| General anesthesia can cause abnormal phosphorylation of Tau protein and long-term cognitive dysfunction in the developing brain of young mice,but the specific mechanism has not been clarified.It is suggested that apolipoprotein E(Apo E)may play a protective role in the neuronal activity and repair of Alzheimer disease(AD),and its toxic fragments may cause neurodegeneration and neurocognitive impairment in AD.Based on previous studies,this paper aims to explore the role of Apo E and its toxic fragments in the phosphorylation of Tau protein and the difference of neurocognitive function in the hippocampus of young mice induced by sevoflurane anesthesia.In this study,wild-type(WT),Apo E knockout(Apo E-KO),Apo E3 targeted replacement(Apo E3,expressing full-length Apo E and Apo E fragment)and Apo E2 targeted replacement(Apo E2,expressing only full-length Apo E)mice were anesthetized with sevoflurane for several times.In addition,neurons with different gene phenotypes were also used for research.The results showed that the expression of Apo E m RNA,total Apo E,full-length Apo E,Apo E fragments and phosphorylated Tau protein(AT8 and PHF1)were up-regulated by sevoflurane anesthesia compared with the Control group,and the long-term cognitive dysfunction was also induced,but there was no significant effect on the adult mice.Compared with the Control group,sevoflurane stimulation can significantly increase the expression of phosphorylated Tau protein in WT and Apo E-KO mice and immature neurons,and sevoflurane multiple anesthesia can simultaneously cause long-term cognitive dysfunction in WT and Apo E-KO mice.Compared with the Control group,sevoflurane stimulation significantly increased the expression of phosphorylated Tau protein in Apo E3 mice and immature neurons,but not in Apo E2 mice and neurons.Furthermore,sevoflurane anesthesia can only cause long-term cognitive impairment in Apo E3 mice,but not cognitive impairment in Apo E2 mice.These data suggest that the up-regulation of Apo E fragments level,rather than the increase of full-length Apo E,may be one of the potential mechanisms of age-dependent Tau protein phosphorylation and neurocognitive impairment in young mice induced by sevoflurane anesthesia.Experiment 1: The role of apolipoprotein E in the brain damage induced by sevoflurane anesthesia Objective: To investigate the effects of sevoflurane anesthesia on the expression of Apo E in hippocampus and long-term cognitive function of young(P6)and adult(P60)mice.Methods: 6-day-old(P6)mice and 60-day-old(P60)mice were randomly divided into four groups: infant control group(P6+Control),infant sevoflurane group(P6+Sevoflurane),adult control group(P60+Control)and adult sevoflurane group(P60+Sevoflurane).The mice in the sevoflurane group were anesthetized with 3% sevoflurane + 60% oxygen 2 h/day for 3 days.The mice in the Control group were only treated with 60% oxygen in the same environment.After anesthesia,10 mice in each group were randomly selected and placed in a standardized feeding environment.They were raised to 30 days after birth(P30)and 84 days after birth(P84).The long-term cognitive function of mice in each group was tested by the Morris water maze test(MWM test).The hippocampus of the remaining mice was extracted for Western Blot,ELISA and immunofluorescence to detect the total tau(Tau5),Tau-PS202/PT205(AT8),Tau-PSer396/404(PHF1),full-length Apo E and Apo E fragments.The Apo E m RNA transcription level was measured by real-time quantitative reverse transcription(RT-PCR).Results: 1.Sevoflurane anesthesia could increase the expression levels of total Apo E,full length Apo E and Apo E fragments in hippocampus of young mice(all P < 0.05),but had no effect on adult mice(P > 0.05);2.Sevoflurane anesthesia could increase AT8 and PHF1 expression levels in hippocampus of young mice(all P < 0.05)but not of adult mice(P > 0.05);3.Repeated sevoflurane anesthesia could cause long-term cognitive impairment in young mice(P < 0.05),but not in adult mice(P > 0.05).Conclusion: Repeated sevoflurane anesthesia can increase Tau phosphorylation level in hippocampus and cause long-term cognitive dysfunction of young mice,which may be related to the increased Apo E level.Experiment 2: The relationship between increased expression of total Apo