Alpha-7 Nicotinic Acetylcholine Receptor Signaling Pathway Participates In The Neurogenesis Induced By ChAT-positive Neurons In The Subventricular Zone

Background and ObjectiveWith the increasing morbility of stroke and high possibility of neurologic impairment left,therapy for stroke has attracted the overwhelming attention of the public.Choline acetyltransferase(ChAT)-positive neurons in subventricular zone(SVZ)can promote neurogenesis via regulating the synthesis of acetylcholine(ACh)under normal and pathological condition.Alpha7 nicotinic acetylcholine receptor(?7 nAChR)has been reported to have protective role in the recovery of stroke.To explore the mechanism of ChAT~+ neurons-induced neurogenesis after stroke,we induced middle cerebral artery occlusion(MCAO)on mice to explore the effect of ischemic stroke on cholinergic activity and explored whether ChAT-positive neurons can promote neurogenesis via ?7nAChR signaling pathway.MethodsMice were operated to occlude the middle artery via suture method.The expression of ChAT,acetylcholinesterase(AChE)and ?7nAChR were measured by Western blot.Mice were randomly divided into three groups: Sham group,MCAO group and MCAO~+methyllycacotine(MCAO~+MLA)group.Mice were induced MCAO and injected with vehicle or MLA via lateral ventricle.The neurological defects of mice were assessed by investors blinded to the group according to modified neurological severity scores(mNSS).Western blot and immunofluorescence(IF)were performed to evaluate the effect of MLA on the distribution of membrane and nucleus fibroblast growth factor receptor 1(FGFR1).They were also used to evaluate the expression of corresponding downstream proteins such as PI3 K,pAkt and so on.Results1.The expression levels of ChAT and AChE were lower in MCAO group than those in Sham group while the level of ChAT/AChE was higher in MCAO group.The level of ?7nAChR expression was also higher in MCAO group than that in Sham group.2.Mice in MCAO group exhibited higher brain water content,worse neurological deficits and lower weight.Compared with MCAO group,brain water content was increased and neurologic deficit was worse in MCAO~+MLA group.No statistical difference was found in the weight between MCAO group and MCAO~+MLA group.3.Compared with Sham group,expression of membrane FGFR1,phosphatidylinositol-3-kinase(PI3K)and pAkt was increased after ischemic stroke and the number of glial fibrillary accidicprotein/5-bromo-2'-deoxyuridine(GFAP/BrdU)-positive cells was also increased.MLA administration upregulated the expression of membrane FGFR1,PI3 K and pAkt.The number of GFAP/BrdU-positive cells was higher in MCAO~+MLA group than that in MCAO group.4.Mice in MCAO group exhibited higher levels of nucleus FGFR1,doublecortin(DCX),mammalian achaete scute homolog-1(mash1)and phosphorylated-neural cell adhesion molecule(PSA-NCAM)expression than those in Sham group.The increase was also observed in the number of DCX-positive cells after ischemic stroke.Mice in MCAO~+MLA group showed lower expression levels of nucleus FGFR1,DCX,mash1 and PSA-NCAM and less DCX-positive cells.Conclusion1.The activity of cholinergic system in SVZ was increased after ischemic stroke.2.ChAT-positive neurons within SVZ can promote stroke-induced neurogenesis via ?7nAChR signaling pathway.