Background Postsurgical secondary lymphedema is usually a progressive and lifelong condition lacking any curative treatment. The aim of this study was to develop new, simple surgical mouse models of chronic lymphedema, better simulating chronic nature of human postsurgical lymphedema. Methods Two experimental mouse models of secondary lymphedema were created surgically without radiation by modifications of the previously described methods: the tail model and the hind limb model. Lymphedema formation was clinically assessed and quantitatively evaluated by measuring circumferences and limb volumes. Postmortem specimens were assessed histologically to examine the efficacy of the models. Results In the tail models, although a substantial frequency of tail necrosis (30.0%) was noted and the increase in circumference was maintained for only limited times postoperatively depending on the particular tail model, the overall success rate was 65.0%. In the mouse hind limb model, the overall success rate was 88.9%, and the increased circumference and limb volume were maintained over the entire study period of 8 weeks. The overall success rate of the mouse hind limb model was significantly higher than that of the mouse tail model(s). Conclusions We have successfully established modified mouse tail and hind limb lymphedema models via only surgical techniques without radiation, which have characteristics of chronic secondary lymphedema. The mouse hind limb model has a higher success rate than the mouse tail model and has advantages of having the healthy contralateral hind limbs as an internal control.
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Lymphedema is a progressive and lifelong pathologic condition in which microcirculatory imbalances of tissue fluid equilibrium are created by loss of lymphatic-transport capacity. 6–13 Unfortunately, no curative treatment exists for this potentially debilitating condition, and current treatments for chronic lymphedema, which include conservative treatments such as lifelong physiotherapy, surgical removal of edematous tissue (reducing operation), or lymphovenous microsurgical bypass, can only slow its progression. 14,15 For these reasons, there has been substantial interest in new therapeutic development such as gene-mediated or cell-mediated lymphangiogenesis, and these new approaches may ultimately reverse the clinical course of chronic lymphedema. 16–19 However, such approaches will require suitable preclinical animal models of chronic lymphedema to precisely test their therapeutic responses and their safety. Previously, several animal models of chronic secondary lymphedema have been developed for studying pathophysiology and therapy for lymphedema. While studies used a large animal model for induction of chronic lymphedema, mice are still preferable for research for many reasons: (1) mice are easy to handle and breed, and there is a wide range of inbred, transgenic, and knockout strains available; (2) they have the advantage of cost-effectiveness; and (3) there is a great range of research tools available for mice studies, such as antibodies and databases. 4 The mouse tail lymphedema model has provided a straightforward and reproducible system for lymphatic researchers and has been used widely to advance our understanding of surgically induced lymphedema, although it is to some extent different from the human disease from an anatomical and physiological standpoint. 5,7,11 Recently, to overcome the limitations of tail models, lymphedema has been induced in the mouse hind limb by preoperative or postoperative radiation and lymphatic vessel ablation, which recapitulate the human disease more precisely compared with the mouse tail model. The mouse hind limb model would be more useful to enhance our understanding of molecular biology and pathogenesis of this disease and to test emerging therapies such as gene-mediated or cell-mediated lymphangiogenesis due to its longer duration of lymphedema and its similarity to human counterparts such as arm or leg lymphedema. 4,5 However, surgery with preoperative or postoperative radiation is too complicated to be widely applied in conventional laboratory settings. Thus, in this study, we sought to create lymphedema models in mice only by surgery without radiation, which are sustainable, easy to create, allows repeated physiologic measurement. According to the existing methods, 7,10 we created a mouse tail model using 8-week-old BALB/C female mice and a mouse hind limb model using 28-week-old C57BL/6 male mice. However, in our pilot study, these models gave unsatisfactory long-term results. Several authors have reported that the mouse tail model of lymphedema is reproducible and reliable and has many characteristics of chronic secondary lymphedema. 