International Journal of Neuroscience

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Protective effect of MitoTEMPO against cardiac

dysfunction caused by ischemia–reperfusion:
MCAO stroke model study

Ahmet Akkoca, Belgin Büyükakıllı, Ebru Ballı, Burcu Gültekin, Erkan Özbay,
Hatice Oruç Demirbağ & Çağatay Han Türkseven

To cite this article: Ahmet Akkoca, Belgin Büyükakıllı, Ebru Ballı, Burcu Gültekin, Erkan Özbay,
Hatice Oruç Demirbağ & Çağatay Han Türkseven (24 Oct 2023): Protective effect of MitoTEMPO
against cardiac dysfunction caused by ischemia–reperfusion: MCAO stroke model study,
International Journal of Neuroscience, DOI: 10.1080/00207454.2023.2273768

To link to this article: https://doi.org/10.1080/00207454.2023.2273768

Published online: 24 Oct 2023.

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International Journal of Neuroscience
https://doi.org/10.1080/00207454.2023.2273768

Research Article

Protective effect of MitoTEMPO against cardiac dysfunction caused by

ischemia–reperfusion: MCAO stroke model study
Ahmet Akkocaa , Belgin Büyükakıllıb , Ebru Ballıc , Burcu Gültekind , Erkan Özbaye , Hatice
Oruç Demirbağc and Çağatay Han Türksevenb
a
Department of Occupational Health and Safety, Taşkent Vocational School, Selcuk University, Konya, Türkiye; bDepartment of Biophysics,
Faculty of Medicine, Mersin University, Mersin, Türkiye; cDepartment of Histology and Embryology, Faculty of Medicine, Mersin
University, Mersin, Türkiye; dDepartment of Histology and Embryology, Faculty of Medicine, Necmettin Erbakan University, Konya,
Türkiye; eDepartment of Medical Services and Techniques, Health Services Vocational School, Karamanoğlu Mehmetbey University,
Karaman, Türkiye

ABSTRACT ARTICLE HISTORY

Purpose: Neurological impairments are the leading cause of post-stroke mortality, while Received 16 August 2023
stroke-related cardiovascular diseases rank second in significance. This study investigates the Revised 6 October 2023
Accepted 17 October
potential protective effects of MitoTEMPO (2,2,6,6-tetramethyl-4-[[2-(triphenylphosphonio) acetyl] 2023
amino]-1-piperidinyloxy, monochloride, monohydrate), a mitochondria-specific antioxidant, against
cardiac and neurological complications following stroke. The objective is to assess whether KEYWORDS
MitoTEMPO can be utilized as a protective agent for individuals with a high risk of stroke. Cardiovascular disease;
Materials and methods: Seventeen-week-old male Wistar Albino rats were randomly assigned to ischemic stroke; middle
cerebral artery occlusion
three groups: SHAM, ischemia–reperfusion and MitoTEMPO + ischemia–reperfusion (MitoTEMPO
injection 0.7 mg/kg/day for 14 days). The SHAM group underwent a sham operation, while the
ischemia–reperfusion group underwent 1-h middle cerebral artery occlusion followed by
three days of reperfusion. Afterwards, noninvasive thoracic electrical bioimpedance and
electrocardiography measurements were taken, and sample collection was performed for
histological and biochemical examinations.
Results: Our thoracic electrical bioimpedance and electrocardiography findings demonstrated
that MitoTEMPO exhibited a protective effect on most parameters affected by ischemia–reperfusion
compared to the SHAM group. Furthermore, our biochemical and histological data revealed a
significant protective effect of MitoTEMPO against oxidative damage.
Conclusions: The findings suggest that both ischemia–reperfusion-induced cardiovascular
abnormalities and the protective effect of MitoTEMPO may involve G-protein coupled
receptor-mediated signaling mechanisms. This study was conducted with limitations including a
single gender, a uniform age group, a specific stroke model limited to middle cerebral artery, and
pre-scheduled only one ischemia–reperfusion period. In future studies, addressing these limitations
may enable the implementation of preventive measures for individuals at high risk of stroke.

