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Protective Effect of Total Flavones of Abelmoschus manihot L. Medic Against Poststroke Depression Injury in Mice and Its Action Mechanism

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THE ANATOMICAL RECORD 292:412–422 (2009)
Protective Effect of Total Flavones of
Abelmoschus manihot L. Medic Against
Poststroke Depression Injury in Mice
and Its Action Mechanism
MEI LIU, QIU-HONG JIANG, JI-LI HAO, AND LAN-LAN ZHOU*
Department of Pharmacology, Basic Medical College, Anhui Medical University,
Hefei 230032, People’s Republic of China
ABSTRACT
Total flavones of Abelmoschus manihot L. Medic (TFA) is the major
active component isolated from the traditional Chinese herb Abelmoschus
manihot L. Medic. We investigated the protective effect of TFA against
poststroke depression (PSD) injury in mice and its action mechanism. A
mouse model of PSD was induced by middle cerebral artery occlusion
(MACO) 30 min/reperfusion, followed by isolation feeding and chronic
unpredictable mild stress for 2 weeks. Treatment groups received TFA
at three different doses (160, 80, and 40 mg/kg, p.o.) or fluoxetine (Flu,
2.5 mg/kg, p.o.) daily for 24 days. Change in behavior, brain tissue malondialdehyde (MDA) levels, and the activity of superoxide dismutase (SOD)
and glutathione peroxidase (GSH-Px) were measured. The expression of
brain-derived neurotrophic factor (BDNF) was detected by immunohistochemistry, and mRNA expression of BDNF and cAMP response elementbinding protein (CREB) analyzed by reverse transcription-polymerase
chain reaction (RT-PCR). Treatment with TFA (160, 80, and 40 mg/kg)
significantly ameliorated mice escape-directed behavioral impairment
induced by PSD, markedly reduced MDA levels, and increased the activity of SOD, GSH-Px close to normal levels. TFA administration also attenuated PSD-induced neuronal death/losses, upregulated expression of
BDNF both at mRNA and protein levels, as well as CREB mRNA levels.
TFA had a protective effect against PSD injury in mice. Cardioprotection involves the inhibition of lipid peroxidation and upregulation of
BDNF-CREB levels in the hippocampus, which may also be important
mechanism of its antidepressants. This potential protection makes TFA a
promising therapeutic agent for the PSD. Anat Rec, 292:412–422,
2009. Ó 2009 Wiley-Liss, Inc.
Key words: poststroke depression; total flavones of Abelmoschus manihot L. Medic; brain-derived neurotrophic factor; cAMP response element-binding
protein; lipid peroxidation
Grant sponsor: Annual Research Project of Anhui Provincial
Natural Science Foundation; Grant number: 05021005; Grant
sponsor: Doctoral Foundation of Anhui Medical University;
Grant number: xj2004005.
*Correspondence to: Lan lan Zhou, Department of Pharmacology, Basic Medical College, Anhui Medical University, Hefei
Ó 2009 WILEY-LISS, INC.
230032, People’s Republic of China. Fax: 186-5515161133.
E-mail: lanlzhou@yahoo.com.cn
Received 18 August 2008; Accepted 3 December 2008
DOI 10.1002/ar.20864
Published online in Wiley InterScience (www.interscience.wiley.
com).
POSTSTROKE DEPRESSION
Poststroke depression (PSD) is a common mood disturbance in stroke patients that potentially affects survivors and their recovery, and its estimated frequency
ranges from 25%–79% (Spalletta et al., 2005). It is associated with excess disability, increased cognitive impairment, poor rehabilitation outcomes, worse quality of life,
and increased suicidal tendencies and mortality (Ramasubbu et al., 1998; Kauhanen et al., 1999; Kimura et al.,
2000). The pathophysiology of PSD remains elusive and
appears to be multifactorial rather than purely biological
or psychosocial in origin; psychological and social stressors associated with stroke are therefore considered to
be the primary cause of depression (Gainotti et al., 1999;
House, 1996). Recognition and treatment of the depression in stroke survivors is very important. An effective
neuroprotective therapy for PSD has not been postulated
until now.
Recent studies have shown that major depression is
associated with a significant excess of free radicals and/
or lipid peroxidation, The involvement of oxidative stress
in neuronal loss after PSD is well established. Several
components of reactive oxygen species (ROS), i.e., superoxide, hydroxyl radical, hydrogen peroxide and peroxynitrite radical have been found to be generated after ischemia reperfusion injury, and play an important part in
the neuronal loss after stoke. Inhibition of formation of
free radicals during lipid peroxidation prevents development of brain edema and neuronal damage, which may
be an important antidepression mechanism. Several
enzymes (e.g., superoxide dismutase (SOD), glutathione
peroxidase (GSH-Px)) are important in the antioxidant
defense system because they metabolize free radicals or
reactive oxygen intermediates to nonradical products.
Dysfunction of SOD and GSH-Px may result in the loss
of protective activity exerted by these enzymes, and has
manifested as increased infraction in many studies.
