Embryonic Neural Stem Cell Mproved Neuron Survival In The Mouse Brain Slice By Regulation Of Microglia Activity

Neuronal loss is one of the hallmarks in most neurodegenerative diseases (ND), such as Alzheimer’s disease and Parkinson’s disease. Studies in patients with PD have provided proof that neuronal replacement using neural stem cells (NSCs) for the source to generate specialized cells is believed to be a promising therapy method. However, for the ND which massive neuronal loss occurs in large parts of the brain, the NSCs cell replacement therapy is considered to be less positive. Further, many studies showed that the neuron metabolic changes are prior to the neuronal loss. Thus, the therapy to rescue neurons from degeneration and loss might be more attractive and prospective. It is interesting that the neuroprotective effect of NSCs was observed in many neurodegenerative model. Activated microglia are present in large numbers in CNS tissue from patients with ND, therefore, whether neural stem cells play an important role in inducing microglia activation without inflammation?In this study, first, the prefrontal cortex tissue slices (n=16) in experimental mice will be separation cultured with the mouse embryonic neural stem cells (14days after pregnancy) in vitro. The results show that:Neural stem cells improved the neuron survival in the brain sliceIn contrast with control groups, the number of the survival cell increased significantly (p<0.001), and the number of the death cell reduced significantly (p <0.001) in the NSCs co-culture group, also the total number of the cell decreased significantly (p<0.001); the number of the dead cell and the leaky cell among the total cell decreased with the time. The ratio of survival cell among the total cell increased with the time.Neural stem cell promote microglia activity in the brain slice①The beneficial effect of NSCs on neuronal survival was abrogated by a microglial inhibitor minocycline, while was mimicked by a microglial agonist, Toll-like receptor9(TLR9) ligand CpG-ODN,②In the stem cell co-cultured group, the ratio of Locomotory microglia was higher than in control group, but the ratio of the motile microglia was less that the control group.③The activity of Marker IBA1was measured by Real-time Quantitative PCR and Western blotting. In stem cell co-cultured groups, the protein and the mRNA level of IBA1were significantly higher than the control group; Immunohistochemistry results showed that more IBA1positive microglia cells were observed in the stem cell co-cultured brain slices; the phosphorylation levels of ERK1/2in co-cultured group was significant higher than in control group.④Real time PCR revealed that NSCs inhibited the expression of proinflammatory molecules, whereas significantly increased the expression of molecules associated with a neuroprotective phenotype such as CX3CR1, triggering receptor expressed on myeloid cells-2(TREM2) and insulin growth factor1(IGF-1).NSCs induced microglial activation via TLR9-ERK1/2pathway①Real time quantitative PCR revealed that TLR9mRNA level but not TLR4, TLR2was significantly higher in the NSCs co-cultured brain slices compared with that in the mock-treated group②Western analyses indicated that the ERK1phosphorylation level was increased significantly in the NSCs co-cultured group③Interestingly, a significant correlation was found between TLR9and IBA-1(pearson r2=0.5486,*p<0.05, Fig.5C), p-ERK1and IBA-1(pearson r2=0.845,**p<0.01, Fig.5D) respectively.④The results displayed that3-days NSCs co-culturing increased TLR9and IBA-1protein expression, and enhanced ERK1phosphorylation of BV2cells, whereas,5μM chloroquine treatment abrogated this effect of NSCs,10μM U0126treatment decreased NSCs-induced ERK1/2activation and IBA-1expression, but not the TLR9expression. Furthermore, incubation with5μM TLR9ligand CpG-ODN1668for24h significantly increased the protein expression of TLR9, phosphorylated ERK1and IBA-1in primary microglia cells. CpG-ODN1668and5μM chloroquine simultaneous treatment decreased CpG-ODN1668induced ERK1/2activation, IBA-1and TLR9expression, whereas, CpG-ODN1668and10μM U0126simultaneous treatment decreased CpG-ODN1668induced ERK1/2activation and IBA-1expression, but not the TLR9expression. The same result was found in BV2cell line.In this study, particular isolated co-cultured method is used in the cultured process of the brain tissue in vitro, which helps to avoid the effect of cell transformation in transplantation experiment. Our study indicated that the neural stem cells could promote the neuron survival of the cultured brain slice cells, and the function is related to the activity of the microglia. The detailed mechanism under this phenomenon needs further study and this result will provide a new insight in the research of neuronal cell survival under neurodegenerative disease.