Protective Actions of Ghrelin on Global Cerebral Ischemia-Induced Memory Deficits
In our study, we investigated transient global cerebral ischemia (TGCI) -induced changes of spatial memory and motor activity together with apoptotic, oxidant, and NO/NOS signaling parameters in rats and the effects of treatment of the animals with ghrelin. The TGCI-induced deficiencies of spatia...
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Інститут фізіології ім. О.О. Богомольця НАН України
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| Cite this: | Protective Actions of Ghrelin on Global Cerebral Ischemia-Induced Memory Deficits / G. Basaranlar, N. Derin, R. Tan, G. Tanriover, N. Demir // Нейрофизиология. — 2014. — Т. 46, № 4. — С. 381-390. — Бібліогр.: 58 назв. — англ. |
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nasplib_isofts_kiev_ua-123456789-1483082025-02-09T22:06:27Z Protective Actions of Ghrelin on Global Cerebral Ischemia-Induced Memory Deficits Протективний вплив греліну на розлади пам’яті у щурів, зумовлені глобальною церебральною ішемією Basaranlar, G. Derin, N. Tan, R. Tanriover, G. Demir, N. In our study, we investigated transient global cerebral ischemia (TGCI) -induced changes of spatial memory and motor activity together with apoptotic, oxidant, and NO/NOS signaling parameters in rats and the effects of treatment of the animals with ghrelin. The TGCI-induced deficiencies of spatial memory and motor activity in the Y-maze and open field tests were attenuated by ghrelin treatment. Furthermore, ghrelin administration lowered the levels of caspase-3 and iNOS elevated by TGCI in the hippocampus. Thus, we conclude that ghrelin exerts the neuroprotective action against hippocampal TGCI injury via influencing apoptotic, oxidant, and/or NO/NOS pathways. If underlying mechanisms of the action of this agent will be fully clarified, ghrelin might be a candidate drug for treatment of TGCI-induced memory impairments. У наших дослідах ми вивчали зміни просторової пам’яті та моторної активності, викликані в щурів транзієнтною глобальною церебральною ішемією (ТГЦІ). Використовували тести в Y-подібному лабіринті та відкритому полі; оцінювали також параметри процесу апоптозу, оксидативного стресу та сигнального шляху оксиду азоту. Розлади просторової пам’яті та моторної активності під впливом греліну (ендогенного ліганда рецепторів гормону росту) зменшувалися. Крім того, в результаті введень греліну знижувалися рівні каспази-3 та індуцибельної NO-синтази в гіпокампі, що були підвищеними після ТГЦІ. Зроблено висновок, що грелін має нейропротективні властивості в умовах ушкодження гіпокампа в наслідок ТГЦІ, впливаючи на процес апоптозу, оксидативний стрес та стан сигнального шляху оксиду азоту. Якщо механізми дії цього агента будуть докладно з’ясовані, грелін може виявитися перспективним фармакологічним засобом при лікуванні розладів пам’яті, пов’язаних з ТГЦІ. This work was supported by a grant from the Research Foundation of the Akdeniz University, Turkey (project No. 2009.02.0122.017). This study was carried out as a part of the MSc thesis by G. Başaranlar presented to the Akdeniz University Health Sciences Institute. 2014 Article Protective Actions of Ghrelin on Global Cerebral Ischemia-Induced Memory Deficits / G. Basaranlar, N. Derin, R. Tan, G. Tanriover, N. Demir // Нейрофизиология. — 2014. — Т. 46, № 4. — С. 381-390. — Бібліогр.: 58 назв. — англ. 0028-2561 https://nasplib.isofts.kiev.ua/handle/123456789/148308 616.005.4+616.89-008.46 en Нейрофизиология application/pdf Інститут фізіології ім. О.О. Богомольця НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
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English |
| description |
In our study, we investigated transient global cerebral ischemia (TGCI) -induced changes of
spatial memory and motor activity together with apoptotic, oxidant, and NO/NOS signaling
parameters in rats and the effects of treatment of the animals with ghrelin. The TGCI-induced
deficiencies of spatial memory and motor activity in the Y-maze and open field tests were
attenuated by ghrelin treatment. Furthermore, ghrelin administration lowered the levels of
caspase-3 and iNOS elevated by TGCI in the hippocampus. Thus, we conclude that ghrelin
