Location of injury influences the mechanisms of both regeneration and repair within the MRL/MpJ mouse

Abstract

The adult MRL/MpJ mouse regenerates all differentiated structures after through-and-through ear punch wounding in a scar-free process. We investigated whether this regenerative capacity was also shown by skin wounds. Dorsal skin wounds were created, harvested and archived from the same animals (MRL/MpJ and C57BL/6 mice) that received through-and-through ear punch wounds. Re-epithelialization was complete in dorsal wounds in both strains by day 5 and extensive granulation tissue was present by day 14 post-wounding. By day 21, wounds from both strains contained dense amounts of collagen that healed with a scar. The average wound area, as well as alpha-smooth muscle actin expression and macrophage influx were investigated during dorsal skin wound healing and did not significantly differ between strains. Thus, MRL/MpJ mice regenerate ear wounds in a scar-free manner, but heal dorsal skin wounds by simple repair with scar formation. A significant conclusion can be drawn from these data; mechanisms of regeneration and repair can occur within the same animal, potentially utilizing similar molecules and signalling pathways that subtly diverge dependent upon the microenvironment of the injury.

Fig. 1

Day 3 wound sections showing different regions of the wound: (a) MRL/MpJ wound margin, (b) MRL/MpJ wound centre, (c) C57BL/6 wound margin, (d) C57BL/6 wound centre. GT, granulation tissue; E, epithelium; D, dermis; M, muscle. Arrows denote wound margins. Scale bar = 1 mm.

Fig. 2

Day 5 wound sections showing different regions of the wound: (a) MRL/MpJ wound margin, (b) MRL/MpJ wound centre, (c) C57BL/6 wound margin, (d) C57BL/6 wound centre. GT, granulation tissue; E, epithelium; D, dermis; M, muscle. Arrows denote wound margins. Scale bar = 1 mm.

Fig. 3

Day 7 wound sections showing different regions of the wound: (a) MRL/MpJ wound margin, (b) MRL/MpJ wound centre, (c) C57BL/6 wound margin, (d) C57BL/6 wound centre – arrowheads denote areas of collagen deposition within the granulation tissue. GT, granulation tissue; E, epithelium; D, dermis; M, muscle. Arrows denote wound margins. Scale bar = 1 mm.

Fig. 4

Sections of the wound centres for the two strains at different time points. Day 14 (a – MRL/MpJ wound centre, b – C57BL/6 wound centre), day 21 (c – MRL/MpJ wound centre, d – C57BL/6 wound centre), day 28 (e – MRL/MpJ wound centre, f – C57BL/6 wound centre) wound sections. E, epithelium; D, dermis; M, muscle. Arrows denote wound margins. Scale bar = 0.5 mm.

Fig. 5

Sections of the wound centres for the two strains at different time points. Day 56 (a – MRL/MpJ wound centre, b – C57BL/6 wound centre) and day 84 (c – MRL/MpJ wound centre, d – C57BL/6 wound centre) wound sections. HF, hair follicle; E, epithelium; D, dermis; M, muscle. Arrows denote wound margins. Scale bar = 0.5 mm.

Fig. 6

Graph of collated wound surface areas. Total wound areas were calculated using data collected from the evenly spaced wound sections. Note that the MRL/MpJ wounds are slightly larger than the C57BL/6 wounds at the early time points, although not significantly so (P > 0.05). Also note that the day 84 wound areas are the same in both groups, indicative of similar sized scars, n = 3.

Fig. 7

Mac-3 staining for macrophages at the wound edge (a,d), wound centre (b,e) and in the normal skin to the side of the wound (c,f) in MRL/MpJ (a–c) and C57BL/6 (d–f) sections at 3 days post-injury. Note that there are few positive cells in the wound centre and margin at this time, but more in the normal skin away from the wound. WE = wound edge, HF = hair follicle, arrows denote positively stained cells. Insets (a,b,d,e) are a slightly higher magnification of the stained cells. Scale bar = 0.25 mm.

Fig. 8

Mac-3-positive cell counts expressed as a percentage of the total cell count in both MRL/MpJ (closed boxes) and C57BL/6 (open boxes) wounds. Cells were counted at the wound centre (a), wound edge (b) and in the normal dermis away from the wound (c). There was no significant difference in the percentage of Mac-3-positive cells between the two strains in any region at any time point. Total cell counts at the wound edge (d) demonstrate that no significant difference existed between the two strains of mice, suggesting that percentage cell counts are truly representative. Results shown are mean ± SEM, n = 3.

Fig. 9

Alpha smooth muscle actin immunostaining at days 5 (a,b) and 7 (c–f) post-injury in MRL/MpJ (a,c,e) and C57BL/6 (b,d,f) wound sections. Note that the majority of positive staining occurs as a dense band stretching from the cut panniculus to the epidermis at the wound edge, between the dermis and granulation tissue (a–d). By contrast, there is much less staining in the granulation tissue in the centre of the wound (e,f). In this area minimal differentiation of fibroblasts into myofibroblasts has occurred. Also note that the blood vessel smooth muscle cells have stained strongly for α-SMA, as was expected. HF = hair follicle. Scale bar = 0.15 mm.

Fig. 10

The histological differences between repair and regeneration in the MRL/MpJ mouse shown at two different time points during healing. Back skin wounds at day 14 post-wounding show full re-epithelialization and maturing granulation tissue (a). By day 84 post-wounding a scar can be seen at the wound centre, devoid of differentiated structures and showing a parallel arrangement of collagen bundles (b). Towards the left-and right-hand wound margins the collagen is more loosely arranged and contains hair follicles (HF), sweat and sebaceous glands as well as adipose tissue (A). (c) A transverse section through an ear, 14 days post-wounding. Two opposing blastema-like structures can be seen with thickened apical epithelial tips and downgrowths projecting into the mesenchymal space (arrowed). The regenerated tissue can be seen from the cut cartilage edge. By day 84 post-wounding in the ear, the two opposing blastema-like structures fuse (d). Cartilage islands are present (C), skeletal muscle is regenerating (M) and the epithelial edges are seen to fuse together (arrow). SG, sebaceous gland; E, epithelium; D, dermis. Scale bar = 0.5 mm.