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Brain Research Bulletin 170 (2021) 264–273

Contents lists available at ScienceDirect

Brain Research Bulletin

journal homepage: www.elsevier.com/locate/brainresbull

Effects of vanillic acid on Aβ1-40-induced oxidative stress and learning and

memory deficit in male rats
Nesa Ahmadi a, b, Samaneh Safari a, b, Naser Mirazi b, Seyed Asaad Karimi a, c,
Alireza Komaki a, c, *, 1
a
Neurophysiology Research Center, Hamadan University of Medical Sciences, Hamadan, Iran
b
Department of Biology, Faculty of Basic Sciences, Bu-Ali Sina University, Hamadan, Iran
c
Department of Neuroscience, School of Science and Advanced Technologies in Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: Alzheimer’s disease (AD) is a neurodegenerative disease, in which the accumulation of β-amyloid (Aβ) peptide in
β-Amyloid the extracellular space causes a progressive reduction in cognitive performance. Aβ stimulates active oxygen
Vanillic acid species generation leading to oxidative stress and neural cell death. Vanillic Acid (VA) is the oxidant form of
Oxidative stress
vanillin widely found in vanilla beans. VA has many properties, such as suppressing apoptosis and eliminating
Hippocampus
the harmful effects of oxidative stress in animal models. The VA effects on impaired learning and memory in Aβ
Learning and memory
Alzheimer’s disease rats were assessed. Forty adults male Wistar rats were assigned to the following five groups in random: the
control, sham (received saline (vehicle) via intracerebroventricular (ICV) injection), Aβ (received Aβ1–40 via ICV
injection), VA (50 mg/kg by oral gavage once a day through four weeks), and Aβ + VA (50 mg/kg) groups. Open
field test, novel object recognition (NOR) test, Morris water maze (MWM) test, and passive avoidance learning
(PAL) task were performed, and finally, we determined the malondialdehyde (MDA), total antioxidant capacity
(TAC) and total oxidant status (TOS) levels. Aβ decreased the cognitive memory in NOR, spatial memory in
MWM, and passive avoidance memory in PAL tests. In contrast, VA improved learning and memory in the treated
group. Aβ significantly increased MDA and TOS and decreased TAC levels, whereas VA treatment significantly
reversed TAC, TOS and MDA levels. In conclusion, VA decreased the Aβ effects on learning and memory by
suppressing oxidative stress and can be regarded as a neuroprotective substance in AD.

1. Introduction neurofibrillary tangles within select regions including hippocampus and

Alzheimer’s disease (AD) is an age-dependent and progressive
neurodegenerative disease, affecting the central nervous system (CNS),
and eventually leading to cognitive and memory impairment (Burns and
Iliffe, 2009; Mendiola-Precoma et al., 2016). Considering the increasing
rate of older people worldwide, the number of AD cases it is estimated at
114 million by 2050 (Querfurth and LaFerla, 2010). Increased in the
prevalence of AD can lead to medical costs and death in cases over 65
years (Alzheimer’s; Prince et al., 2015). AD is mainly characterized by
the presence of neurofibrillary tangles and β-amyloid (Aβ) plaques in the
cortex and hippocampus (Samandouras et al., 2006; Tanzi and Bertram,
2005). Abnormal phosphorylation and hyperphosphorylation of the
cytoskeletal protein tau within neurons leading to the formation of
cortex (Braak and Braak, 1990). The disease is divided into familial cases
and sporadic cases (Piaceri et al., 2013). In both cases, Aβ peptides,
produced from amyloid precursor protein (APP) by β and γ-secretases,
are major contributors to the pathology of the disease (Palop and Mucke,
2010). Severe atrophy of the hippocampus due to neuronal cell death, is
one of the major hallmarks of AD progression (Wirths and Bayer, 2010).
In addition to neurofibrillary tangles and Aβ plaques, deficits in
cholinergic function (Giacobini, 2003), neuroinflammation (Heneka
et al., 2015), sex steroid hormones deficiencies (Vest and Pike, 2013),
excitotoxicity (Hynd et al., 2004), altered synaptic function (Bell and
Claudio Cuello, 2006), abnormal mitochondrial dynamics and synaptic
degeneration (Reddy et al., 2012), and oxidative stress (Markesbery,
1997) are considerate to contribute to the pathology of AD.

* Corresponding author at: Department of Physiology, School of Medicine, Hamadan University of Medical Sciences, Shahid Fahmideh Street, Hamadan, 65178/
518, Iran.
E-mail address: Komaki@umsha.ac.ir (A. Komaki).
1
URL:umsha.ac.ir

https://doi.org/10.1016/j.brainresbull.2021.02.024
Received 7 October 2020; Received in revised form 14 February 2021; Accepted 24 February 2021
Available online 27 February 2021
0361-9230/© 2021 Elsevier Inc. All rights reserved.
