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Summary. Objectives: Study of the effects of olive leaf
extract on antioxidant enzyme activities in midbrain and
dopaminergic neurons of Substantia Nigra in young and
old rats. Methods: Male wistar rats age 4 and 18 months
were randomized into control and experimental groups.
A single daily dose of 50 mg/kg of olive leaf extract was
administered orally by gavage to each rat for 6 months.
The control group received only distilled water. All rats
were sacrificed 2 hours after the last gavage and their
midbrains were separated for Malondialdehyde (MDA)
and antioxidant enzyme activitiy analysis. TUNEL assay
and immunohistochemical (IHC) staining were used for
evaluation of the number of neurons in the Substantia
Nigra. Resu lts : The level of Catal ase , Glut hatione
Peroxidase and Superoxide Dismutase enzyme activity
were significantly increased in experimental young and
old groups compared to their control groups. However
the level of Superoxide Dismutase enzyme activity was
significantly increased in experimental old group when
co m pared t o co n trol g roup ( P <0.01) , the lev e l of
Superoxide Di s m u t a s e enzyme ac t i v i t y was no t
significantly changed in young groups. MDA level was
decreased significantly in experimental young and old
rats compared to their control groups . Histological
analysis demonstrated that the number of neurons in
Substantia Nigra of experimental old group was more
than t h e control group (P < 0 . 0 1 ) . The number of
apoptotic cells was si g n i f i ca n t l y d e c r e a s e d i n
experimental old group compared to the corresponding
control group (P<0.05). In IHC and TUNEL assay, no
change was observed in the number of neurons between
experimental and control young groups. Conclusion:
Long term treatment with olive leaf extract increases
antioxidant enzyme activity and protects the neurons in
Substantia Nigra against oxidative stress.
Ke y wo r d s: Ol i ve l e af ex tract , Sub s tantia Nig r a,
Midbrain
Introduction
Oxidative stress i s a ma j o r f a c t o r fo r l i p i d
peroxidation in the brain. Excessive production of
Reactive Oxygen Species (R O S ) an d decline of
antioxidant enzyme activity result in oxidative stress.
Elevated oxidative stress interferes with the function of
neurons (Floyd and Carney, 1 992; Dringen, 2000 ).
Recently, a ttention has been drawn towar d d iet ary
strategies that can protect brain tissue against oxidative
stress damage. Po l y p h e n ol compounds po s s e s s
antioxidant and anti-inflammatory properties (Youdim et
al., 2002). The neuroprotective effect of olive phenols
upon oral administration in mice has been shown and
olive leaf extract is rich in phenols including Oleuropein
and Hydroxy Tyrosol (HT) (Visioli et al., 1999). Its
medicinal use in ancient Egypt was reported (Durlu-
Ozkaya and Ozkaya, 2011). Oleuropein is the main
glycoside present in olive leaf and Hydroxy tyrozol the
Different effects of olive leaf extract on antioxidant
enzyme activities in midbrain and dopaminergic
neurons of Substantia Nigra in young and old rats
Fereshteh Mehraein1,2, Maryam Sarbishegi3and Zoleikha Golipoor4
1Department of Anatomy, Faculty of Medicine, Iran University of Medical Sciences, Tehran, 2Minimally Invasive Surgery Research
Center, Iran University of Medical Sciences, Tehran, 3Department of Anatomy, Faculty of Medicine, Zahedan University of Medical
Sciences, Zahedan and 4Department of Anatomy, Faculty of Medicine, Hamedan University of Medical Sciences, Hamedan, Iran
Histol Histopathol (2016) 31: 425-431
http://www.hh.um.es
Offprint requests to: Dr. Fereshteh Mehraein, Department of Anatomy,
Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran.
e-mail: femehra@yahoo.com
DOI: 10.14670/HH-11-687
Histology and
Histopathology
From Cell Biology to Tissue Engineering
derivative of oleuropein is effective in prevention of
complications associated with oxidative stress (Visioli et
al., 2002). Recent studies have also indicated that HT
exerts its neuroprotection effect after long term oral
administration of olive leaf extract in mice (Gonzalez-
Correa et al., 2008). Therefore, it is believed that HT
reduces the neuronal damage induced by oxidative stress
(Schaff e r et al., 2007). In th e pr e s e n t study, we
investigated the neuroprotective effect of olive leaf
extract on midbrain by measuring lipid peroxidation
level, antioxidant e n z y m e a c ti v i t y an d a l s o by
histologica l e xam ination of Subs tantia Ni gra (S N)
tissues in young and old rats.
