Animal Study and Cadaver Dissection of Lymphedema

Introduction

The lymphatic system is essential to maintaining tissue-fluid homeostasis and aids in immune surveillance by trafficking lymphocytes and antigen-presenting cells to the lymph nodes. Dysfunction in the lymphatic system leads to lymphedema, a chronic and progressive disease where regional accumulation of interstitial fluid, macromolecules, and cellular debris. This leads to regional compromise of immune function and irreversible changes to the affected tissue, skin thickening, subcutaneous fibrosis, and recurrent infections.

Lymphedema is a chronic and debilitating disease. In developed and developing countries, it is commonly a delayed sequelae of cancer treatment secondary to lymph node extirpation and/or radiation therapy. Effective pharmacologic and surgical therapies remain a challenge and currently there is no cure.

Overview of Lymphedema Research

This chapter highlights the historical and current basic research in lymphedema. The development of animal models to study the lymphatic system has resulted in better understanding of the genetic, molecular and pathophysiology of both inherited and acquired lymphedema. Animal models with chronic acquired lymphedema has allowed for refinement of surgical techniques and evidence-based assessment of treatment outcomes.

Molecular Investigation

The mechanisms of lymphangiogenesis were first described by Florence Sabin in the early 1900s based on experiments in pigs describing lymphatic development arising from primary lymph sacs that emerge from embryonic veins. Subsequent studies using genetic mouse models described early lymphatic vascular development as arising from signal-dependent differentiation of embryonic venous endothelial cells. Rickson et al. described the progression of lymphangiogenesis as four distinct stages: lymphatic competence, lymphatic commitment, lymphatic specification, and lymphatic maturation.

Lymphatic competence occurs between embryonic day 8.5 and 9.5, where all venous endothelial cells express vascular endothelial growth factor receptor-3 (VEGFR-3) and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). Endothelial cells can be stimulated to undergo lymphatic differentiation by vascular endothelial growth factor C (VEGF-C) and its receptor (VEGFR-3). Studies of VEGFR-3 knockout mice showed early death from cardiovascular failure and VEGF-C knockout mice showed complete cessation of lymphatic formation.

Currently, the Chy mouse model that expresses a heterogeneous inactivation of VEGFR-3 mutation is used as a model for human primary hereditary lymphedema, Milroy’s disease. In this model, lymphatic function is restored after treatment with a virus-mediated delivery of recombinant VEGF-C. Crossing a Chy mouse with a mouse strain that overexpresses the VEGFR-3–specific ligand VEGF-C156S results in lymphatic function restoration in the double-transgenic offspring. These studies indicate VEGF-C as a promising potential molecular therapy for lymphatic insufficiency.

Lymphatic commitment occurs at embryonic day 9.5–10.5 and is the stage where subpopulations of embryonic venous endothelial cells are marked for lymphatic differentiation via expression of transcription factor prospero-related homeobox (PROX-1). An unknown signal from surrounding mesenchymal cells is thought to stimulate PROX-1 transcription factor. Studies in support of PROX-1 knockout mice demonstrate the absence of lymphatic development, which indicates PROX-1 role in lymphatic-specific differentiation.

Embryonic days 10.5–11.5 mark lymphatic specification and are characterized by the expression of additional lymphatic-specific markers and downregulation of vascular endothelial-specific markers.

Rudimentary lymphatic sacs start to form on embryonic days 11.5–12.5 in a process called lymphatic maturation. By embryonic day 14.5, these lymphatic vessels’ maturation is nearly complete. The process of lymphatic vessel maturation and organization continues until the early period after birth, forming the adult lymphatic network.