Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.

The Mas-related G protein-coupled receptor d (Mrgprd) mediates pain hypersensitivity in painful diabetic neuropathy.

ABSTRACT: Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. Painful diabetic neuropathy is characterized by neuropathic pain accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability, axonal degeneration, and changes in cutaneous innervation. However, the complete molecular profile underlying the hyperexcitable cellular phenotype of DRG nociceptors in PDN has not been elucidated. This gap in our knowledge is a critical barrier to developing effective, mechanism-based, and disease-modifying therapeutic approaches that are urgently needed to relieve the symptoms of PDN. Using single-cell RNA sequencing of DRGs, we demonstrated an increased expression of the Mas-related G protein-coupled receptor d (Mrgprd) in a subpopulation of DRG neurons in the well-established high-fat diet (HFD) mouse model of PDN. Importantly, limiting Mrgprd signaling reversed mechanical allodynia in the HFD mouse model of PDN. Furthermore, in vivo calcium imaging allowed us to demonstrate that activation of Mrgprd-positive cutaneous afferents that persist in diabetic mice skin resulted in an increased intracellular calcium influx into DRG nociceptors that we assess in vivo as a readout of nociceptors hyperexcitability. Taken together, our data highlight a key role of Mrgprd-mediated DRG neuron excitability in the generation and maintenance of neuropathic pain in a mouse model of PDN. Hence, we propose Mrgprd as a promising and accessible target for developing effective therapeutics currently unavailable for treating neuropathic pain in PDN.

Recommendations For a Standardized Approach to Histopathologic Evaluation of Synovial Membrane in Murine Models of Experimental Osteoarthritis.

Background: Synovial pathology has been linked to osteoarthritis (OA) pain in patients. Microscopic grading systems for synovial changes in human OA have been described, but a standardized approach for murine models of OA is needed. We sought to develop a reproducible approach and set of minimum recommendations for synovial histopathology in mouse models of OA. Methods: Coronal and sagittal sections from male mouse knee joints subjected to destabilization of medial meniscus (DMM) or partial meniscectomy (PMX) were collected as part of other studies. Stains included Hematoxylin and Eosin (H&E), Toluidine Blue (T-Blue) and Safranin O/Fast Green (Saf-O). Four blinded readers graded pathological features (hyperplasia, cellularity, and fibrosis) at specific anatomic locations in the medial and lateral compartments. Inter-reader reliability of each feature was determined. Results: There was acceptable to very good agreement between raters. After DMM, increased hyperplasia and cellularity and a trend towards increased fibrosis were observed 6 weeks after DMM in the medial locations, and persisted up to 16 weeks. In the PMX model, cellularity and hyperplasia were evident in both medial and lateral compartments while fibrotic changes were largely seen on the medial side. Synovial changes were consistent from section to section in the mid-joint area mice. H&E, T-blue, and Saf-O stains resulted in comparable reliability. Conclusions: To allow for a standard evaluation that can be implemented and compared across labs and studies, we recommend using 3 readers to evaluate a minimum set of 3 pathological features at standardized anatomic areas. Pre-defining areas to be scored, and reliability for each pathologic feature should be considered.

Machine Learning Reveals Synovial Fibroblast Genes Associated with Pain Affect Sensory Nerve Growth in Rheumatoid Arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We identified a module of 815 genes associated with pain, using a novel machine learning approach, Graph-based Gene expression Module Identification (GbGMI), in samples from patients with longstanding RA, but limited synovial inflammation at arthroplasty, and validated this finding in an independent cohort of synovial biopsy samples from early, untreated RA patients. Single-cell RNA-seq analyses indicated these genes were most robustly expressed by lining layer fibroblasts and receptor-ligand interaction analysis predicted robust lining layer fibroblast crosstalk with pain sensitive CGRP+ dorsal root ganglion sensory neurons. Netrin-4, which is abundantly expressed by lining fibroblasts and associated with pain, significantly increased the branching of pain-sensitive CGRP+ neurons in vitro . We conclude GbGMI is a useful method for identifying a module of genes that associate with a clinical feature of interest. Using this approach, we find that Netrin-4 is produced by synovial fibroblasts in the absence of inflammation and can enhance the outgrowth of CGRP+ pain sensitive nerve fibers. One Sentence Summary: Machine Learning reveals synovial fibroblast genes related to pain affect sensory nerve growth in Rheumatoid Arthritis addresses unmet clinical need.

