ELSEVIER

Journal of Neuroscience Methods 610995) 23-32

Microtubule-associated protein 2 (MAP-2) : a sensitive marker

of seizure-related brain damage It2
G.P.H. Ballough , L.J. Martin b, F.J. Cann a, J.S. Graham a, C.D. Smith a, C.E. Kling ,
J.S. Forster a, S. Phann a, M.G. Filbert a~*
Pathophysiology
Division,
U. S. Army Medical Research Institute of Chemical Defense,
Aberdeen Proving Ground,
MD 21010-5425,
USA
of Pathology and Neuroscience,
The Johns Hopkins
University
School of Medicine, Baltimore,
MD 21205-2196,
Department
of Biology, La Salle Universiiy,
Philadelphia,
PA 19141-I 199, USA

a Neurotoxicology
h Departments

Branch,

USA

Received 30 August 1994; revised and accepted 19 January 1995

Abstract
We have assessed
the efficacy of MAP-2 immunohistochemistryasa marker of seizure-relatedbrain damageand its suitability
for quantitation of the damage using densitometric and morphometric image analysis.Seizures were produced in rats by
administrationof 1.5 LD,, soman,an irreversible AChE inhibitor. Our resultsdemonstratethat neuronal damage,assessed
using
hematoxylin and eosin,and cresylviolet staining, wascolocalized on adjacent serial sectionswith clearly demarcatedreductions
in MAP-2 staining. The most severely damaged brain regions were devoid of MAP-2 staining. Reductions in MAP-2
immunostainingwere found to be exceptionally well suited for quantitation using densitometric and morphometric image
analysis.This study representsthe first demonstrationof seizure-inducedexcitotoxic alterations in MAP-2.
Keywords:

Microtubule-associatedprotein-2; Seizure; Neurotoxicity; Soman;GFAP; Cresyl Violet; Hematoxylin; Rosin

1. Introduction
Cytoskeletal proteins have key roles in a variety of
neuronal functions such as intracellular
transport,
maintenance of nerve cell shape and neurotransmitter
release (Shepherd, 1988). Microtubule-associated
proteins (MAPS) comprise a class of cytoskeletal proteins
that are essential for the functions of microtubules and
are generally believed to serve as microtubule-connecting links to organelles, vesicles and other cytoskeletal
elements. The most abundant MAP in the mammalian
brain is MAP-2. It is found almost exclusively in the

Corresponding author. CDR, USAMRICD, Attn: SGRD-UVYN, Dr. Filbert, APG MD 21010-542.5.
The opinions or assertions contained herein are the private views
of the authors and are not to be construed as official or as the views
l

of the Army of the Departmentof Defense.

In conducting the research described in this report, the investigators adheredto the Guide for the Care and Use of Laboratory
Animals as adopted and promulgated by the National Institutes of
Health publication 86-23.
Elsevier Science B.V.
SSDI 0165-0270(95)00019-4

somatodendritic
compartments
of neurons, and has
recently received attention due to its exquisite sensitivity to many influences. Studies have reported that
alterations in MAP-2 expression are of key importance
in differentiation,
growth and plasticity of neurons
(Wiche, 1989; Johnson and Jope, 1992). Moreover, it
has been demonstrated
that neurotoxicity
resulting
from ischemic brain damage and acrylamide poisoning
results in near-total loss of MAP-2 immunoreactivity
in
affected brain regions (Kitagawa et al., 1989; Chauhan
et al., 1993a,b). We hypothesize that the cytoskeletal
protein MAP-2 is vulnerable to seizure activity, and its
loss is a marker of seizure-induced brain damage.
Brain damage resulting from seizure activity is
brought on by exposure to soman (pinacolylmethylphosphonofluoridate)
and occurs as a result of irreversible inhibition of acetylcholinesterase (AChE) and
the ensuing cholinergic crisis. Once initiated, seizure
activity is sustained by an excitotoxic mechanism that is
mediated by the excitatory amino-acid
neurotransmitter glutamate (Olney, 1983; Braitman and Sparenborg, 1989; Sparenborg et al., 1992). This seizure-re-

G.P.H.

