Chronic pain conditions are often driven by hyperexcitability of nociceptors, the peripheral sensory neurons that detect noxious stimuli. Recent single-cell transcriptomic studies have begun to clarify the molecular identity of distinct peripheral sensory neuron subtypes, but tools that restrict transgene expression to nociceptors while sparing other dorsal root ganglion (DRG) subtypes remain limited. Here, we combined single-nucleus multi-omic profiling of human and mouse DRG with in vivo AAV enhancer screening to identify cis-regulatory elements that drive biased AAV expression in mouse DRG nociceptors and human iPSC-derived nociceptors. We then validated that an enhancer AAV designed to express Kir2.1 preferentially in nociceptors reduces DRG neuronal excitability. Leveraging these multi-omic datasets, we trained a sequence-based model to decode the cis-regulatory logic of nociceptors, enabling both the prioritization of native candidate elements and the design of synthetic enhancers with a range of nociceptor targeting properties. Together, these cross-species multi-omic resources define conserved DRG regulatory programs and provide a viral toolkit for pain research with potential translational applications for patients with refractory pain.

INTRODUCTION:Targeted Muscle Reinnervation (TMR) can prevent and treat neuropathic pain in amputees, but the degree of success varies. This study developed a Machine Learning (ML) model to predict the likelihood of sustained pain mitigation following primary and secondary TMR based on patient characteristics. METHODS:Patients who underwent TMR at a tertiary care center (2017-2024) were included. Patients were categorized as achieving good or poor pain outcomes based on predefined criteria: ≥3/10-point reduction (Numeric Rating Scale) for secondary TMR, or pain scores ≤3/10 for ≥3 months for primary TMR. Three ML architectures (lasso logistic regression, random forest classifier, and relevance vector machine (RVM)) were tested. Model performance was evaluated using area under the receiver operating characteristic (AUROC) curve; feature importance was quantified using Shapley additive explanations (SHAP). RESULTS:In total, 77 primary TMR and 101 secondary TMR patients were included (median follow-up: 2.0 years). The RVM model achieved test prediction accuracy scores of 0.74±0.12 for both primary and secondary TMR, with AUROC scores of 0.78±0.13 and 0.80±0.05, respectively. For primary TMR, pre-operative opioid use, male sex, and history of depression showed strong negative impacts. For secondary TMR, pre-operative smoking, elevated pain scores, and history of anxiety were strong negative predictors. The model significantly outperformed traditional statistical approaches. DISCUSSION:This novel custom ML model achieved strong predictive capability for TMR outcomes, demonstrating proof of concept of a practical tool for surgical planning and patient selection. The identification of several key modifiable risk factors suggests opportunities for pre-operative optimization to improve surgical outcomes.

Human dermal sleeping nociceptors display ongoing activity in neuropathic pain, affecting 10% of the population. Despite advances in rodents, a molecular marker for these mechano-insensitive C-fibers (CMis) in human skin remains elusive, preventing targeted therapy. Using a Patch-seq approach, we combined single-cell transcriptomics, following electrophysiological characterization, with single-nucleus and spatial transcriptomics from pigs and integrated our findings with cross-species and human transcriptomic data. We functionally identified CMis in pig sensory neurons with patch clamp, using adapted protocols from human microneurography. We identified oncostatin M receptor (OSMR) and somatostatin (SST) as marker genes for CMis. Following dermal injection in healthy human volunteers, oncostatin M, the ligand of OSMR, exclusively modulates CMis. Our findings characterize the molecular architecture of human dermal sleeping nociceptors, providing a framework for mechanistic insight into neuropathic pain and potential therapeutic strategies.

Neurons extend long axons that traverse distinct microenvironments, yet how these subcellular compartments acquire and maintain specialized identities remains unclear. Here, we use spatial translatomics to define the local translatomes of somatosensory dorsal root ganglion (DRG) neurons. Translating Ribosome Affinity Purification and RNA sequencing (TRAP-seq) reveal thousands of mRNAs preferentially translated within central axons, peripheral axons, or DRG somata, establishing compartment-specific translational programs. Many of these transcripts encode ion channels and neurotransmitter receptors that may confer distinct electrophysiological and regenerative properties to each axon. Integration of the TRAP-seq data with published RNA-seq identify locally translated components that change following neuropathic injury and could thereby adjust neuronal activity. We identify RNA regulons coordinated by RNA-binding proteins (RBPs) SFPQ and SRSF10, which preferentially bind and traffic mRNAs to peripheral or central axons, respectively. These findings indicate that RBP-guided RNA sorting and local translation enable the establishment and dynamic local modulation of somatosensory function. Here authors define compartment-specific translatomes in somatosensory dorsal root ganglion neurons and identify distinct local translational programs across central axons, peripheral axons and neuronal somata. They show that the RNA-binding proteins SFPQ and SRSF10 coordinate the selective trafficking and local translation of mRNAs in peripheral and central axons.