exerts the neuroprotective action against hippocampal TGCI injury via influencing apoptotic,
oxidant, and/or NO/NOS pathways. If underlying mechanisms of the action of this agent will
be fully clarified, ghrelin might be a candidate drug for treatment of TGCI-induced memory
impairments. |
| format |
Article |
| author |
Basaranlar, G. Derin, N. Tan, R. Tanriover, G. Demir, N. |
| spellingShingle |
Basaranlar, G. Derin, N. Tan, R. Tanriover, G. Demir, N. Protective Actions of Ghrelin on Global Cerebral Ischemia-Induced Memory Deficits Нейрофизиология |
| author_facet |
Basaranlar, G. Derin, N. Tan, R. Tanriover, G. Demir, N. |
| author_sort |
Basaranlar, G. |
| title |
Protective Actions of Ghrelin on Global Cerebral Ischemia-Induced Memory Deficits |
| title_short |
Protective Actions of Ghrelin on Global Cerebral Ischemia-Induced Memory Deficits |
| title_full |
Protective Actions of Ghrelin on Global Cerebral Ischemia-Induced Memory Deficits |
| title_fullStr |
Protective Actions of Ghrelin on Global Cerebral Ischemia-Induced Memory Deficits |
| title_full_unstemmed |
Protective Actions of Ghrelin on Global Cerebral Ischemia-Induced Memory Deficits |
| title_sort |
protective actions of ghrelin on global cerebral ischemia-induced memory deficits |
| publisher |
Інститут фізіології ім. О.О. Богомольця НАН України |
| publishDate |
2014 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/148308 |
| citation_txt |
Protective Actions of Ghrelin on Global Cerebral Ischemia-Induced Memory Deficits / G. Basaranlar, N. Derin, R. Tan, G. Tanriover, N. Demir // Нейрофизиология. — 2014. — Т. 46, № 4. — С. 381-390. — Бібліогр.: 58 назв. — англ. |
| series |
Нейрофизиология |
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Article
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4 381
UDC 616.005.4+616.89-008.46
G. BASARANLAR,1 N. DERIN,1 R. TAN,2 G. TANRIOVER,1 and N. DEMIR1
PROTECTIVE ACTIONS OF GHRELIN ON GLOBAL CEREBRAL ISCHEMIA-
INDUCED MEMORY DEFICITS
Received 25.11.2013
In our study, we investigated transient global cerebral ischemia (TGCI) -induced changes of
spatial memory and motor activity together with apoptotic, oxidant, and NO/NOS signaling
parameters in rats and the effects of treatment of the animals with ghrelin. The TGCI-induced
deficiencies of spatial memory and motor activity in the Y-maze and open field tests were
attenuated by ghrelin treatment. Furthermore, ghrelin administration lowered the levels of
caspase-3 and iNOS elevated by TGCI in the hippocampus. Thus, we conclude that ghrelin
exerts the neuroprotective action against hippocampal TGCI injury via influencing apoptotic,
oxidant, and/or NO/NOS pathways. If underlying mechanisms of the action of this agent will
be fully clarified, ghrelin might be a candidate drug for treatment of TGCI-induced memory
impairments.
KEYWORDS: transient global cerebral ıschemia, ghrelin, hippocampus, caspase-3,
total oxidant status, nitric oxide.
1 Medical School of the Akdeniz University, Antalya, Turkey.
2 Medical School of the Near East University, North Nicosia, Northern Cyprus.
Correspondence should be addressed to N. Derin
(e-mail: nderin07@gmail.com or narinderin@akdeniz.edu.tr).
INTRODUCTION
Transient global cerebral ischemia (TGCI), usually
stemming from a severe arrhythmia, cardiac arrest,
or hemorrhagic shock, is a leading cause of death
and adult disability in industrialized countries, and
the respective treatment options are limited [1].
Even though several agents have been found to be
neuroprotective in animal models, most of them
have failed to be transferred from the laboratory to
the clinics [2]. Thus, there is a huge medical need to
develop novel therapies for TGCI.
Neurophysiological dysfunct ions, including
learning and memory disabilities, are major outcomes
of TGCI, mediated through delayed neuronal death
(DND) starting 2-3 days after reperfusion mostly
in the cerebral cortex and hippocampus [1]. The
latter is the brain structure greatly vulnerable to
neurodegenerative effects of TGCI in humans [3-
5] and animals [6]. Hence, behavioral and cognitive
disturbances, particularly within the learning and
memory domains, are the most apparent symptoms
of TGCI, and loss of spatial learning and memory
abilities are the parameters usually described in rodent
models of TGCI [7].
