Role And Mechanism Of IL-17A In Developmental Neurotoxicity Induced By Repeated Sevoflurane Exposure

Sevoflurane is one of the most widely used halogenated inhaled anesthetics in the induction and maintenance of general anesthesia in infants and young children.It has always been a hot topic of medical and social debate whether inhaling anesthetics repeatedly during infancy and childhood will affect long-term learning and cognitive abilities.However,the specific mechanism of sevoflurane’s neurotoxicity to the developing brain is far from clear.Interleukin 17A（IL-17A）is mainly secreted by Th17 cells.It is the first and most important member of the IL-17 family and plays an important role in host defense reaction and neuroinflammation.We recently reported that IL-17A gene knockout can inhibit hippocampal neuroinflammatory response level and alleviate the developmental neurotoxicity of sevoflurane.In addition,recent studies have shown that IL-17 can cause cognitive and synaptic defects in the early stage of Alzheimer’s disease.Although the immunopathological role of IL-17A has been explored,its potential role in the neurotoxicity of sevoflurane to the developing brain has not been fully clarified.Therefore,in this study,we used WT mice and IL-17A gene knockout mice to explore the mechanism of IL-17A in the long-term cognitive impairment of neonatal mice induced by repeated sevoflurane exposure through transcriptome sequencing,GO and KEGG enrichment analysis,PPI network construction and Hub gene extraction,immunofluorescence,Golgi staining and other technical methods.Its research results are expected to reveal the new mechanism of sevoflurane-induced developmental neurotoxicity,Provide new targets for clinical prevention.Part one Effect of repeated sevoflurane exposure on long-term cognitive function and gene expression profile of hippocampal CA1 region in neonatal miceObjective:The aim of this study was to investigate the effect of repeated sevoflurane exposure on long-term cognitive function and gene expression profile of hippocampal CA1 region in neonatal mice.Method:Twelve 6-day-old healthy male wild-type（WT）mice,weighing6-8g,were divided into two groups:control group（Group WT）and sevoflurane group（Sev group）（n=6）.Twelve 6-day-old healthy male IL-17A knockout（KO）mice,weighing 6-8g,were divided into two groups:IL-17KO+sevoflurane group(IL-17A^-/-+Sev)and IL-17KO group(IL-17A^-/-group)（n=6）.Sev and IL-17A^-/-+Sev group mice were exposed to 3%sevoflurane for 2 hours on the 6th,7th and 8th day after birth for 3consecutive days.Group C and IL-17A^-/-group mice were exposed to 30%humidified oxygen.The spontaneous motor function of mice was detected by open-field test on the 29th day after birth.Morris water maze was used to detect the long-term cognitive function of mice from 30 to 36 days after birth.After the experiment,the tissues in the CA1 area of mouse hippocampus were taken for transcriptome analysis.Results:At 29 days after birth,there were no differences in spontaneous motor function between the WT and Sev group,as well as between the IL-17A^-/-+Sev and IL-17A^-/-group.Compared with group WT,the escape latency of Sev group mice was extended at 32-36 days after birth,and the number of crossing platforms was decreased.The results of transcriptome sequencing showed that there were 131 differential genes between group WTand group Sev,such as chemokine activity,immune response,extracellar region,extracellar space and inflammatory response,and other biological processes were significantly enriched.The pathways such as IL-17 signaling pathway,chemokine signaling pathway,cyclokine-cytokine receptor interaction,ECM-receptor interaction and influenza A were significantly enriched.Compared with IL-17A^-/-+Sev group,the escape latency of IL-17A^-/-group mice was shortened 33-36 days after birth,and the number of times of crossing the platform was increased.Transcriptome sequencing results showed that biological processes and pathways related to neuroinflammatory response and synaptic plasticity were not enriched.Conclusion:Repeated sevoflurane exposure during development can lead to long-term cognitive dysfunction in newborn mice,and its mechanism may be related to IL-17A-mediated neuroinflammatory response and synaptic plasticity changes in hippocampal CA1 region.Part two Effect of IL-17A knockout on long-term cognitive impairment in neonatal mice induced by repeated sevoflurane exposure and the role of NF-κB signal pathway in itObjective:The purpose of this study was to explore the effect of IL-17A knockout on long-term cognitive impairment of neonatal mice induced by repeated sevoflurane exposure and the role of NF-κB signal pathway in it.Method:Sixty 6-day-old healthy male wild-type（WT）mice weighing6-8g were divided into two groups:control group（WT group）and sevoflurane group（Sev group）（n=30）.Thirty 6-day-old healthy male IL-17A knockout（KO）mice weighing 6-8g were assigned to the IL-17KO+sevoflurane group(IL-17A^-/-+Sev).Sev and IL-17A^-/-+Sev group mice were exposed to 3%sevoflurane for 2 hours on the 6th,7th and 8th day after birth for 3consecutive days.Mice in WT group were exposed to 30%humidified oxygen.The spontaneous motor function of mice was detected by open-field test on the 29th day after birth.Morris water maze was used to detect the long-term cognitive function of mice 30-36 days after birth.After the experiment,the tissues in the CA1 area of the mouse hippocampus were taken,and the number of Nissl corpuscles in the hippocampal neurons was measured by Nissl staining,and the pathological changes in the CA1 area of the hippocampus were evaluated by HE staining.The expression of NF-κB signal pathway