Mechanism Research Of Anesthesia And Surgery Induced Neurotoxicity In Developing Mouse Brain

During the past decades,a lot of studies focused on anesthesia and surgery induced neurotoxicity in infants and chilidren.It's clear that multiple anesthesia and surgery can lead to long-term cognitive dysfuntion in children,however,whether single exposure to anesthesia and surgery will cause cognitive dysfuntion still have no conclusion.Moreover,Tau protein phosphorylation is one of the key point in anesthesia and surgery induced learning impairment.Extracellular ATP has a regulatory effect on neuronal excitability and synaptic plasticity.In this study,we discuss the interaction between extracellular ATP,Tau and the cognitive function in young mice.Our aim is to provide a theoretical support for anesthesia and surgery brain protection.PART 1: Effects of single time sevoflurane anesthesia and surgery on extracellular ATP and cognitive function in young miceObjective: Single anesthesia and surgery may affect young mice long-term learning and memory function.This part experiment was designed to study the effects of sevofluane plus surgery on extracellular ATP level and long-term cognitive function in young(P8)mice.Method: Postnatal(P)8 day-old(P8)mice were randomLy divided into 2 groups: Control group and Sevoflurane+Surgery group.Sevoflurane+Surgery group mice received 3% sevoflurane with 60% oxygen for 2 hours and during the 2 hours,mice received exploratory laparotomy.Control mice were hold in box with 60% oxygen flow for 2 hours.Immediately after sevoflurane anesthesia and surgery,collect cortex and blood samples doing ATP detection.Also harvest cortex mitochondria doing the mitochondria resparation function detection.Usinng microdialysis collcet P8 mice ISF samples when the mice awake,under anesthesia and during surgical procedure.Using Morris water maze to detect the learning and memory functions of mice in postnatal 30 days.Results: 1.Sevoflurane anesthesia and surgery could cause cognitive impairment in P8 mice;2.Sevoflurane anesthesia and surgery slightly increased cortex ATP level;3.Mice blood ATP level didn't changed after Sevoflurane anesthesia and surgery;4.Mitochondria respararion rate had no difference between two groups;5.When mice exposed to single sevoflurane anesthesia,ISF ATP level won't be affect,but sevoflurane anesthesia plus surgery significantly increased ISF ATP level in the same mice.Conclusion: P8 mice exposed to sevoflurane anesthesia and surgery can result in long-term learning and memory dysfunction,and this effects may relate to ISF ATP increasing.PART 2: Key Role of extracellular ATP in the neurotoxicity caused by sevoflurane anesthesia and surgery in the developing brain of young miceObjective: According to first part result,sevoflurane anesthesia and surgery can cause ISF ATP increase in young mice.In this experiment,our aim is to demonstrate the relationship of increased extracellular ATP ang Tau phosphorylation both in vivo and in vitro.Methods: 1.Dividing postnatal(P)8 day-old(P8)mice into 3 different groups,including Control group,ATP group and Sevoflurane+Surgery group.ATP group were treated with 250mg/kg/BW ATP by intraperitoneal injection(i.p.)right before sevoflurane anesthesia,and Control group and Sevoflurane+Surgery group treated with same volum saline.After sevoflurane anesthesia and surgery,harvest cortex protein and measureTau-PS202/PT205(AT8)?total Tau(Tau46)expression using Western blot.2.Cultured SH-SY5 Y cell were divided into two groups,Control group and ATP group.ATP group were treated with 200 nM or 2?M ATP for 2 hours,then collceted protein sample and measured Tau-PS202/PT205(AT8)?total Tau(Tau46)expression level.3.Given primary neuron cell 100?M or 1mM ATP stimulation for 2 hours,then detected Tau-PS202/PT205(AT8)?total Tau(Tau46)expression level.Results: 1.Both ATP injection and sevoflurane anesthesia plus surgery increased Tau-PS202/PT205 level without total Tau change;2.2?M ATP treated SH-SY5 Y cell expressed higher Tau-PS202/PT205,total Tau had no difference compared with