| Background and Objective:Every year,millions of children are exposed to general anesthetics.Infants and young children are the critical and sensitive period for brain development,which are easily affected by general anesthesia.Clinical studies have shown that both of infants and children may suffer neurological dysfunction after long-term or repeated general anesthesia exposure,and neurodevelopmental retardation learning disabilities,and inattention may occur in adulthood.This phenomenon is often referred to as "neurotoxic effects of general anesthetics during development".Brain development,synaptogenesis and neural circuit formation occurs mainly in the last three months of fetal development and several years after birth.If general anesthetics are used in this period,synaptic plasticity,neuronal apoptosis,degenerative neuropathy and adult learning/memory will be damaged to some degree.Studies have shown that general anesthetics can destroy the neurotransmitters balance of Glutamatergic and γ-aminobutyric acid(GABA),interfere with the stability of cytoskeleton,induce mitochondrial toxicity,and interfere with the membrane structure and lipid composition of neurons during synaptogenesis.Sevoflurane,a commonly used inhalation general anesthesia drug in clinic,is widely used in pediatric surgery.Sevoflurane is a GABA receptor agonist and/or N-methyl-D-aspartate(NMDA)receptor antagonist,and plays a key role in regulating neurotransmission through a variety of ligand gated channels.Numerous studies have shown that GABA and NMDA,the important neurotransmitters in the central nervous system,are closely related to depression,learning and memory.Clinical studies have indicated that sevoflurane exposure during childhood development may affect children’s emotion,cognition,learning and memory.Experimental studies have shown that sevoflurane exposure can cause apoptosis and degeneration of cerebral nerve cells during the window period of neural development,and induce the changes in synaptic plasticity in hippocampus.However,these results cannot completely explain the developmental neurotoxicity of sevoflurane,especially the molecular mechanism of the abnormal changes in learning and memory.In addition,the long-term effect of sevoflurane anesthesia on cognitive function of newborn animals is not clear,and the underlying mechanism is not well understood.Lipid molecules are the important material basis of neurons.The changes of lipid structure,composition,distribution and lipid raft micro domain will affect the structure and function of neurons,neurotransmitter transmission and nerve signal transduction.The brain expresses hundreds of enzymes,which are related to lipid metabolism,and are involved in the synthesis and metabolism of thousands of lipid molecules.Various lipid molecules transform each other,so a complex regulatory network of lipid molecules and participates is formed in complex neural functions.A large number of basic and clinical studies have shown that abnormal lipid metabolism is closely related to Alzheimer’s disease,depression,anxiety and other neurological diseases.Due to the continuous development in high-throughput mass spectrometry detection technology,more and more attention has been paid to the exploration of the occurrence and development mechanism of the diseases by lipidomics and proteomics.High throughput mass spectrometry has become an important technology to study the pathogenesis of diseases and find potential targets of diseases.Therefore,this study proposed a new strategy for the integration of lipidome and proteome to study the interaction between lipid molecules and their metabolic networks,and to analyze the hippocampal proteins(metabolic enzymes)and lipid metabolism molecule.This study aimed to reveal the molecular mechanism of sevoflurane developmental neurotoxicity,by comparing the changes of lipid metabolism profile,key lipid molecules and key metabolic enzymes(proteins)in hippocampus after sevoflurane exposure.Materials and Methods:1.Ninety-six newborn Sprague-Dawley rats(postnatal days 7,P7)were randomly divided into control group(40% oxygen partial pressure exposure for 2hours)and sevoflurane group(3% sevoflurane,40% oxygen partial pressure exposure for 2 hours).On the 49 th day after exposure(P56),neurobehavioral tests were performed,including social test,open field test,forced swimming test,object recognition memory,object location memory,elevated plus maze,Morris water maze,and T maze.These behavioral studies were performed to elucidate the long-term neurobehavioral effects,including learning memory,anxiety,depression and social interaction after sevoflurane exposure.2.Based on liquid chromatography tandem mass spectrometry(LC/MS-MS),the changes of the hippocampal lipidomics were detected to find the key lipid molecules differentially expressed and their related metabolic pathways.We further integrated the proteomics of hippocampus after sevoflurane exposure,and screened the key differential proteins by Gene Ontology(GO)terms and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway database.Finally,Westernblotting and immunofluorescence were used to screen and identify the key metabolic enzymes or regulatory proteins after sevoflurane exposure.3.This study further investigated the effects of lysophosphatidylcholine acyltransferase 1(Lpcat1)re-expression in hippocampus after sevoflurane exposed on learning memory