Moderate Hypoxia Followed by Reoxygenation Results in Blood-Brain Barrier Breakdown via Oxidative Stress-Dependent Tight-Junction Protein Disruption

Abstract

Re-canalization of cerebral vessels in ischemic stroke is pivotal to rescue dysfunctional brain areas that are exposed to moderate hypoxia within the penumbra from irreversible cell death. Goal of the present study was to evaluate the effect of moderate hypoxia followed by reoxygenation (MHR) on the evolution of reactive oxygen species (ROS) and blood-brain barrier (BBB) integrity in brain endothelial cells (BEC). BBB integrity was assessed in BEC in vitro and in microvessels of the guinea pig whole brain in situ preparation. Probes were exposed to MHR (2 hours 67-70 mmHg O2, 3 hours reoxygenation, BEC) or towards occlusion of the arteria cerebri media (MCAO) with or without subsequent reperfusion in the whole brain preparation. In vitro BBB integrity was evaluated using trans-endothelial electrical resistance (TEER) and transwell permeability assays. ROS in BEC were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (DCF), MitoSox and immunostaining for nitrotyrosine. Tight-junction protein (TJ) integrity in BEC, stainings for nitrotyrosine and FITC-albumin extravasation in the guinea pig brain preparation were assessed by confocal microscopy. Diphenyleneiodonium (DPI) was used to investigate NADPH oxidase dependent ROS evolution and its effect on BBB parameters in BEC. MHR impaired TJ proteins zonula occludens 1 (ZO-1) and claudin 5 (Cl5), decreased TEER, and significantly increased cytosolic ROS in BEC. These events were blocked by the NADPH oxidase inhibitor DPI. MCAO with or without subsequent reoxygenation resulted in extravasation of FITC-albumin and ROS generation in the penumbra region of the guinea pig brain preparation and confirmed BBB damage. BEC integrity may be impaired through ROS in MHR on the level of TJ and the BBB is also functionally impaired in moderate hypoxic conditions followed by reperfusion in a complex guinea pig brain preparation. These findings suggest that the BBB is susceptible towards MHR and that ROS play a key role in this process.

Cytokine-mediated dysregulation of zonula occludens-1 properties in human brain microvascular endothelium

“…Moreover, our data confirm for the first time that the cytokine-dependent activation of NADPH oxidase leading to ROS generation contributes in-part to these events. These findings are consistent with, and add mechanistic depth to, recent in vivo models in which ZO-1 dysregulation and BBB disruption are shown to accompany cytokine-associated neuroinflammatory injuries such as stroke (Huang et al, 2013;Zehendner et al, 2013), intracerebral haemorrhage (Ding et al, 2014), and cerebral infection (Zhou et al, Fig. 3.…”

“…Some evidence of the corelationship between ZO-1 levels and ROS signalling can also be found in the literature. A recent study by Zehendner et al (2013) for example has demonstrated how disruption of cell border ZO-1 in murine bEnd.3 Fig. 2.…”

“…This cytosolic scaffolding protein is associated with both adherens and tight junction complexes, where it serves to intracellularly tether transmembrane junction proteins to the actin cytoskeleton (Fanning and Anderson, 2009). Using the transient middle cerebral artery occlusion (tMCAO) model, researchers have recently demonstrated in vivo how ischemic stroke conditions disrupt ZO-1 expression and localization at the cell-cell border in cerebral microvessels, correlating with barrier failure and extravasation (Huang et al, 2013;Zehendner et al, 2013), an inflammatory process in which multiple cytokines are implicated. Consistent with this notion, the ability of TNF-α to disrupt ZO-1 localization to the cell-cell border in microvascular endothelial cells in vitro for example, has previously been reported (Lutgendorf et al, 2014), whilst other recent microvascular endothelial studies have demonstrated how ZO-1 dysregulation may accompany injury-specific induction of IL-6 (Chaudhuri et al, 2008;Labus et al, 2014).…”

Osthole, a Natural Coumarin Improves Cognitive Impairments and BBB Dysfunction After Transient Global Brain Ischemia in C57 BL/6J Mice: Involvement of Nrf2 Pathway

“…* P \ 0.05, ** P \ 0.01 compared with sham-operated group, # P \ 0.05, ## P \ 0.01 compared with model group. Sham: sham-operated animals, Model: animals of vehicle treatment and ischemia, osthole: animals of osthole treatment and ischemia oxidative stress induced by moderate hypoxia can breakdown the BBB integrity in brain endothelial cells (BEC) [38]. Another study showed that peroxidation of membrane polyunsaturated fatty acids induced by ROS may affect BBB function by active aldehydes, such as 4-hydroxynonenal (4-HNE), which can significantly increase endothelial monolayer permeability in experimental studies [1].…”

The in vitro isolated whole guinea pig brain as a model to study epileptiform activity patterns

“…BBB integrity was also analyzed by measuring the TJ (ZO-1) protein arrangement. BBB damage is also associated with ZO-1 down-regulation in endothelial cells Zehendner et al, 2013). BBB permeability during electrophysiological experiments in the IWB can be monitored by evaluating changes in the extracellular K + concentration in the brain parenchyma (Librizzi et al, 2001;Gnatkovsky et al, 2008;Kuhlmann et al, 2009).…”