Brain Research 1040 (2005) 55 – 63
www.elsevier.com/locate/brainres
Research report
Effect of chronic intermittent restraint stress on hippocampal expression
of marker proteins for synaptic plasticity and progenitor cell
proliferation in rats
Holger RosenbrockT, Eliza Koros, Anita Bloching, Jana Podhorna1, Franco Borsini2
Department of CNS research, Boehringer-Ingelheim Pharma GmbH and Co KG, Birkendorfer Strasse 65, D-88397 Biberach, Germany
Accepted 14 January 2005
Available online 11 March 2005
Abstract
Chronic restraint stress may change hippocampal mRNA levels of markers for synaptic plasticity such as synaptophysin, growthassociated protein 43 (GAP-43), and brain-derived neurotrophic factor (BDNF). In order to examine the relation between that stressor and
those biochemical markers on protein level as well as the Ki-67 protein, a marker of progenitor cell proliferation, we subjected rats to
chronic intermittent restraint stress for 6 h per day for 14 days excluding the weekends. This stress intensity caused a significant increase
in adrenal gland weight and decrease in body weight gain. However, we did not find significant alteration of protein expression levels for
synaptophysin, GAP-43, and BDNF by using Western blot analysis. Unlike these findings, the hippocampal protein expression of Ki-67
was significantly reduced by using both Western blot and immunohistochemical analyses. This reduction of Ki-67 expression in
chronically stressed rats was correlated with increased adrenal gland weight and decreased body weight gain. All marker proteins used did
not show any changes of hippocampal expression level after a single restraint stress session of 3 h. In conclusion, chronic intermittent
restraint stress caused changes in the physiological stress response in rats, and a decrease of hippocampal progenitor cells using the Ki-67
protein as marker which indicates a suppression of adult neurogenesis. The results might contribute to understand the relationship between
stress and cellular neurobiology of depression, since chronic antidepressant treatment have been shown to increase adult neurogenesis in
the rat hippocampus.
D 2005 Elsevier B.V. All rights reserved.
Theme: Neural basis of behavior
Topic: Stress
Keywords: Restraint stress; Hippocampus; Neurogenesis; Ki-67; BDNF
1. Introduction
Preclinical studies have shown that acute and chronic
stress, dependent on the species and the stressor (immobilization, restraint, psychosocial conflict), produces structural
T Corresponding author. Fax: +49 7351 54 98928.
E-mail address: [email protected]
(H. Rosenbrock).
1
Current address: H. Lundbeck A/S, DK-2500 Valby, Denmark.
2
Current address: Sigma-Tau S.p.A., I-00040 Pomezia, Italy.
0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2005.01.065
and functional changes in the brain, especially in the
hippocampus [17,24]. The hippocampal structural changes,
after chronic stress, include atrophy of apical dendrites of
CA3 pyramidal neurons which can be prevented by
antidepressant or benzodiazepine treatment [21,38]. The
functional changes may include decreased mRNA levels of
specific proteins linked to neural and synaptic plasticity,
such as growth-associated protein 43 (GAP-43), brainderived neurotrophic factor (BDNF), and synaptophysin
[18,25,36]. Such structural and functional changes have also
been shown in subjects suffering from psychiatric illnesses,
like major depression and bipolar disorders [5,10,13,28,32].
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H. Rosenbrock et al. / Brain Research 1040 (2005) 55–63
Therefore, such findings may indicate a link between stress
burden, synaptic plasticity, and mental disorders.
