Effects Of Sevoflurane On Learning And Memory Of Neonatal Rats And Its Underlying Gene Regulation Mechanism

Background:Whether general anesthetic was of adverse effects to learning and memory of infants and preschool children or not has always been a hot issue in medical research and a focus for social concern.As early as 2008,America Society of Anesthesiologist once published three clinical survey reports on mid-long-term cognition changes after pediatric general anesthesia operation.It’s specifically pointed out in the reports that risks for long-term cognition changes would increase if the patients received general anesthesia in neonatal period or infancy for multiple times(over two times).Therefore,in-depth exploration of general anesthetic was of great significance to the learning and memory of preschool children.Sevoflurane was an inhaled general anesthetic commonly used in clinical practice and was suitable for anesthetic induction and maintenance for each age and surgeries on each part.As sevoflurane induction was featured by rapidity,stable process,no irritation and easy acceptance,it’s especially suitable for child patients who were unable to cooperate.With widespread use of sevoflurane in pediatric surgery,some researchers found that sevoflurane was of potential neurotoxicity to the brains of mammals in developmental stage and long-term learning and memory impairment would be caused.However,the detailed mechanism was unclear.It’s showed in retrospective clinical studies that children who received general anesthesia before age 1 would suffer from memory impairment when they were 6 to 11 years old.It’s indicated in animal experiments that obvious spatial learning and memory impairment were seen at 7th week after the 7-day old mice inhaled 2.4%sevoflurane for 4 hours.A research was performed on rhesus monkeys in which sevoflurane was inhaled for 4 hours in 6-10 day-old rhesus monkeys and then inhaled again after 14 days and 28 days,and the examination at the age of 6 months showed that memory was markedly impaired,and decline of memory would last till 1-2 years old.Clinical researches and animal experiments all indicated that sevoflurane was of toxicity to brains in developmental stage.However,as influencing factors were complicated and changeable,especially exposure age,administration mode,route and concentration varied,there was still controversy regarding impairment and extent of sevoflurane to brain learning and memory in developmental stage.As one of the most basic functions of brain,learning and memory was a dynamic and complex process and its formation and expression involved multiple molecular biological mechanisms.At present,researches on effects of sevoflurane to learning and memory mechanism mainly focused on regulating learning and memory related-protein expression,affecting plasticity of neuronal synapse and regulating apoptosis.Firstly,sevoflurane played its anesthetic function through activatingγ-aminobutyric acid receptor and/or N-methyl-D-aspartic acid.NMDA receptor mediated slow components during excitatory synapse transmission and mainly involved regulating long-term potentiation and long-term depression.It’s an important molecule related to learning and memory.As utmost inhibitory neurotransmitter in central nervous system,GABA was correlated to learning and memory as well.Then,sevoflurane could regulate expression of related molecules and thus disturb learning and memory process,for example elevating expression of β-amyloid protein and down-regulating expression of brain-derived neurotrophic factor,etc.However,further exploration was required for detailed mechanism.Moreover,plasticity of synapse was a significant mechanism for learning and memory,including LTP and LTD.It’s indicated in researches that sevoflurane could inhibit LTP formation,which might be possibly one of the mechanisms for learning and memory impairment caused by sevoflurane.Finally,sevoflurane could also lead to apoptosis of nerve cells,especially that in developmental stage,thud leading to the reduction of learning and memory capabilities.miRNA was a kind of non-coding single-stranded micro RNA molecule whose encoding length of endogenous gene was approximately 21 to 23 nucleotides widely existed in eucaryotic organism and it also participated in regulation of gene expression after transcription.Researches verified that miRNA could degrade or inhibit mRNA transcription through