Rodent Model Of Osteoarthritis Research Articles

Comparison of nociceptor properties using electrophysiology in preclinical models of osteoarthritis

Osteoarthritis (OA) pain originates in the joint by sensitization of articular nociceptors. While behavioural assessments provide valuable information regarding pain symptoms, the techniques are subjective and open to interpretation by the experimenter. This study used in vivo electrophysiological approaches to measure objectively joint nociceptor properties in three rodent models of OA. Single unit extracellular recordings of joint mechanosensitive afferents were carried out in male and female rats following either (1) transection of the medial meniscus (MMT: post-traumatic OA), (2) intra-articular injection of sodium monoiodoacetate (MIA: chemically-induced OA), or (3) intra-articular injection of lysophosphatidic acid (LPA: neuropathic OA). In naïve male control rats, the mechanical threshold of joint mechanonociceptors (23.5 ± 1.8 mNm) was significantly reduced with MMT (9.4 ± 1.1 mNm) and MIA (15.1 ± 1.6 mNm). In females, the mechanical threshold of naïve rats (23.2 ± 3.1 mNm) was reduced following induction of MMT (8.3 ± 1.0 mNm) and LPA (10.6 ± 2.2 mNm). Afferent firing frequency increased in male MMT (∼275 %), LPA (∼175 %), MIA (225 %), and female MMT (∼146 %), LPA (∼200 %), and MIA (∼192 %). Mechanical threshold and evoked firing were negatively correlated in all models for both sexes except LPA rats (male + female) and female MMT. These data indicate that MMT, MIA, and LPA induce peripheral sensitization of joint afferents thereby validating their use in OA pain studies.

Uncoupling the CRMP2-CaV2.2 interaction reduces pain-like behavior in a preclinical osteoarthritis model.

Osteoarthritis (OA) represents a significant pain challenge globally, as current treatments are limited and come with substantial and adverse side effects. Voltage-gated calcium channels have proved to be pharmacologically effective targets, with multiple FDA-approved CaV2.2 modulators available for the treatment of pain. Although effective, drugs targeting CaV2.2 are complicated by the same obstacles facing other pain therapeutics-invasive routes of administration, narrow therapeutic windows, side effects, and addiction potential. We have identified a key regulator of CaV2.2 channels, collapsing response mediator protein 2 (CRMP2), that allows us to indirectly regulate CaV2.2 expression and function. We developed a peptidomimetic modulator of CRMP2, CBD3063, that effectively reverses neuropathic and inflammatory pain without negative side effects by reducing membrane expression of CaV2.2. Using a rodent model of OA, we demonstrate the intraperitoneal administration of CBD3063 alleviates both evoked and non-evoked behavioral hallmarks of OA pain. Further, we reveal that CBD3063 reduces OA-induced increased neural activity in the parabrachial nucleus, a key supraspinal site modulating the pain experience. Together, these studies suggest CBD3063 is an effective analgesic for OA pain.

TMT quantitative proteomics reveals key proteins relevant to microRNA-1-mediated regulation in osteoarthritis

Osteoarthritis (OA) is the second-commonest arthritis, but pathogenic and regulatory mechanisms underlying OA remain incompletely understood. Here, we aimed to identify the mechanisms associated with microRNA-1 (miR-1) treatment of OA in rodent OA models using a proteomic approach. First, N = 18 Sprague Dawley (SD) rats underwent sham surgery (n = 6) or ACL transection (n = 12), followed at an interval of one week by randomization of the ACL transection group to intra-articular administration of either 50 µL placebo (control group) or miR-1 agomir, a mimic of endogenous miR-1 (experimental group). After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and immunohistochemically stained for the presence of MMP-13. Second, N = 30 Col2a1-cre-ERT2 /GFPf1/fl -RFP-miR-1 transgenic mice were randomized to intra-articular administration of either placebo (control group, N = 15) or tamoxifen, an inducer of miR-1 expression (experimental group, N = 15), before undergoing surgical disruption of the medial meniscus (DMM) after an interval of five days. After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and underwent differential proteomic analysis. Specifically, tandem mass tagging (TMT) quantitative proteomic analysis was employed to identify inter-group differentially-expressed proteins (DEP), and selected DEPs were validated using real-time quantitative polymerase chain reaction (RT-qPCR) technology. Immunohistochemically-detected MMP-13 expression was significantly lower in the experimental rat group, and proteomic analyses of mouse tissue homogenate demonstrated that of 3526 identified proteins, 345 were differentially expressed (relative up- and down-regulation) in the experimental group. Proteins Fn1, P4ha1, P4ha2, Acan, F2, Col3a1, Fga, Rps29, Rpl34, and Fgg were the *top ten most-connected proteins, implying that miR-1 may regulate an expression network involving these proteins. Of these ten proteins, three were selected for further validation by RT-qPCR: the transcript of Fn1, known to be associated with OA, exhibited relative upregulation in the experimental group, whereas the transcripts of P4ha1 and Acan exhibited relative downregulation. These proteins may thus represent key miR-1 targets during OA-regulatory mechanisms, and may provide additional insights regarding therapeutic mechanisms of miR-1 in context of OA.

