Zeinab SafialHosseini1, Mohammadreza Bigdeli2,3, Sepideh Khaksar4, Abbas Aliaghaei5. 1. Department of Physiology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran. 2. Department of Physiology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran. Electronic Address: bigdelimohammadreza@yahoo.com. 3. Institute for Cognitive and Brain Science, Shahid Beheshti University, Tehran, Iran. 4. Department of Herbal Science, Faculty of Biological Sciences, Alzahra University, Tehran, Iran. 5. Department of Anatomy and Cell Biology, School of Medicine, Shahid Beheshti University of Medical Science, Tehran, Iran.
Cerebral ischemia is the third reason of death and
physical impairment in the world caused by the blood
vessel blockage, through a blood clot or rupture of a
vessel, responsible for the supply of a part of brain tissue
(1). About 85% of stroke cases are caused by ischemia and
15% by a brain hemorrhage. The best way for controlling
the stroke is the early prevention of ischemic damage
expansion and thrombotic therapy (2). During cerebral
ischemia, due to lack of oxygen and ATP, the ion pumps
that are dependent on ATP, such as sodium-potassium and
calcium pumps suffer from functional impairment (3). So,
the excessive release of glutamate into the synaptic space
leads to excitotoxicity.Consequently, the extreme influx of extracellular
calcium causes an imbalance in cellular homeostasis.
An increase in the concentration of calcium inside
the cell can activate the caspase enzymes that are in
charge of inducing cell death and damages to ischemic
cells. Moreover, intracellular calcium can increase the
production of free radicals and cause more damages to
ischemic cells (4). It should also be emphasized that the
main reason for ischemic tissue damage is excitotoxicity.
Therefore, a decrease in the concentration of glutamate
in the synaptic space can significantly reduce ischemic
damages (5).One of the reasonable choice for reducing glutamate
effects in synaptic space is blockage of the N-methyl-D
aspartate (NMDA) receptor. NMDA is a receptor for
stimulant neurotransmitters, called glutamate, which
is a mediator of stimulant neural transmissions in the
central nervous system. The excessive activity of this
receptor leads to an increase in calcium intake, which
provokes excitotoxicity and ultimately death of cells (6).
In physiological conditions, the coupling of Mg2+ ion with
the NMDA receptor prevents over-depolarization of the
nerves, and in pathological conditions, lack of binding
of Mg2+ ion to the NMDA receptor stimulates the nerve
extremely (7). Memantine, as a non-competitive antagonist
of the NMDA receptor, has a significant role in decreasing
the destructive cytotoxicity of the stroke. Memantine is
prescribed for the treatment of dementia, Alzheimer’s disease, and Parkinson’s disease. The advantage of
this drug in comparison with other glutamate receptor
antagonists is that it blocks the NMDA receptor without
affecting on the natural activity of the receptor, leading to
the reduction in neuronal function and excitotoxicity (8).
This drug prevents the toxic interactions of free radicals,
such as nitric oxide (NO) and reactivity oxygen groups
(ROS) with vital macromolecules and also prevents
the stimulation and activation of apoptosis-stimulating
proteins, such as caspases, neural NO synthase (nNOS),
and cytochrome C (9). In another report, it was proved
that memantine alone and in combination with melatonin
reduced brain damages due to the reduction of P38,
ERK-1/2, and inducible NO synthase (iNOS) (10). Also,
memantine ameliorated the pathogenesis of Alzheimer’s
disease in animal models via blockage of the NMDA
receptor and reduction of glutamate excitability (11).In the brain, non-fatal ischemia can induce protective
responses against subsequent intensive ischemic injury,
called ischemic tolerance (12). The cerebral ischemia
is common in people who are susceptible to cerebral
ischemia, including patients with a history of heart attack
and aneurysm. Accordingly, our purpose is the induction
of ischemic tolerance by pretreatment of rats with Sertoli
cells (SCs) and memantine.Along with drug treatment, new strategies, such as cellbased therapy is used for the treatment of stroke. The most
of cell resources, including fetal neural cells, stem cells,
and SCs (as somatic cells) are suggested as an effective
way for the management of some neurodegenerative
diseases such as Alzheimer’s disease, amyotrophic lateral
sclerosis, Huntington’s disease, and stroke. These cells
are viable and can be replaced with the cells that reside in
damaged tissues. They are also capable of reconstructing
neural circuits and reducing functional impairment in the
brain of patients with the above disorders (13). In the current
research, transplanted SCs, as a pretreatment option, for
brain ischemia were used. These cells are found in testicular
tissues, possessing a high antigenic property, and providing a
proper environment for the development of germ cells (14).
