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Research Article
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Cerebroprotective Effect of Date Seed Extract (Phoenix dactylifera) on Focal Cerebral Ischemia in Male Rats
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T.P. Kalantaripour,
M. Asadi-Shekaari,
M. Basiri
and
A. Gholaamhosseinian Najar
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ABSTRACT
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In the present study, we investigate the role of Date Seed Extract (DSE) in protection against cerebral ischemic damages. Focal cerebral ischemia was induced by using Middle Cerebral Artery Occlusion (MCAO) method in male rats. DSE was administrated at the dose of 80 mg kg-1 i.p. 30 min after MCAO. Superoxide Dismutase (SOD), Malondialdehyde (MDA) and Total antioxidant Activity (TAS) were measured. Morphological studies and behavioral activity were also done. MCAO significantly decreased SOD and TAS activities. MDA content significantly was elevated by MCAO. Motor coordination was also decreased due to transient ischemia. Treatment with DSE attenuated all of alterations and neuronal damage induced by MCAO in male rats. The data showed that treatment with DSE could protect cortical neurons against cerebral-induced injuries most probably due to its antioxidant properties.
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Received:
December 08, 2011; Accepted: March 12, 2012;
Published: June 12, 2012 |
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INTRODUCTION
The date palm (Phoenix dactylifera) is a palm in the genus Phoenix,
widely cultivated for edible fruit. Date seed constitutes approximately 15%
of date fruit (Hussein et al., 1998). At the
present time, date seeds are used mostly for animal feed, while most regarded
as waste. Use of such waste is very important to date agriculture and to increase
the income to this part. Date seed (pit) contains relatively high amounts of
fat (9.0 g/100 g) and protein (5.1 g/100 g) compared to date fruit. They are
a very rich source of antioxidants (80400 μmol 100 g), phenolics (3942
mg/ 100 g) and dietary fiber (73.1 g/100 g). The seed could potentially be considered
as an economical source of natural antioxidants and dietary fiber (Al-Farsi
and Lee, 2008).
Iranian date seeds are potent antioxidants and strong free radical scavengers
(Ardekani et al., 2010). They contain several
antioxidant components including phenolic compounds (Phenolic acids, anthocyanins
and flavonoids), selenium (coenzyme of GPx), vitamin C, Oleic acid and carotenoids
(Al-Farsi and Lee, 2008; Al-Qarawi
et al., 2004). A recent study showed that the antioxidants of DSE
are approximately 27 fold of date fruit (Al-Farsi and Lee,
2008). It supports its noticeable antioxidant activity.
Stroke is a leading cause of death in developed countries. Current therapeutic
strategies for stroke have been largely unsuccessful (Caplan,
2004). Many studies have shown the neuroprotective effects of herbal extracts
on different animal models (Waggas, 2009; Tehranipour
and Javaheri, 2009; Tehranipour and Ghadamyari, 2010).
Therefore, there is a great interest in prevention and treatment of the disease.
In the present study, we investigate the cerebroprotective effect of DSE in
male rats.
MATERIALS AND METHODS Animals: Healthy NMRI male rats (weighing 225-280 g) were used in the study. This study was performed in accordance with neuroscience research center guidelines for care and use of laboratory animals (EC/KNRC/87-29). All of efforts were taken to minimize distress, discomfort, and pain to the animals.
Date seed extract preparation: Date fruits (Bamy Mozafaty rutab) were
purchased from authenticated market. Date seeds were isolated from date fruit
and were soaked in distilled water, washed to get rid of any adhering flesh
and dried at room temperature. The Seeds were milled in a heavy-duty grinder.
The fine powder (50 g) was extracted with distilled water (1: 3 ratio, w/v)
and centrifuged at 4°C for 20 min at 4000 g and the supernatant was collected,
lyophilized and stored at -20°C until use.
Experimental design: Rats were randomly divided into 3 groups: The ischemic group suffered 30 min MCAO followed by 48 h reperfusion (MCAO) (n = 9), the sham group suffered the same as ischemic group without MCAO (control) (n = 10) and the experimental group suffered 30 min MCAO treated with the dose of DSE 80 mg kg-1 i.p. 30 min after MCAO induction and 48 h reperfusion (DSE) (n = 10). The dose of DSE was selected based on a pilot study.
Middle cerebral artery occlusion: The right middle cerebral artery occlusion
was induced using intraluminal filament model (Longa et
al., 1989). In brief, the animals were anesthetized with chloral hydrate
(360 mg kg-1). In supine position, a midline ventral incision was
made the right Common Carotid Artery (CCA) was exposed and separated carefully
from Vagus nerve. All of External Carotid Artery (ECA) branches and extra cranial
of Internal Carotid Artery (ICA) were blocked. Then 4-0 nylon suture was introduced
into ICA and advancing in intracranially to block blood flow into MCA. After
thirty min ischemia, suture was withdrawn to restore the blood flow (reperfusion).
