Neurobiology of Pain

Nociceptive pain is the most common pain condition, and moderate-to-severe nociceptive pain substantially impacts daily functioning, constituting a significant public health burden. Despite this, most studies investigating the neural mechanisms underlying somatosensory processing and inhibition have focused on other pain conditions (e.g., neuropathic, nociplastic, or mixed pain). Thus, the extent to which neural aberrancies detected in these other populations extend to or differentiate from nociceptive pain conditions remains largely unknown. In this study, 29 individuals with moderate-to-severe nociceptive pain (MSNP) and 47 pain-free (PF) controls underwent magnetoencephalography (MEG) alongside a paired-pulse somatosensory stimulation paradigm to examine somatosensory cortical processing and functional inhibition. Pain status and intensity were determined using validated pain questionnaires, painDETECT and PROMIS-29, respectively. MEG oscillatory responses were source localized via a beamformer to the primary somatosensory cortex (S1) and time series data were extracted from the peak voxel to quantify the dynamics of somatosensory gating (SG; index of cortical inhibitory processing), oscillatory response power, and spontaneous power. We found that adults with MSNP exhibit aberrant theta SG in contralateral S1 compared to PF controls, reflecting reduced functional inhibition of innocuous stimulus processing in this region. Additionally, individuals with MSNP demonstrated exaggerated gamma responses but blunted alpha responses in contralateral S1 to innocuous stimulation. Finally, individuals with MSNP were characterized by weaker spontaneous alpha in contralateral S1 that scaled with self-reported pain intensity. Together, these findings suggest that experiencing MSNP is associated with disrupted somatosensory and cortical inhibitory processing.

Spinal cord injury (SCI) results in dysfunction of both motor and sensory systems, which can be characterized by neuropathic pain, hypersensitivity, muscular spasticity and rigidity. Most SCIs result from incidents such as vehicle accidents or falls, resulting in polytraumatic SCI that includes peripheral injuries in addition to direct CNS damage. Recent findings suggest that spinal cord synaptic plasticity plays a crucial role in neuropathic pain pathophysiology, specifically in association with spinal sensitization and the consequent onset of AMPA-related maladaptive plasticity. Further findings have demonstrated that nociceptive peripheral stimulation in the acute phase of SCI results in maladaptive spinal synaptic plasticity by overdriving GluA2-lacking calcium-permeable AMPARs (CP-AMPARs). Here, we investigated the effect of a spared nerve injury (SNI) in conjunction with SCI to determine the effect of polytraumatic SCI on maladaptive plasticity in the spinal cord. Near-IR quantitative Western blot analysis demonstrated that SCI+SNI increases spinal GluA1 expression, but not GluA2. Patch-clamp confirmed that AMPAR currents in spinal motorneurons increase after SCI with SNI, and decrease after the administration of NASPM, a CP-AMPAR antagonist. Data-driven analysis using non-linear principal components analysis (NL-PCA) also demonstrated that SCI with SNI produces a multivariate signature of AMPAR plasticity that is observed in other forms of nociceptive peripheral input, indicating a general mechanism for maladaptive plasticity in spinal motor systems in response to polytraumatic SCI.

