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. 2012;7(11):e49215.
doi: 10.1371/journal.pone.0049215. Epub 2012 Nov 13.

Effects of hydrogen peroxide on wound healing in mice in relation to oxidative damage

Alvin Eng Kiat Loo  1 , Yee Ting Wong, Rongjian Ho, Martin Wasser, Tiehua Du, Wee Thong Ng, Barry Halliwell
Affiliations

Effects of hydrogen peroxide on wound healing in mice in relation to oxidative damage

Alvin Eng Kiat Loo et al. PLoS One. 2012.

Abstract

It has been established that low concentrations of hydrogen peroxide (H(2)O(2)) are produced in wounds and is required for optimal healing. Yet at the same time, there is evidence that excessive oxidative damage is correlated with poor-healing wounds. In this paper, we seek to determine whether topical application of H(2)O(2) can modulate wound healing and if its effects are related to oxidative damage. Using a C57BL/6 mice excision wound model, H(2)O(2) was found to enhance angiogenesis and wound closure at 10 mM but retarded wound closure at 166 mM. The delay in closure was also associated with decreased connective tissue formation, increased MMP-8 and persistent neutrophil infiltration. Wounding was found to increase oxidative lipid damage, as measured by F(2)-isoprostanes, and nitrative protein damage, as measured by 3-nitrotyrosine. However H(2)O(2) treatment did not significantly increase oxidative and nitrative damage even at concentrations that delay wound healing. Hence the detrimental effects of H(2)O(2) may not involve oxidative damage to the target molecules studied.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Low concentrations of H2O2 promoted wound closure but high concentrations of it delay wound closure.
(A) Four full thickness excision wounds were created on each mouse and 15 µL of H2O2 was added into the wound cavity as shown. The left panel is a representative picture of the wounds generated. The right panel shows how the H2O2 is applied onto the wounds. (B) Effects of different concentrations of H2O2 on wound closure rate. The wound size of 6 to 8 mice was monitored for 6 and 10 days respectively before they were euthanized. The graph shown is the mean ± S.E.M. of the pooled results. 1-way ANOVA was used to analyze wound size. The differences between 166 mM H2O2 and control (p<0.05) or 10 mM H2O2 (p<0.01) were statistically significant on day 6. The differences between 10 mM H2O2 and control (p<0.05) were statistically significant on day 8 and 10. The differences between 10 mM and 166 mM H2O2 were also statistically significant on day 8 (p<0.05) but not on day 10.
Figure 2
Figure 2. High concentrations of H2O2 retard connective tissue formation.
Paraffin sections from day 6 wounds were stained with the Masson-Goldner trichrome stain as described in material and method. Connective tissues are stained green. Fibrin, eschar and cytoplasm are stained red. Nuclei are stained dark brown. Representative images for control (A,D) 10 mM (B, E) and 166 mM (C, F) treated wounds are shown. Images A-C are at 100X magnification while D-F are at 200X magnification. (G) Quantification of the fraction of pixels that are stained green. The number of pixels stained green within the neodermis at 100X magnification was quantified using a custom software. The area quantified is outlined with the dashed line. Results were analyzed using 1-way ANOVA followed by Dunnett’s multiple comparison test with control. Graph shown is the mean ± S.E.M. n = 6–7, *** p<0.001.
Figure 3
Figure 3. Low concentrations of H2O2 increased wound angiogenesis.
Paraffin sections from day 6 wounds were stained for CD31 using an immunohistochemical method. Representative photomicrograph of (A) control, (B) 166 mM H2O2 and (C) 10 mM H2O2 treated wounds are shown. (D) The number of brown lumen-like structures within the neodermis was counted in a single blinded fashion and analyzed using 1-way ANOVA followed by Dunnett’s multiple comparison test with control. Graph shown is the mean ± S.E.M, n = 6–7, *** p<0.001.
Figure 4
Figure 4. High concentrations of H2O2 increase levels of MMP-8 in wounds.
Western blot analysis of wound tissues lysate collected 6 days after wounding. Each lane represents a sample from a different animal. (A) Representative blot of MMP-8. (B) Densitometry analysis of MMP-8 normalized against α-tubulin re-probed from their respective blot. Results are mean ± S.E.M. (n = 4) and were analyzed using 1-way ANOVA followed by Tukey multiple comparison among all the columns. ** p<0.01 (C) Representative blot of MMP-9. (D) Densitometry analysis of MMP-9 normalized against α-tubulin re-probed from their respective blot. Results are mean ± S.E.M. (n = 4), p values for 1-way ANOVA is p = 0.13.
Figure 5
Figure 5. 166 mM H2O2 increased neutrophil infiltration in day 1 and 6 wounds.
Fluorescence intensity of the neodermis was quantified using ImageJ. The area quantified is outlined with the dashed line. Results shown are mean ± S.E.M, n = 6-7. A representative section from each treatment is shown. ES – Eschar; HE – Hyper-proliferating epidermis; ND – neodermis.*p<0.05.
Figure 6
Figure 6. Wounding increases ERK1/2 and p38 phosphorylation which can be further increased by 166 mM H2O2 treatment.
(A) Representative blots of wound tissues lysate collected 30 min after wounding. Skin denotes skin from non-wounded animals while control refers to wounds treated with PBS. (B) The density of phosphorylated ERK and pan-ERK and (C) phosphorylated p38 and pan p38 were normalized against α-tubulin re-probed from their respective blot. Results shown are mean ± S.E.M. (n = 4). Densitometry results were analyzed by 1-way ANOVA and test of significance between all column was determined using Tukey’s post hoc test. Only the comparison between 166 mM treated wounds and skin was statistically significant for both B and C. ** p<0.01.
Figure 7
Figure 7. Wounding increased lipid peroxidation and nitrative damage but not protein carbonylation.
Levels of F2-isoprostanes levels in skin and wounds were compared by normalizing against arachidonic acid (A) or tissue weight (B). Results shown are mean ± S.E.M, n = 5. Wounds were compared to skin using 1-way ANOVA with Dunnett’s post-hoc test. Asterisks denote level of significance when compared to skin. Control and H2O2 wounds were also compared against each other using 2-way ANOVA but the differences was not statistically significant. (C) Levels of arachidonic acid in skin and wound tissues. Results shown are mean ± S.E.M, n = 5. Wounds were compared to skin using 1-way ANOVA with Dunnett’s post-hoc test. Asterisks denote level of significance when compared to skin. Control and H2O2 wounds were also compared against each other using 2-way ANOVA but the differences was not statistically significant. (D) Levels of protein carbonyls in wounds were compared to intact skin and expressed as fold change. The results shown are the mean fold change ± S.E.M. No difference in the levels of protein carbonyl was observed in control wounds and 166 mM H2O2 treated wounds. (E) Comparison of 3-nitrotyrosine level in skin and wounds. Results shown are mean ± S.E.M., n = 5. The 3-nitrotyrosine levels of skin were compared to control wounds or H2O2 treated wounds and analyzed with 1-way ANOVA followed by Dunnett’s post-hoc test. Levels of 3-nitrotyrosine were significantly higher at day 6 after wounding. Levels of 3-nitrotyrosine in control and 166 mM H2O2 treated wounds were also compared using 2-way ANOVA and the differences were not statistically significant. *p<0.05, ** p<0.01, ***p<0.001.
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