Download The fate of Nissl-stained dark neurons following

Acta Neuropathol (2006) 112:471–481
DOI 10.1007/s00401-006-0108-2
O RI G I NAL PAPE R
The fate of Nissl-stained dark neurons following traumatic brain
injury in rats: diVerence between neocortex and hippocampus
regarding survival rate
Hidetoshi Ooigawa · Hiroshi Nawashiro · Shinji Fukui ·
Naoki Otani · Atsushi Osumi · Terushige Toyooka ·
Katsuji Shima
Received: 13 April 2006 / Revised: 20 June 2006 / Accepted: 26 June 2006 / Published online: 21 July 2006
© Springer-Verlag 2006
Abstract We studied the fate of Nissl-stained dark
neurons (N-DNs) following traumatic brain injury
(TBI). N-DNs were investigated in the cerebral neocortex and the hippocampus using a rat lateral Xuid
percussion injury model. Nissl stain, acid fuchsin stain
and immunohistochemistry with phosphorylated extracellular signal-regulated protein kinase (pERK) antibody were used in order to assess posttraumatic
neurons. In the neocortex, the number of dead neurons
at 24 h postinjury was signiWcantly less than that of
the observed N-DNs in the earlier phase. Only a few
N-DNs increased their pERK immunoreactivity. On
the other hand, in the hippocampus the number of
dead neurons was approximately the same number as
that of the N-DNs, and most N-DNs showed an
increased pERK immunoreactivity. These data suggest
that not all N-DNs inevitably die especially in the neocortex after TBI. The fate of N-DNs is thus considered
to diVer depending on brain subWelds.
Keywords Dark neuron · Nissl stain · Traumatic brain
injury · Extracellular signal-regulated protein kinase
Introduction
Dark neurons are traditionally known to represent a
typical morphological change of injured neurons following many kinds of insults, and have been observed
H. Ooigawa · H. Nawashiro (&) · S. Fukui · N. Otani ·
A. Osumi · T. Toyooka · K. Shima
Department of Neurosurgery, National Defense Medical
College, 3-2 Namiki, Tokorozawa, Saitama, 359-8513, Japan
e-mail: [email protected]
using various staining methods, such as hematoxylin
and eosin stain, Nissl stain and silver stain [17, 19, 25,
29, 33, 41]. Since dark neurons show massive shrinkage
and abnormal basophilia, they can be clearly distinguished from normal neurons. Some studies have
shown the following electron microscopic characteristics of dark neurons: (1) a marked condensation of
both karyoplasm and cytoplasm, (2) an intact nuclear
membrane, cytoplasmic membrane and cytoplasmic
organelles, (3) mitochondrial swelling, (4) a dilatation
of the Golgi cisternae, (5) a decrease in the rough
endoplasmic reticulum, and polyribosomes, and (6) an
aggregation of the nuclear chromatin [2, 3, 11, 39],
which resembles the appearance of necrotic neurons.
Similarly, in the Weld of traumatic brain injury (TBI),
dark neurons have been observed as one kind of feature
of damaged neurons. The regions where dark neurons
appear at a high rate after TBI, such as neocortex, CA3
subWeld and dentate hilus, coincide with the regions
where subsequent neuronal death is induced. These
facts appear to indicate that dark neurons inevitably die
[10, 25, 33]. On the other hand, some reports have
described the possibility that dark neurons recover
from such a damaged status [11, 15, 16].
In the rat lateral Xuid percussion injury (FPI) model,
numerous dark neurons are observed in the neocortex
and the hippocampus in the early phase after TBI [10,
17, 29]. However, it remains unclear whether all dark
neurons result in subsequent neuronal cell death. In
this study we addressed a numerical relationship
between the emergence of Nissl-stained dark neurons
(N-DNs) and subsequent neuronal cell loss in the neocortex and the hippocampus after TBI. We counted
total neuronal number and N-DNs in the neocortex
and the hippocampus from immediately to 24 h after
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TBI. Neuronal death was calculated from the subtraction of total neuronal number in sham brains minus
total neuronal number in injured animals at 24 h after
TBI. Since at least some of the observed neuronal cell
death is due to apoptosis [4, 5, 8, 22], we ensured the
neuronal death using terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling
(TUNEL) stain. Nissl/acid fuchsin stain was also performed as another staining method to conWrm the presence of damaged neurons [6]. Furthermore, we
immunohistochemically studied phosphorylated extracellular signal-regulated protein kinase (pERK) on
dark neurons, which plays an important role in signal
transduction after TBI [31], and shows a diVerential
immunoreactivity between the cortex and hippocampus regarding duration and magnitude [35, 36, 43].
