The Modulation Of ATP On Microglia Polarization In Neuropathic Pain

Part 1 The contribution of microglia polarization in neuropathic painAim The change and contribution of spinal microglia polarization in neuropathic pain(1) To investigate the alteration of spinal microglia polarization in SNL-induced neuropathic pain rats;(2) To investigate the alteration of spinal microglia polarization in STZ-induced diabetic neuropathic pain rats;(3) In vitro, to investigate the change of BV2 microglia polarization induced by LPS or IL-4;(4) In vitro, to investigate the change of primary microglia polarization induced by LPS or IL-4;(5) To observe the change of rat pain behavior after spinal intrathecal injection of primary microglia pre-induced by LPS or IL-4.Methods(1) The first series of experiments were performed to examine the painbehavior and spinal microglia polarization of SNL rats. Rats’ mechanical allodynia was assessed using the 50% paw-withdrawal threshold(PWT). Flow cytometry was used to assess the protein level of M1 marker CD86.(2) The second series of experiments were performed to examine the pain behavior and spinal microglia polarization of STZ diabetic rats. Rats’ mechanical allodynia was assessed using the 50% paw-withdrawal threshold(PWT). Flow cytometry was also used to assess the protein level of M1 marker CD86.(3) The BV2 microglia was incubated with LPS or IL-4. Westernblot was used to examine the protein level of M1 marker i NOS and M2 marker Arg. Flow cytometry was also used to assess the protein level of M1 marker CD86 and M2 marker CD206. Confocal immunofluorescence was used to examine the level of i NOS and Arg.(4) The primary microglia was gained and incubated with LPS or IL-4. Westernblot was used to examine the protein level of M1 marker i NOS and M2 marker CD206. Confocal immunofluorescence was used to examine the level of CD86 and Arg.(5) After spinal intrathecal injection of microglia pre-stimulated by LPS or Il-4, the na?ve and neuropathic pain rats were examined by PWT test.Result(1) SNL produced a rapid development of mechanical allodynia with the up-regulation of M1 marker CD86 in rats’ spinal microglia.(2) The administration of STZ also produced the development of mechanical allodynia with the up-regulation of M1 marker CD86 in rats’ spinal microglia.(3) BV2 microglia were incubated with LPS. M1 marker i NOS increased and the M2 marker Arg decreased, which were evidenced by Westernblot, flow cytometry and confocal immunofluorescence. Then, BV2 microglia were incubated with IL-4. M1 marker decreased and the M2 marker increased, which were also evidenced by Westernblot, flow cytometry and confocal immunofluorescence.(4) Primary microglia were gained and incubated with LPS. M1 marker increased and the M2 marker decreased, which were evidenced by Westernblot and confocal immunofluorescence. Then, they were incubated with IL-4. M1 marker decreased and the M2 marker increased contrarily, which were evidenced by Westernblot and confocal immunofluorescence.(5) After intrathecal administration of microglia pre-incubated with LPS, the na?ve rats’ PWTs decreased, which suggested that LPS-induced M1 microglia would strengthen pain behavior.(6) After intrathecal administration of microglia pre-incubated with IL-4, the pain rats’ PWTs increased, which suggested that IL-4-induced M2 microglia would relieve pain behavior.Conclusion The change of spinal microglia polarization contributes to neuropathic pain. The M1 type can strengthen pain behavior,but M2 type can relieve pain behavior.Part 2 the modulation of ATP on microglia polarization in neuropathic painAim The modulation of ATP on microglia polarization in neuropathic pain(1) In vitro, to investigate to modulation of ATP on polarization in BV2 microglia;(2) To observe the change of rat pain behavior after intrathecal injection of primary microglia pre-induced by different dozens of ATP.Methods(1) The BV2 microglia was incubated with different dozens of ATP. The M1 and M2 markers were examined by Western blot, flow cytometer and confocal immunofluorescence separately.(2) The BV2 microglia was incubated with low dozen ATP plus LPS. The M1 and M2 markers were examined by Western blot, flow cytometer and confocal immunofluorescence separately(3) After intrathecal injection of microglia pre-stimulated by different dozens of ATP, the na?ve and neuropathic pain rats were examined by PWT test.Result(1) The BV2 microglia was incubated with different dozens of ATP. In the low dozens of ATP groups, the M1 and M2 markers do not change, which were examined by Westernblot, flow cytometry and confocal immunofluorescence. But in the high dozen ATP group, M1 markerdecreased and M2 marker increased, which were evidenced by Westernblot, flow cytometry and confocal immunofluorescence.(2) The BV2 microglia was incubated with low dozen ATP plus LPS. In the single low dozen ATP group, the M1 and M2 markers do not change. But in the low dozen ATP plus LPS group, M1 marker was higer and M2 marker was lower, campared with the single ATP group,(3) After intrathecal administration of microglia pre-incubated with low dozen ATP plus LPS, the na?ve rats’ PWTs decreased, which suggested that low dozen ATP could facilitate the ability of LPS-inducing M1 microglia in neuropathic pain.(4) After intrathecal administration of microglia pre-incubated with high dozen ATP, the pain rats’ PWTs increased, which suggested that high dozen ATP-induced M2 microglia would relieve pain behavior.Conclusion ATP cans modulation microglia polarization in neuropathic pain. The low dozen can facilitate LPS-inducing M1 microglia, which might strengthen pain behavior, and the high dozen can induce M2 microglia directly, which might relieve pain behavior contrarily.