Quantitative assessment of the normal cerebral microvasculature by endothelial barrier antigen (EBA) immunohistochemistry: application to focal cerebral ischemia
Quantitative assessment of the normal cerebral microvasculature by endothelial barrier antigen (EBA) immunohistochemistry: application to focal cerebral ischemia
复制标题
DOI：
10.1016/s0006-8993(00)02228-9
复制
发表时间：
2000-05-26
期刊：
影响因子：
2.9
通讯作者：
Ginsberg, MD
中科院分区：
文献类型：
--
作者：
Lin, BW;Ginsberg, MD
脑血管内皮在缺血性损伤的病理生理学中发挥重要作用。内皮屏障抗原（EBA）是一种位于正常中枢和周围神经系统内皮细胞腔质膜中的蛋白质。在本研究中，我们评估了 EBA 作为正常内皮定量标志物的敏感性和特异性，并表征了局灶性脑缺血后 EBA 免疫组织化学的变化。对麻醉的非缺血对照大鼠（N=6）进行了研究。其他动物 (N=5) 接受 90 分钟的大脑中动脉闭塞 (MCAo)，然后存活 3 天。通过灌注固定和石蜡包埋制备脑。对于 EBA 免疫组织化学，使用单克隆抗体（1:2000 稀释）。通过同工凝集素免疫化学对相邻切片进行活化的小胶质细胞反应。形态测量图像分析是在标准化显微视野中进行的。在对照大脑中，各种大小的软脑膜和实质血管均被明显且选择性地免疫标记为 EBA；不存在背景染色。 EBA 阳性血管分布占显微镜视野的 4.3 +/- 0.36%（平均值+/-S.D.）。每个识别轮廓的平均面积为 51 +/- 13 μm(2)。剖面数量 (17%) 和分数面积 (8%) 的低变异系数表明动物间的高度一致性。在患有 MCAo 的大脑中，与对侧半球相比，梗塞皮层和纹状体中 EBA 免疫反应性血管分布的数量分别减少了 39% 和 46%，其分数面积分别减少了 63% 和 76%。活化的小胶质细胞在明显梗塞区域和邻近的旁正中皮层中很明显；然而，后一个区域显示出正常的 EBA 免疫染色。 EBA-免疫组织化学提供了各种大小的正常脑血管内皮结构的敏感且特异的指数。该技术非常适合定量形态测量，并且适用于灌注固定的石蜡包埋材料。缺血性梗塞区域的 EBA 免疫反应性下降。 (C) 2000 Elsevier Science B.V. 保留所有权利。
Cerebrovascular endothelium participates importantly in the pathophysiology of ischemic injury. Endothelial barrier antigen (EBA) is a protein located in the luminal plasma membrane of normal central and peripheral nervous-system endothelium. In this study, we assessed the sensitivity and specificity of EBA as a quantitative marker of normal endothelium and characterized alterations of EBA immunohistochemistry following focal cerebral ischemia. Anesesthetized, non-ischemic control rats (N=6) were studied. Other animals (N=5) received 90 min of middle cerebral artery occlusion (MCAo) followed by 3-day survival. Brains were prepared by perfusion-fixation and paraffin-embedding. For EBA immunohistochemistry, a monoclonal antibody (1:2000 dilution) was used. Adjacent sections were reacted for activated microglia by isolectin immunochemistry. Morphometric image-analysis was carried out in standardized microscopic fields. In control brains, pial and parenchymal blood vessels of all sizes were distinctly and selectively immunolabeled for EBA; background staining was absent. EBA-positive vascular profiles occupied 4.3 +/- 0.36% (mean+/-S.D.) of the microscopic field. The mean area of each identified profile was 51 +/- 13 mu m(2). The low coefficients of variation for both numbers of profiles (17%) and fractional areas (8%) denoted high inter-animal consistency. In brains with prior MCAo, numbers of EBA-immunoreactive vascular profiles in infarcted cortex and striatum were reduced by 39 and 46%, respectively, and their fractional areas were decreased by 63 and 76%, respectively, compared to contralateral hemisphere. Activated microglia were prominent in zones of frank infarction and in adjacent paramedian cortex; the latter region, however, showed normal-appearing EBA-immunostaining. EBA-immunohistochemistry provides a sensitive and specific index of normal cerebrovascular endothelial structures of all sizes. The technique lends itself well to quantitative morphometry and is applicable to perfusion-fixed paraffin-embedded material. EBA immunoreactivity declines in zones of ischemic infarction. (C) 2000 Elsevier Science B.V. All rights reserved.