The integrity of BBB was evaluated with a modified Evans blue extravasation method before sacrifice [23 (link)]. Briefly, 70 h post-ICH, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and infused via the right femoral vein with 37 °C Evans blue dye (2% in 0.9% normal saline, 4 ml/kg) over 5 min. Two hours later, the rats were perfused with 300 ml normal saline to wash out any remaining dye in the blood vessels, and then the brains were removed and sectioned to 2 mm thickness with a rodent brain matrix. Coronal brain sections were taken starting at +2 mm and ending at −2 mm from bregma. BBB permeability was evaluated in the ipsilateral and contralateral striatum, and cerebellum. The cerebellum was used as an internal control. Each portion was weighed immediately and placed in 1 ml of 0.9% normal saline for homogenization of the sample. For protein precipitation, 1 ml of 60% trichloroacetic acid solution was added and vortexed for 2 min. Subsequently, the mixture was cooled for 30 min and centrifuged (1500 × g at 4 °C) for another 30 min. The absorbance of Evans blue in the supernatant was then measured with a spectrophotometer (Molecular Devices OptiMax, USA) at 610 nm. Dye concentration was expressed as μg/g of tissue weight and calculated from a standard curve obtained from known amounts of dye.
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