Blood-spinal cord barrier leakage was assessed by EB extravasation (Li et al., 2014 (link); Zhou et al., 2017 (link)). EB solution (3%, 45 mg/kg, Sigma-Aldrich) was administrated through the femoral vein after anesthetization. One hour later, spinal cord samples of 1.0-cm long, where the injured site was centered, were obtained after transcardial perfusion with 0.1M PBS. The sample was homogenized in 3 mL 50% trichloroacetic acid and centrifuged at 12,000 × g at 4°C for 20 min. Next, 1 mL of the supernatant was mixed with 1 mL of ethanol and trichloroacetic acid solution (1:3) and incubated at 4°C overnight. The sample was then centrifuged at 15,000 × g at 4°C for 30 min. The supernatant was examined at an emission wavelength of 680 nm and excitation wavelength of 620 nm with a microplate reader (Bio-Tek Instruments, Winooski, VT, United States). The fluorescence value was converted to an EB content (μg) according to the standard curve. The researcher was blinded to the group assignments of the rats. Each test was conducted twice, and the average value was taken as the result. The results were expressed as the EB content (μg) in unit tissue (g).
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