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Involvement Of Spinal Glia In Tolerance To Morphine Analgesia: A Study On P2X7 Receptor

Posted on:2011-04-29Degree:DoctorType:Dissertation
Country:ChinaCandidate:M L ChenFull Text:PDF
GTID:1100360305497602Subject:Neurobiology
Abstract/Summary:PDF Full Text Request
Opioid substances, like morphine, are the most effective analgesics used to treat moderate to severe pain. However, their use is limited by the development of tolerance, which is manifested clinically by the need for increasing opioid dosages over time to maintain the same level of pain relief. Recently, the role of microglia in the central nervous system in the development of analgesic tolerance has been studied. Although considerable progress has been made, the mechanism underlying microglial contribution to morphine analgesic tolerance is not fully explored.P2X7 receptor (P2X7R) is a subtype of the ATP-gated non-selective ion channel family P2X. It has a widespread tissue distribution and its expression is particularly strong in the immune system. In spinal cord, P2X7R is predominately present in microglia. P2X7R plays an important role in the production of proinflammatory cytokines and the regulation of glutamate transporter efficiency. Recently, mounting evidence indicates that P2X7R is involved in multiple neuropathic models. Given that morphine analgesic tolerance and neuropathic pain are supposed to share some common mechanisms, it is reasonable to hypothesize that spinal P2X7R contributes to morphine analgesic tolerance.Here, we tested our hypothesis in rats using experimental techniques including behavioral tests, RNA interference, Western blot analysis, immunofluorescence and cell culture. The results were as follows:1. Spinal microglia-expressed P2X7R contributed to the development of chronic morphine tolerance but not the maintenanceP2X7Rs were exclusively localized in OX42-labeled microglia in spinal dorsal horn revealed by immunofluorescence, which was performed on lumbar spinal cord sections from normal and morphine tolerated adult rats. The cellular localization of P2X7R was confirmed in the mixed-glia culture from 17-day-old embryonic (E17) SD rats. The expression of spinal P2X7R was upregulated gradually during the development of morphine analgesic tolerance; and the significant increase occurred since D4 (after 3 days'morphine injection), at which tolerance was just generated. Pretreatment with P2X7R antagonists [oxidized ATP (oxATP) or brilliant blue G (BBG)] or P2X7R-targetting siRNA intrathecally inhibited the development of chronic morphine tolerance significantly. However, intrathecal administration of BBG failed to reverse the established tolerance to morphine analgesia.2. Spinal P2X7R mediated the activation of glia and neurons in the development of morphine analgesic toleranceThe expressions of microglial marker Ibal, the neuronal kinase PKCy and astroglial marker GFAP in rat lumbar spinal cord were upregulated upon the development of chronic morphine tolerance; and the significant increase began at D2, D4 and D6 respectively. Pretreatment with P2X7R antagonist BBG or P2X7R-targetting siRNA intrathecally not only inhibited the upregulation of Ibal and p-p38 expression in microglia, but also suppressed the upregulation of GFAP expression in astroglia and PKCy expression in neurons of the spinal cord treated by chronic morphine.3. Spinal proinflammatory cytokine interleukin 18 (IL-18) as one downstream of P2X7R was involved in chronic tolerance to morphine analgesiaThe expressions of proinflammatory cytokine IL-18 and its corresponding receptor IL-18 receptor (IL-18R) in rat lumbar spinal cord were upregulated upon the development of chronic morphine tolerance; and the significant increase began at D4 and D6 respectively. IL-18 was mainly localized in OX42-positive microglia of rat spinal cord, whereas IL-18R majorly distributed in GFAP-labeled astroglia. Pretreatment with IL-18 antagonists IL-18BP or IL-18Ab intrathecally inhibited the development of morphine tolerance significantly. Pre-administration of P2X7R antagonist BBG or P2X7R-targetting siRNA intrathecally suppressed the upregulation of spinal IL-18, IL-1βand pNFκB induced by repeated morphine injection remarkably. Pretreatment with IL-18BP intrathecally suppressed the upregulation of spinal GFAP and PKCy significantly, but not affected the upregulation of either Ibal or pNFκB induced by chronic morphine.Taken together, we demonstrated that the expression of spinal microglia-expressed P2X7R was increased after chronic exposure to morphine, which facilitated the development but not the maintenance of morphine analgesic tolerance. Moreover, as one of P2X7R downstream targets, pro inflammatory cytokine IL-18 was involved in the generation of morphine tolerance, possibly by mediating the activation of microglia-astroglia-neurons cascade. Although NFκB was involved in the downstream of P2X7R, it might not be the predominant pathway through which IL-18 mediated the activation of astroglia. These findings highlight the possibility of a new clinical strategy to improve morphine tolerance.
Keywords/Search Tags:P2X7 receptor, Morphine tolerance, Spinal cord, Glia, RNA interference, Brilliant blue G, Oxidized ATP, Interleukin 18, NFκB, Rat
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