Ultrastructural demonstration of peroxidative activity and peroxidation in ischaemic and ischaemic-reperfused rabbit hearts.

OBJECTIVE: The aim was to characterise subcellular histochemical evidence of the involvement of peroxidation and peroxidases in myocardial reperfusion injury. The histochemical technique involved the use of 3,3'-diaminobenzidine (DAB), which reacts with peroxides and proteins with peroxidase activity to form an electron dense polymer. METHODS: Isolated rabbit hearts were perfused (Langendorff method) for 30 min with oxygenated physiological saline solution. Some were subjected to 30 min of normothermic global ischaemia, with or without 30 min reperfusion. Non-ischaemic control hearts were perfused continuously for 90 min. Hearts were fixed with glutaraldehyde and cut into 100-150 microns sections that were incubated for 1 h in buffered DAB (1 mg.ml-1) with or without added KCN or H2O2. They were processed further for transmission electron microscopy. Planimetry was done on micrographs taken from random fields (approximately 500 photos). RESULTS: The total amount of DAB polymer in non-ischaemic control heart sections incubated with DAB alone occupied 1.19(SEM 0.44) micron 2 x 1000 micron-2 total cell area. For ischaemic-nonreperfused hearts, the value was 2.32(0.90) micron 2 x 1000 micron-2 (p = 0.223 v control); DAB occupied 7.49(1.42) micron 2 x 1000 micron-2 in ischaemic-reperfused hearts (p = 0.001 v control). DAB positive staining of mitochondria and lipid droplets, but not of peroxisomes, was significantly increased in reperfused hearts compared with non-ischaemic controls. CONCLUSIONS: Reperfusion, but not ischaemia, was associated with increased DAB staining. This suggests a reperfusion induced increase in myocyte peroxidation. Increased staining may be due to the actions of haem proteins with peroxidase activity on peroxidized lipid.

Superoxide dismutase plus catalase enhances the efficacy of hypothermic cardioplegia to protect the globally ischemic, reperfused heart.

Experiments were conducted in an attempt to improve upon the contractile and metabolic protection of globally ischemic and reperfused isolated hearts provided by hypothermic (27 degrees C) cardioplegia. Hypoxic substrate-free test solutions were perfused through isolated rabbit hearts for 5 minutes before and after an uninterrupted 2 hour period of global ischemia. Test solutions included a modified physiological saline solution (PSS) (not cardioplegic); a potassium- and magnesium-enriched cardioplegia solution; or a PSS or cardioplegic solution supplemented with superoxide dismutase (SOD) plus catalase (150,000 units/L each). Postreperfusion contractile and biochemical function was compared to preischemic function or that of nonischemic control hearts. On the basis of measurements of left ventricular pressure development, left ventricular compliance, spontaneous heart rate, coronary vascular resistance, and isolated mitochondrial oxidative phosphorylation, we concluded that supplementing hypothermic cardioplegia solution with enzymes gave protection which was significantly better than that obtained with the other interventions, with values of these indicators not significantly different from those of nonischemic perfused controls. The results indicate that SOD plus catalase significantly enhances the protection afforded by hypothermic cardioplegia. They also implicate cytotoxic oxygen radicals as important contributors to "ischemic" damage and suggest that this component of damage can be prevented effectively.

Effects of supplementing hypothermic crystalloid cardioplegic solution with catalase, superoxide dismutase, allopurinol, or deferoxamine on functional recovery of globally ischemic and reperfused isolated hearts.