Another feature of stress is the regulation of adult
neurogenesis in animals [9,11,14]. The process of neurogenesis occurs mainly in the subventricular zone adjacent to
the lateral ventricles and in the subgranular zone of the
dentate gyrus of the hippocampus where the progenitor cells
are located. In the dentate gyrus, after proliferation and
differentiation, the new cells become incorporated into the
granule cell layer and develop the morphological characteristics of mature granule neurons able to form functional
synapses [37]. It has been shown in the rat that adrenal
steroid hormones, chronic psychosocial stress, inescapable
foot-shock stress, and chronic restraint stress lead to a
suppression of this adult hippocampal neurogenesis, measured by the BrdU labeling method [6,8,22,27]. Furthermore,
antidepressant treatment increases neurogenesis in animals
[23], and depressed subjects exhibit a reduction of hippocampal volume [5,32]. Thus, it was hypothesized that the
waning and waxing of adult hippocampal neurogenesis are
important factors in the precipitation of and the recovery
from episodes of depression [14].
Since none of the studies about the effect of chronic and
acute restraint stress on hippocampal gene expression of the
mentioned marker proteins (synaptophysin, GAP-43, and
BDNF) provided results on their protein level, in this study,
we aimed analyzing their protein expression level after
restraint stress by using the Western blotting approach.
Additionally, we also assessed the Ki-67 protein as a
proliferation marker by using both methods, Western
blotting and (double) immunofluorescence staining. The
nuclear protein Ki-67, which is expressed in all phases of
the cell cycle except the resting phase, can be used for
determination of progenitor cells of adult neurogenesis,
since it was shown that this protein mimicks the expression
pattern of BrdU-labeled cells when examined soon after the
BrdU injection [15]. This was corroborated by a recent
study showing increased Ki-67-positive cells in the hippocampus after chronic antidepressant treatment similar to
what was found by using the BrdU labeling method [23,30].
GnostR) from Merck KGaA (Darmstadt, Germany). Ketamin
(KetanestR) was purchased from Albrecht GmbH and Co KG
(Aulendorf, Germany) and Xylazin (RompunR) from Bayer
AG (Leverkusen, Germany). All other chemicals were
purchased in highest quality from common suppliers.
For Western blotting, primary and secondary antibodies
were diluted in tris buffered saline (TBS) containing
0.1% (w/v) Tween-20, 2% (w/v) skim milk powder and
0.1% (w/v) sodium azide. For immunohistochemistry, primary and secondary antibodies were diluted in phosphatebuffered saline (PBS) containing 0.1% (w/v) Triton X-100,
1% (w/v) BSA and 0.1% (w/v) sodium azide. The following
primary antibodies were used: polyclonal rabbit anti-BDNF
(dilution 1:500 for Western blotting, Santa Cruz Biotechnology, Heidelberg, Germany); polyclonal rabbit anti-glial
fibrillary acidic protein (GFAP) (1:2000 for immunohistochemistry; Sigma-Aldrich Chemie GmbH, Taufkirchen,
Germany); polyclonal rabbit anti-GAP-43 (1:10,000 for
Western blotting, Chemicon Int., Hofheim, Germany);
polyclonal rabbit anti-Ki-67 (1:200 for immunohistochemistry, Biotrend GmbH, Cologne, Germany); monoclonal
mouse anti-Ki-67 (1:100 for Western blotting, 1:200 for
immunohistochemistry, BD Biosciences, Heidelberg, Germany); monoclonal mouse anti-Cd11b (clone Ox-42, 1:500
for immunohistochemistry, BD Biosciences); monoclonal
mouse anti-synaptophysin (1:5000 for Western blotting,
Sigma-Aldrich Chemie GmbH); monoclonal mouse antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(1:4000 for Western blotting, Biotrend GmbH); monoclonal
mouse anti-neuronal nuclear protein (NeuN) (1:100 for
immunohistochemistry, Chemicon Int.). As secondary antibodies for immunohistochemistry, goat-anti-mouse (GAM)IgG conjugated with rhodamine red-Xk (1:600, Dianova,
Hamburg, Germany) and goat-anti-rabbit (GAR)-IgG conjugated with CY-2k (1:600, Dianova) were applied. As
secondary antibodies for Western blotting, biotinylated
GAR-IgG (1:1000, Vector Laboratories, Burlingame, CA,
USA), biotinylated horse-anti-goat (HAG)-IgG, and biotinylated horse-anti-mouse (HAM)-IgG (rat-adsorbed,
1:1000, Vector Laboratories) were used.