The rectal temperature was maintained at 37±0.5°C with a thermistor
coupled to a heating blanket during surgery. After recovery from the anesthesia,
the animals were returned to their cages. After full recovery, neurological
examination (Garcia et al., 1995) was performed
to ensure MCAO occurred and the animals without clinical signs were excluded
from the experiment.
Sample preparation method
Light microscopy: Under deep anesthesia, the animals were perfused intracardially
with 10% formalin in phosphate buffered saline (pH 7.4, M 0.1%). Brains were
removed and immersed in the same fixative overnight at 4°C. After, the brains
were processed for light microscopy studies according to routine procedures.
Coronal sections 1.6-2.8 mm posterior to bregma were cut at a thickness of 5
μm using a microtome. Neuronal damage in the cortex was assessed by staining
sections with hematoxylin and eosin using the standard method (Sakurai-Yamashita
et al., 2006).
Electron microscopy: Rats were killed by perfusion transcardially with
4% paraformaldehyde in 0.1 M phosphate buffer solution (pH 7.4). The brains
were removed and immersed in buffered 2.5% glutaraldehyde overnight. A small
piece of cortical area was dissected and fixed in the same fixative for an additional
24 h. Specimens were postfixed in 1% (w/v) OsO 4/1% (w/v) of phosphate
buffer. After dehydration in ethanol, slices were embedded in Epon 812 resin.
A section (300 nm) was stained with toluidine blue to locate areas of interest.
Subsequently, 70-80 nm sections were cut and stained with uranyl acetate and
lead citrate stain. The sections were examined with a Philips (EM 300, Eindhoven,
Netherlands) transmission electron microscope (Zangiabadi
et al., 2011a).
Biochemical analysis: Forty-eight h after reperfusion onset, the rats
were sacrificed and brains removed immediately. Homogenization was performed
according to Aydin et al. (2002) with some modification.
Briefly, piece of the cortical tissue was removed and washed with saline and
homogenized by sonication (4 °C) in 0.15 M NaCl for lipid peroxidation and
antioxidant parameters. Homogenate was centrifuged at 5000 rpm for 15 min. aliquots
for each sample were poured in small Eppendorf tubes and kept frozen at -70°C
until use. Malondialdehyde (MDA) levels were determined spectrophotometrically
by modified method of Uchiyama and Mihara (1997). Superoxide
Dismutase (SOD) activity and Total antioxidants level (TAS) were measured by
spectrophotometric methods using Randox assay kits (Randox Laboratories, Antrim,
UK) based on the manufacturer protocols. In order to compensate the effect of
size and amount of used sample in the homogenization step, the protein content
of each preparation was measured by Folin phenol reagent of Lowry
et al. (1951).
Rotarod activity: The animals were evaluated for balance and grip strength
using the rotarod. Each animal was given a former training session before beginning
of therapy to adapt them on a rotarod apparatus (Technical and Scientific Equipment,
GmbH, Germany). Rats were placed on the rotating rod with a diameter of 7 cm
(speed 20 rpm). Three trials were given to each animal at 10 min interval and
cut off time (300 sec) was maintained throughout the trial. The average results
were recorded as fall of time (Gaur and Kumar, 2010a).
Statistical analysis: The statistical analysis was done using SPSS software
v13.5. All values are expressed as Mean±SEM.
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Fig. 1: |
Cerebroprotective effect of treatment with DSE on ischemic-reperfusion
injury in male rat, Neuronal damage in the cerebral cortex 48 h after reperfusion
was assessed using hematoxylin and eosin (H and E), Results are expressed
as Mean±SE and data were analyzed by one-way ANOVA |
Differences in measured parameters among different groups were analyzed by
one-way ANOVA followed by post hoc Tukeys test. A statistical difference
was determined by a value of p<0.05.
RESULTS Effects of DSE on neuronal damage: According to the obtained data_ 30 min MCAO followed by 48 h reperfusion induced 89.37% neuronal death in MCAO group. Treatment with DSE (80 mg kg-1) significantly decreased the neuronal damage (30.33%) (Fig. 1). Electron microscopy: Cortical neurons were examined by transmission electron microscope 48 h after 30 min MCAO. Morphology of neurons in control group was normal. Degenerative changes including darkening of nucleus, chromatin aggregation, organelles swelling were observed in MCAO group. The ultrastructure of most cortical neurons was preserved in DSE group (Fig. 2). Effect of DSE on fall-off time in rotarod after MCAO in rats: The fall-off time is measured for rotarod evaluation to measure motor in coordination. A significant decrease observed in fall-off time in MCAO group as compared to control group which show motor in coordination and muscle weakness. DSE significantly (p<0.05) improved the fall-off latency time as compared to MCAO group (Fig. 3).
Effect of DSE on oxidative stress in brain after MCAO in rats: MDA as
an oxidative marker was significantly increased after ischemia reperfusion damage
in MCAO group as compared to control group.
