Effect And Related Mechanism Of Mid-trimester Sevoflurane Exposure On Off-spring Rat Neural Stem Cell Differentiation

Objectives:With the constant development of surgery technologies and anesthesiology,the number of surgery during pregnancy is constantly increasing and the demand of anesthesia during pregnancy is continuously growing.Inhalational anesthetics is the primary choice of intrauterine surgery and the topic of anesthesia-induced neurotoxicity is the focus of anesthesiology and society.Mid-trimester used to be the relatively safe period of surgery that most of non-obstetric surgery during pregnancy was performed during midtrimester to perform some fetal interventions and treatments.But with numerous animal studies showing that inhalational anesthetics can influence the developing brain and cause long-term neurocognitive impairment,people gradually realized that the relatively safe period of non-obestetric surgery during pregnancy is not equally to the safe period of anesthesia.Mid-trimester is the key period of central nervous system development,which means that nervous system is proceeding proliferation,differentiation and migration.In this period,minor influences such as drugs and environment may cause severe neurocognitive impairment.Recently,several studies showed that exposure to anesthetics during mid-trimester can affect neural stem cells in off-spring,but the mechanism is not explicit yet.Our previous studies has shown that exposures to 3% sevoflurane 2 h for 3 days can cause learning and memory impairment in the offspring,in the mean time that single 3%sevoflurane exposure can not cause the same impairment.Exposure to 3.5% sevoflurane for 2 h can cause excessive autophagy which can cause the apoptosis and suppressed selfrenewal of neural stem cell in off-spring,and 2% sevoflurane exposure for 2 h cannot cause the same results.All these results indicated that sevoflurane-induced neurotoxicity is related to drug concentration,exposure duration and exposure times.Recently,numerous studies revealed that anesthetics can cause abnormal neural stem cell differentiation.Cell cycle timing dictates cell fate.Once the setting cell cycle timing being broken,it may cause severe consequences.Neural differentiation is the crucial period of nervous system development.Premature or suppressed NSC differentiation may have long-term negative effects on developing brain.There are several genes which participate in the NSC maintenance during the nervous system development lead NSC to differentiate on set.ATN1 is a crucial gene for NSC maintenance according to the reference.Our preexperiment results also showed that multiple exposures can downregulate ATN1 expression in off-spring and primary cultured NSCs.There is not a study that revealed different sevoflurane exposure times affecting NSC differentiation and its mechanism at present.Thus,this study investigated whether sevoflurane affects NSC differentiation by regulating ATN1 expression.Micro RNAs(mi RNAs)is coded by endogenous genes,highly conserved,single stranded non-coding RNA of 20-24 nucleotides long which plays a crucial role in vivo.Several studies revealed that mi RNAs play an important role in the development of nervous system.We used TARGETSCAN database to detect that ATN1 is a direct target of mi R-410-3p and confirmed using dual-luciferase reporter assay.Our results revealed that after multiple sevoflurane exposure,the expression of mi R-410-3p in primary cultured NSCs was upregulated.This study investigated whether sevoflurane affects NSC differentiation by regulating mi R-410-3p expression.In summary,this study explicated whether single or multiple exposures to sevoflurane affects NSC differentiation,and sevoflurane affects NSC differentiation by regulating the mi R-410-3p and ATN1 expression.Materials and methods: 1.Adult Sprague-Dawley rats were chosen,caged to free mating and marked gestational time.Pregnancy rats were exposed to 3% sevoflurane for 2 h in G14(Gestational,G)or G13,G14,G15.The fetal brain tissues were obtained after cesarean section at 24 h,72 h,and postnatal day 28,using Western Blot and Immunofluorescence staining to detect NSC marker Nestin,neuron marker ?