Polyherbal formulation PL02 alleviates pain, inflammation, and subchondral bone deterioration in an osteoarthritis rodent model.

Osteoarthritis (OA) is a debilitating disease with significant personal and socioeconomic burdens worldwide. To address this, we developed a multitargeted formulation called PL02, which includes standardized extracts of Rosa canina L, Hippophae rhamnoides, and collagen peptide. We tested the pharmacological efficacy of PL02 in a rodent model of OA induced by Monosodium iodoacetate (MIA). Our results demonstrate that oral administration of PL02 has antioxidant effects by down-regulating NOS, reduces pain-related behavior, and mitigates inflammation by inhibiting IL-1b and TNF-α production, as well as downregulating CGRP1 and COX-II. PL02 also exhibits anti-catabolic and chondroprotective activity by significantly downregulating MMP13 and upregulating BCL2. Additionally, PL02 demonstrates chondrogenic activity by significantly upregulating SOX-9 (a master regulator of chondrogenesis), Coll-I, and aggrecan, which are major components of articular cartilage. Furthermore, PL02 prevents microarchitectural deterioration of subchondral bone. Overall, PL02 is an orally active, multi-targeted therapy that not only alleviates pain and inflammation but also effectively halts cartilage and subchondral bone deterioration. It represents a safe and promising candidate for the treatment and management of OA.

Endothelial PDGF-BB/PDGFR-β signaling promotes osteoarthritis by enhancing angiogenesis-dependent abnormal subchondral bone formation

The mechanisms that coordinate the shift from joint homeostasis to osteoarthritis (OA) remain unknown. No pharmacological intervention can currently prevent the progression of osteoarthritis. Accumulating evidence has shown that subchondral bone deterioration is a primary trigger for overlying cartilage degeneration. We previously found that H-type vessels modulate aberrant subchondral bone formation during the pathogenesis of OA. However, the mechanism responsible for the elevation of H-type vessels in OA is still unclear. Here, we found that PDGFR-β expression, predominantly in the CD31hiEmcnhi endothelium, was substantially elevated in subchondral bones from OA patients and rodent OA models. A mouse model of OA with deletion of PDGFR-β in endothelial cells (ECs) exhibited fewer H-type vessels, ameliorated subchondral bone deterioration and alleviated overlying cartilage degeneration. Endothelial PDGFR-β promotes angiogenesis through the formation of the PDGFR-β/talin1/FAK complex. Notably, endothelium-specific inhibition of PDGFR-β by local injection of AAV9 in subchondral bone effectively attenuated the pathogenesis of OA compared with that of the vehicle-treated controls. Based on the results from this study, targeting PDGFR-β is a novel and promising approach for the prevention or early treatment of OA.

