Microglial activation and blood-brain barrier function in ischemia-related neuronal death

本研究探讨了血脑屏障功能和小胶质细胞活化在迟发性神经元死亡动物模型中的病理生理学意义，提出了血脑屏障损伤的小胶质细胞是由血脑屏障的主要成分脑毛细血管内皮细胞产生的单核细胞趋化蛋白L激活的假设，损伤的血脑屏障通过内皮素ET_B受体刺激小胶质细胞分泌血管内皮生长因子，血管内皮生长因子是血脑屏障修复因子。该定量受体成像系统包括三种实验技术，1)体外放射配基结合技术检测受体的功能部位，1^2^51-mCP-I和1^2^5I-hVEGF2)受体蛋白与抗MCP-1和抗hVEGF2抗体成熟部位的定量放射免疫组织化学技术，3)受体产生部位的原位杂交技术与mCP-L和血管内皮生长因子受体基因的^3^5S-cRNA探针。在短暂性前脑缺血后7天，即海马区CA1区锥体细胞层神经元死亡时，我们采用的定量受体放射自显影方法显示^1^2^5I-MCP-L结合位点的数目显著增加。新生结合部位出现在与锥体细胞层解剖相对应的区域，伴有神经元死亡和小胶质细胞聚集。闭塞后第2天和第5天无明显变化。在短暂性前脑缺血后7d，在CA I区毛细血管丰富的海马区可检测到新的1^2^5I-hVEGF结合位点。结合MCP-I和VEGF受体mRNA的表达，我们初步得出结论：BBB功能障碍发生在神经元死亡前，并与MCP-1激活小胶质细胞密切相关。

We studied the pathophysiological significance of the blood-brain barrier function and microglial activation on our animal model of delayed neuronal death, based on the hypothesis that microglia across the damaged BBB is activated by monocyte chemotactic protein (MICP)-l produced by brain capillary endothelial cells, a main composer of the BBB, and the damaged BBB stimulates microglia with endothelin ET_B receptor to secrete vascular endothelial growth factor (VEGF) , a repair factor for the BBB.To test the hypothesis, we used our newly-developed method for analyzing the receptor dynamics ; the quantitative receptor-imaging system with three experimental techniques, 1) in vitro radioligand-binding technique for functional sites of receptors with ^1^2^51-MCP-I and ^1^2^5I-hVEGF.2) quantitative radioimmunohistochemical technique for sites of receptor proteins maturation with anti-MCP-1 and anti-hVEGF antibodies, and 3) in situ hybridization technique for production sites of receptors with ^3^5S-cRNA probes of MCP-l and VEGF receptors genes. At 7 days after transient forebrain ischemia, a time when neuronal death in the hippocampus CAl pyramidal cell layer occurred, the quantitative receptor autoradiographic method we used revealed a dramatic increase in number of ^1^2^5I-MCP-l binding sites. The de novo binding sites appeared in the area corresponding anatomically to the pyramidal cell layer with neuronal death and microglial aggregation. No changes were observed at 2 and 5 days after occlusion. Also, the de novo ^1^2^5I-hVEGF binding sites were detected in the area with a rich capillary in the CA I hippocampus area at 7 days after a transient forebrain ischemia. Taken together with the expression of MCP- I and VEGF receptor mRNA, we tentatively conclude that a dysfunction in the BBB occurs just before neuronal death and is closely related to microglial activation due to MCP-1.