Intrathecal Injection NMDA Receptor Agonist NMDA Up-regulates COX-2 Expression In The Spinal Dorsal Horn In Rats

Objective: Prostaglandins (PGs), especially the PGE2 and PGI2, are important mediators of inflammation, pain and fever. Cyclooxygenas (COXs) are the key enzyme catalyzing the synthesis of PGs. COX-2 and PGs play an important role in the development and maintenance of pain and hyperalgesia induced by neciceptive stimulation. For example, oral or intrathecal administration of COX-2 inhibitors produced equally antinociceptive effect in a dose-dependent manner. It was showed by immunohisto- chemistry and /or Western blotting analysis that the COX-2 expression in the dorsal horn of the spinal cord was up-regulated in many pain models such as the formalin induced inflammatory pain and hyperalgesia, etc. These results evidently suggested the involvement of spinal COX-2 in the nociceptive transmission. However, the mechanisms mediating the up-regulation of spinal COX-2 during nociceptive inputs is still not completely understood.A variety of evidence indicated the important role of neurotransmitters of excitatory amino acids (EAAs) and their receptors, especially the N-methyl-D-aspartate (NMDA) receptor in transmission of nociceptive information in the spinal cord. Furthermore, several lines of evidence suggested that the up-regulation of spinal COX-2 expression and release of PGs elicited by nociceptive inputs might be resulted from the activation of spinal NMDA receptor. For instance, the release of glutamate and PGE2 was increased during many pain models such as the kaolin/carrageenan and formalin elicited pain and hyperalgesia. Both the antagonist of NMDA receptor and selective inhibitor of COX-2 could attenuate pain related behaviors induced in the formalin test. The nociceptive behavioral responses induced with intrathecal injection of NMDA could be inhibited with intrathecal or systemic administration of COX-2 inhibitors.Recently, we used immunohistochemistry showed that intrathecal injection of NMDA receptor agonist NMDA up-regulated expression of COX-2 in the spinal dorsal horn, and that the up-regulation 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 definitely 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 further observe, using western blotting and RT-PCR analysis, the expression of COX-2 protein and mRNA, respectively, in the dorsal horn of the spinal cord after intrathecal injection of NMDA in rats.Methods: One hundred and ten male Spague-Dawley rats were divided randomly into the following groups:Sham group: Normal saline (NS) in volume of 10μl was intrathecally injected. Observations were performed at time points corresponding with the following treatment groups.NMDA I group: NMDA solution in volume of 10μl was intrathecally injected. According to doses of NMDA used, this group was further divided into 0.1, 1 and 10 nmol subgroups in order to examine the dose–dependence of NMDA in up-regulating the expression of COX-2. The expression of spinal COX-2 was assayed at 4 h after the intrathecal injection. NMDA+MK-801 group: MK-801 solution in volume of 10μl (50 nmol) was intrathecally injected at 15 min prior to NMDA injection 10μl (10 nmol). The expression of spinal COX-2 was assayed at 4 h after the intrathecal injection of NMDA.NMDA II group: NMDA solution in volume of 10μl (10 nmol in dose) was intrathecally injected. The examination of COX-2 expression was assayed at time points of 1 h, 2 h, 4 h, 24 h, 36 h (only for Western blot), and 3 d (only for Western blot) after the NMDA injection. In order to observe the relationship between COX-2 expression and hyperalgesia after intrathecal NMDA, the tail-flinck latency, thermal withdrawal latency, and the mechanical withdrawal threshold were examined before decapitation of the animals at the corresponding time points above.Data were expressed as mean±SD ( x±s) and analyzed by One-Way ANOVE and paired t-test using SPSS statistical software. P<0.05 was regarded significant.In each group above, ten rats were included, 5 of them were used for Western blotting analysis of COX-2 protein and another 5 were used for RT-PCR analysis of COX-2 mRNA.Results:1 Nociceptive behavioral responses. Intrathecal injection of NMDA elicited a characteristic, caudally directed, nociceptive behavioral response consisting of intense biting and scratching episodes, etc.1.1 The tail-flinck and the thermal withdrawal latency. Compared with sham group, the tail-flinck latency tested to hot water immersion significantly decreased at time points of 1 h, 2 h, 4 h, and 24 h after the intrathecal NMDA (P<0.05). The results indicated that the thermal hyperalgesia developed at earlier time and maintained a relative long period after the intrathecal NMDA. The tail-flick latency returned to sham level at the time points of 36 h after the intrathecal NMDA, indicated the thermal hyperalgesia recovered. The characteristic in the thermal withdrawal latency of hind paws to heat radiant were similar with those in the tail-flinck latency.1.2 Mechanical withdrawal threshold. The mechanical withdrawal threshold of hind paws to von Frey fibers decreased at time point as early as 1 h after the intrathecal NMDA (P<0.05), and the decrease continued to the time point of 36 h after the intrathecal NMDA. The results indicate that the mechanical hyperalgesia developed at earlier time and maintained a relative long period after the intrathecal NMDA. The mechanical withdrawal threshold returned to sham level at the time points of 3 d after the intrathecal NMDA, indicated the thermal hyperalgesia recovered.2 Intrathecal injection of NMDA up-regulates COX-2 expression in the dorsal horn of the spinal cord. The expression of COX-2 in the dorsal horn of the spinal cord was steadily up-regulated along with the increase in the dose of NMDA used (0.1 nmol, 1 nmol, and 10 nmol) (P<0.05). The most intensive up-regulation induced by 10 nmol NMDA was significantly inhibited by prior intrathecal administration of MK-801 (P<0.05).3 The characteristics in the time course of the up-regulation of COX-2 expression are consistent with those of nociceptive behavioral responses induced by intrathecal NMDA. The intrathecal NMDA induced an up-regulation in COX-2 protein expression as early at 2 h (P<0.05), and reached peak at 4 h, and returned to the sham level on 3 d after the intrathecal NMDA. The expression of COX-2 mRNA is similar with the expression of COX-2 protein, except for the initiation and the returned time of the up-regulation moved up to 1 h and 24 h after the intrathecal NMDA (P<0.05). The characteristics in the time course of the up-regulation of COX-2 were consistent with those of nociceptive behavioral responses mentioned above.Conclusion:1 Intrathecal administration of NMDA, a selective agonist of NMDA receptor, up-regulated the expression of COX-2 in the dorsal horn of the spinal cord in a dose-dependent manner.2 The up-regulation could be inhibited by intrathecal injection of MK-801, a noncompetitive inhibitor of NMDA receptor, and was consistent in characteristics of the time course with those of nociceptive behavioral responses.3 These results suggested that the activation of NMDA receptor is one of the mechanisms for the up-regulation of COX-2 expression in the spinal dorsal horn during nociceptive inputs.