Immunohistochemistry of matrix metalloproteinases in reperfusion injury to rat brain: activation of MMP-9 linked to stromelysin-1 and microglia in cell cultures
Immunohistochemistry of matrix metalloproteinases in reperfusion injury to rat brain: activation of MMP-9 linked to stromelysin-1 and microglia in cell cultures
复制标题
DOI：
10.1016/s0006-8993(00)03294-7
复制
发表时间：
2001-03-02
期刊：
影响因子：
2.9
通讯作者：
Gearing, A
中科院分区：
文献类型：
--
作者：
Rosenberg, GA;Cunningham, LA;Gearing, A
再灌注损伤血脑屏障(BBB)。基质金属蛋白酶(MMPs)与血脑屏障的开放有关，但其细胞定位和激活机制尚不清楚。用免疫组织化学方法检测MMPs在再灌流大鼠脑内的细胞定位，并用细胞培养法研究其活性。自发性高血压大鼠(SHR)阻断大脑中动脉(MCAO)90min，再灌注3h~21d。冰冻切片进行明胶酶A(MMP2)、基质分解酶1(MMP3)和明胶酶B(MMP9)免疫组化染色。假手术对照组大鼠在血管旁的星形细胞突起可见基质金属蛋白酶-2免疫染色。再灌流3h后，星形胶质细胞基质金属蛋白酶-2免疫染色增强。24小时出现基质金属蛋白酶-3和基质金属蛋白酶-3免疫反应阳性。MMP3与激活的小胶质细胞(Ox-42+)和缺血神经元(Neun+)共定位。48h内皮细胞、中性粒细胞和神经元均可见基质金属蛋白酶-9免疫染色。在第5天和第21天，在缺血核心周围的反应性星形胶质细胞中可见较强的基质金属蛋白酶-2染色。在脂多糖(LPS)刺激的星形胶质细胞和小胶质细胞培养中对基质金属蛋白酶的激活进行研究。受刺激的星形胶质细胞产生一种激活形式的基质金属蛋白酶-2。当小胶质细胞受到刺激时，它们会激活基质金属蛋白酶-9。免疫组织化学染色显示富集小胶质细胞培养上清液中有基质金属蛋白酶-3的表达。在混合胶质细胞培养中，羟基型基质金属蛋白酶抑制剂BB-1101可阻断脂多糖对基质金属蛋白酶-2和基质金属蛋白酶-9的激活。我们认为基质金属蛋白酶-2在星形细胞的末端足中正常存在，并且在缺血期间产生基质金属蛋白酶-9和基质金属蛋白酶-3。小胶质细胞/巨噬细胞中的基质金属蛋白酶-3可能激活了基质金属蛋白酶原9。我们的结果表明，星形胶质细胞、小胶质细胞和血管内皮细胞对MMPs的差异表达参与了血脑屏障的蛋白分解破坏。(C)2001 Elsevier Science B.V.保留所有权利。
Reperfusion damages the blood-brain barrier (BBB). Matrix metalloproteinases (MMPs) are associated with the opening of the BBB, but their cellular localization and activation mechanisms are uncertain. We used immunohistochemistry to determine the cellular localization of the MMPs in reperfused rat brain, and cell cultures to study their activation. Spontaneously hypertensive rats (SHR) had a 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion for times from 3 h to 21 days. Frozen sections were immunostained with antibodies to gelatinase A (MMP-2), stromelysin-1 (MMP-3), and gelatinase B (MMP-9). Sham-operated control rats showed MMP-2 immunostaining in astrocytic processes next to blood vessels. After 3 h of the onset of reperfusion MMP-2 immunostaining increased in astrocytes. At 24 h immunoreactivity for MMP-3 and MMP-3 appeared. MMP-3 co-localized with activated microglia (Ox-42+) and ischemic neurons (NeuN+). MMP-9 immunostaining was seen at 48 h in endothelial cells, neutrophils, and neurons. At 5 and 21 days intense MMP-2 staining was seen in reactive astrocytes around the ischemic core. Studies of activation of the MMP were done in lipopolysaccharide (LPS)-stimulated astrocyte and microglia cultures. Stimulated astrocytes produced an activated form of MMP-2. When microglia were stimulated, they activated MMP-9. Immunostaining showed MMP-3 in cultures of enriched microglial cells. The hydroxymate-type, MMP inhibitor, BB-1101, blocked the activation of MMP-2 and MMP-9 by LPS in mixed glial cultures. We propose that MMP-2 is normally present in astrocytic end feet, and that during ischemia MMP-9 and MMP-3 are produced. MMP-3 in microglia/macrophages may be activating proMMP-9. Our results show that a differential expression of MMPs by astrocytes, microglia, and endothelial cells at the blood vessels is involved in the proteolytic disruption of the BBB. (C) 2001 Elsevier Science B.V. All rights reserved.