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Legal Medicine 7 (2005) 81–88
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Quantitative morphometry of granular ‘dot-like’
ubiquitin-immunoreactivity in the crus cerebri
in asphyxiation and fire fatalities
Li Quan*, Takaki Ishikawa, Tomomi Michiue, Dong-Ri Li, Dong Zhao,
Bao-Li Zhu, Hitoshi Maeda
Department of Legal Medicine, Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, 545-8585 Osaka, Japan
Received 1 July 2004; received in revised form 12 August 2004; accepted 23 August 2004
Available online 11 November 2004
Abstract
In the central nervous system (CNS), a variety of ubiquitinated structures have been reported, usually as pathological
alterations of the brain related to degenerative diseases or aging. However, previous studies showed an increase in the ubiquitin
(Ub)-immunoreactive intranuclear inclusion of the pigmented neurons of the substantia nigra in the midbrain in asphyxiation
and fire fatalities in the adult subjects. The aim of the present study was to examine granular ‘dot-like’ Ub-immunoreactivity in
the crus cerebri (cortico-spinal tracts) in related fatalities (over 35 years of age, nZ169), including fatal asphyxiation (nZ27),
drownings (nZ14), fire fatalities (nZ60), and control groups (nZ68). Dot-like Ub-immunoreactivity was clearly observed in
the descending tract of the crus cerebri. Morphometric analysis of the positive granular area (dot-like Ub-area) showed a higher
value in strangulation and fire fatalities and a lower value in hemorrhagic and head injury deaths, as was observed for the
inclusion-type neuronal Ub-positivity. However, there was a difference between those markers: a low value was seen for the
inclusion-type neuronal Ub-positivity in hanging and drownings, and a difference in the dot-like Ub-area was detected between
fire fatalities with lower and higher COHb levels. Our findings suggested the possible usefulness of these markers for
examination of CNS stress responses in traumas, at least in middle-aged and elderly victims and a partial difference in stress
reaction between the cortico-spinal tracts and dopaminergic neurons.
q 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Ubiquitin; Immunohistochemistry; Human midbrain; Stress; Asphyxia; Fire death
1. Introduction
* Corresponding author. Tel.: C81 6 66453767; fax: C81 6
66343871.
E-mail address: [email protected] (L. Quan).
Ubiquitin (Ub; shock protein) is a common
immunohistochemical marker of neurodegeneration
in the central nervous system (CNS) [1–3]. A variety
of ubiquitinated structures have been reported, usually
as pathological alterations of the brain related to
1344-6223/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.legalmed.2004.08.007
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L. Quan et al. / Legal Medicine 7 (2005) 81–88
degenerative diseases or aging [4–10]. However, the
Ub-system can very rapidly respond to various kinds
of stress [11–17]. Previous studies showed an increase
in the inclusion-type intranuclear Ub-immunoreactivity, closely related to Marinesco bodies, of the
pigmented dopaminergic neurons of the substantia
nigra in the midbrain (inclusion-type neuronal Ubindex) in acute deaths in fires and also in those from
asphyxiation in adult subjects, suggesting the possibility of evaluation of the stress on the CNS resulting
from physical activity before death [18,19]. Meanwhile, granular ‘dot-like’ Ub-immunoreactive structures have been described as most consistent with
dystrophic neurites and are mostly composed of focal
swellings in myelin lamellae containing heterogenous
dense material [20]. However, there appears to have
been no report on the relation of these structures to the
traumatic stress.
The present study was focused on the evaluation of
granular dot-like Ub-immunoreactivity in the crus
cerebri (cortico-spinal tracts) of the midbrain as a
marker of neuronal stress in fatal asphyxiation,
drownings and fire fatalities, in comparison with the
inclusion-type neuronal Ub-index.
