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

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.

NADPH Oxidase as a Therapeutic Target for Neuroprotection against Ischaemic Stroke: Future Perspectives

“…We too have recently shown that treatment with a high dose of apocynin reduces cortical damage after endothelin-1-induced stroke and reperfusion in rats with a 3 day recovery, but we showed no effect of apocynin treatment on striatal infarcts, as previously reported [217]. This lack of effect was correlated with increased neuronal ROS generation within the penumbra of apocynin-treated rats and we proposed that treatment with apocynin merely delayed the progression of injury over time [217].…”

“…We too have recently shown that treatment with a high dose of apocynin reduces cortical damage after endothelin-1-induced stroke and reperfusion in rats with a 3 day recovery, but we showed no effect of apocynin treatment on striatal infarcts, as previously reported [217]. This lack of effect was correlated with increased neuronal ROS generation within the penumbra of apocynin-treated rats and we proposed that treatment with apocynin merely delayed the progression of injury over time [217]. Indeed previous reports showing protective actions of apocynin within the striatum measured infarcts only up to 24 h post-reperfusion, at times when the infarct may not have fully matured in the models used for assessment [154,160,167,176,177].…”

Apocynin attenuates testicular ischemia–reperfusion injury in rats

“…In this study we created two doses of apocynin since in literature both the low (20 mg/kg) and high doses (50-100 mg/kg) were used against oxidative stress in different tissues and plasma [9,10,34]. Furthermore in the study of Li et al, apocynin, administered at a dose of 16 mg/kg for 4 weeks, protected against diabetes-induced testicular damage [35].…”

Pathophysiology and Treatments of Oxidative Injury in Ischemic Stroke: Focus on the Phagocytic NADPH Oxidase 2

“…Among these, a large numbers of studies used the 4-hydroxy-3methoxy-acetophenone (apocynin), a natural, powerful antioxidant compound used in traditional Ayurvedic medicine (1). Although its molecular mechanism is not fully understood, apocynin would impair NOX2 assembly and activation (242), thus resulting as a NOX2-targeted therapeutic strategy against stroke (227). Experimental models of either focal or global cerebral ischemia have supported the neuroprotective role of apocynin.…”