Analysis of matrisome expression patterns in murine and human dorsal root ganglia.

The extracellular matrix (ECM) is a dynamic structure of molecules that can be divided into six different categories and are collectively called the matrisome. The ECM plays pivotal roles in physiological processes in many tissues, including the nervous system. Intriguingly, alterations in ECM molecules/pathways are associated with painful human conditions and murine pain models. Nevertheless, mechanistic insight into the interplay of normal or defective ECM and pain is largely lacking. The goal of this study was to integrate bulk, single-cell, and spatial RNA sequencing (RNAseq) datasets to investigate the expression and cellular origin of matrisome genes in male and female murine and human dorsal root ganglia (DRG). Bulk RNAseq showed that about 65% of all matrisome genes were expressed in both murine and human DRG, with proportionally more core matrisome genes (glycoproteins, collagens, and proteoglycans) expressed compared to matrisome-associated genes (ECM-affiliated genes, ECM regulators, and secreted factors). Single cell RNAseq on male murine DRG revealed the cellular origin of matrisome expression. Core matrisome genes, especially collagens, were expressed by fibroblasts whereas matrisome-associated genes were primarily expressed by neurons. Cell-cell communication network analysis with CellChat software predicted an important role for collagen signaling pathways in connecting vascular cell types and nociceptors in murine tissue, which we confirmed by analysis of spatial transcriptomic data from human DRG. RNAscope in situ hybridization and immunohistochemistry demonstrated expression of collagens in fibroblasts surrounding nociceptors in male and female human DRG. Finally, comparing human neuropathic pain samples with non-pain samples also showed differential expression of matrisome genes produced by both fibroblasts and by nociceptors. This study supports the idea that the DRG matrisome may contribute to neuronal signaling in both mouse and human, and that dysregulation of matrisome genes is associated with neuropathic pain.

Sexual dimorphism of the synovial transcriptome underpins greater PTOA disease severity in male mice following joint injury.

OBJECTIVE: Osteoarthritis (OA) is a disease with sex-dependent prevalence and severity in both human and animal models. We sought to elucidate sex differences in synovitis, mechanical sensitization, structural damage, bone remodeling, and the synovial transcriptome in the anterior cruciate ligament rupture (ACLR) mouse model of post-traumatic OA (PTOA). DESIGN: Male and female 12-week-old C57/BL6J mice were randomized to Sham or noninvasive ACLR with harvests at 7d or 28d post-ACLR (n = 9 per sex in each group - Sham, 7d ACLR, 28d ACLR). Knee hyperalgesia, mechanical allodynia, and intra-articular matrix metalloproteinase (MMP) activity (via intravital imaging) were measured longitudinally. Trabecular and subchondral bone (SCB) remodeling and osteophyte formation were assessed by µCT. Histological scoring of PTOA, synovitis, and anti-MMP13 immunostaining were performed. NaV1.8-Cre;tdTomato mice were used to document localization and sprouting of nociceptors. Bulk RNA-seq of synovium in Sham, 7d, and 28d post-ACLR, and contralateral joints (n = 6 per group per sex) assessed injury-induced and sex-dependent gene expression. RESULTS: Male mice exhibited more severe joint damage at 7d and 28d and more severe synovitis at 28d, accompanied by 19% greater MMP activity, 8% lower knee hyperalgesia threshold, and 43% lower hindpaw withdrawal threshold in injured limbs compared to female injured limbs. Females had injury-induced catabolic responses in trabecular and SCB, whereas males exhibited 133% greater normalized osteophyte volume relative to females and sclerotic remodeling of trabecular and SCB. NaV1.8+ nociceptor sprouting in SCB and medial synovium was induced by injury and comparable between sexes. RNA-seq of synovium demonstrated similar injury-induced transcriptomic programs between the sexes at 7d, but only female mice exhibited a transcriptomic signature indicative of synovial inflammatory resolution by 28d, whereas males had persistent pro-inflammatory, pro-fibrotic, pro-neurogenic, and pro-angiogenic gene expression. CONCLUSION: Male mice exhibited more severe overall joint damage and pain behavior after ACLR, which was associated with persistent activation of synovial inflammatory, fibrotic, and neuroangiogenic processes, implicating persistent synovitis in driving sex differences in murine PTOA.

Sensory Profiling in Classical Ehlers-Danlos Syndrome: A Case-Control Study Revealing Pain Characteristics, Somatosensory Changes, and Impaired Pain Modulation.

