Animals: Experiments were performed on the 50 adult male rats. Wistar rats weighing between 200 and 250 g. Animals were placed in a neutral temperature environment (22±2 °C) on a 12/12-h photoperiod and were fed with a standard diet. The study was approved by the Institutional Review Board and the animal care complied with the quide for the care and use of laboratory animals.
Experimental Groups and Surgical Procedures: All surgical procedures were performed while the rats were under anesthesia achieved by intraperitoneal injection of ketamine and xylazine cocktail (0.5 mL/kg + 1 mg/kg, i.p.). An additional dose of anesthesia cocktail was repeated at 30±45 min for maintenance. After induction, rats were placed with a preselected 37 °C temperature maintained during the whole surgery protocol.
Rats were randomized into five groups, each of which contained 10 rats. Sham operation, only an inguinal incision without femoral artery occlusion, was performed in sham-operated group. All rats in group 2 were exposed to 2 h ischemia period followed by 1 h reperfusion. After incision of the right inguinal region, the right femoral artery was dissected to free from the femoral vein and nerve. Then the femoral artery was occluded with microvascular clamp. After 2 h ischemia, the microvascular clamp was removed. All rats in group 3 were preconditioned with one-cycle of 10 min ischemia/10 min reperfusion before the long I/R protocol. The rats were subjected to 2 h ischemia and 1 h reperfusion, but 1 h after placing an occlusive vascular clamp across the right femoral artery. As indicated in the literature 14, KO (200 mg/kg) was injected into the peritoneum in group 4. The last group included not only IPC strategy, but also drug-reinforcement protocol. The rats were subjected to one-cycle of 10 min I/R episodes. After this, the specific occlusion and reperfusion periods (2 h/1 h) were performed.
Collection of Blood and Tissue Samples: At the end of the experiments, rats were sacrified by taking blood from intracardiac area. Blood samples were collected into polyethylene tubes and centrifuged (3000 x g for 15 min at 4 °C). The right gastrocnemius muscle was also harvested and immediately stored at –80 °C until further biochemical analysis. The remaining muscle samples were firstly fixed in a 10% formaldehyde solution for histopathological analysis and reached the histology laboratory for processing.
Biochemical Analysis
Preparation of Tissue Sample: The extracted gastrocnemius muscle was washed three times in cold isotonic saline. After drying the gastrocnemius muscle, tissues were then weighed and homogenized in a four volumes of ice-cold Tris-HCI buffer (50 mM, pH 7.4) using a homogenizer for 3 min at 5000 g.
Level of MDA and NO were measured from a part of the homogenate. The other part of the homogenate was centrifuged at 3500 x g for 30 min to obtain supernatant. The supernatant was used to determine CAT and GSH-Px activity. SOD activity was assessed in the ethanol phase of the supernatant after ethanol/chloroform mixture.
Tissue MDA concentration was measured by a method based on the ability of reaction with thiobarbituric acid (TBA) at 90 °C-100 °C 15.
Tissue nitrite (NO2-) and nitrate (NO3-) were assessed as an indicator of tissue NO level. The NO levels were measured using the Griess reaction assay 16, which is based on the spontaneous oxidation of NO to nitrite (NO2-), and subsequently to nitrate (NO3-).
Tissue CAT activity was measured according to the method of Aebi 17. The principle of the method was that the rate constant k of hydrogen peroxide decomposition determined at 240 nm. The absorbance changes per minute was measured, and then the rate constant (k) of the enzyme was detected.
Total SOD activity was determined in the tissue homogenates using the method described by Sun et al 18. This method is based on the inhibition of nitroblue tetrazolium (NBT) reduction by a superoxide generator system (xanthine-xanthine oxidase).
Tissue GSH-Px activity was measured spectrophotometrically by the method of Paglia and Valentine 19. The amount of protein in the samples was measured by the Lowry method 20.