Materials and methods
Olive leaf extract was provided from Razi herbal
medicine research center (Lorestan, Iran). Male Wistar
rats age 4 months (220-240 gr) and 18 months (450-550
gr) were housed in a temperature controlled room at
23°C and 12 hours light and dark cycle. The animals
were f e d with p e l l e t s during t h e experiment a n d
randomized into four groups, each containing 10 rats.
The four groups were as follows; 1- Control young rats
2- Ex peri mental young rats 3- Control o ld r ats 4-
Experimental old rats. All animal work was approved by
the ethical guidelines for the care of laboratory animals
of the research center of Iran University of Medical
Sciences (Tehran, Iran).
The experimental groups received a daily single
dose of 50 mg/kg of olive leaf extract by oral gavage for
6 months. The control groups received only distilled
water. Two hours after the last oral gavage half of the
rats from each group were anesthetized and decapitated,
their brains were removed and immersed in cold PBS
(0.1 M, pH: 7.4) Then their midbrains were separated
and homogenized in ice cold 10nmol/l Tris –HCl. The
homogenate was centrifuged at 12000 x g at 4°C for 20
minutes. The supernatant was collected for antioxidant
enzyme activity assay. Lipid peroxidation level was
me asur ed b y Th ioba rbitu ric aci d (TBA) using the
method of Satoh (Satoh, 1978). This method was used to
measure the color development that results from the
reaction of TBA with Malondialdehyde. Superoxide
Dismutase (SOD) activity was measured by the method
of Misra and Fridovich (Misra and Fridovich, 1972).
Catalase (CAT) activity was measured based on the
ability of the enzy me to break down th e h ydr oge n
peroxide (H2O2). This was performed acco rding to
modified version of Aebi me t h o d (A e b i , 19 8 4) .
Glutathione Peroxidase (GPx) activity was determined
using a kit from R a n d o x ( U K ) following the
manufacturer’s instruction. The other half of the rats
were anesthetized and perfused transcardially with PBS
(pH: 7.4) followed by fixation in 4% paraformaldehyde.
Their brains were removed and further fixed in the same
fixative overnight. After incubation, the midbrains were
separated and dehydrated in graded concentration of
alcohol, cleared in xylene, infiltrated with paraffin and
finally embedded in paraffin. The 5 µm coronal sections
were serially collected from bregma-4.52 mm to -6.04
mm of midbrains (Paxinos and Watson, 2006) with a 30
µm interval between each consecutive section. Half of
the sections were stained for TUNEL assay using a
detection k i t from R o c h e , according t o the
manufacturer’s instructions (Roche, Germany). Briefly,
the sections were rehydrated, incubated in 3% H2O2for
10 minutes and then incubated in proteinase –K (20
µg/ml in 10 mM Tris/HCL, pH: 7.6) for 30 minutes.
After incubation, TUNEL reaction mixture was added to
the sections and incubated for 1 hour. Each 5 µm section
was incubated further with antibody conjugated
horseradish peroxidase (Roch, Germany) for 30 minutes
an d de v eloped wit h 0. 0 5% 3, 3-Dia m inoban zidine
(DAB) fo r 1 - 2 mi n and counterstained with
Hematoxylin. The cells with brown stained nuclei were
counted at a magnification of × 400. For positive control
the 5 µm sections were incubated in DNAase (3000
U/ml in 50 mM Tris-HCl, pH: 7.5, 1 mg/ml BSA) for 10
min t o induce DNA st rand br eak prior to labeling
pr oced ure. For ne gati ve c ontrol t he s ectio ns w ere
incubated with labeling buffer only.
For i mmun ohis tochemi stry st aini ng (IHC ), t he
sections were dehydrated in graded concentration of
al c o h ol, i m m e rsed i n 10% H2O2/m e t h anol f o r 10
minutes and washed in 0.1 Tris wash buffer (TBS). For
retrieving the antigens, the sections were kept in citrate
buffer and boiled for 11 minutes. After cooling, the
sections were washed in Tris buffer and incubated in
Bovine Serum Albumine (BSA) for 10 minutes. After
antigen retrieval, the sections were incubated in the
pr imary ant ibody (mo use m onocl onal ant ibody to
tyrosine hydroxylase (1:80, Abcam, UK) for 1 hour at
room temperature. The sections were washed in Tris
buffer (pH: 7.4) and i ncubated in HRP conj uga ted
secondary antibody (1:100, Abcam, UK) for 1 hour.