Pain is one of the most important yet poorly understood complaints in heritable connective tissue disorders (HCTDs) caused by monogenic defects in extracellular matrix molecules. This is particularly the case for the Ehlers-Danlos syndrome (EDS), paradigm collagen-related disorders. This study aimed to identify the pain signature and somatosensory characteristics in the rare classical type of EDS (cEDS) caused by defects in type V or rarely type I collagen. We used static and dynamic quantitative sensory testing and validated questionnaires in 19 individuals with cEDS and 19 matched controls. Individuals with cEDS reported clinically relevant pain/discomfort (Visual Analogue Scale ≥5/10 in 32% for average pain intensity the past month) and worse health-related quality of life. An altered somatosensory profile was found in the cEDS group with higher (P = .04) detection thresholds for vibration stimuli at the lower limb, indicating hypoesthesia, reduced thermal sensitivity with more (P < .001) paradoxical thermal sensations (PTSs), and hyperalgesia with lower pain thresholds to mechanical (P < .001) stimuli at both the upper and lower limbs and cold (P = .005) stimulation at the lower limb. Using a parallel conditioned pain modulation paradigm, the cEDS group showed significantly smaller antinociceptive responses (P-value .005-.046) suggestive of impaired endogenous pain modulation. In conclusion, individuals with cEDS report chronic pain and worse health-related quality of life and present altered somatosensory perception. This study is the first to systematically investigate pain and somatosensory characteristics in a genetically defined HCTD and provides interesting insights into the possible role of the ECM in the development and persistence of pain. PERSPECTIVE: Chronic pain compromises the quality of life in individuals with cEDS. Moreover, an altered somatosensory perception was found in the cEDS group with hypoesthesia for vibration stimuli, more PTSs, hyperalgesia for pressure stimuli, and impaired pain modulation.

Notch signaling is activated in knee-innervating dorsal root ganglia in experimental models of osteoarthritis joint pain.

BACKGROUND: We aimed to explore activation of the Notch signaling pathway in knee-innervating lumbar dorsal root ganglia (DRG) in the course of experimental osteoarthritis (OA) in mice, and its role in knee hyperalgesia. METHODS: Cultured DRG cells were stimulated with the TLR4 agonist, lipopolysaccharide (LPS). Notch signaling in the cells was either inhibited with the γ-secretase inhibitor, DAPT, or with soluble Jagged1, or activated through immobilized Jagged1. CCL2 production was analyzed at mRNA and protein levels. In in vivo experiments, knee hyperalgesia was induced in naïve mice through intra-articular (IA) injection of LPS. The effect of inhibiting Notch signaling was examined by pre-injecting DAPT one hour before LPS. OA was induced through surgical destabilization of the medial meniscus (DMM) in male C57BL/6 mice. Gene expression in DRG was analyzed by qRT-PCR and RNAscope in situ hybridization. Activated Notch protein (NICD) expression in DRG was evaluated by ELISA and immunofluorescence staining. DAPT was injected IA 12 weeks post DMM to inhibit Notch signaling, followed by assessing knee hyperalgesia and CCL2 expression in the DRG. RESULTS: In DRG cell cultures, LPS increased NICD in neuronal cells. Inhibition of Notch signaling with either DAPT or soluble Jagged1 attenuated LPS-induced increases of Ccl2 mRNA and CCL2 protein. Conversely, activating Notch signaling with immobilized Jagged1 enhanced these LPS effects. In vivo, IA injection of LPS increased expression of Notch genes and NICD in the DRG. Pre-injection of DAPT prior to LPS alleviated LPS-induced knee hyperalgesia, and decreased LPS-induced CCL2 expression in the DRG. Notch signaling genes were differentially expressed in the DRG from late-stage experimental OA. Notch1, Hes1, and NICD were increased in the neuronal cell bodies in DRG after DMM surgery. IA administration of DAPT alleviated knee hyperalgesia post DMM, and decreased CCL2 expression in the DRG. CONCLUSIONS: These findings suggest a synergistic effect of Notch signaling with TLR4 in promoting CCL2 production and mediating knee hyperalgesia. Notch signaling is activated in knee-innervating lumbar DRG in mice with experimental OA, and is involved in mediating knee hyperalgesia. The pathway may therefore be explored as a target for alleviating OA pain.

