Bilgehan et al.indd 249 Correspondence: Bilgehan Erkut, Assist Prof, Atatürk Bulvari, Eda Apartmani Palandoken Polikliniği Üstü, Kat: 3, No: 3, 25080 Yenişehir, Erzurum, Türkiye. Tel: (+90 533) 745 10 06; Fax: (+90 442) 316 63 40; Email: bilgehanerkut@yahoo.com Copyright in this article, its metadata, and any supplementary data is held by its author or authors. It is published under the Creative Commons Attribution By licence. For further information go to: http://creativecommons.org/licenses/by/3.0/. Effects of Ascorbic Acid, Alpha-Tocopherol and Allopurinol on Ischemia-Reperfusion Injury in Rabbit Skeletal Muscle: An Experimental Study Bilgehan Erkut1, Ahmet Özyazıcıoğlu2, Bekir Sami Karapolat1, Cevdet Uğur Koçoğulları3, Sait Keles4, Azman Ateş1, Cemal Gundogdu5, Hikmet Kocak1 1Department of Cardiovascular Surgery, Atatürk University Medical Faculty, Erzurum, Turkey. 2Department of Cardiovascular Surgery, Yüksek İhtisas Hospital, Bursa, Turkey. 3Department of Cardiovascular Surgery, Afyon Kocatepe University Medical Faculty, Afyon, Turkey. 4Department of Biochemistry, Atatürk University Medical Faculty, Erzurum, Turkey. 5Department of Pathology, Medical Faculty of Atatürk University, Erzurum, Turkey. Abstract Purpose: Ischemia reperfusion injury to skeletal muscle, following an acute arterial occlusion is important cause of morbid- ity and mortality. The aim of the present study was to determine and evaluate the effects of ascorbic acide, alpha-tocopherol and allopurinol on ischemia reperfusion injury in rabbit skeletal muscle. Methods: Forty-eight New Zealand white rabbits, all male, weighing between 2.5 to 3.0 (mean 2.8) kg, were used in the study. They were separated into four groups. Group I was the control group without any drugs. The other groups were treat- ment groups (groups II, III, and IV). Group II rabbits administrated 50 mg/kg ascorbic acide and 100 mg/kg alpha-tocoph- erol 3 days prior to ischemia, group III rabbits received 50 mg/kg allopurinol 2 days prior to ischemia, and group IV rabbits were administrated both 50 mg/kg ascorbic acide, 100 mg/kg alpha-tocopherol 3 days prior to ischemia and 50 mg/kg allopurinol 2 days prior to ischemia. Two hours ischemia and 2 hours reperfusion were underwent to the treatment groups. At the end of the reperfusion periods, muscle samples were taken from rectus femoris muscle for determination of superoxide dismutase, catalase and glutathione peroxidase activities as antioxidant enzymes, and malondialdehyde as an indicator of lipid peroxidation and xanthine oxidase levels as source hydroxyl radical. Besides, histopathological changes (edema, infl ammation, ring formation and splitting formation) were evaluated in the muscle specimens. Results: In the treatment groups; superoxide dismutase (U/mgprotein), catalase (U/mgprotein), and glutathione peroxidase (U/mgprotein) levels increased, malondialdehyde (nmol/mgprotein) and xanthine oksidase (mU/mgprotein) levels decreased compared to control I ( p � 0.05). Increase of superoxide dismutase, catalase, and glutathione peroxidase levels were the highest and decrease of malondialdehyde and xanthine oxidase levels were the highest in group IV compared to groups II and III, but no signifi cant as statistically. Also amount of cellular injury in group II, III, and IV were lower than group I. Conclusions: Antioxidant medication may help lowering ischemia reperfusion injury. In our study, all drug medications are shown to be able to have an effective role for preventing ischemia reperfusion injury. Moreover, ascorbic acide + alpha- tocopherol + allopurinol group (group IV) may have a benefi cial effect to decrease the local and systemic damage due to ischemia-reperfusion injury. Keywords: Ischemia-reperfusion injury, antioxidant agents, ascorbic acid, alpha-tocopherol, allopurinol Introduction Ischemia-reperfusion (I/R) injury is an important adverse clinical outcome in a wide range of vascular conditions and surgical interventions including stroke, transplantation, cardiopulmonary bypass, trauma, and abdominal aortic aneurysm repair. It is a complex and serious condition and may life-threatening (1). Experimental studies have shown that the tissue-destructive effects of I/R injury are mediated by free oxygen radicals (H2O2, O2 −, OH−), which damage cellular components and they cause lipid per- oxidation of cellular membranes and generate more free radicals (FRs) in a self-propagating cycle, leading to cell death by necrosis (1–3). Drug Target Insights 2007:2 249–258 ORIGINAL RESEARCH http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ 250 Erkut et al Drug Target Insights 2007:2 The physiopathology