Baliough

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of Neuroscience

Merhotls

61 (1995)

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lated damage is bilaterally symmetrical and is most

severe in the thalamus, piriform and entorhinal cortices, amygdala and hippocampus. It has been reported
that thalamic damage stems primarily from vasogenic
edema associated with disruption of the blood-brain
barrier (Lemercier et al., 1983; Carpentier et al., 1990;
Petrali et al., 1991). Damage to the remaining structures is predominantly
excitotoxic in origin (Braitman
and Sparenborg, 1989; Sparenborg et al, 1992; McDonough and Shih, 1993).
In the present study, we examined the utility of
MAP-2 immunohistochemistry
as a marker of seizurerelated brain damage, and its suitability for quantitation using densitometric
and morphometric
image
analysis.

protect against the peripheral effects of soman and to

reduce mortality without affecting seizures. The following treatment regimen was employed. Thirty min prior
to 1.5 LD,, soman administration
(i.e., 100 pg/kg,
i.m.>, rats received an injection of pyridostigmine (0.13
mg/kg, i.m.1. This was followed by AMN (4 mg/kg,
i.m.) 5 min prior to soman and 2-PAM (25 mg/kg, i.m.)
l-2 min after soman administration.
At the end of the
6 h observation period, all surviving rats received supplementary injections of AMN (4 mg/kg, i.m.> and i.p.
injections of 5% glucose in isotonic saline (5 ml). The
latter was given to provide sustenance and prevent
dehydration. Only the soman-treated subjects which
showed evidence of sustained status epilepticus (> 1 h)
and remained alive for at least 24 h were considered to
have reached criterion.

2. Materials

2.4. Tissue processing

and Methods

2.1. Animals

Eighteen male Sprague-Dawley rats (CRL: CD[SD]BR), weighing 250-300 g, served as subjects. Animals
were housed individually in polycarbonate cages under
conditions of constant temperature (23C) and illumination (12 h light-dark cycle). Throughout the experiment, food and water were available ad libitum except
during the observation period beginning 1.5 h prior to
and ending 6 h following soman administration.
2.2. Surgeries
To document the development of seizures and status epilepticus, rats were anesthetized with sodium
pentobarbital
(50 mg/kg, i.p.) and chronically instrumented for electrocorticogram
(ECoG) recordings in
accordance with the procedure of Braitman
and
Sparenborg (1989). Following a postoperative delay of
11-12 days, unanesthetized rats were placed into individual Plexiglass cubicles (30 cm/side)
for E&G
recording and drug administration.
ECoG recordings
were monitored for 1.5 h prior to and 6 h following
soman administration;
additional recordings were obtained at 24 h from surviving animals.
2.3. Drug administration

Rats were randomly assigned to 2 treatment groups:

a control group (n = 6) which received either saline
only or quaternary drug treatment and a group which
received the quaternary drugs plus soman (n = 12).
Quaternary-drug-treated
rats received pyridostigmine,
atropine methylnitrate (AMN) and pralidoxime-Zchloride (2-PAM) in accordance with the regimen described by Sparenborg et al. (1992). These drugs which
do not cross the blood-brain barrier were employed to

Twenty-seven h after soman or saline/quaternary

drug treatment, rats were placed under deep pentobarbital anesthesia and euthanized via transcardial
perfusion with ice-cold 4% paraformaldehyde
in 0.1 M
phosphate buffer (PB, pH 7.4). Brains were immediately excised and longitudinally
divided into left and
right hemispheres. Alternate (left or right) hemispheres were postfixed by immersion in a second solution of ice-cold 4% paraformaldehyde
in 0.1 M PB for
4-6 h. They were subsequently placed into a solution
containing 30% sucrose in 0.1 M PB where they remained for 48 h. Sucrose saturated hemispheres were
coronally sectioned at 40 pm and serial sections were
collected directly onto gelatin-coated slides and dried
(for AChE and cresyl violet histochemi&)
or placed
into 0.1 M PB. The latter sections were subsequently
cryoprotected by immersion in a 0.1 M PB solution
containing 30% sucrose + 30% ethylene glycol (Watson
et al., 1986) and stored at -20C pending immunostaining. Contralateral hemispheres were paraffin processed, sectioned at 4 pm and stained with hematoxylin and eosin (H&E).
2.5. H&E,

AChE and cresyl violet histochemistry

Paraffin-processed brain sections were stained with

H&E to assess regional neuropathology. Damage was
scored on a scale of 0 to 4. To verify the central effects
of soman, frozen brain sections were stained for AChE
reactivity using the procedure of Karnovsky and Roots
(1964). Cresyl violet staining for Nissl substance was
performed according to the following modifications of
the procedure recommended
by Powers and Ctark
(1955): slides were immersed in distilled water (dH,O)
for 30 s, then into a solution containing 0.8% cresyl
violet acetate in 0.1 M acetate buffer for 7 min at 60C.
They were subsequently placed in dH,O for 30 s, and

G.P.H.

