Immunohistochemical analysis of brain lesions using S100B and glial fibrillary acidic protein antibodies in arundic acid- (ONO-2506) treated stroke-prone spontaneously hypertensive rats.

Abstract

Stroke-prone spontaneously hypertensive rats (SHRSP) used as a model of essential hypertension cause a high incidence of brain stroke on the course of hypertension. Incidences and sizes of brain lesions are known to relate to the astrocyte activities. Therefore, relation between brain damage and the expression profile of the astrocytes was investigated with morphometric and immunohistochemical analyses using astrocyte marker antibodies of S100B and glial fibrillary acidic protein (GFAP) with or without arundic acid administration, a suppressor on the activation of astrocytes. Arundic acid extended the average life span of SHRSP. An increase in brain tissue weight was inhibited concomitant with a lower rate of gliosis/hemosiderin deposit/scarring in brain lesions. S100B- or GFAP-positive dot and filamentous structures were decreased in arundic acid-treated SHRSP, and this effect was most pronounced in the cerebral cortex, white matter, and pons, and less so in the hippocampus, diencephalon, midbrain, and cerebellum. Blood pressure decreased after administration of arundic acid in the high-dose group (100 mg/kg/day arundic acid), but not in the low-dose group (30 mg/kg/day). These data indicate that arundic acid can prevent hypertension-induced stroke, and may inhibit the enlargement of the stroke lesion by preventing the inflammatory changes caused by overproduction of the S100B protein in the astrocytes.

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BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - ORIGINAL ARTICLE
Immunohistochemical analysis of brain lesions using S100B
and glial fibrillary acidic protein antibodies in arundic
acid- (ONO-2506) treated stroke-prone spontaneously
hypertensive rats
Hideaki Higashino Æ Æ Atsuko Niwa Æ Æ Takao Satou Æ Æ
Yoshio Ohta Æ Æ Shigeo Hashimoto Æ Æ Masaki Tabuchi Æ Æ
Kana Ooshima
Received: 22 May 2009/Accepted: 17 July 2009/Published online: 6 August 2009
? The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract
(SHRSP) used as a model of essential hypertension cause a
high incidence of brain stroke on the course of hyperten-
sion. Incidences and sizes of brain lesions are known to
relate to the astrocyte activities. Therefore, relation
between brain damage and the expression profile of the
astrocytes wasinvestigated
immunohistochemical analyses using astrocyte marker
antibodies of S100B and glial fibrillary acidic protein
(GFAP) with or without arundic acid administration, a
suppressor on the activation of astrocytes. Arundic acid
extended the average life span of SHRSP. An increase in
brain tissue weight was inhibited concomitant with a lower
rate of gliosis/hemosiderin deposit/scarring in brain
lesions. S100B- or GFAP-positive dot and filamentous
structures were decreased in arundic acid-treated SHRSP,
and this effect was most pronounced in the cerebral cortex,
white matter, and pons, and less so in the hippocampus,
diencephalon, midbrain, and cerebellum. Blood pressure
decreased after administration of arundic acid in the
Stroke-prone spontaneously hypertensive rats
withmorphometricand
high-dose group (100 mg/kg/day arundic acid), but not in
the low-dose group (30 mg/kg/day). These data indicate
that arundic acid can prevent hypertension-induced stroke,
and may inhibit the enlargement of the stroke lesion by
preventing the inflammatory changes caused by overpro-
duction of the S100B protein in the astrocytes.
Keywords
S100B ? SHRSP
Arundic acid ? Astrocyte ? Brain ? GFAP ?
Introduction
Stroke-prone spontaneously hypertensive rats (SHRSP)
(Okamoto et al. 1974) developed from SHR (Okamoto and
Aoki 1963) by the selective crossbreeding are widely used
for the investigations of hypertension and stroke as a model
of human essential hypertension, which cause a high
incidence of brain stroke more than 90% concomitant with
myocardial fibrosis and arterionecrosis in the kidney during
stage of severe hypertension. S100B and glial fibrillary
acidic protein (GFAP) are considered to be relatively
astrocyte-specific and astrocyte-specific marker proteins,
respectively (Steiner et al. 2007). S100B specificity varies
between animal species (Boyes et al. 1986). This protein is
an acidic calcium-binding protein that is produced mainly
by astrocytes. Depending on its concentration, S100B has
two opposing effects, trophic and toxic (Rothermundt et al.
2003). At nanomolar concentrations, S100B stimulates
neurite outgrowth and enhances survival of neurons.
