Legal Medicine 9 (2007) 109–114

www.elsevier.com/locate/legalmed

Timing of skin wounds

Toshikazu Kondo *

Department of Forensic Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama, Japan

Abstract

Wound examination is indispensable in forensic practice. It is always necessary to determine wound vitality or wound age to correctly
evaluate the relationship between death and any wounds. Thus, the determination of wound vitality or wound age is a classic but still
modern theme in forensic pathology. Skin wound healing is a primitive but well orchestrated biological phenomena consisting of three
sequential phases, inflammation, proliferation, and maturation. Many biological substances are involved in the process of wound repair,
and this short and simplified overview of wound healing can be adopted to determine wound vitality or wound age in forensic medicine.
With the development of immunohistochemistry and chemical analyses, the scientific field of wound age determination has advanced
progressively during recent years. In particular, it has been demonstrated that collagens, cytokines, and growth factors are useful can-
didates and markers for the determination of wound vitality or age. In this review article, some interesting and instructive results are
presented, contributing to the future practice of every forensic pathologist.
Ó 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Wound healing; Wound age; Immunohistochemistry; Cytokines; Extracellular matrix

1. Introduction adopted to determine wound vitality or wound age in

Skin wound healing starts immediately after injury and
consists of three phases: inflammation, proliferation, and
maturation. These phases proceed with complicated but
well-organized interaction between various tissues and cells
[1,2]. During the inflammatory phase, platelet aggregation
at the injury site is followed by the infiltration of leukocytes
such as neutrophils, macrophages, and T-lymphocytes into
the wound site. In the proliferative phase, reepithelializa-
tion and newly formed granulation tissue begin to cover
the wound area to complete tissue repair. Angiogenesis is
indispensable for sustaining granulation tissue. As shown
in Fig. 1, advanced cell biological studies demonstrated
that many cytokines, growth factors, proteases, and so
on are closely involved in the wound healing process to
complete normal tissue repair after damage [1,2]. This
short and simplified overview of wound healing can be
*
Tel./fax: +81 73 441 0641.
E-mail address: kondot@wakayama-med.ac.jp
forensic medicine.
In forensic practice, it is needless to say that wound
examination is one of the most important and indispens-
able areas for forensic pathologists. During the process of
wound examination, to evaluate the causal relationship
between death and any wounds, forensic pathologists are
always required to discriminate antemortem wounds from
postmortem damage. Moreover, when the wound is vital,
it is necessary to determine how long before death it was
sustained. In daily life, anybody can see that the colors of
subcutaneous hemorrhages change in accordance with the
post-infliction interval. However, objective scientific
evidence is required in forensic practice. Historically,
Walcher [3] and Orsos [4] first discussed, based on their
practical experiences without scientific evidence, that the
determination of wound vitality or wound age was indis-
pensable in forensic practice. For example, chronological
histopathological alterations characterizing the different
phases of wound healing can be applied to wound age
determination. In particular, upon injury, neutrophils are
recruited at the injury site, followed by macrophages in
accordance with the post-infliction interval. In the 1960’s,

1344-6223/$ - see front matter Ó 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.legalmed.2006.11.009
110 T. Kondo / Legal Medicine 9 (2007) 109–114

Table 1
Wound age and extracellular matrices
Markers Earliest Routine Latest
Fibronectin 10–20 min 4h Months
Collagen III 2–3 days 6 days Months
Collagen V 3 days 6 days Months
Collagen VI 3 days 6 days Months
Collagen I 5 days 6 days Months
Laminin in mf 1.5 days 6 days Months
HSPG in mf 1.5 days – Months
Collagen in mf 4 days – Months
a-Actin in mf 5 days – Months
mf, myofibroblast.

of the basement membrane components, laminin and hep-

arin soleplate proteoglycan (HSPG), which are useful for
wound age determination, and basement membrane frag-
ments associated with replacement of the epidermis were
present at the earliest about 4 days after infliction with
complete restoration of the epidermal basement membrane
occurring 8 days after injury.

Fig. 1. Schema fro skin wound healing.