Low back pain is a prevalent issue with few reliable treatments. Although there is great variation in clinical presentation within the low back pain population, little is known about the neurobiological mechanisms underlying these differences. In this study, we sought to stratify chronic low back pain patients (N = 275) into phenotypes characterized by correlated patterns of resting-state brain activity and sensory abnormalities (pain, numbness, and pins and needles) indicated on hand-drawn body maps. Our cross-decomposition analysis yielded phenotypes that resemble previously documented mechanistic pain types, revealing distinct brain connectivity patterns associated with different clinical presentations. Our model was then used to predict pain body maps from fMRI data in a small novel dataset of chronic pain subjects, suggesting that these relationships may generalize to other chronic pain conditions. Our results support the utility of resting-state fMRI in understanding the heterogeneity of chronic pain, which may be leveraged to develop more targeted pain treatments.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common and painful side effect of paclitaxel (PTX) treatment. The most common measures of painful neuropathy focus on evoked mechanical hypersensitivity, but clinically relevant ongoing pain remains understudied in preclinical models. Automated machine learning methods for pose estimation and behavioral classification have been proposed to capture non-evoked pain-like behaviors, though these approaches have primarily been applied to unilateral injury models such as spared nerve injury or unilateral inflammatory compound injection. Here, we evaluated the extent to which paclitaxel-induced CIPN affects the posture and spontaneous behavior of freely moving mice using a commercially available automated recording system (BlackBox). We found that paclitaxel-treated mice develop a broad and reproducible behavioral and postural phenotype relative to vehicle-treated controls, characterized by reduced front paw luminance and print size, increased front paw lifting, and altered body measurements consistent with a guarded posture. This phenotype was replicated across two independent cohorts and was detectable at both day 2 and day 6 following the final paclitaxel injection. To identify behavioral features specific to CIPN, we administered gabapentin, an analgesic often used to treat neuropathic pain in patients, to determine whether paclitaxel-induced behavioral changes could be attenuated. Gabapentin reduced several behavioral features in both paclitaxel-treated and vehicle-treated animals, suggesting that its effects on posture and gait are not specific pain in CIPN. These findings demonstrate that automated behavioral recording captures a robust paclitaxel-induced postural phenotype but question whether captured behaviors are indicative of ongoing pain as alleviated by gabapentin.

Cerebral Palsy (CP) is the most common motor disability in childhood, and the most frequent comorbidity is pain. Rabbit kits subjected to prenatal hypoxia-ischemia (HI) exhibit allodynia and an expansion of nociceptive afferents in the lumbar spinal cord at postnatal day (P5). In this study, we examined how HI alters the development of multiple sensory modalities and its effect on psychosocial measures and C-fiber distribution in the spinal cord. To do this, we performed an HI surgery to occlude blood flow to fetal New Zealand White rabbits for 40 minutes, or a sham surgery. We performed von Frey, Hargreaves, and a cold allodynia test at P1, P5, P11, and P18. Additionally, we performed open field, a two-texture preference test, and immunofluorescence assays at P18. HI kits exhibit altered development and allodynia in von Frey and Hargreaves and minor decreased sensitivity in cold allodynia. HI kits spend less time on the aversive side of the two-texture preference apparatus and more time in the center of an open field but a higher ratio of that time immobile. This is accompanied by changes in the distribution of C-fibers in the dorsal horn of the cervical and lumbar spinal cord. A principal components analysis revealed altered nociception and psychosocial changes are important for differentiating between control and HI kits but not distribution of C-fibers. Overall, HI rabbits kits exhibit altered sensory development, allodynia, anxiety-like behavior, and changes to the distribution of nociceptive afferents in the dorsal horn of the spinal cord.

The excitability of afferents involved in nociceptive signaling reflects the interaction of several co-expressed membrane receptors. Current studies have shown that Toll-like receptor-4 (TLR-4) signaling can exacerbate excitation evoked by transient receptor potential vanilloid type 1 (TRPV1) activity, and this interaction plays a key role in driving and sustaining facilitated pain states. The mechanism by which this potentiated TRPV1 activity secondary to TLR-4 agonism occurs in sensory neurons remains unknown, although intracellular kinase activity is a strong candidate. To address this hypothesized linkage, neuronal cell cultures prepared from dorsal root ganglia (DRG) of male wildtype (WT) and Tlr4-/- mice were used to evaluate calcium transients of neurons after capsaicin administration in culture, pre-treated for 30 minutes with the TLR-4 agonist, lipopolysaccharide (LPS). TRPV1 protein expression at the neuron surface in cultured DRG cells with or without LPS treatment was quantified by flow cytometry assay. The roles of protein kinase A (PKA) and C were assessed using selective inhibitors (KT5720 for PKA and Chelerythrine chloride for PKC) applied to WT-DRG neurons or administered in vivo by intraplantar or intrathecal injection, prior to LPS and capsaicin administration. Behavioral effects of in vivo TRPV1 activation were assessed through paw flinch responses evoked by intraplantar capsaicin injection and by hind paw tactile thresholds measured by von Frey filaments. LPS incubation in cultured DRG neurons enhances the intensity of calcium influx following TRPV1 activation in WT but not Tlr4-/ cells. The augmented calcium influx evoked by capsaicin was prevented by the inhibition of PKA but not PKC. Similarly, mice treated with LPS in the hind paw displayed greater nociceptive responding after capsaicin and increased tactile allodynia. The facilitated component was prevented by the local pre-treatment with the PKA inhibitor. Correspondingly, lumbar spinal blockade of PKA resulted in temporary reversal of hyperalgesia induced by intrathecal LPS injection in mice. Together, these results demonstrate the relevance of TLR-4 in modulating the excitability of nociceptor signaling by regulating TRPV1, thereby influencing pain transmission through PKA signaling.