Application of Formononetin for the Treatment of Knee Osteoarthritis Induced by Medial Meniscectomy in a Rodent Model

Formononetin suppresses catabolic effects in primary rat chondrocytes induced by IL-1β, which makes it a promising candidate for in vivo studies on the treatment and prevention of osteoarthritis (OA). The goal of this study is to investigate the effects of the oral administration of formononetin in a rodent model of OA. OA was induced by medial meniscectomy in the right knee joint of rats. The animals were assigned into four groups (n = 6): Vehicle (treated with saline), FNT10 (formononetin, 10 mg/kg), Ibuprofen (10 mg/kg), and Sham (simulated surgery, treated with saline). The treatment of the animals was performed daily by the oral route. After six weeks, the knee joints were removed and histologically processed. Histological sections stained in Safranin-O were used to assess the histological grading of the articular cartilage damage. An analysis of the immunohistochemical expression of type II collagen and IL-1β was also performed. The oral administration of formononetin significantly reduced cartilage-matrix-loss width (p < 0.01), degeneration scores (p < 0.05), and the total articular cartilage-wear depth (p < 0.01) in comparison with Group Vehicle. Type II collagen immunoexpression was intense and homogeneous in FNT10, comparable to that of Sham, scarce and irregularly distributed in Vehicle, and homogeneous but less intense in Ibuprofen. Furthermore, formononetin significantly reduced the immunohistochemical expression of IL-1β in joint chondrocytes (p < 0.01), but ibuprofen did not (p > 0.05). From this study, the oral administration of formononetin was found to attenuate OA-associated pathological damage in rodents, likely because of IL-1β expression downregulation in chondrocytes. These findings suggest that formononetin is a potential therapeutic for treatment.

Age alters gait compensations following meniscal injury in male rats.

With age, susceptibility to osteoarthritis (OA) and OA-related pain and disability increases. Like in OA patients, gait patterns in rodent OA models shift to protect the injured limb during loading. However, unlike in OA patients, it is unknown how age affects gait changes in rodent OA models. In this study, gait compensations following meniscal injury in 3-, 6-, and 9-month-old rats were evaluated to examine age-effects of OA-related joint dysfunction. Rats 3, 6, and 9 months received medial collateral ligament transection plus medial meniscus transection (MCLT + MMT) surgery (n = 8/age group) or a skin incision (n = 8/age group). Postsurgery, rats underwent gait testing at 2, 4, 6, and 8 weeks. Postmortem, joints were processed for histology to assess cartilage damage. MCLT + MMT rats walked with reduced vertical loading in their injured limbs immediately after injury and throughout OA progression. Compared to sham-operated limbs, 6-and 9-month MCLT + MMT animals reduced loading in their injured limbs while 3-month MCLT + MMT animals did not. MCLT + MMT rats also increased stance time on the injured limb compared to the contralateral limb. Additionally, for the MCLT + MMT animals, 6-and 9-month animals had significantly worse cartilage damage compared to 3-month animals. These data indicated age at injury onset affects how animals load the OA-affected joint, with older animals developing gait compensations that more markedly reduce weight on the injured limb during walking.

Dorsal Root Ganglia Macrophages Maintain Osteoarthritis Pain.

Pain is the major debilitating symptom of osteoarthritis (OA), which is difficult to treat. In OA patients joint tissue damage only poorly associates with pain, indicating other mechanisms contribute to OA pain. Immune cells regulate the sensory system, but little is known about the involvement of immune cells in OA pain. Here, we report that macrophages accumulate in the dorsal root ganglia (DRG) distant from the site of injury in two rodent models of OA. DRG macrophages acquired an M1-like phenotype, and depletion of DRG macrophages resolved OA pain in male and female mice. Sensory neurons innervating the damaged knee joint shape DRG macrophages into an M1-like phenotype. Persisting OA pain, accumulation of DRG macrophages, and programming of DRG macrophages into an M1-like phenotype were independent of Nav1.8 nociceptors. Inhibition of M1-like macrophages in the DRG by intrathecal injection of an IL4-IL10 fusion protein or M2-like macrophages resolved persistent OA pain. In conclusion, these findings reveal a crucial role for macrophages in maintaining OA pain independent of the joint damage and suggest a new direction to treat OA pain.SIGNIFICANCE STATEMENT In OA patients pain poorly correlates with joint tissue changes indicating mechanisms other than only tissue damage that cause pain in OA. We identified that DRG containing the somata of sensory neurons innervating the damaged knee are infiltrated with macrophages that are shaped into an M1-like phenotype by sensory neurons. We show that these DRG macrophages actively maintain OA pain remotely and independent of joint damage. The phenotype of these macrophages is crucial for a pain-promoting role. Targeting the phenotype of DRG macrophages with either M2-like macrophages or a cytokine fusion protein that skews macrophages into an M2-like phenotype resolves OA pain. Our work reveals a mechanism that contributes to the maintenance of OA pain distant from the affected knee joint and suggests that dorsal root ganglia macrophages are a target to treat osteoarthritis chronic pain.

