Ginkgo Biloba of Rotenone in Adult Male Rats

Journal of Applied Sciences Research, 5(6): 622-635, 2009
© 2009, INSInet Publication
Protective Effect of Ginkgo Biloba Extract and Pumpkin Seed Oil Against Neurotoxicity
of Rotenone in Adult Male Rats
Hanaa H. Ahmed, Mona Abdel-Rahman, Rania S. Ali, Ahmed E. Abdel Moniem
Department of Hormone, Medical Research Division, National Research Centre, Dokki-Cairo, Egypt
Department of Zoology & Entomology, Faculty of Science, Helwan University.
Abstract: The principal goal of the current work was to investigate the protective effect of Ginkgo biloba
extract and pumpkin seed oil in separate against rotenone induced neurotoxicity in male rats. Adult male
albino rats were orally treated with Ginkgo biloba extract (EGb) at a dose of 150 mg/kg body weight or
with pumpkin seed oil (PSO) at a dose of 40 mg/kg body weight once a day for 70 days and on the 21 st
day, rotenone (2.5 mg/kg body weight) was given orally for 50 days. Dopamine "DA" and norepinephrine
"NE" contents in striatum, cerebellum and cerebral cortex were determined on 40, 55 and 70 days of
treatment. Also, the striatum Na + /K + -ATPase activity, serum and striatum lipid peroxidation, nitric oxide
(NO), reduced glutathione (GSH), total antioxidant capacity (TAC), and serum testosterone level were
evaluated with histological investigation of striatum. Results revealed that rotenone administration for 50
days produced significant increase in striatum and serum lipid peroxidation and NO levels, while,
significant decrease in DA in striatum and cerebral cortex was detected. Also, striatum Na + /K + -ATPase
activity, serum and striatum GSH, TAC levels and serum testosterone level were significantly decreased
as a result of rotenone administration. On the other hand, the administration of EGb or PSO resulted in
marked improvement in the all studied parameters. Noteworthy, EGb produced more pronounced protective
effect against rotenone-induced neurotoxicity than PSO. In conclusion, the present study provided clear
evidence that EGb possesses a promising activity against rotenone-induced neurodegeneration. Thus, it may
be useful against neurotoxicity induced by environmental neurotoxins. Our study also suggested the
possibility of PSO usefulness in limiting toxicant-induced oxidative stress.
Key words: G inkgo biloba extract; Pumpkin
Norepinepherine; Oxidative stress.
seed
oil;
Rotenone; N eurotoxicity; Dopamine;
onset form but the occurrence of the more prevalent
late onset form does not have an established genetic
basis.[4 ]
A reduction of complex I activity has been
demonstrated in the mitochondria of PD patients,[6 ] and
the complex I inhibitors such as environmental toxins
are involved in some cases of PD. Factors such as
drinking water from wells and exposure to agricultural
chemicals have been investigated and claimed as
support for an association between pesticide and
increased PD. [4 ] It is well known that the dopaminergic
neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MTPP) after its conversion to one of the most
prominent mitochondrial complex I inhibitors 1-methyl4-phenylpyridinium (M PP + ) produces PD symptoms
with severe motor symptoms, striatal dopamine
d ep letio n a nd lo ss o f tyrosine hyd ro xylase
immunoreactivity in humans, monkeys and various
other species.[7 ,8 ] Previous studies demonstrated that
chronic systemic exposure to rotenone (an inhibitor of
mitochondrial NADH dehydrogenase, a naturally
occurring toxin and a commonly used pesticide)
INTRODUCTION
Brains of patients suffering from neurodegenerative
diseases undergo many changes, such as the
degradation of neural membrane glycerophospholipids,
the disruption of protein synthesis and degradation, and
the generation of reactive oxygen species (ROS) and
reactive nitrogen species. Among these, oxidative stress
and nitrosative stress are major factors that affect the
death process.[1,2 ,3 ].
Parkinson’s disease (PD) is regarded as the second
most common neurodegenerative disorder in humans
and it affects about 2% of the population over the age
of 60 years. Clinically, PD is a disorder of motor
function characterized by tremor, slow and decreased
movement (bradykinesia), muscular rigidity, poor
balance and problems in gait.[4 ] Pathologically, PD
patients possess loss of dopaminergic neurons in the
substantia nigra (SN) pars compacta and frequently
have Lewy bodies, eosinophilic intracellular inclusions
composed of amyloid-like fibers and á-synuclein. [5 ] PD
may have a genetic basis for susceptibility for an early
Corresponding Author: Hanaa Hamdy Ahmed, Department of Hormone, Medical Research Division, National Research
Centre, Dokki-Cairo, Egypt
E-mail: [email protected]
622
J. Appl. Sci. Res., 5(6): 622-635, 2009
reproduces many features of PD in rats, including
nigrostriatal dopaminergic degeneration and formation
of alpha-synuclein-positive cytoplasmic inclusions in
nigral neurons. [6,9 ].
Rotenone, a naturally occurring compound derived
from the roots of several tropical and subtropical plant
species b elo nging to the genus D erris and
Lonchorcarpus, is the active ingredient in hundreds of
pesticide products widely used as a household
insecticide and as a tool for eradicating nuisance fish
populations in lakes and reservoirs. [9 ] Rotenone is
extremely hydrophobic, and it crosses biological
membranes easily for access to the cytoplasm of
dopaminergic neurons. [9 ,1 0 ].
Rotenone has been shown to lead to selective
dopaminergic cell death in vivo [9 ,1 1 ] and selective
nigrostriatal dopaminergic degeneration in rats infused
intravenously or subcutaneously, as well as it induced
degeneration of non-dopaminergic neurons in both the
basal ganglia and the brainstem.[1 2 ] In vitro studies,
revealed that the dopaminergic neurons were more
sensitive to rotenone-induced toxicity than other
neuronal cells and glial cells.[1 3 ,1 4 ,1 5 ,1 6 ] The possible
result of complex I inhibition by rotenone is the
increased formation of ROS, creating oxidative damage
within the cell. Oxidative stress has been implicated in
PD.[1 7 ] Moreover, increased oxidative damage to
lipids,[1 8 ,1 9 ] DNA,[2 0 ,2 1 ] and proteins [2 2 ] has been observed
in PD substantia nigra pars compacta, along with
decreased levels of reduced glutathione.[23 ].
