Leukocyte Mobilization, Chemiluminescence Response, and Antioxidative Capacity of the Blood in Intestinal Ischemia and Reperfusion

Intestinal ischemia and reperfusion elicits changes in leukocyte counts and increased production of reactive oxygen species (ROS). The purpose of this study was to investigate whether these changes were followed by and/or connected with changes in the extracellular antioxidative capacity in a rat superior mesenteric artery (SMA) occlusion/reperfusion model. The SMA was occluded for 45 min and then allowed to be reper-fused. Changes of leukocyte, polymorphonuclear (PMN), and lymphocyte counts, chemiluminescence (CL) of whole blood samples as a marker of ROS production, and the total antioxidative capacity of the serum were quantified at the end of ischemia and in 1 h intervals during the postischemic period up to 4 h. The myeloperoxidase (MPO) activity in the serum and intestinal tissue samples was also determined. The MPO activity in the intestinal tissue samples was significantly elevated at the end of ischemia, and this elevation lasted for the whole postischemic period. The oxidative challenge to the body induced a fast mobilization of extracellular antioxidative mechanisms already at the end of ischemia, which was followed by a significant increase in PMN counts and whole blood CL starting at the 2nd hour after reperfusion. The increased CL activity of whole blood was attributed to the increase of the circulating PMNs. No significant changes were observed in leukocyte and lymphocyte counts. It is concluded that compensatory mechanisms of the oxidative-antioxidative balance of the body react very quickly if challenged.     

Role of Oxygen-Derived Free Radicals in Gastric Mucosal Injury Induced by Ischemia or Ischemia-Reperfusion in Rats

Oxygen-derived free radicals have been implicated as possible mediators in the development of tissue injury induced by ischemia and reperfusion. Clamping of the celiac artery in rats reduced the gastric mucosal blood flow to 10% of that measured before the clamping. The area of gastric erosions and thiobarbituric acid (TBA) reactants in gastric mucosa were significantly increased 60 and 90 min after clamping. These changes were inhibited by treatment with SOD and catalase. Thirty and 60 min after reoxyganation, produced by removal of the clamps following 30 min of ischemia, gastric mucosal injury and the increase in TBA reactants were markedly aggravated compared with those induced by ischemia alone. SOD and catalase significantly inhibited these changes. The serum a-tocopherol/cholesterol ratio, an index of in vivo lipid peroxidation, was significantly decreased after long periods of ischemia (60 and 90 min), or after 30 and 60 min of reperfusion following 30 min of ischemia. These results indicated that active oxygen species and lipid peroxidation may play a role in the pathogenesis of gastric mucosal injury induced by both ischemia alone and ischemia-reperfusion. Although, allopurinol inhibited the formation of gastric mucosal injury and the increase in TBA reactants in gastric mucosa, the depletion of polymorphonuclear leukocytes (PMN) counts induced by an injection of anti-rat PMN antibody did not inhibit these changes. As compared with the hypoxanthine-xanthine oxidase system, PMN seem to play a relatively small part in the formation of gastric mucosal injury induced by ischemia-reperfusion.     

兔急性肠道缺血再灌注损伤模型的建立

目的:建立小肠急性缺血再灌注损伤模型,确定合适的肠系膜上动脉阻断时间.方法:将70只新西兰兔随机按不同的肠系膜缺血时间(0、15、30、45、60min)分为A、B、C、D、E5组,每组14只,取8只用于恢复血供2h后留取各组兔下腔静脉血标本及肠组织,检测血清中MDA含量的变化,光镜下观察小肠组织形态学变化并对小肠黏膜损伤程度进行评分.另6只用于术后24h、 48h及72h生存率的观察.结果:A、B、C组的术后生存率均>83.3%.C、D、E组的MDA含量及肠黏膜损伤评分与A组比较,差异均有显著性(F=12.13～280.24,p<0.01).结论:肠系膜缺血30min,再灌注2h是建立兔小肠急性再灌注损伤的合适时间.     

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.     