An emerging hypothesis suggests that impaired neuroprotection is closely involved associated with the pathogenesis of major depression. For example, decreased
neuronal survival and disordered neurogenesis in the
hippocampus have been repeatedly found in patients
with major depression. They and are considered to be
potential pathophysiological factors and therapeutic targets for major depression. The hippocampus is an important limbic structure that modulates mood, anxiety,
learning and memory, all of which are commonly disrupted in depression (Fumagalli et al., 2005). Preclinical
and clinical studies have demonstrated that major
depression results in a reduction in the total number of
neurons in the hippocampus by inducing a prolonged
reduction in the rate of cell proliferation (WarnerSchmidt and Duman, 2006). Neurotrophins are known
to be critical for the survival of neurons in the hippocampus during development in vivo and in culture
(Duman et al., 1997; McAllisler et al., 1996; Thoenen,
1995). Brain-derived neurotrophic factor (BDNF) is the
most widespread growth factor in the brain. It has
diverse functions in the adult brain as a regulator of
neuronal survival, proliferation and differentiation of
cells, fast synaptic transmission, and activity-dependent
synaptic plasticity. The BDNF plays a central part in
the development and plasticity of the brain by opposing
neuronal damage, and promoting neurogenesis and cell
survival (Lewin and Barde, 1996; Blum and Konnerth,
2005). The transcription factor cAMP-response element-
413
binding protein (CREB) is an important regulator of
BDNF-induced gene expression, and has a central role
in mediating neurotrophin responses (Finkbeiner et al.,
1997). Studies have shown that long-term exposure to
stress is accompanied by neuronal atrophy of the hippocampus, and a decrease in the expression of BDNF
(Smith et al., 1995). Brain imaging studies have demonstrated that the volume of the hippocampus is reduced
in patients with chronic depression (Sapolsky, 2001).
Long-term administration of antidepressants causes an
increase in the expression of BDNF and CREB, and
upregulation of CREB and BDNF occurs in response to
several different classes of antidepressant treatment,
including norepinephrine reuptake inhibitors, serotoninselective reuptake inhibitors, and electroconvulsive seizure. This indicates that the BDNF–CREB cascade is a
common postreceptor target of these therapeutic agents.
At present, there are three main kinds of classical
antidepressants for PSD in clinical practice, including
tricyclic antidepressants, selective reuptake inhibitors
and monoamine oxidase inhibitors. Most of these drugs,
however, have undesirable side effects, and their mechanism of action incompletely understood. Seeking safe
and effective antidepressant agents from traditional
herbs may uncover novel treatments for depressive disorders and reveal unknown mechanisms by which
depressive symptoms can be alleviated.
Abelmoschus manihot L. Medic is a plant rich in flavones such as quercetin, hyperin and rutin. Total flavone
of Abelmoschus manihot L. Medic (TFA) is the major
active component of this plant. TFA has been reported to
have a neuroprotective effect against cerebral ischemia
injury in rats and rabbits (Wen and Chen, 2007; Guo
and Chen, 2002). Previous studies from our research
group demonstrated that TFA treatment produced a
protective effect against injury due to PSD in rats by
normalizing hyperactivity in the hypothalamic–pituitary–adrenal axis (Hao et al., 2007). However, the mechanisms underlying the antidepressant effect are still
obscure. Therefore, in the present study, we used a PSD
model in the mouse to further explore the neuroprotective effect of TFA and its mechanism. We focused mainly
on the antioxidant defense system and hippocampal neurogenesis, which are deeply involved in the etiology and
treatment of depression.
MATERIALS AND METHODS
Animals
This investigation conformed to the regulations stipulated by Anhui Medical University Animal Care Committee, which follows the protocol outlined in The Guide
for the Care and Use of Laboratory Animals published
by the US National Institute of Heath (NIH publication
number 85-23; revised 1996). Experimental protocols
described in this study were approved by the Ethics
Review Committee for Animal Experimentation of Anhui
Medical University.
Healthy male mice (25 6 3.0 g) were housed in a room
at a constant temperature of 228C 6 38C with 40% humidity under a 12-hr light-dark cycle. They had free
access to tap water and food. They were allowed 1 week
to acclimatize before experimentation.
414
LIU ET AL.
Drugs and Reagents
TFA (yellow powder; content of flavones >60%; batch
number, 20050809) was obtained from the Institute of
Medicine of Anhui Province. Fluoxetine (Flu) was provided by the Eli Lilly and Drug Company Limited
(Suzhou, China). Malondialdehyde (MDA), GSH-Px and
SOD test kits were purchased from Nanjing Jiancheng
Bioengineering Institute (Nanjing, China). The antibody
of BDNF (rabbit anti-mouse), Streptavidin–Biotin Complex (SABC) kit and diaminobenzidine (DAB) staining
kit were purchased from Boster Biological Engineering
Company (Wuhan, China). Primer DNA was synthesized
by the Shanghai Bioengineering Technical Service Limited Company (Shanghai, China). Trizol reagent and
reverse transcription-polymerase chain reaction (RTPCR) Test Kits were obtained from Biotechnology Company (TakaRa, Biotechnology Company, Limited, Beijing,
China). Drugs were dissolved in distilled water and all
other chemicals were of the highest analytical grade
available.
Experimental Model of PSD
Treatment of animals. Mice were randomly divided into seven groups: sham, ischemia, model, TFA
(160, 80, and 40 mg/kg) and Flu (2.5 mg/kg). Drugs were
given via the oral route, once a day for 24 consecutive
days. Sham, ischemia, and model groups were given an
equal volume of vehicle. Experiments were conducted on
day-3, 60 min after the designated amount of TFA, Flu
or vehicle had been given. Ischemia/stroke was induced
in anesthetized (4% chloral hydrate, 350 mg/kg, i.p.)
mice. After the stroke, mice in model and drug treatment groups were housed individually in metabolic
cages; sham and ischemia mice were assigned randomly
to two groups of five: maintained at a room temperature
of 238C 6 28C, on a light-dark cycle of 12 hr/12 hr, with
free access to water and food. On day-8, mice in the
model and drug treatment groups were exposed to stress
for 14 days. On day-24, behavioral changes were evaluated, and mice were killed for biochemical study. The
design of the time course of the experiment and sampling times are shown in Fig. 1.