Ballough

et al. /Journal

of Neuroscience

destained in 70% ethanol for 2 min, 95% ethanol for 2

min, and 2.4% acetic acid in 95% ethanol for 1 min.
They were then dehydrated, cleared and mounted.
2.6. MAP-2 and GFAP immunohistochemistry

Immunohistochemistry
employed separate monoclonal antisera, raised in mice, against MAP-2 and
GFAP (Sigma, St Louis, MO, USA), and utilized the
avidin-biotin-peroxidase
method of Hsu et al. (1981);
reagents were obtained from Vector Labs (Burlingame,
CA, USA). Since the host species for both monoclonal
antisera were the same, the procedures for MAP-2 and
GFAP staining were identical with the exception of
primary antisera dilutions (see below). Free-floating
sections were removed from the cryoprotectant solution and rinsed several times in 0.1 M PB. To block
nonspecific staining, sections were incubated in 0.05 M
Tris buffer (TB, pH 7.6) containing 5% normal horse
serum and 0.1 M n,t,-lysine. Sections were incubated
for 18 h at 4C in primary antiserum diluted 1:4000 for
MAP-2 and 1:1600 for GFAP with 0.05 M TB containing 1% normal horse and 0.1% sodium azide. Primary
antibodies were omitted for MAP-2 and GFAP negative controls. Following rinsing in 0.05 M TB, sections
were incubated for 1.5 h at room temperature in biotinylated secondary antiserum (i.e., horse-anti-mouse,
diluted 1:200) containing 1% normal horse serum and
1% normal rat serum in 0.05 M TB. Sections were then
rinsed in several changes of 0.05 M TB and incubated
for 1 h in the avidin-biotin peroxidase complex (diluted
1:25 in 0.05 M TB). Again, sections were rinsed in
several changes 0.05 M TB. Sections were preincubated in 3,3-diaminobenzidine
tetrahydrochloride
(0.05% DAB, Sigma) for 5 min, at which time H,O,
was added; sections remained in the resultant solution
containing 0.048% DAB and 0.03% H,O, for 2.5 min.
The reaction was stopped by several rinses in 0.05 M
TB. Sections were then mounted onto gelatin-coated
slides, dried, dehydrated, cleared and mounted. For
MAP-2 staining, homologous brain sections from all
animals were processed simultaneously
in order to
minimize individual variations associated with tissue
handling and the histochemical procedure.

Methods

61 (1995)

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25

pixels lighter than 168 (on a scale of O-256, with 0

being black and 256 being white). This sensitivity
threshold was determined following examination of the
digitized gray images of control brain sections and
selecting the average value which provided the greatest
contrast between gray and white matter; the latter is
largely devoid of MAP-2 (Tucker et al., 1989; Wiche,
1989). The detection level was selected by increasing
the threshold until the size of the binary image accurately reflected non-immunoreactive
tissue seen both
in the microscope (25 X magnification) and on the digitized image. MAP-2 loss was represented as an increase in the percentage of detected pixels contained
within a mask of predefined area (i.e., area fraction or
density of detected pixels). Subsequently, the area fraction of undetected pixels was then calculated by subtraction. This subtraction method was preferred over a
direct detection procedure for determining the density
of MAP-2 immunostaining
when it was ascertained
that the former was less prone to observer error (i.e.,
inconsistency during the initial visual inspection and
contrast determination).
A circular field-delimiting
mask with a diameter of
20 pixels (i.e., 340 pm) was used to sample the density
of MAP-2 immunostaining
in various brain regions
known to be affected by soman-induced seizures (e.g.,
Lemercier et al., 1983). For each animal, 3 to 7 samples
were obtained from each of the following regions (regional nomenclature from Paxinos and Watson, 1982):
primary olfactory (piriform) cortex/endopiriform
nucleus (Pir/En),
entorhinal (Ent) and frontoparietal
(FrPaSS) cortices, hippocampal fields CA1 and CA3,
lateral (La), basolateral (BL), central (Ce> and posteriolateral cortical (PLCo) amygdaloid nuclei, and laterodorsal (LD), mediodorsal (MD) and ventromedial
(VM) thalamic nuclei. The number of samples taken
per region varied inversely with region size and homogeneity of MAP-2 staining. Adjacent subcortical structures served as landmarks for neuroanatomical
considerations.
In brain regions exhibiting necrosis, morphometry
was used to determine lesion size (i.e., cross-sectional
area); MAP-2-negative
areas were interactively outlined using a mouse, and area determinations automatically made by the image analysis system.

