The Role Of Tau Phosphorylation And α₂ Adrenergic Receptor In Sevoflurane-induced Developmental Neurotoxicity

Background:Repeated or prolonged exposure to sevoflurane has been shown in animal studies to be neurotoxic in the developing brain.The clinical results also show that there are subtle declines in processing speed,fine-motor coordination,problem-solving and social skills in adolescence after sevoflurane exposure.Tau phosphorylation is the pathological change of a variety of cognitive dysfunction.The phosphorylated Tau protein forms fibrous tangles,leading to neurocognitive dysfunction.A number of studies have proved that Tau phosphorylation can cause cognitive dysfunction.Sevoflurane is the most-used anesthetic in infants and young children.Recent studies have shown that repeated sevoflurane anesthesia during the infant stage can lead to long-term cognitive dysfunction,but the specific mechanism remains unclear.Based on our previous study,we hypothesized that Tau protein phosphorylation is one of the important mechanisms of long-term cognitive dysfunction caused by repeated anesthesia in developing young mice.To explore the prevention scheme and the mechanism of multiple sevoflurane-induced long-term cognitive function of young mice,which can provide theoretical basis for clinical optimization of anesthesia scheme for infants and young children.Dexmedetomidine is a highly selectiveα₂ adrenergic receptor agonist commonly used in clinical anesthesia and has been shown to have neuroprotective effects in many studies,but whether it can reduce the neurotoxic of sevoflurane-induced in the developing brain remains unclear.Based on the results of the preliminary experiment,we speculated that dexmedetomidine could alleviate repeated sevoflurane-induced long-term cognitive function of young mice,and the mechanism may be related to the reduction of Tau phosphorylation by stimulatingα₂adrenergic receptor.The study was divided into four parts,Firstly,we explored whether the mechanism of developmental neurotoxicity induced by repeated sevoflurane anesthesia was related to Tau phosphorylation;Secondly,we observed whether dexmedetomidine inhibited sevoflurane-induced Tau phosphorylation and prevented the developmental neurotoxicity of sevoflurane-induced.Finally,we explored the role and mechanism ofα₂ adrenergic receptor in the prevention of sevoflurane induced neurotoxicity during development.Part I The effects of repeated sevoflurane anesthesia on long-term cognitive function and the role of Tau phosphorylation in developing miceObjective:To investigate whether repeated sevoflurane anesthesia causes long-term cognitive dysfunction and whether it is related to Tau phosphorylation induced by sevoflurane.Methods:In the experiment,newborn healthy C57BL/6 wild-type six-day-old mice from both sexes were randomly divided into two groups:control group（Con group）:P6,P9 and P12 were given 40%oxygen for 2 hours each day,Sevoflurane general anesthesia group（Sevo group）:inhaled 3%sevoflurane plus 40%oxygen anesthesia for 2 hours on days P6,P9 and P12.Immediately after anesthesia,hippocampal tissue was harvested.WB was used to detect the expression of AT8（Tau-PS202 and Tau-PT205）in the hippocampal tissues of mice in the two groups（n=6）.The other mice were returned to the cage after anesthesia,and The New Object Recognition Test（NOR）was performed on days P29-30.Morris Water Maze（MWM）were performed on days P31-37（n=12）.After the MWM test,the expression of PSD-95 in the hippocampal tissues of the two groups was detected by WB.Cell part experiment:HT22 cell line was used in the cell part experiment,which was also divided into two groups.The control group（Con）was given 40%oxygen for 24h,and the sevoflurane group（Sevo）was given 3%sevoflurane plus 40%oxygen for 24h.The expression of Tau protein in the two groups was detected by WB.Results:1.Compared with the Control group,WB results showed that the Sevoflurane group increased the phosphorylation of Tau AT8 in the hippocampus（P<0.05）,but had no effect on the expression of Tau-PS262 and total Tau protein.