Molecular And Cellular Mechanisms Of 5-HT_2A-Cx43 Signaling Pathway Mediating Mirror Image Pain

Pain is an intractable biomedical scientific and clinical problem to human health. Pain could be induced by many kinds of reasons including tissue injury, inflammation, emotion-dysfunction, sensorimotor disorders, genetic mutations, cancer and so on. Persistence, paroxysmal, chronicalization, diversities, personalization of pain profiles are involved in detection, transmission, synaptic integration of noxious stimuli for individual and adaptive plasticity. It thus leads the occurrence of spontaneous pain, primary and secondary, and also mirror image pain hypersensitivity. However, the understanding of pain mechanisms and control, especially of mirror image pain, is still far from preventation, diagnosis, translational medical treatment, although more and more rapid development of pain researches and controls, novel discovery of anti-pain drugs and strategies has been found.Many evidences have been demonstrated the 5-HT2 A receptor(5-HT2A), gap junction intercellular communication, glia may participate in the regulation of pain through different molecular and cellular mechnisams. However, whether their interactions are underlying or not remains to be a necessary. In this paper, based on our well-established the experimental animal mirror image pain model mimicking natural scorpion sting inducing pain, the single side of rat hindpaw plantar subcutaneous injection of specific sodium channel activator Bm K I, one of main algogenic peptidergic components from the venom of Chinese scorpion Buthus Martensi Karsch(Bm K), was applied as pain-evoking stimulus. Combining with behavioral observation, real-time quantitative PCR, immunoblotting, immunofluorescence, live cell imaging and other experimental techniques, the dynamic expression in peripheral and central 5-HT2 A, connexin 43(Cx43), glia were detected to build novel 5-HT2A- Cx43 signaling pathways regulating mirror image pain. The main research contexts and results were as follows:(i) The synergetic inhibitive enhancement of Bm K I-induced pain by 5-HT2 A antagonist and gap junction blockersThe unilateral plantar subcutaneous injection of Bm K I could provoke long term persistent spontaneous pain, thermal hyperalgesia and bilateral mirror mechanical hyperalgesia. Pre-treatment with intrathecal administration of 5-HT2 A antagonist, ketanserin, or gap junction blocker carbenoxolone(CBX) could significantly inhibit Bm K I induced-pain behaviors. Moreover, the effects of ketanserin were enhanced strongly by CBX when injected together. These results demonstrate that the spinal and dorsal root ganglia(DRG) 5-HT2 A and connexins are involved in Bm K I-induced pain, implying that 5-HT2A- connexins signaling pathway may underlie the induction, development and maintenance of mirror image pain.(ii) The differential expression of dorsal root ganglion 5-HT2 A –Cx43 and mirror image painq RT-PCR and Western blotting results showed that Bm K I could induce 5-HT2 A m RNA slightly down-regulated ipsilaterally but significantly up-regulated contralaterally, Cx43 m RNA and microglia Iba1 increased bilaterally, and however make satellite astrocyte GFAP elevated ipsilaterally but decreased contralaterally. Moreover, immunofluorescent detection demonstrated that 5-HT2 A co-localized abundantly with Cx 43, GFAP, Iba1, large-diameter neuron marker NF200 but only few with small-diameter neuron marker IB4. The co-localization of 5-HT2 A with these cellular markers was elevated after Bm K I injection. On the contrary, the expression of 5-HT2A-Cx43 itself and co-localization with other marker proteins was significantly reversed by pretreatment with intrathecal ketanserin. These results suggest that dynamic and diverse expression of 5-HT2A-Cx43 may play key role in inductive and transmission integration underlying mirror image pain.(iii) Expression profiles of spinal 5-HT2 A –Cx43 pathway mediating mirror image painCompared with DRG, spinal 5-HT2 A –Cx 43 showed a different expression pattern. It demonstrated that spinal 5-HT2 A mainly co-localized with nerurons extensively, with astrocyte GFAP and Cx43 in deeper laminar, but few with microglia OX42. The amount of co-localization changed well-fit with time-development of the induction, maintenance and recovery of Bm K I-induced spontaneous pain and mirror mechanical pain hypersensitivity. Identically to DRG, the reverse effects of pre-treatment with intrathecal clonidine were also clearly found in spinal cord. These results revealed some direct opposite, if any, cellular effects of 5-HT2A-Cx43 signaling on peripheral detection, transmission and central transformation of pain modulation.(iv) Live cell imaging studies on 5-HT2A-Cx43 cellular signaling pathwayWith the isolated and acute subcultured spinal astrocytes, the live cell confocal microscopy imaging results verified that neurotransmitter 5-HT activated and opened gap junctional channels, which allowed fluorescent yellow dye transferring from extracellular bath into the intracellular cytoplasm. Importantly, the 5-HT-drived dye movement was blocked evidently by 5-HT2 A antagonist ketanserin or gap junction inhibitor CBX, individually. The results finally provide a direct proof supporting that 5-HT2A-Cx43 signaling pathways mediated the induction, development, maintenance and recovery of pain responses.We conclude here that the novel 5-HT2A-Cx43 signaling pathway is different between peripheral and central induction, transmission, modulation and cellular integration of mirror image pain. The findings would help to elucidate the persistence, chronicalization, diversities, personalization of pain mechanisms, and further provide a more effective anti-pain strategy combing with 5-HT2 A and connexins.