Melatonin-pretreated adipose-derived mesenchymal stem cells efficeintly improved learning, memory, and cognition in an animal model of Alzheimer's disease

Abstract

Currently, mesenchymal stem cells (MSCs) based therapy has extensive attraction for Alzheimer’s disease (AD). However, low survival rate of MSCs after transplantation is a huge challenging. The current study aimed to improve adipose-derived MSCs (AD-MSCs)-based therapy by their pre-treatment with melatonin (MT) ‘a well-known antioxidant’ in an animal model of AD. In this study, after isolating rat AD-MSCs from the epididymal white adipose tissues, the cells were pretreated with 5μM of MT for 24 hours. Forty male Wistar rats were randomly allocated to control, sham, amyloid-beta (Aβ) peptide, AD-MSCs and MT-pretreated ADMSCs groups. The novel object recognition, passive avoidance test, Morris water maze and open field test were performed two months following the cell transplantation. The rats were sacrificed 69 days following cell therapy. The brain tissues were removed for histopathological analysis and also immunohistochemistry was performed for two Aβ1-42 and Iba1 proteins. It has been revealed that both AD-MSCs and MT-AD-MSCs migrated to brain tissues after intravenous transplantation. However, MT-ADMSCs significantly improved learning, memory and cognition compared with AD-MSCs (P<0.05). Furthermore, clearance of Aβ deposition and reduction of microglial cells were significantly increased in the MT-ADMSCs compared with AD-MSCs. Although stem cell therapy has been introduced as a promising strategy in neurodegenerative diseases, however, its therapeutic properties are limited. It is suggested that pretreatment of MSCs with melatonin partly would increase the cells efficiency and consequently could decrease AD complication including memory and cognition.

Full text

ORIGINAL ARTICLE
Melatonin-pretreated adipose-derived mesenchymal stem cells
efficeintly improved learning, memory, and cognition in an animal
model of Alzheimer's disease
Ebrahim Nasiri
1
&Akram Alizadeh
2
&Amaneh Mohammadi Roushandeh
3
&Rouhollah Gazor
1
&
Nasrin Hashemi-Firouzi
4
&Zoleikha Golipoor
1,4
Received: 4 January 2019 /Accepted: 17 April 2019 /Publ ished online: 25 May 201 9
#Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Currently, mesenchymal stem cells (MSCs) based therapy has extensive attraction for Alzheimer’s disease (AD). However, low
survival rate of MSCs after transplantation is a huge challenging. The current study aimed to improve adipose-derived MSCs
(AD-MSCs)-based therapy by their pre-treatment with melatonin (MT) ‘a well-known antioxidant’in an animal model of AD. In
this study, after isolating rat AD-MSCs from the epididymal white adipose tissues, the cells were pretreated with 5μMofMTfor
24 hours. Forty male Wistar rats were randomly allocated to control, sham, amyloid-beta (Aβ) peptide, AD-MSCs and MT-
pretreated ADMSCs groups. The novel object recognition, passive avoidance test, Morris water maze and open field test were
performed two months following the cell transplantation. The rats were sacrificed 69 days following cell therapy. The brain
tissues were removed for histopathological analysis and also immunohistochemistry was performed for two Aβ1-42 and Iba1
proteins. It has been revealed that both AD-MSCs and MT-AD-MSCs migrated to brain tissues after intravenous transplantation.
However, MT-ADMSCs significantly improved learning, memory and cognition compared with AD-MSCs (P<0.05).
Furthermore, clearance of Aβdeposition and reduction of microglial cells were significantly increased in the MT-ADMSCs
compared with AD-MSCs. Although stem cell therapy has been introduced as a promising strategy in neurodegenerative
diseases, however, its therapeutic properties are limited. It is suggested that pretreatment of MSCs with melatonin partly would
increase the cells efficiency and consequently could decrease AD complication including memory and cognition.
Keywords Alzheimer’sdisease .β-amyloid .AD-MSCs .Melatonin
Introduction
Alzheimer’s disease (AD), which is recognized as a neurode-
generative disorder, is associated with the progressive loss of
neurons, leading to memory impairment and interference in
daily activities (Tong et al. 2015). Extracellular deposition of
amyloid-β(Aβ) and Tau hyperphosphorylation, as two major
factors in neuronal loss (Ruzicka et al. 2016;Zhaoetal.2017),
reduce the size of frontal and temporal lobes, which are in-
volved in memory and learning. Therefore, abnormalities,
such as progressive learning dysfunction, memory decline
and cognitive impairment occur in patients with AD
(Lemmens et al. 2011; Yan et al. 2014).
There are only a few therapeutic strategies for AD, which
just relieve the symptoms with no effects on the disease pro-
gression or factors involved in its pathogenesis (Ager et al.
2015; Choi et al. 2014; Tong et al. 2015). Cell therapy is one
of the most advanced and novel approaches that have been
suggested in treatment of neurodegenerative disorders.
According to recent researches, mesenchymal stem cells
(MSCs) transplantation improves cognitive performance in
*Zoleikha Golipoor
masoomeh_golipoor@yahoo.com
1
Cellular and Molecular Research Center, Faculty of Medicine,
Guilan University of Medical Sciences, Rasht, Iran
2
Department of Tissue Engineering and Applied Cell Sciences,
Faculty of Medicine, Semnan University of Medical Sciences,
Semnan, Iran
3
Medical Biotechnology Department, Paramedicine Faculty, Guilan
University of Medical Sciences, Rasht, Iran
4
Neurophysiology Research Center, Hamadan University of Medical
Sciences, Hamadan, Iran
Metabolic Brain Disease (2019) 34:1131–1143
https://doi.org/10.1007/s11011-019-00421-4

