World Journal of Neuroscience, 2011, 1, 9-18
doi:10.4236/wjns.2011.12002 Published Online August 2011 (http://www.SciRP.org/journal/wjns/
WJNS
).
Published Online August 2011 in SciRes. http://www.scirp.org/journal/WJNS
Neurogenesis-enhancing effect of sodium ferulate and its role
in repair following stress-induced neuronal damage
Lijian Yu1*, Yongping Zhang1, Mingneng Liao1, Yanping W ang1, Rundi Ma1, Xiaoyu Zhang1,2, Tingxi Yu1,3*
1Key Laboratory of Marine Materia Medica, Guangdong Ocean University, Zhanjiang, China;
2Department of Otorhinolaryngology-Head and Neck Surgery, University of Maryland School of Medicine, Baltimore, USA;
3Cell Biology Group, Department of Surgery, Department of Pathology, University of Maryland School of Medicine and Baltimore
Veterans Affairs Medical Center, Baltimore, USA.
Email: *yutingxi@yahoo.com, *ywyj9578@sohu.com
Received 22 April 2011; revised 11 May 2011; accepted 18 June 2011.
ABSTRACT
Ferulic acid (FA) is a ubiquitous phenolic acid of low
toxicity, and sodium ferulate (SF) is its sodium salt.
Our previous studies have revealed that FA shows
neuroprotective effect and significant antidepressant-
like effect. The aim of this study was to investigate its
potential neurogenesis-enhancing effect and its role
in repair following stress-induced neuronal damage.
MTT assay was performed to measure the effect of
SF on the growth of rat pheochromocytoma (PC12)
cells; morphological and immunocytochemical meth-
ods were used for assessing its differentiation-induc-
ing action. Chronic mild stress (CMS) tests were per-
formed to establish rat model of depression. The
histopathology of animal brains was studied to ana-
lyze CMS-induced morphological changes and the
effect of SF on the repair of CMS-induced brain in-
jury. The expressions of nerve growth factor (NGF)
and brain-derived neurotrophic factor (BDNF) and
the proliferation of neural stem cell/neural progenitor
cells were assessed in the hippocampi of chronic mild
stress (CMS)-induced depression-like model rats by
immunohistochemistry and bromodeoxyuridine (BrdU)-
incorporation assays, respectively. Our in vitro tests
showed that SF promoted the proliferation of PC12
cells in the concentration range of 5 - 320 µM, and
induced PC12 cells to differentiate to more mature
cells with the morphological characteristics and mo-
lecular marker of neuronal-like cells. In vivo tests
showed that SF up-regulated the expressions of NGF
and BDNF, and induced the proliferation of neural
stem cell/neural progenitor cells in the hippocampi of
CMS-induced depression-like model rats. This study
provides evidences that SF shows neurogenesis-en-
hancing effect, and its antidepressant-like effect of SF
may be related directly and closely to its above-men-
tioned effect.
Keywords: Sodium Ferulate; Neurogenesis-Enhancing
Effect; Rat Pheochromocytoma (PC12) Cells; Stress-
induced Neuronal Damage
1. INTRODUCTION
Today, preclinical and clinical investigations have shown
the involvement of dysregulation of hypothalamic-pi-
tuitary-adrenal (HPA) axis in the pathogenesis of de-
pression. Excessive release of glucocorticoid is closely
related to the hippocampus atrophy. Hypercortisolemia
and the associated hippocampal atrophy were observed
in patients with depression, which could be ameliorated
by the treatment with antidepressants [1]. Moreover, the
volumes of the double-side hippocampus were reduced
in the major depressive patients compared to the healthy
control and there was a positive correlation between the
hippocampus atrophy and the time course of the depress-
sion [2,3]. Consequently, it is reasonable that, in some
cases, psychopathology arises as a consequence of al-
tered morphological structure of particular brain. There
is growing evidence that adult animals continue to pro-
duce new neurons in the dentate gyrus of hippocampus;
neurogenesis in the adult dentate gyrus has been ob-
served in all mammalian species examined to date, in-
cluding humans [4]; stress causes a decrease of neuro-
genesis in the dentate gyrus; and antidepressant treat-
ment in turn stimulates the cell proliferation in the den-
tate gyrus. Furthermore, the waning and waxing of neu-
rogenesis in brain areas such as the dentate gyrus is
proposed as a key factor in the descent into and recovery
from clinical depression, respectively, and a decrease in
neurogenesis could occur due to genetic factors and
stress (especially because of the involvement of adrenal
corticoids) [5].
