Moderate hypoxia followed by reoxygenation results in blood-brain barrier breakdown via oxidative stress-dependent tight-junction protein disruption.

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

MHR disrupts BEC integrity on the level of tight junction proteins.

<p>BEC displayed a physiological arrangement of TJ proteins Claudin 5 (<b>A</b>) and zonula occludens 1 (<b>B</b>) in a co-localizing manner (<b>C</b>) with only few TJ gaps (arrowheads). MHR disrupted the continuous arrangement of both junctional proteins in BEC (arrowheads <b>D-F</b>). Here, DPI blocked TJ injury in MHR, only few TJ disruptions were detected in BEC pre-treated with the NADPH oxidase inhibitor DPI (arrowheads <b>G-I</b>). Representative images from 3 experiments per group are shown. Quantitative analyses of Cl5 (Cl5 normoxia: 5.85 <u>+</u> 0.33 vs. Cl5 MHR: 2.72 <u>+</u> 0.54, P < 0.0001; n = 12 cells per group from 3 experiments, <b>J</b>) and ZO-1 (ZO-1 normoxia: 12.53 <u>+</u> 1.27 vs. ZO-1 MHR: 3.98 <u>+</u> 0.67, P < 0.0001; n = 12 cells per group from 3 experiments, <b>K</b>) integrity on cell membranes revealed that both junctional proteins are significantly perturbed in MHR. DPI pre-treatment significantly restored TJ integrity at cellular membranes under MHR (Cl5 MHR: 2.72 <u>+</u> 0.54 vs. Cl5 MHR+DPI: 6.57 <u>+</u> 0.69, P = 0.0002; ZO-1 MHR: 3.98 <u>+</u> 0.67 vs. ZO-1 MHR+DPI 10.55 <u>+</u> 1.21, P < 0.0001; n = 12 cells per group from 3 experiments). </p

MHR impairs TEER in Brain endothelial cells and increases transcellular permeability towards high molecular mass molecules.

<p>Trans-endothelial resistance values detected with ECIS (<b>A</b>) were significantly lower in BEC after MHR than in normoxic conditions (normoxia 1 <u>+</u> 0.004 vs. MHR no inhibitor 0.77 <u>+</u> 0.02, P < 0.0001; n = 169 - 288 TEER measurements from 3 experiments). DPI prevented the decrease of TEER significantly (MHR+DPI 0.98 <u>+</u> 0.01 vs. MHR no inhibitor 0.77 <u>+</u> 0.02, P < 0.0001; n = 168 - 192 TEER measurements from 3 experiments). MHR resulted in an increase of FITC-Dextran (150,000 Dalton) permeability in bEnd.3 monolayers (normoxia 1 <u>+</u> 0.09 vs. MHR 2.27 <u>+</u> 0.75, P < 0.05; n = 7-15 permeability assays, <b>B</b>). </p

MCAO followed by reperfusion leads to the generation of ROS in cerebral microvessels.

<p>Images from a representative control (<b>A</b>) hemisphere and its contralateral tMCAO hemisphere (<b>B</b>) of the neocortex are shown. MAP 2 (red), Fluorescein isothiocyanate –albumin (FITC-albumin; green) and anti-nitrotyrosine (blue) are displayed (n = 3 brains). Perfused control hemispheres (contralateral to tMCAO) displayed an intact MAP2 immunoreactivity, no FITC-albumin extravasation and absence of nitrotyrosine formation (<b>A</b>). In the penumbra of corresponding tMCAO hemispheres MAP2 damage was present as well as large and diffuse FITC-albumin extravasation. Here we found that cerebral microvessels (indicated by arrows and inset) were immunoreactive for nitrotyrosine indicating the formation of ROS (<b>B</b>). Note the cytosolic staining pattern of nitrotyrosine indicating the cytosolic localization of ROS.</p