Also, SCs express neurotrophic and growth factors, such as
glial cell-derived neurotrophic factor (GDNF), insulin-like
growth factor (IGF), transforming growth factor (TGF),
vascular endothelial growth factor (VEGF), and fibroblast
growth factor (FGF) (15). The reason for choosing the SCs
for this research is the dominant properties of SCs compared
with other somatic cells, including the inhibition of the
immune system by SCs preventing the rejection of organ in
a recipient.Furthermore, SCs suppress the immune system
by forming the tight connections around the nerve
cells, thereby the production of interleukin-2 (IL-2)
inhibitory factor, and inhibition of expression of major
histocompatibility complex (MHC) (16). In another study,
it was found that the co-culture of embryonic stem cells
with SCs led to the differentiation of embryonic stem
cells into dopaminergic neurons as a result of the presence
of GDNF factor that is secreted from SCs and acts as a
dopaminergic inducer in stem cells (17). Moreover, it
was indicated that transplantation of SCs reduced the
ischemic damages, such as infarction, brain edema, and
the breakdown of the blood-brain barrier (BBB) (18).
Concerning the characteristics mentioned about SCs,
they could be applied as competent candidates for the
amelioration of ischemic injuries.In the current research, we focused on the effect
of transplanted SCs in combination with the use of
memantine in an animal model of cerebral ischemia.
Materials and Methods
Animal assignment and experimental protocol
In this experimental study, 98 adult male rats (with the
weight range between 250 and 350 g, age range between
5 to 7 weeks) were procured from the Pasteur Institute.
The water and food were given to all rats without
restriction, and they were kept in standard conditions at
the temperature of 22 ± 2˚C and 12:12 light-dark (LD)
cycle. Rats were randomly divided into five groups: sham,
control, allograft SC transplant recipient, memantinetreated group, and SC- and memantine-treated groups.
The control group (n=21) consisted of rats that underwent
ischemia following MCAO surgery. The control group
was classified into three sub-groups to evaluate infarction
(n=7), the BBB permeability (n=7), and brain edema
(n=7). The sham group (n=14) consisted of rats that were
suffered from the same surgery procedure of MCAO
without importing suture and subdivided to assess the
BBB permeability (n=7) and cerebral edema (n=7). The
animals belonging to the allograft SC transplantation group
received SCs from another rat tissue and were induced by
MCAO surgery. The allograft SC transplantation group
(n=21), the memantine-treated group (n=21), and the
SC and memantine-treated group (n=21), divided into
subgroups, as well as the control group. All rats were
examined for the neurological deficit.In this research, the two groups were selected for receiving
SC culture medium and SCs without ischemic surgery. The
comparison between these two groups and allograft SC
transplantation did not indicate any significant difference.
Also, the experimental methods were accomplished on
vehicle (solvent of memantine) group. There was no
remarkable difference between the vehicle group and other
groups. So, the data of these groups are not shown.
Ethical statement
This study was designed according to the rules of the
National Institutes of Health and Care Guidance, the use
of the Animal Laboratory (NIH Publications) revised
in 2011, and the Ethics Committee of Shahid Beheshti
University (No. 1012.667). Our whole effort was to use
the lowest number of animals in this research.
Pharmaceutical compound
Memantine, as a NMDA receptor blocker was used in this study was injected with different doses (1 mg/kg and
20 mg/kg; intraperitoneally). A week after stereotaxic
surgery and SC transplantation, the injection of the drug
was performed. Memantine was solved in saline. First,
the high dose of 20 mg/kg and 12 hours later a low dose
(1 mg/kg) was injected. One hour after the final injection,
middle cerebral artery occlusion (MCAO) surgery was
started. The minimum dose of memantine for each rat
was 1 mg/kg, and the minimum toxic dose was 25 mg/
kg; so, the most appropriate dose used as the therapeutic
dose was 20 mg/kg (19, 20). According to the previous
studies, the dose of 20 mg/kg was selected in this
research. By injecting this dose, the concentration of the
drug in the brain tissue reaches 1-10 micromolar that this
concentration is suitable for blocking a large number of
NMDA receptors.Moreover, the half-life of the drug is 12 hours. Therefore,
for keeping the levels of the drug at 1-10 micromolar in
the brain, 12 hours after the first injection, the preservative
dose of 1 mg/kg was administered (19, 20).
Isolation and culture of sertoli cell
At first, rats were sacrificed, and their testis removed and
transferred into a falcon containing the culture medium and
antibiotics. Under sterile conditions, the small pieces were
separated from testicular tissue. Tissues were transmitted
to tubes containing trypsin (0.25%, Gibco, USA) and
were incubated at 37˚C for 15 minutes. First, the nephrotic
tubes were separated; then, trypsin was aspirated, and 1%
collagenase was added to the tubes consisting of tissues,
and the samples were incubated at 37˚C for 15 minutes.
