Investigating the Role of Heme in Acute and Chronic Sickle Cell Disease Pain

项目摘要 患有镰状细胞病(SCD)的人患有复杂的刺激诱发的持续慢性疼痛，即 部分是由其外周感觉神经元的过度兴奋所介导的。然而，潜在的 疾病病理导致持续的炎症和神经病理性疼痛仍然知之甚少。在过去的几年里 十年来，临床和临床前研究表明，血红素升高，这是糖尿病的一个关键病理特征 SCD，可能与SCD疼痛有关。我的初步数据显示，野生型(WT)小鼠接受足底注射 注射氯化高铁血红素时，会出现急性机械过敏和爪子反应 出勤行为。我的初步数据进一步表明，减少SCD小鼠的血红素负担 腹膜内注射结合珠蛋白，一种血红素清除剂，可以降低机械过敏。 考虑到这些观察，我建议调查是否需要升高的血红素来维持 在唐斯SCD的疼痛行为。在目标1中，我假设升高的血红素导致持续的、非诱发的 外周伤害性感觉失调对WT小鼠疼痛行为的影响。我会给C57BL/6注射氯化血红素 并进行面部鬼脸和条件性位置厌恶，以确定血红素对非 诱发疼痛行为(目标1A)。在治疗窗口期后，我将收获背根神经节(DRGs) 并使用膜片钳电生理学来确定是否由血红素诱导 疼痛行为是由外周神经元过度兴奋性驱动的(目标1B)。在一项平行研究中，我将确定 在培养的WT背根节神经元中急性应用氯化血红素是否会导致神经元活动异常，如果是这样 依赖于血红素的典型受体，Toll样受体4(TLR4)(目标1C)。在目标2中，我假设 慢性升高的血红素是维持SCD疼痛所必需的。我将利用唤起和非唤起 疼痛行为分析以确定血红素清除剂结合珠蛋白是否能减少镇上的疼痛行为 SCD小鼠(Aim 2A)。此外，我将使用体外胫骨神经纤维记录(目标2B)和全细胞贴片 钳夹电生理学(目标2C)确定结合珠蛋白是否降低SCD感觉神经元 这些小鼠的兴奋性和机械过敏性。这些目标加在一起将决定血红素是否 通过外周敏化驱动诱发和非诱发疼痛行为，如果血红素可能被靶向 缓解SCD慢性疼痛。

Project Summary Individuals with sickle cell disease (SCD) suffer from complex stimulus-evoked and ongoing chronic pain that is mediated in part by the hyperexcitability of their peripheral sensory neurons. However, how the underlying disease pathology leads to ongoing inflammatory and neuropathic pain remains poorly understood. Over the last decade, clinical and pre-clinical research has demonstrated that elevated heme, a key pathological feature of SCD, may contribute to SCD pain. My preliminary data suggest that wildtype (WT) mice receiving intraplantar injections of hemin chloride, the common salt of heme, display acute mechanical hypersensitivity and paw attending behavior. My preliminary data further suggest that reducing the heme burden in SCD mice using intraperitoneally administered haptoglobin, a heme scavenger, reduces mechanical hypersensitivity. Considering these observations, I propose to investigate whether elevated heme is required for the maintenance of pain behavior in Townes SCD. In Aim 1, I hypothesize that elevated heme causes ongoing, non-evoked pain behavior in WT mice by dysregulation of peripheral nociception. I will administer hemin to C57BL/6 mice and conduct facial grimace and conditioned place aversion to determine the contribution of heme to non- evoked pain behavior (Aim 1A). Following the treatment window, I will harvest the dorsal root ganglia (DRGs) sensory neurons from these mice and use patch clamp electrophysiology to determine whether heme-induced pain behavior is driven by peripheral neuron hyperexcitability (Aim 1B). In a parallel study, I will determine whether acute application of hemin to cultured WT DRG neuron induces aberrant neuronal activity and if this dependent on heme's canonical receptor, Toll-like Receptor 4 (TLR4) (Aim 1C). In Aim 2, I hypothesize that chronically elevated heme is required for the maintenance of SCD pain. I will utilize evoked and non-evoked pain behavior assays to determine whether the heme scavenger haptoglobin reduces pain behavior in Townes SCD mice (Aim 2A). Additionally, I will use ex vivo tibial nerve fiber recordings (Aim 2B) and whole-cell patch clamp electrophysiology (Aim 2C) to determine whether the haptoglobin reduces SCD sensory neuron hyperexcitability and mechanical hypersensitivity in these mice. Together these aims will determine if heme drives evoked and non-evoked pain behavior through peripheral sensitization and if heme may be targeted to alleviated SCD chronic pain.