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N. Ahmadi et al.
Clinically AD is characterized by progressive cognitive decline and
abnormalities in behavior (Larson et al., 1992; Wilson et al., 2011) and is
the leading cause of dementia in the elderly, presenting itself clinically
by progressive loss of memory and learning (Larson et al., 1992). The
involvement of subcortical structures, including the "hippocampus" in
learning and memory processes has been well documented (Bird and
Burgess, 2008; Jarrard, 1993).
Oxidative stress reflects the imbalance between the oxidant/antiox­
idant status, with progressive generation and accumulation of reactive
oxygen species (ROS) in healthy cells. Oxidative stress is involved in
many pathological conditions, such as nervous system diseases (Jenner,
2003; Sayre et al., 2001). Evidence suggest that oxidative stress is an
early event in AD, occurring prior to plaques or tangle pathology, and
therefore may play a major pathogenic role in the disease. The AD brain
also shows evidence of ROS-induced damage (Pereira et al., 2012), and
patients with AD have damaged, oxidized DNA, RNA, protein and lipid
products caused by ROS that can be used as possible disease progression
markers (Ahmad et al., 2017). Considering the well-known association
between oxidative stress and learning and memory deficit (Alzoubi
et al., 2018; Su et al., 2008), it seems that therapeutic strategies directed
at controlling the excessive production of pro-oxidant factors may be
valuable to control neurodegeneration in AD and dementia (Agostinho
et al., 2010). Therefore, there is a lot of attention to the use and devel­
opment of antioxidant therapies to manage cognitive decline during AD.
Vanillic acid (VA) appears to have considerable antioxidant activity
(Kumpulainen, 1999). Vanillic acid (VA, a famous flavonoid, is abun­
dant in multifarious nuts, fruits, and herbs, like honey, and its crude type
is taken from the roots of an indigenous herb called Angelica Sinensis
(Fig. 1) (Singh et al., 2015). It is also detected in black rice, where it
peaked at maturity. It is normally used as a food additive (Huang et al.,
2019; Shao et al., 2014). Several pharmacologic effects, including
inhibiting the snake venom activity, as well as antimicrobial,
anti-inflammatory, and antioxidant properties have been reported for
VA (Mattila and Kumpulainen, 2002; Tai et al., 2012). The antioxidant
mechanisms of VA include exacerbating antioxidative status and ATPase
activity, free radical scavenging activity, reducing power, the inhibition
of lipid peroxidation, and increase in catalase and superoxide dismutase
(SOD) activity while inhibiting cholinergic enzymatic activity and
reactivating oxidative-deactivated purine metabolism and pentose
phosphate pathways (Chou et al., 2010; Salau et al., 2020). Vanillic acid
has been reported to have cognitive enhancing effects against
streptozotocin-induced neurodegeneration by downregulation of neu­
roinflammatory process and exerting specific anti-inflammatory and
antioxidant effects (Singh et al., 2015).
Although AD symptoms can temporarily be controlled by some
medications and therapeutic methods, no permanent treatment is
available. Accordingly, identifying new therapeutic options to slow
down or prevent the AD progression is of great importance. Given the
Fig. 1. Chemical structure of the vanillic acid.
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antioxidant effects of VA and the association between AD and
enhancement in oxidative stress and a decrease in the antioxidant status,
we hypothesized that VA will act as a neuroprotective factor against
Aβ1− 42 induced memory impairment. Accordingly, we designed the
present study for evaluating the possible therapeutic effectiveness of VA
as a therapeutic or protective agent in Aβ1− 42 -related memory and
oxidative stress deficit in male rats.
2. Materials and methods
2.1. Ethics statement
The experiments on rats were performed under animal care and use
guidelines proved by the institutional ethics committee, Hamadan
University of Medical Sciences, according to the National Institutes of
Health Guide for Care and Use of Laboratory Animals. Minimize
suffering was considered.
2.2. Animals and experimental design
Forty male Wistar rats (200–220 g) provided by the laboratory ani­
mal house of Hamadan University of Medical Sciences were studied.
Animals (four rats per cage) were housed an animal house provided with
controlled temperature (20–25 ◦ C/ a 12-h light/dark cycle/50–70 %
humidity) and water and food were available. After one week of adap­
tation, animals were randomly divided into the following groups (n =
6–8 animals in each group): group 1 (control; treated with normal saline
by oral gavage once a day for 4 weeks), group 2 (sham; intra­
cerebroventricular (ICV) injection of phosphate-buffered saline (PBS)
and receiving normal saline by oral gavage once a day for 4 weeks),
group 3 (Aβ; ICV Aβ1− 42 injections), group 4 (VA; receiving 50 mg/kg of
VA by oral gavage once a day for 4 weeks), and group 5 (Aβ + VA; ICV
Aβ1− 42 injections and receiving VA by oral gavage once a day for 4
weeks). Administration of VA was done by oral gavage, once a day
through four weeks after the Aβ injection, followed by performing the
considered tests. Dose of VA was selected according to the previous
studies (Leal et al., 2011; Singh et al., 2015). The experimental design
and schedule of the behavioral tests are shown in Fig. 2. Images of
behavioral tests are shown in Fig. 3.