animal AD models (Eftekharzadeh et al. 2015; Kan et al. 2011;
Munoz et al. 2005). MSCs produce neurotrophic factors and
neuroprotective cytokines, leading to improve dentate gyrus
proliferation of endogenous neural stem cells and enhancement
of behavioral outcomes (Kan et al. 2011; Munoz et al. 2005).
Also, they have pivotal effects on Aβclearance in animal AD
models (Eftekharzadeh et al. 2015). Adipose-derived MSCs
(AD-MSCs) is promising among other sources of MSCs be-
cause of their easy access, culture and expansion and also
immunemodulatory Properties. It is noteworthy noted that
AD-MSCs promote neurogenesis in vitro (Yan et al. 2014).
Transplantation of AD-MSCs reduces Aβdeposits and re-
stores memory in AD models (Kim et al. 2012; Lee et al. 2010).
Nevertheless, considering the noxious microenvironment of
the brain, viability reduction of transplanted cells is a potential
problem in AD (Rockenstein et al. 2015). Direct MSCs trans-
plantation is not efficacious, and nearly 80-90% of cells die
following transplantation within three days (Liu et al. 2009).
Multiple factors may be involved in the early eradication of
transplanted cells, such as inflammation, hypoxia, thermal
stress, and free radical activity (Khatibi et al. 2017;
Roushandeh et al. 2017;Tangetal.2005). Consequently, es-
tablishing a strategy to improve cell survival and homing is
necessary in cell therapy. Cell preconditioning is a promising
approach for increasing cell survival and homing after trans-
plantation; it also represents multiple therapeutic benefits
(Haider and Ashraf 2012;Tangetal.2014;Yuetal.2013).
Melatonin (MT), a pineal gland hormone, regulates multi-
ple physiological processes. It also controls different regula-
tory functions of the cells, including cell signaling, immune
responses, and inhibition of nuclear DNA damage and oxida-
tion of fatty acids. MT induces antiapoptotic effects on normal
cells and exhibits significant anti-aging properties (Esteban-
Zubero et al. 2016; Gholami et al. 2014). It is also a powerful
free radical scavenger, which activates cellular antioxidants in
different cell types (Carpentieri et al. 2016; Liu et al. 2016).
Pretreatment with MT can enhance the homing of MSCs after
transplantation (Mortezaee et al. 2016). Evidence suggests
that MT also protects human AD-MSCs against oxidative
stress and apoptosis (Rafat et al. 2018; Tan et al. 2016).
Therefore, The purpose of the present study was to inves-
tigate the antioxidant potential of MTand AD-MSC-base ther-
apy on the histological properties, cognition and memory in a
rat model of Alzheimer's disease.
Materials and Methods
AD-MSCs isolation and expansion
Epididymal white adipose tissues were removed aseptically from
six-week-old male Wistar rats. Phosphate-buffered saline (PBS;
Invitrogen, USA), containing 1% penicillin/streptomycin
(Invitrogen, USA), was used twice to wash the tissues at 37°C.
Adipose tissues were then treated with 0.1% collagenase type I
(Gibco, USA), which was dissolved in 1% bovine serum albu-
min (BSA) in warm PBS. The samples were kept in a water bath
for half an hour to achieve total digestion and homogenization.
The supernatants were removed after five minutes of cen-
trifugation at 1200 rpm, and the pellets were resuspended in
1% BSA. This step was repeated in order to remove red blood
cells (RBCs) using RBC lysis buffer. Finally, the harvested
cells were cultured in DMEM/Ham F-12 medium at 37°C,
consisting of 1% penicillin/streptomycin, besides 10% fetal
bovine serum (FBS). The medium was changed every three
days, and the cells sub cultured with trypsin/EDTA (Sigma,
USA) after 90% confluences.
Flow cytometry
AD-MSCs between 3–5 passages were harvested and probed
for 20 minutes with CD44-FITC (550974), CD90-PerCPCY5
(557266), CD45-FITC (554877), and CD34-PE (551387) an-
tibodies (BD bioscience, USA). After rinsing the cells twice in
PBS, an NxT Flow Cytometer (Attune™; Thermo Fisher
Scientific, USA) was used.
Preconditioning with MT
The passage 4 AD-MSCs with concentration of 10
6
cells /flask
were cultured with 5μM of MT (Sigma, USA) for 24 hours.
Animals and experimental design
The adult male Wistar rats (250-300 g) were purchased from
Pasteur Institute of Iran, which were kept in a 12:12 h light/
dark cycle with free access to food and water at 22°C. The
Research Committee of Guilan University of Medical
Sciences (IR.GUMS.REC.1396.544) approved all the proce-
dures, which were in line with the NIH guidelines. In a ran-
dom manner, the rats were classified into five groups (N=8):
control; sham; Aβ; experimental group 1 receiving AD-MSCs
through the tail vein one week post-Aβinjection; and exper-
imental group 2 receiving MT-ADMSCs from the tail vein
one week post-Aβinjection. In the sham group, a Hamilton
syringe needle was stereotaxically inserted in the brain and
removed without any injections (Fig. 1).
AD induction with Aβinjection
After Aβ1-42 dissolving (Tocris Bioscience, UK) in 100μLof
PBS, incubation was performed at 37°C for seven days before
application. For inducing deep anesthesia, ketamine (87 mg/kg),
along with xylazine (13 mg/kg), was injected intraperitoneally
(Van 1977). Bilateral intracerebroventricular injections of Aβ
(5μg) were performed using a 5-μL Hamilton microsyringe
1132 Metab Brain Dis (2019) 34:1131–1143

