Life Sciences 249 (2020) 117482

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Life Sciences
journal homepage: www.elsevier.com/locate/lifescie

Treadmill exercise enhances the promoting effects of preconditioned stem T

cells on memory and neurogenesis in Aβ-induced neurotoxicity in the rats
Rokhsareh Abshenasa,b, Tayebe Artimanib,c, Siamak Shahidia, Akram Ranjbard, Alireza Komakia,
Iraj Salehia, Iraj Amirib,c, Sara Soleimani Asla,b,
⁎

a
Neurophysiology Research Centre, Hamadan University of Medical Sciences, Hamadan, Iran
b
Department of Anatomy, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
c
Endometrium and Endometriosis Research Centre, Hamadan University of Medical Sciences, Hamadan, Iran
d
Department of Pharmacy, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran

ARTICLE INFO ABSTRACT

Keywords: Aims: Improving the environment of the injured area and the preconditioning of mesenchymal stem cells (MSCs)
Treadmill exercise are promising approaches to optimize the therapeutic properties of transplanted MSCs. Herein we investigated
Beta-amyloid the synergistic effects of treadmill exercise and dimethyloxalylglycine (DMOG)-preconditioned stem cells in an
MSCs preconditioning Alzheimer's disease (AD) animal model.
Neuroprotection
Materials and methods: The MSCs were treated with DMOG for 24 h and transplanted in the AD model in-
travenously. In addition to cell transplantation, the rats went on treadmill exercise for one month. Memory
function, BDNF expression, neurogenesis, apoptosis, and antioxidant capacity were assessed using shuttle box
and Morris water maze tasks, ELISA, immunohistochemistry, western blot, and biochemical methods.
Key findings: Transplantation of DMOG-treated cells caused a memory improvement compared to the AD group
via an increase in neurogenesis and expression of nestin, Sox-2, and NeuroD. Moreover, the injection of pre-
conditioned cells was more effective in increasing the total antioxidant capacity and the BDNF level and de-
creasing the MDA and caspase-3 than the non-treated cells. Treadmill exercise improved spatial memory and
learning through an increase in BDNF and neurogenesis. Finally, treadmill exercise and transplantation of the
treated cells together had the most neuroprotective effects.
Significance: It seems that the transplantation of DMOG-treated cells besides exercise may have protective effects
in the AD model via an increase in BDNF, antioxidants, and neurogenesis and a decrease in apoptosis.

1. Introduction cells, and the increase in the secretion of brain-derived neurotrophic

The incidence of Alzheimer's disease (AD) is about 6% of the po-
pulation aged over 65 years old. The progressive deposition of Amyloid-
β (Aβ) and hyperphosphorylation of Tau protein are the key events that
lead to AD. As a result, there will be a loss of neuronal cells of the brain
cortex leading to learning and memory impairment. A number of
therapeutic approaches, including the administration of chemical and
herbal medicine and physical activity are used to inhibit AD progress
[1,2]. Despite years of research to develop a treatment against AD, an
effective treatment still does not exist. The recent advances in re-
generative medicine have drawn attention to the transplantation of
mesenchymal stem cells (MSCs) for the treatment of neurodegenerative
diseases [3,4]. They have a great potential for clinical applications. Due
to their high proliferation activity, differentiation capacity into nerve
factor (BDNF) with regenerative impacts, the administration of MSCs is
under extensive research in neurodegenerative diseases. An increase in
BDNF and functional recovery following the transplantation of bone
marrow-derived MSCs into the injured spinal cord of rats has been
shown [5]. The transplantation of MSCs into the brain could alleviate
pathological deficit, enhance the level of Acetylcholine, and improve
the cognitive ability and physical activity in aged mice [6] and AD rats
[7]. Nevertheless, poor migration and high death rate of the trans-
planted cells in sites of damage are the major impediments in stem cell-
based therapy [8]. It has been shown that MSCs are short-lived
and < 1% of the transplanted cells migrate and survive after 4 days
following injection [9]. Therefore, considerable efforts have been made
to improve the efficacy of stem cell-based therapy. Hypoxic pre-
conditioning has been suggested as a promising strategy that can

⁎
Corresponding author at: Neurophysiology Research Centre, Hamadan University of Medical Sciences, Hamadan, Iran.
E-mail address: s.soleimaniasl@umsha.ac.ir (S. Soleimani Asl).

