Figure 4. Astrocyte-derived VEGF mediates HS-induced BBB breakdown. (A) Representative double immunofluorescence staining of ZO-1+ bEnd.3 endothelium. Endothelial cells were cultured alone, or co-cultured with primary rats′ astrocytes, and treated with NS, HS, conditioned medium, or VEGF neutralizing antibody (NA). (B, C) Western blotting analysis of Occludin, Claudin-5, and ZO-1 in endothelial cells. (D) Permeability of tight junctions measured using NaF; *P<0.05, **P<0.01, compared with EC+NS. At least three separate experiments were conducted; means ± SD.