Middle cerebral artery occlusion in mice. Middle cerebral artery occlusion (MCAO) was induced in
mice using the intraluminal filament method as previously described (Kilic et al., 2001) with mild modification. Briefly, anesthesia was induced by injection of 4%
chloral hydrate (350 mg/kg, i.p). Under an operating
microscope, a mid-line incision was made on the ventral
surface of the neck, and the right common carotid
arteries and external carotid artery isolated and ligated
with an 8.0 silk suture. A polyamide monofilament
(Ethilon, Johnson and Johnson International, Belgium)
coated to a round tip with silicone resin (thread thickness; Xantopren, Bayer Dental, Germany) was introduced into the intracranial internal carotid artery (ICA)
through an incision in the common carotid artery. The
monofilament was carefully advanced approximately 11–
13 mm distal to the carotid bifurcation (which was
beyond the origin of the middle cerebral artery). After
occlusion was achieved, the monofilament was secured
Fig. 1. Outline of the design of the time course of the experiments
and the sampling times.
in place with ligature. The incision was sutured. Occlusion was done for a period of 30 min. After 30 min of
MCAO, the filament was completely withdrawn from the
ICA to allow reperfusion. Sham-operated mice underwent the same surgical procedure except that their common carotid arteries were not ligated and occluded. The
rectal temperature was maintained at 378C 6 0.58C
throughout surgery using a heating pad (Biomed S.L,
Spain). The heart rate and electrocardiogram were
monitored throughout the procedure (Surgi Vet, USA).
Neurological status. Neurological function was
evaluated 2 hr, 1, 3, 5, 7, 14, 21 days after MCAO 30min/reperfusion using a modified seven-point behavioral
rating scale (Longa et al., 1989). Neurological scores
were defined as: 0 5 no neurological deficit; 1 5 failure
to fully extend left forepaw; 2 5 reduced grip of left
forelimb while the tail was gently pulled; 3 5 circling
toward the contralateral side if tail was pulled when
moving spontaneously; 4 5 circling or walking to the
left; 5 5 walking only if stimulated; 6 5 unresponsive
to stimulation with a depressed level of consciousness;
7 5 death.
Mice with neurological score of 1–6 were included in
the current study for further establishment of a depression model; mice displaying subarachnoid hemorrhage
and deep anesthesia signs were excluded.
Chronic unpredictable mild stress model. On
the day-8, poststroke mice in drug treatment and model
groups were subjected to different stressors per day randomly over 14 days between 8:00 AM to noon. The procedure was a minor modification of the method described
previously (Willner et al., 1987). The order of stressors
used was: high-speed agitation for 1 min; day and night
reversal for 24 hr; cold stress at 48C for 5 min; overhang
for 1 min; heat stress at 458C for 5 min; food deprivation
for 24 hr; and water deprivation for 24 hr. Each stressor
was repeated twice during the 14-day stress procedure.
After 24 hr, mice underwent behavioral tests.
Behavioral Tests
The forced swimming
Forced swimming test.
test employed was similar to that described elsewhere
(Porsolt et al., 1977). Briefly, mice were individually
forced to swimming inside a rectangular glass jar (25 cm
3 12 cm 3 25 cm containing 15 cm of water maintained
at (238C–258C) for 6 min. After the initial 2–3 min of
vigorous activity, they showed immobility by floating
POSTSTROKE DEPRESSION
415
with minimum movements. A mouse was considered to
be immobile when it ceased struggling and remained
floating motionless in the water, making only small
movements necessary to keep its head above water. The
duration of immobility was recorded during the last
4 min of the 6-min testing period.
fixed for 24 hr with 10% paraformaldehyde PBS. They
were embedded in paraffin, and coronal sections (thickness, 5 mm) cut on a paraffin microtome (Leica RM2135,
Germany) for histological analysis. Paraffin sections
were stained with hematoxylin and eosin for morphological evaluation.
Passive avoidance task. Twenty-four hours after
the last time stress, mice were trained individually in a
step-through passive avoidance apparatus termed a
‘‘shuttle-box.’’ A straight wooden case (40 cm 3 12 cm 3
18 cm) had a removable cover. The alley was divided
into two equal-length compartments (20 cm 3 12 cm 3
18 cm) by a partition with a rotundum. A light bulb
(40 W) was positioned in the corner on the left side of
the apparatus. The left compartment was bright and the
right compartment was dark. The floor of the apparatus
was made of copper grid (length, 12 cm; diameter, 2 cm).
The method was similar to a previously described
method (Simona and Claudio, 1997; Zhou et al., 2000).
On the learning day, each mouse was placed in the
bright compartment, facing away from the dark compartment. When the mouse turned around, the door
leading to the dark compartment was opened. When the
mouse had stepped with four paws into the dark side, a
foot shock (40 V) was delivered and the time (latency, L)
taken to move to the dark compartment was recorded.
Numbers (number of errors, NR) for the mice to enter
the darkroom to suffer electric shock in 5 min were also
recorded. Mice were returned immediately to their
cages. Retention was tested 24-hr later following the
same procedure, after which mice were killed. Brain and
hippocampal tissues were isolated for biochemical studies and histopathological examination.