E and brain damage induced by sevoflurane Objective: To investigate whether sevoflurane anesthesia is related to the increase of total Apo E in hippocampus.Methods:(1)Cell experiment: the primary neurons extracted from the hippocampus of wild-type and Apo E-KO fetal mice were cultured to the 5th day.They were randomly divided into wild-type control group(WT+Control),wild-type sevoflurane group(WT+Sevoflurane),knock-out wild group(Apo E-KO+Control),knock-out sevoflurane group(Apo E-KO+Sevoflurane).On the fifth day of culture,neurons in the sevoflurane group were treated with 60% oxygen + 5% carbon dioxide + 4.1% sevoflurane for 4 hours,while neurons in the control group were treated with 60% oxygen + 5% carbon dioxide for 4 hours.At the end of modeling,the neurons were collected to evaluate the levels of Tau5,AT8,PHF1 and Apo E.The transcription level of Apo E m RNA was measured by RT-PCR.(2)Animal experiment: P6 wild-type female mice and P6 Apo E-KO female mice were randomly divided into four groups: wild-type control group(WT+Control),wild-type sevoflurane group(WT+Sevoflurane),knock-out wild group(Apo E-KO+Control)and knock-out sevoflurane group(Apo E-KO+Sevoflurane).The mice in the sevoflurane group were anesthetized with 3% sevoflurane + 60% oxygen for 2 hours a day for 3 days.The mice in the control group were only treated with 60% oxygen in the same environment.At the end of anesthesia,10 mice in each group were randomly selected and placed in standardized feeding environment until P30.MWM test was used to detect the long-term cognitive function of mice in each group.The expression levels of Tau5,AT8,PHF1,total Apo E,full-length Apo E and Apo E fragments were evaluated by WB,ELISA and IF.The transcription level of Apo E m RNA was evaluated by RT-PCR.Results:(1)Cell experiment:(1)The neurons in WT+Control group expressed little Apo E m RNA,total Apo E,full-length Apo E,and almost no Apo E fragments,while sevoflurane anesthesia significantly increased the levels of Apo E m RNA,total Apo E,full length Apo E and Apo E fragments in WT + sevoflurane group(all P < 0.05).(2)Western Blot results showed that the AT8 and PHF1 levels in Apo E-KO+Control group were significantly higher than those in WT+Control group(both P < 0.05).Sevoflurane induced up-regulation of AT8 and PHF1 in WT neurons was not improved in Apo E-KO neurons(both P > 0.05).(3)The results of immunofluorescence showed that the expression levels of Apo E and AT8 in WT neurons were significantly higher than those in the Control group(all P < 0.05).(2)Animal experiments:(1)In WT mice,compared with the Control group,Apo E m RNA and total Apo E levels in sevoflurane group were significantly higher(both P < 0.05).(2)WB results showed that compared with the Control group,the expression levels of full-length Apo E and Apo E fragment in WT + Sevoflurane group were significantly increased(both P < 0.05).(3)In addition,in the Control group,the levels of AT8 and PHF1 in hippocampus of Apo E-KO mice were significantly higher than those of WT mice(all P < 0.05),and sevoflurane anesthesia could not increase the expression levels of AT8 and PHF1 in hippocampus of Apo E-KO mice(both P > 0.05).(4)Compared with the Control group,sevoflurane induced cognitive impairment in WT,but not in Apo E-KO,mice(P < 0.05),and even in the Control group,Apo E-KO mice were more "stupid" than WT mice(P < 0.05).Conclusion: Multiple sevoflurane anesthesia can increase the expression of total Apo E,but the lack of Apo E can lead to the up-regulation of Tau protein phosphorylation in immature neurons and developing hippocampus.Inhibition of the expression of total Apo E can not improve the abnormal phosphorylation of Tau protein and long-term cognitive impairment induced by multiple sevoflurane anesthesia.Experiment 3: The relationship between the increased expression of Apo E fragment and the brain damage induced by sevoflurane Objective: To investigate the relationship between apolipoprotein E(Apo E)toxic fragments and sevoflurane induced brain injury in developmental stage.Methods:(1)Cell experiment: the primary neurons extracted from the hippocampus of Apo E3 and Apo E2 fetal mice were cultured to the 5th day,and randomly divided into Apo E3 control group(Apo E3+Control),Apo E3 sevoflurane group(Apo E3+Sevoflurane),Apo E2 control group(Apo E2+Sontrol),Apo E2 sevoflurane group(Apo E2+Sevoflurane).On the fifth day of culture,neurons in the sevoflurane group were treated with 60% oxygen + 5% carbon dioxide + 4.1% sevoflurane for 4 hours,while neurons in the control group were treated with 60% oxygen + 5% carbon dioxide for 4 hours.At the end of modeling,the neurons were collected for Western Blot,If,Tau5,AT8,PHF1 and Apo E levels.The Apo E m RNA transcription level was measured by RT-PCR.