https://doi.org/10.1016/j.lfs.2020.117482
Received 5 January 2020; Received in revised form 26 February 2020; Accepted 28 February 2020
Available online 02 March 2020
0024-3205/ © 2020 Elsevier Inc. All rights reserved.
R. Abshenas, et al.
Fig. 1. The graphical timeline of the study design. The rats were divided into control or Alzheimer (ALZ) groups. ALZ group received an ICV injection of Aβ. Fourteen
days following injection, the exercise groups (TRD) went on a treadmill for one month and MSCs groups received 1 × 106 MSCs. The learning and memory were
assessed on day 44 in all groups. Finally, the rats were killed to histopathological, biochemical, western blotting, and ELISA studies.
overcome this challenge and improve the survival of the transplanted
cells [10,11]. MSCs preconditioning using various agents such as
growth factors, hypoxia, thermal shock, cytokines, and chemokines has
been reported in different cell lines for cell therapy (for more details see
[12]). We recently reported that the preconditioning of MSCs with di-
methyloxalylglycine (DMOG) enhanced cell viability and migration in
vitro. Furthermore, the transplantation of DMOG-treated MSCs en-
hanced the level of BDNF and total antioxidant capacity in the hippo-
campus of Aβ-injected rats compared to untreated MSCs [13]. It has
been suggested that DMOG, as a cell-permeable oxoglutarate analog,
inhibits prolyl hydroxylase and subsequently stabilizes HIF-1α expres-
sion that plays an important role in the maintenance of oxygen home-
ostasis in the cells [14]. As there is a crucial relationship between the
microenvironment of the injured area (niche) and the efficacy of MSCs,
the strategies that enhance the niche of MSCs improve the function of
the transplanted cells. One of the most important factors in the survival
and differentiation of neuronal cells and tissues is BDNF that acts on
receptor kinase [15]. It is believed that marked changes in BDNF gene
expression in cortical and hippocampal neurons represent neuropro-
tective responses [16,17]. Exercise has been reported to enhance the
BDNF level in the brain and induce the plasticity of hippocampal trans-
synaptic circuitry [18]. Although there are many studies about stem
cell-based therapy in the AD model, the effects of combination therapies
with exercise and preconditioned stem cells have not yet been studied.
Therefore, in this study, it is aimed to examine the synergic effects of
DMOG-preconditioned MSCs and treadmill exercise on Aβ-induced
neurotoxicity in male rats.
2. Materials and methods
2.1. Animals' cohorts and treatment
Adult Wistar male rats (250–300 g) were obtained from the
breeding center of the animal facility of Hamadan University of Medical
Sciences (UMSHA). All experimental protocols were approved by the
Ethics Committee of UMSHA (IR.UMSHA.REC.1396.99) and conducted
during the light phase (9 AM–5 PM). No blinding was performed in this
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Life Sciences 249 (2020) 117482
study. The rats were housed in a temperature-controlled room main-
tained at 25 ± 1 °C and 55- ± 1% humidity with free access to food
and water ad libitum. The rats were randomly (simple randomization)
divided into 9 groups (n = 7 per each group): Intact (control), sham-
operated, and exercise groups (TRD), Aβ-injected (ALZ) group that re-
ceived untreated MSCs (ALZ + MSC) or DMOD-preconditioned MSCs
(ALZ + MSC + DMOG) and exercised for one month (ALZ + TRD)
following the transplantation of untreated (ALZ + MSC + TRD) or
DMOG–treated MSCs (ALZ + MSC + DMOG+TRD) (Fig. 1).
2.2. Injection of Aβ
Studies have shown that the formation of the Aβ plaque and gliosis
are necessary for the induction of cognitive impairment in the models of
AD [19]. In this study, we used the intracerebroventricular injection of
Aβ (1–40) that aggregates contained fibrillar structures similar to the
amyloid fibrils of AD [20]. After anesthesia using a mixture of ketamine
(100 mg/kg) and xylazine (10 mg/kg) that produces short-term surgical
anesthesia with good analgesia, the rats were located on a stereotaxic
instrument (Stoelting, USA). An incision was made in the scalp and the
skull was exposed. To induce AD, 5 μg Aβ (1–40), was injected into the
right lateral ventricle [13] according to Paxinos and Watson rat brain
atlas (coordinates: 0.9 mm posterior, 1.6 mm lateral, and 2.0 mm
dorsal, relative to Bregma) [21]. The injection was performed using a
30-gauge Hamilton syringe (0.1 μl/min) and the syringe was left in
place for an additional 5 min. The rats recovered on a heating pad and
received meloxicam (1.0 mg/kg s.c.) for analgesia [22]. Two weeks
after Aβ injection, the exercise groups were scheduled to run on the
treadmill (Tajhiz Gostare Omide Iranian, Model: 2011- Iran) for one
month (5 days/week), 30 min daily at a speed of 25 m/min [23].
2.3. Preconditioning and transplantation of MSCs
The isolation, culture, identification, and preconditioning of MSCs
with DMOG (CAT: D3695, Sigma-Aldrich) have been described in our
previous study [13]. In brief, the rats were exposed to Isoflurane and
MSCs were isolated from femur and tibia of male Wistar rats (70–80 g,
R. Abshenas, et al. Life Sciences 249 (2020) 117482

