50_jc_neuroprotective effect of curcumin in mca occlusion in rats

8/19/2019 50_JC_Neuroprotective Effect of Curcumin in MCA Occlusion in Rats

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Meenakshisundaram Thiyagarajan, Shyam S. Sharma

Life Sciences 74 (2004) 969-985

Tidarat Yameobsilp6/06/2007

NeuroprotectiveNeuroprotective effect ofeffect of curcumincurcumin ininmiddle cerebral artery occlusion (MCAO)middle cerebral artery occlusion (MCAO)

induced focal cerebral ischemia in ratsinduced focal cerebral ischemia in rats


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•• Oxidative stress was suggested to be involved inOxidative stress was suggested to be involved in

the pathogenesis of ischemia/ reperfusion injury.the pathogenesis of ischemia/ reperfusion injury.

IntroductionIntroduction


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OxidantsOxidants

Oxidative Stress

Oxidative Protection Oxidative Stress

Oxidative Protection

AntioxidantsAntioxidants

Oxidants:

• SuperoxideSuperoxide, Hydrogen peroxide,, Hydrogen peroxide,

•• hydroxyl radical, nitric oxide,hydroxyl radical, nitric oxide,peroxynitriteperoxynitrite

•• can damage to lipids, protein,can damage to lipids, protein,DNADNA

Antioxidant :

Enzymatic (endogenous)Enzymatic (endogenous)

-- enzyme e.g.enzyme e.g. superoxidesuperoxide

dismutasedismutase (SOD), glutathione(SOD), glutathioneperoxidaseperoxidase ((GPxGPx),), catalasecatalaseNon-enzymatic

- vitamin E,C

- beta-carotene- flavonoid

BalanceBalance


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Oxidants

Antioxidants

After ischemic After ischemic--reperfusion injuryreperfusion injury

I m b a l a n

c e

I m b a l a n

c e

Cell damage and deathCell damage and death

resulted inresulted in

Oxidative Stress

Oxidative Protection Oxidative Stres

Oxidative Protection


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XanthineXanthine

dehydrogenasedehydrogenase (XD)(XD)

XanthineXanthineoxidaseoxidase (XO)(XO)

irreversibleirreversible

ATP breakdown ATP breakdown ADP ADP AMP AMP Adenosine AdenosineInosineInosine

Hypoxanthine andHypoxanthine and xanthinexanthine

substratesubstrate


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At present, At present, therethere isis nonoeffectiveeffective neuroprotectiveneuroprotective

therapytherapy forfor treatmenttreatment

ischemiaischemia--reperfusionreperfusion


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Introduction

Many antioxidants have been reportedMany antioxidants have been reported

to beto be neuroprotectiveneuroprotective in experimentalin experimentalmodel of cerebral ischemiamodel of cerebral ischemia


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Antioxidant

Reduction ofReduction of ROSROS mediatedmediated reactionsreactions

wouldwould rescuerescue thethe neuronsneurons fromfromreperfusionreperfusion inducedinduced neuronalneuronal lossloss inin

animalanimal model of cerebral ischemiamodel of cerebral ischemia

Clemens et al.(1991), Cuzzocrea et al.(2000),

Fuson et al.(1999), Soehle et al.(1998)


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CurcuminCurcumin

CurcuminCurcumin isis the mostthe most activeactive ingredientingredient amongamong

thethe threethree curcuminoidscurcuminoids that found in curcumathat found in curcumalongalonga Linn. (or turmeric or KaLinn. (or turmeric or Ka--MinMin--Chan)Chan)

CurcuminCurcumin


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CurcuminCurcumin

antioxidantantioxidant andand neuroprotectiveneuroprotective propertiesproperties

potentpotent scavenger scavenger of of oxygenoxygen radicalsradicals

inhibitor inhibitor of of lipidlipid peroxidationperoxidation andand

xanthinexanthine oxidaseoxidase--induced Oinduced O22--

productionproduction


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KunchandyKunchandy andand RaoRao (1990),(1990),ShaliniShalini andand SrinivasSrinivas