Introduction hypertrophy and obesity [4]. In addition, it is known

Stroke is the second leading cause of death worldwide
and a major contributor to disability, primarily occur-
ring due to ischemia [1,2]. Clinical data indicate that
ischemic stroke most frequently occurs in the middle
cerebral artery (MCA), which supplies blood to volu-
metric and functional large part of the brain [3]. The
risk of stroke varies depending on factors such as age,
gender, tobacco and alcohol use, as well as the pres-
ence of diseases such as hypertension, diabetes, coro-
nary heart disease, atrial fibrillation, left ventricular
CONTACT Belgin Büyükakıllı bbuyukakilli@mersin.edu.tr
Yenişehir, Mersin, Türkiye
© 2023 Informa UK Limited, trading as Taylor & Francis Group
that 17.4% of patients who had a transient ischemic
attack had an acute ischemic stroke in the first
3 months following the transient ischemic attack [5].
This information emphasizes the significance of imple-
menting protective measures to minimize post-stroke
symptoms in individuals with a high risk of experienc-
ing a stroke. On the other hand, when reviewing stud-
ies related to stroke, it becomes evident that unlike
ours, researchers tend to focus on the treatment of
post-stroke complications. The novelty of our study lies
Department of Biophysics, Mersin University Medical Faculty, Çiftlikköy Campus, 33343
2 A. AKKOCA ET AL.
in its design, which primarily concentrates on preven-
tive measures for individuals at high risk of stroke.
Although most of the deaths after stroke are due to
neurological defects, cardiovascular complications are
the second leading cause of death. Cardiac injuries
after stroke can cause death as well as lifelong heart
problems such as heart failure, abnormal ECG findings,
or mild and curable cardiovascular diseases such as
neurogenic stress cardiomyopathy and Takotsubo car-
diomyopathy [6,7]. Research investigating the impor-
tance of preventive measures against cardiovascular
problems that may occur after a stroke will make sig-
nificant contributions to clinical practice.
In this study, it was aimed to investigate the pro­
tective effect of mitochondria-specific antioxidant
(MitoTEMPO) against cardiac dysfunctions that may
occur due to stroke. Many studies in the literature
show that MitoTEMPO is highly effective against sys-
temic inflammation and the increase in the amount of
reactive oxygen species (ROS) resulting from various
pathologies [8–11]. Our study was performed with the
MCA occlusion (MCAO) model, which is known to best
mimic human ischemic stroke and allows highly con-
trollable reperfusion [12,13]. With the MCAO model
applied in our study, the protective effect of MitoTEMPO
against ischemia–reperfusion (IR)-induced cardiac dys-
function was investigated. Our results put forward that
MitoTEMPO has shown a great protective effect against
electrophysiological, biochemical and histological find-
ings after IR. These data can be considered as a prom-
ising development for the discovery of protective
measures in people who are known to have a higher
incidence of stroke than other individuals.
Materials and methods
Experimental animals and procedures
Seventeen-week-old adult Wistar Albino rats were used
in the experiments. The initial body weights of the rats
were in the range of 250–304 g, and only male experi-
mental animals were used to eliminate physiological
variations due to sex. The rats were housed at a con-
stant temperature of 23 °C, on a 12/12 h light/dark cycle,
without any restrictions on their access to feed and
water. The procedures applied in this study were
approved by Mersin University Animal Experiments
Local Ethics Committee (approval nos.: 2021/35, 2022/8).
The rats were randomly selected into three different
groups as SHAM, IR and MitoTEMPO + IR (MT + IR)
according to the experimental procedures. MitoTEMPO
(C29H35N2O2P•Cl [H2O]; Cayman Chemical Comp., cata-
log no.: 16621, USA) dissolved in distilled water was
injected intraperitoneally for 0.7 mg/kg/day × 14 days
to the MT + IR group [14]. Intraperitoneal injections of
distilled water were applied to the animals in the
SHAM and IR groups (distilled water injection group)
at the same dose and duration. SHAM and IR surgery
were conducted as described in our previous study
[15]. Briefly, in SHAM surgery, the internal carotid
artery (ICA) and external carotid artery (ECA) bifurca-
tion was reached and then the opened tissues were
closed again. In the IR and MT + IR groups, MCAO was
applied to the right MCA of rats in accordance with
the procedures described by Koizumi et al. [16].
Monofilaments were prepared by us using 4-0 Ethilon
Nylon Suture (Ethicon Comp., G7143, Somerville, NJ) as
previously described in the literature [17]. Prepared
monofilaments were photographed under a 2× objec-
tive with millimetric paper using a surgical microscope,
and the tip diameters of the monofilaments were mea-
sured from these photographs using a digital image
analysis software (ImageJ, 1.53k, Bethesda, MD) [18].
The tip diameters of the monofilaments used through-
out the experiments were determined as
0.308 ±0.029 mm (mean ± standard deviation (SD)).
Thoracic electrical bioimpedance (TEB) and
electrocardiography (ECG) measurements
TEB and ECG electrodes and heart sound microphone
were placed as previously described by Buyukakilli
et al. [19]. Electrical potential differences accompany-
ing bioimpedance changes in the thorax were recorded
using the EBI100C module via the BIOPAC MP160
Acquisition System (Biopac Systems, Inc., MP160WSW,
Goleta, CA). ECG data were recorded using the
ECG100C module. In addition, heart sounds were
recorded with a microphone (Biopac Systems, Inc.,
TSD108, Goleta, CA) on the DA100C module.
Body surface area (BSA) was calculated by Mosteller’s
formula [20], stroke volume (SV) was calculated by
Bernstein’s equation [21] and corrected QT (QTc) was
calculated by Bazett’s equation [22]. Heart rate (HR)
was determined by reading the RR interval value from
ECG recordings [23]. All data were determined by aver-
aging 10 different values from each rat.
Biochemical experiments
For biochemical studies, the thorax of the animals was
opened by sternotomy under anesthesia (80 mg/kg
ketamine + 7 mg/kg xylazine intraperitoneally) and
blood samples were taken from the left ventricle of
the heart and serum samples were obtained by