Background. Pain influences motor function and has been proposed to reduce corticospinal and intracortical excitability. At the same time, performance can be maintained during pain, at the cost of increased perceived effort, a centrally generated signal reflecting resource engagement. Here, we tested whether contralateral thermal heat pain-related changes in corticospinal and intracortical excitability contribute to increased effort perception. Methods. In this preregistered transcranial magnetic stimulation (TMS) study, twenty-one healthy participants received single and paired pulse TMS at rest and during submaximal isometric right wrist flexions performed at 20% maximal peak force. Trials were conducted under a control condition or during contralateral thermal stimulation (painful or non-painful warm) applied to the left forearm. After each contraction, participants rated the intensity of their perceived effort. Corticospinal and intracortical excitability of the right wrist flexor was assessed at rest and during submaximal contractions. Results. Contralateral heat pain significantly increased perceived effort compared with the control and warm conditions. Contralateral heat pain did not reduce corticospinal or intracortical excitability. Conversely, contralateral heat pain increased corticospinal excitability, reflected primarily in decreased cortical silent period duration. Perceived effort was associated with the subjective experience of pain rather than with TMS-derived variables. Conclusions. These findings suggest that increased effort during contralateral heat pain cannot be attributed to inhibition of the primary motor cortex or the corticospinal pathway. The higher perceived effort in the presence of contralateral heat pain likely reflects the cognitive cost of pain rather than alterations in the transmission of the motor command.

Evaluation of the effectiveness of therapeutic interventions for dentine hypersensitivity is limited by a lack of standardisation and objectivity in measuring the associated pain. To address this, we investigated whether electroencephalography (EEG) can provide an objective, quantitative measure of the condition. Participants with and without dentine hypersensitivity underwent evaporative (air puff) and thermal (cooling probe) tooth stimulation during continuous recording of EEG activity. Sensitivity scores (Schiff Sensitivity score for air puff stimuli, and Visual Analogue Scale score (VAS) for thermal stimuli) were recorded, and participants' responses to the Dentine Hypersensitivity Experience Questionnaire (DHEQ) collected. There were strong positive correlations between the Schiff and VAS scores, and also between both sensitivity scores and the impact of dentine hypersensitivity on quality of life (DHEQ). Additionally, EEG data analysis revealed significant differences in event-related potentials (ERP) following evaporative stimulation between participants with different Schiff scores, and in cortical activity between traces where participants indicated discomfort and those where participants did not indicate discomfort during thermal stimulation trials. Topographical maps of EEG band power during thermal stimulation showed progressive cortical recruitment and focal activation emerging in the 3 seconds prior to indication of discomfort. Comparison of EEG band power between response and no response trials to thermal stimulation showed significantly higher delta frequency band power in response trials than in no-response trials. Peak-to-peak amplitude of cortical response during thermal stimulation correlated with DHEQ and VAS scores, and the probe temperature at which participants indicated discomfort. These findings suggest that components of EEG responses align with other measures of dentine sensitivity (DHEQ, Schiff and VAS scores) and can serve as objective neurophysiological markers for evaluating the severity of dentine hypersensitivity.

