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

Abstract

Secondary lymphedema is swelling of upper or lower extremity caused by physical resection of lymphatic system, resulting in drainage dysfunction of lymph flow. Most prominent case is caused by unsuccessful recovery from surgical procedure of cancer patients (breast cancer or ovarian cancer) after removal of lymphatic vessels and/or lymph nodes during cancer resection surgical procedure to prevent cancer metastasis. When reconnection of lymphatic system fails and transport capacity is not sufficiently recovered, accumulation of unsuccessfully drained lymph flow triggers irreversible changes and secondary lymphedema occurs. Number of clinical therapies were invented and practiced, but all of them are either palliative or conservative treatments so therapeutic efficacy is significantly below its expectation. For this reason, standardized secondary lymphedema animal model with high correlation to the clinical patient is necessary in order to achieve further in-depth studies for understanding the disease and its effective therapeutic strategies but previously established animal model has major limitations both in severity and duration of secondary lymphedema, making it significantly difficult for further translational and preclinical studies. Therefore, the purpose of this study was to establish long-term secondary lymphedema animal model in a mouse hindlimb which shares high correlation with clinical lymphedema patients. As a method, combination of novel surgical procedure, controlled radiation exposure, and immobilization technique were thoroughly designed and applied to create lymphedema hindlimb model. By applying newly developed, novel surgical procedure called folding suture, both superficial lymphatic system and deep lymphatic system were successfully disrupted and lymphedema was effectively created and controlled radiation exposure was practiced to inhibit lymphangiogenesis in an early stage of lymphedema to induced and maintained severity of lymphedema. Moreover, immobilization technique significantly decreased lymphatic pumping capability and encouraged its severity, causing irreversible, severe, and chronic lymphedema as a result. As a result, hindlimb paw thickness of a newly established model was significantly higher and edema was observed over six weeks. Also, evaluation of immunofluorescence images showed that both number of regenerated lymphatic vessels and their diameter were significantly lower than previously established model. Moreover, both FITC-Dextran lymphography and live-time ICG lymphangiography, enabling structural visual mapping, evaluation of regeneration of lymphatic system and lymph flow analysis, revealed that both structural regeneration of lymphatic vessels (lymphangiogenesis) and flow of the lymphatics were significantly decreased, validating edema as indeed secondary lymphedema, caused by disruption of lymphatic system and its flow capacity. For further evaluation of lymphedema severity, LDB Stage evaluation, one of the most widely used methods for assessment of clinical lymphedema patients, was performed for in-depth, thorough evaluation of severity of lymphedema and its physiological condition and severity of lymphedema was much significant in newly established lymphedema hindlimb model. Based on this findings, newly established lymphedema hindlimb model has high correlation with clinical patients and may serve as a newly standardized animal model for further studies. Moreover, it may warrant further studies of secondary lymphedema not only for understanding its pathophysiology but also for developing its therapeutic strategies.