Biochemical and Biophysical Research Communications 678 (2023) 62–67
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Dose-dependent neuroprotective effects of adipose-derived mesenchymal
stem cells on amyloid β-induced Alzheimer’s disease in rats
Hossein Babaei a, Alireza Kheirollah a, Mina Ranjbaran b, Alireza Sarkaki c, **,
Maryam Adelipour a, c, *
a
Department of Clinical Biochemistry, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Department of Physiology, School of Medicine, Persian Gulf Physiology Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of
Medical Sciences, Ahvaz, Iran
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Alzheimer’s disease
Mesenchymal stem cells
Neuroprotective effect
Dose-dependent
Aim: Mesenchymal stem cells (MSCs) have emerged as an intriguing candidate in cell therapy for treating
neurodegenerative diseases, including Alzheimer’s disease (AD). To achieve the maximum efficiency of cell
therapy, determining the optimal dose of MSCs is essential. This study was conducted to assess the dosedependent therapeutic response of MSCs against pathological and behavioral AD-associated alterations.
Methods: Aβ1-42 was injected intrahippocampally to establish an AD rat model. The MWM test was utilized to
evaluate the animal’s behavioral functions after receiving low and high doses of MSCs in the hippocampus re­
gion. ELISA and RT-qPCR were also employed to assess the concentration of markers related to antioxidant
activity and inflammation and the gene expression related to apoptosis in the hippocampus region, respectively.
Results: Low-dose MSC transplantation by increasing the concentrations of the antioxidant GSH, the antiinflammatory cytokine IL-10, as well as by lowering the concentrations of TNF-α, and the expression levels of
apoptotic factors (Bax and caspase 3), exerted a neuroprotective effect in the hippocampus of AD rats and
relatively ameliorated spatial learning and memory impairments. However, increasing the dose of MSCs
decreased the therapeutic benefits of these cells and had no significant effect on the recovery of behavioral
disorders.
Conclusion: Our findings reveal the dose-dependent neuroprotective effect of MSCs in AD. The therapeutic
response of MSCs to ameliorate the pathological and behavioral alterations associated with AD is attenuated
when the dosage of MSCs is increased.
1. Introduction
Alzheimer’s disease (AD), a progressive neurodegenerative disorder,
is distinguished by memory loss, cognitive decline, aberrant behavior,
and so forth [1]. The emergence of neurofibrillary tangles and the
development of amyloid plaques are among the most significant
neuropathological signs of this disease [2]. The induction of Aβ1-42
causes the first metabolic alteration, oxidative stress, which ultimately
leads to neuronal inflammation and death [3]. Apoptosis is a major
result of Aβ-induced cytotoxicity, which accelerates the disease pro­
gression [4].
Mesenchymal stem cells (MSCs), owing to their unique therapeutic
characteristics, are known as superb candidates for cell therapy against
neurodegenerative disorders, including AD [5]. Studies have shown that
MSCs can promote neuronal regeneration, modulate neuro­
inflammation, and inhibit oxidative stress and apoptosis of neuronal
tissue [6]. Factors such as timing and dose of administration of MSCs
have been demonstrated to affect the therapeutic efficacy of these stem
cells [7]. Hence, it was suggested that different doses of MSCs could have
distinct protective effects [8]. Furthermore, there is great variation
among clinical trials and experimental models of disease in the injected
dosage of MSCs [9,10], suggesting that MSCs can effectively treat dis­
eases in a dose-dependent manner [11,12]. Finding the optimal MSC
dosage for transplantation also provides advantages of a lower risk of
* Corresponding author. Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
** Corresponding author. Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
E-mail addresses: sarkaki_a@ajums.ac.ir (A. Sarkaki), adelimaryam@rocketmail.com, Adelipour-m@ajums.ac.ir (M. Adelipour).
https://doi.org/10.1016/j.bbrc.2023.08.041
Received 8 August 2023; Accepted 17 August 2023
Available online 19 August 2023
0006-291X/© 2023 Elsevier Inc. All rights reserved.
H. Babaei et al.
Biochemical and Biophysical Research Communications 678 (2023) 62–67
cell mutation and production costs, as well as requiring less tissue for
MSC expansion and reducing the possibility of accumulation and em­
bolism at the transplant site [11].
Despite plenty of research demonstrating the neuroprotective effect
of MSCs in AD, no investigations have examined the relationship of dose
with the therapeutic response of MSCs against pathological and behav­
ioral alterations caused by amyloid-β. In this research, we investigated
the effect of low- and high-dose MSCs on pathological and behavioral
features associated with AD. To clarify the correlation between dose and
therapeutic response of MSCs, behavioral indicators and factors related
to oxidative stress, inflammation, and apoptosis were evaluated in a rat
model of Aβ-AD.