Chronic postsurgical pain (CPSP) prevalence after total knee arthroplasty (TKA) is >20%. Circulating immune biomarkers are known factors of musculoskeletal pain but poorly understood as CPSP predictors. This prospective, longitudinal study of 203 patients s/p TKA tested presurgical plasma biomarkers associated with 6-month CPSP, using promising approaches from geriatrics biomarker research: expected recovery differential (ERD; resilience outcome) and penalized, machine-learning regularization modeling (elastic net and LASSO regression). Forty-nine presurgical candidate biomarkers were considered. CPSP was operationalized using ERDs built around PROMIS pain intensity and pain interference, which quantified the difference between observed and expected recovery after accounting for demographic, comorbidity, reserve, and perioperative factors. Plasma/ERDs from ~130 patients revealed 13 biomarkers with the highest selection stability criteria, and either positive or negative (+/-) associations with ERDs. Interleukin (IL) 5 (-) and Lipopolysaccharide-Binding Protein (LBP; +) were associated with both ERDs. Unique associations with pain intensity ERD included Cytomegalovirus-Specific IgG Negative (CMV IGg-; -), Macrophage Inflammatory Protein-1 Beta (MIP1b; -), IL12p70 (-, Cluster of Differentiation 30 (sCD30;-), Interferon alpha 2a (IFN2a;+), and Leukemia Inhibitory Factor (LIF;+). Unique associations with pain interference ERD included Lipopolysaccharide (LPS;-), Activin A (-), IL8 (-), Serum Amyloid A (SAA;-), and IL7 (+). Protein-protein interaction analyses and topology motifs suggest a centralized network with higher-than-expected connectivity, involving IL5, IL7, IL8, MIP1{beta}, and IFN2a, among others. This study proposes rigorous yet feasible approaches to expedite pain biomarker research, and introduces presurgical biomarkers t0 consider in future TKA-CPSP biosignature derivation.

The kappa opioid receptor (KOR) has emerged as a promising, nonaddictive analgesic target, yet the neural mechanisms underlying KOR inhibition of pain are not entirely understood. Here, we provide converging evidence that KOR expressed on spinal neurons inhibits acute pain. We demonstrate that pharmacological inhibition of KOR-expressing neurons in the spinal cord blocks nocifensive behaviors. Conversely, chemogenetic activation of KOR-expressing spinal neurons elicits nocifensive behaviors. We then perform a series of molecular characterizations and show that excitatory KOR spinal neurons are recruited by noxious stimuli and coexpress pain-promoting neuropeptides such as Tac1 in both mouse and human. Together, these data suggest that kappa opioids inhibit pain by reducing the release of pain-promoting neuropeptides from a translationally relevant population of spinal neurons.

Background: Chronic pain is associated with impaired muscle health, but whether these changes reflect site-specific factors, broader systemic factors, or both remains unclear. The purpose of this study is to determine whether normative markers of muscle health derived from MRI show site-specific patterns in chronic pain. Methods: UK Biobank participants who underwent whole-body MRI from 2006 to 2010 were included in this retrospective cross-sectional study. The MuscleMap Toolbox quantified volume and intramuscular fat (IMF) in 42 muscles of the abdomen, pelvis, and thigh. Normative models trained on a no pain group generated muscle-specific deviations from normal (i.e., Z-scores) for single- and multi-site chronic and acute pain. Results: Of 17,843 participants, the primary site-specific analysis included 9,704 no pain, 885 single-site chronic back pain (CBP), 438 single-site chronic hip pain (CHP), and 1,315 single-site chronic knee pain (CKP) participants (n=12,342; mean age 63.7{+/-}7.5 years; 52.7% female). Additional analyses included single-site chronic neck/shoulder pain, acute pain, and multi-site chronic pain groups. In CBP, deviations were localized to abdominal muscles, with decreased volume in 6/8 and increased IMF in 6/8. In CHP, deviations were broad, with decreased volume in 3/8 of the abdominal and 14/26 of the thigh muscles, and increased IMF in 6/8 of the abdominal, 5/8 of the pelvic, and 4/26 of the thigh muscles. In CKP, deviations were localized to thigh muscles, with decreased volume in 8/26 and increased IMF in 6/26. Acute pain groups showed no significant differences except for decreased volume in one thigh muscle in acute knee pain. With each additional chronic pain site, volume decreased ({beta}=-.078;IQR:-0.100-0.051), and IMF increased ({beta}=.085;IQR:0.066-0.101). Combined Z-scores classified chronic pain groups better than chance (accuracy: 48.6%;p<.001), but not acute pain groups (accuracy: 39.0%;p=.20). Conclusions: Whole-body MRI combined with AI-driven muscle segmentation and normative modeling revealed site-specific patterns of muscle health in single-site chronic pain.