Causes of death were fatal asphyxiation (nZ27:
hanging, nZ8; strangulation, nZ15; aspiration,
nZ4), drownings (nZ14: fresh water, nZ8; salt
water, nZ6), fire fatalities (nZ60) including those
with a blood carboxyhemoglobin (COHb) level lower
than 60% (nZ37) and higher than 60% (nZ23), as
shown in Table 1. Younger subjects were not included
because a sufficient number of relevant cases were not
available. Control groups consisted of those with
acute cardiac death (nZ28), fatal hemorrhages from
chest/abdominal stab wounds (nZ24) and acute head
injury death (nZ16). The above-mentioned causes of
death were classified on pathological and toxicological bases, excluding cases with complications which
may have contributed to the dying process. The acute
cardiac death group consisted of cases that showed
macro- and microscopical findings of acute ischemic
heart diseases without any evidence of cause of death
other than a cardiac attack [21,22].
2.2. Methods
2.1. Materials
2.2.1. Tissue sections
Serial sections (5 mm thick) were prepared from
the formalin-fixed paraffin-embedded tissue specimens of the brain: the frontal, temporal, parietal and
occipital lobes, capsula interna, midbrain, pons and
medulla oblongata. The tissue sections were used for
hematoxylin-eosin (HE) and immunostaining.
Formalin-fixed paraffin-embedded brain tissue
specimens of forensic autopsy cases (over 35 years
of age, nZ169) at our institute were examined.
2.2.2. Immunostaining
A polyclonal rabbit anti-ubiquitin serum (Dako
A/S, Glostrup) at a 100-fold dilution and a mouse
2. Materials and methods
Table 1
Case profiles (nZ169)
Cause of death
Male/female
Age (mean, years)
Survival time
(median, h)
Postmortem time
(median, h)
Acute cardiac death (nZ28)
Hemorrhages (nZ24)
Head injuries (nZ16)
Hanging (nZ8)
Strangulation (nZ15)
Aspiration (nZ4)
Drowning (nZ14)
Fire fatalities
COHb !60% (nZ37)
COHb O60% (nZ23)
21/7
20/4
15/1
6/2
5/10
4/0
8/6
39–84 (61.6)
38–85 (53.6)
37–88 (54.7)
35–68 (54.1)
43–85 (63.5)
51–75 (61.8)
35–88 (61.6)
!0.5–15.0 (!0.5)
!0.5–7.0 (!0.5)
!0.5–1.0 (!0.5)
!0.5
!0.5
!0.5
!0.5
6.0–96.0 (19.0)
6.2–106.5 (18.9)
6.0–38.0 (22.0)
12.0–58.5 (31.3)
10.5–52.0 (25.0)
7.2–20.5 (11.0)
7.5–84.0 (28.7)
27/10
16/7
42–89 (65.8)
35–87 (64.2)
!0.5
!0.5
6.5–36.0 (13.5)
6.0–35.0 (13.0)
COHb, blood carboxyhemoglobin level.
L. Quan et al. / Legal Medicine 7 (2005) 81–88
monoclonal anti-human amyloid precursor protein
(APP) reagent (MBL, Nagoya) at a 50-fold dilution
were used, with 3 h incubation at 37 8C, on a
Vectastain Universal Elite ABC Kit (DAB) (Vector
Laboratories, Burlingame, Calif.) according to the
manufacturer’s instructions (counterstaining with
hematoxylin). Endogenous peroxidase was inactivated
by incubation with 3% hydrogen peroxide for 5 min.
For the control study to confirm the specificity of
immunostaining, phosphate buffered saline or normal
rabbit serum was substituted for the primary antibody.
2.2.3. Quantitative analysis of ubiquitinimmunoreactive dot-like area in the crus cerebri
Quantitation of the Ub-immunoreactive granular
dot-like area was performed on the horizontal sections
of the crus cerebri in the midbrain (Fig. 1a and b)
using a two-dimensional evaluation: the number and
area of Ub-positive granular structures larger than
83
0.5 mm in diameter per mm2 (dot-like Ub-area) were
measured using a color image processor (IPAP;
Sumica Technos, Osaka).