2.7. Image analysis

2.8. Statistical analysis

Densitometric
evaluation of MAP-2 immunohistochemistry was performed using a Quantimet 970 Image
Analysis System (Leica Cambridge, Cambridge, UK)
equipped with an Olympus BH-2 Biological Microscope (Olympus, Tokyo, Japan). To compare the DAB
of
reaction products (i.e., MAP-2 immunoreactivity)
homologous brain regions between animals, the sensitivity on this system was arbitrarily preset to detect

For each brain region/nucleus,

MAP-2 sample densities were averaged and a mean density obtained.
Respective means were grouped according to treatment and comparisons made between control and soman-treated groups using one-way analysis of variance.
P values < 0.05 were considered significant; values
where 0.05 < P < 0.10 were considered tendencies.
The relationship between soman-induced alterations

G. P. H. Ballough

ei al. /Journal

of Neuroscience

in MA P-2 immunostaining
densities and damage scores
obtain, ed from H&E-stained
contralateral hemispheres
was te.sted using correlation analysis. For each brain
region /nucleus of each rat, MAP-2 immunostaining

Methods

61 (1995)

23-32

density values were represented as a percent age of

appropriate control means; these values were subIsequently paired with respective H&E damage SC0res
and a Pearson correlation coefficient was obtain led.

2mm
Fig. 1. Photomicrographs (10 X ) of MAP-Zstained brain sections. (A) control, showing normal pattern of MAP-2 immunoreactivity.
soman treated: arrows indicate total MAP-2 loss in (B) Pir cortex, (0 LD thalamic nuclear group, and (D) En nucleus.

(B,C,D)

G. P.H. Bnllough

et nl. /Journal

of Neuroscience

3.1. MAP-2 immunostaining

Consistent with the findings of Bernhardt and Matus (1984), MAP-2 immunoreactivity
was localized in
neuronal perikarya, proximal dendrites and neuropil. It
was not observed in areas composed of white matter,
except in small numbers of scattered neurons. Moreover, negative control sections for which non-immunized serum was used showed no MAP-2 immunoreactivity.
Pronounced and clearly demarcated reductions in
MAP-2 immunostaining
were observed in the following
brain regions/nuclei
of rats which underwent somaninduced seizures (i.e., reached criterion; Fig. lB-D):
Pir cortex, En nucleus, FrPaSS (perirhinal area), Ent
cortex, PLCo amygdaloid nucleus, LD, LP, MD and
VM thalamic nuclei. The most severe lesions were
typified by an almost total absence of MAP-2 immunoreactivity. In the Pir cortex, lateral and ventral borders
of MAP-Znegative
lesions directly abutted, but did not
include, the primary olfactory cortical neurons of piriform cortical layer 2. Pir cortical lesions often extended
dorsally to include ventral portions of the FrPaSS
cortex (perirhinal area), and lesions were occasionally
seen in the dorsal FrPaSS cortex; these were not contiguous with the former. In many cases, when Pir
cortical damage extended to the ventral FrPaSS cortex,
a band of large projection neurons remained MAP-2
positive. Medially, the area of damage consistently

Cortical

27

23-32

3.2. MAP-2 image analysis

The results of MAP-2 densitometric image analysis

are summarized in Table 1. These results corroborate
our visual observations and indicate that soman-induced seizures significantly reduced MAP-2 immuno-

densities and H&E damage ratings

Pooled controls
Soman-treated
MAP-2
MAP-2
(l-100)
(l-100)

Soman-treated
MAP-2
(% of controls)

Soman-treated
H&E
(Ratinn O-4)

39.9 -f 17.6
63.3 + 14.4
59.3 * 22.6

2.71 + 0.64
not examined
f2.14 of:0.60

107.9 f 9.9
73.9 f 12.0
102.1 f 12.0
41.3 rf: 15.4

2.20 + 0.73
2.00 f 0.71
0.80 -f 0.20
1.67 + 0.56

areas

Pir (PO/En)
FrPaSS (PRh)
Ent
Amygdaloid

Hippocampal

96.9 * 2.1
95.3 * 1.4
80.4 + 8.5

38.7 * 17.1 = *
60.3 f 13.7
47.7 rt 18.2 *

80.1
86.7
83.9
83.9

86.4 f
64.0 f
85.6 *
34.6 +

nuclei

BL
La
Ce
PLCO

f 11.4
f 5.6
f 8.5
f 11.7

8.0
10.4 *
10.1
12.9 * *

Fields

CA1
CA3
Thalamic

61 (1995)