After pipetting the samples and addition of the serum, the
samples were centrifuged, and the resultant pellet was
transferred into the culture flasks containing Dulbecco’s
Modified Eagle’s Medium (DMEM/F12, Gibco, USA)
culture medium supplemented with 10% fetal bovine
serum (FBS, Gibco, USA) and antibiotics. After 48 hours
of the culture period, the culture medium was replaced
by the new one. When the cells reached to appropriate
density, they were passaged with trypsin (18).
Immunocytochemistry of Sertoli cells
SCs were transferred into the plates for 24 hours, and
after the cells reached appropriate density, the whole
culture medium was removed. Then, it was washed
with phosphate buffered saline (PBS), and the cells
were fixed by 4% paraformaldehyde solution. The cells
were permeabilized by 0.3% Triton X-100 solution
(Sigma Aldrich, USA), and then incubated in blocking
solution. Afterward, the cells were incubated with the
primary antibody (anti-GATA4, Abcam, USA) overnight.
Subsequently, the cells were washed with PBS and
incubated with the second antibody (goat conjugatedFITC anti-mouse antibody). The nuclei of the cells were
stained by Hoechst and were observed under a fluorescent
microscope. The presence of SCs was performed by the
immunocytochemistry technique, as GATA4 was defined
as a marker.
Stereotaxic surgery and injecting Sertoli cells
After SCs reached to appropriate density, the cells
were located in a suspension of a 2 μl DMEM aliquot.
Then, they were separated with a trypsin solution. After
centrifuging, the cells were counted with trypan blue
staining method, and 500,000 cells were selected for the
injection. Rats were anesthetized, and their heads were
fixed in a stereotaxic device. After cleaning the skull
surface, the distance between the Lambda and Bregma
was found, and injection of the cells into the right striatum
was carried out by a Hamilton micro-syringe, according
to the coordinates of the brain atlas specified for rats:
Bregma: +0.5 mm AP; ± 2.6 mm ML; -5 mm DV (21).The injected cells were labeled with DiI (5 μg/ml for
20 minutes before the injection) (Sigma Aldrich, USA)
and Hoechst staining. After 7 days, rats were sacrificed,
and the brain was prepared for the determination of the
survival and distribution of transplanted cells. The cells
were detected by fluorescing microscopy.
Induction of focal ischemic stroke
Anesthesia of rats was performed with chloral hydrate
(400 mg/kg). According to the method explained by
Lange et al. (22), MCAO surgery was performed. In this
surgery, a 0-3 nylon suture was inserted to the internal
artery through the trunk of the common carotid artery and
to the anterior cerebral artery. It continued through the
internal carotid artery. Due to the insertion of the suture
and blockage of arterial blood flow, the blood flow was
not able to reach MCA. After 60 minutes, the suture was
withdrawn and the right hemisphere blood flow restored
through the Willis Ring.
Neurobehavioral evaluation
Behavioral evaluation of neurological defects was
conducted 24 hours after the reperfusion process. The
neurological finding was categorized into 5 parts,
including motor function, sensory function, beam test,
raise the Tail, and reflex activity. The maximum score for
each animal was 18 (23).
Infarction volume assessment
After 24 hours of the reperfusion process, rats were
sacrificed with a high dose of an anesthetic drug, their
brains were isolated, and kept at 4˚C for 10 minutes. Then,
brains were coronally sectioned at a thickness of 2 µm.
After that, 2% triphenyl tetrazolium chloride solution was
poured on the slides, and incubated at 37˚C for 15 minutes.
The summed infarct volume from all brain sections was
calculated as the total infarct volume. The contribution
of the edema to the infarct volume was modified with
the formula mentioned below. The evaluation of infarct
volume in the cortex, striatum, and the Piriform cortexamygdala regions was also accomplished separately (24).
The corrected volume of damaged area=left hemisphere
volume - (left hemisphere volume- damaged area
volume).
Assessment of brain edema
After removing the brains from the skulls, the weights
of various areas including the striatum, cortex, and cortex
Piriform-amygdala as the wet weight were measured and
after incubation at 120˚C for 24 hours, the dry weight
(DW) was also measured. Finally, the content of brain
water was estimated according to the following formula
(24). [(WW-DW)/WW]×100
Measuring the permeability of the blood-brain barrier
After 30 minutes of the induction of ischemia, the
injection of 2% Evans Blue solution (in the amount
of 4 ml/kg) was carried out through the blood vein.