Immunohistochemistry for BDNF. Paraffin sections from mice brains were used for immunohistochemical analysis. In brief, 5-mm sections were deparaffinized
in xylene and dehydrated through graded ethanol, and
washed thrice with PBS (0.02 mmol/L, pH 7.4) for
5 min. Sections were incubated with 3% H2O2 for
10 min at room temperature to inactivate native peroxidase. After rinsing thrice with PBS for 5 min, they were
heated in a microwave oven at a low power for 5 min in
0.01 mmol/L citrate buffer (pH 6.0). After rinsing thrice
with PBS for 5 min, sections were kept in 5% BSA for
30 min, followed by incubation with a polyclonal antibody to BDNF (diluted 1:150; Boster) overnight at 48C
(PBS buffer solution was used to replace antibody-BDNF
in the negative control group). Sections were rinsed for
5 min three times with PBS before incubation with biotinylated goat anti-rabbit IgG antibody for BDNF for
60 min, and then incubated with streptavidin-biotin
complex for 60 min at 378C after rinsing three times
with PBS. Sections were rinsed for another 5 min three
times with PBS before the reaction with DAB solution
for 5 min. Sections were dehydrated through graded
ethanol. They were enveloped with gelatin and observed
under a bright-field microscope. Analyses were carried
out with an observer blinded to the experimental protocol. Six sections per animal and three fields per section
of the same magnification were utilized for quantitative
analysis. Expression of BDNF-positive neurons in the
CA1, CA3, and DG of hippocampus was observed under
the microscope. The mean optical density of BDNF-positive neurons was measured using Image J 1.34 analysis
system in each section.
Biochemical analysis. Six mice from each group
were decapitated at the end of the behavioral test.
Brains were quickly removed and the hippocampus
excised on an ice-plate. It was washed with ice-cold sterile physiological (0.9%) saline, blotted dry on filter paper
and weighed before 10% (wt/vol) homogenates were
made in ice-cold saline. Homogenates were centrifuged
at 1000g for 10 min, and the supernatant stored at
2808C for biochemical study.
Measurement of MDA levels, SOD, and GSH-Px
activity in brain tissue. Brain homogenates were
used for estimation of MDA levels, as well as measuring
the activity of SOD and GSH-Px. The MDA levels were
measured at 523 nm by the thiobarbituric acid method
(Wong et al., 1987). The activity of SOD and GSH-Px
was detected by spectrophotometry at 550 nm (Sun
et al., 1998) and 423 nm (He et al., 2008), respectively.
Protein concentration was estimated using bovine serum
albumin as a standard (Lowry et al., 1951).
Histological examination of hippocampus
specimens. At the end of behavioral test, mice were
anesthetized with 4% chloral hydrate (350 mg/kg, i.p)
Intracardiac perfusion with cold saline was done, followed by 4% paraformaldehyde in phosphate-buffered
saline (PBS) (0.1 M; pH 7.4). Brains were removed and
Analysis of BDNF mRNA and CREB mRNA
expression levels by RT-PCR. The levels of BDNF
mRNA and CREB mRNA were determined by RT-PCR.
Mice were decapitated and whole brains quickly
removed on an ice-plate. Tissues were washed with cold
saline. Hippocampal extracts were carefully excised and
stored at 2808C until RNA extraction. Levels of BDNF
and CREB transcripts were analyzed by RT-PCR in
studies in which total RNA from hippocampal tissues
(30–50 mg) were isolated using an RNA isolation kit
(TakaRa) according to manufacturer’s instructions.
cDNA was reverse transcribed from 1 mL of total RNA
with a RNA PCR Kit (AMV) Ver. 3.0. Briefly, reverse
transcription was done using 1 mL total RNA with 5 U/mL
AMV Reverse Transcriptase 0.5 mL, 40 U/mL RNase Inhibitor 0.25 mL, 103 RT Buffer 1 mL, Oligod T-Adaptor Primer
0.5 mL, 10 mM dNTP Mixture 1 mL, 25 mM MgCl2 2 mL,
RNase Free dH2O 3.75 mL in a reaction volume of 10 mL.
Then incubation at 558C for 30 min, 998C for 5 min, and
58C for 5 min was done.
Template cDNA were amplified according to manufacturer’s instructions: 53 PCR Buffer 10 mL, TaKaRa
ExTaqHS 0.25 mL, Template cDNA 10 mL Sense Primer
(10 mM) 0.5 mL, Anti-Sense Primer (10 mM) 0.5 mL then
416
LIU ET AL.
TABLE 1. Effect of TFA on recovery of neurological function in PSD mice
Group
Sham
Ischemia
Model
Flu
TFA
Postoperative
Dose /
mg kg21
–
–
–
2.5
160
80
40
2.50
1.67
1.78
1.73
1.63
1.40
2h
d1
d3
0
6 1.35
6 1.32D
6 1.09
6 0.82
6 0.80
6 0.84
0
6 0.71
6 0.76D
6 0.87
6 0.71
6 0.64
6 0.67
0
6 0.64
6 0.76D
6 0.60*
6 0.43*
6 0.46
6 0.60*
1.50
1.56
1.33
1.64
1.25
1.3
0.90
1.56
0.89
0.80
1.00
0.90
d5
0.70
1.67
0.67
0.58
0.88
0.60
0
6 0.45
6 0.93D
6 0.36**
6 0.52**
6 0.49*
6 0.42**
d7
0.50
1.33
0.56
0.20
0.50
0.58
0
6 0.41
6 1.00D
6 0.53*
6 0.10**
6 0.38*
6 0.42*
d14
0.40
0.78
0.44
0.10
0.38
0.36
0
6 0.35
6 0.69D
6 0.26
6 0.10*
6 0.37
6 0.25
d21
0.40
0.56
0.23
0.09
0.10
0.20
0
6 0.35
6 0.44D
6 0.20*
6 0.07**
6 0.10**
6 0.20*
Note: Neurological function was evaluated postoperatively using a seven-point behavioral rating scale as described in the
‘‘Materials and Methods’’ section. Data are mean 6 SD (n 5 15).