Endocytosis replenishes synaptic vesicle (SV) pools that are required for persistent transmission of chronic pain signals within nociceptive spinal circuits. The nociceptor-specific contribution of SV endocytosis to pain and the therapeutic potential of endocytosis inhibitors are unclear. We identified SV endocytosis in nociceptors as a critical driver of ongoing pain and developed a gene-based strategy to target this mechanism. Nociceptor-specific adeno-associated virus-mediated knockdown of adaptor-associated kinase 1 (AAK1) or dynamin 1 (Dnm1) in dorsal root ganglia Nav1.8-positive neurons inhibited postoperative and neuropathic hypersensitivity without affecting baseline mechanical or thermal sensitivity, locomotion or spontaneous behavior. Electrophysiological recordings from spinal neurons combined with optogenetic activation of nociceptor afferents showed that AAK1 or Dnm1 downregulation blocked the sustained synaptic transmission between nociceptors and dorsal horn neurons by disrupting SV recycling and reducing neurotransmitter release probability. Lipid nanoparticle (LNP)-encapsulated CRISPR/dCas9-repressor mRNA constructs (dCas9-R) were engineered to achieve sustained and reversible transcriptional and epigenetic repression of Aak1 or Dnm1 following intrathecal delivery. LNP-mediated gene modulation produced sustained downregulation of Aak1 or Dnm1 mRNA in sensory neurons and resulted in robust and long-lasting analgesia in preclinical models of postoperative, inflammatory, neuropathic and osteoarthritis pain without impairing acute nociception or locomotor activity. Mechanistically, targeting endocytic machinery disrupted SV recycling at nociceptor terminals, thereby reducing excitatory neurotransmission within spinal pain circuits. Together, these findings establish presynaptic endocytic regulation as a convergent mechanism underlying chronic pain and demonstrate the translational potential of LNP-delivered CRISPR/dCas9-R as a durable, non-opioid pain therapy that surmounts inherent redundancy of pain signaling mechanisms. One Sentence SummarySynaptic vesicle endocytosis in nociceptors is a critical mechanism driving ongoing pain and targeting this process with intrathecal LNP-delivered CRISPR/dCas9-mediated gene repression produces durable, non-opioid analgesia across multiple chronic pain models.

Background: Growing evidence implicates the hypothalamus as a key structure in migraine pathophysiology; however, our understanding of its precise role and of the specific nuclei involved remains limited. We combined MRI data from our laboratory with publicly available MRI datasets from OpenNeuro to examine hypothalamic subunit volumes in episodic migraine and assess the specificity of these alterations relative to chronic pain conditions. Methods: Structural MRI combined with an automated atlas-based segmentation algorithm and a discovery-replication design was employed to investigate cross-sectional volumetric differences across 5 bilateral hypothalamic subunits in two independent migraine cohorts: DS1-MIG (DS1-MIG-base, n = 111 patients, n = 35 controls) and DS2-MIG (n = 27 patients, n = 31 controls). The adjusted volumes were compared between groups using MANOVA as an omnibus test, followed by Welch t-tests to test univariate follow-up. Longitudinal volumetric changes were additionally assessed in DS1-MIG participants with available follow-up scans using linear mixed models. To assess the specificity of findings to migraine, the same pipeline was applied to two chronic pain datasets, one including patients with fibromyalgia (DS-FM, n = 33 patients, n = 33 controls) and the other including patients with trigeminal neuralgia (n = 119 patients, n = 55 controls). Results: MANOVA revealed significant multivariate group differences in the discovery and replication migraine cohorts (DS1-MIG-base: = .006; DS2-MIG: = .008). Follow-up univariate analyses identified a consistent enlargement of the left anterior-superior subunit across both cohorts (FDR = .023 in DS1-MIG-base and FDR = .046 in DS2-MIG), representing the only cross-cohort replication finding. Beyond this shared signature, DS2-MIG exhibited additional significant enlargements of the right anterior-inferior and right tubular-inferior subunits. Longitudinal analyses in DS1-MIG showed that hypothalamic subunit volumes remained broadly stable over time within both migraine patients and control participants. No significant volumetric alterations were detected in the fibromyalgia or trigeminal neuralgia cohorts, either in multivariate or univariate analyses, underscoring migraine-specific findings. Conclusions: These findings provide evidence for subunit-specific hypothalamic structural alterations in migraine localized in the left anterior hypothalamic subunit. The stability of these differences over time and their absence in other chronic pain conditions suggest a migraine-specific structural organisation of hypothalamic circuitry.

