Skeletal Muscle Ischemia-reperfusion Injury Research Articles

Partial hexokinase II knockout results in acute ischemia–reperfusion damage in skeletal muscle of male, but not female, mice

Cellular studies have demonstrated a protective role of mitochondrial hexokinase against oxidative insults. It is unknown whether HK protective effects translate to the in vivo condition. In the present study, we hypothesize that HK affects acute ischemia–reperfusion injury in skeletal muscle of the intact animal. Male and female heterozygote knockout HKII (HK+/-), heterozygote overexpressed HKII (HKtg), and their wild-type (WT) C57Bl/6 littermates mice were examined. In anesthetized animals, the left gastrocnemius medialis (GM) muscle was connected to a force transducer and continuously stimulated (1-Hz twitches) during 60 min ischemia and 90 min reperfusion. Cell survival (%LDH) was defined by the amount of cytosolic lactate dehydrogenase (LDH) activity still present in the reperfused GM relative to the contralateral (non-ischemic) GM. Mitochondrial HK activity was 72.6 ± 7.5, 15.7 ± 1.7, and 8.8 ± 0.9 mU/mg protein in male mice, and 72.7 ± 3.7, 11.2 ± 1.4, and 5.9 ± 1.1 mU/mg in female mice for HKtg, WT, and HK+/-, respectively. Tetanic force recovery amounted to 33 ± 7% for male and 17 ± 4% for female mice and was similar for HKtg, WT, and HK+/-. However, cell survival was decreased (p = 0.014) in male HK+/- (82 ± 4%LDH) as compared with WT (98 ± 5%LDH) and HKtg (97 ± 4%LDH). No effects of HKII on cell survival was observed in female mice (92 ± 2% LDH). In conclusion, in this mild model of acute in vivo ischemia–reperfusion injury, a partial knockout of HKII was associated with increased cell death in male mice. The data suggest for the first time that HKII mediates skeletal muscle ischemia–reperfusion injury in the intact male animal.

Protective Effect of Zinc Aspartate on Long-Term Ischemia–Reperfusion Injury in Rat Skeletal Muscle

The present study investigated the protective effect of zinc aspartate, in connection with reactive oxygen species and nitric oxide, on long-term ischemia-reperfusion injury (IRI) in rat skeletal muscle. Following ketamine anesthesia, 24 rats were randomly assigned to four groups: groups 1 and 2, each without tourniquet application, received no drug and zinc, respectively; groups 3 and 4, each subjected to tourniquet-induced IRI (3 + 24 h), received no drug and zinc, respectively. IRI was achieved by the application of an elastic rubber band in the left hind limb of the anesthetized rats. Gastrocnemius muscle samples were obtained for biochemical measurements. Malondialdehyde levels were lower in group 2 and higher in group 3 than those seen in group 1. However, zinc aspartate (group 4) totally reversed malondialdehyde levels to control levels. Superoxide dismutase activity was increased in group 2 compared with group 1; however, there was no difference between groups 1 and 3, and Zn injection (group 4) increased superoxide dismutase activity. While catalase values were similar in groups 1 and 2, significant increments were observed in 3 and 4. A similar enhancement in glutathione levels were observed in groups 2 and 4 compared with group 1. Nitric oxide levels were lower in group 2 than 1, and no difference between groups 1 and 3 was demonstrated. In conclusion, zinc seems to be an effective treatment option against IRI.

The Role of Nitric Oxide Synthase and Heme Oxygenase in the Protective Effect of Hypothermia in Ischemia-Reperfusion Injury