Perfusion was performed using 250 ml saline, 24 hours
after the withdrawal of suture. The different parts of the
brain, including the striatum, cortex, and the Piriform
cortex-amygdala were separated from each other, and
each of them was weighed, separately. Then, it was
homogenized with phosphate buffered solution, and
after mixing with 60% acid trichloroacetic acid, it
was agitated by a vortex for 3 minutes. Then, microtubes were kept at 4˚C for 30 minutes. In the next
step, the samples were centrifuged at 1000 rpm for
30 minutes. Therefore, the light absorbance of brain
solution was measured at the wavelength of 610 nm
and compared with the standard concentration curve,
and concentration of Evans Blue dry was calculated as
μg/g of the brain tissue (24).
Statistical analysis
All of the statistical analyses were conducted by the
SPSS software version 22 (SPSS Inc, Chicago, IL,
USA). The data from NDS were quantified by the nonparametric test (Kruskal-Wallis), followed by the Dunn
test. The volume of tissue damage (infarction) was
determined by the ImageJ software (Version 1.50), and
the obtained findings were analyzed by one-way ANOVA.
The comparison of the BBB integrity and brain edema
were calculated by one-way ANOVA, followed by the
Bonferroni post hoc test. The results were expressed as
mean ± SEM. The level of significance was set at P<0.05.
Results
Confirming the presence of Sertoli cells in testiclederived cells
The immunocytochemistry analysis of SCs against antiGATA4 antibody showed that the cultured cells expressed
the GATA4 marker. GATA4 is expressed in SCs (green
color). The nuclear staining conducted by Hoechst
confirmed that the blue nuclei belonged to SCs (Fig .1A).
Fig 1
The identification and presence of Sertoli cells in testicle-derived
cells. A. Sertoli cells expressed GATA4 marker that is shown in green.
Staining the cultured cells with Hoechst showed that the Sertoli cells with
blue nuclei (scale bar: 200 µm, ×20). The immunocytochemistry proves
that the cultured cells are Sertoli cells. B. Staining the injected Sertoli
cells with DiI. By using fluorescent microscopy, it was illustrated that the
injected cells are alive after 7 days (scale bar: 200 µm, ×20).
Survival of injected cells in the striatum after 7 days
Staining injected SCs with DiI (a fluorescent lipophilic
cationic indocarbocyanine dye) confirmed the survival of
injected cells in the striatum after 7 days (Fig .1B).The identification and presence of Sertoli cells in testicle-derived
cells. A. Sertoli cells expressed GATA4 marker that is shown in green.
Staining the cultured cells with Hoechst showed that the Sertoli cells with
blue nuclei (scale bar: 200 µm, ×20). The immunocytochemistry proves
that the cultured cells are Sertoli cells. B. Staining the injected Sertoli
cells with DiI. By using fluorescent microscopy, it was illustrated that the
injected cells are alive after 7 days (scale bar: 200 µm, ×20).
The effect of Sertoli cell transplantation and injection
of memantine on neurological deficits
The analysis of the total score of the behavioral assessment
showed that in the allograft SC transplantation, memantine,
and allograft SC transplantation+memantine a significant
decrease groups was observed in neurological deficits
compared with the control group. Moreover, the total
score obtained from the neurological examinations in the
sham group had a significant decrease compared with the
MCAO surgery or control group (Fig .2). The partial tests
were statistically analyzed separately in the experimental
groups. The results revealed that SCs, memantine, and the
simultaneous administration SCs with memantine exerted a
reduction in neurological tests such as reflex activity, sensory
functions, and motor functions (Table 1).
Fig 2
The effect of the Sertoli cell transplantation, memantine, and
allograft transplantation of Sertoli cells plus memantine on total
neurological deficit scores. Values are expressed as the mean ± SEM
(n=10). P˂0.05 compared with the control group (Nonparametric KruskalWallis analysis). *; P<0.05 and **; P<0.01.
