Microglial activation protects ischemia-related blood-brain barrier dysfunction

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The question whether microglia activated in ischemia-related neural tissue damage initiates neuronal cell death by promoting excitotoxicity, or it can protect neurons against toxic insults is still under debate. In this study, the in vitro effect of microglia on the non-specific transport mechanisms of the blood-brain barrier (BBB) was investigated after hypoxia and reoxygenation. We constructed a heterogenous co-culture system, in which murine N9 microglia were cultivated on one surface of a porous membrane and GP8 cells on the opposite surface (the in vitro-BBB system). Cultures of GP8 rat brain endothelial cell line immortalized by a temperature-sensitive SV40 large T were used between passage 15 and 22. GP8 cells were plated on rat tail collagen- and fibronectin-coated 24-well plates or glass cover slips at a density of 10^5 cells/cm^2. After the passage, cells were removed to the in vitro-BBB system. To check the BBB function, after cells became confluent, we measured the electrical resistance across the cell monolayer. Cells were subjected to severe hypoxia for 1 to 6 h using Anaerocult A mini kits (Merck) at 37℃. Reoxygenation (30 min, 3 h, 18 h) was accomplished by removing the cells from the bags and changing hypoxic buffer to normoxic one. Hypoxia and reoxygenation resulted in time-dependent increase in Lucifer yellow uptake by brain endothelial cells. The most profound elevations (22-82% increase vs. Control) were seen after 3 h of posthypoxic reoxygenation. Concomitantly, an increase in LDH release was also seen. Co-culture with N9 microglial cells significantly decreased the non-specific transport under normoxic conditions. This effect of microglia was significantly blocked by serine protease antagonist (200 μM AEBSF) pretreatment.Thus, we obtained evidence supporting the idea that microglial activation prevents the in vitro hypoxia-reoxygenation induced increase in the BBB permeability.