response to experimental stroke is inhibited by eriodictyol

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ContentslistsavailableatScienceDirect

Behavioural

Brain

Research

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / b b r

Research

report

Neuroinflammatory

response

to

experimental

stroke

is

inhibited

by

eriodictyol

Emerson

de

Oliveira

Ferreira

a

_,

_Mara

_Yone

_Soares

_Dias

_Fernandes

b

_,

Neila

Maria

Rocha

de

Lima

a

_,

_Kelly

_Rose

_Tavares

_Neves

b

_,

_Marta

_Regina

_Santos

_do

_Carmo

b

_,

Francisco

Arnaldo

Viana

Lima

b

_,

_Analu

_Aragão

_Fonteles

b

_,

_Ana

_Paula

_Fontenele

_Menezes

a

_,

Geanne

Matos

de

Andrade

a,b,c,∗

a_{Post-Graduate}_Programme_in_Medical_Sciences,_Department_of_Clinical_Medicine,_Faculty_of_Medicine,_Federal_University_of_Ceará,_Brazil b_{Post-Graduate}_Programme_in_{Pharmacology,}_Department_of_Physiology_and_{Pharmacology,}_Faculty_of_Medicine,_Federal_University_of_Ceará,_Brazil c_Institute_of_Biomedicine_of_Brazilian_Semi-arid,_Brazil

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•Neuroprotectiveeffectoferiodictyol inbrainischemia.

•Eriodictyol ameliorates memory impairmentinmice.

•Anti-inflammatoryactivityof eriod-ictyol.

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Articlehistory: Received27March2016

Receivedinrevisedform21June2016 Accepted24June2016

Availableonline25June2016

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Background:Cerebralischemiaisacommondiseaseandoneofthemostcommoncausesofdeathand disabilityworldwide.Thelackofglucoseandoxygeninneuronaltissueleadstoaseriesof inflamma-toryevents,culminatinginneuronaldeath.EriodictyolisaflavonoidisolatedfromtheChineseherb Dracocephalumrupestrethathasbeenproventohaveanti-inflammatoryproperties.

Abbreviations:BDNF,brain-derivedneurotrophicfactor;CREB,cAMP-response-element-bindingprotein;DAB,3,3′_{-diaminobenzidine;}_ERK,_{extracellular-signal-regulated} kinase;Etyol,eriodictyol;GFAP,glialfibrillaryacidicprotein;HTAB,hexadecyltrimethylammoniumbromide;iNOS,induciblenitricoxidesynthase;MAPK,mitogen-activated proteinkinases;MPO,myeloperoxidase;NF-kB,nuclearfactorkappaB;NO,nitricoxide;Nrf2,nuclearfactor(erythroid-derived2)-like2;PBS,phosphatebufferedsaline; PKB,proteinkinaseB;pMCAO,permanentmiddlecerebralarteryocclusion;RT,roomtemperature;SO,sham-operated;TNF␣,tumornecrosisfactoralpha;t-PA,tissue plasminogenactivator;TTC,2,3,5-triphenyltetrazolium.

∗ Correspondingauthorat:DepartmentofPhysiologyandPharmacology,FacultyofMedicine,Federal,UniversityofCeará,RuaCel.NunesdeMelo1127,Porangabussu, Fortaleza,CE,60430-270,Brazil.

E-mailaddresses:emersonoliveira.shalom@hotmail.com(E.d.O.Ferreira),maraiony@hotmail.com(M.Y.S.D.Fernandes),neilamrl@hotmail.com(N.M.R.d.Lima), kelly.rose@hotmail.com(K.R.T.Neves),martacarmo@yahoo.com.br(M.R.S.d.Carmo),arnviana@hotmail.com(F.A.V.Lima),analufonteless@gmail.com(A.A.Fonteles), apfontenele@yahoo.com.br(A.P.F.Menezes),gmatos@ufc.br,geannecunha@yahoo.com.brr(G.M.d.Andrade).

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Keywords: Eriodictyol Flavonoids Memory Neuroprotection pMCAO

Neuroinflammation

Hypothesis/Purpose:Thus,thepresentstudywasdesignedtoexplorewhethereriodictyolhas neuropro-tectiveeffectsagainsttheneuronaldamage,motorandmemorydeficitsinducedbypermanentmiddle cerebralarteryocclusion(pMCAO)inmice.

StudyDesign:Animalswereorallytreatedwitheriodictyol(1,2and4mg/kg)orvehicle(saline)30min beforepMCAO,2hafter,andthenoncedailyforthefollowingfivedays.

Methods:Theparametersstudiedwereneuronalviability,braininfarctedarea;sensorimotordeficits; exploratoryactivity;workingandaversivememory;myeloperoxidase(MPO)activity;TNF␣,iNOSand GFAPimmunoreactivity.

Results:Thetreatmentwitheriodictyolpreventedneuronaldeath,reducedinfarctareaandimproved neurologicalandmemorydeficitsinducedbybrainischemia.TheincreaseofMPOactivityandTNF-␣, iNOSandGFAPexpressionwerealsoreducedbyeriodictyoltreatment.

Conclusion:Thesefindingsdemonstratethateriodictyolexhibitpromisingneuroprotectioneffectsagainst thepermanentfocalischemiacerebralinjuryinthemiceexperimentalmodelandtheunderlying mech-anismsmightbemediatedthroughinhibitionofneuroinflammation.

