Search

Lymphangiogenesis, the formation of lymphatic vessels from preexisting ones, is an important process in wound-healing physiology. Deregulation of lymphangiogenesis and lymphatic vascular remodeling have been implicated in a range of inflammatory conditions, such as lymphedema, lymphadenopathy, tumor growth, and cancer metastasis. Any attempt in understanding various parameters of the lymphangiogenic process and developing desirable therapeutic targets requires recapitulating these conditions in in vivo models. One pitfall with some experimental models is the absence of immune response, an important regulatory factor for lymphangiogenesis. We overcome this issue by using immune competent mice. In this chapter, by using Angiopoietin-2 (Ang2), a protein that belongs to the Ang/Tie signaling pathway, we describe the ear sponge assay with important adaptations, highlighting a reproducible and quantitative tool for assessment of in vivo lymphangiogenesis.

Re-epithelialization after cutaneous injury is a complex and multifaceted process that incorporates numerous cellular components interacting in a myriad of pathways. One of the most crucial aspects of this process is the initiation of keratinocyte migration to fill the wound bed. Re-epithelialization involves both the individual and collective movement of epidermal cells under the control of integrated signaling paradigms. It is therefore essential to develop a simple methodology to dissect the basic movement of epidermal cells in vitro. Scratch assays are relatively simple experiments in which a single layer of cells are plated onto a prepared dish with multiple furrows created in the cell bed. The resulting cellular migration to fill the wound bed can then be imaged and processed quantitatively to investigate migration rates and other factors of interest. Here, we provide important adaptations to the classic scratch assay to make it a robust, reproducible, and quantitative tool for the evaluation of epidermal cell migration.