However, at micromolar concentrations, S100B stimulates
the expression of proinflammatory cytokines such as IL-6
and induces apoptosis. S100B protein, previously called
S100b protein, exists as a bb dimer in the cytoplasm of
astrocytes (Boyes et al. 1986). GFAP is an intermediate
H. Higashino (&) ? A. Niwa ? M. Tabuchi ? K. Ooshima
Department of Pharmacology, Kinki University School
of Medicine, Osaka-Sayama, Osaka 589-8511, Japan
e-mail: higasino@med.kindai.ac.jp
T. Satou
Department of Pathology, Kinki University School of Medicine,
Osaka-Sayama 589-8511, Japan
Y. Ohta
Department of Clinical Examinations, Kinki University
Nara Hospital, Nara 630-0293, Japan
S. Hashimoto
Department of Clinical Pathology, PL Hospital,
Osaka 584-8585, Japan
123
J Neural Transm (2009) 116:1209–1219
DOI 10.1007/s00702-009-0278-x

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filament protein found in reactive astrocytes (Reymond
et al. 1996; Ridet et al. 1996). The roles and precise
localization of each protein are not fully understood.
Up-regulation of S100B protein synthesis and leakage of
S100B from damaged astrocytes that express GFAP in the
glial scar can be induced by transient middle cerebral
artery occlusion in rats. These events lead to an increase in
the infarct volume (Matsui et al. 2002; Yasuda et al. 2004).
The Ono Pharmaceutical Co. Ltd (Osaka, Japan) has dis-
covered that arundic acid ((2R)-2-propyloctanoic acid,
ONO-2506) is a potent inhibitor of the production and
release of S100B protein from astrocytes (Shimoda et al.
1998). During development of this drug, it was discovered
that administration of arundic acid to experimental animals
reduced the expression of S100B protein and GFAP in
activated astrocytes, and inhibited the increase in the
infarct volume of brain injured animals (Tateishi et al.
2002; Mori et al. 2004).
Since brain injuries such as cerebral hemorrhage,
infarction, and subarachnoidal hemorrhage spontaneously
occur with high frequency in stroke-prone spontaneously
hypertensive rats (SHRSP) during the advanced stages of
hypertension we used these animals as a hypertension-
inducing brain lesion model. Our study examined the
effects of arundic acid on brain damage and the profiles of
S100B and GFAP, expressed by astrocytes. We used
morphometry and immunohistochemistry to examine brain
lesions on the course of hypertension.
Materials and methods
Animals and experimental design
Male SHRSP (Okamoto et al. 1974) and Wistar Kyoto rats
(WKY) aged 6 weeks and with normal blood pressure were
purchased from Kinki University Animal Center (Osaka,
Japan) and maintained under lighting control (light and
dark phases for 12 h each) at a temperature of 15 ± 2?C
and in 60% humidity. Arundic acid was provided by Ono
Pharmaceutical Co. Ltd (Osaka, Japan) as a candidate for
suppression of the activation of astrocytes (Tateishi et al.
2002). Three SHRSP groups of rats (n = 6 each) were
studied. Each group initially consisted of ten rats to survey
the process of cerebral lesions. Brains were obtained from
one rat in each group every 4 weeks for 12 weeks by
infusion of 4% paraformaldehyde buffer solution (pH 7.0–
7.5) into the cerebral artery under sodium pentobarbital
anesthesia. After the 12th week of the experiment, there
were no significant morphological findings in the brains of
rats in any group, and so the remaining six rats in each
group were morphologically investigated after that. No
arundic acid (control), 30 mg/kg/day arundic acid (low
dose) mixed with SP-2 chow (Funabashi Farm, Chiba,
Japan), and 100 mg/kg/day arundic acid (high dose) mixed
with SP-2 chow were given to three groups of rats,
respectively. Experiments were continued until 61 weeks
of age, at which time animals were killed if they had not
already died. Doses were determined by Ono Pharmaceu-
tical Co. Ltds information that dosages of 3, 10, and
30 mg/kg p.o. prevented S100B release into the cerebral
spinal fluid of rats during experimental cerebral ischemia.
WKY rats were fed with SP-2 chow without arundic acid.
