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•Translational efforts can be facilitated by investigations of human nervous system tissues.•Putative preclinical analgesic targets mustbe expressed in the relevant human cells.•Establishing human relevance enhances the value of mechanistic animal studies.•Multi-omic databases of human tissue data are growing rapidly.•Human tissue investigations require sustained research support and planning.

•Temporal gene regulatory programs with emphasis on the “inflammatory secretome.”•Sequential waves of leukocytes and antecedent patterns of the chemoattractants.•Subcellular localization of differentially expressed genes providing new targets.•Unique expression patterns of collagen-encoding genes related to tissue remodeling.

•The biochemical pathways related to oxylipins were outlined.•Genes encoding oxylipin related enzymes are expressed in the pain-circuit.•Gene expression and oxylipin concentrations were modified by inflammation.•Oxylipin injections evoked thermal nociceptive hypersensitivity in rats.

•Dynorphin and enkephalin are opioid peptides involved in spinal nociceptive modulation.•Peptide and mRNA were measured by Radioimmunoassay and RNA-Seq after peripheral inflammation.•Marked upregulation of prodynorphin mRNA and dynorphin A 1-8 occurred.•In situ hybridization localized the neurons mainly to medial dorsal horn.•RNA-Seq data mining of dynorphin in dorsal root ganglion revealed a role in nerve injury.

Pain from advanced cancer is often intractable leading to significant impairment of quality of life. The standard of care for relieving cancer pain often relies on opioids, which can be highly or incompletely efficacious, and cause adverse effects such as addiction and respiratory suppression. A major priority of the pain field is the development of novel non-opioid analgesic strategies to improve patient care. In this study we correlate the clinical presentation of a single well-characterized patient treated with lumbar intrathecal RTX to a molecular phenotype from postmortem DRG and SC tissue collected from this patient at autopsy (approximately 4-5 months after RTX injection). This patient was enrolled in a Phase 1 Study evaluating safety and efficacy of intrathecal RTX treating pain in advanced cancer (NCT00804154). We stained the DRG and SC with markers of nociceptive sensory afferents such as calcitonin gene-related peptide (CGRP) and Substance P (SP) to map the denervation and potential cell loss at different spinal levels. In the SC, a marked decrease in primary afferent-derived SP and CGRP was observed at all rostro-caudal levels, consistent with the clinical presentation showing decreased sensation to heat to all extremities and torso. In the DRG, quantitative cell counts for mRNA expression and nociceptive protein markers showed minimal cell loss consistent with our previous work. Numerical pain rating scorThis clinical observation formed the basis for a reverse translational effort to assess the effects of dose, rate, and volume on RTX spread in the intrathecal space of rats and pigs.

Patients suffering from peripheral neuropathic pain often experience inadequate pain relief. Morton’s neuroma (MN), which is caused by a localized nerve lesion in the forefoot, represents an excellent model to study the pathology and treatment of peripheral neuropathic pain. Patients suffer from burning or stabbing pain upon weight-bearing, which limits their mobility. A significant fraction of patients continues to experience pain after non-surgical treatments. We aim to evaluate analgesic efficacy of long-term inactivation of a specific peripheral axon population that expresses TRPV1. We hypothesize that a perineural injection of the super-potent TRPV1 agonist resiniferatoxin (RTX) will selectively lesion these heat- and inflammation sensitive afferent fibers. This strategy is based on preclinical studies and successful clinical trials for patients with refractory pain. In order to investigate the involvement of these and other fiber types in MN pain, we performed quantitative sensory testing. We observed individual patterns of heat hyperalgesia across several testing locations, indicating variable nerve sensitization in the patient population. In a subset of patients heat hyperalgesia included an area outside the territory of the affected nerve and implicating processes of spinal secondary sensitization. Most MN patients showed hyperalgesia to mechanical deep tissue stimulation in both the affected and healthy foot, but not at a remote testing site. Overall, these findings suggest both sensitization of local peripheral fibers and central segmental circuits. The prevalent symptom of burning pain in MN patients reinforces the idea that RTX may provide effective pain relief in Morton’s neuroma patients.

