The Effect And Mechanism Of FNS In Proliferation And Differentiation Of Neural Stem Cell After Focal Cerebral Ischemia/Reperfusion Injury

BackgroudIschemic stroke is the disease of nervous system,which is commonly seen and frequently occurs. Among all the types of stroke,ischemic stroke occupies the stroke disease above 75%. Most of the stroke patients have some scrious sequelas,such as paralysis,aphasia,and et al. It not only torments the stroke patients,but also bring the society and the patients, families heavy burden. Cerebral ischemia/reperfusion cause the cerebral functional impairment and structure damage become worse,which degrades the therapeutic efficacy obviously.The appropriate methods of prevention and therapy for ischemia/reperfusion to restore the function of damaged neurons and to reduce disability and mortality remain a subject of hottest focuses in recent years. Cerebral ischemia/reperfusion has complicated pathophisiological process and the specific mechanism. Functional impairment could be caused neuron disorganization and depletion which performed important pathological significance in cerebral ischemicin jury. At present,there is no satisfactal treatment of cerebral ischemia/reperfusion jury in study and clinical application. Studies showed that FNS has exact neuroprotective effects against damage of nervous system diseases, include anti-inflammatory,supress cell apoptosis and promoting nerve tissue re- construction. Cerebellum electric stimulation devices which start from experimental fastigial nucleus stimulation(FNS) and minic biological electric current have applied in clinic for treating some disease and showed the satisfied clinical effects on several central nervous system diseases recently. The signal pathway of Notch1 can control destiny of the cell. The neural stem cell carries on proliferation after Notch1 is activated and the neural stem cell enters the procedure of differentiation when the activeness of Notch1 is suppressed. The basic helix-loop-helix factor of bHLH gene richly contains an sequence of alkalinity amino acid in its upstream and regulate transcription of many genes. The research indicates that the Hes5 signal passage can inhibit differentiation of neural stem cell to the neuron and promote proliferation of neural stem cell. Mash1 may promote the differentiation of neural stem cell when the expression of Mash1 is high and cause condition of proliferation through the suppression of differentiation of nerve stem cell when the expression of Mash1 is low. There is a very complex mechanism of NF-κB in regulation proliferation and differentiation of neural stem cell which involves the aspect of inflammation and immune response. Based on the past clinical and experimental achievements, this project is to proceed with how FNS influence the endo- genesis neural stem cell and research the proliferation and differentiation of endogenous neural stem cell and mechanism with FNS. With the project carried out,we can get the experimental basis for the clinical treatment of ischemic stroke.Objectives(1) The protection of FNS was observed from the aspects of neural function comprehend evaluation, brain tissue water content and cerebral infarction size.(2) To identify proliferation and differentiation of neural stem cells in the adult rats which were treated with FNS at each time point of ischemia/reperfusion respectively and to investigate the possible effect of FNS on the plasticity of nerve of the brain.(3)The change of mRNA and protein expression of Notch1, Hes5, Mash1 and NF-кB p65 in each group was assessed to study on the mechanism of proliferation and differentiation of neural stem cells in focal cerebral ischemia/reperfusion rats.Methods(1) Focal cerebral ischemia/reperfusion injury was induced by intraluminal middle cerebral artery occlusion (MCAO) method using a monofilament thread in rats and and treated with FNS after the reperfusion was performed. Adult SD rats were randomly devided into five groups: normal control group(N group),sham-operation control grop(S group),ischemia/reperfusion group(I/R group),ischemia/ reperfusion treated with FN sham-stimulation group(SF group) and ischemia/reperfusion treated with FNS group(F group). Each group contained three time points of 1d,3d and 7d. The protective effect of FNS was observed from the aspects of neural function comprehend evaluation, brain tissue water content and cerebral infarction size.(2) Animal model was made by ligating external carotidartery and inserting a piece of nylon thread into the internal carotidarter. Adult SD rats were randomly devided into four groups: normal control group(N group),sham-operation control group(S group),ischemia/reperfusion group(I/R group)and ischemia/reperfusion treated with FNS group(F group). Each group contained four time points of 3d,7d,14d and 28d . There were seven rats in each group at each time point. Single and double immunofluorescence histochemical method was used to identify proliferation and differentiation of neural stem cells in the adult rats which were treated with FNS after ischemia/reperfusion at each time point.