A mouse model of chronic primary pain that integrates clinically relevant genetic vulnerability, stress, and minor injury.

Chronic primary pain conditions (CPPCs) affect over 100 million Americans, predominantly women. They remain ineffectively treated, in large part because of a lack of valid animal models with translational relevance. Here, we characterized a CPPC mouse model that integrated clinically relevant genetic (catechol-O-methyltransferase; COMT knockdown) and environmental (stress and injury) factors. Compared with wild-type mice, Comt+/- mice undergoing repeated swim stress and molar extraction surgery intervention exhibited pronounced multisite body pain and depressive-like behavior lasting >3 months. Comt+/- mice undergoing the intervention also exhibited enhanced activity of primary afferent nociceptors innervating hindpaw and low back sites and increased plasma concentrations of norepinephrine and pro-inflammatory cytokines interleukin-6 (IL-6) and IL-17A. The pain and depressive-like behavior were of greater magnitude and longer duration (≥12 months) in females versus males. Furthermore, increases in anxiety-like behavior and IL-6 were female-specific. The effect of COMT genotype × stress interactions on pain, IL-6, and IL-17A was validated in a cohort of 549 patients with CPPCs, demonstrating clinical relevance. Last, we assessed the predictive validity of the model for analgesic screening and found that it successfully predicted the lack of efficacy of minocycline and the CB2 agonist GW842166X, which were effective in spared nerve injury and complete Freund's adjuvant models, respectively, but failed in clinical trials. Yet, pain in the CPPC model was alleviated by the beta-3 adrenergic antagonist SR59230A. Thus, the CPPC mouse model reliably recapitulates clinically and biologically relevant features of CPPCs and may be implemented to test underlying mechanisms and find new therapeutics.

Biopsychosocial Factors Associated With Pain and Pain-Related Outcomes in Adults and Children With Sickle Cell Disease: A Multivariable Analysis of the GRNDaD Multicenter Registry.

Pain is the primary symptomatic manifestation of sickle cell disease (SCD), an inherited hemoglobinopathy. The characteristics that influence pain experiences and outcomes in SCD are not fully understood. The primary objective of this study was to use multivariable modeling to examine associations of biopsychosocial variables with a disease-specific measure of pain interference known as pain impact. We conducted a secondary analysis of data from the Global Research Network for Data and Discovery national SCD registry. A total of 657 children and adults with SCD were included in the analysis. This sample was 60% female with a median age of 34 (interquartile range 26-42 years) and a chronic pain prevalence of 64%. The model accounted for 58% of the variance in pain impact. Low social (P < .001) and emotional (P < .001) functioning, increasing age (P = .004), low income (P < .001), and high acute painful episodes (P = .007) were most strongly associated with high pain impact in our multivariable model. Additionally, multivariable modeling of pain severity and physical function in 2 comparable samples of registry participants revealed that increasing age and low social functioning were also strongly associated with higher pain severity and low physical functioning. Overall, the results suggest that social and emotional functioning are more strongly associated with pain impact in individuals with SCD than previously studied biological modifiers such as SCD genotype, hemoglobin, and percentage fetal hemoglobin. Future research using longitudinally collected data is needed to confirm these findings. PERSPECTIVE: This study reveals that psychosocial (ie, social and emotional functioning) and demographic (ie, age) variables may play an important role in predicting pain and pain-related outcomes in SCD. Our findings can inform future multicenter prospective longitudinal studies aimed at identifying modifiable psychosocial predictors of adverse pain outcomes in SCD.

Higher Cardiovagal Baroreflex Sensitivity Predicts Increased Pain Outcomes After Cardiothoracic Surgery.