Materials and methods
Animal experimental procedure
Adult male Sprague-Dawley rats weighing from 300 to
400 g were used. The rats were housed in individual
cages under controlled environmental conditions (12/
12 h light/dark cycle, 20–22°C; room temperature) with
food and water available for at least 1 week before
undergoing experimental surgery. The Animal Care
and Use Committee of the National Defense Medical
College approved all animal procedures. All surgical
procedures were performed under aseptic surgical conditions. The rats were anesthetized with isoXurane in
30% oxygen and 70% nitrous oxide gas mixture
through a facemask. The rats were Wxed in a stereotaxic Xame. A 4.8 mm craniectomy was made over the
right parietal cortex (3.8 mm posterior and 2.5 mm lateral to the bregma). A plastic Luer-Loc was placed
over the craniectomy site with dental acrylic cement.
The next day, the rats were anesthetized and a PE
catheter was implanted in the right femoral artery. The
rats were intubated with a 14-gauge angiocatheter and
maintained on a mechanical ventilator after the infusion of pancronium bromide (tidal volume: 2.5–3.0 ml/
kg; respiratory rate: 70 per min). The rectal temperature was measured with a rectal probe and then it was
maintained at a constant level of around 37.0°C using a
heating pad. Arterial blood samples were analyzed
intermittently. The rats were subjected to FPI at a
moderate severity (1.8–2.2 atmosphere, 12–14 ms in
duration) using a DragonXy Xuid percussion device
(model HPD-1700, DragonXy R&D, Silver Spring,
MD) as previously described [27]. The pressure pulse
was measured using a pressure transducer (Model 211
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Acta Neuropathol (2006) 112:471–481
B4, Kistler Instrument Corp., Amherst, NY) and it was
recorded on a digital storage computer (MacLab, AD
Instruments, NSW, Australia). Following the induction
of the injury, the connection cap was removed and the
scalp was sutured. The rats were placed on the heating
pad to maintain their temperature around 37.0°C until
they could move independently. The sham-operated
rats (n = 6) were subjected to all the same procedures
except for an actual insult, and then were sacriWced
15 min after pseudoinjury. After completing the surgical procedures, the rats were returned to their home
cage with food and water available ad libitum.
Tissue preparation
Under the intrapenetneal injection of pentobarbital
sodium, the rats were transcardially perfused with normal saline followed by 4% buVered paraformaldehyde
after predetermined survival times (0, 15, 30, 60 min; 3,
6 and 24 h; n = 6–8 per each time point). The brains
were removed just after perfusion and immersed in the
4% buVered paraformaldehyde overnight, and then
were embedded in paraYn. Three series of 5-m-thick
coronal sections were cut every 100 m from 3.3 to
4.3 mm posterior to the bregma. Each series of sections
was used for Nissl stain, Nissl/acid fuchsin stain and
immunostain against pERK. The existence of morphological changes, such as a gliding contusion, was conWrmed in all sections of the traumatized animals.
Combined Nissl/acid fuchsin staining
For combined Nissl/acid fuchsin staining, the sections
were stained with 0.1% cresyl violet. The sections were
then placed in 0.1% acid fuchsin fortiWed with a few
drops of acetic acid for 20 s, and then they were subsequently washed with distilled water, dehydrated with
ethanol and lemosol, and Wnally coverslipped.
