Assessing Blood-Brain Barrier Permeability

To investigate the effect of CypA on blood–brain barrier function in vivo, CypA or its vehicle for negative control was injected intravenously once into mice (200 μg/kg), and the permeability of retinal vasculature was determined 3, 6 and 24 h after the injection of CypA as described previously^{5 (link)–7 (link)}. Briefly, 500 μl of PBS containing 100 μg/ml Hoechst stain H33258 (molecular mass, 534 Da; Sigma-Aldrich) and 1 mg/ml tetramethylrhodamine-conjugated lysine-fixable dextran (molecular mass, 10,000 Da; Thermo Fisher Scientific) were injected into the left ventricle. After the injection of fluorescent dyes, eyes were enucleated and immediately fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature under protection from light, and retinal flat mounts were prepared. They were mounted in fluorescent mounting medium and observed under a Zeiss LSM510 META laser confocal microscope (Carl Zeiss). Experiments were performed independently at least 3 times.