We evaluated whether supplemental pharmacologic interventions that altered formation or degradation of reactive oxygen metabolites, when added to hypothermic crystalloid cardioplegic solution (procaine-free St. Thomas' Hospital solution), alter postischemic function of isolated rabbit hearts. Hypoxic, substrate-free cardioplegic solutions cooled to 27 degrees C were perfused through isolated rabbit hearts for 5 minutes before and after an uninterrupted 2 hour period of global ischemia at 27 degrees C. Hearts were then reperfused with standard buffer for 1 hour at 37 degrees C. In some experiments, the cardioplegic solution was supplemented with the following: superoxide dismutase (30 micrograms/ml; degrades superoxide anion); catalase (1.7 micrograms/ml; degrades hydrogen peroxide); allopurinol (1 mmol/L; inhibits xanthine oxidase); or deferoxamine (Desferal, 0.5 mmol/L; selectively chelates ferric iron). Postreperfusion contractile parameters of supplemented hearts, including left ventricular pressure development and its first derivative, left ventricular compliance, spontaneous heart rate, and coronary vascular resistance, were statistically compared to data obtained from hearts arrested with unsupplemented cardioplegic solution. Catalase supplementation provided statistically significant improvement of most functional parameters; somewhat less protection was obtained with allopurinol. Deferoxamine provided little added protection except for the ability to prevent ischemia-induced increases of coronary vascular resistance. There was no evidence of added protection by superoxide dismutase. The data suggest that an important component of ischemia-induced cardiac cell damage in an asanguineous setting is hydrogen peroxide-dependent, and interventions that either inhibit production of superoxide anion or degrade hydrogen peroxide offer best protection. They may be clinically efficacious additives to crystalloid cardioplegic solutions.

The effect of hypothermic ischemia on recovery of left ventricular function and preload reserve in the neonatal heart.

Neonatal and adult myocardium respond differently to ischemia. In addition, the neonatal heart possesses a limited preload reserve. The effect of uninterrupted hypothermic ischemia on recovery of left ventricular function and preload reserve was studied in two groups of isolated rabbit hearts: group 1 (neonates, n = 8), 7 to 10 days old; group 2 (adults, n = 15), 6 to 12 months old. Peak left ventricular systolic pressure, the first derivative of left ventricular systolic pressure, and heart rate were measured at left ventricular pressures of 0, 5, 10, and 15 mm Hg before and after 120 minutes of global ischemia at 27 degrees C. Before ischemia, left ventricular systolic pressure increased significantly at each increment of left ventricular end-diastolic pressure for both groups of hearts. After hypothermic ischemia, recovery of left ventricular systolic pressure was significantly reduced at each level of left ventricular end-diastolic pressure among neonatal hearts (range 75% to 79% of control values). The postischemic recovery of left ventricular systolic pressure in the adult hearts was markedly reduced from baseline values (range 43% to 53% of control values) and was significantly worse than that of neonatal hearts at each level of left ventricular end-diastolic pressure (p less than 0.001). Both groups were able to respond to increasing preload after ischemia. The slope of the curve describing the relationship between left ventricular end-diastolic pressure and percent recovery of left ventricular systolic pressure was not different from zero for neonatal hearts but was significantly greater than zero among the adults (0.22 +/- 0.21 versus 0.73 +/- 0.07, p = 0.0056). After ischemia, the first derivative of left ventricular systolic pressure fell significantly from control values among neonatal hearts (71% of control values). The reduction was considerably greater, however, among the adult hearts (54% of control values). These data indicate that the neonatal heart recovers systolic function better than the adult heart after global ischemia with moderate hypothermia.

Protective effects of beta-adrenergic blockade in isolated ischemic hearts.

The protective effects of the beta-adrenergic blocking drugs, propranolol and atenolol, were tested in a model of global ischemia and assessed electron microscopically. Cats isolated hearts were perfused retrogradely with arterial blood drawn from donor cats. After a period of equilibration, isolated hearts were rendered globally ischemic for 1 h and subsequently reperfused for another hour. Hearts were then flushed with physiological salt solution followed by perfusion-fixation with cacodylate-buffered glutaraldehyde, containing ionic lanthanum. Lanthanum was included as a probe of myocardial membrane integrity. Left ventricular subendocardial samples were processed and examined electron microscopically. Nontreated hearts, which underwent normothermic ischemia and reperfusion, displayed extensive ultrastructural damage. Nonischemic and donor cat control myocardial tissue appeared normal in all respects. Hearts that received either propranolol or atenolol maintained their ultrastructural integrity, resembling controls. Ionic lanthanum proved to be reliable as a marker of membrane integrity and permeability, as nontreated hearts displayed intracellular deposition of the marker, indicating that deteriorations of membrane integrity occurred. The results suggest that beta-adrenergic blockade may be valuable in preserving myocardium subjected to ischemia and reperfusion.

The effect of pass/fail grading and weekly quizzes on first-year students' performances and satisfaction.