2. Materials and methods
2.2. Animals, restraint stress procedure, and preparation
of brain tissue
2.1. Materials and antibodies
Amersham Biosciences (Freiburg, Germany) provided the
enhanced chemiluminescence (ECL)-Plus detection system
and Millipore GmbH (Schwalbach, Germany) the PVDF
membrane Immobilon P. Bovine serum albumin (BSA) (IgGfree, protease-free) was from Jackson ImmunoResearch Lab.
(West Grove, PA, USA), the SuperFrostR-slides were from
Carl Roth AG (Karlsruhe, Germany), mowiol was from
Calbiochem-Novabiochem (Schwalbach, Germany), the
cryomedium Tissue-TekR from Sakura (Zoeterwoude, the
Netherlands), and streptavidin-peroxidase solution (Tissue-
Adult male rats Sprague–Dawley, weighing 300–350 g at
the beginning of the stress procedure were used (Harlan
Bioservice for science GmbH, Walsrode, Germany). They
were housed individually with controlled temperature (22 F
1 8C) and light/dark cycle (light from 6 AM to 6 PM).
Standard rodent chow and tap water were available ad
libitum. After arrival, rats were left in the cages for 10 days
in order to adjust to new environment until the beginning of
experiment. During these 10 days, rats were daily habituated
to experimental handling. All animal experiments were conducted in accordance with the European Union guidelines
(European Communities Council Directive 86/609/ECC)
H. Rosenbrock et al. / Brain Research 1040 (2005) 55–63
on the use of laboratory animals. This study was approved by
the Ethical Committee of the regional council of Upper
Swabia (Tqbingen, Germany).
Rats were exposed to restraint stress between 8 AM and
2 PM in the experimental room. Restraint stress was
performed using a rodent restrainer made in Plexiglas
(Harvard Apparatus GmbH, March-Hugstetten, Germany)
that allowed for a close fit to rats. For chronic restraint
stress, rats were put into the restrainers for 6 h every day for
14 days (excluding the weekends), starting on Monday and
were killed 1 day after the last session. Since the weekends
were excluded during the chronic stress procedure, this
treatment is described as chronic intermittent restraint stress.
Weight gain was monitored on a 3-day basis throughout the
experiment and, upon termination, adrenal glands were
removed and weighed (see below for anesthesia condition).
For the acute restraint stress, rats were put into the
restrainers for 3 h (8 AM – 11 AM), and were killed immediately after the session, since it was shown that hippocampal BDNF protein expression was altered at least after
3 h of immobilization stress [35]. Control animals were
handled daily and were killed at the same time as the
stressed ones. Animals were deeply anesthetized by using
intraperitoneal administration of a mixture of ketamin
(70 mg/kg) and xylazin (6 mg/kg). Ten minutes after
transcardial perfusion with Ringer solution, brains and
adrenal glands were immediately removed and brains were
cut into two halves sagittally. For immunohistochemistry,
one half was frozen in sagittal position in Tissue TekR
cryomedium on cork supports by immersion in isopentane
precooled in liquid nitrogen and stored at 70 8C until use.
For Western blotting, the hippocampus of the other brain
half was removed, frozen in liquid nitrogen, and stored at
70 8C until use.
2.3. Homogenization, SDS-gel electrophoresis, and
Western blotting
Homogenization procedure was carried out on ice or at
4 8C. Rat hippocampi were homogenized in approximately
5 volumes of 100 mM Tris/HCl (pH 7.4) containing 150 mM
NaCl, 1.0% (w/v) Triton X-100, 0.1% (w/v) SDS, 5 mM
EDTA, 1.0 mM PMSF and 10 Ag/ml aprotinin by using a
sonifier (4–6 bursts for 1 s each, Branson Cell Disrupter,
Dunbury, CC, USA). Then, protein concentration was
determined according to Bradford [4] with BSA as standard.