Table 1
The partial neurologic deficit scores in each experimental group (n=10)
Experimental groups
Rats
Raise the tail
Motor function
Sensory function
Beam test
Reflex activity
Sum
Average
Survey result
Control (MCAO)
1
2
4
2
2
1
11
13.2
Meaningful to the sham group (P<0.001)RT: 1-2 (P=0.002)MF: 1-2 (P=0.001)SF: 1-2 (P=0.002)BT: 1-2 (P=0.003)RA: 1-2 (P=0.001)
2
2
4
2
2
2
12
3
1
5
2
3
2
13
4
3
5
2
3
2
15
5
3
4
2
2
3
14
6
3
6
2
2
2
15
7
2
5
2
2
2
13
8
2
4
2
3
3
14
9
2
5
2
2
2
13
10
2
5
2
2
1
12
Sham
1
1
1
2
1
1
6
5
2
0
2
1
0
1
4
3
0
2
0
2
2
6
4
0
2
1
1
0
4
5
1
3
1
0
1
6
6
0
1
1
2
0
4
7
0
1
2
1
1
5
8
0
1
1
1
1
4
9
1
2
1
1
0
5
10
0
1
1
2
2
6
Sertoli cells(SCs cells)
1
0
3
2
1
1
7
8
Meaningful to the control group (P=0.04)RT: 3-1 (P=0.01)MF: 3-1 (P=0.03)SF: 3-1 (P=0.03)BT: 3-1 (P=0.05)RA : 3-1 (P=0.03)
2
1
5
2
1
1
10
3
1
3
1
1
1
7
4
2
5
1
1
2
11
5
0
4
2
2
2
10
6
1
4
0
1
1
7
7
1
2
1
1
1
6
8
1
5
1
1
1
9
9
1
3
2
0
2
8
10
0
2
1
1
1
5
Memantine
1
1
3
1
1
1
7
7.1
Meaningful to the control group (P=0.01)RT: 4-1 (P=0.03)MF: 4-1 (P=0.01)SF: 4-1 (P=0.03)BT: 4-1 (P=0.02)RA: 4-1 (P=0.001)
2
2
2
1
1
2
8
3
1
3
2
0
1
7
4
0
5
1
1
1
8
5
1
4
1
0
1
7
6
1
3
2
0
0
6
7
1
3
1
1
1
7
8
1
4
1
1
1
8
9
0
3
1
2
1
7
10
1
2
2
0
1
6
SCs+Memantine
1
2
2
1
2
2
9
6.1
Meaningful to the control group (P<0.001)RT: 5-1 (P=0.01)MF: 5-1 (P=0.001)SF: 5-1 (P=0.003)BT: 5-1 (P=0.04)RA: 5-1 (P=0.02)
2
2
1
1
2
2
8
3
2
3
0
0
1
6
4
1
2
1
1
2
7
5
1
1
1
1
0
4
6
0
3
1
0
1
5
7
0
2
1
1
2
6
8
1
2
1
1
2
7
9
1
1
1
1
1
5
10
1
1
0
1
1
4
MCAO; Middle Cerebral Artery Occlusion, SCs; Sertoli cells, RT; Raise the tail, MF; Motor function, SF; Sensory function, BT; Beam test, RA; Reflex action,
1: Control group, 2; Sham group, 3, Sertoli cell group, 4; Memantine group, and 5; The Sertoli cell transplantation+memantine (Nonparametric KruskalWallis analysis).
The effect of the Sertoli cell transplantation, memantine, and
allograft transplantation of Sertoli cells plus memantine on total
neurological deficit scores. Values are expressed as the mean ± SEM
(n=10). P˂0.05 compared with the control group (Nonparametric KruskalWallis analysis). *; P<0.05 and **; P<0.01.The partial neurologic deficit scores in each experimental group (n=10)MCAO; Middle Cerebral Artery Occlusion, SCs; Sertoli cells, RT; Raise the tail, MF; Motor function, SF; Sensory function, BT; Beam test, RA; Reflex action,
1: Control group, 2; Sham group, 3, Sertoli cell group, 4; Memantine group, and 5; The Sertoli cell transplantation+memantine (Nonparametric KruskalWallis analysis).
The effect of Sertoli cell transplantation and injection
of memantine on infarct volume
The effect of SCs and memantine on infarction volume
was evaluated 24 hours after reperfusion. The results
showed that pretreatment of allograft SCs, memantine, and
allograft transplantation of SCs plus memantine caused
a reduction in infarct volume in the total, the striatum,
the cortex, and the Pir-Amy regions compared with the
control group (Fig .3). Analysis of the total infarct volume
in the experimental groups revealed that the memantine
(95.48 ± 7.70 mm3, P=0.001), allograft SC transplantation
(142.69 ± 5.74 mm3, P=0.03), SC transplantation plus
memantine (59.24 ± 9.59 mm3, P=0.001) groups indicated
a significant reduction in comparison with the control
group (213.86 ± 13.42 mm3). The administration of
memantine (51.11 ± 6.31 mm3, P=0.003), allograft SCs
(86.38 ± 9.80 mm3, P=0.03), and SC transplantation plus
memantine (25.09 ± 7.40 mm3, P=0.001) caused a decrease
in the cortex infarct volume in comparison with the
control group (116.98 ± 9.26 mm3). The Pir-Amy infarct
volume in the memantine (17.32 ± 2.69 mm3, P=0.01), the
allograft SC transplantation (21.05 ± 3.32 mm3, P=0.04),
and SC transplantation plus memantine (12.21 ± 2.03
mm3, P=0.001) groups was diminished compared with
the control group (37.34 ± 3.63 mm3). Furthermore, the
infarct volume of the striatum in the memantine (26.07± 3.52 mm3, P=0.02), the allograft SC transplantation
(33.64 ± 3.80 mm3, P=0.04), and SC transplantation plus
memantine (17.75 ± 4.04 mm3, P=0.006) groups showed
a remarkable decrease in comparison with the control
group (61.37 ± 6.95 mm3).
