NMDA Receptor Antagonist MK-801 Inhibits The Activation Of Astrocyte And Microglia In The Spinal Dorsal Horn During Bee Venom-induced Inflammatory Pain And Hyperalgesia

Objective: Spinal cord is the primary center to transmit and integrate the information of pain. At the level of the spinal cord, pathological pain is classically viewed as being developed and maintained solely by neurons. Glia (astrocyte and microglia) only play a nutritional and supporting role to neurons, because it does not have the function to transmit the synaptic information between cells. However, more and more evidence indicate that the activation of glia in the spinal cord is closely related to the production and maintenance of pathologic pain and hyperalgesia. A variety of nociceptive stimuli can activate the astrocytes and microglia in the spinal level. The activated glia can release many types of cytokine such as tumor necrosis factors, interleukins, etc., to accelerate the development and maintenance of pathologic pain. Administration of drugs such as fluorocitrate, CNI-1493, minocycline etc, which can inhibit the function or metabolism of glia, can inhibit the development and maintenance of pathologic pain. These findings indicate that the activation of glia in the spinal cord plays an important role in the generation and maintenance of the pathological pain and hyperalgesia. However, the activating mechanism of glia in spinal cord during pathologic pain has not been well understood.Excitatory amino acids (EAAs, including glutamic acid and aspartic acid) are important excitatory neurotransmitter to transmit nociceptive information in the spinal cord. Available information indicates that N-methyl-D-aspartate (NMDA) receptor is a subtype of EAAs receptors and belongs to ionotropic receptors. It is widely distributed in the central nervous system, and is involved in many complex physiological responses and pathological processes. A large number of studies have shown that NMDA and its receptor play an important role in the transmission of nociceptive information in the spinal cord. Well-developed NMDA receptor system is essential to the transmission of peripheral nociceptive stimuli to the center, central sensitization and the development pathologic pain. It has been demonstrated that NMDA receptor takes part in the activation of glia in the spinal dorsal horn in neuropathic pain models. However, whether NMDA receptor is involved in the activation of glia in peripheral inflammatory pain and hyperalgesia has not been reported.Bee venom test is a new pain model with multi-phenotypes of pain. Subcutaneous injection of bee venom into plantar surface of the hind paw of rats can induce inflammatory manifestations such as edema and redness in the injected region. Following the inflammatory manifestations, monophasic and long lasted for 1~2 h spontaneous flinches of the injected paw, and primary heat and mechanical hyperalgesia in the injected area can be induced after the bee venom injection. Therefore, the bee venom test shows characteristics more similar to clinical pathological pain and is believed to be more suitable for study in inflammatory pathological pain. Recently, using immunohisto- chemical techniques, we have preliminarily observed the effect of NMDA receptor on the activation of astrocytes in the dorsal horn of the spinal cord.In order to further study the role of NMDA receptors in activation of spinal glia during nociceptive stimulation, we use western blot analysis techniques in the present study to observe the expressions of glial fibrilary acidic protein (GFAP), a labeling protein of astrocytes, and CD11b/c(OX-42), a labeling protein of microglia, in the rat spinal dorsal horn during bee venom-induced inflammatory pain and hyperalgesia, and the effect of dizocipine maleate (MK-801), a non-selective NMDA receptor antagonist, on the expressions. This study will provide further experimental evidence to clarify the role of spinal glia in the transmission of nociceptive information, and provide new ideas and clues for the study in prevention and treatment of pathological pain.1 Spinal astrocytes and microglia are activated during bee venom-induced peripheral inflammatory pain and hyperalgesia Methods: Twenty five male Sprague-Dawley rats weighing 200-220 g were randomly divided into sham group (n=5) and bee venom group (n=20). The bee venom group was further divided into 1 h, 1 d, 2 d and 4 d subgroups (n=5 in each group). The rats in sham group were subcutaneously injected with normal saline (NS) of 100μl into the plantar surface of the right hind paw, while the rats in bee venom groups were injected with bee venom solution of 100μl (4g·L-1) in the same route to that in the sham group to produce peripheral inflammatory pain and hyperalgesia. Spontaneous pain behavioral responses within 60 min after injection of bee venom were rated with the weighted pain score of Dubuisson and Dennis in each group. After measurement of the thermal withdrawal latency and mechanical withdrawal threshold at the corresponding time point after subcutaneous injection of bee venom solution in bee venom groups, the rats were sacrificed and the lumbar 5(L5) segment of the spinal cord was dissected out. The sham group was sampled on 1 h after the plantar injection. The expressions of GFAP in astrocytes and CD11b/c (OX-42) in microglia in the dorsal horn of L5 spinal cord were measured using Western blot analysis to reflect the activation of astrocytes and microglia, respectively.Data were expressed as mean±SD ( x±s) and statistically treated with statistical software of Statistical Program for Social Sciences 13.0. One-Way ANOVA was used to compare between groups. If it had significantly difference, least significant difference was used to test difference between groups. P<0.05 was considered to be significant.Result: Subcutaneous injection of bee venom into the plantar surface of the right hindpaw produced a monophasic nociception characterized by continuously flinching reflex and lifting/licking the injected hindpaw, which could last for 60 min after injection. Compared with the sham group, the weighted pain score was