Quantitative assessment of the normal cerebral microvasculature by endothelial barrier antigen (EBA) immunohistochemistry: application to focal cerebral ischemia

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-sy stem endothelium. In this study, we assessed the sensitivity and specificit y of EBA as a quantitative marker of normal endothelium and characterized a lterations of EBA immunohistochemistry following focal cerebral ischemia. A nesesthetized, non-ischemic control rats (N=6) were studied. Other animals (N=5) received 90 min of middle cerebral artery occlusion (MCAo) followed b y 3-day survival. Brains were prepared by perfusion-fixation and paraffin-e mbedding. For EBA immunohistochemistry, a monoclonal antibody (1:2000 dilut ion) was used. Adjacent sections were reacted for activated microglia by is olectin immunochemistry. Morphometric image-analysis was carried out in sta ndardized 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-immunor eactive vascular profiles in infarcted cortex and striatum were reduced by 39 and 46%, respectively, and their fractional areas were decreased by 63 a nd 76%, respectively, compared to contralateral hemisphere. Activated micro glia were prominent in zones of frank infarction and in adjacent paramedian cortex; the latter region, however, showed normal-appearing EBA-immunostai ning. EBA-immunohistochemistry provides a sensitive and specific index of n ormal cerebrovascular endothelial structures of all sizes. The technique le nds 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.