2.12. In vitro BBB model and permeability assay

In vitro BBB models were established to measure permeability, as previously described [51]. Transwell inserts (pore size 0.4 µm, Falcon, Corning, NY) were placed into 24-well plates to divide each well into top (luminal) and bottom (abluminal) compartments. This apparatus allows for free passage of nutrients while cellular trafficking across the compartments is blocked. After OGD treatment, FITC-dextran (4 kDa, Sigma-Aldrich, St. Louis, MO) was added into the top compartment at a final concentration of 0.05 mg/mL for the indicated durations, and fluorescence intensity was measured in an aliquot of 50 µL media from the bottom compartment. For the monoculture model, MBMECs were plated into the inserts and incubated until confluence was achieved. For the co-culture model, inserts were reversed, and astrocytes were plated onto the reversed surface for 20 min in the incubator, which is sufficient for astrocyte adherence. Next, inserts were placed into the 24-well plates in the presence of culture media. MBMECs were then plated into the insert.