Glial-cytokine-neuronal interactions in neuropathic pain

描述（由申请人提供）：在神经性疼痛中，功能失调的神经胶质细胞如何导致脊髓背角的异常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和信号转导通路激活的关键步骤，导致病理性疼痛的开始和维持。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）去除谷氨酸的速率和突触后谷氨酸受体的特性。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阻断谷氨酸从突触前末端释放或突触后水平阻断谷氨酸受体和突触后神经元中谷氨酸激活的信号转导通路。以谷氨酸受体为靶点纠正谷氨酸受体异常活化的研究还很少。神经胶质细胞调节谷氨酸能突触信号传递的一个关键作用是神经胶质转质细胞清除突触谷氨酸。对前脑的研究表明，神经胶质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，神经胶质GTs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，我们假设神经损伤诱导的神经胶质细胞激活导致神经胶质gt功能障碍，从而导致脊髓背角嗜离子性谷氨酸受体异常激活和行为超敏反应。这一假设将通过三个具体目标进行检验。特异性目的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的发生、胶质细胞的激活和促炎细胞因子的释放之间的关系。特异性目的2将确定神经胶质gt对谷氨酸摄取不足对神经症大鼠脊髓背角AMPA和NMDA受体异常激活和行为过敏的贡献。特异性目的3将确定神经胶质gt恢复谷氨酸摄取对脊髓背角嗜离子性谷氨酸受体“正常化”激活的贡献，以及对神经病变大鼠在接受促炎细胞因子胶质抑制剂或拮抗剂治疗后的“正常化”伤害性行为的贡献。实验将在对照（正常和假手术）大鼠和部分坐骨神经结扎诱导的神经病鼠中进行，已知部分坐骨神经结扎可以模拟患者部分神经损伤引起的神经性疼痛。多学科技术，包括脊髓切片的可视化全细胞电压钳记录，药理学，免疫组织化学和行为测试将被应用于验证我们的假设。该研究将为神经性疼痛中神经胶质-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，并可能导致使用谷氨酸受体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：在神经性疼痛中，功能失调的神经胶质细胞如何导致脊髓背角的异常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和信号转导通路激活的关键步骤，导致病理性疼痛的开始和维持。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巨噬细胞功能障碍，从而导致脊髓背角嗜离子性谷氨酸受体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