Popliteal Vascular Lymph Node Resection in the Rabbit Hindlimb for Secondary Lymphedema Induction
Summary
Here, the surgical induction of stable acquired lymphedema in the rabbit hindlimb is described. This experimental animal can be used to further investigate the effect of lymphedema treatment by microsurgical techniques.
Abstract
Lymphedema is a common condition often associated with cancer and its treatment, which leads to damage to the lymphatic system, and current treatments are mostly palliative rather than curative. Its high incidence among oncologic patients indicates the need to study both normal lymphatic function and pathologic dysfunction. To reproduce chronic lymphedema, it is necessary to choose a suitable experimental animal. Attempts to establish animal models are limited by the regenerative capacity of the lymphatic system. Among the potential candidates, the rabbit hindlimb is easy to handle and extrapolate to the human clinical scenario, making it advantageous. In addition, the size of this species allows for better selection of lymphatic vessels for vascularized lymph node resection.
In this study, we present a procedure of vascular lymph node resection in the rabbit hindlimb for inducing secondary lymphedema. Anesthetized animals were subjected to circumferential measurement, patent blue V infiltration, and indocyanine green lymphography (ICG-L) using real-time near-infrared fluorescence, a technique that allows the identification of single popliteal nodes and lymphatic channels. Access to the identified structures is achieved by excising the popliteal node and ligating the medial and lateral afferent lymphatics. Special care must be taken to ensure that any lymphatic vessel that joins the femoral lymphatic system within the thigh without entering the popliteal node can be identified and ligated.
Postoperative evaluation was performed at 3, 6, and 12 months after induction using circumferential measurements of the hindlimb and ICG-L. As demonstrated during follow-up, the animals developed dermal backflow that was maintained until the 12th month, making this experimental animal useful for novel long-term evaluations in the management of lymphedema. In conclusion, the approach described here is feasible and reproducible. Additionally, during the time window presented, it can be representative of human lymphedema, thus providing a useful research tool.
Introduction
Lymphedema is a chronic condition that deserves special attention, owing to its worldwide incidence, lack of curative and standardized treatment, and serious impact on patients' quality of life1,2.
In developed countries, lymphedema is mainly acquired and is secondary to breast cancer, owing to the high prevalence of this malignancy; the cumulative incidence of breast cancer-related lymphedema 10 years after surgery can reach up to 41.1%3. However, diseases such as melanoma, gynecological cancers, genitourinary tumors, and head and neck neoplasms are also associated with a high incidence of this disease4. Regional lymph node resection, as part of the necessary oncological treatment to increase survival rates, leads to the disruption of functional lymphatic drainage. In some cases, this results in compensatory mechanisms that prevent or delay the onset of lymphedema5. However, when chemotherapy and radiotherapy are administered, these mechanisms are not able to compensate for the change, and lymphedema resultantly occurs. This has a negative impact on patients' quality of life, affecting their functional, social, and psychological well-being6,7.
The need for an effective cure for lymphedema requires understanding of the physiopathology of the lymphatic system, as well as a deep insight into the complex cellular mechanisms and their responses in both normal and dysfunctional lymphatic systems8,9,10. Such insights can be obtained initially from experimental animal models that can reproduce chronic human diseases11.
Many attempts have been made to replicate lymphedema in experimental animal models; however, most of them have been hindered by some limitations, including the inability to reproduce chronic lymphatic insufficiency in a stable animal model, the costs of the study, and most importantly, the great regenerative capacity of the lymphatic system, which enables it to restore circulation12,13.
This study presents the experimental approach for surgically inducing stable acquired lymphedema using the rabbit hindlimb. Based on literature review, this animal can be considered optimal for the development of lymphedema because of the consistent anatomy of its hindlimb lymphatic system, which includes a single popliteal node that drains the hindlimb and reaches the main femoral lymphatic system in the leg14,15.
The specific anatomy of the rabbit's hindlimb allows for the reproduction of the surgical procedures performed in humans to induce secondary lymphedema. Therefore, this procedure can be used for microsurgical training and preclinical research to extrapolate the results to human medicine.
Protocol
Representative Results
Discussion
Resection of the PLN in an experimental animal is a relatively new procedure that can induce secondary lymphedema in the limbs for assessment and study. After lymph node resection, there is a period of alteration of lymphatic system functionality, lymph accumulation, and histological changes of lymphatic vessels that appear dilated. When this lymph accumulation reaches adequate levels, the characteristic dermal backflow of lymphedema, similar to that observed in humans, can be observed using objective techniques such as …
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research project was performed at the Jesús Usón Minimally Invasive Surgery Center (CCMIJU), which is part of the ICTS Nanbiosis. The study was performed with the assistance of the following Nanbiosis units: U21, experimental operating room, and U22, animal housing. This work was supported by Hospital de la Santa Creu i Sant Pau. This work has been partially funded by the Junta de Extremadura, the European Regional Development Fund (Grant Number GR21201). The funder played a role in the study design, data collection, analysis, decision to publish, and manuscript preparation. Special thanks are extended to María Pérez for preparing the figures and to the Microsurgery Department of JUMISC for providing constant encouragement.
Materials
| Bleu Patente V sodique (Guerbet) | Guerbet. Villepinte, France | 2.5 g/100 mL | |
| Buprenorphine (Bupaq) | Richter Pharma. Wels, Austria | 0820645AA | 3 mg/10 mL |
| Fluobeam | Fluoptics. Grenoble, France | Fluorescence imaging | |
| IBM SPSS software | IBM | version 21.0 | |
| Indocyanine green (Verdye, Diagnostic Green GmbH) | Diagnostic Green GmbH. Aschheim-Dornach, Germany | 5 mg/mL | |
| Ketorolaco (Normon) | Normon, S.A. Madrid, Spain | T01H | 30 mg/mL |
| Microsoft Excel | Microsoft | version 16.66.1 | |
| Midazolam (Normon) | Normon, S.A. Madrid, Spain | T35M | 15 mg/3 mL |
| Pentero 800 microscope, fluorescence module | Carl Zeiss Meditec AG. Goeschwitzer Strasse 51-52. Jena, Germany | 302581-9245-000 | |
| Potassium chloride (Braun) | B.Braun. Barcelona, Spain | 19262010 | 20 mmol/10 mL |
| Propofol (Propomitor, Orion Pharma) | Orion Pharma. Spoo, Finland | 20R039B | 200 mg/20 mL |
| RÜSCH endotracheal tubes | Teleflex Medical IDA Business and Technology Park. Athione, Ireland. | 12CE 12 | Size Tube 4.0 I.D. mm |
| Sevoflurano (SevoFlo, Zoetis) | Zoetis Belgium. Luvain-la-Neuve, Belgium | 6093559 | 1000 mg/g (250 mL) |
| Tramadol (Normon) | Normon, S.A. Madrid, Spain | T08U | 100 mg/2 mL |
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