Quantitative Analysis of Microvascular Alterations in Traumatic Brain Injury by Endothelial Barrier Antigen Immunohistochemistry

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Abstract
Endothelial barrier antigen (EBA) is a protein triplet located in the plasma membrane of microvascular endothelium and selectively expressed in the normal nervous system. In this study, microvascular alterations following traumatic brain injury were studied using EBA immunohistochemistry. Anesthetized, physiologically regulated, normothermic Sprague-Dawley rats received moderate (1.5-2.0 atm) parieto-occipital parasagittal fluid-percussion traumatic brain injury (TBI). Control rats were subjected to similar anesthesia and physiological monitoring. Seven days after operative procedures, brains were perfusion-fixed, and coronal sections were reacted for EBA immunohistochemistry using a monoclonal antibody to rat EBA. Selected sections were reacted for isolectin B4 histochemistry. Computerized image analysis was used to compute numbers of EBA-immunopositive vascular profiles and mean vascular profile areas. In control brains, virtually all brain microvessels were clearly and positively immunostained, and antibody binding was specific for blood vessels. In rats with TBI, EBA immunoreactivity was greatly reduced in the zone of cortical contusion. Within the core contusion, fractional areas occupied by vascular profiles were markedly reduced (on average, by 57%), vascular profile counts were diminished, and lectin histochemistry revealed a robust inflammatory response with abundant macrophages. Taken together, these findings were thought to indicate frank microvascular destruction. At adjacent peri-contusional sites, the intensity of EBA immunostaining was also diminished; and vascular profile counts were reduced at adjacent cortical sites and homologous contralateral sites. The latter findings were interpreted as sublethal microvascular alterations possibly related to cerebral edema. The present results confirm that EBA is a specific immunohistochemical marker of normal central nervous system microvessels; that it is suitable for use in formaldehyde-fixed material; and that it is useful in quantitatively assessing microvascular alterations observed at contusional, peri-contusional and more remote sites following traumatic brain injury.