Calcium-dependent blood-brain barrier breakdown by NOX5 limits postreperfusion benefit in stroke
Ana I. Casas, Pamela W.M. Kleikers, Eva Geuss, Friederike Langhauser, Thure Adler, Dirk H. Busch, Valerie Gailus-Durner, Martin Hrabê de Angelis, Javier Egea, Manuela G. Lopez, Christoph Kleinschnitz, Harald H.H.W. Schmidt
Ana I. Casas, Pamela W.M. Kleikers, Eva Geuss, Friederike Langhauser, Thure Adler, Dirk H. Busch, Valerie Gailus-Durner, Martin Hrabê de Angelis, Javier Egea, Manuela G. Lopez, Christoph Kleinschnitz, Harald H.H.W. Schmidt
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Concise Communication Neuroscience

Calcium-dependent blood-brain barrier breakdown by NOX5 limits postreperfusion benefit in stroke

Abstract

Ischemic stroke is a predominant cause of disability worldwide, with thrombolytic or mechanical removal of the occlusion being the only therapeutic option. Reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain barrier. Its mechanism, however, is unknown. Here, we identified type 5 NADPH oxidase (NOX5), a calcium-activated, ROS-forming enzyme, as the missing link. Using a humanized knockin (KI) mouse model and in vitro organotypic cultures, we found that reoxygenation or calcium overload increased brain ROS levels in a NOX5-dependent manner. In vivo, postischemic ROS formation, infarct volume, and functional outcomes were worsened in NOX5-KI mice. Of clinical and therapeutic relevance, in a human blood-barrier model, pharmacological NOX inhibition also prevented acute reoxygenation-induced leakage. Our data support further evaluation of poststroke recanalization in the presence of NOX inhibition for limiting stroke-induced damage.

Authors

Ana I. Casas, Pamela W.M. Kleikers, Eva Geuss, Friederike Langhauser, Thure Adler, Dirk H. Busch, Valerie Gailus-Durner, Martin Hrabê de Angelis, Javier Egea, Manuela G. Lopez, Christoph Kleinschnitz, Harald H.H.W. Schmidt

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Figure 2

Increased in vitro ROS production is related to Ca2+ overload and NOX5.

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Increased in vitro ROS production is related to Ca2+ overload and NOX5. ...
(A) In vitro, mouse OHCs were subjected to OGD, followed by the reoxygenation period. (B) ROS production measured after OGD in OHCs was higher in NOX5-KI mice (blue, n = 6) versus NOX5 WT mice (black, n = 6), starting directly from the OGD period until 4 hours after OGD. **P < 0.01, Bonferroni’s multiple comparison test. (C) Difference in ROS production between NOX5-KI (blue, n = 6) and WT (black, n = 6) mice showed a NOX5-dependent effect within the first hour after OGD (0, 0.25, 0.5, and 1 hour). (D) Addition of the ionophore A23187 (10 μM) showed a Ca2+-dependent increase in ROS formation in NOX5-KI mice (blue, n = 3, Bonferroni’s multiple comparison test), while no changes were detected in WT mice (black, n = 3). *P < 0.05. (E) Difference in ROS production between NOX5-KI (blue, n = 3) and WT (black, n = 3) mice showed a direct link between Ca2+ overload and NOX5 induction at 15 and 30 minutes after A23187.