Excessive postoperative pain can lead to extended hospitalization and increased expenses, but factors that predict its severity are still unclear. Baroreceptor function could influence postoperative pain by modulating nociceptive processing and vagal-mediated anti-inflammatory reflexes. To investigate this relationship, we conducted a study with 55 patients undergoing minimally invasive cardiothoracic surgery to evaluate whether cardiovagal baroreflex sensitivity (BRS) can predict postoperative pain. We assessed the spontaneous cardiovagal BRS under resting pain-free conditions before surgery. We estimated postoperative pain outcomes with the Pain, Enjoyment, and General Activity scale and pressure pain thresholds on the first (POD1) and second (POD2) postoperative days and persistent pain 3 and 6 months after hospital discharge. We also measured circulating levels of relevant inflammatory biomarkers (C-reactive protein, albumin, cytokines) at baseline, POD1, and POD2 to assess the contribution of inflammation to the relationship between BRS and postoperative pain. Our mixed-effects model analysis showed a significant main effect of preoperative BRS on postoperative pain (P = .013). Linear regression analysis revealed a significant positive association between preoperative BRS and postoperative pain on POD2, even after adjusting for demographic, surgical, analgesic treatment, and psychological factors. Moreover, preoperative BRS was linked to pain interfering with general activity and enjoyment but not with other pain parameters (pain intensity and pressure pain thresholds). Preoperative BRS had modest associations with postoperative C-reactive protein and IL-10 levels, but they did not mediate its relationship with postoperative pain. These findings indicate that preoperative BRS can independently predict postoperative pain, which could serve as a modifiable criterion for optimizing postoperative pain management. PERSPECTIVE: This article shows that preoperative BRS predicts postoperative pain outcomes independently of the inflammatory response and pain sensitivity to noxious pressure stimulation. These results provide valuable insights into the role of baroreceptors in pain and suggest a helpful tool for improving postoperative pain management.

Immunotherapies in chronic pain through modulation of neuroimmune interactions.

It is generally believed that immune activation can elicit pain through production of inflammatory mediators that can activate nociceptive sensory neurons. Emerging evidence suggests that immune activation may also contribute to the resolution of pain by producing distinct pro-resolution/anti-inflammatory mediators. Recent research into the connection between the immune and nervous systems has opened new avenues for immunotherapy in pain management. This review provides an overview of the most utilized forms of immunotherapies (e.g., biologics) and highlight their potential for immune and neuronal modulation in chronic pain. Specifically, we discuss pain-related immunotherapy mechanisms that target inflammatory cytokine pathways, the PD-L1/PD-1 pathway, and the cGAS/STING pathway. This review also highlights cell-based immunotherapies targeting macrophages, T cells, neutrophils and mesenchymal stromal cells for chronic pain management.

Intrathecal administration of conditioned serum from different species resolves Chemotherapy-Induced neuropathic pain in mice via secretory exosomes.

Chemotherapy-induced peripheral neuropathy (CIPN) is the most prevalent neurological complication of chemotherapy for cancer, and has limited effective treatment options. Autologous conditioned serum (ACS) is an effective biologic therapy used by intra-articular injection for patients with osteoarthritis. However, ACS has not been systematically tested in the treatment of peripheral neuropathies such as CIPN. It has been generally assumed that the analgesic effect of this biologic therapy results from augmented concentrations of anti-inflammatory cytokines and growth factors. Here we report that a single intrathecal injection of human conditioned serum (hCS) produced long-lasting inhibition of paclitaxel chemotherapy-induced neuropathic pain (mechanical allodynia) in mice, without causing motor impairment. Strikingly, the analgesic effect of hCS in our experiments was maintained even 8 weeks after the treatment, compared with non-conditioned human serum (hNCS). Furthermore, the hCS transfer-induced pain relief in mice was fully recapitulated by rat or mouse CS transfer to mice of both sexes, indicating cross-species and cross-sex effectiveness. Mechanistically, CS treatment blocked the chemotherapy-induced glial reaction in the spinal cord and improved nerve conduction. Compared to NCS, CS contained significantly higher concentrations of anti-inflammatory and pro-resolving mediators, including IL-1Ra, TIMP-1, TGF-ß1, and resolvins D1/D2. Intrathecal injection of anti-TGF-ß1 and anti-Il-1Ra antibody transiently reversed the analgesic action of CS. Nanoparticle tracking analysis revealed that rat conditioned serum contained a significantly greater number of exosomes than NCS. Importantly, the removal of exosomes by high-speed centrifugation largely diminished the CS-produced pain relief, suggesting a critical involvement of small vesicles (exosomes) in the beneficial effects of CS. Together, our findings demonstrate that intrathecal CS produces a remarkable resolution of neuropathic pain mediated through a combination of small vesicles/exosomes and neuroimmune/neuroglial modulation.