They were incubated with DAB for 10 minutes, washed
in Tris bu f f e r (pH: 7 . 4 ) and c o un t e r s t a i n e d with
hematoxyline, the sections were washed under tap water,
dehydrated in graded concentration of alcohol, cleared in
Xylol and covered with a cover slip. The cells were
counted in five coronal sections of substantia nigra from
each animal in five separate microscopic fields randomly
with ×400 magnification using a microscope (Olympus
AX70), Olympus DP11microscope digital camera and
OLYS IA au tobi orep ort soft ware (Olymp us o ptic al
Co.Ltd.Japan). A g r i d wa s sup e r i m p o s e d on the
photographs and the cells with obvious nucleus were
counted, then a cross was placed on the counted cells to
prevent recounting (Mehraein et al., 2011). Statistical
anal yse s w ere perform ed using SPSS by t-te st and
ANOVA. The data are expressed as mean ± standard
deviation (SD).
Results
Fig. 1A shows SOD activity in all groups. SOD
ac t i vity w a s in c reased in e xperime n t al ol d gro u p
426
Olive leaf extract and mid brain of rats
compared to corresponding control group (P<0.01). An
increase of SO D a c t i v i t y w a s a l s o o b s e r v e d i n
experimental young group compared to their control,
which statistically was not significant. Administration of
olive leaf extract caused a significant increase in CAT
activity in midbrain tissues of experimental old rats
when compared to their contro l gro up (P<0 .01), as
shown, the level of CAT enzyme activity in experimental
ol d group reached th e same level of CAT enzy me
activity in control young rats (P<0.05) (Fig. 1B). Also, a
si g n i f i cant i n c r e a se of CAT e n z y m e act i v i t y was
observed in experimental young group compared to
control young group (P<0.05) (Fig. 1B). A marked
incre ase of GP X enzyme a ctivity wa s obser ved in
experimental old group when compared to their
corresponding control group (P<0.01) as well as in
experimental and control young groups (P<0.05) (Fig.
1C). The level of MDA in midbrain tissues of control old
rats was signifi cantly higher than the other groups
(P<0.05), while the level of MDA in experimental old
rats wa s s ign ifi cantly de cre ase d c omp ared to t hei r
control group (P<0.01). There was also a significant
reduction of the level of MDA in experimental young
group in comparison to control young group (P<0.05)
(Fig. 1D). Analysis of TUNEL assay revealed a small
number of TUNEL positive ne u r o n s in SN of
experimental young group compared to control young
group but the difference was not significant (Fig. 2C,D).
427
Olive leaf extract and mid brain of rats
Fig. 1. Antioxidant enzyme activities in control and experimental young and old groups. A. Superoxide dismutase (SOD). B. Catalase (CAT).
C. Glutathione peroxidase GPx. D. The levels of malondialdehyde (MDA). Values are expressed as mean ± SD. *P<0.01 vs. Control old, **P<0.05 vs.
control young.
428
Olive leaf extract and mid brain of rats
Fig. 2. Photomicrographs of Tunel staining of SN in the
co n t rol an d ex per i m ent a l youn g and ol d gr oup s .
A. Positive control staining. B. Negative control staining.
C. Control young group. D. Experimental young group. E.
Control old group. F. Experimenta l old group. G. The
hi sto gra m sho ws the diffe ren ces in the num b er of
apoptotic neurons between control and experimental old
groups which was significant (*P<0.05). x 400
429
Olive leaf extract and mid brain of rats
Fig. 3. Photomicrographs of IHC staining of SN in the control
and experimental young and old groups. A. Photomicrograph
of boundaries of the midbrain in coronal section. B. Control
young group. C. Experimental young group. D. Control old
group. E. Experimental old group. F. Columns represent the
nu m b er of d opa m i ner g i c ne uro n s . The n umb e r of
dopaminergic neurons in control old group was significantly
less than experimental old group (*P<0.01). A, x 100; B-E,
x 400
TUN E L s t a i n i n g al s o show e d tha t the number of
apoptotic neurons in experimental old group was less
than t h e number in c o r r e s p o n d i n g c o n t r o l group
(P<0.05) (Fig. 2E,F.G). IHC staining of the SN tissues
revealed that the differences of the number of TH+
neurons between experimental and control young groups
were not significant (Fig. 3B,C), while experimental old
group had significantly more neurons compared to their
control group (P<0.01) (Fig. 3D,E,F).
