Fig. 4.

Removal of astrocytes abolishes the impact of OGD-treated neurons on occludin and claudin-5 protein levels in endothelial cells. Neuronal cells (CATH.a cells) grown on inserts were exposed or not to OGD for 3 hours and then co-cultured with astrocytes (C8-D1A cells) /or not for 24 hours. After that, the neurons were replaced by endothelial monolayer and incubated for another 24 hours before assessing occludin and claudin-5 protein levels by western blot. (A) Representative blots for occludin, claudin-5 and β-actin. The relative quantity of occludin protein (B) or claudin-5 protein (C) was calculated after normalization to β-actin. Data are expressed as mean ± SD, n = 3. *P < 0.05 vs W/Astro/N-Neu, ^#P < 0.05 vs W/Astro/OGD-Neu. W/Astro/N-Neu: astrocytes were first co-cultured with normal neurons and then with endothelial monolayer; W/N-Neu: incubating with normal neuron-conditioned media; W/Astro/OGD-Neu: astrocytes were first co-cultured with OGD-treated neurons and then with endothelial monolayer; W/OGD-Neu: incubating with conditioned media collected from OGD-treated neurons