Oxytocin effects on experimental skin wound healing

Abstract

Objective: Oxytocin (OXY) has significant effects on mammalian behavior. Next to its role in lactation and social interactions, it is described to support better wound healing as well. However, direct OXY effects on wound healing and the regeneration of the microvascular network are still not clarified. We therefore examined the effects of OXY and an OXY receptor antagonist [atosiban (ATO)] on skin wound healing, focusing on epithelialization and neovascularization.

Methods: Skin wound healing has been assessed using intravital fluorescence microscopy in a model of full dermal thickness wounds in the dorsal skin fold chamber of hairless mice. Animals received repetitive low or high doses of OXY or ATO. Morphological and cellular characterization of skin tissue repair was performed by histology and in vitro cell assays.

Results: The assessment of skin tissue repair using this therapy regimen showed that OXY and ATO had no major influence on epithelialization, neovascularization, wound cellularity, or inflammation. Moreover, OXY and ATO did neither stimulate nor deteriorate keratinocyte or fibroblast migration and proliferation.

Conclusion: In summary, this study is the first to demonstrate that OXY application does not impair skin wound healing or cell behavior. However, until now, the used transmitter system seems not to be clarified in detail, and it might be proposed that it is associated with the stress response of the organism to various stimuli.

Keywords: atosiban; emotion; epithelialization; intravital fluorescence microscopy; skin fold chamber.

Figure 1:

Representative intravital fluorescence microscopic images of microcirculation on day 9 of wound healing in an HD OXY-treated animal (A–C). The process of wound healing shows distinct patterns of newly formed microvascular networks (A). At first, the regeneration of the microvascular network creates an inner circular ring of vessels at the direct wound margin (A; higher magnification in B). The inner ring is surrounded by outer radially localized vessels, supplying the vasculature within the inner circular ring (A; higher magnification in C). Dashed line (A) marks the edge of the wound at the day of wounding; continuous line indicates border between radial and circular microvessels. Scale bar, 200 μm (A) and 80 μm (B and C).

Figure 2:

Analysis of wound epithelialization. (A) Photomacroscopic images and quantitative planimetric analysis of wounds during regeneration, displaying the continuous process of wound closure with complete epithelialization on day 12. Left, skin fold chamber directly after wounding of the control group; right, wounds of the HD ATO group (dotted line, initial wound area; continuous line, wound area on day 6). (B) Quantitative analysis of wound epithelialization on days 3, 6, 9, and 12 in mice treated daily with saline (control; 0.9% NaCl; 12.5 mL/kg bw; n=8), LD OXY (1 mg/kg bw; n=9), HD OXY (10 mg/kg bw; n=6), LD ATO (1 mg/kg bw; n=7), or HD ATO (10 mg/kg bw; n=6). Data are means±SEM. No statistically significant differences.

Figure 3:

Quantitative analysis of diameters (μm) and FMD (cm/cm²) in circular vessels (A and C) and radial vessels (B and D). Animals were treated daily with saline (control; 0.9% NaCl; 12.5 mL/kg bw; n=8), LD OXY (1 mg/kg bw; n=9), HD OXY (10 mg/kg bw; n=6), LD ATO (1 mg/kg bw; n=7), or HD ATO (10 mg/kg bw; n=6). Data are means±SEM. *p<0.05 vs. control; #p<0.05 vs. HD OXY.

Figure 4:

Quantitative analysis (A) and representative images (B–D) of H&E staining for cellularity in wound tissue specimens on day 12 after wounding. Animals were treated daily with saline (control; 0.9% NaCl; 12.5 mL/kg bw; n=8), LD OXY (1 mg/kg bw; n=9), HD OXY (10 mg/kg bw; n=6), LD ATO (1 mg/kg bw; n=7), or HD ATO (10 mg/kg bw; n=6). Data are means±SEM. No statistically significant differences. Scale bar, 80 μm.

Figure 5:

Quantitative analysis (A) and representative images (B–D) of leukocyte infiltration (AS-D CAE) in wound tissue specimens on day 12 after wounding. Animals were treated daily with saline (control; 0.9% NaCl; 12.5 mL/kg bw; n=8), LD OXY (1 mg/kg bw; n=9), HD OXY (10 mg/kg bw; n=6), LD ATO (1 mg/kg bw; n=7), or HD ATO (10 mg/kg bw; n=6). Data are means±SEM. No statistically significant differences. Scale bar, 80 μm.

Figure 6:

Representative images (A) and quantitative analysis (B) of CD31-stained endothelial lining to determine microvessel density in wound tissue specimens after wounding. Animals were treated daily with saline (control; 0.9% NaCl; 12.5 mL/kg bw; n=8), LD OXY (1 mg/kg bw; n=9), HD OXY (10 mg/kg bw; n=6), LD ATO (1 mg/kg bw; n=7), or HD ATO (10 mg/kg bw; n=6). Data are means±SEM. *p<0.05 vs. HD OXY; #p<0.05 vs. LD ATO. Scale bar, 120 μm.

Figure 7:

Quantitative assessment of in vitro cell proliferation by the WST-1 assay of (A) fibroblasts (L929) and (B) keratinocytes (HaCaT) over an observation period of 240 min. Cells (4×10³ cells/well) were treated with DMEM supplemented with 10% FCS, bFGF (20 ng/mL), or OXY in three different concentrations with 10 nmol/mL (OXY 10), 100 nmol/mL (OXY 100), and 1000 nmol/mL (OXY 1000). After incubation for 48 h, cells were washed with PBS and the cell proliferation reagent WST-1 was added to the cell culture medium followed by incubation for 4 h with repetitive measurements of optical density every 30 min. All experiments were performed in triplicate. Data are means±SEM. *p<0.05 vs. bFGF, OXY 100, and OXY 1000.

Figure 8:

Quantitative assessment of in vitro cell migration using the wound scratch assay with (A) fibroblasts (L929) and (B) keratinocytes (HaCaT) over an observation period of 72 h. Cells were grown to confluence in 10% FCS on Petri dishes and then scratched with a pipette tip (C; scratch 0 h). Cells were treated with DMEM supplemented with 10% FCS, bFGF (20 ng/mL), or OXY in three different concentrations with 10 nmol/mL (OXY 10), 100 nmol/mL (OXY 100), and 1000 nmol/mL (OXY 1000). Cells were photographed immediately and 24, 48, and 72 h after the scratch, as representatively shown for fibroblasts among groups at the time point of 72 h after scratching (C). Scale bar, 400 μm. Data are means±SEM. *p<0.05 vs. control; #p<0.05 vs. bFGF; ßp<0.05 vs. OXY 10; §p<0.05 vs. OXY 10; $p<0.05 vs. OXY 100.