Ischemia-reperfusion injury plays an important role in limb salvage following limb ischemia. The purpose of the present study was to evaluate the effect of local hypothermia and chemical modulators on microvascular permeability following ischemia-reperfusion injury in skeletal muscle. Sprague-Dawley rats were randomized into nine groups. Postcapillary venules of the extensor digitorum longus muscle were visualized with use of intravital microscopy. Following an intravenous bolus of fluorescein isothiocyanate-labeled albumin, the intravascular and extravascular space was examined for leak. Rats in the sham group underwent a one-hour mock ischemic period without the application of a femoral artery tourniquet, followed by one hour of mock reperfusion. The treatment groups (n = 5 in each group) had the tourniquet applied for one hour, followed by one hour of reperfusion at 10 degrees C (cold) alone, at 10 degrees C with nitric oxide synthase inhibitor, at 10 degrees C with heme oxygenase inhibitor, at 10 degrees C with a combination of inhibitors, at 34 degrees C (warm) alone, at 34 degrees C with a heme oxygenase inducer, at 34 degrees C with a nitric oxide synthase inducer, or at 34 degrees C with a combination of inducers. Rats in the sham group did not show a significant increase in microvascular permeability. Rats in the warm ischemia/reperfusion group displayed significant increases in microvascular permeability, as did the rats that received inhibitors of heme oxygenase and nitric oxide synthase at 10 degrees C. No significant increase in microvascular permeability was observed in the animals in the cold ischemia/reperfusion group or in animals that received inducers of heme oxygenase and nitric oxide synthase at 34 degrees C. Local hypothermia protects skeletal muscle from increased microvascular permeability following ischemia-reperfusion injury. This protective effect is also seen with the induction of the nitric oxide synthase and heme oxygenase systems at physiologic temperature. We also have shown that the protective effects of hypothermia are blocked by giving heme oxygenase and nitric oxide synthase inhibitors while keeping the muscle hypothermic. These findings demonstrate that heme oxygenase and nitric oxide synthase play a combined role in ischemia-reperfusion injury, suggesting possible pathways for clinical intervention to modulate injury seen following trauma, tourniquet use, vascular surgery, and microvascular surgery.

The effects of ischemic postconditioning against ischemic reperfusion injury of rat skeletal muscle

Objective To investigate the effects of ischemic posteondifioning on ischemic repeffusion injury of rat skeletal muscle and the timing of applying isehemie posteondifioning. Methods 32 Wistar rats were randomly divided into 4 groups. To create a skeletal muscle ischemia reperfusion injury model, the skin,muscles and nerves of the hind limb were severed while the femoral vessels were left intact and damped and released at designated time. By measuring serum malondialdehyde (MDA) level and skeletal muscle myeloperoxidase (MPO) level after 4 hours of isehemia and 1 hour of teperfusion and observing the exteny of skeletal muscle death 6 hours following repeffusion, the effects of posteonditioning on skeletal muscle isehemia repeffusion injury were evaluated. The effect of ischemia postconditioning applied 5 minutes following repeffusion on the ischemia repeffusion injury of skeletal muscles were also studied. Results Skeletal muscles that sustained 4 hours of isehemia and 6 hours of reperfusion could be protected by immediate isehemie postconditioning with three cycles of alternating 30 seconds of ischemia and 30 seconds of reperfusion. The extent of skeletal muscle isehaemie reperfusion injury was reduced as shown by the decreased level of neutrophil infiltration (MDA) in the repeffused areas, the decreased level of serum free radicals (MPO) and the reduced extent of muscle death. Posteonditioning applied 5 minutes after repeffusion did not reduce MDA level in the repeffused areas and serum MPO level, neither did it reduce the extent of muscle death. It therefore did not protect skeletal muscles from isehemia repeffusion injury. Conehtsion Isehemie posteanditioning at the beginning of reperfusion can protect skeletal muscle against isehemie reperfusion injury. Timing of applying posteonditioning is critical. If applied 5 minute after reperfusion, posteonditioning lost its protective effects. Key words: Muscle,skeletal; Repeffusion injury; Ischemia

Vascular Anatomy of the Hamster Retractor Muscle with Regard to Its Microvascular Transfer

Background: The hamster retractor muscle (RET) is used as an in vivo model in studies of skeletal muscle ischemia-reperfusion injury. The RET is unique in that the muscle can be isolated while preserving the primary vascular supply so that its contractile function can be measured simultaneously with local microvascular responses to experimental interventions. The goal of this study was to understand the anatomical origin of the vascular supply to the RET and determine whether the RET can be used as a free flap after surgical isolation of the thoracodorsal vessels. Methods: Microdissection was performed to determine the anatomy of the vasculature that supplies and drains the RET. Results: Distinct numbers and patterns of feed arteries (2–4) and collecting veins (1–3) were identified (n = 26 animals). Dye injection (n = 8) of the thoracodorsal artery demonstrated that the RET remains perfused following its isolation on the thoracodorsal pedicle. Heterotopic allograft transplantation of the RET (n = 2) was performed by anastomosing the thoracodorsal vessels to the femoral vessels using the end-to-side technique. Conclusions: The anatomical relationships indicate that the RET can be used as a free flap model for evaluating the effect of preservation strategies and transplantation on skeletal muscle microcirculation and contractile function.