D
P < 0.01, versus the Sham; *P < 0.05 and **P < 0.01 versus the model.
sterile water was added to a total volume of 40 mL.
Primer sequences for the RT-PCR primers were: (forward: 50 -GACAAGGCAACTTGGCCTAC-30 and reverse:
50 -CTGTCACACACGCTCAGCTC-30 , BDNF, 352 bp,
accession number, NM007540); (forward: 50 -TACCCAGG
GAGGAGCAATAC-30 and reverse 50 -GAGGCAGCTT
GAACAACAAC-30 , CREB, 183 bp accession number,
NM009952);
(forward:
50 -GCTGAGAGGGAAATCGT
0
0
GCGT-3 and reverse: 5 -GAAGCATTTGCGGTGCAC
GATG-30 ,
b-actin,
516
bp,
accession
number,
NM007393).
PCR was done by MyCyclerTM Thermal cycler (BioRad, USA) under the following amplification conditions:
PCR cycles consisted of 3 min at 948C, 33 cycles 948C for
20 sec, 558C for 20 sec, 728C for 30 sec and a final extension at 728C for 4 min (BDNF, 352bp); 45 cycles 948C for
20 sec, 558C for 20 sec, 728C for 30 sec and a final extension at 728C for 4 min (CREB, 183 bp); 26 cycles 948C
for 20 sec, 558C for 20 sec, 728C for 30 sec and a final
extension at 728C for 4 min (b-actin, 516bp). After
amplification, samples underwent electrophoresis on 2%
agarose gels, stained with ethidium bromide and photographed under UV light. The intensity of the PCR products generated by the b-actin and target sequences
(BDNF, CREB) was quantified by a computer-assisted,
linear scanning densitometric analysis of the photograph
in reflectance mode. A ratio of target to b-actin PCR
product units was used to calculate the relative increase
of BDNF mRNA and CREB mRNA expression.
Statistical analysis. Statistical analysis was done
by SPSS 11.5. Results were mean 6 SD significant differences were assessed by one-way analysis of variance
(ANOVA) for comparisons among groups with least significant difference (LSD) test. Differences between
groups were deemed significant at P < 0.05.
score of 0). All ischemic mice showed prominent neurological function deficits 2 hr after stroke, indicated by
physical signs. Over a period of 14 days after surgery,
the neurological function of all ischemic mice recovered
to some extent. In comparison with the sham or ischemia groups, recovery of neurological function in the
model group were significantly slower (P < 0.05, P <
0.01), indicating that comprehensive separate feeding
plus chronic unpredictable mild stress (CUMS) after
stroke seriously affected the recovery of neurological
function of mice. Treatment with TFA (160, 80, and 40
mg/kg) or Flu (2.5 mg/kg) clearly improved the impairment in neurological function in PSD mice compared
with model group (P < 0.05, P < 0.01).
Effect of TFA on Immobility Time in PSD Mice
The immobility time in the forced swimming test was
significantly longer in the PSD group compared with the
sham group (Fig. 2). TFA treatment at 160, 80, and 40
mg/kg significantly reduced the total immobility time.
This suggested that TFA could reverse the behavior despair induced by PSD, similar to effect of Flu.
Effect of TFA on Learning and Memory Deficits
in PSD Mice
The passive avoidance test is a standard test used in
passive learning and memory research. Twenty-four
hours after the last time stress, the passive avoidance
test was conducted. The results showed that mice in the
model group developed a remarkable loss of learning
and memory compared with the sham-operated (P <
0.01; Table 2). After administration of various doses of
TFA, L was clearly increased, and NR was significantly
reduced (P < 0.01, P < 0.05). This result suggested that
the impairment in learning and memory of PSD mice
were partly restored by TFA treatment.
RESULTS
Effect of TFA on Neurological Status
Effect of TFA on MDA Levels, SOD and GSH-Px
Activity in PSD Mice
Mice usually exhibited some neurological function deficits after cerebral ischemia (e.g., hemiplegia, loss of balance, no spontaneous motor activity). Two hours after
MCAO 30 min/reperfusion, we immediately noted the
neurological function score (Table 1). Sham-operated
mice did not show neurological deficits (neurological
MDA levels were elevated, and the activity of SOD
and GSH-Px was significantly decreased by 61% and
69.8%, respectively, in PSD mice (Table 3). Treatment
with TFA (160, 80, and 40 mg/kg) or Flu (2.5 mg/kg)
could markedly reduce lipid peroxidation, and elevate
the activity of the two enzymes to near control values.