(USA) according to the stereotaxic coordinates (AP: 1.2 mm;
ML: 2 mm; DV: 4 mm from the Bregma) on a stereotaxic device
(Stoelting Co., USA). The animals recovered in cages after su-
turing the skin (Cetin et al. 2013).
AD-MSC transplantation
In line with the manufacturer’s protocol, the fluorescent Dil
Stain (Invitrogen) was used to label AD-MSCs and MT-
ADMSCs. Incubation of the cells was performed for 20 mi-
nutes in DiI solution (1μg/mL) at 37°C. After washing, the
cells were resuspended in PBS. Seven days following the
injection of Aβ, AD-MSCs or MT-ADMSCs (10
6
cells/200
μL) were injected in the tail vein of rats.
Behavioral studies
Different tests including the novel object recognition (NOR)
test, open field (OF) test, Elevated plus maze (EPM) test pas-
sive avoidance learning (PAL) test, and Morris water maze
(MWM) were conducted two months after cell transplantation
(Kim et al. 2015;Kommaddietal.2018).
OF test This test was used for evaluating the animals’loco-
motor activity. It uses a white acrylic field (surface area,
50×50 cm; height of walls, 38 cm) with low ambient light
and records the time spent in the peripheral and central zones.
After placing the rats in the middle of open field, they were
given time to explore for 10 minutes. A video camera was
used to record the number of animals’entries to the center,
spent time in the peripheral and central regions, and total trav-
eled distance (Etaee et al. 2017).
Elevated plus maze (EPM) test: This test was desined to
measure anxiety behavior in rodent. Briefly, the maze
consisted of two open arms (50 × 10 cm each), two enclosed
arms (50 × 10 × 50 cm each) and a central platform (10× 10
cm). The maze was elevated 100 cm above the floor and was
illuminated with two 100 W lamps. During the 10 min test
period, each rat was allowed to explore the maze, and their
behavior was checked by a digital camera above the maze.
The time spent in the open arms, the number of open and
enclosed arms entries, and time spent in open and enclosed
arms were recorded. The number of entries and time spent in
the closed arms indicate anxiety. The time spent and number
of entries in the open arms show anxiolytic behavior in rodent
(Campos et al. 2013;Lister1987).
Novel object recognition test One day before the test, the
animals were put in an apparatus (70×50×40 cm) for 20 mi-
nutes so that their exploratory behaviors would not interrupt
their object interactions. Two round or square bowls (familiar
objects) were placed in the box on the following day (familiar-
ization phase). After placing the rat next to the front wall and at
the midpoint for 10 minutes (in front of the objects), it was
moved back to its cage. One hour later in the testing phase, a
novel object replaced a familiar object, and the animal was
given time to explore for five minutes. The distinction ratio
was determined as the ratio of spent time with the novel object
to the total spent time on either object. Using 70% ethanol, all
the areas and objects were cleaned between trials so that the rats
could perceive the olfactory cues (Komaki et al. 2014).
MWM test This test examines the animals’reference and spatial
working memory. Briefly, a circular black pool (diameter, 180
Histological
Study
PAL
Test
MWM
NOR
Test
Open
Field
Surgery and
AD Induction
Habituation
Recovery
Cell
Transplantation
2 mounts
14
7
174
75-
76
77-
81
81-
82
Day
83
Fig. 1 Study design diagram; Alzheimer was induced by ICV injection of Aβ. Cell transplantation was done 7 days after the injections of Aβ. Finally,
behavioral studies (Open field, NOR test, MWM, PAL test) were performed and brain tissue samples were studied histologically
Metab Brain Dis (2019) 34:1131–1143 1133