SDS-gel electrophoresis was carried out under reducing
conditions using 7.5% (w/v), 10%, or 14% (w/v) polyacrylamide gels [19] using Mini-Protean 3 cell system (Bio-Rad
Laboratories GmbH, Mqnchen, Germany). The separated
proteins (20–50 Ag homogenate per lane) were electrophoretically transferred to PVDF membrane, and the membrane was blocked with TBS containing 5% (w/v) skim
milk powder and 0.1% (w/v) NaN3 for 1 h at room
temperature (RT). After 3 5 min washing with TBS
containing 0.1% (w/v) Tween-20 (TBS/Tween), the mem-
57
brane was probed with primary antibodies for 1 h at room
temperature (RT) or overnight at 4 8C. Then, after washing,
the membrane was incubated with the biotinylated secondary antibodies for 1.5 h at RT. Subsequently, after washing,
the membrane was incubated with streptavidin–peroxidase
diluted 1:1000 in TBS/Tween containing 2% (w/v) skim
milk powder for 20 min at RT. After once washing with
TBS/Tween and subsequent washing with TBS, peroxidase
activity was visualized with the ECL-Plus detection system
according to the manufacturer’s procedure and recorded
using the ImageMaster VDS-CL system including a CCD
camera (Amersham Biosciences). Quantification of optical
density of bands was carried out using ImageMaster 1DElite software (version 3.01, Amersham Biosciences). Since
multiple gels were analyzed for quantification of marker
proteins, immunopositive bands of the control protein
GAPDH were used for normalization of optical densities
of marker protein bands of each probe. Data are expressed
as normalized optical density. Negative controls were
performed by omitting the first antibody and did not show
any signals.
2.4. (Double) immunofluorescence labeling
For analyzing the proteins of interest in the hippocampus,
consecutive slices from 0.5 mm to 1.0 mm lateral (according
to Paxinos and Watson [26]) were used. Frozen, TissueTekR
embedded brain halves were cut parasagittally in 12 Am thin
sections ( 18 8C chamber temperature, 14 8C object
holder temperature) and removed from the knife by attachment to SuperFrostR slides. After 1 h drying at RT and
subsequent fixation in acetone/methanol (1:1, v/v) for
15 min at 20 8C, the brain slices were dried at RT and
then stored at 20 8C until use. These frozen brain slices
were rehydrated in PBS 3 5 min at RT. Unspecific protein
binding sites were blocked with PBS containing 3% (w/v)
BSA, 0.1% (w/v) NaN3 for 1 h at RT. Then, brain sections
were directly incubated with primary antibodies over night
at 4 8C. After 3 5 min washing with PBS, the slices were
incubated with secondary antibodies for 2 h at RT to
visualize the binding sites of the primary antibodies. Then,
brain slices were rinsed for 15 min in triple changes of
PBS, subsequently washed with water and embedded in
0.2 mM Tris/HCl (pH 8.5) containing 12% (w/v) mowiol
and 1.5% 1,4-diazoabicyclo[2.2.2]octane as anti-fading
agent. Fluorescent signals were analyzed using a fluorescence microscope (Axioplan 2 Imaging, Carl Zeiss
Gfttingen, Gfttigen, Germany) with a CCD camera
(AxioCam s/w, Carl Zeiss Gfttingen) and AxioVision 2.05
software to grasp and handle the digitized images. Double
fluorescence staining was analyzed using a confocal laser
scanning microscope (Leica TCS SP, Leica Biosystems,
Heidelberg, Germany). Quantification of digitized images
was carried out using Halcon imaging software (MVTech
Software GmbH, Mqnchen, Germany). For quantification
of Ki-67-immunopositive cells, three slices per animal with
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H. Rosenbrock et al. / Brain Research 1040 (2005) 55–63
24 Am space in-between were used in order to have a
reliable estimation of cell numbers per region of interest
which represents the subgranular zone and the hilus of the
dentate gyrus. The median of these three values per animal
was used for further statistical analysis. Negative controls
were performed by omitting the primary antibodies and did
not show any fluorescent signals.
