Single and combined impacts of irradiation and surgery on lymphatic vasculature and fibrosis associated to secondary lymphedema

Abstract

Lymphedema (LD) refers to a condition of lymphatic dysfunction associated with excessive fluid accumulation, fibroadipose tissue deposition and swelling. In industrialized countries, LD development mainly results from a local disruption of the lymphatic network by an infection or cancer-related surgery (secondary LD). In the absence of efficient therapy, animal models are needed to decipher the cellular and molecular mechanisms underlying LD and test putative drugs. In this study, we optimized and characterized a murine model of LD that combines an irradiation of the mice hind limb and a radical surgery (lymph node resection associated to lymphatic vessel ligation). We investigated the respective roles of irradiation and surgery in LD formation by comparing their impacts, alone or in combination (with different intervention sequences), on eight different features of the pathology: swelling (paw thickness), indocyanine green (ICG) clearance, lymphatic vasculature remodeling, epidermal and dermal thickening, adipocyte accumulation, inflammatory cell infiltration and collagen deposition. This study supports the importance of radiation prior to surgery to experimentally induce a rapid, severe and sustained tissue remodeling harboring the different hallmarks of LD. We provide the first experimental evidence for an excessive deposition of periostin (POSTN) and tenascin-C (TNC) in LD. Through a computerized method of digital image quantification, we established the spatial map of lymphatic expansion, as well as collagen, POSTN and TNC deposition in papillary and reticular dermis of lymphedematous skins. This mouse model is available to study the patho-physiology of LD and test potential therapeutic targets.

Keywords: collagen; fibrosis; irradiation; lymphatic; lymphedema; periostin; tenascin-C.

FIGURE 1

Schematic representation of the mice experimental model of secondary hind limb lymphedema (LD) induced by surgery and/or irradiation (alone or combined, in two sequences of intervention). Animals were separated into four groups: post-Surgery (n = 8), post-Irradiation (n = 7), post-Surgery/Irradiation (n = 11), post Irradiation/Surgery (n = 13). For each animal, the left hind limb was subjected to LD induction, while the right hind limb was used as control. The chronology of the interventions is detailed for each group as well as the different analyses performed.

FIGURE 2

Macroscopic and histological characterization of lymphedema. Experimental groups are defined in Figure 1. (A) Representative images of control (right paw) and LD (left paw) limbs at day 28. (B) Quantification of the relative volume of LD to control limbs for the four groups over time. (C) Bar graph showing relative paw volume at D28 (statistical test: one-way ANOVA Kruskal–Wallis test, p < 0.05: *, p < 0.01: **, p < 0.001: ***, p < 0.0001: ****). (D) Representative image of a paraffin-embedded control skin section stained with Masson’s trichrome. The epidermis, papillary dermis (PD), reticular dermis (RD) and hair follicles are delineated. Scale bar = 100 µm. (E) Representative images of paraffin-embedded LD skin sections stained with Masson trichrome for each group. Scale bar: 250 µm. The black rectangle represents the magnification (×30) on adipocytes. Graphs on the right correspond to epidermal (top) and dermal (bottom) thicknesses in control and LD skin from each group (Statistical test: two-way ANOVA with multiple comparison Turkey’s test, p < 0.05: *, p < 0.01: **, p < 0.001: ***, p < 0.0001: ****). (F) Presence of CD45 positive inflammatory cells within the LD tissue of each group (scale bar: 50 µm).

FIGURE 3

Impact of the procedures on lymphatic vasculature. (A) Representative images of Lyve-1 positive vessels in control and LD skins. * White asterisk highlights large lymphatic vessels in the reticular dermis (Scale bar: 100 µm). (B) Spatial distribution of Lyve-1 positive lymphatic vessels within the dermis, 0 on the x-axis corresponds to the junction between dermis and epidermis. The dotted line delineates the junction between papillary (PD) and reticular (RD) dermis. (C) Comparison of lymphatic vessel distribution curves between the different experimental groups. (D) Quantification of total labeling area in the dermis determined by measuring the area under the distributions curve (“Integrated Area”) (Statistical test: two-way ANOVA with multiple comparison Turkey’s test, p < 0.05: *, p < 0.01: **, p < 0.001: ***, p < 0.0001: ****).

FIGURE 4

Tracking of ICG fluorescence resorption over time in lymphedema. (A) ICG is accumulated in the LD of the post-Surgery/Irradiation and Irradiation/Surgery groups. (B) Comparison of areas under the curve between 1 h (onset of clearance) and 5 h between the different groups. (Statistical test: two-way ANOVA with multiple comparison Turkey’s test, p < 0.05: *, p < 0.01: **, p < 0.001: ***, p < 0.0001: ****).

FIGURE 5

Impact of the procedures on collagen deposition. (A) Representative images of red Sirius visualized through polarized light in control and LD skins (Scale bar: 200 µm). (B) and (C) Scar Index (red/green staining’s) calculated through computerized method in PD (B) and RD (C). The dotted line delineates the junction between PD and RD.

FIGURE 6

Impact of the procedures on POSTN deposition. (A) Representative images of POSTN area staining in control and LD skins (Scale bar: 100 µm). (B) Distribution curves of POSTN (X axis correspond to the distance from the epidermis/dermis junction). The dotted line delineates the junction between papillary and reticular dermis (PD and RD, respectively). (C) Comparison of the distribution curves of POSTN staining in the different groups. (D) Quantification of total labeling area in the dermis determined by measuring the area under the distributions curve (“Integrated Area”) (Statistical test: two-way ANOVA with multiple comparison Turkey’s test, p < 0.05: *, p < 0.01: **, p < 0.001: ***, p < 0.0001: ****).

FIGURE 7

Impact of the procedures on TNC deposition. (A) Representative images of TNC area staining in control and LD skins (Scale bar: 100 µm). (B) Spatial distribution of TNC within the dermis (0 on the x-axis is the junction between dermis and epidermis). The dotted line delineates the junction between papillary (PD) and reticular (RD) dermis. (C) Comparison of the distribution of TNC staining area between experimental groups. (D) Quantification of total labeling area in the dermis determined by measuring the area under the distributions curve (“Integrated Area”) (Statistical test: two-way ANOVA with multiple comparison Turkey’s test, p < 0.05: *, p < 0.01: **, p < 0.001: ***, p < 0.0001: ****).