Immunohistochemistry
Immunohistochemistry was performed using a HISTOFINE SAB-PO (R) Kit (NICHIREI Co, Tokyo,
Japan). After deparaVinization and hydration, the sections were boiled in 10 mmol/l sodium citrate buVer
(pH 6.0) for 10 min at 95°C, and were cooled for
20 min. Endogenous peroxidase was blocked with 3%
hydrogen peroxidase. The sections were incubated with
PBS containing 10% normal goat serum at room temperature to eliminate any nonspeciWc binding, and then
they were incubated overnight at 4°C with polyclonal
antibody against pERK1/2 (Cell Signaling Technology,
Danvers, MA1, 1:100). The sections were incubated
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with biotinylated secondary antibody and horseradish
peroxidase-linked streptavidin for 1 h at room temperature. Peroxidase was then demonstrated with DAB.
TUNEL staining
APOPTOSIS in situ Detection Kit (WAKO, Osaka,
Japan) was used for TUNEL staining, according to the
manufacturer’s instruction. After deparaVinization and
hydration, sections were incubated with protein digestion enzyme solution for 5 min at 37°C, The sections
were treated with TdT solution and with 3% hydrogen
peroxide for 5 min to block endogenous peroxidase
activity, and then treated with peroxidase-conjugated
antibody for 10 min at 37°C. Nick end labeling was
visualized by DAB solution. For the positive control,
sections were treated with DNase I before treatment
with TdT. For negative control sections were incubated
with TdT buVer that did not contain the enzyme.
Counting of neurons
Three periodic Nissl-stained sections at the coronal
level of 3.8 mm posterior to the bregma were used for
cell counts and the average was calculated. In the
neocortex, neurons were counted in two diVerent
regions circumscribed with a rectangle measuring
0.47 £ 0.35 mm2 (Fig. 1). The centers of both counting boxes were 5.0 mm far from each other. The one
counting box was located just under the craniectomy,
in which no cerebral contusion was seen. The center
of the box was situated in the cortical layer V and
2.5 mm lateral to the midline. The other box was
placed in the region close to the gliding contusion.
This area is located on the boundary between the area
1 of temporal cortex and the area 1 of parietal cortex
[37]. In the hippocampus, neurons were counted in
the pyramidal cell layer of the CA3 and the dentate
hilus (Fig. 1). The CA3 subWeld was determined as a
territory from the CA3/2 border to a point where the
pyramidal cells enter the hilus as deWned by the lateral aspect of the granule cell layer’s dorsal and ventral leaves. The dentate hilar neurons were counted as
those cells existing within the region deWned by the
superior and inferior granular cell layers, thus excluding the CA3 pyramidal cell neurons and transitional
neurons within approximately 20 m of the pyramidal
neurons [25]. The specimens were scanned at a magniWcation of £200 within the identical areas in the
neocortex and the hippocampus, using a microscope
(Olympus Optical Co, LTD, Japan) equipped with a
digital camera system (Pixera 600CL-CU, Pixera Corporation, Japan). The intact neurons were deWned as
Fig. 1 Schematic illustration of regions that were selected for
neuronal cell counting (colored with blue). The area colored with
red indicates the lesions of a gliding contusion
nonbasophilic neurons with both the pale nuclei and
the discrete nucleoi. Any neurons that had nucleolar
fragments larger than one-half of the average nucleolar diameter were included in the count as well as the
neurons that had intact neuronal bodies [23]. The
N-DNs were deWned as neurons with abnormal morphologies of massive shrunken, hyperbasophilic features,
and corkscrew-like dendrites. No correlation factor
was applied because the somata were smaller than the
distance between the two sections stained by the same
method [23].
In the Nissl/acid fuchsin sections, intensely acidophilic cells were counted.
The pERK-positive cells were also counted in the
aforementioned areas. All cells were counted as
pERK-positive cells regardless of the density of labeling by DAB [30]. We used sections taken by 1 h after
TBI for cell counting of pERK-positive cells, because
ERK is activated predominantly in non-neuronal cells
as early as 2 h postinjury [43]. Fragments of somata
that could be clearly identiWed as cells were also
counted [30]. All neuronal counting was performed
blinded to the treatment conditions.
