To Access the Efficacy of Rutin on 6-Hydroxydopamine induced Animal
Model of Memory Impairment in Parkinson’s Disease.
V.P. Kahale, P.R. Upadhay*, A.J. Mhaiskar, P.S. Shelat, D. R. Mundhada.
Department
of Pharmacology, Agnihotri College of Pharmacy, Wardha.
ABSTRACT:
The core finding of the present study is
that administration of relevant doses of Ruin is remarkably neuroprotective in
rats against 6-Hydroxydopamine induced neurotoxicity. Parkinson's disease (PD)
is a chronic and progressive neurodegenerative disease with multiple motor and
non-motor features that contribute to the impairment of health-related quality
of life (QOL) .It is characterized by reduced movement, rigidity, and tremor.
It is characterized by a preferential loss of the dopaminergic neurons of the
substantia nigra pars compacta. Rutin (3,3′,4′,5,7-pentahydroxyflavone-3-rhamnoglucoside)
was a citrus flavonoid glycoside. Flavonoids are polyphenolic compounds that
occur ubiquitously in foods of plant origin. It act as a antioxidant and can prevent injury caused by reactive oxygen
species (ROS) in various ways. Rutin was found to be a neuroprotective agent.
Rutin was identified as the major LDL antioxidant compound of mulberry in an in
vitro study. Rutin acts as a memory enhancer and an anti-oxidant Rutin
treatment protects behavioral changes, and significantly attenuated oxidative
damage and improved mitochondrial complexes enzyme activities in different
regions (striatum, cortex and hippocampus) of rat brain against 6-OHDA induced
neurotoxicity. I.C.V. administration of 6-Hydroxydopamine is known to produce
hypoactivity that resembles juvenile onset and advanced Parkinson's disease in
rats. The results show that Rutin treatment is effective in various behavioural
models, thus it could be used as an effective therapeutic agent in the management
of Parkinson's disease and related conditions. We attempted to investigate the
neuroprotective effect of Rutin in animal model of Parkinson's disease, and
thus it shows the effect of Rutin on 6-hydroxydopamine onduced memory impairment
in Parkinson’s disease in Rodents.
KEYWORDS:
6- OHDA, I.C.V., Parkinson Disease, Memory impairment, Rutin.
1. INTRODUCTION:
1.1 Parkinson Disease:
Parkinson's
disease (PD) is a chronic and progressive neurodegenerative disease with
multiple motor and non-motor features that contribute to the impairment of
health-related quality of life (QOL) (1). It is characterized by reduced
movement, rigidity, and tremor (2). It is
characterized by a preferential loss of the dopaminergic neurons of the substantia nigra pars compacta.
Parkinson's
disease (PD) was first associated with the loss of the brown pigment
neuromelanin from the substantia nigra. Later, it was postulated that the
progressive loss of dopamine-producing cells in the substantia nigra pars compacta
of the ventral midbrain caused PD symptomatology. In addition, PD is also
associated with the presence of intra-cytoplasmatic inclusions known as Lewy
Bodies (LBs), which are composed largely of alpha-synuclein (alpha-syn) (3).
In pathogenesis of Parkinson's disease
includes abnormilities in cellular protein transport, interaction between
proteins & protein aggregation. The neurochemistry have shown involvement
of excitotoxicity & oxidative stess in cell death. Parkinson's Disease is
pathologically characterized by loss of chatecholaminergic neurons in brain
stem (4). There was a tight correlation between cognitive impairment in PD and
cholinergic deficit (5).
A) Genetics:
Approximately
15 percent of people with Parkinson disease have a family history of this
disorder. Familial cases of Parkinson disease can be caused by mutations in the LRRK2, PARK2, PARK7, PINK1, or SNCA gene, or by alterations in genes that
have not been identified.
B) Inheritance:
Among
familial cases of Parkinson disease, the inheritance pattern differs depending
on the gene that is altered. If the LRRK2 or SNCA gene is involved, the disorder is
inherited in an autosomal dominant pattern, which means one copy of an altered
gene in each cell is sufficient to cause the disorder.
2. DRUG PROFILE:
2.1 INDUCER DRUG:
A)
6-Hydroxydopamine:
6-hydroxydopamine
neurotoxicity is initiated via extracellular auto-oxidation and the induction
of oxidative stress from the oxidative products generated. 6- OHDA shares some
structural similarities with dopamine and norepinephrine, exhibiting a high
affinity for several catecholamininergic plasma membrane transporter such as
the dopamine (6).
B) Stereotaxic Surgery:
Anesthetised the
rat with 10 mg/kg ketamine + 3 mg/kg zylaxine (i.p.) each animal was mouted on
stereotaxic apparatus (5) with the nose oriented 11º below the horizontal plane
(7). A 2 cm midsaggital skin incision is made on scalp & skin overlying the
skull was cut to expose the skull & coordinates for substantia nigra (SNPC)
was measured accurately (Anteroposterior-0.5mm from bregma, mediolateral-2.1mm
from midline, and dorsoventral-7.7 mm from the skull). An infusion cannula
consting of a sterilized length of 30 guage stainless steel tubing is
stereotaxically placed via a hole in skull & the internalised tip is
located within the nigrostrital pathway. A Right-unilateral lesion was made
& the solution of 6-µg of 6-OHDA in 2 µL 0.2% ascorbic acid saline were
infused into SNPC through 30 guage stainless needle (5) at a rate of 1µL/min
for 4.50 min., the syring was left in place for 5 min. then slowly withdrown
and skin incision closed with stainless steel wound clips (7).
