrocyte Cu,Zn-superoxide dismutase (SOD) did not exhibit significant changes during the interval studied in perfused patients, probably due to the stability of erythrocyte metabolism. In non-reperfused patients, a decrease of SOD was found during prolonged hypoxia. These results help to elucidate the mechanisms of fast activation of plasma antioxidant system during the reperfusion after myocardial infarction.     

Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs

Transient episodes of ischemic preconditioning (PC) render myocardium protected against subsequent lethal injury after ischemia and reperfusion. Recent studies indicate that application of short, repetitive ischemia only during the onset of reperfusion after the lethal ischemic event may obtain equivalent protection. We assessed whether such ischemic postconditioning (Postcon) is cardioprotective in pigs by limiting lethal injury. Pentobarbital sodium-anesthetized, open-chest pigs underwent 30 min of complete occlusion of the left anterior descending coronary artery and 3-h reflow. PC was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. Postcon was elicited by three cycles of 30-s reperfusion, followed by 30-s reocclusion, after the 30-min occlusion period and before the 3-h reflow. Infarct size (%area-at-risk using triphenyltetrazolium chloride macrochemistry; means +/- SE) after 30 min of ischemia was 26.5 +/- 5.2% (n = 7 hearts/treatment group). PC markedly limited myocardial infarct size (2.8 +/- 1.2%, n = 7 hearts/treatment group, P < 0.05 vs. controls). However, Postcon had no effect on infarct size (37.8 +/- 5.1%, n = 7 hearts/treatment group). Within the subendocardium, Postcon increased phosphorylation of Akt (74 +/- 12%) and ERK1/2 (56 +/- 10%) compared with control hearts subjected only to 30-min occlusion and 15-min reperfusion (P < or = 0.05), and these changes were not different from the response triggered by PC (n = 5 hearts/treatment group). Phosphorylation of downstream p70S6K was also equivalent in PC and Postcon groups. These data do not support the hypothesis that application of 30-s cycles of repetitive ischemia during reperfusion exerts a protective effect on pig hearts subjected to lethal ischemia, but this is not due to a failure to phosphorylate ERK and Akt during early reperfusion.     

Apomorphine-induced myocardial protection is due to antioxidant and not adrenergic/dopaminergic effects

Apomorphine (Apo), a dopaminergic agonist used for treatment of Parkinson disease, is a potent antioxidant. In addition to its antioxidative effects, the dopaminergic and adrenergic effects of Apo were studied. Isolated perfused rat hearts were exposed to 25 min of no-flow global ischemia (37 degrees C) and 60 min of reperfusion (I/R, control). Drugs were introduced for the first 20 min of reperfusion. The LVDP of the control group recovered to 54.6 +/- 3.3%. Apo-treated hearts had significantly improved recovery (61.6 +/- 5%, p < 0.05). The recovery of the work index LVDP x HR was even bigger: 67.8 +/- 3.7% (Apo treatment) vs 41.7 +/- 4.6% (control, p < 0.001). Haloperidol, a dopaminergic antagonist, did not affect the recovery with Apo. Propranolol, a beta-adrenergic blocker, initially inhibited the effect of Apo. However, the recovery of the combined group (Apo + propranolol) increased and reached significance (LVDP, p < 0.05 vs control group) after cessation of propranolol perfusion. At 60 min of reperfusion this group was superior to Apo-treated hearts (LVDP, p < 0.05). Propranolol (without Apo) did not improve the hemodynamic recovery. The same pattern of recovery applies also to the recovery of the +dP/dt during the reperfusion. L-DOPA was less effective than Apo. I/R caused significant increase in carbonylation of proteins. Apomorphine inhibited the increase in carbonylation. Haloperidol did not affect this beneficial effect of Apo. L-DOPA significantly decreased the carbonylation of proteins. We conclude that the antioxidative effect of Apo is its main mechanism of cardioprotection.     

Hepatic ischemia/reperfusion in rats induces iNOS gene transcription by activation of NF-kappaB.

