Evans Blue Dye Extravasation for BBB Damage

The brain tissue Evans blue (EB) dye extravasation method was applied to determine the extent of blood-brain barrier (BBB) damage on the 3rd day after ICH. EB (E8010, Solarbio, Beijing, China) with a final concentration of 2% was administered intraperitoneally (4 ml/kg) in anesthetized mice and allowed to circulate in vivo for 3 h (Manaenko et al., 2011 (link)). After transcardiac perfusion with phosphate buffer saline (PBS, P1010, Solarbio, Beijing, China), the right hemisphere was collected and homogenized in a glass homogenizer with PBS (1,100 μL). The brain tissue homogenates were then sonicated and centrifuged (30 min, 15,000 g, 4°C). The resulting supernatant was collected and mixed with an equal amount of 50% trichloroacetic acid (T104261, Aladdin, Shanghai, China). After incubation overnight at 4°C, the mixture was centrifuged at 15,000 g for 30 min at 4°C. Finally, the absorbance of the supernatant at 610 nm was determined using a microplate reader. The extent of BBB damage in each group (vehicle, ATO, DXM, and ATO-DXM) was evaluated by comparing the experimental group to the OD₆₁₀ of the sham group.