cm; height, 60 cm) was divided into four quadrants (north, east,
west, and south), and at the center of the north quadrant, a black
platform was placed. The pool was placed in the room with low
light and sound insulated. The geometric shapes were pasted at
the walls for visual cues. Also, the room has various cues such as
racks, sweep, sink, water hoses and shelves.
The animals were assessed by a five-day protocol at the
same time (10:00 and 12:00). They were placed in a quadrant
and given time to find the platform in 90 seconds during the
first four days. If the animal did not find the platform at the set
time, it handler directed to the platform in training. The plat-
form was removed on day five (probe trial), and the rats were
allowed 60 seconds to swim in order to evaluate their refer-
ence memory. Thirty seconds of rest was determined between
the two trials, and a five-minute rest was allowed between the
two blocks. Then, escape latency, time in the target quadrant,
average swimming speed and travelled distance were mea-
sured (Asadbegi et al. 2017).
PAL test A step-through apparatus was usedfor the PAL test. It
consisted of two compartments (20×20×30 cm), including a
transparent plastic compartment and a dark compartment of
opaque plastic, separated by a rectangular opening (6×8 cm).
Stainless steel rods were applied on the floor of compartments,
enabling the delivery of an electric shock by the generator
(Behbood Pardaz, Iran).
For training, the door was lifted after placing the rats in the
light chamber for five seconds. As soon as the rat entered the
dark chamber, the guillotine door was closed, and they were
kept in the chamber for 30 seconds; this trial was repeated
after a 30-minute interval. The entrance latency to the dark
chamber (STLa) was also measured. The guillotine door was
shut for 30 seconds after the animal’s entry into the dark com-
partment; then, an electric shock was induced for three sec-
onds (0.5 mA). After two minutes, this procedure was repeat-
ed. The training ended after the rat’stwo-minutestayinthe
light chamber. In addition, the number of entries to the dark
chamber was determined.
Moreover, in the retention test, the guillotine door was lifted
after putting the rats in the light chamber for five seconds. The
time spent in the dark compartment (TDC), as well as the step-
through latency in retention (STLr), was recorded for five mi-
nutes. The retention test ended if the rat failed to enter the dark
chamber in this period (Hasanein and Shahidi 2011).
Histopathological and immunohistochemistry studies
Sixty-nine days after cell therapy, the animals were
sacrificed, and the brain tissues were stained using an-
tibodies against Aβ1-42 and IBA1. Using xylazine and
ketamine, deep anesthesia was induced, and then, 150-
200 mL of PBS and 4% paraformaldehyde were used
for perfusion. The brain tissues were then harvested and
fixed using 4% paraformaldehyde. After tissue process-
ing, they were paraffin-embedded (Merck, Germany).
Using a microtome, 5μmtransversesectionsofbraintis-
sues were prepared. When the sections were cleared and
dehydrated, antigen retrieval was performed. Incubation was
performed overnight at 4°C using a blocking solution, follow-
ed by anti-beta-amyloid (ORB10087) and anti–Iba1
(ORB336635) primary antibodies. Afterwards, the sections
were washed with PBS. Incubation was performed for two
hours using a secondary anti-rabbit antibody (406403) at room
temperature. DAPI was used for counterstaining the nuclei for
45 minutes after PBS washing; the sections were then
mounted.
Data analysis
Kolmogorov–Smirnov test was used to analyze normality of
data in SPSS v 16. Non_parametric Kruskal–Wallis H_test
was used to assess the significance among non_normality of
data for some variables. One-way ANOVA and Tukey’stests
were applied for the analysis of normal data. All data were
presented as the mean ± S.E.M. The significance level was
0.05 in all analyses.
Results
AD-MSCs were expanded easily in culture
and presented their surface markers
The cells from rat adipose tissues were attached to a flask
during 2-3 weeks and formed a spindle shape (Fig. 2a). The
flow cytometry findings confirmed the positivity of cells for
CD44 and CD90 markers and their negativity for CD45 and
CD34 (Fig. 2b, c, e, and f).
Behavioral studies
MT pretreatment of AD-MSCs had no effects on behavioral
parameters in the OF test:
Kolmogorov–Smirnov test indicated nonparametric of data
for the total traveled distance (p<0.001) and moving velocity
(p<0.01). Kruskal–Wallis test revealed no significant differ-
ences between groups for total traveled distance (H value =
4.476, P = 0.214>0.05; Fig. 3a) and moving velocity (H value
= 6.021, P = 0.111>0.05; Fig. 3b).
MT pretreatment of AD-MSCs had no effects on anxiety
behavioral parameters in the EPM test:
Kolmogorov–Smirnov test indicated nonparametric of data
for the time spent in open and closed arms, and number of
1134 Metab Brain Dis (2019) 34:1131–1143