(1987),(1987),SubramanianSubramanian etet alal (1994)(1994)

Curcuma longa was demonstrated to haveantioxidant potential

DikshitDikshit etet alal (1995),(1995),JonesJones andand ShoskesShoskes (2000)(2000)

Curcumin have cytoprotective activity

in cardiac and renal ischemia


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Aim of study

To evaluate the neuroprotective potential of

curcuminin middle cerebral artery occlusion

induced focal cerebral ischemia

in rat model


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Experimental studyExperimental study

Reperfusion injuryReperfusion injuryinduced by MCAOinduced by MCAO

genaratedgenarated ROSROS

assessedassessed by measuring these parametersby measuring these parameters

••LipidLipid peroxidationperoxidation

••SuperoxideSuperoxide dismutasedismutase (SOD)(SOD)activityactivity

••GlutathioneGlutathione peroxidaseperoxidase ((GPxGPx))

activityactivity

••PeroxynitritePeroxynitrite

••Tyrosine nitrationTyrosine nitration

••Infarct and edema volumeInfarct and edema volume

••Neurological deficitNeurological deficit


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Materials and methods


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•• 92 Male Sprague92 Male Sprague--DawleyDawley ratsrats

•• weight 240weight 240--260 g.260 g.

Preparation of the animalsPreparation of the animals

••The rats wereThe rats were housed in room at 22housed in room at 22±±11ººCC

••12 h. dark and light cycle12 h. dark and light cycle

••Standard rat chow pellets and waterStandard rat chow pellets and water

was allowed adwas allowed ad libitumlibitum


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Vehicle

(NaCMC+tween 80)

Curcumin

30 mg/kg

Curcumin

100 mg/kg

Curcumin

300 mg/kg

92 rats after induced MCAO

temporally, were randomly

divided into vehicle andcurcumin treated groups

Treatment ProtocolTreatment Protocol


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Vehicle

(CMC)

Curcumin

30 mg/kg

Curcumin

100 mg/kg

Curcumin

300 mg/kg

Brain infarct andBrain infarct and

edema volume,edema volume,neurological deficitneurological deficit

Treatment Protocol

LipidLipid

peroxidationperoxidation

, SOD,, SOD,

GPxGPx,, peroxynitriteperoxynitrite, tyrosine, tyrosinenitrationnitration

were measuredwere measured


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MaterialsMaterials andand methodsmethods

Preparations of animalsPreparations of animalsTemporally focal cerebral ischemiaTemporally focal cerebral ischemia

induced byinduced byMiddle cerebral artery occlusionMiddle cerebral artery occlusion

Measure all parameters of ischemic reperfusion injuryMeasure all parameters of ischemic reperfusion injury


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PreanaestheticPreanaesthetic medication :medication :atropineatropine sulphatesulphate

Anaesthetic Anaesthetic medication :medication :chloral hydratechloral hydrate

Midline incision of the neck to expose trachea and leftMidline incision of the neck to expose trachea and left

common carotid arterycommon carotid artery

Cerebral ischemia/reperfusion injuryCerebral ischemia/reperfusion injury


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Cerebral ischemia/reperfusion injuryCerebral ischemia/reperfusion injury

•• Respiratory rateRespiratory rate

•• Rectal temperatureRectal temperature

•• Blood pressureBlood pressure

•• Heart rateHeart rate

Investigate effect ofInvestigate effect of

curcumincurcumin 300 mg/kg300 mg/kg

Monitored pMonitored physiologicalhysiologicalparametersparameters

InIn shamsham operatedoperated groupgroup,, thethe filamentfilament waswas introducintroducee intointo thethe

externalexternal carotidcarotid arteryartery onlyonly butbut notnot advancedadvanced further further ..

Middle cerebralartery

Internal carotid

artery

External carotidartery

Commoncarotid artery


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2 h.2 h. 4 h.4 h.

MCAOMCAO

reperfusionreperfusion

24 h.24 h.

