The Role Of DNA Methylation In The Pathogenesis Of Neuropathic Pain

BackgroudsNeuropathic pain is caused by primary or secondary damage or disfunction of the nervous system, and is characterized by hyperalgesia, allodynia and spontaneous pain. Neuropathic pain is a chronic pain, with the increased incidence year by year. Pathogenesis of neuropathic pain is complex and not discovered clearly, and the conventional painkillers such as opioids and non-steroidal anti-inflammatory drugs have poor effecacy for it. It's urgent to explore the pathogenesis of neuropathic pain clearly and find better therapic drugs for it. Recent evidence suggests that transcription and expression level of pro-or antinociceptive genes in sensory neurons, immune cells and glial cells are highly involved in the generation and maintenance of neuropathic pain.Epigenetic mechanisms which induce heritable changes in gene expression without alterations in the DNA sequence can regulate gene transcription and expression. Epigenetic mechanisms mainly include the DNA methylation, chromatin remodeling, genomic imprinting, X chromosome inactivation, and non-coding RNA regulation and so on. DNA methylation is one of the earliest discovered and most characteristic epigenetic mechanisms in mammals. DNA hypermethylation can turn off gene expression, and DNA demethylation can reactive gene expression. Therefore we speculate that epigenetic mechanisms, in particular DNA methylation mechanism are likely involved in the development and maintenance of neuropathic pain. There are few studies about the involvement of DNA methylation in neuropathic pain, and successful completion of the study may give some new ideas in neuropathic pain research.ObjectivesTo observe global DNA methylation level changes in the lumbar spinal cord in rats following sciatic nerve chronic constriction injury (CCI); to observe DNA methyltransferases (DNMTs) DNMT1, DNMT3a, DNMT3b and methyl-CpG-binding protein2(MeCP2) expression changes in the lumbar spinal cord in CCI rats; to observe glutamate decarboxylase67(GAD67) expression changes and promotor methylation changes of its encoding gene GAD1in the lumbar spinal cord in CCI rats; to observe effects of intrathecal5-azacytidine (5-AZA) on global DNA methylation, DNMTs and MeCP2expression, GAD67expression, GAD1promotor methylation in the lumbar spinal cord, and neuropathic pain in CCI rats.Methods1. Adult male Sprague-Dawley (SD) rats were randomly divided into the sham operation group and CCI group. After measurement of base values of mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL), a modified CCI to the sciatic nerve was performed on the left side according to Bennett and Xie, and a sham operation was just exposed the nerve but not ligated under deep anesthesia. MWT and TWL of each rat were measured3,5,7,10and14days after surgery. On day14after surgery, all the rats were killed under deep anesthesia and their lumbar spinal cord were dissected freshly to observe global DNA methylation level changes in the lumbar spinal cord.2. On day14after surgery, sham operation rats and CCI rats were killed under deep anesthesia and their lumbar spinal cord were dissected to observe DNMT1, DNMT3a, DNMT3b and MeCP2expression changes with immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis.3. On day14after surgery, sham operation rats and CCI rats were killed under deep anesthesia, and their lumbar spinal cord were dissected freshly to observe GAD67mRNA expression changes with real-time quantitative RT-PCR (qRT-PCR) and promotor methylation changes of its encoding gene GAD1with pyromark CpG assays.4. Chronic lumbar intrathecal catheter of the subarachnoid space was implanted in all rats. Adult male SD rats with successful implantment were randomly divided into3groups:sham operation+NS group, CCI+NS group, and CCI+5-AZA group. Sham operation rats received NS, and CCI rats received either NS or10μM·d-1demethylation reagent5-AZA (dissolved in NS) by spinal once daily from3days to14days after surgery. MWT and TWL of each rat were measured3,5,7,10and14days after CCI surgery. On day14after CCI surgery, all the rats were killed under deep