NADPH oxidase Nox1 contributes to ischemic injury in experimental stroke in mice

doi:10.1016/j.nbd.2010.05.023

Neurobiology of Disease

Volume 40, Issue 1, October 2010, Pages 185-192

https://doi.org/10.1016/j.nbd.2010.05.023 Get rights and content

Abstract

Reactive oxygen species (ROS) are mediators of brain injury in ischemia/reperfusion. An involvement of the NADPH oxidase Nox2 has been demonstrated. In contrast, only little is known about the contribution of the Nox1 homologue in this context. Thus, we studied the role of Nox1 in early cerebral reperfusion injury in the middle cerebral artery filament occlusion model using Nox1 knockout mice. Genetic deletion of a functional Nox1 lead to a 55% attenuation in lesion size at 24 h after induction of 1 h ischemia (p < 0.05). This result was paralleled by a significant improvement of neurological outcome, preservation of blood–brain barrier integrity and reduced cerebral edema in Nox1^y/⁻ compared to WT mice. Interestingly, no difference in infarct size between WT and Nox1^y/⁻ was observed with an occlusion time of 2 h and longer. Apoptosis rate as measured by TUNEL staining was similar between the groups. Moreover, infusion of the antioxidant TEMPOL as well as of the unspecific NO-synthase inhibitor l-NAME elicited similar changes with respect to ischemic tissue damage between WT and Nox1-deficient mice. In conclusion, Nox1 is involved in the pathophysiology of cerebral ischemia. Our data however indicate that ROS-mediated direct cellular injury is unlikely to explain the protective effect achieved by genetic deletion of the enzyme.

Introduction

Early restoration of cerebral blood flow is the main approach to limit brain injury in ischemic stroke. Upon reperfusion, however, ischemic tissue is further damaged by the reperfusion injury (Aronowski et al., 1997), which is accompanied by an opening of the blood–brain barrier (BBB) through endothelial cell contraction and disassembly of tight junctions. Moreover, reperfusion induces excessive generation of reactive oxygen species (ROS), which contribute to BBB opening (Heo et al., 2005) and the reperfusion injury. Recently, several studies demonstrated an important role for NADPH oxidases of the Nox family in ischemia/reperfusion (I/R) tissue injury of several organs, including the brain. Indeed, under certain conditions, Nox proteins can generate high amounts of ROS and several enzymes of this protein family as well as their cytosolic activator proteins are expressed in the central nervous system (Bedard and Krause, 2007).

The family of Nox proteins currently consists of seven members, but only the role of Nox2 for I/R-induced brain injury has extensively been studied to date (Chen et al., 2009, Kahles et al., 2007, Walder et al., 1997). Interestingly, Nox1 has some similarities to Nox2. Its activity depends on cytosolic activator proteins and requires an interaction with Rac1 for ROS formation (Bedard and Krause, 2007). Some observations even suggest that Nox1 can be activated by cytosolic activator proteins required for Nox2 and, thus, Nox1-dependent ROS formation might parallel that of Nox2. The expression of Nox1 and Nox2 differs between cells with Nox1 being highly expressed in epithelial cells and smooth muscle cells, whereas Nox2, in addition to the expression in leukocytes, is also expressed in fibroblasts and endothelial cells (Sorce and Krause, 2009). Despite this difference in expression, the function of Nox1 and Nox2 at least in the cardiovascular system appears to be similar. Both proteins contribute to acute agonist-stimulated ROS formation and thus agonist signalling, and are mediators of endothelial dysfunction (Matsuno et al., 2005).