BACKGROUND: In 1992-93 the University of Michigan Medical School revised its first-year curriculum. An evaluation system using honors, high-pass, pass, and fail grading and only two examinations (a midterm and a final) was replaced with a system using pass/fail grading and weekly quizzes in addition to the two examinations. The objective was to increase students' satisfaction while maintaining a high level of achievement. METHOD: Students' performance scores and survey data from the final year of the former system (1991-92, 222 students) and the first year of the new system (1992-93, 195 students) were used to investigate whether overall performance decreased and whether the students liked the new approach to grading. Statistical methods used were one-sample t-tests, Student's t-test, and Fisher's Z-test. RESULTS: Under the new system, the average scores for courses remained well above passing, and no evidence was found that the students achieved at lower levels than had their predecessors with the former, more traditional grading system. Also, higher cumulative pre-final scores (i.e., scores on the weekly quizzes as well as the midterm) did not predict lower, "just passing" achievement on final examinations. The students' responses to the surveys included comments that pass/fail grading eased anxiety and reduced competition while encouraging the students' co-operation. CONCLUSION: Despite concerns that implementing pass/fail grading for all first-year courses would result in lower overall performance and decreased motivation among students, during the first year of implementation these fears proved to be unfounded as the students continued to perform well and reported greater satisfaction with the new system.

Human neutrophil hydrogen peroxide generation following physical exercise.

The effects of prolonged, submaximal exercise by seven healthy, untrained individuals on the generation of H2O2 by neutrophils was studied. Hydrogen peroxide generation by neutrophils isolated from pre-exercise (control) and post-exercise blood samples was measured 10, 15 and 20 minutes following stimulation with phorbol myristate acetate (PMA). Exercise was associated with a significant elevation in the number of circulating neutrophils and a diminished capacity for neutrophil H2O2 generation following PMA stimulation. Addition of post-exercise plasma to neutrophils isolated from pre-exercise blood caused a small reduction in H2O2 generation, suggesting the presence of an inhibitory factor(s) in the plasma during physical exercise. These results support the concept that exercise may contribute to an attenuation of oxygen-dependent neutrophil killing.

Subcellular actions and potential adverse cardiac effects of the cardiotonic ionophore monensin.

Monensin concentrations between 0.5 and 30 microM produced dose-dependent positive inotropy when administered to normal, electrically driven rabbit left atria. These doses did not produce contracture. When monensin was combined with very low concentrations of ouabain (10 or 50 nM), irreversible contracture, irregular responses to electrical stimulation, or both, occurred in a significant number of atria treated with both drugs. This was apparently due to intracellular Na+ overload produced by both drugs, with secondary elevations of intracellular Ca++ concentrations. Monensin (17 nM, 1.7 or 170 microM) did not inhibit canine myocardial Na+mK+-adenosine triphosphatase activity. When administered to mitochondria isolated from normal rabbit hearts, monensin concentrations greater than 10 nM significantly depressed ADP-stimulated (State 3) respiratory rates and calculated respiratory control ratios. Maximum inhibition of the respiratory control ratio (85% decrease) occurred with monensin concentrations of 1 microM or more. These concentrations also significantly decreased calculated ADP:0 ratios. The data show that monensin concentrations required to increase contractility of isolated myocardium can produce marked inhibitory effects on isolated mitochondria, but apparently do not do so when the drug is administered to intact, normal cardiac tissue.

A method to reduce interference by sucrose in the detection of thiobarbituric acid-reactive substances.

A thiobarbituric acid (TBA) reaction for measuring lipid peroxidation products was evaluated for interference by several ingredients commonly used in solutions to prepare or analyze tissue homogenates or subcellular organelles. These included sucrose (up to 100 mM final concentration in the assay medium), Tris-maleate (up to 40 mM), imidazole (up to 20 mM), inorganic phosphate (up to 10 mM), and 4- morpholinepropanesulfonic acid (up to 20 mM). When the samples were heated at 95 degrees C as recommended in some procedures, only sucrose significantly affected color development. Sucrose concentrations as low as 10 mM significantly increased absorbance at 532 nm of aqueous tetramethoxypropane (TMP) standards, and so the assay could not be applied reliably to tissue samples prepared in sucrose. Sucrose interference was only partially reduced by subsequent organic extraction (n-butanol plus pyridine), with measured absorbances remaining significantly greater (50-100%) than sucrose-free controls at sucrose concentrations of 20 mM or more. Modifying the assay to include sucrose in blanks and TMP standards failed to adequately correct for interference when the absorbance of unextracted (aqueous) solutions was measured. Further modification by adding sucrose to blanks and TMP standards, followed by butanol-pyridine extraction, gave standard curves that were linear, through the origin, and had slopes equivalent to those of sucrose-free standards. This modification enabled almost complete recovery (average 2% error) of known amounts of TMP added to aliquots of tissue homogenates containing amounts of sucrose that otherwise significantly interfered. Also, with the modified method the content of TBA-reactive substances in tissues homogenized in sucrose was found to be not significantly different from that measured in tissues homogenized in a noninterfering substance, KCl.