centrifugation at 5000 revolutions per minute (rpm) for
10 min. Afterwards, tissues of the heart left ventricle
and brain right cerebral hemisphere were quickly
collected and stored for further studies. Cyclic adenos-
ine monophosphate (cAMP; Nepenthe Research
Technologies, NE010029803, Gebze, Turkey), inositol
triphosphate (IP3; Elabscience Biotechnology Inc., E-EL-
0059, Houston, TX), catalase (with 1/5 dilution ratio –
Fine Biotech Co., Ltd., ER0264, Wuhan, China), cardiac
troponin I (cTnI; Nepenthe Research Technologies,
NE010063903, Turkey), total antioxidant status (TAS;
Elabscience Biotechnology Inc., E-BC-K801-M, Houston,
TX) and total oxidant status (TOS; Elabscience
Biotechnology Inc., E-BC-K802-M, Houston, TX) levels
were measured with Enzyme-Linked ImmunoSorbent
Assay (ELISA) kits according to manufacturer’s proto-
cols. Oxidative stress index (OSI) was calculated as fol-
lows [24]:
 µmol H 2 O2 Equiv. 
TOS  
OSI ( arbitrary unit ) =  L  ×100
 µmol Trolox. Equiv. 
TAS  
 L  Body and heart weights of experimental animals
Histological experiments
To perform electron microscopic examinations, sam-
ples of heart left ventricle and brain right cerebral
hemisphere tissues were fixed in glutaraldehyde and
osmium tetroxide solutions. These tissues were then
processed, embedded in epoxy resin, and thin sections
were prepared. These sections were contrasted and
examined using a transmission electron microscope
(Jeol® JEM1011, Akishima, Japan) with digital photogra-
phy (Megaview III, Olympus GmbH, Hamburg, Germany)
for analysis.
In light microscopic histological experiments, heart
left ventricle and brain right cerebral hemisphere
tissues were fixed with 10% formaldehyde. After
Figure 1. (A) Body weights of experimental animals, (B) heart weights and (C) heart weight/body weight. Values were given as
mean ± SEM. +p < .05 vs. distilled water group, *p < .05 vs. SHAM group and #p < .05 vs. IR group.
International Journal of Neuroscience 3
dehydration, they were embedded in paraffin, cut into
5-μm sections, and stained with hematoxylin–eosin
(H&E), Masson’s trichrome (MT) and Toluidine Blue (TB).
Photographs of the preparations obtained as a result
of staining were taken with a light microscope (Carl
Zeiss AG, Axio Lab. A1, Oberkochen, Germany) and
structural changes were examined.
Statistical analysis
Our ECG and TEB data were presented as mean ± stan-
dard error of the mean (SEM), while all other data were
presented as mean ± SD. The normal distribution of
data was analyzed with Shapiro–Wilk test for normal-
ity, and the unpaired Student’s t-test was used to com-
pare rat weight data. One-way ANOVA and post hoc
test Tukey were used to compare all other data.
Changes at the p < .05 level were considered statisti-
cally significant. GraphPad Prism (GraphPad Prism
Software Inc., 5 Demo, La Jolla, CA) software was used
for statistical analysis.
Results
The initial body weights of the experimental animals
were 271.13 ± 12.48 g in the distilled water injection
group, 280 ± 15.42 g in the MitoTEMPO injection
group, and the body weights at the end of the
two-week injection period were 285.75 ± 11.31 g in
the distilled water injection group, 302.57 ± 17.55 g in
the MitoTEMPO injection group (mean ± SD). While
there was no statistically significant difference between
the two groups in the body weights of the experimen-
tal animals at the beginning, MitoTEMPO caused a sta-
tistically significant increase in the body weights of the
experimental animals at the end of the injection period
(Figure 1(A)). The heart weights of the SHAM, IR and
MT + IR group rats were measured as 844.18 ± 29.91 mg,
978.2 ± 68.76 mg and 926.42 ± 78.13 mg, respectively,
4 A. AKKOCA ET AL.

