Neuroprotective effect of curcumin in

middle cerebral artery occlusion (MCAO)

induced focal cerebral ischemia in rats
Meenakshisundaram Thiyagarajan, Shyam S. Sharma
Life Sciences 74 (2004) 969-985

6/06/2007

1
Tidarat Yameobsilp

Introduction
Oxidative stress was suggested to be involved in
the pathogenesis of ischemia/ reperfusion injury.

Oxidative Protection

Balance

Oxidants
Oxidative Stress
Oxidants:
Superoxide, Hydrogen peroxide,
hydroxyl radical, nitric oxide,
peroxynitrite
can damage to lipids, protein,
DNA

Oxidative Stress

Antioxidants
Oxidative Protection
Antioxidant :
Enzymatic (endogenous)
- enzyme e.g. superoxide
dismutase (SOD), glutathione
peroxidase (GPx), catalase
Non-enzymatic
- vitamin E,C
- beta-carotene
3
- flavonoid

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Oxidative Protection

After ischemicreperfusion injury

Oxidative Stress

Antioxidants

Oxidants
Oxidative Stress

Oxidative Protection

resulted in

Cell damage and death

4

Inosine

Adenosine

AMP

ADP

ATP breakdown

Hypoxanthine and xanthine

Xanthine
dehydrogenase (XD)
irreversible

substrate

Xanthine
oxidase (XO)

At present, there is no
effective neuroprotective
therapy for treatment
ischemia-reperfusion

Introduction

Many antioxidants have been reported

to be neuroprotective in experimental
model of cerebral ischemia

Antioxidant
Reduction of ROS mediated reactions
would rescue the neurons from
reperfusion induced neuronal loss in
animal model of cerebral ischemia
Clemens et al.(1991), Cuzzocrea et al.(2000),
Fuson et al.(1999), Soehle et al.(1998) 8

Curcumin
Curcumin

Curcumin is the most active ingredient among

the three curcuminoids that found in curcuma
longa Linn. (or turmeric or Ka-Min-Chan)

Curcumin
antioxidant and neuroprotective properties
potent scavenger of oxygen radicals
inhibitor of lipid peroxidation and
xanthine oxidase-induced O2- production

10

Kunchandy and Rao (1990),Shalini and Srinivas

(1987),Subramanian et al (1994)

Curcuma longa was demonstrated to have

antioxidant potential
Dikshit et al (1995),Jones and Shoskes (2000)

Curcumin have cytoprotective activity

in cardiac and renal ischemia
11

Aim of study
To evaluate the neuroprotective potential of

curcumin

in middle cerebral artery occlusion

induced focal cerebral ischemia
in rat model

12

Experimental study

induced by MCAO

Reperfusion injury
genarated ROS
assessed by measuring these parameters
Lipid peroxidation
Superoxide dismutase (SOD)
activity
Glutathione peroxidase (GPx)
activity

Peroxynitrite
Tyrosine nitration
Infarct and edema volume
Neurological deficit13

Materials and methods

14

Preparation of the animals

92 Male Sprague-Dawley rats

weight 240-260 g.

The rats were housed in room at 221C

12 h. dark and light cycle
Standard rat chow pellets and water
was allowed ad libitum

15

Treatment Protocol
92 rats after induced MCAO
temporally, were randomly
divided into vehicle and
curcumin treated groups

Vehicle

Curcumin

Curcumin

Curcumin

(NaCMC+
tween 80)

30 mg/kg

100 mg/kg

300 mg/kg
16

Treatment Protocol

Vehicle

Curcumin

Curcumin

Curcumin

(CMC)

30 mg/kg

100 mg/kg

300 mg/kg

Brain infarct and

edema volume,
neurological deficit

Lipid peroxidation, SOD,

GPx, peroxynitrite, tyrosine
nitration

were measured

17

Materials and methods

Preparations of animals
Temporally focal cerebral ischemia
induced by
Middle cerebral artery occlusion
Measure all parameters of ischemic reperfusion injury

