Cell apoptosis assay

Cell death of nerve cells in the periventricular cerebral mass, corpus callosum region, SVZ and hippocampal dentate gyrus brain tissue was assessed using the immunofluorescent terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) in the ApopTag Fluorescein In Situ Apoptosis Detection kit (cat. no. S7110; Chemicon; Merck Millipore, Temecula, CA, USA) according to the manufacturer's instructions. The nucleus presenting yellow or light yellow bulk or appearing granular under the light microscope was considered as TUNEL-positive. A blinded evaluator counted the number of TUNEL-positive nuclei in the brain tissue samples from the periventricular cerebral mass, corpus callosum region, SVZ and hippocampal dentate gyrus. A total of 10 random non-overlapping fields of view from each brain were counted.