Ferulic acid (FA), 3-(4-hydroxy-3-methoxyphenyl)-2-
L. J. Yu et al. / World Journal of Neuroscience 1 (2011) 9-18
10
propenoic acid (Figure 1(a)), is one of the most com-
mon phenolic acids with low toxicity [6]. FA is waterin-
soluble, but its sodium salt, sodium ferulate (SF) (Figure
1(b)), is water-soluble, stable, and can be prepared by
chemical synthesis [7]. FA shows a lot of biological ac-
tivities, including antioxidant [8,9]), anti-inflammatory
[10,11], and hypotensive effect [12,13].
Our previous work demonstrates that SF shows obvi-
ous neuro-protective effect [14]. The simultaneous ad-
ministration of SF with monosodium glutamate (MSG)
reverses the effects of MSG on behavior and histopa-
thology in mice [15-17]; intracerebroventricularly ad-
ministered SF mediates brain repair following MSG-
induced exitotoxic neuronal damage in adult mice [18,
19]; and administration of SF also shows antidepressant-
like effects in animal models of depression [14]. There-
fore, it is interesting and reasonable to further investigate
the neurogenesis-enhancing effect of SF in vitro and in
vivo and to discuss their relationship with its antide-
pressant-like effects.
2. MATERIALS AND METHODS
2.1. Drugs and Chemicals
SF was purchased from Yaoyou Pharmaceutic Co. Ltd
(Chongqing, China); fluoxetine (FLU) hydrochloride
purchased from Eli Lilly and Company Limited (USA).
Mouse anti-neuronfilament-200 (NF-200) monoclonal
body was purchased from Wuhan Boster Biological
Technology LTD (China), and TRICH-anti-mouse IgG
purchased from Beijing Zhong Shan—Golden Bridge
Biological Technology Co. LTD (China). Rabbit anti-rat
polyclonal antibodies (anti-NGF and anti-BDNF) were
purchased from Beijing Boisynthesis Biotechnology
LTD (Beijing, China). 5-Bromo-2’-deoxyuridineBrdU)
was purchased from Roche Diagnostics GmbH (Boeh-
ringer-Mannheim, Indianapolis, IN), and rat anti-BrdU
monoclonal antibody purchased from Abcam (Hong
Kong) Ltd. Biotinyllated horse anti-mouse antibody,
avidin-biotin complex were purchased from Beijing
Figure 1. Chemical structure of ferulic acid (a) and
sodium ferulate (b).
Biosynthesis Biotechnology Co. (China). DAB substrat
kit was purchased from Invitrogen corporation. Dulbec-
co’s Modified Eagle Medium (DMEM) and 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
were purchased from Sigma Chemical Co.. NCS was
obtained from Sijiqing Biological Material Co. (Hang-
zhou, China). Horse serum was purchased from Hyclone.
All other chemicals used were of reagent grade.
2.2. Cell Line and Cell Culture
A clonal cell line derived from a pheochromocytoma
(PC12) of the rat adrenal medulla [20] was purchased
from Shanghai Institute of Biochemistry and Cell Bio-
logy, Chinese Academy of Sciences. Cells were grown in
DMEM supplemented with 10% newborn calf serum
(NCS), 5% horse serum and antibiotics (100 units/ml
penicillin, 100 μg/ml streptomycin) in flasks precoated
with collagen [21]. The culture was replaced after 2
months of passage by thawing a fresh aliquot of frozen
cells. The culture were maintained in a humidified atmos-
phere containing 5% CO2 at 37˚C. Cells in log phase
growth were used in the experiments.
