Figure 1.
Wound healing assay design and morphology of Xenopus tropicalis and human skin.
(A) Diagram of the ‘punch within a punch’ biopsy injury inflicted on both X. tropicalis and human skin (not to scale). (B) X. tropicalis punch-in-a-punch biopsy. (C) Human skin punch-in-a-punch biopsy. Full thickness X. tropicalis (D) and human skin (F) with white lines demarcating epidermis (Ep), dermis (De) and hypodermis/subcutis (Hd). Black arrows in (D) indicate the margins of the stratum spongiosum (SSp) stratum compactum (SCo). A smaller mucous (Mu) and larger granular/poison (Gr) gland are indicated (D). Human sebaceous glands (SG) and sweat glands (SW) and hair follicle (HF) are indicated (F). Higher magnification images (E & G) display the epidermis, specifically indicating Stratum germinativum (SGe), Stratum spinosum (SSp) and Stratum corneum (SCo) of frog epidermis (C) and Stratum basale (Sba) stratum spinosum (SSp), Stratum granulosum (SGr), Stratum Lucidum (SLu) and Stratum corneum (SCo) of human epidermis (G). A white arrow shows the location of a melanocyte (Mel) at the dermal-epidermal junction. (E). Scale bars in D and F = 300 µm and in E and G = 50 µm.
Figure 2.
Frog serum and estrogen promote re-epithelialisation and proliferation in Xenopus tropicalis skin.
(A) Representative images of re-epithelialisation sheet in control, 5% frog serum and 100 nM 17β-estradiol (Est)-treated punch wounds at day 7 in culture (scale bars: 0.5 mm). White-hatched lines indicate the leading edge of new epithelial sheets. (B) Representative images of PH3-positive cells in control, 5% frog serum and 100 nM 17β-estradiol-treated punch wounds at day 7 in culture. The white-hatched line demarcates new epithelial tongue. (C) Representative images of TUNEL-positive cells in control, 5% frog serum and 100 nM 17β-estradiol-treated punch wounds at day 7 in culture. The white-hatched line demarcates new epithelial tongue. Scale bars in (B) and (C) are 100 µm. (D) The graph shows the percentage reduction in wound-area of Xenopus tropicalis punch wounds in control, 5% frog serum (FS) and 100 nM 17β-estradiol-treated skin. (E) Percentage of proliferative (PH3-positive) cells present in the new epithelial tongue during re-epithelialisation. (F) Percentage of apoptotic (TUNEL-positive) cells present in the new epithelial tongue during re-epithelialisation. Data are mean ± SEM of 4–5 frogs (2 male and 3 female). Significance relative to control data at the same time-point denoted by *P<0.05, **P<0.01, ***P<0.001.
Figure 3.
Xenopus tropicalis wound closure: promotion of re-epithelialisation and proliferation by TRH.
(A) Representative images of the re-epithelialisation sheet in control, 10 nM and 10 µM TRH-treated punch wounds at day 7 in culture (scale bars: 0.5 mm). White-hatched lines indicate the leading edge of new epithelial sheets. (B) Representative images of PH3-positive cells in control, 10 nM and 10 µM TRH-treated punch wounds at day 7 in culture. The white-hatched line demarcates new epithelial tongue. (C) Representative images of TUNEL-positive cells in control, 10 nM and 10 µM TRH-treated punch wounds at day 7 in culture. The white-hatched line demarcates new epithelial tongue. Scale bars in (B) and (C) are 100 µm. (D) The graph shows the percentage reduction in wound-area of X. tropicalis punch wounds in control, 10 nM and 10 µM TRH-treated skin. (E) Percentage of proliferative (PH3-positive) cells present in the new epithelial tongue during re-epithelialisation. (F) Percentage of apoptotic (TUNEL-positive) cells present in the new epithelial tongue during re-epithelialisation. Data are mean ± SEM of 16 frogs (8 male and 8 female). Significance relative to control data at the same time-point denoted by *P<0.05, ***P<0.001.
Figure 4.
Xenopus tropicalis mitosis: TRH increases epidermal mitosis.
(A–C) Representative images of Weigert’s stained X. tropicalis epidermis with black arrows indicating mitotic cells. (D) The graph shows the percentage of mitotic figures identified by Weigert’s staining in the X. tropicalis epidermis. 200 nuclei were analysed per skin section, with 3 sections per animal counted. (E) The graph displays the total number of DAPI+ nuclei in the new epithelial tongues. Data are mean ± SEM of 4 frogs (2 male and 2 female). Significance relative to control data at the same time-point denoted by **P<0.01, ***P<0.001.
