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

Summary

Here, a three-dimensional visualization of the lymphatic vessel structure in human skin was achieved using confocal or light-sheet microscopy after the application of three distinct tissue-clearing techniques. Combinations of these methods with immunohistochemical staining provide insight into functional changes in lymphatic vessels during the progression of lymphedema.

Abstract

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.

Introduction

There is increasing interest in the function of lymphatic vessels since the recognition of their role in maintaining skin homeostasis, beyond their well-established involvement in cancer metastasis^1,2. Lymphatic vessels are structurally divided into capillaries and collecting vessels, each possessing distinct characteristics. Lymphatic capillaries consist of a single-layered structure formed by lymphatic endothelial cells and serve as initial collection sites for interstitial fluid, waste products, and immune cells. In contrast, collecting lymphatic vessels have a bilayered structure with smooth muscle cells surrounding the lymphatic endothelial layer, enabling active transport of lymph fluid collected by the capillary network toward the central circulation³. Mouse models have been utilized to elucidate the structural features, particularly of bronchial lymphatic vessels, and have revealed how inflammatory conditions induce structural remodeling⁴. Previous work demonstrated that ultraviolet-induced inflammation causes abnormal dilation of lymphatic capillaries, which compromises their fluid collection function. The molecular mechanisms underlying this dysfunction were identified, specifically showing that increased VEGF-A expression coupled with decreased VEGF-C expression drives these structural alterations^5,6,7.

While three-dimensional visualization techniques have significantly advanced understanding of the lymphatic network in mouse models^8,9, comparable structural analysis of human lymphatic vessels has remained challenging. This analytical gap arises from the difficulties of working with human tissues, including limited sample availability and the technical issues involved in achieving comprehensive three-dimensional imaging. In particular, the skin, characterized by a three-layer structure comprising the epidermis, dermis, and subcutaneous fat, and functioning as a barrier to the external environment, presents greater challenges than other soft organs. Furthermore, human skin presents distinct analytical challenges relative to mouse models, including greater tissue thickness, hampering deep imaging penetration. In addition, the abundant extracellular matrix components, particularly collagen, along with their age-related structural alterations, create optical barriers that reduce the efficacy of emerging tissue-clearing techniques that have revolutionized lymphatic imaging in mice¹⁰.

Various tissue-clearing agents were previously tested to visualize human skin capillaries, demonstrating their tissue compatibility¹¹. Following that work, tissue-clearing reagents were successfully used to visualize human skin lymphatic vessels in three dimensions¹². By focusing on the adherens junctions of lymphatic endothelial cells, structural changes in lymphatic vessels associated with the progression of lymphedema were identified¹³. The purpose of this article is to describe in detail the procedures for three-dimensional visualization of human skin lymphatic vessels using combinations of three skin-clearing techniques, along with confocal microscopy and light-sheet microscopy, and immunohistochemical staining tailored to specific research objectives. In this study, although we focus solely on fresh human skin tissues that were fixed on the day of excision and have not undergone freeze-thaw cycles, these techniques provide insights into not only the structural changes of lymphatic vessels during disease and aging, but also the associated functional alterations.

Protocol

Skin samples of human subjects were acquired from the Chiba University Hospital (Chiba, Japan). All procedures involving human subjects were approved by the Institutional Review Board of the Chiba University Hospital and the Shiseido Global Innovation Center, and all subjects provided written informed consent. The reagents and the equipment used are listed in the Table of Materials.

1. Pretreatment of human skin samples

1. Remove the subcutaneous tissue of excised specimens upon acquisition.
   NOTE: The interval between skin excision and acquisition was approximately h. During transport, the tissue was immersed in DMEM and kept on ice packs.
2. Place the samples with the epidermal side down on a 60 °C hot plate for 45 s to separate the epidermis and dermis.
3. Immediately immerse the specimens in ice-cold PBS.
4. Separate the epidermis and dermis using tweezers.
5. Fix the dermal parts in 4% paraformaldehyde at 4°C for 16 h, and subsequently wash with PBS prior to use.
   NOTE: After fixation, the samples were stored in PBS at 4 °C, and the immunostaining steps were initiated within months.
   CAUTION PFA solution is toxic. All preparation and handling must be performed in a fume hood while wearing appropriate personal protective equipment.