（P>0.05）;Compared with the Control group,Sevoflurane group reduced the expression of PSD-95 protein in adult hippocampus（P<0.05）;2.The results of MWM showed that there was no change in escape latency time between two groups.Compared with Control group,the number of platform crossing of Sevoflurane group was significantly decreased（P<0.05）;3.Compared with the Control group,the results of NOR showed that the ability of new object discrimination of Sevoflurane group decreased significantly（P<0.05）;4.Compared with the Control group,the cell part of the experiment showed that the Sevoflurane group increased the phosphorylation of Tau protein AT8 in HT22 cell line（P<0.05）,but had no effect on the expression of total Tau protein.Conclusion:3%sevoflurane anesthesia for 2 hours on P6,P9 and P12 days induced long-term cognitive dysfunction,which may be related to Tau hyperphosphorylation in the hippocampus induced by sevoflurane.PartⅡ The effects of dexmedetomidine on long-term cognitive dysfunction and Tau phosphorylation in developing mice induced by repeated sevoflurane anesthesiaObjective:To investigate whether dexmedetomidine can attenuate the cognitive dysfunction caused by repeated sevoflurane anesthesia in young mice,and whether this effect is related to the reduction of Tau phosphorylation.Methods:In the experiment,newborn healthy C57BL/6 wild-type six-day-old mice from both sexes were randomly divided into four groups:control group（Con+Veh group）:P6,P9 and P12 were given 40%oxygen for 2 hours each day,and the same amount of normal saline was injected intraperitoneally 30 minutes before oxygen inhalation.Dexmedetomidine control group（Con+Dex group）:P6,P9 and P12 were given 40%oxygen for 2h,and 10μg/kg dexmedetomidine was intraperitoneally injected30 minutes before oxygen inhalation.Sevoflurane general anesthesia group（Sevo+Veh group）:inhaled 3%sevoflurane plus 40%oxygen anesthesia for 2 hours on days P6,P9and P12,and intraperitoneal injection of the same amount of normal saline 30 minutes before anesthesia;Dexmedetomidine+Sevoflurane general anesthesia group（Sevo+Dex group）:P6,P9 and P12 were anesthetized with 3%sevoflurane plus 40%oxygen for 2 hours,and 10μg/kg dexmedetomidine was intraperitoneally injected 30minutes before anesthesia.Immediately after anesthesia,hippocampal tissue was harvested.WB was used to detect the expression of AT8 in the hippocampal tissues of mice in the four groups（n=6）.The other mice were returned to the cage after anesthesia,and The NOR test was performed on day P 29-30.MWM were performed on days P31-37（n=12）.After the MWM test,the expression of PSD-95 in the hippocampal tissues of the four groups was detected by WB.HT22 cell line was used in the cell part experiment,which was also divided into 4 groups:Control group（Con）was given 40%oxygen for 24h,Sevoflurane group（Sevo）was given 40%oxygen plus 3%sevoflurane for 24h,Dexmedetomidine group（Dex）was given 40%oxygen for 24h,1μmol dexmedetomidine was added into medium 30min before oxygen inhalation;Dexmedetomidine+Sevoflurane（Dex+Sevo）medium was added with 1μmol dexmedetomidine,and 40%oxygen plus 3%sevoflurane were administered 30min later for 24h.Tau protein expression in cells of all four groups was monitored by WB.Results:1.Compared with the Control group,WB results showed that group Con+Dex had no effect on the phosphorylation of Tau AT8 in the hippocampus（P>0.05）,the phosphorylation of Tau AT8 in hippocampus was increased in Sevo+Veh group（P<0.05）,compared with Con+Dex group,Sevo+Dex group had no significant effect on Tau phosphorylation,compared with Sevo+Veh group,Sevo+Dex group attenuated Tau phosphorylation induced by sevoflurane（P<0.05）;Compared with Control group,Con+Dex group had no effect on the expression of PSD-95 in adult hippocampus（P>0.05）,Sevo+Veh group reduced the expression of PSD-95 protein in adult hippocampus（P<0.05）,compared with Con+Dex group,Sevo+Dex group had no significant effect on the expression of PSD-95 in adult hippocampus,and compared with Sevo+Veh group,Sevo+Dex group reduced the expression of PSD-95 in adult hippocampus induced by sevoflurane;2.The results of MWM showed that there was no change in escape latency time among groups.Compared with Control group,the number of platform crossing of Con+Dex group was not significantly decreased（P>0.05）,the number of platform crossing of Sevo+Veh group was significantly decreased（P<0.05）,compared with the Con+Dex group,there was no significant change in the