These time points were chosen in order to estimate the impact of the treatment on acute/necrotic and late/apoptotic cell death (Fujikawa, 1996 and Weise et al., 2005). Rats were anaesthetized see more with chloral hydrate and transcardially perfused with a solution of paraformaldehyde (PF 4%) in phosphate buffer (PB 0.1 M). The brains were removed immediately after perfusion and post-fixed with a solution of PF 4% and sucrose
(30%) in PB 0.1 M. Fifty-micron coronal sections through the entire extension of the hippocampus were obtained using a cryostat (−18 °C), mounted on glass slides, and stained with cresyl violet (Nissl). The estimative of total number of neurons in the CA1 hippocampal sub-region and the hilus of the dentate gyrus was obtained in five animals per group using the stereological method optical fractionator (West et al., 1991). Briefly, every fifth section was selected, resulting in a section sampling fraction of 0.2 (ssf = 0.2). In each section, the hippocampal subfield CA1 and the hilus of the dentate gyrus were identified according to a brain atlas ( Paxinos and Watson, 1982). Disector counting probes (25 μm × 25 μm) were uniform and randomly distributed through
the hippocampus (right and left). Each disector correspond to an area (a) of 625 μm2 and the distance between counting frames (x,y step) was 250 μm, resulting in an area sampling fraction of 0.01 (asf = 0.01). Neuronal cell bodies (tops) were counted through the entire thickness of each section, resulting in a thickness sampling fraction of 1 (tsf = 1). Olaparib nmr The estimative of total neuronal cell number (N) for each region was calculated using the formula ( West et al., 1991): N=∑Q−⋅1ssf⋅1asf⋅1tsfwhere ΣQ− is the number of counted neurons, tsf is the thickness sampling fraction, asf is the area sampling fraction, and ssf is the
section sampling fraction. A pilot study showed that this sampling scheme produced acceptable coefficients of error (CE) and variance (CV) ( West et al., 1991 and Keuker et al., 2001). Caspase-1 and -3 activities isothipendyl were studied in five animals per group using the method described by Thornberry et al. (1997) and modified by Belizario et al. (2001). Rats were killed, hippocampi were dissected at 4 °C and added to 20 mM HEPES buffer (pH 7.4) that contained 2 mM EDTA, 0.1% CHAPS, 10% sucrose, 0.1% PMSF, 0.1% benzamidin, 0.1% antipain, 0.1% TLCK, 0.1% chemostatin and 0.1% pepstatin (5 μl homogenization buffer/mg tissue). Homogenates were obtained by mechanically disrupting the tissue three times on dry-ice, with thawing in an ice bath, interpolated by 1 min of moderate vortex shaking. Samples were centrifuged at 12,000 × g for 40 min at 4 °C to remove cellular debris. Total proteins were determined in the supernatants using the Bio-Rad Protein Assay (Bio-Rad Labs, Germany).