Delayed neuronal death is suggested to involve
apoptosis, the programmed cell death essential for
eliminating damaged or aged cells and maintaining
tissue integrity [8]. The specific morphological
changes associated with apoptosis are cell shrinkage,
c h roma t i n conden s a t i on , i n t e r nuc l eo soma l
deoxyribonucleic acid (DNA) fragmentation, and
formation of apoptotic bodies [9]. The majority
of morphological and biochemical changes stem
from various enzymatic reactions most of which are
catalyzed by caspases playing essential roles during
apoptotic cell death [10]. There are two types of
caspases: initiator caspases (caspases 8, 10, 9, and
2) and effector caspases (caspases 3, 7, and 6); the
latter are activated by initiator ones [11]. Among all
members of the caspase family, caspase-3 is the most
abundant in the brain, and its activation is considerably
implicated in ischemic cell death [12].
Oxidative stress, stemming from distortion of the
equilibrium between production and scavenging of
free radicals, is one of the most important pathological
mechanisms leading to DND following TGCI [13].
Beside oxidative stress, nitrosative stress is another
pathological mechanism significantly contributing
to DND induced by excessive nitric oxide (NO)
production through nitric oxide synthases (NOSs)
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4382
G. BASARANLAR, N. DERIN, R. TAN et al.
enzymes [14]. The role of NO in pathogenesis of
ischemic brain damage is controversial. Some evidence
has been accumulated that NO plays both neurotoxic
and neuroprotective roles during the process of
cerebral ischemia and reperfusion [15, 16]. Moreover,
modulation of different NOSs, such as neuronal NOS
(nNOS), inducible NOS (iNOS), and endothelial NOS
(eNOS), have been shown to produce a variety of
outcomes in ischemic brain injury [17]. Neuronal NOS
and especially iNOS activities have been proposed to
be detrimental to the ischemic brain [18-20]. Hence,
agents preventing iNOS overexpression, such as
ghrelin, might be potential therapeutic means in TGCI
[21].
Ghrelin is a 28-amino acid peptide esterified with
octanoic acid on serine 3. It is mainly released from
oxyntic cells of the stomach mucosa. The ability of
ghrelin to stimulate growth hormone (GH) release by
activating GH secretagogue receptors (GHSRs 1a) in
the pituitary was discovered [22]. Ghrelin has several
other peripheral actions, including direct effects on
the systemic vascular resistance, gastric secretion,
stomach motility, sleep, learning, and memory [23-27].
Additionally, recent studies highlighted that ghrelin
also exerts neuroprotective actions in ischemic models
of stroke both in vivo and in vitro [28, 29].
Therefore, our study was conducted to search the
effects of ghrelin on TGCI-induced hippocampal
damage and memory impairments together with its
relation to apoptotic, oxidant, and NO/NOS signaling
in rats.
METHODS
Animals. Forty-five adult male Wistar rats weighing
300-350 g were used for the experiments. The animals
were housed at room temperature (23 ± 1°C) and 12-h
day/night cycle and supplied with standard laboratory
chow and water ad libitum.
Experimental Procedures. Rats were randomly
divided into control (C), ischemia/reperfusion (IR)
and ischemia/reperfusion+ghrelin (IR+G) groups (n
= 15 in each group). Rats of the C group underwent
sham surgery, i.e., without vessel occlusion, and
received saline via intraperitoneal (i.p.) daily
injections of vehicle after reperfusion for three days.
The IR-group rats were exposed to global cerebral
ischemia/reperfusion and received daily saline via i.p.
injections of vehicle also for three days of reperfusion.
Rats of the IR+G group, underwent global cerebral
ischemia/reperfusion surgery and received daily
ghrelin injections (80 µg/kg·day, i.p.) as treatment
for three post-reperfusion days [28]. The Y-maze
and open field tests were carried out on the first and
second post-reperfusion days. The Y-maze test was
followed by the open field test with a 1-h-long time
interval. On the third day after reperfusion, rats were
perfused transcardially with heparinized saline under
deep urethane anesthesia; the brains were removed
immediately, and the extracted hippocampi were stored
at –80°C for biochemical assays.
Drug Preparation. Rat ghrelin, purchased from
GenScript Inc. (USA) was dissolved in distilled
water (1 mg/ml), and stored at –20°C until the time
of preparation for administration. Immediately before
administration by i.p. injection, ghrelin was diluted
again with 0.9% saline to the final concentration of
0.1 mg/ml. [30].
Global Cerebral Ischemia/Reperfusion. Global
ischemia was induced by four-vessel occlusion
(4-VO), as previously described by Pulsinelli and
Brierley [31], with a modification. In brief [32],
an incision was made 2.5 cm dorsal to the cervical
midline under deep ketamine/xylazine anesthesia
(100/10 mg/kg, i.p.), and vertebral arteries were
irreversibly occluded by electrocoagulation through
the alar foraminae of the 1st cervical vertebra.