2.3. ICV injection of Aβ1− 42 and neurosurgical procedure
We dissolved Aβ1− 42 (1 mg; Tocris Bioscience Bristol, UK) in 1 mL of
PBS as a vehicle and incubated for 7 days at 37 ◦ C before use that results
in producing amyloid fibrils (with neurotoxic activity) (Asadbegi et al.,
2018; Lorenzo and Yankner, 1994). Before transferring animals to a
stereotaxic apparatus (Stoelting Co., USA), anesthetization was done
through i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/ kg)
(Karimi et al., 2019). The skull was exposed over the ventricular area
according to the coordinates as follows: 2 mm lateral to the midline, 1.2
mm posterior to bregma, and 4 mm ventral to the cortex surface (Pax­
inos and Watson, 2005). The microsyringes (Hamilton Laboratory
Products, USA) (5 μL) were used for injections through 5 min and per­
formed gently (1 μL/ min). The syringes were kept in the injection site
for 5 min following the injection and then gently removed. PBS was
injected into the Sham-operated group. The rats were recovered for 7
days (Asadbegi et al., 2017). In our experiment, Congo Red staining was
performed to confirm the formation of Aβ plaque in the rats’ brain. For
this purpose, serial coronal sections of 5 μm thickness were obtained at
the level of the hippocampus. Then the slides were studied by optic
microscope and Image J software. Fig. 4 shows the Aβ plaques (red
spots) in the coronal sections of the hippocampus region.
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N. Ahmadi et al. Brain Research Bulletin 170 (2021) 264–273

Fig. 2. Experimental design and schedule of the behavioral tests.

Fig. 3. Images of behavioral tests. The Open-Field test (A) was used to assess locomotor activity. The Novel Object Recognition (B) task was used to evaluate
recognition memory. Morris water maze (C), and step-through apparatus (D) were used to evaluate the spatial and passive learning and memory, respectively.

2.4. Behavioral tests
2.4.1. Open field test (locomotion)
The open-field test (OFT) measures locomotor activity, motor
impairment, and anxiety in rodents. OFT was first developed to measure
emotions in rats (Prut and Belzung, 2003). The rats were transferred to
the testing room in their home cages and allowed to habituate through
30 min prior to testing. The apparatus was made of a black round arena
(48 × 41.5 × 36 cm) elevated 60 cm above the floor. The floor can be
seen with a grid of 16 squares. Using a video camera, the paths traveled
by animals were recorded within 10 min. The numbers of squares
crossed using four paws were calculated. The OFT box was washed using
a 70 % alcohol solution before placing other rats for preventing olfactory
perception by other animals (Asadbegi et al., 2016; Ganji et al., 2017).
2.4.2. Novel object recognition test (NOR)
NOR was first developed by Ennanceur and Delacour, according to
the spontaneous behavior of animals for recognizing a novel object in a
familiar environment (Ennaceur and Delacour, 1988). Recognition
memory as a type of declarative memory evaluates an animal’s capa­
bility for judging or discriminating between objects, considering visual
and tactile data (Antunes and Biala, 2012; Ennaceur and Delacour,
1988). In this research, the NOR test was used for evaluating the VA
effects on cognitive flexibility in a rat model of Aβ neurotoxicity. In brief,

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Fig. 4. Photomicrographs of Congo Red staining in rat hippocampus sections. Staining the Congo Red was done to confirm the presence of Aβ plaque in the
hippocampus (straight arrows) from studied groups. The scale bar is 100 μm.
the apparatus (a black wooden box) (48 × 41.5 × 36 cm) was elevated
60 cm above the floor. During the habituation (before performing the
test), the rats were allowed to freely explore the box with no object
through 10 min, and then it was subjected to a training phase (30 min
following habituation), in which two identical plastic cubbies (length: 7
cm, width: 3.5 cm, height: 8.5 cm) were located in the box. Twenty-four
hours later, the animals were subjected to the retention test, where a
novel (unfamiliar) object (length: 4 cm, width: 5.5 cm, height: 8.5 cm)
was replaced with one of the objects. Using a video camera, the rats’
behaviors were recorded, and the time taken to explore each object was
determined within 10 min. Exploration was considered as the rat placing
its nose within 2 cm from the object. The discrimination index (DI) was
determined as the time taken to explore the novel object, divided by the
whole exploration time (Takuma et al., 2014). At the end of each ses­
sion, using a wet tissue paper the box and objects were cleaned (10 %
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ethanol solution) for eliminating the remaining odors (Hashemi-Firouzi
et al., 2018).