Elucidating the molecular and regulatory processes underlying tissue damage and repair has critical implications for pain management and postoperative care. Earlier published work from our laboratory exploring the anatomical and spatial landscape of the surgical wound edge in the rat uncovered candidate genes involved in immune cell recruitment, nociceptive signaling pathways, and re-epithelialization. However, the translation and validation of these findings to human models requires greater exploration. The present study investigates molecular events following tissue injury in a human surgical incision model using transcriptomics and multiplex fluorescence in situ hybridization to localize gene-level transcriptional changes. Tissue was collected from surgery patients (n=12; 58% Female) at surgery onset, prespecified timepoints during surgery, and at closure. RNA-Seq was performed to determine and quantify gene expression patterns. Anatomical localization of the most highly induced transcripts was examined through RNAscope. Our findings revealed the induction of secreted signaling molecules including interleukins 6 and 8, metallothionein 2A, oncostatin M, and tissue growth factors. In situ hybridization localized the transcripts superficially in the keratinocyte layer and in deeper follicular structures and gland tissues. We further analyzed our transcriptomic data to assess the ability of these molecules to signal to dorsal root ganglion neurons and to drive pathways involving immuno-chemoattraction, inflammation, nerve terminal sensitization, and algesia mediation. Ultimately, illuminating the network of the nociceptive secretome can inform analgesic development. This abstract is based on a clinical trial registered at ClinicalTrials.gov (NCT04224870).

Adenosine receptors are a family of purinergic G-protein coupled receptors (GPCR) widely expressed in the peripheral and central nervous systems. Recently, studies done in model organisms suggest analgesic action for the adenosine A3(ADORA3) and its agonists have been proposed as new neuropathic pain treatments. Given this conclusion, we hypothesized that this receptor may be expressed in nociceptive primary afferent neurons. However, when further investigated using multiplex fluorescence in situ hybridization, ADORA3transcripts were found in the ring of support cells around the neuron, but absent from the neuron itself. To determine the identity of these support cells, the expression of ADORA3was analyzed alongside markers for macrophage and satellite glial cells (SGC). Our results clearly showed ADORA3expression exclusively in perineuronal macrophages. In the spinal cord, ADORA3was expressed exclusively in microglia. These results suggest that, if translated to human clinical trials, the potential analgesic action of the adenosine A3receptor agonism may result from a more complex or indirect mechanism involving modulation of macrophages and/or microglia. To further elucidate the precise molecular identity of these macrophage-like perineural cells we will evaluate ADORA3expression alongside other non-neuronal molecular markers. This will allow for a better understanding of the functional implications of this cell and its proximity to the neuron. Additionally, further research may investigate the relationship between the neuron and its support cells, specifically the mechanisms underlying the intercell communication and its role in pain modulation.

Nociceptive input to the spinal cord is transmitted by primary afferent neurons in the dorsal root ganglia (DRG). A subset of DRG neurons has the ability to attenuate nociceptive transmission through expression of the µ-opioid receptor. The relationship between algesic and analgesic properties of individual DRG neurons has not yet been evaluated in the human. By using a combination of six different 4-Plex in-situ hybridization experiments, we were able to identify three different main nociceptive populations. First, we detected a population poly-responsive, poly-modulated small-diameter nociceptors that co-express TRPV1, OPRM1, and SCN11A, and in most cases additionally SCN10A and P2RX3 and in subpopulations additionally TAC1, TRPA1, TRPM8, OPRD1, PIEZO2, and/or OPRL1. Second, we detected a medium-sized set of multimodal putative nociceptors with a very similar molecular profile, apart from a lack of expression of TRPA1. Both nociceptor types do express the µ-opioid receptor (encoded by OPRM1) and their activity and input into the spinal cord likely can be attenuated by clinically used µ-receptor agonists. The third population consists of small-diameter neurons that have a molecular expression profile characteristic for nociceptive neurons, such that they express TRPV1 together with SCN11A, SCN10A, P2RX3, PIEZO2, OPRD1, and partially TRPA1. The most distinguishable characteristic of this population is the lack of expression of the µ-opioid receptor and thus are non-susceptible to clinically used opioids. Further molecular characterization of these nociceptive populations might reveal attractive molecular candidates for targeting different types of pain indications.

