Evans blue staining was applied to evaluate the blood–brain barrier integrity. 2% EB solution (8 mL/kg, Sigma–Aldrich) was intra-peritoneally injected after anesthetization. After 24 h, the rats received trans-cardiac perfusion with 0.1M PBS. Next, the brain was removed and homogenized in 50% trichloroacetic acid. The sample was incubated in a water bath (50°C) for 48 h and centrifuged at 15,000 × g for 30 min. Afterward, the supernatant was detected through spectro-fluorophotometry at 620 nm (Zhao et al., 2016 (link)).
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