Three-Dimensional Visualization of Lymphatic Vessels in Human Skin and Their Structural Changes During Lymphedema

The lymphatic vessels of the skin, comprising capillaries and collecting vessels, have key roles in waste removal and immune responses, and are essential for maintaining tissue homeostasis. However, the human skin, with its three layers -- epidermis, dermis, and subcutaneous fat -- contains a dense extracellular matrix rich in collagen, so that visualizing lymphatic networks in three dimensions is challenging. To address this limitation, this paper introduces three distinct methods, each tailored to specific molecules of interest and experimental objectives, for the three-dimensional visualization of human skin lymphatics using tissue clearing reagents in combination with confocal or light-sheet microscopy. By targeting adherens junction molecules in lymphatic endothelial cells through immunohistochemistry, structural changes with clear functional implications, particularly the characteristic button-to-zipper-like remodeling of capillary junctions, were evaluated in skin tissues derived from patients with secondary lymphedema. This three-dimensional visualization technique reveals vascular structures that are not discernible through conventional histological tissue section analysis, thereby enhancing the understanding of lymphatic vessel structure and function.