DeWnition of the number of dead neurons
The number of dead neurons was deWned as (neuronal
cell number in sham-operated rats) ¡ (neuronal cell
number in injured rats sacriWced 24 h after TBI)
Statistical analysis
All data are expressed as mean § SD. The physiological parameters were analyzed using one-way analysis
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Acta Neuropathol (2006) 112:471–481
of variance. Cell counting was analyzed using Kruskal–
Wallis test. If any signiWcance was noted, individual
comparisons were made using Mann–Whitney’s U test.
A P value of less than 0.05 compared to sham values
was considered statistically signiWcant.
Results
Physiological data
There were no signiWcant diVerences in the physiological data between each group (Table 1).
Table 1 Summary of physiological parameters
Preinjury
After 5 min
After 15 min
7.43 § 0.03
38.2 § 2.4
128.4 § 15.6
108.7 § 8.9
7.41 § 0.02
38.6 § 3.1
125.8 § 15.4
110.0 § 9.4
TBI group sacriWced immediately after TBI
pH
7.44 § 0.02
7.43 § 0.03
pCO2
36.1 § 2.8
37.9 § 4.1
pO2
127.0 § 15.1
122.1 § 10.8
MABP
114.2 § 10.7
121.1 § 13.2
7.42 § 0.04
38.9 § 3.4
119.8 § 20.6
115.1 § 12.2
TBI group sacriWced 15 min after TBI
pH
7.42 § 0.02
7.42 § 0.04
pCO2
37.8 § 2.2
38.4 § 4.0
pO2
129.4 § 5.4
129.0 § 15.9
MABP
111.4 § 8.2
113.9 § 7.6
7.43 § 0.03
36.8 § 4.8
123.0 § 24.4
111.5 § 8.9
TBI group sacriWced 30 min after TBI
pH
7.44 § 0.04
7.44 § 0.04
pCO2
38.2 § 2.7
38.9 § 5.2
pO2
128.3 § 13.6
129.4 § 15.0
MABP
112.1 § 10.2
114.7 § 13.8
7.43 § 0.04
39.6 § 4.8
125.5 § 19.3
113.6 § 10.3
TBI group sacriWced 1 h after TBI
pH
7.43 § 0.03
7.42 § 0.04
pCO2
35.6 § 1.1
38.7 § 4.5
pO2
127.2 § 13.5
124.8 § 21.1
MABP
107.9 § 9.2
108.5 § 10.2
7.43 § 0.03
37.9 § 3.8
121.0 § 13.6
106.9 § 10.8
TBI group sacriWced 3 h after TBI
pH
7.44 § 0.02
7.43 § 0.03
pCO2
37.1 § 1.4
39.1 § 3.4
pO2
126.5 § 16.6
123.1 § 9.7
MABP
109.9 § 9.4
112.3 § 12.6
7.45 § 0.04
41.0 § 5.0
113.0 § 18.9
108.6 § 13.0
TBI group sacriWced 6 h after TBI
pH
7.44 § 0.03
7.42 § 0.02
pCO2
36.4 § 3.6
39.0 § 2.7
pO2
126.8 § 7.8
122.7 § 17.6
MABP
112.2 § 13.9
114.0 § 11.0
7.43 § 0.03
39.1 § 2.8
119.3 § 16.5
114.2 § 10.7
TBI group sacriWced 24 h after TBI
pH
7.43 § 0.03
7.42 § 0.05
pCO2
36.5 § 2.6
37.0 § 2.5
pO2
126.3 § 11.5
123.4 § 17.2
MABP
112.1 § 10.4
110.3 § 6.6
7.45 § 0.04
37.1 § 2.8
120.4 § 13.7
111.2 § 8.3
Sham-operated group
pH
7.43 § 0.03
pCO2
36.6 § 3.0
PO2
132.1 § 15.6
MABP
112.1 § 10.2
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Dark neurons in the neocortical region distant from
the contusion
Nissl-stained dark neurons were not detected in the
sham-operated rats (Fig. 2b, c). N-DNs began to
appear immediately after TBI, and kept at maximum
number for up to 30 min postinjury (Fig. 2b, d).