WNK1 (With No Lysine(K) 1) is a ubiquitously expressed serine/threonine kinase containing several splice variants of clinical significance. These splice variants are known to have tissue-specific expression patterns. In particular, the large exon, called HSN2, is selectively expressed in neuronal tissues, and during development, and is named because mutations in the HSN2 exon are associated with hereditary sensory and autonomic neuropathy, type II (HSAN2). The HSN2-containing WNK1 transcripts are enriched in primary afferent dorsal root ganglion neurons, which are the sensory neurons that become damaged and dysfunctional during HSANII disease progression. This observation implies that expression of HSN2-containing WNK1 transcript variants in DRG neurons is required for survival and/or function of these neurons. Using long-read RNA-Sequencing we further refined the exon-level expression of WNK1 in human DRG. The expression of HSN2-containing WNK1 splice variants in DRG was similar to total WNK1 expression but was not uniform across all neurons. We hypothesized enrichment of HSN2-containing transcripts in nociceptive subpopulations, examining HSN2 expression in combination with nociceptive neuronal markers. However, overall, HSN2 was observed at lower levels in neurons encoding the precursor for Substance P peptide (TAC1). Conversely, HSN2 is enriched in large diameter human sensory neurons expressing the mechano-transducing ion channel gene PIEZO2. These results clarify the expression pattern of HSN2-containing WNK1 splice variants in human nociceptive tissues, and indicate that their expression, while ubiquitous, is enriched in proprioceptive large fibers. These data clarify the underlying molecular-cellular basis for HSANII neuropathology. The Intramural Research Program at the NIH Clinical Center.

Autofluorescence in human tissue impedes optimal multiplex fluorescence imaging and accurate quantification of signal. One pervasive source of autofluorescence is lipofuscin, an age-associated agglomeration of oxidized lipoprotein, which absorbs and emits strongly at 400-650 nm, which are critical wavelengths for multiplex imaging. This study aimed to reduce the presence of autofluorescence using broad spectrum LED white light exposure to photobleach lipofuscin across the full spectral range. Deparaffinized formalin-fixed human dorsal root ganglion tissue (sourced from AnaBios Corp.) was prepared for in situ hybridization using standard antigen retrieval procedures, and exposed to high intensity focused beams. We sought to determine the time to complete photobleaching by examining time points from 1 – 72 hours of light exposure. After bleaching, multiplex fluorescence in situ hybridization was completed in photobleached sections to assess improvement in signal to noise. The optimal light exposure was 24 hours with little to no difference between 24 and 72 hours. This resulted in an approximately 5-fold decrease in autofluorescent signal in several wavelengths compared to untreated controls. This reduction allows for higher quality RNA fluorescence images and more accurate signal data. Similar photobleaching methods have been used with lower light units/mm2 and/or different spectral peaks using exposures up to 72 hours. Our usage of high intensity white light with a spectral intensity maximum nearer to the maximal absorbance wavelength of lipofuscin (∼400-420 nm) provides a rapid and effective method for removing interfering autofluorescence, enhancing multiplex staining and imaging in highly autofluorescent human tissues. Funding to the Intramural Research Program of the National Institutes of Health Clinical Center ZIACL090033, ZIACL090034, ZIACL090035.

•We present the molecular response in dorsal spinal cord to peripheral inflammation.•Identify ALK as an induced neuronal tyrosine kinase receptor in dynorphinergic neurons.•Distinguish interferon immune responses from afferent driven complement increase.•Identify targetable analgesic signaling molecules induced by peripheral injury.•Articulate application of RNA-Seq to analgesic drug discovery.