Preventive Role of Magnesium on Skeletal Muscle Ischemia–Reperfusion Injury—an Experimental Study

The present study aims to explore whether Mg infusion has a preventive effect on ischemia-reperfusion injury in rats. A total of 20 Sprague-Dawley-type adult male rats were used. In group 1 (control), 0.9% isotonic solution was administered. In group 2 (experiment), magnesium sulfate (0.5 mg per 100 g) was administered. Ischemia was induced for 15 min for the two groups. Magnesium (Mg), interleukin 8 (IL-8), and malondialdehyde levels were analyzed in blood, while edema, neutrophil infiltration, eosinophilia, loss of striation, and nucleolization were evaluated in histopathological examination. Mg levels in the experiment group were higher than those in the control group after ischemia-reperfusion (p < 0.05). In the control group, postischemia and postreperfusion IL-8 values were higher than preoperative values (p < 0.05). As for eosinophilia and loss of striation values, these were higher in the experiment group after ischemia-reperfusion than the values in the control group (p < 0.05). Histopathologically, Mg infusion cannot prevent the tissue injury triggered in ischemia-reperfusion periods. Eosinophilia can be one of the major and earliest markers of ischemia-reperfusion injury.

Ischemia-Reperfusion Injury in Skeletal Muscle: Comparison of the Effects of Subanesthetic Doses of Ketamine, Propofol, and Etomidate

Interest in the effects of anesthetics in skeletal ischemia-reperfusion injury (IRI) has recently emerged. Thus, the objective was to compare the effects of subanesthetic doses of ketamine, propofol, and etomidate in a skeletal IRI model. IRI was applied to rats by tourniquet method. Under thiopental anesthesia, five experimental groups were established as follows: (1) sham-control, (2) IRI, (3) IRI+Ketamine, (4) IRI+Propofol, and (5) IRI+Etomidate. Malondialdehyde, superoxide dismutase, catalase, and glutathione peroxidase were measured in skeletal muscle via a spectrophotometer. Zinc, iron, copper, and selenium were evaluated by atomic absorption spectrophotometer. While the values of malondialdehyde and glutathione peroxidase were higher and lower, respectively, those of superoxide dismutase and catalase were identical in IRI group in comparison with sham-control. Zinc displayed a decrease in IRI group; however, no differences in iron and copper levels were determined. In rats treated with subanesthetic doses of ketamine, elevated malondialdehyde levels in IRI group were reversed to control levels by each dose. While each dose of ketamine decreased superoxide dismutase activities in comparison with IRI group, a reduction in catalase activity was only seen in 3mg/kg ketamine-treated rats. The attenuated glutathione peroxidase activity seen in IRI was greatly reversed by ketamine administration in all doses. No differences in zinc, copper, and iron levels were detected between IRI and ketamine-treated groups. Similar results were obtained either by the administration of propofol or etomidate. Subanesthetic doses of ketamine, propofol and etomidate displayed beneficial effects in IRI.