of I/R injury is a complex cascade of events starting from the point of release of FRs followed by lipid peroxidation that ends by procuding substances such as malonyldialdehyde (MDA) (4,5). The determination of MDA can be used as a marker of FR formation (6). During ischemia, ATP production is diminished related to the limited oxygen availability. Secondly, the changes in membrane ion gradients cause an infl ux of calcium in damaged membranes. This leads to an elevation of the cytosolic calcium concentration that activates proteases capable of transforming xanthine dehydrogenase to xanthine oxidase (XO). During reperfusion, the provided molecular oxygen is converted to superoxide radicals by XO (6). There is evidence that XO levels are elevated during ischemia (6,7). The fi rst line defence mechanism includes anti- oxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) which they are the enzymatic part repre- sented by FR scavenger enzymes (8). These enzymes catalyse the conversion of FRs into less reactive species. Another mechanism is the non- enzymatic part including a large number of natural or synthetic antioxidant compounds, which have the ability to inhibit the oxidative damage by scav- enging the highly destructive FR species (9). Ascorbic acide is a well-known antioxidant agent and can protect the endothelium from direct injury by oxidants, including H2O2, and prevent microvascular dysfunction (10,11). We and others have also shown that ischaemia-reperfusion injury is reduced by the administration of ascorbic acide (10–12). Alpha-tocopherol is a potent antioxidant and shown that it prevents reperfusion injury in miscellaneous tissue (13). Allopurinol is a XO inhibitor that prevents the generation of FRs and may play a role in the protection of the cells dur- ing cerebral ischemia. Previous studies have indicated that allopurinol can improve the tissue energy metabolism during reperfusion after isch- emia (14,15). A number of authors have examined the role of FRs species in ischemic damage to skeletal muscle and experimentally, and the effect of FR scavengers have been evaluated in skeletal muscle (16,17). Besides, in many studies, the antioxidant activity was shown the roles of ascorbic acide, alpha- tocopherol and allopurinol in I/R injury through biochemical enzyme studies in addition to histo- pathological studies (18–21). But, according to our knowledge, there was no report to show the effects of ascorbic acide, alpha-tocopherol and allopurinol on reducing reperfusion injury in muscle of rabbit. The aim of this study was to determine the protective effect of the ascorbic acide, alpha- tocopherol and allopurinol as antioxidant agents against FRs in extremity ischemia and reperfusion. For this purpose, we measured SOD, CAT, GPx and MDA, XO levels, and evaluated as histo- pathologically in rabbit muscle in ischemia reper- fusion model. Our fi ndings suggest that all drug groups, especially group IV, reduces I/R injury. Materials and Methods The experiment was performed in compliance with the Principles of Laboratory Animal Care formu- lated by the National Institutes of Health. The experiment and animal care protocol, and all pro- cedures were approved by the Local Ethics Com- mittee in Animal Experiments. Animals Fourty-eight rabbits were used as subjects in our study. Male adult New Zealand type rabbits weigh- ing 2,500–3,000 g (2,610 ± 1,122 g) were kept in a light-controlled room with a 12:12-h light–dark cycle; temperature (22 ± 0.5 °C) and relative humid- ity (65%–70%) were kept constant. Animals received a standard rabbit diet and water and libi- tum. They had not been used in priory another study and they had not been given a drug regularly, in addition they had not a disease, previously. The rats were deprived of food for 12 h before the experi- ment but had free access to water. The subjects were inserted 22 no branul through ear veins. Experi- ments were carried out under sterile conditions and antibiotic prophylaxis with cefazolin sodium (30 mg/kg intramuscularly, single preoperative dose) was given. Isotonic NaCl solution was given intravenously at the rate of 3 ml/kg/h. During all experimental manipulations, to prevent the effects of hypothermia and to provide the stability of hemodynamic parameters, the body temperature was maintained at 37.2 °C with a rectal probe. For this, animals were placed on an operating table with thermoregulatory, and was used heat pad. Artifi cial respiration The rabbits were shaved from abdomen to leg. The surgical area was painted with batticon. Surgical 251 Ischemia reperfusion injury Drug Target Insights 2007:2 area was