1 ml. of 5%1 ml. of 5%dextrose solutiondextrose solution

pulled outpulled out the filamentthe filament

Summarized diagram of the experimentSummarized diagram of the experiment

0 12 h.12 h.

Sacrifice rats andSacrifice rats andbrains were removedbrains were removed

For measure all parameters ofFor measure all parameters of

oxidative stressoxidative stress


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Parameters measuredParameters measured

Infarct and edema volumeInfarct and edema volume

Neurological deficitNeurological deficitlipidlipid peroxidationperoxidation

superoxidesuperoxide dismutasedismutase (SOD) activity(SOD) activity

glutathioneglutathione peroxidaseperoxidase ((GPxGPx) activity) activity

peroxynitriteperoxynitrite

tyrosine nitration bytyrosine nitration by immunofluorescenseimmunofluorescenseTheThe ratsrats,, notnot showshownn neurologicalneurological deficitsdeficits iimmediatelymmediately after after

reperfusionreperfusion ((neurologicalneurological scorescore


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InfarctInfarct andand edemaedema volumevolume

10 min. 3710 min. 37°°CC

2%2% triphenyltriphenyl tetrazoliumtetrazolium

chloridechloride ((TTCTTC)) solutionsolutioninin salinesaline

10%10% formalformal salinesaline

overnightovernight

22 mmmm.. thickthick

StainedStained FixedFixed

MMeasureeasuredd tthehe areaarea of of infarctioninfarction byby usingusing

‘‘‘‘Leica QwinLeica Qwin’’’’ imageimage analysisanalysis softwaresoftware


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= total infarct area= total infarct area ×× thickness of brain sectionthickness of brain section

Infarct volume (mmInfarct volume (mm33

))

Edema volume (mmEdema volume (mm33))

= infarct volume of= infarct volume of ipsilateralipsilateral sideside××volumevolume ofof contralateralcontralateral sideside

Volume ofVolume of IpsilateralIpsilateral sideside

InfarctInfarct andand edemaedema volumevolume

(in occlude side)


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NeurologicalNeurological deficitdeficitss

scoredscored onon aa 55--pointpoint scalescale

0 =0 = NoNo neurologicalneurological deficitdeficit

1 =1 = failurefailure toto extendextend rightright pawpaw fullyfully2 =2 = circlingcircling toto rightright

3 =3 = fallingfalling toto rightright

4 =4 = diddid notnot walkwalk spontaneouslyspontaneously andand

hadhad depresseddepressed levelslevels of of cconsciousnessonsciousness


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EstimationEstimation

of of

lipidlipid

peroxidationperoxidation

Lipid peroxidation wasmeasured in the formed

malondialdehyde (MDA)

by spectrophotometer

Quantification based on

generate the standard curve

by using authentic MDA


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EstimationEstimation of of lipidlipid peroxidationperoxidation

Contralateral brain

homoganate

ipsilateral brain

homoganate

5% aqueous

trichloroacetic acid

0.1 ml. of methanolic butylated

hydroxy toluene solution Boiling water

30 min.

spinning

supernatant1ml. of thiobarbituricacid reagent

Boiling water

30 min.

spectrophotometer

532 nM.


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EstimationEstimation of of superoxidesuperoxide dismutasedismutase

((SODSOD)) activityactivity

SOD activity was estimatedSOD activity was estimated

in brain homogenate byin brain homogenate bycalculated the rate inhibitioncalculated the rate inhibition

of nucleotide oxidationof nucleotide oxidation


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EstimationEstimation of of superoxidesuperoxide dismutasedismutase

((SODSOD)) activityactivity

800 μl of triethanolamine-diethanolamine

buffer (pH 7.4), 40 μl of 7.5 mM NADH, 25 μl

of 100-50 mM EDTA-MnCl2 (pH 7),

100 μl of brain homogenate

homogenate

control 800 μl of triethanolamine-diethanolamine

buffer (pH 7.4), 40 μl of 7.5 mM NADH, 25

μl of 100-50 mM EDTA-MnCl2 (pH 7),

50 mM phosphate buffer

100 μl of 10 mMmercaptoethanol Spectrophotometer

340 nm

calculatedcalculated raterate of of nucleotidenucleotide oxidationoxidation ((inin controlcontrol)) andand

inhibitioninhibition of of nucleotide oxidation in homogenatenucleotide oxidation in homogenate..


