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Introduction
Alzheimer¡¯s disease (AD) is a neurodegenerative disorder that is clinically characterized by progressive memory loss and
other cognitive impairments. The neuropathological hallmarks of AD include the formation of senile plaque of beta-amyloid
(Ab), neurofibrillary tangles, and synapse and neuron loss in several areas (hippocampus, cortex, amygdale, and others) of
the brain[1]. Ab has been thought to be a critical factor in the pathogenesis of AD. Many studies have found
that the accumulation of Ab in the brain is associated with progressive neuronal death and cognitive
deficits[2-4]. Therefore, preventing the neurotoxicity induced by
Ab might be an optimal strategy for treatment of AD.
Ab25-35 is considered the shorter toxic
fragment exerting neurotoxic effects similar to those produced by
Ab1-40/42, such as learning and memory impairment, neuronal
apoptosis, cholinergic dysfunction, and oxidative
stress[5,6], thus Ab25-35 is
usually used to establish the AD model for study of
the neurotoxic properties of Ab and drug screening.
Because memory loss and cognitive dysfunction are the main clinical
symptoms of AD patients, any therapy of AD
requires identification of the factors that can confer protection against learning and memory impairment. Compound FLZ
(Figure 1) is a novel synthetic cyclic analogue of natural squamosamide from a Chinese
herb[7]. Our previous studies have demonstrated that compound FLZ has a strong
antioxidant property, and that FLZ protected against the
damage and apoptosis of primary cultured rat brain
neurons and PC12 cells exposed to hydrogen peroxide, glutamate,
N-methyl-D-aspartate (NMDA), dopamine,
MPP+, and
ischemia-reoxygenation. FLZ also improved abnormal
behavior due to the functional disturbance of dopaminergic and cholinergic neurons in mice, indicating that FLZ possesses
a neuroprotective property[8]. It is very interesting to study whether FLZ can attenuate learning and memory impairment, and
pathological and biochemical damages in the hippocampus induced by
icv injection of Ab25-35 in mice.
Materials and methods
Reagents Compound FLZ was kindly provided by Professor Xiao-tian LIANG in the Department of Pharmaceutical
Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences. FLZ, a white powder with 99% purity, was
suspended in 0.5% (w/v) sodium carboxymethyl cellulose (CMC-Na) for oral administration. Tacrine (Sigma
Chemical, St Louis, MO, USA) was dissolved in saline.
Ab25-35 (Sigma) was dissolved in sterile double-distilled water at a concentration of
2 g/L, and incubated at 37 °C for 4 d for aggregation, and then stored at -20 °C for
use[9]. Anti-acetylcholinesterase (AChE)
polyclonal antibody was purchased from Boster Biotechnology (Boster, Wuhan, China). Bcl-2 and Bax polyclonal antibodies,
second antibody and diaminobenzidine tetrahydrochloride (DAB) were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). Other reagents were of analytical grade from Beijing Chemical Company (Beijing, China).
Animals Male ICR mice weighing 23-27 g were obtained from the Center of Experimental Animals, Chinese Academy of
Medical Sciences (Grade II, Certificate No SCXKJing2004-0001). The mice were housed 5 or 6 per cage in a thermoregulated
environment (23±1 °C, 50%±5% humidity) with free access to food and water, under a 12 h light/dark cycle. All animal
experiments followed the instructions of the Committee for Care and Welfare of Laboratory Animals in Chinese Academy of
Medical Sciences and Peking Union Medical College.
Ab25-35 injection and drug
treatment The icv injection of Ab25-35
was performed as described in a previous
study[6]. Mice were lightly anesthetized with ether. The aged
Ab25-35 or saline at a volume of 7.5 µL was gradually injected into the right
ventricle by a 28-gauge stainless-steel needle, with the following stereotaxic coordinates (in mm) from the bregma: A: -0.22, L:
1.0, V: 2.5. The injection point had already been confirmed at
ventricle by injection of Indian ink in stead of
Ab25-35 peptide in preliminary experiments. The day after icv injection, mice were randomly divided into groups including:
control, Ab25-35 model, FLZ (75 mg/kg, 150 mg/kg), and tacrine (15 mg/kg). The FLZ, tacrine and vehicle (0.5% CMC-Na) were administered
by gavage to mice once a day for 16 d. To observe the memory and learning function of mice, the step-down test was started
on d 8 and the Morris water maze test on d 11 after the injection of
Ab25-35. The learning and memory tests were carried out
between 9:00 AM and 18:00 PM.