It has been known that many immediately early genes are expressed during ischemia/reperfusion (I/R) injury. Here, employing a model of hepatic I/R, we show that inducible nitric oxide synthase (iNOS) is induced via the activation of nuclear factor kappaB (NF-kappaB) after I/R in rat liver. When liver was subjected to ischemia followed by reperfusion, but not ischemia alone, an NF-kappaB complex composed of p50/p65 heterodimer and p50 homodimer was rapidly activated within 1 h and remained elevated for up to 3 h, and then tended to decline after 5 h of reperfusion. Also, the expression of iNOS mRNA was initiated after 1 h and continued to increase after 5 h of reperfusion during the time course studied. This upregulated iNOS mRNA expression coincides with increased iNOS enzyme activity and NF-kappaB binding activity after hepatic I/R. Administration of N-acetylcysteine (NAC, 20 mg/kg i.v. 10 min before reperfusion), an antioxidant, not only significantly inhibited the expression of iNOS mRNA but also blocked upregulated NF-kappaB binding activity after reperfused liver. These results suggest that NF-kappaB is activated by oxidative stress during hepatic I/R and may play a significant role in the induction of the iNOS gene.     

Salvia miltiorrhiza treatment during early reperfusion reduced postischemic myocardial injury in the rat

Oxidative stress may play a causative role in myocardial ischemia-reperfusion injury. However, it is a relatively understudied aspect regarding an optimal timing of antioxidant intervention during ischemia-reperfusion. The present study investigates the effect of different treatment regimens of Salvia miltiorrhiza (SM) herb extracts containing phenolic compounds that possess potent antioxidant properties on postischemic myocardial functional recovery in the setting of global myocardial ischemia and reperfusion. Langendorff-perfused rat hearts were subjected to 40 min of global ischemia at 37 degrees C followed by 60 min of reperfusion, and were randomly assigned into the untreated control and 2 SM-treated groups (n = 7 per group). In treatment 1 (SM1), 3 mg/mL of water soluble extract of SM was given for 10 min before ischemia and continued during ischemia through the aorta at a reduced flow rate of 60 microL/min, but not during reperfusion. In treatment 2 (SM2), SM (3 mg/mL) was given during the first 15 min of reperfusion. During ischemia, hearts in the control and SM2 groups were given physiological saline at 60 microL/min. The SM1 treatment reduced the production of 15-F2t-isoprostane, a specific index of oxidative stress-induced lipid peroxidation, during ischemia (94 +/- 20, 43 +/- 6, and 95 +/- 15 pg/mL in the coronary effluent in control, SM1, and SM2 groups, respectively; p < 0.05, SM1 vs. control or SM2) and postponed the onset of ischemic contracture. However, SM2, but not the SM1 regimen, significantly reduced 15-F2t-isoprostane production during early reperfusion and led to optimal postischemic myocardial functional recovery (left ventricular developed pressure 51 +/- 4, 46 +/- 4, and 60 +/- 6 mmHg in the control, SM1, and SM2 groups, respectively, at 60 min of reperfusion; p < 0.05, SM2 vs. control or SM1) and reduced myocardial infarct size as measured by triphenyltetrazolium chloride staining (26% +/- 2%, 22% +/- 2%, and 20% +/- 2% of the total area in the control, SM1, and SM2 groups, respectively, p < 0.05, SM2 vs. control). It is concluded that S. miltiorrhiza could be beneficial in the treatment of myocardial ischemic injury and the timing of administration seems important.     

Equal reduction in infarct size by ethylisopropyl-amiloride pretreatment and ischemic preconditioning in the in situ rabbit heart

Inhibition of Na⁺/H⁺ exchange with amiloride analogues has been shown to provide functional protection during ischemia and reperfusion and to reduce infarct size in isolated rat hearts. In rat hearts, treatment with ethylisopropyl-amiloride (EIPA, a selective Na⁺/H⁺ exchange inhibitor) was additive to the protection afforded by ischemic preconditioning. In addition, such compounds have been demonstrated to reduce infarct size in in situ rabbit hearts. The aim of the present study was to determine to what extent preischemic treatment with EIPA could reduce infarct size in an in situ rabbit model of regional ischemia and reperfusion. We also wished to determine if this effect was additive to the infarct reducing effect of ischemic preconditioning. Anaesthetized, open chest rabbits, were subjected to 45 min of regional ischemia and 150 min of reperfusion. The risk zone was determined by fluorescent particles and infarct size was determined by TTC staining. Four groups were investigated: control, ischemic preconditioned (IP) (5 min of ischemia followed by 10 min reperfusion), EIPA (0.65 mg/kg iv given preischemically) and EIPA + IP. The main results expressed as percent infarction of the risk zone ± S.E.M. for the different groups were: control 59.2 ± 3.3% (n = 6), IP 16.3 ± 2.1% (n = 6) (p < 0.001 vs. control), EIPA 16.9 ± 4.1% (n = 5) (p < 0.001 vs. control), EIPA + IP 22.5 ± 9.5% (n = 6) (p < 0.001 vs. control). In conclusion: EIPA, when administered prior to ischemia, caused a reduction in infarct size in the in situ rabbit heart which was similar to that seen with ischemic preconditioning, however, the effect was not additive to ischemic preconditioning.     

