Defining Wound Healing Progression in Cetacean Skin: Characteristics of Full-Thickness Wound Healing in Fraser's Dolphins (Lagenodelphis hosei)

Abstract

Cetaceans are tight-skinned mammals that exhibit an extraordinary capacity to heal deep soft tissue injuries. However, essential information of large full-thickness wound healing in cetaceans is still lacking. Here, the stages of full-thickness wound healing were characterized in Fraser's dolphins (Lagenodelphis hosei). The skin samples were collected from normal skin and full-thickness cookiecutter shark (Isistius brasiliensis)-bite wounds of stranded carcasses. We defined five stages of wound healing according to macroscopic and histopathological examinations. Wounds in Stage 1 and 2 were characterized by intercellular and intracellular edema in the epidermal cells near the wound edge, mixed inflammatory cell infiltration, and degradation of collagen fibers. In Stage 3 wounds, melanocytes, melanin granules, rete and dermal ridges were noticed in the neo-epidermis, and the adipose tissue in adjacent blubber was replaced by cells and fibers. Wounds in Stage 4 and 5 were characterized by gradual restoration of the normal skin architecture including rete and dermal ridges, collagen bundles, and adipose tissue. These phenomena were quite different from previous studies in terrestrial tight-skinned mammals, and therefore, further in-depth research into the mechanisms of dolphin wound healing would be needed to gain new insights into veterinary and human regenerative medicine.

Keywords: adipose tissue; dolphins; melanocytes; regenerative medicine; rete ridges; wound healing.

Figure 1

Normal skin of Fraser’s dolphin. H&E staining of Fraser’s dolphin normal skin. (A) Low power view of epidermis, dermis, and blubber. Scale bar = 500 µm. (B) High power view of epidermis and dermis. Flattened cells with pyknotic nuclei were observed in the stratum externum. Melanin granules were distributed in all layers of the epidermis. Prominent rete ridges and dermal papillae formed an interdigitated interface between epidermis and dermis. Scale bar = 100 µm.

Figure 2

Reference macroscopic and low power microscopic images of each wound healing stage. (A,F) Stage 1 wound. (B,G) Stage 2 wound. (C,H) Stage 3 wound. (D,I) Stage 4 wound. (E,J) Stage 5 wound.

Figure 3

Stage 1 wound of Fraser’s dolphin. H&E staining of Fraser’s dolphin Stage 1 wound. (A) Low power view of Stage 1 wound. There was no obvious change in the dermis and blubber. A few collagen fibers at the wound edge were necrotic. Black arrow: edge of wound. Scale bar = 500 µm. (B) High power view of Stage 1 wound epidermis. Epidermal cells were pale, enlarged and edematous. Karyolysis (red arrowhead) and pyknosis (black arrowhead) were observed. Scale bar = 100 µm.

Figure 4

Stage 2 wound of Fraser’s dolphin. H&E staining of Fraser’s dolphin Stage 2 wound. (A) Low power view of Stage 2 wound. Scale bar = 1 mm. (B) High power view of wound edge epidermis. Near the wound edge, keratinocytes were edematous with pyknotic nuclei or even without nuclei. Scale bar = 50 µm. (C) High power view of blubber on the wound surface. The collagen and adipose tissue were necrotic. Scale bar = 50 µm. (D) High power view of blubber nearby the wound edge. Inflammatory cells infiltrated in the blubber. Scale bar = 20 µm. (E) High power view of blubber away from the wound edge. Hyperemia and hemorrhage occurred in the blubber, mainly in the middle and bottom layer of blubber. Scale bar = 50 µm.

Figure 5

Stage 2 wound of Fraser’s dolphin. H&E staining of Fraser’s dolphin Stage 2 wound. (A) Low power view of Stage 2 wound epidermis and dermis. Degraded collagen fibers (arrowhead) in the dermis close to the wound edge. Black arrow: rete ridges. Scale bar = 200 µm. (B) High power view of degraded collagen fibers (arrowhead). Scale bar = 20 µm. (C) Low power view of blubber nearby the wound edge. Scale bar = 200 µm. (D) A loose fibrin network (asterisks) composed of fibrin, erythrocytes, and inflammatory cells replaced adipose tissue in the blubber. Scale bar = 20 µm.

Figure 6

Stage 3 wound of Fraser’s dolphin. (A,B,D,E): H&E staining of Fraser’s dolphin Stage 3 wound. (A) Low power view of Stage 3 wound. Black arrow: wound center. Scale bar = 1 mm. (B) High power view of migrating epithelial tongue. Elongated epithelial strands appeared in the migrating epithelial tongue. Scale bar = 100 µm. (C) Fontana-Masson staining of Fraser’s dolphin Stage 3 wound. Melanocytes and melanin granules were present in the neo-epidermis. Scale bar = 100 µm. (D) High power view of Stage 3 wound dermis. Thick collagen fibers broke into pieces (arrowhead), and regions of adipose tissue were replaced by numerous cells and thin fibers (asterisks). Scale bar = 50 µm. (E) High power view of Stage 3 wound center. Wound center granulation tissue consisted of cells, extracellular matrix, and numerous microscopic blood vessels. Scale bar = 50 µm. (F,G): Trichrome staining of Fraser’s dolphin Stage 3 wound. (F) Low power view of Stage 3 wound. Block arrow: wound center. Scale bar = 1 mm. (G) High power view of migrating epithelial tongue. Neo-epidermis showed a reticular structure. Thin collagen fibers and blood vessels were present in the papillary dermis connected to the neo-epidermis. Scale bar = 100 µm.

Figure 7

Stage 4 wound of Fraser’s dolphin. H&E staining of Fraser’s dolphin Stage 4 wound. (A) Low power view of Stage 4 wound. Scale bar = 500 µm. (B) High power view of Stage 4 wound dermis. Numerous blood vessels, cells, and thin collagen fibers existed in the reticular dermis of Stage 4 wounds. Scale bar = 50 µm. (C) High power view of Stage 4 wound blubber. There were a few thick collagen fibers (asterisks) in the lower part of the blubber. Scale bar = 50 µm.

Figure 8

Stage 5 wound of Fraser’s dolphin. H&E staining of Fraser’s dolphin Stage 5 wound. (A) Low power view of Stage 5 wound. Normal skin structure including adipose tissue was noticed in the Stage 5 wound. Scale bar = 500 µm. (B) High power view of Stage 5 wound epidermis. Epidermis showed nearly normal rete and dermal ridges. Scale bar = 100 µm. (C) High power view of Stage 5 wound blubber. Adipocytes appeared in the perivascular region (asterisks). Scale bar = 50 µm.