Peripheral sensory neurons, long appreciated for their role in detecting environmental stimuli, also play an active role in local and systemic immune responses. In response to tissue damage, the coordinated activation of peripheral sensory neurons and immune cells facilitates tissue repair. When dysregulated, however, these neuroimmune interactions can become maladaptive and contribute to chronic disorders such as chronic pain or headache. Recent single-cell transcriptomic studies have uncovered multiple distinct sensory neuron subtypes, each with unique molecular profiles, anatomical niches, and immune functions. In this review, we examine how distinct sensory neuron subtypes coordinate immune responses in barrier and internal tissues, forming cell type- and context-specific neuroimmune circuits. We also discuss how disruption of these circuits contributes to neuroinflammatory diseases such as atopic dermatitis and neuropathic pain. Clarifying these subtype-specific neuroimmune mechanisms offers a path toward more precise and effective therapies that target maladaptive neuroimmune circuitry in multiple disorders.

Neuropathic pain encompasses a spectrum of conditions that significantly impact quality of life. In case of nerve injuries, when regenerating axons fail to reach their target organs, they can form painful neuromas, yet the mechanisms underlying their formation remain unclear. In this exploratory study, we analyzed the neural components of painful neuromas by examining the expression patterns of key nociceptor markers. Neural components within neuromas exhibited up-regulated growth-associated protein 43, indicating their regenerative potential. Calcitonin gene-related peptide was expressed in 84% of axons, versus 3% in healthy nerves, suggesting a phenotypic shift toward a nociceptive profile. We also observed increased expression of the mechanosensitive channel Piezo2 in 78% of axons and up-regulation of the low-threshold sodium channel Nav1.3, consistent with heightened mechanosensitivity and hyperexcitability. These observations identify candidate molecular alterations in painful neuromas and may provide a guide for future development of targeted therapies for neuroma-related pain.

Chronic pain accounts for nearly half of owner-reported canine euthanasia decisions, yet dogs remain underutilized as a large-animal model for studying pain and developing translational therapeutics. Here, we present a canine dorsal root ganglion (DRG) cell atlas generated from six donors, representing five breeds, both sexes, and three spinal segments. Our dataset comprises 3,026 neurons and 11,734 non-neuronal cells and resolves 15 neuronal subtypes that map cleanly onto A- and C-fiber classes. We further identify eight major non-neuronal subtypes, including glial, vascular, and immune populations and characterize neuronal and non-neuronal expression of physiologically relevant neuropeptides, receptors, and ion channels. We identify region-specific differences in subtype composition between lumbar and sacral DRGs, with transcriptional programs suggestive of enhanced tactile-associated signaling in lumbar DRGs and heightened nociception-associated signaling in sacral DRGs. Cross-species comparisons reveal that canine DRG subtypes are broadly conserved with human and mouse, while also exhibiting canine-specific and canine-human shared molecular features relevant for translation. Together, this atlas serves as a valuable resource for understanding canine sensory neurobiology, comparing DRG organization across mammals, and leveraging dogs as a translational model for pain research and therapeutic development.

