Abstract PR317

Abstract

Background & Objectives: Our previous study showed that results showed that MBD1 knockout mice showed dramatically higher pain thresholds in Mechanical, Thermal, and Cold test in acute pain and SNL-pain models compared to wild-type mice. However, it remains unknown how MBD1 to regulate genesis of acute pain and chronic pain. This observation led us to hypothesize that MBD1 might regulate some related factors of pain. So, we first investigated both the mRNA and protein level of pain-related opioid receptors and potassium channels in the dorsal root ganglia, spinal cord and cortex, and determined whether MBD1 targeted tissue-specific subtypes of opioid receptors and/or potassium channels. Second, we used MBD1-AAV5 overexpression viral to treat those specific tissue sites and observed pain behavior and related expression of pain related mRNA and proteins. Finally, we determined whether MBD1 co-localization with opioid receptors or potassium channels gene in a single DRG cell or not. Materials & Methods: All experiments were carried out with the approval of the Animal Care Committee at Rutgers New Jersey Medical School. For detected opioid receptors or potassium channels mRNA, we used RT-qPCR to detect Oprm1, Oprk1, Oprd1, Kcna1 and Kcna2 mRNA gene expression and used western blot to detected mu, kappa, delta, Kv1.1 and Kv1.2 protein expression on the level of dorsal root ganglia, the spinal cord and cortex. For in vivo experiments, wild-type mice were microinjected with GFP-AAV5 viral (1 µl) or MBD1-AAV5 viral (1 µl) in DRG. CPP test was carried out to test the spontaneous pain behavior. Overexpression of MBD1 in wild-type mice induced spontaneous pain behavior after 6 weeks. On week 8, DRG was collected. For in vitro experiments, cultured DRG neurons were exposed to GFP or MBD1-AAV5 viral and proteins were assessed by Western blot. Single cell RT-PCR was carried out to test whether MBD1 co-localization with opioid receptors or potassium channels gene in a single DRG cell or not. Results: Oprm1, Oprk1, and Kcna2 mRNA gene expressed at high levels in DRG, spinal cord and cortex, but not midbrain. Interestingly, only mu opioid receptor and Kv1.2 receptor protein significantly increased in DRG, other proteins and sites were not altered. These increases were blocked by MBD1 over-expression in DRG, and MBD1 over-expression can cause spontaneous pain behavior. Furthermore, MBD1 overexpression in cultured DRG neurons down-regulate mu opioid receptor and Kv1.2 receptor protein expression. Single cell RT-PCR assay revealed that MBD1 co-localization with mu opioid receptor and Kv1.2 receptor gene in a single DRG cell. Conclusion: Our findings indicate that Expression of mu opioid receptor and Kv1.2 controlled by MBD1 in the dorsal root ganglion. Disclosure of Interest: None declared