18

Cerebral ischemia/reperfusion injury

Preanaesthetic medication :
atropine sulphate
Anaesthetic medication :
chloral hydrate

Midline incision of the neck to expose trachea and left

common carotid artery
19

Cerebral ischemia/reperfusion injury

Middle cerebral
artery
Internal carotid
artery
External carotid
artery
Common
carotid artery

Monitored physiological
parameters
Respiratory rate
Rectal temperature
Blood pressure
Heart rate
Investigate effect of
curcumin 300 mg/kg

In sham operated group, the filament was introduce into the

external carotid artery only but not advanced further. 20

Summarized diagram of the experiment

0

2 h.

4 h.

12 h.

MCAO

24 h.

Sacrifice rats and

brains were removed

reperfusion
pulled out the filament

For measure all parameters of

1 ml.oxidative
of 5%
stress
dextrose solution

21

Parameters measured
Infarct and edema volume
Neurological deficit
lipid peroxidation
superoxide dismutase (SOD) activity
glutathione peroxidase (GPx) activity
peroxynitrite
tyrosine nitration by immunofluorescense
The rats, not shown neurological deficits immediately after
reperfusion (neurological score < 3)
22
were excluded from the study.

Infarct and edema volume

10 min. 37C

Stained

2 mm. thick

overnight
Fixed

2% triphenyl tetrazolium
chloride (TTC) solution
in saline

10% formal saline

Measured the area of infarction by using

Leica Qwin image analysis software
23

Infarct and edema volume

Infarct volume (mm3) (in occlude side)
= total infarct area thickness of brain section
Edema volume (mm3)
= infarct volume of ipsilateral sidevolume of contralateral side
Volume of Ipsilateral side
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Neurological deficits
scored on a 5-point scale
0 = No neurological deficit
1 = failure to extend right paw fully
2 = circling to right
3 = falling to right
4 = did not walk spontaneously and
had depressed levels of consciousness
25

Estimation of lipid peroxidation

Lipid peroxidation was
measured in the formed
malondialdehyde (MDA)
by spectrophotometer
Quantification based on
generate the standard curve
by using authentic MDA

26

Estimation of lipid peroxidation

Contralateral brain

30 min.

homoganate

spinning

ipsilateral brain
homoganate

0.1 ml. of methanolic butylated

hydroxy toluene solution

Boiling water

5% aqueous
trichloroacetic acid
532 nM.

30 min.

Boiling water
spectrophotometer

1ml. of thiobarbituric
acid reagent

supernatant

27

Estimation of superoxide dismutase

(SOD) activity

SOD activity was estimated

in brain homogenate by
calculated the rate inhibition
of nucleotide oxidation

28

Estimation of superoxide dismutase

(SOD) activity
homogenate
800 l of triethanolamine-diethanolamine
340 nm
control
of triethanolamine-diethanolamine
buffer (pH800
7.4),l40
l of 7.5 mM NADH, 25 l
buffer mM
(pH 7.4),
40 l of 27.5
of 100-50
EDTA-MnCl
(pHmM
7), NADH, 25
mM EDTA-MnCl2 (pH 7),
100llofof100-50
brain homogenate
50 mM phosphate buffer
100 l of 10 mM
mercaptoethanol

Spectrophotometer

calculated rate of nucleotide oxidation (in control) and

inhibition of nucleotide oxidation in homogenate.
29

Estimation of superoxide dismutase

(SOD) activity
One unit of the SOD activity

amount of enzyme required to inhibit the rate

of NADH oxidation of the control 50 %

30

Estimation of glutathione peroxidase

(GPx) activity

GPx activity was estimated

in brain homogenates and
measured the change in
NADH absorbance by
using spectrophotometer

31

Estimation of glutathione peroxidase

(GPx) activity
1 mM of glutathione,
0.2 mM of NADH,
1.4 U glutathione reductase,
brain homogenate equivalent to
0.1 mg of protein
in 50 mM phosphate buffer.
0.25 mM hydrogen peroxide

340 nm

Spectrophotometer

measure the change in NADH absorbance

32

Estimation of glutathione peroxidase

(GPx) activity
One unit of GPx activity

amount of sample required to oxidize 1 M

of NADH per min. based on the molar absorbance
of 6.22106 for NADH.
All values which get from this experiment was subtracted
with the basal reaction rate which used PBS instead of
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brain homogenate.