Sensory Neuron-TRPV4 Modulates Temporomandibular Disorder Pain Via CGRP in Mice.

Temporomandibular disorder (TMD) pain that involves inflammation and injury in the temporomandibular joint (TMJ) and/or masticatory muscle is the most common form of orofacial pain. We recently found that transient receptor potential vanilloid-4 (TRPV4) in trigeminal ganglion (TG) neurons is upregulated after TMJ inflammation, and TRPV4 coexpresses with calcitonin gene-related peptide (CGRP) in TMJ-innervating TG neurons. Here, we extended these findings to determine the specific contribution of TRPV4 in TG neurons to TMD pain, and examine whether sensory neuron-TRPV4 modulates TMD pain via CGRP. In mouse models of TMJ inflammation or masseter muscle injury, sensory neuron-Trpv4 conditional knockout (cKO) mice displayed reduced pain. Coexpression of TRPV4 and CGRP in TMJ- or masseter muscle-innervating TG neurons was increased after TMJ inflammation and masseter muscle injury, respectively. Activation of TRPV4-expressing TG neurons triggered secretion of CGRP, which was associated with increased levels of CGRP in peri-TMJ tissues, masseter muscle, spinal trigeminal nucleus, and plasma in both models. Local injection of CGRP into the TMJ or masseter muscle evoked acute pain in naïve mice, while blockade of CGRP receptor attenuated pain in mouse models of TMD. These results suggest that TRPV4 in TG neurons contributes to TMD pain by potentiating CGRP secretion. PERSPECTIVE: This study demonstrates that activation of TRPV4 in TG sensory neurons drives pain by potentiating the release of pain mediator CGRP in mouse models of TMJ inflammation and masseter muscle injury. Targeting TRPV4 and CGRP may be of clinical potential in alleviating TMD pain.

Rationale and design of a multicenter randomized clinical trial of vestibulodynia: understanding pathophysiology and determining appropriate treatments (vestibulodynia: UPDATe).

BACKGROUND: Limited data are available to establish evidence-based management protocols for vestibulodynia (VBD), a chronic vulvar pain condition that affects approximately 14 million women in the U.S. For the purposes of the study, our group subdivided VBD subtypes that may benefit from different types of treatment: 1) VBD peripheral (VBD-p), characterized by pain localized to the vulvar vestibule and 2) VBD central (VBD-c), characterized by VBD alongside one or more other chronic overlapping pain conditions (e.g. irritable bowel syndrome, temporomandibular disorder, and fibromyalgia syndrome) that affect remote body regions. Here, we describe the rationale and design of an NIH-funded multicenter clinical trial comparing the effectiveness of topical and/or systemic medication for alleviating pain and normalizing pain- relevant biomarkers among women with VBD-p and VBD-c. METHODS: Participants will be randomly assigned to one of four parallel arms: peripheral treatment with 5% lidocaine + 0.5 mg/ml 0.02% oestradiol compound cream + oral placebo pill, 2) central treatment with the tricyclic antidepressant nortriptyline + placebo cream, 3) combined peripheral cream and central pill treatments, or 4) placebo cream and placebo pill. The treatment phase will last 16 weeks, with outcome measures and biomarkers assessed at 4 time points (0, 8, 16, and 24 weeks). First, we will compare the efficacy of treatments in alleviating pain using standardized tampon insertion with a numeric rating scale and self-reported pain on the short form McGill Pain Questionnaire. Next, we will compare the efficacy of treatments in improving perceived physical, mental, and sexual health using standardized questionnaires. Finally, we will measure cytokines and microRNAs in local vaginal and circulating blood samples using multiplex assays and RNA sequencing, and determine the ability of these biomarkers to predict treatment response. CONCLUSION: This is the first multicenter randomized controlled trial to evaluate the efficacy of peripherally and centrally acting medications currently used in clinical practice for treating unique VBD subtypes based on distinct clinical and biological signatures. ADMINISTRATIVE INFORMATION: Vestibulodynia UPDATe is a multi-centre, two-by-two factorial designed randomized, double-blind, placebo-controlled trial registered at clinical trials.gov (NCT03844412). This work is supported by the R01 HD096331 awarded to Drs. Nackley, Rapkin, Geller and Carey by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD).Key messagesPeripheral lidocaine and oestradiol and centrally-targeted nortriptyline medications are used for the treatment of pain in women with VBD, but there is a lack of data from well-powered RCTs.This two-by-two factorial RCT will test the efficacy of these medications in VBD subtypes characterized by distinct clinical characteristics and biomarker profiles.We hope that results will provide clinicians with scientific evidence of therapeutic efficacy in distinct VBD subtypes in an effort to direct and optimize treatment approaches.