and social behavior,by the doxycycline controlled Tet-On gene expression systems with adeno-associated virus(AAV)virus vector,which was used to re-express Lpcat-1 protein in rat hippocampus.Neonatal rats were exposed to sevoflurane on day 7th,and AAV carrying Lpcat1 gene was injected into hippocampus on day 35 th,and the expression of Lpcat1 protein was induced by drinking water containing doxycycline from day 49 th.The neurobehavioral tests were detected on day 56 th,the expression of exogenous Lpcat1 protein in hippocampus was confirmed by Western blot,and the changes of lipidomics were also detected in hippocampus by LC/MS-MS.Results:1.Sevoflurane exposure caused long-term memory impairment,anxiety-like behavior and social disorder in rats.The results of neurobehavioral experiments were as follows: in the elevated plus maze,the movement distance and time of sevoflurane-exposed rats in the open arm were significantly lower than those in the control group(p < 0.001);In the social test,the target quadrant times of sevoflurane exposure rats were significantly lower than those in the control group(p < 0.01);In the object recognition memory test,the recognition ability of sevoflurane exposed rats was significantly lower than that of the control group(p < 0.01);In the object location memory,the memory ability of sevoflurane exposed rats was significantly lower than that of the control group(p < 0.05);In the T-maze test,the times and distances of sevoflurane exposed rats entering the new open arm were significantly lower than those in the control group(p < 0.05);In Morris water maze test,the latencies of finding platform in sevoflurane exposed rats were significantly longer than those in control group,but the times of crossing platform,total swimming distance and target quadrant time in sevoflurane exposed rats were less than those in control group(p < 0.05).2.The study of hippocampal lipidomics after sevoflurane exposure showed that a total of 130 differentially expressed lipid molecules were identified,mainly including phospholipid,glyceride and sphingolipid.Among them,the abnormal of phospholipid metabolism is the most significant,and sevoflurane exposure significantly affected the length and unsaturation of phospholipid carbon chain.These results suggested that sevoflurane could remodel the phospholipid metabolism in hippocampus.Further comparative analysis the proteomics of the hippocampus,the bioinformatics analysis found that there were 70 differentially expressed proteins.Of them,42 proteins increased and 28 proteins decreased significantly compared with the control group.GO and KEGG analysis showed that the protein variances were mainly enriched in lipid metabolism pathway,which was consistent with the results of lipidomics.Western blot and immunofluorescence were used to screen and identify the protein variances related to phospholipid metabolism.The expression levels of Lpcat1 and phosphatidylinositol 4-Kinase β(Pi4kb)were significantly decreased in the hippocampus of sevoflurane exposed rats.Immunofluorescence study showed that Lpcat1 and Pi4 kb were mainly located in CA1 and CA2 regions of hippocampus,and mainly located in astrocytes and neurons.The results of Western blot showed that the expression of Lpcat1 in hippocampal CA1 and CA2 decreased significantly after sevoflurane exposure.These results suggested that the down-regulation of Lpcat1 and/or Pi4 kb expression in hippocampus may be the important target(s)of sevoflurane,and might further induce the phospholipid metabolism abnormality and developmental neurological dysfunction.3.Because sevoflurane downregulated Lpcat1 expression in hippocampus,the study further used the Tet-on systems for doxycycline-inducible gene expression of AAV vector to re-express Lpcat1 in hippocampus,and investigated the effects of sevoflurane exposure on memory function and social behavior in rats.The results showed that the doxycycline Tet-On system successfully re-expressed Lpcat1 protein in hippocampus,and the level of Lpcat1 in hippocampus was significantly increased.The re-expression of Lpcat1 protein could partially reverse the abnormal phospholipid metabolism in adult hippocampus,showing a nearly normal phospholipid metabolism group.Importantly,the impaired long-term memory function and social behavior induced by sevoflurane were reversed(p < 0.05).The results further demonstrated that the down-regulation of Lpcat1 in hippocampus was an important molecular mechanism of sevoflurane induced developmental neurotoxicity.Conclusion:Sevoflurane exposure in neonatal rats caused long-term developmental neurotoxicity,which shows decreased memory function,anxiety-like behavior,and decreased social behavior ability.Sevoflurane significantly downregulated the expression of Lpcat1,which is a key enzyme in hippocampal phospholipid metabolism,and sevoflurane could reconstructe hippocampal phospholipid metabolism.Re-expression of Lpcat1 protein in hippocampus reduced the long-term developmental neurotoxicity of sevoflurane and normalized the phospholipid metabolism affected by sevoflurane in hippocampus.Collectively,this study shows that the down-regulation of Lpcat1 is an important mechanism underlying sevoflurane developmental neurotoxicity and provides new experimental evidence for the prevention and treatment of clinical neurotoxicity of sevoflurane. |
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