Mechanism Research Of Sevoflurane-induced Neurotoxicity In Developing Mouse Brain

Sevoflurane is the general anesthetic which can be most commonly used in newborns and children.Recently,studies have shown that children who exposed to anesthesia and surgery may cause neurotoxicity in their developing brain,which may affect their long-term learning ability,however,the specific mechanism is still unclear.Moreover,Tau protein phosphorylation played an important role in the development of learning impairment.And,some researches have reported that microtubule-unbound Tau(MUT)in the developing brain may be the main reason of the neurodevelopmental toxicity caused by sevoflurane,which can be regulated by Nuak1 and energy deprivation.In this study,we discuss the mechanism of sevoflurane-induced neurotoxicity in the developing brain of young mice through Nuak1 / Tau pathway.Our aim is to provide a relevant theoretical basis for the age selectivity of neurotoxicity induced by multiple sevoflurane anesthesia,and to provide a new idea to explore effective methods of brain protection in pediatric anesthesia as well.PART 1: Effects of multiple sevoflurane anesthesia on Tau expression and cognitive impairment in mice of different agesObjective: Sevoflurane anesthesia may induce long-term cognitive dysfunction in young mice.Tau protein phosphorylation can cause neurotoxicity,and microtubule-unbound Tau may perform as the marker of early metabolism of Tau protein.The present study was designed to investigate the effects of sevofluane multiple anesthesia on long-term cognitive function and also on the expression of different types of Tau protein in young(P6)and adult(P60)mice in vivo.Method: Postnatal(P)6 day-old(P6)and 60 day-old(P60)mice were random Ly divided into 4 groups: P6 + Control,P6 + Sevoflurane,P60 + Control and P60 + Sevoflurane.Mice in sevoflurane groups were anesthetized with 3% sevoflurane plus 60% oxygen 2 hours daily for 3 days,and control mice only received 60% oxygen 2 hours daily for 3 days.22 days after sevoflurane was used(P30,P84),Morris water maze was performed to detect the cognitive functions of mice;0 days after treatment with sevoflurane and oxygen,mice were killed,their cortex and hippocampus were harvested,and RT-PCR,ELISA,Western blot,immunofluorescence were used to determine the Tau m RNA,total Tau,Tau-PS356,Tau-PS202/PT205(PHF-Tau)and T22(oligomers)expression.Moreover,microtubule binding protein spin-down kits and tandem mass spectrometry(MS/MS)were also use to measure the different types of total Tau and different sites of phosphorylated Tau.Results: 1.Sevoflurane anesthesia could cause cognitive impairment in P6 mice,but not in P60 mice;2.P6 mice expressed higher levels of Tau m RNA and Tau protein than P60 mice;3.T22 and Tau-PS356 expression in P6 mice were higher than those in P60 mice and sevoflurane treatment could increase the Tau-PS202/PT205 level in P6 mice;4.Microtubule-unbound Tau(MUT)was mainly expressed in P6 mice,but microtubule-bound Tau(MBT)was mainly expressed in P60 mice;5.We found some T22 early aggregation in the cortex in P6 mice,but not in P60 mice;6.Quantitative mass spectrometry showed that P6 mice had more numbers and higher levels of phosphorylated Tau than those in P60 mice.Conclusion: Young mice,compared with adult mice,their developing brains are more vulnerable to multiple anesthesia with sevoflurane,which can result in the increasing of Tau phosphorylation and may affect their long-term cognitive function,and all these may relate to the expression of microtubule-unbound Tau.PART 2: Role of Nuak1 in the neurotoxicity caused by sevoflurane multiple anesthesia in the developing brain of young miceObjective: According to a new study which published in 2016,Nuak1(Nuak family DNF1-like kinase 1,also known as AMPK related protein 5,ARK5)can regulate the metabolism of Tau protein by selectively phosphorylate Tau on the site of Ser356.Through using the specific inhibitor of Nuak1(HTH-01-015),this experiment is to demonstrate the key role of Nuak1 in the neurotoxicity caused by sevoflurane multiple anesthesia in young mice’s developing brain.Methods: Postnatal(P)6 day-old(P6)and 60 day-old(P60)mice were random Ly divided into 4 groups: P6 + Control,P6 + Sevoflurane,P60 + Control and P60 + Sevoflurane.Treatments in different groups were as same as what we said in Part 1.0 days after treatment with sevoflurane and oxygen,mice were killed,their cortex and hippocampus were harvested,and RT-PCR,Western blot were used to determine the Nuak1 m RNA and Nuak1 protein expression.Moreover,we also used tandem mass spectrometry(MS/MS)to measure the Nuak1 phosphorylation.At the same time,another P6 mice were randomly arranged into 4 groups: P6 + Control + Vehicle,P6 + Sevoflurane + Vehicle,P6 + Control + HTH-01-015,P6 + Sevoflurane + HTH-01-015.The treatments of each group are as same as what we mentioned above.HTH-01-015(a Nuak1 inhibitor)were administered 30 minutes before each of sevoflurane anesthesia or control to demonstrate the cause-effect relationship.Morris water maze was used to detect the cognitive function,ELISA,Western blot