2,7,11,12 However, in our pilot study, this model gave satisfactory results only up to 10 postoperative days. Furthermore, after 10 days, there was a substantial rate of tail necrosis probably due to dry-up followed by thrombosis of the extremely small tail vessels and deterioration of the circulation ( Supplemental Fig. 5 ). To reduce the incidence of tail necrosis and induce longer duration of lymphedema, we modified the model with a skin bridge, 2-mm in width, to improve circulation in the tail and the circumferential incision site was protected with a rubber band to prevent restoration of the superficial lymphatics. However, in spite of acceptable results in clinical, microlymphangiographic, and histologic studies, a high rate of tail necrosis still occurred. We thus decided to develop another model, the mouse hind limb model. Our initial results of the mouse hind limb model were satisfactory in the early postoperative period. However, during the follow-up, there was rapid healing of the area of skin and subcutaneous excised tissue in some cases, followed by the development of collateral lymphatic flow and a return to normal limb size ( Supplemental Fig. 6 ). We therefore modified the mouse hind limb model as follows: (1) after lymphatic vessel ablation, the skin wound edges were continuously sutured to the underlying muscle leaving a gap of 5 mm between the 2 sides of wound edges to block the rapid regrowth of superficial lymphatics and prevent the development of collateral lymphatic flow; and (2) after the operation, the circumferential incision sites were protected with rubber bands to prevent restoration of the superficial lymphatics and infection. Our modified hind limb model gave satisfactory results in clinical and histologic studies over the entire study period of 8 weeks. Intriguingly, in histologic examination of our mouse hind limb model, we encountered interesting incidental findings in a tissue sample at the postoperative week 8. Light microscopic assessment of the postmortem specimen revealed a high-grade spindle cell sarcoma compatible with lymphangiosarcoma as well as typical findings of chronic lymphedema. In humans, lymphangiosarcoma is the only malignant disease of the lymphatics, and the etiology is largely unknown. 20–22 Chronic lymphedema is a widely recognized risk factor for lymphangiosarcoma. 21 Typically, lymphangiosarcoma occurs in patients who have been treated for breast cancer with mastectomy and/or radiotherapy and who have developed a significant degree of chronic edema for many years. 20–22 Other chronic lymphedema resulting from a congenital, idiopathic, traumatic, or infectious cause also predisposes the patients to lymphangiosarcoma. 21 Although the true prevalence of the condition is not known, Mulvenna et al. 23 found only 6 cases of lymphangiosarcoma in more than 5,000 patients with lymphedema over 30 years. Consequently, this finding points to the reliability of our mouse hind limb model as a chronic secondary lymphedema model. Several limitations should be noted. Although clinical and histologic studies showed that our models are reliable and consistent for investigating chronic secondary lymphedema, we were unable to perform fluorescence microlymphangiography studies in the hind limb model due to the large size of limbs that do not allow microscopic examination to better characterize the lymphatic networks. In the mouse tail models, despite our modification, there was a substantial occurrence of tail necrosis in all 3 types of models, and we could not adjust for the increase in circumference in the controls due to age-appropriate growth. In conclusion, despite the aforementioned potential limitations, our modified mouse tail and hind limb lymphedema models via only surgical techniques without radiation are simple, reproducible, and reliable, with many of the characteristics of chronic secondary lymphedema; however, in the mouse tail model, there was a substantial frequency of tail necrosis. On the other hand, the mouse hind limb model had a higher success rate and adequate internal controls in healthy contralateral hind limbs. This work was supported in part by grants from NIH ( HL127759 , DP3DK094346 , and DP3DK 108245 to Y-.S.Y.), the Bio & Medical Technology Development Program of the NRF funded by the Korean government (MSIP) (No 2015M3A9C6031514), and the Korea Health Technology R&D Project through the KHIDI, funded by the Ministry of Health & Welfare, Republic of Korea (No HI15C2782).
© 2017 Elsevier Inc.
All Science Journal Classification (ASJC) codes
- Cardiology and Cardiovascular Medicine