Immediately after electrocoagulation, bitemporal
subdermal EEG needle electrodes were placed in
reference to a frontal subdermal electrode, and an
incision was made 2.5 cm with respect to the ventral
cervical midline. After a baseline EEG level had
been estimated, both common carotid arteries were
exposed and occluded with micro aneurysm clips
for 8 min, and this was followed by reperfusion.
The experiments were performed only if the animals
showed a completely flat bitemporal EEG during
the carotid artery occlusion. Sham-operated rats
underwent all procedures except the latter bilateral
common carotid artery occlusion. A heating pad was
applied in order to maintain the rats’ body temperature
at 37 ± 0.5°C. Each group of animals received the
same degree of surgical manipulations and the same
recovery paradigms, to minimize variations that could
be induced by surgical procedures.
Behavioral Tests
The Y-maze Test. Spatial memory was evaluated
using a Y-maze test paradigm [33] at 24 and 48 h after
ischemia. The maze was constructed of grey Plexiglas
and consisted a stem arm (42 cm long) and two
identical arms (37 cm long, diverging at a 45° angle
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4 383
PROTECTIVE ACTIONS OF GHRELIN ON GLOBAL CEREBRAL ISCHEMIA-INDUCED MEMORY DEFICITS
from the stem arm) surrounded by extra-maze cues of
varying shapes and sizes. During 15-min information
trial, rats were free to explore the stem arm and one
of the second arms, but access to the third arm (novel
arm) was blocked by a plastic partition. Following the
initial information trial, rats were returned to their
home cages for a 4-h-long interval. After the delay,
each rat re-entered the Y-maze in the stem arm and
was allowed to explore all three arms during a 5-min
retention trial. Retention trials were video-recorded
(Noldus Ethovision XT System, country?) for scoring
the arm entries. Arm entry was defined as all four
paws crossed the dividing line between the arm and
the central area. The maze was cleaned thoroughly
with 70% ethanol and air-dried after each trial to
remove olfactory cues. Spatial memory was indicated
by increased entries into the novel arm during the
retention trial.
Open Field. Exploratory behavior was tested
in an open field constructed of black Plexiglas
(100 cm × 100 cm × 45 cm) arena. Each rat was placed
in the center of the open field and allowed a single
exploration trial of 3 min. The floor and walls were
cleaned thoroughly with 70% ethanol and allowed to
air-dry after each trial, to remove olfactory cues. The
activity was monitored by a video camera and recorded
to allow for later analysis. The field was virtually
divided into 16 equal-sized squares. Entry into a
square was scored when the rat crossed the borders of
a designated square with all four paws. Exploratory
behavior and locomotor activity were evaluated by
calculating the total number of squares entered and
total distance moved, respectively [33].
Biochemical Assays
Estimation of the Total Oxidant Status (TOS).
Total oxidant status was measured by a commercially
available TOS assay kit (cat. No. RL0024, Rel Assay
Diagnostics, Turkey). The assay was calibrated with
hydrogen peroxide, and the results are expressed
below as µM H2O2/g protein.
NO Metabo l i t e s (N i tr i t e+Ni tra te ) . The
nitrite+nitrate level was measured using a commercial
nitrate/nitrite colorimetric assay kit (cat. No. 78001;
Cayman Chemical, USA) in accordance with the
manufacturer’s instructions. Briefly, the hippocampi
were dissected on ice, homogenized in PBS, and
centrifuged at 10,000g for 20 min. The supernatant
was centrifuged at 100,000g for 30 min and then
filtered through a 10-kDa molecular mass cutoff filter.
The nitrate+nitrite level was measured in duplicate on
96-well plastic microplates. The absorbance was read
at 570/620 nm using a multiwell microplate reader.
Immunohistochemistry of iNOS, nNOS, and
Caspase-3. Under deep anesthesia, animals were
perfused transcardially with 200 ml of saline followed
by 300 ml of 4% paraformaldehyde solution. Brains were
removed and postfixed in the same paraformaldehyde
solution for 24 h and then embedded in paraffin. Five-
μm-thick coronal sections were cut, collected on poly-
L-lysine coated slides (Sigma–Aldrich, USA) and
incubated overnight at 56°C. For immunohistochemical
assays, brain sections were deparaffinized in xylene and
rehydrated in a graded ethanol series. Sections were
then treated with 10 mM citrate buffer (pH 6.0) for 5
min twice in a microwave oven and left to cool down
for 20 min. After three washes in phosphate-buffered
saline (PBS), sections were incubated in a universal
blocking reagent (BioGenex, USA) for 7 min at room
temperature in order to block nonspecific binding.