and immunofluorescence staining were used to measure total Tau,Nuak1,Tau-PS356,Tau-PS202/PT204 and T22 expression,microtubule-binding test was used to determine different types of Tau protein as well.Results: 1.The expression of Nuak1 in P6 mice was significantly higher than that of P60 mice;2.P6 mice expressed less phosphorylated sites of Nuak1(Ser389,Ser446)than that of P60 mice;3.Sevoflurane multiple treatment could not cause cognitive impairment in P6 mice after HTH-01-015 injected for 3 days;4.There were no significant difference of total Tau expression between P6 + Vehicle group and P6 + HTH-01-015 group,but,compared with P6 + Vehicle group,Tau-PS356 and Tau-PS202/PT205 were obviously decreased in P6 + HTH-01-015 group,and sevoflurane treatment had no effect in P6 + Sevoflurane + HTH-01-015 group compared with P6 + Control + Vehicle group;5.After the administration of HTH-01-015,the microtubule-unbound Tau protein in the cortex of young mice was significantly reduced and the early aggregation of Tau protein was decreased as well.Conclusion: The higher brain Nuak1 levels in the young mice induce Tau phosphorylation in Ser356,causing higher total Tau levels and higher levels of microtubule-unbound Tau than those in adult mice.Sevoflurane only acts on the microtubule-unbound Tau to induce Tau phosphorylation and aggregation,finally leading to cognitive impairment.PART 3: Effects of energy deprivation on sevoflurane-induced neurotoxicity in young mice’s developing brainObjective: Studies have pointed out that energy deprivation and oxidative stress may be the main cause of Tau phosphorylation and aggregation.In this study,we used Vitamin K2—a kind of energy supplement,to demonstrate the effect of energy on the neurotoxicity induced by sevoflurane multiple anesthesia in young mice’s developing brain.Methods: Postnatal(P)6 day-old(P6)and 60 day-old(P60)mice were randomly divided into 4 groups: P6 + Control,P6 + Sevoflurane,P60 + Control and P60 + Sevoflurane.Treatments in different groups were the same as what we said in Part 1.ATP kits were used to determine the ATP levels each group.At the same time,another P6 mice were randomly arranged into 4 groups: P6 + Control + Corn oil,P6 + Sevoflurane + Corn oil,P6 + Control + Vitamin K2,P6 + Sevoflurane + Vitamin K2.The treatments of each group are as same as what we mentioned above.Vitamin K2(an energy supplement)were administered 30 minutes before each of sevoflurane anesthesia or control to demonstrate the cause-effect relationship.Morris water maze was used to detect the cognitive function,ELISA,Western blot and immunofluorescence staining were used to measure total Tau,Nuak1,Tau-PS356,Tau-PS202/PT204 and T22 expression,microtubule-binding test was used to determine the different types of Tau protein as well.Results: 1.ATP levels in P6 mice was significantly lower than that of P60 mice;2.Sevoflurane multiple treatment could not cause cognitive impairment in P6 mice after Vitamin K2 injected for 3 days;3.Compared with P6 + Corn oil groups,Nuak1,Tau-PS356 and Tau-PS202/PT205 were obviously decreased in P6 + Vitamin K2 groups,and sevoflurane treatment had no effect in P6 + Sevoflurane + Vitamin K2 group compared with P6 + Control + Corn oil group;4.After the administration of Vitamin K2,the microtubule-unbound Tau protein in the cortex of young mice was significantly reduced and the early aggregation of Tau protein was decreased as well.Conclusion: Nuak1,which is activated by energy deprivation,can phosphorylate Tau protein at the site of Ser356 and make Tau detached from microtubules.Sevoflurane anesthesia then acts on the microtubule-unbounded Tau to induce Tau super-phosphorylation and to increase early stage of Tau aggregation,finally leading to cognitive impairment in the young mice.Summary1.Multiple anesthesia with sevoflurane can induce long-term cognitive impairment in young mice’s developing brain,but has no effect on adult mice;2.Microtubule-unbound Tau was mainly expressed in the cortex of young mice,however,microtubule-bound Tau was mainly expressed in adult mice;3.Sevoflurane anesthesia then acts on the microtubule-unbounded Tau to induce Tau super-phosphorylation and to increase early stage of Tau aggregation,finally leading to cognitive impairment in the young mice;4.Nuak1 can regulate the metabolism of Tau protein by selective phosphorylation of Tau-PS356,which plays a key role in the neurotoxicity induced by sevoflurane multiple treatment in young mice’s developing brain.5.The content of ATP in young mice are far lower than that of adult mice,which can cause energy deprivation in young mice;6.These data suggest that young(6 day-old)mice have lower brain energy levels,which causes higher Nuak1 levels as compared to adult mice(60 day-old)mice.The higher brain Nuak1 levels in the young mice induce Tau phosphorylation in serine 356,causing higher total Tau levels(due to less degradation of Tau)and higher levels of microtubule-unbound Tau than those in the adult mice.Sevoflurane only acts on the microtubule-unbound Tau to induce Tau phosphorylation and aggregation,finally leading to cognitive impairment.