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-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 micromter(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.