3. Fibronectin

Raekallio, one of the most famous forensic scientists, tried
to resolve the problem scientifically. He investigated the
activity of several enzymes at a wound site by enzyme his-
tochemistry [5]. Some years later, Berg et al. [6,7] evaluated
the levels of serotonin and histamine at the wound edges,
which gave evidence of vital phenomena. Thereafter, with
the advance of immunohistochemistry, the application of
immunohistochemical techniques opened up a new field
of wound age investigation by forensic pathologists, begin-
ning with Eisenmenger et al. [8] and Oehmichen [9]. During
the past 10 yr, wound age determination has been one of
the most popular themes in forensic research. In particular,
growth factors, cytokines, extracellular matrices, and so on
have been intensively investigated, resulting in significant
advancement of the determination of wound vitality or
wound age.
2. Collagens
Collagens are the main extracellular component of the
skin. During the proliferative phase of skin wound healing,
the synthesis of different proteins of particularly collagen
subtypes within wounds increases to replace necrotic tissue.
Betz and co-workers [10–13] performed a series of immu-
nohistochemical studies on various collagens for wound
age determination (Table 1). In their study, the typical
morphological feature of collagen in the form of strongly
staining network-like structures associated with fibroblasts
is to be expected for collagen III about 2–3 days after inju-
ry, for collagen V and VI in lesions older than 3 days, and
for collagen I 5 days after infliction. Moreover, they exam-
ined basement membrane collagens IV and VII and those
Fibronectin is a multifunctional cell adhesion protein
found in blood and in a variety of tissues [14]. In wound
healing, fibronectin can support the adhesion and move-
ment of fibroblasts, keratinocytes, and endothelial cells.
Previous literature reports indicated that fibronectin could
be a marker of vitality for wounds with a survival time of
more than a few minutes [15]. Fibronectin is presumed to
be the most sensitive marker for wound age determination,
which is supported by evidence that some wounds a few
minutes old showed an immunopositive reaction for fibro-
nectin (Table 1). However, Grellner [16] suggested that a
fibronectin-positive reaction found in post-mortem-incised
wounds of porcine skin was similar to those occurring in
vital wounds. Thus, we have to be aware of the evaluation
of fibronectin immunoreactions.
4. Adhesion molecules
In the inflammatory phase of skin wound healing, leu-
kocytes such as neutrophils and macrophages are recruited
at the injury site. For the migration of leukocytes, the inter-
action between leukocytes and endothelial cells is the most
important event [1,2]. This interaction is mediated by
adhesion molecules. Dreßler and his colleagues [17–19]
Table 2
Wound age and adhesion molecules
Markers Earliest Latest
P-selectin A few min 7h
E-selectin 1h 17 days
ICAM-1 1.5 h 3.5 days
VCAM-1 3h 3.5 days
 T. Kondo / Legal Medicine 9 (2007) 109–114
demonstrated that these adhesion molecules were sensitive
markers for wound age determination (Table 2). In partic-
ular, a strong immunopositive reaction for P-selectin could
be observed 3 min at the earliest and 7 h at the latest after
injury. The strong expression of intercellular adhesion mol-
ecule (ICAM)-1, vascular endothelial adhesion molecule
(VCAM)-1 and E-selectin could be detected 1–3 h at the
earliest after injury.
5. Inflammatory cytokines
Cytokines are multifunctional glycoproteins, which are
closely involved in various biological events including the
immune, central nervous, endocrine, and hematopoietic
systems. Interleukin (IL)-1, IL-6, and tumor necrosis
factor (TNF)-a are representative pro-inflammatory cyto-
kines. These cytokines constitutively exist in keratinocytes
and sweat glands of the uninjured skin [20]. Our experi-
mental study using mice demonstrated that these pro-in-
flammatory cytokines were up-regulated at both protein
and mRNA levels at the injury sites, suggesting that they
could become useful markers for wound age determina-
tion [20–23]. Several groups including ourselves have
investigated the expression of these cytokines in human
skin wounds of different ages. We immunohistochemically
examined IL-1a expression and evaluated IL-1a-positive
