Intravital insights in skin wound healing using the mouse dorsal skin fold chamber

Abstract

The skin fold chamber is one of the most accepted animal models for studying the microcirculation both in health and disease. Here we describe for the first time the alternative use of the skin fold chamber in mice for intravital microscopic investigation of skin regeneration after creating a full dermal thickness wound. The dorsal skin fold chamber was implanted in hairless SKH1-hr mice and a full dermal thickness wound (area approximately 4 mm2) was created. By means of intravital fluorescence microscopy, the kinetics of wound healing were analyzed for 12 days post wounding with assessment of epithelialization and nutritive perfusion. The morphology of the regenerating skin was characterized by hematoxylin-eosin histology and immunohistochemistry for proliferation and microvessel density. The model allows the continuous visualization of wound closure with complete epithelialization at day 12. Furthermore, a sola cutis se reficientis could be described by an inner circular ring of vessels at the wound margin surrounded by outer radial passing vessels. Inner circular vessels presented initially with large diameters and matured towards diameters of less than 15 microm for conversion into radial spreading outer vessels. Furthermore, wound healing showed all diverse core issues of skin repair. In summary, we were able to establish a model for the analysis of microcirculation in the healing skin of the mouse. This versatile model allows distinct analysis of new vessel formation and maturation in regenerating skin as well as evaluation of skin healing under different pathologic conditions.

Fig. 1

(A,B) Observation window of the mouse dorsal skin fold chamber at day 0 after creation of a circular full-thickness wound with an area of ~5 mm². Bars: 0.5 cm (A), 625 µm (B). Schematic illustration (C) of an implanted dorsal skin fold chamber with its different skin layers after creation of a full dermal thickness wound.

Fig. 2

Photomacroscopic images (A) and quantitative planimetric analysis (B) of wounds during regeneration, displaying the continuous process of wound closure with complete epithelialization at day 12. Images of the upper panel in (A) display the skin fold chamber prior to (d 0_pre) and directly after wounding (d 0_post). Values are given as means ± sem; one-way repeated measures anova with *P < 0.05. Scale bar in d 0_pre = 0.2 cm; Scale bar in d 0_post–d 12 = 625 µm.

Fig. 4

Representative intravital fluorescence microscopic images of radial (A) and circular (B) arranged vessels in the wound re-surfacing skin. Quantitative analysis of their diameters (µm) during regeneration at days 3, 6, 9 and 12 post wounding shows a continuous decrease in caliber, indicating vessel maturation. Values are given as means ± sem; one-way repeated measures anova with *P < 0.05. Bar = 150 µm.

Fig 3

Schematic illustration (A) and representative intravital fluorescence microscopic images (B–D) of the regenerating skin. The sola cutis se reficientis (A,B) consists of an inner ring of circular arranged vessels round the wound margin (C) and an outer pattern of radially arranged vessels (D) supplying the inner ones. The radial vessels represent the sun rays (red) whereas the circular ring of vessels (dark red) round the wound margin represents the sun itself. (B) scale bar = 500 µm, (C,D) scale bar = 150 µm.

Fig. 5

Representative histological H&E-stained sections of skin repair in the mouse dorsal skin fold chamber at day 3 (A,C) and day 12 post wounding (B,D). Whereas the day 3 wound (A,C) is incompletely filled with a fibrin-like granulocyte-rich layer and is beginning to be covered by an epithelial tongue (C, asterisk), the day 12 wound presents with a cellular and highly vascularized granulation tissue and a complete reconstitution of epidermal covering (B,D). Arrows (A,C) point to the tip of epithelial tongues, double-lined arrows (B,D) indicate wound edges defined by the clear cut between the subcutaneous tissue in non-wounded skin and the granulation tissue (D, arrowheads). Bars, 850 µm (A,B); 170 µm (C,D).

Fig. 6

Representative PCNA-immunohistochemistry in sections of skin repair in the mouse dorsal skin fold chamber at day 3 (A,C) and day 12 post wounding (B,D). PCNA staining demonstrated high proliferative activity in the basal layers of the epithelial tongue (C, asterisk) as well as in the granulation/connective tissue, demarcating the skin defect towards normal subcutaneous tissue (B,D, double-lined arrows and arrowheads). Arrows (A,C) point to the tip of epithelial tongues. Bars (A,B), 850 µm; (C,D) 170 µm.

Fig. 7

Representative CD31-immunohistochemistry of the mouse dorsal skin fold chamber at day 12 post wounding (A). High magnification images of the wound granulation tissue display a high density of microvessels, presenting with CD31-positive endothelial lining (B,C, arrows). Bar, 70 µm (A), 30 µm (B,C).