2.4.3. Morris water maze (MWM) task
The MWM task was applied for measuring learning and spatial
memory. It includes a water-filled (to a depth of 25 cm/ 22 ± 1 ◦ C) black
circular pool (180 cm in diameter and 60 cm in height. The apparatus
was placed in a soundproof, dim lighted room; however, there were
different visual cues. The pool has four quadrants: north (N), south (S),
east (E), and west (W), as well as a fixed hidden platform just below (i.e.,
~ 1.0 cm) the surface of the water. The training trial took for 4 days at
roughly the same time each day with two blocks with four trials (90 s). A
30 s interval was considered between two trials on the platform. The
animals could rest through 5 min between two consecutive blocks. A
video camera (Nikon, USA) attached to a computer above the pool was
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N. Ahmadi et al.
used for recording the time taken to reach the hidden platform (the
escape latency). One day following the spatial acquisition phase (fifth
day), the retention phase, the probe trial was conducted, and during the
trial, the platform was removed from the pool, and animals were
allowed to swim for 60 s. The swim speed, escape latency, time spent in
the target quadrant, and distance traveled were recorded in each trial, In
the visible platform trials, the covering of the platform was done using a
sheet of aluminum foil (Moridi et al., 2020; Rezvani-Kamran et al., 2017;
Zarrinkalam et al., 2016).
2.4.4. Passive avoidance learning (PAL) task
2.4.4.1. Apparatus. The step-through apparatus was applied to estimate
passive avoidance learning and memory. It a two-compartment box
(bright and dark) with the same size (20 × 20 × 30 cm) isolated through
a guillotine door. The bright compartment was made of transparent
plastic, whereas the dark one made from dark opaque plastic. A shock
generator was used for electrifying the floor of the dark chamber. An
insulated stimulator delivered electric shocks (50 Hz, 1 mA, 1.5 s) to the
grid floor of the dark chamber (Barzegar et al., 2015; Komaki et al.,
2015).
2.4.4.2. Training. Habituation was done 30 min prior to the experi­
ments. The groups had two trials of habituation to the apparatus.
Accordingly, an animal was located in the light chamber facing away
from the door, followed by raising the door after 30 s. The rats prefer
dark environments compared to lit places. Following entering the dark
chamber, the door was closed, and 30 s later, the rat was taken from the
dark chamber placing in the home cage. After an interval of 30 min, the
habituation was repeated. At the same interval, the first acquisition trial
was conducted.
In the training trial, each rat was located in the light chamber, and 30
s later, the guillotine door was opened. The rat was free for entering the
dark chamber, and 2 min later, a mild electrical shock was applied and
the animal was returned to the home cage. We recorded entrance latency
to the dark compartment (step-through latency, STLa) when the rat put
four paws on the grid floor of the dark chamber. The experiment was
repeated after 2 min. Finally, a foot shock was received by the rat when
reentered the dark chamber; however, training was finished as the rat
remained in the light chamber through 120 s (consecutive). The entries
number into the dark compartment was noted (Habibitabar et al., 2020;
Khodamoradi et al., 2015).
2.4.4.3. Retention. In this step, 24 h following the training trial, the
retention test was conducted. Each rat was placed in the light chamber,
followed by raising the door 5 s later, and the step-through latency in the
retention trial (STLr), as well as the time spent in the dark compartment
(TDC) were noted for 300 s. The test was finished as the rat either
entered the dark chamber or stayed in the light chamber for 300 s,
showing retention of the passive avoidance response. The retention trial
was free of applying electric shock (Barzegar et al., 2015; Shekarian
et al., 2020).
2.5. Biochemical parameters
After the experiments, anesthetization was done through ketamine
(100 mg/kg)/ xylazine (10 mg/ kg) i.p. injection (ethyl carbamate, 1.8
g/kg; i.p.). Blood sampling (4 mL) was done from the vena cava vein and
transferred into heparinized tubes. The samples were centrifuged (3500
rpm/ 10 min 4 ◦ C), the serum was frozen at − 80 ◦ C and sent for
biochemistry assessment. In the end, malondialdehyde (MDA), total
antioxidant capacity (TAC) and total oxidant status (TOS) levels were
also measured (Habibitabar et al., 2020; Komaki et al., 2019; West,
1993).
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2.6. Statistical analysis
The GraphPad Prism software v. 8.0 was applied for statistical ana­
lyses using one-way and two-way analysis of variance (ANOVA) fol­
lowed by Tukey’s post-hoc test for multiple comparisons. The results are
presented as the mean ± S.E.M at a probability level of <0.05.