anesthesia, and their lumbar spinal cord were dissected to observe global DNA methylation, DNMT1, DNMT3a, DNMT3b and MeCP2expression, GAD67expression, and GAD1promotor methylation changes in the lumbar spinal cord.Results1. Before surgery there was no significant difference in MWT and TWL among all the rats (P>0.05). On the third day MWT and TWL of CCI rats decreased which was obviously lower than that of sham operation rats (P<0.05). After surgery, MWT and TWL obviously reduced in CCI rats from day3to day14(P<0.05), with the lowest point on day7(P<0.05). On day14after surgery, global DNA methylation increased in the lumbar spinal cord in CCI rats than sham operation rats (P<0.05).2. On day14after surgery, RT PCR results showed that DNMT1didn't change significantly (P>0.05), and DNMT3a, DNMT3b and MeCP2mRNA expression increased significantly in the lumbar spinal cord dorsal horn in CCI rats than sham operation rats (P<0.05). Results of western blot analysis showed that DNMT1, DNMT3a, DNMT3b and MeCP2protein expression increased significantly in the nucleus in the lumbar spinal cord dorsal horn in CCI rats than sham operation rats (P<0.05). Immunohistochemistry results showed that DNMT1, DNMT3a, DNMT3b and MeCP2were expressed mainly in gray matter, especially in the spinal dorsal horn. And positive particles were mainly located in the nucleus, little in the cytoplasm. DNMT1, DNMT3a, DNMT3b and MeCP2-positive cells increased significantly in the lumbar spinal cord dorsal horn in CCI rats than the sham operation rats.3. On day14after surgery, qRT-PCR results showed that GAD67mRNA decreased significantly in the lumbar spinal cord dorsal horn in CCI rats than sham operation rats (P<0.05). Pyromark CpG assays results showed that GAD1promotor methylation increased significantly in the lumbar spinal cord dorsal horn in CCI rats than sham operation rats (P<0.05).4. After surgery, MWT and TWL reduced in all NS-treated CCI rats from day3to day14(P<0.05). Mechanical allodynia and thermal hyperalgesia were constantly attenuated in5-AZA-treated CCI rats from day5to day14(P<0.05).5-AZA-treated CCI rats showed lower global DNA methylation than NS-treated CCI rats in the lumbar spinal cord dorsal horn (P<0.05).5-AZA-treated CCI rats showed lower DNMT1, DNMT3a, DNMT3b and MeCP2expression than NS-treated CCI rats in the lumbar spinal cord dorsal horn (P<0.05);5-AZA-treated CCI rats showed higher GAD67expression and lower GAD1promotor methylation than NS-treated CCI rats in the lumbar spinal cord dorsal horn (P<0.05). HE staining showed that intrathecal10μM5-AZA did not produce significant damage in the spinal cord.Conclusions1. Up-regulated global DNA methylation was clearly detected in the lumbar spinal cord in CCI rats14days after surgery, accompanying with mechanical allodynia and thermal hyperalgesia. That indicates up-regulated global DNA methylation may be highly involved in the neuropathic pain in CCI rats.2. Up-regulated DNMT1, DNMT3a, DNMT3b and MeCP2expression were clearly detected in the lumbar spinal cord in CCI rats14days after surgery, accompanying with mechanical allodynia and thermal hyperalgesia. That indicates increased DNMT1, DNMT3a, DNMT3b and MeCP2expression may play an important role in the development and maintenance of neuropathic pain.3. Down-regulated GAD67expression and up-regulated GAD1promotor methylation were clearly detected in the lumbar spinal cord in CCI rats14days after surgery, accompanying with mechanical allodynia and thermal hyperalgesia. That indicates decreased GAD67expression and increased GAD1promotor methylation may play an important role in the development and maintenance of neuropathic pain.4. Mechanical allodynia and thermal hyperalgesia induced by CCI were markedly attenuated by intrathecal5-AZA from day5to day14in CCI rats. Meanwhile, the changes of global DNA methylation, DNMT1, DNMT3a, DNMT3b and MeCP2expression, GAD67expression, and GAD1promotor methylation in the lumbar spinal cord in CCI rats were significantly reversed by5-AZA, which further suggest DNA methylation may play an important role in neuropathic pain, and5-AZA may become a potential therapeutic drug for neuropathic pain.