Establishment of long-term secondary lymphedema animal model in a rodent hindlimb

Sung-Hwan Yoon¹Hyun Suk Peter Suh²Jin-Hui Yoon³Hayeong Cho¹Yujin Myung^1*Jae Yong Jeon^4*

• ¹Department of Plastic and Reconstructive Surgery, Seoul National University Bundang Hospital, Seongnam, Republic of Korea
• ²Department of Plastic and Reconstructive Surgery, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul, Republic of Korea
• ³Institute for Basic Science Center for Vascular Research, Daejeon, Republic of Korea
• ⁴Department of Rehabilitation Medicine, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul, Republic of Korea

Secondary lymphedema is a chronic swelling of the upper or lower extremities caused by surgical disruption of the lymphatic system, resulting in impaired lymph drainage. It most commonly occurs in breast or gynecologic cancer patients following lymph node or lymph vessel removal during cancer resection to prevent metastasis. When reconnection of the lymphatic network fails and lymph transport capacity remains insufficient, accumulated lymph fluid induces irreversible tissue remodeling and the onset of secondary lymphedema. Although various clinical therapies have been developed, most are palliative or conservative, providing limited therapeutic benefit. Therefore, establishing a standardized secondary lymphedema animal model that closely reflects clinical pathology is essential for advancing mechanistic and therapeutic research. However, existing models exhibit limited severity and duration of edema, restricting their translational value. In this study, we developed a long-term mouse hindlimb model of secondary lymphedema that closely mimics the clinical condition. A combination of a novel folding-suture surgical procedure, controlled radiation exposure, and limb immobilization was applied to induce and sustain lymphedema. This approach effectively disrupted both superficial and deep lymphatic systems, inhibited early lymphangiogenesis, and reduced lymphatic pumping, resulting in irreversible, severe, and chronic edema. The model exhibited significantly increased paw thickness and persistent swelling for over six weeks. Immunofluorescence analysis revealed a marked reduction in both the number and diameter of regenerated lymphatic vessels compared with previously reported models. Furthermore, FITC-Dextran and real-time ICG lymphangiography demonstrated diminished lymphatic regeneration and flow, confirming the presence of secondary lymphedema due to disrupted lymphatic drainage. Lymphedema severity assessed by the LDB staging method—a clinically relevant evaluation system—was also significantly higher in this model. Collectively, these findings demonstrate that our newly established mouse hindlimb model closely replicates the clinical progression and pathology of secondary lymphedema. This model provides a robust and standardized platform for elucidating disease mechanisms and developing effective therapeutic strategies.