Early life stress (ELS), modeled in rodents through neonatal maternal separation (NMS), induces lasting behavioral and molecular alterations including pain hypersensitivity, anxiety-like behaviors, and cognitive deficits. While NMS disrupts the oxytocinergic system, the specific contribution of oxytocin receptor (OTR) dysfunction during critical neurodevelopmental periods remains unclear. Here, we investigated whether neonatal OTR blockade alone could recapitulate key features of the NMS phenotype. Control rats received daily injections of the selective OTR antagonist d(CH2)5-Tyr(Me)-[Orn8]-vasotocin (dOVT) during postnatal days 2-12, matching the NMS period. At adulthood, behavioral assessments revealed that control+dOVT animals exhibited mechanical and cold thermal hypersensitivity similar to NMS rats, though hot thermal sensitivity was unaffected. Anxiety-like behaviors observed in NMS animals were not reproduced by dOVT treatment. Notably, sex-specific spatial memory deficits emerged: male NMS and female control+dOVT rats showed impaired object location recognition, while females and males in their respective opposite groups remained unaffected. Molecular analyses of spinal cord tissue revealed significant downregulation of GAD65, BDNF, and CD11b in control+dOVT animals. Chloride cotransporters NKCC1 and KCC2 exhibited sexual dimorphism with opposite changes in NMS males versus females and different responses to dOVT. These expressions yet converged on an elevated NKCC1/KCC2 ratio in both sexes, indicating compromised chloride homeostasis despite sex-divergent molecular pathways. These findings demonstrate that developmental OTR dysfunction likely contributes to nociceptive and cognitive consequences of ELS, while anxiety-like phenotypes probably involve additional mechanisms. This work highlights OTR as a critical mediator of neurodevelopmental programming and a potential therapeutic target for mitigating ELS-related disorders.

Chronic pain is a major risk factor for depression, yet the molecular mechanisms underlying this comorbidity remain poorly understood, particularly in women. To address this gap, we systematically investigated sex differences in the epigenomic adaptations associated with chronic pain-induced depressive-like behaviors. Neuropathic pain was induced in the mouse using the sciatic nerve cuff model, and molecular analyses were performed in the anterior cingulate cortex (ACC), a key brain region implicated in both pain and affective processing. We profiled genome-wide DNA methylation, three histone modifications (H3K27ac, H3K4me1, and H3K27me3), and gene expression using EM-seq, Cut&Tag sequencing, and RNA-seq, respectively. Differential analyses were conducted for each molecular layer and integrated through gene co-expression network analysis. We found that chronic pain induced extensive remodeling of DNA methylation and histone modification landscapes in both sexes. Strikingly, these changes occurred at largely distinct genomic loci in males and females, revealing pronounced sex-specific epigenetic responses. Despite this divergence, the affected regions displayed similar regulatory organization, including enrichment at shared genic features, transcription factor binding sites, and chromatin profiles. Importantly, these adaptations converged on partly overlapping genes, biological pathways, and co-expression modules across sexes. The most affected gene modules were predominantly associated with synapse-related processes, consistent with previous knowledge, and were closely connected to modules enriched for epigenetic regulatory functions. Together, these findings indicate that chronic pain engages sex-specific epigenetic mechanisms that ultimately converge on common functional outcomes. Such convergence highlights the potential value of targeting sex-specific epigenetic substrates in future therapeutic strategies.

