ORIGINAL ARTICLE
The Importance of Tenascin and Ubiquitin
in Estimation of Wound Age
Hulya Guler, MD, Ekin O. Aktas, MD, PhD, Huseyin Karali, MD, and Safiye Aktas, MD, PhD
Abstract: Human wound age determination is of prime importance in
forensic sciences. Ubiquitin is a small protein required for ATP-dependent,
nonlysosomal intracellular protein degradation, which eliminates most of
the intracellular defective proteins. Tenascin is an extracellular matrix
glycoprotein. The aim of this study was to determine the importance of
ubiquitin and tenascin in wound age. Ubiquitin and tenascin were immu-
nohistochemically applied on formalin-xed parafn-embedded tissues
of 170 wound biopsies taken from 89 autopsy cases with known wound
age. Pearson correlation analysis was performed for statistical analysis.
The mean age was 39.44 years. Tenascin was negative in 123 cases
(72.4%) in all series, and it was negative in 98.3% in cases with wound
age under 24 hours. It was positive in 91.8% in cases with wound age
over 24 hours. Mean number of cells which expressed ubiquitin was
10.56%, while it was 4.25% in cases with wound age under 24 hours,
and it was 26.14% in cases with wound age over 24 hours. The correla-
tion analysis showed that both tenascin and ubiquitin were positively
correlated with wound age. But in cases with wound age over 40 days,
tenascin was found to be negative and ubiquitin was still expressed in
broblasts. We concluded that tenascin and ubiquitin together was useful
in determining wound age semiquantitatively.
Key Words: wound age, ubiquitin, tenascin
(Am J Forensic Med Pathol 2011;32: 83Y89)
W ound age determination includes understanding of com-
plex organization of cutaneous healing.1 This leads to
using substances whose amount and localization change during
healing.2 Human wound age determination is of prime impor-
tance in forensic sciences. The different reactions of tissues to
different natures of wounds require modern diagnostic methods
to determine wound age. The methods usable for this are his-
tochemical methods dening wound age according to the
changes in enzyme activity in the wound area, biochemical
methods which determine the values of histamine and serotonin,
and histologic methods which determine the age of the wound
according to the chronological order of appearance of the cel-
lular elements.3 The use of immunohistochemistry in estimating
of wound age has been used in recent years.4,5 Only one marker
has not been reported to be the determinant of wound age yet.
Fibronectin was found to be effective to determine vitality after
severe trauma by showing expression in early stages of the
wound healing.5 The efciency of immunohistochemical tech-
niques for the diagnosis of vitality of wounds decreases for
Manuscript received August 3, 2009; accepted April 6, 2010.
From the Dokuz Eylul University Institute of Oncology, Izmir, Turkey.
Supported by Ege University Research Foundation.
Correspondence: Safiye Aktas, MD, PhD, Dokuz Eylul University Institute
of Oncology, Mithatpasa cad Inciralti, Izmir, Turkey 35340. E-mail:
safiyeaktas@yahoo.com.
Copyright * 2011 by Lippincott Williams & Wilkins
ISSN: 0195-7910/11/3201Y0083
DOI: 10.1097/PAF.0b013e3181edf2c0
Am J Forensic Med Pathol & Volume 32, Number 1, March 2011
Copyright 2011 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
lesions occurred fairly close to death and within 10 days after
death.6,7
Ubiquitin is a small protein required for ATP-dependent,
nonlysosomal intracellular protein degradation, which elimi-
nates most of the intracellular defective proteins with a rapid
turnover. It has been suggested to play a key role in essential
intracellular functions, such as heat shock response, protein
breakdown, and regulation of immune responses.8,9 It is a shock
protein and a common immunohistochemical marker. It is in-
duced very rapidly by various types of traumatic stress, and this
is why it is of great concern in forensic pathology especially
neurodegenerative conditions.10 It is expressed both in intracel-
lular and extracellular areas. Extracellular ubiquitin acts as a
cytokine-like protein with anti-inammatory properties.9 It is
expressed in the nuclei of the neutrophile leukocytes, macro-
phages, and broblasts in wound area.
Tenascin is an extracellular matrix glycoprotein.11 It is
expression is restricted in adult tissues, although it is widely
distributed in embryonic tissues.12,13 Although Mackie et al
suggested that tenascin has an important and unique role in the
wound healing process, it can not determine the wound age over
9 days.7 It has different isoforms which might be expressed as
a result of alternative splicing of the premRNA, but the isoforms
are distributed in a similar way in tissues.14 In early stages of
wounds (3 minutesY8 hours), tenascin was found to be negative
in 58 cases in their series.2
The aim of this study was to determine the importance of
ubiquitin and tenascin in wound age estimation and form a
semiquantitative scale for their expression according to wound
age intervals.
MATERIALS AND METHODS
This study was approved by the Ethics Committee of Ege
University Faculty of Medicine.
Samples
This study included 170 wound biopsies prospectively ob-
tained from 89 forensic autopsy cases with known wound age
(Figs. 1Y4). Cases which were autopsied 24 hours after death
time were not included in the study. Median postmortem time
was 18 hours (4Y24 hours). Age, sex, reason of death, and loca-
lizations, diameters, type, and morphologic description of the
wounds were recorded. The wounds were examined morpholog-
ically on photographs, histologically and immunohistochemi-
cally. After external autopsy evaluation was done, the wounds
were digitally photographed. After autopsy procedures were n-
ished, biopsy samples 1 to 3 cm in length and at least 7 mm in
depth were obtained from wound sites. All biopsy samples in-
cluded normal adjacent skin at 1 edge. The tissues were xed in
formalin for 12 hours. After tissue processing in routine pathol-
ogy they were embedded in parafn. One slide was stained with
hematoxylin eosin and 4 micrometer sections were obtained on
polylysine-coated slides for immunohistochemistry.