and a statistically significant increase was detected in
the heart weight of the IR group (mean ± SD). The
heart weight data of the MT + IR group were mea-
sured as a value between the SHAM and IR groups
(Figure 1(B)). In addition, heart weight data normalized
to body weights (SHAM: 2.98 ± 0.14 mg/g, IR:
3.39 ± 0.12 mg/g and MT + IR: 3.01 ± 0.17 mg/g;
mean ± SD) showed a statistically significant increase
in the IR group (Figure 1(C)).
Thoracic electrical bioimpedance and
electrocardiography parameters
The data obtained from TEB recordings and the
changes in the hemodynamic parameters of the heart
between the groups were compared. Accordingly, the
volume of electrically participating tissue (VEPT)
value decreased in the IR group, and in the MT + IR
group that received protective MitoTEMPO injection,
this value did not differ significantly compared to the
data of the SHAM group. In addition, HR increased in
the IR group after stroke and this value was deter-
mined at the level of the SHAM group in the MT + IR
group. Left ventricular ejection time (LVET) value,
which shows the time between S1 and S2 sounds of
the heart, was significantly shortened in the IR group,
and this value was measured at the level of the
SHAM group in the MitoTEMPO group. Other mea-
sured and calculated parameters (BSA, thoracic basal
impedance – Z0, maximum rate of change of the tho-
racic impedance (dZ/dt)max, thoracic fluid content –
TFC, SV, cardiac output – CO, myocardial contractility
index – IC, thoracic fluid content index – TFCI, SV
index – SI and cardiac index – CI) did not show sta-
tistical significance between the groups. On the other
hand, the p value of the SV parameter was deter-
mined as 0.0564 and it is the closest value to statis-
tical significance (Table 1).
The ECG data in Table 1 showed that P-R interval,
QRS complex time, QTc, T-wave time, T-wave repolar-
ization time and R-R interval values were significantly
changed in the IR group compared to the SHAM
group. The mentioned changes occurred in the direc-
tion of increase in P-R interval, QTc, T-wave duration
and T-wave repolarization time, and decrease in QRS
complex time and R-R interval values compared to
SHAM group. When the data in the MT + IR group
were examined, it was seen that MitoTEMPO had a
protective effect against these changes. It is notewor-
thy that the prolonged T-wave duration in the IR group
is due to the repolarization phase. In addition, it was
found that the P-wave amplitude value decreased in
the IR group and the T-wave amplitude value increased
compared to the SHAM group (Table 1).

Table 1. Thoracic electrical bioimpedance (TEB) and electrocardiography (ECG) data.

Order Parameter (unit) SHAM (n = 7) IR (n = 7) MT + IR (n = 6) p
Thoracic electrical bioimpedance (TEB) data
1 BSA (m2) 0.0422 ± 0.0001 0.0420 ± 0.0004 0.0433 ± 0.0004 .0565
2 Z0 (Ω) 160.31 ± 16.54 167.51 ± 8.73 151.36 ± 10.22 .6752
3 (dZ/dt)max (Ω/s) 7.02 ± 0.69 9.44 ± 0.71 8.81 ± 1.26 .1537
4 VEPT (cm3) 33.76 ± 2.65 24.37 ± 1.45* 35.51 ± 2.89# .0081
5 LVET (s) 0.084 ± 0.001 0.068 ± 0.002* 0.082 ± 0.001# <.0001
6 HR (beat/min) 283.02 ± 16.78 341.13 ± 7.52* 283 ± 22# .0066
7 TFC (kΩ–1) 7.03 ± 0.60 6.08 ± 0.36 6.75 ± 0.45 .3423
8 SV (ml/beat) 0.142 ± 0.019 0.098 ± 0.013 0.167 ± 0.025 .0564
9 CO (l/min) 0.031 ± 0.007 0.033 ± 0.004 0.048 ± 0.009 .2817
10 IC (s–1) 0.046 ± 0.006 0.058 ± 0.007 0.059 ± 0.008 .4203
11 TFCI (kΩ–1/m2) 167.23 ± 13.98 144.78 ± 8.72 155.97 ± 9.30 .3631
12 SI (ml/beat/m2) 3.39 ± 0.46 2.34 ± 0.31 3.90 ± 0.65 .0852
13 CI (l/min/m2) 0.85 ± 0.18 0.79 ± 0.10 1.13 ± 0.23 .3567
Electrocardiography (ECG) data
14 P-wave amplitude (mV) 0.0309 ± 0.0010 0.0225 ± 0.0019* 0.0279 ± 0.0006 .0278
15 P-R interval (s) 0.0304 ± 0.0008 0.03506 ± 0.0012* 0.0303 ± 0.0011# .0118
16 QRS complex time (s) 0.0420 ± 0.0005 0.0400 ± 0.0006* 0.0393 ± 0.0003* .0103
17 Q-T interval (s) 0.0769 ± 0.0018 0.0788 ± 0.0046 0.0724 ± 0.0008 .3577
18 QTc (s) 0.162 ± 0.004 0.193 ± 0.010* 0.157 ± 0.006# .0098
19 T-wave amplitude (mV) 0.0476 ± 0.0009 0.0801 ± 0.0079* 0.0640 ± 0.0022 .0018
20 T-wave time (s) 0.0309 ± 0.0006 0.0442 ± 0.0018* 0.0318 ± 0.0007# <.0001
21 T-wave depolarization 0.0107 ± 0.0004 0.0113 ± 00004 0.0103 ± 0.0005 .2364
time (s)
22 T-wave repolarization 0.0202 ± 0.0004 0.0329 ± 0.0019* 0.0215 ± 0.0005# <.0001
time (s)
23 R-R interval (s) 0.2171 ± 0.0131 0.1767 ± 0.0038* 0.2153 ± 0.0120# .0154
Values were given as mean ± standard error of the mean (SEM).
*
p < .05 vs. SHAM group.
#
p < .05 vs. IR group.
 International Journal of Neuroscience 5