Fig. 2. A. Spatial memory assessing. The mean latency time to the escape platform (a: p < 0.001 vs. the control, sham, and exercise groups, b: p < 0.001 vs. ALZ
group, c: p < 0.05 vs. ALZ+ TRD group, d: p < 0.001 vs. ALZ + TRD and ALZ + MSCs groups); B: distance traveled (a: p < 0.001 vs. the control, sham, and
exercise groups, b: p < 0.001 vs. the ALZ group, c: p < 0.05 vs. the ALZ + MSCs group, d: p < 0.001 vs. ALZ + MSCs group, e: p < 0.001 vs. ALZ + TRD and
ALZ + MSCs groups, f: p < 0.01 vs. ALZ + MSCs+DMOG group); and C: percent of total time spent in the target quadrant (a: p < 0.001 and b < 0.05 vs. the
control, sham, and exercise groups, c: p < 0.001 and d < 0.001 vs. the ALZ group) in the Morris water maze. Each value is mean ± S.E.M. (n = 7 rats per group).

4–5 weeks old) and cultured with DMEM containing 10% FBS at a 5%
CO2 and in a 37 °C incubator. Cells at passages four to six were treated
with 750 μm DMOG for 24 h. Next, the preconditioned and un-pre-
conditioned cells were incubated in CM-Dil (a green fluorescent dye) for
20 min at 37 °C and transplanted with a density of 1 × 106 via the tail
vein. MSC transplantation was performed two weeks following the in-
duction of the AD model [13].
2.4. Learning and memory assessing
One month after cell transplantation and 44 days after AD induc-
tion, passive avoidance learning and spatial memory were assessed in
all groups in order that shown in animal classification.
To examine the passive avoidance test, an apparatus consisting of
two dark and white chambers that were separated by a guillotine door
was used. There was an intermittent electric shock on the floor of the
dark compartment. The rats were placed in the white compartment and
when they entered into the dark module, the door was closed and
100 V, 0.3 mA electric shocks were delivered for 5 s [24]. Twenty-four
hours later, the experiment was repeated again and the animals were
placed in the white chamber but there was no electric shock in the dark
area. The step-through latency (STL) and the total time spent in the
dark compartment (TDC) were recorded [25]. The time between the
placement in the white chamber and the entry into the dark chamber