Antioxidants, free radical scavengers and other
such agents have the potential for therapeutic
development for PD.[2 4 ] Ginkgo biloba is a plant with
a high antioxidant power capable of scavenging various
reactive oxygen species, including superoxide, peroxy
radical, and hydroxyl radical, [2 5 ] and inhibiting or
reducing the functional and morphologic impairments
observed after lipid peroxidation. [2 6 ] Ginkgo biloba has
been reported to enhance the activities of superoxide
dismutase and catalase and to decrease lipid
peroxidation in striatum, substantia nigra, and
hippocampus, the major sites damaged in Parkinson’s
and Alzheimer’s diseases. [2 7 ] Ginkgo biloba extract
(EGb) exhibited protective properties against animal
models of hypoxia,[2 8 ] excitotoxicity and focal as well
as global cerebral ischemia.[2 9 ] EGb is a potent inhibitor
of brain monoamine oxidases. [3 0 ] Some studies [3 1 ] have
shown antiapoptotic properties of EGb. Some other
studies reported that Ginkgo biloba, possibly through
the antioxidant properties of its flavonoids, was able to
protect hippocampal cells against toxic effects induced
by amyloid beta peptides [3 2 ] and possesse protective
effect on the PD in vitro. [3 3 ].
Pumpkin seed oil (PSO) is a natural product
commonly used in folk medicine for hypertension and
atherosclerosis treatments. [3 4 ] It is rich in many
antioxidants and beneficial nutritional supplements such
as essential fatty acids, amino acids (especially tyrosine
and L phenylalanine) and phytosterols (e.g. betasitosterol), â-carotenes, lutein and selenium. [3 4 ] The
seeds also contain L-tryptophan and omega (-6 and -3)
fatty acids which help to alleviate the symptoms of
PD. [3 5 ] Pumpkin seeds have a very high concentration
of the antioxidant vitamin E.[36 ,3 7 ].
The principal goal of this study was to evaluate the
protective role of extract of Ginkgo biloba or pumpkin
seed oil against rotenone induced neurotoxicity in rats.
This could be fulfilled through the determination of
dopamine (DA) and norepinephrine (NE) levels in
different brain regions (striatum, cerebellum and
cerebral cortex), and Na + /K + -ATPase activity in
striatum. Lipid peroxidation, nitric oxide (NO), reduced
glutathione (GSH) levels and total antioxidant capacity
(TAC) in striatum and serum have been estimated.
Serum testosterone level has been also assayed. The
study was also extended to investigate striatum
histologically.
M ATERIALS AND M ETHODS
Experimental Design: Adult male albino rats were
obtained from The Holding Company for Biological
Products and Vaccines, VACSERA, Cairo, Egypt. The
range of animals weight was 120-150g. The animals
were kept in wire bottomed cages in a room under
standard condition of illumination with a 12-hour lightdark cycle at 25±1 o C. They were provided with tap
water and balanced diet ad libitum. Animals were
divided into 6 groups each was comprised of thirty rats
as follows: The first group was served as control which
was received corn oil as vehicle (CON group) for 70
days. The second group was orally administrated corn
oil which repeated every day for 20 days and on the
21 st day the animals were daily orally administrated
with rotenone (Sigma) in a dose of 2.5 mg/Kg b.wt.
dissolved in corn oil[3 8 ,3 9 ] (ROT group) for 50 days. The
third group was orally received Ginkgo biloba extract
(EGb) ''Arab Company for Pharmaceutical and
M edicinal Plants, MEPACO'' at a dose of 150 mg/kg
b.wt. dissolved in water [2 6 ] for 70 days. The fourth
group was orally administrated with EGb (150 mg/Kg
b.wt.) for 70 days and on the 21 st day they were orally
administrated with rotenone (2.5 mg/Kg b.wt.) for 50
days (EGb & ROT). The fifth group was orally
received pumpkin seed oil (PSO) ''Arab Company for
Pharmaceutical and Medicinal Plants, MEPACO'' at a
dose of 40 mg/kg b.wt.[3 4 ] for 70 days. The sixth group
was orally administrated with PSO (40 mg/Kg b.wt.)
for 70 days and on the 21 st day they were orally
administrated with rotenone (2.5 mg/Kg b.wt.) for 50
days (PSO & ROT). The animals were decapitated
after 40, 55 and 70 days post-treatment (n=10).
After decapitation of animals (eight from each
subgroup), the blood samples were collected, allowed
to stand for half an hour and then centrifuged at 3000
rpm for 15 min. under cooling to separate serum. Each
623
J. Appl. Sci. Res., 5(6): 622-635, 2009
brain was carefully removed and vertically divided into
two portions. Dissection of each brain portion was
performed on an ice-cold glass plate for the separation
of three brain regions (striatum, cerebellum and
cerebral cortex) according to the method described by
Glowinski and Iversen.[4 0 ] The three regions of the first
portion of each brain were blotted and frozen for
further determination of DA and NE. Striatum of the
2 n d portion of each brain was weighed and
homogenized immediately to give 50% (w/v)
homogenate in ice-cold medium containing 50 mM
Tris-HCl and 300 mM sucrose.[4 1 ] The homogenate was
centrifuged at 3000 rpm for 10 min in cooling
centrifuge at 4°C. The supernatant (10%) was used for
the various biochemical determinations.
RESULTS AND DISCUSSION
Effect of EGB and PSO on Dopamine (DA) Content
of Striatum, Cerebellum and Cerebral Cortex of
Adult M ale Albino Rats Treated with Rotenone: The
results in Table (1) showed that administration of
rotenone (ROT) caused significant decline in DA level
of both striatum and cerebral cortex after 55 and 70
days. These results are in agreement with those of Di
Monte; [5 0 ] Hirata and Nagatsu [5 1 ] and Ren et al.[5 2 ].
