2.9. Evans blue dye extravasations

Briefly, Evans blue (EB, 2% in PBS, 4 mL/kg), as BBB permeability tracer, was injected into the tail vein of the rats on third day after TBI. One hour after EB injection, the rat was perfused by 200 mL heparinized saline under deep anaesthesia. The brain tissue was then obtained, weighted and homogenized in formamide (1 mL) and incubated at 60°C overnight and then centrifuged (30 minute at 22 000 x g) to get the supernatant. Using a spectrophotometer (UV/VIS, Spectrometer, UK) to measure the optical density of the supernatant at 625 nm. The amount of extravasated EB dye (μg/g wet weight) was quantified according to a linear standard curve.