DOI: 10.5061/dryad.69p8cz8xw
title: DNA methylation manipulation of memory genes is involved in sevoﬂurane induced cognitive impairments in aged rats

description: 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.
description: <b>MassARRAY EpiTYPER Assay</b> 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
 H<sub>2</sub>O, and the amplification was carried out with an
 initial denaturation step at 94 <sup>o</sup>C for 4 min
 followed by 45 cycles of 94 <sup>o</sup>C for 20 s, 56
 <sup>o</sup>C for 30 s and 72 <sup>o</sup>C for 1
 min, then final extension at 72 <sup>o</sup>C 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 Genes Orientation Sequence (5’ to 3’) Location CpG sites <i>Arc</i> Forward aggaagagagGGTAGAGGAGAGTGT TTTTGGTTTT -274 to +318 42 Reverse cagtaatacgactcactatagggagaaggctACA CTTACCAATCTACAAAATCACATT <i>Bdnf</i> <i>Primer I</i> Forward aggaagagagATTGTGATTTTTTTGGT AAAAAGGA -644 to -99 16 Reverse cagtaatacgactcactatagggagaaggctCCA AAACCCACCTTCTAAAACTTAT <i>Bdnf</i> <i>Primer II</i> Forward aggaagagagTTATTTTTTAGTATTTG TTGGGGAGA -634 to -36 6 Reverse cagtaatacgactcactatagggagaaggctCCTT TCCATATATAAAAACATTACCCA <i>Bdnf</i> <i>Primer III</i> Forward aggaagagagTGTTTATTTATAATGAA ATGGGTAATGT -1216 to -730 18 Reverse cagtaatacgactcactatagggagaaggctACC AAAAATCTATTCCAACCTACAC <i>Bdnf</i> <i>Primer IV</i> Forward aggaagagagTTGTTGTTGTTTAGATG ATGAAAGG -245 to +346 18 Reverse cagtaatacgactcactatagggagaaggctACC CACCTTTTTCAATCACTACTTA <i>Bdnf</i> <i>Primer VI</i> Forward aggaagagagGAGTTTTGGGGTTAAG TAGTTGGTT -192 to +349 35 Reverse cagtaatacgactcactatagggagaaggctCCT CAAAATCCACACAAAACTCTC <i>Reln</i> Forward aggaagagagGTAGTTAGGTTGAAA GGGAGATTGG -1383 to -834 17 Reverse cagtaatacgactcactatagggagaaggctTAA TACCCTTTTCCCAAACTCAAAC