| Objective: To investigate the role of Apo E gene in sevoflurane-induced cognitive dysfunction in aging mice and explore the possible mechanism.Methods: A total of 8-week-old 60 mice(24 Apo E-/-mice and 36 C57BL/6)were used in the study.Half of them were male while the other half were female.Modal of aging mice: 1% D-galactose(120mg/Kg·d)was injected subcutaneously on the back of the mices’ s neck for 6 consecutive weeks.The same amount was given to the mice of the control group(wild C57BL/6)at the same injection site for 6 consecutive weeks.After modeling,the Y-maze and open field experiments were performed.Then the mice were divided into five groups according to the random number grouping method: CON group,C57-AO group,C57-SEV group,Apo E-KO-AO group and Apo E-KO-SEV group.Next,the mice continuously inhaled 1.3MAC sevoflurane(3.2%)+ air-oxygen mixed gas(carrier gas)for 4 hours(h)while the mice of the control group continuously inhaled the carrier gas for 4h.Sampling and testing: test of Apo E knockout mice:(1)Apo E transcription level in mice tail was detected by RT-PCR;hippocampal Apo E protein expression was detected by Western-Blot;(2)test of aging modal: Y maze and open field experiments were performed to detect mice’s ethology after D-galactose modeling;ELISA was used to detect serum SOD and MDA levels;(3)Cognitive changes and protein expression in brain tissue of mice after sevoflurane anesthesia: Y maze and open field experiments were carried out on the 3rd day and 7th day after sevoflurane anesthesia;ELISA was used to detect Aβ,Aβ1-40,Aβ1-42 protein levels in the cerebral cortex of mice in each group;hippocampal Aβ,Aβ1-40,Aβ1-42 transcription levels were detected by RT-PCR;hippocampal Aβ protein expression was detected by Western-Blot;hippocampal Aβ,Aβ1-40,Aβ1-42 expression were detected by immunofluorescence.Results:1.Test of Gene knockout: the mice that were used in the experiment were identified as Apo E-KO homozygotes by RT-PCR.2.Test of aging modal: after modaling,by comparing the C57 model group and the Apo E-KO model group with the control group,it was found that alternation rate and SOD decreased(P<0.05)and that the MDA increased(P<0.05).3.Ethology:(1)y-maze:(1)Within the group: the alternation rate of C57-SEV group3 days(d)after sevoflurane anesthesia was less than that before sevoflurane anesthesia(P<0.05).The alternation rate of Apo E-KO-SEV group 3d after sevoflurane anesthesia was less than before sevoflurane anesthesia and 7d after sevoflurane anesthesia(P<0.05).(2)Between groups: 3d after sevoflurane anesthesia,the alternation rate of C57-SEV and Apo E-KO-SEV group was lower than that of C57-AO group(P<0.05).(2)Open field experiment:(1)Within group: peripheral velocity and total distance in Apo E-KO-SEV and C57-SEV groups 3d after sevoflurane anesthesia were less than those before sevoflurane anesthesia(P<0.05).The proportion of central area distance in C57-SEV group 3d after sevoflurane anesthesia was less than that before sevoflurane anesthesia(P<0.05).Peripheral velocity and total distance in Apo E-KO-SEV group 7d after sevoflurane anesthesia were lower than those before sevoflurane anesthesia(P<0.05).In the C57-SEV group,the peripheral velocity and the proportion of distance in the central area 7d after sevoflurane anesthesia were lower than those before sevoflurane anesthesia(P<0.05).(2)Between groups: 3d after sevoflurane anesthesia,the peripheral velocity of the C57-SEV group was lower than that of the CON and Apo E-KO-SEV groups;the proportion of distance in the central area of the C57-SEV group was less than that of the CON and Apo E-KO-AO groups(P<0.05).7d after sevoflurane anesthesia,the peripheral velocity in the C57-SEV group was lower than that in the control group(P<0.05).4.Difference in protein expression in brain tissues:(1)ELISA results:(1)Within group: Aβ,Aβ1-42 in C57-SEV group 3d after sevoflurane anesthesia was greater than after 7d(P<0.05).(2)Between groups: 3d after Aβ sevoflurane anesthesia,the C57-SEV group was greater than the C57-AO,Apo E-KO-AO,and control groups(P<0.05).The Apo E-KO-SEV group was greater than the Apo E-KO-AO and control groups(P<0.05).3d after Aβ1-40 sevoflurane anesthesia,the C57-SEV group was greater than the C57-AO and Apo E-KO-AO groups(P<0.05).3d after Aβ1-42 sevoflurane anesthesia,the C57-SEV group was greaterr than the C57-AO and control groups(P<0.05).The Apo E-KO-SEV group was greater than the Apo E-KO-AO and control groups(P<0.05).(2)RT-PCR results: comparison between groups at each time point showed that there was no significant difference in m RNA in each rgoup(P>0.05).(3)Western-Blot results:(1)Within group: 7d after sevoflurane anesthesia,Aβ in C57-SEV and Apo E-KO-SEV groups decreased compared with 3d after sevoflurane anesthesia(P<0.05).(2)Between groups: 3d after sevoflurane anesthesia,Aβ in C57-SEV group increased compared with the control and Apo E-KO-SEV groups(P<0.05).7d after sevoflurane anesthesia,Aβ in C57-SEV group increased compared with Apo E-KO-AO and Apo E-KO-SEV groups(P<0.05).(4)Immunofluorescence results:(1)Aβ: 3d after sevoflurane anesthesia,the size of CA3 and CA1 areas in the C57-SEV group increased compared with those in the control and C57-AO groups(P<0.05).Compared with that in the Apo E-KO-SEV group,the size of CA1 area in the C57-SEV group increased(P<0.05).7d after sevoflurane anesthesia,the size of CA3 and CA1 areas in the C57-SEV groups increased compared with those in the control and Apo E-KO-SEV groups(P<0.05).The size of CA1 area in the C57-SEV group increased compared with that in the C57-AO group(P<0.05).(2)Aβ1-40: 3d after sevoflurane anesthesia,the size of CA3 and CA1 areas in the C57-SEV group increased compared with those in the control group(P<0.05).The size of CA1 area in the C57-SEV group increased compared with that in the Apo E-KO-SEV group(P<0.05).(3)Aβ1-42: 3d after sevoflurane anesthesia,the size of CA3 and CA1 areas in the C57-SEV group increased compared with those in the control and C57-AO groups(P<0.05).The size of CA3 area in the Apo E-KO-SEV group increased compared with that in the control group(P<0.05).The size of CA1 area in the C57-SEV group increased compared with that in the Apo E-KO-SEV group(P<0.05).7d after sevoflurane anesthesia,the size of CA3 area in the C57-SEV group increased compared with that in the control group(P<0.05).Conclusion: Sevoflurane anesthesia could cause cognitive dysfunction in aging mice,which was more obvious on the 3d after anesthesia.The mechanism was related to the increase of Aβ expression in brain tissues.Knockout of Apo E gene could down-regulate Aβ expression in brain tissues of sevoflurane-induced aging mice with postoperative cognitive dysfunction. |
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