1. Introduction

Strokeisadebilitatingdiseasethataccountsforbehavioraland cognitivedisturbances,especiallythoseinvolvinglearning,motor andmemorydeficits[1,2].Strokeoccursafterasuddenblockof bloodflowtothebrain,deprivingthetissueofoxygenandglucose

[3,4].Clearly,glutamate excitotoxicity,calciumoverload, oxida-tivestress,inflammationandapoptosisareimportantcontributing factorsinthepathophysiologyofcerebralischemia[5]. Inflamma-tionisresponsibleformuchofcerebralischemictissueinjury[6,7]. Togetherwithmicroglia,astrocytescontributetotheproductionof inflammatorymediators,suchasTNF-␣andiNOS,whichare trig-geredbypro-inflammatorygenes,andNF-kB,whichisdrivenby astrogliosisandmicrogliosis[8].Anti-inflammatoriesconstitutean importanttherapeuticstrategyforstroke[2,9]becausecurrently, theonlyavailabletreatmenttoreducebraindamageafterstrokeis the“clot-buster,”tissueplasminogenactivator(t-PA).Thus,thereis agreatneedtodevelopnoveltherapiesforcerebrovasculardiseases

[10].

A diet rich in vegetables and fruits with polyphenols is knowntopromotefunctionalbenefitsforhealth[11].Amongthe polyphenols,flavonoidsarecompoundsthatarecommonin nat-urally occurring plants and vegetables [12]. Eriodictyol (Etyol) isaflavonoid,“flavanone”,(3′_,4′_{,5,7-tetrahydroxyflavanone)} iso-latedfromtheChineseherb(Dracocephalumrupestre)andcitrus fruits.Thereisevidenceforitsanti-inflammatory,anti-allergenic, antimicrobial,anti-cancer[13]andantioxidantproperties[14,15]. Eriodictyol reduced nitric oxide (NO) and pro-inflammatory cytokines in LPS-stimulated Raw 264.7 cells and suppressed the phagocytic activity of activated macrophages. This anti-inflammatory effect of eriodictyol wasrelated to the blockade ofnuclear factorkappa B(NF-␬B)[13].In a rat modelof tran-sientfocalcerebralischemia,eriodictyolreducedbraindamageand neurologicaldeficits[9].However,theeffectsoferiodictyolon neu-roinflammationandmemorydeficitsafterfocalcerebralischemia havenotbeenreported.Thus,theobjectiveofthepresentstudy wastoinvestigatetheeffectsoferiodictyolonthememorydeficits inducedbypermanentmiddlecerebralarteryocclusion(pMCAO) inmiceandtheprobablemechanismsofactioninvolved.

2. Materialandmethods

2.1. Drugs

Eriodictyol (Sigma, USA), xylazine (2%, Kensol®

, König, Argentina)andketamine(5%,Vetanarcol®

,König,Argentina).All otherreagentswereofanalyticalgrade.

2.2. Animals

MaleSwissmiceweighing25–30gobtainedfromtheCentral AnimalHouseofthePhysiologyandPharmacologyDepartmentof theFederalUniversityofCearáwereused.Animalswerehoused undera12-hlight,12h-darkcycleandallowedaccesstofoodand wateradlibitum.Allproceduresinthisstudywereinagreement withtheGuidefortheCareandUseofLaboratoryAnimalsfrom theUSHealthandHumanServicesDepartmentandwereapproved bytheethicscommitteeonanimalexperimentationoftheFederal UniversityofCeará,undertheregistrationnumber90/2013.

2.3. Inductionofpermanentmiddlecarteryocclusion(pMCAO)

pMCAOwasproducedbyelectrocoagulation oftheleft mid-dlecerebralartery asreported previously[16].Briefly, animals wereanaesthetizedwithxylazine(10mg/kg, i.p.)andketamine (90mg/kg,i.p.),anincisionwasmadeonthelefttemporo-parietal region,andthetemporalismusclewaspartiallyremoved.Aburr holewasdrilledintotheskulloverthemiddlecerebralarteryand thevesselwasoccludeddirectlyproximaltothelateral lenticu-lostriatebranchesusingelectrocoagulationwithamicro-unipolar coagulator.Thecompleteinterruptionofbloodflowwasconfirmed byvisualinspection.Bodytemperaturewaskeptnear37◦_C. Sham-operatedanimals(SOgroup)underwentthesameprocedurewith theexceptionofcauterizationofthemiddlecerebralartery.

2.4. Experimentalprotocols

The animals were divided in groups and submitted to oral administration of 5% Tween 80 and saline (pMCAO group) or eriodictyoland saline(1,2, and 4mg/kg,by mouth,orallyp.o.) thirtyminutesbeforepMCAOandtwohoursafter;thetreatment continued oncedaily for the following five days. At 24h post-ischemia, a sub-group of animals (n=6/group) were tested for neurologicdeficitsand euthanizedforischemicdamage evalua-tion.At72and96hpost-ischemia,anothersub-groupofanimals (n=8/group)weretestedforlocomotoractivityandworkingand aversivememories,immunohistochemistryandhistology.At24h post-ischemia,athirdsub-group,animalswereevaluatedforthe MPO(n=6/group).

2.5. Neurologicalevaluation

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describedbyGarciaetal.[17].Sixitemsweremeasured,andthe totalscorerangedfrom3to18:thehigherthescore,thebetter themotorperformance.Items1–4(spontaneousactivity, symme-tryofmovements,symmetryofforelimbsandclimbingthewall ofthewirecage)measuredmotorperformance;items5–6 (reac-tiontotouchonandresponsetovibrissaetouch)measuredsensory function(Table1).