Fig. 2 Undifferentiated ADSCs (a) under phase contrast microscopy displayed a flattened fibroblast-like morphology. Flow cytometric analysis of
ADSCs showed that they were positive for CD44 and CD90 (band c, respectively) and negative for CD34 and CD45 (dand e, respectively)
Metab Brain Dis (2019) 34:1131–1143 1135

entries into open and closed arms (p>0.001). Kruskal–
Wallis test revealed no significant differences between
groups in time spent in the open arms (H value = 2.72, P
= 0.436>0.05) and number of entries into open arms (H
value = 5.217, P = 0.157>0.05). Also, Kruskal–Wallis test
showed that there was no significant difference in the time
spent in closed arms [H value =0.149, P = 0.985>0.05] and
number of entries into closed arms [H value = 1.691, P =
0.639>0.05] between groups (Fig. 4).
ADSCs and MT-ADSCs had positive effects on the novel object
recognition test:
Kolmogorov–Smirnov test showed normality of data
(p=0.20>0.05). One-way ANOVA showed that there was sig-
nificant difference in discrimination index between groups [F
(4,39) =10.259, p> 0.001]. Compared to the Aβ+ADMSC,
Aβ+MT-ADMSC, sham, and control groups, the results of
this index were lower in the AD group (P< 0.01). Both AD-
MSC and MT-ADMSC transplantations had positive effects
on discrimination indices in contrast with the Aβgroup (P<
0.05). Interestingly, MT-ADMSC-based therapy showed more
ameliorative activities in contrast with the AD-MSC therapy
(P< 0.05) (Fig. 5).
Transplantation of MT-ADSCs ameliorated memory
impairments, as well as context and spatial learning
Based on the MWM test, the effects of Aβinjection and cell
transplantation on spatial memory were examined. Data on ac-
quisition trials are listed in Table 1.TheAβgroup spent less time
in the target quadrant in comparison with the controls in the probe
trial; also, it took more time for this group to find the platform.
Transplantation of MT-ADMSCs majorly increased the spent
time in the target quadrant, while it reduced the escape latency
in contrast with Aβ(P< 0.05). Nevertheless, the swimming speed
was not significantly different between the test groups (Fig. 6).
MT-ADSCs effect on the swimming speed at MWM test
The swimming speed data on acquisition trials are showed in
Fig. 7. One-way ANOVA showed that there was significant
difference between groups at first day [F (4,39) =77.854, p<
0.001]. Tukey post revealed that control group has more speed
compare to sham, Aβ,Aβ+ADMSC and Aβ+MT-ADMSC
groups (p< 0.001). The Aβanimals had more speed time com-
pare to sham group (p< 0.001). One-way ANOVA analysis in
second day showed that there was significant difference be-
tween groups [F (4, 39) =75.308, p< 0.001]. Tukey post
Fig. 3 Effect of cell
transplantation in Aβrat model
on distance move (a) and velocity
(b) in open field test. Data are
expressed as mean ± SEM.
1136 Metab Brain Dis (2019) 34:1131–1143

