Measuring Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) permeability was measured by the extravasation of Evans Blue dye on days 1 and 3 post-ICH [22 (link)]. In brief, a 2% solution of Evans Blue in sterile saline (4 ml/kg of body weight, Servicebio, China) was injected into the tail vein 3 h before mice were sacrificed. Then, mice were transcardially perfused with 40 ml of 0.9% cold saline. Afterward, brain tissues were quickly removed, weighed, and homogenized in 1100 μl PBS. After sufficient grounding, the samples were centrifuged at 6000×g for 30 min. The supernatant was collected and mixed with an equal amount of 50% trichloroacetic acid (Sigma-Aldrich, St Louis, MO). Samples were incubated overnight at 4 °C and centrifuged for 30 min (6000×g, 4 °C). Evans Blue dye were measured by a spectrophotometer (Thermo Spectronic Genesys 10 UV, Thermo Fischer Scientific Inc., Waltham, MA, USA) at 610 nm and quantified from a standard curve. The results are presented as (microgram of Evans Blue dye)/(gram of tissue).