Effect of NFκB Inhibition by CAPE on Skeletal Muscle Ischemia-Reperfusion Injury

Nuclear factor kappa B (NFkappaB) plays important role in the pathogenesis of skeletal muscle ischemia/reperfusion (I/R) injury. Caffeic acid phenyl ester (CAPE), a potent NFkappaB inhibitor, exhibits protective effects on I/R injury in some tissues. In this report, the effect of CAPE on skeletal muscle I/R injury in rats was studied. Wistar rats were submitted to sham operation, 120-min hindlimb ischemia, or 120-min hindlimb ischemia plus saline or CAPE treatment followed by 4-h reperfusion. Gastrocnemius muscle injury was evaluated by serum aminotransferase levels, muscle edema, tissue glutathione and malondialdehyde measurement, and scoring of histological damage. Apoptotic nuclei were determined by a terminal uridine deoxynucleotidyl transferase dUTP nick end labeling assay. Muscle neutrophil and mast cell accumulation were also assessed. Lipoperoxidation products and NFkappaB were evaluated by 4-hydroxynonenal and NFkappaB p65 immunohistochemistry, respectively. Animals submitted to ischemia showed a marked increase in aminotransferases after reperfusion, but with lower levels in the CAPE group. Tissue glutathione levels declined gradually during ischemia to reperfusion, and were partially recovered with CAPE treatment. The histological damage score, muscle edema percentage, tissue malondialdehyde content, apoptosis index, and neutrophil and mast cell infiltration, as well as 4-hydroxynonenal and NFkappaB p65 labeling, were higher in animals submitted to I/R compared with the ischemia group. However, the CAPE treatment significantly reduced all of these alterations. CAPE was able to protect skeletal muscle against I/R injury in rats. This effect may be associated with the inhibition of the NFkappaB signaling pathway and decrease of the tissue inflammatory response following skeletal muscle I/R.

Influence of fiber-type composition on recovery from tourniquet-induced skeletal muscle ischemia–reperfusion injury

This study was designed to determine if previously reported differences in the functional impairment of muscles composed of predominantly different fiber types occurs following extended periods of ischemia. We hypothesized that the soleus (Sol) muscle, a predominantly slow-twitch muscle, would be less vulnerable to tourniquet-induced ischemia-reperfusion than the plantaris (Plant), a predominantly fast-twitch muscle, as determined by the assessment of isometric contractile function. Male Sprague-Dawley rats were assigned to one of the following groups to undergo tourniquet application (TKA) (n = 6/group): 2 h TKA, 2 d recovery; 4 h TKA, 2 d recovery; 2 h TKA, 14 d recovery; or 4 h TKA, 14 d recovery. In situ isometric contractile properties were assessed in the predominantly slow-twitch Sol and the predominantly fast-twitch Plant; the contralateral muscle served as the internal control. At 2 d, muscle contraction could not be elicited via neural stimulation, but muscles did contract with direct stimulation, which indicates neural injury. This condition was resolved by day 14. At this time point, tetanic tension (Po) in the Plant was reduced by 45% and 69% in the 2 and 4 h groups, respectively. Po for the Sol was unaffected in the 2 h group, but was reduced by 30% in the 4 h group. The fatigue resistance of the Plant was increased 2 fold in the 4 h group and was unchanged in all other groups. These results demonstrate that vulnerability to tourniquet-induced ischemia-reperfusion injury is dramatically different with respect to muscle fiber-type composition.

Hypertonic saline reduces skeletal muscle injury and associated remote organ injury following ischemia reperfusion injury

Background and purpose Pharmacological modulation of skeletal muscle reperfusion injury after traumaassociated ischemia may improve limb salvage rates and prevent the associated systemic sequelae. Resuscitation with hypertonic saline restores the circulating volume and has favorable effects on tissue perfusion and blood pressure. We evaluated the effects of treatment with a bolus of hypertonic saline on skeletal muscle ischemia reperfusion (IR) injury and the associated end-organ injury.Methods Adult male Sprague-Dawley rats (n = 27) were randomized into 3 groups: (1) a control group, (2) an IR group treated with normal saline, and (3) an IR group treated with hypertonic saline. Bilateral hindlimb ischemia was induced by application of a rubber band proximal to the level of the greater trochanters for 2.5 h. The treatment groups received either normal saline (4 mL/kg) or hypertonic saline (4 mL/kg) prior to tourniquet release. Following 12 h of reperfusion, the tibialis anterior muscle was dissected and muscle function was assessed electrophysiologically. The animals were then killed, and skeletal muscle and lung tissue were harvested for evaluation.Results Hypertonic saline significantly attenuated skeletal muscle reperfusion injury, as shown by reduced myeloperoxidase content, wet-to-dry ratio, and electrical properties of skeletal muscle. There was a corresponding reduction in lung injury, as demonstrated by reduced myeloperoxidase content and reduced wet-to-dry ratio.Interpretation Treatment with hypertonic saline attenuates skeletal muscle ischemia reperfusion injury and its associated systemic sequelae.

