This study investigates the role of lymphatic vessels in the skin dermis and their involvement in lymphedema development. It outlines a method for inducing lymphedema in murine models through surgical intervention.
Lymphatic vessels are present in the skin dermis and their function is to remove excess interstitial fluid from the tissue. Damage to lymphatic vessels causes excess fluid accumulation in the interstitial space and an increase in subcutaneous deposition of fibrous and adipose tissue. These changes result in lymphedema.
To generate lymphedema in the murine tail, take an anesthetized mouse and place it in the prone position. Make a full-thickness circumferential skin excision at an appropriately distant site, distal to the base of the tail. Incise another site on the tail inferior to the first cut.
Make a vertical skin incision connecting the two pre-made incisions. Using this cut, excise the overlaying skin patch to expose the lateral veins and lymphatic vessels.
Take a syringe containing colored tracking dye and inject it subcutaneously near the end of the tail, making the lymphatic vessels appear blue. Transect the lymphatic vessels, wrap the wound site, and monitor the mouse for the desired time duration.
Microdissection arrests the flow of lymph in the vessels and fluid collects in tissue space. Consequently, sustained lymphedema is observed as swelling of the mouse tail dorsal to the surgical site.
To begin, position the 8-week-old sedated mouse sternally, and prep the tail with 70% isopropyl alcohol. Use a caliper to measure the tail diameter at 5-millimeter increments starting 20-millimeters from the base of the tail. Mark a 3-millimeter circumferential incision on the tail 20-millimeters from the base.
Under surgical microscopic magnification, perform a meticulous 3-millimeter full-thickness skin excision, leaving all the underlying vasculature intact. Incise a superior circumferential mark, first through the dermis, followed by a circumferential full-thickness incision 3-millimeters distal to the first incision. Make a perpendicular full thickness vertical incision to connect the two incisions, then use a toothed fine pickup to grasp a leading edge and dissect deep within the avascular plane to the dermis and superficial to the vein adventitia, with microscissors.
Inject 0.1 milliliter of 1% isosulfan blue subcutaneously proximal to the tip of the tail. Identify the two lymphatic channels appearing blue due to the injection, adjacent to the lateral tail veins. Transect the lymphatics carefully, dissecting a plane between the lateral vein and the lymphatic with straight microsurgical scissors. Pass the tip of one scissor blade between the lymphatic vessel and the lateral vein, and close the blades to transect the lymphatic vessel. When finished, dress the tail wound with a sterile adherent clear dressing.
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