number of platform crossing of Sevo+Dex group（P>0.05）,compared with Sevo+Veh group,Sevo+Dex group increased the number of platform crossing（P<0.05）;3.Compared with the Control group,the results of NOR showed that the new object discrimination ability of the Con+Dex group did not change significantly（P>0.05）,the ability of new object discrimination of Sevo+Veh group decreased significantly（P<0.05）,Compared with the Con+Dex group,Sevo+Dex group had no significant change in the ability to distinguish new objects（P>0.05）,compared with Sevo+Veh group,the ability of Sevo+Dex group to distinguish new objects was significantly increased（P<0.05）;4.Compared with the Control group,the cell part of the experiment showed that the Sevoflurane group increased the phosphorylation of Tau protein AT8 in HT22 cell line（P<0.05）,group Dex and group Dex+Sevo had no effect on the phosphorylation of Tau AT8 in HT22 cell line（P>0.05）;compared with Dex group,Dex+Sevo group had no significant effect on Tau phosphorylation;compared with Sevo group,Dex+Sevo group attenuated Tau phosphorylation induced by sevoflurane（P<0.05）;but there were no effect on the expression of total Tau protein among the four groups.Conclusion:Dexmedetomidine ameliorated long-term cognitive dysfunction induced by repeated sevoflurane anesthesia in young mice via reducing Tau phosphorylation and synaptic loss in the hippocampus.PartⅢ The role ofα₂ adrenergic receptors in dexmedetomidine alleviating long-term cognitive dysfunction induced by repeated sevoflurane anesthesiaObjective:To investigate the effect of dexmedetomidine on Tau phosphorylation and cognitive dysfunction in young mice induced by repeated sevoflurane anesthesia whether throughα₂ adrenergic receptor.Methods:In the experiment,newborn healthy C57BL/6 wild-type six-day-old mice from both sexes were randomly divided into four groups:Control group（Con+Veh group）:P6,P9 and P12 were given 40%oxygen for 2 hours each day,and the same amount of normal saline was injected intraperitoneally 40 minutes and 30 minutes before oxygen inhalation.Dexmedetomidine+Yohimbine control group（Con+Yoh+Dex group）:P6,P9 and P12 were given 40%oxygen for 2h,1mg/kg yohimbine and 10μg/kg dexmedetomidine were intraperitoneally injected at 40 minutes and 30 minutes before oxygen inhalation.Sevoflurane general anesthesia group（Sevo+Veh group）:inhaled 3%sevoflurane plus 40%oxygen anesthesia for 2 hours on days P6,P9 and P12,and intraperitoneal injection of the same amount of normal saline40 minutes and 30 minutes before anesthesia;Dexmedetomidine+Yohimbine+Sevoflurane general anesthesia group（Sevo+Yoh+Dex group）:P6,P9 and P12 were anesthetized with 3%sevoflurane plus 40%oxygen for 2 hours,and 1mg/kg yohimbine and 10μg/kg dexmedetomidine was intraperitoneally injected at 40 minutes and 30minutes before anesthesia.Immediately after anesthesia,hippocampal tissue was harvested.WB was used to detect the expression of AT8 in the hippocampal tissues of mice in the four groups（n=6）.The other mice were returned to the cage after anesthesia,and The NOR test was performed on days P29-30.MWM were performed on days P31-37（n=12）.After the MWM test,the expression of PSD-95 in the hippocampal tissues of the four groups was detected by WB.Results:1.Compared with Control group,WB showed that Con+Yoh+Dex group had no effect on Tau AT8 phosphorylation in hippocampus region（P>0.05）,Sevo+Veh group increased the phosphorylation of Tau AT8 in hippocampus of young mice（P<0.05）;Compared with Con+Yoh+Dex group,Sevo+Yoh+Dex group increased Tau phosphorylation（P<0.05）,Sevo+Yoh+Dex group had no effect on Tau phosphorylation compared with SV group（P>0.05）;Compared with the Control group,Con+Yoh+Dex group had no effect on the expression of PSD-95 in adult hippocampus（P>0.05）,Sevo+Veh group reduced the expression of PSD-95 protein in adult hippocampus（P<0.05）,Compared with Con+Yoh+Dex group,Sevo+Yoh+Dex group reduced the expression of PSD-95 in adult hippocampus（P<0.05）;Compared with Sevo+Veh group,Sevo+Yoh+Dex group had no effect on the expression of PSD-95 in hippocampus（P>0.05）;2.The results of MWM showed that there was no change in escape latency time among groups.Compared with Control group,the number of platform crossing of Con+Yoh+Dex group was not significantly