2.3. Animals
Adult male Sprague-Dawley (SD) strain rats weighing
220 - 280 g (specific pathogen free) purchased from the
experimental animal center of Guangdong Medical Col-
lege (experimental animal license SCXKyue 2007-2008
A034, No.0001909; Zhanjiang, China), were used across
all the experiments. They had free access to tap water
and standard laboratory food unless otherwise stated.
Housing conditions were controlled, temperature was
maintained at 22˚C ± 1˚C with approximately 60% rela-
tive humidity. They were kept on a reversed 12/12 h
light/dark cycle (light 07:00 - 19:00 h). Animals were
acclimated to the animal quarters for 1 week before any
experimental procedure. All the animals were treated in
compliance with “Guidance Suggestions (Instructions)
for the Care and Use of Laboratory Animals” Issued on
September 30, 2006 by The Ministry of Science and
Technology of the People’s Republic of China.
2.4. MTT Assay
The mitochondrial metabolism of MTT to its insoluble
blue formazan was used for enumerating cells to assess
the effects of SF, H2O2, and glucocorticoid on the growth
of PC12 cells and the protective effects of SF against
oxidative damage and glucocorticoid-induced neurotox-
icity according to the methods of Hansen et al. [22].
Briefly, Single-cell suspensions were prepared and seeded
onto 96 well microculture plates at 1.0 × 105 cells/ml (90
µl/well). Cells were cultured for 12 h before addition of
drugs. Drugs were diluted into DMEM and added to
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each well in a volume of 10 µl. Cells were incubated at
37˚C for the time indicated. MTT solution (5 mg/ml)
was aliquoted to each well in a volume of 20 µl, and 5 h
later 100 µl of the solubilization solution (10% SDS-5%
isobutyl alcohol-0.012 M HCl (w/v/v)) was added into
each well. The plates were allowed to stand overnight in
the incubator in a humidified atmosphere. Absorbance at
570 nm was determined for each well using an ELISA
reader. Control wells contained all of the agents pre-
sented in the treated wells except the drug(s). Each ex-
perimental point was performed in three replicates. Data
were expressed as a percentage of untreated control cul-
tures.
2.5. Effect of SF on the Growth of PC12 Cells
PC12 cells were cultured with different concentrations
of SF for 24 h. Cell viability was examined by MTT
assay.
2.6. Differentiation Induction of PC12 Cells
PC12 cells were cultured in DMEM with SF for the time
indicated, and the effects of different concentrations (10
µM, 20 µM, 40 µM, 80 µM, 160 µM) of SF on morpho-
logical and molecular changes were investigated at dif-
ferent time points in PC12 cells. Cells were analyzed
throughout the differentiation process (6 h and 1, 2, 3, 4,
5, 6, 7, 8, and 9 days), for neurite outgrowth, and for
molecular marker NF-200.
For neurite outgrowth, visual fields of 1 cm2 were
randomly selected in each dish of SF-induced differenti-
ated PC12 cells. Photographed images were generated
for all fields at each time point (6 h and 1, 2, 3, 4, 5, 6, 7,
8, and 9 days) using Leica DM IRB photomicroscope
[23].
For molecular marker NF-200 [24], the cells were
submitted to the immunocytochemistry of the NF-200
[25,26] on the 7th day. Briefly, cells in the wells of the
24-wells plates were washed with PBS. Cells then were
fixed at room temperature for 30 min. Nonspecific bind-
ing sites were blocked with 5% NCS, 5% goat serum
and 0.5% triton X-100 all in PBS for 1 hour. The pri-
mary antibody was added to the cells after being appro-
priately diluted in PBS and incubated overnight at +4˚C.