3. Result
3.1. Aβ and VA effects on locomotion (in OFT)
The experimental groups were found with no significant difference in
locomotor activity. There were not significant differences in distance
traveled [F (4, 36) = 2.417, P = 0.0665, one-way ANOVA, Fig. 5A] and
mean velocity [F (4, 39) = 2.155, P = 0.0923, one-way ANOVA, Fig. 5A]
among experimental groups. Thus, these results confirmed that treat­
ment with the VA did not affect locomotion.
3.2. Aβ and VA effects on recognition memory (in NOR test)
Fig.6 indicates DI related to the experimental groups. According to
the one-way ANOVA results, the groups were found with a significant
difference in the DI [F (4, 43) = 38.78, P < 0.0001, Fig. 6]. Based on the
Tukey’s post-hoc test results, the DI of Aβ rats was significantly lower
compared with the control and sham groups (P < 0.0001). post hoc
comparisons showed the memory restoring effect of VA agent against
Aβ. DI increased in the Aβ + VA group compared to the Aβ group (P <
0.0001). No significant differences were observed in the DI in the Aβ +
VA group and the control and sham groups based on the one-way
ANOVA results (P = 0.8953 and P = 0.0852, respectively).
3.3. Aβ and VA effects on memory acquisition (in MWM task training)
The escape latency (i.e., time to reach the hidden platform) and
swimming distance were used to assess the acquisition of the MWM task.
There were significant differences in escape latency between experi­
mental groups (Treatment effect: F (2.423, 16.96) = 29.28, P < 0.0001,
Day effect; F (2.148, 15.04) = 42.32, P < 0.0001, repeated measures
two-way ANOVA, Fig. 7). According to the results obtained from four
training days, the escape latency increased in the Aβ group compared
with the other experimental groups. VA improved Aβ-induced impair­
ment in memory acquisition. The escape latency decreased in the VA +
Aβ group compared with the Aβ group.
In addition, there were statistically significant differences in traveled
distance between experimental groups (Treatment effect: F (3.258,
91.22) = 27.72, P < 0.0001, Day effect; F (3, 28) = 159.8, P < 0.0001,
repeated measures two-way ANOVA, Fig. 8). Total traveled distance to
reach platform during four days of hidden platform training reveals Aβ
animals travel further before reaching the platform. The traveled dis­
tance decreased in the VA + Aβ group compared with the Aβ group.
3.4. Aβ and VA effects on spatial reference memory (in MWM task probe
test)
To assess reference memory at the end of learning, a probe trial was
conducted 24 h after the last training trial on day 5. During this test, the
platform was removed and the time spent in each quadrant of the MWM
was recorded. There were significant differences in the time spent in
target zone between the experimental groups [F (4, 35) = 8.387, P <
0.0001, one-way ANOVA, Fig. 9]. Aβ animals spent less time in the
target zone in compare with other animals. As shown in Fig. 9, animals
in Aβ + VA group spent more time in the target zone in compare with Aβ
animals.
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Fig. 5. Effect of Vanillic acid and Aβ on locomotion in open field test. The experimental groups were found with no significant difference in locomotor activity.
There were not significant differences in distance traveled (A) and mean velocity (B) among experimental groups. Data are expressed as mean ± SEM.
Fig. 6. Effect of Vanillic acid on Aβ-induced impairment in novel object-
recognition test. Discrimination index of Aβ rats was significantly lower
than the other groups. Vanillic acid increased the discrimination index of Aβ
rats. Data are expressed as mean ± SEM. *** P < 0.001.
Fig. 7. Aβ and VA effects on escape latency (during four days of hidden
platform training in MWM task). There were significant differences in latency
to find the hidden platform between the experimental groups. The escape la­
tency increased in the Aβ group compared with the other experimental groups,
and decreased in the VA + Aβ group compared with the Aβ group. Data are
presented as mean ± SEM. On the first day: Aβ vs. control *** (P < 0.001) and
sham && (P < 0.01), on the second day: Aβ vs. control *** (P < 0.001) and
sham && (P < 0.01), on the third day: Aβ vs. control *** (P < 0.001) and sham
&&&& (P < 0.0001), and on the fourth day: Aβ vs. control **** (P < 0.0001)
and sham &&&& (P < 0.0001). Also, on the first day: Aβ vs. VA # (P < 0.05),
second day: ## (P < 0.01), third day ### (P < 0.001), and fourth day ###
(P < 0.001). A significant difference was detected between the Aβ vs. Aβ + VA
on the first day # (P < 0.05), second day ## (P < 0.01), third and fourth days
### (P < 0.001).
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Fig. 8. Aβ and VA effects on traveled distance (during four days of hidden
platform training in MWM task). There were significant differences in trav­
eled distance to find the hidden platform between the experimental groups. The
traveled distance increased in the Aβ group compared with the other experi­
mental groups, and decreased in the VA + Aβ group compared with the Aβ
group. Data are presented as mean ± SEM. Aβ vs. control on third day * (P <
0.05) and fourth day **** (P < 0.0001), and vs. sham on third day && (P <
0.01) and fourth day &&&& (P < 0.0001). Aβ group vs. VA on the third (P <
0.05) and fourth days ## (P < 0.0001), and Aβ vs. Aβ + VA group on third and
fourth days #### (P < 0.0001).