Subsequently, sections were incubated overnight at
4 °C with rabbit anti-iNOS (1:200; ab3523), rabbit
anti-nNOS (1:100; ab76067, both from Abcam, USA)
and cleaved caspase-3 (1:100, Cell Signaling No.
9661) primary antibodies. After several washes in
PBS, sections were incubated with goat anti-rabbit-
biotin secondary antibody (1:400; Vector Labs, USA)
for 60 min at room temperature and rinsed with PBS.
Visualization was performed via a streptavidin-
peroxidase complex (Dako, USA) and diaminobenzidine
(BioGenex). Sections were counterstained with Mayer’s
hematoxylin (Dako), mounted with Kaisers Glycerol-
Gelatin (Merck Kaisers Glycerin Gelatine, Cat No:
1.09242.0100, , Germany), and were examined using a
light microscope.
Statistical Analysis. The obtained numerical data
were treated by SPSS (version 18.0) software package
for Windows. Since the numbers of entries to the novel
arm in the Y-maze did not follow a normal distribution,
and their variance did not fit the assumption of
homoscedasticity, the relevant data are expressed
as medians (min-max), while the remaining results
are expressed as means ± s.e.m. All variables were
analyzed with the non-parametric Kruskal-Wallis test,
and all pairwise multiple comparisons were performed
by the Mann-Whitney U test; differences with P values
smaller than 0.05 were considered significant.
RESULTS
TGCI-Induced Spatial Memory Changes and
Effects of Ghrelin Treatment. Table 1 shows number
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4384
G. BASARANLAR, N. DERIN, R. TAN et al.
of entries to the novel arm and total distance moved
in the Y-maze. The number of entries to the novel arm
and total distance moved were smaller in the IR group
with respect to the C group on both post-reperfusion
day 1 and day 2. On day 1, ghrelin administration
resulted in an increase of the number of entries to the
novel arm, as compared to the IR group; however, on
day 2 no significant effect of the drug was observed.
Ghrelin treatment did not cause significant changes in
the total distance moved in the IR+G group neither on
day 1 nor on day 2.
TGCI-Induced Motor Activity Changes and
Effects of Ghrelin Treatment. The number of squares
entered and total distance moved in the open field test
F i g. 1. Total oxidant status (µmol H2O2/g protein) in the hippo-
campi of rats of the C (n = 12), IR (n = 6), and IR+G (n = 5) groups.
Means ± s.e.m. are shown; * indicates a significant difference
(P < 0.05) with respect to group C.
Р и с. 1. Загальний оксидантний статус (мкмоль H2O2/г
протеїну) в гіпокампах щурів груп контролю (C, n = 12), ішемії-
реперфузії (IR, n = 6) та ішемії-реперфузії+грелін (IR+G, n = 5).
F i g. 2. Nitrite+nitrate levels (µmol/g protein) in the hippocampi
of the C, IR, and IR+G groups. Designations are similar to those in
Fig. 1.
Р и с. 2. Рівні нітритів+нітратів (мкмоль/г протеїну) в гіпо кам-
пах щурів груп C, IR та IR+G.
0
C IR
*
*
IR+G
5
0
5
10
15
20
25
30
35
40
45
10
15
20
25 µmol/g protein
µmol/g protein
C IR IR+G
T a b l e 1. Indices observed in the Y-maze test.
Т а б л и ц я 1. Результати тестування у Y-подібному лабіринті.
Groups Number of entries to the novel arm Total distance moved (cm)
Day1 Day 2 Day1 Day 2
C (n = 15) 2.00 ± (0.00-8.00) 1.50 ± (0.00-4.00) 717.6 ± 46.2 744.2 ± 55.0
IR (n = 9) 0.00 ± (0.00-1.00)* 0.00 ± (0.00-2.00)* 467.6 ± 39.9* 367.3 ± 67.8*
IR+G (n = 8) 1.00 ± (0.00-2.00)+ 0.00 ± (0.00-6.00) 378.1 ± 33.6* 396.3 ± 24.8*
Footnotes. Numbers of entries to the novel arm are expressed as medians ± (min-max), while other results are expressed as means ± s.e.m.
*P < 0.05 compared to the C group, +P < 0.05 compared to the IR group.
T a b l e 2. Indices observed in the open field test.
Т а б л и ц я 2. Результати тестування у відкритому полі.
