Involvement Of Spinal Microglia-expressed P2X₇ Receptor In Tolerance To Morphine Analgesia In Rats

Involvement of spinal microglia-expressed P2X7 receptor in tolerance to mprphine analgesia in ratsMorphine is a highly potent opiate analgesic drug and it is clinically used in the treatment of moderate to severe pain. However, its analgesic potency fades rapidly upon frequent administration so that larger dose of morphine is required to maintain the same effect. Morphine analgesic tolerance greatly hampered its usage in pain management. We have previously demonstrated that spinal glia contributes to the de-velopment of morphine analgesic tolerance. Although considerable progress has been made, the mechanism underlying glial contribution to morphine analgesic tolerance is not fully clear.P2X7 receptor (P2X7R) is a subtype of the ATP-gated non-selective ion channel fam-ily P2X. It has a widespread tissue distribution and its expression is particularly strong in the immune system. The activation of P2X7R requires a high concentration of ex-tracellular ATP and prolonged activation leads to the formation of transmembrane pore permeable to large molecular weight molecules. P2X7 receptor plays an impor-tant role in the production of proinflammatory cytokines, and the regulation of gluta-mate transporter efficiency. Recently, mounting evidence indicates that P2X7R is in-volved in multiple neuropathic models. Given that morphine analgesic tolerance and neuropathic pain are supposed to share a common mechanism, it is reasonable to hy-pothesize that spinal P2X7R contributes to the development of morphine analgesic tolerance.Here, we tested our hypothesis using experimental techniques including behav-ioral tests, immunohistochemistry, western blotting as well as cell culture. The results are as follows:1. Spinal P2X7R was specifically expressed on microgliaSpinal cells from 17-day-old embryonic (E17) SD rats were harvested and cul-tured by a standard enzyme treatment protocol for further immunohistochemical labeling. P2X7R immunoreactivity (IR) was colocalized with microglial marker 0X42 but not astrocytic marker GFAP nor neuronal marker NeuN. Furthermore, on dorsal horn regions of lumbar spinal cord sections from NS treated rats and morphine tolerated rats, similar distribution pattern of P2X7R expression was ob-served.2. Chronic morphine exposure significantly upregulated spinal P2X7RAfter twice daily subcutaneous morphine injection for at most 9 days, significant tolerance to the antinociception effect of morphine was established in behavioral tests. Parallel to the behavioral results, marked upregulation of the spinal P2X7R protein level was detected in semi-quantificational western blotting, suggesting a possible functional role for spinal P2X7R in the development of morphine analge-sic tolerance.3. Intrathecal P2X7R antagonists attenuated morphine analgesic toleranceWe applied intrathecal P2X7R antagonists 30 minutes before daily subcutaneous morphine to investigate the contribution of spinal P2X7R. The behavioral results demonstrated that both antagonists [oxidized ATP (oxATP) and brilliant blue G (BBG)] significantly preserved morphine analgesic potency after chronic mor-phine administration.4. Intrathecal P2X7R antagonist did not reverse established morphine analgesic toleranceWe continued to administrate subcutaneous morphine for 3 days after the estab-lishment of morphine analgesic tolerance. Intrathecal BBG was delivered 30 min-utes before regular subcutaneous morphine. The behavioral results demonstrated that BBG treatment failed to produce any statistically significant effect on mor-phine antinociception compared with control group, indicating a limited involve-ment of spinal P2X7R in the maintenance of morphine analgesic tolerance.5. Spinal P2X7R knockdown attenuated morphine analgesic toleranceTo corroborate the results obtained by pharmacologically antagonizing spinal P2X7R, we genetically knockdown the spinal P2X7R by applying intrathecal small interfering RNA (siRNA). Similarly, morphine analgesic potency was sig-nificantly higher in the P2X7R knockdown group, suggesting a specific contribu- tion of spinal P2X7R to the development of morphine analgesic tolerance.6. Disruption of spinal P2X7R function inhibited activation of microgliaUpregulation of protein level of microglial marker Ibalin the lumbar spinal cord tissue induced by chronic morphine was inhibited by either pharmacologically or genetically interfering P2X7R function. Parellel to Ibal expression, p38 mito-gen-activated protein kinase (MAPK) was also significantly activated during morphine analgesic tolerance. Intefering P2X7R function diminished upregulation of phosphorylated p38MAPK induced by chronic morphine. These findings sug-gest that P2X7R's effect on morphine analgesic tolerance is at least partially achieved by activatiing spinal microglia.Taken together, we demonstrated that the spinal immunoreactivity (IR) of P2X7R was colocalized with microglial marker 0X42 but not astrocytic marker GFAP or neuronal marker NeuN. The Protein level of spinal P2X7R was upregulated after chronic ex-posure to morphine. Selective P2X7R inhibitors significantly attenuated the loss of morphine analgesic potency to both mechanical and thermal stimuli. In addition, RNA interference (RNAi) against P2X7R in the spinal cord exhibited a similar toler-ance-attenuating effect. However, intrathecal BBG showed no effect on established analgesic tolerance. Our results suggest that P2X7R is involved in the induction but not maintenance of morphine analgesic tolerance and it may be a promising target for improving the clinical use of morphine.