DNA Methylation Is Involved In Neonatal Sevoflurane Exposure-induced Cognitive Impairments In Rats

Objective With the advances in medical and surgical techniques, millions of children receive surgical operation with general anesthesia each year all over the world. The early life is the peak development of the central nervous system, and in comparison with the adult brain, the neonates are more susceptible to anesthetics. General anesthetics-induced neurocognitive impairment have been linked to neuron apoptosis, inflammation, oxidative stress, Aβ aggregation and so on. Hemodynamic stability, less irritation to the airway, and short-lasting action make sevoflurane one of the most widely used general anesthetics, especially for the neonates and pediatrics. Unfortunately, like other anesthetics, sevoflurane exposure can induce widespread neuronal apoptosis and cognitive impairments. Yet, the mechanisms underlying sevoflurane-mediated neuronal degeneration and delayed functional abnormalities remain largely to be determined. Epigenetics is a quickly growing field encompassing mechanisms regulating gene expression that do not involve changes in the genotype, and include DNA methylation, histone modifications and non-coding RNAs. Much attention has been focused on DNA methylation, a covalent chemical modification of DNA catalyzed by DNA methyltransferases (DNMTs) and leading to a long-term shutdown of the associated genes. It is well established that DNA methylation plays an important role in the process of memory. Synaptic plasticity is considered to be the important neurochemical basis on learning and memory, whose damage can lead to cognitive impairments. In recent years, the mechanism of synaptic plasticity was focused on synaptic plasticity-related proteins. Brain-derived neurotropic factor (BDNF) belongs to the neurotrophin family that plays a key role in development, differentiation, and survival of neurons. Reelin is an extracellular glycoprotein which performs diverse roles in the developing brain, including regulation of neuronal migration and positioning. By contrast, serine/threonine protein phosphatase 1 (PP1) is a negative factor regulating synaptic plasticity and memory. However, the roles of BDNF, Reelin, and PP1 genes methylation in sevoflurane exposure-induced cognitive impairments remain unclear. Therefore, we suppose that dysregulation of the hippocampal DNA methylation of synaptic plasticity-related genes, including BDNF, Reelin, and PP1, might be a molecular mechanism responsible for neonatal sevoflurane exposure-induced cognitive impairments in rats. We will investigate the DNA methylation pattern of synaptic plasticity-related genes, the expression of BDNF, Reelin, and PP1, and the the number of dendritic spines in the neonatal sevoflurane exposure rats to reveal the new mechanism underlying sevoflurane-induced cognitive impairments in developing brain. This work will provide the theoretical basis and interventional targets for the prophylaxis and treatment of cognitive impairments induced by neonatal sevoflurane.Method On postnatal day 7 (P7), Sprague-Dawley male rats were exposed to 3 % sevoflurane or 30% oxygen/air for 2 h daily for three consecutive days, and were treated with DNA methyltransferases (DNMTs) inhibitor 5-aza-2’-deoxycytidine (5-AZA; 1 mg/kg,2 μl) or dimethyl sulphoxide (DMSO) 1 h before the first sevoflurane exposure. We used two sets of rats for the following experiments. The first set of rats (n= 60) were randomized into one of the following five groups (n= 12):Control group, Control+ 5-AZA group, Sevoflurane group, Sevoflurane+ DMSO group, or Sevoflurane+ 5-AZA group for behavioral tests. The second set of rats (n= 176) were used for the biochemical study in the Control group, Sevoflurane group, Sevoflurane+ DMSO group, and Sevoflurane+5-AZA group (n= 4 for each biochemical assay at each time point). Cognitive functions were evaluated by the open field (OF), fear conditioning (FC), and Morris water maze (MWM) tests on P39, P41-43, and P50-57, respectively. The second set of rats were euthanized 1,6,24 h, and 30 d after the last sevoflurane exposure, and the brain tissues were harvested and then subjected to Golgi staining to detect the number of dendritic spines in the pyramidal neurons of hippocampus; Quantitative real-time polymerase chain reaction (qPCR) and western blotting to measure the expression of DNMTs, Methyl-CpG binding protein 2 (MeCP2), BDNF, Reelin, and PP1; methylation-specific PCR (MSP) to assess the methylation levels of BDNF and Reelin promoter regions.Results In the FC test, the freezing time to context was decreased in the Sevoflurane group compared with the Control group (P< 0.05).5-AZA pretreatment increased the freezing time to context compared with the Sevoflurane+DMSO group (P< 0.05). In the MWM test, the rats had a longer escape latency in the Sevoflurane group than in the Control group from day 3 to day 7 during the training sessions (P< 0.05). The percent of time in the target quadrant was decreased in the Sevoflurane group compared with the Control group (P< 0.05).5-AZA pretreatment increased the escape latency from day 5 to day 7 (P< 0.05) and the percent of time in the target quadrant (P< 0.05) compared with the Sevoflurane+DMSO group. In the Golgi staining, the number of dendritic spines decreased in the Sevoflurane group compared with the Control group (P< 0.05).5-AZA pretreatment reversed the sevoflurane-induced decrease of dendritic spines compared with the Sevoflurane+ DMSO (P< 0.05). The mRNA and protein levels of DNMT3a and DNMT3b were significantly increased 6,24 h, and 30 d after the last sevoflurane exposure in the Sevoflurane group compared with the Control group (P< 0.05). The mRNA levels of MeCP2, BDNF, and Reelin were decreased 6 h,24 h, and 30 d after the last sevoflurane exposure (P< 0.05), and the protein levels were decreased 24 h and 30 d after the last sevoflurane exposurecompared with the Control group (P< 0.05). However,5-AZA pretreatment restored the sevoflurane induced up-regulation of DNMT3a and DNMT3b, and down-regulation of MeCP2, BDNF and Reelin mRNA as well as proteins in the Sevoflurane+ 5-AZA group compared with the Sevoflurane +DMSO group (P< 0.05). The methylation levels of BDNF and Reelin promoter regions were up-regulated 6 h and 30 d after the last sevoflurane exposure in the Sevoflurane group compared with the Control group (P< 0.05).5-AZA pretreatment abrogated the sevoflurane-induced hypermethylation of BDNF and Reelin compared with the Sevoflurane+DMSO group (P< 0.05).Conclusion Repeated neonatal sevoflurane exposure induced significant cognitive impairments later in life, which was accompanied with persistent hypermethylation of the neuroplasticity-related genes BDNF and Reelin, and their consequent down-regulation. Sevoflurane also reduced the number of dendritic spines in the pyramidal neurons of hippocampus. By contrast, pre-administration of 5-AZA reversed these abnormal biochemistry parameters and ameliorated the cognitive impairments induced by repeated sevoflurane exposure. These results suggested that the hypermethylation of hippocampcal BDNF and Reelin might contribute to neonatal sevoflurane exposure-induced cognitive impairments.