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Guler et al
FIGURE 1. Examples for gunshot wounds (AYF).
Immunohistochemistry
Immunohistochemistry was performed manually. Ubiq-
uitin and tenascin were immunohistochemically applied on
formalin-xed parafn-embedded tissues. The protocol was
applied according to the manufacturer_s recommendations
at room temperature. The slides were dewaxed with xylene
15 minutes; dehydrated in decreasing concentrations of al-
cohol (99%, 95%, 80%, 70%; each 5 minutes). The sections
were immersed in 3% H2O2 in distilled water for 10 minutes at
room temperature to block endogenous peroxidase activity. After
washing in distilled water, heat induced antigen retrieval was
applied in citrate buffer (pH 5.5) at 400 watt for 20 minutes.
After cooling at room temperature, the slides were washed in
distilled water and then in PBS (pH 7.4). PBS was used for all
subsequent washes. After nonspecic binding was blocked with
blocking reagent for 5 minutes, primary antibodies were applied
at 1:100 dilutions at room temperature for 60 minutes (mono-
clonal antibodies to ubiquitin Novocastra NCL-Ubi-1 moue
monoclonal 137705 and tenascin Novocastra mouse monoclonal
110406). Universal kit was used by rabbit/mouse streptavidin
biotin technique, (Novostain Universal Detection Kit NCL-
RTU-D). Visualization of the bound primary antibodies was
performed using diaminobenzidine solution (Liquid DAB-Plus
Substrate Kit 12 mL Zymed) as a chromogene and counter-
stained with Mayer_s hematoxylin. One of the slides was in-
cubated with PBS without the primary antibody as negative
control. Positive control was evaluated on normal skin.
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Am J Forensic Med Pathol & Volume 32, Number 1, March 2011
Evaluation
The expression intensity was nearly the same in all posi-
tive areas. The intensity was not taken into consideration. The
staining percentage, localization, and pattern were evaluated.
For ubiquitin expression, the expression positivity in inam-
matory cells and/or broblasts was scored as percentage. For
ubiquitin evaluation, the wounds of the cases were grouped
according to wound age as; 0 to 12 hours: group 1; 24 to
120 hours (1Y5 days): group 2; 168 to 336 hours (7Y14 days):
group 3; and 408 to 504 hours (17Y21 days): group 4. For
ubiquitin evaluation, the wounds of the cases were groups ac-
cording to wound age as: group 0: no wound (which might be
considered as control group); group 1: 0 to 12 hours; group 2: 24
to 120 hours (1Y5 days); group 3: 168 to 336 hours (7Y14 days);
and group 4: 408 to 504 hours (17Y21 days) (Fig. 5). Positivity
in epidermis was considered as positive control. For ubiquitin;
group 0 indicated no expression; group 1: G10% positivity in
inammatory cells and broblasts (0Y12 hours); D: group 2: 11%
to 20% positivity (1Y5 days); E: group 3: 21% to 50% positivity
(7Y14 days); F: group 4: 15% to 25% positivity (17Y21 days).
For tenascin expression; pattern 0 demonstrated cases with
no expression, pattern 1 dermoepidermal junction positivity,
pattern 2 positivity in both dermoepidermal junction and around
dermoepidermal junction, pattern 3 positivity in granulation
tissue, pattern 4 positivity around granulation tissue and in deep
reticular dermis, and pattern 5; positivity in deep dermis but not
in granulation tissue.
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Am J Forensic Med Pathol & Volume 32, Number 1, March 2011
FIGURE 2. Examples for sharp weapon injuries (AYF).
Statistical Analysis
Pearson correlation analysis was performed on SPSS
9.05. P values smaller than 0.05 was considered statistically
signicant.
RESULTS
Seventy-four cases (83.15%) were male and 15 cases
(16.85%) were female. The mean age was 39.44 years (range:
15Y85 years). Among 170 wound samples 74 (43.5%) were
gunshot wounds, 20 (11.8%) were blunt injuries, and 76 (44.7%)
were sharp weapon injuries or surgical excisions. The study
included all the cases between the periods of the study so that
it included broad range of wound types. Tenascin was negative
in 123 cases (72.4%) in all series, while it was negative in
98.3% in cases with wound age under 24 hours. It was positive
in 91.8% in cases with wound age over 24 hours. Pattern 0
indicates wound age approximately around day 0; pattern 1:
day 1; pattern 2: day 3; pattern 3: day 6; pattern 4: day 10; and
pattern 5: day 22 (Fig. 6).
Mean number of cells which showed ubiquitin expression
was 10.56%, while it was 4.25% in cases with wound age under
24 hours, and, it was 26.14% in cases with wound age over
24 hours. In correlation analysis, both tenascin (P = 0.0001) and
ubiquitin (P = 0.0001) were positively correlated with wound
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Tenascin and Ubiquitin
age. But in cases with wound age over 40 days tenascin was
found to be negative, and while ubiquitin still showed expression
in broblasts. The wound age matching with studied parameters
did not change with wound type (P 9 0.05).
Comparison of wound ages according to months for
tenascin and ubiquitin is shown in Figure 7. Comparison of
tenascin and ubiquitin expression according to wound times is
shown in Tables 1 and 2, respectively.
DISCUSSION
To exactly diagnose the cause and manner of death, it is
essential to describe the ndings of wounds correctly and ob-
jectively.15 Determining wound time as if it was caused before,
at the time or after death is an important question in forensic
wound examination besides the other questions such as how it
was caused; what caused it; what amount of force was required
to produce it; what degree of injury has resulted from it and
whether it has inuenced death or caused disability.15 If there
are multiple wounds, it must be claried whether they were at
the same time or not. Wound healing is an interactive process
that involves soluble mediators, extracellular matrix compo-
nents, keratinocytes, broblasts, endothelial cells, nerve cells,
and leukocytes (whether the wound is sterile or not).16,17 Lit-
erature review showed us that there have been studies about
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Guler et al Am J Forensic Med Pathol & Volume 32, Number 1, March 2011