Fig 3
The effect of the Sertoli cell transplantation on infarction volume. A.
The graph shows the effect of Sertoli cell transplantation, memantine, and
allograft transplantation of Sertoli cells plus memantine on infarction volume
in the total, cortex, striatum, and Piriform cortex-amygdala (Pir-Amy). Each
column illustrates the mean ± SEM of the infarct volume (n=7). P˂0.05
compared with the control group (One-way ANOVA test). *; P<0.05 and
***; P<0.001. B. The Sertoli cell transplantation and injection of memantine
reduced infarct volume in comparison with the control. Each column displays
coronal sections of the control, Sertoli cell transplantation, memantine, and
the Sertoli cells+memantine groups. Strained or red parts, as well as unstained
or white parts of the brain tissue, are considered as normal and damaged
areas, respectively.
The effect of the Sertoli cell transplantation on infarction volume. A.
The graph shows the effect of Sertoli cell transplantation, memantine, and
allograft transplantation of Sertoli cells plus memantine on infarction volume
in the total, cortex, striatum, and Piriform cortex-amygdala (Pir-Amy). Each
column illustrates the mean ± SEM of the infarct volume (n=7). P˂0.05
compared with the control group (One-way ANOVA test). *; P<0.05 and
***; P<0.001. B. The Sertoli cell transplantation and injection of memantine
reduced infarct volume in comparison with the control. Each column displays
coronal sections of the control, Sertoli cell transplantation, memantine, and
the Sertoli cells+memantine groups. Strained or red parts, as well as unstained
or white parts of the brain tissue, are considered as normal and damaged
areas, respectively.
The effect of Sertoli cell transplantation and injection
of memantine in brain water content
The results of brain edema also revealed the reduction
of brain edema in the right striatum, cortex, and Pir-Amy
regions of the brain compared with the control group
(Fig .4). The beneficial effect of SC transplantation and
the injection of memantine on brain edema in the cortex
of the memantine- (77.99 ± 0.75%, P=0.005), allograft
SC transplantation (78.14 ± 0.52%, P=0.03), and SC
transplantation plus memantine (76.64 ± 0.65%, P=0.003)
groups was demonstrated in comparison with the control
group (82.45 ± % 0.79). Brain edema in the Pir-Amy
of the memantine (76.58 ± 0.66%, P=0.01), allograft
SC transplantation (77.10 ± 0.9%, P=0.01), and SC
transplantation plus memantine (76.33 ± 0.53%, P=0.004)
groups were minimized compared with the control
group (80 ± 0.93%). The memantine (79.92 ± 0.67%,
P=0.004), allograft SC transplantation (80.31 ± 1.02%,
P=0.04), and SC transplantation plus memantine (77.96
± 0.75%, P=0.001) groups showed the protective role
of our therapeutic strategy (memantine and SC) on the
brain water in comparison with the control group (84.18
± 1.02%). Moreover, the brain edema was enhanced in
the control group compared with the sham group in three
areas of the brain, as expected (data not shown).
Fig 4
Brain edema in experimental groups, including ischemic hemisphere
(Total. R), non-ischemic hemisphere (Total. L), striatum (right and left),
cortex (right and left), and Piriform cortex-amygdala (Pir-Amy) (right and
left) areas of the control, sham, Sertoli cells transplant, memantine, and
allograft Sertoli cell transplantation plus memantine groups. Values are
expressed as the mean ± SEM (n=7). P˂0.05 (One-way ANOVA test). *;
P<0.05 and ***; P<0.001.
Brain edema in experimental groups, including ischemic hemisphere
(Total. R), non-ischemic hemisphere (Total. L), striatum (right and left),
cortex (right and left), and Piriform cortex-amygdala (Pir-Amy) (right and
left) areas of the control, sham, Sertoli cells transplant, memantine, and
allograft Sertoli cell transplantation plus memantine groups. Values are
expressed as the mean ± SEM (n=7). P˂0.05 (One-way ANOVA test). *;
P<0.05 and ***; P<0.001.