Groups Number of squares entered Total distance moved (cm)
Day 1 Day 2 Day1 Day 2
C (n = 15) 28.79 ± 4.01 27.67 ± 3.73 679.8 ± 90.2 691.8 ± 101.0
IR (n = 9) 8.67 ± 1.04* 8.83 ± 1.56* 386.2 ± 38.6* 436.5 ± 63.3*
IR+G (n = 8) 19.00 ± 4.16+ 22.33 ± 4.04+ 515.9 ± 75.6+ 612.1 ± 74.1+
Footnotes. Means ± s.e.m are shown, *P < 0.05 compared to the C group, + P < 0.05 compared to the IR group.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4 385
PROTECTIVE ACTIONS OF GHRELIN ON GLOBAL CEREBRAL ISCHEMIA-INDUCED MEMORY DEFICITS
A
B
C
B
C
A
F i g. 3. iNOS expression in hippocampal slices of rats of the C, IR,
and IR+G groups (A-C, respectively); 40x.
Р и с. 3. Експресія iNOS у зрізах гіпокампа щурів груп C, IR та
IR+G (A-C відповідно).
F i g. 4. nNOS expression in hippocampal slices of rats of the C, IR,
and IR+G groups (A-C, respectively); 40x.
Р и с. 4. Експресія nNOS у зрізах гіпокампа щурів груп C, IR та
IR+G (A-C відповідно).
for all experimental groups are shown in Table 2. Both
these indices were significantly lower in the IR group
compared to the C group on both day 1 and day 2.
Ghrelin administration significantly increased both
the number of squares entered and the total distance
moved on day 1 and day 2 in the IR+G group with
respect to the IR group.
Hippocampal TOS Levels. Total oxidant status
results are illustrated by Fig. 1. The respective index was
significantly greater in the IR group (18.41 ± 1.14 µM
H2O2/g protein) in reference to the C group (12.19 ±
± 0.67 µM H2O2/g protein). Ghrelin treatment lowered
the TOS level in the IR+G group (16.19 ± 2.16 µM
H2O2/g protein) with respect to the IR group; however,
this effect was not statistically significant.
Hippocampal Nitrite+Nitrate Levels. Nitrite and
nitrate levels in the hippocampus are shown in Fig.
2. These levels were higher in both the IR (33.61 ±
± 4.59 µM/g protein) and IR+G groups (25.05 ± 3.16
µM/g protein) than in C group (18.17 ± 0.92 µM/g
protein). Although a decrease in the hippocampal
nitrite+nitrate levels was observed in the IR+G group
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G. BASARANLAR, N. DERIN, R. TAN et al.
compared to the IR group, the difference did not rich
the level of significance.
Hippocampal iNOS Expression. Hippocampal
iNOS immunoreactivity (brown straining) was
evaluated by immunohistochemistry (Fig. 3). The
immunoreactivity with respect to iNOS in the
hippocampus was significantly higher in the IR group
(Fig. 3B) with respect to the C group (Fig. 3A).
Ghrelin treatment noticeably decreased the number of
iNOS-positive neurons in the IR+G group (Fig. 3C) in
reference to the IR group.
Hippocampal nNOS expression. Hippocampal
nNOS immunoreactivity (brown staining) was also
evaluated by immunohistochemistry (Fig. 4). The
nNOS immunostaining was mostly observed in the
hippocampi in the IR group (Fig. 4B). There was weak
nNOS immunolabelling found in the IR+G group
(Fig. 4C). No significant difference was observed
between the nNOS immunoreactivity in the C and
IR+G groups.
Hippocampal Cleaved Caspase-3 Expression.
Immunohistochemical analysis of the hippocampal
region demonstrated that the caspase-3 level was
elevated after TGCI in the IR group (Fig. 5B). Ghrelin
caused a strong decrease in the caspase-3 level in
the IR+G group (Fig. 5C) compared to the above-
mentioned IR group. No caspase-3 expression was
observed in the C group (Fig. 5A).
DISCUSSION
Even though TGCI is one of the leading causes of
human death and disability across the world [34],
there are still very limited therapeutic options [35].
Hence, this dramatic disorder remains a demanding
clinical problem for which the development of new
therapies is necessary.
Patients who experienced TGCI usually suffer
from various degrees of memory impairment and
learning dysfunction due, to a significant extent, to
the hippocampal damage [36, 37]. Circulating ghrelin
in the hippocampus was shown to cross the blood-
brain barrier, to enhance long-term potentiation
(LTP), and to improve spatial memory [38]. Based on
these results, we investigated the possible therapeutic
effects of ghrelin on TGCI-induced deterioration
of spatial memory and exploratory behavior in rats.
