| Objective: Many studies have reported sevoflurane can negatively impact adult neurogenesis in hippocampal dentate gyrus and result in cognitive deficits in developing brain in rodents.After sevoflurane,widely used volatile anesthetic,exposure in infants,how the neurogenesis of the surviving late progenitor cells was ongoing,and whether the recovery of the surviving immature and mature cells were affected by the apoptosis by anesthetics,were uncertain.We queried whether the differentiation of the surviving late progenitor granule cells after exposure to sevoflurane might be affected by apoptosis.Meanwhile,We plan to observe the changes of autophagy in the hippocampal dentate gyrus after sevoflurane exposure,in order to explore the relationship between autophagy and apoptosis and whether they both participate the process of regulating the neural differentiation of late progenitor granule cells.And the exact mechanism was explored in the current study.Methods: Rats used for double fluorescence staining were injected subcutaneously with 5-bromo-2-deoxyuridine(Brd U)on 13,14,15 days before sevoflurane exposure to label the late progenitor cells.On postnal 21 days after birth,rats were treated with 3% sevoflurane for 4 hours.The animals were randomly divided into two groups due to the time window points(2h,24 h and 4d)after anesthesia or air exposure: control group(Ctl)and 3% sev group(3% sev)(N = 24,n = 8 each time point,48 in total);and rats harvested on 4d after anesthesia or air exposure were randomly divided into eight groups:(1)Saline control group(ctl);(2)Caspase-3 inhibitor control group(ctl + Q-VD-OPh);(3)LC3B inhibitor control group(ctl + 3-MA);(4)NF-κB inhibition control group(ctl+ BAY 11-7085);(5)sevoflurane group(3% sev);(6)caspase-3 inhibitor sevoflurane group(3% sev+Q-VD-OPh)(7)LC3B inhibitor sevoflurane group(3% sev+3-MA);(8)NF-κB inhibitor sevoflurane group(3% sev + BAY 11-7085).Rats in these eight groups were injected with LC3 B inhibitor 3-MA,caspase-3 inhibitor Q-VD-OPh,NF-κB inhibitor BAY 11-7085,or an equal volume of normal saline into the lateral ventricle 1 hour before anesthesia.(n = 8,64 in total).Take three(3 samples)dentate gyrus of rat hippocampus in each group for Western Blot protein determination(including LC3 BII,P62 / SQSTM1,Caspase-3,P65,P-P65,IκB).Five(5 samples)brain tissues of rat hippocampus were used for immunofluorescence section staining(including Tunel,LC3 B,and Brd U/Neu N or Brd U /DCX double staining).The rats to be tested in the Morris water maze test were also divided into five groups:(1)saline control group(Ctl);(2)saline sevoflurane group(3% sev);(3)caspase-3 inhibitor sevoflurane group(3%sev+Q-VD-OPh);(4)LC3B inhibitor sevoflurane group(3%sev+3-MA);(5)NF-κB inhibitor sevoflurane group(3% sev+BAY 11-7085).On postnal 21 days after birth(P21)received 3% sevoflurane for 4 hours,and starting at postnal 28 days(P28),rats received Morris water maze test to determine the spatial memory abilities.(n = 10,50 in total).Results: After anaesthesia on P21 by 3% sevoflurane for 4 hours,compared with the ctl group,the neuronal differentiation of late progenitor cells in the dentate gyrus(DG)to immature and mature cells was significantly reduced compared in 3%sev group.There was statistically significance(P <0.01);Start from P28,compared with the ctl group,the escape latencies in 3% sev group rats on the first,second,third,and fourth days were similar,the escape latency in 3% sev group was significantly prolonged on the fifth day,and the difference was statistically significant(P <0.01);compared with the ctl group,the number of platform crossing in 3% sev group was significantly reduced,and the difference was statistically significant(P<0.01).After 3% sevoflurane for 4 hours at 2h,24 h,4d,and 7d,the neural apoptosis and neural autophagy in DG area were increased significantly,and the differences were statistically significant at each time point(P <0.05);In 3%sev+Q-VD-OPh group and 3%sev+3-MA group,compared with 3%sev group,the differentiation of late progenitor cells in DG to newborn immature and mature cells were significantly increased.The difference was statistically significant(P <0.05);compared with 3%sev group,3%sev + 3-MA group had significantly lower protein level of caspase-3(P <0.01),however,compared with 3%sev group,the protein levels of LC3 BII and P62 in 3%sev + Q-VD-OPh group were similar;Among 3%sev group,3%sev +3-MA group and 3%sev +Q-VD-OPh group, there were no significant difference in escape latency on the first,second,third,and fourth days.Compared with 3%sev group,the escape latencies were significantly shortened,and the difference was statistically significant(P <0.01)in3%sev +3-MA group and 3%sev Q-VD-OPh group;Compared with ctl group,the expression levels of P65 in DG at 2h,24 h,4d,were significantly increased,the differences were statistically significant(P <0.05),but there was no significant change at 7d(P> 0.05);Compared with the ctl group at 2h,24 h,4d,and 7d after 4h anesthesia,the expression level of P-P65 in DG increased significantly,and the differences were statistically significant(P<0.05),while the expression of IκB decreased,and the differences were statistically significant(P <0.05);Compared with the 3%sev group,the late progenitor cells in DG in 3%sev +BAY 11-7085 group,had significantly increased differentiation into newborn immature and mature granulocytes,and the difference were statistically significant(P <0.05);compared with 3%sev group,the levels of LC3 BII and caspase-3 in DG region in 3%sev + BAY 11-7085 group were significantly reduced,and the differences were statistically significant(P<0.05),however,the level of P62 was significantly increased,and the difference was statistically significant(P<0.05);Compared with 3%sev group,there was no significant difference in escape latency in 3%sev+ BAY 11-7085 group on the first,the second,the third,and the fourth day,but compared with 3%sev group on the fifth day,the escape latency in 3%sev+ BAY 11-7085 group was significantly shortened,and the difference was statistically significant(P <0.01);Compared with sev group,the number of plate crossing in 3%sev + BAY 11-7085 group was significantly increased,and the difference was statistically significant(P <0.05).Conclusion: 1)3% sevoflurane 4h could inhibit the differentiation of late progenitor cells into immature and mature neurons in DG region,and caused short term impairment of spatial memory function;2)there were increased apoptosis of granule cells in DG caused by 3% sevoflurane 4h,and apoptosis inhibitors can reverse the decline of late progenitor cells to immature and mature neurons;3% sevoflurane for 4h could increase autophagy in DG,and autophagy inhibitors can reverse the decreased differentiation of late progenitor cells towards immature and mature neurons;the increased autophagy after anesthesia indirectly decreased the differentiation of late progenitor cells into neurons by affecting apoptosis;the increased neuronal apoptosis and autophagy in DG region were involved in affecting the recent spatial memory dysfunction in rats.3)IκB / NF-κB / P65 pathway could affect the differentiation of late progenitor cells into immature and mature neurons;3% sevoflurane 4h increases autophagy and apoptosis in DG by IκB / NF-κB / P65 signaling pathways;IκB / NF-κB / P65 pathways are involved in a decrease in spatial memory function caused by 3% sevoflurane 4h in young rats. |
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