The Pain Neural Transcriptome

Clinical Center

Investigators

Linked publications & trials

Abstract

Overview: The objectives of this project are to understand (a) the molecular biology of pain-sensing neurons and damaged peripheral tissues at the transcriptome level, (b) the modulation of transcriptomic parameters in acute and chronic pain models and (c) to extend and verify the models with samples obtained from human surgical patients or post-mortem tissues. Beyond these questions, the empirical framework we are developing forms the foundational knowledge for translational projects (CL090033-07 Integrative and Molecular Studies of Pain and Pain Control and CL090034-07: Mechanisms of Pain and Immune Processes) and clinical trials of resiniferatoxin (RTX). To meet these objectives, we established research protocols, hardware and software infrastructure, analytical pipelines, and collaborative arrangements for data analysis and experimental models. We utilize cell biological and in vivo behavioral measurements in combination with deep RNA-Seq, multiplex fluorescent in situ hybridization and single nucleus sequencing to probe basic nociceptive mechanisms in multiple species. The resulting pain transcriptome encompasses primary afferent and spinal cord neurons and peripheral tissues during inflammation, surgical incision or other models of nociception. All have been intensively analyzed in a series of publications. Another objective is to extend our studies to include human nociceptive neurons. In this cycle we performed single nucleus sequencing of human spinal cord and dorsal root ganglia. This has been extremely informative and meshes well with the whole tissue RNA-Seq and corresponding high resolution in situ hybridization. What emerges is the entire molecular repertoire of the nociceptive system in both basal and pathological states. This extensive foundational data greatly facilitates formation of incisive hypotheses regarding pain physiology and the choice and design of effective interventions. Our understanding of how pain is generated, transmitted, processed, and modulated in animal models and humans forms a strong and multilayered basis for generation of new, non-opioid analgesics. The TRPV1 Transcriptome: One important focus is the subpopulation of DRG neurons expressing the thermo-, chemo-, pH-, and lipid-responsive ion channel called TRPV1. This ion channel is also gated by capsaicin, the active ingredient in hot pepper. We have demonstrated that the potent capsaicin analog resiniferatoxin (RTX) can control cancer pain in canine and human patients by inactivating TRPV1 axons or nerve terminals, indicating a crucial role for TRPV1+ neurons in transmission of clinically relevant pain. We published several comprehensive transcriptomic profiles of these clinically important nociceptive neurons and extended the analysis to DRGs obtained at autopsy from two of our human cancer pain patients who had been treated with RTX. The data demonstrate that, with intrathecal administration, the centrally projecting axon is the most sensitive neuronal component whereas the cell bodies in DRG are comparatively resistant to RTX. This important mechanistic insight was used, in part, to fine-tune drug administration our human clinical trial (low volume, slow rate of infusion). The strong efficacy of RTX implicates TRPV1+ DRG neurons as a crucial population of nociceptive neurons for transmitting human clinical pain. We are presently using single nucleus sequencing to further examine the TRPV1 population. The objective is to identify new molecular routes to control a specific subpopulation of TRPV1+ DRG neurons, which we now call tissue damage nociceptors, to obtain effective non-opioid analgesia. The Spinal Pain Transcriptome: In this cycle we delved into the spinal dynorphinergic neurons more thoroughly. Prodynorphin mRNA upregulation is a strong molecular signature in both inflammation and incision models. Multiplex in situ hybridization showed that upregulation occurred in a population of glutamatergic dorsal horn neurons that we hypothesize regulates spinal cord hyperexcitability and resolution of hyperalgesia and that activation of this circuit is of adaptive significance, such that reduction of tonic nociceptive hyperexcitability allows an injured organism to continue to forage for food while protecting the injured limb from further damage. In our published report we also discriminate two categories of immune gene signatures. One signature was lateralized to dorsal horn receiving innervation from the incised or inflamed hind paw and is composed of complement genes necessary for microglial-mediated synaptic remodeling. The second immune signature occurred bilaterally, indicating delocalization from the afferent input, and subserves generalized immune defensive priming. These findings bring clarification to previously fragmented observations based on individual gene studies. Peripheral Inflammatory and Surgical Incision Transcriptomes: Our objective is to understand peripheral processes following tissue damage at a molecular level. By conducting RNA seq on periphera tissue after inflammation or incision, we delineated the tissue damage secretome and multiple, temporally distinct gene inductions and cellular recruitment responses related to resident cells and infiltrating leukocytes. We also initiated a human study to extend and verify the rat results. Working with a team of surgeons from the NCI we obtained skin from the incision at various intervals out to closure (between 6 and 8 hours) and have performed RNA-Seq, in situ hybridization and LC-MS-MS lipidomic profiling on the samples. The results provide a basis for targeted interventions, and a transcriptomic roadmap of objective biochemical readouts that may inform a patients pain and wound healing status which can guide regarding therapeutic interventions. Anesthesia Transcriptome: In this cycle we completed assessments of an inhalation anesthetic and ketamine infusion on cortical, hippocampal and amygdala transcriptomes. In humans, general anesthesia can be deleterious to cognitive function. We hypothesize that mechanistic insight into the defect state induced by anesthesia and the capacity for recovery can be obtained by understanding molecular-level transcriptome changes. Novel insights into both anesthetics were obtained with implications for improving recovery. The results have been assembled into manuscripts for submission.