Tap water was available ad libitum. Once a week, all rats
were weighed and their systolic arterial blood pressure was
measured with the tail-cuff method using a photoelectric
detector (UR-5000, Ueda, Tokyo, Japan). The brains from
rats that died spontaneously during the experimental period
and from rats that were killed with euthanasia using an
overdose of sodium pentobarbital (50 mg/kg i.p.) at the
61st week were collected for analysis. To avoid post-
mortem organ changes, brains were removed as soon as
possible after discovery of a dead animal during the regular
rounds that occurred four times a day. All animals used in
this experiment were handled with due care according to
the guidelines established by the Japanese Association for
Laboratory Animal Science, which complies with interna-
tional rules and policies.
Morphometric and immunohistochemical analyses
The brains were carefully resected and post-fixed in 4%
paraformaldehyde solution in 0.1 M phosphate-buffer (PB,
pH 7.0–7.5) for 12 h, and then rinsed with PB. After fixed
tissues were embedded in paraffin wax, 3-lm-thick serial
coronal sections were obtained and mounted on poly-L-
lysine-coated glass slides. For morphometric analysis,
hematoxylin–eosin staining was performed according to
standard protocols. For immunohistochemical analysis,
paraffin-embedded sections were dewaxed in xylene and
hydrated. S100B antigen was retrieved by heating sections
in 10 mM sodium citrate buffer (pH 6.0) in a microwave
oven for 20 min, and sections were then cooled in
de-ionized water. For GFAP staining, the sections were
incubated in buffer solution containing 0.1% trypsin for
5 min at room temperature. Endogenous peroxidase
activity was quenched by incubation of the sections for
5 min with 3% hydrogen peroxide. Sections were incu-
bated overnight at 4?C in primary antibodies for S100
rabbit polyclonal antibody (Code No. Z 0311; 1:800;
DAKO, Glostrup, Denmark), or GFAP rabbit polyclonal
antibody (Code No. Z 0334; 1:500; DAKO). The speci-
ficity of the S100B antibody was determined by western
blotting of purified human recombinant S100 protein (Ilg
et al. 1996). The antibody labels S100B strongly, S100A1
weakly, and S100A6 very weakly. The anti-GFAP
1210 H. Higashino et al.
123

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antibody does not cross-react in immunocytochemical
assays with vimentin or other intermediate filament pro-
teins, as described in a previous report (Rettig et al. 1986).
Staining was detected using biotin-labeled anti-rabbit sec-
ondary antibodies and streptavidin conjugated to horse-
radishperoxidase(DAKO
Cytomation, Carpinteria, CA, USA), according to the
manufacturer’s instructions. Reaction products were visu-
alized using diaminobenzidine (Sigma-Aldrich Co., St.
Louis, MO, USA). Nuclei were stained with hematoxylin.
Rabbit immunoglobulin (DAKO, Glostrup, Denmark) and
secondary antibodies were used for negative controls.
Sections were examined with microscope (ECLIPSE E800;
Nikon, Tokyo, Japan) using computer-assisted image
analysis software (Mac Scope Version 2.5 software; Mitani
Co., Fukui, Japan). The areas of the tissue examined by
microscopewere136 lm 9 96 lm
els) = 1.3 9 104lm2
(3.42 9 105pixels)
investigation, and 200 lm 9 150 lm (1,024 9 768 pix-
els) = 3.0 9 104lm2
(7.86 9 105pixels)
investigation. Depending on the size of the area, four to
five areas from each specimen were observed by micro-
scope. Average values were calculated using the data
obtained from 22–42 areas in SHRSP, and 15–21 areas in
WKY. Color-staineddots
(0.38 lm2) that were associated with antibody staining
were counted as the least fragment of astrocytes, and the
total dot area in the specimen was calculated using com-
puter-assisted image analysis software, and compared
among the four groups of rats.
LSAB2 System; DAKO
(696 9 492 pix-
for S100B
forGFAP
greaterthan 10 pixels
Statistical analysis
All results were expressed as the mean ± SEM. Compar-
isons between means of groups were made using a one-way
analysis of variance (ANOVA) and a Scheffe multiple
comparisons test, or a two-way ANOVA test for the
comparison between S100B or GFAP immunostaining
specimens. Survival was analyzed using standard Kaplan–
Meier analysis with a Mantel–Cox log-rank test. For all
tests, differences were considered statistically significant at
a value of p\0.05.
