Glutamate-evoked calcium signaling in spinal cord after nerve injury

神经损伤后脊髓中谷氨酸诱发的钙信号传导

• 批准号: 8445756
• 负责人: Suzanne Doolen
• 金额: $ 7.43万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8445756
• 关键词: 6-Cyano-7-nitroquinoxaline-2,3-dione; AMPA Receptors; Action Potentials; Adult; Adverse effects; Analgesics; Animals; Astrocytes; Attenuated; Behavior; Behavioral; Calcium; Calcium Signaling; Cells; Chemosensitization; Chronic; Data; Development; Dose; Frequencies; Fura-2; Glutamate Receptor; Glutamates; Goals; Healthcare; Hyperalgesia; Hypersensitivity; Image; Injury; Knowledge; Label; Lead; Ligation; Measures; Mediating; Mission; Modeling; Mus; Nerve; Neurons; Operative Surgical Procedures; Pain; Pain Measurement; Pain management; Peripheral Nerves; Peripheral nerve injury; Physiological; Population; Posterior Horn Cells; Receptor Signaling; Relative (related person); Research; Signal Transduction; Skin; Slice; Specificity; Spinal Cord; Stimulus; Substantia Gelatinosa; Tactile; Techniques; Testing; Time; Variant; allodynia; base; cell type; central pain; central sensitization; chronic pain; clinically relevant; dorsal horn; inhibitor/antagonist; injured; innovation; kainate; nerve injury; neurotransmission; painful neuropathy; response; sciatic nerve; spinal nerve posterior root; therapeutic target

DESCRIPTION (provided by applicant): A fundamental gap exists in understanding the cellular mechanisms that initiate and maintain neuropathic pain. This gap represents an important problem because current analgesic drugs rarely provide sufficient efficacy without serious side effects. The long-term goal is to understand the mechanisms that lead to injury-induced central sensitization and establish clinically relevant therapeutic targets for chronic pai. The objective in this application is to evaluate the contribution glutamate receptor subtypes to dorsal root stimulation (DRS)-evoked Ca2+ transients in the dorsal horn, and correlate enhanced Ca2+ responses with the magnitude of pain-like behavior. Based on preliminary data suggesting that glutamate-evoked Ca2+ responses in mouse spinal cord slices are potentiated after nerve injury, the central hypothesis is that nerve injury increases AMPA receptor signaling in the dorsal horn, leading to increases in [Ca2+]i that results in central sensitization and neuropathic pain. The rationale for the proposed project is that [Ca2+]i in dorsal horn neurons is essential for central sensitization and pain hypersensitivity. The central hypothesis will be teste by pursuing three specific aims: AIM 1 tests the hypothesis that glutamate-mediated activation of neuronal ionotropic AMPA receptors drives Ca2+ signaling. Electrophysiological recordings and real-time fluorescent labeling of astrocytes will be used to evaluate the cell types that respond to dorsal root stimulation (DRS) with a rise in [Ca2+]i. Next, the relative contribution of glutamate receptor subtypes will be determined by quantifying DRS-evoked [Ca2+]i transients in the presence of selective antagonists. AIM 2 tests the hypothesis that peripheral nerve injury potentiates DRS-evoked Ca2+ responses, and this will correlate with the magnitude of hyperalgesia. To allow for a correlation analysis between behavior and [Ca2+]i, a variant model of nerve injury has been developed that gradually elicits robust allodynia in 1 week and then resolves in 4 weeks. Behavioral hyperalgesia will be evaluated and compared to DRS-evoked [Ca2+]i in spinal cord slices from sham, traditional and variant nerve injured animals sacrificed at 7, 14 and 21 d after injury. AIM 3 tests the hypothesis that PKM¿ mediates SNI-induced increases in hyperalgesia, Ca2+ signaling and AP frequency in dorsal horn. We will administer multiple PKM¿ inhibitors to sham and SNI mice and measure pain- like behavior, Ca2+ transients and/or AP frequency. Based on our preliminary results, we predict that PKM¿ blockade will reverse injury-induced hyperalgesia, increases in [Ca2+]i and AP frequency. This project employs innovative wide-field calcium imaging simultaneously from numerous cells in spinal cord slices from adult mice. The proposed research is significant because it reveals the Ca2+ channels that regulate DRS-evoked Ca2+ transients, and is a critical first step in understanding nerve injury-induced potentiation of neuronal Ca2+ signaling. Ultimately, this knowledge will establish clinically relevant therapeutic targets for alleviating chronic pain. PUBLIC HEALTH RELEVANCE: Chronic pain management is a major scientific and health care challenge, as current analgesic drugs rarely provide sufficient efficacy in the absence of serious side effects. This project is relevant to NINDS's mission because it will 1) determine mechanisms that lead to injury-induced central sensitization and 2) establish clinically relevant therapeutic targets for alleviating chronic pain. This Research Plan employs highly innovative wide-field calcium imaging from numerous cells in a single spinal cord slice from adult mice.