In which period of skeletal muscle ischemia-reperfusion injury is local hypothermia more effective?

We investigated the potential beneficial effects of local hypothermia applied during different periods of ischemia-reperfusion injury in a rat model. An isolated gracilis muscle model of ischemia-reperfusion injury was used consisting of four groups, each with six rats. Ischemic injury was induced by clamping the femoral artery for six hours. Local hypothermia at 10 degrees C was applied during only ischemia (IH), during reperfusion (RH) for four hours, and during both ischemia and reperfusion (IRH). The control group remained untreated. After 24 hours of perfusion, the rats were sacrificed and the gracilis muscles were removed to determine muscle edema (wet-to-dry weight ratio), polymorphonuclear leukocytes (PMNL), and the percentage of necrosis. Compared to the control group (193.7 +/-38.9), the PMNL count was significantly lower in the IH, RH, and IRH groups (111.5+/-36.2, p<0.004; 82+/-18.6, p<0.002; 54.5+/-21.8, p<0.002, respectively). The only significant difference in the PMNL count was between the IH and IRH groups (p<0.004). In all the hypothermia groups, the percentage of necrosis was found significantly less than that of the control group (22.5+/-5.2% vs 11.7+/-5.2% in IH, p<0.004; 10.8+/-3.8% in RH, p<0.004; 6.7+/-2.6% in IRH, p<0.002).Similarly, muscle edema was significantly decreased in the study groups (control, 2.89+/-0.46 vs 2.01+/-0.26 in IH, p<0.001; 1.98+/-0.34 in RH, p<0.001; 1.97+/-043 in IRH, p<0.001). There were no significant differences between the three hypothermia groups with respect to the percentage of necrosis and muscle edema. Our results show that local hypothermia applied during ischemic and reperfusion periods is significantly effective in reducing ischemia-reperfusion injury.

Development of reproducible histologic injury severity scores: Skeletal muscle reperfusion injury

Ischemia-reperfusion injury of skeletal muscle is a common clinical and experimental problem. To date, there has been no uniform and reproducible method to assess the extent of histologic injury. In this study, we developed a novel statistical methodology for evaluating injury in individual myocytes and 3 distinct methods for the interpretation of this data. C57/BL6 mice underwent 2 h of hindlimb ischemia followed by reperfusion for 3 (n = 11), 24 (n = 12), or 48 (n = 10) h. The gastrocnemius muscles were harvested, stained, and evaluated under microscopy. Standardized criteria were applied to score individual myocytes as healthy or injured, and injury score was expressed as injured fibers/total fibers %. Three methods of analyzing myocyte data were developed and evaluated with statistical Block-Random Sampling to determine the number of counted fibers required to represent accurately the total injury. The Full-Frame Counting, Fourfold Divided Counting, and Stratified Individual Counting methods differ in the random order in which fibers or microscopic fields are scored. The 3 methods were found to be statistically sound at all experimental time points. Using the Full-Frame, Fourfold, and Stratified methods, the maximum number of required fibers at all time points was 600, 300, and 100, respectively, to obtain an estimation of injury with a 95% confidence interval. These criteria and statistical methods for histologic evaluation of ischemia-reperfusion injury in skeletal muscle are accurate and reproducible. The Fourfold method is the most practical and technically efficient method of assessing injury. Such a quantitative, direct assessment of injury is important and will be useful for future studies.