Results
Effects of arundic acid on physical indices
The blood pressure levels of the three SHRSP groups were
the same at the beginning of the experiment. In the high-
dose group (100 mg/kg/day arundic acid), the rise in blood
pressure was significantly decreased after the eighth week
until around the 36th week of the experiment. There were
no differences in the blood pressure of the low dose
(30 mg/kg/day arundic acid) or control groups of SHRSP
from the beginning of the experiment until their death at
week 36 (Fig. 1). No significant differences were noted in
body weight or heart rate during the experiment among the
SHRSP groups as follows: for example, at 24 weeks when
all rats were still alive and in good condition, body weight
was 224.5 ± 2.7 g in the SHRSP control group, 224.0 ±
3.4 g in the SHRSP low-dose group, and 234.2 ± 2.7 g in
the SHRSP high-dose group. Heart rate at this time was
451 ± 6 bpm in the SHRSP control group, 410 ± 5 bpm
Fig. 1 Effects of arundic acid on blood pressure in SHRSP and WKY
rats. Blood pressure was measured by the tail-cuff method. Each
group consisted of six rats. Filled circle SHRSP: control, filled
triangle SHRSP: 30 mg/kg/day arundic acid, filled square SHRSP:
100 mg/kg/day arundic acid, open circle WKY. * and **indicate
significant differences of p\0.05 and p\0.01, respectively,
compared with control SHRSP
Fig. 2 Comparison of survival rates in the SHRSP and WKY groups
with or without oral administration of arundic acid. Each group
consisted of six rats. Time 0 is the start point of the experiments.
Filled circle SHRSP: control, filled triangle SHRSP: 30 mg/kg/day
arundic acid, filled square SHRSP: 100 mg/kg/day arundic acid, filled
circle WKY. *indicates significant difference of p\0.05 compared
with control SHRSP
Immunohistochemical analysis of brain lesions1211
123

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in the SHRSP low-dose group, and 418 ± 5 bpm in the
SHRSP high-dose group.
Survival rates and average life spans
of the SHRSP groups
The survival rate in the control SHRSP group rapidly
declined after the 30th week, and the average life span
(273.2 ± 10.5 days) was the shortest among the groups
(Fig. 2). The low-dose group showed an upper shift in
the survival rate, and the average life span ([392.3 ±
18.1 days) was longer compared to control. The survival
rate of the high-dose group showed a strong upper shift
compared with the low-dose group, and the average life
span ([473.3 ± 7.4 days) was much longer compared with
the control and low-dose groups. WKY rats given no
arundic acid lived more than 483 days, longer than the end
of the experimental period (Table 1).
Cerebral morphometric findings, incidences
of morphological changes, and brain wet weights
at the time of natural death or killing at the 61st week
in SHRSP groups
Cerebral autopsy was done at the time of natural death or
killing at the 61st week in SHRSP (Table 1). The SHRSP
control group showed five cerebral thromboses and one
hemorrhage in a total of six rats. On the other hand, the
low- and high-dose groups showed three cerebral throm-
boses, one subarachnoidal hemorrhage, and three heart
failures in a total of six rats, and one cerebral thrombosis,
two subarachnoidal hemorrhages, and three survivors at
61 weeks in the six total rats, respectively. On the other
hand, neither death nor brain lesions were noted in WKY
animals. As shown in Table 2, brain softening, bleeding,
angionecrosis, and gliosis/hemosiderin deposit/scarring
changes were observed in 5/6, 2/6, 5/6, and 6/6 control
SHRSP, respectively. Thus, these lesions occurred in
almost all rats. All abnormal morphological changes were
decreased by the administration of arundic acid as shown in
Fig. 3. In particular, gliosis/hemosiderin deposit/scarring
changes were completely eliminated in high-dose animals
(p\0.05 vs. control SHRSP). No abnormal morphological
changes were found in WKY rats. Brain tissue extending
from the cerebrum to the medulla oblongata was isolated
from the skull and weighed immediately after autopsy
(Table 3). In the low- and high-dose groups, the brain
tissue weight and the ratio of brain/body weight were
significantly decreased compared with control SHRSP, and
were similar to that seen in WKY rats. However, these
preferable effects caused by the administration of arundic
acid were not observed in the heart and kidney (Fig. 3).