2.5. Statistical analysis
Comparisons between control and stressed group were
performed by two-tailed unpaired Student’s t test, if the
values were parametrically distributed. In the case of nonparametrical distributed values, Wilcoxon rank sum test
was used. Correlation analyses were assessed with Spearman’s correlation coefficient (r s). P b 0.05 was considered
as significant.
3. Results
3.1. Impact of chronic intermittent restraint stress on body
and adrenal weights
To confirm the physiological efficacy of the stress procedure, we measured body and adrenal weights. The control
and stressed group of animals did not differ significantly in
body weight at the beginning of the experiment (324 F 12 g
and 321 F 9 g for the control and experimental group, respectively). Chronic intermittent restraint stress for 6 h per
day for 14 days radically reduced the weight gain, expressed
as the difference between the end and the beginning of the
experiment ( P b 0.001, Table 1). In the same animals, the
relative weights of the left adrenal gland was found to be
increased ( P b 0.05, Table 1). The relative weight of the
right adrenal gland was not significantly increased ( P N 0.1).
3.2. Western blot analysis of expression of marker proteins
for synaptic plasticity and proliferation in the rat
hippocampus after restraint stress
Fig. 1 shows representative immunoblots of used marker
proteins (BDNF, synaptophysin, GAP-43, Ki-67). This also
illustrates the high specificity of the antibodies used and,
Table 1
Body weight gain and relative weight of adrenal glands after chronic
intermittent restraint stress
Group
Control
Stress
Body weight gain (g)
55.6 F 4.7
3.0 F 4.1**
Adrenal weight (mg)/body weight (g)
Left
Right
0.078 F 0.03
0.088 F 0.03*
0.073 F 0.03
0.083 F 0.05
Rats were exposed to restraint stress for 6 h per day for 14 days. Data
expressed as mean F SEM, n = 7–8. **P b 0.001, *P b 0.05 vs. control
(Student’s t test).
Fig. 1. Representative immunoblots show the expression level of used
marker proteins in the hippocampal homogenate of control (C) and
chronic restraint (6 h per day for 14 days) rats (S). GAPDH was used as
loading control.
therefore, the usefulness of the Western blotting approach
for quantitative analysis of various proteins in rat hippocampal homogenates. The protein expression level of the
so-called house-keeping gene GAPDH was used for normalization of optical densities of marker proteins, since its
expression was not changed after stress treatment. This
was proven in preliminary Western blotting analyses of
GAPDH-immunopositive bands from control and stressed
animals using exact the same protein load per probe which
was checked by ponceau S protein staining on the PVDF
membrane (data not shown).
Chronic intermittent restraint stress for 6 h per day for
14 days caused a significant decrease of the expression level
of Ki-67 in the rat hippocampus ( P b 0.01, Fig. 2A). However, only a trend towards a decrease of expression level
in the stressed animals was found for BDNF ( P b 0.1,
Fig. 2A). Synaptophysin and GAP-43 expression levels
were not significantly changed ( P N 0.4 for both, Fig. 2A).
The expression level of hippocampal Ki-67 per animal
was significantly positively correlated with the body weight
gain (r s = 0.69, P b 0.005) and significantly negatively
correlated with the relative weight of the left adrenal gland
(r s = 0.65, P b 0.01).
In order to check if acute restraint stress causes changes
in the expression levels of these marker proteins, rats were
restrained for 3 h (Fig. 2B). All marker proteins used for
the analysis of chronic intermittent restraint stress did not
show any significant changes of expression level in the rat
hippocampus ( P N 0.2 for all).
