Male Wistar-Albino rats weighing 250-300 g were used in this study. All animals were housed in a light-controlled room with a 12 hours light/dark cycle and were allowed free access to food and water. Animals were housed in the animal facility for at least 7 day prior to use to stabilize their intestinal flora. The operative procedure, use of anesthesia, and animal care methods in the experiments were consistent with the National Institutes of Health Guidelines on the Care and Use of Laboratory Animals (NIH publication No.86-23, revised 1985, Bethesda, MD) and also approved by the Experimental Animal Committee of Pamukkale University School of Medicine.
Operative details
After fasting overnight, the rats were anesthetized by an intramuscular injection of ketamine 50 mg/kg (Ketalar; Parke Davis, Eczacibasi, Istanbul, Turkey) and xylazine 10 mg/kg (Rompun; Bayer AG, Leverkusen, Germany). Animals were allowed to breathe spontaneously during the surgery. A heating lamp was used to preserve the body temperature at approximately 37°C. At the end of the operation, 10 ml Ringer’s Lactated solution was administered subcutaneously, to prevent dehydration of the rats. The abdomen was shaved and twice soaked with 10% povidone-iodine solution before rats were aseptically operated using sterile instruments. After a midline laparotomy, the superior mesenteric artery (SMA) was exposed. At this stage of the experiment, rats were randomly divided into three groups, each having 10 animals. In group I (sham operated group), the superior mesenteric artery (SMA) was isolated but not occluded. In group II (intestinal I/R only group) and group III (intestinal I/R + melatonin treated group), the SMA was gently isolated, and occluded immediately distal to the aorta with collateral interruption for 60 minutes with atraumatic microvascular clamps as described elsewhere [10]. This procedure provided ischemia in small intestine, cecum and right colon which was confirmed when the mesenteric pulsation was lost and the intestines became pale. After 60 minutes of ischemia, the clamp was removed and the reperfusion period began. In group III, 30 minutes before the start of reperfusion, 10 mg/kg melatonin was administered intraperitonally. Subsequently, the abdominal incisions were closed in two layers with 3/0 polyglactin suture (Vicryl, Ethicon, UK). Animals were fed with standard rat chow and water postoperatively. All animals were anesthetized and euthanized 24 hours after reperfusion. Tissue samples were collected to evaluate the I/R-induced intestinal injury and bacterial translocation (BT). Using sterile technique and instruments, a midline laparotomy was performed to yield biopsies of liver, spleen, mesenteric lymph nodes, terminal ileum for quantitative culture of aerobic and anaerobic organisms. A 1 ml sample of blood from vena cava was taken and cultured in appropriate media for aerobic and anaerobic organisms. The segments of ileum were removed and frozen in liquid nitrogen and stored at –80°C for further biochemical analysis of lipid peroxidation, myeloperoxidase activity, and tissue glutathione (GSH) levels, and were also fixed in 10% formaldehyde solution for further histo-pathological examination.
Microbiological analysis
Microbiological analysis was performed as described previously [11]. Blood (0.5 mL) samples were cultured in 5 mL of brain heart infusion broth for 7 days at 37°C. The cultures were inspected daily and subcultured on blood agar and eosine methylene blue (EMB) agar plates. Samples of mesenteric lymph nodes, liver, spleen and ileal contents were weighed and placed in a sterile grinding tube. Tissues were homogenized in 1 mL of saline for quantitative cultures. This preparation was plated on blood agar and EMB agar and was incubated at 37°C for 24-48 h in ambient air. The identification of bacterial species was performed by standard microbiologic methods. Colonization was expressed as the number of colony-forming units (CFU) per gram of tissue homogenate (CFU/g).
Histopathological examinations
The sections of ileum embedded in Paraffin and they were stained with hematoxylin and eosin (H & E). A pathologist blinded to the experimental groups examined these sections under a light microscope. Intestinal mucosal lesions were graded on a scale from 0 to 5 as described by Chiu et al. [12]: grade 0, normal mucosal villi; grade 1, development of subepithelial Gruenhagen’s space, usually at the apex of the villus, often with capillary congestion; grade 2, extension of the subepithelial space with moderate lifting of the epithelial layer from the lamina propria; grade 3, massive epithelial lifting with a few denuded villi; grade 4, denuded villi with exposed dilated capillaries; and grade 5, digestion and disintegration of lamina propria, hemorrhage, and ulceration.
Estimation of neutrophil accumulation
The levels of myeloperoxidase (MPO) activity in the segment of ileum were determined as an indicator of neutrophil accumalation. Tissues were homogenized in 50 mM phosphate buffer (pH 7.4) (1/10, w/v) containing protease inhibitor, 0.2 μM phenylmethanesulphonyl fluoride (PMSF) and 1 mM Ethylenediamin tetra acetic acid (EDTA), at 4°C for 30 s using a homogenizer (Potter S, B. Braun, Germany). Then, appropriate volume of homogenate was used MPO determination.
The method of Suzuki et al. was used with a slight modification [13]. This method is based on the oxidation of the synthetic substrate 3,3’,5,5’-tetramethyl benzidine (TMB) by MPO. The standard reaction mixture consisted of 500 μl detergent-containing buffer (160 mM potassium phosphate buffer, pH 5.4, 1% hexadecyltrimethylammonium bromide), 100 μl TMB (16 mM, dissolved in dimethylformamid), 50 μl homogenate and 300 μl water. The reaction was started by the addition of 50 μl H2O2 (diluted 0.003%) at 37°C. The rate of MPO-catalyzed oxidation of TMB was followed by recording the increase in absorbance at 655 nm. Considering the initial and linear phase of the reaction, we measured the absorbance change per minute, and one enzyme unit was defined as the amount of enzyme producing one absorbance change per minute under assay conditions. Enzyme activity was calculated as units per gram of wet weight tissue.
Determination of lipid peroxidation
The levels of malondialdehyde (MDA) in ileal tissue specimens were determined as an indicator of lipid peroxidation. The MDA production and hence lipid peroxidation were assessed in the tissues by the method of Ohkowa. MDA forms a colored complex in the presence of TBA, which is detectable by measurement of absorbance at 532 nm. Absorbance was measured with Shimadzu UV-160 spectrophotometer. 1,1′,3,3′ -Tetraethoxypropane was used as a standard and the results were expressed in tissue as μmol/g protein [14].
Determination of glutathione
The concentrations of glutathione (GSH) in tissue samples were measured by the method of Ellman [15]. One ml tissue homogenate precipitated by 2 ml of 5% TCA was taken and 0.5 ml of Ellman’s reagent (0.0198% DTNB in 1% sodium citrate) and 3 ml of phosphate buffer (pH 8.0) were added. The color developed was read at 412 nm. The concentration was expressed in mg/g protein in tissue samples.
Statistics
The results were expressed as mean ± SEM. Statistical evaluation for proportional comparisons for positive cultures of tissues was made with Chi-square (Fisher’s Exact test) analysis. Comparisons for quantitative culture and ileal MDA, MPO, GSH levels were analyzed with Kruskal-Wallis test, and multiple comparisons between the groups were performed with Mann Whitney-U test. Differences were considered statistically significant when P <0.05. Data were analyzed by statistical software (SPSS for Windows 11.5; SPSS, Chicago, Illinois).
