| In the United States,about 1.5 million newborns and infants are subjected to general anesthesia every year.Considering the huge population of China,the demands of anesthesia for infants and young children are increasing rapidly.A large number of reports have pointed out that general anesthesia will affect the development of nervous system in rodents and non-human primates,and induce the dysfunction of long-term cognitive.The FDA has cautioned about the potential harmful effects of repeated general anesthetics on brain development of children less than 3 years old.However,several subsequent large scale clinical trials showed that the use of general anesthesia did not cause significant decline in long-term learning and memory.Different research results lead to the controversy on whether general anesthetics can cause neurodevelopmental abnormalities.These disputies will delay the implementation of protective measures that could have been carried out among children,resulting in a series of serious consequences.Sevoflurane is one of the most widely used inhaled anesthetics in children.Many research results derived from animal testings,including our previous study,have shown that repeated or lengthy exposure to sevoflurane in the neonatal period can lead to a variety of neurotoxicities,such as apoptosis,ROS stress,neurogenesis disorder,synaptic injury and inflammation.In related human studies,some reports pointed out that exposure to sevoflurane during development had no significant effect on long-term learning ability and intellectual development,while others pointed out that learning and memory ability were impaired after sevoflurane exposure in early childhood.Due to the particularity of the use of anesthetics,most human anesthetic neurotoxicity studies are retrospective, resulting in no definitive answer to the neurotoxicity of sevoflurane.In view of the limitations of clinical researches,more and more experts and scholars realize the importance of the application of animal research results in clinical practice.In the study of anesthetic neurotoxicity,due to there are too many uncertain factors in directly transforming the experimental results of animal researches into clinical application,it is of great significance to directly use human cells or tissues for experiments rather than animal models for the study of anesthetic neurotoxicity.In order to reveal the toxic effect of sevoflurane on human neural cells,we used human embryonic stem cell to study the toxic effect of sevoflurane exposure on human neural precursor cells in vitro and in vivo.This study consists of the following four parts.Part Ⅰ:Induction and differentiation of human neural precursor cells and construction of chimeric mice implanted with human neural precursor cellsObjective:To establish a technical method for studying the neurotoxicity of anesthesia on human neural cells in vivo and in vitro.Methods:Human embryonic stem cells were cultured in m Te SR medium without feeder layer.Embryonic stem cells were induced to differentiate into neural precursor cells by dual inhibition of SMAD signaling for 14 days in vitro.The induction efficiency of neural precursor cells was evaluated by immunofluorescence and Western Blot.In in vivo study,neural precursor cells were injected into immunodeficient Nod Scid mice to obtain human neural cell chimeric mice.The distribution and differentiation of human neural precursor cells in the mouse brain were observed by fluorescence tracing and immunofluorescence staining.Meanwhile,the molecular mechanism of the neurotoxicity of sevoflurane on the migration ability of human neural precursor cells was investigated by high-throughput sequencing,Western Blot,RT-PCT and other methods.Results:(1)Under maintenance culture conditions,human embryonic stem cell line H9 and GFP~+human embryonic stem cell line H8 keep good cell viability,and the efficiency of embryoid body formation is high.After 14 days of neural induction,more than 80%of human embryonic stem cells expressed Pax6,Nestin and Sox2,while the positive rate of embryonic stem cell marker protein Oct4 decreased to 13%.After 28 days of induction,more than 80%were tuj-1 positive,VGLUT2 positive and Ca MKII positive excitatory neurons.(2)Neural precursor cells were successfully transplanted into neonatal immunodeficient Nod Scid mice.Human neural precursor cells integrated,migrated and differentiated into neurons in the mouse brain.Adult human neurons of chimeric mice were widely distributed in various brain regions such as mouse cortex and hippocampus.Conclusion:Human neural precursor cell induction were successfully established and human neural precursor cell chimeric mice were obtained,which can be used to study the effects of anesthetics in vitro and in vivo.Part Ⅱ:The neurotoxicity of sevoflurane on human neural cells in vitroObjective:To study the neurotoxicity of sevoflurane on human neural precursor cells in vitro and determine the sensitive stage of human neural precursor cells to sevoflurane during