The effect of Sertoli cell transplantation and injection
of memantine on blood-brain barrier permeability
These results showed a reduction in the BBB breakdown
in the right striatum, cortex, and Pir-Amy areas in
comparison with the control group (Fig .5). The BBB
breakdown in the right cortex of the memantine (3.11 ±
0.06, P=0.02), allograft SC transplantation (3.42 ± 0.21,
P=0.03), and SC transplantation plus memantine (3.01 ±
0.09, P=0.01) groups was decreased in comparison with
the control group (4.31 ± 0.12). The reduction of BBB
permeability in the Pir-Amy of memantine (2.76 ± 0.16,
P=0.002), the allograft SC transplantation (2.81 ± 0.18,
P=0.04), and transplantation of SCs plus memantine
(2.54 ± 0.10, P=0.003) was observed compared with
the control group (3.33 ± 0.28). Analysis of Evans blue
concentration in the striatum of the memantine (3.44 ±
0.12, P=0.001), allograft SC transplantation (4.21 ± 0.08,
P=0.02), and SC transplantation plus memantine (3.04 ±
0.20, P=0.001) groups illustrated the reduction of BBB
permeability in comparison with the control group (5.01 ±
0.11). Furthermore, the increase in the BBB permeability
was observed in the control group compared with the sham group in three areas of the brain, as expected (data
not shown).
Fig 5
The Evans Blue extravasations in ischemic hemisphere (Total. R),
non-ischemic hemisphere (Total. L), cortex (right and left), striatum (right
and left), Piriform cortex-amygdala (Pir-Amy) (right and left) areas of the
control, shame, Sertoli cell transplantation, memantine, and allograft
Sertoli cell transplantation plus memantine groups. Values are expressed
as the mean ± SEM (n=7). P<0.05 compared with the control group (Oneway ANOVA test). *; P<0.05 and ***; P<0.001.
The Evans Blue extravasations in ischemic hemisphere (Total. R),
non-ischemic hemisphere (Total. L), cortex (right and left), striatum (right
and left), Piriform cortex-amygdala (Pir-Amy) (right and left) areas of the
control, shame, Sertoli cell transplantation, memantine, and allograft
Sertoli cell transplantation plus memantine groups. Values are expressed
as the mean ± SEM (n=7). P<0.05 compared with the control group (Oneway ANOVA test). *; P<0.05 and ***; P<0.001.
Discussion
As a pioneer, we focused on investigating the effect
of the combination of SC and memantine on ischemic
damages. The result of the study demonstrated that
allograft transplantation of SC, the injection of memantine,
and simultaneous administration of SCs plus memantine
reduced ischemic damages in ischemic rats. Ischemic
injuries occur in forms of neurological deficits, infarction,
cerebral edema, and increased permeability of the bloodbrain barrier. Transplanted SCs, along with memantine
injection, significantly ameliorated these injuries in the
striatum (transplantation area), cortex, and Piriform
cortex-amygdala.Evaluating sensory and motor behaviors in stroke-related
research are conventional methods to identify the severity
of the injury or the recovery process after treatment.
The present result shows that memantine could decrease
neurological deficits. According to the previous studies,
the significant effect of memantine on the improvement
of the neurological deficits in experimental and clinical
observations was reported (11, 25, 26). With reference to
the reported findings, transplantation of SCs reduced the
severity of neurological function (18). For the first time,
the notable reduction was also observed in the neurological
deficits scores in the SCs+memantine recipient group.
Probably, improvement of the neurological function is
associated with attenuation of damages in related brain
areas.The other considerable result of this work is the decrease
of infarction volume in both memantine-treated and SCtransplanted groups compared with the control group.
Based on the preliminary reports, the positive effect of
memantine on infarction volume was confirmed (11,
25, 27). In the past investigation, the SC transplantation
could result in a significant reduction in infarction (18).