Reversibility of ultrastructural, contractile function and Ca2+ transport changes in guinea pig hearts after global ischemia

To understand the subcellular basis of contractile failure due to ischemia-reperfusion injury, effects of 20, 60, and 90 min of global ischemia followed by 30 min of reperfusion were examined in isolated guinea pig hearts. Cardiac ultrastructure and function as well as Ca2+ transport abilities of both mitochondrial and microsomal fractions were determined in control, ischemic, and reperfused hearts. Hearts were unable to generate any contractile force after 20 min of ischemia and showed a 75% recovery upon reperfusion. However, there were no significant changes in the subcellular Ca2+ transport in the 20-min ischemic or reperfused hearts. When hearts were made ischemic for 60 and 90 min, the recovery of contractile force on reperfusion was 50 and 7%, respectively. There was a progressive decrease in mitochondrial and microsomal Ca2+ binding and uptake activities after 60 and 90 min of ischemia; these changes were evident at various times of incubation period and at different concentrations of Ca2+. Mitochondrial Ca2+ transport changes were only partially reversible upon reperfusion after 60 and 90 min of ischemia, whereas the microsomal Ca2+ binding, uptake and Ca2+ ATPase activities deteriorated further upon reperfusion of the 90-min ischemic hearts. Ultrastructural changes increased with the duration of the ischemic insult and reperfusion injury was extensive in the 90-min ischemic hearts. These data show that the lack of recovery of contractile function upon reperfusion after a prolonged ischemic insult was accompanied by defects in sarcoplasmic reticulum Ca2+ transporting properties and structural damage.     

Protective effects of leukopenia and tissue plasminogen activator in microvascular ischemia-reperfusion injury

Ischemia shifts the anticoaugulant/procoagulant balance of the endothelium in favor of activation of coagulation. We studied whether cheek pouch microcirculation of leukopenic hamsters was protected by tissue plasminogen activator (tPA) (50 microg/100 g body wt) against ischemia-reperfusion injury. Adherent leukocytes, total perfused capillary length (PCL), permeability increase, and arteriolar and venular red blood cell (RBC) velocity were investigated by fluorescence microscopy. Measurements were made at control, 30 or 60 min of ischemia, and at 30 or 60 min of reperfusion. Hamsters were made leukopenic by treatment with cyclophosphamide (20 mg/100 g body wt ip, 4 days before the experiment), which decreased circulating leukocyte count by 85-90%. Leukopenic hamsters undergoing 30 min of ischemia followed by 30 min of reperfusion showed no significant decrease in PCL or increased permeability. Leukopenic hamsters undergoing 60 min of ischemia followed by 60 min of reperfusion presented a significant decrease in microvascular perfusion where PCL was 28 +/- 7% of baseline, low-flow conditions, and increased permeability. In leukopenic hamsters treated with tPA there was complete protection of capillary perfusion with no significant changes in permeability or arteriolar and venular RBC velocity. In conclusion, thrombus formation may be an additional and independent factor that with leukocyte-mediated mechanisms determines ischemia-reperfusion injury.     

Biphasic changes in phospholipid hydroperoxide levels during renal ischemia/reperfusion.

The involvement of lipid peroxidation in renal ischemia/reperfusion was explored by measuring changes in the cortical content of specific primary lipid hydroperoxides (using chemluminescent detection with HPLC) following ischemia and reperfusion and by correlating the changes in hydroperoxide content with measurements of renal blood flow. Phosphatidylcholine and phosphatidylethanolamine hydroperoxide concentrations were significantly lowered during 30 or 60 min of ischemia (to levels less than 50% of control at 60 min). Following 30 min of renal ischemia, reperfusion resulted in a rebound of phospholipid hydroperoxide tissue content to levels higher than controls. Increased phospholipid hydroperoxide formation was not, however, observed in response to reperfusion following long-term (60 min) ischemia. In separate animals it was demonstrated that following 30 min ischemia and reperfusion, renal blood flow recovers to about 65% of control in 1 h. In contrast, following 60 min ischemia and reperfusion, the renal blood flow remains more highly impaired (less than 25% recovery for periods up to 24 h). These results imply that phospholipid hydroperoxides are produced and accumulate in the kidneys under normal aerobic conditions and that lipid peroxidative activity increases during renal ischemia/reperfusion to an extent dependent on the degree of local blood perfusion.     

