Lymphatic and Blood Network Analysis During Obesity

Summary

Obesity is a growing global public health issue. It has been previously associated with lymphatic dysfunction, suggesting a vital crosstalk between the adipose tissue and the lymphatic system. Here, we propose an accessible methodology allowing the distinct labeling of blood and lymphatic vasculatures within the subcutaneous adipose tissue.

Abstract

Lymphatic collecting vessels and lymph nodes are inevitably embedded in adipose tissue. The physiological significance of this observation remains still not elucidated. However, obesity is characterized by impaired lymphatic function and increased vessel permeability. Inversely, lymphatic dysfunction induces obesity in mice, suggesting a significant interplay between lymphatic vessels and the adipose tissue. Therefore, understanding factors leading to lymphatic dysfunction might open new therapeutic windows to prevent obesity and associated comorbidities. The first step in this process requires a precise and detailed visualization of the lymphatic network in healthy and inflamed adipose tissue. Here, we describe a rapid, inexpensive, and efficient method that allows to label and analyze lymphatic and blood vessels. This approach takes advantage of the skin-draining brachial lymph node localization within the subcutaneous adipose tissue. The lymphatic arborization of this tissue can be revealed by injecting fluorochrome-conjugated lectins subcutaneously. Moreover, the in vivo labeling approach provides a way to evaluate lymphatic vessel density and functions. Coupled to blood vessel, adipocyte and immune cell staining, the protocol allows for high-resolution mapping of the subcutaneous adipose tissue by 3D imaging.

Introduction

The lymphatic circulatory system plays a crucial role in the maintenance of tissue homeostasis and the induction of efficient immune responses. Lymphatic vessels run parallel to blood vessels and carry interstitial fluid, metabolites, and immune cells to the local draining lymph node (LN) and finally towards the venous circulation¹. Dysfunctional lymphatic drainage has been observed during infection, inflammation and metabolic diseases^2,3,4,5. The lymphatic vasculature is composed of small size vessels named lymphatic....

Protocol

All animal experimentation was performed in accordance with local ethical committees.

NOTE: Prox1-cre-ERT2 (Prox1tm3(cre/ERT2)Gco/J, Jax #022075) and Rosa26-LSL-tdTomato (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J, Ai9, Jax #007914) were obtained from The Jackson Laboratory and crossed to obtain the inducible lymphatic reporter mouse line Prox1-cre-ERT2::tdTomato. Mice were backcrossed to C57BL/6 background for 10 generations. Six-week-old Prox1-cre-ERT2::tdTomato male mice received tamoxifen d.......

Discussion

This approach provides efficient and robust labeling of the blood and lymphatic vasculatures of the subcutaneous adipose tissue. The separate analysis of blood and lymphatic endothelial networks might unravel pathological mechanisms affecting one or both of the circulatory systems during obesity or other pathological conditions. This protocol aims to analyze the architecture of the vascular systems, their interaction with stromal and immune cells, and their functionality during health and disease.

Materials

Name	Company	Catalog Number	Comments
Lectin DyLight 649	Vector Labs	DL-1178-1	Described in protocol
Lectin DyLight 488	Vector Labs	DL-1174	Described in protocol
Paraformaldehyde	VWR Chemicals	9713.1000
Sucrose	Euromedex	CAS Number 57-50-1
Anti-Podoplanin	AngioBio	11-033	Dilution : 1/50
Lectin DyLight 594	Vector Labs	DL-1177	Described in protocol
Anti-MHCII (Clone M5/114.15.2)	Biolegend	107618	Dilution : 1/100
Anti-CD11b (Clone M1/70)	Biolegend	101218	Dilution : 1/100
Anti-CD68 (Clone FA.11)	Biolegend	137004	Dilution : 1/100
Anti-B220 (Clone RA3-6B2)	Biolegend	103225	Dilution : 1/100
Anti-Perilipin (Clone PERI 112.17)	Progen	651156	Dilution : 1/50
Anti-CD3 (Clone 17A2)	Biolegend	100210	Dilution : 1/100