Objective The aim of this study was to determine whether selected damage-associated molecular patterns (DAMPs) present in osteoarthritic (OA) joints excite nociceptors through toll-like receptor (TLR)-4. Methods The ability of S100A8 and α2-macroglobulin to excite nociceptors was determined by measuring: (1) Release of monocyte chemoattractant protein (MCP)-1 by cultured dorsal root ganglion (DRG) cells; (2) Intracellular calcium (Ca)i levels in cultured DRG neurons from naïve mice or mice 8 weeks after destabilization of the medial meniscus (DMM). The role of TLR4 was assessed using Tlr4−/− cells or a TLR4 inhibitor. (Ca)i levels in neurons within ex vivo intact DRG were measured using Pirt-GCaMP3 mice. Neuronal Tlr4 expression was determined by in situ hybridization. DMM surgery was performed in wild-type and Tlr4−/− mice; mechanical allodynia was monitored, and joint damage was assessed histologically after 16 weeks. Results Both naïve and DMM DRG neurons expressed Tlr4. Both S100A8 and α2-macroglobulin stimulated release of the pro-algesic chemokine, MCP-1, by DRG cultures and neurons rapidly responded to S100A8 and α2-macroglobulin with increased (Ca)i. Blocking TLR4 inhibited these effects. Neurons within intact DRG responded to the TLR4 agonist, lipopolysaccharide. In both calcium-imaging assays, it was primarily the nociceptor population of neurons that responded to TLR4 ligands. Tlr4−/− mice were not protected from mechanical allodynia or from joint damage associated with DMM. Conclusion Our experiments suggest a role for TLR4 signaling in the excitation of nociceptors by selected DAMPs. Further research is needed to delineate the importance of this pathway in relation to OA pain.

Objective The primary goal of this study was to test the disease-modifying effect of blocking a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 with a neutralizing monoclonal antibody (mAb) starting 4 weeks after destabilization of the medial meniscus (DMM) in the mouse. We also investigated whether ADAMTS-5 blockade reversed mechanical allodynia and decreased monocyte chemoattractant protein (MCP)-1 production by dorsal root ganglia (DRG) cells. Methods Ten-week old male C57BL/6 mice underwent DMM surgery and were either left untreated or treated with anti-ADAMTS-5 mAb or IgG2c isotype control mAb starting 4 weeks after surgery. Knees were collected for histopathology 4 or 12 weeks later. Mechanical allodynia was monitored biweekly in the ipsilateral hind paw through 16 weeks. DRG were collected and cultured 8 weeks after DMM for analysis of MCP-1 production. Results By 4 weeks after DMM, mild cartilage degeneration was evident in the medial compartment, small osteophytes were present, and subchondral bone sclerosis was established. By 16 weeks after surgery, significant cartilage deterioration was apparent on the medial tibial plateaux and medial femoral condyles, osteophyte size had increased, and subchondral bone sclerosis was maintained. Treatment with ADAMTS-5 mAb from week 4-16 after surgery slowed cartilage degeneration and osteophyte growth but did not affect subchondral bone sclerosis. Moreover, ADAMTS-5 blockade resulted in temporary reversal of mechanical allodynia, which correlated with decreased MCP-1 production by cultured DRG cells. Conclusions This study suggests therapeutic efficacy of an ADAMTS-5 mAb in the DMM model, when therapy starts early in disease.