2.2.4. Quantitative analysis of ubiquitinimmunopositivity in the nuclei of pigmented neurons
of the substantia nigra
The intranuclear Ub-immunostaining of the pigmented neurons was classified into two patterns, as
previously reported: inclusion-type and diffuse type
(Fig. 1c and d) [18,19]. The total number of
pigmented neurons and numbers of inclusion- and
diffuse type Ub-positive pigmented neurons were
quantitatively analyzed in the horizontal sections:
the number of neurons with nuclei in which
Ub-immunoreactivity was detected was counted in
10 fields under 200! magnification, and the percentage of nuclear Ub-positivity (neuronal Ub-index) was
estimated.
Fig. 1. Ubiquitin-immunoreactivity in the midbrain. Horizontal (a) and longitudinal (b) sections of the crus cerebri of the midbrain, showing a
large number of granular ‘dot-like’ positivity (arrowheads), in a fire victim with 28.7% COHb, 42-year-old male, survival time !30 min, 12 h
postmortem. (c) The inclusion-type intranuclear ubiquitin-positivity (arrowheads) in the pigmented neurons of the substantia nigra in a ligature
strangulation case, a 49-year-old female, 11 h postmortem; (d) the diffuse type ubiquitin-positivity (arrowhead) in a freshwater drowning case, a
50-year-old male, 26 h postmortem. Bar, 10 mm.
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2.2.5. Chemical analysis
Blood %COHb saturation was analyzed on a COoximeter system (Ciba-Corning 270, New York).
Blood cyanide and alcohol levels were determined by
head-space gas chromatography/mass spectrometry
[23]. Drug analyses were performed by gas chromatography/mass spectrometry.
2.2.6. Statistical analyses
A regression equation analysis was used to
examine the relationship of the dot-like Ub area and
neuronal Ub-indices with the age of victims and blood
COHb level. Comparisons between groups were
performed using Student’s t-test and the Mann–
Whitney U-test. The logistic regression and stepwise
regression tests were used in the multivariate
analyses. These analyses were performed using
Microsoft Excel and Statview (version 5.0, SAS
Institute Inc.), and a P value less than 0.05 was
considered statistically significant.
3. Results
3.1. Distribution of ubiquitin-immunoreactive
dot-like structures
Granular dot-like Ub-immunoreactivity was
observed mainly in the white matter of the parietal
lobe, internal capsule, crus cerebri in the midbrain, and
the ventral portion of the pons and medulla oblongata,
showing a distribution most consistent with the
cortico-spinal tracts (Fig. 2) and a case-to-case
difference. These structures were negative for APP.
Such granular Ub-immunoreactivity was observed
only in a few numbers in the ascending tract zone.
Fig. 2. Distribution of ubiquitin-immunoreactive granular ‘dot-like’ structures in the cortico-spinal tracts. The parietal gray matter (a), internal
capsule (b) and the ventral portion of the pons (c), showing a large number of granular ‘dot-like’ positivity. In the medial lemniscus (d),
containing the ascending nerve tracts, ubiquitin-immunoreactivity was sporadic. A fire victim with 28.7% COHb, 42-year-old male, survival
time!30 min, 12 h postmortem.
L. Quan et al. / Legal Medicine 7 (2005) 81–88
85
3.2. Quantitative analysis of ubiquitinimmunoreactive dot-like area in the crus cerebri:
dot-like ubiquitin-area
Quantitative analysis of the dot-like Ub-area was
performed in the crus cerebri of the midbrain, where
the densest distribution was observed. The analysis
showed a significantly higher value in strangulation
and fire fatalities, whereas the value was low in fatal
hemorrhages and head injuries (Fig. 3a). The values
were intermediate in the other groups. There was a
significant difference between fire fatalities with
lower and higher COHb levels and an inverse relation
of the dot-like *Ub-area (y) to the blood COHb level
(x): yZK26.7 xC6470.2; nZ60, rZ0.37, PZ0.004.
3.3. Quantitative analysis of ubiquitinimmunopositivity in the nuclei of pigmented neurons
of the substantia nigra: neuronal ubiquitin-index
The inclusion-type neuronal Ub-index showed a
significantly higher value in strangulation and fire
fatalities, whereas the value was low in hangings,
drownings, fatal hemorrhages and head injuries
(Fig. 3b). An increase was observed in aspiration
and acute cardiac death. The diffuse type Ub-index
was significantly high in drownings and fire fatalities,
and a high value was also observed in aspiration
(Fig. 3c). There was no significant relation to the
postmortem interval, the age and gender of the
subjects, or blood alcohol (!2.5 mg/ml) or cyanide
level (!0.06 mg/ml).
