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

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：在理解引发和维持神经性疼痛的细胞机制方面存在根本性的差距，这是一个重要的问题，因为目前的镇痛药物很少能提供足够的功效而不产生严重的副作用。导致损伤诱导的中枢敏化的机制并建立慢性pai的临床相关治疗靶点本申请的目的是评估谷氨酸受体亚型对背根刺激的贡献。 (DRS) 诱发背角 Ca2+ 瞬变，并将增强的 Ca2+ 反应与疼痛样行为的程度相关联。初步数据表明，神经损伤后，小鼠脊髓切片中谷氨酸诱发的 Ca2+ 反应增强。假设是神经损伤增加了背角的 AMPA 受体信号传导，导致 [Ca2+]i 增加，从而导致中枢敏化和神经性疼痛。该项目的基本原理。背角神经元中的 [Ca2+]i 对于中枢敏化和疼痛超敏反应至关重要。中心假设将通过追求三个特定目标进行测试：AIM 1 测试谷氨酸介导的神经元离子型 AMPA 受体激活驱动 Ca2+ 信号传导的假设。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（DRS）做出反应的细胞类型，接下来，[Ca2+]i 的相对贡献。谷氨酸受体亚型将通过在选择性拮抗剂存在下量化 DRS 诱发的 [Ca2+]i 瞬变来确定，AIM 2 测试周围神经损伤增强 DRS 诱发的 Ca2+ 反应的假设，这将与痛觉过敏的程度相关。为了进行行为与 [Ca2+]i 之间的相关性分析，开发了一种神经损伤的变体模型，该模型在 1 周内逐渐引起强烈的异常性疼痛，然后在4 周后，将评估行为痛觉过敏，并与受伤后 7、14 和 21 天处死的假手术、传统和变异神经损伤动物的脊髓切片中的 DRS 诱发的 [Ca2+]i 进行比较，AIM 3 测试了 PKM¿介导 SNI 诱导的背角痛觉过敏、Ca2+ 信号传导和 AP 频率增加。根据我们的初步结果，我们预测 PKM¿阻断将逆转损伤引起的痛觉过敏、[Ca2+]i 和 AP 频率的增加。该项目同时采用创新的广域钙成像技术，对成年小鼠脊髓切片中的大量细胞进行成像。这项研究意义重大，因为它揭示了 Ca2+ 通道。调节 DRS 诱发的 Ca2+ 瞬变，是了解神经损伤诱导的神经元 Ca2+ 信号传导增强的关键第一步，最终，这一知识将为缓解慢性疼痛建立临床相关的治疗目标。公共健康相关性：慢性疼痛管理是一项重大的科学和卫生保健挑战，因为目前的镇痛药物很少能在没有严重副作用的情况下提供足够的疗效。该项目与 NINDS 的使命相关，因为它将 1) 确定导致损伤的机制。 -诱导中枢敏化；2）建立临床相关的治疗目标，以减轻慢性疼痛。该研究计划采用高度创新的广域钙成像技术，对成人的单个脊髓切片中的大量细胞进行成像。老鼠。