Cerebral Microvessel Isolation

Cerebral microvessels were isolated from rat brains as previously described (Zhang L. et al., 2014). In brief, the left brain cortex was dissected, cleared of pia mater, homogenized by hand in a Dounce tissue grinder in 3 vol ice-cold PBS (0.01 mol/L, pH 7.4), and then centrifuged at 1,000 g for 10 min at 4°C. The supernatant of brain homogenates was stored at –80°C for further assays of inflammation status. The final pellet was filtered with a strong stream of cold PBS through a nylon mesh screen (50 mm). The microvessel fraction was removed from the screen, resuspended in PBS, and diluted to 2 mg protein/ml. Protein content was determined utilizing Lowry’s method (Lowry et al., 1951), and the homogenates were used for the assays of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin. Aliquots were separated for microvessel morphologic evaluations on dried smears fixed with 10% formaldehyde and stained with toluidine blue.