2.6. QuantificationofcerebralinfarctsizethroughTTCstaining

Animals were euthanized 24h after ischemia. Brains were removed,and 2mm coronalsectionsweremade fromthe pre-frontal cortex to the midbrain. Slices were immersed in a 2% solutionof TTC (2,3,5-triphenyltetrazolium)in normal salineat 37±◦_C_for₂₀_min._This_salt_accepts_a_proton_from_succinate dehy-drogenase in the inner membrane of the mitochondria, which reducesittoitsredinsolubleformknownasformazan[18].Thus, anareawithinactiveenzymesandtheinfarctionisnotstainedand appearspale.TheunstainedareasweremeasuredbytheOsirisTM software(UniversityofGeneva,Switzerland)andcalculatedasthe percentofthewholecoronalsection.

2.7. Openfieldtest

Allanimalsweretestedforlocomotoractivityusinganopen field apparatus, which consisted of a black acrylic chamber (30×30cm)withthefloordividedinto9squaresofequalareas

[19].Seventy-twohoursaftersurgery,theanimalwaspositioned inthecenterofthearenaandallowedtoexplorefreely.Thenumber ofcrossings(horizontalexploration)andrearings(vertical explo-ration)werescoredfor5min.Thearenawascleanedwith20%ethyl alcoholtoremoveanyodorsbeforethenexttest.

2.8. Y-Mazetest

Onthethirddayaftersurgery,spatialworkingmemorywas assessedbyrecordingspontaneousalternationbehaviorinthe Y-maze[20].Themazewasconstructedofwhitewoodwiththree identicalarms(40×15×4.5cm)positionedatequalangles. Ani-malswereplacedattheendofonearmandallowedtomovefreely throughthemazeduringan8-minsession.Theseriesofarmentries wasrecordedvisually,andarmentrywasconsideredtobe com-pletewhenthehindpawsofthemicewerecompletelyplacedin thearm.Theabilitytoalternaterequiresthemicetoremember whicharmshavealreadybeenvisited.Eachexperimentwasscored;

thepercentageofspontaneousalternationwascalculatedusingthe followingformula:

Spontaneous alternations(%)= alternationbehavior

maximumalternations×100

Alternationbehaviorisdefinedasthenumberofconsecutive entriesintoeach of thethree armswithoutrepetition,and the maximumalternationsarethetotalnumberofarmentriesminus two.

2.9. Passiveavoidancetest

Aversivememorywasassessedat72haftersurgeryasdescribed byGold[21]usingatwo-compartmentapparatus(48×22×22cm; Insight,Brazil).Onecompartmenthadanelectrifiedgridfloorand theotheraplatform.Eachanimalwasplacedontheplatformand allowedtoexploretheapparatusfor1min.Thirtysecondslater it wasplaced oncemore ontheplatformand whentheanimal steppeddownafootshockof0.5mAwasdeliveredfor1sthrough thegridfloor.Thelatencytimetosteponthegridcompartment wasmeasureduptoacut-offtimeof5min(training).Theanimal wasthenremovedfromtheapparatus,andthetrialwasrepeated 15minlater(earlymemory).Theretrievaltrialwasperformedin thesamemanner,24hlater,butnoanimalwasshocked(late mem-ory).

2.10. Enzymaticactivityofmyeloperoxidase(MPO)

Myeloperoxidase(MPO)isanenzymepresentinthegranules ofneutrophils.Thisenzymeisusedasanindicatorof inflamma-tion,morespecifically,asneutrophilmigrationoftissuemarker. Twenty-fourhoursafterinductionofischemia,theanimalswere sacrificed,theirbrainsrapidlyremovedandsamplesofthecortex andstriatumweredissectedandweighed.Theareaswere homog-enized(50mg/ml)inasolutionofhexadecyltrimethylammonium bromide(0.5%HTAB)in50mMphosphatebuffer,pH6.0.Thenthe sampleswerecentrifugedat14,000rpmat4◦_C_for₂_min.₃₀_␮_L_was addedto200␮Lofsamplesupernatantandthesolutioncontaining 0.167mg/mlo-dianisidine hydrochlorideand 0.0005%hydrogen peroxide.Theabsorbancewasmeasuredat0.1timesand3min atwavelength460nm.TheresultswerereportedasunitsofMPO permgoftissue.AndanMPOunitistheamountofenzymewhich degrades1␮mol/minofhydrogenperoxide.

Table1

Usedscoreforneurologicalevaluation(Garciaetal.,1995).

Test Score

0 1 2 3

Spontaneousactivity Nomovement Norearingand barelymoves

Movesbutdoesnot approachatleast3 sidesofthecage

Movesandapproaches atleastthreesidesof thecage

Symmetryof movements(4limbs)

Contraleteral side:no movement Contraleteralside: slightmovement Contraleteralside: slowmovement Bothsides: symmetrical movement Symmetryofforelimbs

(outstretchingwhile heldbytail)

Contraleteral side:no movement Contraleteralside: slightmovement Contraleteralside: slowmovement Bothsides: symmetrical movement Climbingwallofwire

cage

... Failstoclimbor

tendstomovein circles

Contralateralsideis weakerwhengriping

Normalclimbingand gripping

Bodyproprioception ... Noresponseon contralateralside

Weakerresponseon contralateralside

Symmetricalresponse

Responsetovibrissae touch

... Noresponseon

contralateralside

Weakerresponseon contralateralside

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2.11. ImunohistochemistrystainingTNF-˛,iNOSandGFAP

Briefly,theanimalswereanesthetizedwithsodium thiopen-talandtranscardiallyperfusedwithice-coldPhosphateBuffered Saline(PBS)followedby4%paraformaldehydeinPBS.Thebrains wereremoved,post-fixed in4%paraformaldehydefor 24h, and cryoprotectedin30%sucrosefor48hat4◦_C._Coronal_sections_from

cortexandstriatumwerecollectedinaone-in-sixseriesof50␮m (300␮mofinterval)usingacryostat(LeicaCM3050S,Heidelberg, Germany)at−21◦_C.

