Astroglial mechanisms for inflammatory pain

描述(申请人提供)：组织损伤引起的炎性疼痛的特征是由不同的机制介导的机械性痛觉过敏和热痛敏。特别是，持续性疼痛的特征是机械性异位痛觉。我们对机械性异位痛觉如何维持的不完全理解阻碍了慢性疼痛的有效治疗。目前对疼痛的研究主要集中在神经细胞和神经网络。关于神经胶质细胞和神经胶质网络的重要性，尤其是星形胶质细胞网络在慢性疼痛条件下的重要性，人们知之甚少。这项应用的总体目标是研究脊髓星形胶质细胞和星形胶质细胞网络在调节持续性炎性疼痛期间机械性异位痛中的作用。我们的策略是靶向在组织损伤后在脊髓星形胶质细胞中特异性表达和诱导的信号分子。C-jun氨基末端激酶(INK)是MAP激酶家族的一员。我们的初步研究表明：(A)CFA炎症仅诱导脊髓星形胶质细胞中JNK的激活，(B)多肽墨水抑制剂阻断机械痛敏的维持阶段而不影响热痛敏，以及(C)炎症上调星形胶质细胞缝隙连接蛋白-43(Cx43)，缝隙连接阻滞剂抑制机械性痛觉过敏但不影响热痛敏。我们的中心假设是JNK是脊髓星形胶质细胞中的关键信号分子，炎症持续激活JNK在通过星形胶质细胞网络维持机械性痛觉异常中起着重要作用。这一假说将使用行为测试、免疫组织化学、原位杂交、Western blotting和激酶分析等方法进行验证。信号分子的细胞定位将通过免疫荧光双重染色以及荧光原位杂交和免疫荧光双重染色进行检测。我们将使用CFA炎症模型来实现以下三个特定目的：(1)建立炎症诱导脊髓星形胶质细胞持续的JNK激活，这种激活对于维持机械性异常痛是必不可少的；(2)确定Cx43介导的脊髓星形胶质细胞网络在持续性机械性异常痛中的重要性；(3)探讨脊髓JNK和星形胶质细胞网络调节机械性异常痛的分子和细胞机制。这些拟议的研究将(A)确定维持机械性痛觉异常的新的分子和细胞机制，(B)证明脊髓星形胶质细胞和神经胶质网络在慢性疼痛中的重要作用，以及(C)揭示脊髓JNK通路作为治疗慢性疼痛的新靶点。

DESCRIPTION (provided by applicant): Tissue injury-induced inflammatory pain is characterized by mechanical allodynia and heat hyperalgesia that are mediated by distinct mechanisms. In particular, persistent pain is characterized by mechanical allodynia. Our incomplete understanding of how mechanical allodynia is maintained hinders the effective treatment of chronic pain. Current pain research focuses primarily on neuronal cells and neural networks. Less is known about the importance of glial cells and the glial network, in particular the astroglial network in chronic pain conditions. The overall goal of this application is to investigate the role of spinal astrocytes and astroglial network in regulating mechanical allodynia during persistent inflammatory pain. Our strategy is to target signaling molecules that are specifically expressed and induced in spinal astrocytes after tissue injury. c-Jun N-terminal kinase (INK) is a member of MAP kinase family. Our pilot studies have shown that (a) CFA inflammation induces JNK activation exclusively in spinal astrocytes, (b) a peptide INK inhibitor blocks maintenance phase of mechanical allodynia without affecting heat hyperalgesia, and (c) the astroglial gap junction protein connexin-43 (Cx43) is upregulated by inflammation and a gap junction blocker suppresses mechanical allodynia but not heat hyperalgesia. Our central hypothesis is that JNK is a critical signaling molecule in spinal astrocytes, and that persistent JNK activation by inflammation makes important contribution to the maintenance of mechanical allodynia via an astroglial network. This hypothesis will be tested using the methods of behavioral testing, immunohistochemistry, in situ hybridization, Western blotting, and a kinase assay. Cellular localization of signaling molecules will be examined by double staining of immunofluorescence and double staining of fluorescence in situ hybridization and immunofluorescence. We will use the CFA inflammation model to accomplish the following three specific aims: (1) to establish that inflammation induces persistent JNK activation in spinal astrocytes and that this activation is essential for the maintenance of mechanical allodynia, (2) to define the importance of Cx43-mediated spinal astroglial network for persistent mechanical allodynia, and (3) to explore molecular and cellular mechanisms by which spinal JNK and the astroglial network regulate mechanical allodynia. These proposed studies will (a) identify novel molecular and cellular mechanisms underlying the maintenance of mechanical allodynia, (b) document important roles of spinal astrocytes and the glial network for chronic pain, and (c) reveal spinal JNK pathway as a new target for the treatment of chronic pain.