S. Ishihara

Damage-associated molecular patterns generated in osteoarthritis directly excite murine nociceptive neurons through Toll-like receptor 4.

To determine whether selected damage‐associated molecular patterns (DAMPs) present in the osteoarthritic (OA) joints of mice excite nociceptors through Toll‐like receptor 4 (TLR‐4).

Therapeutic effects of an anti-ADAMTS-5 antibody on joint damage and mechanical allodynia in a murine model of osteoarthritis

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 to 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.

The Role of Peripheral Nociceptive Neurons in the Pathophysiology of Osteoarthritis Pain

Knee osteoarthritis is characterized by progressive damage and remodeling of all tissues in the knee joint. Pain is the main symptom associated with knee osteoarthritis. Recent clinical and pre-clinical studies have provided novel insights into the mechanisms that drive the pain associated with joint destruction. In this narrative review, we describe current knowledge regarding the changes in the peripheral and central nervous systems that occur during the progression of osteoarthritis and discuss how therapeutic interventions may provide pain relief.

Chemogenetic Inhibition of Pain Neurons in a Mouse Model of Osteoarthritis

To determine the ability of drugs that activate inhibitory G protein–coupled receptors (GPCRs) expressed in peripheral voltage‐gated sodium channel 1.8 (NaV1.8)–positive sensory neurons to control osteoarthritis (OA)–associated pain.

Spinal microglial activation in a murine surgical model of knee osteoarthritis

OBJECTIVE Microgliosis, the activation of microglial cells, is thought to contribute to synaptic transmission in the dorsal horn and thereby promote chronic pain. The primary aim of this study was to document the temporal profile of dorsal horn microgliosis after destabilization of the medial meniscus (DMM) in wild type (WT) and Adamts5 null mice. Since neuronal fractalkine (CX3CL1) contributes to microgliosis, we assessed its release from dorsal root ganglia (DRG) cultures after DMM. DESIGN DMM or sham surgery was performed in the right knee of 10-week old male WT, CX3CR1-green fluorescent protein (GFP), or Adamts5 null C57BL/6 mice. Hind paw mechanical allodynia was monitored using von Frey fibers. L4 dorsal horn microgliosis was assessed 4, 8 and 16 weeks after surgery, based on the morphology of Iba1-immunoreactive microglia. DRG cells (L3-L5) were cultured and supernatants collected for fractalkine (FKN) ELISA. RESULTS In WT mice, numbers of activated microglia were increased 8 and 16 weeks, but not 4 weeks, after DMM but not sham surgery. DRG cultures showed increased basal FKN release at 8 and 16 weeks. Adamts5 null mice did not develop mechanical allodynia up to 16 weeks after DMM. Accordingly, DRG cultures from these mice did not exhibit increased FKN release and dorsal horn microgliosis did not occur. CONCLUSION DMM surgery leads to late stage dorsal horn microgliosis. The temporal correlation with DRG FKN release suggests it may contribute to microgliosis. Reduced microgliosis in Adamts5 null mice, which are protected from joint damage and associated mechanical allodynia after DMM, suggests that microgliosis is associated with joint damage and accompanying persistent pain.

An aggrecan fragment drives osteoarthritis pain through Toll-like receptor 2

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.

Visualization of Peripheral Neuron Sensitization in a Surgical Mouse Model of Osteoarthritis by In Vivo Calcium Imaging

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).

FRI0002 Spinal Microglial Activation in A Murine Surgical Model of Knee Osteoarthritis

Background Microglial cells are the resident macrophages of the central nervous system (CNS). Their activation (termed “microgliosis”) is thought to modulate synaptic transmission in the dorsal horn, thereby promoting chronic pain. Fractalkine derived from the central termini of primary sensory afferents has been shown to contribute to microgliosis. Objectives The primary aim of this study was to document the temporal profile of dorsal horn microgliosis after destabilization of the medial meniscus (DMM) in the mouse. Further, we assessed fractalkine release from dorsal root ganglia (DRG) after DMM surgery. We evaluated these pathological events in the pain pathway in three strains of mice: wildtypes (WT), which develop slowly progressive osteoarthritis following DMM surgery, associated with sustained pain-related behaviours during the 16-week follow-up period1; Ccr2 null mice, which are protected from persistent pain in spite of joint damage1; and Adamts5 null mice, which are protected from joint damage and associated pain-related behaviours after DMM surgery2. Methods DMM or sham surgery was performed in the right knee of 10-week old male wild type (WT), Ccr2 null or Adamts5 null C57BL/6 mice. Mechanical allodynia in the hindpaw was monitored using von Frey fibers. L4 dorsal horn microgliosis was assessed 4, 8 and 16 weeks after surgery, based on the morphology of Iba1-immunoreactive (ir) microglia: Iba1-ir cells were classified as “resting” if their process length was more than double the soma diameter, and as “activated” if their process length was less than double the soma diameter3. The observer was blinded to the treatment groups. DRG cells (L3-L5) from DMM, sham or naïve control mice were cultured and supernatants collected for fractalkine ELISA. Results Four weeks after surgery in WT mice, the number of activated microglia in the L4 dorsal horn was not different between naïve and DMM mice. In contrast, 8 weeks after DMM, there was a significant increase in the number of activated microglia compared to shams. By 16 weeks after DMM, activated microglia were increased compared to age-matched naïve controls. DRG cultures from WT mice showed increased fractalkine release 8 and 16 weeks, but not 4 weeks, after DMM. In contrast, DRG cultures from Ccr2 null and Adamts5 null mice did not release increased fractalkine and L4 dorsal horn microgliosis was absent in these mice. Conclusions DMM surgery leads to late stage dorsal horn microgliosis, 8–16 weeks after surgery. This suggests that involvement of the CNS is a late event in this experimental model. Further, the temporal correlation with fractalkine release by DRG cells suggests that fractalkine may contribute to microglial activation. Finally, reduced microgliosis in Ccr2 null and Adamts5 null mice, both of which fail to display persistent pain behaviors after DMM, suggests that microgliosis is associated with the persistence of OA pain. Joint protection and prevention of peripheral sensitization may attenuate one aspect of central sensitization. References Miller RE et al, PNAS 2012; Malfait AM et al, Osteoarthritis Cart 2008; Thakur M et al. PLoS One 2012. Acknowledgement NIH, NIAMS R01-AR-064251 and R01-AR-060364 Disclosure of Interest None declared