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R. Abshenas, et al.
Fig. 3. A. Passive avoidance learning assessing. The mean of the step-through latency (a: p < 0.001 vs. the control, sham, and exercise groups, b: p < 0.01 and
c < 0.001 vs. the ALZ group, d < 0.05 vs. the ALZ + MSCs group), and B: time in the dark compartment (a: p < 0.001 vs. the control, sham, and exercise groups,
b: p < 0.01 and c < 0.001 vs. the ALZ group) in the passive avoidance task. Each value is mean ± S.E.M. (n = 7 rats per group).
was recorded as STL. Spatial memory was carried out using the Morris
water maze (MWM) task that consisted of a water-filled black pool
(180 cm in diameter, 60 cm in depth) with a platform 2 cm below the
surface of the water. There were some constant visual cues around the
pool. The rats were trained on the task for 4 consecutive days consisting
of two blocks of four trials. In each trial, the animals were allowed to
swim for 60 s to find the hidden platform. There were 30 s and 5 min
intervals between the trials and blocks, respectively. A video camera
installed above the pool recorded the time to reach the platform (escape
latency) and the length of the swim path (traveled distance). On day 5,
a probe trial was performed for 60 s without a platform and the per-
centage of time spent in the target quadrant was assessed as a para-
meter of memory retrieval.
At the beginning of the study we defined that any animals with an
obvious movement disorder and vision problem will be excluded, so
after probe trial, each rat was given a single 60-s visible test that the
platform was covered with aluminum foil.
2.5. Histological and immunohistochemical examination
After the behavioral test, three rats from each group were an-
esthetized using ketamine (100 mg/kg) and xylazine (10 mg/kg) and
transcardially perfused with 4% paraformaldehyde and post-fixed in
the same solution. After tissue processing, coronal sections (with the
thickness of 10 μm) of the hippocampus were prepared and processed
for immunohistochemical staining of nestin and Sox-2, double im-
munostaining of CM-Dil, and cresyl violet staining. To examine the
expression of nestin and Sox-2 in the hippocampus, five sections of each
rat were blocked in a 10% normal serum with 1% BSA in TBS for 2 h,
and incubated with a rabbit anti-nestin and Sox-2 antibodies (1:100,
Abcam, Cambridge, UK) overnight at 4 °C and a secondary antibody
horseradish peroxidase (HRP)-conjugated anti-rabbit, Abcam,
Cambridge, UK) for 1 h. After incubation of the slides with DAB
(Abcam, Cambridge, UK) as a chromogenic substrate for 20 min, the
nuclei were counterstained with hematoxylin.
To assess cell density in the hippocampus, the slides underwent
cresyl violet staining (0.1% cresyl violet for 3 min), and were photo-
graphed with a digital camera attached to a light microscope. Only
intact neuronal cells with clearly defined cell bodies and nuclei were
counted.
For cell tracking, double immunostaining of CM-Dil was done. After
permeabilization (0.5% Triton X-100) and blocking (1% bovine serum
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Life Sciences 249 (2020) 117482
albumin), the sections were stained with DAPI and visualized using
fluorescent microscopy.
2.6. Western blot analysis
Four rats from each group were killed by exposing to Isoflurane and
decapitation with specialist rodent guillotines and the hippocampi were
dissected out. The left hippocampi were homogenized in lysis buffer
containing Ripa buffer and inhibitor cocktail (1:10) for 1 h and cen-
trifuged at 12,000g for 20 min at 4 °C. After denaturation, total protein
was separated on 12% SDS-PAGE (sodium dodecyl sulfate poly-
acrylamide gel) and transferred to a nitrocellulose membrane. The
membrane was blocked with 5% skim milk and incubated with a rabbit
anti-NeuroD, caspase-3, and b-actin antibodies (1:1000, Abcam,
Cambridge, UK) overnight at 4 °C and HRP-conjugated anti-rabbit
secondary antibody (1:10,000, Abcam, Cambridge, UK) for 1 h. The
bands were detected by incubation with an ECL Western Blotting
Substrate Kit (Abcam, Cambridge, UK) and the densities were measured
by ImageJ software. Experiments were done in triplicate.
2.7. Determination of BDNF concentration using ELISA
The right hippocampi were homogenized in PBS and centrifuged for
10 min at 12,000g and 4 °C. The supernatant was collected for analysis.
Commercial BDNF ELISA kits (ZellBio, Germany) were used to quantify
the concentration of BDNF in each sample according to the manufac-
turer's instructions.
2.8. Measuring the biomarkers of stress oxidative
Previously published protocols were used to assess the mal-
ondialdehyde (MDA, a marker of lipid peroxidation) content, total an-
tioxidant capacity (TAC), and total thiol molecule (TTM) in the super-
natant of the homogenized hippocampi [26].
2.9. Statistical analysis
The data were presented as means ± S.E.M. and the statistical
analyses were performed using SPSS 20 software. The one-way analysis
of variance (ANOVA) and Tukey's multiple comparison test was used to
analyze the significance between the groups. Significance was assumed
at p ≤ 0.05.
R. Abshenas, et al. Life Sciences 249 (2020) 117482

Fig. 4. Tracking transplanted stem cells. Detection of signals of CM-Dil (green color) and DAPI (blue color) in the hippocampus revealing that the injected cells
successfully crossed the BBB and arrived at the hippocampus. Part of this figure has been published in our previous study [13]. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)

3. Results
Considering the fact that there were no significant differences be-
tween the control, sham, and exercise groups, they were all listed as
control groups. The results of the visible trial did not show any move-
ment disorder and vision problem, so all of the rats included in the
study.
3.1. The synergic effects of treadmill exercise and preconditioned MSCs on
memory function in the hippocampus of Aβ-injected rats
The results of the four training days showed that the Aβ-treated
group (ALZ group) spent more time finding the hidden platform in
MWM (p < 0.001) compared to the control groups (Fig. 2A). The la-
tency for the MSC-treated groups was significantly less than that of the
ALZ group, but there was no remarkable difference between DMOG-
treated and non-treated MSC groups. Further analysis showed that
treadmill exercise along with MSC therapy caused a reduction in escape
latency when compared with the ALZ + TRD group (p < 0.05). The
preconditioned MSCs groups that exercised for one month exhibited a
shorter latency compared to the ALZ + MSC or ALZ + TRD groups
(p < 0.05).
According to the results, Aβ-treated groups swam farther to reach
the platform (p < 0.001) compared to the control groups (Fig. 2B).
Intravenous transplantation of DMOG-treated cells resulted in a less
traveled distance compared with the non-treated MSCs group
(p < 0.05). Exercise for one month besides the administration of non-
treated cells, also significantly increased this parameter (p < 0.01).
Ultimately, the greatest effect was observed in the
ALZ + MSC + DMOG+TRD group which was significant compared to
the ALZ + MSC + TRD (p < 0.001) and ALZ + MSC + DMOG
(p < 0.01) groups. The analysis of the probe trial indicated that the
control groups spent less time in the target quadrant compared to the
ALZ group, indicating a deficit in memory retention (p < 0.001,

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R. Abshenas, et al. Life Sciences 249 (2020) 117482