3.3. Immunofluorescence analysis of hippocampal Ki-67
expression after restraint stress
The phenotypic identification of Ki-67-immunopositive
cells using double immunofluorescence staining is shown
in Fig. 3. After confocal laser scanning microscopic
analysis, Ki-67 was not co-localized with either the neuronal marker NeuN (Fig. 3A), the astroglial marker GFAP
(Fig. 3B), or the microglial marker Cd11b (Fig. 3C). Since
both monoclonal and polyclonal antibodies against Ki-67
had to be employed for double labeling studies, their
H. Rosenbrock et al. / Brain Research 1040 (2005) 55–63
59
A
2,50
control
stress
normalized
2,00
optical
density
1,50
1,00
*
0,50
0,00
Synaptophysin
GAP-43
BDNF
Ki- 67
BDNF
Ki-67
B
1,40
normalized
1,20
optical
density
1,00
0,80
0,60
0,40
0,20
0,00
Synaptophysin
GAP-43
Fig. 2. Quantification of marker proteins (expressed as normalized optical density) for synaptic plasticity and progenitor cell proliferation in the adult rat
hippocampus using quantitative immunoblotting after exposure of animals to chronic intermittent restraint stress for 6 h per day for 14 days (A) and acute
restraint stress for 3 h (B). Data expressed as mean F SEM, except for Ki-67 in which is expressed as median F quartile, n = 7–8. *P b 0.01 vs. control
(Wilcoxon rank sum test).
co-localization was checked using confocal laser scanning
microscopy revealing a fully concordant staining pattern
(data not shown). Ki-67-immunopositive cells reside in the
subgranular zone at the border between the granule cell
layer and the hilus showing round- to oval-shaped nuclei
(Figs. 3 and 4). A small number of irregularly shaped Ki-67labeled nuclei organized in clusters were also found in the
subgranular zone which indicates newly born daughter cells
that they have yet to migrate out in the granule cell layer
(Fig. 4A, arrows). Some Ki-67-immunopositive cells were
also present in the crest of the dentate gyrus and throughout
the Ammon’s horn (data not shown) which was previously
shown for proliferating cells of the adult mouse hippocampus [29].
Chronic intermittent restraint stress for 6 h per day for
14 days caused a significant decrease of precursor cell
proliferation as measured by counting the total number of
Ki-67-immunopositive cells (non-clustered and clustered) in
the subgranular zone and the hilus of the dentate gyrus per
section ( P b 0.05, Fig. 4C). The number of Ki-67immunopositive cells per section in the subgranular zone/
hilus per animal was significantly positively correlated with
the expression level of hippocampal Ki-67 analyzed by
Western blotting (r s = 0.57, P b 0.05). Acute restraint stress
for 3 h did not show any changes of the number of Ki-67-
immunopositive cells in the subgranular zone and hilus
(data not shown).
4. Discussion
As a first step, before conducting the study described
here, we performed preliminary tests in order to find out the
chronic intermittent restraint stress procedure of choice.
Thus, rats were subjected to restraint stress for different
intensities and time frames and measured afterwards in
spontaneous motor activity. We found that chronic intermittent restraint stress for 6 h per day for 14 days, but not
less (4 h) or more (8 h) stress duration, caused significant
increases in motor activity, that is, rearing and distance
travelling, compared with controls when measured 1 day
after cessation of stress procedure. Looking at the adrenal
axis, we observed that those chronically stressed rats (6 h
per day for 14 days) exhibited increased relative weight of
adrenal glands and reduced body weight gain. These results
indicate physiological efficacy of the used stress procedure
of 6 h per for 14 days, and they are in accordance with
previous findings which additionally showed increased
plasma corticosterone levels in rats after chronic restraint
stress [3,38].