Step-down test Amnesia in mice was examined through the step-down test on
d 8 after the Ab25-35 peptide injection. The
apparatus (Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China) was a transparent acrylic cage
. An insulated platform was fixed in the center of the grid floor. During the training, each mouse was kept in the cage for 3 min
to adapt to the environment before electricity was delivered to the grid floor for 5 min. The retention test was carried out 24
h after the above training test. The grid floor was delivered with 36 v electricity, individual mice were placed on the insulated
platform and the step-down latency and the number of errors within 5 min was recorded.
Morris water maze test The apparatus of Morris water maze (Institute of Materia Medica, Chinese Academy of Medical
Sciences, Beijing, China) test is a white circular pool, 120 cm diameter and 40 cm deep, randomly divided into 4 quadrants.
Milk powder was put into the pool to render the water opaque. The water temperature was maintained at 23±1
°C. A transparent Plexiglas platform was placed 2 cm below the water surface in the middle of one quadrant. The position of the platform was
unchanged during the training trials. Two training trials per day were conducted for 5 consecutive days from d
11 after the injection of Ab25-35. In each trial, a mouse was placed in the water faced to the pool wall at one starting position. The latency
to find the platform was recorded up to 2 min. Mice that found the platform were allowed to remain on the platform for 30 s,
and were then returned to the home cage. If the mouse could not find the platform within 2 min, it was gently guided to find
the platform by the experimenter, and allowed to remain on the platform for 30 s, and the latency was recorded for 2 min. Data
of each mouse behavior were collected by a video camera linked to a computer through an image analyzer. The total sum of
latency in two trials of each mouse was counted as the individual result of a mouse per day. The mean latency was calculated
by all tested mice per group per day.
At the end of the training period, mice were tested on a spatial probe trial in which the platform was removed, and they
were allowed to swim freely for 2 min. The latency of the location of first crossing the platform and the number of crossings
of the platform were recorded.
Malondialdehyde (MDA) measurement After completion of the Morris water maze behavior observation, 9-10 mice in
every group were killed by decapitation. The hippocampus of the brain was rapidly removed and placed on ice. The
dissected tissue was weighed and homogenized with cold 0.9% physiological saline. MDA content was determined by the
thiobarbituric acid method. 1,1,3,3-tetra-methoxypropane was used as a
standard[10].
Pathology examination of brain tissues After completion of the above behavior tests, 4 mice in every group were
anesthetized with pentobarbital sodium (50 mg/kg, ip), and the brains were transcardially perfused with phosphate buffer
solution (PBS, pH 7.4), followed by 4% paraformaldehyde in PBS (pH 7.4). The brains were removed and kept overnight in
PBS containing 4% paraformaldehyde at 4 °C, and embedded in paraffin. Sections of 5-µm thickness were stained with
hematoxylin and eosin. The lesions of brain tissues were observed with light microscope (NIKON E600, Japan) and the
images were collected by image analysis system (Image Pro-Plus 7200, America, SONY 3CCD, Japan).
Immunohistochemistry assay
of AChE, Bcl-2, and Bax After the brains were fixed as above, they were removed and kept
overnight in PBS containing 20%-30% sucrose and 4% paraformaldehyde at 4 °C. Coronal sections of
35-mm thickness were cut using a cryostat at the hippocampus.
Immunohistochemistry was performed as described in a previous
study[11-13]. The primary antibodies were AChE (1:500),
Bcl-2, and Bax (1:200). Immunostaining was visualized by the peroxidase method with
a biotinylated anti-rabbit secondary antibody and diaminobenzidine oxidation (ABC kit, Santa Cruz). The primary antibody
was replaced with normal serum in negative control.