FIGURE 3. Examples for surgical incisions (AYI).

FIGURE 4. Examples for blunt injuries (AYD).

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Am J Forensic Med Pathol & Volume 32, Number 1, March 2011 Tenascin and Ubiquitin

FIGURE 5. Ubiquitin expression patterns (A) positivity in epidermis considered as positive control; (B) Group 0: no expression;
(C) Group 1: G10% positivity in inammatory cells and broblasts (0Y12 hours); (D) Group 2: 11% to 20% positivity (1Y5 days);
(E) Group 3: 21% to 50% positivity (7Y14 days); (F) Group 4: 15% to 25% positivity (17Y21 days).

wound age determination by tenascin or ubiquitin. This is the
rst study in which the 2 proteins were explored together, and
used in a scale to discriminate late wound ages.
ICAM-1, VCAM-1, and selectins were found to be the in-
dicators of the vitality of wound but they do not determine the
time of wound in days. P-selectin was demonstrated to be ex-
pressed in very early stages of wounds (between 3 minutes and
7 hours after injury).18 Strong positive staining for ICAM-1 was
observed 12 hours after the time of injury and as the earliest
and 32 hours after the time of injury as the latest.19
Oshima and coworkers demonstrated the possible use of
mRNA analysis for forensic wound examination in their study
about interleukin 10, because mRNA was detectable by RT-
PCR over a longer postmortem time course.20 Among chemo-
kines, ratios of 950% for IL-8 indicated a wound age of at least 1
day.21 Apoptotic cell number detected by in situ end labeling
of fragmented DNA was found to indicate a postiniction in-
terval of approximately 3 weeks or more with a value exceeding
3 cells/0.0001 cm2.22
Tenascin is known to be produced by broblasts and mes-
enchymal cells.7 Its localization of expression changes accord-
ing to wound age.7 In normal skin, tenascin has been detected in
the basal lamina of the dermal capillaries and the hair follicles.
In the literature, there have been 3 types of studies about wound
evaluation. These are cell culture studies, animal models and
surgery or autopsy cases. Our study has an adequate number of
cases collected from autopsies with known wound age. The
cases in which the wound age was hesitated were excluded. All
of the cases had detailed clinical data and digital photographs.
Macroscopic examination might be very helpful if a clinico-
pathologic scoring is planned. This study did not include mac-
roscopic evaluation, because all the cases were with known
wound age and the macroscopic views were all in concordance
with the known age. The second important nding of this study
was that tenascin and ubiquitin expression did not change ac-
cording to the wound type.
The wound types included in this study were gunshot
wound, blunt injury, and sharp injury (weapon or surgical exci-
sion). The wound type did not effect the wound age determina-
tion by ubiquitin and tenascin. This nding strongly supports
the possible use of these markers for wound age estimation.
The reasons why tenascin and ubiquitin is chosen in
this study includes their different types of activities. Ubiquitin
is mainly included in inammatory area, while tenascin is a