Influence of Systemic Hypotension on Skeletal Muscle Ischemia-Reperfusion Injury After 4-Hour Tourniquet Application

Tourniquet use for extremity hemorrhage control is common in military trauma. Tourniquet use may be accompanied by systemic hypotension, but this phenomenon has not been studied. We aimed to define the muscle effects of the combined insult of tourniquet-induced skeletal muscle ischemia-reperfusion injury (I-R) and hemorrhagic hypotension. After a 33% carotid arterial hemorrhage, Sprague-Dawley rats underwent 240-min hindlimb ischemia induced by pneumatic tourniquet. Control animals were not hemorrhaged. No resuscitation was given. After tourniquet release, muscles were reperfused for 120 min and then dissected. Weights and mitochondrial viability assays (nitroblue tetrazolium method) were performed on the plantaris (PL), and soleus (SO). Histologic analysis was performed on the PL and SO. Muscle edema is expressed as the ratio of tourniquet limb to contralateral limb muscle weight. Animal laboratories of the United States Army Institute of Surgical Research. STUDY ANIMALS: Twelve Sprague-Dawley rats. The mean arterial pressure of hemorrhaged animals was 38 +/- 3 mm Hg before tourniquet placement and 86 +/- 4 mm Hg before release, both significantly (p < 0.05) lower than controls at the same time points. Pre- tourniquet mortality was 38% with hemorrhage and 0% without. All muscles experienced edema, with weight ratios greater than 1. The PL experienced significantly (p < 0.05) less edema with hemorrhage. Viability was unaffected by hemorrhage in all muscles, as was tissue inflammation. No differences in inflammation were observed with hemorrhage. Systemic hypotension modulates the impact of 4 hours of tourniquet ischemia by decreasing muscle edema but minimally altering measures of muscle viability. Compartment anatomy and muscle fiber type both influence muscle response to the combined insult of hypotension and I-R. In this model, hypotension did not worsen the skeletal muscle I-R observed after the use of a tourniquet for 4 hours.

Statins Inhibit Neutrophil Infiltration in Skeletal Muscle Reperfusion Injury

Neutrophil infiltration is a major determinant of ischemia-reperfusion injury (IRI). Statins improve endothelial function by elevating nitric oxide synthase activity and inhibiting adhesion molecule expression and may, therefore, inhibit IRI-induced neutrophil extravasation. Although statins are protective against myocardial IRI and stroke, a role for statins in ameliorating skeletal muscle IRI has not yet been confirmed. This study, therefore, addressed the hypothesis that simvastatin would attenuate the severity of tissue damage during skeletal muscle IRI. Rats were administered simvastatin for 6 d before 4 h hind limb ischemia and 24 h reperfusion. Neutrophil infiltration was assessed using myeloperoxidase (MPO) assays and tissue damage by quantitative immunohistochemical analysis of collagen IV. The effect of reducing nitric oxide levels on the severity of IRI was assessed by administering the NOS inhibitor, N-Imino-L-ornithine (L-NIO), before ischemia. Simvastatin significantly inhibited IRI-induced MPO activity but not collagen degradation in postischemic skeletal muscle. Inhibition of nitric oxide synthase by L-NIO markedly inhibited neutrophil infiltration and protected against IRI-induced collagen degradation. When both simvastatin and L-NIO were administered before IRI, the IRI-induced elevation in MPO activity was completely inhibited. However, paradoxically, simvastatin counteracted the protective effect of L-NIO against IRI-induced collagen IV degradation. The inhibition by simvastatin of IRI-induced neutrophil infiltration in skeletal muscle suggests that statins may be a useful therapy to attenuate the severity of IRI but their precise mechanisms of action remains to be determined. Nitric oxide also plays a cytotoxic, rather than protective, role in mediating IRI in this model.

Simvastatin pretreatment reduces the severity of limb ischemia in an experimental diabetes model