Four mice were taken from each group for quantitative immunohistochemistry. One in every four sections was taken from
a continuous series of sections prepared from hippocampal tissue. Six sections were selected in each mouse, so 24 sections
of each group were read under a ×10 objective, and the number of positively stained cells in each group was counted. The
mean of the number of positively stained cells was
calculated from 24 sections of each
group[11].
Statistical analysis Data are expressed as mean±SEM. Statistical analysis was performed by one-way ANOVA followed
by least significant difference test. In all tests,
P<0.05 was considered as statistically significant.
Results
Effect of FLZ on the learning and memory impairment induced by icv injection of
Ab25-35 in mice In the step-down test,
the icv injection of aged Ab25-35 (15 nmol/mouse) induced a significant decrease of the avoidance latency and increase of the
number of errors, and also the step-down percentage of mice from the platform increased significantly
(P<0.05). Oral administration of FLZ (75 mg/kg, 150 mg/kg) and tacrine (15 mg/kg) significantly improved the above impairments of learning
and memory induced by Ab25-35 in mice
(P<0.05, Table 1). The latency of FLZ treated groups was restored to near that of the
normal control group. The number of errors and step-down percentage were also improved markedly.
Furthermore, the Morris water maze was used to test the spatial learning and memory of each mouse from d 11 till d 16 after
the icv injection of Ab25-35. There was no difference of the mean latency to find the platform for mice between all groups on
the first training day (d 11 after the icv injection of
Ab25-35). On the second day, the latency of mice in
b-amyloid injected model group was longer than that of the control group, and still significantly longer on d 4 and d 5
(P<0.05). In comparison with
Ab25-35 model group, the latency of mice treated with FLZ (75 mg/kg, 150 mg/kg) significantly shortened on d 4 and d 5
(P<0.05). The latency of mice treated with tacrine also significantly decreased on d 5
(P<0.05,
Figure 2).
The spatial probe trial was performed to examine whether the mice had remembered the position of the platform. In
comparison with the control group, the latency of location for first crossing the platform for mice in the
Ab25-35 model group increased (P<0.05), while the number of crossings of the platform for mice decreased
(P<0.05). The treatment of mice with FLZ
(75 mg/kg, 150 mg/kg) and tacrine (15 mg/kg) significantly shortened the latency of first-crossing the platform and increased
the number of crossings of the platform within 2 min
(P<0.05, Table 2).
All the results of the above two behavioral tests indicated that FLZ protected against the impairment of learning and tion
memory function of mice induced by
Ab25-35.
Effect of compound FLZ on the MDA level in the hippocampus of mice icv injected with
Ab25-35 After completing the behavior
test of Morris water maze test, the MDA level in the cerebral hippocampus of 9-10 mice per group was deter-mined.
The icv injection of Ab25-35 induced a significant increase in MDA level in the hippocampus
(P<0.05). The treatment with FLZ (75 mg/kg, 150 mg/kg) and tacrine (15 mg/kg) markedly decreased MDA levels in the hippocampus of
Ab25-35-treated mice (Table 3).
Effect of FLZ on the pathological injury of the hippocampus induced by icv injection of
Ab25-35 in mice No remarkable neuronal abnormalities in the hippocampus from mice of the normal control group were observed, while all examined brains
of Ab25-35 model group mice showed degenera
of neurons in the hippocampus and disorder of the array of neurons. The body of neuron became short and deeply stained
with dye. Some neurons were shrunk and necrosed. Whereas the neurons in the FLZ (75 mg/kg, 150 mg/kg) and tacrine (15
mg/kg) group mice were close to that of normal control group, indicating that FLZ protected against the
injuries of the hippocampus induced by
Ab25-35 injection (Figure 3).
Effect of FLZ on AChE, Bcl-2, and Bax immunoreactivity in the hippocampus cells of mice icv injected with
Ab25-35 There were few AChE and Bax immunoreactive neuronal cells in the hippocampal CA1 region of control group mice (Figure 4, 5).