cleaned and draped. The rabbits were intubated via endotracheal cannulation (16-G Vasofi x, B. Braun Melsungen, AG, and Germany). Tidal volume and respiratory rate were adjusted to 10 ml/kg (3 ml for an average of 250–300 g subject) and 60 times per minute, respectively. Ventilator (Ugo Basile, Biological Research Apparatus, Comerio, and Varesee, Italy) was used for artifi cial respiration. Experimental groups Before starting the experimental protocols, rabbits were divided into four groups of twelve animals. The fi rst group was the control group (group I) without any treatment, the treatment groups were the group II, which was medicated intravenous with 50 mg/kg ascorbic acide (Redoxan 500 mg ampule, Roche, Germany) and intramuscular with 100 mg/kg alpha-tocopherol (α-T; Evigen 300 mg ampule, Aksu Farma, Turkey) for 3 days prior experiment, the group III, which was medicated with 50 mg/kg allopurinol (Urikoliz 300 mg, Ilsan, Turkey) for 2 days via per oral prior experiment, and the group IV, which was medicated with both ascorbic acide + alpha-tocopherol + allopurinol. The dosage and timing of each antioxidant were based on our preliminary data on the metabolism of the drugs, and on published data, which deter- mined the peak serum concentrations of the drugs at the time reperfusion was initiated (22–25). The protective effects against increasing of lipid per- oxidation caused by I/R were shown in rats treated with different dosages (range, 30–100 mg/kg) of ascorbic acide (22). In light of these past studies, we used 50 mg/kg of ascorbic acide as the dosage in our study. Dosage of alpha–tocopherol and allopurinol were chosed on on the basis of earlier studies (24,26,27). In experiment day, all animals were performed ischemia for 2 hours, and then performed reperfusion for 2 hours. Hoballah’s I/R model was used with direct occlusion of femoral artery and vein occlusions in the medial part of rectus femoris muscle (28). Surgical method The rabbits were anesthetized with intramuscular injection of 15 mg/kg ketamine hydrochloride (Ketalar; Pfi zer, Istanbul, Turkey) and 2 mg/kg xylazine hydrochloride (Rompun, Bayer, Turkey) before the surgical procedure. Longitudinal inci- sion was performed in venteromedial of right thigh. After the femoral area exploration, we reached to the rectus femoris muscle. Heparin of 400 U/kg (Liquemine, Roche, Brazil) was given via ear vein. The medial part of rectus femoris muscle was cut and separated from surrounding tissues. The I/R model was constituted by using the direct occlusion procedure as the study of Hoballah (Fig.1) (28). We exposed the muscle to ischemia for 2 hours. During the ischemic waiting period, incision was closed and the rabbits were freed. At the end of the ischemic period, was applied intraperitoneally ketamine hydrochloride and incision was opened, again. Microvascular clamps were removed and the wounds were closed. It was exposed to reperfu- sion for 2 hours. At the end of the reperfusion, muscle tissue samples were collected for bio- chemical and histopathological examination after the incision was opened. The muscle pieces from proximal and distal were anastomozed with one another. The femoral area was closed with silk suture. During these surgical interventions inter- vals, they were given same sort analgesics. Biochemical assay SOD, CAT, GPx, XO, MDA were detected in muscle tissue cuts. Each tissue was stocked in a separate bowl at −80 °C till analysis. Tris tampon of 10 ml was added into each one gram of frozen tissues. Homogenates are to be centrifuged at 10.000 × G for 10 minutes after homogenization. Supernatants were kept in stock at −80 °C till analysis. Analysis of tissue samples was carried out spectrophotometrically as below. Results were expressed as units per miligram protein for SOD, CAT, GPX, and nanomoles per milligram for MDA and miliunits per milligram for XO. Tissue SOD Assay: The method is based on the inhibition of nitroblue tetrazolium (NBT) reduction by the xanthine-XO system as a superoxide gen- erator by using Yi-Sun method (29). Study solution was prepared by mixing xanthine (0.3 mmol/l), ethylenediaminetetraacetate (EDTA) (0.6 mmol/l), NBT (0.15 mmol/l), sodium carbonate (Na2CO3) (400 mmol/l), bovine serum albumin (1 g/l). Study solution of 2850 ul, 100 UL supernatan, 100 ul distilled water and 50 UL XO (5 U/L) were incu- bated for 25 minutes at 20 °C. Following 30 seconds, absorbance was recorded. One unit is the amount of SOD that inhibits the rate by 50%. Tissue CAT assay: Catalase activity was assayed according to the methods of Cohen et al. (30) 252 Erkut et al Drug Target Insights 2007:2 To a 100 µL aliquot