Fig. 5. Immunohistochemical examination of cell proliferation. Detection of nestin-positive cells in the dentate gyrus (K) of the control (A), sham (B), exercise (C),
ALZ (D), ALZ + TRD (E), ALZ + MSC (F), ALZ + MSC + TRD (G), ALZ + MSC + DMOG (H), and ALZ + MSC + DMOG+TRD (I) groups using im-
munohistochemical staining. Blue arrows show nestin-positive cells (a: p < 0.001 vs. the control and sham groups, b: p < 0.001 vs. the ALZ group, c: p < 0.05 vs.
the exercise group, d: p < 0.05 vs. the ALZ + TRD group, e: p < 0.01 vs. and ALZ + MSCs group). Each value is mean ± S.E.M. (n = 5 slides of three hippocampi
of each group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2C). A slightly greater increase was found in the groups that ex-
ercised for one month or received MSCs but there was no significance.
Treadmill exercise along with the transplantation of un-preconditioned
or preconditioned MSCs was found to improve the deficit remarkably in
the probe trial (p < 0.01 and p < 0.001, respectively) when com-
pared to the ALZ group.
The results of passive avoidance learning test showed that the Aβ-
treated group had less latency to the entrance of the dark chamber
(STL) than the control groups (Fig. 3A, p < 0.001). The injection of
untreated or treated MSCs besides exercise increased the STL
(P < 0.001) relative to the ALZ group. There was also a significant
difference between the ALZ + MSC + DMOG+TRD and ALZ + MSC
groups (p < 0.05).
According to the results, the control groups spent less time in the
dark chamber (TDC) on the second day of the test which was significant
compared to the ALZ group (Fig. 3B, p < 0.001). The increase in TDC
with Aβ injection was significantly attenuated by the treadmill exercise
or injection of DMOG-treated MSCs (p < 0.01). Similar to the results
of MWM, the ALZ + MSC + DMOG+TRD group showed a more sig-
nificant difference than the previous two groups (p < 0.001).
3.2. The synergistic effects of treadmill exercise and preconditioned MSCs
on cell migration and neurogenesis in the hippocampus of Aβ-injected rats
The MSCs were labeled with CM-Dil before transplantation. As
shown in Fig. 4, the transplanted MSCs were distinguishable from the
host cells by green fluorescent labeling cytoplasms and blue nuclei. Our
results showed that the injected cells successfully crossed the brain-
blood barrier (BBB) and arrived at the hippocampus. The highest cell
migration was observed in the groups that received DMOG-treated
MSCs alone or with treadmill exercise.
To evaluate the synergic effects of the preconditioned MSCs and

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R. Abshenas, et al. Life Sciences 249 (2020) 117482

Fig. 6. Immunohistochemical examination of neuronal survival. Detection of Sox2+ cells in the dentate gyrus (K) of the control (A), sham (B), exercise (C), ALZ (D),
ALZ + TRD (E), ALZ + MSC (F), ALZ + MSC + TRD (G), ALZ + MSC + DMOG (H), and ALZ + MSC + DMOG+TRD (I) groups using immunohistochemical
staining. Blue arrows show Sox2+ cells. (a: p < 0.001 and b < 0.05 vs. the control and sham groups, c: p < 0.001 vs. the exercise group, d: p < 0.001 vs. the ALZ
group, e: p < 0.05 vs. the ALZ + TRD and ALZ + MSCs groups). Each value is mean ± S.E.M. (n = 5 slides of three hippocampi per group). (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of this article.)

exercise on neurogenesis, the expression of nestin and Sox-2 in the
dentate gyrus (DG) of the hippocampus was assessed. The results of the
immunohistochemical staining of nestin (Fig. 5) showed that the ex-
pression of this protein remarkably decreased in the Aβ-treated group
(p < 0.001). Running on the treadmill or the transplantation of un-
preconditioned MSCs following the induction of AD slightly increased
the expression of nestin but they were not significantly different from
the ALZ group. However, the percent of nestin-positive cells were
considerably higher in the ALZ + MSC + DMOG+TRD group than the
ALZ + TRD (p < 0.05) as well as the ALZ + MSC groups (p < 0.01).
The neuronal survival and expression of Sox-2 are investigated in
the DG (Fig. 6). Although the expression of this protein decreased fol-
lowing intraventricular injection of Aβ relative to the control groups
(p < 0.001), the transplantation of MSCs along with treadmill exercise
attenuated this reduction (p < 0.001). There was a near significance
between the ALZ group and the ALZ + MSC and ALZ + TRD groups
(p = 0.055). The analysis of the treatment groups showed that tread-
mill exercise besides DMOG-treated MSCs increased the percentage of
Sox2+ cells significantly higher than the ALZ + MSC or
ALZ + MSC + TRD groups (p < 0.05).
To assess neuronal differentiation, NeuroD western blotting of the
hippocampus was used. The quantification of the bands (Fig. 7) re-
vealed the lowest expression of NeuroD in the ALZ group compared to
the control groups (p < 0.01 for the control and sham groups,
p < 0.001 for the exercise group). Administration of MSCs along with
exercise, as well as the transplantation of DMOG-treated cells led to a
significant increase in NeuroD expression when compared with the ALZ
group (p < 0.05). The highest significance was observed in the
ALZ + MSC + DMOG+TRD group (p < 0.001).
3.3. The synergic effects of treadmill exercise and preconditioned MSCs on
apoptosis in the hippocampus of Aβ-injected rats
The results of Nissl staining showed a reduction in the densities of
intact pyramidal cells in the CA1 hippocampus (Fig. 8). A significant
difference existed between the ALZ group and the other treatment