Hirata and Nagatsu [5 1 ] demonstrated that rotenone
inhibits tyrosine hydroxylation (TH), suggesting the
inhibition of biosynthesis responsible for dopamine and
norepinephrine. Another suggested mechanism for
rotenone-induced reduction in dopamine content in the
studied brain areas is the increased the process of
oxidative deamination. [ 5 3 ] O n the other hand,
administration of Ginkgo biloba extract (EGb) or
pumpkin seed oil (PSO) produced significant increase
in DA level in striatum at all time intervals of the
experiment and after 55 and 70 days in cerebellum of
EGb-treated group. The increment in dopamine content
in EGb-treated group may be due to kaempferol, major
ingredient of EGb, which is considered as a potent
monoamine oxidase B inhibitor (MAO-B), [5 4 ,5 5 ] that
prevents the degradation of DA and increases its
availability. Regarding the increasing effect of PSO on
dopamine content in striatum could be due to the high
concentration of vitamin E in this oil. Vitamin E has
been shown to increase dopamine anabolism in the
brain by activating the tyrosine hydroxylase, which is
a rate limiting enzyme for the biosynthesis of the
neurotransmitters in the brain. [5 6 ]
Non-significant
change was detected in DA content of striatum,
cerebellum and cerebral cortex of EGb & ROT-treated
group at all time intervals versus control group.
Similarly, non-significant change was demonstrated in
DA level in cerebellum and cerebral cortex of PSO &
ROT-treated group as compared to control. In
comparison to ROT group, treatment of EGb & ROT
showed significant increase in DA level of striatum at
all time intervals and after 55 and 70 days in PSO &
ROT-treated group. The protective effect of Ginkgo
biloba extract could be attributed to the ability of this
extract to stabilize and protect the mitochondrial
function by improving the mitochondrial membrane
potential and reversing the decrease in ATP production
as well as to its potent effect against oxidative
stress. [5 7 ,5 8 ] Ginkgo biloba extract and its bilobalide
could increase the antioxidant enzyme activities to
overcome the oxidative damage caused by ROT. In
addition, flavonoid (mainly flavonol glycoside), another
pharmacologically active constituent identified in EGb,
has direct free radical scavenging activities that also
play a role in EGb’s wide range of antioxidative
effects.[5 9 ,6 0 ] PSO could reduce the effect of rotenone as
a neurotoxin, due to its high content of vit. E.
Tissue Samples for Histological Investigation: The
brains of the remaining two rats were stored in
formalin buffer (10%) for histological examination
according to the method described by Drury and
W allington. [4 2 ] by using the ordinary hematoxylin and
eosin stain.
Biochemical Determinations: T otal antioxidant
capacity and nitric oxide were assayed colorimetrically
in serum and striatum [4 3 ,4 4 ] respectively by using kits
provided from Biodiagnostic Co., Egypt. Na + / K + ATPase activity in striatum homogenate was assayed
chemically according to method described by Tsakiris
et al. [4 1 ] This method depends on measuring the release
of P i associated with the hydrolysis of ATP. Reduced
glutathione and lipid peroxidation were determined
chemically in serum and striatum homogenate using
Elman's reagent,[4 5 ] and thiobarbituric acid (TBA)
modified Salter's method, [4 6 ] respectively. The procedure
of reduced glutathione is based on the reduction of
Elman's reagent [5, 5 ` dithio bis- (2-nitrobenzoic acid)]
by SH groups to form 1 mole of 2-nitro-5mercaptobenzoic acid per 1 mole of SH. Lipid
peroxidation was determined by using TBA which
reacts with malondialdehyde in acidic medium at
temperature of 95 o C for 30 min. to form thiobarbituric
acid reactive product which can be measured at 534
nm. Quantitative measurement of serum testosterone
was carried out adopting ELISA technique using kits
purchased from Calbiotech Inc., (Austin, Canada)
according to the method described by Joshi et al.[4 7 ]
DA and NE were extracted and estimated in the three
regions of the first half of each brain using
fluorometric technique according to the method of
Chang [4 8 ] modified by Ciarlone.[4 9 ] The fluorescence was
measured in Jenway 6200 fluorometer.
Statistical analysis: The obtained data were presented
in Tables as mean ± standard error. O ne-way ANOVA
was carried out, and the statistical comparisons among
the groups were performed with Duncan 's test using a
statistical package program (SPSS version 10.0).
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J. Appl. Sci. Res., 5(6): 622-635, 2009
Table 1: Effect of G inkgo biloba extract (EGb) and pum pkin seed oil (PSO ) adm inistration on dopam ine content (ng/g tissue) in striatum ,
cerebellum and cerebral cortex of adult m ale albino rats treated with rotenone (RO T).
Groups
D opam ine of striatum (ng/g tissue)
D opam ine of cerebellum (ng/g tissue)
D opam ine of cerebral cortex (ng/g tissue)
---------------------------------------------------------------------------------------------------------------------------------------------------------40 days
55 days
70 days
40 days
55 days
70 days
40 days
55 days
70 days
CO N
1834.7±49.8 1813.9±42.4 1795.6±28.3 620.1±41.8
644.9±54.8 627.1±47.7 1451.6±29.8 1457.4±40.2 1447.1±35.6
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------RO T
1810.4±28.7 1637.0±35.7 a 1411.3±26.3 a 617.3±44.6
617.6±33.4 574.7±33.2 1431.8±41.9 1315.9±33.9 a 1337.3±28.4 a
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EGb
2157.7±48.6 a 2078.5±44.8 a 2487.6±46.3 a 643.2±35.6
740.1±27.3 a 739.1±32.7 a 1467.9±49.8 1511.8±50.5 1487.1±55.4
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EGb &
1950.1±47.7 b 1861.4±50.7 b 1741.6±55.1 b 647.7±30.1
663.0±47.2 656.5±31.9 1454.5±46.7 1436.8±65.1 1447.8±44.8
RO T
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PSO
2076.1±46.6 a 2050.5±47.3 a 2146.6±54.9 a 561.9±37.0
626.9±43.0 663.7±29.8 1476.5±59.1 1441.4±26.9 1437.8±32.7
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------b
ab
PSO &
1947.6±66.8 1852.1±27.6 1604.5±49.7 582.0±26.5
649.3±26.5 589.4±25.1 1368.2±34.5 1398.8±47.4 1351.3±33.8
RO T
The num ber of anim als was 8 in each group. D ata are expressed as m ean ± SE., a: Significant change at p > 0.05 with respect to control
group )CO N(, b: Significant change at p > 0.05 with respect to RO T group.
