Fig 1.
Healing of limb wounds of horses following treatment with viral proteins.
(A) Representative photos of healing limb wounds following a single topical administration of vehicle (HyStem hydrogel) or viral proteins in vehicle (VEGF-E and ovIL-10) prior to bandaging on day 1 and a subcutaneous injection of vehicle (saline) or viral proteins in vehicle at day 8. (B) Surface area and (C) exuberant granulation tissue (EGT) formation of healing wounds at the days indicated. Wound surface area is calculated relative to the original wound area. EGT formation was scored on 50% on protuberance (0 none– 2 marked), 25% colour (0 pink– 1 yellow-red) and 25% on quality (0 smooth—1 rough). Values represent mean ± SEM, n = 4. *p ≤ 0.05 between means of viral protein treated and vehicle control wounds at each time point, as determined using a linear mixed model with a priori contrasts followed by a Benjamini-Hochberg sequential adjustment.
Fig 2.
Epithelialisation of limb wounds of horses following treatment with viral proteins.
(A) Representative photos of wound margin sections taken 7 days post-wounding, stained with MSB trichrome. White and black lines in each photo indicate the length of the epidermal tongue and epidermal coverage, respectively. (B) Epidermal tongue length and (C) epidermal coverage following topical administration of vehicle or viral proteins. (D) Representative photos of healed wounds stained with MSB trichrome. Rete ridges are indicated by white arrows. (E) Epidermal thickness (measured at 5 equidistant points across the neo-epidermis) and (F) number of epidermal rete ridges projecting into the neo-dermis of healed wounds following administration of vehicle or viral proteins. Values represent mean ± SEM, n = 4. P values indicated were determined using a two-tailed ratio paired t-test.
Fig 3.
Inflammation in limb wounds of horses following treatment with viral proteins.
Quantitative PCR was used to measure the expression of (A) equine (e)MCP-1, (B) eIL-6, (C) eIL-10, and (D) eIL-10Rα in wound margin samples taken 2 days after wounding. Expression of mRNA is relative to that of GAPDH and to levels measured in unwounded skin (day 0). (E) Representative photos of skin sections taken 7 days post-wounding stained with DAPI (blue) and an antibody against the inflammatory cell marker calprotectin (MAC387: green). Enlarged images show nucleated inflammatory cells. (F) Number of calprotectin+ve cells in the granulation tissue and surrounding skin of wounds following administration of vehicle or viral proteins. Values represent mean ± SEM, n = 4. P values indicated were determined using a two-tailed ratio paired t-test.
Fig 4.
Vascularisation of limb wounds of horses following treatment with viral proteins.
Quantitative PCR was used to measure the expression of (A) eVEGF-A, (B) eVEGFR2, and (C) ePDGF-β in wound margin samples taken 7 days after wounding. Expression of mRNA is relative to that of GAPDH and to levels measured in unwounded skin (day 0). (D) Representative photos of skin sections taken 14 days post-wounding, stained with DAPI (blue) and antibodies against blood vessel endothelial cells (CD31: pink) and basal lamina (collagen IV: green). Enlargements show single colour images of representative blood vessels. (E) Area of CD31 staining, (F) number of collagen IV+ve blood vessels, (G) lumen area in collagen IV+ve blood vessels and (H) percentage of blood vessels occluded (lumen area less than 7 μm2) in the granulation tissue of wounds following administration of vehicle or viral proteins. Values represent mean ± SEM, n = 4. P values indicated were determined using a two-tailed ratio paired t-test.
Fig 5.
Blood vessel integrity in healed limb wounds of horses following treatment with viral proteins.
(A) Representative photos of healed wounds stained with DAPI (blue) and antibodies against blood vessel endothelial cells (CD31: pink) and basal lamina (collagen IV: green). (B) Number of collagen IV+ve blood vessels (C) lumen area in collagen IV+ve blood vessels and (D) percentage of blood vessels occluded in the scar tissue of healed wounds following administration of vehicle or viral proteins. (E) Representative photos of healed wounds stained with DAPI (blue) and by TdT dUTP Nick-End Labelling (TUNEL: green). Green arrows indicate individual dying cells. Yellow and white arrows indicate a healthy and a dying blood vessel, respectively. Number of TUNEL+ve (F) cells (area of single cell < 50 μm2) and (G) blood vessels (area of 3 or more adjacent cells > 150 μm2) in the scar tissue of healed wounds following administration of vehicle or viral proteins. Values represent mean ± SEM, n = 4. P values indicated were determined using a two-tailed ratio paired t-test.