Inhibiting The Activities Of Striatal Neurons After Ischemic Stroke Using Optogenetics Improves The Survival Of Transplanted Neural Stem Cell And Promote The Recovery

Ischemic stroke is a cerebrovascular disease which can result in motor,sensory,cognitive disorder and even death.The treatment and the rehabilitation of ischemic stroke remain challenges at present.In recent years,numerous studies on animal models showed that transplanting different types of neural stem cells(NSCs)holds promise for the recovery of stroke.In the early phase after brain ischemia,transplanted NSCs can activate endogenous repair pathway such as immunomodulation,angiogenesis,neurogenesis and plasticity through paracrine signaling.In later period after stroke,transplanted NSCs can replace the lost neuron and physically connect with surrounding host cells,which may contribute to long-term recovery.However,the therapeutic efficiency of stem cells is limited by the poor survival of transplanted cells.Previous studies had illuminated the influence on the survival and proliferation of embryonic and adult NSCs by various extrinsic survival factors including growth factors,morphogens,proteoglycans,cytokines and hormones in post-stroke niche.Adult NSCs can additionally respond to a variety of neurotransmitters such as glutamate,dopamine,histamine,GABA,D-Serine,NO and5HT.Given that the occurrence of action potential can lead to the secretion of neurotransmitters,it suggests that the activity of surrounding intrinsic neurons may influent the survival,proliferation and differentiation of transplanted adult NSCs.In this study,we used optogenetics technique to regulate striatal neuronal activity after transient middle cerebral artery occlusion(tMCAO)model and NSC transplantation in mice,in order to investigate whether exciting or inhibiting striatal neurons in peri-infarct region could improve the survival of transplanted NSC and promote the recovery.Further,we explored the mechanism of the influence on NSC survival by neuronal activity in vitro.In the animal experiment of this research,we studied the influence on the survival of transplanted NSC by regulating striatal neuronal activity with optogenetics after ischemic stroke.Before the formal experiment,we had confirmed that the adenovirus-associated virus(AAV)carrying the gene of opsins under the control of CaMKII promotor could transfect most striatal neuron.Further corfirmation was acquired by the method of electrophysiological recording.The striatal neurons expressing ChR2 or ArchT were proved to be able to response to the stimulation of laser with specific wavelength,that established the feasibility and validity of the optogenetics method.In either excitatory stimulating experiment or inhibitory stimulating experiment,animals were randomly grouped in PBS control gourp,PBS administration with laser stimulation group,NSC transplantation group and NSC transplantation with laser stimulation group(NSC-E group or NSC-I group).At two weeks before the middle cerebral artery occlusion(MCAO)model,mice left striatum were injected with AAV-CaMKII-ChR2-mCherry or AAV-CaMKII-ArchT-EGFP.Mice were experienced 60 minutes transient MCAO and received PBS or 3×10~5 mice NSC at ipsilateral striatum at 4 days after tMCAO model.For the NSC-E group or NSC-I group,laser stimulation was daily administrated from day 7 to day 13,in order to excite or inhibit the striatal neuronal activity.Animals were sacrificed and the brain tissue were collected at day 14 for further examination.We found that mice in NSC-E group showed significant larger brain infarct volume,lower transplanted NSC survival,lower vascular density in peri-infart area and higher neurological severity score(NSS)when compared to NSC transplantation group,and NSC-E group showed little difference with PBS control group on these indices.To the contrary,mice in NSC-I group showed significant smaller brain infarct volume,higher transplanted NSC survival,higher vascular density in peri-infart area when compared to NSC transplantation group.The result of cell apoptosis detection showed NSC-E group had a higher number of apoptotic cells in transplanting area when compared to NSC group,while NSC-I group displayed a lower number of apoptotic cells.These data suggested that exciting striatal neuronal activity reduced the survival of transplanted NSC and cancelled the curative effect from transplanted NSC after brain ischemia,while inhibiting striatal neuronal activity enhanced the survival of transplanted NSC and promote the recovery.In the cell experiment,we stimulated neurons expressing ChR2 with blue laser pulse and collected the conditioned medium to culture NSC.We found that the NSC treated with conditioned medium showed lower cell viability and lower expression of mRNA of NGF and GDNF.This result further represented that neuronal activity could influent the survival of neighboring through paracrine pathway.According to above results,we conclude that inhibiting striatal neuronal activity after ischemic stroke can enhance the survival of transplanted NSC and contribute to a better outcome.This research made an important step for revealing the relationship between neuronal activity and NSC survival,and provided a new potential approach for treating ischemic stroke.