| Neuropathic Pain?NPP?is a chronic pain caused by peripheral nerve injury.It is difficult to heal in clinical treatment which is seriously effects the quality of patients'lives.The spinal dorsal horn plays an important role in nociceptive information transmission and modulation from the periphery to central nervous system.Purine P2Y6receptor is selectively expressed in dorsal spinal cord microglia and plays a significant role in regulating the function of microglia.However,it is not clear that whether the P2Y6 receptor can further participates in the modulation of nociceptive information by regulating the function of microglia at the spinal level.It is well known that the abnormal pain behavior such as hyperalgesia,allodynia,spontaneous pain in chronic constriction injury of the sciatic nerve?CCI?model rat are very similar with the chronic neuropathic pain symptoms and behaviors which is induced by peripheral nerve injury.Therefore,we aimed to observe:1)the changes in mechanical allodynia and thermal hyperalgesia and other pain behavior in CCI rats;2)the effects of intrathecal injection of P2Y6 receptor antagonist MRS2578 on the pain behavior and the expression of P2Y6 receptor and Iba-1in dorsal spinal cord in CCI rats;3)the effects of intrathecal injection of p38MAPK inhibitor SB203580 or minocycline on the pain behavior and the expression of P2Y6receptor and Iba-1 in dorsal spinal cord in CCI rats;4)the effects of intrathecal injection of P2Y6 receptor agonist UDP on the pain behavior and the expression of P2Y6 receptor and Iba-1 in dorsal spinal cord in naive rats and CCI rats;5)the effects of intrathecal injection of MRS2578 on the pain behavior in UDP-treated rats.In these experiments,we investigated the modulation effects of P2Y6 receptor on the activity of microglia and explored the corresponding intracellular signal transduction mechanisms from different angles.Our findings may provide a new idea for developing the analgesic drugs.Objective:1.The aim of this study is to explore whether P2Y6 receptor in dorsal horn microglia is involved in the occurrence and maintenance of neuropathic pain in CCI rats.2.The aim of this study is to clear whether inhibiting the activity of microglial cells or p38MAPK phosphorylation can down-regulate the expression of P2Y6 receptor and further produces analgesic effect.Methods:We randomly selected SD male rats with 56 weeks old.All rats should be implantation of intrathecal catheter first.1.All rats were randomized into eight groups?n=8?:?1?sham group?sham operation?,?2?vehicle-treated CCI group?CCI+intrathecal 0.005%DMSO in saline?,?3?8MRS2578-treated group(CCI+intrathecal 10-11-10-4M MRS2578).For the rats after ligation,MRS2578 or 0.005%DMSO in saline?10?L?were intrathecal administered twice a day for 7 days.Then,valuating the TWL?thermal withdrawal latency?and MWT?mechanical withdrawl threshold?at 1 hour after drug administration that before the surgery and on 1,3,5 and 7days after surgery.2.All rats were randomized into four groups?n=8?:?1?sham group?sham operation?,?2?vehicle-treated CCI group?CCI+intrathecal 0.005%DMSO in saline?,?3?SB203580-treated group?CCI+intrathecal 50?M SB203580?,?4?Minocycline-treated group?CCI+intrathecal 0.2?g/?L minocycline?.For the rats after ligation,SB203580 or minocycline or 0.005%DMSO in saline?10?L?were intrathecal administered twice a day for 7 days.The TWL and MWT are evaluated before the surgery and on 1,3,5 and 7days after surgery.The expression of P2Y6 receptor and Iba-1 in dorsal horn was assessed by using quantitative real-time PCR?q-PCR?at 3,7 post-operative days.3.All rats were randomized into four groups?n=8?:?1?control group?intrathecal0.005%DMSO in saline?,?2?4UDP-treated group(intrathecal 10-4-10-6M UDP).For the rats after implantation of intrathecal catheter,UDP or 0.005%DMSO in saline?10?L?were intrathecal administered twice a day for 7 days.The TWL and