Blood-brain barrier breakdown after embolic stroke in rats occurs without ultrastructural evidence for disrupting tight junctions

PLoS One. 2013;8(2):e56419. doi: 10.1371/journal.pone.0056419. Epub 2013 Feb 26.

Abstract

The term blood-brain barrier (BBB) relates to the ability of cerebral vessels to hold back hydrophilic and large molecules from entering the brain, thereby crucially contributing to brain homeostasis. In fact, experimental opening of endothelial tight junctions causes a breakdown of the BBB evidenced as for instance by albumin leakage. This and similar observations led to the conclusion that BBB breakdown is predominantly mediated by damage to tight junction complexes, but evidentiary ultrastructural data are rare. Since functional deficits of the BBB contribute to an increased risk of hemorrhagic transformation and brain edema after stroke, which both critically impact on the clinical outcome, we studied the mechanism of BBB breakdown using an embolic model of focal cerebral ischemia in Wistar rats to closely mimic the essential human pathophysiology. Ischemia-induced BBB breakdown was detected using intravenous injection of FITC-albumin and tight junctions in areas of FITC-albumin extravasation were subsequently studied using fluorescence and electron microscopy. Against our expectation, 25 hours after ischemia induction the morphology of tight junction complexes (identified ultrastructurally and using antibodies against the transcellular proteins occludin and claudin-5) appeared to be regularly maintained in regions where FITC-albumin massively leaked into the neuropil. Furthermore, occludin signals along pan-laminin-labeled vessels in the affected hemisphere equaled the non-affected contralateral side (ratio: 0.966 vs. 0.963; P = 0.500). Additional ultrastructural analyses at 5 and 25 h after ischemia induction clearly indicated FITC-albumin extravasation around vessels with intact tight junctions, while the endothelium exhibited enhanced transendothelial vesicle trafficking and signs of degeneration. Thus, BBB breakdown and leakage of FITC-albumin cannot be correlated with staining patterns for common tight junction proteins alone. Understanding the mechanisms causing functional endothelial alterations and endothelial damage is likely to provide novel protective targets in stroke.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood Vessels / metabolism
  • Blood-Brain Barrier / metabolism
  • Blood-Brain Barrier / pathology*
  • Blood-Brain Barrier / ultrastructure
  • Brain Infarction / pathology
  • Brain Ischemia / pathology
  • Endothelium, Vascular / pathology
  • Endothelium, Vascular / ultrastructure
  • Male
  • Occludin / metabolism
  • Permeability
  • Rats
  • Stroke / metabolism
  • Stroke / pathology*
  • Tight Junction Proteins / metabolism
  • Tight Junctions / ultrastructure*
  • Time Factors

Substances

  • Occludin
  • Tight Junction Proteins

Grant support

Funding was provided for initial experiments by Prof. Dietmar Schneider and Dr. Carsten Hobohm (Department of Neurology, University of Leipzig) and then by the DFG (FOR1336) to Prof. Ingo Bechmann. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.