0.01 M PBS was added to the cells in control group in-
stead of the primary antibody. Cells were washed with
PBS 3 times for 10 minutes and the secondary antibodies
were added after being appropriately diluted in PBS and
left for one hour at room temperature. After adding the
second secondary antibodiy for 1 h, cells were washed
again in PBS 3 times for 10 minutes. Slides were stored
in dark at 4˚C. Slides were later visualized using Leica
inverted confocal microscope and Zeiss laser confocal
microscope.
2.7. CMS Procedures
The animals were assigned randomly into six matched
groups (n = 12 animals in each group) based on sucrose
consumption (1% sucrose solution) before onset of CMS:
control, CMS, fluoxetine control (CMS + FLU), CMS +
SF (10, 20, 40 mg/kg/d) groups. The stressed rats were
ex- posed to CMS for 28 days; The rats in CMS + FLU
group were exposed to CMS and received administration
of FLU (2.0 mg/kg/d, ig, once-daily) for 28 days; the
rats in CMS + SF groups were exposed to CMS and re-
ceived administration of SF (10, 20, 40 mg/kg/d, ip,
once-daily) respectively for 28 days. The control rats
were given ordinary daily care and received ip admini-
stration of normal saline instead of SF and FLU, for 28
days. The stressed and control rats were kept in different
rooms to allow independent manipulation of their envi-
ronments during the duration of the stress procedure.
Control rats were housed together, while the stressed rats
were housed singly.
Most of the stressors were adapted from the procedure
described by Willner and collaborators [27] and some
stressors were included from Moreau and collaborators
(e.g. empty water bottle, restricted food) [28]. Each
week included 2 h of paired caging, 3 h of tilted cage (45
degrees), 18 h of food deprivation immediately followed
by 1 h of restricted access to food (5 micropellets), 2 ×
18 h of water deprivation immediately followed by 1 h
exposure to an empty bottle, 21 h of wet cage (200 ml
water in 100 g sawdust bedding), and 36 h of continuous
light. Stressors were presented both during the rats’ ac-
tive (dark) period and during the inactive (light) period.
The same stressors were used in all experiments.
2.8. Immunohistochemistry
NGF and BDNF levels. For in vivo analysis of NGF and
BDNF levels, the whole brains of 4 unselected rat were
dissected and removed 2 days after the last antidepres-
sant treatment and inmediately fixed in 10% formalin in
0.1 M PBS (pH 7.4) at 4˚C for 2 days, and processed by
paraffin embedding methods. Sections 4 μm thick were
cut and processed for inmunohistochemistry. The im-
munohistochemistry of NGF and BDNF on sections was
performed using a streptavidin-peroxidase conjugate (SP)
method according to the manufacturer’s instructions [29,
30]. Primary antibodies (see Chemicals) were diluted 1:
100. After incubation of primary antibodies at room
temperature overnight, the sections were incubated with
second antibodies 15 min at room temperature, and the
reaction was visualized with DAB. Controls were made
according to the same procedure, but omitting the pri-
mary antibody.
Sections obtained were matched for comparable hip-
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pocampal level. Brain regions were identified via a light
microscope. Rabbit anti-rat polyclonal antibody (anti-
NGF and anti-BDNF) labels were identified by the char-
acteristic yellowish brown stain seen in NGF and BDNF
positive cell bodies. Changes in the number of immuno-
positive cells of the hippocampal DG sector in stained
sections were counted under a light microscope at a
magnification of a × 200 without the examiner knowing
the experimental protocols, and the average of five dif-
ferent areas was determined. All values were expressed
as the means ±SEM and statistical significance was eva-
luated by Dunnet’s multiple comparison test fornon-
parametric analysis.
2.9. In Vivo BrdU-Incorporation Assay
For in vivo BrdU-incorporation assay [31], rats were
administered BrdU (4 × 75 mg/kg every 2 h; Sigma, St.
Louis, MO) 4 days after the last antidepressant treatment.
The 4 days time point was chosen because a similar pa-
radigm has been used in a previous study of chemical-
induced seizures on hippocampal neurogenesis [32].
