DNA methylation is an essential epigenetic mechanism involving in gene transcription modulation. An age-related increase in promoter methylation has been observed for neuronal activity and memory genes, and participates in neurological disorders. However, the position and precise mechanism of DNA methylation for memory gene modulation in anesthesia related cognitive impairment remained to be determined. Here, we tested whether sevoflurane anesthesia would suppress the transcription of memory genes in aged rat hippocampus. Then, we investigated changes in DNA methylation of involved genes and verify whether dysregulated DNA methylation would contribute to anesthesia induced cognitive impairment. The results indicated that sevoflurane anesthesia down-regulated the mRNA and protein levels of three memory genes, Arc, Bdnf and Reln, which were accompanied with promoter hypermethylation and increased Dnmt1, Dnmt3a and Mecp2 expression, and finally impaired hippocampus dependent memory. Furthermore, inhibition of DNA hypermethylation by 5-Aza rescued sevoflurane induced memory gene expression decrease and cognitive impairment. These findings provides an epigenetic understanding for the pathophysiology of cognitive impairment induced by general anesthesia in aged brain.

MassARRAY EpiTYPER Assay

In brief, DNA was extracted from C6 glioma cells and rat Hippocampi using Cell/Tissue DNA Extraction Kit (BioTeke, Beijing, China), then was bisulfite converted using Zymo Research EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA). Primers were designed using the EpiDesigner Software (http://www.epidesigner.com/index.html), and their sequences are listed in table 2. PCR amplification was carried out in a 8 μl reaction volume containing 0.8 μl of 10×PCR Buffer, 0.8 μl of dNTPs, 0.1 μl PCR enzyme, 0.2 μl of each primer, 1.0 μl of bisulfite-converted DNA and H₂O, and the amplification was carried out with an initial denaturation step at 94 ^oC for 4 min followed by 45 cycles of 94 ^oC for 20 s, 56 ^oC for 30 s and 72 ^oC for 1 min, then final extension at 72 ^oC for 3 min. After PCR and Shrimp Alkaline Phosphatase treatment, fragments were ligated to a T7 promoter segment, and then transcribed into RNA. The synthesized RNA was cleaved with RNase A and all cleavage products were analyzed on a mass spectrometer, according to the manufacture’s protocol. The generated mass signal patterns were translated into quantitative DNA methylation levels of different CpG sites of the selected genes by MassARRAY EpiTYPER Analyzer software. The locations of promoter regions encompassing the transcription start and the number of CpG sites assessed in the promoter regions are listed in table 2. The results were processed and analyzed by the MassARRAY Workstation software. All measurements were performed in triplicate and the average was used for statistical analysis.

 Table 2 The primer sequences and assessed locations for Bisulfite sequencing PCR