1 A ROLE FOR CHEMOKINES AND NGF IN PAIN GENERATION IN A MURINE MODEL OF OSTEOARTHRITIS

Abstract

Purpose: Pain is the major symptom in osteoarthritis (OA), yet how this pain develops remains unknown. We recently reported that destabilization of the medial meniscus (DMM) provides a suitable mouse model for studying pain development and progression at different stages of OA. We found that, following DMM in the right knee, male C57BL/6 mice develop progressive mechanical allodynia in the ipsilateral hindpaw, as early as 2 weeks and progressing over 8 weeks post DMM (but not sham) surgery. Additionally, total distance traveled over a specified period was relatively constant over the first 8 weeks, but decreased from 12 to 16 weeks after surgery. The current goal was to identify molecular pathways in the knee joint and its innervating nociceptive system that correlate with these different stages of disease. Methods: DMM or sham surgery was performed in the right knee of 10-week old male C57BL/6 mice. At 8 and 16 weeks post surgery, histopathology of the knees was evaluated according to OARSI recommendations. Additionally, at 4 and 8 weeks after surgery, whole knee joints were collected for protein extraction and ELISA. At 4, 8, or 16 weeks post surgery, innervating dorsal root ganglia (DRG), L3L5, from DMM or sham-operated mice and age-matched naive mice were collected for protein extraction for ELISA or for mRNA extraction for quantitative RT-PCR of nerve growth factor (NGF), monocyte chemoattractant protein (MCP)-1 and its receptor CCR2, and stromal cell-derived factor (SDF)-1. Finally, at 8 weeks post surgery, the response of DRG neurons to chemokines was recorded though intracellular Ca2+imaging, following standard protocols. In brief, DRG neurons were acutely isolated, plated on coverslips, cultured for 3–4 days, and loaded with a calcium indicator dye. The number of cells responding to chemokines was counted. Results: Following DMM (but not sham) surgery, C57BL/6 mice developed cartilage degeneration and osteophyte formation by 8 weeks in the ipsilateral knee only, with progressive cartilage deterioration occurring up to 16 weeks. At 4 weeks post DMM, protein levels of MCP-1 and NGF were elevated in total joint extracts from the operated knee compared to naive age-matched controls (p = 0.025 and p=0.099, respectively); by 8 weeks post DMM, levels of both proteins had returned to baseline. mRNA levels of NGF, MCP-1, CCR2, and SDF-1 in the innervating DRGwere upregulated compared to naive and sham age-matched controls, peaking at 8 weeks post surgery. Protein levels of NGF were also increased in DRG at 8 weeks post surgery (p = 0.03), while MCP-1 protein was below the detection limit at all time points. Exposing isolated DRG neurons from DMM mice at 8 weeks post surgery to MCP-1 or to SDF-1 (200 or 500ng/mL, respectively) resulted in an increased intracellular calcium response compared to naive age-matched controls, indicating an upregulation of their respective receptors, CCR2 and CXCR4, on these cells. Conclusions: In this study, gene and protein expression in the knee joint and innervating DRG of DMM mice reveal temporal changes in NGF, SDF-1 and MCP-1 during the development of painful OA. In addition, Ca-imaging studies suggest a functional role for the chemokines MCP-1 and SDF-1 in pain generation in this model. NGF is known to play a role in sensitization of the nervous system in inflammation-associated pain; recent research has shown that the chemokines MCP-1 and SDF-1 contribute to the development of chronic pain in neuropathic pain models. Therefore, this work supports burgeoning clinical evidence that OA-related pain may include aspects of both inflammatory and neuropathic pain.