Age-Associated Changes in Knee Osteoarthritis, Pain-Related Behaviors, and Dorsal Root Ganglia Immunophenotyping of Male and Female Mice.

OBJECTIVE: Osteoarthritis (OA) is a leading cause of chronic pain, yet OA pain management remains poor. Age is the strongest predictor of OA development, and mechanisms driving OA pain are unclear. We undertook this study to characterize age-associated changes in knee OA, pain-related behaviors, and dorsal root ganglion (DRG) molecular phenotypes in mice of both sexes. METHODS: Male or female C57BL/6 mice 6 or 20 months of age were evaluated for histopathologic knee OA, pain-related behaviors, and L3-L5 DRG immune characterization via flow cytometry. DRG gene expression in older mice and humans was also examined. RESULTS: Male mice at 20 months of age had worse cartilage degeneration than 6-month-old mice. Older female mouse knees showed increased cartilage degeneration but to a lesser degree than those of male mice. Older mice of both sexes had worse mechanical allodynia, knee hyperalgesia, and grip strength compared to younger mice. For both sexes, DRGs from older mice showed decreased CD45+ cells and a significant increase in F4/80+ macrophages and CD11c+ dendritic cells. Older male mouse DRGs showed increased expression of Ccl2 and Ccl5, and older female mouse DRGs showed increased Cxcr4 and Ccl3 expression compared to 6-month-old mouse DRGs, among other differentially expressed genes. Human DRG analysis from 6 individuals >80 years of age revealed elevated CCL2 in men compared to women, whereas CCL3 was higher in DRGs from women. CONCLUSION: We found that aging in male and female mice is accompanied by mild knee OA, mechanical sensitization, and changes to immune cell populations in the DRG, suggesting novel avenues for development of OA therapies.

Piezo2 expressing nociceptors mediate mechanical sensitization in experimental osteoarthritis.

Non-opioid targets are needed for addressing osteoarthritis pain, which is mechanical in nature and associated with daily activities such as walking and climbing stairs. Piezo2 has been implicated in the development of mechanical pain, but the mechanisms by which this occurs remain poorly understood, including the role of nociceptors. Here we show that nociceptor-specific Piezo2 conditional knock-out mice were protected from mechanical sensitization associated with inflammatory joint pain in female mice, joint pain associated with osteoarthritis in male mice, as well as both knee swelling and joint pain associated with repeated intra-articular injection of nerve growth factor in male mice. Single cell RNA sequencing of mouse lumbar dorsal root ganglia and in situ hybridization of mouse and human lumbar dorsal root ganglia revealed that a subset of nociceptors co-express Piezo2 and Ntrk1 (the gene that encodes the nerve growth factor receptor TrkA). These results suggest that nerve growth factor-mediated sensitization of joint nociceptors, which is critical for osteoarthritic pain, is also dependent on Piezo2, and targeting Piezo2 may represent a therapeutic option for osteoarthritis pain control.

Sensory profiling in classical Ehlers-Danlos syndrome: a case-control study revealing pain characteristics, somatosensory changes, and impaired pain modulation.

Pain is one of the most important, yet poorly understood complaints in heritable connective tissue disorders (HCTD) caused by monogenic defects in extracellular matrix molecules. This is particularly the case for Ehlers-Danlos syndromes (EDS), paradigm collagen-related disorders. This study aimed to identify the pain signature and somatosensory characteristics in the rare classical type of EDS (cEDS) caused by defects in type V or rarely type I collagen. We used static and dynamic quantitative sensory testing and validated questionnaires in 19 individuals with cEDS and 19 matched controls. Individuals with cEDS reported clinically relevant pain/discomfort (VAS ≥5/10 in 32% for average pain intensity the past month) and worse health -related quality of life. Altered sensory profile was found in the cEDS group with higher (p=0.04) detection thresholds for vibration stimuli at the lower limb indicating hypoesthesia, reduced thermal sensitivity with more (p<0.001) paradoxical thermal sensations, and hyperalgesia with lower pain thresholds to mechanical (p<0.001) stimuli at both the upper and lower limbs and to cold (p=0.005) stimulation at the lower limb. Using a parallel conditioned pain paradigm, the cEDS group showed significantly smaller antinociceptive responses (p-value between 0.005 and 0.046) suggestive of impaired endogenous central pain modulation. In conclusion, Individuals with cEDS report chronic pain and worse health-related quality of life, and present altered somatosensory perception. This study is the first to systematically investigate pain and somatosensory characteristics in a genetically defined HCTD and provides interesting insights on the possible role of the ECM in the development and persistence of pain.