Myocardial flavin reductase and riboflavin: a potential role in decreasing reoxygenation injury.

Ferrylmyoglobin has been implicated in cardiac reoxygenation damage. Flavin reductase, an enzyme previously isolated from erythrocytes, can reduce ferrylmyoglobin in the presence of sufficient flavin concentrations. Flavin reductase mRNA signals were detected in rabbit heart, lung, liver, kidney, and isolated cardiomyocytes. It was hypothesized that increasing flavin reductase catalysis by administering flavins exogenously could decrease cardiac reoxygenation damage in isolated rabbit hearts. Riboflavin (150 microM) inhibited reoxygenation-induced lactate dehydrogenase release by 57%, an effect prevented by hematoporphyrin, a flavin reductase inhibitor. The results suggest that riboflavin supplementation has cardioprotective effects during reoxygenation and that these effects are mediated by flavin reductase.

Spatial and temporal characteristics of circumferential flow-function relations during acute myocardial ischemia in the conscious dog.

In the anesthetized open-chest dog the ischemic area produced by coronary occlusion is surrounded by an area of nonischemic contractile dysfunction, identified as the functional border zone. To establish whether a similar functional border zone exists in the conscious animal during acute regional ischemia and to determine its spatial dimensions and temporal changes, we performed simultaneous two-dimensional echocardiography and radioactive microsphere studies in nine chronically instrumented dogs. We produced circumferential flow-function maps at 22.5-degree intervals over the full circumference of the left ventricle at the midpapillary muscle level during control conditions, 5 minutes after left circumflex occlusion, and 2.5 hours after left circumflex occlusion. After occlusion there was no change in left ventricular end-diastolic area, an increase in left ventricular end-systolic area (p less than 0.01), and a decrease in left ventricular area ejection fraction (p less than 0.01). The circumferential extent of left ventricular dysfunction was 197 +/- 11 degrees (mean +/- SEM) at 5 minutes of left circumflex occlusion, whereas the extent of subendocardial hypoperfusion was 144 +/- 6 degrees (p less than 0.0005). This produced a functional border zone measuring 54 +/- 8 degrees, or 25% of the nonischemic myocardium, which did not change over the 2.5-hour occlusion period. Despite a modest but significant decrease in wall thickening (70 +/- 6% to 43 +/- 6%; p less than 0.01) in the functional border zone, there was no difference in subendocardial blood flow between the functional border zone and the control nonischemic area. We conclude that a discrete functional border zone exists in the conscious dog during acute regional ischemia produced by circumflex coronary occlusion, which does not change during the early evolution of myocardial infarction. The functional border zone likely contributes to minor overestimation of infarct size in the early hours after myocardial infarction if extent of left ventricular dysfunction is used as an index of infarction in humans.

Mitochondrial hydrogen peroxide generation and activities of glutathione peroxidase and superoxide dismutase following global ischemia.

We used isolated, buffer-perfused rabbit hearts to evaluate whether global, normothermic ischemia altered mitochondrial hydrogen peroxide (H2O2) generation and mitochondrial activities of the major enzymes responsible for degrading H2O2 and superoxide anion (O2-.): glutathione peroxidase (GPD) and superoxide dismutase (SOD), respectively. This preparation lacks exogenous neutrophils and endogenous xanthine oxidase, which are other potential sources of oxygen metabolites. Ischemia depressed mitochondrial oxidative phosphorylation parameters, State 4 succinate-supported H2O2 generation rates, and the relative flux of State 4 oxygen consumption that was diverted to H2O2 formation. The production of H2O2 was not abolished. Ischemia and reperfusion significantly reduced the activities of SOD (by 43%) and GPD (by 39%) in the mitochondrial fraction. Cytosolic GPD activity was also depressed. The results suggest that the myocardial cell's ability to enzymatically degrade H2O2 and O2-. is compromised, particularly in the mitochondrion. Although mitochondrial H2O2 production is decreased, the mitochondria may persist as a source of this oxygen metabolite following ischemia. Collectively, the data may help explain why mitochondria are vulnerable targets of free radical-mediated damage due to ischemia.

