Integrative And Molecular Studies Of Pain And Pain Control

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Summary Overview: This research program addresses basic molecular and physiological processes of nociceptive (pain-sensing) transmission in the peripheral and central nervous systems (CNS) and new ways to effectively control pain. The molecular research is performed using animal and in vitro cell-based models. We concentrate on primary afferent pain-sensing neurons located in dorsal root ganglion (DRG) that send axonal projections to skin and deep tissues and make connections within dorsal spinal cord, which is the first CNS site of synaptic information processing for pain. The mechanisms of transduction of pain stimuli are investigated through models of pathophysiological damage or using reductionist preparations such as primary DRG cultures or heterologous expression systems of ion channels or receptors. Our goals are (a) to understand the molecular and cell biological mechanisms of acute and chronic pain at the initial steps in the pain pathway, (b) to investigate mechanisms underlying human chronic pain disorders, (c) to explore neuronal plasticity and altered gene expression in persistent pain states, and (d) to use this knowledge to devise new treatments for pain. New Treatments for Pain: We address the new treatment goal through translational research coupled with human clinical trials to develop and introduce new molecular interventions for severe pain. Studies with the TRPV1 agonist resiniferatoxin (RTX) have resulted in a Phase I clinical trial for in patients with intractable pain from advanced cancer. RTX activates an influx of sodium and calcium ions and once bound to TRPV1, RTX props open the ion channel causing an intracellular calcium cytotoxicity. Depending on the route of administration RTX disables TRPV1 pain-sensing nerve endings or axons (i.e., the nerve fiber) or deletes the neuron entirely. RTX produces very effective pain control in pre-clinical models. The central route involves administration into the cerebrospinal fluid around the spinal cord (intrathecal). To date, we have treated 19 patients with pain from advanced cancer. This Phase I study is provisionally complete although we plan to examine a higher dose tier. It and being readied for publication. Earlier we published studies of RTX injections around or directly into sensory ganglia which formed the basis of our protocol to treat localized chronic pain by periganglionic RTX injection. Other peripheral routes of RTX administration include injection into skin, joints, nerve bundles, or topically. Preclinical studies show that analgesia by these routes is long-lasting but reversible, since the peripheral nerve endings regrow. Human protocols for post-operative incision and neuroma pain indications were generated in collaboration with the Thoracic and Oncologic Surgery Branch, NCI and local podiatrists, respectively. The initial post-operative study is designed to evaluate preemptive treatment with RTX injected subcutaneously prior to incision. This protocol has passed scientific review and was evaluated by the FDA which asked for certain additional experiments which are pending. The protocol for treating Mortons neuroma will be by perineural injection just proximal to the neuroma. This protocol also has passed scientific review The IND has been prepared and will soon be submitted to the FDA. Early Translational Investigations: In this cycle we extended our systems approach for integrated RNA-Seq and lipidomics to humans through a our human intraoperative tissue biopsy protocol. We obtained samples from surgical wound margins over time. This longitudinal tissue procurement was completed, and we are presently analyzing the samples transcriptomically, anatomically, and lipidomically. During this cycle we have made a strong effort to incorporate direct in human studies of nociceptive molecular biology. This was prompted by our repeated observations of species differences in gene expression in dorsal root ganglion (DRG) and spinal cord. Most of the skin, nerve, DRG and spinal cord tissues are recovered from organ donors or obtained at autopsy of patients in NCI Laboratory of Pathology. These tissues are being analyzed intensively by whole tissue sequencing, single nucleus sequencing, in situ hybridization and immunofluorescence staining of proteins. In collaboration with the Dr. Ashok Kulkarnis lab, we have examined DRG from patients with painful diabetic peripheral neuropathy (DPN). The first paper describing these results was published in this cycle and a second one is about to be submitted. We observe a loss of a select subpopulation of nociceptors suggesting a dynamic interplay occurs between initial hyperexcitability and subsequent neurotoxicity. We emphasize the importance of enhanced focus on human studies in a recent publication in the Journal of Pain (PMID: 35504570). Basic Pain Mechanisms: Underlying the translational and clinical studies are investigations of neuronal function, behavior, and molecular biological mechanisms of pain transduction and wound healing. We systematically investigate molecular alterations at the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal (sensory) spinal cord to obtain a comprehensive, quantitative foundational molecular understanding of nociceptive processes related to inflammation, surgical incision, and nerve injury. One of the main methods we used is called RNA-Seq with which we can sequence all of the mRNAs in a given tissue or cell population. Our work now integrates RNA-Seq as a component in most of our investigations. Another main method is multiplex fluorescence in situ and immunofluorescence anatomical studies of tissues in the nociceptive circuit. Using these techniques we now investigate humans with genetic variations that affect pain sensitivity. At present we are investigating a group of rare individuals with a copy number variant involving a specific gene locus. Individuals with three copies of this region exhibit profound decreases pain sensitivity. Using RNA-Seq we identified one gene in the locus of 25 that is an excellent candidate for mediating the analgesic action when overexpressed. The results are both compelling and informative and define a previously unidentified genetic mechanism for governing pain sensitivity. The RNA-Seq investigations provide new quantitative assessments of neuronal and glial genes as well as immune process related to nociceptive circuit function. Through this basic research we are obtaining a deeper understanding of mechanisms that trigger acute pain and sustain chronic pain and we are identifying molecular components to control pain.