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

Baowan Lin, Myron Ginsberg

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

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 μm2. 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. Copyright (C) 2000 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)237-244
Number of pages8
JournalBrain Research
Volume865
Issue number2
DOIs
StatePublished - May 26 2000

Fingerprint

Microvessels
Brain Ischemia
Immunohistochemistry
Endothelium
Blood Vessels
Middle Cerebral Artery Infarction
Microglia
Infarction
Brain
Perfusion
Paraffin Embedding
rat endothelial barrier antigen
Immunochemistry
Peripheral Nervous System
Lectins
Paraffin
Central Nervous System
Monoclonal Antibodies
Cell Membrane
Staining and Labeling

Keywords

  • Brain
  • Endothelium
  • Middle cerebral artery occlusion
  • Morphometry
  • Rat

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

@article{e2e5c68b911044cf8ec533ad105f5b86,
title = "Quantitative assessment of the normal cerebral microvasculature by endothelial barrier antigen (EBA) immunohistochemistry: Application to focal cerebral ischemia",
abstract = "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 μm2. 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. Copyright (C) 2000 Elsevier Science B.V.",
keywords = "Brain, Endothelium, Middle cerebral artery occlusion, Morphometry, Rat",
author = "Baowan Lin and Myron Ginsberg",
year = "2000",
month = "5",
day = "26",
doi = "10.1016/S0006-8993(00)02228-9",
language = "English",
volume = "865",
pages = "237--244",
journal = "Brain Research",
issn = "0006-8993",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Quantitative assessment of the normal cerebral microvasculature by endothelial barrier antigen (EBA) immunohistochemistry

T2 - Application to focal cerebral ischemia

AU - Lin, Baowan

AU - Ginsberg, Myron

PY - 2000/5/26

Y1 - 2000/5/26

N2 - 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 μm2. 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. Copyright (C) 2000 Elsevier Science B.V.

AB - 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 μm2. 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. Copyright (C) 2000 Elsevier Science B.V.

KW - Brain

KW - Endothelium

KW - Middle cerebral artery occlusion

KW - Morphometry

KW - Rat

UR - http://www.scopus.com/inward/record.url?scp=0034716957&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034716957&partnerID=8YFLogxK

U2 - 10.1016/S0006-8993(00)02228-9

DO - 10.1016/S0006-8993(00)02228-9

M3 - Article

C2 - 10821926

AN - SCOPUS:0034716957

VL - 865

SP - 237

EP - 244

JO - Brain Research

JF - Brain Research

SN - 0006-8993

IS - 2

ER -