Effects of N-dimethyl propranolol (UM-272) on isolated cardiac mitochondria and microsomes.

N-dimethyl propranolol (UM-272) has been shown to protect the heart from injury produced by ischemia. In the present study we examined the effects of UM-272 on the function of isolated rabbit cardiac mitochondria and microsomes. Concentrations of 13 micrometers or below were without effect on these organelles. UM-272 (130 micrometers) significantly decreased respiratory control of mitochondria utilizing glutamate plus malate, or succinate, as substrates. At 1.3 mM, UM-272 increased the initial rate of basal oxygen consumption, and decreased the rate of ADP-stimulated respiration. UM-272 was slightly more potent than d,1-propranolol. At a concentration of 1.3 mM, UM-272 significantly decreased the rate and maximum amount of 45CaCl2 accumulated by microsomes in the presence of ATP and oxalate. Concentrations of drug that suppress cellular metabolism are close to those required to prevent ischemic injury. We suggest that sarcolemmal and intracellular actions of the drug which help to depress oxygen demand and ATP utilization may account for part of the drug's protective effects.

Cardiac chronotropic mechanisms of dimethyl sulphoxide: inhibition of acetylcholinesterase and antagonism of negative chronotropy by atropine.

The chronotropic effects of dimethyl sulphoxide (DMSO) were studied in spontaneously beating rabbit atria. Low concentrations of DMSO (0.14, 0.42 M) produced slight positive chronotropic (+C) responses; 0.84 and 1.41 M DMSO caused significant negative chronotropic (--C) responses. All concentrations decreased contractile strength. Atropine sulphate (10)-7) to 10(-5) M) antagonized the chronotropic effects, but not the inotropic effects. In the presence of atropine all DMSO concentrations produced significant +C responses. Reserpine pretreatment or propranolol did not affect contractile responses to DMSO in the absence or presence of atropine. DMSO produced concentration-dependent inhibition of acetylcholinesterase (AChE) activity of atrial homogenates. The results indicate that the --C responses are due specifically to AChE inhibition by DMSO and resulting cholinergic influences on the atrial pacemaker. Adrenergic mechanisms do not appear to mediate the +C responses. Data presented here provide evidence that a cardioactive effect of DMSO is mediated by a well-defined receptor-linked mechanism, and that this effect can be modified by a specific receptor blocking agent.

Hydrogen peroxide generation by mitochondria isolated from regionally ischemic and nonischemic dog myocardium.

We occluded the left anterior descending coronary artery of anesthetized, open-chest dogs, for 1 or 2 h. Some hearts were reperfused for 1 h after 1 h of ischemia. We isolated mitochondria from the central ischemic zone (CIZ) and a surrounding nonischemic zone (NIZ) of the left ventricle, and assayed H2O2 production using a horseradish peroxidase-dual wavelength spectrophotometric technique. Mitochondria, studied in the absence of exogenous respiratory chain inhibitors, generated H2O2 during State 4 respiration with succinate as the substrate. NIZ mitochondria in all groups produced ca. 1.5 nmols H2O2/min/mg protein (no significant differences between groups). The State 4 O2 consumption rates of NIZ mitochondria from hearts subjected to 1 h ischemia plus reperfusion, or 2 h of ischemia (ca. 30 nmols/min/mg) were significantly higher than that of NIZ mitochondria of hearts subjected to only 1 h of ischemia (23 nmols/min/mg). Thus, the ratio between H2O2 produced and State 4 O2 consumption fell from 6.5% to 5%. Mitochondria from all CIZ samples had State 4 O2 consumption rates that were not different from corresponding NIZ values. However CIZ mitochondria of hearts subjected to 1 h ischemia without reperfusion produced less H2O2 (1.1 +/- 0.1 nmols/min/mg), and had a slightly reduced H2O2/O2 ratio (4.4 +/- 0.7%), compared with their NIZ samples (1.5 +/- 0.1 nmols/min/mg; 5.3%). Reperfusion after 1 h of ischemia abolished these regional differences. The CIZ mitochondria from hearts subjected to 2 h ischemia produced only 0.75 +/- 0.22 nmols H2O2/min/mg (2.5% of State 4 O2 consumption). These values were 50% of corresponding NIZ values, and were significantly less than for any other group or tissue region. If similar phenomena occur in conscious animals subjected to incomplete regional ischemia, especially of relatively brief duration or if accompanied by reduced intracellular defenses against oxidants such as H2O2, they suggest that mitochondria persist as H2O2 sources and so may contribute to the oxidant load and myocardial dysfunction.