complementary matching with target genes mRNA,thus regulating expression of target genes.Complete cycle of proliferation of eucaryotic organism included G0/G1,S,G3 and M phase.Two important nodal points during cell cycle progression were transformation from G1 to S phase and G2 to M phase,which were strictly regulated by different cyclin-dependent kinase and its activated cyclin subunits.Studies showed that cyclin A was of great importance to maintaining beginning and ending of S phase and mitosis,and cyclin B was associated with initiating mitosis.Researches verified that sevoflurane could lead to expression changes of a large amount of genes in hippocampus and pose injuries to spatial learning and memory capabilities of new-born rats.Then,should varied gene expression caused by sevoflurane be associated with targeted regulation of miRNA?Whether sevoflurane could inhibit cell proliferation through affecting cell cycle progression or not?What’s its detailed mechanism?Till now,no related reports were seen.This research would explore inner relationship between behavioral changes of new born rats and expression of cyclin as well as miRNA targeted regulation caused by sevoflurane from multiple angles such as behavioristics,molecular biology and gene regulation as well as illustrate effects and mechanism of sevoflurane to the learning and memory capabilities of new born rats,providing reference to reasonable medication in clinical practice.Part One Effects of sevoflurane on learning and memory of neonatal ratsObjective:To verify the impairment of learning and memory by sevoflurane in neonatal rats.Methods:The 4-week old male SD rats were divided randomly into control group(CTL group)and sevoflurane group(SEVO group),with 10 in each group.CTL group is exposed to normal air for 4 hours and SEVO group to 5%sevoflurane for 4 hours.Fresh gas flow was set at 1.5 L/min.Morris water maze test and PM-DAT were taken to evaluate learning and memory indexes of new born rats.Western blotting was applied to examine expression level of hippocampus BDNF and Ki-67 staining was used to observe proliferation activity of hippocampus nerve cells of rats.Results:Morris water maze test showed that escape latency in the two groups was gradually reduced along with time extension.There was significant difference in two groups(P<0.01).Compared with CTL group,escape latency was obviously extended in SEVO group at 4th week and 6th week testing after sevoflurane exposure(P<0.05).Path length in two groups was gradually shortened along with time extension.Compared with CTL group,path length at 6th week testing in SEVO group-was greatly extended(P<0.05),indicating that sevoflurane exposure could significantly injure spatial memory capabilities of new born rats.In PM-DAT testing,length of stay of close arm in SEVO group was greatly longer than that in CTL group(P<0.05),which meant that after sevoflurane exposure,during-exercise time was longer and performance of new born rats was poorer.Length of stay of open arm in SEVO group was also remarkably longer than that in CTL group(P<0.05),which meant that after sevoflurane exposure,time was longer and performance of new born rats was poorer during testing.Compared with CTL group,BDNF level in cerebral tissues was significantly reduced in SEVO group and number of hippocampus Ki-67 positively proliferated nerve cells were markedly decreased(P<0.05).Conclusion:Sevoflurane could injure learning and memory capabilities of new born rats,and injury function might possibly be related to down regulating expression of intracerebral BDNF protein and inhibiting proliferation of hippocampus nerve cells.Part Two Effects of sevoflurane on proliferation of HCN-2 neurons and its molecular mechanismObjective:To obersve the impacts of sevoflurane exposure on proliferation of neurons and to explore its molecular mechanism.Methods:HCN-2 cell line was applied for in vitro cell experiment to study effects of sevoflurane to proliferation of nerve cells at cellular level.Same amount of HCN-2 cells was divided randomly to sevoflurane group of different concentration(0.5%group,1%group,2%group and 5%group)and control group(air ratio was 5%CO2,21%O2 and N2).After 48 hours,Ki-67 staining and DAPI staining were performed to analyze proliferation activity of nerve cells.Western blotting was taken to examine expression level of cyclin-dependent kinase(CDK)and cyclin(CCNA1,CCNA2,CCNB1,CCNE1,CCNE2,CDK1,CKD2 