Ceramide-induced preconditioning involves reactive oxygen species

INTRODUCTION: Ceramide induces programmed cell death and it is thought to contribute to cardiac ischemia/reperfusion (I/R) injury. In contrast, we have demonstrated that administration of low doses of ceramide engenders cardiac preconditioning (PC). Ceramide is known to generate reactive oxygen species (ROS) in cells. Since mechanisms triggering the ceramide-induced cardioprotection remain unknown, we investigated the role of ROS in the genesis of this protective mechanism. METHODS: Using an isolated Langendorff-perfused rat heart model, four groups (n > or = 6 in each group) were considered: Control hearts underwent 30 min index regional ischemia and 120 min of reperfusion. In the ceramide group, hearts were preconditioned with c2-ceramide 1 microM for 7 min followed by 10 min washout prior to the I/R insult. In additional groups, MPG (1 mM), a synthetic antioxidant was given for 15 min alone or bracketing the ceramide perfusion. In each group, infarct size was determined at the end of the reperfusion period and superoxide dismutases (CuZnSOD and MnSOD) and catalase activities were evaluated. RESULTS: Ceramide preconditioning reduced the infarct/area at risk (I/AAR) ratio (8.3 +/- 1.1% for ceramide vs. 36.4 +/- 1.2% for control, p < 0.001). Perfusion with MPG abolished the preconditioning effect of ceramide (I/AAR ratio = 36.7 +/- 4.9%). Ceramide was also associated with a 29% and 38% increase in catalase and CuZnSOD activities, respectively, compared with control group. CONCLUSION: Production of reactive oxygen species following ceramide preconditioning of the ischemic-reperfused heart appears to play a role in the cardioprotective effect of ceramide.     

Heat Shock Preconditioning Reduces the Formation of 8-hydroxy-2′-deoxyguanosine and 4-hydroxy-2-nonenal Modified Proteins in Ischemia-reperfused Liver of Rats

Heat shock preconditioning (HSPC) is a promising strategy for providing ischemic tolerance. The objective of this study is to investigate the effectiveness of HSPC in preventing oxidative damage of cellular proteins and DNA during ischemia-reperfusion of the liver. Male Wistar rats were divided into a heat shock group (group HS) and control (group C). Forty-eight hours prior to ischemia, rats in group HS received HSPC at 42°C for 15 &#117 min. All rats received hepatic warm ischemia for 30 &#117 min and subsequent reperfusion. The formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE) modified proteins in liver tissue, survival rate of the animals, and changes in biochemical and histological parameters were compared between groups. Heat shock protein 72 was produced only in group HS. The 7-day survival of rats was significantly better in group HS (10/10) than in group C (5/10) ( p <0.01). The serum release of alanine aminotransferase ( n =10, p <0.01) and the concentration of adenosine triphosphate in liver tissue ( n =10, p <0.01) 40 &#117 min after reperfusion was significantly better in group HS than in group C. The formation of 8-OHdG in liver tissue measured by high-performance liquid chromatography was suppressed in group HS ( p <0.01). The production of HNE-modified proteins as determined by Western-blot analysis was also decreased in group HS. These results were also confirmed by immunohistochemical analysis. As determined by levels of 8-OHdG and HNE-modified proteins produced during ischemia-reperfusion of the liver, HSPC reduced the oxidative injury of cellular proteins and DNA in the liver tissue.     

The damage-associated molecular pattern HMGB1 is released early after clinical hepatic ischemia/reperfusion

Objective and backgroundActivation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice.MethodsPlasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury.ResultsIn patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects.ConclusionHMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage.     

