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DOI 10.1007/s11064-007-9547-y
ORIGINAL PAPER
Introduction
It is largely accepted that stroke is a leading cause of
morbidity and poses serious health problems globally. The
affected persons often suffer from neurological deficits and
at times lead a crippled life [1]. Middle cerebral artery
occlusion (MCAO) in rat is widely used to study experimental ischemic stroke and has provided invaluable
understanding of the patho-physiology of focal cerebral
ischemia. Several mechanisms have been suggested to be
involved in the etiology of stroke including NMDA
receptor activation leading to excitotoxicity, excessive
nitric oxide (NO) generation and free radical mediated
oxidative stress [24]. Recently, role of intercellular
adhesion molecule-1 (ICAM-1) protein has been suggested
in stroke since inhibition of ICAM-1 protein was found to
be neuroprotective in MCAO rats [5]. Several studies have
revealed that during ischemia there is excessive generation
of free radicals and the antioxidant defence is impaired
causing more vulnerability and damage to the brain [6].
A number of agents, both synthetic and natural, have
been screened to evaluate their preventive and therapeutic
efficacy against ischemia [710]. Dietary supplementation
with blueberries, spinach and spirulina reduces ischemia/
reperfusion induced apoptosis and cerebral infarction [6].
Turmeric (Curcuma longa rhizomes) has been extensively
used as an effective therapeutic agent since ages [1114].
Turmeric as well as its constituent curcumin has been
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Neurochem Res
Neurological score
Neurological evaluation of rats was carried out after
30 min to verify successful MCAO and immediately before
they were sacrificed after 72 h. An eight point behavioural
rating scale, modified from the scale as described by
Rogers et al. [1] was used to score the neurological deficits.
0 = no neurological deficit.
1 = failure to extend right forepaw fully.
2 = decreased grip of the right forelimb while tail gently
pulled.
3 = spontaneous movement in all direction, contralateral circling only if pulled by the tail.
4 = circling or walking to the right.
5 = walks only when stimulated.
6 = unresponsive to stimulation with a depressed level
of consciousness.
7 = dead.
Sham operated
Middle cerebral artery occlusion (MCAO)
Curcumin treatment and MCAO
Treatment
Curcumin from rhizomes of Curcuma longa (suspended in
2% gum acacia) was used to investigate its protective
potential against MCAO. Rats were pre-treated with curcumin (100 mg/kg, po) for 5 days before MCAO. The
treatment was continued for another 3 days after MCAO
following which behavioural, biochemical and histological
studies were carried out.
Behavioural studies
(i)
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Biochemical estimations
Rats in all groups were sacrificed 72 h after MCAO by
cervical decapitation and the brains were immediately
removed. The brain regions, corpus striatum and cerebral
cortex, were dissected out following the standard procedure
[32] and processed for biochemical assays within 45 min in
each case.
(i)
Lipid peroxidation: As a measure of lipid peroxidation, malonaldialdehyde (MDA) levels were estimated
by measuring thiobarbituric acid reactive substances
(TBARS) following the standard protocol [33].
Briefly, equal volumes (120 ll) of EDTA (10 mM),
ascorbate (10 mM) and mixture of EDTA (16.7 mM)
and FeSO4 (16.7 mM) were mixed and to this
homogenate of different brain regions (0.6 ml) was
added. The reaction mixture was incubated at 37C for
0 and 60 min. Immediately after incubation, the
reaction was stopped by adding 1 ml of ice cold
10% trichloro acetic acid (TCA). The deproteinised
homogenate was centrifuged at 2000g for 10 min and
supernatant was aspirated out. The supernatant was
mixed with an equal amount of 0.67% TBA and kept
in boiling water bath for 1520 min. The intensity of
pink colour developed was read at 532 nm on a
spectrophotometer.
(ii) Superoxide dismutase: Activity of superoxide dismutase (SOD) was measured following the method of
Kakkar et al. [34] using NADH as a substrate.
Briefly, the assay mixture in a final volume of 3 ml
contained sodium pyrophosphate buffer (0.082 M,
pH 8.3), phenazine methosulphate (186 lM), nitro
blue tetrazolium (300 lM), NADH (780 lM),
enzyme preparation and distilled water. The reaction
Histological studies
Five coronal sections (2 mm thick) of the whole brain were
taken from the region beginning 1 mm from the frontal pole
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Neurochem Res
Sham
215 34
Ischemic (MCAO)
77 17*
Behavioural changes
(i)
Initial
Post-treatment Neurological
weight (g) weight (g)
scores
0
Range
Sham (n = 10)
Ischemic
(MCAO)
(n = 6)
4.36 0.7*
00
Curcumin
pre-treated
ischemic
(n = 8)
1.90 0.7** 07
17
165 15**
Biochemical changes
(i)
Total steps/60 s
Errors in placement of
forelimb
Sham
35 2.5
1 0.4
Ischemic (MCAO)
17 1.7*
5 0.8*
Curcumin pre-treated
ischemic
26 1.1**
3 0.4**
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Neurochem Res
Ischemic damage
In ischemic rats, contralateral hemispheric infarction was
well demarcated (33%), 72 h after MCAO, as revealed by
Discussion
An impairment in the sensorimotor functions is well known
following ischemia. In the present study, the neurological
score was found to be increased due to ischemic damage.
Treatment with curcumin resulted in significant decrease in
the neurological score. Neurological score was also corelated with severity of infarction [1]. Grid walking is one
of the sensitive tests to monitor the spontaneous locomotor
activity and muscle co-ordination. Any deficit in placement
of forelimbs and foot-fault error is easily detected through
grid walking test. Rats with ischemic reperfusion injury
following transient and permanent occlusion of middle
cerebral artery have been reported to show an impairment
in grid walking [1]. Errors in placement of foot and
40
35
% change
30
25
20
15
10
5
0
Sham
MCAO
MCAO+Curcumin
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