Brain-specific genes contribute to chronic but not to acute back pain.

Introduction: Back pain is the leading cause of disability worldwide. Although most back pain cases are acute, 20% of acute pain patients experience chronic back pain symptoms. It is unclear whether acute pain and chronic pain have similar or distinct underlying genetic mechanisms. Objectives: To characterize the molecular and cellular pathways contributing to acute and chronic pain states. Methods: Cross-sectional observational genome-wide association study. Results: A total of 375,158 individuals from the UK Biobank cohort were included in the discovery of genome-wide association study. Of those, 70,633 (19%) and 32,209 (9%) individuals met the definition of chronic and acute back pain, respectively. A total of 355 single nucleotide polymorphism grouped into 13 loci reached the genome-wide significance threshold (5x10-8) for chronic back pain, but none for acute. Of these, 7 loci were replicated in the Nord-Trøndelag Health Study (HUNT) cohort (19,760 chronic low back pain cases and 28,674 pain-free controls). Single nucleotide polymorphism heritability was 4.6% (P=1.4x10-78) for chronic back pain and 0.81% (P=1.4x10-8) for acute back pain. Similar differences in heritability estimates between acute and chronic back pain were found in the HUNT cohort: 3.4% (P=0.0011) and 0.6% (P=0.851), respectively. Pathway analyses, tissue-specific heritability enrichment analyses, and epigenetic characterization suggest a substantial genetic contribution to chronic but not acute back pain from the loci predominantly expressed in the central nervous system. Conclusion: Chronic back pain is substantially more heritable than acute back pain. This heritability is mostly attributed to genes expressed in the brain.

Repurposing cancer drugs identifies kenpaullone which ameliorates pathologic pain in preclinical models via normalization of inhibitory neurotransmission.

Inhibitory GABA-ergic neurotransmission is fundamental for the adult vertebrate central nervous system and requires low chloride concentration in neurons, maintained by KCC2, a neuroprotective ion transporter that extrudes intracellular neuronal chloride. To identify Kcc2 gene expression­enhancing compounds, we screened 1057 cell growth-regulating compounds in cultured primary cortical neurons. We identified kenpaullone (KP), which enhanced Kcc2/KCC2 expression and function in cultured rodent and human neurons by inhibiting GSK3ß. KP effectively reduced pathologic pain-like behavior in mouse models of nerve injury and bone cancer. In a nerve-injury pain model, KP restored Kcc2 expression and GABA-evoked chloride reversal potential in the spinal cord dorsal horn. Delta-catenin, a phosphorylation-target of GSK3ß in neurons, activated the Kcc2 promoter via KAISO transcription factor. Transient spinal over-expression of delta-catenin mimicked KP analgesia. Our findings of a newly repurposed compound and a novel, genetically-encoded mechanism that each enhance Kcc2 gene expression enable us to re-normalize disrupted inhibitory neurotransmission through genetic re-programming.

IL-23/IL-17A/TRPV1 axis produces mechanical pain via macrophage-sensory neuron crosstalk in female mice.

