Isoflurane And Sevoflurane Affects Wnt/?-catenin Signaling Pathways In Hippocampal Formation Of Developmental Rats

BackgroundSevoflurane and isoflurane is two commonly used volatile anesthetics and demonstrates numerous advantages over other intravenous or inhalation anesthetics,including a lower blood solubility that facilitates rapid induction.It has been found that inhaled anesthetics can affect the proliferation and differentiation of synapses of neurons in the central nervous system during development,lead to apoptosis of neurons,and even affect their learning and memory ability in adulthood.And the damage caused by different types of inhaled anesthetics may vary.Paediatric neurologist JohnOlney and his colleagues first found that blocking N-methyl-D-aspartate(NMDA)glutamate receptor for only a few hours could cause extensive apoptotic neurodegeneration in the brain of developing rats,suggesting that excitatory neurotransmitter glutamate(Glu)could control the survival of neurons under the action of NMDA receptor.However,some retrospective studies have shown transient neurological sequelae in children after prolonged anesthesia,and more studies confirm long-term neurological damage after neonatal surgery and anesthesia.On Dec.14,2016,the US Food and Drug Administration issued a warning regarding impaired brain development in children following exposure to certain anesthetic agents used for general anesthesia,namely the inhalational anesthetics isoflurane,sevoflurane,and desflurane,and the propofol and midazolam during the first 3 months of pregnancy.There has been growing concern about the detrimental effects of certain anesthetic agents on the developing brain.Human studies in this area are limited and currently inconclusive.The Wnt/?-catenin signaling pathway is a crucial regulator of the growth and proliferation of nerve cells.Wnt signaling is critical for neuronal maturation,apoptosis,dopaminergic and hippocampal neurogenesis.GSK3? and ?-catenin are key molecules in the Wnt pathway.Studies have shown sevoflurane may affect the learning and memory abilities of the developmental rat.Furthermore,the Wnt/?-catenin pathway may be involved in the negative effects of sevoflurane exposure in the developmental rat.However,the relevant literature involves fewer protein expression studies involving signaling channels.Therefore,this topic is proposed to further investigate the specific role of the Wnt/?-catenin signaling pathway in the neurotoxicity caused by inhaled anesthetics.Inhalation anesthetic is the most commonly used inhalation anesthetic in children.Through our study on the neurotoxicity of inhaled anesthetics to the developing brain,We hope to investigate the mechanism of Wnt/?-catenin channel in the neurotoxicity of inhaled anesthetics,and to find the target of inhibiting neurotoxicity.we hope that under the condition of ensuring the anesthetic effect,we can reasonably select different anesthetics and adjust the concentration and time of anesthetics for different patients.Ultimately these efforts would lead to safer anesthesia care and better postoperative outcomes in children.We used animal experimental models and HT22 hippocampal neuronal cell models to observe the effects of commonly used inhaled anesthetics on rodent hippocampal neurons and learning memory,in order to understand the pathway mechanisms to provide a theoretical basis for clinical work.Part one To investigate isoflurane and sevoflurane affects Wnt/?-catenin signaling pathways in hippocampal formation by using animal modelPurpose:Different anesthetic models of rat brain were established and different treatments were carried out in each group to explore the spatial learning and memory ability of neonatal rats damaged by different inhaled anesthetics based on Wnt-catenin signal pathway.Methods:1.SD rats on postnatal day 7 were divided into 5 groups.Control group:it was exposed to air for 6 h;Ioflurane group(ISA):0.75%isoflurane for 6h;Hepaflurane group(SEA):0.85%sevoflurane 6h;The Wnt inhibitor isoflurane group(group ISAI):0.75%isoflurane was inhaled for 6 h;after injection of the Wnt inhibitor;Wnt inhibitor sevoflurane group(group SEAI):0.85%sevoflurane was 6 h.inhaled after injection of the Wnt inhibitor;The rearing was continued until 5 weeks and 10 weeks.2.The spatial learning and memory abilities were observed in different groups of rats using water maze experiments3.Rat hippocampal tissue sections were performed,and hippocampal cell damage was observed under electron microscopy.4.The mRNA expression levels of wnt,?-catenin and gsk-3 ? were measured by quantitative real-time polymerase chain reaction(RT-PCR).5.The protein expression levels of wnt,?-catenin and gsk-3 ? were measured by westernblot.Results:1.Morris water maze results show isoflurane and sevoflurane could impair P7 rats' learning and memory capability,while these effects were reduced over time.When rats were treated with Wnt inhibitor XAV 939,the impairment of brain could relieve.(p<0.05)2.Hematoxylin and Eosin(H?E)results show isoflurane and sevoflurane treated group,the neurons were significant shrinkage,loss of neurons and widespread damage in ISA and SEA group at 5-week after anesthesia.However,P7 rats treated with Wnt inhibitor XAV 939 could markedly relief that damage.Those morphological changes were significantly moderated and reduced neuronal loss at 10-week.3.RT-PCR results show the mRNA expression levels of Wnt3a and ?-catenin were significantly higher in the ISA and SEA groups,while GSK-3? expression level was significantly lower than that of control group(p<0.01).The expression levels of Wnt3a and ?