Microglial activation protects ischemia-related blood-brain barrier dysfunction

在缺血相关的神经组织损伤中激活的小胶质细胞是否通过促进兴奋性毒性而启动神经元细胞死亡，或者它是否可以保护神经元免受毒性损伤的问题仍在争论中。本研究探讨了小胶质细胞在体外对缺氧复氧后血脑屏障（BBB）非特异性转运机制的影响。我们构建了一个异源共培养系统，其中小鼠N9小胶质细胞在多孔膜的一个表面上培养，GP 8细胞在相反表面上培养（体外BBB系统）。在第15代和第22代之间使用通过温度敏感性SV 40大T永生化的GP 8大鼠脑内皮细胞系的培养物。将GP 8细胞以10^5个细胞/cm ^2的密度接种在鼠尾胶原和纤连蛋白包被的24孔板或盖玻片上。传代后，将细胞移至体外BBB系统。为了检查BBB功能，在细胞变得汇合后，我们测量了穿过细胞单层的电阻。使用Anaerocult A mini试剂盒（Merck）在37℃下对细胞进行重度缺氧1至6 h。通过从袋中取出细胞并将低氧缓冲液更换为常氧缓冲液来完成复氧（30 min、3 h、18 h）。缺氧和复氧导致脑内皮细胞对荧光黄的摄取呈时间依赖性增加。最显著的升高（与对照组相比增加22-82%）出现在缺氧后再氧合3小时后。同时，还观察到LDH释放增加。在常氧条件下，与N9小胶质细胞共培养显著降低了非特异性转运。丝氨酸蛋白酶拮抗剂（200 μM AEBSF）预处理可显著阻断小胶质细胞的这种作用，从而证实了小胶质细胞的活化可阻止体外缺氧-复氧诱导的血脑屏障通透性增加。

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.