2. Whole-mount immunostaining combined with clearing reagents

1. iDISCO technique
   1. Trim the specimens to approximately 5 mm × 5 mm × 5 mm in size.
   2. Transfer the specimens into 5% Triton X-100 and 2.5% Tween 20 in PBS on an orbital shaker set to 50 rpm at 24 °C for 16 h for permeabilization.
   3. Wash the specimens with PBS at 24 °C for 1 h, repeating the process three times.
   4. Immerse the specimens in freshly prepared blocking buffer A (1% BSA, 0.1% Tween 20, 0.03% sodium azide in PBS) on an orbital shaker set to 50 rpm at 24 °C for 72 h.
   5. Incubate the specimens with primary antibody against podoplanin (1:100) in a blocking buffer A on an orbital shaker set to 50 rpm at 37 °C for 72 h.
   6. Wash the specimens with washing buffer A (0.1% Tween-20 in PBS) at 24 °C for 1 h, repeating the process three times. Then, maintain the specimens in washing buffer A on an orbital shaker set to 50 rpm at 4 °C for 72 h.
   7. Incubate the specimens with secondary antibody against mouse IgG conjugated to Alexa Fluor 647 (1:1000) in a blocking buffer A on an orbital shaker set to 50 rpm at 37 °C for 72 h.
   8. Repeat step 1.6.
   9. Incubate the specimens with 50% MeOH at 24 °C for 3 h.
   10. Incubate the specimens with 70% MeOH at 24 °C for 3 h.
   11. Incubate the specimens with 100% MeOH at 24 °C for 16 h.
   12. Incubate the specimens with dichloromethane at 24 °C for 15 min, repeating the process twice.
   13. Clear the specimens with dibenzyl ether at 24 °C for 16 h.
       NOTE: If the tissue has not become transparent at this point, further waiting will result in no significant improvement.
   14. Capture images of the specimens with a light-sheet microscope.
       NOTE: Use a Lightsheet with an EC Plan-Neofluar 5×/0.16 objective; set illumination (excitation) at 63 nm and detection at 648 nm; acquire a Z-stack with a step size of 5.8 µm.
       CAUTION: Methanol, dichloromethane, and dibenzyl ether are hazardous. All preparation and handling must be performed in a fume hood while wearing appropriate personal protective equipment.
2. Rapiclear 1.52 reagent
   1. Trim the specimens to approximately 5 mm × 5 mm × 2 mm in size.
   2. Transfer the specimens into 2% Triton-X 100 in PBS on an orbital shaker set to 50 rpm at 24 °C for 16 h for permeabilization.
   3. Immerse the specimens in freshly prepared blocking buffer B (5% donkey serum and 1% Triton-X 100 in PBS) on an orbital shaker set to 50 rpm at 4 °C for 16 h.
   4. Incubate the specimens with primary antibodies against podoplanin and VE-cadherin (both at 1:100) in a blocking buffer B on an orbital shaker set to 50 rpm at 4 °C for 72 h.
   5. Wash the specimens with washing buffer B (3% NaCl and 0.2% Triton-X 100 in PBS) at 24 °C for 1 h, repeating the process three times. Then, maintain the specimens in washing buffer B on an orbital shaker set to 50 rpm at 4 °C for 16 h.
   6. Wash the specimens with PBS at 24 °C for 1 h.
   7. Incubate the specimens with secondary antibodies against mouse IgG and rabbit IgG, conjugated to Alexa Fluor 647 and 568, respectively (both at 1:1000), in blocking buffer B on an orbital shaker set to 50 rpm at 4 °C for 16 h.
   8. Repeat steps 1.5. and 1.6.
   9. Clear the specimens with at least 5 times the sample volume of Rapiclear 1.52 at 24 °C for 16 h.
      NOTE: The clearing of the peripheral regions of the tissue was observed in less than 1 h.
   10. Embed the cleared specimens in a polydimethylsiloxane gel chamber placed on a coverslip with fresh Rapiclear 1.52 reagent.
   11. Image the specimens with a confocal microscope.
       NOTE: Use a confocal microscope with a Plan-Apochromat 10×/0.45 M27 objective; set excitation at 633 nm and 561 nm, and detection range 641-754 nm and 566-628 nm, with a GaAsP detector; acquire a Z-stack with a step size of 3.15 µm.
3. CUBIC technique
   1. Trim the specimens to approximately 5 mm × 5 mm × 3 mm in size.
   2. Immerse the specimens in 50% CUBIC-L at 24 °C for 24 h as a pretreatment for delipidation.
   3. Transfer the specimens into 100% CUBIC-L on an orbital shaker set to 50 rpm at 37 °C for 7 days for delipidation.
   4. Wash the specimens with PBS at room temperature for 24 h.
   5. Immerse the specimens in freshly prepared blocking buffer A on an orbital shaker set to 50 rpm at 24 °C for 72 h.
   6. Incubate the specimens with primary antibody against VE-cadherin, podoplanin, and CD31 (1:100, 1:100, and 1:200, respectively) in blocking buffer A on an orbital shaker set to 50 rpm at 37 °C for 72 h.
   7. Wash the specimens with washing buffer A at 24 °C for 1 h. Repeat the process three times, then maintain the specimens in washing buffer A on an orbital shaker set to 50 rpm at 4 °C for 72 h.
   8. Incubate the specimens with secondary antibodies against mouse IgG, rabbit IgG, and sheep IgG, conjugated to Alexa Fluor 647, 568, and 488, respectively (all at 1:1000), in blocking buffer A on an orbital shaker set to 50 rpm at 37 °C for 72 h.
   9. Repeat step 3.7.
   10. Pre-clear the specimens with 50% CUBIC-R+(M) at 24 °C for 16 h.
   11. Clear the specimens with 100% CUBIC-R+(M) at 24 °C for 16 h.
       NOTE: If the tissue has not become transparent at this point, further waiting will result in no significant improvement.
   12. Image the specimens with a confocal microscope.
       ​NOTE: Use a confocal microscope with a Plan-Apochromat 20×/0.8 M27 objective; set excitation at 633 nm, 561 nm, and 488 nm, and detection range 641-754 nm, 566-628 nm, and 479-550 nm, with a GaAsP detector; acquire a Z-stack with a step size of 0.78 µm.

