Original communication
A fundamental study on the dynamics of multiple biomarkers in mouse excisional wounds for wound age estimation

https://doi.org/10.1016/j.jflm.2016.01.027Get rights and content

Highlights

  • Mouse skin excisional wounds were created to simulate dermal defective injury.

  • Multiple biomarkers were measured and analyzed comprehensively.

  • Fibroblast and fibrocyte transformation were highly discriminative parameters.

Abstract

Wound age estimation is a classic but still modern theme in forensic practice. More experiments on different types of wound are needed to further improve its accuracy. In this study, mouse skin excisional wounds were created to simulate dermal defective injury. The neutrophil and macrophage infiltration, fibroblast and fibrocyte accumulation as well as their myofibroblastic transformation were examined. In addition, some wound healing-related molecules, including IL-1β, IL-6, TNF-α, IFN-γ, MCP-1, CXCL12, VEGF-A, EGF, KGF, pro-col Iα2 and pro-col IIIα1, were quantified by Western blotting and real-time quantitative PCR. Neutrophils and macrophages profoundly infiltrated in the wound at 12 h–1 d and 3 d–10 d respectively. Fibroblasts and fibrocytes accumulated in the wound from 3 d, and transformed into contractile myofibroblasts from 5 d post injury. The transformation ratios of fibroblasts and fibrocytes were highest at 7 d–10 d and 10 d respectively (over 50%). MCP-1 and CXCL12 increased from 12 h to 5 d, and IL-1β, TNF-α and pro-col IIIα1 up to 7 d. IL-6 and VEGF-A increased from 12 h to 1 d–10 d. Pro-col Iα2 increased from 7 d to 21 d. IFN-γ decreased from 12 h to 10 d. By comprehensive analysis of these factors from the perspective of morphometrics, protein and gene expressions, this study provided us with fundamental information for wound age estimation, especially in the wounds with full-thickness defection.

Introduction

Wound age estimation is a classic but still modern theme in forensic pathology, which provides useful information for crime scene reconstruction.1, 2 Skin wound healing is a complicated process that involves coordinated interactions among diverse cells and regulatory mediators in a series of overlapping but distinct stages, including inflammation, new tissue formation, and remodeling.3, 4, 5 Based on this cognition, forensic pathologists carried out extensive experiments to search chronological markers for wound age estimation. The acquired information referring to time-dependent appearances of effector cells6, 7, 8, 9, 10, 11 and expressions of regulatory mediators (cytokines,1, 12, 13, 14, 15 chemokines,14, 16, 17 growth factors,17, 18, 19 proteases,17, 20 members of extracellular matrix,17, 21, 22, 23 etc.), provides valuable experiences for wound age estimation.

Wound age estimation should take into consideration the local factors, e.g. the size and type of lesions.24 In forensic practice, we often encounter some severely injured wounds with full-thickness defection. For these cases, the existing experiences mostly derived from incisional wound studies might not provide accurately chronological information. Therefore, some specific studies in defective wounds should be carried out to improve the accuracy of wound age estimation. Wound contraction is an important characteristic of skin wound healing, which is especially obvious in defective injury. It is well accepted that under mechanical and cytokine stimuli, some fibroblasts in the wound cavity would transform into α-SMA-expressing myofibroblasts, which possess contractile property and contribute to wound contraction.5 Meanwhile, bone marrow-derived mesenchymal progenitor fibrocytes, which co-express leukocyte and mesenchymocyte markers (CD45, CD34, collagen I, pro-collagen I, etc.), also contribute to the fibroblast populations and have potential to differentiate into myofibroblasts.25, 26, 27 Previous studies have testified the appearances of fibroblasts, fibrocytes, and myofibroblasts are good candidates for wound age estimation.7, 10 Therefore, we hypothesize the time-dependent transformation of fibroblast-to-myofibroblast and fibrocyte-to-myofibroblast might also be usefully chronological biomarkers.

On wound age estimation, there is a consensus that various parameters with different, but complementary characteristics should be examined, so that the combination of results minimizes error margin in time calculation.24 Therefore, besides the fibroblast and fibrocyte accumulation and their myofibroblastic transformation, some other biomarkers that have been well accepted being critical for skin wound healing, including the neutrophil and macrophage infiltration, as well as the expressions of chemokines (MCP-1, CXCL12), inflammatory cytokines (IL-1β, IL-6, TNF-α, IFN-γ), growth factors (EGF, KGF, VEGF-A), and matrix proteins (pro-col Iα2, pro-col IIIα1) were measured and analyzed comprehensively, aiming to provide information for chronological estimation in defective wounds.

Section snippets

Animal model of skin wound

A total of 60 male 8-week-old healthy BALB/c mice, each weighing 25–30 g, were used in this experiment. Excisional wounds were created according to the instruction of Birch et al.28 The mice were anesthetized by sodium pentobarbital (i.p. injection). Two full-thickness dermal excisional wounds were created symmetrically over the midline on the dorsal skin with a 6-mm-diameter sterile biopsy punch. After surgery, mice were housed individually in a temperature-controlled sterile environment with

Neutrophil and macrophage infiltration

Immunohistochemical staining showed the infiltration of neutrophils (MPO+) and macrophages (F4/80+) after injury. The neutrophil infiltration was peaked at 12 h, then decreased in the following days and dropped to the normal level at 13 d post-injury (Fig. 1a and c). The average neutrophil number was over 50 per field at 12 h (102.6 ± 9.04, p = 0.001), and 1 d (80.6 ± 6.95, p = 0.002) post-injury. Monocytes were recruited to the wound cavity and matured into F4/80 positive macrophages from 1 d

Discussion

In this fundamental study, we detected the dynamics of multiple biomarkers in mouse excisional wounds, and found them discriminative parameters for wound age estimation.

Previous studies have testified the time-dependent appearances of fibroblasts, fibrocytes, and myofibroblasts are good candidates for wound age estimation.7, 10 In the present study, we detected their dynamics simultaneously and further analyzed the fibroblast-to-myofibroblast and fibrocyte-to-myofibroblast transformation.

Acknowledgment

The study was financially supported in part by grants from projects funded by National Natural Science Foundation of China (81273342), Shenyang Scientific and Technological Plan (F12-277-1-03), and Anshan Scientific and Technological Plan (2060499).

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    These authors contributed equally to the study.

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