Mvs10

Evan’s blue and fluorescent dye were used as measures for BBB permeability in mice. Evans blue (2% in saline; 4 mL/kg, Sigma, CA), or mixture of Texas Red (6 mg/Kg, Alfa Aesar, MA) and rhodamine-123 (6 mg/Kg, Life Technologies, CA) was intravenously administered through tail vein 30 minutes prior to euthanization. Animals were transcardially perfused with saline and brains were sectioned with a 2 mm brain matrix for Evan’s blue extravasation assay. Hemisphere samples were weighed, homogenized with 400 uL PBS, and precipitated with 50% trichloroacetic acid (Sigma, CA) overnight. All samples were centrifuged at 1000 rpm for 30 minutes to separate out the brain tissue in pellet prior to measuring. Absorption was quantified at 610 nm with a plate reader (BioTek, Winooski, VT). To quantify Evans blue in the brain, an Evans blue standardized curve was used. Results were quantified as microgram/gram brain tissue. For Rhodamine and Texas Red detection, all brains were frozen immediately in −80 °C isopentane. Leica CM30505 Cryostat was used to cut brains slices (20 µm). All brain sections were inspected for fluorescence under a microscope (Olympus MVS10, Japan). Every 5 sections between bregma = 0 mm and bregma = −2 mm were photographed for analysis of mean fluorescence intensity using ImageJ software.