Thereafter, the N-DNs began to decrease at 1 h
postinjury and completely disappeared by 24 h after
TBI (Fig. 2b, g). Despite the existence of N-DNs in
the acute phase, there was no signiWcant decrease in
neuronal number at 24 h postinjury in comparison to
the sham values (Fig. 2a). Acid fuchsin-positive neurons were also observed in the acute phase after TBI,
and coincided with the N-DNs (Fig. 2e). No acid fuchsin-stained neurons were detected 24 h postinjury
along with the disappearance of the N-DNs (Fig. 2b,
h). pERK-positive neurons also emerged in this area.
pERK positive neurons were clearly less than N-DNs
in number during the period till 1 h after TBI
(Fig. 2b). In fact, most dark neurons did not have
pERK immunoreactivity (Fig. 2f). No TUNEL-staining cells were detected in this region 24 h after TBI
(Fig. 2i).
Dark neurons in the neocortical region close
to the contusion
Nissl-stained dark neurons were observed from
immediately to 6 h after TBI (Fig. 3b, e). The number of N-DNs observed during this period was signiWcantly higher than that in the region distant from the
contusion (P < 0.01 at 15 and 30 min after TBI;
Figs. 2b, 3b). The number of total neurons at the
point of 24 h after TBI was decreased compared to
that of sham-operated rats (P = 0.02; Fig. 3a, h).
However, the number of dead neurons was signiWcantly less than that of the N-DNs appearing in the
acute phase (P = 0.03, Fig. 3c). The acid fuchsin-positive neurons were consistent with N-DNs in the early
phase after TBI (Fig. 3f). Although there were no
N-DNs in the neocortex close to the contusion at
24 h after TBI (Fig. 3b, h), acid fuchsin-staining cells
could be observed (Fig. 3i). Some pERK-positive
neurons were observed in the acute phase (Fig. 3b),
but most of them were not dark neurons (Fig. 3g). In
spite of the diVerence in the number of N-DNs
between the two cortical areas, almost the same number of pERK-positive neurons was observed
(Figs. 2b, 3b). Many TUNEL-positive cells were
observed 24 h after TBI in contrast to the other neocortical regions (Fig. 2k).
Acta Neuropathol (2006) 112:471–481
Fig. 2 Neuronal counts and typical patterns of brain tissue in
the neocortex distant from the contusion. Bar graphs show the
total neuronal number (a) and the number of Nissl-stained dark
neurons, acid fuchsin-positive neurons and pERK-positive neurons (b), respectively. Photomicrographs of the following were
taken: sham-operated rats (c Nissl stain), injured rats in the
acute phase after TBI (d Nissl stain, e Nissl/acid fuchsin stain, f
pERK immunostaining) and injured rats at 24 h postinjury (g
Nissl stain, h Nissl/acid fuchsin stain, i TUNEL stain) . j indicates a positive control of TUNEL stain. Insets show magniWed
475
views. There were no N-DNs in sham-operated rats (b and c).
Despite emergence of many N-DNs in the early phase after TBI
(b and d), neuronal cell loss was not observed 24 h after TBI (a
and g). Acid fuchsin-positive neurons were also observed in the
acute phase (e), and then disappeared by 24 h after TBI. Some
pERK-positive neurons were seen in this area. No TUNELstaining cell was observed 24 h after TBI (i). S and I indicate
sham-operated and immediately sacriWced groups, respectively.
#
P < 0.05, *P < 0.01 compared with sham-operated rats. Scale
bar = 200 m
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Fig. 3 Neuronal counts and typical patterns of brain tissue in the
neocortex close to the contusion. Bar graphs show the total neuronal number (a), the number of Nissl-stained dark neurons, acid
fuchsin-positive neurons and pERK-positive neurons (b), and
comparison in number between N-DNs appearing 30 min after
TBI and dead neurons (c), respectively. Photomicrographs of the
following were taken: sham-operated rats (d Nissl stain, j TUNEL stain), injured rats in the acute phase after TBI (e Nissl stain,
f Nissl/acid fuchsin stain, g pERK immunostaining) and injured
rats at 24 h postinjury (h Nissl stain, i Nissl/acid fuchsin stain, k
TUNEL stain). Insets show magniWed views. No N-DNs were observed in the sham-operated rats (b and d). Many N-DNs ap-
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Acta Neuropathol (2006) 112:471–481
peared in the early phase after TBI (b and e), SigniWcant neuronal
cell loss was observed 24 h after TBI (a and h). However, the
number of dead neurons was signiWcantly less than that of N-DNs
appeared in the acute phase (c). Acid fuchsin-positive neurons
were observed 24 h after TBI (i) as well as in the acute phase (f).