Thesectionswere washed3 timesfor 10minwithPBS and incubatedwithPBSsupplementedwith10%methanoland1.05% hydrogenperoxidefor40minatroomtemperature(RT),toblock endogenousperoxidase-likeactivities.Afterwashing3timesfor 10minwithPBSandblockingendogenousproteinswith10% nor-malgoatseruminPBSsupplementedwithTritonX-100(blocking solution)fortwohoursatRT,thesectionswereincubatedwith theprimaryantibodies(anti-TNF-␣,1:250 oranti-iNOS,1:400, SantaCruzBiotechnology)dilutedinblockingsolutionat4◦_C_for

48h.Thesectionswerethenwashedthreetimesfor10mininPBS andsubsequentlyincubatedwithavidin–biotin–horseradish per-oxidaseconjugate(ABCStainingSystem,SantaCruzBiotechnology) for30min.Afterwashing,theslideswereincubatedwith biotiny-latedgoatanti-rabbitsecondaryantibody,diluted1:500inblocking solution.The color was developed using DAB as a chromogen. ThesectionsweremountedinEntellan(Merck,Germany),cover slippedandvisualizedunderamicroscope(NikonElipseE200)at 40×zoom.Cellswereconsideredpositivelylabeledwhenabrown colorationwasundoubtlyobservedoverahematoxiline counter-coloration.Threefieldsof each sectionwereselectedrandomly alongthelesionareaandquantificationofimageswascarriedout usingImageJsoftware(NIH,Bethesda,MD,USA).

ForGFAPimmunostaining,sectionswerewashed3timesfor 10minwithPBSandsimultaneouslypermeabilizedandblocked with0.2% Triton-X and 10% horse serum in PBS for 1hat RT. Thesectionswereincubatedfree-floatingwiththeprimary anti-body (anti-GFAP, 1:500, rabbit polyclonal, Sigma-Aldrich, USA) dilutedin blocking solutionat 4◦_C _for ₄₈_h. _The _sections_were

thenwashedthreetimesfor10mininPBSwith0.25%Triton-X, followedbyincubationwiththesecondaryantibody(AlexaFluor 594donkeyanti-rabbit,1:500,Invitrogen,Portugal).Thesections werewashed3timesinPBSandmountedinFluoromount(Sigma), cover slippedand visualized undera microscope(Nikon Elipse E200)at 40× zoom.The meanfluorescence intensitywasused asasemi-quantitativemeasureoftheimmunoreactivityandwas estimatedusingtheprogramImageJsoftware(NIH,Bethesda,MD, USA).Thevaluesofmeanfluorescenceintensityobtainedforthe controlgroup(SO)wereaveragedandallofthemeanfluorescence intensityvalueswerecalculatedasapercentageofthatmean.

2.12. Cellviabilityevaluation

The cresyl-violet staining is used to show the Nissl bodies presentin thecytoplasm of viable neurons[22]. Brainsections (50␮mthickand300␮mapart)weremountedonslideswith5% gelatin,lefttodryandthenincubatedinacetate-buffered0.5% cre-sylvioletsolutionfor10min.Then,theslidesweredehydratedin alcohol(50,70and100%).Theywerethenimmersedinxyleneand mountedwithEntellan(Merck,Germany).Theslideswereviewed underamicroscope(NikonE200Ellipse)with40×magnification. Threeslicesfromeachanimalwererandomlyselectedalongthe lesionareaandquantifyingthecellswasperformedusingImageJ software(NIH,Bethesda,MD,USA)withagride100.Cellswere consideredpositivewhentheyshowedcresylvioletstaininginthe cytoplasmofcells, aswellasnormalmorphology(cellroundor ovalnucleiwithcentralized).Cellswereconsiderednegativewhen

Etyol 4 Etyol 1 Etyol 2 Etyol 4

12 14 16 18 20

*

#

SO

pM CAO

s

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Fig.1.Eriodictyolintakereducestheischemia-inducedneurologicaldeficits.Swiss malemicereceivedeithervehicle(Tween80+saline)oreriodictyol (1,2,and 4mg/kgp.o.,n=6)at30minbeforeandtwohoursaftertopMCAOorasham operation(SO).Theneurologicalscore,calculatedasacompositeofsixmeasured itemsandmeasured24hafter ischemia,rangedfrom3to18andthehigher thescore,thebetterthemotorperformance.Dataareexpressedasthemedian (interquartileranges).*p<0.05vs.SOgroupand#_p_<_0.05_vs._pMCAO_group,_using

aKruskall–WallisandMann–Whitneytests.

noviolet colorationassociated withmorphologicalchanges, i.e. shrunkencellnucleiaccompaniedbypyknoticnuclei,waspresent. Theresultswereexpressedaspercentage ofcresylpositive and cresylnegativecells.

2.13. Statisticalanalysis

Dataareexpressedasmean±S.E.M.orasmean(interquartile range)and statistical differenceswereestimated usingeither a one wayANOVA, Tukeypost hoctest or a Kruskall-Wallis and Mann-Whitneytests. Analyseswere performedwiththeuseof GraphPad Prism software (GraphPad Software Inc., La Jolla,CA, USA).P-values<0.05wereconsideredsignificant.

3. Results

3.1. EriodictyolimprovedneurologicaldeficitsinpMCAOmice

Theneurologicaldeficitsscoreswereassessed24hpoststroke induction.AsshowinFig.1,thepMCAOanimalsshowedsignificant neurologicaldeficits(reducedmotorabilityanddecreasedability torespondtostimulionthesideofthebodycontralateraltothe ischemia)comparedtoshamoperatedanimals(SO:17.8(17–18); SO+Etyol4:17.8(17–18);pMCAO:14.8(14–16),p<0.05). Eriod-ictyol(Etyol)treatmentat4mg/kgdoseimprovedtheneurological deficitsinducedbypMCAO(pMCAO+Etyol4:17(16–18),p<0.05).