Our data suggested that ghrelin has a clear positive
effect on spatial memory impairment induced by
TGCI on the first day after reperfusion. However, its
protective effect mostly vanished on the second post-
reperfusion day. This situation might occur due to an
insufficient dose of ghrelin used in our experiments.
In other words, as hippocampus-dependent memory
impairment progresses within the reperfusion
period, an administered amount of ghrelin could not
be adequate to overcome the damage within later
segments of the post-reperfusion period. In addition,
ghrelin administration resulted in enhancement of
motor activity that was previously deteriorated by
TGCI. The alleviative effect of ghrelin on motor
activity was observed not only on the first day, but
B
C
A
F i g. 5. Caspase-3 expression in hippocampal slices of rats of the
C, IR, and IR+G groups (A-C, respectively); 40x.
Р и с. 5. Експресія каспази-3 у зрізах гіпокампа щурів груп C,
IR та IR+G (A-C відповідно).
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PROTECTIVE ACTIONS OF GHRELIN ON GLOBAL CEREBRAL ISCHEMIA-INDUCED MEMORY DEFICITS
also on the second day after reperfusion. Although
memory- and motor activity-enhancing effects of
ghrelin were reported in the literature, the underlying
mechanism has not been fully understood yet [39].
Therefore, further studies are required to clarify the
mechanisms of action of this agent.
The hippocampus is selectively vulnerable to
TGCI; hippocampal degeneration begins to be
observed histologically 2-3 days after ischemia [6].
In reference to these crucial findings, we evaluated
apoptotic changes in the hippocampus three days
after ischemia via caspase-3 immunostaining, in order
to see is there any correlation between behavioral
and apoptotic changes. Ultimately, higher caspase-3
immunoreactivity was observed in the IR group with
respect to the C group. In the same line with our
findings, Liu et al. [40] showed that 8-min-long TGCI
resulted in dramatic cell loss in the hippocampus
three days after ischemia. Pooling our behavioral and
histological results together, we can suggest that TGCI
causes memory impairment by inducing DND via an
apoptotic pathway in the hippocampus.
At present, a few investigations are being carried
out in order to suggest neuroprotective therapies to
overcome hippocampal degeneration caused by TGCI.
Recent studies highlighted neuroprotective actions
of ghrelin in ischemic models of stroke, both in vivo
and in vitro. In vivo rat IR models showed that ghrelin
provides significant neuroprotection in the forebrain
by reducing an infarct volume and cell death [28, 29].
We also observed that ghrelin treatment (80 µg/kg)
lowered the caspase-3 immunoreactivity in the IR+G
group. Together with behavioral changes, it might
be suggested that ghrelin attenuates TGCI-induced
memory impairment by affecting an apoptotic pathway.
In that respect, ghrelin might be considered as a
potential therapeutic mean with respect to hippocampal
TGCI injury. Therefore, further researches should be
conducted in order to find out the details of molecular
pathway mediating the anti-apoptotic action of ghrelin.
Oxidative stress, the consequence of free radical
overproduction, is a well-identified pathophysiological
mechanism of IR injury [41, 42]. With the beginning
of reperfusion, abrupt oxygenation of the brain tissue
causes excessive production of oxygen radicals
intensely oxidizing membrane lipids, cellular
proteins, and DNA, thus leading to intensification
of apoptosis [43]. In our study, similar to previous
reports, we also observed an elevation of TOS in the
hippocampus after TGCI. We, however, found that
ghrelin administration did not significantly lower the
TOS level in the IR+G group. At the same time, other
authors [44, 45] suggested that ghrelin expression
affects oxidative stress in different cell types. Our
negative result might be related to a dose of ghrelin
insufficient to reduce oxidative stress significantly.
In order to clarify the pathways mediating possible
effects of ghrelin, protein levels, mRNA expression,
and activities of both oxidant and antioxidant enzymes
should be investigated in a dose-dependent manner in
further studies.
Nitric oxide, a highly reactive signaling molecule
in the CNS is one of the free radicals elevated
during ischemic brain injury [46]. Being a gaseous
chemical messenger, NO plays key roles in neuron-
to-neuron communication, synaptic plasticity, and
memory formation [47, 48]. Unless existing in
physiological low concentrations, NO might affect
the tissues through toxic actions mediated mostly by
the peroxinitrite radical (ONOO-) [49]. Although not
being fully understood yet, the mechanism underlying
TGCI injury is suggested to involve NO signaling due
to the induction of excessive NO production [50, 51].
Together with NO, the levels of its metabolites, such as
nitrites and nitrates, are also elevated following TGCI
[52]. In our study, a marker of NO production, the
nitrite+nitrate level, was elevated in the hippocampus
after TGCI. We observed that ghrelin administration
lowered this level in the hippocampi in the IR+G
group, but the effect was not statistically significant.
