LIPUS promotes calcium oscillations to improve lymphatic drainage and alleviates cold-stimulated pain and effusion in osteoarthritis joints

Background Osteoarthritis (OA) patients in remission usually experience acute exacerbations of pain and joint swelling following cold stimulation. This represents one of the characteristic clinical presentations of “cold knees”. In Traditional Chinese Medicine, this phenomenon correlates with “Cold Bi”, reflecting “lingering dampness” and “stagnation of Blood and Qi” in the joints, and is conceptually consistent with dysfunction of synovial lymphatic system (SLS) observed in OA. However, the underlying mechanisms of cold-exacerbated OA and the therapeutic strategies remain poorly explored. Methods OA mouse models were established by destabilization of the medial meniscus (DMM) surgery. The mice were exposed to cold stimulation at 4 weeks post-surgery to mimic OA patients in remission experiencing acute cold stimulation. Joint pain-related behaviors were assessed using multiple approaches including gait analysis and von Frey test. Magnetic Resonance Imaging (MRI) was used to evaluate joint effusion. Near-infrared indocyanine green (NIR-ICG) imaging and intra-articular injections of Fluorescein isothiocyanate-dextran (FITC-dextran) were employed to assess lymphatic drainage. Furthermore, the effects of temperature decrease and low-intensity pulsed ultrasound (LIPUS) on calcium oscillations and nitric oxide (NO) production by lymphatic endothelial cells (LECs) were investigated using live-cell fluorescence imaging. Additionally, the mechanisms through which LIPUS plays therapeutic roles were also explored using transient receptor potential vanilloid 4 ( TRPV4) inhibitor HC-067047. Results Acute cold stimulation aggravated gait abnormality and decreased paw withdrawal threshold in DMM-operated mice. Furthermore, weight-bearing asymmetry and total locomotor distance in the open field were also worsened in these mice. The knee joint effusion was significantly aggravated following acute cold stimulation accompanied by impaired synovial lymphatic drainage. Mechanistically, cold stimulation suppressed calcium oscillations of LECs and decreased their NO production, which were efficiently restored by LIPUS. Additionally, LIPUS treatment improved lymphatic drainage of the knee joint and alleviated cold-stimulated exacerbation of OA phenotypes in mice. These therapeutic effects were significantly suppressed under TRPV4 inhibition as well as hindlimb lymphatic vessel ligation. Conclusion Dysfunction of lymphatic drainage contributes to cold-stimulated exacerbation of OA phenotypes in mice, which could be alleviated by LIPUS-induced NO production through the TRPV4/Ca 2+ /eNOS/NO axis. These findings provide novel insights into the mechanisms of “cold knees” and highlight the therapeutic potential of LIPUS in the treatment of pain and joint effusion in OA. The translational potential of this article: Our study suggests that LIPUS ameliorates cold-induced worsening of pain and joint effusion in OA by improving synovial lymphatic drainage. These findings provide preliminary evidence for the translational potential of LIPUS application in skeletal disorders characterized by lymphatic drainage dysfunction.