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One unit of the SOD activity

amount of enzyme required to inhibit the rate

of NADH oxidation of the control 50 %

EstimationEstimation of of superoxidesuperoxide dismutasedismutase((SODSOD)) activityactivity


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EstimationEstimation of of glutathioneglutathione peroxidaseperoxidase

((GPxGPx)) activityactivity

GPx activity was estimated

in brain homogenates and

measured the change inNADH absorbance by

using spectrophotometer


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((GPxGPx)) activityactivity1 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 peroxideSpectrophotometer

340 nm

measure the change in NADH absorbance


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One unit of GPx activity


((GPxGPx)) activityactivity

amount of sample required to oxidize 1 µM

of NADH per min. based on the molar absorbance

of 6.22×106

for NADH.

All values which get from this experiment was subtracted All values which get from this experiment was subtractedwith the basal reaction rate which used PBS instead ofwith the basal reaction rate which used PBS instead of

brain homogenate.brain homogenate.


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EstimationEstimation of of peroxynitriteperoxynitrite

OONO -

NO•

Nitric Oxide

Peroxynitrite

e-O2

•–

Superoxideanion

O2


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Peroxynitrite was estimated by using a fluorescent

dye dihydrorhodamine 123

Level of rhodamine123 was calculated from

the standard curve obtained from authenticrhodamine123(in a concentration 0-10 nM which prepared in plasma obtained

from the untreated rat.)

oxidized to rhodamine123

PeroxynitritePeroxynitrite dependent manner dependent manner


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1 h. 1 h.1 h.

MCAO reperfusiondye injection

Collect the blood bycardiac puncture


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excitation wavelength

500 nM.

emission wavelength

536 nM.


spectrofluorometer

All values derived from this experiment were subtracted

with the basal reaction rate


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EstimationEstimation of of tyrosinetyrosine nitrationnitrationbyby immunofluorescenceimmunofluorescence

Peroxynitrite mediated tyrosine nitration was

estimated by using anti-nitrotyrosine antibody byimmunofluorescence technique


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EstimationEstimation of of tyrosinetyrosine nitrationnitration

byby immunofluorescenceimmunofluorescence

reperfusion

4 h.

reanaesthetize withchloral hydrate

transcardial

perfusion

Fixed thebrain

Ice-cold PBS embedded10% bufferedformal saline

waxPBS

washedincubated

proteinase K

20 min

washed

distilled water blocking buffer

120 min

incubated incubated

1o Ab

at 4oc

overnight

anti-nitrotyrosine

in blocking buffer

microscopic slides

avidinbiotin

30 minincubated


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1o Ab

at 4oc

overnight

anti-nitrotyrosine

in blocking buffer

washed

PBS

3 timesincubated

2 h.

Fluorescence microscope

EstimationEstimation of of tyrosinetyrosine nitrationnitration

byby immunofluorescenceimmunofluorescence

avidin conjugated FITC system

image analysis software

‘‘Leica Qwin’’.

CCD camera

0.23 S.

biotin

anti-mouse IgG

blocking buffer

Detection

specificlabelling


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StatisticalStatistical evaluationevaluation

Means ± S.E.M

Student t-test

ANOVA followed by a Tukey test

Neurological deficit median±95% CI


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ResultsResults


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PhysiologicalPhysiological parametersparameters

0

10

20

30

40

Rectal

temperature(o C)

After MCAO6 h.

After MCAO9 h.

After MCAO12 h.

Ph i l i lPh i l i l tt


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0

50100

150

200

250

300

350

400450

PhysiologicalPhysiological parametersparameters

Before After 30

min.

After 60

min.