Adachi et al.[5 6 ] demonstrated that the activity of
tyrosine hydroxylase, which is a rate limiting enzyme
for the biosynthesis of the neurotransmitters in the
brain, was significantly lower in the vitamin E deficient
rats than that of the controls. This means that
monoamine anabolism in the vit. E deficient rat brain
is impaired and the supplementation with vitamin E
reversed this effect. In addition, PSO may have the
capability to preserve neuronal resistance and probably
to recover the atrophying neurons. [6 1 ]
groups as compared to control. Administration of ROT
to EGb-treated (EGb & ROT) group or to PSO-treated
(PSO & ROT) group showed significant increase in NE
content of striatum after 70 days as compared with
rotenone group. The protective effect of Ginkgo biloba
and pumpkin seed oil may be attributed to their
antioxidant activity and scavenging capability of
various reactive oxygen species.
Effect of EGB and PSO on the Lipid Peroxidation
Level in Striatum and Serum of Adult M ale Albino
Rats Treated with Rotenone: As shown in Table (3),
the results indicated significant increase in lipid
peroxidation level in striatum and serum of ROT group
at all intervals of experiment with a maximum
elevation after 70 days. Increased lipid peroxidation in
the present study is in agreement with Dexter et al.[6 6 ]
and Good et al.[6 7 ] ROS may attack any type of
molecules, but their main target appears to be
polyunsaturated fatty acids, which is the protector
against lipid peroxide formation. [6 8 ] Jenner et al. [6 9 ] have
also shown elevated levels of lipid hydroperoxide, an
earlier component of the lipid peroxidation cascade in
the parkinsonian SN, indicating the damaging impact
on cell membrane structure due to elevated ROS.[1 7 ,7 0 ]
Thus the increased lipid peroxidation products
suggested that ROS have an important role in the
patho genesis of neuro degenerative diseases. [ 7 1 ]
Administration of EGb or PSO to rats treated with
ROT caused a significant increase in lipid peroxidation
level in striatum after 40 days when compared with
control. After 70 days in PSO & ROT group showed
significant increase in serum lipid peroxidation level as
compared to control. At the same time, the change in
lipid peroxidation in both striatum and serum were
significantly decreased at all experimental intervals as
compared with ROT group in EGb & ROT or in PSO
& ROT group. The decrement in lipid peroxidation in
EGb & ROT is due to that Ginkgo biloba is an
antioxidant capable of scavenging various reactive
oxygen species,
including superoxide, peroxyl
Effect of EGB and PSO on Norepinephrine Content
of Striatum, Cerebellum and Cerebral Cortex of
Adult M ale Albino Rats Treated with Rotenone: As
illustrated in Table (2), ROT administration induced
significant decrease in NE content after 70 days only
in the cerebral cortex area of the brain. This decrement
in NE content is mainly due to that rotenone inhibits
TH.[5 1 ] Mitochondrial dysfunction may also responsible
for this effect of ROT on NE content since
mitochondrial disorder has a major role in the
production of cellular ROS. The drawback of
mitochondrial function induced by ROT is due to the
ability of ROT to inhibit complex I activity in the
mitochondrial respiratory chain which was similarly
observed not only in SN pars compacta of PD
patients [6 2 ] but also in their platelets.[6 3 ] This inhibition
can lead to the generation of ROS, which when
produced in the near vicinity and targeted the
respiratory chain, may lead to further inhibition with
subsequent ROS production which ultimately resulted
in mitochondrial damage.[6 4 ] Mitochondrial related
energy failure may also disrupt the vesicular storage of
neurotransmitters, leading to increased free cytosolic
concentrations of the autooxidizable neurotransmitter.[6 5 ]
Administration of EGb or PSO caused non-significant
change in NE level of all examined brain areas at all
time intervals of the experiment. In addition, nonsignificant change in NE level in each of striatum,
cerebellum and cerebral cortex was noticed at all time
intervals in EGb & ROT or in PSO & ROT-treated
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J. Appl. Sci. Res., 5(6): 622-635, 2009
Table 2: Effect of G inkgo biloba extract (EGb) and pum pkin seed oil (PSO) adm inistration on norepinephrine content (ng/g tissue) in striatum ,
cerebellum and cerebral cortex of adult m ale albino rats treated with rotenone (RO T).
Groups
N orepinephrine of striatum
N orepinephrine of cerebellum
N orepinephrine of cerebral cortex
(ng/g tissue)
(ng/g tissue)
(ng/g tissue)
---------------------------------------------------------------------------------------------------------------------------------------------------40 days
55 days
70 days
40 days
55 days
70 days
40 days
55 days
70 days
CO N
2162.2±35.6 2172.1±57.1 2119.6±32.6 736.2±48.5
741.0±34.7 737.2±38.8 793.2±42.9 803.2±28.6 815.0±29.6
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------RO T
2140.7±53.8 2123.8±65.0 1974.9±69.8 710.0±44.5
689.1±43.2 700.8±35.2 737.4±35.3 755.9±44.1 733.8±55.8 a
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EGb
2288.6±61.0 2223.9±75.3 2285.6±69.8 799.4±35.1
840.5±46.1 871.0±42.4 774.3±47.9 865.3±45.1 910.1±46.3
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EGb & RO T 2211.9±71.5 2112.8±71.5 2238.4±65.7 b 757.5±41.1
773.0±42.7 809.9±55.0 770.0±44.4 774.6±46.2 808.4±39.1
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PSO
2205.6±90.9 2214.2±42.7 2230.8±43.6 793.7±30.6
775.9±45.2 809.0±68.7 816.8±33.8 781.5±37.3 771.0±52.5
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------b
PSO & RO T 2238.8±66.2 2289.1±82.7 2175.3±49.8 753.2±54.9
750.3±54.7 704.1±33.1 807.4±39.9 802.8±44.8 767.8±45.7
The num ber of anim als was 8 in each group. D ata are expressed as m ean ± SE., a: Significant change at p > 0.05 w ith respect to control
group )CO N(, b: Significant change at p > 0.05 with respect to RO T group.
