Targeting Oxidative Stress Injury after Ischemic Stroke in Conscious Rats: Limited Benefits with Apocynin Highlight the Need to Incorporate Long Term Recovery
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. 1. 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); 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
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