描述（由适用提供）：了解启动和维持神经性疼痛的细胞机制的基本差距。该差距代表了一个重要的问题，因为当前的镇痛药很少提供足够的有效性而没有严重的副作用。长期目标是了解导致损伤引起的中枢灵敏度的机制，并为慢性PAI建立临床相关的治疗靶标。本应用中的目的是评估谷氨酸受体亚型对背侧角中诱发的CA2+瞬变的谷氨酸受体亚型，并将增强的Ca2+反应与疼痛样行为相关。基于初步数据表明，在神经损伤后可能提出了小鼠脊髓切片中的谷氨酸诱发的Ca2+反应，中央假设是神经损伤会增加背侧角中的AMPA受体信号传导，从而导致[CA2+] I的增加，导致中枢敏感性和神经病性疼痛。拟议项目的理由是，背角神经元中的[Ca2+] I对于中枢灵敏度和疼痛超敏反应至关重要。中央假设将通过追求三个特定目的来检验：目标1检验了谷氨酸介导的神经元离子型AMPA受体激活的假设驱动Ca2+信号传导。星形胶质细胞的电生理记录和实时荧光标记将用于评估对背根刺激（DR）反应的细胞类型，[Ca2+] i。接下来，相对贡献 谷氨酸受体亚型将通过在存在选择性拮抗剂的情况下量化DRS诱发的[Ca2+] I瞬变来确定。 AIM 2检验了以下假设：外周神经损伤潜力DRS引起的Ca2+反应，这将与Hypergensia的大小相关。为了允许行为和[Ca2+] I之间的相关性分析，已经开发出一种神经损伤的变异模型，该模型逐渐在1周内引起强大的异常性痛，然后在4周内解决。将评估行为痛觉过敏并将其与DRS诱发的[Ca2+] I的[Ca2+] I中的脊髓切片中的脊髓切片中的Sham，传统和变体神经受伤的动物，在受伤后7、14和21 D处置。 AIM 3检验了PKM介导SNI诱导的Hypergeria，Ca2+信号传导和AP频率的假设。我们将对假小鼠和SNI小鼠进行多个PKM抑制剂，并测量类似的行为，Ca2+瞬态和/或AP频率。基于我们的初步结果，我们预测PKM课将逆转损伤引起的痛觉过敏，[Ca2+] I和AP频率增加。该项目从成年小鼠的脊髓切片中的众多细胞中同时采用创新的广阔钙成像。拟议的研究很重要，因为它揭示了调节DRS引起的Ca2+瞬变的Ca2+通道，并且是理解神经损伤引起的神经元CA2+信号传导潜力的关键第一步。最终，这些知识将建立临床相关的治疗靶标，以减轻慢性疼痛。 公共卫生相关性：慢性疼痛管理是一项主要的科学和医疗保健挑战，因为目前的镇痛药在没有严重副作用的情况下很少提供足够的效率。该项目与Ninds的任务相关，因为它将1）确定导致损伤引起的中枢灵敏度的机制； 2）建立临床相关的治疗靶标，以减轻慢性疼痛。该研究计划员工从成年小鼠的单个脊髓切片中的众多细胞中高度创新的广阔钙成像。