Figure 2. Data determined by ELISA technique. (A) Blood serum total antioxidant status (TAS), (B) blood serum total oxidant
status (TOS), (C) oxidative stress index (OSI), (D) blood serum cardiac troponin-I (cTnI), (E) heart left ventricle tissue cyclic adenos-
ine monophosphate (cAMP), (F) heart left ventricle tissue inositol triphosphate (IP3), (G) brain right cerebral hemisphere tissue
cAMP, (H) brain right cerebral hemisphere tissue IP3 and (I) brain right cerebral hemisphere tissue catalase. Values were given as
mean ± SEM. *p < .05 vs. SHAM group and #p < .05 vs. IR group.

Biochemical parameters Histological investigations

TAS and TOS data determined from blood serum
samples showed that MitoTEMPO injection had a
protective effect against decreased antioxidant and
increased oxidant substance amounts in the IR
group as a result of IR injury (Figure 2(A,B)). In addi-
tion, the protective effect of MitoTEMPO was
detected against the increased OSI value in the IR
group (Figure 2(C)). There was no statistically signif-
icant difference between all groups in the cTnI value
(Figure 2(D)). ELISA experiments performed on heart
left ventricular tissue showed a decrease in cAMP
and IP3 levels in the IR group, whereas it was close
to the SHAM group in the MT + IR group (Figure
2(E,F)). Although the amount of cAMP and IP3 sub-
stance in the brain right cerebral hemisphere tissue
decreased in the IR group, the MT + IR group data
were found to be within the range of the values of
the SHAM and IR group (Figure 2(G,H)). MitoTEMPO
showed a protective effect in the MT + IR group
against the increased amount of catalase in the IR
group (Figure 2(I)).
Electron microscopic studies showed that myofibrils in
left ventricle cardiac muscle cells were regular and
their thickness was normal in heart tissues belonging
to the SHAM group. In addition, the structures of
mitochondria and sarcoplasmic reticulum located
between the myofibrils were normal (Figure 3(A–C)).
Although myofibrils with normal morphological fea-
tures were observed in the IR group, irregularity, thin-
ning and marked degenerative changes were observed
in myofibrils in some areas. In addition, enlargement
of the sarcoplasmic reticulum cisternae was observed
in some areas (Figure 3(D–F)). In the MT + IR group,
although degenerative changes were observed in
myofibrils in some areas, in general, cardiac muscle
cells had normal morphological features (Figure 3(G–
I)). Electron microscopic studies of the brain right
cerebral hemisphere tissue showed that neurons, glial
cells, vessels, myelinated nerve fibers, neurite and glial
cell extensions had normal morphological features in
the SHAM group (Figure 4(A–C)). In the IR group,
while the neurons were observed in normal structure,
6 A. AKKOCA ET AL.

Figure 3. Ultrastructural image of the heart left ventricle muscle. (A–C) SHAM group, (D–F) IR group and (G–I) MT + IR group.
Normally structured myofibrils (asterisks), mitochondria (arrowheads), sarcoplasmic reticulum (thin black arrows), enlarged sarco-
plasmic reticulum (thin white arrows), degenerated myofibrils (thick black arrows). A, D, G: ×6000; B, H: ×10,000; C, I: ×20,000;
E, F: ×12,000.