Estimation of peroxynitrite
O2

O2

e-

Superoxide
anion

NO

Nitric Oxide
OONO Peroxynitrite
34

Estimation of peroxynitrite
Peroxynitrite was estimated by using a fluorescent
dye dihydrorhodamine 123
Peroxynitrite dependent manner

oxidized to rhodamine123
Level of rhodamine123 was calculated from
the standard curve obtained from authentic
rhodamine123
(in a concentration 0-10 nM which prepared in plasma obtained
from the untreated rat.)
35

Estimation of peroxynitrite

1 h.

MCAO

1 h.

1 h.

dye injection reperfusion

Collect the blood by
cardiac puncture

36

Estimation of peroxynitrite
excitation wavelength
500 nM.

emission wavelength
536 nM.

spectrofluorometer

All values derived from this experiment were subtracted

with the basal reaction rate

37

Estimation of tyrosine nitration

by immunofluorescence

Peroxynitrite mediated tyrosine nitration was

estimated by using anti-nitrotyrosine antibody by
immunofluorescence technique

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Estimation of tyrosine nitration

by immunofluorescence
4 h.

reperfusion

Fixed the
brain

transcardial
perfusion

reanaesthetize with
chloral hydrate

Ice-cold PBS

10% buffered
formal saline

embedded

20 min
washed

incubated

proteinase K

washed

PBS

120 min
incubated

microscopic slides
30 min

incubated

blocking buffer

overnight
incubated

avidin
distilled water

wax

biotin

1o Ab
at 4oc

anti-nitrotyrosine
in blocking buffer
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Estimation of tyrosine nitration

by immunofluorescence
3 times

overnight
1o Ab
at 4oc

anti-nitrotyrosine
in blocking buffer

washed

PBS

incubated
2 h.

biotin
anti-mouse IgG
blocking buffer
Detection
specific
labelling
avidin conjugated FITC system

image analysis software

Leica Qwin.
CCD camera
0.23 S.
Fluorescence microscope

40

Statistical evaluation
Means S.E.M
Student t-test
ANOVA followed by a Tukey test
Neurological deficit

median95% CI

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Results

42

Physiological parameters
40
30
Rectal
temperature 20
(o C)
10
0

After MCAO
6 h.

After MCAO
9 h.

After MCAO
12 h.
43

Physiological parameters
Arterial blood pressure
(mm.Hg)

Curcumin-treatment
300 mg/kg

Heart rate (beats/min.)

450
400
350
300
250
200
150
100
50
0

Before

After 30
min.

After 60
min.

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Effect of curcumin on cerebral infarction

210.3931.25 mm3

169.8616.13 mm3
37.235.10%
132.0610.73 mm3
46.3910.23%
112.7921.53 mm3

* Represents p < 0.05 of respective group as compared to vehicle treated group

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Effect of curcumin on cerebral infarction

Vehicle

Curcumin
100 mg/kg

Curcumin
300 mg/kg

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Effect of curcumin on cerebral edema

120.599.99 mm3
89.0720.02 mm3
26.1416.60%
85.3912.38 mm3
29.1910.27%
59.147.29 mm3
26.1416.60%

* Represents p < 0.05 of respective group as compared to vehicle treated group

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Effect of curcumin on neurological deficit

V ehicle (5)
C urcum in 30 m g/kg (6)
C urcum in 100 m g/kg (6)
C urcum in 300 m g/kg (7)

Neurological deficits
4

1 = failure to extend right

paw fully
2 = circling to right
3 = falling to right
4 = did not walk spontaneously
and had depressed levels
of consciousness