The staining was light and the immunoreactive cells
had few processes. In the hippocampal CA1 region of
Ab25-35-icv injected mice, the number of AChE
(P<0.05) and Bax (P<
0.01) immunoreactive cells increased significantly in comparison with control group mice. The immunoreactive cells
had long and darkly stained processes. Compared with
Ab25-35-treated mice, the number of AChE and Bax immunoreactive cells in the
hippocampal CA1 region of FLZ (150 mg/kg) and tacrine-treated group mice was significantly reduced
(P<
0.05 or P<0.01), the staining was light, and the marked immunoreactive cells had few processes.
The results of Bcl-2 immunochemical staining showed that the hippocampal CA1 region of control group mice contained
abundant and darkly stained Bcl-2 immunoreactive cells (Figure 4, 5). The marked cells had long processes. In the
hippocampal CA1 region of Ab25-35-treated mice, the number of Bcl-2 immunoreactive cells was less than that of control group mice
(P<0.05), the staining was light, and the marked cells had few processes. There were more Bcl-2
immunoreactive cells in the hippocampal CA1 region of FLZ (150
mg/kg) and tacrine-treated mice in contrast to
Ab25-35-treated mice (P<0.01), and the
immunoreactive cells had long processes. The staining was near to that of control group mice.
Discussion
Animal models are playing a critical role in ongoing attempts to understand the pathology and screen therapeutics of AD.
Although no available model can meet all the full pathologic spectrum of AD disease, the injection of
Ab into the brain was shown to impair learning and memory, and elicit a degree of Alzheimer¡¯s-type
neurodegeneration[2-6]. The important point of
Ab25-35-induced amnesia is the influence of the physical state of the peptide, aggregated or soluble, at the time of administration.
The in vitro incubation of
Ab25-35 leads to the formation of stable oligomeric aggregates, which contain an increased
proportion of b-sheet structure that appears to be an important feature of the
b-amyloid-induced neurotoxicity. This aggregation
mimics in vitro the slow aging process that
in vivo leads to the formation of the senile
plaques[14,15]. In the present study, a
single icv injection of aged Ab25-35 to mice induced a significant impairment of learning and memory in step-down test and
Morris water maze test, and obvious pathological lesions of the hippocampus. In mammals, the hippocampus is a critical
neural structure in the early stage of memory formation. The impairment of memory formation is caused by damage in the
hippocampus and associated areas of the temporal cortex. The deposition of
Ab first forms in temporal cortical regions including the
hippocampus[16]. Some researchers reported that
Ab-induced injury of the hippocampus was associated with
impairment in learning and memory beside biochemical changes and neuronal
degeneration[12,17,18]. Compound FLZ was
shown to prevent the neurotoxicity of Ab25-35,
as it significantly attenuated the learning and memory impairment and
pathological injury of hippocampus in mice injected with
Ab25-35.
Apoptosis is considered as the main cause of the loss of cholinergic neurons in AD. Several authors reported that
Ab25-35 induced the neuronal degeneration through an apoptosis pathway
in vitro and in
vivo[19,20]. Bcl-2 family proteins play a
pivotal role in regulating apoptotic cell death, some of which members, such as Bcl-2 and
Bcl-xL, inhibit apoptosis and others such as Bax induces cell apoptosis. The relative ratio of proapoptotic and antiapoptotic proteins is important to determine
cell survival or death[21]. The authors found that
Ab25-35 increased the expression of proapoptotic protein Bax and decreased
the expression of anti-apoptotic protein Bcl-2 in the CA1 region of hippo-campus, indicating that
Ab25-35-induced pathological injury of the hippocampus was related to cell apoptosis. Compound FLZ exerted a significant anti-apoptotic effect
through modulating the expression of Bcl-2 and Bax protein in the CA1 region of the hippocampus, which resulted in
protection against pathological injury of the hippocampus and impairment of learning and memory.