OBJECTIVE:Neuropathic pain significantly impacts quality of life (QoL), mental health, and function. Targeted muscle reinnervation (TMR) is an intervention that can effectively treat and prevent neuropathic pain, but its effects on psychosocial outcomes remain underexplored. This study evaluates psychosocial outcomes after TMR surgery for neuropathic pain in amputees (both primary [pTMR] and secondary [sTMR]) and non-amputees. METHODS:In this prospective study, 46 patients (15 sTMR, 19 pTMR, 12 non-amputees) who underwent TMR for neuropathic pain management were assessed for psychosocial outcomes. Preoperative and postoperative surveys measured pain catastrophizing (Patient-Reported Outcomes Measurement Information System [PROMIS]), depression (Patient Health Questionnaire-2 [PHQ-2]), anxiety (Generalized Anxiety Disorder-2 [GAD-2]), sleep metrics (PROMIS sleep disturbance, sleep duration), and QoL (World Health Organization Quality of Life assessment [WHOQOL-2]). Mean follow-up duration was 1.5 ± 0.8 years. RESULTS:Pain catastrophizing significantly decreased across all groups (overall from 50.39 ± 6.24 to 42.41 ± 4.40, P < .001). Depression and anxiety scores improved significantly in the non-amputee and pTMR groups but not in sTMR patients. Sleep disturbance decreased significantly in all groups (from 59.67 ± 8.64 to 51.82 ± 8.01, P < .001), whereas sleep duration increased (from 5.32 ± 1.63 to 6.09 ± 1.29 hours, P < .001). QoL scores improved significantly across all groups (from 2.45 ± 0.87 to 3.43 ± 0.62, P < .001). Patients with psychiatric comorbidities (60.9%) showed similar improvements, despite having higher preoperative and postoperative depression and anxiety scores. CONCLUSIONS:Patients who underwent TMR for neuropathic pain management demonstrated improved psychosocial outcomes. Non-amputees and pTMR patients demonstrated greater improvements in depression and anxiety than did sTMR patients. Sleep quality and duration improved substantially, a previously underreported benefit of TMR. Future, larger prospective studies should further validate relationship between neuropathic pain reduction through TMR and psychosocial outcomes.

Spatial transcriptomics has emerged as a powerful tool to define the cellular structure of diverse tissues. One such method is multiplexed error robust fluorescence in situ hybridization (MERFISH). MERFISH identifies RNAs with error tolerant optical barcodes generated through sequential rounds of single-molecule fluorescence in situ hybridization (smFISH). MERFISH performance depends on a variety of protocol choices, yet their effect on performance has yet to be systematically examined. Here we explore a variety of properties to identify optimal choices for probe design, hybridization, buffer storage, and buffer composition. In each case, we introduce protocol modifications that can improve performance, and we show that, collectively, these modified protocols can improve MERFISH quality in both cell culture and tissue samples. As RNA FISH-based methods are used in many different contexts, we anticipate that the optimization experiments we present here may provide empirical design guidance for a broad range of methods.

Spontaneous pain is a common but poorly understood consequence of peripheral nerve injury 1–3 , including injuries that lead to the formation of neuromas 4,5 . We developed a chronic neuroma model for measuring spontaneous pain-related behaviours in mice, which revealed that limb flicks - emerging predominantly 2 months post-injury - reflect spontaneous paroxysmal pain. Ectopic activity of injured dorsal root ganglia (DRG) sensory neurons whose axonal endings terminate within the neuroma drives this spontaneous pain. In vivo imaging showed that a subset of small-diameter DRG sensory neurons are the source of spontaneous neural signals emanating from the neuroma, and these spontaneously active neurons are distinct from the intact larger diameter sensory neurons that mediate stimulus-evoked mechanical allodynia from spared nerves. Cell-type–specific gain- and loss-of-function studies identified a genetically- and functionally-defined subtype of small-diameter C-fibre nociceptors whose injured axons in neuromas drive spontaneous limb flicks/neuropathic pain. These findings establish the neurobiological basis of spontaneous pain enabling targeted pain management strategies and define a cellular and mechanistic separation between spontaneous and evoked neuropathic pain.

Sensory neurons within the dorsal root ganglion (DRG) are the primary trigger of pain, relaying activity about noxious stimuli from the periphery to the central nervous system; however, targeting DRG neurons for pain management has remained a clinical challenge. Here, we demonstrate the use of lipid nanoparticles (LNPs) for effective intrathecal delivery of small interfering RNA (siRNA) to DRG neurons, achieving potent silencing of the transient receptor potential vanilloid 1 (TRPV1) ion channel that is predominantly expressed in nociceptor sensory neurons. This leads to a reversible interruption of heat-, capsaicin-, and inflammation-induced nociceptive conduction, as observed by behavioral outputs. Our work provides a proof-of-concept for intrathecal siRNA therapy as a novel and selective analgesic modality.

The recent approval of suzetrigine for acute pain treatment highlights both the success of targeting peripheral sensory neurons for pain management and the potential of developing new pain therapies primarily in human-based systems. To realize this transformative potential, further research into somatosensation and pain neuroimmunology in human systems is essential.