Most dark neurons do not increase their immunoreactivity for
pERK (g). TUNEL-staining cell was observed 24 h after TBI (k).
In the bar charts, S and I indicate sham-operated group and
immediately sacriWced group, respectively. #P < 0.05, *P < 0.01
compared with sham-operated rats (a and b). #P < 0.05 comparison between two groups (c). Scale bar = 200 m
Acta Neuropathol (2006) 112:471–481
Dark neurons in the hippocampus
Nissl-stained dark neurons were also detected in the
hippocampus in the early phase after TBI both in the
dentate hilus (Fig. 4b, e) and the CA3 subWeld (Fig. 5b,
d). The number of surviving neurons at 24 h postinjury
signiWcantly decreased in comparison to the shamoperated rats in those areas (dentate hilus; P < 0.01,
CA3; P = 0.01, Figs. 4a, 5a). The maximum number of
N-DNs in the acute phase, which were observed immediately after TBI in the dentate hilus, and 15 min after
TBI in the CA3 subWeld (Figs. 4b, 5b), were approximately the same as the number of dead neurons
(Figs. 4c, 5c). The acid fuchsin-positive cells coincided
with the N-DNs in the early phase (Fig. 4f), and could
be detected even after the N-DNs had disappeared
(Figs. 4h, i, 5f). pERK-positive cells could also be
observed in both of hippocampal regions. The number
of the pERK-positive cells reached a maximum 15 min
after TBI in the dentate hilus (Fig. 4b), and 30 min
postinjury in the CA3 subWeld (Fig. 5b). In the pERK
immunostaining, pERK-positive cells are consistent
with dark neurons in the acute phase after TBI
(Figs. 4g and 5e). Although some TUNEL-positive
neurons were observed in the hippocampus 24 h after
TBI, there was no proliferation in comparison to sham
groups (data not shown).
Discussion
Methodological considerations
The Nissl stain is a very popular technique and clearly
supports a neuronal appearance. However, counting of
Nissl-stained neurons is aVected by the characteristics
of the dye used, time of exposure of the section to dye
solution, and the thickness of the section [9, 23]. Many
dyes are available for Nissl staining; cresyl violet, thionin, toluidine blue, and methylen blue. We used cresyl
violet, because the dye has advantages of both the clarity of background and the intensity of staining of the
Nissl substance, nuclei and nucleoi [23]. Among 11
periodic sections, we counted neurons using only three
adjacent sections near the level of 3.8 mm posterior to
the bregma, at which most large gliding contusions
were observed in our model (data not shown). When
attempting to count the absolute neuronal number in
brain tissue, the use of serial thick sections may be
more accurate. However, using serial thick sections
may also result in serious problems, since the same
neurons may be counted more than once or the dye
used does not always inWltrate suYciently in the section
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[9, 18]. We therefore used periodic 5-m sections to
avoid such problems.
When intending to observe damaged neurons, some
staining methods such as hematoxylin and eosin stain,
Nissl stain, acid fuchsin stain, silver stain and FluoroJade stain can be suitable [10, 11, 14–17, 19, 41, 45–47].
The nomenclature of ‘dark neuron’ has been mainly
used for a damaged neuron stained with Nissl stain or
silver stain because of its morphological feature.