Curcumin-treatment300 mg/kg

Arterial blood pressure(mm.Hg)

Heart rate (beats/min.)

Eff tEff t ff ii b lb l i f tii f ti


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EffectEffect of of curcumincurcumin onon cerebralcerebral infarctioninfarction

210.39±31.25 mm3

169.86±16.13 mm3

132.06±10.73 mm3

112.79±21.53 mm3

37.23±5.10%

46.39±10.23%

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

EffectEffect ofof curcumincurcumin onon cerebralcerebral infarctioninfarction


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EffectEffect of of curcumincurcumin onon cerebralcerebral infarctioninfarction

Vehicle Curcumin100 mg/kg

Curcumin300 mg/kg

EffectEffect ofof curcumincurcumin onon cerebralcerebral edemaedema


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EffectEffect of of curcumincurcumin onon cerebralcerebral edemaedema

120.59±9.99 mm3

89.07±20.02 mm3

85.39±12.38 mm3

59.14±7.29 mm3

26.14±16.60%

29.19±10.27%

26.14±16.60%

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

EffectEffect ofof curcumincurcumin onon neurological deficitneurological deficit


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1

2

3

4

Vehicle (5)Curcum in 30 m g/kg (6)Curcum in 100 m g/kg (6)Curcum in 300 m g/kg (7)

N e

u r o l o g i c a l d e f i c i t

EffectEffect of of curcumincurcumin onon neurological deficitneurological deficit

NeurologicalNeurological deficitdeficitss

0 =0 = NoNo neurologicalneurological deficitdeficit

1 =1 = failurefailure toto extendextend rightright

pawpaw fullyfully

2 =2 = circlingcircling toto rightright

3 =3 = fallingfalling toto rightright

4 =4 = diddid notnot walkwalk spontaneouslyspontaneously

andand hadhad depresseddepressed levelslevels

of of cconsciousnessonsciousness

ffEff t ff i li id id ti


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EffectEffect of of curcumincurcumin onon lipidlipid peroxidationperoxidation

0.00.5

1.0

1.5

2.0

2.5

3.0

3.54.0

4.5

ipsilateral sidecontralateral side

MDA level

(nM)

Vehicletreated group

Curcumin300 mg/kg

* *

* p


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pp

dismutasedismutase activityactivity

0

5

10

15


SOD(U)

Vehicletreated group

Curcumin300 mg/kg

EffectEffect of of curcumincurcumin onon glutathioneglutathione


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0

1020

30

40

50

6070

80

90

100

110120


Gpx(mU)

gg

peroxidaseperoxidase activityactivity

Vehicle

treated group

Curcumin

300 mg/kg

*

* p


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EffectEffect of of curcumincurcumin onon peroxynitriteperoxynitrite

(300 mg/kg)


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EffectEffect of of curcumincurcumin onon peroxynitriteperoxynitritemediated tyrosine nitrationmediated tyrosine nitration

vehicle Curcumin 300 mg/kg


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Discussion

DiscussionDiscussion


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DiscussionDiscussion

CCurcuminurcumin--treatreatmenttment ((i.pi.p.).)

showedshowed dosedose ddependentependent

reductionreduction inin infarctinfarct volumevolumeinin MCAOMCAO inducedinduced focalfocal

cerebralcerebral ischemiaischemia modelmodel

of of strokestroke inin ratsrats

CytoprotectiveCytoprotective activity ofactivity of curcumincurcumin is also reported inis also reported in


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y p y pmodel ofmodel of cytotoxicitycytotoxicity

protectiveprotective effecteffect inin isoproterenolisoproterenol inducedinduced

myocardialmyocardial ischemiaischemia inin ratsrats ( Nirmala and Puvanakrishnan;1996 ),

reducesreduces renalrenal damagedamage andand inflammationinflammation inin renalrenal

ischemiaischemia reperfusionreperfusion injuryinjury ( Jones and Shoskes ,; 2000 )