"
above. (b) Inclusion-type neuronal ubiquitin-index in the pigmented
neurons of the substantia nigra. *Significantly high: strangulation
vs. hanging (P!0.05), drowning (P!0.001), fire fatality with a
lower COHb level (!60%) (P!0.01), fatal hemorrhages, head
injuries (P!0.001) and acute cardiac death (P!0.005); fire fatality
with a higher COHb level (O60%) vs. hanging (P!0.05),
drowning, fatal hemorrhages and head injuries (P!0.01). †SignifiSignificantly low: fatal hemorrhages vs. aspiration and fire fatality
with a lower COHb level (!60%) (P!0.05); drowning vs.
aspiration and fire fatality with a lower COHb level (!60%)
(P!0.05); both groups vs. strangulation and fire fatality with a
higher COHb level as mentioned above. (c) Diffuse type neuronal
ubiquitin-index. *Significantly high: drowning vs. the groups other
than aspiration and fire fatalities (P!0.001); fire fatality with a
lower COHb level (!60%) vs. hanging (P!0.05) and fatal
hemorrhages (P!0.005). There was no significant difference in
any marker between fresh- and saltwater drownings.
Fig. 3. Quantitative analysis of ubiquitin-immunoreactivity. The
results of the data analyses are shown in box-plots, in which 50% of
the data are summarized in the box, the line represents the median
and the lines outside of the box represent the 90% confidence
interval. (a) Granular ‘dot-like’ ubiquitin-areas in the crus cerebri.
*Significantly high: strangulation vs. fire fatality with a higher
COHb level (O60%) (P!0.05), fatal hemorrhages, head injuries
and acute cardiac death (P!0.001); fire fatality with a lower COHb
level (!60%) vs. fire fatality with a higher COHb level (O60%)
(P!0.05) and acute cardiac death (P!0.005), fatal hemorrhages
and head injuries (P!0.001). †Significantly low: fatal hemorrhages
vs. fire fatality with a higher COHb level (O60%) (P!0.05); head
injuries vs. hanging, drowning, fire fatality with a higher COHb
level (O60%) and acute cardiac death (P!0.05); both groups vs.
strangulation and fire fatality with a lower COHb level as mentioned
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Fig. 4. Relationship of the granular ‘dot-like’ ubiquitin-areas in the crus cerebri to the inclusion-type neuronal ubiquitin-index in the pigmented
neurons of the substantia nigra.(a) strangulation: yZ99.7 xC8011.5 (nZ15, rZ0.26, PZ0.35). (b) hanging: yZ238.1 xC2695.5 (nZ8, rZ
0.74, P!0.05). (c) drownings: yZ387.9 xC1824.7 (nZ14, rZ0.51, P!0.05). (d) fire fatalities: (1) lower COHb group, yZ87.8 xC6619.4
(nZ37, rZ0.26, PZ0.12); (2) higher COHb group, yZ67.6 xC3462.7 (nZ23, rZ0.23, PZ0.29).
3.4. Comparison of dot-like ubiquitin-area in the crus
cerebri to neuronal ubiquitin-index in the substantia
nigra
Ub-area in the crus cerebri and the diffuse type
neuronal Ub-index in the substantia nigra.
There was a correlation between the dot-like
Ub-area in the crus cerebri (y) and the inclusiontype neuronal Ub-index in the substantia nigra (x) in
hangings (yZ238.1 xC2695.5; nZ8, rZ0.74,
P!0.05) and drownings (yZ387.9 xC1824.7;
nZ14, rZ0.51, P!0.05) (Fig. 4b and c). However,
the relationship between these markers was not clear
in the other groups. A predominant increase in the
neuronal Ub-index was frequently observed in
strangulation, fire fatalities and acute cardiac death
(Fig. 4a and d). Some cases of fire fatality with a lower
COHb level showed an increase in the dot-like
Ub-area with a lower neuronal Ub-index (Fig. 4d).
