The Effect And Mechanism In Sevoflurane-induced Neurotoxicity Due To Brain-derived Neurotrophic Factor (BDNF) Cleavage Caused By Changes In The Balance Of Fibrinolytic System

Objective: Due to the improvement of current pediatric anesthetic technology,the death that the parents of children were concerned about has gradually transformed into more advanced needs: whether it can reduce the impact on the children's learning and memory abilities.Nowadays,sevoflurane is commonly used for pediatric general anesthesia.In recent years,more and more animal experiments have confirmed that sevoflurane can cause extensive neuronal apoptosis in the developing brain,hippocampal synaptic dysfunction,and memory and cognitive impairment.However,the neurotoxicity of sevoflurane and the mechanism of neurodevelopment is still unclear.This makes "the effect of general anesthetics on brain development" and how to avoid neurotoxicity of general anesthetics has become a hot spot in the fields of anesthesiology,neuroscience and other fields.Therefore,we use this as a benchmark to study the mechanism of sevoflurane-induced neurodevelopmental abnormalities,as a target to study how to reduce the occurrence of sevoflurane neurotoxicity.Affecting synaptic plasticity is an important reason for the decline in cognitive ability of the developing brain caused by anesthetics.The tissue-type plasminogen activator(t PA)can convert inactive plasminogen into active plasmin in the central nervous system.Pro BDNF can be cleaved into mature BDNF by plasmin.So t PA has an absolute effect on synaptic plasticity.Type 1 inhibitor of plasminogen activator(PAI-1)is a physiological inhibitor of t PA in the brain.Nowadays,more and more scholars believe that the t PA/PAI-1 cleavage system plays an important role in the process of neuroplasticity and cognitive memory,and can affect the long-term plasticity of the hippocampus.Therefore,we speculate that repeated inhalation of sevoflurane in developing rats can affect the balance of t PA/PAI-1 in the plasmin system in the hippocampus.Then it affects the cleavage of the pro BDNF to the mature body.Therefore,this topic first observes the effects of multiple inhalations of sevoflurane on long-term learning and memory ability and synaptic plasticity in developing rats,and explores the mechanism of t PA/PAI-1 fibrinolytic system changes and the regulation of the Brain-derived neurotrophic factor/Tyrosine kinase receptor B(BDNF/Trk B)signaling pathway.Methods: 1.72 newborn male SD rats aged 6 days,weighing 10-12 g,were randomly divided into 2 groups,each with 36 rats.The control group(Con group)inhaled 40%oxygen and 60% nitrogen for 2 hours a day for three consecutive days.The sevoflurane group(Sev group)inhaled a mixture of 3% sevoflurane,40% oxygen and 60% nitrogen for two hours a day for three consecutive days.During anesthesia,the MAC value of exhaled and end-respiratory carbon dioxide value were monitored,and then returned to the cage and fed by the mother rat.At 24 h,48h and 72 h after the anesthesia,6 rats were randomly selected from each group,anesthetized with sodium pentobarbital(100 mg/kg),decapitated and removed the brain,peeled off the hippocampus on ice,and applied Western-blot to detect PAI-1,t PA,pro BDNF,m BDNF,Trk B,p-Trk B protein expression in hippocampal tissue.At 24 h after the anesthesia,6 rats were randomly selected from each group.4% paraformaldehyde was perfused into the heart and the brains were removed.Immunofluorescence method was used to detect PAI-1,t PA,pro BDNF,m BDNF protein expression in the brain.The remaining rats continued to be reared by their mothers and were separated from the mothers on the 21 th day.On the 28 th day,the remaining rats in the two groups do Morris water maze behavior experiment.After the water maze experiment(6 days),6 rats at the age of 33 days were randomly selected,the brains were decapitated,the hippocampus were peeled off on ice.Western-blot method was used to detect synapse-related protein:SYN,PSD-95 and GAP-43 in the hippocampus.Six rats were randomly selected to determine the dendritic spine density by Golgi-cox staining.2.120 male SD rats on the 5th day of newborn(P5),weighing 10-12 g,were randomly divided into 4 groups:control group(Con group),sevoflurane group(Sev group),sevoflurane + recombinant t-PA(t PA group),sevoflurane + PAI-1 inhibitor TM5275 group(TM5275 group),30 rats in each group.P5: Newborn rats in the TM5275 group were given the PAI-1 inhibitor TM5275(50 mg/kg)by gavage,and the Con group,Sev group and