decreased（P>0.05）,the number of platform crossing of Sevo+Veh group was significantly decreased（P<0.05）,Compared with the Con+Yoh+Dex group,the number of platform crossing of Sevo+Yoh+Dex group was significantly decreased（P<0.05）;Compared with Sevo+Veh group,there was no significant change in the number of platform crossing of Sevo+Yoh+Dex（P>0.05）;3.Compared with Control group,the results of NOR showed that the new object discrimination ability of the Con+Yoh+Dex group did not change significantly（P>0.05）,the ability of new object discrimination of Sevo+Veh group decreased significantly（P<0.05）;Compared with the Con+Yoh+Dex group,the ability of new object discrimination of Sevo+Yoh+Dex group decreased significantly（P<0.05）;Compared with Sevo+Veh group,Sevo+Yoh+Dex group had no significant change in the ability to distinguish new objects（P>0.05）.Conclusion:Dexmedetomidine attenuated cognitive dysfunction induced by repeated sevoflurane anesthesia in developing mice through reducing Tau phosphorylation in the hippocampal via stimulatingα₂ adrenergic receptors.PartⅣ The role of differentα₂ adrenergic receptor agonists in long-term cognitive dysfunction induced by repeated sevoflurane anesthesiaObjective:To investigate whether otherα₂ adrenergic receptor agonists could reduce Tau phosphorylation and cognitive impairment induced by repeated sevoflurane anesthesia in young mice,and to further confirm whetherα₂ adrenergic receptor is a new target for preventing sevoflurane induced neurotoxicity in developing mice.Methods:Experiment one,newborn healthy C57BL/6 wild-type six-day-old mice from both sexes were randomly divided into four groups:Control group（Con+Veh group）:P6,P9 and P12 were given 40%oxygen for 2 hours each day,and the same amount of normal saline was injected intraperitoneally 30 minutes before oxygen inhalation.Clonidine control group（Con+Clo group）:P6,P9 and P12 were given 40%oxygen for 2h,and 1mg/kg clonidine was intraperitoneally injected 30 minutes before oxygen inhalation.Sevoflurane general anesthesia group（Sevo+Veh group）:inhaled 3%sevoflurane plus 40%oxygen anesthesia for 2 hours on days P6,P9 and P12,and intraperitoneal injection of the same amount of normal saline 30 minutes before anesthesia;Clonidine+Sevoflurane general anesthesia group（Sevo+Clo group）:P6,P9and P12 were anesthetized with 3%sevoflurane plus 40%oxygen for 2 hours,and1mg/kg clonidine was intraperitoneally injected 30 minutes before anesthesia.Immediately after anesthesia,hippocampal tissue was harvested.WB was used to detect the expression of AT8 in the hippocampal tissues of mice in the four groups（n=6）.The other mice were returned to the cage after anesthesia,and The NOR test was performed on days P29-30.MWM were performed on days P31-37（n=12）.After the MWM test,the expression of PSD-95 in the hippocampal tissues of the four groups was detected by WB.Experiment 2,newborn healthy C57BL/6 wild-type six-day-old mice from both sexes were randomly divided into four groups:Control group（Con+Veh group）:P6,P9and P12 were given 40%oxygen for 2 hours each day,and the same amount of normal saline was injected intraperitoneally 40 minutes and 30 minutes before oxygen inhalation.Clonidine+Yohimbine control group（Con+Yoh+Clo group）:P6,P9 and P12 were given 40%oxygen for 2h,1mg/kg yohimbine and 1mg/kg clonidine were intraperitoneally injected at 40 minutes and 30 minutes before oxygen inhalation.Sevoflurane general anesthesia group（Sevo+Veh group）:inhaled 3%sevoflurane plus40%oxygen anesthesia for 2 hours on days P6,P9 and P12,and intraperitoneal injection of the same amount of normal saline 40 minutes and 30 minutes before anesthesia;Clonidine+Yohimbine+Sevoflurane general anesthesia group（Sevo+Yoh+Clo group）:P6,P9 and P12 were anesthetized with 3%sevoflurane plus 40%oxygen for 2 hours,and 1mg/kg yohimbine and 1mg/kg clonidine was intraperitoneally injected at 40minutes and 30 minutes before anesthesia.Immediately after anesthesia,hippocampal tissue was harvested.WB was used to detect the expression of AT8 in the hippocampal tissues of mice in the four groups（n=6）.The other mice were returned to the cage after anesthesia,and The NOR test was performed on days P29-30.MWM were performed on days P31-37（n=12）.After the MWM test,the expression of PSD-95 in