(3) Focal cerebral ischemia/reperfusion injury was induced by MCAO method. Adult SD rats were randomly devided into three groups: sham-operation control group(S group),ischemia/reperfusion group(I/R group) and ischemia/reperfusion treated with FNS group(F group). Each group contained two time points of 14d and 28d. There were seven rats in each group at each time point. The mRNA and protein expression of Notch1,Hes5 and Mash1 were assessed by western blotting and RT-PCR and the mRNA and protein expression of NF-кB p65 was assessed by RT-PCR and immunohistochemistry stain.Results(1) The results showed that FNS can increase scores of comprehensive neurological function and decrease brain water content and reduce the volume of infarct volume.(2) Only a small amount of Brdu-labeled cells was found in SVZ and in cortex in the result of immunofluorescence histochemical method in normal control group and sham-operation control group. After three day of focal cerebral ischemia/reperfusion ,t he number of Brdu-labeled cells in SVZ increase significantly(P<0.01) and increased to a peak after seven days of focal cerebral ischemia/reperfusion(P<0.01), then the rate of increase descended after fourteen days and twenty eight days of focal cerebral ischemia/reperfusion(P<0.01). After three day of focal cerebral ischemia/reperfusion, the number of Brdu-labeled cells in cortex increase significantly(P<0.001) and increased to a peak after seven days of focal cerebral ischemia/reperfusion(P<0.001), then the rate of increase descended after fourteen days and twenty eight days of focal cerebral ischemia/reperfusion(P<0.001). Compared with group of focal cerebral ischemia/reperfusion at the time point of three days,seven days,fourteen days and twenty eight days,the number of Brdu-labeled cells in SVZ and in cortex increased more strikingly in the group of focal cerebral ischemia/reperfusion treated with FNS , the difference was significant(P<0.05). There were a very small amount Brdu/DGX- labeled and Brdu/GFAP-labeled cells which were found in normal control group and sham-operation control group and only a small amount of Brdu/DGX- labeled and Brdu/GFAP-labeled cells were found in focal cerebral ischemia/reperfusion group. A large amount of number of Brdu/DGX- labeled and Brdu/GFAP-labeled cells were found in F group. The proliferated neural stems cells were induced to differentiate into glia cells and neurons.(3)Compared with sham operation control group, the mRNA and protein expression of Notch1 , Hes5,Mash1 and NF-кB p65 increased in ischemia/reperfusion group and ischemia/reperfusion treated with FNS group(P<0.01). Compared with ischemia/reperfusion group, the mRNA and protein expression of Notch1,Hes5 and Mash1 increased and the mRNA and protein expression of NF-кB p65 decreased in ischemia/ reperfusion treated with FNS group(P<0.05).Conclusions(1)Focal cerebral ischemia/reperfusion injury model was established successfully. The application of FNS could lower the mortality of rats in the acute stage of ischemia/reperfusion injury.The application of FNS could increase scores of comprehensive neurological function,decrease brain water content and reduce the volume of infarct volume. FNS can protect brain after ischemia/reperfusion injury.(2) There was proliferation of neural stem cells in SVZ and cortex after ischemia/reperfusion injury. Only a small amount of Brdu-labeled cells were found in SVZ and in cortex in adult rats. The proliferation of endogenous neural stem cells in SVZ and cortex gradually increased after ischemia/reperfusion injury,the rate of increase gradually slowed down after the peak of 7d time point. Proliferation of neural stem cells is more obvious after focal cerebral ischemia/reperfusion treated FNS than only focal cerebral ischemia/reperfusion,the rate of increase gradually slowed down after the peak of 7d time point. There were a very small amount Brdu/DGX-labeled and Brdu/GFAP-labeled cells which were found in normal control group and sham-operation control group and only a small amount of Brdu/DGX- labeled and Brdu/GFAP-labeled cells were found in focal cerebral ischemia/reperfusion group. A large number of Brdu/DGX- labeled and Brdu/GFAP-labeled cells were found after focal cerebral ischemia/reperfusion treated FNS. The proliferated neural stems cells were induced to differentiate into neurons and glia cells. FNS could promote the differentiation of neural stem cells to into glial-like cells and neuron-like cells. FNS plays a role in nerve reconstruction of brain tissue by promoting the proliferation and differentiation of neural stem cells.(3) The mRNA and protein expression of Notch1,Hes5,Mash1 and NF-кB p65 was low in adult rats and the mRNA and protein expression of Notch1,Hes5 ,Mash1 and NF-кB p65 increase after ischemia/ reperfusion and ischemia/reperfusion treated with FNS. Compared with only ischemia/reperfusion,there was a significant increase of the mRNA and protein expression of Notch1,Hes5 and Mash1 and a significant de- crease of the mRNA and protein expression of NF-кB p65 after ischemia/ reperfusion treated with FNS. FNS could influence proliferation and differentiation of neural stem cells by promoting the mRNA and protein expression of Notch1,Hes5 and Mash1 and inhibiting mRNA and protein expression of NF-кB p65.