INTRODUCTION: Efforts to treat pain by reducing the excitability of nociceptors (first order pain-sensing neurons), have focused on the NaV1.7 sodium channel, since loss of SCN9A (gene for NaV1.7) leads to congenital insensitivity to pain, whereas NaV1.7 gain-of-function mutation causes the severe painful syndrome familial erythromelalgia. Despite these genetic mutations and their distinct phenotypes, pharmacologic Nav!.7 targeting has not been a successful strategy. Most pain conditions are focal, yet most treatments are systemic and expose patients to potentially unnecessary systemic adverse side effects. METHODS: The effect of nociceptor specific potassium channel expression was confirmed by dissociated dorsal root ganglion (DRG) calcium imaging, and several rodent pain assays: von Frey mechanical threshold (sciatic injection), Hargreaves thermal threshold (sciatic injection), CFA inflammatory paw model (sciatic injection), and trigeminal neuralgia whisker pad mechanical threshold (trigeminal ganglion injection). Animals were either wild-type mice or TrpV1::ChR2-EYFP (expression of Channelorhodopsin-2 in TrpV1 lineage neurons). Treated animals were compared to age-, and sex-matched controls that received GFP-control virus in the same dose RESULTS: Animals treated with therapeutic potassium channels showed increased mechanical and thermal thresholds, decreased inflammatory response (higher mechanical threshold after CFA injection), and increased whisker pad thresholds compared to animals treated with GFP-control viruses. Animals that showed improved behavioral outcomes with potassium channel treatment were sacrificed and the DRGs dissociated. Calcium influx was lower in treated neurons when compared to GFP-controls supporting the hypothesis that hyperpolarization of nociceptors is a viable analgesic strategy. CONCLUSIONS: Our results validate the over-expression of potassium channels as a promising alternative for pain treatment and support the use of spatial restriction to achieve precision in AAV-mediated gene therapies.

BACKGROUND AND OBJECTIVES:Anatomic features of neuromas have been explored in imaging studies. However, there has been limited research into these features using resected, ex vivo human neuroma specimens. The aim of this study was to investigate the influence that time may have on neuroma growth and size, and the clinical significance of these parameters. METHODS:Patients who underwent neuroma excision between 2022 through 2023 were prospectively included in this study. Neuroma specimens were obtained after operative resection. Standardized neuroma size measurements, expressed as a neuroma-to-nerve ratio (NNR), were conducted with ImageJ software. Pain data (numeric rating scale, 0-10) were prospectively recorded during preoperative evaluation, and patient factors were collected from chart reviews. RESULTS:Fifty terminal neuroma specimens from 31 patients were included, with 94.0% of the neuromas obtained from individuals with amputations. Most neuromas were excised from the lower extremities (n = 44, 88.0%). The neuromas had a median NNR of 2.45, and the median injury to neuroma excision interval was 6.3 years. Larger NNRs were associated with a longer injury to neuroma excision interval and with a smaller native nerve diameter. In addition, sensory nerves were associated with a larger NNR compared with mixed nerves. NNR was not associated with preoperative pain or with anatomical nerve distribution. CONCLUSION:This study suggests that neuromas seem to continue to grow over time and that smaller nerves may form relatively larger neuromas. In addition, sensory nerves develop relatively larger neuromas compared with mixed nerves. Neuroma size does not appear to correlate with pain severity. These findings may stimulate future research efforts and contribute to a better understanding of symptomatic neuroma development.

Somatosensory perception largely emerges from diverse peripheral sensory neurons whose cell bodies reside in dorsal root ganglia (DRG). Damage or dysfunction of DRG neurons is a major cause of chronic pain and sensory loss. In mice, deep single-cell transcriptomic profiling and genetically defined models have offered important clues into DRG function, but in humans, the cellular and molecular landscape of DRG neurons remains less understood. Here, we constructed a reference cell atlas of the human DRG by profiling transcriptomes of cells and nuclei from 126 donors sampled across cervical, thoracic, and lumbar DRGs. This atlas resolves 22 neuronal subtypes, including known and previously unrecognized subtypes linked to nociception, mechanosensation, thermosensation, and proprioception, as well as 10 types of non-neuronal cells. Cross-species integration, spatial transcriptomics, and microneurography enabled cell-type-specific comparisons of soma size and conduction velocity between species. Human DRG somata are larger across all cell types than their mouse counterparts, and the conduction velocities of human hair follicle innervating A-fibers are faster than in mice, suggesting a functional shift in rapid mechanical detection in humans. This integrated human DRG reference cell atlas provides a resource for exploring new molecular and physiological features of human DRG, which could help identify new strategies for treating chronic pain and other diseases of the peripheral nervous system.

