Intrathecal Administration Of NMDA, A Agonist Of NMDA Receptor, Activated Spinal Microglia In Rats

Objective: At present, our understanding of pathological pain has evolvedfrom solely neuronal mechanisms to neuron-glial interactions. In particular,microglia have been paid more attention because they are the residentmacrophages and principal immune-response cells in the central nervoussystem. Many studies indicated that after injury of peripheral nerve ornociceptive stimuli, microglia in spinal dorsal horn could be activated. Theactivated microglia synthesize and release various cytokines and chemokines,including IL-1b, tumor necrosis factor-a (TNF-a), PGE2, and nitric oxide, topromote the production of pathological pain and hyperalgesia. Administrationof drugs such as receptor blocker or inhibitor of the function of microglia,could markedly inhibit the development of pathologic pain and hyperalgesia.These studies indicate that the activation of microglia in the spinal cord playsan important role in the regulation of the pathological pain and hyperalgesia.However, the complex activating mechanism of microglia in spinal cordduring nociceptive stimulation has not been well understood.N-methyl-D-aspartate (NMDA) receptor is a subtype of excitatory aminoacid receptors, and belongs to ionotropic receptors. It is widely distributed inthe central nervous system, including microglia. In physiological condition,NMDA receptor is involved in learning, memory and growth of nervoussystem. Under pathological circumstances, activated NMDA receptor couldcause a variety of intra-cellular cascades and up-regulate neuronal synapticexcitability, so play an important role in the production of pathological painand hyperalgesia.Recently, using immunohistochemical techniques and Western blotanalysis techniques, we have observed that intrathecal injection NMDAreceptor antagonist MK-801could inhibit the activation of microglia in spinal dorsal horn caused by bee venom pain model. The finding preliminarilydemonstrated the role of NMDA receptor in the activation of spinal microgliaduring the nociceptive stimulation.In order to further study the role of NMDA and its receptor in theactivation of spinal microglia during nociceptive stimulation, we usedimmunohistochemical techniques in the present study to observe the effect ofintrathecal administration of NMDA, the selective agonist NMDA receptor, onthe activation of microglia in the dorsal horn of the spinal cord. This study willprovide further experimental evidence to clarify the activation mechanism ofmicroglia in the process of pathological pain and hyperalgesia.Methods: Ninety-five male Sprague-Dawley rats weighing260-280g wererandomly divided into following groups (n=5):Sham group: Normal saline (NS) in volume of10μl was intrathecallyinjected. Observations were performed at time points corresponding with thefollowing treatment groups.NMDA I group: NMDA solution in volume of10μl (10nmol in dose) wasintrathecally injected. The examination of CD11b/c（OX-42）expression wasperformed at time points of1h、4h、8h、12h、1d、2d and3d after theNMDA injection. In order to observe the relationship between CD11b/c（OX-42）expression and hyperalgesia after intrathecal NMDA, the thermalwithdrawal latency of the hind paws and the tail-flinch latency, andmechanical withdrawal threshold were examined at the corresponding timepoints above.NMDA II group: NMDA solution in volume of10μl was intrathecallyinjected. According to doses of NMDA used, this group was further dividedinto0.1nmol,1nmol and10nmol subgroups in order to examine thedose–dependence of NMDA in regulating the CD11b/c（OX-42）expression inspinal dorsal horn. The expression of spinal CD11b/c（OX-42）was assayed at8h after the intrathecal injection because it was shown in our preliminaryexperiment that the expression of spinal CD11b after intrathecal NMDA wasmost obvious at that time point. the thermal withdrawal latency of the hind paws and the tail-flinch latency, and mechanical withdrawal threshold werealso examined at8h after the intrathecal injection.NMDA+MK-801group: MK-801solution in volume of10ul (50nmol) wasintrathecally injected15min prior to NMDA injection10μl (10nmol). Othertreatments are the same to those in NMDA II group.All rats in each group above were paraformaldehyde perfusion-fixed at thedesigned ending time point. The L5segment of spinal cord was dissected outand sectioned in series in frozen preparation for CD11b/c （OX-42）immunohistochemistry. The activation status of microglia was assayedaccording to observing the CD11b/c（OX-42）protein expression in the spinaldorsal horn.All data were expressed as mean±SD, and analyzed by using SPSSstatistical software13.0. One-Way ANOVA was used to compare betweengroups. If it had significantly difference, least significant difference was usedto test difference between groups. p<0.05was regarded significant.Results:1Nociceptive behavioral response1.1Spontaneous pain behavior response. Intrathecal injection of NMDAelicited a characteristic nociceptive behavioral response including intensebiting, twisting and scratching episodes, etc, which could be inhibited byNMDA receptor antagonist MK-801.1.2The thermal withdrawal and the tail-flinch latency. In NMDA I group,the thermal withdrawal latency decreased at time points of1h, and thedecrease lasted to12h after the intrathecal NMDA compared with sham group(p<0.05). On1d after the intrathecal NMDA, the thermal withdrawal latencyreturned to sham level. The results showed that the thermal hyperalgesiaoccurred at earlier time and lasted a relative long period after the intrathecalNMDA. In NMDA II group, the changes in the thermal withdrawal latencyafter intrathecal NMDA showed a dose–dependency and could be inhibitedwith prior administration of MK-801, an inhibitor of NMDA receptor (p<0.05).The characteristic in the tail-flinch latency were similar to those in the thermal withdrawal latency.1.3Mechanical withdrawal threshold. In NMDA I group, compared withsham group, the mechanical withdrawal threshold of hind paws to von Freyfibers decreased at time point4h, and the decrease continued to the timepoints of1d after the intrathecal NMDA(p<0.05). On2d after the intrathecalNMDA, the threshold returned to sham level. The results indicated that themechanical hyperalgesia developed at earlier time and lasted for some timeafter the intrathecal NMDA. In NMDA II group, the changes in mechanicalwithdrawal threshold after intrathecal NMDA showed a dose–dependency, andcould be inhibited with prior administration of MK-801, an inhibitor ofNMDA receptor（p<0.05）.2The CD11b/c（OX-42） expression in the spinal dorsal horn. Comparedwith sham group, microglia at the time points of4h after the intrathecalNMDA were activated, which was characterized by hypertrophy withthickened and retracted processes. The expression of CD11b/c (OX-42) wasincreased significantly（p<0.05）.The morphological changes and up-regulationin the expression of CD11b/c (OX-42) of microglia reached peak at the8hafter intrathecal NMDA, and returned to sham level at the time points of3d.The up-regulation of CD11b/c（OX-42） expression in the spinal dorsal hornexhibited a dose-dependent manner, which was represented with gradualup-regulation of the CD11b/c（OX-42） expression along with the increase inthe dose of NMDA used (from0.1nmol, to10nmol). In addition, theup-regulation of CD11b/c (OX-42) induced intrathecal NMDA wassignificantly inhibited by prior intrathecal administration of NMDA receptorantagonist MK-801(p<0.05).Conclusion:1Intrathecal administration of NMDA, the selective agonist NMDA receptor,produced nociceptive responses including spontaneous pain behavioralresponses and thermal and mechanical hyperalgesia. These nociceptiveresponses could be inhibited by NMDA receptor antagonist MK-801.2Intrathecal administration of NMDA induced activation of microglia repr- esented by the up-regulation of CD11b/c (OX-42) expression in the spinaldorsal horn in a dose-dependent manner, and the activation could be inhibitedby intrathecal NMDA receptor antagonist MK-801.3These results indicated that NMDA and its receptor played an important rolein the activation of microglia in the spinal dorsal horn during nociceptivestimulation.