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 12^th 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 life^1,2.

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....

Protocol

All procedures were approved by the ethical committee of the Jesús Usón Minimally Invasive Surgery Center and the welfare guidelines of the regional government, which are based on European legislation.

1. Presurgical and surgical preparation

1. House nine female New Zealand white rabbits weighing 4-4.5 kg and aged 4 months in separate cages maintained at a temperature of 22-25 °C, with free access to food and water. Make sure that the cages contain a polysulfone tray with a surface area of 3 m² and a height of 40 cm, as well as a bed with wood shavings.
   1. Identify the cages wit....

Results

Nine rabbits underwent lymphedema induction in this study, however, three rabbits died during the immediate postoperative period and could not be evaluated. Study data were obtained at 3, 6, and 12 months postoperatively by three independent researchers. Circumferential hindlimb measurements and ICG-L were performed under general anesthesia to assess the lymphatic system function and dermal backflow.

The data obtained by ICG-L at 3 months postoperatively showed dermal backflow and the absence .......

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 .......

Materials

Name	Company	Catalog Number	Comments
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