E2129

Evans blue is widely used to assess the integrity of the BBB. After modeling, the mice were intravenously injected with 4% Evans blue dye (E2129, Sigma-Aldrich) at 100 μL/20 g via the tail vein, and the body quickly turned blue. The vital signs were carefully monitored for 30 min, and then the mice were sacrificed and transcardially perfused with cold PBS to flush away the blood and Evans Blue from the blood vessels. The ischemic lateral brain tissue was quickly removed and placed in a solution of 50% trichloroacetic acid in PBS (1.5 mL). The brains were homogenized, sonicated in trichloroacetic acid, and centrifuged at 12,000 rpm for 20 min. The supernatant was collected, and the fluorescence emission was measured at 680 nm (with an excitation wavelength of 620 nm) using an ultraviolet spectrophotometer.

EB extravasation and fluorescence were measured as reported previously [29 (link), 30 (link)]. After disinfection with iodophor, the mice were injected with 2% EB dye (5 ml/kg; E2129; Sigma‒Aldrich) through the tail vein. The EB dye was allowed to circulate in the mice for 2 h before sacrifice. Under continuous anesthesia with isoflurane (R510-22, RWD Life Sciences Ltd. China), the thorax was opened, and the heart was perfused with NS. The brain was immediately harvested, and the right hemisphere was isolated. Then, the tissue was weighed, and NS (400 μl) was added for homogenization. The samples were centrifuged at 15,000×g for 30 min. Next, an equal amount of 50% trichloroacetic acid was added to the supernatant. The samples were incubated overnight at 4 °C and centrifuged at 15,000×g for 30 min again. The content of EB in the supernatant was then quantified with a spectrophotometer (615 nm; Varioskanfla, Thermo Fisher Scientific) and a standard curve. To analyze EB fluorescence, the brain was harvested promptly after cardiac perfusion with NS and 4% paraformaldehyde, and frozen coronal brain sections (20 μm) were prepared and stored at -20 °C until use. After the sections were stained with 4’-6-diamidino-2-phenylindole (DAPI), EB fluorescence was observed using a confocal laser scanning microscope (mRFP 555; ZISS 880, Carl Zeiss Microscopy Ltd., England).

The permeability of the BBB was evaluated using extravasation of Evans blue dye (EB, E2129, Sigma-Aldrich; Exk = 620 nm, Emk = 680 nm) as previously reported with some modification [10 (link), 31 (link)]. In brief, 1 h after the intravenous injection of 2% EB (5 mL/kg), the mice were intracardially perfused with heparinized 0.9% saline to remove the intravascular dye. The brains were quickly harvested and separated into left and right hemispheres and weighted, homogenized in normal saline, and centrifuged. Then, 1 ml of supernatant was incubated with 50% trichloroacetic acid for 6 h. Then, the optical density of supernatants was measured at 620 nm using a multimode microplate reader (Molecular Devices, USA). The brain content of EB was expressed as the content of EB dye quantity in the brain quantity (μg/g).