Evaluation of electroacupuncture for symptom modification in a rodent model of spontaneous osteoarthritis.

Faced with the frustration of chronic discomfort and restricted mobility due to osteoarthritis (OA), many individuals have turned to acupuncture for relief. However, the efficacy of acupuncture for OA is uncertain, as much of the evidence is inconclusive. The purpose of this study was to evaluate electroacupuncture (EA) in a rodent model of OA such that conclusions regarding its effectiveness for symptom or disease modification could be drawn. Ten 12-month-old male Hartley guinea pigs-which characteristically have moderate to advanced OA at this age-were randomly assigned to receive EA for knee OA (n = 5) or anesthesia only (control group, n = 5). Treatments were performed three times weekly for 3 weeks, followed by euthanasia 2 weeks later. Gait analysis and enclosure monitoring were performed weekly to evaluate changes in movement. Serum was collected for inflammatory biomarker testing. Knee joints were collected for histology and gene expression. Animals receiving EA had significantly greater changes in movement parameters compared to those receiving anesthesia only. There was a tendency toward decreased serum protein concentrations of complement component 3 (C3) in the EA group compared to the control group. Structural and antioxidant gene transcripts in articular cartilage were increased by EA. There was no significant difference in total joint histology scores between groups. This study provides evidence that EA has a positive effect on symptom, but not disease, modification in a rodent model of OA. Further investigations into mechanistic pathways that may explain the efficacy of EA in this animal model are needed.

Measures of cardiovascular function suggest autonomic nervous system dysregulation in the male lewis rat after surgical induction of joint injury

Purpose: A recent review entitled “The brain-joint axis in osteoarthritis: nerves, circadian clocks and beyond” by Prof. Francis Berenbaum provides a framework for the contribution of the autonomic nervous system in osteoarthritis (OA). The autonomic nervous system is comprised of two branches, the parasympathetic (PNS) and sympathetic nervous systems (SNS) with reciprocal effects on involuntary actions throughout the body, such as heart rate and blood pressure. In homeostasis, the PNS and SNS balance these body functions, and serve as important regulators of immune system homeostasis and allostasis. An imbalance between the PNS and SNS functions is known as autonomic dysregulation. Because the two branches have reciprocal effects on heart rate, heart rate parameters may be used as an indicator of autonomic dysregulation. Autonomic balance is dysregulated in chronic inflammatory diseases, such as rheumatoid arthritis, hypertension, and diabetes, among others. To be clear, the etiologies of each of these diseases are different, but the commonality is inflammation that fails to resolve. Similarly, OA presents with chronic inflammation, but OA-related inflammation is low-grade and local, often failing to alter circulating levels of cytokines. Despite the emerging evidence for autonomic nervous system dysregulation in inflammatory joint conditions, changes have not yet been characterized in a rodent model of OA. Methods: Radiotelemetry probes were implanted into the descending aorta to collect blood pressure measurements from which the heart rate was derived from the peaks in pulse pressure. Heart rate was evaluated as an indirect assessment of vagal nerve function, a key part of the autonomic nervous system. Measurements were collected for 5 minutes each hour for 24 hours. Recordings occurred at baseline and every other week for 8 weeks after induction of OA via medial collateral ligament transection plus medial meniscus transection (MCLT+MMT; n=13) or MCLT alone (n=5), with skin incision (n=9) used as a control. Because stress is known to exaggerate autonomic responses, characteristics of heart rate responses to novel environment stress were also assessed (n=4-8/group). A 5-HT3a agonist, 1-phenylbiguanide, with reported vagal activation properties was used to indirectly assess vagal nerve function. Heart rate and blood pressure responses to the 5-HT3a agonist were recorded in anesthetized rats (n=5-8/group). Heart rate responses to mechanical stimuli applied at the knee via a weight and pulley system were used to indirectly assess cardiac vagal responses. Results: Longitudinal heart rate measurements revealed decrease in mean heart rate over time (Fig. 1a; p=0.037, week main effect) in all groups. Additionally, MCLT and MCLT+MMT had a tendency for a more rapid decline in heart rate over the 8 week study period (p=0.082, surgery*week interaction). Introduction to novel environment produced a typical increase in heart rate in all groups, confirming a stressful stimulus. This response was significantly different between groups (Figure 1b; p=0.045, surgery main effect), with a trend of attenuation in MCLT (p=0.067) and MCLT + MMT (p=0.09) groups. Mechanical stimuli at the knee resulted in a sudden drop in heart rate and blood pressure, with no differences seen between groups (Fig. 2a & 2b). However, the 5-HT3a agonist produced a dose-dependent drop in heart rate in all groups (p<0.001; dose main effect) with a larger drop in heart rate in both MCLT (p=0.014) and MCLT+MMT (p=0.010) animals at the highest dose compared to control (Fig. 2c & 2d).Figure 2Heart rate and blood pressure responses to mechanical stimulation at the knee (a,b) and systemic administration of a 5-HT3a agonist (c,d). Sudden drops in heart rate and blood pressure were observed with mechanical stimulation at the knee, with no differences seen between groups. Responses to the 5-HT3a agonist were dose-dependent with MCLT and MCLT+MMT groups having a larger drop in heart rate at the highest dose (bar indicates p<0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conclusions: Measurement of cardiovascular responses to novelty stress and pharmacological stimulation of 5-HT3a receptors, which are reportedly able to induce acute cardiac vagal responses, demonstrate for the first time a shift in ANS function in rodent knee injury models of OA. Our data suggest a sensitization of cardiac vagal activation to acute stress and 5-HT3a agonist in OA groups, which was also supported by the tendency for a more prominent chronic reduction in heart rate in OA groups. Lastly, acute mechanical stimulation of the knee produced an immediate reduction in heart rate, suggesting a direct neural joint-brain connection that may modulate autonomic responses in OA. Although we did not observe a difference in these responses between control and OA groups, further studies should delineate this using direct measurements of vagal nerve activity in response to mechanical stimulus of the joint in OA.