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R. Abshenas, et al.
Fig. 7. Western blot analysis of neuronal differentiation. Mean ± S.E.M. of NeuroD protein concentrations in the hippocampus (a: p < 0.01 vs. the control and
sham groups, b: p < 0.001 vs. the exercise group, c: p < 0.05, and d: p < 0.01 vs. the ALZ group). (n = triplicates of 4 hippocampi per group).
groups that received MSCs (p < 0.001). There was a lower significance
difference between the ALZ and ALZ + TRD groups (p < 0.01). The
transplantation of DMOG-treated cells besides exercise led to a re-
markable increase in cell density relative to the ALZ + TRD group
(p < 0.05).
The injection of Aβ caused an increase in caspase-3 expression
(Fig. 9, p < 0.001) and all treatments attenuated this increase
(p < 0.001). Of course, this reduction was less in the ALZ + TRD
group which resulted in a significant difference relative to the control
groups (p < 0.05).
3.4. The synergic effects of treadmill exercise and preconditioned MSCs on
antioxidant capacity in the hippocampus of Aβ-injected rats
The total antioxidant capacity (TAC) and the total thiol molecule
(TTM) were significantly reduced by the injection of Aβ (P < 0.05 for
TAC, p < 0.001 for TTG) relative to the control and sham groups. The
highest level of TAC was in the exercise group (p < 0.001). As shown
in Fig. 10A, the groups which exercised for one month (with or without
cell transplantation) had a significant increase in the TCA compared to
the ALZ group, but there was more significance in the group receiving
the cell with exercise (p < 0.001) than the group that exercised
without cell transplantation (p < 0.01). Finally, there was a significant
increase in the TAC of the groups that received cell transplantation
along with exercise as compared to the ALZ + MSC group (p < 0.05).
Transplantation of treated or untreated cells did not affect TTG, but
doing treadmill exercise along with untreated MSCs (p < 0.01) and
DMOG-treated cells (p < 0.001) caused a significant increase in TTG
when compared with the ALZ group (Fig. 10B). Furthermore, there was
a significant difference between the ALZ + MSC + DMOG+TRD and
ALZ + MSC groups (p < 0.01). These results indicate the great
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Life Sciences 249 (2020) 117482
importance of exercise in increasing the antioxidant capacity than MSC.
Furthermore, the preconditioning of MSCs has a higher capacity in
increasing TAC. The results of malondialdehyde (MDA) evaluation in-
dicated that the administration of MSCs or treadmill exercise could not
attenuate the increase in MDA following intraventricular injection of
Aβ (Fig. 10C). Though the transplantation of untreated MSCs along
with exercise or treated cells with exercise or alone caused a reduction
in MDA (p < 0.001), there was no significant difference between
various treatment groups.
3.5. The synergic effects of treadmill exercise and preconditioned MSCs on
the BDNF level in the hippocampus of Aβ-injected rats
The results of the ELISA examination showed a reduction in the
BDNF level in the hippocampus of Aβ-treated rats (Fig. 11, p < 0.001).
There was a remarkable difference between the ALZ group and other
treatment groups (p < 0.001). Further analysis revealed a significant
increase in BDNF when the rats were administrated with untreated
MSCs besides treadmill exercise relative to the ALZ + MSC or
ALZ + TRD groups (p < 0.001). The transplantation of untreated cells
along with exercise significantly increased the BDNF level when com-
pared with the ALZ + MSC + DMOG group (p < 0.05) exhibiting the
greater importance of exercise in increasing the BDNF level than pre-
conditioning of MSCs.
4. Discussion
The present study demonstrated the synergistic effects of DMOG-
preconditioned MSCs and treadmill exercise on Aβ-induced memory
impairment and neurotoxicity in the hippocampus. We found that
treadmill exercise along with transplantation of DMOG-treated MSCs
R. Abshenas, et al. Life Sciences 249 (2020) 117482