Table 3: Effect of G inkgo biloba extract (EGb) and pum pkin seed oil (PSO ) adm inistration on lipid peroxidation level in striatum (nm ol/m g
protein) and serum (nm ol/m l) of adult m ale albino rats treated with rotenone (RO T).
Groups
Lipid peroxidation of striatum (nm ol/m g protein)
Lipid peroxidation of serum (nm ol/m l)
-------------------------------------------------------------------------------------------------------------------------------40 days
55 days
70 days
40 days
55 days
70 days
CO N
6.2±0.3
6.5±0.2
6.2±0.3
21.8±0.6
22.3±0.9
22.3±0.9
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------RO T
8.4±0.5 a
9.1±0.4 a
9.3±0.4 a
26.3±1.2 a
29.8±0.8 a
30.3±1.2 a
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EGb
5.9±0.3
5.8±0.3
5.3±0.3
20.4±1.1
22.8±0.7
22.2±0.5
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ab
b
b
b
b
EGb & RO T
7.4±0.3
6.6±0.3
6.1±0.4
21.1±0.5
22.8±0.7
22.3±0.7 b
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PSO
5.9±0.3
6.5±0.2
6.4±0.3
21.5±0.6
21.0±1.0
20.4±1.1
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PSO & RO T
7.6±0.2 ab
6.9±0.5 b
7.1±0.4 b
22.3±0.8 b
24.9±1.1 b
25.4±0.9 ab
The num ber of anim als was 8 in each group. D ata are expressed as m ean ± SE., a: Significant change at p > 0.05 with respect to control
group )CO N(, b: Significant change at p > 0.05 with respect to RO T group.
radical and hydroxyl radical. [2 5 ] Ginkgo biloba has been
reported to enhance the activities of superoxide
dismutase and catalase activities and to decrease lipid
peroxidation in striatum, substantia nigra, and
hippocampus, the major sites damaged in Parkinson’s
and Alzheimer’s diseases.[2 7 ] It is also likely that the
flavonoid fraction of Ginkgo biloba has an important
role in this respect.[2 5 ,2 6 ] W hile, decreased lipid
peroxidation in PSO & ROT group may be due to
carotinoids and vitamin E in pumpkin seeds that protect
against oxidative damage of membranes, organelles and
protein. [72 ,7 3 ,7 4 ] Tocopherols and selenium present in
pumpkin-seed oil might contribute, by their combined
synergistic action to constitute an important antioxidant
defense mechanism against free radical mediated lipid
peroxidation of cell membrane. [7 3 ]
depolarization and contribute to glutamate excitotoxicity
and further free radical-mediated injury involving nitric
oxide and peroxynitrite. [5 ] Microglial activation is
associated with the degeneration of dopaminergic
(DAergic) neurons in PD patients.[7 8 ] Activation of
microglia in response to injury is associated with an
upregulation of inducible nitric oxide synthase (iNOS)
resulting in increased production of NO. Increased
immunostaining for iNOS has been previously detected
in the SN pars compacta of PD brains.[7 9 ,8 0 ] In addition,
administration of EGb induced significant decline in
striatum N O level after 70 days compared to control.
Administration of EGb and ROT caused non-significant
change in NO level of striatum and serum at all
experimental days as compared to control, however
they produced significant decrease in NO level in both
striatum and serum at all time intervals compared with
ROT group. EGb was already described in 1994 as an
NO scavenger. [2 5 ] Beside its NO-scavenging ability,
EGb has been shown to reduce NO release after
ischemia in the brain [8 1 ] and heart after heat stress [8 2 ,8 3 ]
as well as after lipopolysaccharide-stimulated
macrophages by reducing iNOS mRNA and protein
expression. [8 4 ,8 5 ] The ginkgolides A and B, the active
ingredients of EGb, inhibited NO production of
lipopolysaccharide-stimulated microglia. [2 5 ,8 4 ] W hile,
administration of PSO and ROT induced significant
increase in NO level in striatum at all days of
Effect of EGB and PSO on the Nitric Oxide Level
in Striatum and Serum of Adult M ale Albino Rats
Treated with Rotenone: Table (4) showed significant
increase in nitric oxide level of striatum and serum in
ROT group at all experimental days and the maximum
elevation was occurred in striatum and in serum after
70 days. The generation of NO in striatum and serum
in agreement with the studies carried by Bashkatova et
al.[7 5 ]; Li et al. [7 6 ] and Testa et al. [7 7 ] which
demonstrated that this may be due to decrements in
ATP, where it can lead to mitochondrial membrane
626
J. Appl. Sci. Res., 5(6): 622-635, 2009
experiment as compared to control. After 55 and 70
days, a significant increase in NO level was detected
in PSO & ROT group as compared to control. In
comparison with ROT group at all time intervals,
significant decrease in serum NO level was
demonstrated. The protective effect of PSO may be due
to the presence of á-tocopherol and selenium in this oil
which have been shown to scavenge free radicals
including peroxynitrate radicals. [7 3 ]
Effect of EGB and PSO on the Total Antioxidant
Capacity in Striatum and Serum of Adult M ale
Albino Rats Treated with Rotenone: As illustrated in
Table (6) that ROT induced significant reduction in
total antioxidant capacity (TAC) of striatum and serum
at all experimental days. Oral administration of Ginkgo
biloba extract or pumpkin seed oil for 70 consecutive
days showed significant increase in TAC of striatum
after 55 and 70 days regarding to EGb and after 70
days with respect to PSO as compared to control. In
addition, administration of rotenone to rats treated with
EGb or PSO showed non- significant change in TAC
in striatum of EGb & ROT and PSO & ROT groups
and in serum of EGb & ROT group at all experimental
days as compared to control. Significant decrease in
TAC in serum of PSO & ROT group was observed
after 55 and 70 days as compared to control. TAC in
striatum showed significant increase after 55 and 70
days in EGb & ROT group and after 55 days in PSO
& ROT group as compared to ROT. Serum TAC in
EGb & ROT or PSO & ROT group as compared to
ROT recorded significant increase at all corresponding
time intervals. These data supported by the pervious
studies that showed the powerful antioxidant activity of
EGb [32 ,3 3 ] and PSO. [91 ,9 2 ].