Although both of the two staining methods can detect
damaged neurons in the early phase after TBI, the
characteristics of those stainings are somewhat diVerent from each other [11, 15, 16]. The silver method has
been known to be more speciWc to detect damaged
neurons than Nissl method [15, 16, 44]. On the other
hand, the method fails to demonstrate damaged neuron within the territories of contusion, in spite of the
existence of massive shrunken and hyperbasophilic
damaged neurons in Nissl-stain preparations [16]. We
therefore needed to identify the diVerence in characteristics between each staining methods, even if both of
them can show damaged neurons. In the present study,
we used the Nissl method to observe dark neurons,
which is easier and more routinely performed than the
silver method in the Weld of neuropathology.
We also employed acid fuchsin staining as another
method to detect damaged neurons. There was a clear
diVerence in the staining properties between Nissl stain
and acid fuchsin stain at 1 h or later of TBI. Two possible hypotheses have been described previously [17].
One is that some neurons become newly damaged and
stained with acid fuchsin. The other is that many
injured neurons lose their aYnity for Nissl staining by
12–24 h, even though they continue to stain for acid
fuchsin. As a result, acid fuchsin staining was able to
indicate injured neurons at 24 h after injury, in contrast
to Nissl staining. In the neocortex distant from the contusion, no acid fuchsin-positive neurons were detected
at 24 h after injury. These results suggest that the neurons, which recovered from N-DNs, lost their aYnity to
acid fuchsin.
Nissl-stained dark neurons can recover
in the neocortex
The present study indicates that not all N-DNs necessarily die after TBI. Especially in the neocortex, many
damaged neurons, even N-DNs close to the contusion,
were able to survive for 24 h after TBI. However, there
was a discernible diVerence between the two neocortical lesions regarding the survival ratio of the N-DNs.
Almost all N-DNs were able to recover in the neocortex distant from the contusion. On the other hand, in
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Fig. 4 The neuronal counts and typical patterns of brain tissue in
the dentate hilus. Bar graphs show the total neuronal number (a),
the number of Nissl-stained dark neurons, acid fuchsin-positive
neurons and pERK-positive neurons (b), and comparison in
number between N-DNs appeared immediately after TBI and
dead neurons (c), respectively. Photomicrographs of the following were taken: sham-operated rats (d Nissl stain), injured rats in
the acute phase after TBI (e Nissl stain, f Nissl/acid fuchsin stain,
g pERK immunostaining) and injured rats at 24 h postinjury (h
Nissl stain, i Nissl/acid fuchsin stain). Insets show magniWed views.
There was no N-DNs in sham-operated rats (b and d). Many
N-DNs appeared in the early phase after TBI (b and e), SigniWcant neuronal cell loss was observed 3 h and later after TBI (a and
h). The number of dead neurons was approximately the same as
that of N-DNs appeared in the acute phase (c). Acid fuchsin-positive neurons were observed 24 h after TBI (i) as well as in the
acute phase (f). Most dark neurons increased their immunoreactivity for pERK (g). In the bar charts, S and I indicate sham-operated group and immediately sacriWced group, respectively.
#
P < 0.05, *P < 0.01 compared with sham-operated rats (a and b).
Scale bar = 200 m
the neocortex close to the contusion, both the incidence and the death rate of the N-DNs were higher.
This result seems to be consistent with the fact that it is
diYcult to save dark neurons, when they are induced
by severe injury to the brain [19]. In the neocortex, a
contusion or disruption of the vascular permeability
induced by FPI might aVect the diVerence in the recov-
erability of N-DNs. In the hippocampus, the maximum
number of N-DNs observed over each time point
approximately matched the number of dead neurons.
There was an obvious diVerence in the surviving rate of
N-DNs between the neocortex and the hippocampus.
