Research On Effects And Mechanism Of Neural Stem Cell-mediated Hippocampal Synaptic Plasticity Abnormality Induced By Microwave Radiation

Objective:With the popularization of microwave technology,the damage to human health has attracted more and more attention.Brain is one of the sensitive target organs of microwave radiation.The neural stem cells(NSCs)in the subgranular region of the dentate gyrus of the hippocampi can differentiate into neurons and participate in the hippocampal neural circuits.Brain derived neurotrophic factor(BDNF)plays a central role in promoting the proliferation,differentiation and migration of NSCs and regulating synaptic plasticity.Fat mass and obsity-associated gene(FTO)may regulate BDNF protein expression by regulating m RNA m~6A modification.In this study,based on the establishment of a model of NSCS-mediated synaptic plasticity abnormality induced by microwave radiation,FTO and BDNF were used as the breakthrough points to explore its mechanism from the perspective of m RNA m~6A modification,so as to provide a new candidate target for the prevention and treatment of microwave radiation brain injury,in the co-culture model,CCK-8 was used to detect the effect of microwave radiation on the proliferation ability of NSCs cultured in vitro..Methods:Secondary male Wistar rats were randomly divided into two groups:the radiation group and the control group.The S-band radiation simulation source was used for radiation with an average power density of 30m W/cm~2.The rats were irradiated for 4 days,3 times per day,6minute per time,with each interval of 6 minute.Immediately after radiation to the 11th day,Morris water maze and new object recognition test were used to detect the learning and memory ability of rats.At 6h,7d,14d and 28d after radiation,the mossy fibers sprouting in hippocampus was observed by Timm staining,and the ultrastructure of hippocampal synapses was observed by transmission electron microscopy.The proliferation and differentiation ability of hippocampal NSCs were detected by co-labeling immunofluorescence.The m RNA expressions of BDNF and FTO were detected by real-time fluorescence quantitative PCR.The protein expressions of BDNF and FTO were detected by western blot.The methylation level of total m6A in rat hippocampi was detected by colorimetry.The co-culture model of neural stem cells and primary hippocampal neurons in vitro was established by Transwell compartment,CCK8 was used to detect the proliferation ability of cultured NSCs in vitro at 1d,3d and 5d after microwave irradiation.Results:(1)Learning and memory ability:Compared with the control group(0m W/cm~2),in the Morris water maze experiment,the average escape latency of rats in the radiation group was prolonged,while the numbers of platform crossing and the swimming time in the target quadrant were decreased(P<0.05 or P<0.01);the recognition index in the new object recognition test was declined.(2)Synaptic structure:at 14d and 28d after radiation,the hippocampal mossy fiber sprouting were decreased(P<0.05).At 6h and 14d after radiation,the length and thickness of the postsynaptic density in the hippocampus were decreased(P<0.05 or P<0.01).(3)Proliferation and differentiation ability of NSCs:at 6h,7d,14d and 28d after radiation,the number of Brd U and Ki67positive cells decreased(P<0.05 or P<0.01);At 6h after radiation,the proportion of Brd U and Nestin co-labeled positive cells to Brd U positive cells decreased(P<0.01);At 14d and 28d after radiation,the proportion of Brd U and Neu N co-labeled positive cells to Brd U positive cells decreased(P<0.01);At 6h,7d and 14d after radiation,the proportion of Brd U and DCX co-labeled positive cells to Brd U positive cells decreased(P<0.05 or P<0.01).(4)BDNF and FTO expression and m~6A methylation level of total m RNA:m RNA and protein expression of BDNF decreased at 7d and 28d after radiation(P<0.05 or P<0.01);at 14d,BDNF m RNA expression increased(P<0.05).The expression of FTO m RNA and protein increased at 6h after radiation(P<0.05),but FTO m RNA decreased at 14 and 28 days after radiation(P<0.05).At 6h and 14d after radiation,the level of m RNA m~6A methylation in hippocampus decreased(P<0.05 or P<0.01).(5)Co-culture of NSCs and hippocampal neurons:in vitro co-culture,neural stem cells grew normally in a spherical shape,with clear boundaries and good refractive properties.The hippocampal neurons were full and developed,rich synapses forming a dense neural fiber network.at 1d,3d and 5d after microwave radiation,the CCK-8optical density of NSCs in the radiation group decreased significantly(P<0.01).Conclusion:(1)30m W/cm~2microwave radiation can induce spatial learning and memory impairment,synaptic structure abnormity in hippocampal DG region,showing abnormal distribution of postsynaptic dense matter,and inhibition of mossy fiber sprouting due to reduced proliferation and differentiation ability of NSCs.(2)The abnormal expression of BDNF m RNA and protein after 30m W/cm2 microwave radiation is an important reason for the abnormal synaptic plasticity in rat hippocampus mediated by the reduced proliferation and differentiation of NSCs.The decrease of total m RNA m~6A methylation level caused by up-regulation of FTO may be the mechanism of abnormal BDNF expression.(3)The co-culture cell model of NSCs and primary hippocampal neurons was successfully established to provide an in vitro experimental model for the study of the molecular mechanism of NSCs mediated synaptic plasticity abnormality induced by microwave radiation.