differentiation.Methods:Neural precursor cells at different differentiation stages were exposed to sevoflurane(4.1%sevoflurane,6 hours)in vitro.Apoptotic and necrotic cells were detected by TUNEL and PI staining respectively.Immunofluorescence staining and Western Blot were used to observe the neurotoxicity of sevoflurane on the differentiation of neural precursor cells at different developmental stages.After finding the sensitive period of neurotoxicity caused by sevoflurane,immunofluorescence and Western Blot were used to study the neurotoxicity of sevoflurane on the synaptic development of human neurons,and morphological study were used to observe the neurotoxicity of sevoflurane on the migration ability of human neural precursor cells.Results:(1)In the initial stage of neural precursor cells,4.1%sevoflurane exposure did not significantly cause apoptosis and necrosis of human neural stem cells,nor did it significantly affect the differentiation of neural precursor cells into neurons.(2)Compared with the control group,sevoflurane exposure had no significant effect on the apoptosis, necrosis and proliferation of neural precursor cells,but in the experimental group,the number of human neural precursor cells differentiated into neurons decreased significantly,the expression levels of neuronal marker proteins tuj-1 and Ca MKII decreased significantly,the expression of synapse associated protein PSD-95 decreased,and the migration number and distance of human neural precursor cells decreased significantly.The results of transcriptome sequencing showed that the genes related to cell differentiation and migration were significantly changed,and the non-canonical Wnt/β-catenin signal pathway closely related to migration was inhibited.Conclusion:Sevoflurane exposure has no significant effect on the survival of human neural precursor cells,and it can inhibit the differentiation of neural precursor cells into neurons and their migration ability.Part Ⅲ:The neurotoxicity of sevoflurane exposure on human neural precursor cells in vivoObjective:To study the toxic effect of sevoflurane exposure on human neural cells in vivo.Methods:Chimeric mice were exposed to sevoflurane 1 week after birth(3%sevoflurane,2 hours each time for 3 days),or chimeric mice were constructed after sevoflurane exposure of human neural precursor cells in vitro(4.1%sevoflurane,6 hours).The differentiation of human nerve cells in mouse brain after sevoflurane exposure was observed by fluorescence tracing,immunofluorescence staining and Western blot.The effect of sevoflurane exposure on the migration of human nerve cells was studied by fluorescence staining.Results:Compared with the chimeric mice in the control group,at the age of 2 months,there was no significant difference in the differentiation and synaptic formation of human neural cells in the brain of chimeric mice treated with or without sevoflurane,but the migration of human neural precursor cells in the brain of chimeric mice treated with sevoflurane decreased significantly.Conclusion:Sevoflurane exposure can inhibit the migration of human neural precursor cells in chimeric mice,without affecting the differentiation of human neural precursor cells into neurons and the formation of synapses.Part Ⅳ:Behavioral study of sevoflurane exposure on chimeric mice implanted with human neural precursor cellsObjective:To study the effect of sevoflurane exposure on neurocognitive function of chimeric mice implanted with human neural precursor cell.Methods:Chimeric mice were exposed to sevoflurane 1 week after birth(3%sevoflurane,2 hours each time for 3 days),or chimeric mice were constructed after sevoflurane exposure of human neural precursor cells in vitro(4.1%sevoflurane,6 hours).Many behavior tests such as Open Field,Fear Memory and Novel Object Recognition were used to observe the effects of sevoflurane exposure on the learning and memory and anxiety related emotions of adult chimeric mice.Results:In behavioral test,sevoflurane treatment significantly reduced the freezing time in context and clue fear memory test of wild-type mice as well as the novel object exploration time in Novel Object Recognition experiment.However,compared with chimeric mice in the control group,the freezing time and novel object exploration time of sevoflurane-treated chimeric mice did not change significantly.In the open field experiment,compared with wild-type mice,chimeric mice spent significantly longer time in the central area.Sevoflurane treatment significantly reduced the time and distance of wild-type mice in the central area.However,in comparison with those in the chimeric control group,the time and moving distance of chimeric mice in the central area in sevoflurane treatment group did not change significantly.Conclusion:Human neural precursor cell chimerism increases the exploration tendency of mice.Sevoflurane exposure has no significant effect on the learning and memory of adult chimeric mice implanted with human neural cells. |
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