Although sex dimorphism is increasingly recognized as an important factor in pain, female-specific pain signaling is not well studied. Here we report that administration of IL-23 produces mechanical pain (mechanical allodynia) in female but not male mice, and chemotherapy-induced mechanical pain is selectively impaired in female mice lacking Il23 or Il23r. IL-23-induced pain is promoted by estrogen but suppressed by androgen, suggesting an involvement of sex hormones. IL-23 requires C-fiber nociceptors and TRPV1 to produce pain but does not directly activate nociceptor neurons. Notably, IL-23 requires IL-17A release from macrophages to evoke mechanical pain in females. Low-dose IL-17A directly activates nociceptors and induces mechanical pain only in females. Finally, deletion of estrogen receptor subunit α (ERα) in TRPV1+ nociceptors abolishes IL-23- and IL-17-induced pain in females. These findings demonstrate that the IL-23/IL-17A/TRPV1 axis regulates female-specific mechanical pain via neuro-immune interactions. Our study also reveals sex dimorphism at both immune and neuronal levels.

Epithelia-Sensory Neuron Cross Talk Underlies Cholestatic Itch Induced by Lysophosphatidylcholine.

BACKGROUND & AIMS: Limited understanding of pruritus mechanisms in cholestatic liver diseases hinders development of antipruritic treatments. Previous studies implicated lysophosphatidic acid (LPA) as a potential mediator of cholestatic pruritus. METHODS: Pruritogenicity of lysophosphatidylcholine (LPC), LPA's precursor, was examined in naïve mice, cholestatic mice, and nonhuman primates. LPC's pruritogenicity involving keratinocyte TRPV4 was studied using genetic and pharmacologic approaches, cultured keratinocytes, ion channel physiology, and structural computational modeling. Activation of pruriceptor sensory neurons by microRNA-146a (miR-146a), secreted from keratinocytes, was identified by in vitro and ex vivo Ca2+ imaging assays. Sera from patients with primary biliary cholangitis were used for measuring the levels of LPC and miR-146a. RESULTS: LPC was robustly pruritic in mice. TRPV4 in skin keratinocytes was essential for LPC-induced itch and itch in mice with cholestasis. Three-dimensional structural modeling, site-directed mutagenesis, and channel function analysis suggested a TRPV4 C-terminal motif for LPC binding and channel activation. In keratinocytes, TRPV4 activation by LPC induced extracellular release of miR-146a, which activated TRPV1+ sensory neurons to cause itch. LPC and miR-146a levels were both elevated in sera of patients with primary biliary cholangitis with itch and correlated with itch intensity. Moreover, LPC and miR-146a were also increased in sera of cholestatic mice and elicited itch in nonhuman primates. CONCLUSIONS: We identified LPC as a novel cholestatic pruritogen that induces itch through epithelia-sensory neuron cross talk, whereby it directly activates skin keratinocyte TRPV4, which rapidly releases miR-146a to activate skin-innervating TRPV1+ pruriceptor sensory neurons. Our findings support the new concept of the skin, as a sensory organ, playing a critical role in cholestatic itch, beyond liver, peripheral sensory neurons, and central neural pathways supporting pruriception.

Activation of GPR37 in macrophages confers protection against infection-induced sepsis and pain-like behaviour in mice.

GPR37 was discovered more than two decades ago, but its biological functions remain poorly understood. Here we report a protective role of GPR37 in multiple models of infection and sepsis. Mice lacking Gpr37 exhibited increased death and/or hypothermia following challenge by lipopolysaccharide (LPS), Listeria bacteria, and the mouse malaria parasite Plasmodium berghei. Sepsis induced by LPS and Listeria in wild-type mice is protected by artesunate (ARU) and neuroprotectin D1 (NPD1), but the protective actions of these agents are lost in Gpr37-/- mice. Notably, we found that ARU binds to GPR37 in macrophages and promotes phagocytosis and clearance of pathogens. Moreover, ablation of macrophages potentiated infection, sepsis, and their sequelae, whereas adoptive transfer of NPD1- or ARU-primed macrophages reduced infection, sepsis, and pain-like behaviors. Our findings reveal physiological actions of ARU in host cells by activating macrophages and suggest that GPR37 agonists may help to treat sepsis, bacterial infections, and malaria.

