Glial-cytokine-neuronal interactions in neuropathic pain

描述（由申请人提供）：神经病理性疼痛中功能失调的胶质细胞如何导致脊髓背角异常疼痛信号传导仍然是一个谜。谷氨酸激活谷氨酸受体是急性疼痛传递和激活导致病理性疼痛的启动和维持的信号转导途径的关键步骤。谷氨酸受体的激活受三个基本因素支配：突触释放的谷氨酸的量、谷氨酸被谷氨酸转运体（GT）去除的速率和突触后谷氨酸受体的性质。针对疼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末梢的谷氨酸释放或突触后水平以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GT以纠正谷氨酸受体的异常激活的研究要少得多。神经胶质细胞在调节神经元能突触的信号传递中的一个关键作用是通过神经胶质GT去除突触谷氨酸。对前脑的研究表明，胶质细胞GT占所有CNS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，神经胶质GT在调节脊髓背角突触传递中的作用及其对异常疼痛信号的贡献尚未确定。基于我们的初步研究结果，我们假设神经损伤引起的胶质细胞激活导致胶质细胞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超敏反应。将以三个具体目标来检验这一假设。具体目标1将确定神经胶质GT表达与神经损伤诱导的行为超敏反应、神经胶质活化和促炎细胞因子释放之间的关系。具体目标2将确定神经胶质GT摄取谷氨酸不足对脊髓背角AMPA和NMDA受体异常激活和神经病大鼠行为超敏反应的贡献。具体目标3将确定神经胶质GT的恢复谷氨酸摄取对脊髓背角中离子型谷氨酸受体的“正常化”活化以及对神经病大鼠中在神经胶质抑制剂或促炎细胞因子拮抗剂治疗后的“正常化”伤害感受行为的贡献。实验将在对照（正常未手术和假手术）大鼠和通过部分坐骨神经结扎诱导的神经病大鼠中进行，已知其模拟患者中部分神经损伤诱导的神经病性疼痛。多学科的技术，包括可视化的全细胞电压钳记录从脊髓切片，药理学，免疫组织化学和行为测试将被应用于测试我们的假设。这项研究将为神经病理性疼痛中神经胶质细胞-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，并可能导致使用GT作为减少慢性疼痛管理中谷氨酸受体持续和异常激活的新靶点。公共卫生相关性：在神经病理性疼痛中，神经胶质细胞功能失调如何导致脊髓背角疼痛信号异常仍然是一个谜。谷氨酸激活谷氨酸受体是急性疼痛传递和激活导致病理性疼痛的启动和维持的信号转导途径的关键步骤。我们推测神经损伤引起的胶质细胞激活导致胶质细胞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超敏反应。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