Discussion
ROS is produced during normal metabolism which
may lead to oxidative damage to lipids and proteins. The
excess of ROS that is the result of biochemical changes
in neurons may lead to neuronal dysfunction and death
of the neuron (Poon et al., 2004). Brain tissue is rich in
polyunsatured lipids and has high iron content and its
function is dependent on aerobic metabolism, therefore it
is vulnerable to oxidative damage (Beckman and Ames,
1998). Malondialdehyde (MDA) level is a marker of
lipid peroxidation and oxidative stress (Kasapoglu and
Ozben, 2001). Lipid peroxidation caused by ROS is
involved in several brain disorders. In this study, a
comparative analysis of MDA in control young and
control old rats showed a higher level of MDA in control
old rats, indicating that the level of MDA increases by
age, as has been reported by others (Kasapoglu and
Ozben, 2001). In human, the mean MDA values in 50-59
age groups are significantly higher than 30-39 age
groups (Kasapoglu and Ozben, 2001). The data from the
same study also showed that the level of oxidative stress
by aging may be related to a decrease in antioxidant
enzyme activity. Protective antioxidant enzymes reduce
oxidative stress by degrading the ROS. SOD converts
superoxide radicals into hydrogen peroxide and oxygen.
CAT and GPX convert H2O2to water and oxygen (Sies,
1993). In the present study, we showed that the level of
SOD and CAT enzyme activity in control old rats was
less than control young group, which is in agreement
with Head and Kasap oglu studie s (Kasa poglu an d
Ozben, 2001; Head, 2009). Our findings also indicated
that GPX enzyme activity was not different between
young and old control groups, which is consistent with
Holmes et al (Holmes, 1999; Erden-Inal et al., 2002). In
contrast Sandhu reported the decrease of GPX enzyme
ac tivit y in old su bjects co m pared to you ng group
(Sandhu and Kaur, 2002). There are reports that show
the ne u r o p r o t e c t i v e pr o p e r t i e s of olive ph e n o l i c
compounds on reduction of lipid peroxidation in neurons
(De La Cruz et al., 2000). It is believed that a diet rich in
antioxidant may reduce oxidative damage to neurons
during aging (Elmadfa and Meyer, 2008). In this study,
we observed a significant decrease in MDA level in the
ex peri ment al y oung and ol d rats. Our re sult s al so
showed that CAT a n d G P X e n z y me l e v el w e r e
significantly increased in the experimental young and
old groups compared to their c ontrol grou ps. SOD
enzyme activity was increased significantly in
experimental old gr o up w h e n c o m p a r e d t o
corresponding control group; however the differences in
young groups did not reach a statistical significance.
SOD enzyme activity causes morphological and
biochemical changes (Pejic et al., 1999). Studies showed
th a t oliv e leaf extr a c t sca v e nges f r e e rad i c als by
inducing antioxidant enzyme activity (Visioli et al.,
2002). Olive phenolic compound primarily may increase
the activity of a ntioxidant enzymes in the brain to
prevent free radical oxidative damage (Servili et al.,
2014). We observed that apoptotic cell numbers in
control old rats are significantly more than control young
group. The results from IHC staining also showed that
control old rats had less dopaminergic neurons than
control young rats in SN of their midbrain as reported
before by Gao (Gao et al., 2011). Analysis of TUNEL
assay revealed that the number of apoptotic neurons was
reduced in experimental old group compared to their
control. These data was supported by IHC staining
which showed that the number of TH positive neurons in
experimental old group was significantly more than
control old group while the differences of the number of
TH positive neurons in experimental and control young
groups were not significant.
Conclusion
Dietary supplementation of olive leaf extract plays
an important role in prevention of neuron loss in SN and
in crease s th e ant i oxida n t en zyme a ctivi t y ag ainst
oxidative damage.
Acknowledgements. This research was financially supported by Iran
University of Medical Sciences, Tehran, Iran (grant number: 12988).
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Accepted November 12, 2015
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