56 × 105 M−1 cm−1. Reduced glutathione (GSH) GSH content was determined by the method of Jollow et al. (1974). Ten percent tissue homogenate was mixed with 4.0% sulphosalicylic acid (w/v) in a 1:1 ratio (v/v). The samples were incubated at 4°C for 1 h. The assay mixture contained 0.1 mL of supernatant, 1.0 mM DTNB, and 0.1 M PB (pH 7.4). The yellow #this website randurls[1|1|,|CHEM1|]# color developed was read immediately at 412 nm in a spectrophotometer (BioRad). The GSH content was calculated as Inhibitors,research,lifescience,medical nmol GSH mg−1 protein, using molar extinction coefficient of 1.36 × 103 M−1 cm−1. Myeloperoxidase (MPO) MPO was evaluated by the method of Bradley et al. (1982). A total of 0.1 mL of tissue homogenate was added to 1.45 mL of
o-dianisidine in 1 Inhibitors,research,lifescience,medical mL methyl alcohol, 98 mL 50 mM phosphate buffer (pH 6.0), and 1 mL of 0.05% H2O2 solution as a substrate for MPO enzyme. The change in absorbance was measured at 460 nm using BioRad spectrophotometer. One unit of MPO activity was defined as the quantity able to convert 1 μmol H2O2 per min at 25°C and was expressed as unit per gram tissue. TTC staining A 1.5-mm-thick cross-section of spinal cord tissue was rapidly cut after completion of hypoxia or hypoxia + treatment in various groups. The sections were stained by incubating them in
a solution of 2% of 2,3,5-triphenyltetrazolium chloride (TTC) at 37 ± 0.5°C for 15 min. For imaging, the Inhibitors,research,lifescience,medical sections were scanned by a high-resolution scanner (CanoScanLiDE25). The total mean infract area of each section was measured. Infarct size of the different groups was normalized to the sham and expressed as a percentage and an average value from Inhibitors,research,lifescience,medical the four slices was presented. Statistical analysis of data The statistical analysis of data was done using analysis of variance (ANOVA) with post hoc analysis. The Tukey–Kramer post hoc test was applied to serve as significant among groups. The significance of results was ascertained at P < 0.05. All the data are presented as
mean ± SE (n = 6) of the means. Results ATP quantitation Mitochondrial ATP content decreased significantly (P < 0.001) in hypoxic group Inhibitors,research,lifescience,medical by 30.64% when compared with sham group values. However, as a result of drugs treatment, a significant (P < 0.05–0.01) restoration was seen in ATP level in the hypoxic groups treated with FK-506 (FK-506 + Hypoxic) and CsA (CsA + Hypoxic) by 11.19% and 16.14%, whatever respectively, when compared to hypoxic group (Fig. 1). Figure 1 ATP content in various groups. Values are expressed as pmol ATP per mg protein (mean ± SE, n = 6). Significant difference §P < 0.001 when compared with sham; *P < 0.05, **P < 0.01 when compared with hypoxic group. … Calcium uptake in mitochondria Hypoxia/reperfusion resulted in significant mitochondrial swelling (P < 0.001) evident by a decrease in absorbance (90.9%) in the hypoxic group (Fig. 2). It was observed that FK-506 treatment at a concentration 0.1 μM caused a significant decrease (P < 0.