Murine Tail Lymphedema Model: An In Vivo Technique to Generate Sustained Lymphedema in Murine Tail

Overview

This video describes an in vivo technique to generate murine tail lymphedema, an abnormal fluid accumulation in the interstitial tissue space leading to swelling in the tail. The model is applied to study mechanisms and therapy for lymphedema.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

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-release (SR) buprenorphine subcutaneously for pain control.
   NOTE: Additional analgesic drugs administered postop: Carprofen once every 24 h for at least 48 h and Bupivacaine once either after the incision was made or before closing the incision, applied by dripping onto skin edges (lasts up to 4 – 6 h).
4. Position the mouse dorsally and prep the tail with 70% isopropyl alcohol.
5. Measure the tail diameter prior to the procedure at 5 mm increments starting 20 mm from the base of the tail using a caliper. These measurements will be used to calculate volume using the truncated cone equation.
6. Mark a 3 mm circumferential excision on the tail 20 mm from the base.
7. Perform a meticulous 3 mm full-thickness skin excision with a sterile surgical blade (size 15), leaving all the underlying vasculature intact under surgical microscopic magnification. Incise the superior circumferential mark (20 mm from tail base) first through the dermis followed by a circumferential full thickness incision 3 mm distal to the first incision
   1. Make a perpendicular full thickness vertical incision to connect the two incisions. Use a toothed fine pickup to grasp a leading edge and use microscissors to carefully dissect deep within the avascular plane to the dermis and superficial to the vein adventitia.
8. Inject 0.1 mL of isosulfan blue (1%) subcutaneously proximal to tip of the tail.
9. Identify the two lymphatic channels adjacent to the lateral tail veins under the surgical microscope. The lymphatics will appear blue because of isosulfan injection. Transect the lymphatics using straight microsurgical scissors. Use the scissors to carefully dissect a plane between the lateral vein and the lymphatic. Then pass the tip of one scissor blade between the lymphatic vessel and the lateral vein and close the blades to transect the lymphatic vessel.
10. Dress the tail wound with a sterile adherent clear dressing. Check post-op incisions daily to ensure that they are not infected or bleeding and provide wound care for 2 weeks.
11. House the animals singly to prevent any further injury to the tail and to prevent the animals from biting each other, which would lead to surgical complications.

Materials

Name	Company	Catalog Number	Comments
Surgical Microscope	Leica, Wetzlar, Germany	MSV266
Adherent Dressing (Tegaderm)	3M, St. Paul, Minn.	1626W
C57BL/6 mice	Jackson Laboratories	664
Micro-Adson Forceps - 1x2 Teeth	Fine Science Tools (USA) Inc.	11019-12
Disposable Needle 30GX1	Fischer Scientific	305128
Operating Scissors	Fischer Scientific	12-460-796
IsosulfanBlue (Lymphazurin) 50mg/5ml	Mylan	67457-220-05