Using lightsheet microscopy to investigate the initial lymphatic network in the murine knee joints

Introduction. Lymphatic vasculature in mouse joints is difficult to study due to its vast anatomical variation. We reported increased lymphatic capillaries in post-traumatic OA (PTOA) murine joint based on 2D immunostaining. However, a gap of knowledge with this approach is that histology highly subjected to histological sampling, which might not be appropriate for a highly variable system such as the lymphatic vasculature. We hypothesize that using lightsheet microscopy would reveal the detailed structure of the initial lymphatic network. Methods. We established lightsheet imaging protocol and four quantifiable outcome parameters for visualizing and analyzing the initial lymphatic network in young C57/B6 wild type mouse knees. Lymphatic vessels were identified by staining samples with lymphatic endothelial marker LYVE1. We validated our lightsheet imaging protocol in mouse knee joints following PTOA surgery and knee joints received sham operation. Results. We used the histological landmarks of growth plates and menisci shape for consistent data collection for the 3D reconstruction of initial lymphatic network. Comparing to sham joints, PTOA joints have less mean branches length (58.91{+/-} 6.50 vs. 146.29 {+/-} 2.48 m in sham, p<0.0001), and mean branch diameter (12.25{+/-} 0.80] vs. 28.51 {+/-} 4.99 m in sham, p=0.0051), but higher total branching points (378{+/-} 239 vs. 69 {+/-} 17 m in sham, p=0.020), and total initial lymphatic network volume (51247{+/-} 14239 vs. 1019289 {+/-} 544458 m3 in sham, p=0.037). Conclusion. Lightsheet imaging of murine knee joints is a powerful and promising tool to study the joint lymphatic system and arthritis. Similar to previous report, the PTOA joint has increased branching with smaller and shorter branches.