of tissue extract, ethanol was added to a concentration of 0.17 mol/L (10 µL ethanol/mL) and samples were incubated in an ice bath for 30 min. After 30 min, 10% Triton X–100 was added to a fi nal concentration of 1% and samples were kept at room temperature. Reactions were performed at room temperature. The enzyme-catalysed decomposition of H2O2 was measured. In a tube containing 200 µL phosphate buffer and 50 µL tissue extract, 1 mL of 6.0 mmol/L H2O2 (in phosphate buffer) was added and mixed thoroughly. The reaction was stopped after exactly 3 min by the addition of 100 µL of 6 mol/L H2SO4. The excess H2O2 was measured by reacting it with a standard excess of KMnO4 and then measuring the residual KMnO4 spectrophotometrically at 480 nm within 30–60 s using 1.0 absorbance unit for standard KMnO4. Tissue GPx assay: GPx catalyzes oxidation of glutathione (GSH) by using hydrogen peroxide. The activity of GPx was determined by the Beutler method (31). Briefly, solutions of tris-HCl (1000 mmol/l), EDTA (5 mmol/l) (pH = 8.0), GSH (100 mmol/l), GSH reductase (10 U/ml), nicotin- amide adenine dinucleotide phosphate (NADPH) (2 mmol/l), and t-butil hydroperoxide (7 mmol/l) were incubated with 10 UL hemolysate for Figure 1. Occlusion appearance of two vascular systems of rectus femoris muscle (direct occlusion) as described by Hoballah. 253 Ischemia reperfusion injury Drug Target Insights 2007:2 10 minutes at 37 °C. Decrease in NADPH was foolewed spectrophotometically at 340 nm. Tissue MDA assay: Thiobarbituric acid (TBA) reactive substances, MDA and product of fatty acid peroxidation, reacts with TBA to form a colored complex that has maximum absorbance at 532 nm by Ohkawa method (32). 200 ul sodium dodecyl sulfate (8.1%), 1.5 ml acetic acid (20%), 1.5 ml TBA (0.8%), 0.6 ml distilled water were mixed by 200 UL tissue homogenate (10%) and heated in boiling water for 60 minutes. After cooling, 1 ml distilled water and 5 ml butanol/pyrimidine (15:1) were added. After centrifuging at 4000 rpm for 10 minutes, absor- nance of the supernatant was read at 532 nm. Tissue XO assay: XO activity was determined spectrophotometrically by the method of Hashi- moto (33), based on the formation of uric acid from xanthine at 293 nm. Histopathological examination The muscle biopsy samples of the subjects were fi xed in neutral formalin solution (3%) for 24 hours. Paraffi n blocks were prepared after biopsy samples were exposed to routine tissue examina- tion. Later 1 to 2 micro hematoxylin-eosin (HE) cuts of paraffin blocks were prepared. Light microscopy fi ndings were scored as 0 to + 3 which corresponds to no change, mild, moderate and severe changes, respectively. Assessments were made for interstitial edema, infl ammation, splitting formation and ring formation in tissues through the use of Nikon optiphot-2 microscope. Statistical analysis All the results were obtained as mean ± SEM for each study group. All statistical analyses were car- ried out using SPSS 10.0 statistical software (SPSS Inc, Chicago, IL). The signifi cance of differences between the groups was assessed using the Non- parametric analyses with Mann-Whitney U-test, and The Kruskal-Wallis test was used to compare group medians for histopathological. Statistical signifi cance was set up p � 0.05. Results Biochemical evaluation SOD, CAT, and GPx levels were measured in skeletal muscle after 2 hours reperfusion, and the levels increased in the treatment groups compared to control group (from 0,17 ± 0,02 to 0,23 ± 0,02, 0,23 ± 0,03 and 0,25 ± 0,04, respectively for SOD; from 6,74 ± 1,00 to 9,02 ± 0,60, 8,22 ± 1,35 and 9,09 ± 1,16, respectively for CAT; from 18,52 ± 2,26 to 22,48 ± 2,38, 23,06 ± 2,34 and 25,67 ± 3,72, respectively for GPx) (Fig. 2), and it was signifi cant as statistically (p � 0.05). However, there was no a statistically signifi cant among group II, III, and IV in terms of the increasing in enzymes levels. XO levels in the skeletal muscle were found to be higher in the control group. However, in treatment groups lowered (from 2,86 ± 0,35 to 2,19 ± 0,34, 1,94 ± 0,41 and 1,02 ± 0,32, respectively) the levels of XO during ischemia and reperfusion (Fig. 3). Tissue MDA levels were decreased (from 0,38 ± 0,13 to 0,12 ± 0,04, 0,13 ± 0,07 and 0,10 ± 0,02, respec- tively) in the treatment group compared to the control group. The results of MDA assays are shown in Figure 3. Histopathological evaluation Cell infi ltration, edema, splitting and ring forma- tions were evaluated for 4 groups. There was a signifi cant difference in terms of pathological parameters between treatment groups and control group as histopathological scores ( p � 