infiltrating cells such as neutrophils, macrophages, and
fibroblasts. As shown in Table 3, a ratio of IL-1a-positive
cells considerably exceeding 30% indicates a post-infliction
interval between 4 h and 1 day [24]. On the other hand,
Grellner et al. [25,26] emphasized that IL-1, IL-6, and
TNF-a were available for the determination of wounds
aged 30–90 min. This discrepancy can be explained by
the fact that they evaluated the staining intensity in
keratinocytes and sweat glands where these cytokines exist
constitutively.
6. Chemokines
In the process of wound healing, the recruitment of leu-
kocytes such as neutrophils and macrophages is a charac-
teristic trait. The trafficking of leukocytes into injury sites
is regulated by chemotactic cytokine, a so-called chemo-
kine. IL-8/CXCL8 has chemotactic activity against neutro-
phils [27], T-lymphocytes [28] and keratinocytes [29] as well
as promoting epidermal proliferation [29]. Moreover,
Table 3
Summary for our previous studies
Markers Positive ratio (%) Wound age
IL-1a >30 4 h–1 days
IL-8 >50 1–4 days
MCP-1 >30 1–7 days
MIP-1a >40 1–9 days
VEGF >50 7–14 days
Ubiquitin >30 7–21 days
c-Fos >20 >1 days
c-Jun >10 >1 days
111
monocyte chemoattractant protein (MCP)-1/CCL2 and
macrophage inflammatory protein (MIP)-1a/CCL3 pre-
dominantly recruit macrophages/monocytes. The gene
expression of these chemokines is enhanced in murine skin
wounds [21–23], and these chemokines are indispensable
for normal wound repair. Our immunohistochemical study
suggested that IL-8, MCP-1 and MIP-1a are available as
markers of wound age determination (Table 3). In particu-
lar, a ratio of >50% for IL-8 indicates a wound age of 1–4
days. A ratio of >30% for MCP-1 or >40% for MIP-1a
could be detected in skin wounds with a wound age of at
least 1 day [30]. Moreover, it is considered that the com-
bined evaluation of these three chemokines can determine
wound age with a high degree of accuracy and objectivity.
7. Angiogenic cytokines
In skin wound healing, angiogenesis is a crucial event in
the formation of new granulation tissue in the proliferative
phase [1,2]. Vascular endothelial growth factor (VEGF)
and basic fibroblast growth factor (bFGF) are potent
angiogenic factors. Takamiya and his colleagues [31,32]
demonstrated the time-dependent expression of both
VEGF and bFGF in a murine skin model, suggesting that
these growth factors can become useful markers for wound
age determination. However, they did not conduct a prac-
tical study using human skin-wound samples. Hayashi and
his colleagues [33] showed that VEGF was expressed on
macrophages and fibroblasts in human skin wounds. In
their study, morphometrical analysis demonstrated that a
VEGF-positive ratio of >50% possibly indicates a wound
age of 7–21 days (Table 3).
8. Transforming growth factor (TGF)
TGF-a and TGF-b1 are involved in wound healing [1,2].
However, TGF-a has a different biological role from TGF-
b1. TGF-a promotes the proliferation of epidermal cells
such as epidermal growth factor (EGF) [1,2], whereas
TGF-b1 is a potent fibrogenic growth factor, essential for
the deposition of extracellular matrices such as collagen
fibers. According to the semiquantitative evaluation of
immunostaining intensity for both TGF-a and TGF-b1,
their expression was enhanced within the first hour after
an injury, suggesting that they can be used in the determi-
nation of wound vitality and wound age [34]. This particu-
larly applies to TGF-b1, because it is easy to evaluate and
is up-regulated rapidly.
9. Stress proteins
Ubiquitin (Ub) is a highly conserved protein with a
molecular weight of 8500 [35] and is present in all eukary-
otes [36]. This protein mediates non-lysosomal protein deg-
radation in eukaryotic cells by covalently attaching to
various proteins by the Ub-protein ligase system [37]. Ub
is a member of the heat shock protein family, which is
112 T. Kondo / Legal Medicine 9 (2007) 109–114
rapidly induced by various types of stimuli such as hyper-
thermia, chemical or mechanical stress [38]. Recently, Ub
expression in various human organs was examined from
a forensic aspect [39–41]. In animal experiments, Ub
expression was more intense in the proliferative rather than
the inflammatory phase of wound healing, suggesting that
Ub expression in skin wounds was likely to be useful for
wound age determination. From the viewpoint of a foren-