The Genesis of Pain in Osteoarthritis: Inflammation as a Mediator of Osteoarthritis Pain.

Chronic pain is a substantial personal and societal burden worldwide. Osteoarthritis (OA) is one of the leading causes of chronic pain and is increasing in prevalence in accordance with a global aging population. In addition to affecting patients' physical lives, chronic pain also adversely affects patients' mental wellbeing. However, there remain no pharmacologic interventions to slow down the progression of OA and pain-alleviating therapies are largely unsuccessful. The presence of low-level inflammation in OA has been recognized for many years as a major pathogenic driver of joint damage. Inflammatory mechanisms can occur locally in joint tissues, such as the synovium, within the sensory nervous system, as well as systemically, caused by modifiable and unmodifiable factors. Understanding how inflammation may contribute to, and modify pain in OA will be instrumental in identifying new druggable targets for analgesic therapies. In this narrative review, we discuss recent insights into inflammatory mechanisms in OA pain. We discuss how local inflammation in the joint can contribute to mechanical sensitization and to the structural neuroplasticity of joint nociceptors, through pro-inflammatory factors such as nerve growth factor, cytokines, and chemokines. We consider the role of synovitis, and the amplifying mechanisms of neuroimmune interactions. We then explore emerging evidence around the role of neuroinflammation in the dorsal root ganglia and dorsal horn. Finally, we discuss how systemic inflammation associated with obesity may modify OA pain and suggest future research directions.

Mitochondrial calcium uniporter deletion prevents painful diabetic neuropathy by restoring mitochondrial morphology and dynamics.

ABSTRACT: Painful diabetic neuropathy (PDN) is an intractable complication affecting 25% of diabetic patients. Painful diabetic neuropathy is characterized by neuropathic pain accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability, resulting in calcium overload, axonal degeneration, and loss of cutaneous innervation. The molecular pathways underlying these effects are unknown. Using high-throughput and deep-proteome profiling, we found that mitochondrial fission proteins were elevated in DRG neurons from mice with PDN induced by a high-fat diet (HFD). In vivo calcium imaging revealed increased calcium signaling in DRG nociceptors from mice with PDN. Furthermore, using electron microscopy, we showed that mitochondria in DRG nociceptors had fragmented morphology as early as 2 weeks after starting HFD, preceding the onset of mechanical allodynia and small-fiber degeneration. Moreover, preventing calcium entry into the mitochondria, by selectively deleting the mitochondrial calcium uniporter from these neurons, restored normal mitochondrial morphology, prevented axonal degeneration, and reversed mechanical allodynia in the HFD mouse model of PDN. These studies suggest a molecular cascade linking neuropathic pain to axonal degeneration in PDN. In particular, nociceptor hyperexcitability and the associated increased intracellular calcium concentrations could lead to excessive calcium entry into mitochondria mediated by the mitochondrial calcium uniporter, resulting in increased calcium-dependent mitochondrial fission and ultimately contributing to small-fiber degeneration and neuropathic pain in PDN. Hence, we propose that targeting calcium entry into nociceptor mitochondria may represent a promising effective and disease-modifying therapeutic approach for this currently intractable and widespread affliction. Moreover, these results are likely to inform studies of other neurodegenerative disease involving similar underlying events.

Pain in the Ehlers-Danlos syndromes: Mechanisms, models, and challenges.

Chronic pain is one of the most common, yet poorly studied, complaints in people suffering from Ehlers-Danlos syndromes (EDS). This heterogeneous group of heritable connective tissue disorders is typically characterized by skin hyperextensibility, joint hypermobility, and generalized connective tissue fragility. Most EDS types are caused by genetic defects that affect connective tissue biosynthesis, thereby compromising collagen biosynthesis or fibrillogenesis and resulting in a disorganized extracellular matrix. Even though chronic pain is a major source of disability, functional impairment, and psychosocial suffering in EDS, currently used analgesics and other treatment strategies provide inadequate pain relief and thus represents an important unmet medical need. An important contributor to this is the lack of knowledge about the underlying mechanisms. In this narrative review, we summarize the current understanding of pain and the associated mechanisms in EDS based on clinical studies focusing on questionnaires and experimental pain testing as well as studies in animal models of EDS. In addition, we highlight the challenges, gaps, and opportunities in EDS-pain research.