417
POSTSTROKE DEPRESSION
Effect of TFA on Histopathological Response of
Hippocampal Tissues in PSD Mice
H&E staining revealed that in the sham-operated
group the morphology of neurons in CA1, CA3, and DG of
hippocampal tissues were normal, which manifested as
intact cellular bodies, clear structure, light-red cytoplasm
and deep-blue nucleus (Fig. 3A). In the model group, pyra-
mid neurons of the CA1, CA3, and granule neurons in
dentate gyrus were severely degenerative, including cellular swelling, loss or death of neurons, partial karyopyknosis in neurons, and proliferation of microglial cells (Fig.
3C). Treatment with TFA at different doses or Flu clearly
improved pathomorphological change of hippocampal neurons, such as reduction of neuronal degeneration and loss
or death of neurons (Fig. 3E–G).
Effect of TFA on Hippocampal BDNF
Immunoreactivity in PSD Mice
Immunohistochemical staining demonstrated normalsized BDNF-positive neurons arranged closely in the
sham-operated group that were mainly in pyramid cell
layer of the CA1 and CA3, and in the granule neuron
layer in DG (Fig. 4A). Neurons of model mice were in a
loose arrangement; some had degenerated into a
vacuole- or atrophic-like shape, and the relative number
of BDNF-positive neurons was significantly reduced
(Figs. 4C, 5). Long-term TFA treatment at 160, 80,
or 40 mg/kg or Flu (2.5 mg/kg) significantly improved
pathomorphological change induced by PSD and increased the relative number of BDNF-positive neurons
(Figs. 4E–G, 5).
Fig. 2. Effect of TFA on the immobility time in PSD mice in the
forced swimming test. Procedures are described in ‘‘Materials and
Methods’’ section. Data are mean 6 SD of 15 mice per group. DP <
0.05 versus the sham; *P < 0.05 and **P < 0.01 versus the model. S,
sham; I, ischemia; M, model.
Effect of TFA on the Expressions of BDNF
mRNA and CREB mRNA in PSD Mice
We examined the levels of BDNF mRNA and CREB
mRNA by RT-PCR to further investigate the protective
TABLE 2. Effect of TFA on impairment of learning and memory in PSD mice
Learning test
Group
Sham
Ischemia
Model
Flu
TFA
21
L (S)
Dose/mg kg
2.5
160
80
40
80.8
71.7
14.3
69.2
70.1
71.6
72.0
6
6
6
6
6
6
6
Memory test
NR
44.4
54.9
7.8D
63.7**
54.8**
58.8**
66.8**
3.8
4.8
10.6
6.0
5.6
5.7
6.3
6
6
6
6
6
6
6
L(S)
2.2
4.4
5.3D
3.3*
4.4*
5.9*
3.6*
241.9
225.3
99.8
213.0
219.9
190.5
217.4
6
6
6
6
6
6
6
NR
93.5
96.7
62.4D
110.5**
95.6**
120.6*
104.2**
1.0
1.4
2.4
1.5
1.3
1.4
1.6
6
6
6
6
6
6
6
0.5
0.7
1.0D
0.6**
0.8**
0.5**
0.9*
Note: Twenty-four hours after the last time stress, mice were trained individually in a step-through passive avoidance apparatus termed a ‘‘shuttle-box’’. A foot shock (40 V) was delivered, and the L and NR for the mice to move from the light
compartment to the dark compartment was recorded. Data are mean 6 SD (n 5 15).
D
P < 0.01 versus the Sham; *P < 0.05 and **P < 0.01 versus the model.
TABLE 3. Effect of TFA on MDA levels, and the activity of SOD and GSH-Px in brain tissue of PSD mice
Group
Sham
Ischemia
Model
Flu
TFA
Dose/ mgkg21
2.5
160
80
40
MDA/Ug21pr
2.57
2.88
4.56
3.40
2.52
2.78
3.70
6
6
6
6
6
6
6
0.57
0.88
0.93DD
0.63**
0.64**
0.87**
0.62**
SOD/NUmg21pr
56.81
47.28
34.70
56.00
89.00
55.16
39.53
6
6
6
6
6
6
6
15.88
15.28
10.45DD
12.89**
15.60**
17.78**
16.53
GSH-PX/activity U
22.79
19.8
15.90
21.86
26.95
21.92
23.15
6
6
6
6
6
6
6
7.81
8.93
5.96D
4.43*
6.39**
3.50*
5.63*
Note: brain samples were collected after the passive avoidance task and 10% (w/v) homogenates were prepared as described
in the ‘‘Materials and Methods’’ section. MDA level, and the activity of SOD and GSH-Px was measured. Data are mean 6
SD (n 5 8).
D
P < 0.05 and DDP < 0.01 versus the sham. *P < 0.05 and **P < 0.01 versus the model.
418
LIU ET AL.
Fig. 3. Effect of TFA on the histopathology of the hippocampus of
PSD mice. Mice were decapitated and hippocampus tissue quickly
removed for histological analysis after the passive avoidance task.
Sections (5 mm) were cut. Paraffin sections were stained with hematoxylin and eosin and observed with a microscope for conventional
morphology (Original magnification A–G, 3400, scale bar 5 50 mm).
(A) Sham, (B) Ischemia, (C) Model, (D) Flu 2.5 mg/kg, (E) TFA 160 mg/
kg, (F) TFA 80 mg/kg, and (G) TFA 40 mg/kg.
effect of TFA on hippocampal neurons. Densitometric
analysis of the levels of BDNF mRNA and CREB mRNA
(which were normalized to expression levels of the
housekeeping gene b-actin and expressed as the ratio of
b-actin) revealed a significant increase of the levels of
BDNF mRNA and CREBmRNA in PSD mice upon treatment with TFA. A significant reduction in BDNF mRNA
and CREB mRNA levels in the hippocampus were
observed after the PSD procedure compared with the
sham-operated or ischemia group. TFA (160, 80 mg/kg)
or Flu (2.5 mg/kg) could clearly increase the expression
of BDNF mRNA and CREB mRNA in the hippocampus
(Fig. 6).