Fig. 9. Measurement of the spatial reference memory in the probe test. Aβ
animals spent less time in the target zone in compare with other animals. An­
imals in Aβ + VA group spent more time in the target zone in compare with Aβ
animals. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, **** p
< 0.0001.
3.5. Aβ and VA effects on the PAL acquisition and retention
There was no significant difference in the STLa among the experi­
mental groups in the acquisition trial (before receiving the electrical
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N. Ahmadi et al.
shock) [F (4, 35) = 0.6970, P = 0.5992, one-way ANOVA, Fig. 10A].
This result indicates that the exploratory behavior of the different
groups of rats in the dark did not differ. Also, significant differences were
not observed among the different experimental groups with respect to
the number of trials to acquisition criterion [F (4, 35) = 2.333, P =
0.0748, one-way ANOVA, Fig. 10B].
The retention test was conducted 24 h after the training. It revealed a
significant difference in the STLr among the groups [F (4, 35) = 39.94, P
< 0.0001, One-way ANOVA, Fig. 10C]. Specifically, STLr in the Aβ
group was significantly less than the control group (P < 0.0001). STLr
increased in the Aβ + VA group compared to the Aβ group (P < 0.0001).
In addition, there was also a statistically significant difference in TDC
between experimental animals [F (4, 35) = 13.28, P < 0.0001, one-way
ANOVA, Fig. 10D]. Aβ group spent more time in the dark compartment
in comparison with the control animals (P < 0.0001). TDC decreased in
the Aβ + VA group compared to the Aβ group (P < 0.0001). All results
are summarized in Fig. 10.
3.6. Aβ and VA effects on biomarkers of oxidative stress
MDA is the most common indicator of oxidative stress in various
health conditions (Habibitabar et al., 2020; Khoubnasabjafari et al.,
2015; Weber et al., 2017). The impact of Aβ and VA administration on
MDA concentration in serum was evaluated. There was a significant
difference in the case of MDA among the experimental groups of rats [F
(4, 34) = 9.370, P < 0.0001, One-way ANOVA, Fig. 11A]. As illustrated
in Fig. 11A, Aβ increased MDA concentration in serum compared with
the control group (P < 0.0001, Fig. 11A). Treatment of Aβ animals with
VA decreased MDA concentration (P < 0.01). In addition, there was a
significant difference in the case of TAC among the experimental groups
of rats [F (4, 28) = 6.239, P = 0.0010, One-way ANOVA, Fig. 11B). Aβ
decreased TAC (P = 0.0018, Fig. 11B). Treatment of Aβ animals with VA
increased total TAC (P = 0.0146). Furthermore, there was a significant
difference in the case of TOS among the experimental groups of rats [F
Fig. 10. Aβ and VA effects on the PAL acquisition and retention. There was no significant difference in the STLa (A) and number of trials to acquisition criterion
(B) among the experimental groups in the acquisition trial (before receiving the electrical shock). But Aβ decreased STLr (C) and increased TDC (D) in the retention
test. VA reversed the effects of Aβ on STLr and TDC. Data are presented as mean ± SEM. **** p < 0.0001.
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(4, 24) = 26.35, P < 0.0001, One-way ANOVA, Fig. 11C). Aβ increased
TOS in compare with control group (P < 0.0001). Treatment of Aβ an­
imals with VA decreased TOS (P < 0.0001).
4. Discussion
The major findings of the present study are as follows: In Aβ rats, VA
1) decreased plasma oxidative stress, 2) improved impairment in
acquisition and retrieval phases of reference memory (in MWM test), 3)
ameliorated the impairment in the PAL acquisition and retention. Aβ
and treatment with the VA did not affect locomotion in OF test.
It has been shown that oxidative stress is involved in the neuropa­
thology of AD (Markesbery, 1997). Heat shock proteins (HSPs) are
abnormally present in the neuropathological lesions of AD, suggesting
that oxidative stress may play significant roles in this disorder (Cam­
panella et al., 2018). HSPs are one of the main markers of oxidative
stress. Pappolla et al. by immunohistochemistry revealed a very pro­
nounced increase of superoxide dismutase (SOD) and hemoxygenase-1
(HO-1) in neurons surrounding amyloid deposits in transgenic mouse
model of AD (Pappolla et al., 1998). Also, inducing oxidative stress in
primary chick brain neurons by exposure to sublethal doses of H2O2
increases levels of total secreted endogenous Aβ (Goldsbury et al., 2008).
