Protective effect of sildenafil citrate on contralateral testis injury Yıldız H et al. such as pulmonary hypertension, cardiovascular, spinal cord injury and type II diabetes).7 Several studies have indicated that sildenafil may exert a powerful protective effect against ischemia and reperfusion (I/R) injury in the testis in rat models.11,12 Furthermore, it has been shown that administration of sildenafil may also be useful against ischemic injury in other organs such as the liver, colon and brain.13–15 In the present study, we investigated the protective effect of sildenafil citrate on the contralateral testis after testicular I/R injury, and we discuss the mechanisms underlying the effects of sildenafil on these processes. MATERIAL AND METHODS The experimental protocols were approved by the local Animal Use Committees of Firat University (Elazig, Turkey) (No: 13/62 (03/09/2008). Animal care and experimental protocols complied with the NIH Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1985). This study was carried out at the Fırat University Experimental Medical Research Center. During the study period, rats were fed with a standard laboratory diet and allowed water. A total of 37 male Wistar Albino rats, weighing between 220 and 250 g were housed in a climate-controlled (relative humidity of 40– 60% and temperature of 21–24˚C) animal care facility, with a 12-h light/dark cycle. Before surgical procedures, the animals were provided with standard rat chow and water ad libitum. The rats were anesthetized with intramuscular ketamine (Ketalar; Eczacıbas¸ı, I˙stanbul, Turkey) at 85 mg/ kg body weight and xylazine (Rompun Vet; Bayer AG, I˙stanbul, Turkey) at 6 mg/kg body weight). Following anesthesia, the skin of the scrotal area was shaved and antisepsis was obtained using 10% povidone iodine solution. The rats were divided into four groups. A midline incision was made in the scrotum. In all the groups except for the sham-operated group, torsions were created by rotating the right testis 720˚ clockwise for 2 h. The torsion was maintained by fixing the testis in the scrotum with a 4-0 silk suture, and the incision was closed. After a 2-h torsion period, the suture was removed, and the right testis was then detorted and replaced in the scrotum for 2 h. In group 1 (sham-operated group, n = 7), the testes were taken out through the incision lines then replaced, and a silk suture was placed through the tunica albuginea. The rats were maintained in an ambient temperature of 24˚C. Group 2 (torsion/detorsion (T/D) + saline, n = 10) was used as the control group. Saline (2 ml of 0.9% NaCl) was injected intraperitoneally 1 h before the detorsion. In group 3 (T/D + 0.7 mg/kg sildenafil citrate group, low dose, n = 10), the treatment with intraperitoneal sildenafil citrate (Deva Holding A.S., Degra 50 mg), 2 ml from 0.7 mg/ kg dissolved in 0.9% NaCl, was carried out 1 h before the detorsion. In group 4 (T/D + 1.4 mg/kg sildenafil citrate group, high dose, n = 10), treatment with intraperitoneal sildenafil citrate, 2 ml from 1.4 mg/kg dissolved in 0.9% NaCl, was carried out 1 h before detorsion. The torsion lasted for 2 h followed by a detorsion period of 2 h, after which the rats were killed and bilateral orchiectomy performed. Contrala- teral testis in all groups were evaluated for the biochemical assay and histopathological examinations. 138 CLINICS 2011;66(1):137-142 Homogenate preparation Tissues were washed twice with cold saline solution, placed in glass bottles, labeled and stored in a deep freeze (–30˚) until processing (maximum 10 h). After weighing, the tissue (1 g) was placed on dry ice, cut into small pieces with scissors, and homogenized (2 min at 3000 6 g) in five volumes (1:5, w/v) of ice-cold Tris-HCl buffer (50 mM, pH 7.4), using a glass Teflon homogenizer ( C¸aliskan Cam Teknik, Ankara, Turkey). All the procedures for preparation were performed at 4˚C. After addition of butylhydroxyto- luol (4 ml per ml), the tissue homogenate samples were used for immediate NO, malondialdehyde (MDA), glulathione peroxidase (GPx), catalase (CAT) and GSH measurement. Biochemical assay Lipid peroxidation (as malondialdehyde) levels in tissue homogenate were measured with the thiobarbituric acid reaction by the method of Placer et al.16 The quantification of thiobarbituric acid-reactive substances was determined by comparing the absorption to the standard curve of malondialdehyde equivalents generated by acid-catalyzed hydrolysis of 1,1,3,3 tetramethoxypropane. The values of MDA were expressed as mmol/g protein. The GSH content of the tissue homogenate was measured at 412 nm using the method of Sedlak and Lindsay.17 The solution was kept at room temperature for 5 min, and then read at 412 nm on the spectrophotometer. Results were expressed as mmol/g protein. GSH-Px activities of the tissue were spectrophoto- metrically measured at 37˚C and 412 nm according to Lawrence and Burk.18 Results were expressed as mmol/g protein. The protein content in the tissue was measured by the method of Lowry et al.19 with bovine serum albumin as the standard. CAT activity was assayed in tissue homo- genate by the Aebi20 method. The principle of the assay is based on the determination of the rate constant (s – 1, k) for H2O2 decomposition at 240 nm using a spectrophotometer. Results were expressed as unit/g protein. The NO content of the tissue was determined according to method of Green et al.21 Results were expressed as mmol per gr protein. Histopathologic evaluation The testicular tissues from rats for histopathological examination were collected. The tissue samples were fixed in 10% neutral buffered formalin embedded in paraffin wax and cut into 5-mm sections and stained with hematoxylin and eosin. The tissues were examined under a light microscope and the microscopic scoring was graded on a scale of mild (+), moderate (++) and severe (+++). Spermatogenesis was assessed histopathologically using Johnsen’s mean testicular biopsy score (MTBS) criteria.22 All cross-sectioned tubules were evaluated systematically, and a score between 1 (very poor) and 10 (excellent) was given to each tubule according to Johnsen’s criteria. Light microscopy with an ocular micro- meter was used to measure mean values of seminiferous tubule diameter (MSTD) and germinal cell layer thicknesses (GCLT) and to evaluate the damage to testicular tissue. Twenty-five tubules were evaluated for each animal. Statistical analysis Differences between group means were estimated using a one-way analysis of variance (ANOVA) and the Duncan test was carried out for multiple comparisons using 11.0 for