3.2. EriodictyolreducedcerebralinfarctsizeinpMCAOmice

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Fig.2. Eriodictyolintakereducestheischemia-inducedinfarctarea.Swissmalemicereceivedeithervehicle(Tween80+saline)oreriodictyol(1,2,and4mg/kgp.o.,n=6) at30minbeforeandtwohoursafterpMCAOorashamoperation(SO).Thepercentageofinfarctareaat24hafterpMCAO(B),wascalculatedfromcoronalsectionsstained with2%2,3,5-triphenyltetrazolium,asshownintherepresentativesetofimages(A).Thepale-coloredregionshowstheinfarctregion,whereasthereddishcoloredregion showstheviabletissue.Dataareexpressedasmean+SEM.*p<0.05vs.SOgroupand#_p_<_0.05_vs._pMCAO_group,_using_a_{Kruskall–Wallis}_and_{Mann–Whitney}_tests.

Etyol 4 Etyol 1 Etyol 2 Etyol 4 0

20 40 60 80 100

Crossings Rearings

*

# #

SO pM CAO

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Fig.3.Eriodictyolattenuatestheischemia-inducedlocomotormodifications.Swiss malemicereceivedeithervehicle(Tween80+saline)oreriodictyol (1,2,and 4mg/kgp.o.,n=8)at30minbeforeandtwohoursafterpMCAOorasham oper-ation(SO)andonceadayinthefollowingdays.Miceweretestedforhorizontal explorationandverticalexplorationinanopenfieldat72haftersurgery.Dataare expressedasmean+SEM.*p<0.05vs.SOgroupand#_p_<_0.05_vs._pMCAO_group,

usingaKruskall–WallisandMann–Whitneytests.

3.3. EriodictyolimprovedlocomotoractivitydeficitsinpMCAO mice

The locomotor activity was evaluated 72h after ischemia. Nosignificantdifferencewasfoundin thenumber ofcrossings between groups. However, the number of rearings was lower in the pMCAOgroup compared to thesham (SO: 11.88±1.76; SO+Etyol4:13.38±1.63;pMCAO:5.25±0.88,p<0.05). Eriodic-tyoltreatmentatdosesof2and4mg/kg,significantlypreventedthe reductioninthenumberofrearings(pMCAO+Etyol1:8.87±2.33; pMCAO+Etyol 2: 18.75±2.34; pMCAO+Etyol 4: 19.00±2.08, p<0.05)(Fig.3).

3.4. Eriodictyolimprovedworkingandaversivememorydeficits inpMCAO

TheanimalssubjectedtopMCAOshowedsignificantdeficitsin workingmemoryat72hafterischemia;thiseffectwasprevented by eriodictyol (spontaneous alternations−SO: 79.73±2.47; SO+Etyol 4:78.75±1.04; pMCAO: 50.88±3.39; pMCAO+Etyol 1:73.50±2.67;pMCAO+Etyol2:76.13±0.95;pMCAO+Etyol4: 77.75±4.29,p<0.05)(Fig.4A).

The step-downlatency of animalssubjected topMCAO was significantly shorter compared to the sham-operated group 72 and96haftertheinductionofischemia(Latency–earlymemory: SO:244±18.81;SO+Etyol4:265±16.90;pMCAO:31.75±5.15; lateMemory:SO:270±16.28;SO+Etyol4:261±14.89;pMCAO: 26.63±9.45, p<0.05). These findings demonstrated significant deficitsinearlyand late memories.Eriodictyol atdose4mg/kg showedprotectionagainstdeficits(pMCAO+Etyol4:212.6±26.64, p<0.05)(Fig.4B).

3.5. Eriodictyoladministrationdecreasedmyeloperoxidase activityandpro-inflammatorymediatorsinpMCAOmice

pMCAOincreasedenzymaticactivityofmyeloperoxidase(MPO) intheipsilateralcortexbutnotinstriatum24haftersurgery com-pared to sham-operated animals (SO: 5.35±1.57; SO+Etyol 4: 7.24±2.50;pMCAO:43.45±2.73u/mgtissue,p<0.05).Treatment witheriodictyol at doseof 4mg/kg significantly decreased this effect(pMCAO+Etyol4:5.00±0.97u/mgtissue,p<0.05)(Fig.5). OurfindingsalsodemonstratedthatthenumberofTNF-␣-positive (Fig.6AandB)andiNOS-positivecells(Fig.7AandB)were sig-nificantlyloweronischemicpenumbraincortexandstriatumof animalstreatedwitheriodictyolcomparedwithpMCAOgroup.

3.6. EriodictyolattenuatedastroglialcellsactivationinpMCAO mice

Theischemicbraindamagepromotedtheactivationof astro-cytesdemonstratedbyanincreasedglialfibrillaryacidicprotein (GFAP). Astrogliosiswasevidenced incortex andstriatum from pMCAOanimals.GFAPimunoreactivitywassignificantlydecreased with the highest eriodictyol dosage (cortex: pMCAO+Etyol 4: 200.2±28.9;striatum:pMCAO+Etyol4:232.1±42.82)(Fig.8Aand B).

