Optogenetic Stimulation Reduces Neuronal Nitric Oxide Synthase Expression After Stroke

Abstract

Abstract INTRODUCTION Poststroke optogenetic stimulation has been shown to enhance neurovascular coupling and functional recovery. Neuronal nitric oxide synthase (nNOS) has been implicated as a key regulator of neurovascular response in acute stroke but its role in subacute recovery remains unclear. Here we investigate nNOS expression in stroke mice undergoing optogenetic stimulation of the contralesional lateral cerebellar nucleus (cLCN). We also examine the effects of nNOS inhibition on functional recovery using a pharmacological inhibitor targeting nNOS. METHODS Transgenic Thy1-ChR2-YFP male mice (10-12 wk) were used. Stereotaxic surgery was performed to implant a fiber cannula in the cLCN and animals underwent intraluminal middle cerebral artery suture occlusion (30 min). Optogenetic stimulation began at poststroke (PD) day 5 and continued until PD14. Sensorimotor tests were used to assess behavioral recovery at PD4, 7, 10, and 14. At PD15, primary motor cortex from both ipsi- and contralesional motor cortex (iM1, cM1) were dissected. nNOS mRNA and protein levels were examined using quantitative polymerase chain reaction and western blot. In another set of studies, nNOS inhibitor ARL 17477 dihydrochloride (10 mg/kg, intraperitoneally) was administered daily between PD5-14 and functional recovery was evaluated using sensorimotor tests. RESULTS cLCN stimulated stroke mice demonstrated significant improvement in speed (cm/s) on the rotating beam task at PD10 and 14 day (P < .05, P < .001 respectively). nNOS mRNA and protein expression was significantly and selectively decreased in cM1 of cLCN stimulated mice (P < .05). The reduced nNOS expression in cM1 was negatively correlated with improved recovery (R2 = −0.839, Pearson P = .009). nNOS inhibitor-treated stroke mice exhibited a significant functional improvement in speed at PD10, when compared to stroke mice receiving vehicle (saline) (P < .05). CONCLUSION Our results suggest that nNOS may play a maladaptive role in poststroke recovery. Optogenetic stimulation of cLCN and systemic nNOS inhibition produce functional benefits after stroke.