Glial-cytokine-neuronal interactions in neuropathic pain

描述(申请人提供)：在神经病理性疼痛中，功能障碍的神经胶质细胞如何导致脊髓背角异常的疼痛信号仍然是一个谜。谷氨酸激活谷氨酸受体是急性疼痛传递的关键步骤，也是启动和维持病理性疼痛的信号转导通路的关键步骤。谷氨酸受体的激活受三个基本因素的控制：突触释放谷氨酸的量、谷氨酸转运体(GTS)清除谷氨酸的速率和突触后谷氨酸受体的特性。针对痛信号传递的现代疼痛治疗主要集中在突触前水平以阻断突触前终末的谷氨酸释放，或在突触后水平阻断突触后神经元中的谷氨酸受体和谷氨酸激活的信号转导通路。靶向GTS以纠正谷氨酸受体的异常激活的研究要少得多。胶质细胞在调节谷氨酸能突触的信号传递中的一个关键作用是胶质细胞GT清除突触的谷氨酸。对前脑的研究表明，胶质GT占中枢神经系统突触谷氨酸摄取的94%以上，是调节突触信号传递和可塑性的关键机制。然而，胶质GT在调节脊髓背角突触传递中的作用以及它们在异常疼痛信号中的作用尚不清楚。根据我们的初步发现，我们假设神经损伤引起的神经胶质细胞的激活导致神经胶质GT功能障碍，从而导致脊髓背角离子型谷氨酸受体的异常激活和行为过敏。这一假设将通过三个具体目标进行检验。具体目的1将确定神经损伤诱导的行为超敏反应、神经胶质细胞激活和促炎细胞因子释放与神经胶质GT表达的关系。特异性目标2将确定神经胶质细胞谷氨酸摄取不足在神经病大鼠脊髓背角AMPA和NMDA受体异常激活和行为过敏中的作用。具体目标3将确定胶质GT恢复谷氨酸摄取对脊髓背角离子型谷氨酸受体的“正常化”激活的贡献，以及在神经病变大鼠接受神经胶质抑制药或促炎细胞因子拮抗剂治疗后对“正常化”的伤害性行为的贡献。实验将在对照组(正常大鼠和假手术大鼠)和部分坐骨神经结扎诱导的神经病理性大鼠身上进行，这是已知的模拟患者部分神经损伤引起的神经病理性疼痛。多学科技术，包括可视化的脊髓切片全细胞电压钳记录，药理学，免疫组织化学和行为测试将被应用来检验我们的假设。这项拟议的研究将为神经病理性疼痛中神经胶质-细胞因子-神经元相互作用的突触和分子机制提供新的见解，并可能导致将GTS用作减少谷氨酸受体在慢性疼痛治疗中持续和异常激活的新靶点。公共卫生相关性：神经病理性疼痛中，神经胶质细胞功能障碍如何导致脊髓背角异常疼痛信号仍是一个谜。谷氨酸激活谷氨酸受体是急性疼痛传递的关键步骤，也是启动和维持病理性疼痛的信号转导通路的关键步骤。我们推测，神经损伤引起的神经胶质细胞激活导致神经胶质GT功能障碍，从而导致脊髓背角离子型谷氨酸受体的异常激活和行为过敏。1

Description (provided by applicant): How dysfunctional glial cells lead to abnormal pain signaling in the spinal dorsal horn in neuropathic pain remains a mystery. Activation of glutamate receptors by glutamate is a key step for acute pain transmission and activation of signal transduction pathways leading to initiation and maintenance of pathological pain. Activation of glutamate receptors is governed by three essential factors: the amount of synaptically released glutamate, the rate at which glutamate is removed by glutamate transporters (GTs) and the properties of postsynaptic glutamate receptors. Modern pain treatments directed at the transmission of pain singals have mainly focused either on the presynaptic levels to block glutamate release from presynaptic terminals or on the postsynaptic levels to block glutamate receptors and glutamate activated signal transduction pathways in postsynaptic neurons. Targeting at GTs to correct abnormal activation of glutamate receptors has been much less investigated. One key role for glial cells in regulating signal transmission at the glutamatergic synapse is the removal of synaptic glutamate by glial GTs. Studies on the forebrain have demonstrated that glial GT accounts for more than 94% of all CNS synaptic glutamate uptake and is a key machinery regulating synaptic signal transmission and plasticity. However, the role of glial GTs in regulating synaptic transmission in the spinal dorsal horn and their contribution to abnormal pain signaling have not been established. Based on our preliminary findings, we hypothesize that activation of glial cells induced by nerve injury results in dysfunction of glial GTs, which leads to abnormal activation of ionotropic glutamate receptors in the spinal dorsal horn and behavioral hypersensitivity. This hypothesis will be tested with three specific aims. Specific Aim 1 will determine the relationships between glial GT expression and development of behavioral hypersensitivity, glial activation and release of pro-inflammatory cytokines induced by nerve injury. Specific Aim 2 will determine the contribution of deficient glutamate uptake by glial GTs to abnormal activation of AMPA and NMDA receptors in the spinal dorsal horn and behavioral hypersensitivity in neuropathic rats. Specific Aim 3 will determine the contribution of recovered glutamate uptake by glial GTs to the "normalized" activation of ionotropic glutamate receptors in the spinal dorsal horn and to the "normalized" nociceptive behaviors in neuropathic rats after treatments of glial inhibitors or antagonists for pro-inflammatory cytokines. Experiments will be conducted in control (normal naove and sham operated) rats and neuropathic rats induced by partial sciatic nerve ligation, which is known to mimic neuropathic pain induced by partial nerve injury in patients. Multi-disciplinary- techniques, including visualized whole cell voltage clamp recordings from spinal slices, pharmacology, immunohistochemistry and behavioral tests will be applied to test our hypothesis. The proposed study will provide new insights into the synaptic and molecular mechanisms underlying glial-cytokine-neuronal interactions in neuropathic pain and may potentially lead to the use of GTs as a new target for reducing persistent and abnormal activation of glutamate receptors in the management of chronic pain. PUBLIC HEALTH RELEVANCE: How dysfunctional glial cells lead to abnormal pain signaling in the spinal dorsal horn in neuropathic pain remains a mystery. Activation of glutamate receptors by glutamate is a key step for acute pain transmission and activation of signal transduction pathways leading to initiation and maintenance of pathological pain. We hypothesize that activation of glial cells induced by nerve injury results in dysfunction of glial GTs, which leads to abnormal activation of ionotropic glutamate receptors in the spinal dorsal horn and behavioral hypersensitivity. 1