Results

Difference in light transmittance of cleared human skin tissue with and without epidermis
Figure 1 shows representative photographs of human skin specimens with and without epidermis, following permeabilization and clearing with Rapiclear 1.52 reagent. Epidermal removal enhances tissue transparency by eliminating melanin, a potent light absorber, as confirmed by the enhanced visibility of the black line beneath the samples.

Three-dimen...

Discussion

Three-dimensional visualization of lymphatic networks in biological tissues is critically important for understanding their structural characteristics and physiological functions, as well as pathological changes. This protocol presents three distinct tissue clearing methods optimized for human skin tissue, each tailored for specific observational objectives.

Epidermal removal is considered a critical step for eliminating melanin, which reduces tissue transparency and interferes with histologic...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-CD31 antibody	R&D Systems	AF806	dilution at 1:200
Anti-Podoplanin antibody	Dako	M3619	dilution at 1:100
Anti-VE-cadherin antibody	Cell Signaling Technology	#2500	dilution at 1:100
CUBIC-L	Tokyo Chemical Industry	T3740
CUBIC-R+(M)	Tokyo Chemical Industry	T3741
DMEM, low glucose, pyruvate	Thermo Fisher SCIENTIFIC	11885084
Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647	Thermo Fisher SCIENTIFIC	A-31571	dilution at 1:1000
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568	Thermo Fisher SCIENTIFIC	A10042	dilution at 1:1000
Donkey anti-Sheep IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	Thermo Fisher SCIENTIFIC	A-11015	dilution at 1:1000
Rapiclear 1.52	Sunjin Lab	RC152001
ZEISS Lightsheet Z.1 Microscope	Carl Zeiss
ZEISS LSM 880 Confocal Laser Scanning Microscope	Carl Zeiss