NMDA, A NMDA Receptor Agonist, Up-regulates Astrocyte-specific Protein GFAP And Microglia-specific Protein CD11b Expression In The Spinal Dorsal Horn In Rats

Objective: Recent studies showed that spinal glial cells play an importantrole in the development and maintenance of neuropathologic pain. Glial cellscan be activated during nociceptive input and the activated glial cells releasepro-inflammatory substances, such as interleukins, tumor necrosis factor, etc.These cytokines can enhance neuronal excitability and pain sensitivity.Administration of glial inhibitor such as minocycline, tetracyclines can inhibitnervous inflammation, prevent the activation of microglia and astrocytes.However, mechanisms underlying the activation of the spinal glial cells in theprocess of development and maintenance of neuropathologic pain are not fullyunderstood.A variety of evidence indicated the important role of excitatory aminoacids and their receptors, especially the N-methyl-D-aspartate acid (NMDA)and its receptor in the transmission of nociceptive information in the spinalcord. NMDA receptor is a subtype of excitatory amino acid receptors andbelongs to ionotropic receptors. It is widely distributed in the central nervoussystem, and is involved in central sensitization in the spinal level, which isinvolved in the hyperalgesia, spontaneous pain and pain hypersensitivityformation. Some researches suggest that NMDA receptor is involved in theactivation of the spinal glial cells in the neuropathologic pain model. However,whether the NMDA receptor is also associated with the activation of spinalglia in the development and maintenance of peripheral inflammatory pain andhyperalgesia is not fully understood. Recently, we showed using western blotanalysis and immunohistochemistry techniques that the expressions of glialfibrilary acidic protein (GFAP), a labeling protein of astrocytes, and CD11b/c(OX-42), a labeling protein of microglia, were increased in the rat spinal dorsal horn during bee venom-induced inflammatory pain and hyperalgesia.And the upregulation of the above labeling protein could be inhibited bydizocipine maleate (MK-801), a non-selective NMDA receptor antagonist.These results preliminarily suggested the involvement of activation of NMDAreceptor in the activation of spinal glia in inflammatory pain and hyperalgesiaprocess.Therefore, in order to definitely illustrate the role of activation of NMDAreceptor in the up-regulation of spinal GFAP and CD11b resulted fromnociceptive inputs, the present study was undertaken to further observe effectsof intrathecal administration of NMDA, the agonist of NMDA receptor, on theexpression of GFAP and CD11b protein in rat spinal dorsal horn using westernblotting analysis.Methods: One hundred and twenty male Spague-Dawley rats weredivided randomly into the following groups:①S ham group: Normal saline(NS) in volume of10μl was intrathecallyinjected. Observations were performed at time points corresponding with thefollowing treatment groups.②N MDA time-dependent group: NMDA solution in volume of10μl (10nmol in dose) was intrathecally injected. The examination of GFAP andCD11b expression was performed at time points of1h、4h、8h、12h、1d、2d and3d after the NMDA injection. In order to observe the relationshipbetween GFAP and CD11b expression and hyperalgesia after intrathecalNMDA, the tail-flinch latency, thermal withdrawal latency, and themechanical withdrawal threshold were examined at the corresponding timepoints above.③N MDA dose-dependent group: NMDA solution in volume of10μlwas intrathecally injected. According to doses of NMDA used, this group wasfurther divided into0.1nmol,1nmol and10nmol subgroups in order toexamine the dose–dependence of NMDA in up-regulating the expression ofGFAP and CD11b. The expression of spinal GFAP and CD11b was assayed at8h after the intrathecal injection because it was shown in our preliminary experiment that the expression of spinal GFAP and CD11b after intrathecalNMDA was most obvious at that time point. The tail-flinch latency, thermalwithdrawal latency, and the mechanical withdrawal threshold were examinedas well.