revealed that control group has higher speed compare to sham,
Aβ,Aβ+ADMSC and Aβ+MT-ADMSC groups (p< 0.001).
Furthermore, One-way ANOVA showed that there was signifi-
cant difference between groups at third day [F (4,39) =68.936,
p< 0.001] and forth day [F (4,39) =144.482, p< 0.001]. Tukey
post showed that control group has higher speed compare to
sham, Aβ,Aβ+ADMSC and Aβ+MT-ADMSC groups at third
and fourth days (p< 0.001).
The results of the swimming speed in the control group by
one-way ANOVA showed that there was significant difference
between groups on the different days [F (3,27) =11.15, p<0.001].
Tukey post showed that first day of learning day has high speed
Fig. 5 Effect of cell
transplantation in Aβrat model
on the discrimination index in
new object recognition test (*P
<0.01 when compared with the
control group, **P <0.05 when
compared with Aβgroup, #P
<0.05 when compared with Aβ+
ADSC group). Data are expressed
as mean ± SEM
0
5
10
15
20
25
30
35
Control Sham AβAβ+ADSC AD+MT-ADSC
Time spent in open arms (sec)
Group
a
0
2
4
6
8
10
12
Control Sham AβAβ+ADSC AD+MT-ADSC
Number of entries in Closed arms
Group
b
0
100
200
300
400
500
600
700
Control Sham AβAβ+ADSC AD+MT-ADSC
Time spent in closed arm (sec)
Group
c
0
0.5
1
1.5
2
2.5
3
Control Sham AβAβ+ADSC AD+MT-ADSC
Number of open arms entries
Group
d
Fig. 4 Effect of MT pretreatment of AD-MSCs on elevated plus maze
test performance: (a) Time spent in the open arms; (b)numberofentries
in closed arms; (c) Time spent in the close arms; and (d) number of total
entries into the open arms. Data were analyzed by Kruskal–Wallis test.
Data are means ± SEM (n= 8 per group)
Metab Brain Dis (2019) 34:1131–1143 1137

compare to other days (p<0.001). There was not significant dif-
ference between second, third and fourth days.
The results of the swimming speed in the sham group by one-
way ANOVA showed that there was significant difference be-
tween different days [F (3,27) =23.47, p<0.001]. Tukey post
showed that first day of learning day has high speed compare
to third and fourth days (p<0.01). Also, second day of learning
day has high speed compare to third and fourth days (p<0.01).
The results of the swimming speed in the Aβgroup by one-
way ANOVA showed that there was significant difference
between days [F (3,27) =12.2, p<0.001]. The second day of
learning day has high speed compare to fourth days (p<0.01).
One-way ANOVA showed that there was not significant
difference between acquisition days in the ADMSC group [F
(3,27) =2-.04, p= 0.131>0.05].
The results of the swimming speed in the Aβ+MT-
ADMSC group by one-way ANOVA showed that there was
significant difference between different days [F (3,27) =10.92,
p<0.001]. Tukey post showed that first day of learning day has
low speed compare to third and fourth days (p<0.01and
p<0.05, respectively). Also, second day of learning day has
low speed compare to third day (p<0.05).
MT-ADMSCs enhanced memory in the PAL test
The results of the PAL learning phase are shown in Fig.
7. During the first acquisition trial, the test groups were
not significantly different regarding STLa (Fig. 8a). The
number of acquisition trials was different between the
test groups. The frequency of trials was higher in the
Aβgroup versus the sham, control, and experimental
groups (P< 0.05; Fig. 8b). Fig. 6.C depicts the PAL
retention phase. The test groups were significantly differ-
ent in terms of STLr; in comparison with the sham and
control groups, the Aβgroup had lower STLr (P< 0.05).
However, the AD-MSC and Aβgroups were not signif-
icantly different.
In comparison with the Aβand AD-MSC groups, MT-
ADMSC transplantation significantly increased STLr (P<
0.01; Fig. 8c). Furthermore, the MT-ADSC group spent less
time in the dark chamber in contrast with the AD-MSC and
Aβgroups (Fig. 8d). On the other hand, TDC was longer in
the Aβgroup, compared to the MT-ADMSC, control, and
sham groups. TDC was not significantly different between
the Aβgroup and AD rats receiving AD-MSCs (P <0.05;
Fig. 8d).
MT-AD-MSCs-based therapy considerably decreased Aβ
deposit in comparison with the AD-MSCs group
The AD-MSCs and MT-ADMSCs reduced the brain Aβ
deposition. However, reduction was significant only in
the MT-ADMSC group (Fig. 9a-d;P<0.05).
Table 1 Effect of cell transplantation in Aβrat model on scape latency during acquisition trials. A) Comparison between groups in each day B)
Comparison between the performances of each group in four days
A
Day 1 Day 2 Day 3 Day 4
Control vs. Sham Ns Ns Ns Ns
Control vs. AβNs Ns Ns Ns
Control vs. Aβ+ ADSC * **** Ns *
Control vs. Aβ+ MT-ADSC **** Ns Ns Ns
Sham vs. Aβ* NsNsNs
Sham vs. Aβ+ADSC ** **** * *
Sham vs. Aβ+ MT-ADSC **** Ns Ns Ns
Aβvs. Aβ+ ADSC Ns ** Ns Ns
Aβvs. Aβ+ MT-ADSC **** Ns Ns Ns
Aβ+ ADSC vs. Aβ+ MT-ADSC **** **** Ns Ns
B
Control Sham AβAβ+ADSC Aβ+MT-ADSC
Day-1 vs. Day 2 *** ** **** Ns Ns
Day-1 vs. Day 3 **** **** **** **** Ns
Day-1 vs. Day 4 **** **** **** **** Ns
Day 2 vs. Day 3 Ns Ns Ns *** Ns
Day 2 vs. Day 4 Ns * ** **** Ns
Day 3 vs. Day 4 Ns Ns Ns Ns Ns
Ns not significant
1138 Metab Brain Dis (2019) 34:1131–1143

