For measurements of BBB permeability, extravasation of mouse IgG into brain parenchyma was assessed. Mice were perfused and brains removed. Coronal brain sections were cut at 25 μm and blocked in 5% bovine serum albumin for 1 h. Sections were immunostained with biotinylated horse anti-mouse IgG (1:500, Vector Laboratories, Burlingame, CA), followed by incubation in streptavidin/horseradish peroxidase reagents (Vectastain Elite ABC; Vector Laboratories) and then 0.5 mg/ml diaminobenzidine with 0.05% H2O2. Seven sections encompassing the MCA territory were quantified for cross-sectional area of IgG staining. These data were summed and multiplied by the distance between sections (1 mm) to yield volume in mm3.
As a second measurement of BBB integrity, the blood brain transfer coefficient (Ki) for fluorescein isothiocyanate (FITC)-albumin was measured as previously described [31 (link)]. FITC-albumin was injected into the femoral vein and arterial blood was collected at 5 min intervals for 20 min to measure FITC-albumin in plasma. Mice were then decapitated and hemispheres weighed. Brains were homogenized in 50 mmol/L Tris buffer and centrifuged at 3000 rpm for 30 min. Methanol was added to the supernatant at a ratio of 1:1 and the mixture centrifuged. The Ki was assessed according to the equation: Ki=(Cbr–VoCbt)/∫Cpl·dt, where Cbr is the concentration of FITC-albumin in brain at decapitation (ng/g), Cbl is the concentration of FITC-albumin (ng/mL) in the final blood sample, Vo is regional blood volume (mL/g), and ∫Cpl·dt is the integral of the arterial concentration of FITC-albumin over time t. Vo was determined in separate mice decapitated 1 minute after injection of FITC-albumin.
As a second measurement of BBB integrity, the blood brain transfer coefficient (Ki) for fluorescein isothiocyanate (FITC)-albumin was measured as previously described [31 (link)]. FITC-albumin was injected into the femoral vein and arterial blood was collected at 5 min intervals for 20 min to measure FITC-albumin in plasma. Mice were then decapitated and hemispheres weighed. Brains were homogenized in 50 mmol/L Tris buffer and centrifuged at 3000 rpm for 30 min. Methanol was added to the supernatant at a ratio of 1:1 and the mixture centrifuged. The Ki was assessed according to the equation: Ki=(Cbr–VoCbt)/∫Cpl·dt, where Cbr is the concentration of FITC-albumin in brain at decapitation (ng/g), Cbl is the concentration of FITC-albumin (ng/mL) in the final blood sample, Vo is regional blood volume (mL/g), and ∫Cpl·dt is the integral of the arterial concentration of FITC-albumin over time t. Vo was determined in separate mice decapitated 1 minute after injection of FITC-albumin.