3.7. Eriodictyolincreasedthenumberofviablecellsincortexand striatumfrompMCAOmice

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Etyol 4 Etyol 1 Etyol 2 Etyol 4 0 20 40 60 80 100

*

# # #

SO pM CAO

) %( s n oi t a nr et l a s u o e n at n o p S

Etyol 4 Etyol 1 Etyol 2 Etyol 4 0 100 200 300 Training Early Memory Late Memory

* *

# # SO pMCAO La te n cy (s )

A

B

Fig.4.Eriodictyolattenuatestheischemia-inducedmemorydeficits.Swissmalemicereceivedeithervehicle(Tween80+saline)oreriodictyol(1,2,and4mg/kgp.o.,n=8) at30minbeforeandtwohoursafterpMCAOorashamoperation(SO)andonceadayinthefollowingdays.Miceweretested72haftersurgeryforworkingmemoryina Y-mazetest[A,wherethepercentageofalternationwascalculatedaccordingtotheformula:totalofalternations/(totalarmentries−2)×100]andforaversivememory[B, estimatedattwodifferenttimeintervals(15minand24h)].Dataareexpressedasmean+SEM.*p<0.05vs.SOgroup,#_P_<_0.05_vs._pMCAO_group,_using_a_{Kruskall–Wallis}

andMann–Whitneytests.

Etyol 4 Etyol 4 0 10 20 30 40 50

Cortex

Striatum

*

#

SO pM CAO

) e u s si t g m/ U( yti vi t c a O P M

Fig.5. Eriodictyol(4mg/kg)preventstheincreaseofMPOactivityinthecortexafter pMCAO.Swissmalemicereceivedeithervehicle(Tween80+saline)oreriodictyol (4mg/kgp.o.,n=6)at30minbeforeandtwohoursafterpMCAOorashamoperation (SO).MPOactivity,asaneutrophilinfiltratemarker,wasevaluatedincorticaland striataltissue.Dataareexpressedasmean+SEM.*p<0.05vs.groupand#_p_<_0.05

vs.pMCAOgroup,usingaKruskall–WallisandMann–Whitneytests.

cellswithcentralizednuclei.Inanimalstreatedwitheriodictyol, thenumber and cellular morfology wassignificantly preserved (Fig.9A–C).

4. Discussion

Thisstudyprovidedthefirstreportofpreclinicalevidenceofthe neuroprotectiveeffectoftheflavonoideriodictyol(Etyol)on mem-orydeficitsandneuroinflammationinmicesubjectedtopermanent middle cerebralartery occlusion (pMCAO). Etyol is a flavonoid isolatedfromtheChineseherb Dracocephalumrupestrethathas anti-inflammatory[13]andantioxidantproperties[14,15].

The permanent middle cerebral artery occlusion (pMCAO) modelby electrocoagulationin miceprovedextremely reliable, inducingneuronaldamage[23]andcognitivedeficits[24,25].Thus, thismodelwaschosenforthestudyofprotectiveeffectonmemory impairmentfromeriodictyolduringtheischemicevent.

Aspreviouslyreported[26,27]infarctsmodeledbythedistal middlecerebralarteryocclusionmodelinmiceencompassabout 10–15%ofthehemisphere,therebymimickingamajorityofhuman strokelesionswhichare locatedinthecorticalmiddlecerebral arteryterritory[28,29].Ourresultsshowed24hafterthepMCAO

a lesionsize around12%that wasconsistentwitha previously publishedreport[26].Wefoundthattheoraladministrationof Etyolimprovedneurologicalfunctionandreducedinfarctareain theischemicanimals.ThesefindingsareconsistentwithJingetal.

[9],which showedthateriodictyol-7-O-glucoside(E7G) ina rat modeloffocalcerebralischemia,significantlyreducedbrain dam-ageandamelioratedneurologicaldeficits.Otherflavonoidssuch as(−)-epicatechin[30],luteolin[31]andquercetin[32]havealso shown neuroprotective effects against ischemia-induced injury. Themechanismsofflavonoidsneuroprotectionmayinvolvetheir interactionwithneuronalandglialsignalingcascadesinthebrain leadingto apromotion of neuronal survivaland differentiation

[33,34],theincreaseofperipheralandcerebralvascularbloodflow thatmayleadtotheinductionofangiogenesis[35],andby react-ingdirectlywithandscavengespeciesandproinflammatoryagents

[36].

Evidencehasshownthat thereissignificantlossofneuronal viabilityinanimalssubjectedtopMCAOandthatthemostaffected areasarethecortexandthestriatum[37,38].Inthepresentstudy, weobservedsignificantlossofneuronalviabilitybothincortexand striatumofischemicanimals5daysafterpMCAO.Etyoltreatment significantlyprevented this celldeathin both structures.These resultsareconsistentwithSunil etal. [39]study thatreported theneuroprotectiveactionoftheflavonoidsobtainedfrom Cype-rusrotundusonthecelldeathinducedbyexperimentalcerebral ischemiainrodents.

Humandeficitsafterstrokeoccurwithdamagetospecific cir-cuits,suchasinmotorcortexmaps[40]and recoveryfollowsa reproduciblydeterminedfunctionalreorganization inthebrain. Motor,sensory,andlanguagerecoveryinvolvesaprogressive reor-ganizationandrecoveryofactivationinperi-infarctandipsilateral connectedcorticalsitesafterstroke[41,42].Inthis study,Etyol treatmentprotectedanimalsfrommotorimpairment,probablyby reducingtheinfarctedarealocatedinthemotorcortexand stria-tum.Flavonoislikerutin [43],andsilymarin[44]alsoprotected animalsfrommotorimpairmentinducedbybrainischemia.

The cortex, striatum and hippocampus are brain structures related tocognition and memory functions[45]. These regions are themost strongly affectedby MCAO [46].Previous studies using pMCAO-induced neurological deficits have demonstrated mnemonicdeficitsinrodents[24,47–49].