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Fig. 2 (a-c): |
Electron micrograph of cortical neurons from control, MCAO
and DSE groups, (a) The normal ultrastructure is visible in intact neuron,
(b) Ischemia reperfusion results in severe degenerative changes in cortical
neuron. (c) Cortical neurons in DSE group have some degenerative changes
like extracellular edema, chromatin aggregation but the whole ultrastructure
was maintained (part c), Magnification in (a) and (b) is 8900x and in (c)
is 3900 |
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Fig. 3: |
Effect of DSE on fall-off time from rotarod in ischemic-reperfusion
injury in male rat, ***p<0.001 as compared to control group, #p<0.05
as compared to MCAO |
Table 1: |
*Levels of MDA, SOD and TAS in control, MCAO and MCAO + DSE
groups in rats |
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*p<0.05 as compared to MCAO, *ap<0.001 as
compared to MCAO, ***p<0.001 as compared to control, MDA: Malondialdehyde,
SOD: Superoxide dismutase, TAS: Total Antioxidant Status, MCAO: Middle cerebral
artery occlusion |
DSE administration (80 mg kg-1) significantly decreased MDA level
after ischemia reperfusion as compared to MCAO group (p<0.05).
Effect of DSE on SOD activity and total antioxidants level in brain after MCAO in rats: Antioxidant enzyme activity (SOD) and total antioxidants level were attenuated after ischemia reperfusion damage in MCAO group. A significant increase in SOD activity and TAS level was observed in DSE treated group (p<0.05) (Table 1). DISCUSSION
Oxidative stress is involved in the pathophysiology of stroke. A large number
of studies have shown that oxidative stress contributes to brain injury due
to ischemia reperfusion (Ikeda and Langn, 1990; Yavuz
et al., 1997; Cuzzocrea et al., 2001;
Guizzo et al., 2005; Oboh,
2009). Ischemia reperfusion causes a significant increase in oxidative stress
markers such as: nitrite, MDA, and reactive oxygen species concentration. In
addition, there is a considerable decrease observed in antioxidant enzymes such
as catalase and SOD activity, in the brain (Levine, 2004;
Siesjo, 2008; Gaur and Kumar, 2011;
Nandagopal et al., 2011). In the present study
we have evaluated oxidative stress parameters (MDA, SOD and TAS) in the brain.
However, a marked increase in MDA concentration was observed in MCAO group.
A decrease in SOD as well as TAS activity was also seen 48 h after reperfusion
indicating oxidative stress caused by ischemic-reperfusion damage. DSE significantly
attenuated the oxidative stress and restored the antioxidant enzyme activity.
Motor disorders are one of the most destructive outcomes of cerebral ischemia
due to MCAO because most of the pyramidal tract and motor cortex lie inside
territory supplied by MCA. Motor disorder may arise from failure of cortical
excitability and/or inhibition of electrical impulses at the subcortical area.
After ischemia and reperfusion, axonal conduction readily recovers. However,
a constant failure at cortical synapses leads to motor dysfunction (Bolay
and Dalkara, 1998). Several studies have shown that the neurological and
locomotor deficits after ischemia in rats (Gaur and Kumar,
2010b; Aggarwal et al., 2010; Gupta
et al., 2005). The fall-off time from rotarod was significantly decreased
when compared to control group verifying the deficit in muscle co-ordination
and grip strength. Decrease in muscle co-ordination and grip strength has also
been shown by various studies in MCAO (Jafari et al.,
2011; Maheshwari et al., 2011) as well as
the BCCAO (bilateral common carotid artery occlusion) model of cerebral ischemia
(Gaur et al., 2009; Gaur
and Kumar, 2010c). Treatment with DSE attenuated muscle weakness. This supports
its protective effect against ischemia reperfusion damage.
On the other hand, histological and ultrastructural findings showed that DSE
could protect cortical neurons against ischemia reperfusion induced insults.
According to our data, 30 min MCAO followed by 48 h reperfusion results in severe
damage to neurons (89.37%) in MCAO group. This finding is in agreement with
other studies (Asadi-Shekaari et al., 2010; Asadi-Shekaari
et al., 2008). DSE treatment can protect them against ischemic injury.
In line with above findings, electron microscopy examination showed degenerative
changes in cortical neurons as shown in Fig. 3 after ischemia
reperfusion and DSE had protective effects against these changes.
Previously we demonstrated that aqueous extract of date fruit has neuroprotective
action against cerebral ischemic injuries and diabetic neuropathy most probably
due to its antioxidant effects (Asadi-Shekaari et al.,
2008; Panahi et al., 2008; Zangiabadi
et al., 2011b). Here we showed the DSE significantly improved the
outcome in male rats after ischemia reperfusion. At the present time, the exact
mechanism by which the DSE induces its cerebroprotective activity against ischemia
reperfusion injury is not known. But it is possible that its antioxidant components
present in the DSE are responsible for this protection.
In conclusion, the present study demonstrated that DSE has a cerebroprotective role for the period of brain ischemia followed by reperfusion in male rats. This implies that use of DSE may have beneficial effects in cerebral ischemia. Further studies are needed to be able to propose the potential therapeutic use of DSE in preventing the brain from ischemic-induced oxidative damage. ACKNOWLEDGMENT This study was supported by the research grant from Kerman Neuroscience Research Center (KNRC).
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