included the En nucleus. MAP-2 loss in the Pir cortex

and contiguous regions/nuclei
was surrounded by enhanced immunostaining
in the penumbra. In the amygdaloid nuclear complex, MAP-2 loss was consistently
observed in the PLCo amygdaloid nucleus only. In
many instances, extensive Pir cortical/En nuclear damage appeared to circumscribe the La, BL and medial
amygdaloid nuclei. However, in the most severe cases,
reduced MAP-2 staining was evident in these nuclei.
Reduced MAP-2 immunoreactivity
was never observed
in the Ce amygdaloid nucleus. Marked reductions in
MAP-2 immunoreactivity
were observed in various thalamic nuclei (e.g., LD, LP, MD and VM), but the most
marked loss was seen in the LD thalamic nucleus. No
reductions in MAP-2 immunoreactivity
were visually
discernible in any of the hippocampal fields; however,
MAP-2 loss was seen in the hilus of the dentate gyrus.
In general, MAP-2 loss was region and nucleus specific
with internuclear divisions well demarcated (e.g., between the LD and reticular thalamic nuclei, and delineation of the En nucleus). No differences between
control rats were discerned from microscopic examination of MAP-2 immunohistochemically
stained sections.

3. Results

Table 1
MAP-2 immunostaining
Brain region

Methods

82.0 f 7.9
74.1 k 15.2

72.9 f 13.2
54.6 + 14.4

88.9 f 16.1
73.7 + 19.4

0.86 f 0.14
0.86 rfr0.14

89.4 f 6.9
46.0 f 11.2
59.6 f 17.2

6.9 + 0.3 * *
18.3 f 8.2 *
19.3 * 4.8 *

7.7 f 0.33
39.7 f 17.8
32.4 i 8.0

2.80 f 0.73
3.00 f 0.78
1.80 f 0.58

nuclei

LD
MD
VM

MAP-2 immunoreactivities were calculated by subtraction (i.e., area fractions of undetected pixels). Values were obtained using a field-delimiting
mask (diameter = 0.34 mm), and represent the mean percentages of MAP-2-positive staining contained within the mask. Also depicted are mean
damage ratings (O-4) assessed on H&E stained brain sections from contralateral hemispheres.
Significantly different from respective controls
(P < 0.051, using AOV. Shows tendency to differ from respective controls (0.05 < P < 0.10).
l

28
Table 2
Cross-sectional
determinations)

G.P.H.

areas of MAP-Z

contiguous
region
Pir/En/PRh
Ent/PLCo
BL/La
LD/LP
MD/MDL
VM/G

(FrPaSS)

loss following

Ballough

soman

et al. /Journal

(morphometric

Atlas
coordinate

Circumscribed
area of MAP-2
loss (mm2)

Percentage
of total
area

B - 3.3
B-4.8
B-3.3
B-2.8
B-2.8
B-2.8

2.44 + 0.70
2.10+0.55
0.27kO.16
0.92+0.25
0.52+0.25
0.40+0.19

30.7
32.7
17.1
60.1
44.1
30.8

mm
mm
mm
mm
mm
mm

of Nruroscirme

Cross-sectional
areas of regional
necrosis (means + SEM), obtained
morphometricaily
from digitized
images of MAP-Zstained
brain
sections.
MAP-2-negative
areas were interactively
outlined
using a
mouse and area measurements
obtained
using calibrated
image
analysis. Atlas coordinates
were standardized
in order
to reduce
intrastructural
variability
in morphometric,
as well as densitometric
measurements.
Coronal
sections obtained
at these levels contained
the greatest number of affected brain regions.

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61 (1995)

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neuronal loss) and 4, severe (> 45% neuronal loss).