Myofascial pain syndrome (MPS) is a major cause of chronic neck pain, with tissue ischemia implicated as a contributing factor. This prospective, single-arm interventional study evaluated the analgesic effect of ultrasound-guided fascia hydrorelease (US-FHR) performed around arteries supplying the neck in patients with chronic neck MPS. Thirteen adults (median age 53.0 years; 38.5% female) underwent US-FHR targeting the perivascular fascia of either the transverse cervical or dorsal scapular artery using 2 mL of normal saline. Pain intensity was assessed by visual analog scale (VAS) at rest and during movement; disability by the 5-item Pain Disability Index, Japanese version (PDI-5-J); and arterial blood flow volume before and after the procedure. The primary outcome, pain VAS during movement, decreased from 49.0 mm (interquartile range [IQR], 44.5-64.0) at baseline to 22.0 mm (IQR, 14.5-31.5) at 15 min and 22.0 mm (IQR, 14.0-34.0) at 1 week (Hodges&-Lehmann median difference, 30.5 mm [95% CI, 24.5 to 36.5] and 28.5 mm [95% CI, 18.5 to 37.0]; both P < 0.001). Pain VAS at rest improved from 21.0 mm (IQR, 13.0-43.5) to 8.0 mm at 15 min and 1 week (median difference, 14.5 mm [95% CI, 9.0 to 24.0; P = 0.001] and 13.5 mm [95% CI, 6.0 to 21.0; P = 0.007]). PDI-5-J decreased from 17.0 (IQR, 10.5-23.0) to 13.0 (IQR, 4.0-17.5) at 1 week (median difference, 5 [95% CI, 2 to 8; P = 0.004]). Blood flow volume increased from 11.2 mL/min (IQR, 4.5-14.4) to 17.2 mL/min (IQR, 6.1-23.7) immediately after US-FHR (median difference, +4.1 mL/min [95% CI, +2.5 to +8.9; P = 0.001]), although transient. One patient experienced transient bleeding that was promptly controlled. In this single-arm feasibility study, US-FHR around the target artery was simple and safe to perform and was associated with reduced neck pain. Because the study lacked a control group, these preliminary findings should be regarded as hypothesis-generating and require confirmation in controlled trials; they may also inform the future evaluation of MPS in other anatomical regions. Trial registration: UMIN Clinical Trials Registry, UMIN000053612.

Individuals with sickle cell disease (SCD) experience intense acute episodic pain associated with vaso-occlusive events and persistent, often daily, chronic pain. Triggers for acute episodic pain include cold exposure, strenuous exercise, and hypoxia. The molecular mechanisms underlying acute pain in SCD are poorly defined. We asked whether acute pain was associated with an altered metabolomic profile in individuals with SCD. We performed untargeted metabolomics on plasma from 25 children with SCD obtained during two disease states: 1) during an acute pain episode, and 2) during baseline state of health ("baseline health"). Control plasma was analyzed from 25 race-matched healthy controls. We identified 318 dysregulated metabolites in SCD patients during baseline health relative to healthy controls. Baseline health SCD samples had elevated pyrimidine, polyamine, and methionine metabolites, whereas arginine and sphingomyelin metabolites were decreased. During acute pain, we identified 448 dysregulated metabolites relative to baseline health conditions in the same SCD patients. We found decreased amino acid metabolites and acyl-carnitines, consistent with hypoxia. Network analysis revealed eight metabolic modules that were significantly differentially correlated to healthy controls, baseline health, or SCD acute pain. Modules enriched for porphyrin metabolism were correlated with SCD during acute and baseline health conditions. Other modules identified prominent dyslipidemia during acute pain in SCD relative to baseline health and healthy controls. Furthermore, we identified a metabolic module characterized by multiple sphingomyelins that were reduced in SCD and correlated with acute pain. Our findings identify dyslipidemia and impaired oxidative metabolism as potential drivers of acute pain in SCD.

