Evans blue assay

BBB integrity was determined by Evans blue exclusion assay as described 10 w.p.i⁵⁹. Briefly, mice were injected intraperitoneally with a 2% solution of Evans blue (4 mL/kg body weight, catalog#E0197; TCI America, Portland, OR). The dye was allowed to circulate in the blood stream for 2 hours. Subsequently, mice were anaesthetized and perfused with 30 mL cold PBS through the left ventricle. After perfusion, mouse brains were collected, weighed, and homogenized in cold PBS (1:10 weight per volume). Brain homogenates were centrifuged for 25 minutes at 12,000 × g at 4 °C. Following this, an equal amount of 50% trichloroacetic acid was added to a 500 μl aliquot of brain homogenate supernatant. The samples were then incubated at 4 °C for overnight followed by centrifugation at 12,000 × g at 4 °C. The absorbance was measured at 610 nm using spectrophotometer (Spectramax M3 Multimode Microplate Reader; Molecular Devices, Sunnyvale, CA).