2.3.2. Intrahippocampal injection of aggregated Aβ1–42 peptide and MSC
administration
The rat model of AD was developed by stereotaxic injection of the
aggregated form of the Aβ1–42 into the hippocampus as per a former
described protocol [15]. To this end, we used a 5-μl Hamilton micro­
syringe for bilateral injection of Aβ1–42 peptide (5 μg) dissolved in PBS
(5 μl) in the hippocamps at a speed rate of about 1–2 μl/min. PBS was
also given to the sham group. The injection of MSCs, performed 17 days
after injecting Aβ1–42, was carried out in the same site of amyloid
injection.
2. Materials and methods
2.3.3.1. Assessment of spatial learning and memory by MWM test. Twelve
days following treatment with Aβ1-42 and almost one month after the
administration of MSC, using the MWM test, we analyzed the spatial
learning and memory in rats based on a five-day protocol considering
the time spent in the target quadrant, speed of swimming, and delay to
discover the platform [16].
2.3.3. Behavioral analysis
The experimental methods employed in this study were authorized
by the Ethics Committee of Ahvaz Jundishapur University of Medical
Sciences, Ahvaz, Iran (ethical code: IR. AJUMS.ABHC.REC.1400.104)
adhered to the National Research Council’s Guide for Animal Research.
The design diagram of the present research work is illustrated in Fig. 1.
2.3.4. Sample collection
On the following day after the behavioral test, all the animals were
sacrificed by the intraperitoneal injection of a high-dose anesthetic. The
brains were promptly removed, hippocampal tissues were separated on
dry ice, and the tissues were maintained at − 80 ◦ C for chemical and
molecular assay [17].
2.1. Chemicals and laboratory kits
Aβ1–42 (Sigma, USA), Dulbecco’s modified eagle Medium (DMEM),
phosphate-buffered saline (PBS) (Gibco, USA), TRIzol™ reagent (Sigma,
Pool, UK), Master Mix and oligo (dT) primers and transcriptase kit
(Roche, Germany), TNF-α and IL-10 ELISA kit (ZellBio, Germany), GSH
ELISA kit (Mybiosource, USA), and Protein Assay kit (Bio-Rad, USA)
were the materials and kits used in the current study.
2.3.4.1. RNA extraction and RT-qPCR. For RNA extraction, a piece of
hippocampal tissue was homogenized on ice, in conformity with the
protocols recommended by the manufacturer. Then the total RNA was
extracted from the hippocampus using TRIzol™ reagent. After analyzing
the purity and concentration of the extracted RNA, according to the
standard guidelines of the RT-qPCR method and the primer sequences
utilized in the previous study, the gene expression levels of apoptosisassociated factors (Bax and caspase 3) were determined in the hippo­
campal tissue [18].
2.2. In vitro investigation
2.2.1. Isolation and recognition of MSCs
The isolation of MSCs from epididymal white adipose tissues, which
were harvested under aseptic conditions, was carried out in accordance
with the protocol of a previous study [13]. We examined cell surface
antigens for MSC phenotypic features (CD34, CD44, CD45, and CD90).
2.3.4.2. Sample preparation for biochemical assays. A portion of the
collected sample was homogenized with a 0.1 M of PBS (pH 7.4) solution
to prepare a 10% brain homogenate. According to the protocol of the
previous study, the supernatant was separated and utilized for the
subsequent chemical analyses [19].
2.3.4.2.1. Estimation of biochemical parameters. According to the
manufacturer’s instructions, we determined the protein content in the
tissue homogenate supernatant using the Bradford protein assay kit and
the quantities of GSH, TNF-α, and IL-10 using a high-sensitivity colori­
metric kit (Sandwich Elisa).
2.3. In vivo investigation
2.3.1. Study groups
Considering the effect of gender on the accumulation of Aβ1–42, male
rats were used for the study [14]. Before entering the experiments, an­
imals were located in a Morris water maze (MWM) for 2 min to assess
their eyesight, swimming ability, and normal behavioral patterns.
Overall, 40 male Wistar rats (six months old weighing 270 ± 30 g) were
selected for further investigation. Following random selection, animals
were allotted to five equal experimental groups (n = 8 in each group):
(1) control (completely healthy rats), (2) sham-operated (rats receiving
only 5 μl of PBS in both surgeries), (3) Aβ-induced AD (rats treated with
Aβ), (4) AD + Low-dose MSCs (Aβ-treated rats received 25,000 MSCs),
and (5) AD + High-dose MSCs (Aβ-treated rats received 50,000 MSCs).
