NMDA Receptor Antagonist MK-801 Attenuates COX-2 Protein And MRNA Expression In The Spinal Dorsal Horn During Formalin-induced Inflammatory Pain And Hyperalgesia In Rats

Objective: Prostaglandins (PGs) play an important role in the nociceptive transmission in the spinal cord. Cyclooxygenases (COXs) are the key enzyme catalyzing the synthesis of PGs. Three isozymes of COX have been identified. Several lines of evidence indicate that up-regulation of COX-2 expression and production of PGs are important in the transmission of nociceptive information in the spinal cord. However, the mechanisms mediating the up-regulation of COX-2 during nociceptive inputs are still not completely understood.A variety of evidence showed that excitatory amino acids (EAAs) and NMDA receptor are important neurotransmitters in the transmission of nociceptive information in the spinal cord. Furthermore, many investigations suggested that the increase of spinal COX-2 expression might be related to the activation of NMDA receptor in the spinal cord during pain and hyperalgesia. For example, the release of glutamate and PGE2 was increased during the pain and hyperalgesia. Intrathecal injection of selective COX-2 inhibitor could block the development of thermal hyperalgesia normally induced by intrathecal administration of NMDA in a dose-dependent manner. These results suggested that the increase of spinal COX-2 expression might be resulted from the activation of NMDA receptor during pain and hyperalgesia.Recently, we used immunohistochemistry showed that intrathecal injection of NMDA receptor agonist NMDA upregulated expression of COX-2 in the spinal dorsal horn, and that the upregulation of the spinal COX-2 expression in the formalin test was significantly inhibited by intrathecal injection of MK-801, a noncompetitive antagonist of NMDA receptor.These results preliminarily suggested the involvement of activation of NMDA receptor in the up-regulation of COX-2 in the spinal dorsal horn during nociceptive inputs.Therefore, in order to provide further evidence to illustrate the role of activation of NMDA receptor in the up-regulation of spinal COX-2 resulted from nociceptive inputs, the present study was undertaken to observe, using Western blotting and RT-PCR analysis, the influence of MK-801, a noncompetitive NMDA receptor antagonist, on the up-regulation of COX-2 protein and mRNA expression in the spinal dorsal horn using rat formalin test model.Methods: One hundred and seventy male Spague-Dawley rats weighing 250-280g were divided randomly into the following groups:Sham group: Rats were subcutaneously injected with normal saline (NS) 0.15 ml in the plantar surface of the right hind paw, and according to times for observations after the injection, were divided into 1 h, 2 h (RT-PCR only) and 1 d subgroups.Formalin groups: Rats were subcutaneously injected with formalin solution (5%) 0.15 ml in the plantar surface of the right hind paw, and according to times for observations after the formalin injection, were divided into 1 h, 2 h (RT-PCR only), 4 h, 1 d and 3 d groups.Formalin+NS groups: NS in volume of 10μl was intrathecally injected at 15 min prior to the formalin injection. Others were the same as those in sham group.Formalin+MK-801 group: MK-801 solution in 10μl was intrathecally injected at 15 min prior to the formalin injection. According doses of MK-801 used, this group was further divided into 2 nmol, 20 nmol and 100 nmol subgroups. Others were the same as those in Formalin+NS group.The dorsal horn of lumbar 5 segment (L5) of spinal cord of the rats in each group above was dissected out after sacrificing the animals with decapitation at the designed ending time point for assaying COX-2 protein and mRNA expression using Western blotting (n=5 in each time point) and RT-PCR (n=5 in each time point) analysis, respectively. The weighted pain score of the injected paw within 60 min after the formalin injection were measured before the sampling. The thermal withdrawal latency and mechanical withdraw threshold of the injected area and the corresponding area in uninjected hind paw were measured on 1 d before the sampling.Results:1 Pain behavioral responses1.1 Spontaneous pain behavioral responses The rats showed characteristic flinch reflexes in the injected paw after the formalin injection. The weighted pain score of the injected paw within 60 min after the formalin injection showed a clear biphasic and was significantly suppressed by intrathecal injection of MK-801 in a dose-dependent manner (P<0.05).1.2 The thermal withdrawal latency and mechanical withdraw threshold In contrast to sham group, the thermal withdrawal latency and mechanical withdraw threshold tested with heat radiant and von-Fery fibers, respectively, were increased in the injected area, while decreased in the uninjected hind paw at 1 d after the formalin injection (P<0.05), which indicated that the injection of formalin induced the development of thermal and mechanical hyperalgesia (uninjected hind paw). Intrathecal injection of MK-801 significantly suppressed the thermal and mechanical hyperalgesia induced by the formalin injection represented with increases in the thermal withdrawal latency and mechanical withdraw threshold in the corresponding area in the uninjected hind paw in Formalin+MK-801 group along with the increase in the dose of MK-801 used compared with Formalin+NS group (P<0.05).2 Up-regulation of COX-2 protein and mRNA expression in the spinal dorsal horn during pain and hyperalgesia induced by formalin There were constitutive expression of COX-2 protein and mRNA in the dorsal horn of the L4~L5 spinal segment in sham group. The expression of COX-2 protein in the spinal dorsal horn of injection side increased significantly at 1 h and reached peak on 1 d, returned to sham level on 3 d after the formalin injection (P<0.05). The characteristic in time course of the up-regulation of COX-2 protein was similar with that in pain behavioral responses. The time course of expression of COX-2 mRNA was similar with that of COX-2 protein, except for the peak of the upregulation shifted to a earlier time at 2 h after the formalin injection.3 Inhibition of MK-801 on the up-regulation of COX-2 expression in the spinal dorsal horn during formalin test induced-pain and hyperalgesia In formalin+MK-801 each groups, COX-2 protein and mRNA expression in the spinal dorsal horn in both 1 h and 1 d groups was significantly downregulated after the administration of MK-801compared with formalin+NS group (P<0.05). In addition, the upregulation in COX-2 mRNA at 2 h after the formalin injection, when the upregulation of the mRNA was peaked, was also downregulated after the administration of MK-801. The downregulation showed a clear dose-dependence with the doses of MK-801 used. These results indicated that MK-801 significantly suppressed the upregulation of COX-2 protein and mRNA expression in the spinal dorsal horn during formalin-induced inflammatory pain and hyperalgesia in rats.Conclusion: The increase of COX-2 protein and mRNA expression in the dorsal horn of the spinal cord and pain behavior induced by formalin were significantly inhibited by intrathecal injection NMDA receptor antagonist MK-801 in a dose-dependent manner. These results indicated that the activation of NMDA receptor is one of the mechanisms for the up-regulation of the expression of COX-2 protein and mRNA in the spinal dorsal horn during formalin-induced rat inflammatory pain and hyperalgesia in rats.