MWT are evaluated on the day before drug administration and on 1,3,5 and 7 days after drug administration.The expression of P2Y6 receptor and Iba-1 in dorsal horn was assessed by using q-PCR at 3,7 days post-drug administration treatment.4.All rats were randomized into five groups?n=8?:?1?control group?intrathecal0.005%DMSO in saline?,?2?UDP-treated group(intrathecal 10-4M UDP),?3?5MRS2578-treated group(intrathecal 10-4M-10-6M MRS2578+10-4M UDP).For the rats after implantation of intrathecal catheter,MRS2578 or UDP or 0.005%DMSO in saline?10?L?were intrathecal administered twice a day for 7 days.TWL and MWT are evaluated on the day before drug administration and on 1,3,5 and 7 days after drug administration.5.All rats were randomized into three groups?n=8?:?1?sham group?sham operation?,?2?vehicle-treated CCI group?CCI+intrathecal 0.005%DMSO in saline?,?3?UDP-treated group(CCI+intrathecal 10-4M UDP).For the rats after ligation,UDP or 0.005%DMSO in saline?10?L?were intrathecal administered twice a day for 7days.Then,TWL and MWT are evaluated before the surgery and on 1,3,5 and 7 days after surgery.The expression of P2Y6 receptor and Iba-1 in dorsal horn was assessed by using q-PCR at 3,7 post-operative days.Results:1.Compared with sham group,CCI rats displayed significantly decreased TWL and MWT on day 1 after nerve injury?TWL:P<0.05;MWT:P<0.05?,and the allodynia was sustained for 7 days.Compared with vehicle-treated CCI group,MRS2578 at 10-9-10-4?suppressed CCI-induced mechanical allodynia and thermal hyperalgesia with a concentration-dependent manner?TWL:P<0.05;MWT:P<0.05?.2.Compared with vehicle-treated CCI group,intrathecal administration of SB203580 or minocycline significantly suppressed CCI-induced mechanical allodynia and thermal hyperalgesia?TWL:P<0.05;MWT:P<0.05?);q-PCR results showed that the expression of P2Y6 receptor and Iba-1 were markedly enhanced in the ipsilateral dorsal horn on 3,5,7 days after nerve injury?P<0.05?.Compared with vehicle-treated CCI group,intrathecal administration of SB203580 or minocycline significantly reduced CCI-induced increase in P2Y6 receptor and Iba-1 at the mRNA level on days 3 and 7?P<0.05?.3.Compared with control group,UDP(10-4-10-5 M)-treated rats displayed significantly decreased MWT and TWL on 3 days,and the allodynia was sustained for 7 days with a concentration-dependent manner?MWT:P<0.05;TWL:P<0.05?.Compared with control group,P2Y6 receptor and Iba-1 expression at mRNA levels in UDP-treated rats was significantly increased at 3,7 days after UDP administration?P<0.05?.4.Compared with control group,UDP?10-44 M?-treated rats displayed significantly decreased TWL and MWT on days 3,5 and 7?TWL:P<0.05;MWT:P<0.05?;Compared with UDP-treated rats,pretreated with MRS2578 at 10-6-10-4M significantly inhibited the reduced TWL and MWT on days 3,5 and 7?TWL:P<0.05;MWT:P<0.05?.5.Compared with sham group,CCI rats displayed significantly decreased TWL and MWT on day 1 after nerve injury?TWL:P<0.05;MWT:P<0.05?,and the allodynia was sustained for 7 days.Compared with vehicle-treated CCI group,intrathecal administration of UDP at 10-4M obvious enhances nociceptive responses induced by nerve injury in CCI rats?TWL:P<0.05;MWT:P<0.05?,and the allodynia was sustained for 7 days.Compared with vehicle-treated CCI group,UDP(10-4M,10?l)application obvious enhances the increased expression of P2Y6 receptor and Iba-1 mRNA at dorsal spinal cord of CCI rats at 3,7 days?P<0.05?.Conclusions:1.These observations suggested that P2Y6 receptor in dorsal spinal cord contributes to occurrence and maintenance of neuropathic pain in CCI-and UDP-treated rats.2.Enhanced the activity of microglia and p38MAPK phosphorylation in the dorsal spinal cord can up-regulate the expression of P2Y6 receptor and further promoting the occurrence and maintenance of neuropathic pain induced by CCI. |
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