2.2 Drug profile:
A) Rutin
Rutin ((3, 3′, 4′, 5, 7-pentahydroxyflavone-3-rhamnoglucoside)
was a citrus flavonoid
glycoside. Flavonoids are
polyphenolic compounds that occur ubiquitously in foods of plant origin.
Flavonoids act as a antioxidant and can prevent injury caused by ROS in various
ways . One way is the direct scavenging of free. Rutin was found to be a
neuroprotective agent. Rutin was
identified as the major LDL antioxidant compound of mulberry in an in vitro
study Katsuaki Suzuki,
Kyoko Okada, Tomoyasu Wakuda, Chie Shinmura,
Yosuke Kameno;
2010 February 17, Destruction of Dopaminergic Neurons in the Midbrain by 6-Hydroxydopamine
Decreases Hippocampal Cell Proliferation in Rats: Reversal by Fluoxetine.
3. MATERIALS AND
METHODS:
3.1 Animals:
Male Wistar rats
weighing between 250–300 g were used in this study. (six per cage).They were
acclimatized to condition in the animal housing unit at 23±2ºC under 12:12 hrs
light/dark cycle. The approval of the Institutional Animal Ethics Committee,
was taken prior to the experiments. (Proposal no.03 at dated 27/12/2012).Constituted
for the purpose of control and supervision of experimental animals by Ministry
of Environment and Forests, Government of India, New Delhi, India.
3.2 Drugs and Chemicals:
Analytical grade Chemicals & reagents were used. Rutin
sulphate (5,10,50 mg/kg) (Sigma-Aldrich Labs, Bangalore, India) & 6-OHDA
(6-µg of 6-OHDA in 2 µL 0.2% ascorbic acid saline) (Sigma -Aldrich Labs,
Bangalore, India).Other chemicals, reagents & dietary supplements were used
in the present investigation were of analytical grade & provided by
college/university.
3.3 Tretment
schedule:
Animals were randomly divided into six
groups of 6 animals in each.
Group—1 vehicle treated, received normal
saline (i. p.);
Group—2 received 6-OHDA (6-µg in 2 µL 0.2%
ascorbic acid saline i.c.v.) for14days;
Group—3 received Rutin (50mg/kg i.p.) per
se;
Group—4, 5, 6, received Rutin (5, 10,
50mg/kg i.p.) + 6-OHDA (6-µg of in 2 µL 0.2% ascorbic acid saline) for 14 days.
Rutin was administered 1 h prior to 6-OHDA administration.
3.4 Behavioral tests:
3.4.1 Morris water
maze test:
To
determine if the Rutin treatment affects hippocampus dependent memory function
in animals given 6-OHDA, we used a Morris water maze (MWM) task for animals in
the differentiation paradigm. The animals were given a session of the task for
3 days. Each session consisted of six trials lasting 60 s each, separated by a
60 s inter-trial interval. At the start of each trial, rats were placed at one
of four start locations at the limb of a circular pool (150 cm in diameter,
water temperature at 26°C) with their face toward the wall. Animals were
required to escape to an invisible platform (10 cm in diameter, 1 cm below the
water surface) fixed at a predetermined location. If animals could not reach
the platform within 60 s, an experimenter gently led them onto the platform.
Once animals got upon the platform, they were left on it for 15 s and then
returned to a waiting cage.
3.4.2
Locomotor activity:
Locomotor activity was assessed in actophotometer with an open-field
activity monitoring system (VJ Instruments, Amravati, India), having a circular
arena of 40 cm, equipped with three infrared beam cells connected to digital
counter. Locomotor activity was expressed in terms of total number of counts of
beams interruptions. Each animal was observed over a period of 5 min and values expressed as counts per 5 min.
3.4.5 Elevated Plus Maze:
The EPM was made of
dark gray PVC consisting of two opposite open arms (50
cm×12cm) and two opposite closed arms surrounded by 50cm high walls of the same
dimensions. The middle section that allows the animal to transit from arm to
arm consisted of a square with dimensions of 12×
12cm. The maze was elevated 50cm above ground and the open arms were equipped with 0.5×0.5cm ledges to ensure that no animals would fall off the maze. The
apparatus could be moved between rooms and it was made sure that placement and
lighting conditions were identical for each trial. The trials were video
recorded and computer analyzed with the ethological software viewer (Biobserve GmbH, Bonn, Germany) to
measure time spent in and visits to the arms of the EPM.