significant degenerative changes in some glial cells
and their extensions, enlargement of mitochondria
and loss of cristae were observed. In addition, enlarge-
ment of astrocyte extensions was observed in some
perivascular areas (Figure 4(D–F)). In the MT + IR
group, neurons and some glial cells were observed in
a normal structure, while degenerative changes in
some glial cells and enlargement of astrocyte exten-
sions in the perivascular area were observed (Figure
4(G–I)).
In addition, histopathological changes in the iso-
lated heart left ventricle and brain right cerebral
hemisphere tissues were also examined on light
microscopic images with histological staining tech-
niques. H&E staining in the heart left ventricle tissue
showed that the cell nuclei were located in the
periphery and the branching in the muscle fibers was
irregular in the IR group. In addition, MT staining
showed collagen accumulation around the cardiac
fibrils and vessels and increased interfibrillary spaces
in the IR group. MitoTEMPO showed a large protective
effect on these changes in the IR group (Figure 5).
H&E staining of the brain right cerebral hemisphere
tissue showed red neurons, presence of dilated ves-
sels, perineuronal edema and vacuolization in the IR
group. Moreover, TB staining of the brain right cere-
bral hemisphere tissue showed neurons with homog-
enized cytoplasm in the IR group. MitoTEMPO showed
a large protective effect on these changes. As a result
of MT staining in the brain right cerebral hemisphere
tissue, no difference was found between the groups
in terms of the amount of collagen substance
(Figure 6).
Discussion
Neurological, cardiological and systemic complications
following stroke significantly increase mortality and
morbidity rates [25–27]. In addition, cardiac damage is
mentioned as the biggest cause of death in the first
month after stroke in some patients [28–30]. Moreover,
the increase in ROS levels originating from IR is closely
associated with the pathophysiology of many diseases
[31]. Therefore, the importance of preventive measures
against IR-induced damage is emphasized, especially
in individuals with a high incidence of stroke.
 International Journal of Neuroscience 7

Figure 4. Ultrastructural image of brain right cerebral hemisphere tissue. (A–C) SHAM group, (D–F) IR group and (G–I) MT + IR
group. Neuron (n), glial cells (g), normal mitochondria (thin black arrows), perivascular space (black arrowhead), myelinated nerves
(thick black arrow), degenerated mitochondria (thin white arrows), degenerated glial cells and their extensions (thick white arrow),
enlargement of astrocyte extensions in the perivascular space (white arrowheads). A, D, G: ×4000; B, E, F, H: ×5000; I: ×6000;
C: ×7500.

Figure 5. Histological images of heart left ventricle tissue with hematoxylin–eosin (H&E) and Masson’s trichrome (MT) staining at
×40 magnification. Scale bar is 10 µm. Thin arrow: location of cell nuclei (central in SHAM and MT + IR, peripheral in IR); triangle:
branching of muscle fibers (regular in SHAM and MT + IR, irregular in IR); thick arrow: collagen substances (increased in IR); star:
interfibrillar spaces (increased in IR).

In our study, which began with eight rats in each anesthesia injection. Additionally, one experimental
group, unfortunately, one rat from the SHAM group animal from the MT + IR group died due to bleeding
and one rat from the IR group died after the during the surgical procedures. Moreover, the weight
8 A. AKKOCA ET AL.

Figure 6. Histological images of brain right cerebral hemisphere tissue with hematoxylin–eosin (H&E) staining at ×40 magnifica-
tion. Scale bar is 10 µm. Masson’s trichrome (MT) staining at ×10 magnification. Scale bar is 100 µm. Toluidine Blue (TB) staining
at ×20 magnification. Scale bar is 20 µm. Triangle: view of the vein (dilated in IR, normal in other groups); thick arrow: collagen
substances; thin arrow: view of neurons (red neuron in IR, normal in SHAM and MT + IR group); star: perineuronal edema;
V: vacuolization.

of the experimental animals in the MT + IR group
increased by approximately 8.06% after 14 days of
MitoTEMPO injection, except for one animal with a
weight gain of 1.77%. This rat was excluded from the
MT + IR group. Consequently, the measurement and
sample collection processes were completed with the
following group sizes: SHAM group (n = 7), IR group
(n = 7) and MT + IR group (n = 6). In another study
conducted on the same model as ours, the reported
mortality rate for the IR group was 42.86% [32], while
in our study, it was 12.5% for the same group. Hence,
we believe that we have successfully carried out the
experimental procedures and post-operative care.
In parallel with the results of this study, in another
study, we conducted earlier, it was observed that the
administration of MitoTEMPO at the same protective
dose to experimental animals for 28 days led to
weight gain [11]. Contrary to our findings, Liu et al.
reported that they did not observe weight gain in the
MitoTEMPO injection group (Figure 1(A)) [33]. When
examining other studies, the question regarding the
relationship between mitochondria-specific antioxi-
dants and mechanisms of weight gain or loss arises,
awaiting further explanation. Upon evaluating Figure
1(B,C) together, it can be observed that MitoTEMPO
exhibited a protective effect against the increase in
heart weight in the IR group when the data were nor-
malized by heart weight to body weight (Figure
1(B,C)). Although some studies reported post-stroke
cardiac hypertrophy similar to our data [34,35], Chen
et al. detected cardiac atrophy after MCAO [36].
In their study on cardiac functions after stroke,
Zheng et al. highlighted the development of left ven-
tricular diastolic dysfunction [37]. Biering-Sørensen
et al. conducted a clinical study demonstrating that a
shortened LVET was associated with higher systolic
blood pressure, higher HR and poor fractional shorten-
ing [38]. In another clinical study conducted by Verma
et al. on patients with ischemic stroke in MCA, the HR
value was measured statistically significantly higher
than in patients without a history of stroke [39]. In line
with these studies, our findings also indicated that the
LVET value was shortened, and the HR was higher in
the IR group. Furthermore, Kodama et al. reported that
ischemic stroke has an impact on HR variability, which
reflects the activities of autonomic nerve function. This
suggests that monitoring HR changes could potentially
detect ischemic stroke at an acute stage [40]. Moreover,
by applying ischemia and/or reperfusion periods at
different time intervals, it will be possible to induce a