④M K-801+NMDA group: MK-801solution in volume of10μl (50nmol) was intrathecally injected at15min prior to NMDA injection10μl (10nmol). Other treatments are the same to those in NMDA dose-dependentgroup.In each group above, ten rats were included,5of them were used forGFAP expression assay and another5were used for CD11b assay to reflectactivation state of the spinal astrocytes and microglia, respectively. Themethod of the assay was western blotting analysis.Data were expressed as mean±SD (±s) and statistically treated withstatistical software of Statistical Program for Social Sciences13.0. One-WayANOVA was used to compare between groups. If it had significantlydifference, least significant difference was used to test difference betweengroups. P<0.05was considered to be significant.Result:1Nociceptive behavioral responses1.1Spontaneous pain behavior response Intrathecal injection ofNMDA elicited a characteristic, caudally directed nociceptive behavioralresponse consisting of intense biting and scratching episodes, etc.1.2Mechanical withdrawal threshold The mechanical withdrawalthreshold of hind paws to von Frey fibers decreased at time point of4h(P<0.05), and the decrease lasted to24h after the intrathecal NMDA. On2dafter the intrathecal NMDA, the threshold returned to sham level. The resultsindicate that the mechanical hyperalgesia developed at earlier time andpersisted for some time after the intrathecal NMDA. The changes in thethreshold after intrathecal NMDA showed dose dependency and could beinhibited with prior administration of MK-801, an inhibitor of NMDAreceptor. 1.3The thermal withdrawal and the tail-flinch latency The thermalwithdrawal and the tail-flinch latency decreased at time point of1h (P<0.05),and the decrease lasted to12h after the intrathecal NMDA. On1d after theintrathecal NMDA, the thermal withdrawal and the tail-flinch latency returnedto sham level. The results indicate that the thermal hyperalgesia developed atearlier time and persisted for some time after the intrathecal NMDA. Thechanges in the latencies after intrathecal NMDA showed dose dependency andcould be inhibited with prior administration of MK-801, an inhibitor ofNMDA receptor.2GFAP expression in spinal dorsal hornThe intrathecal NMDA (10nmol in dose) induced an up-regulation inGFAP protein expression as early at1h (P<0.05), and reached peak at8h, andreturned to the sham level on3d after the intrathecal NMDA. Thecharacteristics in the time course of the up-regulation of GFAP were consistentwith those of nociceptive behavioral responses mentioned above. Theupregulation of GFAP expression induced by NMDA exhibited clear dosedependency, which represented with the steadily up-regulation along with theincrease in the dose of NMDA used (0.1nmol,1nmol, and10nmol).Moreover, the up-regulation was significantly inhibited by prior intrathecaladministration of MK-801(P<0.05).3CD11b expression in spinal dorsal hornThe intrathecal NMDA solution in volume of10μl (10nmol in dose)induced an up-regulation in CD11b protein expression as early at1h (P<0.05),gradually increased at4h and reached peak at8h, and returned to the shamlevel on3d after the intrathecal NMDA. The characteristics in the time courseof the up-regulation of CD11b were consistent with those of nociceptivebehavioral responses mentioned above. The expression of CD11b in the dorsalhorn of the spinal cord was steadily up-regulated along with the increase in thedose of NMDA used (0.1nmol,1nmol, and10nmol), which indicated thedose-dependency of the NMDA. The up-regulation induced by NMDA wassignificantly inhibited by prior intrathecal administration of MK-801 (P<0.05).Conclusion:1Intrathecal administration of NMDA, a selective agonist of NMDAreceptor, up-regulated the expression of GFAP and CD11b in the dorsal hornof the spinal cord in a dose-dependent manner. The characteristics in the timecourse of the up-regulation of GFAP and CD11b were consistent with those ofnociceptive behavioral responses.2The up-regulation of GFAP and CD11b could be inhibited byintrathecal injection of MK-801, a noncompetitive inhibitor of NMDAreceptor.3These results suggest that the activation of NMDA receptor is one ofthe mechanisms for the activation of astrocytes and microglia in the spinaldorsal horn during nociceptive inputs.