Use of fabricated microparticles to achieve a slow and constant inhibition of the chemokine receptor 2 to decrease cartilage and bone damage in a mouse ptoa model

Purpose: Nearly half of subjects sustaining significant damage to ligaments, menisci, or articular surfaces will develop osteoarthritis (OA). After injury, clinically measurable OA can require decades to become symptomatic, therefore early diagnosis and intervention is thought to be crucial to slow or prevent subsequent OA. CCR2 has been recognized as an important potential target in OA: CCR2, its ligands CCL12 (a.k.a. MCP-5) and CCL2 (a.k.a. MCP-1, the major human circulating ligand), are significantly increased in both rodent models of OA and in humans with OA.

Cartilage specific deletion of chemokine receptor 2 has a temporal action on joint damage during injury induced osteoarthritis

Purpose: Increased expression of chemokines in cartilage, synovial membrane and subchondral bone are believed to be linked to osteoarthritis (OA). CCR2 has been recognized as an important potential target in OA: CCR2, its ligands CCL12 (a.k.a. MCP-5) and CCL2 (a.k.a. MCP-1, the major human circulating ligand), are significantly increased in both rodent models of OA and in humans with OA. In addition, they have been shown to mediate OA pain. In previous clinical studies, we determined that CCL2 serum levels at baseline were significantly associated with radiographic knee OA progression and joint space narrowing at 5-year follow up.

Kruppel-like factor 4 upregulates matrix metalloproteinase 13 expression in chondrocytes via mRNA stabilization.

Matrix metalloproteinase 13 (MMP13) is indispensable for normal skeletal development and is also a principal proteinase responsible for articular joint pathologies. MMP13 mRNA level needs to be tightly regulated in both positive and negative manners to achieve normal development and also to prevent joint destruction. We showed previously that Kruppel-like factor 4 (KLF4) strongly induces the expression of members of the MMP family of genes including that for MMP13 in cultured chondrocytes. Through expression-based screening of approximately 400 compounds, we identified several that efficiently downregulated MMP13 gene expression induced by KLF4. Compounds grouped as topoisomerase inhibitors (transcriptional inhibitors) downregulated MMP13 expression levels, which proved the validity of our screening method. In this screening, trichostatin A (TSA) was identified as one of the most potent repressors. Mechanistically, increased MMP13 mRNA levels induced by KLF4 were not mainly caused by increased rates of RNA polymerase II-mediated MMP13 transcription, but arose from escaping mRNA decay. TSA treatment almost completely blunted the effect of KLF4. Importantly, KLF4 was detected in chondrocytes at the joint destruction sites in a rodent model of osteoarthritis. Our results partially explain how KLF4 regulates numerous proteinase gene expressions simultaneously in chondrocytes. Also, these observations suggest that modulation of KLF4 activity or expression could be a novel therapeutic target for osteoarthritis.