Animal Models of Ehlers-Danlos Syndromes: Phenotype, Pathogenesis, and Translational Potential.

The Ehlers-Danlos syndromes (EDS) are a group of heritable connective tissues disorders mainly characterized by skin hyperextensibility, joint hypermobility and generalized tissue fragility. Currently, 14 EDS subtypes each with particular phenotypic features are recognized and are caused by genetic defects in 20 different genes. All of these genes are involved in the biosynthesis and/or fibrillogenesis of collagens at some level. Although great progress has been made in elucidating the molecular basis of different EDS subtypes, the pathogenic mechanisms underlying the observed phenotypes remain poorly understood, and consequentially, adequate treatment and management options for these conditions remain scarce. To date, several animal models, mainly mice and zebrafish, have been described with defects in 14 of the 20 hitherto known EDS-associated genes. These models have been instrumental in discerning the functions and roles of the corresponding proteins during development, maturation and repair and in portraying their roles during collagen biosynthesis and/or fibrillogenesis, for some even before their contribution to an EDS phenotype was elucidated. Additionally, extensive phenotypical characterization of these models has shown that they largely phenocopy their human counterparts, with recapitulation of several clinical hallmarks of the corresponding EDS subtype, including dermatological, cardiovascular, musculoskeletal and ocular features, as well as biomechanical and ultrastructural similarities in tissues. In this narrative review, we provide a comprehensive overview of animal models manifesting phenotypes that mimic EDS with a focus on engineered mouse and zebrafish models, and their relevance in past and future EDS research. Additionally, we briefly discuss domestic animals with naturally occurring EDS phenotypes. Collectively, these animal models have only started to reveal glimpses into the pathophysiological aspects associated with EDS and will undoubtably continue to play critical roles in EDS research due to their tremendous potential for pinpointing (common) signaling pathways, unveiling possible therapeutic targets and providing opportunities for preclinical therapeutic interventions.

Neuroimmune interactions and osteoarthritis pain: focus on macrophages.

Bidirectional interactions between the immune system and the nervous system are increasingly appreciated as playing a pathogenic role in chronic pain. Unraveling the mechanisms by which inflammatory pain is mediated through communication between nerves and immune cells may lead to exciting new strategies for therapeutic intervention. In this narrative review, we focus on the role of macrophages in the pathogenesis of osteoarthritis (OA) pain. From regulating homeostasis to conducting phagocytosis, and from inducing inflammation to resolving it, macrophages are plastic cells that are highly adaptable to their environment. They rely on communicating with the environment through cytokines, growth factors, neuropeptides, and other signals to respond to inflammation or injury. The contribution of macrophages to OA joint damage has garnered much attention in recent years. Here, we discuss how macrophages may participate in the initiation and maintenance of pain in OA. We aim to summarize what is currently known about macrophages in OA pain and identify important gaps in the field to fuel future investigations.

The role of intra-articular neuronal CCR2 receptors in knee joint pain associated with experimental osteoarthritis in mice.

BACKGROUND: C-C chemokine receptor 2 (CCR2) signaling plays a key role in pain associated with experimental murine osteoarthritis (OA) after destabilization of the medial meniscus (DMM). Here, we aimed to assess if CCR2 expressed by intra-articular sensory neurons contributes to knee hyperalgesia in the early stages of the model. METHODS: DMM surgery was performed in the right knee of 10-week-old male wild-type (WT), Ccr2 null, or Ccr2RFP C57BL/6 mice. Knee hyperalgesia was measured using a Pressure Application Measurement device. CCR2 receptor antagonist (CCR2RA) was injected systemically (i.p.) or intra-articularly (i.a.) at different times after DMM to test its ability to reverse knee hyperalgesia. In vivo Ca2+ imaging of the dorsal root ganglion (DRG) was performed to assess sensory neuron responses to CCL2 injected into the knee joint cavity. CCL2 protein in the knee was measured by ELISA. Ccr2RFP mice and immunohistochemical staining for the pan-neuronal marker, protein gene product 9.5 (PGP9.5), or the sensory neuron marker, calcitonin gene-related peptide (CGRP), were used to visualize the location of CCR2 on intra-articular afferents. RESULTS: WT, but not Ccr2 null, mice displayed knee hyperalgesia 2-16 weeks after DMM. CCR2RA administered i.p. alleviated established hyperalgesia in WT mice 4 and 8 weeks after surgery. Intra-articular injection of CCL2 excited sensory neurons in the L4-DRG, as determined by in vivo calcium imaging; responses to CCL2 increased in mice 20 weeks after DMM. CCL2, but not vehicle, injected i.a. rapidly caused transient knee hyperalgesia in naïve WT, but not Ccr2 null, mice. Intra-articular CCR2RA injection also alleviated established hyperalgesia in WT mice 4 and 7 weeks after surgery. CCL2 protein was elevated in the knees of both WT and Ccr2 null mice 4 weeks after surgery. Co-expression of CCR2 and PGP9.5 as well as CCR2 and CGRP was observed in the lateral synovium of naïve mice; co-expression was also observed in the medial compartment of knees 8 weeks after DMM. CONCLUSIONS: The findings suggest that CCL2-CCR2 signaling locally in the joint contributes to knee hyperalgesia in experimental OA, and it is in part mediated through direct stimulation of CCR2 expressed by intra-articular sensory afferents.