Assessments of neuronal loss were based on the presence of necrotic neurons and/or the absence of a
defined neuronal population; shrunken neurons were
considered the result of artifactual change. Damage to
the neuropil was progressively greater as ratings increased from mild to severe, and was characterized
by increasingly severe malacia and hyalinization typical
of necrosis. Using this method, it was determined that
the following brain regions averaged no damage to
minimal
damage: hippocampal fields CA1 and CA3,
and the Ce amygdaloid nucleus. The PLCo amygdaloid
nucleus and VM thalamic nucleus averaged minimal
to mild damage, and the Pir cortex/En nucleus, Ent
cortex, BL and La amygdaloid nuclei, and LD and MD
thalamic nuclei averaged mild to moderate damage.
Only the MD thalamic nucleus was determined to have
an averaged damage rating in the moderate to severe
range (i.e., 3.00 f 0.78). No evidence of damage was
seen in H & E-stained contralateral
hemispheres of
control rats, i.e., all regions/nuclei
received ratings of
0.

staining
densities
within
the following
brain
regions/nuclei:
Pir cortex/En
nucleus (- 60%), FrPaSS cortex (i.e., perirhinal area; -37%), PLCo amygdala ( - 58%), LD (- 92%) and VM (- 68%) thalamic
nuclei. (Differentiations
were not made between the
Pir cortex and En nucleus during densitometric sampling; however, it should be noted that the severity of
MAP-2 loss seen in the En nucleus was similar in
magnitude to that observed in the LD thalamic nucleus.) Statistical tendencies for reduced MAP-2 immunoreactivity
were seen in the Ent cortex (-41%),
La amygdala (-26%)
and MD thalamic nucleus
(-60%).
In the penumbra surrounding Pir cortical
necrosis, a slight increase in MAP-2 immunostaining
was observed (+3%); however, this was statistically
discernible (as a tendency) only when animal numbers
were increased by combining the present data with that
of soman-treated rats from a similar study (total n =
15). No statistically significant differences between
control and soman animals were measured in the BL,
La and Ce amygdaloid nuclei. In addition, no somaninduced changes in MAP-2 staining were observed in
hippocampal fields CA1 and CA3. Cross-sectional areas of regional necrosis, i.e., exhibiting profound loss
of MAP-2 immunostaining
are reported in Table 2.

A weak but significant inverse correlation (P < 0.05;

r = - 0.61) was found between MAP-2 immunostaining
density and H&E damage ratings in homologous brain
regions/nuclei
of contralateral
hemispheres. It was
also determined that the average variabilities (i.e., coefficients of variation) obtained using the MAP-2 densitometric image analysis and H&E
damage rating
methods were 0.61 and 0.64, respectively. H&E damage ratings and their corresponding MAP-2 immunostaining densities (mean + SEM) are as follows: 0 =
99.6k4.5;
l= 79.1 ,5.5; 2=73.5 k 11.6; 3= 61.4
+ 14.3; 4= 23.9 + 4.1. It is noteworthy that none of
the examined brain regions/nuclei
received mean
damage ratings above 3, despite the fact that their
contralateral
homologues often showed MAP-2 immunostaining
densities in the moderate and severe
ranges. For example, the LD thalamic nucleus received
a mean damage rating of 2.8 + 0.7, while the MAP-2
density was only 7.7 + 0.3 (i.e., 92% reduction).

3.3. Contralateral

3.5. Cresyl violet and AChE

damage ratings (H&E)

Evaluation
of H&E-stained
contralateral
hemispheres confirmed that soman-induced neuropathology
is, in general, bilaterally symmetrical. The results of
regional damage assessments on serial brain sections
(rated 0 to 4) are summarized in Table I. Damage
ratings are interpreted as follows: 0, no histologic lesion; 1, minimal damage (l-10%
neuronal loss); 2,
mild (ll-25%
neuronal loss); 3, moderate (26-45%

3.4. MAP-2
ratings

immunostaining

density vs. H&E

damage

Examination
of cresyl-violet-stained
brain sections
revealed no evidence of neuropathology
in animals
belonging to the control group (i.e., receiving either
saline or quaternary drug treatments). In seven out of
12 soman-treated rats that reached criterion, soman-induced seizures produced extensive damage in the Pir
cortex, En nucleus, Ent cortex, PLCo amygdatoid nucleus and various thalamic nuclei (e.g., LD, MD, VM,

G.P.H.

Ballough

et al. /Journal

of Neuroscience

late :ral posterior, ventrolateral

and posterior thalamic
nut .lear group). Damage was often observed in the
FrP aSS cortex (specifically, the perirhinal area), amyg-

Methods

61 (1995)

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29

dala (e.g., BL, La, basomedial and posteromedia ~1cortical amygdaloid nuclei) and hippocampal
field Is CA1
and CA3. Depending upon the severity, typic :al evi-

2mm
Fig. 2. Photomicrographs (10 X ) of cresyl-violet- and GFAP-stained brain sections. (A) cresyl violet control; (B) cresyl violet soman-treated:
arrows indicate chromatolysis in dorsal thalamic nuclear group and piriform cortex. (C) GFAP control; (D) GFAP soman-treated: arrows indicate
reactive astrocytosis in dentate gyrus, posterior thalamic nuclear group and amygdala. Stereotaxic coordinates for subhippocampal structures are
comparable in each of these photomicrographs (hippocampi not withstanding).