The purpose of this study was to examine the effects of simvastatin pretreatment in the setting of acute limb ischemia-reperfusion injury in an experimental diabetes model that is associated with a high risk for limb loss. Adult male Sprague-Dawley rats were randomized into two groups. Diabetes was induced in the first group by intravenous streptozotocin injection. The second group served as the nondiabetic group. Eight weeks after the streptozotocin injection, half of the rats in the diabetic and the nondiabetic groups were further randomized to receive either intraperitoneal simvastatin (1 mg/kg per day) or saline treatment for 6 weeks. Bilateral hind-limb ischemia was induced for 4 hours by the tourniquet method. After 24 hours of reperfusion, tissue samples were collected from the gastrocnemius and anterior tibial muscles bilaterally for measurement of muscle edema, percentage of necrosis, and malondialdehyde (MDA), glutathione, and myeloperoxidase (MPO) levels. Ischemic injury was more prominent in diabetic animals. The diabetic animals with limb ischemia exhibited a 7% increase in tissue edema, a 47% increase in muscle necrosis and MPO level, and a 15% reduction in glutathione levels compared with the nondiabetic animals (P < .05). Simvastatin treatment with 1 mg/kg for 6 weeks reduced the ischemic injury. Simvastatin pretreatment led to a 71% reduction in muscle necrosis in diabetic animals (P < .001). The protective effects of simvastatin pretreatment also correlated with a 23% improvement in tissue edema, a 75% reduction in tissue myeloperoxidase content, and a 71% increase in glutathione levels in diabetic animals (P < .01). Furthermore, skeletal muscle injury, characterized by tissue edema and leucosequestration, was significantly less severe with simvastatin pretreatment compared with the nondiabetic animals (P < .01). Simvastatin pretreatment reduced limb ischemia-reperfusion injury in diabetic and nondiabetic animals. We conclude that simvastatin pretreatment may be a potential therapeutic intervention for skeletal muscle ischemia-reperfusion injury in the clinical setting.

Effect of different periods of hyperbaric oxygen on ischemia‐reperfusion injury of rat skeletal muscle

The effect of hyperbaric oxygen (HBO) on ischemia-reperfusion injury of skeletal muscle, applied during different periods, was studied in 56 male rats. Animals were subjected to 6-h ischemia by a tourniquet over the major femoral trocanter and 4 (A) or 24 (B) h of reperfusion. HBO was carried out during 1 h in an acrylic chamber at a pressure of 2.0 ATA (100% oxygen): in the last 60 min of ischemia (II), after ischemia, during 1-h reperfusion time (III), and during the last hour of ischemia plus 1-h reperfusion (IV). Group I was the control group. After 4- or 24-h reperfusion, samples of the soleus muscle were stained by H&E and analyzed immunohistochemically. No interstitial hemorrhage, neutrophil infiltrate, or cellular necrosis were induced by HBO. The apoptosis index did not differ among the groups. HBO reduced morphologic alterations and promoted better results when administered in the ischemia plus reperfusion period (GIV).

Ischemia-Reperfusion Injury in Rat Skeletal Muscle is Attenuated by Zinc Aspartate

Oxygen-derived free radical-induced cell injury has been suggested to have a pivotal role in the etiology of ischemia-reperfusion injury. Thus, several lines of evidence indicate that antioxidant agents may be useful therapeutics in this condition. In this regard, the effect of zinc aspartate on ischemia-reperfusion injury was investigated in skeletal muscle. Tourniquet ischemia-reperfusion injury method was applied to Sprague-Dawley rats. Experimental groups were as follows: 1) sham control, 2) rats received zinc aspartate, 3) rats received hind limb tourniquet operation (left side), and 4) rats received hind limb tourniquet operation and zinc aspartate. Viability of muscle was evaluated by triphenyltetrazolium chloride dye method by using a spectrophotometer. Malondialdehyde, superoxide dismutase, catalase, glutathione, and glutathione peroxidase were measured in muscle, heart, lung, and blood via a spectrophotometer. The viabilities of ischemic limbs, percentage of the contralateral control muscle, in group 1, 2, 3, and 4 were 114 +/- 12%, 87% +/- 5%, 20% +/- 2%, and 95 +/- 10%, respectively. In muscle, increased malondialdehyde and decreased superoxide dismutase, catalase, and glutathione levels in group 3 were normalized by zinc aspartate in both left and right limbs. While malondialdehyde levels in heart and blood increased in group 3, the levels of superoxide dismutase, catalase, glutathione, and glutathione peroxidase were lower in group 3 than those in group 1. All these alterations were prevented by zinc aspartate. Malondialdehyde level of lung in group 3 was significantly higher than group 1 and 2. However, this augmentation was halted by zinc aspartate. The decrease in superoxide dismutase levels in group 3 was statistically reversed by the administration of zinc aspartate. Zinc aspartate seems to be an effective treatment option against ischemia-reperfusion injury.