Suggested mechanism ofSuggested mechanism of cytoprotectivecytoprotective effect::effect::

inhibitinhibit activityactivity over over oxidativeoxidative stressstress inducedinduced activationactivation of of AP AP11andand NFNF--kBkB,, upregulationupregulation of of cc--fosfos,, cc-- jun jun andand phosphorylationphosphorylation of of

cc-- jun jun proteinsproteins.( Luo et al.;1999 )

inhibit JNK, PKC, iNOS and COX-2 enzymes(Huang et al.,1991; Rao et al.,1999)


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Cerebral edema occurred as a result of Cerebral edema occurred as a result of

Ionic imbalanceIonic imbalance

ImmuneImmune--activation (inflammation)activation (inflammation)

infiltration of macrophageinfiltration of macrophage

secondary reactionsecondary reaction

iNOSiNOS and COXand COX--22

CurcuminCurcumin

RaoRao et al.;1999et al.;1999

Reduction of cerebral edemaReduction of cerebral edema

lance lanc e Oxidative Protection

Oxidative Stres


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Oxidants

Antioxidants

After ischemic After ischemic--reperfusion injuryreperfusion injury

I m b a l

a n c

I m b a l

a n c

Cell damage and deathCell damage and death

resulted inresulted in

Oxidative Stress Oxidative Protection


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Oxidants Antioxidants

lipidlipid peroxidationperoxidation

0.0

0.5

1.0

1.52.0

2.5

3.0

3.5

4.0

4.5ipsilateral sidecontralateral side

MDA level

(nM)

Vehicle treated

group

Curcumin

300 mg/kg

* *


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CurcuminCurcumin reduce oxidative stress inducedreduce oxidative stress induced

lipidlipid peroxidationperoxidation reactionreaction (Reddy and(Reddy and LokeshLokesh;1992,Shalini and;1992,Shalini and SrinivasSrinivas ;1987,;1987, SreejayanSreejayan and Rao;1994)and Rao;1994)

CurcuminCurcumin (Kunchandy and Rao.1990)


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peroxynitriteperoxynitrite formationformation

in form the level ofin form the level of rhodaminerhodamine123 and123 and immunofluorescenseimmunofluorescensemethodmethod

C iC i


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CurcuminCurcumin

(Lin and(Lin andShih,1994)Shih,1994)

CurcuminCurcumin

CurcuminCurcuminCurcuminCurcumin

(Kunchandy andRao.1990)

(Kunchandy and Rao.1990)


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endogenousendogenous antioxidantantioxidant

defensedefense enzymesenzymes

SODSOD

GPxGPx

ROSROS scavengingscavenging activityactivity andand enhancedenhancedtranscriptiontranscription ofof SODSOD andand GPxGPx..


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CurcuminCurcumin

transcriptiont a sc pt o of o SODSO anda d GPxG ..( ( Piper Piper etet alal..;;1998,1998, ShahedShahed etet alal..20012001 ). ).

increasedincreased thethe activityactivity of of SODSOD andandGPxGPx inin liver liver hhomogenateomogenate

( ( ReddyReddy andand LokeshLokesh;;19941994 ) )

upup--regulatesregulates thethe SODSOD genegene expressionexpression ininratrat kidneykidney after after ischemiaischemia--reperfusionreperfusion injuryinjury( ( ShahedShahed etet alal..;;20012001 ) )

inincreasedcreased glutathioneglutathione peroxidaseperoxidase( ( OkadaOkada etet alal..;;2001,2001, Piper Piper etet alal..;;1998,1998,VenkatesanVenkatesan etet alal..;;20002000 ) )


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ConclusionConclusion

CCurcuminurcumin offeredoffered significantsignificant neuroprotectionneuroprotection inin

middlemiddle cerebralcerebral arteryartery occlusionocclusion inducedinduced focalfocal

cerebralcerebral ischemiaischemia..


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endogenousendogenous antioxidantantioxidant

defensedefense enzymesenzymes

SODSOD

GPxGPx


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Thank you for your attention..

50_jc_neuroprotective effect of curcumin in mca occlusion in rats

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