Myoelectric bowel activity in ischemia/reperfusion damage. Role of sensory neurons.

The present knowledge indicates that afferent sensory neurons (C-fibres) play an important role in the relationship between intestinal myoelectric activity (IMA) and blood flow (LDBF). The aim of this study was to evaluate the role of C-fibers in myoelectric activity of small intestine during its ischemia and reperfusion. A neurotoxin-capsaicin (CAP) was used to induce functional ablation of afferent sensory neurons. Experiments were performed on 6 groups of anesthetized rats. In the I, II, III group of rats IMA and LDBF were recorded during 100% ischemia induced by AMA 15, 30 and 60 min total occlusion and during 60 min reperfusion period. In group V and VI, IMA and LDBF were registered after intrajejunal placement of 1% CAP. In group IV we measured effects of intraluminal instillation of CAP alone. Intraluminal placement of CAP induced an early increase in slow wave amplitude SWA and slow wave frequency SWF by 35+/-11% and 19+/-10% (p<0.05) with the subsequent decrease in both by 25+/-6 and 24+/-8% (p<0.05) respectively. Short 15 min lasting ischemia induced by 100% occlusion of AMA evoked only a slight increase of SWA. During reperfusion period SWA and SWF returned to the baseline values after 15 min. Total 30 min occlusion decreased SWA and SWF by 25+/-9 and 24+/-6% (p<0.05) respectively. During reperfusion period recovery of IMA parameters to preocclusion values were slower. Intestinal hyperemia was smaller than in previous group. After 60 min lasting intestinal ischemia SWA and SWF were decreased by 58+/-7 and 40+/-6% (p<0.01) respectively. There was no return of IMA parameters to control values. These data demonstrated that intestinal ischemia induces typical changes in the bowel myoelectric activity. These changes possess their own electrical characteristics which can be used in clinical practice for evaluation of the degree ischemically-induced intestinal injury. Capsaicin pretreatment significantly decreased SWA and SWF and LDBF in comparison with those observed in group II and III during 30 and 60 min occlusion and reperfusion period. We conclude that afferent neurons C activated during mesenteric ischemia/reperfusion play an important role in protecting ischemic bowel viability.     

Ischemia-reperfusion injury in the spinal cord of rabbits strongly enhances lipid peroxidation and modifies phospholipid profiles

The effect of spinal cord ischemia (10, 20, and 40 min) and post-ischemic reperfusion (10, 30, and 60 min) on lipid peroxidation and phospholipids was investigated. Spinal cord ischemia was accompanied by lipolytic processes with significant changes in concentration of lipid peroxidation products (LPP). Reestablishment of the blood supply after 10 min ischemia was accompanied by significantly increased levels of thiobarbituric acid reactive substances (TBA-RS) after 10 and 30 min of reperfusion. Following 20 and 40 min ischemia a significant increase was observed at all reperfusion periods. Ischemia itself significantly reduced the concentration of phosphatidyl inositol (IP), phosphatidyl ethanolamine (EP) and ethanolamine plasmalogens (Epls). Significant changes were observed in concentration of phosphatidyl serine (SP) too, but only after 20 and 40 min of ischemia. The concentration of phosphatidic acid (PA) was significantly reduced only after 10 min of ischemia. The onset of reperfusion after ischemia was accompanied by a diverse pattern of changes in PA, IP, Epls and SP, while the concentration of EP remained at the above mentioned ischemic intervals.     

Effects of preceding ischemic time on the recovery course of energy metabolism in rat liver

Effects of the duration of preceding ischemia on the recovery of liver energy metabolism after reperfusion were investigated. Liver ATP level was depleted after the first 30 min of ischemia, and the decrease remained steady thereafter. Recovery of ATP depended on the preceding ischemic time, i.e., 81.5%, 66.4% and 39.5% recovery of the control level were observed after 60 min of reperfusion following 30 min, 60 min and 120 min of ischemia, respectively. Ischemia-induced mitochondrial dysfunction depended on the duration of ischemia. Mitochondrial function was recovered fully after 60 min of reperfusion following both 30 min and 60 min of ischemia. However, deterioration of mitochondrial function did not recover significantly after 60 min of reperfusion following 120 min of ischemia. Similar decreases in adenylate energy charge were observed irrespective of the duration of ischemia, and it recovered fully after 60 min of reperfusion following 30 min, 60 min and 120 min of ischemia. These results suggest that not the energy charge but ATP level itself is a reliable marker of liver energy status.     