Twenty-four hours after the last BrdU injection, rats
were killed and transcardially perfused (0.1 M cold PBS
for 5 min followed by 4% cold paraformaldehyde for 17
min). After perfusion, all brains were post-fixed over-
night in paraformaldehyde (with shaking) at 4˚C and
stored at 4˚C in 30% sucrose. Serial sections of the
brains were cut (35 mm sections through the entire hip-
pocampus [33] on a freezing microtome, and sections
were stored in PBS/NaN3.
Free-floating sections were used in the determination
of BrdU labeling. DNA denaturation was conducted by
incubation for 2 h in 50% formamide/2 × SSC at 65˚C,
followed by several PBS rinses. Sections were then in-
cubated for 30 min in 2 N HCl and then 10 min in H2O2
to eliminate endogenous peroxidases. After blocking
with 3% normal horse serum in 0.01%, triton X-100,
cells were incubated with anti-rat BrdU(1:1000) over
night at 4˚C. Sections were then incubated for 1 h with
secondary antibody (biotinylatedhorseanti-mouse) fol-
lowed by amplification with an avidin-biotin complex
and cells were visualized with DAB.
Sections obtained were matched for comparable hip-
pocampal level. Brain regions were identified via a light
microscope. Anti-BrdU labels were identified by the
characteristic yellowish brown stain seen in BrdU-posi-
tive cell bodies. Changes in the number of immunoposi-
tive cells of the hippocampal CA3 sector in stained sec-
tions were counted under a light microscope at a magni-
fication of a × 200 without the examiner knowing the
experimental protocols, and the average of five different
areas was determined. All values were expressed as the
means ±SEM and statistical significance was evaluated
by Dunnet’s multiple comparison tests for nonparametric
analysis.
2.10. Statistical Analysis
Values are expressed as the means ±SEM. Data were
analyzed with SPSS 10.0 software. A probability of p <
0.05 was considered significant.
3. RESULTS
3.1. Promotion Effect of SF on the Proliferation
of PC12 Cells
PC12 cells were cultured with different concentrations
of SF for 24 h. Figure 2 showed the concentration-de-
pendent promotion effect of cell proliferation by SF. At
concentrations higher than 5 M, the cell proliferation
climbed up slowly, and SF showed significantly promo-
tion effect on the cell proliferation at those from 80 to
320 M.
PC12 cells were cultured with different concentrations
of SF for 24 h. Cell viability was examined by MTT
assay. The data of one representative experiment from
three independent experiments were expressed as mean±
SEM (n = 4). *P < 0.05, **P < 0.01 as compared with
control (without SF).
3.2. Induction of PC12 Cell Differentiation by
SF
In order to study the effect of SF on PC12 cell differen-
tiation, PC12 cells were treated without or with SF (10-
160 μM). There was significant neurite length growth in
the 160 μM SF-supplemented group on the 3rd day, in the
80 μM SF-supplemented group on the 4th day, and in the
40 μM SF-supplemented group on the 7th day. The
SF-supplemented cells showed significantly accelerated
neurite outgrowth compared to the control cells (Figure
3(A-B)). The cells acquired advanced neuronal pheno-
types with long axons structures connecting cells to each
other formed potential complex neural networks (Fig-
ures 3A(c) and B(b-c)).
Figure 2. Effect of SF on the cell viability in
PC12 cells.
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L. J. Yu et al. / World Journal of Neuroscience 1 (2011) 9-18 13
Figure 3. Neurite outgrowth in the presence of SF.
PC12-cells were grown in the absence (control) or presence of SF (80, 160
µM). Neurite outgrowth was monitored for 9 days, starting 6 h post-sup-
plementation of SF. Representative micrographs of PC12-cell culture are
shown (×200). A. 4rd day: a. control, b. 80 M, c. 160 M; B. 7th day: a.
control, b. 80 M, c. 160 M.