Periostin interaction with discoidin domain receptor-1 (DDR1) promotes cartilage degeneration.

Osteoarthritis (OA) is characterized by progressive loss of articular cartilage accompanied by the new bone formation and, often, a synovial proliferation that culminates in pain, loss of joint function, and disability. However, the cellular and molecular mechanisms of OA progression and the relative contributions of cartilage, bone, and synovium remain unclear. We recently found that the extracellular matrix (ECM) protein periostin (Postn, or osteoblast-specific factor, OSF-2) is expressed at high levels in human OA cartilage. Multiple groups have also reported elevated expression of Postn in several rodent models of OA. We have previously reported that in vitro Postn promotes collagen and proteoglycan degradation in human chondrocytes through AKT/β-catenin signaling and downstream activation of MMP-13 and ADAMTS4 expression. Here we show that Postn induces collagen and proteoglycan degradation in cartilage by signaling through discoidin domain receptor-1 (DDR1), a receptor tyrosine kinase. The genetic deficiency or pharmacological inhibition of DDR1 in mouse chondrocytes blocks Postn-induced MMP-13 expression. These data show that Postn is signaling though DDR1 is mechanistically involved in OA pathophysiology. Specific inhibitors of DDR1 may provide therapeutic opportunities to treat OA.

In-vivo inducible tissue-specific deletion of chemokine receptor-2 in mouse cartilage decreases joint damage during injury-induced osteoarthritis

Purpose: Increased expression of chemokines in cartilage, synovial membrane and subchondral bone are believed to be linked to osteoarthritis (OA). CCR2 has been recognized as an important potential target in OA: CCR2, its ligands CCL12 (a.k.a. MCP-5) and CCL2 (a.k.a. MCP-1, the major human circulating ligand), are significantly increased in both rodent models of OA and in humans with OA. In addition, they have been shown to mediate OA pain. In previous clinical studies, we determined that CCL2 serum levels at baseline were significantly associated with radiographic knee OA progression and joint space narrowing at 5-year follow up. Using the destabilization of medial meniscus (DMM) murine model of injury-induced OA, we demonstrated that targeting the CCR2 signaling at early OA stages had beneficial effects on OA progression and that there was a disconnect between structure and pain that has been noted in human OA. We therefore hypothesize that alterations in joint mechanics upregulate the CCR2 pathway in cartilage and bone in a time-dependent manner altering the structural organization of both tissues, ultimately leading to OA and pain. In the present study, by using a temporal and tissue specific inactivation of CCR2 in cartilage, we determine the role of CCR2 signaling in-vivo at sequential times during injury-induced OA in order to establish the contribution of CCR2 to the development of whole joint degeneration. Methods: We obtained an inducible tissue-specific CCR2 deletion in chondrocytes by combining chondrocyte-specific expression of CreER with Tamoxifen (Tam) injections in CCR2flx/flxeGFP mice crossed with Aggrecan-CreERT2 (CCR2AggERKO). The Cre-mediated recombination deletes CCR2 exon3, generating a functional knockout, and activates the expression of eGFP under the CCR2 locus. Therefore, recombination can be validated by GFP expression. CCR2AggERKO and CCR2flx/flx mice were injected with Tamoxifen (40μg/g) for 5 days and subjected to DMM 1 week after the last injection. Mice were euthanized at 2, 4, 8 and 12 weeks after DMM and knees were prepared for histological evaluation (Safranin-O/Fast Green, H&E) and immunohistochemical (IHC) analyses with anti-GFP, anti-Collagen10 and anti-osterix antibodies). Results: We successfully inactivated the CCR2 receptor in the cartilage tissue, as shown by the intense GFP expression in the cartilage tissues of CCR2AggERKO mice compared to controls. The expression of CCR2 in other tissues is not altered by the recombination. Histological evaluation shows that deletion of CCR2 in cartilage tissue decreases articular cartilage structure score and Safranin-O staining score at 4, 8 and 12 weeks post-surgery compared to controls. In addition, at both 8 and 12 weeks post surgery, protein levels of Collagen 10 are significantly higher in DMM-CCR2AggERKO mice while almost absent in DMM-controls, were the whole cartilage is replaced by bone. Conclusions: Our results show that we were able to selectively inhibit the CCR2 expression in chondrocytes during OA progression, while its expression was unchanged in other joint tissues. We demonstrated that the CCR2 deletion in cartilage slows cartilage damage at the mild, moderate and severe OA stage compared to controls. Other studies are currently in progress in our lab to determine whether CCR2 deletion in cartilage indirectly affect the bone tissue as well. Behavioral pain analyses are also in progress to determine whether CCR2 cartilage deletion alleviate pain responses.

