Background/Purpose: The aim of this study was to explore pain-related behaviors and associated cellular changes in the pain pathway in experimental osteoarthritis (OA) induced by destabilization of the medial meniscus (DMM). In wild-type (WT) mice, DMM results in slowly progressive knee OA, associated with pain behaviors, including mechanical allodynia and decreased locomotion. We have previously reported that CCR2 signaling is a key mediator for persistence of pain after DMM. Specifically, 8 weeks after DMM, L3-L5 dorsal root ganglia (DRG) neurons show increased expression of CCR2 gene and protein. Ccr2 null mice develop initial mechanical allodynia following DMM, but the allodynia is not maintained (unlike in WT mice). WT and Ccr2null mice develop the same extent of joint damage after DMM.

Methods: DMM or sham surgery was performed in the right knees of 10-week old male WT or Ccr2 null C57BL/6 mice. The elevated plus maze was employed as a method for assessing anxiety-related responses (time spent grooming; time spent immobile) over a period of 5 minutes. To assess cellular changes in the peripheral and central nervous system, we quantified (1) macrophages in the L4 DRG, by immunostaining for F4/80 and (2) activated microglia in the L4 dorsal horn of the spinal cord, based on the morphology of Iba1-immunoreactive microglia, using established methods.

Results: Eight weeks after sham or DMM surgery, WT mice showed no behavioral changes in the elevated maze plus test. However, by 16 weeks after surgery, DMM (but not sham) operated mice showed behaviors indicative of anxiety, including increased periods of immobility (p=0.002, n=5-13) and increased time spent grooming (p=0.0073, n=6-15). These behaviors did not develop in Ccr2 null mice (p>0.9, n=8-14).

In WT mice, L4 DRG were strongly infiltrated with F4/80 expressing macrophages, 8 and 16 weeks after DMM. In contrast, in Ccr2 null mice, L4 DRG showed some macrophage infiltration at 8 weeks, but to a lesser extent than in WT mice. Furthermore, in Ccr2 null mice, F4/80 levels returned to baseline by week 16. The L4 dorsal horn in WT mice showed a clearly increased number of activated microglia 8 and 16 weeks after DMM but not sham surgery (wk 8: p=0.015; wk 16: p=0.039, n=3-6/group/time point). In Ccr2 null mice, activated microglia were increased 8 wks after DMM compared to baseline (p<0.05, n = 3-9/group/time point), but this returned to baseline by week 16.

Conclusion: We describe a novel behavior that specifically develops during the chronic stage of the DMM model: increased grooming, which is considered an indicator of anxiety. Increased grooming may be indicative of pain-related anxiety, since it coincides with the chronic pain phase of the disease. Ccr2 null mice did not develop this grooming behavior, confirming our previous findings that these mice do not develop chronic pain after DMM. Overall, these data support our previous work suggesting that CCR2 activation is important in the maintenance of chronic OA pain, perhaps by mediating neuro-immune interactions in both the DRG and the spinal cord. The fact that Ccr2 null mice developed initial macrophage infiltration and microgliosis that subsequently resolved suggests that these pathways may be targetable.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-role-for-ccr2-in-chronic-behavioral-and-neuroimmune-changes-in-the-dmm-model-of-osteoarthritis/