DISCUSSION
Experimental models of depression are essential for
investigating the psychobiology of depression and for
screening new antidepressants. Having access to highly
Fig. 4. Effect of TFA on the immunohistochemical examination of
BDNF in the hippocampus of PSD mice. Mice were decapitated and
hippocampus tissue quickly removed for immunohistochemical analysis after the passive avoidance task. Expression of BDNF protein in
the hippocampal CA1, CA3, and DG regions was evaluated by mean
optical density values with an Olympus microscope. Scale bar 5 50
mm. Original magnification A–G, 3400. (A) Sham, (B) Ischemia, (C)
Model, (D) Flu 2.5 mg/kg, (E) TFA 160 mg/kg, (F) TFA 80 mg/kg, and
(G) TFA 40 mg/kg.
valid animal models is very important (Willner, 1991).
Separate feeding plus stressors based on cerebral ischemia was developed as a well-validated animal models of
depression for mimicking biological and psychosocial
syndrome resemblances between the animal model and
clinical depressive condition in humans (Liu et al., 2004;
POSTSTROKE DEPRESSION
419
Fig. 6. Effect of TFA on the levels of hippocampal BDNF mRNA
and CREB mRNA expression in PSD mice. BDNF mRNA and CREB
mRNA expression was detected by RT-PCR. (A), (B): PCR products
were separated on a 2% agarose gel and stained with ethidium bromide. (C), BDNF mRNA expression, and (D), CREB mRNA expression,
upper panel shows RT-PCR example from three experiments and
lower panel shows relative ratios of BDNF mRNA and CREB mRNA to
b-actin, respectively. Data are mean 6 SD of BDNF mRNA and CREB
mRNA to b-actin from at least three experiments. DP < 0.01 versus
the Sham; *P < 0.05 and **P < 0.01 versus the model. S, sham; I, ischemia; M, model. M, Marker; 1, sham; 2, ischemia; 3, model; 4, flu;
5, TFA 160 mg/kg; 6, TFA 80 mg/kg; and 7, TFA 40 mg/kg.
Fig. 5. Effect of TFA on mean optical density of BDNF immunoreactivity in DG and CA1, CA3, regions in PSD mice. The mean optical
density value was analyzed in three fields of the same magnification
with Image J 1.34 analysis system software. Data are mean 6 SD of
six mice per group. DP < 0.01 versus the sham; *P < 0.05 and **P <
0.01 versus the model. S, sham; I, ischemia; M, model.
Tang et al., 2004). We think that the key feature of
a PSD model is to first set-up a model of cerebral
ischemia. Garcia (Garcia, 1984) thought that the ideal
cerebral ischemia model should meet following requirements: (a) a single artery that can be occluded reproducibly; (b) that vascular occlusion results in predictable
changes in blood flow, i.e., focal or regional ischemia;
and (c) reperfusion is possible after cerebral ischemia.
The MCAO model produced by an intraluminal thread
not only meets these requirements, but also has other
advantages: (i) the skull is not opened, thereby avoiding
a change in the internal environment of the skull and
intracranial infection; (ii) the model is reliable, causes
little trauma, and is stable; and (iii) mortality is very
low and neurological function recovers 1 week after surgery. We chose a MCAO 30 min/reperfusion procedure to
produce a model of cerebral ischemia in the mouse.
There were no neurological deficits in the sham-operated
group; and all ischemia-operated mice displayed high
neurological function scores 2 hr after surgery.
Stress is considered to be important for the genesis of
depression, as well as a risk factor in depressive disorder development (Barden, 2004). The CUMS regimen,
an experimental model of depression with good validity,
was employed in conjunction with single rearing to
mimic psychosocial stress including ‘‘daily hassles’’ and
the social isolation of stroke survivors (Willner, 1991).
Separate feeding may cause social memory impairment,
and a reduction in social interaction (Von Frijtag et al.,
2000). The PSD mouse model was set-up on the basis of
focal cerebral ischemia by means of a MCAO 30 min/
reperfusion procedure followed by separate feeding plus
CUMS. Mice were then subjected to forced swimming
420
LIU ET AL.
and a passive avoidance test to screen behavioral
change. The forced swimming test was considered a relatively quick and simple test to the predict antidepressant activity of drugs (Borsini and Meli, 1998; David
et al., 2003; Redrobe and Bourin, 1999). The immobility
in this test reflected a failure of persistence in escapedirected behavior, or the development of passive behavior that disengages the animal from active forms of coping with stressful stimuli, and this behavior is sensitive
for antidepressants. The passive avoidance test was considered to reflect hippocampus-dependent learning and
memory ability. Consistent with previous reports, the
present study showed that PSD animals failed to acquire
a normal escape response as demonstrated by the
increased immobility time in forced swimming test, and
an increased number of errors and reduced latency to a
darkened environment in the passive avoidance task,
which simulated core symptoms in PSD patients. These
behavioral abnormalities induced by PSD were reversed
by long-term treatment with TFA at 160, 80, and 40 mg/
kg or Flu (2.5 mg/kg). Taken together, the results from
the current study confirmed and extended previous findings that TFA could effectively inhibit depression behaviors, and showed a potential protective effect against
injury by PSD insult, similar to the classical antidepressant Flu. Full understanding of the mechanisms responsible for the protective effect of TFA is of great relevance
with respect to its therapeutic potential.
