Evaluating BBB Integrity via Evans Blue Assay

BBB integrity was assessed by measuring the leakage of Evans Blue (EB; Sigma-Aldrich Corp., St. Louis, MO, USA) as previously reported [41 (link)], with some modifications. Briefly, a 2% EB solution in physiological saline (4 mL·kg⁻¹ body weight) was injected at 4 or 24 h following reperfusion. The route of EB injection is tail vein injection. After the dye circulated for 1 h, the brain tissues were homogenized in 50% trichloroacetic acid (TCA) solution. The supernatant was diluted with a 3x volume of 100% ethanol and analyzed in a spectrofluorometer (Thermo Fisher Scientific, Waltham, MA, USA) at an excitation wavelength of 620 nm and an emission wavelength of 680 nm. The EB concentration was normalized to tissue weight (ng·g⁻¹).