Protective effects of caffeic acid phenethyl ester on skeletal muscle ischemia-reperfusion injury in rats

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

Effect of Fluid Resuscitation on Acute Skeletal Muscle Ischemia-Reperfusion Injury after Hemorrhagic Shock in Rats

Severe extremity wounds with vascular injury are common in military trauma, and tourniquets are commonly used for hemorrhage control. The complications of tourniquet use in the setting of trauma are not well studied. This study investigated the combined effect of hemorrhagic shock and fluid resuscitation with Hextend (HX; BioTime, Inc) or lactated Ringer's (LR) on skeletal muscle subjected to tourniquet-induced ischemia-reperfusion injury. Thirty male Sprague-Dawley rats underwent 33% arterial hemorrhage followed by 3 hours of tourniquet application. Before reperfusion, 10 animals each were resuscitated with lactated Ringer's (3 times shed volume) or HX (shed volume). Ten control animals received no resuscitation. Rats were euthanized 2 hours after tourniquet release and the tibialis anterior and medial gastrocnemius muscles were examined for edema (muscle wet weight) and viability (nitroblue tetrazolium reduction). Contralateral muscles served as controls for each animal, with results expressed as the ratio of the tourniquet limb to contralateral limb values. The tibialis anterior and medial gastrocnemius muscles in all groups experienced edema, with all weight ratios greater than one. Resuscitation with HX resulted in significantly (p < 0.05) greater edema than did no resuscitation in both muscles and greater edema than with lactated Ringer's in the medial gastrocnemius. All groups experienced a loss of viability as well, with nitroblue tetrazolium reduction ratios less than one. Resuscitation with HX resulted in significantly less viability loss than did no resuscitation in the medial gastrocnemius. No significant differences in viability were seen in the tibialis anterior. Resuscitation with HX or lactated Ringer's does not adversely affect muscle viability in ischemia-reperfusion injury. HX may be a better clinical choice when skeletal muscle ischemia-reperfusion injury is a risk, despite greater edema.

Combined Enzymatic and Antioxidative Treatment Reduces Ischemia-Reperfusion Injury in Rabbit Skeletal Muscle

Ischemia/reperfusion (I/R) injury is characterized by the production of oxygen-free radicals leading to disturbances in vasomotility (microvascular constriction) and microvascular permeability (interstitial edema formation). The objective was to evaluate the effect of the combined antioxidative and enzymatic preparation Phlogenzym on I/R injury of skeletal muscle. A rabbit hindlimb model of I/R (2.5/2 h) was used (IR group). Phlogenzym, containing rutin, trypsin, and bromelain, was applied enterally (60 mg/kg body weight) as a bolus 30 min prior to ischemia (Ph group). Sham-operated animals served as controls (CO group). Plasma malondialdehyde, potassium, and microvascular perfusion (monitored by laser flowmetry) were assessed. Histomorphometry and electron microscopy were performed from major adductor muscles. Two hours after reperfusion, potassium levels were significantly elevated in IR compared to Ph group (6.7 +/- 1.2 versus 4.9 +/- 0.9 mmol/l, P < 0.006). Enhanced lipid peroxidation, apparent by increased plasma malondialdehyde levels, was ameliorated in the Ph group (1.0 +/- 0.1 versus 0.7 +/- 0.1 nmol/ml, P < 0.0001). No-reflow (reduction of blood flow by 62% in IR group) was not observed in the Ph group (P < 0.004). Phlogenzym treatment prevented microvascular constriction (17.6 +/- 2.3 versus 12.6 +/- 1.1 microm(2), P < 0.0001) and mollified interstitial edema (21.5 +/- 2.0 versus 26.0 +/- 3.7%, P < 0.017), resulting in mild ultrastructural alterations in contrast to pronounced sarcolemmal and mitochondrial damage in untreated rabbits. Phlogenzym had a protective effect on skeletal muscle during I/R injury expressed by prevention of no-reflow and preservation of muscle tissue.