| Background: In recent years,the neurotoxic effects of anesthetics on the nervous system of infants have received extensive social attention.The rapid developmental period of human nervous system is from the third trimester of pregnancy to 2-3 years after birth.During this period,the immature nervous system is very sensitive to the neurotoxic agents.Animal studies have shown that a variety of anesthetics can be neurotoxic to the developmental brain during this period.As one of the most commonly used inhaled general anesthetics,sevoflurane is widely used in the induction and maintenance of pediatric anesthesia due to its unique physical and chemical properties.Currently,many studies have shown that neonatal animals receiving repeated sevoflurane anesthesia can cause learning disabilities and abnormal social behaviors when they grow up,but the specific mechanisms remain unclear.In many large sample of clinical retrospective studies,whether the anesthetic drugs can cause damage to the nervous system in infants is still controversial.This may be related to the type,dosage,time and frequency of anesthetic drugs and also the state of brain development.In addition,some experimental studies also found that the nervous system contains abundant miRNA,which is closely related to the occurrence and development of neural system,neural stem cell differentiation,synaptogenesis and dendritic spines formation.In this experiment,the effect of a brief exposure to sevoflurane anesthesia on the brain development of children with cleft palate was investigated by clinical observation.After that,we investigated the role of miR-27a-3p/PPAR gamma signaling pathway in hippocampal neuronal apoptosis induced by repeated sevoflurane anesthesia during brain development in vitro and in vivo.The purpose of this study is to answer whether sevoflurane may have neurotoxic effects on the developmental brain and its specific mechanism.This study is composed of four parts as follows:Part 1: The preliminary study of neurodevelopmental outcomes after a brief exposure to sevoflurane anesthesia in infants Objective: To observe the impact of a brief exposure to sevoflurane anesthesia on the neurodevelopment in infants.Methods: Young children(less than 2 years old)scheduled for palatoplasty were enrolled in this study and sevoflurane was used as the only anesthetic drug for induction and maintenance of anesthesia.In this study,we allowed each patient to serve as their own control.The levels of plasma NSE and S-100β before and after the surgery were compared.The Bayley Scales of Infant Development-Second Edition(BSID-II)scores before,6 months and 18 months after surgery were also compared to assess neurodevelopmental outcomes.Results: A total of 101 children completed 6 months’ follow-up and 86 children completed 18 months’ follow-up.There was no statistically significant difference in plasma NSE,S-100β and BSID-II scores before and after the operation.Conclusion: A brief exposure(less than 2 hours)to sevoflurane anesthesia had no obvious negative effects on the development of the central nervous system in infants.But its longterm effects remain to be further examined.Part 2: Primary culture and purity identification of mice hippocampal neurons Objective: In vitro the primary hippocampal neurons of mice were cultured and the purity were identified which lay the foundation for further investigating the mechanism of sevoflurane-induced apoptosis in hippocampal neurons.Methods: The hippocampal neurons were isolated and cultured from embryonic day 14 C57BL/6 mice embryos.Cell morphology was observed by light microscope and the purity of cultured mouse neurons was identified by immunofluorescence cytochemistry(beta III tubulin antibody and DAPI double fluorescence staining).Results: The purity of primary cultured hippocampal neurons of mice in vitro was 94.3 +1.9%.Conclusion: In this part of experiment,we successfully obtained the primary hippocampal neurons of mice and completed cell culture in vitro.We got the high purity hippocampal neurons by identification which laid a solid foundation for further experiments.Part 3: The regulatory role of miR-27a-3p/PPARγ in sevoflurane-induced cell damage of mice hippocampal neurons.Objective: To explore the regulatory role of miR-27a-3p/PPARγ in sevoflurane-induced cell damage of mice hippocampal neurons in vitro.Methods: The hippocampal neurons were cultured for seven days in vitro and then randomly divided into four groups: Control,4%Sevo,4%Sevo+inhibitor,4%Sevo+RSG.The treatment duration of sevoflurane was six hours and the final concentration of rosiglitazone was 5μM.The apoptosis rate of hippocampal neurons was detected by flow cytometry and ROS were measured using DHE fluorescent probe.The expressions of NOX1 and NOX4 proteins were analyzed by western blot and the miR-27a-3p was measured using real-time PCR.The content of IL-6,IL-1β and TNF-α in supernatant of culture medium was detected by ELISA method.The targeting relationship between miR-27a-3p and PPAR gamma was detected by double luciferase.Results: Compared with the Control group,4% sevoflurane treated neurons 6h could increase cell apoptosis rate,intracellular ROS content,miR-27a-3p,NOX1 and NOX4 expression levels,as well as the inflammatory factors IL-6,IL-1β and TNF-α.With adding miR-27a-3p inhibitor or rosiglitazone,these changes could be partially inhibited.But rosiglitazone treatment did not inhibit the increase of miR-27a-3p expression in neurons induced by sevoflurane.In addition,the targeting relationship between miR-27a-3p and PPAR gamma did exist.Conclusion: The miR-27a-3p/PPAR gamma signaling pathway mediated the damage of hippocampal neurons induced by sevoflurane treatment and the effect may be through increasing oxidative stress and inflammatory response.Part 4: The regulatory role of miR-27a-3p/PPARγ in sevoflurane-induced hippocampal nerve cells damage of mice during the brain growth spurt Objective: To explore the regulatory role of miR-27a-3p/PPARγ signal pathway in sevoflurane-induced hippocampal nerve cells damage of mice during the brain growth spurt in vivo.Methods: Postnatal day 6 mice pups were randomly divided into four groups: Control,simiR-27a-3p,Sevo,simiR-27a-3p+Sevo.The concentration of sevoflurane was 2.2% and the duration of anesthesia was 2h per day for 3 consecutive days.On postnatal day10 and 45,the apoptosis rate of hippocampal nerve cells was detected by TUNEL detection and ROS were measured using DHE fluorescent probe.The miR-27a-3p of each group was measured using real-time PCR and the expression of PPARγ protein was analyzed by western blot.The content of IL-6,IL-1β and TNF-α in hippocampal tissue homogenate was detected by ELISA.The learning and memory ability of mice in each group was compared by Morris water maze in their adulthood.Results: Compared with the Control group,repeated sevoflurane anesthesia could increase the apoptosis of hippocampus and the expression of miR-27a-3p and ROS,decrease the expression level of PPAR gamma protein and decrease the ability of learning and memory in adult mice.The inflammatory factors IL-6,IL-1β and TNF-α release were also increased after multiple exposure to sevoflurane.By interfering of miR-27a-3p,these changes could be partially suppressed and the learning and memory ability after multiple sevoflurane anesthesia could be improved.Conclusion: Repeated sevoflurane anesthesia could increase the expression of miR-27a-3p which inhibited the expression of PPAR gamma protein in the hippocampus of mice,cause the enhancement of oxidative stress and inflammatory response and lead to the decline of the learning and memory ability in their adulthood. |
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