The Role Of Histone Acetylation In Sevoflurane Anesthesia-induced Long-term Cognitive Impairments In Neonatal Rats And The Underlying Mechanism

Objective:Millions of embryos,infants,and children will receive anesthesia for operation at home and abroad annually.Sevoflurane is widely used as prevalent inhalation anesthetic in pediatric anesthesia due to its small pungent odor,fast inducing effect and rapid recovery characteristics.Extensive bodies of animal studies have suggested that sevoflurane exposure induces cognitive impairments such as learning and memory dysfunction in neonatal rodents.However,the exact mechanisms remain unclear.Inhibitors of histone deacetylases?HDACs?can ameliorate cognitive impairments by restoring hippocampal histone acetylation in neurodegenerative diseases including Huntington disease?HD?,local cerebral ischemia,and Alzheimer's disease?AD?,etc.However,little is known about whether the changes of histone acetylation and synaptic plasticity are involved in the pathogenesis of sevoflurane anesthesia-induced long-term cognitive impairments.The present study aimed to investigate the expression changes of histone acetylation and synaptic plasticity in the brain via establishing the animal model of sevoflurane anesthesia-induced long-term cognitive impairments in neonatal rats,and to explore the role of histone acetylation in sevoflurane anesthesia-induced long-term cognitive impairments in neonatal rats and the underling mechanism.Methods:Forty-eight male Sprague Dawley?SD?rats at postnatal day 6?P6?were divide into the following four groups?n = 12?:oxygen?O₂?+ Saline group,O₂ +sodium butyrate?NaB,a HDACs inhibitor?group,sevoflurane anesthesia + normal saline group?Sev + Saline group?,and sevoflurane anesthesia + NaB group?Sev +NaB group?.Animals in Sev groups were subjected to 3%sevoflurane plus 100%O₂?the flow was 2 L/min?2 h daily for 3 consecutive days from P6-8.And animals in O₂ groups were only subjected to 100%O₂ of the same ages at the same time.Animals in different groups were chronically given intraperitoneal injections of NaB?250 mg/kg?or isometric saline from P6-50,respectively.The open field,Morris water maze,and fear conditioning tests were performed at P33,P36-42,and P48-50,respectively.The brain and the hippocampus were harvested 1 h after the last behavioral test for further examination.The determination of the contents of hippocampal HDAC1,HDAC2,HDAC3,HDAC8,histone H3 and H4,histone H3 lysine 9 acetylation?acetyl-H3K9?,histone H3 lysine 14 acetylation?acetyl-H3K14?,histone H4 acetylation?acetyl-H4?,histone H4 lysine 5 acetylation?acetyl-H4K5?,histone H4 lysine 12 acetylation?acetyl-H4K12?,cAMP-response element binding protein?CREB?,phosphorylated CREB?p-CREB?,brain-derived neurotrophic factor?BDNF?,microtubule associated protein-2?MAP2?,synapsin 1,and postsynaptic density 95?PSD 95?were detected by Western Blot.The fluorescence intensities of acetyl-H3K14 and acetyl-H4K12 in hippocampal subregions were detected by immunofluorescence.The number of dendritic spine in hippocampal CA1 region was detected by golgi staining.Results:In the Morris water maze test,the escape latency at P38-39,the time spent in the target quadrant,and the times across the original platform were decreased in the Sev + Saline group compared with the O₂ + Saline group?P<0.05?;whereas the escape latency at P38-39,the time spent in the target quadrant,and the times across the original platform were increased in the Sev + NaB group compared with the Sev +Saline group?P<0.05?.In fear conditioning test,the percent of the freezing time to context was decreased in the Sev+ Saline group commpared with the O₂ + Saline group?P<0.05?;whereas the percent of the freezing time to context was increased in the Sev + NaB group compared with the Sev + Saline group?P<0.05?.In the Western Blot,the contents of hippocampal HDAC3 and HDAC8 were increased,and the contents of hippocampal acetyl-H3K9,acetyl-H3K14,acetyl-H4,acetyl-H4K5,acetyl-H4K12,p-CREB/CREB,BDNF,MAP2,synapsin 1,and PSD 95 were decreased in the Sev + Saline group compared with the O₂ + Saline group?P<0.05?;whereas the contents of hippocampal HDAC3 and HDAC8 were decreased,and the contents of hippocampal acetyl-H3K9,acetyl-H3K14,acetyl-H4,acetyl-H4K5,acetyl-H4K12,p-CREB/CREB,BDNF,MAP2,synapsin 1,and PSD 95 were increased in the Sev + NaB group compared with the Sev + Saline group?P<0.05?.In the immunofluorescence and golgi staining,the intensities of acetyl-H3K14 and H4K12,and the number of dendritic spine in hippocampal CA1 region were decreased in the Sev + Saline group compared with the O₂ + Saline group?P<0.05?;whereas the intensities of acetyl-H3K14 and H4K12,and the number of dendritic spine in hippocampal CA1 region were increased in the Sev + Saline group compared with the O₂ + Saline group?P<0.05?.However,no difference was detected in the open field test,the speed in the MWM test,the percent of the freezing time to tone in the fear conditioning test,and the contents of hippocampal HD AC 1 and HD AC2 among the four groups?P>0.05?.Conclusion:Sevoflurane anesthesia for neonatal rats may lead to hippocampus-dependent spatial and associative long-term cognitive impairments followed by the down-regulations of hippocampal histone H3 and H4 acetylation,and synaptic plasticity.However,HDACs inhibitor NaB can alleviate these disorders.The results in this study showed that the decreased hippocampal histone acetylation and synaptic plasticity were associated with the long-term cognitive impairments induced by sevoflurane anesthesia in neonatal rats.