Effect of EGB and PSO on the Reduced Glutathione
Level in Striatum and Serum of Adult M ale Albino
Rats Treated with Rotenone: It is clear from the data
in Table (5) that ROT administration caused significant
decrease in striatum glutathione (GSH) level at all
experimental days and in serum glutathione level after
55 and 70 days. Oxidative stress has been widely
implicated to play a key role in rotenone-induced
dopaminergic neuron injuries.[8 0 ] GSH is a well-known
endogenous antioxidant. From the present results and
previous studies, it was shown that rotenone reduced
GSH levels.[8 6 ,8 7 ] GSH normally acts through a
combination of various reduction and conjugation
reactions to protect cells against both exogenous
toxicants and the reaction of endogenous compounds.[8 8 ]
One possible explanation for the decreased level of
GSH is the defective synthesis, utilization, and
degradation of GSH as well as changes in the activity
of glutathione peroxidase and glutathione reductase that
could also lead to its reduction. [7 1 ] EGb or PSO
administration showed significant elevation in striatum
and serum glutathione level at all time intervals as
compared to control. In addition, compared to control,
administration of EGb & ROT or PSO & ROT showed
non-significant change in GSH level in striatum after
40 and 55 days however after 70 days, significant
decrease in GSH of striatum was observed. W hile
serum GSH showed non-significant change at all
experimental days in the last two groups as compared
to control. Significant increase in striatum and serum
glutathione levels in EGb & ROT group at all
experimental days as compared with ROT group.
Moreover, significant increase in GSH level of striatum
at all time intervals and after 70 days only in serum
was noticed in PSO & ROT group as compared with
ROT group in both groups. The protective effect of
Ginkgo biloba extract has been shown to be due to
increase in the protein level and activity of antioxidant
enzymes such as superoxide dismutase and catalase as
well as of glutathione (GSH) reductase in mouse
liver. [8 9 ] Similarly, the activity of ã-glutamylcysteinyl
synthetase, the rate limiting enzyme of GSH synthesis,
was enhanced by EGb. [2 5 ,9 0 ] The protective effect of
PSO may be due to the tocopherols and selenium
contents.[73 ].
Effect of EGB and PSO on the Na + / K + -atpase
Activity of Striatum of Adult M ale Albino Rats
Treated with Rotenone: According to the data in
Table (7), significant decrease in brain Na + /K + -ATPase
activity of ROT group at all experimental days.
Rotenone as a well known neurotoxin has been shown
to reduce Na + /K + -ATPase activity and level which may
be due to complex I inhibition of the mitochondrial
electron transport chain, which in turn reduces the
ATP-producing capacity of cells. [9 3 ,9 4 ] Significant
increase in striatum Na + /K + -ATPase activity of EGb or
PSO group was occurred at all experimental days as
compared to control. Also, striatum Na + /K + -ATPase
activity in EGb & ROT group was significant by
increased at all experimental days, while in PSO &
ROT, striatum N a + /K + -AT Pase activity showed
significant increase at the beginning (40 days), then
returned to the normal value after 55 days and finally
it decreased significantly at the end (70 days) as
compared to control. In comparison with ROT group,
EGb & ROT or PSO & ROT showed significant
elevation in striatum Na + /K + -ATPase activity at all
experimental days. The protective effect of the present
medicinal plants could be due to the antioxidant
properties of Ginkgo biloba extract[32 ,3 3 ] and pumpkin
seed oil, [91 ,9 2 ] since Nicolson [95 ]. demonstrated that
administeration of antioxidants can prevent excess
oxidative membrane damage, restore mitochondrial and
other cellular membrane functions.
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J. Appl. Sci. Res., 5(6): 622-635, 2009
Table 4: Effect of G inkgo biloba extract (EGb) and pum pkin seed oil (PSO ) adm inistration on nitric oxide level in striatum (ìm ol/m g protein)
and serum (ìm ol/l) of adult m ale albino rats treated with rotenone (RO T).
Groups
N itric oxide of striatum (ìm ol/m g protein)
N itric oxide of serum (ìm ol/l)
------------------------------------------------------------------------------------------------------------------------------40 days
55 days
70 days
40 days
55 days
70 days
CO N
33.4±3.0
34.9±2.8
36.9±3.6
53.4±0.4
52.3±0.3
52.9±0.9
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------RO T
40.7±2.2 a
41.2±1.9 a
44.1±1.7 a
58.6±1.2 a
64.1±1.2 a
69.5±1.9 a
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EGb
28.26±2.5
31.88±2.4
30.89±1.1 a
52.8±1.3
50.6±1.9
51.4±1.4
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EGb & RO T
34.76±1.5 b
34.34±2.6 b
38.31±2.3 b
52.6±2.1 b
54.3±1.7 b
54.9±1.4 b
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PSO
31.43±1.1
32.91±1.1
34.1±1.3
53.7±1.7
53.4±1.1
52.9±1.2
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PSO & RO T
38.23±1.7 a
37.9±1.1 a
42.3±1.8 a
54.3±1.9 b
58.6±1.4 ab
60.1±1.4 ab
The num ber of anim als was 8 in each group. D ata are expressed as mean ± SE., a: Significant change at p > 0.05 with respect to control
group )CO N(, b: Significant change at p > 0.05 with respect to RO T group.
Table 5: Effect of G inkgo biloba extract (EGb) and pum pkin seed oil (PSO ) adm inistration on reduced glutathione level in striatum (ìm ol/m g
protein) and serum (ìm ol/m l) of adult m ale albino rats treated with rotenone (RO T).