Subsequently, the simultaneous use of transplanted SCs
and memantine exerted more neuroprotective effect than
the other two groups. In line with this finding, there is no
report. A direct relationship was proved between infarction
and cellular death (28). One of the pathophysiological
processes in neurodegenerative diseases, such as cerebral
ischemia, is excitotoxicity that causes severe and
destructive damages.Excitotoxicity is also defined by increased NMDA
activity, glutamate level, and intracellular calcium. The
extracellular glutamate levels have a direct effect on
cellular death. Memantine, as a NMDA receptor antagonist,
exclusively protects neurons against glutamate-induced
neurotoxicity and reduces calcium intake. Consequently, it
is expectable that memantine prevents excitotoxicity-related
cellular loss (8). With regard to the mentioned properties of
memantine, the previous report showed that the reduction
of glutamate levels in the cortex, hippocampus, and
striatum could be exerted following the pre-ischemic use of
memantine (29). Cell therapy has attracted much attention
since it could be used for the compensation of damages
caused by cerebral ischemia. Transplanted SCs induce
their efficacy via the improvement in ischemic damages,
through various mechanisms. SCs can also be able to secrete
factors that protect the brain against the injuries induced by
ischemia, including GDNF and VEGF (15). It should also
be stated that the increased expression of GNDF and VEGF attenuated the infarction in cerebral ischemia (30, 31).On the other hand, the elevation of GDNF and VEGF
was demonstrated by the injection of memantine in the
striatum and cortex. Moreover, memantine stimulated
capillary formation around the infarct area (11).
Eventually, the neuroprotective effect of the combination
of SCs and memantine probably was enhanced through
the secretion of GDNF and VEGF.It is worth noting that memantine and transplanted
SCs could induce a remarkable effect on reducing BBB
breakdown and brain edema. Similarly, other investigations
confirmed the protective effect of memantine on these
ischemic injuries (27, 32). Only Milanizadeh and
colleagues studied the protective effect of SCs on the
brain water content and permeability of the BBB in
cerebral ischemia (18). On the other hand, memantine,
by blocking the NMDA receptor, reduces the expression
of matrix metalloproteinases (MMPs), triggering the
breakdown of the BBB (33). During ischemia, the NOS
enzyme increases the production of oxygen free radicals
(such as ROS), impairs the cellular energy system, and
finally induces DNA damage (34). Considering the
antioxidant properties of memantine, as inhibitory agent
against NOS activity, it can be useful in maintaining
BBB integrity, as well as reducing the cerebral edema
(35). The distinguished neuroprotective mechanisms
of memantine are the attenuation of inflammation
through decreasing activated microglia and inhibition of MAPK p38 (and NF-kB) activity (32, 36). Referring to the
detrimental role of inflammation in BBB breakdown and
induction of edema, memantine can prevent these damages
following stroke. Oxidative stress is a pathophysiological
process that happens by an imbalance in the amount of
free radicals and antioxidants. So, when oxidative stress is
inhibited, free radicals are reduced, and eventually, cell death
is oppressed. In this respect, it was reported that oxidative
stress increases the secretion of inflammatory factors, such
as IL-6, IL-5, and TNF-α, triggering the breakdown of the
BBB integrity and edema (37). SCs can protect the BBB
through the secretion of antioxidant enzymes, including
superoxide dismutase (SOD), glutathione transferase (GT),
and glutathione reductase (GR). Hence, the ability of these
cells in the inhibition of vasogenic edema by preventing
permeability of BBB is expected (38). Furthermore, SCs can
cause detrimental effects on inflammatory factors, including
TNF-a, that could result in a decrease in permeability of
the BBB and brain water content (39). Accordingly, the
protective effect of the administration of SC plus memantine
on the BBB permeability and edema is probably associated
with the exclusive capabilities in the suppression of free
radicals and inflammatory factors.Referring to the previous study, the combination of the
drug and cell therapy in cerebral ischemia treatment (40)
can open up a new horizon for clinical research.
Conclusion
According to the observed results in this study, the
administration of memantine or SCs caused a reduction
in ischemic injuries. Furthermore, the neuroprotective
findings were apparent in the transplanted SCs plus
memantine group. The neuroprotective effects of SCs
and memantine are probably mediated by the blockage
of NMDA receptor, decreasing intracellular calcium, the
release of antioxidant enzymes, secretion of growth and
neurotrophic factors, reducing inflammatory factors, and
inhibiting apoptosis.
Authors: Mehmet U Aluclu; Seyfi Arslan; Abdullah Acar; Aslan Guzel; Selen Bahceci; Mehmet Yaldiz Journal: Neurosciences (Riyadh) Date: 2008-04 Impact factor: 0.906
Authors: Rachna S Pandya; Lijuan Mao; Hua Zhou; Shuanhu Zhou; Jiang Zeng; A John Popp; Xin Wang Journal: Cent Nerv Syst Agents Med Chem Date: 2011-06-01
Authors: Paul S Baxter; Karen F S Bell; Philip Hasel; Angela M Kaindl; Michael Fricker; Derek Thomson; Sean P Cregan; Thomas H Gillingwater; Giles E Hardingham Journal: Nat Commun Date: 2015-04-09 Impact factor: 14.919
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