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Fig.6. Eriodictyol(4mg/kg)attenuatestheincreaseofTNF-␣expressioninthecortexandstriatumofmicesubmittedtopMCAO.Swissmalemicereceivedeithervehicle (Tween80+saline)oreriodictyol(4mg/kgp.o.,n=4)at30minbeforeandtwohoursafterpMCAOorashamoperation(SO)andonceadayinthefollowingdays.TNF-␣ immunostainingwasperformedinseriesofcoronalsections(50␮mthickand300␮mapart)representativeoftheipsilateralcortexandstriatum(A).Thebargraphs(B) showthequantificationofthenumberofimmune-positivecellsforTNF-␣.Dataareexpressedasmean+SEM.*p<0.05vs.SOgroup,#_p_<_0.05_vs._pMCAO_group,_using_a

Kruskall–WallisandMann–Whitneytests.

information.Thedorsolateralprefrontalcortexhasbeenknownto participateinworkingmemory[50].Wecanassesstheworking memoryusingtheYmazetest.Thistestisbasedonthetendency of animalstoexplore newenvironments withouttheinfluence of emotional or motivational tricks [51]. Additionally, this test providesdissociation betweenlearning and memorybecauseit doesnotrequirerules tobelearned[52].Inanimals,the explo-rationofanewenvironmentdependsontheintegrityoflimbic andnon-limbicsystems,suchasthehippocampus,basalforebrain, prefrontalcortex,thalamus,dorsalstriatumbeyondthe vestibu-larsystemand thecerebellum [51].Carmoet al.[49] observed deficitsinworkingmemoryinmiceafterpMCAO.Inthepresent study, the animals that underwent pMCAO showed deficits in workingmemory72haftertheinductionofischemia,andEtyol treatmentpreventedthesedeficits. Therearereports inthe lit-eraturethat showthat phenolic compoundssuchas flavonoids are abletoimprove performance of animalssubjected to tran-sientcerebralhypoperfusion-inducedontheY-maze;thishasbeen

demonstratedfororoxylinA,aflavonoidderivedfromthe Scutel-lariabaicalensisGeorgiplant[53].

Step-down-type passiveavoidanceparadigmis basedonthe naturalbehavior ofanimalstoexplorebeyondtheplatformbut alsotodeviatefromaversivesituationgeneratedbytheshockin theelectrifiedpartoftheapparatus.Therefore,thismodelcanbe usedtostudyaversivememory[54].AnimalssubjectedtoMCAO havedeficitsinrecentandlatememory,asevaluatedinthe pas-siveavoidancetest[55].Inthisstudy,weobservedthatthepMCAO groupshoweddeficitsinearlyandlateaversivememory,and treat-mentwithEtyolprotectedtheanimalsfromthisdeficit.Thisresult corroboratesthefindings of Wuet al. [56],which showedthat theisoflavonepuerarincouldimprovethelearning-memoryability afterglobalcerebralischemiaandreperfusioninratsevaluatedby thepassiveavoidancetest.

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Fig.7.Eriodictyol(4mg/kg)attenuatestheincreaseofiNOSexpressioninthecortexandstriatumofmicesubmittedtopMCAO.Swissmalemicereceivedeithervehicle (Tween80+saline)oreriodictyol(4mg/kgp.o.,n=4)at30minbeforeandtwohoursafterpMCAOorashamoperation(SO)andonceadayinthefollowingdays.iNOS immunostainingwasperformedinseriesofcoronalsections(50␮mthickand300␮mapart)representativeoftheipsilateralcortexandstriatum(A).Thebargraphs(B) showthequantificationofthenumberofcellsimmune-positiveforiNOS.Dataareexpressedasmean+SEM.*p<0.05vs.SOgroup,#_p_<_0.05_vs._pMCAO_group,_using_a

Kruskall–WallisandMann–Whitneytests.

exertpharmacologicalactivityonreceptors,kinasesand transcrip-tionfactors[36].Flavonoidsmayinteractthroughthemodulation ofkinaseactivity,includingMAPKkinase,MAPKkinaseorMAPKs; through ATP-binding enzymes and receptors; by changing the roleof phosphatasesthat actin opposition tokinases;through activationoftheconnectionbetweentranscriptionfactorsand pro-motersequences;bymaintainingthehomeostasisof Ca++_,_thus preventingthekinase-dependentinfluxofCa++_into_neurons_[36]_. Whenthesepathwaysare activated, thesynthesis of new pro-teinsisinducedinneurons,promotingneuroplasticityandcausing morphologicalchangesthatdirectlyinfluencetheacquisition, con-solidationandstorageofmemory[34].Thus,itispossiblethatEtyol, beinga flavonoid,haspreventedworkingandaversivememory deficitsbyactingonsomeofthesepathways,butthemechanism hasnotbeeninvestigatedinthiswork.

Studieshave shownthatacuteneuroinflammatory processes areresponsibleformuchofcerebralischemictissueinjury[6,7]. Neutrophilsaretheearliestleukocytestomigratetotheischemic

focus.Myeloperoxidase(MPO)isanenzymepresentinthegranules ofneutrophils[59].HighMPOactivitywasestablishedasa quanti-tativeindicatorofleukocyteinfiltrationduetoneuroinflammation inischemicanimals[60].

In ourstudy, a significantincrease in the MPOactivity was observed24hafterpMCAOinthetemporalcortexbutnotinthe striatumofischemicanimals.InagreementtothisresultTuetal.

