Optogenetic Stimulation Reduces Neuronal Nitric Oxide Synthase Expression After Stroke

Abstract

Post-stroke 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 the neurovascular response in acute stroke; however, its role in subacute recovery remains unclear. We investigated the expression of nNOS in stroke mice undergoing optogenetic stimulation of the contralesional lateral cerebellar nucleus (cLCN). We also examined the effects of nNOS inhibition on functional recovery using a pharmacological inhibitor targeting nNOS. Optogenetically stimulated stroke mice demonstrated significant improvement on the horizontal rotating beam task at post-stroke days 10 and 14. nNOS mRNA and protein expression was significantly and selectively decreased in the contralesional primary motor cortex (cM1) of cLCN-stimulated mice. The nNOS expression in cM1 was negatively correlated with improved recovery. nNOS inhibitor (ARL 17477)-treated stroke mice exhibited a significant functional improvement in speed at post-stroke day 10, when compared to stroke mice receiving vehicle (saline) only. Our results show that optogenetic stimulation of cLCN and systemic nNOS inhibition both produce functional benefits after stroke, and suggest that nNOS may play a maladaptive role in post-stroke recovery.

Keywords: Brain stimulation; Functional recovery; Nitric oxide synthase; Optogenetics; Stroke.

Fig. 1

Optogenetic neuronal stimulation of the cLCN promotes behavioral recovery after stroke. a Thy1 mice-expressing channelrhodopsin 2 (ChR2) were used in this study. Blue laser stimulation activates ChR2 and causes neuronal excitation. b Schematic depicting the optogenetic laser stimulation site (blue bar) in the cLCN. Efferent projections travel through the superior cerebellar peduncle, decussate in the midbrain tegmentum and terminate in the ipsilesional ventrolateral thalamus (blue). Second-order neurons then project to multiple cortical regions including prefrontal, premotor, motor, and posterior parietal cortex. c Schematic illustrating the cortical and striatal infarction produced by transient middle cerebral artery occlusion. d Experimental paradigm. Mice were pre-trained (H) on the rotating beam test prior to the pre-fiber implant baseline (b) and the pre-stroke baseline data (day 0). Each mouse in the treatment group received one session of stimulations daily, from post-stroke day 5 and continued until day 14. Behavior tests were performed at post-stroke days 4, 7, 10, and 14. Mice were sacrificed at day 15 for qPCR and Western blot analysis. e Optogenetic neuronal stimulation paradigm. Each stimulation session consists of three 1-min stimulations with 3-min rest periods in between. Laser ON periods (blue) and laser OFF periods (black) are indicated. f Repeated neuronal stimulations of cLCN produced post-stroke recovery. cLCN-stimulated mice demonstrated significant improvement in speed (cm/s) on the rotating beam task at 10 and 14 days post-stroke. n = 4 for stroke + no stim, n = 4 for stroke + stim. Two-way ANOVA with Bonferroni’s post hoc test, *P < 0.05, ***P < 0.001. Data are expressed as mean ± SD

Fig. 2

Validation of cLCN targeting using photothrombotic lesion model. Representative images depict LCN in the ipsilesional and contralesional cerebellar coronal sections (dotted line outlines the location of LCN). Brain structures were visualized by immunostaining with neuronal marker MAP2 (green). Note that LCN on the ipsilesional side is intact, while LCN on the contralesional side where the fiber was implanted (blue) has been lesioned after the photothrombotic model. Int, interposed nuclei; LCN, lateral cerebellar nuclei; 4 , 4th ventricle. Scale bar = 500 μm

Fig. 3

nNOS mRNA is selectively decreased in cM1 after optogenetic stimulation of the cLCN. a Schematic depicting the location of iM1 and cM1 within tissue sections selected for RNA and protein analysis. mRNA expression of NOS isoforms (nNOS, iNOS, eNOS) was quantified in iM1 and cM1 of cLCN-stimulated and non-stimulated mice at post-stroke day 15. b Bar graphs with scatter dot plot shows nNOS mRNA expression was significantly decreased in cM1 of cLCN-stimulated animals. Data are expressed as mean ± SD. There was no significant difference in nNOS mRNA expression in iM1 between stimulated and non-stimulated mice. Similarly, there were no differences in iNOS or eNOS between groups in iM1 or cM1. n = 4 for each group. *P < 0.05, significant difference between stimulated and non-stimulated stroke mice, one-way ANOVA with Fisher’s LSD

Fig. 4

nNOS mRNA expression in cM1 is negatively correlated with behavioral recovery. Correlation analysis between speed (cm/s) across the rotating beam at post-stroke day 14 and nNOS mRNA in a iM1 and b cM. Lower nNOS mRNA levels in cM1 correlated with increased speed (n = 8; Pearson r = − 0.839; p = 0.0092). There was no correlation between iM1 nNOS mRNA expression and speed in the rotating beam

Fig. 5

Optogenetic stimulation of the cLCN decreases nNOS protein expression in cM1. Bar graphs with scatter dot plot shows the results of western blot analysis in a iM1 and b cM1 mice brain sacrificed on post-stroke day 15. Data are expressed as mean ± SD. a In iM1, both cLCN-stimulated and non-stimulated mice demonstrated an increase in nNOS protein levels compared to sham animals. There was no difference in nNOS protein expression between stimulated or non-stimulated mice. b In cM1, cLCN-stimulated mice exhibited a decreased nNOS protein level compared to non-stimulated animals. There was no difference in nNOS protein levels between non-stimulated stroke animals and sham mice. nNOS protein levels are expressed as percentage optimal density measurement over GAPDH. n = 4 for each group. *P < 0.05; significant difference between stimulated and non-stimulated groups, Student’s t test

Fig. 6

Systemic nNOS inhibition produces behavioral recovery after stroke. a Molecular structure of ARL 17477 dihydrochloride which was used for the systemic nNOS inhibition studies. Experimental paradigm depicting mice undergoing baseline behavioral testing followed by transient middle cerebral artery occlusion. Mice received intraperitoneal vehicle (saline) or 10 mg/kg nNOS inhibitor (ARL 17477 dihydrochloride) on post-stroke days 5–14. Behavior on the rotating beam test was performed on post-stroke days 7, 10, and 14. b Mice receiving nNOS inhibition after stroke experienced a significant improvement over vehicle treated animals at post-stroke day 10. Data are expressed as mean ± SD. n = 9 for stroke + vehicle; n = 7 for stroke + nNOS inhibitor; two-way ANOVA with Bonferroni’s post hoc test, *P < 0.05. c Representative coronal brain sections at striatum (Str) and hippocampus (Hpx) level in vehicle and inhibitor groups. The infarct area was identified by CD68-positive activated monocytes/macrophages (red) and neuronal MAP2-negative areas (green). Scale bar = 1 mm. Bar graphs with scatter dot plot show that d % infarct area at the Str and Hpx level and e total infarct area are similar between vehicle and inhibitor group. n = 9 for vehicle group, n = 7 for inhibitor group