Transplanted AD-MSCs and MT-AD-MSCs were detected
in the brain and differentiated to microglia
The histological examination demonstrated the migration
of Dil-labeled AD-MSCs and MT-ADMSCs from the
delivery site to the brain. The MT-ADMSC group had
a significantly higher number of transplanted cells in
comparison with the AD-MSC group. The immunohis-
tochemistry study using Iba1 antibody showed that
transplanted AD-MSCs and MT-ADMSCs differentiated
to microglia in the brain. The microglia count in the
brain significantly decreased in both experimental
groups in comparison with the controls. However, the
number of microglia was significantly lower in the
MT-ADMSC group, compared to the AD-MSC group
(Fig. 9e-h;P<0.05).
Discussion
Stem cell therapy, especially MSC-based therapy, is
promising for AD (Bali et al. 2017). The current study
took some initial steps towards the improvement of
MSC-based cell therapy for AD. The current study, for
the first time, pretreated AD-MSCs with MT and then
transplanted MT-AD-MSCs in an experimental AD
model and investigated the healing effects. It was ob-
served that MT improved ADMSC-based therapy in
AD. Also, MT-ADMSCs improved the learning ability,
memory function, and cognition significantly more than
AD-MSCs. These beneficial effects were showed with-
out change in the locomotion activity or anxiety behav-
ior in the experimental groups.
The avoidance and spatial memory of all rats were
examined by PAL and MWM test. Also, the MWM test
is a negatively reinforced task and reaching the platform
is considered rewarding (Paul et al. 2007). The average
swimming speed is best variable for measured of animal
motivation to reach reward in the water tank of MWM.
The AD rat exhibited cognition and memory impairment
without higher speed and more time of swimming in
acquisition days of MWM test. This result is accordance
to previous study in which Aβmicro-injection into brain
has been considered as suitable model for induction of
amnesia and reward learning impairment (Malin et al.
2001). The AD rats unable to distinguish various cues
to reaching to platform as reward in learning. Current
result is similar to reported study while the behavioral
task and method was different (Malin et al. 2001;Shi
et al. 2015; Shi et al. 2012). The Aβ+ADSC, Aβ+MT-
ADSC groups presented better cognition and memory in
NOR, PAL and MWM tests. The potential effect of MT
and MT-ADSC did not affect in locomotion and anxiety
behavior. However, the shorter distances have specials
for β+ADSC and Aβ+MT-ADSC groups in the open
field test (no significant). Therefore, the EPM test was
chosen for to better understanding the effect of MT and
MT-ADSC in anxiety. The result of EPM test revealed
that the lower speed of β+ADSC and Aβ+MT-ADSC
rats did not cause by anxiety. Also, the AD rats did not
exhibit anxiolytc or anxiogenic behavior in EPM test.
Fig. 6 Effect of cell transplantation in Aβrat model on mean escape
latency to find the hidden platform during acquisition trials (a), time
spent in target quadrant (b), and mean escape latency to find the hidden
platform at probe trial (c) in the Morris water maze (*P <0.05 when
compared with the control group in B, # P <0.05 when compared with
the Aβgroup, *P <0.05 when compared with the Aβgroup in C). Data
are expressed as mean ± SEM
Metab Brain Dis (2019) 34:1131–1143 1139

