Measurement of BBB Permeability

Blood–brain barrier permeability was assessed using sodium fluorescein (NaF) as a tracer molecule according to a previously described technique (Phares et al., 2007; Luo et al., 2018). Mice were administered intravenously with 100 μl of 10% NaF in phosphate-buffered saline (PBS), and after 10 min to allow circulation of NaF, peripheral blood was collected, and the mice were transcardially perfused with PBS after anesthesia. Cerebrum and cerebellum were separated after brain tissues were harvested. The serum was mixed with an equal volume of 15% trichloroacetic acid (TCA) and was centrifuged at 10,000 rpm for 10 min; the supernatant was then collected and mixed with 5 M NaOH to 150 μl. Homogenized brain samples in 7.5% TCA were centrifuged at 10,000 rpm for 10 min, and the harvested supernatant was mixed with 5 M NaOH to 150 μl. The fluorescein of brain and serum samples was measured using BioTek spectrophotometers (BioTek, Winooski, VT, United States) with excitation at 485 and emission at 530 nm. NaF uptake into brain tissue was regarded as the BBB permeability, which was normalized with fluorescence from peripheral blood; the result was expressed as (μg fluorescence brain tissue/mg tissue)/(μg fluorescence sera/ml blood).