was evaluated by western blot and immunofluorescence,and the level of IL-1βand IL-6 was evaluated by ELISA.Results:There was no difference in spontaneous motor function among the three groups on the 29th day after birth.Compared with WT group,the escape latency of Sev group mice was prolonged 32-36 days after birth,and the number of crossing platforms was reduced.The degree of pathological damage in hippocampal CA1 area was aggravated,the number of Nissl body was significantly reduced,and the protein associated with NF-κB signal pathway,the expression level of IL-1βand IL-6 increased significantly.Compared with Sev group,the escape latency of IL-17A^-/-+Sev group mice was shortened 32-36 days after birth,and the number of crossing platforms was increased.The degree of pathological damage in hippocampal CA1 area was reduced,the number of Nissl body was significantly increased,and the protein associated with NF-κB signal pathway,the expression level of IL-1βand IL-6 was significantly decreased,the difference is statistically significant（P<0.05）.Conclusion:IL-17A knockout can improve long-term cognitive impairment of neonatal mice induced by repeated sevoflurane exposure,and its mechanism may be related to inhibiting the activation of NF-κB signal pathway and reducing neuroinflammatory reaction.Part three Effects of IL-17A knockout on long-term cognitive dysfunction in neonatal mice induced by repeated sevoflurane exposure and the role of hippocampal synaptic plasticity in itObjective:To investigate the effects of IL-17A knockout on long-term cognitive dysfunction in neonatal mice induced by repeated sevoflurane exposure and the role of hippocampal synaptic plasticity in it.Method:Fourty neonatal male WT mice were divided into control group（WT group）and sevoflurane group（Sev group）and twenty IL-17A knockout mice were assigned to an IL-17KO+sevoflurane group(IL-17A^-/-+Sev group).Cognitive impairment models were prepared by exposing to 3%sevoflurane for 2 hours on the 6th,7th,and 8th days after birth for 3 consecutive days.The spontaneous movement of rats was evaluated through open field experiments;Morris water maze assessed changes in learning and memory abilities;Western blot was used to evaluate the expression of IL-17A,PSD-95,and synaptophysin proteins;Golgi staining was used to detect the synapses in the hippocampus of mice.Results:On the 29th day after birth,there was no difference in spontaneous motor function among the three groups of mice.Compared with WT group,mice in Sev group had an extended escape latency at 32-36 days after birth,a reduced number of times of crossing the platform,a decreased dendritic length,dendritic ridge density,and the number of intersections between dendrites and concentric circles in the hippocampal CA1 region.The expression level of IL-17A protein was significantly upregulated,while the expression levels of PSD-95 and synaptophysin protein were significantly downregulated.Compared with Sev group,mice in IL-17A^-/-+Sev group had shorter escape latency,increased number of crossing platforms,and increased dendritic length,dendritic ridge density,and the number of intersections between dendritic cells and concentric circles in hippocampal CA1 region.IL-17A protein expression level was significantly decreased,while PSD-95and synaptophysin protein expression levels were significantly increased in IL-17A^-/-+Sev group.Conclusion:IL-17A knockout can improve long-term cognitive impairment in newborn mice induced by repeated sevoflurane exposure,and its mechanism may be related to improving synaptic plasticity in the CA1region of the hippocampus.Part four TLR3 knockout can inhibit the production of IL-17A and improve long-term cognitive impairment caused by repeated sevoflurane exposure in neonatal miceObjective:To investigate the effects of TLR3 knockout on long-term cognitive impairment and IL-17A expression levels in the hippocampus of neonatal mice induced by repeated sevoflurane exposure.Method:Fifty neonatal male WT mice were divided into control group（WT group）and sevoflurane group（Sev group）and twenty five TLR3knockout mice were assigned to an tlr3+sevoflurane group(TLR3^-/-+Sev group).Cognitive impairment models were prepared by exposing to 3%sevoflurane for 2 hours on the 6th,7th,and 8th days after birth for 3consecutive days.Morris water maze assessed changes in learning and memory abilities;Nissl staining was used to detect the morphology of hippocampal neurons,HE staining was used to evaluate pathological changes,transmission electron microscopy was used to detect the ultrastructure of neurons;Western blot was used to evaluate the expression of TLR3、p-TRIF、SHP2 and p-RIP3;ELISA was used to detect the IL-17A level.Results:Compared with WT group,mice in Sev group had an extended escape latency at 32-36 days after birth,a reduced number of times of crossing the platform.The pathological damage in hippocampal CA1 region was worsen,with an increase in the number of atrophic and degenerated cells.The expression levels of LR3,p-TRIF,and p-RIP3 were increased,while the expression level of SHP2 was decreased.Compared with Sev group,mice in TLR3^-/-+Sev group had shorter escape latency,increased number of crossing platforms.The degree of pathological damage in the CA1 region of the hippocampus is reduced,the number of atrophic and degenerated cells is reduced,the expression levels of LR3,p-TRIF,and p-RIP3 are significantly reduced,and the expression level of SHP2 is increased.Conclusion:TLR3 knockout can inhibit the phosphorylation of RIP3,reduce the expression level of IL-17A,and improve long-term cognitive impairment in neonatal mice induced by repeated sevoflurane exposure.