sic pathological application, this study showed that Ub is
available as a marker for wound age determination (Table
3). A Ub-positive ratio considerably exceeding 30% possi-
bly indicates a wound age of 7–14 days [42].
Oxygen-regulated protein (ORP)-150 has been identified
from rat astrocytes, which can be induced under hypoxic
conditions as a molecular chaperone [43–45]. Several lines
of accumulating evidence suggest that immunohistochemi-
cal and morphometrical analyses of ORP-150 in the brain
may be very useful to determine the duration of brain
ischemia before death in forensic autopsy cases [46]. In par-
ticular, ORP-150 may be useful to discriminate between
mechanical asphyxia and sudden infant death syndrome
[47]. Moreover, this protein can modulate the intracellular
transport of VEGF from the endoplasmic reticulum to the
Golgi apparatus, eventually contributing to wound healing
[48]. An ORP-150-positive ratio of significantly more than
40% possibly indicates a wound age of 7 days or more (our
unpublished data). We consider that the combined exami-
nation of ORP-150 and VEGF as mentioned above may
be more reliable for wound age determination.
10. Cell proliferation-related proteins
In wound healing, cell proliferation and cell apoptosis
are well harmonized. Betz et al. demonstrated that the
number of Ki-67-positive fibroblastic cells increased at
the earliest in skin wounds aged 1.5 days [49]. Moreover,
they [50,51] investigated the expression of the p53 gene,
which arrests the cell cycle during the G1 or G2 phase to
enable DNA repair as well as apoptotic fibroblasts. In their
study, morphometrical analysis revealed that the number
of p53-positive or apoptotic fibroblasts increased after a
post-infliction interval of approximately 1–2 days. We
examined the products of proto-oncogenes such as c-fos
and c-jun, which also act as transcriptional factors. The
products of these genes are constitutively expressed in the
basal layer of the epidermis. As shown in Table 3, in
human skin wounds aged 1 day or more, macrophages
and fibroblasts expressed these gene products [52].
11. Eicosanoids
Eicosanoids, which are metabolites derived from arachi-
donic acid (AA), are inflammatory mediators with chemo-
tactic activity in wound healing. He and Zhu [53] examined
the levels of several leukotriene subtypes in human skin
wounds by HPLC. They demonstrated that detecting
LTB4 but not LTD4 or LTC4, is a useful method to distin-
guish antemortem from postmortem injuries. In particular,
in their method, samples were fixed in formalin for less
than 10 days. Prostaglandins (PG) are also derived from
arachidonic acid by cyclooxygenase. Although Hernan-
dez-Cueto and colleagues studied the viability of PGF2a
as a vitality marker in skin wounds, their results showed
that PGF2a is not suitable to diagnose the vitality of
wounds because of its irregular behavior [54]. On the other
hand, immunohistochemical analyses of cyclooxygenase-2
implied its possible use for the determination of wound
vitality (our unpublished data).
12. Future of wound age determination
During the first half of the 1990s, DNA research for
forensic identification flourished in forensic medicine; how-
ever, the determination of wound vitality or wound age is
an archaic but still popular theme in forensic pathology.
We think that this subject has also significantly advanced
scientifically. Many bioactive substances are essentially
involved in skin wound healing, suggesting that the deter-
mination of wound vitality or wound age can advance fur-
ther. Furthermore, experimental studies have tried to apply
gene expressions for wound age determination [55]. In the
future, a system for the determination of wound vitality
or wound age will be established at the molecular as well
as protein level.
Acknowledgements
I am grateful to Prof. Dr. Wolfgang Eisenmenger (Insti-
tute for Legal Medicine, University of Munich, Germany)
and Prof. Dr. Peter Betz (Institute for Legal Medicine,
University of Erlangen-Nürunberg, Germany) for their
critical and instructive supports to our scientific research.
I express my sincere thanks to Prof. Dr. Akihiro Takatsu
(Department of Forensic Medicine, The Jikei University
School of Medicine) for giving me the opportunity for writ-
ing this review article.
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