The Study Of Treatment For Radiation-Induced Cognitive Dy Sfunction Through Brain-Derived Neurotrophic Factor-Overexpressing Neural Stem Cells Transplantation

Part I.Isolation and culture,lentivirus infection and pre-transplant identification of fetal rat neural stem cellsPurpose/Objective(s):Transplantation of neural stem cells(NSCs)has great potential in the treatment of radioactive cognitive dysfunction.Isolation and culture of neural stem cells remains the basis for transplantation therapy.Lentiviral infection with a brain-derived neurotrophic factor(BDNF)gene is used for the overexpression of brain-derived neurotrophic factors to prepare for subsequent studies.Materials/methods:Neural stem cells from the cerebral cortex of Sprague Dawley fetal rats aged 14 to 15 days were cultured,and then cells were infected with a lentivirus carrying green fluorescent protein(GFP)and BDNF gene.The cultured neural stem cells were identified using nestin immunofluorescence staining technique.Results:Cultured neural stem cells were successfully labeled with GFP after infection with two kinds of lentiviruses(GFP-lentivirus,GFP-BDNF-lentivirus).Moreover,the expression of BDNF protein in neural stem cells infected with GFP-BDNF-lentivirus was significantly increased.These neural stem cell spheres were positive for nestin immunofluorescence staining.Conclusion:Neural stem cells overexpressing BDNF can be successfully obtained after infection with GFP-BDNF-lentivirus,which makes preparations for the next part of the experiment.Part II.Study of migration,differentiation and integration into neural circuits after neural stem cells transplantationPurpose/Objective(s):To what extent the neurons derived from transplanted neural stem cells can be integrated into the hippocampal neural circuit remains unclear.In this part of the research,we will investigate whether changes in the microenvironment of rat hippocampus after radiation exposure affect the migration,differentiation and integration of BDNF-overexpressing neural stem cells.Materials/methods:1).Rats received a single dose of 20 Gy whole brain irradiation.After 4 weeks,neural stem cells overexpressing BDNF were transplanted into rat hippocampus for observation of the migration and differentiation of transplanted neural stem cells.2).Four weeks after the rats were delivered a single whole brain irradiation for 20 Gy,the neural stem cells transfected with helper lentivirus were transplanted into the hippocampus of the rats.Ten days after transplantation,rabies virus was injected into the left hippocampus of rats for retrograde monosynaptic tracing experiment.The integration of transplanted nerve cells into the preexisting neural circuit was evaluated.Results:1).The GFP-NSCs transplanted into the hippocampus of the control group migrated and colonized in the subgranular zone of dentate gyrus.The GFP-BDNF-NSCs transplanted into the hippocampus of the whole brain irradiation group migrated to the ubgranular zone.The GFP-NSCs transplanted into the hippocampus of the whole brain irradiation group were disordered distributing in irradiated hippocampus,and showed more astroglia differentiation tendency.2).BDNF-NSCs can establish synaptic connections with the original neural circuit after transplantation into the hippocampus of the whole brain irradiation group The integration efficiency is significantly higher than that of the R+GFP-NSCs group.Conclusion:Neural stem cells overexpressing BDNF can migrate and colonize in the subgranular zone of dentate gyrus after whole brain radiation,and establish synaptic connection with the preexisting neural circuit.Part II.Study on the changes of cognitive function and hippocampus microenvironment in rats after neural stem cell transplantationPurpose/Objective(s):There is an interaction between the neural stem cell transplantation and the microenvironment of the host.In this part of the study,we will investigate the effects of BDNF-NSCs transplanted into the brain of rats after radiation exposure on the hippocampal microenvironment.At the same time,the changes of cognitive function in rats after neural stem cell transplantation will be detected.Materials/methods:1).Four weeks after a single brain irradiation of 20 Gy,neural stem cells overexpressing BDNF were transplanted into the hippocampus of rats.The expression of BDNF,nerve growth factor and glial cell line-derived neurotrophic factor were detected in the hippocampus of the transplanted rats.The number of activated microglia was also examined.2).Four weeks after a single brain irradiation of 20 Gy,,neural stem cells overexpressing BDNF were transplanted into the hippocampus of rats,and Morris water maze test was used to detect the changes of cognitive function in rats after transplantation.Results:Compared with the whole brain irradiation group,the expression of BDNF and NGF in the hippocampus of R+GFP-BDNF-NSCs group was significantly increased.No significant reduction of the number of activated microglia was observed.There was no significant improvement in cognitive function after neural stem cell transplantation.Conclusion:Transplantation of neural stem cells overexpressing BDNF into the hippocampus of rats exposed to whole brain radiation can increase the expression level of trophic factors in hippocampus,but it can not significantly improve the cognitive function of rats.