Of noted, the histopathological study showed more
migration and presence of MT-AD-MSCs in brain tis-
sues. Pretreatment of AD-MSCs with MT resulted in the
significant clearance of Aβdeposition (Jahnke et al.
1999; Sánchez-Barceló et al. 2010). According to previ-
ous studies, MT treatment could improve MSC treat-
ment for different diseases. In a study by Lee et al.,
treatment with MT improved MSC mobility in the um-
bilical cord blood, besides skin wound healing (Lee
et al. 2014).
Based on several studies, MT treatment improves AD-
MSCs treatment for sepsis-induced kidney damage, acute
ischemia-reperfusion damage, and acute interstitial cystitis
(Chen et al. 2014a;Chenetal.2014b; Yip et al. 2013).
Moreover, MT, which can promote the osteogenic differ-
entiation of bone marrow MSCs, is applied in the treat-
ment of osteoporosis (Amstrup et al. 2013; Zhang et al.
2010). As previously reported, MT exhibits major ROS
elimination potential, inhibits the p53 pathway (Zhang
and Zhang 2014), and regulates intracellular signaling
Fig. 8 Effect of cell transplantation in Aβrat model on the step-through
latency in acquisition trial (a), the number of trials to acquisition (b), the
step-through latency in recognition trial (c), and the time spent in the dark
compartment (d) in passive avoidance learning task (*P <0.01 when
compared with the control group, **P <0.05 when compared with the
Aβgroup). Data are expressed as mean ± SEM
***
!! !! !!
## !!^^ !!^^ !!^^
!!^
^^
^
&&
0
10
20
30
40
50
60
70
80
90
1234
Swimming speed
Day
Control Sham AβAβ+ADSC Aβ+MT-ADSC
Fig. 7 Effect of cell
transplantation in Aβrat model
on mean of swimming speed
(Cm/S) to find a hidden platform
in the Morris Water Maze. Data
are expressed as mean ± SEM.
***= P<0.001 as compare control
group. ##= P<0.01 as compare
sham group. &&= P<0.01 as
compare Aβgroup. !!= P<0.01 as
compare to first day. ^^= P<0.01
as compared to second day. ^=
P<0.05 as compared to second
day
1140 Metab Brain Dis (2019) 34:1131–1143

(Luchetti et al. 2010). In addition, its effects on immune
regulation and senescence delay have been reported (Bali
et al. 2017; Cardinali et al. 2008; Espino et al. 2012;
Koyama et al. 2002; Son et al. 2014).
In the current study, homing of both AD-MSCs and MT-
ADMSCs in the brain was confirmed by an immunohisto-
chemical study 69 days after intravenous cell transplantation.
The results showed that preconditioning with MT increased
the transplanted cell count in the brain after this period; this
increase can be due to the greater migration or survival of
these cells in the brain (or both).
The transplanted cells were detected in the Aβdepo-
sition site. The Aβplaque studies showed that both
AD-MSCs and MT-ADMSCs could clear Aβdeposits,
which consequently led to the improvement of cognitive
performance in AD rat models. However, the effect of
a
b
c
e
f
g
hd
Fig. 9 Effect of cell transplantation on Iba1-positive cells and Aβde-
posits in the brain of Aβrat model detected by immunohistochemistry.
ADSC (A, E), MT-ADSC (B, F), AD (C, G), percentage of amyloid beta
protein (D) and Iba1 protein (H). a: compared with the control group; b:
compared with the Aβgroup; c: compared with the ADSC group (P <
0.05)
Metab Brain Dis (2019) 34:1131–1143 1141

MT-ADMSCs was significantly greater on the clearance
of Aβdeposits than that of AD-MSCs. Therefore, pre-
treatment with MT can increase all the outcomes of AD-
MSC transplantation (Liu et al. 2016).
The current immunohistochemical study analyzed the dif-
ferentiation of transplanted cells to microglia using Iba1. The
histological results showed the differentiation of AD-MSCs
and MT-ADMSCs into microglia at the site of Aβdeposits.
The number of microglia cells was majorly lower in MT-
ADMSCs versus AD-MSCs (Ohsawa et al. 2004). Microglia
activation can have a dual role in the AD pathogenesis. On
one hand, acute microglial activation may reduce the accumu-
lation of Aβ, and on the other hand, chronic microglia activa-
tion may lead to synapse loss and neurotoxicity through stim-
ulation of some proinflammatory cascades (Sarlus and
Heneka 2017).
It has been demonstrated that bone marrow MSCs can pre-
serve the resting microglia phenotype and control the activa-
tion of microglia (Yan et al. 2013). Also, the number of mi-
croglia depends on the plaque and Aβcount. Therefore, the
current findings regarding the few number of microglia in
MT-ADMSCs can be related to the enhanced regulatory im-
pact of AD-MSCs and the reduced amount of Aβ
(Mandrekar-Colucci and Landreth 2010).
Conclusion
The current study employed a simple, non-genetic engineer-
ing, and versatile strategy to improve AD-MSCs-based thera-
py, especially for AD. AD-MSCs were pretreated with one of
the well-known antiinflammatory and antioxidant agents’
Melatonin’and transplanted in a rat AD model. Consistent
with our assumption, MT pretreatment increased the survival
of AD-MSCs, improved learning, memory, and cognition, and
reduced Aβplaques, highlighting the positive effects of MT.
Nevertheless, future comprehensive research is essential to
clarify the mechanisms through which MTinduces its positive
effects on ADMSC-based therapy.
Acknowledgments Guilan University of Medical Sciences granted this
study (No., 96120508).
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