Oxidative stress has emerged as a potentially important factor in the pathogenesis of PSD. The brain is
entirely dependent upon oxidative metabolism for cell
survival, and is particularly sensitive to oxidative damage because of its high content of iron, polyunsaturated
fatty acids, catecholamines and excitatory amino acids,
all of which may mediate oxidative stress and ROS
production (e.g., superoxide, hydroxyl ion, nitric oxide)
(Marciniak and Petty, 1996; Juurlink and Sweeney,
1997). Recognition of the involvement of oxidative
stress in neurodegenerative diseases has generated
substantial interest in investigating the ability of naturally occurring antioxidants (photochemical) to ameliorate neuronal damage associated with neurodegeneration and aging (Youdim and Joseph, 2001; Joseph
et al., 2005). MDA, the product of lipid peroxidation of
the cell membrane, indirectly represents the degree of
free-radical production and lipid peroxidation. SOD
and GSH-Px, indicators of the activity of antioxidative
enzymes, are very important metabolic enzymes in
neurons, and directly reflect the scavenging capacity of
some ROS. We measured MDA levels, and the activity of
SOD and GSH-Px to show the extent of peroxidation and
neuronal damage in PSD mice. TFA treatment (160, 80,
and 40 mg/kg) inhibited the increases in levels of brain
MDA and elevated the activity of SOD and GSH-Px. This
may be an important mechanism by which TFA exerts an
antidepressant effect.
Hippocampal formation is a major target tissue in
neurodegenerative disorders and is particularly susceptible to the deleterious effect of stressors (Smith et al.,
1991; Smith et al., 1996). Recent evidence suggests that
depression also has a close association with neuroplasticity and cellular adaptation which may be because of the
downstream events beyond the receptors at the levels of
intracellular signaling molecules (Duman et al., 2000). It
is therefore of great significance to study and develop
antidepressant drugs that can promote hippocampal
neurogenesis. Neurotrophic factors such as BDNF are
known to be crucial for the survival of neurons during
development. The BDNF gene contains a cAMP response
element to which phosphorylated CREB binds and
thereby enhances transcription (Korte et al., 1995).
CREB is a transcription factor regulating expressions of
several genes involved in neuroplasticity, cell survival,
and cognition; CREB can be modulated by BDNF. The
AMPc-MAPkinase-CREB-BDNF cellular cascade may
have a significant role in the mechanisms of dendritic
restructuring, increase in hippocampal neurogenesis and
survival of nerve cells, and may serve as protective factor in neuronal development. Recent basic and clinical
studies provide evidence for a neurotrophic hypothesis of
depression and antidepressant action. According to this
hypothesis, reduced expression of BDNF or CREB could
contribute to the atrophy of hippocampal neurons in
response to stress in stroke patients, and the upregulation of BDNF and CREB could help to protect neurons
from damage and death, which may be the mechanism
of antidepressants (Nestler et al., 2002a; Nestler et al.,
2002b). Cerebral ischemia/reperfusion and stress resulting in reduction of hippocampal BDNF levels are accompanied by structural change and neuronal damage in
certain brain areas (e.g., hippocampus, frontal cortex)
(Vollmayr et al., 2001). Long-term administration of various antidepressants increases the expression, phosphorylation and function of CREB and its downstream target gene BDNF in rat hippocampus, and other limbic
brain regions are thought to be involved in depression
(Thomel et al., 2000; Duman, 1996). Some molecular
events involved in PSD pathology are known to affect
the process of neurogenesis entirely or partially, for
example, exposure to psychosocial or stress decreases
the number of new neurons in the DG, CA1, and CA3
hippocampus subfields by downregulating the proliferation of granule cell precursors. Histological analysis in
our study indicated that many cells characterized by
pyknosis or karyorrhexis were most frequently seen in
the hippocampal CA1, CA3, and DG subfields (H&E
staining) in the model groups. Expression of BDNF
mRNA and CREB mRNA were reduced in PSD mice by
RT-PCR. Long-term TFA treatment (80, 160 mg/kg)
could effectively ameliorate neuron injury, and increase
expression of BDNF mRNA and CREB mRNA. These
results are in accordance with studies showing that certain classical antidepressants (e.g., Flu) can increase
BDNF and CREB levels in rat hippocampus (Tiraboschi
et al., 2004). These observations suggest that TFA can
upregulate BDNF-CREB levels, and may have an important role in protecting hippocampal neurons against
PSD-induced injury.
In summary, these findings raise the possibility that
TFA treatment, via antilipid oxidation, and upregulation of BDNF and CREB levels, produces a protective
effect against injury in response to chronic stress or
other environmental stimuli after stroke, thereby exerting an antidepressant-like effect. These findings shed
light on the mechanisms by which TFA acts as a neuroprotective agent, particularly as an antioxidant and
modulator of the hippocampal neurotrophin and transcription factors.
These data may help to support future development of
TFA as a potential treatment for PSD.
POSTSTROKE DEPRESSION
ACKNOWLEDGMENTS
The author is grateful to De-yi Cen (Department of
Pharmacology, Basic Medical College, Anhui Medical
University, Hefei, China) and Hai-yan Yan (Department
of Pharmacology, Basic Medical College, Anhui Medical
University) for their assistance in this study.
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