Statistical Analysis: Statistical analysis of biochemical data was performed using commercial software (IBM SPSS Statistics 19, SPSS inc., an IBM Co., Somers, NY). Kolmogorov-Smirnov test was used to analyze the distribution of the parameters. When the data were not normally distributed, Kruskal Wallis analysis of variance was used. Mann-Whitney U-test with Bonferroni adjustment was used for interval data. When the continuous variables followed a normal distribution, parametric test was used to analyze the data. One-way analysis of variance (ANOVA) was performed. In the ANOVA test, for post hoc multiple comparison, the Least Significant Difference test was used. Results were expressed as mean±SD. P value of <0.05 was considered to be statistically significant.
Histopathological Analysis: The fixed gastrocnemius muscle samples were passed through an increasing alcohol series. They were embedded in paraffin following treatment with xylene and parafin. The parafin-embedded blocks were cut in 5-μn-thick sections, and the sections were stained with hematoxylin and eosin (H&E). The photomicrographs of the relevant stained sections were taken with the aid of a light microscope (Nikon Eclipse 200 Serial No:T1al 944909, Japan).
The following scores were used to grade the degree of histological damage observed in the gastrocnemius muscle tissue: no any structural damage (0), mild damage (1), moderate damage (2) and severe damage and degeneration (3). These scores were computed as the average damage scores for each group by finding out the average of scoring coefficients. All groups were evaluated by muscle fiber disorganization and inflammatory cell infiltration.
Immunohistochemical Analysis
Immunohistochemistry Staining Protocol: The principal aim of this analysis was to immunohistochemically investigate the detection of beta-actin protein (β-actin protein) in the muscle tissue. Sections from representative regions of the gastrocnemius muscle tissue were immunohistochemically labeled for β-actin protein, and were microscopically examined to determine the distribution of specific immunoreactivities generated by these antibodies. An immunohistochemistry staining protocol was used for detecting the presence of β-actin proteins. Sections of 5 μm were cut from parafin blocks and were placed onto polylysine-covered slides and incubated overnight at 56 °C. The tissue sections were dehydrated with a series of alcohol washes, placed in distilled water. Following this the sections were microwaved in 360 W microwave oven in citrate buffer, for 5 minutes twice and allowed to cool. The sections were then incubated for 10 min in 3% H2O2, washed (3x5 min) in phosphate-buffered saline (PBS). Sections were treated with a blocking serum and incubated overnight in a cold room with primary antibodies. The Mouse monoclonal beta Actin antibody (ab8226; Abcam) was used at a dilution 1:500. The sections were washed off (3x5 min; PBS), incubated overnight in biotinylated secondary antibodies for 30 min at room temperature. The slides were reacted with horse-radish peroxidase for 30 min, and treated with 3-amino-9-ethyl. The slides were reacted with horse-radish peroxidase (HRP) for 30 min, and treated with 3-amino-9-ethylcarbazole (AEC) chromogen. They were counter-stained with Mayer’s hematoxylin solution for 3-5 min. The tissues were washed thoroughly with PBS, rinsed with distilled water and covered using a water-based sealing solution. After the all protocol, photomicrophs were taken with a research microscope.
Scoring of Immunoreactivity: The results of immunohistochemical staining were assessed by semiquantitative scoring. The most common method used in the analysis of images is the semiquantitative scoring system in mouse models 21. For semiquantitative analysis of immunoreactivity of β-actin protein, the histological score (H-SCORE) (22) was used in this study. The intensity of immunoreactivity was evaluated using four intensity categories for each component: 0 (not present), 1+ (weak but detectable), 2+ (middle or distinct), and 3+ (intense). The H-SCORE is obtained by the formula: Σ Pi (i+1), where "i" represents the intensity of the stained cells. The score was obtained by 2 investigators after determining the areas of immunostained slides using a light microscope (x40 objective). The H-SCORE value for each tissue sample and the mean H-SCORE value for each group was calculated and tabulated.
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