Very little is known regarding the function of Nox1 in the central nervous system. A recent study showed an involvement of NADPH oxidase generated ROS in apoptotic cerebellar granule neurons death and speculated about a role for Nox1 (Coyoy et al., 2008). Moreover, Nox-1 appears to promote the neurotoxic microglial activation in vitro (Cheret et al., 2008). Just recently, Jackman et al. (2009) investigated a possible contribution of Nox1 to ischemic injury in the middle cerebral artery filament occlusion (MCAO) mouse model and discovered no significant difference regarding total lesion volume for very short ischemic periods (30 min) and basal superoxide production in cerebral arteries. Regarding the cardiovascular system Nox1 plays a critical role in neointima formation by mediating VSMC migration, proliferation, and extracellular matrix production (Lee et al., 2009) as well as Nox1 deficiency protects from aortic dissection in response to Ang II possibly mediated through TIMP-1 (Gavazzi et al., 2007). Given the great similarities between Nox1 and Nox2 in the vascular system, we hypothesized that Nox1 still contributes to brain injury in experimental stroke and studied the role of this NADPH oxidase during increasing ischemic periods by the MCAO model in Nox1 knockout (Nox1^y/⁻) and their wild-type littermates (WT).

Section snippets

Animal model of focal cerebral ischemia

All experimental procedures were approved by the local governmental authorities (approval number: F28/09) and were performed in accordance with the animal protection guidelines.

Nox1^y/⁻ mice previously characterized (Gavazzi et al., 2006) were kindly provided by Karl-Heinz Krause, University of Geneva and bred at the local animal facility. With regard to physiological parameters, baseline mean blood pressure was reported to be similar in Nox1^y/⁻ and WT mice (Matsuno et al., 2005). A special

NADPH oxidase Nox1 is expressed in cell culture of the murine brain

On the basis of mRNA expression studies from different regions, it has previously been reported that Nox1 is expressed in the brain (reviewed by (Sorce and Krause, 2009)). Up to now the specific cell types contributing to Nox1 expression have not been characterized in detail. To address this point quantitative RT-PCR was performed for cultures of the main cell types of the brain (for gel documentation see Fig.1). RT-PCR rather than western blot or immuno-histochemistry was used as so far no

Discussion

In the present study, we observed that genetic deletion of Nox1 leads to smaller brain infarcts in the middle cerebral artery filament occlusion model selectively with 1 h of ischemia and 23 h of reperfusion. In addition, we could show that longer ischemic periods (2 h and more) did not result in different lesion volumes. Interestingly, Jackman et al. just recently found similar total lesion volumes in Nox1^y/⁻ mice compared to WT at 24 h of reperfusion following very short periods of ischemia (30

Disclosures

The authors declare that they have no conflicts of interest and related to this work.

Acknowledgments

We are grateful to Susanne Schütz for excellent technical assistance. We thank Karl-Heinz Krause for providing Nox1-deficient mice.

This study was supported by grants from the Deutsche Forschungsgemeinschaft to R.P.B. (BR1839/2-3 and Excellence Cluster Cardio-Pulmonary System ECCPS) and by the European Vascular Genomic Network, a Network of Excellence supported by the European Community's sixth Framework Program (Contract LSHM-CT-2003-503254) as well as by the Goethe-University, Frankfurt.

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NADPH oxidase Nox1 contributes to ischemic injury in experimental stroke in mice

Abstract

Introduction

Section snippets

Animal model of focal cerebral ischemia

NADPH oxidase Nox1 is expressed in cell culture of the murine brain

Discussion

Disclosures

Acknowledgments

Free Radic. Biol. Med.

Trends Neurosci.

Eur. J. Cell Biol.

FEBS Lett.

Prog. Neurobiol.

Free Radic. Biol. Med.

Trends Neurosci.

Free Radic. Biol. Med.

Brain Res.

Brain Res.

Free Radic. Biol. Med.

Three distinct mechanisms generate oxygen free radicals in neurons and contribute to cell death during anoxia and reoxygenation

J. Neurosci.

Reperfusion injury: demonstration of brain damage produced by reperfusion after transient focal ischemia in rats

J. Cereb. Blood Flow Metab.

NADPH oxidase 1 deficiency alters caveolin phosphorylation and angiotensin II receptor localization in vascular smooth muscle cells

Antioxid. Redox Signal.

The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology

Physiol. Rev.

Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination

Stroke

A tissue-culture approach to demyelinative disorders

Natl Cancer Inst. Monogr.

Differential vascular functions of Nox family NADPH oxidases

Curr. Opin. Lipidol.