Objective. To develop a method for analyzing sensory neuron responses to mechanical stimuli in vivo, and to evaluate whether these neuronal responses change after destabilization of the medial meniscus (DMM).Methods. DMM or sham surgery was performed in 10-week-old male C57BL/6 wild-type or Pirt-GCaMP3 +/-mice. All experiments were performed 8 weeks after surgery. Knee and hind paw hyperalgesia were assessed in wild-type mice. The retrograde label DiI was injected into the ipsilateral knee to quantify the number of knee-innervating neurons in the L4 dorsal root ganglion (DRG) in wild-type mice. In vivo calcium imaging was performed on the ipsilateral L4 DRG of Pirt-GCaMP3 +/-mice as mechanical stimuli (paw pinch, knee pinch, or knee twist) were applied to the ipsilateral hind limb.Results. Eight weeks after surgery, mice subjected to DMM had more hyperalgesia in the knee and hind paw compared to mice subjected to sham surgery. Intraarticular injection of DiI labeled similar numbers of neurons in the L4 DRG of mice subjected to sham surgery and mice subjected to DMM. Increased numbers of sensory neurons responded to all 3 mechanical stimuli in mice subjected to DMM, as assessed by in vivo calcium imaging. The majority of responses in mice subjected to sham surgery and mice subjected to DMM were in small to medium-sized neurons, consistent with the size of nociceptors. The magnitude of responses was similar between mice subjected to sham surgery and mice subjected to DMM.Conclusion. Our findings indicate that increased numbers of small to medium-sized DRG neurons respond to mechanical stimuli 8 weeks after DMM surgery, suggesting that nociceptors have become sensitized by lowering the response threshold.Nervous system sensitization, as determined by quantitative sensory testing, is associated with osteoarthritis (OA) and has been shown to correlate with symptom severity (1,2). A recent large cohort study demonstrated an association between the presence of inflammation in the knee and sensitization (3). In addition, a number of studies in small cohorts have suggested that following hip or knee joint replacement, sensitization is often reversed, and this is associated with symptom relief (4-6).Sensitization is also a feature of experimental OA and can be detected by evaluating pain-related behaviors in animals, including mechanical allodynia and mechanical hyperalgesia of the hind limb (for review, see ref. 7). Previous work has shown that after destabilization of the medial meniscus (DMM), mice develop slowly progressive joint damage concurrent with pain-related behaviors indicative of sensitization, including hind paw mechanical allodynia and knee hyperalgesia, which develop prior to the manifestation of spontaneous pain behaviors such as locomotive deficits (8-11). In order to develop targeted analgesic strategies, the neuronal mechanisms that mediate this sensitization to mechanical stimuli need to be defined. Therefore, we sought to develop a method for analyzing peripheral sensory neuron activity in response t...

Pain is the predominant symptom of osteoarthritis, but the connection between joint damage and the genesis of pain is not well understood. Loss of articular cartilage is a hallmark of osteoarthritis, and it occurs through enzymatic degradation of aggrecan by cleavage mediated by a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS-4) or ADAMTS-5 in the interglobular domain (E373-374A). Further cleavage by MMPs (N341-342F) releases a 32-amino-acid aggrecan fragment (32-mer). We investigated the role of this 32-mer in driving joint pain. We found that the 32-mer excites dorsal root ganglion nociceptive neurons, both in culture and in intact explants. Treatment of cultured sensory neurons with the 32-mer induced expression of the proalgesic chemokine CCL2. These effects were mediated through TLR2, which we demonstrated was expressed by nociceptive neurons. In addition, intra-articular injection of the 32-mer fragment provoked knee hyperalgesia in WT but not Tlr2-null mice. Blocking the production or action of the 32-mer in transgenic mice prevented the development of knee hyperalgesia in a murine model of osteoarthritis. These findings suggest that the aggrecan 32-mer fragment directly activates TLR2 on joint nociceptors and is an important mediator of the development of osteoarthritis-associated joint pain.

Objective The purpose of this study was to determine the ability of drugs that activate inhibitory G-protein coupled receptors (GPCRs) expressed in peripheral NaV1.8-positive sensory neurons to control osteoarthritis associated pain. Therefore, we used Designer Receptors Exclusively Activated by a Designer Drug (DREADD) technology, which utilizes engineered GPCRs to activate or inhibit neurons upon binding the synthetic ligand, clozapine-N-oxide. Methods NaV1.8-Pdi C57BL/6 mice were created to express the inhibitory DREADD receptor, Pdi, in NaV1.8-expressing sensory neurons. Destabilization of the medial meniscus (DMM) was performed in 10-week old male mice. Four, 8, 12 or 16 weeks after surgery, knee hyperalgesia or hindpaw mechanical allodynia were tested. Subsequently, clozapine-N-oxide or vehicle was administered and the effect on behaviors was measured by a blinded observer. Morphine was used as a control. Results Immunohistochemistry and electrophysiology confirmed functional expression of the Pdi receptor by NaV1.8-positive sensory neurons. Acute inhibition of NaV1.8-expressing neurons in mice treated with clozapine-N-oxide reduced knee hyperalgesia 4 weeks after DMM and mechanical allodynia 8 weeks after DMM. Inhibition had no effect on behaviors 12 and 16 weeks after DMM. Morphine, a drug that activates GPCRs in the peripheral and central nervous systems, was still effective in the later stage. Conclusions Chemogenetic inhibition of NaV1.8-expressing neurons blocks knee hyperalgesia and mechanical allodynia in early experimental osteoarthritis, but is no longer efficacious in the later stages. These data indicate that activation of inhibitory GPCRs located solely outside the central nervous system may be ineffective in treating chronic osteoarthritis pain.