There was no relation between the the dot-like
4. Discussion
The granular dot-like structures in the white matter
and nuclear inclusions in the pigmented neurons of the
substantia nigra have been described as a part of the
spectrum of ubiquitinated structures in normal brains
[20]. Although previous studies suggested an agedependent increase in ubiquitination in these brain
structures [7–10,18,19], there was no significant agedependence of the dot-like Ub-area in the crus cerebri,
which contains myelinated nerve fibers in the corticospinal tracts, and the inclusion-type neuronal
Ub-index in the substantia nigra (dopaminergic
neurons) in the adult subjects (over 35 years of age)
L. Quan et al. / Legal Medicine 7 (2005) 81–88
in the present study. Postmortem interference was also
not observed. Meanwhile, those ubiquitinated structures showed an increase closely related to the cause
of death, although they were sparse in the child
subjects [7,20] and the inclusion-type neuronal
Ub-index of the pigmented neurons may be mildly
enhanced in older victims [9,10,18,19].
The relationship of the dot-like Ub-area to the
inclusion-type neuronal Ub-index varied, depending
on the cause of death. In strangulation and fire
fatalities, there was an increase in both markers,
usually with a predominant elevation in the neuronal
Ub-index. Acute cardiac death cases showed a similar
finding, although their increases were milder. A
parallel increase, showing a relatively low neuronal
Ub-index was observed in hanging, aspiration and
drowning. These findings suggested a different stress
reaction between the cortico-spinal tracts and dopaminergic neurons: neuronal stress to the CNS may be
the most intense and greater in the dopaminergic
system in strangulation and fire fatalities, and similar
stress can be caused in acute cardiac death, whereas
neuronal stress may usually be milder in hanging,
aspiration and drowning. However, in drownings, an
increase in the diffuse type nuclear Ub-immunoreactivity, overlapping the intranuclear inclusions, may
have reduced the inclusion-type neuronal Ub-index
[19]. For the diffuse type neuronal Ub-index, which
was elevated in drownings, aspiration and fire fatalities and usually low in the other groups, another type
of stress, e.g. metabolic alteration, may be considered
[24–26].
Low values of both the dot-like Ub-area and the
inclusion-type neuronal Ub-index in hemorrhagic and
head injury deaths suggested that cerebral ischemia
and/or CNS dysfunction (loss of consciousness) may
be a contributory factor to a reduced neuronal stress
reaction. A low neuronal Ub-index in hanging
suggested a greater contribution of cerebral ischemia
to reducing the stress to the dopaminergic system.
Although a lower value of dot-like Ub-area in a higher
COHb group of fire fatalities suggested neurotoxicity
of carbon monoxide, no influence of alcohol was
observed. The above-mentioned Ub-positivity may
appear within minutes depending on the intensity
of neuronal stress and further develop during
survival [11–15,27,28], suggesting the possibility of
evaluation of the stress on the CNS resulting from
87
the physical activity before death [18,19]. However, a
moderate elevation in the dot-like Ub-area with a low
neuronal Ub-index in hanging suggested that convulsions may be involved in the factors related to the
stress to the cortico-spinal system. For these hypotheses and also for the neuronal ubiquitination in
younger victims, further investigation is necessary.
In conclusion, the present study suggested an
increase in the dot-like Ub-area in the crus cerebri
(cortico-spinal tracts) depending on the cause of death
in adult subjects and a partial difference in stress
reaction between the cortico-spinal tracts and dopaminergic neurons. These markers may be useful for
examining CNS stress responses in traumas, at least in
middle-aged and elderly victims, suggesting that the
neuronal stress may be very intense in strangulation
and fire deaths.
Acknowledgements
This study was supported in part by Grants-in-Aid
for Scientific Research from Japan Society for the
Promotion of Science (JSPS) (Grant Nos. 08307006,
15390217 and 15590585) and a Grant-in-Aid for JSPS
Fellows (14002532).
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