A standardized approach to evaluation and reporting of synovial histopathology in two surgically induced murine models of osteoarthritis.

OBJECTIVE: 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 reporting of 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. Inter-reader agreement of each feature score was determined. RESULTS: There was acceptable to very good agreement when using 3-4 individual readers. After DMM and PMX, expected medial predominant changes in hyperplasia and cellularity were observed, with fibrosis noted at 12 weeks post-PMX. Synovial changes were consistent from section to section in the mid-joint area. When comparing stains, H&E and T-blue resulted in better agreement compared to Saf-O stain. CONCLUSIONS: To account for the pathologic and anatomic variability in synovial pathology and allow for a more standardized evaluation that can be compared across studies, we recommend evaluating a minimum set of 3 pathological features at standardized anatomic areas. Further, we suggest reporting individual feature scores separately before relying on a single summed "synovitis" score. H&E or T-blue are preferred, inter-reader agreement for each feature should be considered.

Clearing-enabled light sheet microscopy as a novel method for three-dimensional mapping of the sensory innervation of the mouse knee.

A major barrier that hampers our understanding of the precise anatomic distribution of pain sensing nerves in and around the joint is the limited view obtained from traditional two dimensional (D) histological approaches. Therefore, our objective was to develop a workflow that allows examination of the innervation of the intact mouse knee joint in 3D by employing clearing-enabled light sheet microscopy. We first surveyed existing clearing protocols (SUMIC, PEGASOS, and DISCO) to determine their ability to clear the whole mouse knee joint, and discovered that a DISCO protocol provided the most optimal transparency for light sheet microscopy imaging. We then modified the DISCO protocol to enhance binding and penetration of antibodies used for labeling nerves. Using the pan-neuronal PGP9.5 antibody, our protocol allowed 3D visualization of innervation in and around the mouse knee joint. We then implemented the workflow in mice intra-articularly injected with nerve growth factor (NGF) to determine whether changes in the nerve density can be observed. Both 3D and 2D analytical approaches of the light sheet microscopy images demonstrated quantifiable changes in midjoint nerve density following 4 weeks of NGF injection in the medial but not in the lateral joint compartment. We provide, for the first time, a comprehensive workflow that allows detailed and quantifiable examination of mouse knee joint innervation in 3D.

Reduced Capsaicin-Induced Mechanical Allodynia and Neuronal Responses in the DRG in the Presence of Ptpn6 Overexpression.

Transient Receptor Potential Vanilloid 1 (TRPV1) is a nonselective cation channel expressed by pain-sensing neurons and has been an attractive target for the development of drugs to treat pain. Recently, Src homology region 2 domain-containing phosphatase-1 (SHP-1, encoded by Ptpn6) was shown to dephosphorylate TRPV1 in dorsal root ganglia (DRG) neurons, which was linked with alleviating different pain phenotypes. These previous studies were performed in male rodents only and did not directly investigate the role of SHP-1 in TRPV-1 mediated sensitization. Therefore, our goal was to determine the impact of Ptpn6 overexpression on TRPV1-mediated neuronal responses and capsaicin-induced pain behavior in mice of both sexes. Twelve-week-old male and female mice overexpressing Ptpn6 (Shp1-Tg) and their wild type (WT) littermates were used. Ptpn6 overexpression was confirmed in the DRG of Shp1-Tg mice by RNA in situ hybridization and RT-qPCR. Trpv1 and Ptpn6 were found to be co-expressed in DRG sensory neurons in both genotypes. Functionally, this overexpression resulted in lower magnitude intracellular calcium responses to 200 nM capsaicin stimulation in DRG cultures from Shp1-Tg mice compared to WTs. In vivo, we tested the effects of Ptpn6 overexpression on capsaicin-induced pain through a model of capsaicin footpad injection. While capsaicin injection evoked nocifensive behavior (paw licking) and paw swelling in both genotypes and sexes, only WT mice developed mechanical allodynia after capsaicin injection. We observed similar level of TRPV1 protein expression in the DRG of both genotypes, however, a higher amount of tyrosine phosphorylated TRPV1 was detected in WT DRG. These experiments suggest that, while SHP-1 does not mediate the acute swelling and nocifensive behavior induced by capsaicin, it does mediate a protective effect against capsaicin-induced mechanical allodynia in both sexes. The protective effect of SHP-1 might be mediated by TRPV1 dephosphorylation in capsaicin-sensitive sensory neurons of the DRG.

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.

Charge-Reversed Exosomes for Targeted Gene Delivery to Cartilage for Osteoarthritis Treatment.