2.4. Statistical analysis
All the data used in the present investigation were presented as mean
± SEM and tested for normal distribution. The MWM test results
(swimming speed and escape latency) were analyzed by a two-way
analysis of variance (ANOVA). Also, the results of the Probe trial and
other experimental parameters were analyzed by one-way ANOVA, and
comparisons among the groups were performed by Tukey’s post-hoc test
using Graph Pad Prism 8.00 (GraphPad Software Inc., San Diego, CA,
USA). Probability level (5%) was considered to be statistically signifi­
cant (p < 0.05).
Fig. 1. Three rat groups underwent stereotactic surgery and injection of Aβ1-42
to create an Alzheimer’s disease (AD) model. The sham-operated group also
received PBS. The AD model was validated using the MWM test after 12 days.
MSC transplantation was then performed, and after one month, behavioral tests
were performed again to evaluate the treatment results. Finally, animals were
sacrificed for molecular and biochemical assays.
3. Results
3.1. Features of stem cells isolated from rat adipose tissue
The results of flow cytometry displayed strong expression of the
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Biochemical and Biophysical Research Communications 678 (2023) 62–67
mesenchymal markers CD44 and CD90, but the hematopoietic lineage
markers CD34 and CD45 showed extremely weak expression (Fig. 2).
during all test days, but this difference was insignificant among the
groups (intergroup; p > 0.05), showing the insignificant effect of motor
ability on the test results.
3.2. Effects of low- and high-dose MSCs on spatial learning and memory
3.3. Effects of low- and high-dose MSCs on antioxidant activity
During days 53–56 of training, the mean ELT decreased gradually in
all the experimental groups (Fig. 3A). However, Aβ-treated rats,
compared to those in the control or sham group, showed impaired
spatial learning, as evinced by prolonging ELT during training days (p <
0.0001). During training days, MSC transplantation significantly
decreased the increased ELT in the AD + low-dose MSCs group
compared to the AD group (p = 0.0014 for all days). Contrary to our
hypothesis, the AD + high-dose MSC group, in comparison to the AD
group, exhibited insignificant variation in the spatial learning function
(p > 0.05 for all days), which implies that the high dose of transplanted
cells probably has a reverse effect on improving the learning impairment
of Aβ-treated rats. This outcome was further confirmed by a significant
increase in the mean ELT in the high-dose MSCs group compared to the
treatment group with low-dose MSCs (p < 0.05 for all days). When
compared to the control or sham groups, the rats treated with Aβ spent
less time in the target quadrant during probe analysis, demonstrating
that Aβ caused a failure in spatial memory (p < 0.0001 vs. sham and
control groups; Fig. 3B). In comparison to the AD group, those rats
receiving 25,000 MSCs spent considerably more time in the target
quadrant (p < 0.0001), which is the indication of relatively improved
spatial memory. In addition, an insignificant difference was observed in
the mean time spent in the target quadrant between the AD + high-dose
MSCs and AD groups of rats (p = 0.0982), suggesting that the increase of
transplanted cells may have a reverse effect on the function of MSCs.
This finding was further verified by the significant decrease in the mean
elapsed time in the rat group receiving high-dose MSCs relative to the
treatment group with low-dose MSCs (p = 0.0010). Based on Fig. 3C, the
average swimming speed was different for each group (intragroup)
According to Fig. 4A, the mean concentration of GSH was found to be
considerably lower in the hippocampus of Aβ-treated rats than that of
the sham rat group and control group (26.1400 ± 1.1180 for AD,
86.2100 ± 4.4670 for control, and 83.5900 ± 4.6500 for sham groups;
p < 0.0001 vs. sham and control groups). Low-dose MSC transplantation
protected the brains of the AD model rats from oxidative stress injuries,
which was shown by a considerable rise in the essential antioxidant
enzyme GSH level (58.1200 ± 0.5797; p < 0.0001 vs. AD group). Highdose MSC transplantation also markedly enhanced the antioxidant
response in the hippocampus of Aβ-treated rats as compared to the AD
group (GSH: 38.3000 ± 0.7486; p = 0.0464 vs. the AD group). Never­
theless, increasing the dose of transplanted MSCs had an adverse effect
on this therapeutic feature, as evidenced by a significant decrease in
GSH levels in the hippocampus of rat groups with AD + high-dose MSCs
compared to AD + low-dose MSCs (p = 0.0003 vs. AD + low-dose MSCs
group).
