The Pain Neural Transcriptome

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Overview: The objectives of this project are to understand the molecular biology of pain-sensing neurons and peripheral tissues at the transcriptome level and modulation of transcriptomic parameters in acute and chronic pain models and 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 a foundational knowledge base for our translational projects (CL090033-07 Integrative and Molecular Studies of Pain and Pain Control and CL090034-07: Mechanisms of Pain and Immune Processes). To meet the objectives of understanding and translation, we have established research protocols, hardware and software infrastructure, analytical pipelines, and collaborative arrangements for data analysis. We utilize cell biological and in vivo behavioral measurements in combination with deep RNA-Seq and multiplex fluorescent in situ hybridization to probe basic pain mechanisms and circuits in multiple species. The resulting pain transcriptome encompasses physiologically and genetically characterized pain neurons, and tissues comprising the nociceptive circuit in combination with tissue inflammation, surgical incision and axotomy. All have been intensively analyzed in a series of publications. In this cycle, the use of newer high-throughput sequencing devices has enhanced our ability to assess the evolution and resolution of interventions, make valid statistical comparisons between manipulations, and avoid batch effects. 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 indicating a crucial role for TRPV1+ neurons in transmission of clinical pain. Because of the efficacy of manipulations aimed at the TRPV1-expressing DRG neurons, we performed deep RNA sequencing (RNA-Seq) on mouse, rat, canine, and human ganglionic preparations targeting TRPV1 neurons. We published several comprehensive transcriptomic profiles of these clinically important nociceptive neurons and extended the analysis to DRGs obtained at autopsy from one of our human cancer pain patients who had been treated with RTX. The data demonstrate that the centrally projecting axon is most sensitive neuronal component whereas the cell bodies in DRG are comparatively resistant to RTX. This important mechanistic insight was gained from transcriptomic analyses and is currently being used to fine-tune the administration protocol in our human clinical trial (low volume, slow rate of infusion). The strong efficacy of RTX implicates TRPV1+ DRG neurons as a crucial population for transmitting human clinical pain. Consequently, we plan to examine the human TRPV1 population in further detail. The objective is to find additional molecular routes to control the activity of TRPV1+ DRG neurons for effective non-opioid analgesia. The Spinal Pain Transcriptome: In the previous cycle we examined regulation of the endogenous opioid peptide precursors proenkephalin and prodynorphin in dorsal spinal cord during experimental inflammation. The transcriptome sequencing showed that dynorphin was very prominently upregulated. Therefore, in this cycle we delved into the spinal dynorphinergic neuronal population more thoroughly in both inflammation and incision models. Dynorphin was a strong neuronal signature in both models. Transcriptome analysis also showed upregulation of anaplastic lymphoma kinase (ALK) in the spinal cord sample. In situ hybridization revealed that ALK and dynorphin were co-localized and that these neurons constituted a population of glutamatergic dorsal horn neurons. The results identify a critical spinal neuronal population, the dynorphinergic-glutamatergic excitatory dorsal horn neurons, that participate in the regulation of spinal cord hyperexcitability and resolution of the hyperalgesic state. Sequencing shows that this is the main population transcriptionally regulated in persistent pain states. We hypothesize that these neurons are of adaptive significance, such that they reduce tonic hyperexcitability and allow an injured organism to continue to forage for food while protecting the injured limb from further damage. Immune-related genes were also regulated in the persistent pain state. We were able to discriminate two categories of immune gene signatures in dorsal horn. One signature was lateralized to the dorsal horn receiving innervation from the incised or inflamed hind paw and was composed of complement genes necessary for microglial-mediated synaptic remodeling. The second immune gene group was upregulated bilaterally, indicating delocalization from the afferent input. This group subserves generalized immune defensive priming that occurs in response to the xenobiotic inflammatory agent carrageenan, which has characteristics of a cell wall component of an infectious agent. Pathway analysis indicates the bilaterally induced genes are downstream to interferon-gamma signaling. We also detected a neutrophil signature which verifies our earlier report documenting arrest of these leukocytes bilaterally on the luminal side of spinal vasculature. Thus, transcriptomic analysis resolves two immune-related pathways regulated in CNS after peripheral tissue injury and brings clarification to previously fragmented observations based on individual gene studies. Peripheral Inflammatory and Surgical Incision Transcriptomes: The origin of pain starts with tissue damage. In this cycle we conducted a finely divided longitudinal transcriptional assessment of the inflamed hind paw to obtain a detailed profile of peripheral inflammatory gene regulation and changes in cellular composition in the widely used carrageenan model of persistent pain. We delineated the inflammatory secretome and multiple, temporally distinct gene inductions and cellular recruitment responses related to resident cells and infiltrating leukocytes. The results provide a basis for hypothesis generation, for targeted interventions, and to build sets of objective biochemical readouts that may inform a patients pain status which, in turn, may guide clinical decisions regarding therapeutic interventions. In this cycle we initiated a human clinical protocol to obtain skin samples longitudinally during surgical procedures. The protocol was approved, and we have recruited 5 patients to date. The human research will extend and verify animal observation of critical therapeutic potential. Anesthesia Transcriptome: We are a department of anesthesiology and in this cycle we completed transcriptomic assessments of general anesthesia and ketamine on cortical, hippocampal and amygdala. 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.