Assessment of Evans blue extravasation

Blood–brain barrier (BBB) disruption was evaluated using Evans blue dye (Sigma Aldrich, St Louis, MO, USA) extravasation. Briefly, at 24 h post-CCI, Evans blue dye (2 %, 4 mL/kg) was injected and administered over 2 min into the left internal jugular vein, after which it was allowed to circulate for 2 h. Under anesthesia, mice were perfused with phosphate-buffered saline through the left ventricle, then the brains were removed and divided into two hemispheres to evaluate the dye extravasation. Each sample was immediately weighed, homogenized in 1 mL 50 % trichloroacetic acid solution, and centrifuged at 15,000g for 30 min. Then, the supernatant was diluted to 1:3 with ethanol, and its absorbance was determined at 610 nm using a spectrophotometer (BioTek, Winooski, VT, USA). The amount of Evans blue dye was calculated using a standard curve and expressed as micrograms per gram of brain tissue.