Extract of grapefruit-seed reduces acute pancreatitis induced by ischemia/reperfusion in rats: possible implication of tissue antioxidants.

Grapefruit seed extract (GSE) has been shown to exert antibacterial, antifungal and antioxidant activity possibly due to the presence of naringenin, the flavonoid with cytoprotective action on the gastric mucosa. No study so far has been undertaken to determine whether this GSE is also capable of preventing acute pancreatic damage induced by ischemia/reperfusion (I/R), which is known to result from reduction of anti-oxidative capability of pancreatic tissue, and whether its possible preventive effect involves an antioxidative action of this biocomponent. In this study carried out on rats with acute hemorrhagic pancreatitis induced by 30 min partial pancreatic ischemia followed by 6 h of reperfusion, the GSE or vehicle (vegetable glycerin) was applied intragastrically in gradually increasing amounts (50-500 microl) 30 min before I/R. Pretreatment with GSE decreased the extent of pancreatitis with maximal protective effect of GSE at the dose 250 microl. GSE reduced the pancreatitis-evoked increase in serum lipase and poly-C specific ribonuclease activity, and attenuated the marked fall in pancreatic blood flow and pancreatic DNA synthesis. GSE administered alone increased significantly pancreatic tissue content of lipid peroxidation products, malondialdehyde and 4-hydroxyalkens, and when administered before I/R, GSE reduced the pancreatitis-induced lipid peroxidation. We conclude that GSE exerts protective activity against I/R-induced pancreatitis probably due to the activation of antioxidative mechanisms in the pancreas and the improvement of pancreatic blood flow.     

Rotenone,a mitochondrial electron transport inhibitor,ameliorates ischemia–reperfusion-induced intestinal mucosal damage in rats

Abstract

In ischemia–reperfusion (I/R)-induced tissue injury, oxygen radicals can be generated by several mechanisms. One of the important sources of oxygen radicals is thought to be mitochondrial respiration. The aim of this study was to investigate the antioxidative defense effect of the mitochondrial electron transport inhibitor, rotenone using the I/R-induced rat intestinal mucosal injury model in vivo. Intestinal ischemia was induced for 30 min by applying a small clamp to the superior mesenteric artery in rats. Rotenone at a dose of 100 mg/kg was given to rats orally 2 h before the ischemia. Intraluminal hemoglobin and protein levels, the mucosal content of thiobarbituric acid-reactive substances (TBARS), the mucosal myeloperoxidase activity, and the content of inflammatory cytokines (CINC-1, TNF-α) were all significantly increased from mean basal levels after 60 min of reperfusion. These increases after I/R were inhibited by treatment with rotenone at a dose of 100 mg/kg. Co-administration with succinate (100 mg/kg), a substrate of the mitochondrial electron transport system, cancelled significant reduction of intraluminal hemoglobin and mucosal TBARS treated with rotenone alone. The results of the present study indicate that rotenone inhibited lipid peroxidation and reduced development of the intestinal mucosal inflammation induced by I/R in rats. This investigation suggests that rotenone has potential as a new therapeutic agent for reperfusion injury.     