60
H. Rosenbrock et al. / Brain Research 1040 (2005) 55–63
A
Ki-67
NeuN
merge
GFAP
Ki-67
merge
Ki-67
Cd11b
merge
gcl
hi
B
hi
gcl
C
gcl
hi
Fig. 3. Ki-67 (green, arrows) and NeuN (red) (A), GFAP (green) and Ki-67 (red, arrow) (B), as well as Ki-67 (green, arrows) and Cd11b (red) (C) double
fluorescence labeling in representative tissue sections from the rat dentate gyrus. Confocal laser scanning microscopic superimposed images illustrate that Ki67 and these different marker proteins (NeuN for mature neurons, GFAP for astrocytes, and Cd11b for microglial cells) were present in different cells. hi—
hilus, gcl—granule cell layer, scale bars = 20 Am.
Preclinical studies have suggested that restraint stress
may change brain structure and activity [7,21,24]. These
changes may depend on stress intensity and/or on de novo
gene transcription and synthesis of proteins involved in
neuronal and synaptic plasticity. In fact, it has been shown
that chronic restraint stress for 45 min per day for 10 days,
but not for 6 h per day for 21 days decreases hippocampal
mRNA levels of BDNF, a neurotrophic factor which is
important for maintenance of neuronal function [18,25].
Using in situ hybridization histochemistry, mRNA expression levels of synaptophysin and GAP-43, markers for
synaptic plasticity, were shown to be slightly decreased in
specific areas of the hippocampal formation after chronic
restraint stress for 1 h per day for 5 days and 6 h per day for
21 days, respectively [18,36]. Differentially from these
findings, we could not observe significant changes in
hippocampal expression of those markers on their protein
levels, either after acute or chronic intermittent restraint
stress. One explanation might be that the findings regarding
synaptophysin and GAP-43 showed differences of their
mRNA levels after stress in specific hippocampal regions,
whereas in this study, the entire hippocampus was used for
H. Rosenbrock et al. / Brain Research 1040 (2005) 55–63
A
B
gcl
gcl
hi
hi
200 µm
200 µm
C
number of Ki-67
immunolabeled
cells per section
61
25,0
20,0
*
control
15,0
10,0
stress
5,0
0,0
Fig. 4. Immunohistochemical staining of Ki-67 as progenitor cell marker in tissue sections from the rat dentate gyrus of control (A) and experimental animals
after exposure to chronic intermittent restraint stress for 6 h per day for 14 days (B). The granule cell layer is marked by dotted lines. Note the decreased Ki-67immunopositive cells as indication of diminished proliferative activity after chronic stress treatment. (C) Quantification of Ki-67-immunopositive cells in the
subgranular zone and hilus of the dentate gyrus of control and stressed rats. Data expressed as median F quartile, n = 7–8. *P b 0.05 vs. control (Wilcoxon
rank sum test). hi—hilus, gcl—granule cell layer.
Western blot analysis which possibly led to a dilution of
existing small changes of mRNA levels. Alternatively, the
strength of the used restraint stress procedure of 6 h per day
for 14 days was different compared to the other studies (see
above) which maybe is a critical factor for detection of
changes of expression levels for a given protein. However,
the used stress intensity caused a significant adrenal
response and, at least, a trend towards a decreased BDNF
protein level in the hippocampus (Fig. 2, P b 0.1) which is
in the range of the reported decreased mRNA level of
BDNF [25]. These together with the decreased Ki-67
protein level (Figs. 2 and 4, P b 0.05) show that the
animals were definitely affected by the stress procedure
used in this study.
Another important effect of chronic stress treatment of
rats is its suppressing influence on adult neurogenesis in the
hippocampus. This was demonstrated after chronic psychosocial and chronic restraint stress by using the BrdU
labeling method [8,27]. In addition to this, after chronic
but not acute restraint stress, we could show a marked
decrease of the proliferation marker Ki-67 which can be
used as a valuable alternative to BrdU labeling for
determination of brain progenitor cells linked to adult
neurogenesis [12,15]. Confocal microscopic analysis verified that the Ki-67-positive cells were most likely progenitor
cells, since Ki-67 was not co-localized with markers for
mature neurons (NeuN), astrocytes (GFAP), and microglia
(Cd11b) (Fig. 3). The Ki-67 reduction in stressed rats was
demonstrated by means of two independent methods of
protein expression analysis (Western blotting and immunohistochemistry). The putative difference in the magnitude of
the Ki-67 decrease between the Western blotting and the
immunohistochemical analysis is probably due to the
relatively high biological variability of the stressed animal
group leading to non-parametrical distributed values.