30

G.P.H.

Ballough

et al. /Journal

of Neuroscience

dence of neuropathology in cresyl-violet-stained

sections included chromatolysis, cell loss and tissue necrosis (Fig. 2A,B).
All soman-treated animals that reached criterion
exhibited a profound and global reduction in AChE
reactivity (not shown). A normal pattern and intensity
of AChE reactivity was observed in both saline and
quaternary drug-treated rats.
3.6. GFAP immunostaining

All brain regions showing histological evidence of

seizure-induced
damage (i.e., in cresyl-violet-stained
sections) also exhibited increased GFAP immunostaining characteristic of reactive astrocytosis, except in
areas of necrosis (Fig. 2C,D). Coincident with subnecrotic damage, reactive astrocytosis was especially
prevalent in the following brain regions/nuclei:
Pir
cortex, En nucleus, Ent cortex, BL, La and PLCo
amygdaloid nuclei, LD, LP and PO thalamic nuclei;
however, it was often the case that widespread necrosis
was present in layer 3 of the Pir cortex, as well as the
En nucleus, and the LD and LP thalamic nuclei. When
necrosis was present in the Pir cortex, GFAP immunoreactivity was especially high on the border (i.e.,
penumbra); this was not the case for the LD and LP
thalamic nuclei. Reactive astrocytosis was frequently
observed in the FrPaSS (cortical layer 21, Ce amygdaloid nucleus, fundus striati, dentate gyrus, subiculum
and hippocampal fields CAl, CA2 and CA3. No reactive astrocytosis was observed in control brain sections.
In general, GFAP immunostaining
provided clear evidence of subnecrotic neuropathology;
however, difficulties were encountered when interpreting these results in brain regions having multifocal necrosis.

4. Discussion
Seizure-related brain damage resulting from soman
intoxication
has been well characterized in previous
reports (Lemercier et al., 1983; McLeod et al., 1984;
Pazdernik et al., 1985; Carpentier et. al., 1990). These
studies have shown that damage resulting from somaninduced seizures is most severe in the Pir and Ent
cortices, amygdala, hippocampus and thalamus. Overall, the present findings are in good agreement with
these reports. The focus of the present study was to
evaluate the usefulness of MAP-2 immunostaining
as a
more accurate and reproducible alternative to standard
histochemical and immunohistochemical
methods for
assessing seizure-induced brain damage.
Our results indicate that all brain regions, in which
a significant reduction in MAP-2 immunostaining
(or
statistical tendency for reduced MAP-2
immuno-

h4erhods

61 (1995)

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staining) was measured showed strong evidence of neuronal damage with cresyl violet staining (i.e., vacuolization, chromatolysis, cell loss and/or widespread necrosis). These findings are consistent with previous reports
of reduced MAP-2 immunoreactivity
following neurotoxic insult (i.e., Kitagawa et al., 1989; Chauhan et al.,
1993a,b). The most severely damaged brain regions
(e.g., LD thalamic nucleus and Pir cortex) were often
totally devoid of MAP-2 immunostaining,
while less
severely damaged regions (e.g., VM thalamic nucleus)
showed significant reductions. In general, areas of
damage were marked by pronounced and clearly demarcated reductions in MAP-2 immunostaining.
It is speculated that the MAP-2 loss that occurs in
association with excitotoxic brain damage, stems from
proteolytic degradation by calcium activated proteases.
Soman-induced seizures produce brain damage primarily through an excitotoxic mechanism that is mediated
by the excitatory amino-acid neurotransmitter
glutamate. Integral to the excitotoxic mechanism is a prolonged elevation in intraneuronal
calcium concentration that leads to activation of various calcium-dependent enzymes (Schanne et al., 1979; Choi, 1987; Nicotera
et al., 1990). There is strong evidence that one such
enzyme, calpain I, is requisite to the development of
excitotoxicity (Siman and Noszek, 1988; Siman et al.,
1989). It has been reported that MAP-2 is one of the
most susceptible cytoskeletal proteins to calpain I-induced hydrolysis (Johnson et al., 1991; Johnson and
Jope, 1992). In light of this evidence, and considering
the cytoarchitectural compartmentalization
of MAP-2,
seizure-induced reductions in MAP-2 immunoreactivity
seen in the present study are likely an indication of a
partial or complete disruption of the neuronal cytoskeleton, resulting from a shrinkage of dendritic arborizations and/or neuronal loss. These effects are
necessarily restricted to neuronal soma and postsynaptic terminals; no information regarding presynaptic terminals can be inferred. Interestingly, an elevation
in MAP-2
immunoreactivity
was observed in the
penumbra surrounding piriform cortical damage; studies to confirm this observation and investigate the
underlying mechanism(s) are currently underway.
A significant inverse correlation was obtained between reduced MAP-2 immunostaining
density and
H&E scores in homologous brain regions/nuclei
of
contralateral
hemispheres. This supports the conclusion that the former is an index of seizure-related brain
damage. On the other hand, the strength of this correlation is less than compelling. The high extraneous
variability observed between these two neuropathology
assessment methods may have resulted from any number of factors. For example, it is likely that regionspecific brain damage resulting from soman exposure
did not exhibit perfect bilateral symmetry. However, it
is speculated that the greatest source of extraneous