Groups
Reduced glutathione of striatum (ìm ol/m g protein)
Reduced glutathione of serum (ìm ol/m l)
----------------------------------------------------------------------------------------------------------------------------------40 days
55 days
70 days
40 days
55 days
70 days
CO N
1.16±0.05
1.16±0.06
1.15±0.05
0.37±0.013
0.40±0.006
0.38±0.011
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------a
a
a
a
RO T
1.08±0.04
0.98±0.06
0.88±0.07
0.36±0.011
0.30±0.010
0.27±0.006 a
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EGb
1.22±0.01 a
1.38±0.02 a
1.33±0.04 a
0.44±0.017 a
0.48±0.025 a
0.50±0.031 a
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EGb & RO T
1.14±0.05 b
1.14±0.06 b
1.05±0.06 ab
0.41±0.020 b
0.45±0.028 b
0.42±0.024 b
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PSO
1.22±0.03 a
1.29±0.04 a
1.30±0.03 a
0.42±0.010 a
0.47±0.030 a
0.45±0.029 a
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PSO & RO T
1.16±0.05 b
1.13±0.04 b
0.97±0.06 ab
0.39±0.010
0.38±0.045
0.33±0.016 b
The num ber of anim als was 8 in each group. D ata are expressed as m ean ± SE., a: Significant change at p > 0.05 with respect to control
group )CO N(, b: Significant change at p > 0.05 with respect to RO T group.
Table 6: Effect of G inkgo biloba extract (EG b) and pum pkin seed oil (PSO ) adm inistration on total antioxidant capacity in striatum (m M /m g
protein) and serum (m M /l) of adult m ale albino rats treated with rotenone (RO T).
Groups
Total antioxidant capacity of striatum (m M /m g protein)
Total antioxidant capacity of serum (m M /l)
------------------------------------------------------------------------------------------------------------------------------------40 days
55 days
70 days
40 days
55 days
70 days
CO N
9.97±0.43
9.66±0.78
9.61±0.62
0.71±0.026
0.71±0.021
0.73±0.020
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------RO T
8.27±0.34 a
7.09±0.50 a
5.93±0.53 a
0.52±0.036 a
0.48±0.060 a
0.44±0.020 a
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EGb
11.83±1.02
11.62±0.69 a
12.03±0.42 a
0.73±0.026
0.75±0.027
0.76±0.020
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------b
b
b
b
EGb & RO T
8.59±0.57
9.88±0.43
10.71±0.44
0.68±0.027
0.66±0.026
0.68±0.014 b
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PSO
10.23±0.82
11.19±0.79
12.79±1.25 a
0.71±0.024
0.74±0.025
0.78±0.035
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PSO & RO T
9.59±0.32
9.57±0.75 b
8.37±0.83
0.67±0.055 b
0.59±0.025 ab
0.59±0.035 ab
The num ber of anim als was 8 in each group. D ata are expressed as m ean ± SE., a: Significant change at p > 0.05 with respect to control
group )CO N(, b: Significant change at p > 0.05 with respect to RO T group.
testosterone as well as ATP levels in the peripheral
organs, such as the adrenal glands and testis. The
depletion of energy levels and excess oxidative stress
due to complex I inhibition could be responsible for
decreasing testosterone level in the current study.
Significant elevation of serum testosterone level was
detected in EGb group after 55 and 70 days and at all
experimental days in PSO group as compared to
control. Similarly, administration of rotenone to rats
Effect of EGB and PSO on Testosterone Level in
Serum of Adult M ale Albino Rats Treated with
Rotenone: The data in Table (7) showed that, oral
administration of rotenone to rats produced significant
decrease in testosterone level after 55 and 70 days. Our
findings are in accordance with those of Alam and
Schmidt, [9 6 ] who hypothesized that Complex I
inhibitors, which are responsible for reproducing
symptoms of PD in rats, also deplete serum
628
J. Appl. Sci. Res., 5(6): 622-635, 2009
testosterone synthesis in Leydig cells.[9 7 ] Regarding the
increased testosterone level due to PSO administeration
could be explained by the presence of fatty acids
which able to inhibit 5á reductase that convert
testosterone into dihydrotestosterone and consequently
leading to increase testosterone level. [9 2 ] Also, PSO
contains zinc in appreciable level. [9 8 ] Zinc may play a
role in regulating testosterone secretion and its
supplementation increased serum testosterone levels.
Zinc enhances human chorionic gonadotropin-induced
production of cAMP and consequently testosterone in
rat testis. Additionally, zinc increases the conversion of
androstenedione to testosterone in the periphery. [1 0 2 ]
treated with EGb (EGb & ROT) group revealed
significant increase in testosterone after 70 days, while
it significantally increased in all experimental days in
PSO & ROT group as compared to control. Also,
significant increase was found in testosterone level as
compared to ROT group after 55 and 70 days in EGb
& ROT group and at all corresponding time intervals
in PSO & ROT group. The elevated testosterone level
in the present study may be due to quercetin (an active
ingredient of EGb) which could increase testosterone
production through increasing intracellular cAMP levels
and steroidogenic protein expression in testis, which is
responsible for facilitating
Table 7: Effect of G inkgo biloba extract (EGb) and pum pkin seed oil (PSO ) adm inistration on N a + / K + -ATPase activity (ìm ol pi/h/m g protein)
of striatum and testosterone level (ng/m l) in serum of adult m ale albino rats treated with rotenone (RO T).