To determine whether the PC12 cells can synaptically
integrate and mature in the culture with SF, we examined
the expression of NF-200, marker of neuronal differen-
tiation. After 3 - 4 days of incubation cells lost their per-
fect round shape and small projections start to grow
from these cells. After a week of differentiation in the
presence of SF, cells showed polar morphology, axons
and dendrites structures were clearly noticed and poten-
tial simple neural network was noticed (Figures 3A(c)
and B(b-c)). The differentiation process was accom-
panied with apoptosis of some differentiation cells. The
differentiating cells formed a much complex potential
neural networks (Figure 4(c-d)). NF-200 (Red) highly
expressed and was highly concentrated along the axon
structures of the neuronal cells (Figure 4(d)). Staining
with anti-NF-200 antibody showed that NF-200 expre-
ssion was evident and PC12 cells underwent a process of
maturation.
The results revealed that under the action of SF, PC12
cells could be induced to differentiate to more mature
cells with the morphological characteristics and molecu-
lar marker of neuronal-like cells.
3.3. Protective Effect of SF against
CMS-Induced Reduction of
Hippocampal NGF and BDNF Levels
Hippocampal NGF and BDNF levels were obviously
reduced in the CMS mice (Figures 5A(a-b) and B(a-b)).
In contrast, SF treatment reversed the effects of CMS on
the hippocampal NGF and BDNF levels in a dose-de-
pendent manner (Figures 5A(a, d, e, f) and B(a, d, e, f)),
and the effect of SF(40 mg/kg/d) was comparable with
that of FLU (2.0 mg/kg/d) control (Figures 5A(c, e, f)
and B(c, e, f)). Consequently, the results suggest that SF
Figure 4. Neuronal-like differentiated and
maturated cells (×200).
PC12-cells were grown in the absence (control) or
presence of SF (80, 160 µM) for 9 days. For molecu-
lar marker NF-200, the cells were submitted to the
immunocytochemistry of the NF-200 on the 7th day
as described in “Materials and methods”. (a) control;
(b) control, using PBS instead of primary antibody
(NF-200 monoclonal body); (c) PC12 cells treated
with 160 μM SF for 7 days. The cells acquired ad-
vanced neuronal-like phenotypes with long axons
structures connecting cells to each other forming po-
tential complex neural networks; (d) PC12 cells
treated with 160 μM SF for 7 days were submitted to
the immunocytochemistry of the NF-200. The cells
with neuronal-like morphology after differentiation
and maturation highly expressed NF-200 (red).
treatment partially reverses the effects of CMS on hip-
pocampal NGF and BDNF.
The whole brains of unselected rat (n = 4) were dis-
sected and removed 2 days after the last antidepressant
treatment and inmediately fixed in 10% formalin, and
processed by paraffin embedding methods. Sections 4
μm thick were cut and processed for inmunohistochem-
istry. The immunohistochemistry of NGF and BDNF on
sections was performed using a streptavidin-peroxidase
conjugate (SP) method according to the manufacturer’s
instructions. Rabbit anti-rat polyclonal antibody (anti-
NGF and anti-BDNF) labels were identified by the char-
acteristic yellowish brown stain seen in NGF and BDNF
positive cell bodies. Changes in the number of im-
munopositive cells of the hippocampal DG sector in
stained sections were counted under a light microscope
at a magnification of a × 200, and the average of five
different areas was determined. All values were ex-
pressed as the means ±SEM, and statistical significance
was evaluated by Dunnet’s multiple comparison tests for
nonparametric analysis.
3.4. Protective Effect of SF against the
Reduction of Neurogenesis in the
Hippocampal CA3 Sector of
CMS-Induced Depression-Like
Model Rats
Analysis of the number of BrdU-labeled cells demon-
strated that the number of BrdU-labeled cells was obvi-
ously reduced in the CA3 of CMS mice (Figure 6(a-b)).
n contrast, long-term administration of SF (10, 20, 40 I
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L. J. Yu et al. / World Journal of Neuroscience 1 (2011) 9-18
Copyright © 2011 SciRes.
