Microglial activation protects ischemia-related blood-brain barrier dysfunction

小胶质细胞激活可保护缺血相关的血脑屏障功能障碍

• 批准号: 11670092
• 负责人: NIWA Masami
• 金额: $ 2.3万
• 依托单位: NAGASAKI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670092/
• 关键词: blood-brain barrier; microglia; ischemia-related neuronal death; brain capillary endothelial cell; ischemic reperfusion injury; astrocyte; セリンプロテアーゼ; 組織型プラスミノゲーン・アクチベーター

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.

小胶质细胞在缺血相关神经组织损伤中的激活是否通过促进兴奋性毒性而启动神经细胞死亡，或者它是否可以保护神经元免受毒性损伤，这一问题仍然存在争议。本研究探讨了小胶质细胞在体外低氧复氧后对血脑屏障(BBB)非特异性转运机制的影响。我们构建了一种异种共培养体系，将小鼠N9小胶质细胞培养在多孔膜的一侧表面，将GP8细胞培养在另一表面(体外-血脑屏障系统)。在第15代至第22代之间，使用温度敏感型SV40 Large T永生化的GP8大鼠脑内皮细胞系培养。将GP8细胞以10 5个/cm2的密度接种于大鼠尾胶原和纤维连接蛋白包被的24孔板或玻片上，传代后，将细胞移入体外血脑屏障系统。为了检查血脑屏障功能，在细胞融合后，我们测量了细胞单层的电阻。在37℃条件下，用A微型无氧试剂盒(MERK)将细胞置于严重缺氧状态下1~6h。复氧(30min、3h、18h)是通过从袋子中取出细胞并将低氧缓冲液改为常氧缓冲液来完成的。缺氧和复氧导致脑内皮细胞对荧光黄摄取的时间依赖性增加。缺氧后复氧3h后最明显的升高(与对照组相比增加22%-82%)。与此同时，乳酸脱氢酶的释放也有所增加。与N9小胶质细胞共培养可显著降低常氧条件下的非特异性转运。小胶质细胞的这种作用可被丝氨酸蛋白酶拮抗剂(200μM AEBSF)显著阻断。因此，我们获得了证据，支持小胶质细胞的激活阻止了体外缺氧-复氧引起的血脑屏障通透性的增加。

项目成果

Juraj Kopacek, Suehiro Sakaguchi, Kazuto Shigematsu, Noriyuki Nishida, Ryuichiro Atarashi, Ryota Nakaoke, Ryoozo Moriuchi, Masami Niwa and Shigeru Katamine: "Upregulation of the genes encoding lysozomal hydrolases, a perforin-like protein, and peroxides i

Juraj Kopacek、Suehiro Sakaguchi、Kazuto Shigematsu、Noriyuki Nishida、Ryuichiro Atarashi、Ryota Nakaoke、Ryoozo Moriuchi、Masami Niwa 和​​ Shigeru Katamine：“编码溶菌体水解酶、穿孔素样蛋白和过氧化物的基因上调

Yuji Nagayama: "Enhancement of efficacy of transcriptionally targeting suicide glne/producing therapy for Shysoid carcinoma with Cre-Ioxpoystem"Cancer Research. 59. 3049-3052 (1999)

Yuji Nagayama：“使用 Cre-Ioxpoystem 增强转录靶向自杀基因/产生针对鞘状癌的治疗效果”癌症研究。

Hirotomo Shibaguchi, Akihiko Himeno, Kazuto Shigematsu, Yasufumi Kataoka and Masami Niwa: "Transient hypoxia/hypoglycemia upregulates endothelin B receptors in rat cultured astrocytes"Glia. 31(1). 91-94 (2000)

Hirotomo Shibaguchi、Akihiko Himeno、Kazuto Shigematsu、Yasufumi Kataoka 和 Masami Niwa：“短暂缺氧/低血糖上调大鼠培养的星形胶质细胞中的内皮素 B 受体”神经胶质细胞。

Bela Kis, Csongor S.Abraham, Maria A.Deli, Hideyuki Kobayashi, Akihiko Wada, Masami Niwa, Hiroshi Yamashita, Yoichi Ueta: "Aderenomedullin in the cerebral circulation"Peptides. (in press). (2001)

Bela Kis、Csongor S.Abraham、Maria A.Deli、Hideyuki Kobayashi、Akihiko Wada、Masami Niwa、Hiroshi Yamashita、Yoichi Ueta：“大脑循环中的腺髓质蛋白”肽。

Maria A.Deli: "Pentosan in transmissible Spongiform encyhalopathies"Lancet. 353・9160. 1272 (1999)

Maria A.Deli：“传染性海绵状包膜病中的戊聚糖”《柳叶刀》353・9160。