Multi-ethnic GWAS and meta-analysis of sleep quality identify MPP6 as a novel gene that functions in sleep center neurons.

Poor sleep quality can have harmful health consequences. Although many aspects of sleep are heritable, the understandings of genetic factors involved in its physiology remain limited. Here, we performed a genome-wide association study (GWAS) using the Pittsburgh Sleep Quality Index (PSQI) in a multi-ethnic discovery cohort (n = 2868) and found two novel genome-wide loci on chromosomes 2 and 7 associated with global sleep quality. A meta-analysis in 12 independent cohorts (100 000 individuals) replicated the association on chromosome 7 between NPY and MPP6. While NPY is an important sleep gene, we tested for an independent functional role of MPP6. Expression data showed an association of this locus with both NPY and MPP6 mRNA levels in brain tissues. Moreover, knockdown of an orthologue of MPP6 in Drosophila melanogaster sleep center neurons resulted in decreased sleep duration. With convergent evidence, we describe a new locus impacting human variability in sleep quality through known NPY and novel MPP6 sleep genes.

Phenotypic profile clustering pragmatically identifies diagnostically and mechanistically informative subgroups of chronic pain patients.

ABSTRACT: Traditional classification and prognostic approaches for chronic pain conditions focus primarily on anatomically based clinical characteristics not based on underlying biopsychosocial factors contributing to perception of clinical pain and future pain trajectories. Using a supervised clustering approach in a cohort of temporomandibular disorder cases and controls from the Orofacial Pain: Prospective Evaluation and Risk Assessment study, we recently developed and validated a rapid algorithm (ROPA) to pragmatically classify chronic pain patients into 3 groups that differed in clinical pain report, biopsychosocial profiles, functional limitations, and comorbid conditions. The present aim was to examine the generalizability of this clustering procedure in 2 additional cohorts: a cohort of patients with chronic overlapping pain conditions (Complex Persistent Pain Conditions study) and a real-world clinical population of patients seeking treatment at duke innovative pain therapies. In each cohort, we applied a ROPA for cluster prediction, which requires only 4 input variables: pressure pain threshold and anxiety, depression, and somatization scales. In both complex persistent pain condition and duke innovative pain therapies, we distinguished 3 clusters, including one with more severe clinical characteristics and psychological distress. We observed strong concordance with observed cluster solutions, indicating the ROPA method allows for reliable subtyping of clinical populations with minimal patient burden. The ROPA clustering algorithm represents a rapid and valid stratification tool independent of anatomic diagnosis. ROPA holds promise in classifying patients based on pathophysiological mechanisms rather than structural or anatomical diagnoses. As such, this method of classifying patients will facilitate personalized pain medicine for patients with chronic pain.

Sex- and age-specific genetic analysis of chronic back pain.

ABSTRACT: Sex differences for chronic back pain (cBP) have been reported, with females usually exhibiting greater morbidity, severity, and poorer response to treatment. Genetic factors acting in an age-specific manner have been implicated but never comprehensively explored. We performed sex- and age-stratified genome-wide association study and single nucleotide polymorphism-by-sex interaction analysis for cBP defined as "Back pain for 3+ months" in 202,077 males and 237,754 females of European ancestry from UK Biobank. Two and 7 nonoverlapping genome-wide significant loci were identified for males and females, respectively. A male-specific locus on chromosome 10 near SPOCK2 gene was replicated in 4 independent cohorts. Four loci demonstrated single nucleotide polymorphism-by-sex interaction, although none of them were formally replicated. Single nucleotide polymorphism-explained heritability was higher in females (0.079 vs 0.067, P = 0.006). There was a high, although not complete, genetic correlation between the sexes (r = 0.838 ± 0.041, different from 1 with P = 7.8E-05). Genetic correlation between the sexes for cBP decreased with age (0.858 ± 0.049 in younger people vs 0.544 ± 0.157 in older people; P = 4.3E-05). There was a stronger genetic correlation of cBP with self-reported diagnosis of intervertebral disk degeneration in males than in females (0.889 vs 0.638; P = 3.7E-06). Thus, the genetic component of cBP in the UK Biobank exhibits a mild sex- and age-dependency. This provides an insight into the possible causes of sex- and age-specificity in epidemiology and pathophysiology of cBP and chronic pain at other anatomical sites.