The oxygen consumption paradox of "stunned myocardium" in dogs.

The contractile state of the heart is a major determinant of myocardial oxygen consumption. Since regional myocardial contractility can be severely impaired following a transient coronary occlusion, post-ischemic myocardium is frequently assumed to consume less oxygen. To test this assumption, regional myocardial function and oxygen consumption were studied in anesthetized dogs during 2 h of myocardial reperfusion following either a 15-min (Group I) or 4-h (Group II) left anterior descending coronary artery occlusion. Both groups developed similar post-ischemic regional dysfunction characterized by paradoxical motion (negative shortening). Measured as a percent of baseline segment shortening, anterior wall function in Group I (n = 8) and Group II (n = 5) at 30 min of reperfusion was -33 +/- 11% and -34 +/- 16% (p = NS) and at 120 min was -23 +/- 9% and -40 +/- 16% (p = NS). However, the two groups showed a marked difference in regional myocardial oxygen consumption during reperfusion. Despite the abnormal wall motion, regional oxygen consumption in Group I at 30 and 120 min of reperfusion was unchanged from pre-ischemic levels as measured as a percent of baseline: 104 +/- 20% (p = NS) and 111 +/- 21% (p = NS). In contrast, regional oxygen consumption in Group II was markedly depressed from baseline at 30 and 120 min of reperfusion: 42 +/- 7% (p less than .01) and 40 +/- 8% (p less than .01). To determine whether the dissociation between regional myocardial oxygen consumption and function in Group I was related to mitochondrial uncoupling, six additional dogs were studied. Tissue samples were obtained from post-ischemic myocardium after 120 min of reperfusion following a 15-min coronary artery occlusion, and compared to non-ischemic myocardium. There were no differences in the in vitro mitochondrial respiratory rates or oxidative phosphorylation capacity between the post-ischemic and non-ischemic myocardium. Therefore, in the post-ischemic myocardium, significant depressions in regional contractility may not be associated with falls in oxygen consumption. Following a 15-min coronary artery occlusion, the injured myocardium maintains a paradoxically high oxygen consumption with normal mitochondrial function despite decreased contractility and abnormal wall motion.

Effect of superoxide dismutase plus catalase on Ca2+ transport in ischemic and reperfused skeletal muscle.

Cytotoxic oxygen metabolites may contribute to skeletal muscle damage associated with ischemia and reperfusion. This study utilized a rat hindlimb ischemia model to investigate the effect of pretreatment with oxygen free radical scavengers superoxide dismutase (SOD) and catalase (CAT) on skeletal muscle Ca2+ uptake by sarcoplasmic reticulum (SR) in limbs subjected to periods of ischemia and reperfusion. SOD and CAT were conjugated to polyethylene glycol to prolong their half lives. Anesthetized rats (ca. 350 g) received an iv injection of either conjugated SOD (2 mg/kg) plus CAT (3.5 mg/kg) (n = 6, Treated Group) or 0.9 saline (4 ml/kg) (n = 6, Control Group) 5 min before unilateral hindlimb tourniquet ischemia of 3 hr duration. After 19 hr of reperfusion, muscle from each lower leg was excised and homogenized. Skeletal muscle SR was isolated by differential centrifugation. ATP-dependent Ca2+ uptake by the SR was then measured with dual wavelength spectrophotometry and used as an index of muscle function. Pretreatment with SOD and CAT maintained higher rates of Ca2+ uptake by SR of skeletal muscle from postischemic reperfused limbs (Treated Group 2.29 +/- 0.21 vs Control Group, 1.61 +/- 0.06 mumole Ca2+/mg protein/min). These results implicate cytotoxic oxygen metabolites in the pathogenesis of ischemic reperfusion skeletal muscle injury.