Basic Mechanisms of Pain in Osteoarthritis: Experimental Observations and New Perspectives.

The specific changes in the peripheral neuronal pathways underlying joint pain in osteoarthritis are the focus of this review. The plasticity of the nociceptive system in osteoarthritis and how this involves changes in the structural, physiologic, and genetic properties of neurons in pain pathways are discussed. The role of the neurotrophin, nerve growth factor, in these pathogenic processes is discussed. Finally, how neuronal pathways are modified by interaction with the degenerating joint tissues they innervate and with the innate immune system is considered. These extensive cellular interactions provide a substrate for identification of targets for osteoarthritis pain.

Pain-related behaviors and abnormal cutaneous innervation in a murine model of classical Ehlers-Danlos syndrome.

Classical Ehlers-Danlos syndrome (cEDS) is a connective tissue disorder caused by heterozygous mutations in one of the type V collagen-encoding genes, COL5A1 or COL5A2. cEDS is characterized by generalized joint hypermobility and instability, hyperextensible, fragile skin, and delayed wound healing. Chronic pain is a major problem in cEDS patients, but the underlying mechanisms are largely unknown, and studies in animal models are lacking. Therefore, we assessed pain-related behaviors in haploinsufficient Col5a1 mice, which clinically mimic human cEDS. Compared to wild-type (WT) littermates, 15 to 20-week-old Col5a1 mice of both sexes showed significant hypersensitivity to mechanical stimuli in the hind paws and the abdominal area, but responses to thermal stimuli were unaltered. Spontaneous behaviors, including distance travelled and rearing, were grossly normal in male Col5a1 mice, whereas female Col5a1 mice showed altered climbing behavior. Finally, male and female Col5a1 mice vocalized more than WT littermates when scruffed. Decreased grip strength was also noted. In view of the observed pain phenotype, Col5a1 mice were crossed with NaV1.8-tdTomato reporter mice, enabling visualization of nociceptors in the glabrous skin of the footpad. We observed a significant decrease in intraepidermal nerve fiber density, with fewer nerves crossing the epidermis, and a decreased total nerve length of Col5a1 mice compared to WT. In summary, male and female Col5a1 mice show hypersensitivity to mechanical stimuli, indicative of generalized sensitization of the nervous system, in conjunction with an aberrant organization of cutaneous nociceptors. Therefore, Col5a1 mice will provide a useful tool to study mechanisms of pain associated with cEDS.

Targeting neurotrophic factors: Novel approaches to musculoskeletal pain.

Chronic pain represents a substantial unmet medical need globally. In recent years, the quest for a new generation of novel, safe, mechanism-based analgesic treatments has focused on neurotrophic factors, a large group of secreted proteins that control the growth and survival of different populations of neurons, but that postnatally are involved in the genesis and maintenance of pain, with biological activity in both the periphery and the central nervous system. In this narrative review, we discuss the two families of neurotrophic proteins that have been extensively studied for their role in pain: first, the neurotrophins, nerve growth factor (NGF) and brain-derived growth factor (BDNF), and secondly, the GDNF family of ligands (GFLs). We provide an overview of the pain pathway, and the pain-producing effects of these different proteins. We summarize accumulating preclinical and clinical findings with a focus on musculoskeletal pain, and on osteoarthritis in particular, because the musculoskeletal system is the most prevalent source of chronic pain and of disability, and clinical testing of these novel agents - often biologics- is most advanced in this area.