Advanced oxidation protein products increase TNF-α and IL-1β expression in chondrocytes via NADPH oxidase 4 and accelerate cartilage degeneration in osteoarthritis progression

Interleukin (IL)-1β and tumor necrosis factor (TNF)-α, in particular, control the degeneration of articular cartilage, making them prime targets for osteoarthritis (OA) therapeutic strategies. Advanced oxidation protein products (AOPPs) are prevalent in numerous diseases. Our previous work demonstrates that intra-articular injections of AOPPs accelerate regression of cartilage in OA models. Whether AOPPs exist in the course of OA and their effects on TNF-α and IL-1β expression in chondrocytes are still unclear. This study confirmed that AOPPs levels in human synovial fluid were positively associated with severity of OA. We also found AOPPs deposition in articular cartilage in anterior cruciate ligament transection (ACLT) induced rodent OA models. AOPPs increased expression of TNF-α and IL-1β in chondrocytes in vitro, which was inhibited by pre-treatment with SB202190 (p38-MAPK inhibitor) or apocynin (NADPH oxidase inhibitor) or NOX4 knockdown by siRNAs. Subsequently, we further verified in vivo that exogenous injection of AOPPs in OA mice up-regulated expression of TNF-α and IL-1β in cartilage, which was blocked by treatment with apocynin. In parallel, apocynin attenuated articular cartilage degeneration resulting in substantially lower OARSI scores. Specifically, apocynin reduced NOX4, p-P38, TNF-α and IL-1β and increased collagen II and glycosaminoglycan (GAG). This study demonstrated that AOPPs increased expression of TNF-α and IL-1β in chondrocytes via the NADPH oxidase4-dependent and p38-MAPK mediated pathway, and accelerated cartilage degeneration in OA progression. These findings suggest an endogenous pathogenic role of AOPPs in OA progression. Targeting AOPPs-triggered cellular mechanisms might be a promising therapeutic option for patients with OA.

The polyadenylation inhibitor cordycepin reduces pain, inflammation and joint pathology in rodent models of osteoarthritis

Clinically, osteoarthritis (OA) pain is significantly associated with synovial inflammation. Identification of the mechanisms driving inflammation could reveal new targets to relieve this prevalent pain state. Herein, a role of polyadenylation in OA synovial samples was investigated, and the potential of the polyadenylation inhibitor cordycepin (3’ deoxyadenosine) to inhibit inflammation as well as to reduce pain and structural OA progression were studied. Joint tissues from people with OA with high or low grade inflammation and non-arthritic post-mortem controls were analysed for the polyadenylation factor CPSF4 and inflammatory markers. Effects of cordycepin on pain behavior and joint pathology were studied in models of OA (intra-articular injection of monosodium iodoacetate in rats and surgical destabilisation of the medial meniscus in mice). Human monocyte-derived macrophages and a mouse macrophage cell line were used to determine effects of cordycepin on nuclear localisation of the inflammatory transcription factor NFĸB and polyadenylation factors (WDR33 and CPSF4). CPSF4 and NFκB expression were increased in synovia from OA patients with high grade inflammation. Cordycepin reduced pain behaviour, synovial inflammation and joint pathology in both OA models. Stimulation of macrophages induced nuclear localisation of NFĸB and polyadenylation factors, effects inhibited by cordycepin. Knockdown of polyadenylation factors also prevented nuclear localisation of NFĸB. The increased expression of polyadenylation factors in OA synovia indicates a new target for analgesia treatments. This is supported by the finding that polyadenylation factors are required for inflammation in macrophages and by the fact that the polyadenylation inhibitor cordycepin attenuates pain and pathology in models of OA.