Figure 5.
Increased re-epithelialisation and proliferation in human skin following 17β-estradiol treatment.
(A) Representative H&E stained sections of human skin punch-wounds following 6 days culture with either vehicle control, 100 nM or 1 µM 17β-estradiol (Est). New epithelial tongue indicated by red lines, scale bar represents 50 µm. (B) Representative images of Ki-67-TUNEL double-stained sections of human skin punch wounds following 6 days culture with either vehicle control, 100 nM or 1 µM 17β-estradiol. The white-hatched line demarcates new epithelial tongue. (C) The graph displays length measurements of the new epithelial tongue (as demarcated by the red lines in A) as analysed from H&E stained human skin sections following treatment with vehicle control, 100 nM and 1 µM 17β-estradiol. (D) Percentage of proliferative (Ki67-positive) cells in the new epithelial tongue of vehicle control, 100 nM and 1 µM 17β-estradiol-treated human skin wounds. (E) Percentage of apoptotic (TUNEL-positive) cells in the new epithelial tongue of vehicle control, 100 nM and 1 µM 17β estradiol-treated human skin wounds. Data are mean ± SEM of 4 female donors. Significance relative to control data denoted by *P<0.05, **P<0.01, ***P<0.001.
Figure 6.
Cytokeratin 6 and Involucrin expression in human skin punch wounds following 17β-estradiol treatment.
Representative images indicating cytokeratin 6 (CK6) expression in control, 100 nM and 1 µM 17β-estradiol (Est)-treated human skin punch wound sections, 6 days post-wounding. The white-hatched line demarcates new epithelial tongue. (B) Representative images indicating involucrin (Inv) expression in control, 100 nM and 1 µM 17β-estradiol-treated human skin punch wound sections, 6 days post-wounding. The white-hatched line demarcates new epithelial tongue. (C) Quantification of CK6 immunoreactivity in the new epithelial tongues indicated (A). (D) Quantification of involucrin immunoreactivity in the new epithelial tongues indicated in (B). Data are mean ± SEM of 4 female donors. Significance relative to control data denoted by **P<0.01.
Figure 7.
TRH stimulates re-epithelialisation and enhances proliferation in wounded human skin.
(A) Representative H&E stained sections of human skin punch-wounds following 6 days culture with either vehicle control or 10 ng/mL TRH. New epithelial tongues indicated by the white-hatched lines. Scale bars represent 50 µm. (B) Representative images of Ki-67-TUNEL double-stained sections of human skin punch wounds following 6 days culture with either vehicle control or 10 ng/mL TRH. The white-hatched line demarcates new epithelial tongue. (C) The graph displays length measurements of the new epithelial tongue (as demarcated by the white-hatched lines in A) as analysed from H&E stained human skin sections following treatment with vehicle control, 5 ng/mL and 10 ng/mL TRH. (D) Percentage of proliferative (Ki67-positive) cells in the new epithelial tongue of vehicle control, 5 ng/mL and 10 ng/mL TRH-treated human skin wounds. (E) Percentage of apoptotic (TUNEL-positive) cells in the new epithelial tongue of vehicle control, 5 ng/mL and 10 ng/mL TRH-treated human skin wounds. Data are mean ± SEM of 4 female donors. Significance relative to control data at the same time-point denoted by *P<0.05, **P<0.01, ***P<0.001.
Figure 8.
TRH increases cytokeratin 6 but not involucrin expression in human skin punch wounds.
(A) Representative images indicated cytokeratin 6 (CK6) expression in control, 5 ng/mL and 10 ng/mL TRH-treated human skin punch wound sections, 6 Days post-wounding. The white-hatched line demarcates new epithelial tongue. (B) Representative images indicating involucrin (Inv) expression in control, 5 ng/mL and 10 ng/mL TRH-treated human skin punch wound sections, 6 Days post-wounding. The white-hatched line demarcates new epithelial tongue. (C) Quantification of involucrin immunoreactivity in the new epithelial tongues indicated in (A). (D) Quantification of CK6 immunoreactivity in the new epithelial tongues indicated in (B). Data are mean ± SEM of 4 female donors. Significance relative to control data at the same time-point denoted by **P<0.01, ***P<0.001.