Alterations in the nitrite+nitrate concentration might
be explained by changes in the hippocampal NOS
levels.
Nitric oxide is a “double-edged sword” in IR
injury; in addition to the amount, the source of NO
is another crucial factor dominating its effects in
cerebral ischemia. Nitric oxide produced by eNOS
has been suggested to exert beneficial effects during
cerebral ischemia-reperfusion; nNOS and, especially,
iNOS are considered to be detrimental since they
produce a considerable amount of NO resulting in
DNA damage [53]. We found that iNOS and nNOS
immunoreactivities were raised in the hippocampus
after TGCI. Therefore, the high nitrite+nitrate level
in the IR group can be attributed to increases in the
iNOS and nNOS levels. The latter effect (increase of
iNOS and nNOS expressions) was reported by other
authors in focal cerebral ischemia models in mice and
rats [54-56]. Ghrelin treatment, on the other hand,
lowered iNOS and nNOS immunoreactivities in the
IR+G group, as compared with the IR group. Hence,
we can conclude that the lower nitrite+nitrate level
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4388
G. BASARANLAR, N. DERIN, R. TAN et al.
was a consequence of decreases in iNOS and nNOS
expressions in the IR+G group with respect to the
IR group. Indeed, ghrelin has in several reports been
suggested to modulate NO/NOS signaling [57, 58].
Brzozowski et al. [58] showed that ghrelin inhibits
expression of iNOS mRNA under stress conditions
and upregulates constitutive NOS (eNOS and nNOS)
mRNA. Additionally, Carlini et al. [57] suggested that
ghrelin affects memory consolidation and reduces the
LTP threshold through the NO/NOS signaling pathway.
Similarly to previous studies, the observed effect of
ghrelin on TGCI-induced memory changes in our
study might also to a noticeable extent be attributed to
NO/NOS pathway modulation.
Thus, we examined the effects of ghrelin on TGCI-
induced behavioral impairment and accompanying
changes in the apoptotic, oxidant, and NO/NOS
pathways in the rat hippocampus. Pooling all results
together, ghrelin might be a candidate drug for
treatment of TGCI-induced memory impairment
provided that the underlying mechanisms should be
fully clarified in further studies.
Acknowledgements. This work was supported by a grant
from the Research Foundation of the Akdeniz University,
Turkey (project No. 2009.02.0122.017). This study was carried
out as a part of the MSc thesis by G. Başaranlar presented to the
Akdeniz University Health Sciences Institute.
All experimental procedures were carried out in agreement
with the internationally accepted regulations and approved by
the Animal Care and Use Committee of the Akdeniz University.
The authors, G. Basaranlar, N. Derin, R. Tan, G. Tanriover,
and N. Demir, have no conflict of interests.
Г. Базаранлар1, Н. Дерін1, Р. Тан2, Г. Танріовер1, Н. Демір1
ПРОТЕКТИВНИЙ ВПЛИВ ГРЕЛІНУ НА РОЗЛА-
ДИ ПАМ’ЯТІ У ЩУРІВ, ЗУМОВЛЕНІ ГЛОБАЛЬНОЮ
ЦЕРЕБРАЛЬНОЮ ІШЕМІЄЮ
1 Університет Акденіз, Анталія (Туреччина).
2 Близькосхідний Університет, Північна Нікозія (Північний
Кіпр).
Р е з ю м е
У наших дослідах ми вивчали зміни просторової пам’яті та
моторної активності, викликані в щурів транзієнтною гло-
бальною церебральною ішемією (ТГЦІ). Використовува-
ли тести в Y-подібному лабіринті та відкритому полі; оці-
нювали також параметри процесу апоптозу, оксидативного
стресу та сигнального шляху оксиду азоту. Розлади просто-
рової пам’яті та моторної активності під впливом греліну
(ендогенного ліганда рецепторів гормону росту) зменшува-
лися. Крім того, в результаті введень греліну знижувалися
рівні каспази-3 та індуцибельної NO-синтази в гіпокампі,
що були підвищеними після ТГЦІ. Зроблено висновок, що
грелін має нейропротективні властивості в умовах ушкод-
ження гіпокампа в наслідок ТГЦІ, впливаючи на процес
апоптозу, оксидативний стрес та стан сигнального шляху
оксиду азоту. Якщо механізми дії цього агента будуть до-
кладно з’ясовані, грелін може виявитися перспективним
фармакологічним засобом при лікуванні розладів пам’яті,
пов’язаних з ТГЦІ.
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