and CDK4).qPCR was used to examine expression level of CCNA2 mRNA.Then bioinformatics was used to analyze miRNA of targeted CCNA2 and check expression changes in HCN-2 cells after sevoflurane exposure.Plasmid transfection was adopted to verify miRNA regulation on CCNA2 expression.Results:Ki-67 staining and DAPI staining showed that sevoflurane could obviously reduce number of Ki-67 positive cells and total cell number and was of concentration-dependence(0%vs 0.5%,0.5%vs 1%,1%vs 2%and 2%vs 5%)(P<0.05).Western blotting was used to examine expression of CCNA1,CCNA2,CCNB1,CCNE1,CCNE2,CDK1,CKD2 and CDK4 protein and the results demonstrated that after sevoflurane exposure only expression level of CCNA2 protein was greatly decreased and was of concentration-dependence(0%vs 0.5%,0.5%vs 1%,1%vs 2%and 2%vs 5%)(P<0.05).However,further experiment demonstrated that expression level of CCNA2 mRNA in HCN-2 cells showed no changes(P>0.05).Bioinformatics analysis showed that rats CCNA2 has 18 kinds of targeted miRNAs.Compared with CTL group,sevoflurane could significantly up regulate miR-19-3p level among the 18 targeted miRNA.Plasmid with antisense miR-19-3p which were transfected in HCN-2 cells could greatly lower miR-19-3p level(P<0.05),while plasmid with antisense miR-19-3p which were transfected in HCN-2 cells could remarkably enhance miR-19-3p level(P<0.05),and luciferase activity of CCNA23’-UTR was markedly lowered(P<0.05).In addition,over-expressed miR-19-3p could significantly decrease protein expression level of CCNA2(P<0.05)and after miR-19-3p knockdown,protein expression level of CCNA2 was significantly enhanced(P<0.05).Conclusion:(1)Sevoflurane would pose injury to the proliferation of HCN-2 neurons,and the mechanism might possibly be related to expression of CCNA2 protein after transcription.(2)Sevoflurane could up regulate miR-19-3p level in HCN-2 cells.(3)In HCN-2 cells,over-expressed miR-19-3p could reduce level of CCNA2 protein.On the contrary,level of CCNA2 protein was increased.Part Three Molecular mechanism of sevoflurane on learning and memory impairment in neonatal ratsObjective:Molecular biology was applied to illustrate molecular mechanism of sevoflurane injuring learning and memory capabilities of new born rats and explore regulation role of miRNA-19-3p in it.AAV with antisense miR-19-3p was constructed and injection to bilateral hippocampus was performed to verify its protective role to proliferation of nerve cells and learning and memory injuries after down regulated expression of miR-19-3p in bilateral hippocampus.Methods:AAV-as-miR-19-3p was constructed and AAV control and HCN-2 cells transfection were performed.AAV-as-miR-19-3 transfection cells,AAV-null transfection cells and null transfection cells(Unt)were observed with immunofluorescence.Then,intracranial AAV-as-miR-19-3p or AAV-null was injected to bilateral hippocampus of four-week old rats,with 10 in each group.After a-week injection,these rats were exposed to 5%sevoflurane.Morris water maze test and PM-DAT were used to assess learning and memory capabilities.Western blotting was applied to examine expression level of BDNF in hippocampus and Ki-67 staining was taken to observe proliferation activity of neurons in rat hippocampus.Results:AAV-as-miR-19-3p was constructed and HCN-2 cells were transfected.The results showed that there was no obvious difference regarding miR-19-3p level in AAV-null transfection cells and Unt cells(P<0.05),while miR-19-3p level in AAV-as-miR-19-3p transfection cells was obviously lower than that in the other two groups(P<0.05)and miR-19-3p level was reduced by about 80%.AAV-as-miR-19-3p or AAV-null was injected to bilateral hippocampus of the rats and the results indicated that escape latency,path length and other indexes of rats in Morris water maze test and PM-DAT testing could be greatly improved in AAV-as-miR-19-3p group(P<0.05).Meanwhile,miR-.19-3p knockdown could significantly elevate BDNF in cerebral tissues and increase positive proliferation number of hippocampus Ki-67(P<0.05).Conclusion:(1)Sevoflurane-would injure learning and memory capabilities through up regulating miR-19-3p to inhibit and transcribe expression of CCNA2 protein.(2)AAV-as-miR-19-3p injection to bilateral hippocampus could inhibit up-regulation of miR-19-3p expression as well as protect proliferation of nerve cells and learning and memory impairment of new born rats after sevoflurane exposure.