142. Optimization of the Murine Hindlimb Lymphedema Model

PURPOSE: Secondary lymphedema is limb swelling from lymphatic injury. It frequently occurs following lymph node dissection and radiation during the treatment of malignancies such as breast cancer or melanoma. The murine tail is the most commonly used model to study secondary lymphedema and involves full thickness tail skin excision and lymphatic vessel disruption. The murine hindlimb model, which has been less frequently used in the literature, offers a more clinically translatable method. However, there is inconsistency and variability, including the benefit of radiation, which have contributed to the model being less widely adapted than the tail model. The purpose of this study is to 1) optimize the murine hindlimb lymphedema to achieve consistent results and 2) assess the effect of radiation on outcome in the murine hindlimb model. METHODS: C57BL/6 mice either underwent 20 Gy irradiation of one hindlimb seven days prior to surgery (n=11) or no preoperative radiation (n=9). For all mice, a circumferential skin incision was created at the proximal hindlimb exposing the subcutaneous soft tissues. Lymphatics were identified with isosulfan blue dye injection into the paw and disrupted. Popliteal lymph nodes were excised. The skin was sutured leaving a 3 mm gap. The contralateral hindlimb served as the control. Paw thickness and calf thickness measurements were obtained at weekly intervals and indocyanine green (ICG) near-infrared laser lymphangiography was used to assess lymphatic function. RESULTS: For the irradiated mice, the average paw thickness of the operated hindlimb on postoperative day (POD) 14 was 3.5±0.3 cm compared to 2.1±0.05 cm on the contralateral limb (p=0.0001). At POD-90, the average paw thickness of the irradiated, operated hindlimb was 2.4±0.1 cm compared to 2.1±0.1 cm for the contralateral limb (p=0.01). ICG lymphangiography at 24-hours postinjection on POD-42 demonstrated an average signal intensity of 97.7±28.5 arbitrary fluorescent units (AFU) in the operated hindlimb compared to 33.6±6.2 AFU in the non-operated hindlimb (p=0.003). In the mice that did not undergo radiation, the average paw thickness was 2.5±0.2 cm on POD-42 was greater than the contralateral limb (2.1±0.1 cm) (p=0.0002) but smaller than hindlimbs that underwent radiation (3.2±0.1 cm) (p=0.0002). The nonradiated mice had greater paw thickness than the contralateral control until POD-56 whereas the radiated mice sustained significant paw thickness until Day 90. CONCLUSION: Radiation of the murine hindlimb model results in sustained lymphedema compared to non-irradiated mice. The murine hindlimb lymphedema model is clinically more translatable than the murine tail model and includes limb lymphatic vessel disruption, and popliteal lymphadenectomy and ideally radiation for consistent results with lymphedema sustained for 90 days.