Histology and TUNEL staining assay

Rats were deeply anesthetized and sacrificed by intracardial perfusion with PBS and 4% ice-cold paraformaldehyde (pH 7.4). The brain was quickly removed, immersed in 30% sucrose solution for at least 48 h, and then cut into 6μm thick coronal sections. Apoptotic cell death was detected using a TUNEL staining Kit (Roche Applied Science, Nutley, NJ, USA). Briefly, brain sections were incubated with 3% hydrogen peroxide (10 min), permeabilization solution (2 min), and TUNEL reaction mixture (60 min) at 37°C. Cell nuclei were stained with DAPI (1lg/ml, Roche Applied Science). Cell counting was performed in the ipsilateral basal cortex under a fluorescent microscope (Olympus). The total number of cells (DAPI+) and the TUNEL-positive cells were counted in 10 separate fields in 5 different slices. The data were expressed as the number of TUNEL-positive neurons per total number of neurons.