Several studies with AD models demonstrated that
Ab increases AChE expression and AChE activity not only in cell
culture but also in the intact
brain[22,23]. AChE may promote beta-amyloid plaque formation and also increase the
Ab toxicity as reported by Rees et
al[24]. Also, there is evidence to indicate that AChE is an exacerbating factor in the apoptosis of
neurons, and plays a key role in the procession of apoptotic cell death. The apoptotic neurons can then secrete higher levels
of AChE into the brain. This cascade amplification leads to progressive neuronal loss, which is the hallmark of
AD[25,26]. In addition, cholinergic system dysfunction of AD is correlated with cognitive impairment. It was reported that acetylcholine
(ACh) levels in the cerebral cortex and hippocampus were significantly decreased by centrally administered
Ab in animals[9,27]. AChE is a key
enzyme in the catabolic metabolism of ACh.
Ab25-35-enhanced AChE expression and AChE activity might decrease the ACh
level. In the present study, the icv injection of
Ab25-35
resulted in a significant increase of AChE expression, and compound FLZ markedly attenuated the increase of the number of
AChE immunopositive cells in the CA1 region of the mouse hippocampus induced by icv injection of
Ab25-35. So, we considered that the inhibition of FLZ on AChE expression may be involved in anti-apoptosis and improvement of amnesia in
mice injected with Ab25-35. We also speculated that the inhibitory effect of FLZ on AChE expression might lead to an increase
in acetylcholine level through inhibition of acetylcholine hydrolysis, which thereby enhanced the cholinergic function and
improved the impairment of learning and memory. To confirm this speculation, direct measurement of hippocampal
acetylcholine level and AChE activity is needed in future experiments.
Multiple lines of evidence indicated that oxygen free radicals (ROS) were involved in
Ab-induced neuronal apoptosis and death. Ab can induce intracellular ROS production, which causes peroxidation of protein and lipid in
neurons[28]. Ab itself can transform into a radical state and further interacts with neuronal
membranes[29]. MDA is an end product of lipid peroxidation
of biomembranes, and the MDA content usually reflects the level of lipid peroxidation and indirectly reflects the extent of
injury. The icv injection of Ab25-35
induced a significant increase of MDA content in the mouse brain hippocampus, indicating that
Ab25-35 induced lipid peroxidation
of neurons. FLZ was shown to have obvious antioxidant property, and also protected against oxidative
damage and apoptosis of neurons induced by many
toxins[8]. In the present study, we confirmed that FLZ significantly decreased the
MDA content in the hippocampus of
Ab25-35-treated mice, suggesting that FLZ has a protective action against oxidative
damage of neurons induced by Ab25-35. Some antioxidants such as vitamin E were reported to eliminate
Ab-induced apoptosis and slow the progression of
AD[30,31]. Xiao et al reported that the protective action of huperzine A and B and tacrine (AChE inhibitor) against
Ab toxicity to PC-12 cells was not cholinesterase-dependent but might be through an antioxidant
pathway[32]. So, it appears that therapeutic efforts aiming at the removal of free radicals formation or preventing their damage to related neurons may be
beneficial in pharmacol-therapy of AD.
The reason why tacrine, an AChE inhibitor, was selected as a positive control in the present study should be made clear.
This was because tacrine was the first drug approved to treat AD by the FDA in the
USA[33], and tacrine was known to attenuate the impairment of leaning and memory induced by icv injection of
Ab25-35[9]. Tacrine also showed neuropro-tective
action against Ab25-35-induced oxidative injury and
H2O2-induced
apoptosis[32,34]. The present results showed that both
tacrine and FLZ have similar neuroprotective
effect on icv injection of the Ab25-35 model in mice. However, tacrine has been known to cause liver toxicity in AD patients by
approximately 50% and has peripheral side
effects[35]. Compound FLZ of the studied dosages had no such effects in mice.
The acute oral LD50 of FLZ was over 5 g/kg body, indicating FLZ is a low toxicity
compound. Moreover, not like tacrine, FLZ is not an inhibitor of AChE
activity[8].
In summary, FLZ significantly attenuated learning and memory deficits as well as pathological and biochemical injuries of
hippocampus in mice induced by icv injection of aged
Ab25-35 through the neuroprotective pathway.
Acknowledgements
We thank Prof Shu-li SHENG and Jian-jun ZHANG for their kind suggestions on this work.
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