Fig. 8. Histological examination of cell density. Light micrographs of CA1 pyramidal cells (K) of the control (A), sham (B), exercise (C), ALZ (D), ALZ + TRD (E),
ALZ + MSC (F), ALZ + MSC + TRD (G), ALZ + MSC + DMOG (H), and ALZ + MSC + DMOG+TRD (I) groups using Nissl staining. The white and black arrows
show the intact pyramidal and the dark pyknotic cells, respectively. (a: p < 0.001, b < 0.01, and c < 0.05 vs. the control, sham, and exercise groups, d:
p < 0.001 and e: p < 0.001 vs. the ALZ group, f: p < 0.05 vs. the ALZ + TRD group). Each value is mean ± S.E.M.

increased the quality of cell therapy in the ALZ model rats so that
learning and memory impairment were attenuated more in the groups
that received treated MSCs besides exercise than the groups which
underwent a single therapeutic approach likely through a decrease in
apoptosis, and an increase in neurogenesis, antioxidant capacity, and
neurotrophic factor. Furthermore, the results of this study showed that
cell transplantation resulted in a greater apoptosis reduction than ex-
ercise, while treadmill exercise had a greater effect on BDNF levels,
antioxidant capacity, and neurogenesis. In recent years, using stem cell
therapy has increased in the treatment of various diseases, including
neurodegenerative disorders [7,13]. Consistent with the results of an-
other study, the results of this study showed that the transplantation of
MSCs attenuated Aβ-induced memory impairment by inhibiting
apoptosis and enhancing the BDNF level, nestin expression, and cell
proliferation in the hippocampus ([27]). In this study, although MSC
transplantation had been associated with cell proliferation, it had no
significant effect on cell survival (Sox2) and neuronal differentiation
(NeuroD). Despite years of research, poor cell migration and survival
after transplantation were one of the prime challenges in the MSCs-
based therapy, therefore, strategies which can enhance the efficacy of
transplanted cells before engraftment is of significant interest [13]. Wei
et al. reported that the transplantation of hypoxia preconditioned MSCs
enhanced neurogenesis and angiogenesis in a stroke model in rats [28].
They found better locomotion recovery following MSCs preconditioning
and suggested that hypoxia preconditioning is an effective tool for
promoting the regenerative capability and therapeutic potential of

9
R. Abshenas, et al.
Fig. 9. Western blot analysis of apoptosis. Mean ± S.E.M. of caspase-3 protein density in the hippocampus (a: p < 0.001 vs. the control, sham, and exercise groups,
b: p < 0.001 vs. the ALZ group, c: p < 0.05 vs. the control and sham groups). (n = triplicates of 4 hippocampi per group).
MSCs. Preconditioning with hypoxic media was proposed to enhance
BDNF expression compared to normoxic media in a rat model of spinal
cord injury [29]. In another study, the preconditioning of human adi-
pose tissue-derived MSCs with deferoxamine led to the upregulation of
the proangiogenic factors like vascular endothelial growth factor alpha
and angiopoietin 1 and increased the expression of nerve growth factor,
glial cell-derived neurotrophic factor, and neurotrophin-3 in an in-vitro
model of diabetic neuropathy [30]. Based on a previously published
study in which the preconditioning of MSCs using DMOG as an inhibitor
of prolyl hydroxylase was able to stabilize HIF-1α and promote MSCs
proliferation, migration, and upregulation of Nrf2 [13], in the present
study, MSCs were preconditioned with DMOG before engraftment and
an improvement in learning and memory through increased neuro-
genesis and decreased apoptosis was observed. HIF-1 is one of the most
important factors that mediate the effects of hypoxia on the cell,
thereby improving the formation of blood vessels and cell migration
[31]. Therefore, the use of factors that contribute to the stability of HIF-
1 can increase the quality of MSCs following transplantation. In line
with this research, it seems that DMOG, as a prolyl hydroxylase in-
hibitor, causes HIF-1 stability and thereby decreases mitochondrial
cytochrome c and apoptosis of stem cells [13]. In addition to stabilizing
HIF-1, DMOG preconditioning improves memory impairment by in-
creasing angiogenesis and decreasing apoptosis. In the present study,
although MSCs transplantation led to improved learning and memory,
this effect was more pronounced in the DMOG-treated group. In line
with this study, preconditioning with resveratrol facilitated umbilical
cord-derived mesenchymal stem cell engraftment and enhanced the
therapeutic effects of these cells as demonstrated by improved learning
10
Life Sciences 249 (2020) 117482
and memory, enhanced neurogenesis, and alleviated neural apoptosis in
the hippocampus of the AD mice [32]. As previously mentioned, un-
preconditioned MSCs increased cell density and proliferation but did
not affect cell survival and differentiation. The transplantation of pre-
conditioned MSCs, in addition to cell proliferation (nestin), increased
the Sox-2 and NeuroD levels, indicating cell survival and neuronal
differentiation, respectively. The transplantation of induced-MSCs acts
as a source of neurotrophic factors and, in addition to facilitating ax-
onal regeneration, protects the remaining cells in the injured area [33].
It seems that the increase in the BDNF level and neurogenesis following
DMOG-preconditioning improves the MSC niche, thereby increasing the
survival and cell differentiation in the hippocampus. Furthermore, the
transplantation of preconditioned MSCs caused a reduction in oxidative
stress and apoptosis in the hippocampus that led to increased phos-
phorylation of tyrosine kinase, which can reduce the degeneration of
cholinergic nerve cells and improve cognitive behavior [34]. There is
some evidence suggesting that non-pharmacological interventions such
as exercise enhance the neuroprotective effects of transplanted MSCs
[35]. Exercise is involved in glucose metabolism and also stimulates
angiogenesis in the brain [36]. Some studies attribute the protective
and therapeutic effects of exercise to the increase of the antioxidant
capacity so that Radak et al. reported that regular exercise decreases the
rate of ROS generation and prevents the age-related increase in NF-kB
signal [37]. This supports the results of this study that treadmill ex-
ercise for one month increased total antioxidant capacity and improved
learning and memory. Additionally, we observed that exercising on a
treadmill alone or besides MSCs transplantation attenuated the Aβ-in-
duced apoptosis in the hippocampus. Furthermore, it inhibited caspase-
R. Abshenas, et al. Life Sciences 249 (2020) 117482

