Sections were stained with DAB (Roche Diagnostics, Mannheim, Germany) and counterstained selleck chemical U0126 with mayer hematoxylin and analyzed by using a light microscope. The apoptotic index was defined as the number of apoptotic TUNEL-positive cells in 20 circular seminiferous tubule cross-sections per testis section. Each section was examined by two persons blind to the treatments and the average was taken [9].Immunohistochemistry procedure for active caspase-3 (AB3623, Millipore, Temecula, CA, USA, and Polyclonal antibody) was also performed. After deparaffinization and rehydration, sections were then treated with 10mM citrate buffer (Cat No. AP-9003-125 LabVision) (pH 6) in a microwave oven for 5minutes. Then sections were washed with PBS and incubated in a solution of 3% H2O2 for 5min at room temperature to inhibit endogenous peroxidase activity.

After washing with PBS sections were incubated with normal serum blocking solution at 37��C for 30min. Sections were again incubated in a humid chamber for 18h at +4��C with antibody active caspase-3 (1/100); thereafter with biotinylated IgG, and then with streptavidin conjugated to horseradish peroxidase at 37��C for 30min each prepared according to kit instructions (Invitrogen-Plus Broad Spectrum 85-9043). Sections were finally stained with DAB (Roche Diagnostics, Mannheim, Germany) and counter-stained with mayer hematoxylin and analyzed by using a light microscope. 2.5. Statistical AnalysisAll data were analyzed by Kruskal-Wallis test using SPSS 15.0 for Windows. Values are presented as mean �� SD.

Differences between the two groups were examined with Brefeldin_A the Mann-Whitney U-test. P < 0.05 is considered statistically significant.3. Results3.1. Biochemical AnalysisFigure 1 presents the GPx enzyme activities in testis tissue. When we analyzed GPx enzyme activities, we observed that I/R significantly decreased the GPx enzyme activities in the testis compared to control and sham groups (P < 0,005 and P < 0,003, resp.). However pretreatment with LA increased the GPx enzyme activities as compared to I/R group (P = 0,007). There was no significant difference observed between control and sham groups (Figure 1).Figure 1Effects of ischemia/reperfusion (2h torsion 720�� in a clockwise direction and 2h detorsion of the testis) and LA (100mg/kg ip, 30minutes prior to detorsion) on GPx levels of rat testes. Data are mean �� …Testis SOD enzyme activities decreased significantly in I and I/R groups when compared to control and sham groups (P < 0,05 and P < 0,01, resp.). LA pretreatment increased SOD enzyme activities significantly compared to I and I/R groups (P < 0,01 and P < 0,01, resp.). There was no significant difference observed between control and sham groups (Figure 2).