Oxidative stress and amyloid toxicity leave neurons chemically vulner­
able (Verri et al., 2012), and plaque formation in certain areas of the
CNS causes neuroinflammation and the induction of free radicals, which
leads to the destruction of areas such as the amygdala, hippocampus and
cortex (Mancuso et al., 2007). Reducing oxidative damage and con­
trolling the excessive production of ROS can be considered a promising
strategy for therapeutic intervention in AD and control neuro­
degeneration (Dumont et al., 2009). The antioxidative activity of
vanillic acid is well documented (Sang et al., 2002; Shyamala et al.,
2007).
Our data in the present study showed that Aβ1− 42-treated rat
exhibited a significant alteration in MDA, TAC, and TOS. Our results are
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N. Ahmadi et al.
Fig. 11. Effect of Aβ and VA on serum levels of malondialdehyde (MDA)
(A), total antioxidant capacity (TAC) (B), total oxidant status (TOS) (C). Aβ
increased MDA and TOS and decreased TAC. While treatment with VA signif­
icantly reversed the effects of Aβ on these parameters. Data presented as means
± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
consistent with other previous reports (Abdul et al., 2008). High levels
of MDA have been suggested as an essential factor in the pathogenesis
and neuronal damage of AD patients (Aybek et al., 2007). Here, in our
work Aβ increased MDA concentration, and treatment of Aβ animals
with VA decreased MDA concentration. The study by Amin et al. showed
that the increase in oxidative stress in the brains of Aβ1− 42-treated mice
was significantly reduced by VA treatment (Amin et al., 2017). VA
treatment reduces ROS induction and prevents the depletion of endog­
enous reduced glutathione (GSH) levels (Verri et al., 2012).
271
Brain Research Bulletin 170 (2021) 264–273
We used OFT for locomotor activity, NOR test for cognitive memory,
MWM test for spatial memory, PAL task for passive avoidance learning
and memory, and finally, oxidative stress biomarkers as possible
mechanisms in the memory-enhancing effects of VA against Aβ. Before
behavioral tests, the motor activity of rats in OFT was evaluated. VA and
surgery did not affect locomotor activity. Therefore, it can be concluded
that the results of behavioral tests are independent of the rats’ motor
activity. In our research, the ICV injection of Aβ1− 40 reduced spatial
memory in the Aβ group. Choi et al., reported that the ICV injection of
Aβ1− 40 in mice destroyed the spatial memory in the MWM test, which
can be due to the accumulation of these peptides in the brain (Choi et al.,
2001). Besides, it has demonstrated that the administration of Aβ leads
to a decrease in antioxidant capacity leading to impairment in synaptic
plasticity, which occurs during learning and memory (Asadbegi et al.,
2018). Furthermore, it has been reported that the Aβ1− 40 injection can
induce memory impairment detected by the PAL task (Jellinger, 2002).
In other word, VA administration in Aβ rats improved their passive
avoidance memory, which can because of the antioxidant properties of
VA.
Consistent with the results of our study, Amin et al. reported that VA
significantly ameliorated cognitive deficits with increased glutathione
(GSH) levels in brain tissues and increased nuclear factor erythroid 2-
related factor 2 (Nrf2) / HO-1 expression in Aβ1− 42-treated mice
(Amin et al., 2017). It has been suggested that the beneficial therapeutic
effects of vanillic acid are through the positive regulation of Akt /
GSK-3β / Nrf2 signaling pathways (Amin et al., 2017). Recent studies
have suggested that Akt/Nrf2 signaling pathways play a central role in
the development process of AD (Hong and An, 2018; Wang et al., 2013),
and activation of Nrf2 provides neuroprotection in AD (Khodagholi
et al., 2010). Previous works have shown a reduction in Nrf2 levels in AD
(Choudhry et al., 2012; Kanninen et al., 2009), and reported that
intrahippocampal injection of a lentiviral vector expressing Nrf2 im­
proves spatial learning in a mouse model of AD (Kanninen et al., 2009).
It has been shown that VA treatment significantly increases Nrf2 protein
expression in the brains of Aβ1− 42-treated mice (Amin et al., 2017).
Based on the above, it can be concluded that one possible mechanism in
the memory-enhancing effects of VA against Aβ, is an increase in Nrf2
protein expression. On the other hand, activation of Akt / Nrf2 signaling
has been reported to reduce learning and memory deficits in APP/PS1
mice (Hong and An, 2018). VA-induced activation in the Akt pathway
can also be considered as one of the possible mechanisms in the
memory-enhancing effects of VA against Aβ (Amin et al., 2017).