0.05). Figure 4 shows markedly ring formation, splitting formation, interstitial edema, and neutrophil cell infi ltration in control group after 2 hour reperfu- sion. In treatment groups ring formation, splitting formation, interstitial edema, and neutrophil cell infi ltration decreased markedly compared to con- trol group. Although ring formation was rare in treatment groups, there was not a difference among 3 treatment groups ( p � 0.05). Splitting formation, interstitial edema and neutrophil cell infi ltration were less determined in treatment groups ( p � 0.05). When treatment groups were compared to each other, it was found that splitting formation, interstitial edema and neutrophil cell infi ltration was less in group IV ( p � 0.05). Discussion Because skeletal muscle is more resistant to the ischemia, we applied total skeletal muscle ischemia model in this study. The transformation of xanthine dehydrogenase into XO during FRs constitution is slower in skeletal muscle, which explains why skeletal muscle is more resistant to ischemia (28,34,35). Recently advances in the understanding of reperfusion injury and the pharmacology of 254 Erkut et al Drug Target Insights 2007:2 antioxidants have made the interruption of reperfusion injury clinically promising and FR mediated tissue injury can be limited by the use of antioxidant therapy and several studies have sug- gested a positive role for antioxidant therapy in skeletal muscle reperfusion injury (36,37). It is well recognized that ischaemia followed by reperfusion in skeletal muscle represents an important clinical problem in many vascular dis- eases and musculoskeletal trauma. The signifi cant mortality and morbidity can be due to compartment syndrome, rhabdomyolysis, renal failure, limb loss, systemic infl ammatory syndrome and respiratory and mesenteric injuries. It has been emphasized the importance of ischaemia duration as a progres- sive increase of ultrastructural lesions takes place between 2- and 7-h ischaemia insult in skeletal muscular tissues (38). Two hours after ischaemia, it is already possible to identify, with histochemi- cal analysis, small muscular lesions which become severe according to the ischaemia duration (39). Regarding reperfusion injury, several reports have Figure 2. Between groups SOD, CAT, and GPx enzyme levels. Increased SOD, CAT and GPx levels in treatment groups compared to control group. Figure 3. Between groups MDA and XO enzyme levels. Decreased MDA and XO levels in treatment groups compared to control group. SOD; superoxide dismutase, CAT; Catalase, GPx; glutathione peroxidase, MDA; malonyldialdehyde, XO; xanthine oxidase, AP: Allopurinol. 255 Ischemia reperfusion injury Drug Target Insights 2007:2 showed that a period over 2 h of reperfusion is enough to establish the muscular lesion (40–42). Most study was made relation to administration time of drugs in order to decrease I/R damage (17–19). These studies were carried out in which antioxidants were given before ischemia. However, antioxidant agents were injected before reperfusion in other study (43). In a study, Feller showed that SOD and dimethylsulfoxide (OH _ radical scavenger) given before reperfusion and decreased reperfusion injury, and in late term, the muscle functions were excellent (44). We administrated antioxidant agents to the drug groups before ischemia in our study. FRs are normal by-products of cellular metabolic processes. The human body has a complex antioxi- dant defense system that includes the antioxidant enzymes (SOD, GPX and CAT) and nonenzymatic antioxidant components such as glutathione, a-tocopherol, ascorbic acide, and b-carotene. These prevent the initiation or propogantation of free radical chain reactions. Post-ischemic reperfusion injury is associated with the generation of FRs which damage cellular components and initiate the lipid peroxidation process. In many studies, antioxidant activity was tried to be shown through biochemical enzyme studies in addition to histopathological studies. We examined that the vitamin combinations given 3 days before the ischemia and allopurinol given 2 days before the ischemia for decreased I/R damage, both histopathological and biochemical. We could not fi nd any report showing the effect of ascorbic acide, alpha-tocopherol, and allopurinol on recuding I/R injury in lower extremity muscle of rabbit as enzymatic and biochemically. SOD is an enzyme which catalyses the trans- formation of the O2 − into H2O2, and it is one of the primary and signifi cant defensive systems against oxidative damage. The physiological function of this enzyme is to protect the cells of oxygen against FRs harmful