statistically significant change in the SV value, which
tends to decrease in the IR group. In summary, our
TEB findings were consistent with other studies, and
the injection of MitoTEMPO demonstrated a protective
effect against stroke-related cardiac dysfunctions
(Table 1).
Changes in ECG parameters following stroke are
commonly reported [41]. In a study conducted by
Kwon et al. on patients who presented to the clinic
with stroke, they reported a relationship between P
wave anomalies and the occurrence of stroke [42].
Conversely, in our study, we observed a statistically
significant decrease in P wave amplitude after per-
forming MCAO in rats without any pre-existing cardiac
pathology. Therefore, it may not always be accurate to
associate the pathological findings in ECG recordings
taken after stroke in clinical settings with the pre-stroke
period. In the ECG study by Kaya et al., ECG abnormal-
ities were observed in 61% of stroke patients, with a
prolonged P-R interval in line with our findings,
although they also reported prolonged QRS and R-R
durations unlike our study [43]. Prolonged QTc is a
common factor that increases the risk of malignant
arrhythmia and death in stroke patients [23]. Togha
et al. reported that the most significant ECG finding
following stroke was prolonged QTc and T wave abnor-
malities [41]. Bilge et al. reported that, similar to our
study, ECG recordings from stroke patients showed
increased QT and QTc values [44]. As a result, our
study showed that MitoTEMPO had a protective effect
on PR interval, QTc, T wave duration and R-R interval
values, which were found to be different compared to
the SHAM group in ECG data (Table 1).
The data from our ELISA studies demonstrated that
MitoTEMPO exhibited a protective effect against
IR-induced changes in molecule levels. The previous
study we conducted showed that after IR, TAS
decreased, TOS increased and MitoTEMPO demon-
strated a protective effect against these alterations
[11]. Similarly, Siotto et al. reported elevated levels of
oxidative substances, reduced levels of antioxidant
substances, and increased OSI values in serum samples
from stroke patients [45], which aligns with the find-
ings of our study (Figure 2(A–C)). Etgen et al. reported
consistent findings with our study, as they observed
no significant changes in cTnI values among patients
with ischemic stroke (Figure 2(D)) [46]. In the heart,
G-protein coupled receptors (GPCR) regulate intra­
cellular Ca2+ concentration through signaling pathways
that mediate cAMP and IP3 synthesis [47]. Similarly,
elevations in Ca2+ concentrations that rely on the
WNT pathway can be accomplished through two
mechanisms: either by suppressing cyclic guanosine
International Journal of Neuroscience 9
monophosphate (cGMP)-dependent protein kinase
(PKG), thereby preventing Ca2+ release in resting cells,
or by triggering phospholipase C (PLC), leading to an
increase in IP3 [48]. In the brain, synaptic transmission
is provided because of phosphorylation of ion chan-
nels and regulation of intracellular Ca2+ concentration
via GPCR-mediated pathways [49]. In our study, the
relationship between GPCR and possible pathophysio-
logical conditions that may develop in the heart and
brain after IR was carried out by assessing the levels of
cAMP and IP3. Choudhary and Dudley stated that in
parallel with our findings, oxidative stress-induced
type-2 phosphodiesterase-mediated cardiac cAMP
level decreased (Figure 2(E)) [50]. Kiselyov and Muallem
emphasized changes in IP3-receptor levels in response
to increased ROS, but our findings showed a decrease
in IP3 levels (Figure 2(F)) [51]. In their study, Ponsaerts
et al. suggested that strategies aimed at increasing
cAMP levels could be a novel treatment approach for
ischemic stroke, owing to the ability of cAMP to reduce
neuroinflammation [52]. Liu et al. conducted the cere-
bral IR model, and in parallel with our findings, they
detected a decrease in brain tissue cAMP levels (Figure
2(G)) [53]. Duan et al. in their MCAO model study
found that the brain tissue IP3 levels had decreased
similarly to ours due to IR (Figure 2(H)) [54]. We believe
that the lack of a complete protective effect of
MitoTEMPO on brain tissue against cAMP and IP3
levels may be related to the dosage and duration
of MitoTEMPO administration (Figure 2(G,H)). As a
result, we hypothesize that the abnormalities observed
in TEB and EKG findings after IR, as well as the pro­
tective effect of MitoTEMPO, are mediated through
GPCR-mediated signal transduction mechanisms.
Furthermore, it is indicated that the levels of catalase,
one of the most important endogenous antioxidant
enzymes, increase in parallel with our findings in vari-
ous metabolic and neurodegenerative diseases (Figure
2(I)) [55]. This result shows that MitoTEMPO injection
does not require additional catalase synthesis and pre-
vents oxidative stress by providing the balance
between antioxidants and the amount of free radical