control group.3.1mM ATP treatment caused Tau-PS202/PT205 expression increasing in primary neuron cell but had no influence on total Tau expression.Conclusion: The higher extracellular ATP levels in the young mice?SH-SY5 Y and primary neuron induce Tau phosphorylation in serine202 and tyrosine205 without change total Tau level.These means extracellular ATP play a key role in sevoflurane anesthesia plus surgery induced cognitive impairment.PART 3: Extracellular ATP induced Tau phosphorylation depends on CDK5Objective: Part 2 result showed that higher extracellular ATP level can reinforce Tau phosphorylation.In this experiment,we used massspec analyze and Western blot to find the key enzyme in extracellular ATP induced Tau phosphorylation.Methods: 1.Dividing postnatal(P)8 day-old(P8)mice into 3 different groups,including Control group,ATP group and Sevoflurane+Surgery group.ATP group were treated with 250mg/kg/BW ATP by intraperitoneal injection(i.p.)right before sevoflurane anesthesia,and Control group and Sevoflurane+Surgery group treated with same volum saline.Aftersevoflurane anesthesia and surgery,harvest cortex protein and do massspec anylyze to find the changed protein belonged to Tau kinase.GSK3??p-GSK3?-ser9?p-GSK3?-tyr216 and CDK5 level were reconfirmed by Western blot.2.Given primary neuron cell1 mM ATP stimulation for 2 hours,then detected GSK3??p-GSK3?-ser9?p-GSK3?-tyr216 and CDK5 level using Western blot.Results: 1.Massspec analyze result find two Tau kinase increased,including GSK3?and CDK5.2.In P8 mice,both sevoflurane plus surgery and ATP administration didn't affect GSK3?level in Western blot result,but p-GSK3-ser9 increased and p-GSK3?-tyr216 decrased in these two group,which means reduced GSK3?activity.CDK5 expression level obviously increased in sevoflurane and surgery group and ATP group 2.ATP treated primary neuron cell had no difference in GSK3??p-GSK3-ser9 and p-GSK3-tyr216 level compared with control group,but had inforced CDK5 expression.These results show that extracellular ATP induced Tau phosphorylation depends on CDK5 but not GSK3?.Conclusion: Extracellular ATP induced Tau phosphorylation depends on CDK5 but not GSK3?.CDK5 is the key enzyme in sevoflurane anesthesia plus surgery induced cognitive impairment.PART 4: Extracellular ATP regulate CDK5 & Tau phosphorylation via GluR2Objective: Both Tau and CDK5 located in cytoplasm but not cell memberane,how extracellular ATP can regulate CDK5 then affect Tau phosphorylation is our aim in this experiment.Analyzing massspec datas find that GluR2 is the most obviously changed memberane receptor.In this part,we use pep2 m,a GluR2 inhibtor to demonstrate if GluR2 is involved in extracellular ATP induced Tau phosphorylation.Methods: 1.36 Postnatal(P)8 day-old(P8)mice were designed to be separated in 6 groups,Control+aCSF group?ATP+aCSFgroup?Sevoflurane+Surgery+aCSF group?Control+ pep2 m group ? ATP+pep2m group ? Sevoflurane+Surgery+pep2m group.pep2 m or aCSF were asministrated using intracerebroventricular injection.After sevoflurane anesthesia and surgery,harvest cortex tissue and determine CDK5 and Tau-PS202/PT205 level by Western blot.Results: 1.Both massspec data and Western blot result show that GluR2 expression level is significantly higher in ATP group and Sevoflurane+Surgery group.2.Consistant with previous data,CDK5 and Tau-PS202/PT205 increased in ATP+aCSFgroup and Sevoflurane+Surgery+aCSF group compared with Control+aCSF group.And these edverse effects can be reversed by pep2 m.Conclusion: Extracellular ATP regulate CDK5 expression and Tau phosphorylation via GluR2.Summary1.Single sevoflurane anesthesia plus surgery in P8 mice can result in long-term learning and memory impairment.2.Sevoflurane anesthesia and surgery can result in higher ISF ATP level in young mice and the increased ATP is from CNS not from blood.3.Increased extracellular ATP can induce Tau phosphorylation both in vivo and in vitro.4.Extracellular ATP induced Tau phosphorylation depends on CDK5 not GSK3?5.Extracellular ATP regulate CDK5 expression and Tau phosphorylation via GluR2.