Migraine is a disabling neurological disorder that frequently co-occurs with depression and anxiety. While prior research suggests a genetic association between these conditions, little is known about the genetic relationships underlying specific migraine subtypes. Bivariate genetic correlations between migraine with depression and anxiety were estimated using Linkage-Disequilibrium Score Regression (LDSC), drawing on publicly available large-scale Genome-Wide Association Study data for these traits. In addition, PGS were constructed using the same data and applied to two target cohorts for out-of-sample prediction of migraine and its subtypes. These were a depression-enriched cohort (Australian Genetics of Depression Study; N=12,601), and an unselected population cohort (QSkin Study of Sun and Health; N=16,532). Migraine subtypes were defined according to standard criteria and comprised a broad migraine without aura phenotype and three nested subtypes: migraine with aura, and chronic migraine with and without aura. We found significant genetic correlations between migraine and depression (rg=0.29), as well as between migraine and anxiety (rg=0.32). Across both cohorts, Migraine (OR{approx}1.35) and Depression PGS (OR{approx}1.12) were significantly associated with all measures of migraine and its subtypes. Depression PGS remained significantly associated with all non-chronic migraine subtypes after controlling for migraine and anxiety PGS, suggesting an independent contribution of depression genetic risk on migraine. Anxiety PGS were significantly associated with all non-chronic migraine subtypes (OR{approx}1.09). However, these associations did not persist after adjustment for migraine and/or depression PGS. These results provide insight into the genetic relationships between migraine and its subtypes with depression and anxiety.

GPR65 is a proton-sensing G protein-coupled receptor implicated in inflammatory pain. In fibroblast-like synoviocytes (FLS), GPR65 activation promotes the release of proinflammatory cytokines capable of sensitizing sensory neurons. Following stimulation by protons, the synthetic agonist BTB09089, and the glycosphingolipid psychosine GPR65 undergoes internalization; however, the contribution of this trafficking to downstream signaling remains unclear. Using heterologous cell systems, the molecular mechanisms governing GPR65 internalization were first defined. Pharmacological and genetic inhibition of internalization revealed that intracellular trafficking is required for activation of extracellular-signal-related kinase (ERK) in the nucleus and transcriptional responses, indicating a spatially restricted signaling program originating from endosomes. The physiological relevance of this pathway was then examined in primary mouse FLS. Inhibition of endogenous GPR65 internalization reduced the ability of the conditioned media from BTB09089 stimulated FLS to sensitize dorsal root ganglia sensory neurons, thus linking receptor trafficking to pro-nociceptive function. Together these findings identify receptor internalization as a key determinant of nuclear ERK signaling and transcription downstream of GPR65 and demonstrate that endosomal signaling is required for pro-nociceptive activity of GPR65 in FLS. One-sentence summaryEndosomal internalization of GPR65 is required to coordinate gene transcription and proinflammatory cytokine production that drive neuronal sensitization.

ObjectiveLateral spinal cord stimulation (LSCS) is a promising approach for restoring somatosensory feedback in lower-limb amputees, but its spatial selectivity remains limited. Percepts often spread to unintended regions of the residual limb, and reducing electrode contact size may not improve focality. This study investigated whether the anatomical organization of lumbar dorsal rootlets (DR) imposes fundamental constraints on LSCS selectivity. ApproachAcute neurophysiology experiments were performed in six adult cats. Both LSCS and individual DR stimulation were conducted in the same animals. For DR stimulation, bipolar hook electrodes were used to stimulate individual DR, while antidromic compound action potentials (CAPs) were recorded from femoral and sciatic nerve branches instrumented with nerve cuffs. For LSCS, custom 32-contact epidural paddle electrodes were placed over the lateral surface of the spinal cord at corresponding vertebral levels. Recruitment thresholds, dynamic ranges, and response patterns were analyzed across spinal levels, and DR recruitment patterns were directly compared to those evoked by LSCS within the same animals. Main resultsA clear rostrocaudal organization was observed across spinal levels during stimulation of individual DR, with femoral branches predominantly recruited at L4-L5 and sciatic branches at L6-L7. However, no somatotopic organization was found across DR within each spinal level; individual DR frequently co-activated multiple branches within the same group, and selective recruitment could only be maintained over a narrow dynamic range (median [~]10 {micro}A). LSCS exhibited even a narrower dynamic range ([~]5 {micro}A) but closely mirrored DR recruitment patterns, indicating that LSCS activates sensory afferents in a manner determined by the organizational structure of the DR. SignificanceThese findings demonstrate that the limited spatial selectivity of LSCS can largely be attributed to the coarse organization of DR within each root level rather than due to limitations of epidural electrode design. Moving electrodes intradurally or reducing contact size further is unlikely to substantially improve focality. Instead, improving paddle stability to ensure consistent placement over the appropriate spinal levels may be a more effective strategy for enhancing percept localization.