Background/Purpose: To investigate the role of MCP-1/CCR2 in the development of pain in osteoarthritis (OA) using a mouse model, destabilization of the medial meniscus (DMM).  The protracted nature of this model enables longitudinal analysis of pain-dependent behaviors and concomitant molecular changes in the innervating dorsal root ganglia (DRG).

Methods: DMM or sham surgery was performed in the right knee of 10-week old male C57BL/6 or Ccr2 null mice (Taconic) (total number used = 225).  Pain-dependent behaviors were assessed at 0, 4, 8, and 16 weeks post surgery.  Mechanical allodynia in the hind paw was assessed with von Frey fibers.  Locomotion was assessed using a LABORAS platform.  At the same time points, innervating dorsal DRG, L3-L5, from DMM or sham-operated and age-matched naïve mice were collected for qRT-PCR of monocyte chemoattractant protein (MCP)-1 and its receptor, CCR2.  The response of DRG neurons to MCP-1 was recorded through intracellular Ca²⁺-imaging.  In brief, neurons were isolated, cultured for 3 days, and loaded with a calcium indicator dye.  The number of cells responding to MCP-1 was counted.  Cell culture supernatants were analyzed for MCP-1 protein via ELISA.   For immunohistochemistry of DRG, mice were perfused transcardially with paraformaldehyde and DRG were collected for staining with anti-F4/80 (macrophage marker).  Histopathology of the knees was evaluated according to OARSI recommendations.  

Results: Joint pathology after DMM progresses slowly over 16 weeks. We documented pain-dependent behaviors longitudinally over this period, and found two stages of OA-associated pain: early-onset mechanical allodynia progressed to week 4, and was maintained for 16 weeks.  Locomotive changes indicative of chronic pain (decreases in distance traveled and climbing) were first apparent 8 weeks after DMM, and maintained up to week 16.  These changes were reversible with buprenorphine; DRG mRNA levels of MCP-1 and CCR2 were increased compared to naïve and sham controls, peaking at week 8 post DMM (p<0.01); Exposure of DRG neurons isolated 8 weeks post DMM to MCP-1 resulted in an increased calcium mobilization response compared to naïve and sham controls, indicating a functional role for MCP-1/CCR2 signaling in DRG neurons (p<0.0001); Protein levels of MCP-1 were increased in the supernatants of these cultured DRG cells compared to naïve and sham (p<0.0001);  DRG may be infiltrated by immune cells, particularly macrophages, which may contribute to pain signaling.  Therefore, we examined changes in the DRG macrophage population following DMM and found that by week 8, macrophages infiltrated the DRG and this was maintained through week 16; Ccr2 null mice developed comparable joint damage 8 weeks post DMM to wild types, but showed altered pain behavior:  i) Similar levels of mechanical allodynia developed up to week 8, but the allodynia completely resolved by week 16 weeks; ii) Ccr2 null mice were protected from decreases in locomotion at 8 and 16 weeks.  Macrophage infiltration was not noted in Ccr2null DRG at 8 weeks post DMM.

Conclusion: These data support a role for MCP-1/CCR2 in the persistence of experimental OA-associated pain.  This pathway merits further exploration as a target for pain in OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/chemokine-c-c-motif-receptor-2-signaling-mediates-persistent-pain-in-experimental-osteoarthritis/