Computer-aided Discovery of a New Nav1.7 Inhibitor for Treatment of Pain and Itch.

BACKGROUND: Voltage-gated sodium channel Nav1.7 has been validated as a perspective target for selective inhibitors with analgesic and anti-itch activity. The objective of this study was to discover new candidate compounds with Nav1.7 inhibitor properties. The authors hypothesized that their approach would yield at least one new compound that inhibits sodium currents in vitro and exerts analgesic and anti-itch effects in mice. METHODS: In silico structure-based similarity search of 1.5 million compounds followed by docking to the Nav1.7 voltage sensor of Domain 4 and molecular dynamics simulation was performed. Patch clamp experiments in Nav1.7-expressing human embryonic kidney 293 cells and in mouse and human dorsal root ganglion neurons were conducted to test sodium current inhibition. Formalin-induced inflammatory pain model, paclitaxel-induced neuropathic pain model, histamine-induced itch model, and mouse lymphoma model of chronic itch were used to confirm in vivo activity of the selected compound. RESULTS: After in silico screening, nine compounds were selected for experimental assessment in vitro. Of those, four compounds inhibited sodium currents in Nav1.7-expressing human embryonic kidney 293 cells by 29% or greater (P < 0.05). Compound 9 (3-(1-benzyl-1H-indol-3-yl)-3-(3-phenoxyphenyl)-N-(2-(pyrrolidin-1-yl)ethyl)propanamide, referred to as DA-0218) reduced sodium current by 80% with a 50% inhibition concentration of 0.74 µM (95% CI, 0.35 to 1.56 µM), but had no effects on Nav1.5-expressing human embryonic kidney 293 cells. In mouse and human dorsal root ganglion neurons, DA-0218 reduced sodium currents by 17% (95% CI, 6 to 28%) and 22% (95% CI, 9 to 35%), respectively. The inhibition was greatly potentiated in paclitaxel-treated mouse neurons. Intraperitoneal and intrathecal administration of the compound reduced formalin-induced phase II inflammatory pain behavior in mice by 76% (95% CI, 48 to 100%) and 80% (95% CI, 68 to 92%), respectively. Intrathecal administration of DA-0218 produced acute reduction in paclitaxel-induced mechanical allodynia, and inhibited histamine-induced acute itch and lymphoma-induced chronic itch. CONCLUSIONS: This study's computer-aided drug discovery approach yielded a new Nav1.7 inhibitor that shows analgesic and anti-pruritic activity in mouse models.

Vitamin D insufficiency increases risk of chronic pain among African Americans experiencing motor vehicle collision.

African Americans experience an increased burden of motor vehicle collision (MVC), post-MVC musculoskeletal pain, and vitamin D insufficiency. In this prospective multicenter study, we tested the hypothesis that African Americans (n = 133) presenting to the emergency department after MVC with low peritraumatic vitamin D levels would have worse chronic musculoskeletal pain outcomes compared to individuals with sufficient vitamin D. Vitamin D levels were assessed in the early aftermath of MVC through enzyme-linked immunosorbent assay, and pain severity was assessed using the 0 to 10 numeric rating scale at 6 weeks, 6 months, and 1 year. In repeated-measures analysis, African American MVC survivors with vitamin D insufficiency experienced more severe chronic pain (ß = 1.18, P = 0.031). In secondary analyses, we assessed for evidence that the effect of vitamin D on post-MVC pain outcomes is mediated, at least in part, by the influence of vitamin D on genetic variants in genes involved in immune system regulation (IL-10 and NLRP3). Genotyping was performed using a genome-wide microarray using collected DNA samples. Secondary analyses suggest that the effect of vitamin D on post-MVC pain outcomes may be influenced by genetic variation in IL-10 and NLRP3. Further studies are needed to assess the impact of vitamin D insufficiency on pain outcomes in African Americans experiencing MVC and other common trauma exposures, to assess factors affecting this relationship, and to assess the efficacy of administering vitamin D in the immediate aftermath of MVC to prevent chronic pain. Such low-cost, nonopioid interventions are urgently needed to address chronic pain development after MVC.