14
(A) (B)
Figure 5. Representative microphotographs and numbers of NGF(A)- and BDNF(B)-immunopositive neurons in the
hippocampal DG sector of CMS-induced depression-like model rats.
(A) Representative microphotographs and numbers of NGF-immunopositive neurons (×200). a. Control; b. CMS; c. CMS + FLU (2.0 mg/kg); d. CMS
+ SF(10 mg/kg); e. CMS + SF(20 mg/kg); f. CMS+; (B) Representative microphotographs and numbers of BDNF-immunopositive neurons (×200). a.
Control; b. CMS; c. CMS + FLU (2.0 mg/kg); d. CMS + SF(10 mg/kg); e. CMS + SF(20 mg/kg); f. CMS + SF(40 mg/kg).
mg/kg/d) significantly increased the number of BrdU-
labeled cells by 8.6%, 27.8%, 47.7% in the CA3 of CMS
mice in a dose-dependent manner (Figure 6(b, d, e, f)),
respectively, whereas FLU (2.0 mg/kg/d) increased the
BrdU labeling by 23.2% (Figure 6(b-c)). Consequently,
the results suggest that SF treatment reverses partially
the effects of CMS on the neurogenesis in the hippo-
campi of the rats, and the effects of SF(20, 40 mg/kg/d)
were comparable with that of FLU (2.0 mg/kg/d) con-
trol.
For in vivo BrdU-incorporation assay, rats were ad-
ministered BrdU(4 × 75mg/kg every 2 hr) 4 days after
the last antidepressant treatment. Twenty-four h after the
last BrdU injection, rats were killed and transcardially
perfused. After perfusion, all brains were post-fixed
overnight in paraformaldehyde (with shaking) at 4˚C and
stored at 4˚C in 30% sucrose. Immunohistochemistry as-
say was performed as “Materials and Methods”. Anti-
BrdU labels were identified by the characteristic yellow-
ish brown stain seen in BrdU-positive cell bodies. Chan-
ges in the number of immunopositive cells of the hippo-
campal CA3 sector in stained sections were counted under
a light microscope at a magnification of a × 400 without
the examiner knowing the experimental protocols, and the
average of five different areas was determined. All values
were expressed as the means ±SEM and statistical sig-
nificance was evaluated by Dunnet’s multiple comparison
tests for nonparametric analysis.
4. DISCUSSION
Our previous investigations reveal that acute administra-
tion of SF significantly decrease the duration of immo-
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L. J. Yu et al. / World Journal of Neuroscience 1 (2011) 9-18 15
Figure 6. SF induces the proliferation of neural stem cell/
neural progenitor cells in the hippocampal CA3 sector of
CMS-induced depression-like model rats (×200).
a. Control; b. CMS; c. CMS + FLU(2.0 mg/kg/d); d. CMS + SF(10 mg/kg/d);
e. CMS + SF (20 mg/kg/d); f. CMS + SF(40 mg/kg/d).
bility during forced-swimming test and tail-supension
test in mice and rats, suggesting that SF has an acute
antidepressant-like effect. However, SF has no any ef-
fects on reserpine-induced hypothermia, 5-HTP-induced
head-twitch response, and potentiation of noradrenaline
toxicity in mice, suggesting that the mechanisms of SF
antidepressant-like effects are different from those of
main kinds of antidepressants that have been employed
in the treatment of depression for several decades. Long-
term ip administration of SF reverses the effects of CMS
on consumption of food and sucrose solution, weight
gain, locomotion and exploration behavior, and signifi-
canly shortens the immobility time during forced-swim-
ming test following CMS in rats, suggesting that SF has
a chronic antidepressant-like effect [14].
The present results obtained from in vitro tests show
that SF treatment promotes the proliferation of PC12
cells in the concentration range of 5 - 320 µmol/L, and
induce PC12 cells to differentiate to more mature cells
with the morphological characteristics and molecular
marker of neuronal-like cells. The present results ob-
tained from in vivo tests show that SF treatment may up-
regulate the expressions of NGF and BDNF, and induce
the proliferation of neural stem cell/neural progenitor
cells in the hippocampi of CMS-induced depression-like
model rats.
