Evaluation of BBB leakage

BBB permeability of large molecules was assessed by the classical EB extravasation assay (19) and a fluorescent tracer assessment. EB (4%) (Sigma-Aldrich) was injected through the femoral vein at 1 hour before mice were sacrificed. Mice were perfused with 0.9% NaCl, and the brains were collected and separated into ipsilateral (MCAo) and contralateral (sham) hemispheres. Mouse brain tissues were homogenized in N,N-dimethylformamide (Sigma-Aldrich) and centrifuged at 25,000 relative centrifugal field for 45 min. The supernatants were collected to detect the absorption of EB in each tissue. EB levels were quantified by the following formula: (A_620nm − (A_500nm + A_740nm)/2)/mg W_Wet. The fluorescent tracer Alexa Fluor 555–conjugated cadaverine (Thermo Fisher Scientific, catalog no. A30677) was injected 30 min before mice were sacrificed. Brain sections were prepared as previously described, and images were acquired using an inverted Nikon Diaphot-300 fluorescence microscope equipped with a SPOT RT slider camera and Meta Series software 5.0 (Molecular Devices). Six sections encompassing the MCA territory were quantified for the cross-sectional area of cadaverine fluorescence. These sections were summarized and multiplied by the distance between sections (1 mm) to yield a leakage volume in cubic millimeters. To measure endogenous IgG leakage, adjacent sections from the same brains used for cadaverine leakage quantification were used. Sections were blocked with avidin followed by biotin and incubated with biotinylated anti-mouse IgG reagent (Vector Laboratories, catalog no. MKB-2225) overnight at 4°C. Sections were then incubated with streptavidin 488 (Jackson ImmunoResearch) at room temperature for 30 min. Images were acquired and quantified as described above.