A study in scarlet: MC1R as the main predictor of red hair and exemplar of the flip-flop effect.

Genetic variation in melanocortin-1 receptor (MC1R) is a known contributor to disease-free red hair in humans. Three loss-of-function single-nucleotide variants (rs1805007, rs1805008 and rs1805009) have been established as strongly correlated with red hair. The contribution of other loss-of-function MC1R variants (in particular rs1805005, rs2228479 and rs885479) and the extent to which other genetic loci are involved in red hair colour is less well understood. Here, we used the UK Biobank cohort to capture a comprehensive list of MC1R variants contributing to red hair colour. We report a correlation with red hair for both strong-effect variants (rs1805007, rs1805008 and rs1805009) and weak-effect variants (rs1805005, rs2228479 and rs885479) and show that their coefficients differ by two orders of magnitude. On the haplotype level, both strong- and weak-effect variants contribute to the red hair phenotype, but when considered individually, weak-effect variants show a reverse, negative association with red hair. The reversal of association direction in the single-variant analysis is facilitated by a distinguishing structure of MC1R, in which loss-of-function variants are never found to co-occur on the same haplotype. The other previously reported hair colour genes' variants do not substantially improve the MC1R red hair colour predictive model. Our best model for predicting red versus other hair colours yields an unparalleled area under the receiver operating characteristic of 0.96 using only MC1R variants. In summary, we present a comprehensive statistically derived characterization of the role of MC1R variants in red hair colour and offer a powerful, economical and parsimonious model that achieves unsurpassed performance.

CACNG2 polymorphisms associate with chronic pain after mastectomy.

Chronic postmastectomy pain (PMP) imposes a major burden on the quality of life of the ever-increasing number of long-term survivors of breast cancer. An earlier report by Nissenbaum et al. claimed that particular polymorphisms in the gene CACNG2 are associated with the risk of developing chronic PMP after breast surgery (Nissenbaum J, Devor M, Seltzer Z, Gebauer M, Michaelis M, Tal M, Dorfman R, Abitbul-Yarkoni M, Lu Y, Elahipanah T, delCanho S, Minert A, Fried K, Persson AK, Shpigler H, Shabo E, Yakir B, Pisante A, Darvasi A. Susceptibility to chronic pain following nerve injury is genetically affected by CACNG2. Genome Res 2010;20:1180-90). This information is important because in principle, it can inform the surgical, radiological, and chemotherapeutic decision-making process in ways that could mitigate the increased risk of chronic pain. In this study, we revisited this claim by independently evaluating the proposed marker haplotype using 2 different patient cohorts recruited in different research settings. Meta-analysis of these new postmastectomy cohorts and the original cohort confirmed significant association of the CACNG2 haplotype with PMP. In addition, we tested whether the same markers would predict chronic postsurgical pain in men who underwent surgery for inguinal hernia repair, and whether there is significant genetic association with cutaneous thermal sensitivity in postmastectomy and postherniotomy patients. We found that the biomarker is selective because it did not predict pain after laparoscopic hernia repair and was not associated with pain sensitivity to experimentally applied noxious thermal stimuli. We conclude that the A-C-C haplotype at the 3 single-nucleotide polymorphisms (rs4820242, rs2284015, and rs2284017) in the CACNG2 gene is associated with increased risk of developing PMP. This information may advance current knowledge on pathophysiology of PMP and serve as a step forward in the prediction of clinical outcomes and personalized pain management.