effects (45,46). When oxidant stress increases in organism, SOD enzyme levels increase (45). The function of CAT is to divide H2O2 into O2 − and H2O by participating into the reaction with H2O2. Through this, it prevents the formation of OH− radicals, which are more toxic. Criado found that CAT enzyme levels increased in 30 minutes after I/R damage (47). For GPx catalyses H2O2 and lipid hydroperoxidase, it protects to cellular mem- branes from damage, prevents the start and devel- opment of lipid peroxidation. Lin showed in a myocardial ischemia reperfusion injury model found that GPx levels increased with the antioxidant Figure 4. The fi gure shows (a) ring formation, (b) splitting formation, (c) interstitial edema, (d) neutrofi l cell infi ltration in control groups (the arrows were depicted pathological changes) × 400 HE. 256 Erkut et al Drug Target Insights 2007:2 agent (48). In our study, SOD, CAT and GPx levels were increased in the treatment groups compared to control group ( p � 0.05). Although there was no signifi cant different between treatment groups, the increase of the enzymes levels was higher in group IV than group II and III. The end production of lipid peroxidation includes aldehydes, hydrocarbon gases, and MDA. It is good markers for increased systemic oxidative stress (48). MDA levels indicate the amount of cellular damage secondary to lipid peroxidation and has been widely adopted as a measure of free radical formation. Lipid peroxidation can cause changer leading to the deaths of cells. Feng found that MDA increase depended on FR appearance (49). A signifi cant elevation of MDA level after the 30 min of ischemia and 45 min of reperfusion was observed in tissues (50). In our study, the MDA levels had signifi cantly increased after ischemia reperfusion in the control group, because of the high level of hydroxyl radicals. In the antioxidant treatment groups, the levels were found have decreased, which may show the effects of anti- oxidant medication on limiting ischemia reperfu- sion injury. XO plays an important role in I/R injury (6,51). There is evidence that XO levels are elevated dur- ing ischemia (52). XO is the fi rst-known O2 − radical source (16). During ischemia, adenosine triphos- phate is degraded to hypoxanthine and xanthine dehydrogenase is converted to XO. During reperfusion, XO catalyzes the conversion of hypoxanthine to uric acid with release of the O2 − radical. Hammerman showed that lipid peroxida- tion was prevented in the group together with the decrease in XO activity (53). Allopurinol is considered to be an XO inhibitor. Smith found that I/R injury were decreased with tungsten and allo- purinol (54). In our study, there were signifi cant differences in the levels of XO between treatment groups and control group ( p � 0.05). Moreover, the decrease was highest in group III and IV admin- istrated allopurinol. Ascorbic acide is a powerful antioxidant agent. It is a critical component of the oxidant shield in skeletal muscle, being actively accumulated by muscle endothelium (55) According to Niki et al. (20), ascorbic acide has an antioxidant effect of the superoxide and hydrophilic radicals. It also acts on limiting lipidic peroxidation and scavenges reac- tive oxidants produced immediately after reperfu- sion (21). In our study, in group II and IV, which was used ascorbic acide, decreased level of XO and MDA enzyme and increased antioxidant enzymes (SOD, CAT and GPx) levels, and these results were supported with histopathological fi nd- ings. Αlpha-tocopherol is an antioxidant, and is protector against FRs. The preventive effects of alpha-tocopherol has been demonstrated in differ- ent experimental models (56,57). It protects cell membrane from oxidative damage and lipid per- oxidation (57). Allopurinol, a specifi c inhibitor of the enzyme XO, blocks the synthesis of xanthine from hypoxanthine and therefore avoids the forma- tion of the free radical superoxide. The studies showed that it is decrease the level of FRs produc- tion and reduce the tissue injury associated with I/R injury (23, 58). It is not only a potent inhibitor of XO but may also be an agent that improves ischemia-induced mitochondrial dysfunction (14,58). Our data show that FR overproduction induced by I/R causes lipid peroxidation of rabbit skeletal muscle. Administration of ascorbic acide, alpha- tocopherol (before 3 days) and allopurinol (before 2 days) skeletal I/R decreased MDA and XO levels and increased in SOD, CAT and GPx enzyme activities in the skeletal muscle and this result was suggested with hystopathological results. 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