NGF is a well known characterized member of the
neurotrophin family that stimulates differentiation, main-
tains phenotype, and ensures the survival of various
populations of neurons in the central nervous system
(CNS) [34,35]. The loss of NGF has been implicated in
the loss of cholinergic tone and function [36-38]. An-
other neurotrophic factor, the BDNF, does not only pro-
mote neuronal survival anddifferentiation, but also regu-
lates synaptic neurotransmission and plasticity in the
CNS [39,40]. Furthermore, BDNF levels are increased in
the hippocampal regions of mice and rats with access to
exercise wheels and trophic factor gene therapy can
ameliorate hippocampal deficits in monkey [41,42].
NGF and BDNF are responsible for the development,
differentiation, maintenance and repair of neurons [43-
45]. Exogenous NGF rescues cholinergic neurons in the
basal forebrain and improves cognitive function in im-
paired, aged or cholinergically lesioned animals [46-48].
A recent report shows that transplantation of fibroblasts
genetically modified tosecrete NGF led to a slowing of
cognitive decline in a Phase I clinical trial performed on
patients suffering from mild Alzheimer’s disease [49].
BDNF is highly present in the dentate gyrus and hippo-
campal CA3 sector, and to a lesser extent in the hippo-
campal CA1 sector [50]. It has been reported that the
extent of dendritic arborization in the hippocampus was
profoundly increased in BDNF transgenic mice com-
pared to their control litter mates [51]. In BDNF gene
knockout mice, furthermore, long-term potential (LTP)
in the hippocampal CA1 region is severely reduced in
hippocampal neurons and spatial learning is significantly
impaired [52,53]. The results obtained from our experi-
ments show that the hippocampal NGF and BDNF levels
were obviously reduced in the CMS mice. Of interest is
that SF treatment reversed the effects of CMS on the
hippocampal NGF and BDNF levels in a dose-dependent
manner. This finding suggests that SF-induced increases
of the hippocampal NGF and BDNF levels may play a
role in the antidepressant-like effect of SF.
The hippocampus is one of only a few brain regions
where production of neurons occurs throughout the life
time of animals, including humans [4]. Hippocampal
C
opyright © 2011 SciRes. WJNS
L. J. Yu et al. / World Journal of Neuroscience 1 (2011) 9-18
16
neurogenesis can be influenced by several environmental
factors and stimuli [54-57]. It has been shown that
stressful experiences, including both physical and psy-
chosocial stress, suppress the formation of hippocampal
granule cells in a number of mammalian species [58-60].
Decreased cell proliferation has also been reported in
response to both acute and chronic stress paradigms [61].
Importantly, it has been shown that chronic antidepres-
sant treatment signicantly increases the number of BrdU-
labeled cells in the dentate gyrus and hilus of the hippo-
campus. BrdU is incorporated into the newly synthesized
DNA of S-phase cells and may provide an estimate for
the fraction of cells in S-phase. A specific marker like
BrdU is not only helpful for gaining further insights into
the genesis of new neurons in the hippocampus, but also
might be applicable to the development of strategies for
therapeutic interventions [62]. Administration of sev-
eral different classes of antidepressant, but not non-an-
tidepressant, agents was found to increase BrdU-labeled
cell number, indicating that this is a common and selec-
tive action of antidepressants [31]. Of interest is that the
results obtained from our experiments show that admini-
stration of SF increase BrdU-labeled cell number in the
hippocampi of CMS-induced depression-like model rats.
This finding raise the possibility that increased cell pro-
liferation and increased neuronal number induced by SF
in CMS-induced depression-like model rats may be a
mechanism by which SF treatment overwhelms the stress-
induced atrophy and loss of hippocampal neurons.
5. ACKNOWLEDGEMENTS
The authors would like to thank Dr. Depu Yu for his great encourage-
ment and continuous promotion.
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