An ear punch model for studying the effect of radiation on wound healing

Abstract

Purpose: Radiation and wound combined injury represents a major clinical challenge because of the synergistic interactions that lead to higher morbidity and mortality than either insult would produce singly. The purpose of this study was to develop a mouse ear punch model to study the physiological mechanisms underlying radiation effects on healing wounds.

Materials and methods: Surgical wounds were induced by a 2 mm surgical punch in the ear pinnae of MRL/MpJ mice. Photographs of the wounds were taken and the sizes of the ear punch wounds were quantified by image analysis. Local radiation to the ear was delivered by orthovoltage X-ray irradiator using a specially constructed jig that shields the other parts of body.

Results: Using this model, we demonstrated that local radiation to the wound area significantly delayed the healing of ear punch wounds in a dose-dependent fashion. The addition of sublethal whole body irradiation (7 Gy) further delayed the healing of ear punch wounds. These results were replicated in C57BL/6 mice; however, wound healing in MRL/MpJ mice was accelerated.

Conclusions: These data indicate that the mouse ear punch model is a valuable model to study radiation and wound combined injury.

Figure 1

Custom jig for irradiation of ear. (A) The components of the shielding jig. (B) A wounded ear exposed for irradiation.

Figure 2

A schema of the dosimetry experiment. A size appropriate phantom was constructed and placed inside the custom jig. MOSFET were placed inside the jig to confirm shielding properties and at the exposed ear position to confirm the dose and rate of irradiation.

Figure 3

Effect of local radiation on wound healing in MRL/MpJ mice. A 2-mm diameter hole was punched in each MRL/MpJ ear using clinical biopsy punch. The mice were irradiated within 2 h after wounding. The holes were imaged once every 7 days. The area of the wound was determined by image analysis. (A) Representative pictures of wound healing (scale bar = 2 mm) at different time points. (B) Summarised data for wound healing. Observed measurements were summarised as mean ± SD. Each group contains four animals. A representative of two similar experiments with similar results is shown. P < 0.01, non-irradiated vs. 7 Gy; P < 0.01, non-irradiated or 7 Gy vs. 10 Gy or 15 Gy; P < 0.01, 10 Gy vs. 15 Gy as analysed by repeated measure ANOVA.

Figure 4

Effect of local radiation on wound healing in C57BL/6 mice. A 2-mm diameter hole was made in each C57BL/6 ear using clinical biopsy punch. The mice were irradiated within 2 h after wounding. Pictures of the holes were taken once every 7 days. The area of the wound was determined by image analysis. Observed measurements were summarised as mean ± SD. Each group contained three animals. A representative of two similar experiments with similar results is shown. P < 0.05, non-irradiated vs. 7 Gy or 10 Gy as analysed by repeated measure ANOVA.

Figure 5

Effect of systemic radiation on wound healing. A 2-mm diameter hole was made in each ear of MRL/MpJ mice using clinical biopsy punch. The mice were irradiated within 2 h after wounding. The ‘local’ group received 10 Gy of radiation to the ear only and the ‘local + systemic’ group received 3 Gy of radiation to the ear plus 7 Gy of whole body radiation (10 Gy total to the ear and 7 Gy to other parts of the body). Pictures of the holes were taken once every 7 days. The size of the hole was determined by image analysis. Observed measurements were summarised as mean ± SD. Each group contains four animals. A representative of two similar experiments with similar results is shown. P < 0.0001, non-irradiated vs. ‘local’ or ‘local + systemic’ or ‘local’ vs. ‘local + systemic’.

Figure 6

Histological analyses. A 2-mm diameter hole was made in each ear of MRL/MpJ mice using clinical biopsy punch. The mice were irradiated within 2 h after wounding. The ‘local’ group received 10 Gy of radiation to the ear only and the ‘local + systemic’ group received 3 Gy of radiation to the ear plus 7 Gy of whole body radiation (10 Gy total to the ear and 7 Gy to other parts of the body). Biopsies were obtained from different animals at different time points after wounding. The samples were stained with H&E stain (A, 10 ×) or Masson's trichrome stain (B, 10 ×) or anti-CD31 antibody (C, 40 ×). Representative pictures from each group at different time points are shown (samples shown in B and C were obtained on day 21 and day 7 after wounding respectively). The arrow shows where the edges of wounds were located. The samples obtained on day 21 after wounding were shown. Histological scores (Mean ± SD of three animals per group per time point) are summarised in D. *P < 0.05, compared with non-irradiated group; #P < 0.05, compared with both non-irradiated and local groups.

Figure 7

Irradiation induces cell apoptosis in the ear skin. A 2-mm diameter hole was made in each ear of MRL/MpJ mice using clinical biopsy punch. The mice were irradiated within 2 h after wounding. The ‘local’ group received 10 Gy of radiation to the ear only and the ‘local + systemic’ group received 3 Gy of radiation to the ear plus 7 Gy of whole body radiation (10 Gy total to the ear and 7 Gy to other parts of the body). Biopsies were obtained at different time points after wounding. The sections were stained for caspase 3 by using immunohistochemistry. Representative pictures from each group are shown.