5. DISCUSSION:
The core finding
of the present study is that administration of relevant doses of Rutin is
remarkably neuroprotective in rats against 6-Hydroxydopamine induced
neurotoxicity. We have chosen the dose of Rutin (5, 10 and 50 mg/kg., i.p.)
according to the previous studies done in our laboratory (7). There are no
previous reports on the protective effect of rutin in 6-Hydroxydopamine induced
neurotoxicity, an animal model for Parkinson's disease. In the present study,
rutin attenuated various behavioral and biochemical alterations due to
6-Hydroxydopamine and thus providing the first evidence regarding its
beneficial effect in Parkinson's disease.
The direct evidence that some single DA
neurons located in the ventral portion of the SN target several structures including
the striatum, the globus pallidus (GP), the frontal cortex and the thalamus has
only been recently demonstrated in rats.(8)
PD is often
complicated by a variety of cognitive symptoms that range from isolated memory
and thinking problems to severe dementia. While the motor symptoms of PD are
well-known (tremor, rigidity, slowness of movement, imbalance). (9)
In the present
study, the administration of 6 µg of 6- OHDA into the right unilateral
ventricle led to a decrease in DA levels of approximately 50–60% in the right
striatum of rats at all ages. Nigral DA levels were reduced to a slightly less
extent. In contrast to more severe bilateral lesions, the pattern of changes in
body weight after surgery were similar between the vehicle and lesioned animals.
(10)
The present study
employed 6-OHDA as animal model of Parkinson disease. 6-OHDA induce
nigrostrital dopaminergic lesion via the generation of hydrogen peroxide and
derived hydroxyl radicals. 6-OHDA could induce catecholaminergic cell death by three
main mechanisms: reactive oxygen species generated by intra or extracellular
auto-oxidation, hydrogen peroxide formation induced by MAO activity or direct
inhibition of the mitochondrial respiratory chain. These events lead to strong
oxidative stress amplified by cytoplasmic free calcium and to a decrease in
cellular ATP avaibility, both leading to cell death. (11).
Impairment of
mitochondrial activity also contributes to both ROS generation and nigral cell
loss. The main mitochondrial defect observed in degenerating PD concerns
complex I (nicotinamide adenine dinucleotide coenzyme Q reductase) of the
mitochondrial respiratory chain. Complex I is located in the inner
mitochondrial membrane and forms a part of the oxidative phosphorylation system
(OXPHOS) responsible for the production of cellular ATP. Decreases in the
activity and immunoreactivity of the reduced form of the complex I were
observed in the SNpc of PD patients.
Unilateral
6-OHDA-induced SNpc degeneration produces an asymetric and quantifiable motor
behavior after unilateral lesion induced by systemic administration of either
DA receptor agonists, l-dopa or dopamine releasing drugs. This allows easy and
reliable control of the extent of the lesion and the potential benefits of
therapeutic treatments. I.C.V. administration of 6-Hydroxydopamine is known to
produce hypoactivity that resembles juvenile onset and advanced Parkinson's
disease in rats. It produces significant motor and behavioral abnormalities
including bradykinesia, muscles weaknesses and rigidity. These findings are in
agreement with earlier reports which also observed a variety of neurobehavioral
abnormalities and motor deficit in rats following 6-OHDA administration.
In the present study, administration of
6-Hydroxydopamine decreased the ambulatory movements (in actophotometer), thus
representing the motor abnormalities. Daily treatment with Rutin for 14 days
dose-dependently attenuated 6-Hydroxydopamine-induced hypolocomotion and motor
incoordination.
I.C.V. administration of 6-Hydroxydopamine
also decreased the SOD levels in the whole brain, suggesting mitochondrial
damage and pretreatment with Rutin attenuated this decrease in SOD levels.
These results show that Rutin may prevent mitochondrial deterioration and
maintain synaptic integrity against damage induced by
6-Hydroxydopamine.Cholinergic neurotransmission is a central process underlying
memory and cognitive function. Cholinergic basal forebrain neurons in the
nucleus basalis magnocellularis innervate the cerebral cortex, amygdaloid
complex and hippocampus, and are essential for learning and memory formation.
One of the most important mechanisms responsible for correct cholinergic
function is performed by enzyme choline esterase (ChE). In the present study,
treatment with Rutin partially decreased the levels of ChE in cerebral cortex
and hippocampus of PD rats.
These findings
demonstrate that daily treatment with Rutin protects against various behavioral
and biochemical alterations induced by 6-Hydroxydopamine in rats. However,
further studies are required to understand the exact mechanism involved in its
neuroprotective role in this animal model of Parkinson's disease.
6. CONCLUSION:
Rutin treatment
protects behavioral changes, and significantly attenuated oxidative damage and
improved mitochondrial complexes enzyme activities in different regions
(striatum, cortex and hippocampus) of rat brain against 6-OHDA induced
neurotoxicity.
The results show
that Rutin treatment is effective, thus it could be used as an effective therapeutic
agent in the management of Parkinson's disease and related conditions. We
attempted to investigate the neuroprotective effect of Rutin in animal model of
Parkinson's disease. To get a detailed account of the Rutin in neuroprotection
further confirmatory studies are required.
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