Nociceptive Mechanisms Underlying Sickle Cell Pain

描述（由申请人提供）： 镰状细胞病伴有慢性和严重的偶发性疼痛，难以治疗，严重影响患者的生活质量。尽管对镰状血红蛋白的遗传学、分子生物学和生物化学有详细的了解，在镰状细胞病中观察到的深度疼痛综合征的发病机制仍不完全清楚，外周和中枢神经系统。本提案的目的是阐明镰状细胞病导致疼痛的机制，重点是初级传入神经末梢的外周机制。使用严重镰状细胞病的小鼠模型，伯克利镰状小鼠，我们证明，这些小鼠表现出显着的机械，热和冷的外周刺激的超敏反应。此外，缺氧诱导急性镰状化特异性地加剧镰状细胞小鼠中正在进行的机械超敏反应。在协议中，从这些小鼠的皮肤神经制备的梳理纤维记录表明，有髓鞘的Ad纤维和无髓鞘的C纤维伤害性感受器对机械刺激敏感。这些发现与镰状细胞病患者报告的机械超敏反应和疼痛平行。因此，这些镰状细胞小鼠代表了一种与人类疾病密切相关的长期慢性疼痛超敏反应的新模型。基于这些发现和我们在其他疼痛模型中对感觉可塑性的观察，我们假设初级传入末梢的敏化有助于镰状细胞疼痛，并且这种敏化是由瞬时受体电位离子通道的功能增加介导的。因此，本项目的具体目的是1）表征镰状细胞病小鼠初级传入纤维对机械、热和冷刺激的致敏状态。2)确定瞬时受体电位（TRP）离子通道TRPA1和TRPV1对镰状细胞病中行为超敏反应和初级传入纤维致敏的作用。3)描述急性血管闭塞如何调节镰状小鼠的机械超敏反应。我们将使用离体和体内电生理记录来表征伯克利镰状细胞小鼠初级传入纤维的致敏状态。接下来，我们将利用遗传（TRP通道无效小鼠诱导镰状细胞病）和药理学方法（选择性TRP通道拮抗剂），以确定特定的TRP家族离子通道在镰状细胞相关的初级传入敏化和疼痛行为中的作用。最后，我们将通过血管阻塞的实验模型诱导急性镰状危象，以研究血管阻塞如何调节镰状小鼠的机械超敏反应。这些相互关联的特定目标提供了一种多方面的，协调的和紧密关注的方法，将阐明初级传入神经元在镰状细胞诱导的血管和器官病理学复杂环境中疼痛综合征发展中的作用，以及提供对镰状细胞疼痛靶向TRP拮抗剂治疗潜在价值的见解。(End摘要）

DESCRIPTION (provided by applicant): Sickle cell disease is accompanied by both chronic and severe episodic pain that is difficult to treat, and profoundly erodes the quality of life of those who suffer from it. Despite a detailed understanding of the genetics, molecular biology and biochemistry of sickle hemoglobin, the pathogenesis of the profound pain syndromes observed in sickle cell disease remain incompletely understood and likely involve complex and heterogeneous steps occurring in both the peripheral and central nervous systems. The goal of this proposal is to elucidate the mechanisms by which sickle cell disease results in pain, focusing on peripheral mechanisms in primary afferent nerve terminals. Using a murine model of severe sickle cell disease, the Berkeley Sickle Mice, we demonstrate that these mice exhibit marked hypersensitivity to mechanical, heat and cold peripheral stimuli. Furthermore, induction of acute sickling with hypoxia specifically exacerbates the ongoing mechanical hypersensitivity in sickle cell mice. In agreement, teased fiber recordings from skin-nerve preparations from these mice indicate that both myelinated Ad fiber and unmyelinated C fiber nociceptors are sensitized to mechanical stimuli. These findings parallel the mechanical hypersensitivity and pain reported by patients with sickle cell disease. Thus, these sickle cell mice represent a novel model of long-lasting chronic pain hypersensitivity that is closely associated with a human disease. On the basis of these findings and our observations with sensory plasticity in other pain models, we hypothesize that sensitization of primary afferent terminals contributes to sickle cell pain and that this sensitization is mediated by increased function of Transient Receptor Potential ion channels. Therefore, the Specific Aims for this project are to 1) Characterize the sensitization state of primary afferent fibers to mechanical, heat and cold stimuli in mice with sickle cell disease. 2) Determine the contribution of the Transient Receptor Potential (TRP) Ion Channels TRPA1 and TRPV1 to both the behavioral hypersensitivity and the sensitization of primary afferent fibers in sickle cell disease. 3) Characterize how acute vaso-occlusion modulates mechanical hypersensitivity in sickle mice. We will use both ex vivo and in vivo electrophysiological recordings to characterize the sensitization state of primary afferent fibers in Berkeley sickle cell mice. Next, we will utilize both genetic (TRP channel null mice induced with sickle cell disease) and pharmacologic approaches (selective TRP channel antagonists) to determine the role of specific TRP-family ion channels in sickle cell-associated primary afferent sensitization and pain behavior. Finally, we will induce acute sickling crises by an experimental model of vaso-occlusion to study how vaso-occlusion modulates mechanical hypersensitivity in sickle mice. These interrelated Specific Aims provide a multifaceted, coordinated and tightly focused approach that will clarify the role of primary afferent neurons in the development of pain syndromes within the complex setting of sickle cell-induced vascular and organ pathologies, as well as provide insight into the potential value of targeted TRP antagonist therapies for sickle cell pain. (End of Abstract)