Histological assessment of hippocampal damage

Histological assessment of hippocampal damage was performed in a blinded manner as described previously⁸. Rats were perfusion-fixed with 0.9% saline and 4% paraformaldehyde under anesthesia after global ischemia followed by 5 d of reperfusion. Brains were removed and further fixed with the same fixation solution at 4 °C overnight. Postfixed brains were embedded with paraffin and then coronal sections (6 μm thick) were prepared using a microtome (Leica, Wetzlar, Germany). The paraffin-embedded brain sections were deparaffinized with xylene and rehydrated in a graded concentration of ethanol, followed by washing with distilled water. The sections were stained with 0.1% Cresyl Violet (Sigma-Aldrich) to assess the neuronal survival in the hippocampus. The numbers of surviving hippocampal CA1 neurons per 1 mm length were counted as the neuronal density.