Anti‐nerve growth factor monoclonal antibodies for the control of pain in dogs and cats

Nerve growth factor (NGF) is essential for the survival of sensory and sympathetic neurons during development. However, in the adult, NGF and its interaction with tropomyosin receptor kinase A receptor (TrkA) has been found to play a critical role in nociception and nervous system plasticity in pain conditions. Thus, various monoclonal antibody (mAb) therapies targeting this pathway have been investigated in the development of new pharmacotherapies for chronic pain. Although none of the mAbs against NGF are yet approved for use in humans, they look very promising for the effective control of pain. Recently, species-specific anti-NGF mAbs for the management of osteoarthritis (OA)-associated pain in dogs and cats has been developed, and early clinical trials have been conducted. Anti-NGF therapy looks to be both very effective and very promising as a novel therapy against chronic pain in dogs and cats. This review outlines the mechanism of action of NGF, the role of NGF in osteoarthritis, research in rodent OA models and the current status of the development of anti-NGF mAbs in humans. Furthermore, we describe and discuss the recent development of species-specific anti-NGF mAbs for the treatment of OA-associated pain in veterinary medicine.

Dopamine-melanin nanoparticles scavenge reactive oxygen and nitrogen species and activate autophagy for osteoarthritis therapy.

Anti-oxidative agents hold great potential in osteoarthritis (OA) therapy. However, most radical scavengers have poor biocompatibility and potential cytotoxicity, which limit their applications. Herein we explore dopamine melanin (DM) nanoparticles as a novel scavenger of reactive oxygen species (ROS) and reactive nitrogen species (RNS). DM nanoparticles show low cytotoxicity and a strong ability to sequester a broad range of ROS and RNS, including superoxides, hydroxyl radicals, and peroxynitrite. This translates to excellent anti-inflammatory and chondro-protective effects by inhibiting intracellular ROS and RNS and promoting antioxidant enzyme activities. With an average diameter of 112.5 nm, DM nanoparticles can be intra-articularly (i.a.) injected into an affected joint and retained at the injection site. When tested in vivo in rodent OA models, DM nanoparticles showed diminished inflammatory cytokine release and reduced proteoglycan loss, which in turn slowed down cartilage degradation. Mechanistic studies suggest that DM nanoparticles also enhance autophagy that benefits OA control. In summary, our study suggests DM nanoparticles as a safe and promising therapeutic for OA.

Differential contributions of peripheral and central mechanisms to pain in a rodent model of osteoarthritis

The mechanisms underlying the transition from acute nociceptive pain to centrally maintained chronic pain are not clear. We have studied the contributions of the peripheral and central nervous systems during the development of osteoarthritis (OA) pain. Male Sprague-Dawley rats received unilateral intra-articular injections of monosodium iodoacetate (MIA 1 mg) or saline, and weight-bearing (WB) asymmetry and distal allodynia measured. Subgroups of rats received intra-articular injections of, QX-314 (membrane impermeable local anaesthetic) + capsaicin, QX-314, capsaicin or vehicle on days 7, 14 or 28 post-MIA and WB and PWT remeasured. On days 7&14 post-MIA, but not day 28, QX-314 + capsaicin signficantly attenuated changes in WB induced by MIA, illustrating a crucial role for TRPV1 expressing nociceptors in early OA pain. The role of top-down control of spinal excitability was investigated. The mu-opioid receptor agonist DAMGO was microinjected into the rostroventral medulla, to activate endogenous pain modulatory systems, in MIA and control rats and reflex excitability measured using electromyography. DAMGO (3 ng) had a significantly larger inhibitory effect in MIA treated rats than in controls. These data show distinct temporal contribtuions of TRPV1 expressing nociceptors and opioidergic pain control systems at later timepoints.