-catenin were significantly reduced after Wnt inhibitor XAV 939 treatment,while GSK-3? expression level was dramatically increased in ISAI and SEAI groups(p<0.05).At 10 weeks,Wnt3a and ?-catenin mRNA were active in the inhaled anesthetics group but reduced(p<0.01)compared with 5 weeks,while the Wnt inhibitor group essentially restored normal expression levels.4.Western Bolt results show the proteins expression levels of Wnt and ?-catenin were significantly up-regulated in ISA and SEA group at 5 weeks post-anesthesia compared to control group,while GSK-3? was markedly decreased in ISA and SEA groups(p<0.01).Wnt inhibitor XAV 939 was significantly inhibited the up-regulation of Wnt and ?-catenin and increased phosphorylated-GSK-3? expression in the hippocampus in ISA and SEA groups,as compared with the control group at 5 weeks(p<0.05).After 10 weeks,Wnt and ?-catenin were also markedly up-regulated,and GSK-3? was dramatically decreased in ISA and SEA group,compared with control grou,but the degree of regulation was much lower than the same group at 5-week.Conclusion:1.Contact to isoflurane and sevaflurane in the developing rodent brain leads to apoptosis in hippocampal neurons,causing memory and learning impairment in rats,and wnt signaling plays an important role.2.Brain exposure to isoflurane and sevoflurane in developmental rodent may be reversible and return to normal over time.Part two To investigate isoflurane and sevoflurane affects Wnt/?-catenin signaling pathways in hippocampal formation by using The mouse hippocampal neuron HT-22 cellsPurpose:In this study,mouse HT22 hippocampal neurons were treated differently per group of neuronal cells to explore the damage to mouse hippocampal neurons with different inhaled anesthetics based on the wnt/?-catenin signaling pathway.Methods:In this study,mouse HT22 hippocampal neurons cells were divided into the control group,the isoflurane group(the ISA group),the sevoflurane group(the SEA group),the Wnt inhibitor isoflurane group(the ISAI group),and the Wnt inhibitor sevoflurane group(the SEAI group).Mouse HT22 hippocampal neuronal cells were exposed to inhaled anesthetic drugs of sevoflurane and isoflurane.1.Mouse HT22 hippocampal neuronal cells apoptosis were detected by TUNEL assay.2.Mouse HT22 hippocampal neuronal cells apoptosis was detected by Hoechst33342/PI fluorescent staining.3.Mouse HT22 hippocampal neuronal cells apoptosis was detected by by Flow Cytometry Using Annexin V and Propidium Iodide Double Staining.4.The mRNA expression levels of wnt,?-catenin and gsk-3? were measured by quantitative real-time polymerase chain reaction(RT-PCR).5.The protein expression levels of wnt,?-catenin and gsk-3? were measured by westernblot.Results:1.Apoptosis of HT22 hippocampal neurons was detected by 1.TUNEL staining:a significant increase in apoptotic cells in the isoflurane and sevoflurane groups(p<0.01)and more pronounced in the isoflurane group than in the sevoflurane group(p<0.05).While the Wnt inhibitor group added had reduced apoptosis(p<0.01)compared to the ISA and SEA groups of neurons.2.Apoptosis and necrosis of HT22 hippocampal neurons were detected by Hoechst33342/PI double staining:late apoptosis and necrosis in the isoflurane and sevoflurane group compared with the control group(p<0.01)and more severe in the isoflurane group than the sevoflurane group(p<0.05).However,the Wnt inhibitor group was added showed reduced neuronal necrosis(p<0.01)compared to the ISA and SEA groups.3.Detection of HT22 hippocampal neurons by flow cytometric analysis:higher HT22 cells(p<0.01)when exposed to isoflurane and sevoflurane and higher than sevoflurane(p<0.05),suggesting that isoflurane showed more significant damage to neurons than sevoflurane.The reduction of apoptosis in the Wnt inhibitor group compared with the ISA and SEA groups(p<0.01),indicating that the Wnt inhibitor improved neuronal apoptosis,suggesting an important role for Wnt signaling in nerve damage induced by inhaled anesthetics.4.The mRNA expression levels of Wnt3a,?-catenin and GSK-3? were determined by qRT-PCR:the mRNA expression levels of Wnt3a and ?-catenin were significantly increased in the ISA,SEA group,but significantly decreased in GSK-3?(p<0.01).mRNA expression levels of Wnt3a and ?-catenin were higher than in the SEA group,while GSK-3? were lower than in the SEA group(p<0.05).The plus Wnt inhibitor group showed significantly lower Wnt3a and ?-catenin mRNA expression levels,whereas GSK-3? was significantly increased compared with the ISA and SEA groups(p<0.01).5.The expression levels of Wnt3a,?-catenin and GSK-3? proteins were determined by Westernblot:Wnt3a and ?-catenin proteins were significantly increased in ISA and SEA,while GSK-3? proteins were significantly decreased(p<0.01).Expression levels of Wnt3a and ?-catenin proteins were significantly higher in the isoflurane group than in the sevoflurane group,while GSK-3? was lower(p<0.05).Wnt inhibitors significantly inhibited the upregulation of Wnt3a and?-catenin proteins and increased GSK-3? expression in hippocampal tissues as compared to the ISA and SEA groups(p<0.01).The results show that isoflurane and sevaflurane can influence the Wnt/?-catenin pathway-related proteins in HT22 hippocampal neurons and cause elevated Wnt3a and ?-catenin protein expression and decreased GSK-3? protein expression.Moreover,the activation of the Wnt/?-catenin pathway was more significant than that of sevoflurane.Conclusion:1.Ioflurane and sevoflurane exert neurodamaging on ht22 hippocampal neuronal cells.2.Ioflurane damage to ht22 hippocampal neurons cells was more pronounced than sevoflurane.3.wnt/?-catenin signaling plays an important role in isoflurane and sevoflurane induced apoptosis in ht22 hippocampal neurons.