Figure 2: Progressive barrier leakage and delayed degradation of endothelial JPs after an ischaemia-like insult in an in vitro BBB model.

(a) Illustration of the in vitro BBB model. An HBMEC monolayer seeded on top of a membrane in the cell culture insert was subjected to 1 h of OGD. Paracellular permeability was determined by measuring the luminal to abluminal diffusion coefficient of a 4.4 kDa TRITC-dextran or a 70 kDa FITC-dextran. (b) The diffusion coefficient of the two fluorescent tracers at 0–6 h after OGD or control non-OGD conditions. Data represent four independent experiments. *P≤0.05, **P≤0.01 versus non-OGD. (c) The MMP inhibitor GM6001 (1 or 3 μM) or vehicle was applied 30 min before, during and after OGD. The diffusion coefficient of the two tracers was measured 0–6 h after OGD. Data represent four independent experiments. *P≤0.05, **P≤0.01 versus vehicle control. (d–g) Cultured HBMECs were subjected to 1-h OGD. (d,e) Expression of TJ proteins occludin, claudin-5 and ZO-1, as well as the AJ protein VE-cadherin was evaluated in HBMECs by western blotting 1–6 h after OGD. β-Actin was used as an internal loading control. Blots were quantified and expressed relative to non-OGD controls (Con). Data represent four independent experiments. *P≤0.05, **P≤0.01 versus Con. (f,g) Western blots showing that the delayed degradation of occludin and VE-cadherin at 4 and 6 h after OGD was prevented by MMP inhibition with GM6001 (3 μM). Data represent four independent experiments. *P≤0.05 versus vehicle.