substances.
In the literature, there was a lack of ultrastructural
research on cardiac muscle tissue following stroke.
However, Wu et al. reported fragmented myofibrils,
damaged mitochondria and vacuoles in their myocar-
dial IR model, which parallel our findings [56]. Our
ultrastructural investigations in the left ventricular tis-
sue of the heart demonstrated morphological changes
in the IR group, which were consistent with our light
microscopic findings. Accordingly, degeneration in the
sarcoplasmic reticulum could potentially disrupt the
10 A. AKKOCA ET AL.
calcium release/reuptake mechanism, leading to dis-
turbances in the electrical activity of the heart. We
believe that this condition could be one of the reasons
for the changes observed in the ECG measurements.
In the MT + IR group, the morphological structure
appeared to be more regular (Figure 3). When ultra-
structural investigations in brain right cerebral hemi-
sphere tissue were examined, Li et al. demonstrated
similar findings to ours in their intracerebral hemor-
rhage model, with expansion and degenerative
changes in mitochondria and specific organelles asso-
ciated with IR [57]. Additionally, Solenski et al. reported
neuronal mitochondrial degeneration following stroke
[58]. Our results also demonstrated similar findings to
previous studies (Figure 4).
Veltkamp et al. stated that in the histological evalu-
ation of the heart tissue after MCAO, they pointed out
the structural changes, but they did not detect an
increase in the amount of collagen substances as a
result of MT staining [59]. However, in our data, there
was an increase in the amount of collagen substance
in the heart tissue (Figure 5). Alrafiah reported degen-
erated neurons, homogenized cytoplasm and struc-
tural changes in nuclei, perineuronal edema and
vacuolization in parallel with our findings in the histo-
logical evaluation of brain tissues after the MCAO
model (Figure 6) [60]. Studies show that Akt and Erk
signaling pathways have an important role in many
neurological diseases and may even be a therapeutic
target for the prevention of oxidative damage [61]. In
our histological studies, the structural changes
observed in the IR group and even the protective
effect of MitoTEMPO may be related not only to the
GPCR-mediated signaling pathways mentioned earlier
but also to pathways associated with Akt and Erk.
This study was conducted with the limitations of a
single gender, a uniform age group, and a specific
stroke model limited to MCA. Furthermore, unlike
patients presenting with stroke complaints at the
clinic, this study incorporated pre-scheduled only one
IR period. Additionally, potential interactions between
the parameters we associated with GPCR-mediated
signaling pathways and other pathways were not elu-
cidated in our study. In this study, which primarily
focuses on the heart and brain, we did not assess the
impact of IR on other organs and systems. We believe
that in future studies aimed at addressing these limita-
tions, implementing preventive measures for individu-
als at a high risk of stroke and improving their quality
of life post-stroke could lead to positive outcomes.
In conclusion, this study utilized the monofilament
insertion technique, which closely mimics human isch-
emic stroke, to investigate both cardiac and brain
tissue in our experimental model. While our primary
focus was on cardiac functions, we also performed
examinations in the brain tissue. The data obtained
from this study, designed with the aim of reducing
post-stroke cardiopathological findings in disease
groups with a higher incidence of stroke, largely sup-
ported our hypothesis. Considering that patients with
transient ischemic attack are at risk of acute ischemic
attack, it is crucial to develop protective measures
against post-stroke pathologies. While our study results
suggest that protective MitoTEMPO injection can miti-
gate many stroke-related findings, further support
from studies conducted by researchers in various fields
with molecular and systemic perspectives is necessary
to strengthen our findings.
Acknowledgements
This study is the PhD thesis of Akkoca A. We extend our
appreciation to Kurul Laboratory Products Company in
Konya, Türkiye, for their valuable contribution to the
­biochemical experiments.
Disclosure statement
The authors report there are no competing interests to
declare.
Funding
This work was supported by the Scientific Research Projects
Department of Mersin University under Grant Number 2021-2-
TP3-4470.
ORCID
Ahmet Akkoca http://orcid.org/0000-0001-8269-9385
Belgin Büyükakıllı http://orcid.org/0000-0003-1153-2558
Ebru Ballı http://orcid.org/0000-0002-9950-5548
Burcu Gültekin http://orcid.org/0000-0001-6461-8123
Erkan Özbay http://orcid.org/0000-0002-8781-3877
Hatice Oruç Demirbağ http://orcid.org/0000-0002-1086-713X
Çağatay Han Türkseven http://orcid.org/0000-0002-0584-0661
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