A Murine Tail Lymphedema Model

Summary

Lymphedema is extremity swelling caused by lymphatic dysfunction. We describe a chronic murine tail model of lymphedema and the novel use of tissue nanotransfection technology (TNT) for genetic cargo delivery to the tail.

Abstract

Lymphedema is extremity swelling caused by lymphatic dysfunction. The affected limb enlarges because of accumulation of fluid, adipose, and fibrosis. There is no cure for this disease. A mouse tail model that uses a focal full thickness skin excision near the base of the tail, resulting in tail swelling, has been used to study lymphedema. However, this model may result in vascular comprise and consequent tail necrosis and early tail swelling resolution, limiting its clinical translatability. The chronic murine tail lymphedema model induces sustained lymphedema over 15 weeks and a reliable perfusion to the tail. Enhancements of the traditional murine tail lymphedema model include 1) precise full thickness excision and lymphatic clipping using a surgical microscope, 2) confirmation of post-operative arterial and venous perfusion using high resolution laser speckle, and 3) functional assessment using indocyanine green near infrared laser lymphangiography. We also use tissue nanotransfection technology (TNT) for novel non-viral, transcutaneous, focal delivery of genetic cargo to the mouse tail vasculature.

Introduction

Lymphedema is extremity swelling caused by lymphatic dysfunction. The affected limb enlarges because of the accumulation of fluid, adipose, and fibrosis¹. Lymphedema affects 250 million people worldwide^2,3,4. It is estimated that 20-40% of patients who undergo treatment for solid malignancies, such as breast cancer, melanoma, gynecological/urologic tumors, or sarcomas, develop lymphedema^2,4,5. Morbidity from lymphedema includes recurrent infections, pain, and ....

Protocol

The protocol follows the guidelines of the institution's animal research ethics committee. All animal experiments were approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee. Animals were housed under a 12-hour light-dark cycle with food and water ad libitum.

1. Surgical Disruption of Mouse Tail Lymphatics

1. Use eight week-old C57BL/6 mice of equal gender distribution.
2. Place a mouse under general anesthesia in an induction chamber with 3-4% isoflurane in 100% oxygen followed by maintenance sedation at 1-3% during the procedure.
3. Administer 0.5 mg/kg sustained-releas....

Results

The technique for the mouse tail model for sustained lymphedema is shown in Figure 1. The figure exhibits the relevant anatomy of the mouse tail model. Figure 2 demonstrates the progressive swelling and sustained persistant lymphedema in the mouse tail after lymphedema induction. The mouse tail volume, as calculated by the truncated cone equation, peaks at week 4 and plateaus to week 6 followed by gradual improvement that is sust.......

Discussion

Lymphedema is categorized as a primary (congenital) or secondary (iatrogenic lymphatic) injury^38,39. Secondary lymphedema comprises 99% of cases³⁹. Secondary lymphedema is most commonly caused by infection (filariasis) or post-oncological treatment with lymphadenectomy or radiation^4,39. A translational animal model is challenging for secondary lymphedema, as 70% of animals treated .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tegaderm Film		1626W
Surgical Microscope	Leica, Wetzlar, Germany	MSV266
Adherent Dressing (Tegaderm)	3M, St. Paul, Minn.	1626W
Laser speckle (Pericam PSI System )	Perimed AB, Stockholm, Sweden)	PSIZ
Near-infrared laser (LUNA)	Stryker (Formerly Novadaq Technologies, Toronto, Canada)	LU3000
C57BL/6 mice	Jackson Laboratories	000664
Micro-Adson Forceps - 1x2 Teeth	Fine Science Tools (USA) Inc.	11019-12
V-Hook	Fine Science Tools (USA) Inc.	18052-12
Scalpel SS NO15	Fischer Scientific	29556
Disposable Needle 30GX1	Fischer Scientific	305128
Operating Scissors	Fischer Scientific	12-460-796
Surgi-Or Jeweler's Forceps, Sklar 4-1/2 in	Fischer Scientific	50-118-4255
Spring Scissors - Straight/Sharp-Sharp/8mm Cutting Edge	Fine Science Tools (USA) Inc.	15024-10
Cardiogreen	Sigma	I2633-25MG
IsosulfanBlue (Lymphazurin)  50 mg/5ml	Mylan	67457-220-05