the hippocampal tissues of the four groups was detected by WB.Results:1.Compared with Control group,WB results showed that group Con+Clo had no effect on the phosphorylation of Tau AT8 in the hippocampus（P>0.05）,Con+Yoh+Clo group also had no effect on Tau AT8 phosphorylation in hippocampus region（P>0.05）,the phosphorylation of Tau AT8 in hippocampus was increased in Sevo+Veh group（P<0.05）;Compared with Con+Clo group,Sevo+Clo group had no significant effect on Tau phosphorylation,compared with Sevo+Veh group,Sevo+Clo group attenuated Tau phosphorylation induced by sevoflurane（P<0.05）;Compared with Con+Yoh+Clo group,Sevo+Yoh+Clo group increased Tau phosphorylation（P<0.05）,Sevo+Yoh+Clo group had no effect on Tau phosphorylation compared with Sevo+Veh group（P>0.05）;Compared with Control group,Con+Clo group had no effect on the expression of PSD-95 in adult hippocampus（P>0.05）,Con+Yoh+Clo group had no effect on the expression of PSD-95 in adult hippocampus also（P>0.05）,Sevo+Veh group reduced the expression of PSD-95 protein in adult hippocampus（P<0.05）;Compared with Con+Clo group,Sevo+Clo group had no significant effect on the expression of PSD-95 in adult hippocampus（P>0.05）;Compared with Sevo+Veh group,Sevo+Clo group reduced the expression of PSD-95 in adult hippocampus induced by sevoflurane（P<0.05）;Compared with Con+Yoh+Clo group,Sevo+Yoh+Clo group reduced the expression of PSD-95（P<0.05）,Con+Clo group had no effect on the expression of PSD-95 in adult hippocampus（P>0.05）;Compared with Sevo+Veh group,Sevo+Yoh+Clo group had no effect on the expression of PSD-95 in hippocampus（P>0.05）;2.The results of MWM showed that there was no change in escape latency time among all groups.Compared with Control group,the number of platform crossing of Con+Clo group was not significantly decreased（P>0.05）,the number of platform crossing of Con+Yoh+Clo group was not significantly decreased（P>0.05）,the number of platform crossing of Sevo+Veh group was significantly decreased（P<0.05）;Compared with the Con+Clo group,there was no significant change in the number of platform crossing of Sevo+Clo group（P>0.05）;Compared with Sevo+Veh group,Sevo+Clo group increased the number of platform crossing（P<0.05）;Compared with the Con+Yoh+Clo group,the number of platform crossing of Sevo+Yoh+Clo group was significantly decreased（P<0.05）;Compared with Sevo+Veh group,there was no significant change in the number of platform crossing of Sevo+Yoh+Clo（P>0.05）;3.Compared with Control group,the results of NOR showed that the new object discrimination ability of the Con+Clo group did not change significantly（P>0.05）,the new object discrimination ability of the Con+Yoh+Clo group did not change significantly also（P>0.05）,the ability of new object discrimination of Sevo+Veh group decreased significantly（P<0.05）;Compared with the Con+Clo group,Sevo+Clo group had no significant change in the ability to distinguish new objects（P>0.05）,Compared with Sevo+Veh group,the ability of Sevo+Clo group to distinguish new objects was significantly increased（P<0.05）;Compared with the Con+Yoh+Clo group,the ability of new object discrimination of Sevo+Yoh+Clo group decreased significantly（P<0.05）;Compared with Sevo+Veh group,Sevo+Yoh+Clo group had no significant change in the ability to distinguish new objects（P>0.05）.Conclusion:Differentα₂ adrenergic receptor agonist could alleviate long-term cognitive dysfunction induced by repeated sevoflurane anesthesia in developing mice through reducing Tau phosphorylation in hippocampus,which suggested thatα₂adrenergic receptor may be an important target for preventing neurotoxicity of sevoflurane during development.Comprehensive Conclusions:1.Repeated sevoflurane anesthesia can induce long-term cognitive dysfunction in developing young mice,which may be related to Tau hyperphosphorylation in hippocampus;2.Dexmedetomidine ameliorated long-term cognitive dysfunction induced by repeated sevoflurane anesthesia in young mice via reducing Tau phosphorylation in the hippocampus and synaptic loss.3.α₂ adrenergic receptor may be involved in sevoflurane-induced Tau phosphorylation in young mice;Both Dexmedetomidine and Clonidine attenuated long-term cognitive impairment induced by repeated sevoflurane anesthesia by stimulatingα2-adrenergic receptors.