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Guler et al Am J Forensic Med Pathol & Volume 32, Number 1, March 2011

FIGURE 6. Tenascin expression patterns (A) positivity in epidermis is considered as positive control. B, Pattern 0; no expression (day 0);
(C) Pattern 1; dermoepidermal junction positivity (day 1); (D) Pattern 2: positivity in both dermoepidermal junction and around (day 3);
(E) Pattern 3; positivity in granulation tissue (day 6); (F) Pattern 4; positivity around granulation tissue and in deep reticular dermis (day 10).

TABLE 1. Comparison of Tenascin Positivity in Each of the

Dened Groups

Wound Type Time Tenascin N Percent

Gunshot wound G24 h Negative 56 88.9
Positive 7 11.1
Total 63 100.0
924 h Negative 4 36.4
Positive 7 63.6
Total 11 100.0
Blunt injury G24 h Negative 14 93.3
Positive 1 6.7
Total 15 100.0
924 h Negative 2 40.0
Positive 3 60.0
Total 5 100.0
Sharp weapon injury or G24 h Negative 40 93.0
surgical excision Positive 3 7.0
Total 43 100.0
924 h Negative 10 30.3
Positive 23 69.7
FIGURE 7. Comparison of wound ages according to months
Total 33 100.0
for tenascin and ubiquitin.

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Am J Forensic Med Pathol & Volume 32, Number 1, March 2011 Tenascin and Ubiquitin

immunohistochemical study of local injuries. Forensic Sci Int. 2003;

TABLE 2. Comparison of Ubiquitin Positivity in Each of the 135:218Y225.
Dened Groups
6. Ortiz-Rey JA, Suarez-Penaranda JM, Munoz-Barus JI, et al. Expression
Wound Type Time Ubiquitin N Percent of bronectin and tenascin as a demonstration of vital reaction in rat
skin and muscle. Int J Leg Med. 2003;117:356Y360.
Gunshot wound G24 h Negative 11 17.5
Positive 52 82.5 7. Mackie EJ, Halfter W, Liverani D. Induction of tenascin in healing
Total 63 100.0 wounds. J Cell Biol. 1988;107:2757Y2767.
924 h Negative 6 54.5 8. Pickart CM, Eddins MJ. Ubiquitin: structures, functions, mechanisms.
Positive 5 45.5 Biochimica et Biophysica Acta. 2004;1695:55Y72.
Total 11 100.0 9. Majetschak M, Krehmeier U, Bardenheuer M, et al. Extracellular
Blunt injury G24 h Negative 4 26.7 ubiquitin inhibits the TNF-alpha response to endotoxin in peripheral
Positive 11 73.3 blood mononuclear cells and regulates endotoxin hyporesponsiveness
Total 15 100.0 in critical illness. Blood. 2003;101:1882Y1890.
924 h Negative 3 60.0 10. Quan L, Zhu BL, Ishida K, et al. Intranuclear ubiquitin immunoreactivity
Positive 2 40.0 of the pigmented neurons of substantia nigra in fatal acute mechanical
Total 5 100.0 asphyxiation and drowning. Int J Leg Med. 2001;115:6Y11.
Sharp weapon injury or G24 h Negative 7 16.3 11. Egging D, van Vlijmen-Willems I, van Tongeren T, et al. Wound healing
surgical excision Positive 36 83.7 in tenascin-X decient mice suggests that tenascin-X is involved in
Total 43 100.0 matrix maturation rather than matrix deposition. Connect Tissue Res.
2007;48:93Y98.
924 h Negative 30 90.9
Positive 3 9.1 12. Odaka K, Uehara T, Arano Y, et al. Noninvasive detection of cardiac
repair after acute myocardial infarction in rats by 111 In Fab fragment
Total 33 100.0
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would not change in different wound types. Tenascin and ubiq- splice variants by human skin cells. Arch Dermatol Res. 2000;292:
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ubiquitin related proteins by quantitative RT-PCR or microarray
16. Grellner W. Time-dependent immunohistochemical detection of
analysis might give more information for time determination in proinammatory cytokines (IL-1A, IL-6, TNF->) in human skin wounds.
wounds in forensic sciences. Forensic Sci Int. 2002;130:90Y96.
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