Therapeutic effects of hyaluronidase on acquired lymphedema using a newly developed mouse limb model

Acquired lymphedema is one of the most dreaded side effects of cancer treatment, such as surgical treatment or irradiation. However, due to the lack of appropriate animal models, there is no effective therapeutic method to cure acquired lymphedema. To develop a reproducible acquired lymphedema animal model, we devised a mouse hind limb model by removing a superficial inguinal lymph node, a popliteal lymph node, a deep inguinal lymph node, and the femoral lymphatic vessel. We measured the volume of lymphedematous leg and observed the change in level of hyaluronic acid (HA) and lymphangiogenic factors after injecting hyaluronidase. Our model showed the distinguishable swelling and the reliable symptoms compared to previously reported models. In the lymphedematous regions of our model, we confirmed that HA, a major component of extracellular matrix, accumulated to higher levels than in a normal mouse. This lymphedema volume was rapidly reduced by treating hyaluronidase. Following hyaluronidase injection, the lymphedematous region of our model resembled a normal hind limb. Our findings indicated that hyaluronidase promoted lymphangiogenesis on the lymphedematous limb. Based on hyaluronidase treatment in the lymphedematous region, this could potentially be a new therapeutic approach for acquired lymphedema mediated through the modification of the size of HA fragments. Impact statement In this manuscript, the essence of the work described in this manuscript involves the development of (1) a mouse limb model showing acquired lymphedema and (2) a potent therapeutic treatment using hyaluronidase to remedy acquired lymphedema in our model. In order to develop a reproducible acquired lymphedema animal model that reflects the most common symptoms experienced by lymphedema patients, we devised a mouse hind limb model by removing lymph nodes and lymphatics. Our model showed the distinguishable swelling and the reliable symptoms compared to previously reported models. In the lymphedematous regions of our model, we confirmed that hyaluronic acid (HA) accumulated to higher levels than in a normal mouse. This lymphedema volume was rapidly reduced by treating the lymphedematous leg with hyaluronidase, which also degraded high molecular weight HA to low molecular weight HA. Immunohistochemical analysis, quantitative real-time PCR analysis and lymphangioscintigraphy showed that hyaluronidase enhanced lymphangiogenesis in the lymphedematous limb.