significantly increased. The subcutaneous injection of bee venom also induced significant decreases of the thermal withdrawal latency and mechanical withdrawal threshold in the injection site (p<0.05), which indicated that the animals appeared thermal and mechanical hyperalgesia. The bee venom-induced hyperalgesia was the most obvious on 1 d, and could last for 4 days after injection, although it gradually eased along with the time went. Western blot analysis showed that, at the time point of 1 h after the injection of bee venom, the expression levels of GFAP and CD11b/c (OX-42) in the L4~L5 spinal segment had no significant changes compared with the sham group (p>0.05). However, at the time point of 1 d after the bee venom injection, the expression levels of GFAP and CD11b/c (OX-42) significantly up-regulated compared with sham group (P<0.05). This up-regulation could last to the forth day observed after the bee venom injection, and there was no significant difference in the expression level among the time points of 1 d, 2 d and 4 d (p>0.05). These results indicated that the bee venom-test induced peripheral inflammatory pain and hyperalgesia, and activated the spinal astrocytes and microglia.2 NMDA receptor antagonist MK-801 inhibits the activation of spinal astrocytes and microglia during bee venom-induced inflammatory pain and hyperalgesiaMethods: Twenty five male Sprague-Dawley rats weighing 200-220 g were randomly divided into the following five groups (n=5):①Sham group: The rats were injected subcutaneously with normal saline (NS) of 50μl into the plantar surface of the right hindpaw.②Bee venom group: The rats were injected subcutaneously with bee venom solution of 50μl (4g·L-1) into the plantar surface of the right hindpaw to induce inflammatory pain and hyperalgesia.③NS+bee venom group: The rats were intrathecally injected firstly with NS in 10μl, and then after 15 minutes they were injected subcutaneously with bee venom solution of 50μl (4g·L-1) in the same route to that in bee venom group.④MK-801+bee venom group: The rats were intrathecally injected firstly with MK-801 solution in 10μl (10 nmol), and the other procedures were the same to those in NS+bee venom group.⑤MK-801+sham group: The rats were intrathecally injected firstly with MK-801 solution in 10μl (10 nmol), and the other procedures were the same to those in sham group.The spontaneous pain behavioral responses within 60 min after the bee venom injection were rated with the weighted pain score of Dubuisson and Dennis in each group. The thermal withdrawal latency and mechanical withdrawal threshold on the first day after the bee venom injection were observed. After the observation, the rats were sacrificed and the dorsal horn of L5 spinal cord was dissected out. The expressions of GFAP in astrocytes and CD11b/c (OX-42) in microglia in the dorsal horn of L5 spinal cord were assayed using westen blot analysis.The first part of this study showed that the rats after bee venom injection exhibited obvious thermal and mechanical hyperalgesia, and the up-regulation in the expression of GFAP and CD11b/c (OX-42) on the first day. This is the reason to select the time point of 1 d in this part of the study.Data were expressed as mean±SD ( x±s) and statistically treated with statistical software of Statistical Program for Social Sciences 13.0. One-Way ANOVA was used to compare between groups. If it had significantly difference, least significant differencewas used to test difference between groups. P<0.05 was considered to be significant.Result:1 Spontaneous pain behavioral responsesThe injection of bee venom produced a monophasic nociceptive behavior responses characterized with continuously flinching and lifting/licking the injected hindpaw. There was no significant difference in the weight pain score between the NS+bee venom and the bee venom groups (P>0.05). However, the weight pain score in the MK-801+bee venom group was decreased significantly compared with bee venom group and NS+bee venom group (P<0.05). The weight pain score of the MK-801+sham group and the sham group was zero.2 Thermal withdrawl latency and mechanical withdrawal thresholdThe thermal withdrawal latency and mechanical withdrawal threshold in the injection site after the bee venom injection in the bee venom group were significantly decreased compared with the sham group (p<0.05), indicating that the animals appeared the thermal and mechanical hyperalgesia. Intrathecal injection of MK-801 in the MK-801+bee venom group significantly prolonged the thermal withdrawal latency and mechanical withdrawal threshold compared with the bee venom group (P<0.05), while intrathecal injection of NS in the NS+bee venom group had no effect on the latency and threshold compared with the bee venom group (P>0.05). Intrathecal injection of MK-801 in MK-801+sham group had no significant effect on the latency and threshold compared with sham group (P> 0.05).3 GFAP and CD11b/c (OX-42) expressionsWestern blot analysis showed that after the injection of bee venom, the expressions of GFAP and CD11b/c (OX-42) were significantly up-regulated compared with the sham group (P<0.05). After intrathecal injection of MK-801 in the MK-801+bee venom group, the expression of GFAP and CD11b/c(OX-42) were significantly down-regulated compared with bee venom group (P<0.05), while intrathecal injection of NS in the NS+bee venom had no effect on the up-regulation of GFAP and CD11b/c (OX-42) induced by bee venom injection. Intrathecal injection of MK-801 in MK-801+sham group had no significant effect on the GFAP and CD11b/c (OX-42) expressions compared with sham group (P>0.05).Conclusion:Subcutaneous injection of bee venom induced characteristic peripheral inflammatory pain and hyperalgesia in the injected site, and activation of spinal astrocytes and microglia. Intrathecal administration of MK-801, a non-selective NMDA receptor antagonist, significantly inhibited the activation of spinal astrocytes and microglia. It indicates that NMDA and NMDA receptor take part in mediation of the activation of spinal astrocyte and microglia during bee venom-induced peripheral inflammatory pain and hyperalgesia.