(2)Animal experiment: The female mice of P6 Apo E3 and P6 Apo E2 were randomly divided into four groups: Apo E3 control group(Apo E3+Control),Apo E3 sevoflurane group(Apo E3+Sevoflurane),Apo E2 control group(Apo E2+Control)and Apo E2 sevoflurane group(Apo E2+Sevoflurane).The mice in the sevoflurane group were anesthetized with 3% sevoflurane + 60% oxygen 4 h/day for 3 days.The mice in the Control group were only treated with 60% oxygen in the same environment.At the end of anesthesia,10 mice in each group were randomly selected and placed in a standardized feeding environment until P30.MWM test was used to test the long-term cognitive function of mice in each group.The expression levels of Tau5,AT8,PHF1,total Apo E,full-length Apo E and Apo E fragments were detected by WB,ELISA and IF,and the transcription level of Apo E m RNA was detected by RT-PCR.Results:(1)Cell experiment:(1)The levels of Apo E m RNA,total Apo E and full-length Apo E in Apo E3 and Apo E2 neurons were significantly higher than those in the Control group(all P < 0.05).In addition,compared with the neurons of Apo E3 + Control group,the level of Apo E fragment in the neurons of Apo E3+Sevoflurane group was significantly higher(P < 0.05),but in Apo E2 neurons,there was no any Apo E fragment in both Control group and Sevoflurane group.(2)After being treated with sevoflurane,the expression levels of AT8 and PHF1 in Apo E3 neurons were higher than that in the Control group(both P < 0.05),but there was no difference in Apo E2 neurons(both P > 0.05).(2)Animal experiments:(1)Sevoflurane anesthesia could increase the Apo E m RNA and total Apo E protein levels of Apo E3 and Apo E2 mice(all P < 0.05).WB results showed that the expression levels of full-length Apo E and Apo E fragments in Sevoflurane group were significantly higher than those in Control group(both P < 0.05),but there was no difference in Apo E2 mice(P > 0.05).In addition,the level of Apo E fragments in hippocampus of the Control group and Sevoflurane group in Apo E2 mice were negligible.(2)The expression levels of AT8 and PHF1 in the Apo E3+Sevoflurane group were higher than those in the Apo E3+Control group(both P < 0.05),but not between Apo E2+Control group and Apo E2+Sevoflurane group.(3)Sevoflurane anesthesia can only cause the long-term cognitive impairment in Apo E3 mice instead of Apo E2 mice.Conclusion: The Apo E toxic fragments,rather than full-length Apo E,is the main reason for the increase of Tau phosphorylation and neurocognitive dysfunction in immature neurons or developing brain after sevoflurane anesthesia.Total conclusion: There may be a balance between full-length Apo E and Apo E fragments in normal brain tissue,in which full-length Apo E plays an important protective role,while fragment Apo E has neurotoxicity.Under normal conditions,Apo E is mainly produced by astrocytes,almost not in neurons,and most of them are full-length Apo E,with few Apo E toxic fragments.When the brain faces harmful stimulation,neurons will be activated to produce full-length Apo E to repair itself.However,once the harmful stimulation continues,the Apo E in neurons will hydrolyze into a large number of toxic fragments and destroy the balance of Apo E in the brain,which may lead to neurotoxicity and neurodegeneration,as well as hyperphosphorylation of Tau protein in the brain and neurocognitive damage.In conclusion,the up-regulation of apolipoprotein E fragments(not full-length apolipoprotein E)after sevoflurane anesthesia may be one of the potential mechanisms of age-dependent Tau protein phosphorylation and neurocognitive dysfunction.Clinically,these findings provide potential targets for the prevention and treatment of postoperative neurocognitive disorders in children. |
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