3.4. Effects of low- and high-dose MSCs on inflammation
Based on Fig. 4B and C, a significant increase was observed in the
levels of TNF-α in the hippocampus of rats treated with Aβ, as compared
to control group and sham group (86.2100 ± 4.4670 for the AD group,
9.5100 ± 0.1074 for the control group, and 11.0400 ± 0.3296 for the
sham group; p < 0.0001 vs. the sham and control groups) and a sig­
nificant decrease in IL-10 (56.6100 ± 1.6150 for the AD group,
104.9000 ± 0.8523 for the control group, and 101.0000 ± 0.9325 for
the sham group; p < 0.0001 vs. the sham and control). Low-dose MSC
Fig. 2. The figure shows that the cells are positive for (A) CD44 and (B) CD90 and negative for (C) CD34 and (D) CD45.
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Fig. 3. Effects of low- and high-dose MSCs on spatial learning and memory. (A and B) ####p < 0.0001 vs. control and sham groups; (A) **p = 0.0014 and (B)
****p < 0.0001 vs. AD group; (A) @p = 0.0133 and (B) @@@p = 0.0010 vs. the low-dose MSCs group. ns: not significant (n = 8 in each group).
Fig. 4. Effects of low- and high-dose MSCs on antioxidant activity, inflammation, and apoptosis (A–E) ####p < 0.0001 vs. control and sham groups; (A–E) ****p <
0.0001, (A) *p = 0.0464, (B) *p = 0.0143, (C) **p = 0.0043, and (E) *p = 0.0133 vs. AD group; (A) @@@p = 0.0003, (B–C) @@@@p < 0.0001, (D) @p = 0.0219,
and (E) @@@p = 0.0004 vs. the low-dose MSCs group. ns: not significant (n = 8 in each group).
transplantation protected the brains of AD model rats from inflamma­
tion, as demonstrated by a substantial reduction in TNF-α (52.2900 ±
2.3850; p < 0.0001 vs. the AD group) and a significant increase in IL-10
(71.1000 ± 0.3325; p < 0.0001 vs. the AD group). Although high-dose
MSC transplantation significantly reduced inflammation in Aβ-treated
rats compared to the AD group (74.9200 ± 1.4450 for TNF-α [p =
0.0143] and 61.8900 ± 0.6447 for IL-10 [P = 0.0043] vs. the AD group),
increasing the dose of transplanted MSCs seems to reduce their thera­
peutic benefits.
weaken the antiapoptotic effect of transplanted MSCs, which was further
strengthened by a substantial increase in mRNA levels of Bax (p =
0.0219) and caspase 3 (p = 0.0004) in the hippocampal of the AD + highdose relative to the AD + low-dose MSC group.
4. Discussion
MSCs have been shown to reduce neuropathological symptoms and
promote the recovery of behavioral disorders in experimental models of
neurodegenerative disorders [20]. They exert neuroprotective and
neurorestorative effects through paracrine mechanisms and neuro­
regulatory molecules [21]. However, there is a dispute over the ideal
dosage of transplanted MSCs to achieve the optimal level of cell therapy
outcomes [22]. While there is no single theory about the dose and fre­
quency of MSC injection, until recently, increasing the dose of MSCs has
been the only choice to achieve the beneficial effects of transplantation
[23]. To this end, we evaluated the clinical influence of low and high
doses of MSCs on the pathological and behavioral changes associated
with AD and found that the protective effect of MSCs is dose-dependent
in an Aβ-AD rat model.
Our findings showed that low-dose MSC transplantation played a
significant role in ameliorating memory and learning deficits in AD
models. Hippocampus is crucial for learning new information, creating
memories, recognizing objects, and developing short-term memory
[24]. Therefore, it is reasonable that alleviation of pathological alter­
ations in a toxic microenvironment induced by amyloid-β could help the
recovery of behavioral disorders. Based on this assumption, in the pre­
sent study, an increase in the antioxidant defense and the reduction of
inflammation and apoptosis in the hippocampus seem logical for the
3.5. Effects of low- and high-dose MSCs on apoptotic markers
The results showed significantly higher mRNA levels of Bax and
caspase 3 in the hippocampus of AD rats when compared to the sham and
control groups. Bax and caspase 3 expression levels were respectively
5.0500 ± 0.0944 and 4.1440 ± 0.0296 folds of change in the AD group
and 1.1750 ± 0.0590 and 1.1290 ± 0.0258 folds of change in the sham
group (P < 0.0001 vs. the control and sham groups; Fig. 4D and E). Lowdose MSC transplantation could inhibit apoptosis, which was found by a
significant reduction in the mRNA levels of Bax and caspase 3 (3.1500 ±
0.0566 and 3.7780 ± 0.0323 folds of change, respectively; P < 0.0001
vs. AD group) in the AD rats treated with low-dose MSCs compared to
the AD group. Furthermore, increasing the dose of MSCs lessened the
mRNA levels of Bax (4.2140 ± 0.5075 folds of change; p = 0.1070) in
AD + high-dose MSC group compared to the AD group. However, this
difference was statistically insignificant and caused a significant
reduction in the mRNA levels of caspase 3 (3.9890 ± 0.0501 folds of
change, p = 0.0133) in the high-dose MSC group compared to the AD
group. This outcome indicates that increasing the dose of MSCs may also
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improvement of animal behavioral disorders in MWM. This result
confirmed and emphasized that the efficacy of MSC in ameliorating the
behavioral deficits associated with AD is directly related to the
improvement of pathological alterations, as demonstrated in extensive
studies in the past [25].