-tubulin III and astrocyte marker glial fibrillary acidic protein(GFAP)to observe NSC differentiation.2.Rat primary cultured NSCs were isolated from the hippocampus of fetal SpragueDawley rats on G14-G16.All experiments were performed on cells from passages 2–4.NSCs were exposed to 4.1% sevoflurane for 2 h or 2 h on 3 consecutive days.The NSCs were collected after sevoflurane exposure at 24 h,72 h and 28 d,using Western Blot and Immunofluorescence staining to detect NSC differentiation.3.Our pre-experiments revealed that ATN1 expression was downregulated after sevoflurane exposure,leading NSC to early differentiation.We used ATN1 overexpression lentivirus to transfect NSCs,lentivirus without ATN1 overexpression as NC group,using RT-q PCR,Western Blot and Immunofluorescence to detect ATN1 expression and NSC differentiation after multiple exposures to sevoflurane at the same collecting time.4.We used TARGETSCAN database to predict the potential binding sites of ATN1,and detected that ATN1 is a direct target of mi R-410-3p which was confirmed using dual-luciferase reporter assay.5.Our preexperiments revealed that the expression of mi R-410-3p in primary cultured NSCs was upregulated after sevoflurane exposure.We used mi R-410-3p suppression lentivirus to transfect NSCs,using RT-q PCR,Western Blot and Immunofluorescence to detect mi R-410-3p,ATN1 expression and NSC differentiation after multiple exposures to sevoflurane.Results: 1.After single maternal exposure to sevoflurane,Nestin,?-tubulin III and GFAP expression,?-tubulin III-positive cells and GFAP-positive cells in off-spring were not significant difference compared to CON group.Multiple exposures to sevoflurane downregulated the expression of Nestin,upregulated the expression of ?-tubulin III and GFAP,upregulated ?-tubulin III-posotive cells and GFAP-positive cells at 24 h and 72 h.The expression of ?-tubulin III and ?-tubulin III-positive cells in hippocampal CA1 region was downregulated,while the expression of GFAP and GFAP-positive cells was upregulated on postnatal 28 d.2.After single exposure to sevoflurane,Nestin,?-tubulin III and GFAP expression,?-tubulin III-positive cells and GFAP-positive cells in primary cultured hippocampal NSCs were not significant difference compared to CON group.Multiple exposures to sevoflurane downregulated the expression of Nestin,upregulated the expression of ?-tubulin III and GFAP,upregulated ?-tubulin III-positive cells and GFAP-positive cells at 24 h and 72 h.The expression of ?-tubulin III and ?-tubulin IIIpositive cells was downregulated,while the expression of GFAP and GFAP-positive cells was upregulated on 28 d.3.Multiple exposures to sevoflurane downregulated the expression of ATN1 in off-spring and primary cultured hippocampal NSCs.After ATN1 overexpression and sevoflurane exposure,compared to SEV×3 group at 24 h and 72 h,the expression of ATN1 and Nestin was upregulated,while the expression of ?-tubulin III and GFAP,?-tubulin III-posotive cells and GFAP-positive cells were downregulated.The expression of ?-tubulin III was upregulated while the expression of GFAP was downregulated on 28 d.4.ATN1 is the direct target of mi R-410-3p.5.Multiple exposures to sevoflurane upregulated the expression of mi R-410-3p in NSCs.After mi R-410-3p suppression and sevoflurane exposure,compared to SEV×3 group at 24 h and 72 h,the expression of mi R-410-3p,?-tubulin III and GFAP,?-tubulin III-posotive cells and GFAPpositive cells were downregulated,while the expression of ATN1 and Nestin was upregulated.The expression of ?-tubulin III was upregulated while the expression of GFAP was downregulated on 28 d.Conclusions: 1.Multiple exposures to sevoflurane led NSCs in off-spring and primary cultured hippocampal NSCs to early differentiation to neurons and astrocytes,and led to long-term neuron reduction and astrocyte proliferation.NSCs after single sevoflurane exposure did not observe the same results.2.Multiple exposures to sevoflurane downregulated the expression of ATN1.ATN1 overexpression can alleviate the effects of sevoflurane on NSC differentiation.Single sevoflurane exposure did not affect the expression of ATN1.3.ATN1 is a direct target of mi R-410-3p.Multiple exposures to sevoflurane upregulated the expression of mi R-410-3p.Mi R-410-3p suppression can alleviate the effects of sevoflurane on NSC differentiation.Single sevoflurane exposure did not affect the expression of mi R-410-3p.