Vanillic acid also reduces β-site APP-cleaving enzyme 1 (BACE-1)
expression in the Aβ1− 42-treated mice (Amin et al., 2017). β-secretase 1,
also known as BACE-1 is the key enzyme responsible for APP processing
and Aβ production (Liang et al., 2010), and is associated with neuronal
loss and dysfunction and memory impairment (Haass and Selkoe, 2007;
Yang et al., 2003).
In AD, neuroinflammation as much as plaques and tangles is
involved in AD pathogenesis (Heneka et al., 2015). Neuroinflammation
often results in enduring memory impairment and has been reported as a
part of the neuropathogenesis of cognitive impairment (Allison and
Ditor, 2014). VA treatment has been shown to reverse the increased
expression of inflammatory markers induced by Aβ1− 42 (Amin et al.,
2017), preventing neuroinflammation and memory impairment in the
Aβ1− 42-treated mice.
VA-induced elevation in the synaptophysin and post-synapse density
95 (PSD95) protein can also be considered as one of the possible
mechanisms in the memory-enhancing effects of VA against Aβ (Amin
et al., 2017). Animal and human studies have shown that PSD95 and
synaptophysin expression decreases in AD (Amin et al., 2017; Canas
et al., 2009; Tu et al., 2014). PSD95 controls AMPA receptor incorpo­
ration during long-term potentiation (LTP) and experience-driven syn­
aptic plasticity (Ehrlich and Malinow, 2004). LTP is a principal form of
synaptic plasticity in the mammalian brain, thought to underlie learning
and memory (Bliss and Collingridge, 1993). Therefore, in mice treated
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N. Ahmadi et al.
with Aβ1− 42, decreased synaptophysin and PSD95 levels were associated
with memory impairment (Amin et al., 2017).
VA has also been shown to improve spatial learning and memory
retention by preventing oxidative stress, and well known to reduce
acetylcholinesterase (AChE), tumor necrosis factor α (TNF-α) and
corticosterone by improving antioxidants to provide neuroprotection
and could be an effective therapeutic agent for the treatment of neuro­
degenerative disorders (Singh et al., 2015).
AD involves a substantial loss of the elements of the cholinergic
system (Kása et al., 1997). Deficits in cholinergic function (Giacobini,
2003) contribute to the pathology of AD, affecting cognition, behavior
and activities of daily living (White and Ruske, 2002). VA has also been
shown to reduce AChE activity in the brain of STZ-treated mice (Singh
et al., 2015). Decreased AChE activity increases acetylcholine levels and
compensates for cholinergic system defects, which ultimately lead to
improved learning and memory (Canas et al., 2009). In the clinic,
pharmacological inhibition of AChE improves memory (Weinstock et al.,
2001) and cholinesterase inhibitors are used as potential anti-Alzheimer
drugs (Weinstock et al., 2001).
In the current study, our results showed a significant reduction in
memory function as evidenced by MWM, PAL and NOR test perfor­
mance. We also observed that VA treatment (30 mg/kg, p.o, 4 weeks)
improved memory, as shown by the reduction in escape latency and
swimming distance, and the increased amount of time spent in the target
quadrant during the probe test. In the PAL test, we observed a lower TDC
and a higher STLr following VA treatment. Vanillic acid treatment
ameliorated the effects of Aβ1− 42 on discrimination index in NOR test.
The observed improvement in memory function associated with VA
treatment demonstrates its neuroprotective effect against Aβ1− 42-
induced memory impairment.
5. Conclusion
In summary, our results demonstrated that the reversal of cognitive
deficits by VA treatment in Aβ1− 42-treated rats might result from the
antioxidant activity of VA; vanillic acid treatment was associated with
an increased in TAC and a decreased in TOS and MDA. Accordingly, VA-
based natural therapeutic agents are applicable for preventing and
treating oxidative stress-associated disorders, like AD. However, more
studies and clinical trials should be conducted to measure the VA
effectiveness in AD in humans.
CRediT authorship contribution statement
Nesa Ahmadi: Conceptualization, Methodology, Investigation.
Samaneh Safari: Formal analysis, Software. Naser Mirazi: Writing -
review & editing, Data curation. Seyed Asaad Karimi: Writing - original
draft, Resources, Validation. Alireza Komaki: Supervision, Conceptu­
alization, Writing - review & editing, Resources.
Declaration of Competing Interest
There are no conflicts of interest to declare.
Acknowledgments
The current study was funded (Grant No.: IR.BASU.REC.1398.030)
by Faculty of Basic Sciences, Bu-Ali Sina University, Hamadan, Iran. The
authors are grateful to the staff of the Neurophysiology Research Center,
Hamadan University of Medical Sciences for supporting this study.
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