We found that low-dose MSC transplantation exerted a significant
part of its neuroprotective effect by increasing antioxidant defense and
ameliorating pathological alterations in the hippocampus of the rat
model of Aβ-AD. Intriguingly, high-dose MSC transplantation failed to
further this positive effect, indicating a dose-dependent effect.
Taken together, Contrary to the results of some researchers who have
shown a positive correlation between the treatment response and the
dose of transplanted cells [26] and also pointed to the beneficial effect of
a high dose of transplanted cells in the early stages of the disease [27], in
the current study, although in terms of quantity, high-dose MSCs con­
tained twice the number of low-dose cells, no advantage was evident in
the beneficial effects of cell therapy, and we found that increasing the
number of transplanted cells may even reverse the therapeutic response
of MSCs. We speculate that this outcome could be due to dosage satu­
ration, which would suggest that even if more cells were transplanted at
the site of damage, only the required number would reside and activate
in the inflamed regions, as demonstrated in a study in the past. That
study has reported that the high dose of MSC prompts cell saturation at
the graft site and could be an ineffective strategy for improving
engraftment [28]. Increasing the repetitions of cultivation over a long
period of time to achieve an appropriate cell number, according to
available evidence, may result in permanent alterations such as senes­
cence and decreased paracrine activity of MSCs [29]. Moreover, satu­
ration of transplanted cells in the target tissue may reduce engraftment
cell survival due to an inadequate supply of nutrients [30]. On the other
hand, high-dose cell transplantation in a single step may lead to
inflammation by activating immune cells [23]. The cited evidence can
be clear reasons for the negative correlation between treatment response
and high doses of MSCs in our research. We speculate that in this study,
the increased pressure from the delivery of a large number of cells sus­
pended in a fixed volume of vehicle solution (versus a small number of
cells) when exiting the needle of the Hamilton syringe may cause the
intensification of stress or death of MSCs. In supporting this concept,
Aguado et al. have reported that the extensional stream at the syringe
needle entrance is the main cause of cell death during cell delivery to the
target region [31]. Furthermore, the variable range of transplanted cells
and the use of immunosuppressive agents in some studies may explain
why our findings differ from those of other studies.
In summary, our study revealed distinct differences in the function of
MSC doses to prevent the progression of AD-related pathological alter­
ations. We found that MSCs, either in low or high dosages, exhibited
positive therapeutic properties in the hippocampus via antioxidant, antiapoptotic and anti-inflammatory activities. However, as evidenced by an
inability to restore behavioral impairments in rats, high-dose MSC was
unable to fully overcome the toxic and apoptotic conditions induced by
the stressed microenvironment. In contrast, the low quantity of MSCs
exhibited a dosage that was adequate and optimal, as clearly evidenced
by the improvement of pathological and behavioral alterations.
Funding information
This research was supported by a fund from Ahvaz Jundishapur
University of Medical Sciences (AJUMS, Ahvaz, Iran; grant number:
APRC-0019). The authors gratefully acknowledge the Physiology
Research Center, Cellular and Molecular Research Center (CMRC), and
Biochemistry Laboratory of Ahvaz Medical School, Ahvaz, Iran, where
the experiments were conducted. This essay is a part of Hossein Babaei’s
Ph.D. dissertation.
Statement of authorship credit
Hossein Babaei, Mina Ranjbaran, Maryam Adelipour, Alireza
Kheyrollah, and Alireza Sarkaki all made significant contributions to the
conceptualization, planning, and preparation of the study’s materials.
Hossein Babaei did data collection and analysis. The final manuscript
was read and approved by all the authors.
Statement of conflicting interest
The authors declare they have no competing interests.
Data availability
The corresponding authors are willing to provide the sets of data
used and/or analyzed during the current investigation under reasonable
request.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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