Fig. 10. A. Measuring the biomarkers of stress oxidative. Mean ± S.E.M. of TAC (a: p < 0.05 vs. the control and sham groups, b: p < 0.001 and d < 0.001 vs. the
exercise group, c: p < 0.01 and e: p < 0.001 vs. the ALZ group, f: p < 0.001 vs. the ALZ + MSCs group), B: TTG (a: p < 0.001 and b: p < 0.05 vs. the control,
sham, and exercise groups, c: p < 0.05 and d: p < 0.001 vs. the ALZ group, e: p < 0.01 vs. the ALZ + MSCs group), and C: MDA (a: p < 0.001 and b < 0.05 vs.
the control, sham, and exercise groups, c: p < 0.001 vs. the ALZ group) in the hippocampus (n = triplicates of 4 hippocampal tissue per group).

3 activity and DNA fragmentation in the hippocampus and subse-
quently improved the memory impairment which confirms the results
of this study [38]. One of the most important results of the present
study was that treadmill exercise had a greater effect on BDNF ex-
pression than MSCs transplantation, and the simultaneous administra-
tion of these showed dramatically protective effects in the hippo-
campus. BDNF affects the neuronal plasticity and neurogenesis in the
hippocampus and aerobic exercise can gradually increase the BDNF
level in the hippocampus [39]. It is likely that in the present study,
treadmill exercise provided a suif environment for the proliferation and
differentiation of transplanted cells by increasing the level of BDNF in
the hippocampus and due to the importance of this protein in neuro-
genesis and cell proliferation, it increased the life span of the injected
cells and reduced their apoptosis. The other mechanisms induced by
exercise are angiogenesis and synaptogenesis [40]. Transplanted MSC
requires the effective blood supply and the formation of a synapse with
other cells for efficient proliferation and differentiation. Treadmill ex-
ercise-induced angiogenesis provides an enriched environment for the
growth of injected stem cells and thereby improves the quality of cell
therapy. Furthermore, the secretion of neurotrophic factors, following
physical activity, not only controls the growth of nerve fibers during
development, but also preserves the proliferation and survival of neu-
rons in adults and improves the synaptic communication in the nervous
system [41,42]. In conclusion, the results of the present study showed
that the preconditioning of stem cells increases the quality of trans-
planted cells and is more effective than untreated cells in improving
memory by reducing apoptosis and oxidative stress and increasing
neurogenesis. On the other hand, exercise and physical activity along
with cell transplantation can increase the effect of cell therapy in
neurodegenerative diseases likely through increased growth factors and
antioxidant capacity.

11
R. Abshenas, et al.
Fig. 11. Determination of BDNF concentration using ELISA. Mean ± S.E.M. of
the BDNF level in the hippocampus (a: p < 0.001 vs. the control, sham, and
exercise groups, b: p < 0.001 vs. the ALZ group, c: p < 0.001 vs. the
ALZ + MSC + DMOG group, d: p < 0.05 and e: p < 0.001 vs. the
ALZ + MSC + DMOG group, and f: p < 0.001 vs. the ALZ + TRD and
ALZ + MSC groups). (n = triplicates of 4 hippocampal tissue per group).
Declaration of competing interest
The authors report no conflicts of interest.
Acknowledgment
This study was supported by the Hamadan University of Medical
Sciences, Iran (9603021342).
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