Groups
N a + / K + -ATPase activity of striatum (ìm ol pi/h/m g protein)
Testosterone level of serum (ng/m l)
----------------------------------------------------------------------------------------------------------------------------------------40 days
55 days
70 days
40 days
55 days
70 days
CO N
6.4±0.6
6.72±0.5
7.2±0.4
1.40±0.01
1.38±0.01
1.42±0.01
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------RO T
5.3±0.2 a
5.3±0.3 a
4.7±0.3 a
1.31±0.01
1.19±0.05 a
0.97±0.06 a
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EGb
10.4±0.2 a
12.6±0.4 a
14.4±0.3 a
1.55±0.04
1.76±0.08 a
3.97±0.07 a
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------EGb & RO T
9.1±0.4 ab
9.9±0.8 ab
9.0±0.5 ab
1.40±0.05
1.50±0.06 b
3.80±0.06 ab
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PSO
7.0±0.3 a
7.2±0.6 a
8.0±0.4 a
3.63±0.08 a
3.98±0.09 a
4.19±0.07 a
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------PSO & RO T
7.0±0.4 ab
6.4±0.6 b
6.4±0.3 ab
1.65±0.05 ab
2.25±0.05 ab
2.37±0.06 ab
The num ber of anim als was 8 in each group. D ata are expressed as m ean ± SE., a: Significant change at p < 0.05 with respect to control
group (CO N), b: Significant change at p < 0.05 with respect to RO T group
The neurons of rotenone treated animals showed
extensive neuronal damage by virtue of loss of
pigmented neurons and the cells appear to be smaller
and shrunken as compared to the control cells,
indicating that apoptosis has occurred (Fig 2a). In
addition, the cells are small in number after 35 days of
rotenone treatment (Figure 2b). Figure (2c; after 50
days of rotenone treatment) shows signs of damage as
evident by disappearance of cells and the presence of
cytoplasmic inclusions of lewy bodies. Moreover,
structural alterations of the surviving neuronal cells can
be also observed. The present histopathological studies
showed that rotenone caused degeneration and
apoptosis of the striatum characterized by the presence
of lewy body. Previous studies indicated that oxidative
stress may interact with other pathophysiologic
mechanisms, including genetic lesions, to create PD
pathology.[9 9 ] For example, oxidative stress may
contribute to á-synuclein aggregation seen in PD and
m itochondrial dysfunction models. á-Synuclein
damaged by free radicals is more prone to
aggregation. [1 0 0 ] In vivo rotenone infusion generates both
increased insoluble protein carbonyls and increased ásynuclein aggregation. [9 ,1 0 1 ] Oxidative modification of ásynuclein
in PD brain can lead to protein aggregation.
[1 0 2 ]
Histological Investigation in Striatum of Different
Treated Groups: Microscopic examination of striatum
section of control rat showed the neuron with the
surrounding supporting cells with normal nuclei which
showed dispersed chromatin and prominent nucleoli.
The cytoplasm of these cells was basophilic (Figure 1).
Fig. 1: M icrographs of striatum of control rat showing
the normal neurons after 40 days (a), 55 days
(b) and 70 days (c) (H & E X 40).
629
J. Appl. Sci. Res., 5(6): 622-635, 2009
Fig. 2: M icrograph of striatum of rat treated with
rotenone (a) for 20 days, (b) for 35 and (c) for
50. Lewy bodies (LB), apoptic (Ap) neurons (H
& E X 40).
Fig. 3: M icrograph of striatum of rat treated with
Ginkgo biloba extract for 40 days (a), 55 days
(b) and 70 days (c) (H & E X 40).
Microscopic investigation of striatum sections of
rats-treated with Ginkgo biloba extract showed normal
structure of neurons and the surrounding cells like
control at all days of investigation (Figure 3).
Examination of striatum sections of rat-treated with
Ginkgo biloba extract and rotenone showed the neurons
more or less like normal (Figure 4a; after 40 days).
Slight degeneration was found after 55 days (Figure
4b). Loss of the number of neurons can be seen after
70 days (Figure 4c). This mean that the neurons of
striatum sections of rats-treated with Ginkgo biloba
extract showed significant protection against rotenone
neurotoxicity.
Microscopic investigation of striatum sections of
rats-treated with pumpkin seed oil showed healthy
neurons as well as the surrounding cells at all days of
investigation (Figure 5).
Examination of straitum sections of rats-treated
with pumpkin seed oil and rotenone showed the
neurons more or less like normal (Figure 6a; after 40
days), while some apoptotic neurons were seen after 55
days (Figure 6b).
More over degenerative neurons and more
apoptotic neurons were noticed after 70 days (Figure
6c). These findings indicated that the treatment with
PSO resulted in some protection against rotenoneinduced neurodegeneration. Generally, the histological
results revealed that the treatment with EGb or PSO
protects the brain against rotenone-induced neurological
apoptosis, where Ginkgo biloba extract or pumpkin
seed oil are heralded as a antioxidant agents.
Noteworthy, EGb appeared to be more neuroprotective
agent than PSO.
It can be concluded that Ginkgo biloba extract has
a potential activity against rotenone- induced
neurodegeneration. Therefore, it may be useful against
neurotoxicity induced by environmental neurotoxins.
Pumpkin seed oil has an effective role in limiting
toxicant-induced oxidative stress.
Fig 4: M icrograph of striatum of rat treated with
Ginkgo biloba extract (a) for 40 days and
rotenone for 20 days, (b) for 55 days from
treatment with Ginkgo biloba extract and for 35
days from rotenone treatment and (c) for 70
days from treatment with Ginkgo biloba extract
and for 50 days from rotenone treatment (H &
E X 40).
Fig 5: M icrograph of striatum of rat treated with
pumpkin seed oil for 40 days (a), 55 days (b)
and 70 days (c) (H & E X 40).
630
J. Appl. Sci. Res., 5(6): 622-635, 2009
8.
9.
10.
11.
Fig 6: M icrograph of striatum of rat treated with
pumpkin seed oil (a) for 40 days and rotenone
for 20 days, (b) for 55 days from treatment
with pumpkin seed oil and for 35 days from
rotenone treatment and (c) for 70 days from
treatment with pumpkin seed oil and for 50
days from rotenone treatment (H & E X 40).
12.
ACKNOW LEDGM ENT
This research was supported by Dr. Abdel Razik
H ussein F arrag, Researcher o f H isto lo gy &
Histochemistry, Department of Pathology, Medical
R esearch D ivision, National Research Centre.
Therefore, the authors of the current study expressed
sincere appreciation to him for his kind cooperation in
conducting histopathological investigations in this
study.
13.
14.
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