Role Of Cyclooxygenase In Neuropathic Pain And Its Mechanisms

Pain remains a significant issue in the modern medicine. Neuropathic pain is another important kind of pain besides inflammatory pain. Neuropathic pain is the result of injury to the peripheral or central nervous system with pretty complex mechanisms. Unlike other pain styles, neuropathic pain will persist even though the injury has healed. In neuropathic pain the peripheral or central nervous systems are malfunctioning and become the cause of the pain. And it often has a long-term process. There are many difficulties in the treatment of neuropathic pain. Poor response to opioid analgesics and addiction by long-term use of them makes it difficult to treat neuropathic pain using opioids. Some agents that can inhibit the abnormal discharge of neurons, such as the tricyclic antidepressants and the anticonvulsants, have been the predominant treatment of neuropathic pain. However, the long-term use of them could lead to great side effects. Furthermore, simply inhibiting the abnormal discharge of neurons is not enough for the treatment of neuropathic pain. Recently, proinflammatory cytokines, suchas IL-6, TNF-a, etc, have been proved to be involved in the process of neuropathic pain, indicating the cross-talking between neuropathic pain and inflammation. Cyclooxygenase (COX) is the key enzyme catalyzing AA to PGs and plays an important part in inflammatory process. Inhibitors of COX have been used in the treatment of inflammatory pain. COX is expected to play a part in the process of neuropathic pain.Three isoforms of COX have been isolated. The former two, COX-1 and COX-2, have got better understanding and the level of these two in the spinal cord has been proved to change after neuropathic pain. However, most studies on the relationship between COX and neuropathic pain up to now were done in peripheral nervous system or the spinal cord while few in the brain. And most studies observed change in a short-term instead of a long-term. Moreover, the post-neuropathic-pain change of COX-3 which has been assumed as the potential target for pain treatment has not been reported.In this study, we compared the distribution of three COX isoforms in some brain areas closely related to pain, observed the change of these isoforms in brain at different time points after neuropathic pain in mouse spared nerve injury (SNI) model, and the analgesic effect of difference COX inhibitors on SNI induced neuropathic pain. No pharmacological study was done on COX-3 due to absence of selective COX-3 inhibitor. Finally, we studied part of the mechanisms of COX-1 and COX-2 participating the process of neuropathic pain.1. The distribution of COX-l,COX-2 and COX-3 in three brain areas related to painThe distribution of COX-1, COX-2 and COX-3 in three pain-related brain areas, PAG, RMg and LC, was observed by immunohistochemical staining. Results showed that physiologically these three isoforms existed in the areas observed with different amount, indicating that the three isoforms were all involved in the process of pain.2. The change of three COX isoforms in brain after SNIThe change of PGFia(catalyzed by COX-l)and PGE2(catalyzed by COX-2) concentration in brain after SNI was measured by radioimmunoassay. The change of COX-1, COX-2 and COX-3 in brain at the level of mRNA was evaluated by RT-PCR and at the level of protein by western blot. Results showed that the expression of COX-2 increased at the early phase after SNI and then gradually decreased. However, the level of COX-1 and COX-3 increased at a later time point (14d). These results indicated that COX-2 play role in the early process of neuropathic pain while COX-1 and COX-3 be involved in the later phase.3. The analgesic effects of different COX inhibitors on SNI induced neuropathic painThe thermal pain threshold of mice was measured by hot-plate test. Effects of different COX inhibitors on SNI induced thermal hyperalgesia were compared. Results showed that administration of NS-398 (selective COX-2 inhibitor) relieved the thermal analgesia significantly but continuing administrating NS-398 didn't lead to better analgesic effect. However, giving SC-560(selective COX-1 inhibitor) at the early phase didn't show significant analgesic effect. Only administration of SC-560 at the late phase increased the thermal pain threshold significantly. o4. The mechanisms of COX-1 and COX-2 being involved in the process of neuropathic painThe cellular change induced by COX-1 and COX-2 were observed at the time point of Id and 60d after SNI respectively. At the time point of Id after SNI, the change of P2X4 receptor expression and the effect of NS-398 on it was checked by RT-PCR, as well as the effect of suramin (inhibitor of P2X receptor) on the expression of COX-2 expression; the effect of NS-398 on expression of pCREB was studied by western blot; the concentration of P-endorphin was measured by radioimmunoassay. At the time point of 60d after SNI, the change of P2X4 receptor expression and the effect of SC-560 on it was checked by RT-PCR, as well as the effect of suramin on the expression of COX-1 expression; the effect of SC-560 on expression of pCREB was studied by western-blot; the concentration of P-endorphin was measured by radioimmunoassay. Results showed that: 1. The level of P2X4 receptor in brain increased significantly at Id after SNI but didn't change at the time point of 60d after SNI; NS-398 significantly decreased the expression of P2X4 receptor while suramin didn't show significant effect on COX-2 expression; SC-560 showed no significant effect on P2X4 expression and suramin didn't affect the COX-1 expression. These results indicated that P2X4 be involved in the early process of neuropathic pain and be a downstream molecule to COX-2. 2.The level of pCREB in brain increased significantly both at the time point of Id and of 60d after SNI; both COX-1 and COX-2 selective inhibitor showed significant effect on the level of pCREB, indicating that CREB play important role in the signal transduction induced by COX in the process of neuropathic pain. 3.