Quantifying Blood-Spinal Cord Barrier Disruption

Evans Blue (EB) dye, 961 Da, was used as a tracer for assessing BSCB disruption. The EB extravasation assay was performed as previously described^{51 (link)–53 (link)}. Briefly, after perfusion, mouse spinal cords were weighed and placed in 50% trichloroacetic acid solution (Sigma). Following homogenization and centrifugation, the supernatant was diluted with ethanol (1:3) and loaded into a 96 well-plate in triplicate. The dye was measured with a spectrofluorometer (Gemini EM Microplate Spectrofluorometer, Molecular Devices) at excitation of 620 nm and emission of 680 nm^{54 (link)}. Calculations were based on external standards in the same solvent. The EB content in tissue was quantified from a linear standard curve derived from known amounts of the dye and was normalized to tissue weight (μg/g). All measurements were performed by two experimenters blinded to the experiment.