Regulation of cardiomyocyte apoptosis in ischemic reperfused mouse heart by glutathione peroxidase

Apoptosis, a genetically controlled programmed cell death, has been found to play a role in ischemic reperfusion injury in several animal species including rats and rabbits. To examine whether this is also true for other animals, an isolated perfused mouse heart was subjected to 30 min of ischemia followed by 2 h of reperfusion. Experiments were terminated before ischemia (baseline), after ischemia, and at 30, 60, 90 and 120 min of reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. The in situ end labeling (ISEL) technique was used to detect apoptotic cardiomyocyte nuclei while DNA laddering was evaluated by subjecting the DNA obtained from the cardiomyocytes to 1.8% agarose gel electrophoresis followed by photographing under UV illumination. The results of our study revealed that apoptotic cells appear only after 60 min of reperfusion as demonstrated by the intense fluorescence of the immunostained genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation showing increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). Since our previous studies showed a role of glutathione peroxidase (GSHPx) in apoptotic cell death, we performed identical experiments using isolated hearts from GSHPx-l knockout mice and transgenic mice overexpressing GSHPx-l. GSHPx-l knockout mice showed evidence of apoptotic cell death even after 30 min of reperfusion. Significant number of apoptotic cells were found in the cardiomyocytes as compared to non-transgenic control animals. To the contrary, very few apoptotic cells were found in the hearts of the transgenic mice overexpressing GSHPx-l. Hearts of GSHPx-l knockout mice were more susceptible to ischemia/reperfusion injury while transgenic mice overexpressing GSHPx- 1 were less susceptible to ischemia reperfusion injury compared to non-transgenic control animals. The results of this study clearly demonstrate a role of GSHPx in ischemia/reperfusion-induced apoptosis in mouse heart.     

Rotenone, a mitochondrial electron transport inhibitor, ameliorates ischemia-reperfusion-induced intestinal mucosal damage in rats

In ischemia-reperfusion (I/R)-induced tissue injury, oxygen radicals can be generated by several mechanisms. One of the important sources of oxygen radicals is thought to be mitochondrial respiration. The aim of this study was to investigate the antioxidative defense effect of the mitochondrial electron transport inhibitor, rotenone using the I/R-induced rat intestinal mucosal injury model in vivo. Intestinal ischemia was induced for 30 min by applying a small clamp to the superior mesenteric artery in rats. Rotenone at a dose of 100 mg/kg was given to rats orally 2 h before the ischemia. Intraluminal hemoglobin and protein levels, the mucosal content of thiobarbituric acid-reactive substances (TBARS), the mucosal myeloperoxidase activity, and the content of inflammatory cytokines (CINC-1, TNF-alpha) were all significantly increased from mean basal levels after 60 min of reperfusion. These increases after I/R were inhibited by treatment with rotenone at a dose of 100 mg/kg. Co-administration with succinate (100 mg/kg), a substrate of the mitochondrial electron transport system, cancelled significant reduction of intraluminal hemoglobin and mucosal TBARS treated with rotenone alone. The results of the present study indicate that rotenone inhibited lipid peroxidation and reduced development of the intestinal mucosal inflammation induced by I/R in rats. This investigation suggests that rotenone has potential as a new therapeutic agent for reperfusion injury.     

Complement inhibitor, complement receptor 1-related gene/protein y-Ig attenuates intestinal damage after the onset of mesenteric ischemia/reperfusion injury in mice.

Complement receptor 1-related gene/protein y (Crry) is a murine membrane protein that regulates the activity of both classical and alternative complement pathways. We used a recombinant soluble form of Crry fused to the hinge, CH2, and CH3 domains of mouse IgG1 (Crry-Ig) to determine whether inhibition of complement activation prevents and/or reverses mesenteric ischemia/reperfusion-induced injury in mice. Mice were subjected to 30 min of ischemia, followed by 2 h of reperfusion. Crry-Ig was administered either 5 min before or 30 min after initiation of the reperfusion phase. Pretreatment with Crry-Ig reduced local intestinal mucosal injury and decreased generation of leukotriene B(4) (LTB(4)). When given 30 min after the beginning of the reperfusion phase, Crry-Ig resulted in a decrease in ischemia/reperfusion-induced intestinal mucosal injury comparable to that occurring when it was given 5 min before initiation of the reperfusion phase. The beneficial effect of Crry-Ig administered 30 min after the initiation of reperfusion coincided with a decrease in PGE(2) generation despite the fact that it did not prevent local infiltration of neutrophils and did not have a significant effect on LTB(4) production. These data suggest that complement inhibition protects animals from reperfusion-induced intestinal damage even if administered as late as 30 min into reperfusion and that the mechanism of protection is independent of neutrophil infiltration or LTB(4) inhibition.     