Neurological deficit

0 = No neurological deficit

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Effect of curcumin on lipid peroxidation

ipsilateral side
contralateral side

4.5

* p<0.05

4.0
3.5
3.0

MDA level
(nM)

2.5

2.0
1.5
1.0
0.5
0.0

Vehicle
treated group

Curcumin
300 mg/kg

49

Effect of curcumin on superoxide

dismutase activity
ipsilateral side
contralateral side
15

10

SOD
(U)
5

Vehicle
treated group

Curcumin
300 mg/kg

50

Effect of curcumin on glutathione

peroxidase activity
ipsilateral side
contralateral side

Gpx
(mU)

* p<0.05

120
110
100
90
80
70
60
50
40
30
20
10
0

Vehicle
treated group

Curcumin
300 mg/kg

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Effect of curcumin on peroxynitrite

(300 mg/kg)

52

Effect of curcumin on peroxynitrite

mediated tyrosine nitration

vehicle

Curcumin 300 mg/kg

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Discussion

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Discussion
Curcumin-treatment (i.p.)
showed dose dependent
reduction in infarct volume
in MCAO induced focal
cerebral ischemia model
of stroke in rats
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Cytoprotective activity of curcumin is also reported in

model of cytotoxicity
reduces renal damage and inflammation in renal
ischemia reperfusion injury (Jones and Shoskes,; 2000)
protective effect in isoproterenol induced
myocardial ischemia in rats (Nirmala and Puvanakrishnan;1996),
Suggested mechanism of cytoprotective effect::
inhibit activity over oxidative stress induced activation of AP1
and NF-kB, upregulation of c-fos, c-jun and phosphorylation of
c-jun proteins.
proteins
(Luo et al.;1999)

inhibit JNK, PKC, iNOS and COX-2 enzymes

(Huang et al.,1991; Rao et al.,1999)

56

Cerebral edema occurred as a result of

Ionic imbalance
Immune-activation (inflammation)
infiltration of macrophage
secondary reaction

Curcumin
iNOS and COX-2
Reduction of cerebral edema
57
Rao et al.;1999

e
c
n
la
a
b
Im

Oxidative Protection

After ischemicreperfusion injury

Oxidative Stress

Antioxidants

Oxidants
Oxidative Stress

Oxidative Protection

resulted in

Cell damage and death

58

Oxidants

Antioxidants

lipid peroxidation
ipsilateral side
contralateral side

4.5
4.0
3.5

3.0

MDA level
(nM)

2.5
2.0

1.5
1.0
0.5
0.0

Vehicle treated
group

Curcumin
300 mg/kg

59

Curcumin (Kunchandy and Rao.1990)

Curcumin reduce oxidative stress induced
lipid peroxidation reaction (Reddy and Lokesh
;1992,Shalini and Srinivas ;1987, Sreejayan and Rao;1994)
60

Oxidants

Antioxidants

lipid peroxidation
peroxynitrite formation
in form the level of rhodamine
123 and immunofluorescense
method

61

Curcumin
(Lin and
Shih,1994)

Curcumin
Curcumin (Kunchandy and Rao.1990)
Curcumin

(Kunchandy and
Rao.1990)

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Oxidants
lipid peroxidation
peroxynitrite formation

Antioxidants

endogenous antioxidant
defense enzymes
SOD
GPx
63

ROS scavenging activity and enhanced

transcription of SOD and GPx.
(Piper et al.;1998, Shahed et al.2001).

increased the activity of SOD and

GPx in liver homogenate
(Reddy and Lokesh;1994)

Curcumin
up-regulates the SOD gene expression in
rat kidney after ischemia-reperfusion injury
(Shahed et al.;2001)

increased glutathione peroxidase64

(Okada et al.;2001, Piper et al.;1998,Venkatesan et al.;2000)

Conclusion
Curcumin offered significant neuroprotection in
middle cerebral artery occlusion induced focal
cerebral ischemia.

65

Oxidants
lipid peroxidation
peroxynitrite formation

Antioxidants

endogenous antioxidant
defense enzymes
SOD
GPx
66

Thank you for your attention..

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