Hippocampal N-DNs died at a higher rate than those
observed in the neocortex. Hippocampal neurons are
123
Acta Neuropathol (2006) 112:471–481
479
Fig. 5 The neuronal counts and typical patterns of brain tissue in
the CA3 subWelds. Bar graphs show the total neuronal number
(a), the number of Nissl-stained dark neurons, acid fuchsin-positive neurons and pERK-positive neurons (b), and comparison in
number between N-DNs appeared 15 min after TBI and dead
neurons (c), respectively. Photomicrographs of the following
were taken: injured rats in the acute phase after TBI (d Nissl
stain, e pERK immunostaining) and injured rats at 24 h postinjury (f Nissl/acid fuchsin stain). Many N-DNs appeared in the early
phase after TBI (b and d); signiWcant neuronal cell loss was observed 24 h after TBI (a). The number of dead neurons was
approximately the same as that of N-DNs appeared in the acute
phase (c). Acid fuchsin-positive neurons were observed 24 h after
TBI (f). Most dark neurons increased their immunoreactivity for
pERK (e). In the bar charts, S and I indicate sham-operated
group and immediately sacriWced group, respectively. #P < 0.05,
*P < 0.01 compared with sham-operated rats (a and b). Scale
bar = 50 m (e) and 100 m (f )
known to be vulnerable against TBI. Owing to the fact
that hippocampal neurons do not tend to recover well
after TBI, such injured hippocampal neurons might
aVect hippocampal vulnerability.
Morphological alterations in neuronal cell after TBI
have been reported to be attributed to the loss of cytoskeletal proteins such as neuroWlament 68 (NF68), neuroWlament 200 (NF200) and microtubule-associated
protein 2 (MAP-2) [20, 42, 44] with activation of calpain [34]. TBI induces neuroWlament compaction
within 5 min after injury, and the compaction persists
for at least 6 h [reviewed in 28, 40]. Thereafter, neuroWlament compaction is thought to demonstrate a state of
neuroWlament proteolysis and Wnally show neuronal
cell death [28]. However, resent studies have shown a
loss of cytoskeletal proteins such as MAP-2 in vivo [49,
50], and neuroWlament proteins in vitro [21] occur in
the absence of neuronal cell death. The present study
also indirectly indicates that neuroWlament compaction
does not inevitably lead to neuronal cell death.
ERK activation in morphologically diVerent type
of neurons
One unique Wnding in the present study is the morphological diVerence in pERK-positive neurons between
the neocortex and the hippocampus. In the neocortex,
ERK was mainly activated in the neurons with a normal feature, but in the hippocampus ERK immunoreactivity was increased in dark neurons. This result
indicates that ERK activation in the neocortical neurons, most of which do not die, may play a survival promoting role, whereas the activation of ERK in the
hippocampus, where most dark neurons die, may play
a death promoting role. ERK activation in the
hippocampus might thus be a sign indicating that the
123
480
circumstances are not favorable for the spontaneous
recovery of the dark neurons.
ERK is known to be activated in response to growth
stimuli for proliferation and survival. In addition, a
recent study revealed that the ERK pathway might
have functions involved in neuronal degeneration and
apoptosis [reviewed in 6, 7, 18, 46], in association with
cerebral ischemia [1, 32], Parkinson’s disease [24, 51]
and Alzheimer disease [13, 38]. Marshall [26] reported
that the short-to-moderate term activation of ERK signaling promoted proliferation and diVerentiation,
respectively, whereas Subramaniam [48] described that
long ERK activation leads to neuronal death. In the
Weld of TBI study, some studies have reported its bilateral characteristics of ERK activation. Mori et al. [31]
reported a deleterious eVect of this pathway using an
inhibitor of ERK pathway, in which the cortical lesion
volume of rats treated with the inhibitor was signiWcantly reduced. In contrast, Dash et al. [12] reported
that the inhibition of this cascade worsened retrograde
amnesia, exacerbated motor deWcit and did not have
any overt eVects on cell survival. The role of ERK activation after TBI is still not fully understood. Further
investigation will be needed to elucidate the function
according to various brain subWelds.
To summarize, the present study investigated the
fate of N-DNs following TBI. N-DNs are observed in
both the neocortex and hippocampus in the early phase
after TBI. However, the fate of N-DNs tended to diVer
depending on the regions of the brain. In the neocortex, most N-DNs appeared to survive for 24 h after
TBI, while N-DNs tended to die in the hippocampus.
Acta Neuropathol (2006) 112:471–481
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Acknowledgment We are grateful to A. Yano, N. Nomura and
T. Suzuki for their valuable technical contributions to this work,
and N. Tsuzuki, H. Katoh, S. Ishihara, T. Miyazawa and A. Onuki
for helpful suggestions.
16.
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