In order to evaluate BBB permeability, mice were injected with 2% Evans blue (EB, 3 ml/kg) (Sigma-Aldrich, St. Louis, MO, USA) via the tail vein at 24 h after TBI. One hour later, the mice were anesthetized with isoflurane and then perfused with phosphate-buffered saline (PBS) to remove the intravascular EB dye. The brains were obtained for imaging and EB content measurement. Briefly, the ipsilateral hemispheres were weighted and homogenized in 0.4 ml of formamide. The samples were incubated in a 37 °C water bath for 24 h. Then, samples were centrifuged at 10000 g at 4 °C for 10 min. The supernatant was collected and EB absorbance was measured by absorbance spectroscopy at 610 nm.

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