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Fig.8.Eriodictyol(4mg/kg)attenuatestheincreaseofGFAPexpressioninthecortexandstriatumofmicesubmittedtopMCAO.Swissmalemicereceivedeithervehicle (Tween80+saline)oreriodictyol(4mg/kgp.o.,n=4)at30minbeforeandtwohoursafterpMCAOorashamoperation(SO)andonceadayinthefollowingdays.The immunoreactivityofGFAPwasperformedinseriesofcoronalsections(50␮mthickand300␮mapart)representativeoftheipsilateralcortexandstriatum(A).Thebar graphs(B)showthequantificationofthemeanfluorescenceintensity(MFI)expressedasapercentageofthemeanMFIoftheSOgroup.Dataareexpressedasmean+SEM. *p<0.05vs.SOgroup,#_p_<_0.05_vs._pMCAO_group,_using_a_{Kruskall–Wallis}_and_{Mann–Whitney}_tests.

theactivation of microglia and astrocytes after brainischemia, whichcontributestotheproductionofvariouspro-inflammatory mediators[65]andanti-inflammatorytherapieshaveshown neu-roprotectiveeffectsinanimalssubjectedtopMCAO[66].Astrocytes playafundamentalroleinmaintainingbrainhomeostasis.They expressglialfibrillaryacidicprotein(GFAP),a astrocyticprotein thatcanbeusedtodistinguishastrocytesfromothercelltypes[67]. Inanytypeofbraininjury,suchasinflammation,surviving astro-cytesintheaffectedareasareactivated.Theybegintoproliferate andtohypertrophy;thiseventiscalledreactiveastrogliosis[68].

In the present study, the animals that underwent pMCAO showedsignificantastrogliosisinthetemporalcortexandstriatum fivedaysaftertheischemicinduction,corroboratingtheresultsof Yangetal.[69]whoobservedGFAP-positiveastrocytesinpMCAO model.WehaveshownthatEtyoltreatmentsignificantlyprevented theincreaseinGFAPimmunoreactivityinthecortexandstriatum ofischemicanimals.

Jingetal.[9]havedemonstratedthateriodictyol-7-O-glucoside (E7G)significantlyprotectsagainst astrocyte-inducedcelldeath

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Fig.9.Eriodictyol(4mg/kg)attenuatestheincreasecelldeathinthecortexandstriatumofmicesubmittedtopMCAO.Swissmalemicereceivedeithervehicle(Tween 80+saline)oreriodictyol(4mg/kgp.o.,n=4)at30minbeforeandtwohoursafterpMCAOorashamoperation(SO)andonceadayinthefollowingdays.Thecresylviolet stainingwasperformedinseriesofcoronalsections(50␮mthickand300␮mapart)representativeoftheipsilateralcortexandstriatum(A).Thebargraphs(BandC)showthe quantificationofthenumberofcellsstainedwithcresylviolet.Dataareexpressedasmean+SEM.*p<0.05vs.SOgroup,#_p_<_0.05_vs._pMCAO_group,_using_a_{Kruskall–Wallis}

andMann–Whitneytests.(Forinterpretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle.).

Inthisstudy,iNOSandTNF-␣immunoreactivityinthecortex andstriatumwassignificantlyhigherintheanimalssubjectedto pMCAOthanintheanimalsoftheSOgroupatfivedaysafterthe ischemicinduction.OtherstudiesreportedanincreaseinTNF-␣ expressionand iNOSinanimalssubjectedtopMCAO[74].Etyol treatmentpreventedtheincreasesinTNF-␣andiNOSexpression onischemicanimals,corroboratingpreviousstudiesthatshowed theability of Etyol tosuppress TNF-␣ and iNOS production in macrophagesstimulatedwithLPS,throughToll-likereceptortype 4blockade[75].Furthermore,Etyolreducedthecytokines(TNF-␣, IL-1␤andIL-6)andNOproductioninRAW264.7cellsstimulated withLPS,bydirectblockadeofNF-kB[13].

Vafeiadouetal.[76]havebeenshownthathesperetinand narin-genin,twoflavonoidsofthesameflavanonegroupthatEtyol,and theflavanols(+)-catechinand(−)-epicatechin,attenuated LPS/IFN-gamma-inducedTNF-alpha productionin glial cells. Naringenin alsoinhibitedLPS/IFN-gamma-inducediNOSexpressionandnitric oxideproduction,andalsoinhibitedLPS/IFN-gamma-inducedp38 mitogen-activated protein kinase (MAPK) phosphorylation and downstream signal transducer and activator of transcription-1 (STAT-1)inLPS/IFN-gammastimulatedglialcells.Itislikelythat theeriodictyol,tobeofthesamechemical groupofhesperetin

and naringenin, has its anti-inflammatory action acting in the p38/MAPKsignalingpathway,butthisroutehasnotbeen inves-tigatedinthiswork.

5. Conclusions

In conclusion, the present study, for the first time, shows neuroprotective potential of eriodictyol in experimental brain ischemia.Eriodictyolmarkedly reducedcerebralinfarction, neu-ronal damage, sensorimotor and memory deficits. We propose thatanti-inflammatory property,at leastinpart,couldaccount forneuroprotectiveactionoferiodictyol.Althoughpriortreatment oferiodictyolamelioratedneuronaldamageinducedbyischemic injury,anassessmentofearly-treatmentefficacyandwindowof opportunityneedtobestudiedinfuturetoidentifyclinical rele-vanceoferiodictyolinstroketreatment.

Conflictofinterests

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financialsupportforthisworkthatcouldhaveinfluencedits out-come.

Acknowledgements

WewishtothanktheBrazilianNationalResearchand Develop-mentCouncil(CNPq),INCT-IBISAB-Capes,andtheResearchSupport FoundationofCeará(FUNCAP)forfinancialsupportintheformof grantsandfellowshipawards.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.bbr.2016.06.046.

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