ChAT~+ Neurons Regulate Stroke-induced Neurogenesis Within Subventricular Zone

Background and ObjectiveNeurons with the marker of choline acetyltransferase, Ch AT+ neurons are newly identified within the subventricular zone(SVZ). These neurons have flat spatial structures, with long axes that parallel to the ependymal surface. It has been proved that Ch AT+ neurons within SVZ can regulate local neurogenesis in a direct and independent way by spontaneously releasing acetylecholine(ACh) under physiological conditions. In this study, we firstly aim to examine the expression or functional changes of SVZ Ch AT+ neurons after ischemic stroke, and to secondly discuss the relationship and possible mechanisms between the Ch AT+ neurons and SVZ neurogenesis under brain ischemia.Materials and methods1 Experimental animals and groupsTwo hundred and twenty-four mice(male, 25~30g, 4~5 weeks old) were randomly assigned to five groups:(1) sham-operated group,n=36,(2)MCAO-operated group,n=49,(3) MCAO+Donepezil group,n=50,(4) MCAO+Vehicle group,n=39,(5) MCAO+Donepezil+PD173074 group,n=50.2 Ischemic stroke model and measurementsA model of transient middle cerebral artery occlusion(t MCAO) of mice was established. Drugs or vehicles administrations were performed until 10 days after the surgery. We analyzed the changes of body weights and m NSS scores of mice, and conducted immunofluorescence(IF) or western blot of the SVZ tissue to examine the changes of Ch AT, VACh T, Mash 1, DCX, and Ki67.3 Statistical analysisStatistical analysis was carried out with SPSS version 21.0. Results were expressed as mean±SD. We used student’s t-test to analyze differences between two groups, and repeated mesures ANOVA followed by least significant difference(LSD)to analyze differences between several groups. p<0.05 was considered statistically significant.Results1 The body weight of all groups decreased rapidly after surgery and started to increase 3 days later. Mice from MCAO+Donenezil group showed higher body weight compared with that of MCAO group and MCAO+Donepezil+PD173074group(p<0.05). Mice from MCAO+Donepezil+PD173074 group had slower recovery of bodyweight than that of MCAO group(p<0.05). No differences were shown among the group of MCAO and MCAO+Vehicle(p>0.05). The neurological functional evaluation showed that mice from MCAO+Donepezil group had lower m NSS scores than that of MCAO group and MCAO+Donepezil+PD173074 group(p<0.05). MCAO group had lower m NSS scores than that of MCAO+Donepezil+PD173074 group(p<0.05).2 On day 10 after surgery, IF showed that Ch AT+ cells did exist within SVZ,and the VACh T+ staining coverage within SVZ of MCAO group mice was larger than that of Sham group animals. The difference was statistically significant(p<0.05);western blot showed the protein levels of Ch AT, VACh T, and DCX within SVZ of MCAO animal were significantly increased versus that of sham group. The difference was statistically significant(p<0.05).3 IF or western blot showed the expressions of Ki67, DCX and Mash 1 within SVZ of MCAO+Donepezil group were significantly increased versus that of MCAO+Vehicle group. The difference was statistically significant(p<0.05).4 IF or western blot showed the expressions of Ki67, DCX and Mash 1 within SVZ of MCAO+Donepezil+PD173074 group were significantly decreased versus that of MCAO+Donepezil group. The difference was statistically significant(p<0.05).5 IF showed the expression of VACh T within SVZ of MCAO+Donepezil+PD173074 group tended to increase compared with that of MCAO+Donepezil group.Conclusions1 The function of Ch AT+ neurons was increased after ischemic stroke, along with more cell proliferation within SVZ.2 SVZ Ch AT+ neurons may participate in the stroke-induced neurogenesis via fibroblast growth factor receptor(FGFR) signaling pathway.