G.P.H.

Ballough

et al. /Journal

of Neuroscience

variability in this correlation derives from the individual variabilities

associated with the methods themselves. Our results demonstrate that the coefficient of
variation was 5% lower and the average error (SEMI
was 8% lower with MAP-2 densitometric image analysis compared to the H&E damage rating method.
Conventional histological techniques, such as H&E
and cresyl violet, are very well adapted for qualitative
morphological
assessments of neurotoxic damage at
the cellular level. However, these methods are relatively inefficient in detecting the kinds of specific damage that characterize most neurotoxins (Switzer, 1991).
The present study demonstrates that mild to severe
brain damage resulting from soman-induced seizures
(i.e., H&E scores 2-4) was easier to detect, localize
and delineate using MAP-2 immunostaining
than with
H&E or cresyl violet histochemical staining. Furthermore, it was ascertained that the error associated with
MAP-2 densitometric image analysis was reduced compared to that of the classical neuropathological
assessment method (i.e., H&E damage ratings).
GFAP immunodetection,
as a marker of reactive
astrocytosis, has become a mainstay of modern neurotoxicology. It is well known that increased GFAP immunostaining is a sensitive, early index of neurotoxic
insult (e.g., OCallaghan
et al., 1992); however, this
procedure also possesses inherent inadequacies. As
was clearly observed in this study, and is well described
in the literature (e.g., Malhotra et al., 19901, enhanced
GFAP immunoreactivity
is not seen in the most severely
damaged (i.e., necrotic) areas. Although this is not
surprising, it does pose a considerable problem for
immunohistochemical
or enzyme-linked
immunosorbent assays (ELISA) of neurotoxicity that rely on
the expression of this or other antigens (e.g., c-Fos
transcription factor, heat-shock protein 70 or peripheral-type benzodiazepine-binding
sites). While the early
detection of GFAP and other proteins can be very
beneficial in pinpointing brain regions which may have
a high probability of sustaining damage (reversible and
irreversible) these markers are not definitive indices of
neuronal injury. In fact, it would seem counter-intuitive
to gauge irreversible excitotoxic or ischemic brain damage by antigen expression.
It is clear that no single histochemical or immunodetection method is sufficient to fully characterize excitotoxic brain damage. However, it is suggested that the
measurable loss of a sensitive neuron-specific marker,
such as MAP-2, is a more reliable indication of irreversible neuropathology than is antigen expression. The
present study demonstrates that MAP-2 densitometric
and morphometric
image analysis offers a direct and
powerful method through which to examine seizure
related brain damage, and it is the first to examine
seizure-induced excitotoxic alterations in MAP-2. As
evidence of the utility of this technique, this is also the

Methods

61 (1995)

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31

first report to identify LD as the most severely affected

thalamic nucleus in soman intoxication.

Acknowledgements
The authors wish to extend deep appreciation to
Ms. D. Tieman, Ms. T. Hamilton, Ms. D. Moltrup and
Mr. D. Carter for their invaluable technical assistance.
The authors are also indebted to Ms. R. Lee, CPT E.
Moore, Dr. J. McDonough, LTC R. Moeller and Dr.
A. Brimfield for insightful discussions. Thanks is also
extended to Dr. J. McDonough and Dr. J. Petrali for
reviewing the manuscript. This research was performed
during a National
Research Council Fellowship
awarded to G.P.H.B.

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