Spontaneous pain is a very common but poorly understood consequence of peripheral nerve injury. We developed a system for measuring spontaneous pain-related behaviors in mice over months, which revealed that limb flicks-emerging predominantly 2 months post-injury-reflect spontaneous pain, and that neuromas are the drivers of this component of neuropathic pain. In vivo dorsal root ganglion imaging showed that small-diameter sensory neurons are the source of spontaneous ectopic neuroma activity and are different from the intact neurons that drive stimulus-evoked pain. Cell-specific optogenetic stimulation studies identified that injured SSTR2 + sensory axons in neuromas are the triggers of spontaneous limb flicks/neuropathic pain. These findings reveal the mechanisms of spontaneous neuropathic pain and open new therapeutic opportunities.

The cornea is an epithelial tissue densely innervated by sensory neurons but devoid of autonomic innervation, lymphatics, and vasculature. The simplicity of the cellular composition suggests that corneal afferents participate in tissue homeostasis by regulating immune cells. Transcriptomic analysis of retrogradely labeled corneal afferents in the trigeminal ganglion (TG) found that they express many immune-related genes. Optogenetic activation of corneal afferents increased neutrophils and monocytes in both the cornea and TG, as well as inducing phenotypic changes in natural killer (NK) cells. Unsupervised pathway analysis indicated neuronally expressed Ccl2 as a modulator of immune cell responses. Selective deletion of neuronal Ccl2 decreased the number of myeloid cells in the cornea and TG in response to herpes simplex virus (HSV) infection, resulting in compromised viral clearance during primary infection. These experiments demonstrate that corneal afferent activation is sufficient to trigger inflammatory responses that can assist the host in initiating anti-viral immunity.

A key concern in early-stage analgesic discovery efforts is the extent to which mechanisms identified in rodents will translate to humans. To evaluate an alternative approach to the use of rodent dissociated DRG neurons for in vitro analyses of nociceptive signaling, we performed a transcriptomic analysis of the HD10.6 human dorsal root ganglion (DRG)-derived immortalized cell line. We conducted RNA-seq on proliferating and mature HD10.6 cells to characterize transcriptional changes associated with maturation. We then compared the transcriptomes of HD10.6 cells and several recently developed lines of human induced pluripotent stem cell-derived sensory neurons (iPSC-SN) to single-nucleus RNA-seq data from human DRGs. HD10.6 cells showed the highest correlation with 3 human sensory neuron subtypes associated with nociception and pruriception. Each of the iPSC-SN lines evaluated showed a distinct pattern of correlation with human sensory neuron subtypes. We identified G protein-coupled receptors (GPCRs) and ion channels that are expressed in both HD10.6 cells and human DRG neurons, as well as numerous genes that are expressed in human DRG but not in rodent, underscoring the need for human sensory neuron in vitro models. Proof-of-concept evaluations of protein kinase A, protein kinase C and Erk signaling provide examples of scalable assays using HD10.6 cells to investigate well-established GPCR signaling pathways. We conclude that HD10.6 cells provide a versatile model for exploring human neuronal signaling mechanisms.

Inflammatory pain results from the heightened sensitivity and reduced threshold of nociceptor sensory neurons due to exposure to inflammatory mediators. However, the cellular and transcriptional diversity of immune cell and sensory neuron types makes it challenging to decipher the immune mechanisms underlying pain. Here we used single-cell transcriptomics to determine the immune gene signatures associated with pain development in three skin inflammatory pain models in mice: zymosan injection, skin incision and ultraviolet burn. We found that macrophage and neutrophil recruitment closely mirrored the kinetics of pain development and identified cell-type-specific transcriptional programs associated with pain and its resolution. Using a comprehensive list of potential interactions mediated by receptors, ligands, ion channels and metabolites to generate injury-specific neuroimmune interactomes, we also uncovered that thrombospondin-1 upregulated by immune cells upon injury inhibited nociceptor sensitization. This study lays the groundwork for identifying the neuroimmune axes that modulate pain in diverse disease contexts.