t PA group were given the same volume of saline by gavage.P6: One day after intragastric administration,the t PA group was given recombinant t-PA intranasally.The t PA was dissolved in PBS(2?g/?L)and administered intranasally at a dose of2mg/kg,3 times per nostril(about 5?L per nostril).The interval is 5 minutes.The Con group,Sev group,and TM5275 group used the same volume of sterile PBS.After 1 hour,the rats in Sev group,t PA group,and TM5275 group inhaled a mixture of 3%sevoflurane,40% oxygen and 60% nitrogen.The rats in Con group inhale a mixture of 40%oxygen and 60% nitrogen.All operations were performed as the same as the sixth day on the seventh day and the eighth day.At 24 h,48h and 72 h after the anesthesia,6 rats were randomly selected from each group.Western-blot method was used to detect the protein expression of PAI-1,t PA,pro BDNF,m BDNF,Trk B,and p-Trk B in the hippocampus.The remaining rats continued to be reared by their mothers and were separated from the mothers on the 21 st day.On the 28 th day,the remaining 12 rats in each of the four groups do the Morris water maze experiment.After the water maze experiment,6 rats were randomly selected at the age of 33 days.The brains were decapitated and the hippocampus was peeled off on ice.Western-blot method was used to detect SYN,PSD-95,GAP-43 proteins in hippocampus.Six brains were randomly selected to determine the dendritic spine density after Golgi-cox staining.3.3.Select 72 SD rats of fifth day old and randomly divide them into six groups:control group(Con group),sevoflurane group(Sev group),sevoflurane + t-PA(t PA group),sevoflurane + PAI-1 inhibitor TM5275 group(TM5275 group),sevoflurane+t-PA+Trk B inhibitor(ANA-12 group),and sevoflurane+TM5275+Trk B inhibitor(TMANA-12 B group),12 rats in each group.The experimental methods in Con group,Sev group,t PA group and TM5275 group are the same as above.The inhibitor group(ANA-12 group)and(TMANA-12 B group)were injected intraperitoneally with ANA-12(0.5mg/kg).Rats in the ANA-12 group and TMANA-12 B group need to be given ANA-12.ANA-12(0.5mg/kg)was intraperitoneally injected.Each group of rats received sevoflurane inhalation or control inhalation 1 hour after the administration of medications.Western-blot method was used to detect SYN,PSD-95,GAP-43 proteins in hippocampus.Six brains were randomly selected to determine the dendritic spine density after Golgi-cox staining.Experimental operations are the same as above.Results: 1.P6,P7,P8 rats daily inhalation of sevoflurane for 2 hours can cause learning and memory dysfunction in P28 rats and decrease in synaptic plasticity in P33.The escape latency of rats in Sev group increased and the number of crossing platforms decreased.In the Sev group,the expression of SYN,PSD-95 and GAP-43 proteins decreased,and the dendritic spine density decreased in Golgi-cox staining.After inhaling,the expression of pro BDNF and PAI-1 protein in hippocampus increased,and the expression of m BDNF,t PA and p-Trk B protein decreased.2.t PA or TM5275 can reverse the learning and memory dysfunction in rats caused by sevoflurane exposure.After the intervention,the learning and memory ability is improved,the synaptic density increases,the expression of pro BDNF and PAI-1 protein is decreased,and BDNF,t PA and p-Trk B are increased.3.After using the Trk B pathway inhibitor ANA-12,the rats after administration of t PA and TM5275 have decreased protection against nerve damage,decreased learning and memory ability,decreased synaptic protein expression,and decreased dendritic spine density.The neuroprotective effects of t PA and TM5275 on rats after repeated inhalation were blocked by ANA-12.Conclusion: 1.In six-day-old newborn rats,inhaling sevoflurane two hours a day for three consecutive days,reduced learning and memory ability and synaptic plasticity of the adult rat(28 days).2.Sevoflurane mediates the imbalance of the t PA/PAI-1 system in the rat central nervous system and inhibits the cleavage of pro BDNF to mature BDNF,blocking the activation of Trk B signaling pathways,reducing hippocampal synaptic plasticity,and leading to long-term impairment of learning and memory.3.Exogenous recombinant t PA and TM5275--an inhibitor of PAI-1 can alleviate the t PA/PAI-1fibrinolytic system abnormality caused by sevoflurane,improve BDNF from precursor to mature form,thereby alleviating the sevoflurane caused learning and memory dysfunction and decreased synaptic plasticity.4.t PA,TM5275 play a role in neurosynaptic protection through the BNDF/Trk B pathway.