Effect And Mechanism Of Mid-gestational Exposure To Sevoflurane On Proliferation Of The Offspring’s Neural Stem Cells In Rats

Objective: With the rapid development of the fetal intervention procedures,more and more pregnant women in the second trimester had received general anesthesia.Anesthesiology focus mainly on: Is there any effect on the development of the fetal nervous system by using anesthetic drugs during the second trimester? Will this influence have any manifestation after birth? What is the possible mechanism? The authors set out to assess the effects of the most widely used anesthetics---sevoflurane,on proliferation of neural stem cells and on learning and memory in fetal and offspring rats,and discuss on the related mechanism.Methods: 1.Pregnant SD rats(gestational day 14)were randomly divided into 3groups: the control group(C GROUP),low concentration group(L GROUP)and high concentration of sevoflurane group(H GROUP).Pregnant rats were treatd with 30%oxygen,2% sevoflurane,3.5 sevoflurane for 2 hours,respectively.Learning and memory function in the offspring rats was determined in the Morris water maze.2.Pregnant SD rats(gestational day 14)were randomly divided into 3 groups: the control group(C GROUP),low concentration group(L GROUP)and high concentration of sevoflurane group(H GROUP).Pregnant rats were treated with 30% oxygen,2%sevoflurane,3.5 sevoflurane for 2 hours,respectively.In six specific time point(6 h,24 h,48 h after treatment,P0,P14 and P28 after birth)fetal brain tissues or offspring rats hippocampal tissue were havested and subjected to western blot and immunofluorescence to assess Nestin,Sox-2,Ki67 level and the number of proliferative and apoptotic NSC.3.Pregnant SD rats(gestational day 14)were randomly divided into 5 groups: control group(C GROUP),low sevoflurane group(L GROUP),high sevoflurane group(H GROUP),sevoflurane+lithium group(H+Li GROUP)and lithium group(Li GROUP).Rats received different concentration for 2h.The rat offspring were weaned at 28 days for Mirror Water Maze behavioral studies and fetal brains or postnatal hippocampal tissues were harvested for immunofluorescence staining,real time PCR,and western blot to detect NSC proliferation and the Wnt/β-catenin signaling levels.Results: 1.There were no significant differences in ABG values and hemodynomic levels of the pregnant rats among the three groups.There was also no statistically significant difference in swimming speed of the offsprings among the three groups.The pups of the H GROUP spent more time to find the platform(escape latency)than those of C GROUP(P<0.05),however,no differences were observed between the C GROUP and L GROUP in comparison of the escape latency.In the probe test,the number of crossing over the former platform location in H GROUP pups was fewer and the time spent in the fouth quadrant where the platform located was shorter than the control animals,while no differences were observed between the C GROUP and L GROUP.2.In the H GROUP pups,the expression of Nestin,Ki67 and Nestin and Ki67 positive cells in the fetal brain or offsprings’ hippocampal area decreased compared with the C GROUP.The TUNEL positive cells displayed a tendency of increase in the fetal brain or offsprings’ hippocampal area in the H GROUP pups.Consistent with the findings of immunofluorescence studies,western blot analysis showed that the levels of Nestin,Sox-2 and Ki67 protein markedly decreased in the fetal brain or offsprings’ hippocampal region of the H GROUP pups when compared with the C GROUP.However,there was no difference between L GROUP and C GROUP.3.The results showed that exposure to 3.5% sevoflurane(H GROUP)in mid-gestational rats increased fetal GSK-3β protein levels as well as reducedβ-catenin protein levels,CD44 and cyclin D1 m RNA levels when compared with control group.However,these effects in fetus were attenuated by lithium chloride.Conversely,2% sevoflurane(L GROUP)did not show the same effects.Conclusions: 1.High concentration sevoflurane anesthesia in pregnant rats of second trimester induced learning and memory impairment in offspring rats,manifesting the prolonged escape latency and reduced the number of passing across platforms the region.While low concentration sevoflurane anesthesia showed no learning and memory impairment,indicating that the toxic effect of anesthetics on the developing brain is concentration-dependent.2.High concentration sevoflurane anesthesia in pregnant rats of second trimester reduced neural stem cell proliferation and induced apoptosis in the fetal brain or hippocampus of the offspring rats.3.Sevoflurane exposure in the second trimester inhibits fetal neural stem cell proliferation possibly via Wnt/β-catenin pathway and impairs postnatal learning and memory function in a dose-dependent manner.