Another factor might be that the entire hippocampal
homogenate was used for Western blotting, but only the
subgranular zone and the hilus of the dentate gyrus was used
for calculating the Ki-67-positive cells after immunohistochemistry. Alternatively, a possible explanation for the
difference in the magnitude of the Ki-67 decrease between
the two approaches used might be, that by using immunohistochemistry, only Ki-67-expressing cells were counted,
whereas by using Western blotting, the entire amount of
hippocampal Ki-67 protein was determined. However, the
immunohistochemical analyses of the hippocampi exhibited
no hints for a change of Ki-67 protein expression within
individual cells after chronic intermittent restraint stress
indicating that decreased Ki-67 expression after this stress
procedure is indeed due to a decreased number of
proliferating cells. In any case, the number of Ki-67immunopositive cells per section in the subgranular zone/
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H. Rosenbrock et al. / Brain Research 1040 (2005) 55–63
hilus per animal was significantly positive correlated with
the hippocampal Ki-67 expression level analyzed by Western blotting. In addition, the decrease of Ki-67 protein
correlated with the physiological stress response of the
animals, that is, body and relative adrenal weight changes.
Taken together, the results as a whole clearly demonstrate
the utility of determination of Ki-67-positive cells for
analysis of hippocampal progenitor cell proliferation. The
reduction of hippocampal progenitor cells after chronic
intermittent restraint stress as measured in this study indicates a suppression of hippocampal neurogenesis in
stressed animals, since it has been shown that the majority
(~70%) of the surviving progenitor cells differentiate into
mature neurons [8,23]. Adult neurogenesis is an extremely
dynamic process that is regulated in both positive and
negative manner by neuronal activity and treatment with
psychoactive drugs or chronic stress [9,11]. Indeed, hippocampal neurogenesis was shown to be increased by chronic
antidepressant treatment, and decreased in heterozygous
BDNF (+/ ) mice as well as by inescapable foot-shock
stress which could be reversed by fluoxetine treatment
[20,22,23]. Furthermore, AMPA receptor potentiators were
not only active in tests for antidepressant activity, but also
revealed enhanced progenitor cell proliferation in the
hippocampus after acute and chronic treatment [2]. These
results together with the antidepressant effect of BDNF
itself in the learned helplessness test and forced swim test
[33] strongly suggest an important role for neurogenesis in
the pathophysiology and treatment of mental illnesses such
as depression as already hypothesized [9,14]. Of course, the
bneurogenic theoryQ of depression is highly speculative and
needs extensive investigations, but nevertheless, the impact
of adult neurogenesis on animal behavior has been described for at least the novelty-suppressed feeding test, the
chronic mild stress model and for some hippocampaldependent learning tasks [1,16,31,34].
In summary, the results presented in this study confirm the
recently reported suppressing effect of chronic restraint stress
on adult neurogenesis by using the BrdU labeling technique
[27]. By using the expression pattern of the alternative
marker of proliferating cells Ki-67, we could show diminished number of progenitor cells in the hippocampus after
chronic intermittent restraint stress by two independent
biochemical methods. Therefore, given the crucial role of
the hippocampus in brain function and the increasing hints
for the role of progenitor cells in the pathophysiology of
mental disorders, understanding the basis and function of
adult hippocampal neurogenesis might contribute to understand the relationship between stress exposure and the
cellular neurobiology of depression.
Acknowledgments
The authors would like to thank Dr. Michael Meyners
for help in statistics and Dr. Gerald Birk for help in
quantification of immunohistochemical stainings, as well
as Carmen Weiss and Michaela Weiss for their excellence
technical assistance.
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