which is why we developed Mrgprd-EGFP reporter mice and investigated if they are present in the murine OA joint. Methods: Mrgprd-EGFPf mice on a C57BL/6 background were used. DMM surgery was performed in the right knee of 10-week old male mice. Knees were harvested from 10-week old naïve mice (n¼5), 26week old naïve mice (n¼3), and mice 16 weeks after DMM (n¼3). Mice were perfused transcardially with paraformaldehyde. Knees were harvested, post fixed, decalcified and cryo-sectioned. Twenty-mm thick coronal sections were collected throughout the knee joint using established methods. Sections were then imaged using laser-scanning confocal microscope (Olympus IX70) and the fluorescence signal was quantified using image J by an observer blinded to the groups. Results: Mrgprdþ signal was present in the knee joint of 10-week old naïve mice, specifically at the insertions of the cruciate ligaments and in bone marrow cavities. By the age of 26 weeks, Mrgprdþ innervation decreased significantly in the cruciate ligament insertions (Figure 1A), which is similar to the age-related decline seen in the cruciate ligaments of Na V 1.8-tdTomato mice. No signal was observed in the medial synovium of young or old naïve mice (Figure 1B). Sixteen weeks after DMM, OA knees showed further decrease in the Mrgprdþ innervation of the cruciate ligament insertions, compared to 26-week old naïve knees (Figure 1A). In contrast to the increase in the medial synovial Na V 1.8þ innervation we previously observed in Na V 1.8-tdTomato mice, no change was observed in the Mrgprdþ innervation of the medial synovium after DMM (Figure 1B). Interestingly, Mrgprd signal was present in channel like structures in the subchondral bone of the medial femoral condyle and tibial plateau of the OA knee (Figure 1C), similar to findings in Na V 1.8-tdTomato mice. Representative confocal images of the medial compartment of the knee 16 weeks after DMM and age matched control are shown in Figure 2A,B. Conclusions: The intra-articular Mrgprd innervation changed markedly with aging and after DMM surgery. Interestingly, the innervation pattern of this mechanosensitive nociceptor subset was different than the pattern we previously observed for Na V 1.8, which marks all nociceptors. Sixteen weeks after DMM surgery, Mrgprd nerve fibers were recruited in medial subchondral bone channels, but not in the synovium, indicating that this subset is selectively innervating the subchondral bone after DMM. In contrast, Mrgprd fibers declined significantly in cruciate ligament insertions with age and after DMM. The biological significance of these findings needs to be explored.
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