Effects of L-NAME and L-arginine on ischemia-reperfusion injury in rat skeletal muscle

The involvement of nitric oxide in ischemia-reperfusion injury remains controversial and has been reported to be both beneficial and deleterious, depending on the tissue and model used. This study evaluated the effects of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine-methyl ester (L-NAME) and the substrate for nitric oxide synthase, L-arginine on skeletal muscle necrosis in a rat model of ischemia-reperfusion injury. The rectus femoris muscle in male Wistar rats (250 to 500 g) was isolated on its vascular pedicle and subjected to 4 hours of complete arteriovenous occlusion. The animals were divided into five groups: (1) sham-raised control, no ischemia, no treatment (n = 6); (2) 4 hours of ischemia (n = 6); (3) vehicle control, 4 hours of ischemia + saline (n = 6); (4) 4 hours of ischemia + L-arginine infusion (n = 6); and (5) 4 hours of ischemia + L-NAME infusion (n = 6). The infusions (10 mg/kg) were administered into the contralateral femoral vein beginning 5 minutes before reperfusion and during the following 30 to 45 minutes. Upon reperfusion, the muscle was sutured in its anatomic position and all wounds were closed. The percentage of muscle necrosis was assessed after 24 hours of reperfusion by serial transections, nitroblue tetrazolium staining, digital photography, and computerized planimetry. Sham (group 1) animals sustained baseline necrosis of 11.9 +/- 3.0 (percentage necrosis +/- SEM). Four hours of ischemia (group 2) significantly increased necrosis to 79.2 +/- 1.4 (p < 0.01). Vehicle control (group 3) had no significant difference in necrosis (81.17 +/- 5.0) versus untreated animals subjected to 4 hours of ischemia (group 2). Animals treated with L-arginine (group 4) had significantly reduced necrosis to 34.6 +/- 7.5 versus untreated (group 2) animals (p < 0.01). Animals infused with L-NAME (group 5) had no significant difference in necrosis (68.2 +/- 6.7) versus untreated (group 2) animals. L-Arginine (nitric oxide donor) significantly decreased the severity of muscle necrosis in this rat model of ischemia-reperfusion injury. L-arginine is known to increase the amount of nitric oxide through the action of nitric oxide synthase, whereas L-NAME, known to inhibit nitric oxide synthase and decrease nitric oxide production, had comparable results to the untreated 4-hour ischemia group. These results suggest that L-arginine, presumably through nitric oxide mediation, appears beneficial to rat skeletal muscle subjected to ischemia-reperfusion injury.     

C-phycocyanin protects against ischemia-reperfusion injury of heart through involvement of p38 MAPK and ERK signaling

We previously showed that C-phycocyanin (PC), an antioxidant biliprotein pigment of Spirulina platensis (a blue-green alga), effectively inhibited doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes. Here we investigated the cardioprotective effect of PC against ischemia-reperfusion (I/R)-induced myocardial injury in an isolated perfused Langendorff heart model. Rat hearts were subjected to 30 min of global ischemia at 37 degrees C followed by 45 min of reperfusion. Hearts were perfused with PC (10 microM) or Spirulina preparation (SP, 50 mg/l) for 15 min before the onset of ischemia and throughout reperfusion. After 45 min of reperfusion, untreated (control) hearts showed a significant decrease in recovery of coronary flow (44%), left ventricular developed pressure (21%), and rate-pressure product (24%), an increase in release of lactate dehydrogenase and creatine kinase in coronary effluent, significant myocardial infarction (44% of risk area), and TdT-mediated dUTP nick end label-positive apoptotic cells compared with the preischemic state. PC or SP significantly enhanced recovery of heart function and decreased infarct size, attenuated lactate dehydrogenase and creatine kinase release, and suppressed I/R-induced free radical generation. PC reversed I/R-induced activation of p38 MAPK, Bax, and caspase-3, suppression of Bcl-2, and increase in TdT-mediated dUTP nick end label-positive apoptotic cells. However, I/R also induced activation of ERK1/2, which was enhanced by PC treatment. Overall, these results for the first time showed that PC attenuated I/R-induced cardiac dysfunction through its antioxidant and antiapoptotic actions and modulation of p38 MAPK and ERK1/2.