Gene therapy has the potential to facilitate targeted expression of therapeutic proteins to promote cartilage regeneration in osteoarthritis (OA). The dense, avascular, aggrecan-glycosaminoglycan (GAG) rich negatively charged cartilage, however, hinders their transport to reach chondrocytes in effective doses. While viral vector mediated gene delivery has shown promise, concerns over immunogenicity and tumorigenic side-effects persist. To address these issues, this study develops surface-modified cartilage-targeting exosomes as non-viral carriers for gene therapy. Charge-reversed cationic exosomes are engineered for mRNA delivery by anchoring cartilage targeting optimally charged arginine-rich cationic motifs into the anionic exosome bilayer by using buffer pH as a charge-reversal switch. Cationic exosomes penetrated through the full-thickness of early-stage arthritic human cartilage owing to weak-reversible ionic binding with GAGs and efficiently delivered the encapsulated eGFP mRNA to chondrocytes residing in tissue deep layers, while unmodified anionic exosomes do not. When intra-articularly injected into destabilized medial meniscus mice knees with early-stage OA, mRNA loaded charge-reversed exosomes overcame joint clearance and rapidly penetrated into cartilage, creating an intra-tissue depot and efficiently expressing eGFP; native exosomes remained unsuccessful. Cationic exosomes thus hold strong translational potential as a platform technology for cartilage-targeted non-viral delivery of any relevant mRNA targets for OA treatment.

Molecular pathogenesis of OA pain: Past, present, and future.

OBJECTIVE: To provide a historical perspective and narrative review on research into the molecular pathogenesis of osteoarthritis pain. DESIGN: PubMed databases were searched for combinations of "osteoarthritis", "pain" and "animal models" for papers that represented key phases in the history of osteoarthritis pain discovery research including epidemiology, pathology, imaging, preclinical modeling and clinical trials. RESULTS: The possible anatomical sources of osteoarthritis pain were identified over 50 years ago, but relatively slow progress has been made in understanding the apparent disconnect between structural changes captured by radiography and symptom severity. Translationally relevant animal models of osteoarthritis have aided in our understanding of the structural and molecular drivers of osteoarthritis pain, including molecules such as nerve growth factor and C-C motif chemokine ligand 2. Events leading to persistent osteoarthritis pain appear to involve a two-step process involving changes in joint innervation, including neo-innervation of the articular cartilage, as well as sensitization at the level of the joint, dorsal root ganglion and central nervous system. CONCLUSIONS: There remains a great need for the development of treatments to reduce osteoarthritis pain in patients. Harnessing all that we have learned over the past several decades is helping us to appreciate the important interaction between structural disease and pain, and this is likely to facilitate development of new disease modifying therapies in the future.

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.

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.

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.

The Evaluation of an Integrated Tobacco Treatment Specialist in Primary Care.

Background: Primary care providers play a key role in screening for tobacco use and assessing desire to quit. Tobacco treatment specialists (TTS) are certified in helping patients who desire tobacco cessation. A primary care nurse practitioner within one Midwestern healthcare organization obtained TTS certification and integrated specialized tobacco cessation visits within a primary care clinic from February 2021 to February 2022. Purpose: To determine the efficiency and effectiveness of an integrated TTS-certified nurse practitioner (TTS-NP) in a primary care setting 1-year postimplementation. Method: This program evaluation utilized retrospective electronic health record review and included thirty-three patients. The logic model served as a framework to define efficiency and effectiveness. Results: Patients were referred by a provider (57.6%), nurse (15.2%), or self (27.3). Patients opted for in-person initial visits (81.8%) more than virtual (18.2%). Of a total of 73 scheduled visits, 8 (11%) were no-showed. Patients who self-referred had the lowest no-show rate (5.6%) compared to those referred by a provider (12.8%) or nurse (12.5%). Of the patients included, 87.9% set a goal quit date. Average time until first and second follow-up was 34.6 and 130.4 days after goal quit date. Follow-up was defined as the date of the patient's first message reply to the TTS-NP, or first visit following the goal quit date. A total of 51.9% (n = 14) and 63% (n = 17) reported cessation at the first and second follow-up. TTS-NP visit's cost, independent of any other coverage, was less than other specialty visits in primary care. Conclusion: TTS-NP visits in primary care enabled patients to benefit from lower cost and longitudinal follow-up within a familiar setting. Over half of patients achieved cessation. Results of this program evaluation suggest support for TTS-certified providers in primary care.

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.

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.

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.

Targeting the tamoxifen receptor within sodium channels to block osteoarthritic pain.

Voltage-gated sodium channels (NaV) in nociceptive neurons initiate action potentials required for transmission of aberrant painful stimuli observed in osteoarthritis (OA). Targeting NaV subtypes with drugs to produce analgesic effects for OA pain management is a developing therapeutic area. Previously, we determined the receptor site for the tamoxifen analog N-desmethyltamoxifen (ND-Tam) within a prokaryotic NaV. Here, we report the pharmacology of ND-Tam against eukaryotic NaVs natively expressed in nociceptive neurons. ND-Tam and analogs occupy two conserved intracellular receptor sites in domains II and IV of NaV1.7 to block ion entry using a "bind and plug" mechanism. We find that ND-Tam inhibition of the sodium current is state dependent, conferring a potent frequency- and voltage-dependent block of hyperexcitable nociceptive neuron action potentials implicated in OA pain. When evaluated using a mouse OA pain model, ND-Tam has long-lasting efficacy, which supports the potential of repurposing ND-Tam analogs as NaV antagonists for OA pain management.

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.