Targeting oxidative stress injury after ischemic stroke in conscious rats: limited benefits with apocynin highlights the need to incorporate long term recovery.

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Abstract
NADPH oxidase is a major source of superoxide anion following stroke and reperfusion. This study evaluated the effects of apocynin, a known antioxidant and inhibitor of Nox2 NADPH, on neuronal injury and cell-specific responses to stroke induced in the conscious rat. Apocynin
treatment (50 mg/kg i.p.) commencing 1 hour prior to stroke and 24 and 48 hours after stroke significantly reduced infarct volume in the cortex by ~ 60 %, but had no effect on striatal damage or neurological deficits. In situ detection of reactive oxygen species (ROS) using dihydroethidium
fluorescence revealed that increased ROS detected in OX-42 positive cells following ischemia was reduced in apocynin-treated rats by ~ 51 %, but surprisingly increased in surrounding NeuN positive cells of the same rats by ~ 27 %, in comparison to the contralateral hemisphere. Reduced
ROS from activated microglia/macrophages treated with apocynin was associated with reduced Nox2 immunoreactivity without change to the number of cells. These findings confirm the protective effects of apocynin and indicate a novel mechanism via reduced Nox2 expression. We
also reveal compensatory changes in neuronal ROS generation as a result of Nox2 inhibition and highlight the need to assess long term individual cell responses to inhibitors of oxidative stress.
Introduction    Oxidative stress contributes to brain reperfusion injury following stroke [1]. Well established sources of reactive oxygen species (ROS) generation in the brain following injury include intracellular organelles (especially mitochondria), invading neutrophils, activated
microglia/macrophages [2] and cerebral blood vessels [3]. Recently we have also shown that neurons themselves generate large amounts of superoxide following transient stroke, an effect that contributes to the progression of injury over time [4].  Several drugs that target oxidative stress have been developed as potential therapies for ischemic stroke. Spin trap free radical inhibitors and antioxidants, including Ebselen (a glutathione peroxidase mimetic); NXY 059 (a nitrone based free radical trapping agent) and edaravone (a free radical scavenger) can reduce infarct volume in rodent reperfusion models of ischemic stroke, supporting the contribution of reactive oxygen species to ischemic damage following reperfusion
[5]. These antioxidants however target reactive oxygen species only after they are formed, and do not address the specific process by which these toxic molecules are generated. This is important for reactive oxygen species can rapidly cause damage before they are inactivated. Novel therapeutic
strategies that target the source of reactive oxygen species generation may offer better neuroprotection.  NADPH oxidase (Nox) is the major enzyme source of reactive oxygen species in the vascular system [6, 7], inflammatory cells [2], cerebral blood vessels [3, 8] and neurons [4, 9]. The NADPH oxidase complex generates the highly reactive free radical, superoxide, via its Nox2 catalytic subunit [6, 10], and is the active NADPH oxidase in inflammatory cells in humans and animals.  There are 2 major isoforms of Nox2, with Nox1 and Nox4 differentially expressed depending on tissue type, and these play different roles in regulating reactive oxygen species production [7]. In the acute phase of stroke damage (1-7 days) following ischemia with reperfusion, we have previously demonstrated the Nox2 catalytic component to be the predominant Nox subtype that is

up regulated and associated with phagocytic microglial cells, indicating this NADPH oxidase to be a potential target for therapeutic intervention [4].
Apocynin (4-hydroxy-3-methoxy-acetophenone) is a reported inhibitor of Nox1 and Nox2 dependent superoxide generation in peripheral inflammatory cells [11] and has been shown to reduce damage in animal models of global and focal cerebral ischemia [12, 13, 14]. Individual cell
responses to stroke injury following apocynin treatment are yet to be fully explored. In the present study we examine the effect of apocynin on cell specific changes in NADPH oxidase and ROS generation in the brain following transient stroke in conscious rats and make the surprising finding
that in addition to attenuating ROS generation in inflammatory cells, Nox2 protein expression is also down regulated, whilst neuronal ROS is conversely increased.