Time course of leukocyte response and free radical release in an early reperfusion injury of the superior mesenteric artery

The sequence of changes in circulating immune cells and in free radical production was studied during the small intestine reperfusion. Rat small intestine ischemia/reperfusion was induced by a 45 min superior mesenteric artery occlusion followed by a 4-hour reperfusion. Samples of peripheral blood were collected every 20 min during reperfusion. While the number of polymorphonuclear leukocytes increased significantly both in the sham-operated controls and the experimental group (about 400 per cent at the end of reperfusion), a decrease in lymphocyte counts to 60 per cent was observed in the experimental group only. Although there were no changes in the counts of total T lymphocytes, a significant reduction in B cell counts was observed. Flow-cytometrical measurements showed no changes in the Tc subpopulation, while the Th subpopulation increased in the experimental group only. Free radical generation in blood (luminometric measurements) increased gradually and reached an eight-fold level by the end of reperfusion in both groups. Thus, it has been shown that the increase in free radical production is mainly due to the increased number of polymorphonuclear leukocytes mobilized already at the initial stages of reperfusion. The reduction in B lymphocyte population is probably due to homing mechanisms     

Superoxide anion production during reperfusion is reduced by an antineutrophil antibody after prolonged cerebral ischemia

Neutrophils may be involved in the pathophysiology of reperfusion injury following cerebral ischemia. One potential mechanism of reperfusion injury by neutrophils is through production of the superoxide anion. We hypothesized that, due to progressive endothelial damage during ischemia, neutrophil activation would be more prominent after longer periods of ischemia prior to reperfusion. Thus, neutrophils would contribute more to pathological processes such as superoxide anion formation after longer than after shorter periods of ischemia. A reversible middle cerebral artery occlusion model in rats was employed and superoxide anion concentration was measured with a cytochrome c coated electrode placed on the cortical penumbral region. Occlusion times were varied from 60 min to 2 h, and neutrophils were inhibited with an antiCD18 antibody administered prior to occlusion. Neutrophil accumulation and reduction with antibody treatment was confirmed immunohistochemically. Superoxide anion (O₂^•−) concentration was detected during the hours following 60 min of occlusion, and increased further with 2 h of occlusion. Treatment with the antiCD18 antibody had no effect on O₂^•− concentration during reperfusion in the 60–90 min occlusion groups, but O₂^•− concentration was significantly lower in the antiCD18 antibody treated group than in the control group during reperfusion after 120 min of ischemia. The antibody also reduced cortical neutrophil accumulation in the 120 min ischemia group. These results indicate for the first time that superoxide production by neutrophils becomes more important with longer periods of ischemia, and other quantitatively less important sources of superoxide predominate with shorter periods of ischemia. This phenomenon may explain some of the variation seen between different models of ischemia with different durations of ischemia when targeting reactive oxygen species, and supports an approach to combination therapy to extend the therapeutic window and reduce the deleterious effects of reperfusion.     

Investigating the dual nature of endothelin-1: ischemia or direct arrhythmogenic effect?

