Abstract

BackgroundC–C chemokine receptor 2 (CCR2) signaling plays a key role in pain associated with experimental murine osteoarthritis (OA) after destabilization of the medial meniscus (DMM). Here, we aimed to assess if CCR2 expressed by intra-articular sensory neurons contributes to knee hyperalgesia in the early stages of the model.MethodsDMM surgery was performed in the right knee of 10-week-old male wild-type (WT), Ccr2 null, or Ccr2RFP C57BL/6 mice. Knee hyperalgesia was measured using a Pressure Application Measurement device. CCR2 receptor antagonist (CCR2RA) was injected systemically (i.p.) or intra-articularly (i.a.) at different times after DMM to test its ability to reverse knee hyperalgesia. In vivo Ca2+ imaging of the dorsal root ganglion (DRG) was performed to assess sensory neuron responses to CCL2 injected into the knee joint cavity. CCL2 protein in the knee was measured by ELISA. Ccr2RFP mice and immunohistochemical staining for the pan-neuronal marker, protein gene product 9.5 (PGP9.5), or the sensory neuron marker, calcitonin gene-related peptide (CGRP), were used to visualize the location of CCR2 on intra-articular afferents.ResultsWT, but not Ccr2 null, mice displayed knee hyperalgesia 2–16 weeks after DMM. CCR2RA administered i.p. alleviated established hyperalgesia in WT mice 4 and 8 weeks after surgery. Intra-articular injection of CCL2 excited sensory neurons in the L4-DRG, as determined by in vivo calcium imaging; responses to CCL2 increased in mice 20 weeks after DMM. CCL2, but not vehicle, injected i.a. rapidly caused transient knee hyperalgesia in naïve WT, but not Ccr2 null, mice. Intra-articular CCR2RA injection also alleviated established hyperalgesia in WT mice 4 and 7 weeks after surgery. CCL2 protein was elevated in the knees of both WT and Ccr2 null mice 4 weeks after surgery. Co-expression of CCR2 and PGP9.5 as well as CCR2 and CGRP was observed in the lateral synovium of naïve mice; co-expression was also observed in the medial compartment of knees 8 weeks after DMM.ConclusionsThe findings suggest that CCL2-CCR2 signaling locally in the joint contributes to knee hyperalgesia in experimental OA, and it is in part mediated through direct stimulation of CCR2 expressed by intra-articular sensory afferents.

Highlights

  • C–C chemokine receptor 2 (CCR2) signaling plays a key role in pain associated with experimental murine osteoarthritis (OA) after destabilization of the medial meniscus (DMM)

  • Wild type mice but not Ccr2 null mice develop primary knee hyperalgesia after DMM surgery Confirming our previous findings [28, 37], WT mice (n = 5) developed knee hyperalgesia 2 weeks after DMM surgery, which gradually recovered by week 16 (Fig. 1a; p < 0.0001 for 4–12 weeks after surgery compared to pre-surgery; p = 0.0041 for 16 weeks after surgery compared to pre-surgery)

  • Ccr2 null mice (n = 5) did not develop hyperalgesia up to 16 weeks after DMM (Fig. 1b). These findings suggest that CCR2 signaling is essential for development of knee hyperalgesia after DMM surgery

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Summary

Introduction

C–C chemokine receptor 2 (CCR2) signaling plays a key role in pain associated with experimental murine osteoarthritis (OA) after destabilization of the medial meniscus (DMM). We aimed to assess if CCR2 expressed by intra-articular sensory neurons contributes to knee hyperalgesia in the early stages of the model. CCR2 receptor antagonist (CCR2RA) was injected systemically (i.p.) or intra-articularly (i.a.) at different times after DMM to test its ability to reverse knee hyperalgesia. In vivo Ca2+ imaging of the dorsal root ganglion (DRG) was performed to assess sensory neuron responses to CCL2 injected into the knee joint cavity. Ccr2RFP mice and immunohistochemical staining for the pan-neuronal marker, protein gene product 9.5 (PGP9.5), or the sensory neuron marker, calcitonin gene-related peptide (CGRP), were used to visualize the location of CCR2 on intra-articular afferents. The disease is characterized by progressive joint damage, associated with chronic pain and disability [7]. Extensive efforts in academia and industry are focused on identifying safe new targets for OA pain [10, 11]

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