Table 1 Cerebral abnormal findings at the time of natural death or killing at the age of 69 weeks in SHRSP and WKY, and average lifespans
with or without arundic acid medication
Rats/treatments Cerebral findings at autopsy in six cases Average life span (days)
SHRSP/control (n = 6)Five cases of cerebral thrombosis, and one case of hemorrhage 273 ± 10.5
SHRSP/30 mg/kg/day
arundic acid (n = 6)
Three cases of cerebral thrombosis, and one case of subarachnoidal
hemorrhage (cf. three cases of heart failures with edema)
392.3 ± 18.1*
SHRSP/100 mg/kg/day
arundic acid (n = 6)
One case of cerebral thrombosis, and two cases of subarachnoidal
hemorrhage (cf. 39 survives)
[473.3 ± 7.4*
WKY No brain lesion (cf. six cases of survives)
[483*
* Represents the significant differences at the level of p\0.05 compared with SHRSP control group
Table 2 Incidences of morphological changes in the brain at the time of natural death or killing at the age of 69 weeks in SHRSP with or
without arundic acid medication
Rats/treatmentsMorphological findings
Softening BleedingAngionecrosis Gliosis/hemosiderin
deposit/scaring
SHRSP/control 5/62/6 5/66/6
SHRSP/30 mg/kg/day arundic acid 2/61/61/62/6
0/6*c
0/6*c
SHRSP/100 mg/kg/day arundic acid2/6
0/6*a
1/6 2/6
0/6*b
WKY0/6
*a, *b, and *crepresent the significant differences at the level of p\0.05 compared with each parameter of SHRSP control group
1212 H. Higashino et al.
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S100B and GFAP immunohistochemical analyses
Cerebral cortex, white matter, and hippocampus
In the cerebral cortex and white matter of control SHRSP,
S100B antibody-reactive dot and filamentous structures
(panels b in Figs. 4, 5) were diffusedly distributed, and the
sum of the areas of the particles was markedly increased or
tended to increase compared with WKY brains (panels a in
Figs. 4, 5). Although the total area of dot and filamentous
structures was less in the low-dose SHRSP group com-
pared with control in the cortex, no difference was
observed between the low- and high-dose groups of
SHRSP (panels c, d in Fig. 4; Fig. 6 top left). In the white
matter, the administration of arundic acid inhibited the
expression of the particles in a dose-dependent manner
(panels c, d in Fig. 5; Fig. 6 row 2, left). The shapes of the
dots immunostained by the S100B antibody were consis-
tent with astrocytic morphology in both of the tissues. In
addition, many round particles, small dots, and circles that
appeared to be blood vessels were also observed. The sum
of the area occupied by the dots encompassed more than
15% of the total specimen area in the cortex. GFAP-
reactive dots and filamentous structures in the cerebral
cortex and white matter of SHRSP were markedly
increased compared with WKY brains, and the abundance
of these GFAP-reactive structures was reduced by arundic
acid in a dose-dependent manner (panels e–h in Fig. 4;
Fig. 6 top right; panels e–h in Fig. 5; Fig. 6 row 2, right).
The shapes of the dots immunostained by the GFAP
antibody showed spider-like astrocytic morphology in both
of the tissues.
Fig. 3 Histological findings of brain, heart, and kidney tissues
stained by hematoxylin–eosin (HE) in SHRSP. a Cerebrovascular
lesions (softening, hemorrhage) in the cortex of the brain were seen in
untreated SHRP. Findings of gliosis/hemosiderin deposit/scaring were
often observed in the cortex and/or white matter close to the stroke
lesions (not shown here). b No cerebrovascular lesions were found in
arundic acid-treated SHRSP, especially in high doses. c Myocardial
necrosis and fibrosis in the surrounding myocardium were noted in
arundic acid-treated SHRSP. d Angionecrosis, collapse of the
glomerulus were noted, and distal and proximal tubules were
enlarged by containing proteinuria and their epithelium was atrophic
and flattened in arundic acid-treated SHRSP. Scale bar equals 50 lm
Table 3 Comparison of brain and body weights, and rates of brain/body weight (%) at the autopsy in WKY and SHRSP groups
Rats/treatmentsBrain weights (g) Body weights (g) Brain/body weights (%)
SHRSP/control 3.33 ± 0.21
2.47 ± 0.04*a
2.53 ± 0.11*a
2.11 ± 0.08*a
244.3 ± 16.9
325.0 ± 36.4*b
322.2 ± 8.8*b
436.4 ± 3.5*b
1.40 ± 0.12
0.81 ± 0.90*c
0.79 ± 0.05*c
0.48 ± 0.01*c
SHRSP/30 mg/kg/day arundic acid
SHRSP/100 mg/kg/day arundic acid
WKY
*a, *b, and *crepresent the significant differences at the level of p\0.001 compared with each parameter of SHRSP control group
Immunohistochemical analysis of brain lesions1213
123