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

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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.