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.
Highlights
Ischemic stroke still represents one of the leading causes of death worldwide [1]
To investigate the fate of endothelial tight junctions in regions of ischemia-related blood-brain barrier (BBB) breakdown, we focused on areas exhibiting fluorescein isothiocyanate (FITC)labeled albumin (FITC-albumin) extravasation
According to our previous study [46], the applied model of embolic middle cerebral artery occlusion (eMCAO) resulted in alterations of the vascular architecture with a clear extravasation of FITC-albumin in respective areas, primarily located in the right striatum
Summary
Ischemic stroke still represents one of the leading causes of death worldwide [1]. On the cellular level, the ischemia-induced breakdown of the blood-brain barrier (BBB), which is known to contribute to an increased risk of hemorrhagic transformation and brain edema with perilous outcome, was often linked to a putative disruption of tight junctions in the endothelial layer of cerebral vessels [2]. In 1900, Max Lewandowski drew the revolutionary conclusion that the morphological correlate of this barrier function must be the capillary wall [4], which was further specified by the groundbreaking paper from Reese and Karnovsky in 1967, who linked this property to belts of endothelial tight junctions in cerebral capillaries [5] These complexes consist of three major transmembrane protein families comprising occludin, claudins and junction associated proteins (JAMs), as well as several cytoplasmic proteins including the zonula occludens (ZO) protein family (ZO-1, ZO-2 and ZO-3), providing the link to the actin cytoskeleton [2,6,7]. Whereas this general composition is known for epithelial tight junctions of peripheral organs, some proteins were found to be specific for brain endothelial cells, such as cingulin, claudin-3 and -5 [8,9]
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