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide, which may also elicit severe ventricular arrhythmias. The aims of our study were to compare the effects of total left anterior descending coronary artery (LAD) occlusion to intracoronary (ic.) ET-1 administration and to investigate the pathomechanism of ET-1 induced arrhythmias in 3 groups of anesthetized, open-chest mongrel dogs. In group A (n=10) a total LAD occlusion was carried out for 30 min, followed by a 60 min reperfusion period. In groups B and C ET-1 was administered into LAD for 30 min at a rate of 30 pmol/min (n=6) and 60 pmol/min (n=8). Epi- and endocardial monophasic action potential (MAP) recordings were performed to detect electrophysiologic changes and ischemia Blood samples for lactate measurements were collected from the coronary sinus (CS) and from the femoral artery. Infrared imaging was applied to follow epimyocardial heat emission changes. At the end of the ET-1 infusion period coronary blood flow (CBF) was reduced significantly in groups B and C (deltaCBF30MIN B: 21+/-2%, p<0.05; C: 35+/-2%, p<0.05), paralleled by a significant epimyocardial temperature decrease in group C (deltaT30MIN: -0.65+/-0.29 degrees C, p<0.05). Two dogs died of ventricular fibrillation (VF) in the reperfusion period in group A. Ventricular premature contractions and non-sustained ventricular tachycardic episodes appeared in group B, whereas six dogs died of VF in group C. Significant CS lactate level elevation indicating ischemia was observed only in group A from the 30th min occlusion throughout the reperfusion period (control vs. 30 min: 1.3+/-0.29 vs. 2.2+/-0.37 mmol/l, p<0.05). Epi- and endocardial MAP durations (MAPD90) and left ventricular epicardial (LV(EPI)) upstroke velocity decreased significantly in group A in the occlusion period. ET-1 infusion significantly increased LV(EPI) MAPD90 in group B and both MAPD90-s in group C. In conclusion, ischemic MAP and CS lactate changes were observed only in group A. Although ET-1 reduced CBF significantly in groups B and C, neither MAP nor lactate indicated ischemic alterations. ET-1 induced major ventricular arrhythmias appeared before signs of myocardial ischemia developed, though reduced CBF presumably contributed to sustaining the arrhythmias.     

Human adrenomedullin combined with human adrenomedullin binding protein-1 is protective in gut ischemia and reperfusion injury in the rat

Previous studies have demonstrated that co-administration of rat adrenomedullin (AM) and human AM binding protein-1 (AMBP-1) has various beneficial effects following adverse circulatory conditions. In order to reduce rat proteins to elicit possible immune responses in humans, we determined the effect of human AM combined with human AMBP-1 after intestinal ischemia and reperfusion (I/R). Intestinal ischemia was induced in the rat by occluding the superior mesenteric artery for 90 min. At 60 min after the beginning of reperfusion, human AM/AMBP-1 at 3 different dosages was administered intravenously over 30 min. At 240 min after the treatment, blood and tissue samples were harvested and measured for pro-inflammatory cytokines (i.e., TNF-alpha and IL-6), myeloperoxidase activities in the gut and lungs, and cleaved caspase-3 expression in the lungs, as well as serum levels of hepatic enzymes and lactate. In additional groups of animals, a 10-day survival study was conducted. Results showed that administration of human AM/AMBP-1 reduced pro-inflammatory cytokines, attenuated organ injury, and improved the survival rate in a seemingly dose-response fashion. Co-administration of the highest dose of human AM/AMBP-1 in this study had the optimal therapeutic effect in the rat model of intestinal I/R.     

Differential changes in phospholipase D and phosphatidate phosphohydrolase activities in ischemia-reperfusion of rat heart

Phospholipase D (PLD2) produces phosphatidic acid (PA), which is converted to 1,2 diacylglycerol (DAG) by phosphatidate phosphohydrolase (PAP2). Since PA and DAG regulate Ca(2+) movements, we examined PLD2 and PAP2 in the sarcolemma (SL) and sarcoplasmic reticular (SR) membranes from hearts subjected to ischemia and reperfusion (I-R). Although SL and SR PLD2 activities were unaltered after 30 min ischemia, 5 min reperfusion resulted in a 36% increase in SL PLD2 activity, whereas 30 min reperfusion resulted in a 30% decrease in SL PLD2 activity, as compared to the control value. SR PLD2 activity was decreased (39%) after 5 min reperfusion, but returned to control levels after 30 min reperfusion. Ischemia for 60 min resulted in depressed SL and SR PLD2 activities, characterized with reduced V(max) and increased K(m) values, which were not reversed during reperfusion. Although the SL PAP2 activity was decreased (31%) during ischemia and at 30 min reperfusion (28%), the SR PAP2 activity was unchanged after 30 min ischemia, but was decreased after 5 min reperfusion (25%) and almost completely recovered after 30 min reperfusion. A 60 min period of ischemia followed by reperfusion caused an irreversible depression of SL and SR PAP2 activities. Our results indicate that I-R induced cardiac dysfunction is associated with subcellular changes in PLD2 and PAP2 activities.