Rosmarinic acid prevents against memory deficits in ischemic mice

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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

Rosmarinic

acid

prevents

against

memory

deficits

in

ischemic

mice

Analu

Aragão

Fonteles

a,c

,

Carolina

Melo

de

Souza

b

,

Julliana

Catharina

de

Sousa

Neves

a

,

Ana

Paula

Fontenele

Menezes

b

_,

_Marta

_Regina

_Santos

_do

_Carmo

a

_,

Francisco

Diego

Pinheiro

Fernandes

b

_,

_Patrícia

_Rodrigues

_de

_Araújo

b

_,

Geanne

Matos

de

Andrade

a,b,c,∗

a_{Post-Graduate}_Programme_in_{Pharmacology,}_Department_of_Physiology_and_{Pharmacology,}_Fortaleza,_Brazil

b_{Post-Graduate}_Programme_in_Medical_Sciences,_Department_of_Medicine,_Faculty_of_Medicine,_Federal_University_of_Ceará,_Fortaleza,_Brazil c_Institute_of_Biomedicine_of_Brazilian_Semi-Arid,_Fortaleza,_Brazil

h

i

g

h

l

i

g

h

t

s

•Rosmarinicacidpreventsmemorydeficitsinducedbypermanentfocalcerebralischemia.

•Rosmarinicacidinducedsynaptogenesisinischemicmice.

•IncreasedBDNFwereobservedinischemicmicetreatedwithrosmarinicacid.

•RosmarinicaciddiminishedMPOactivityandastrogliosisinischemicmice.

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t

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Articlehistory: Received10August2015 Receivedinrevisedform 17September2015 Accepted23September2015 Availableonline9October2015

Keywords: Cerebralischemia Memorydeficits Polyphenols Rosmarinicacid

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Polyphenolshaveneuroprotectiveeffectsafterbrainischemia.Ithasbeendemonstratedthatrosmarinic acid(RA),anaturalphenoliccompound,possessesantioxidantandanti-inflammatoryproperties.To evaluatetheeffectivenessofRAagainstmemorydeficitsinducedbypermanentmiddlecerebralartery occlusion(pMCAO)miceweretreatedwithRA(0.1,1,and20mg/kg/day,i.p.beforeischemiaandduring5 days).Animalswereevaluatedforlocomotoractivityandworkingmemory72hafterpMCAO,andspatial andrecognitionmemories96hafterpMCAO.Inaddition,inanothersetofexperimentsbraininfarction, neurologicaldeficitscoreandmyeloperoxidase(MPO)activitywereevaluates24hafterthepMCAO. Finally,immunohistochemistry,andwesternblot,andELISAassaywereusedtoanalyzeglialfibrillary acidicprotein(GFAP),andsynaptophysin(SYP)expression,andBDNFlevel,respectively.Theworking, spatial,andrecognitionmemorydeficitsweresignificantlyimprovedwithRAtreatment(20mg/kg).RA reducedinfarctsizeandneurologicaldeficitscausedbyacuteischemia.ThemechanismforRA neuro-protectioninvolved,neuronallosssuppression,andincreaseofsynaptophysinexpression,andincrease ofBDNF.Furthermore,theincreaseofMPOactivityandGFAPimmunireactivitywerepreventedinMCAO grouptreatedwithRA.TheseresultssuggestthatRAexertsmemoryprotectiveeffectsprobablydueto synaptogenicactivityandanti-inflammatoryaction.

1. Introduction

Strokeisthemajorcauseofdisabilityandthefourthleading causeofdeathworldwide.Approximately100,000deathsoccurs annuallyduetostroke[1,2,].Ischemicinjury isassociated with

∗Correspondingauthorat:Post-GraduateProgrammeinPharmacology, Depart-mentofPhysiologyandPharmacology,FacultyofMedicine,FederalUniversityof CearáRuaCel.NunesdeMelo1127,PorangabussuFortaleza60430-270,CE,Brazil. Fax:+558533668333.

E-mailaddress:gmatos@ufc.br(G.M.deAndrade).

vascularleakage, inflammation,tissue injury,andcelldeath[3]. Cellularchangesassociatedwithischemiaincludeimpairmentof metabolism,energy failure,free radicalproduction, excitotoxic-ity,alteredcalciumhomeostasis,andproteaseactivation;allthese eventsaffectbrainfunctionandcontributetolongterm disabili-ties[4].Ithasbeenconfirmedthatstrokecanresultincognitive impairment,andtheprevalenceofpost-strokecognitive impair-mentrangesfrom20% to80%[5]and morethana thirdof the patientshavecognitiveimpairmentaftertransientischemicattack [6].Amechanisminvolvedincognitivedeficitsafterstrokeis synap-ticproteinloss[7].Thedisappearanceofsynapticactivityisone

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ofthefirstconsequencesofischemia[8].Abloodflowlessthan 20%leadstoalossofaxonsanddendritesinminutes[9,10]. More-over, inflammation plays a significantrole in the pathogenesis ofischemicstroke[11].Theinflammationresponsesafterbrain ischemiaincludeastrocyteandmicroglialactivation,followedby peripheralinflammatorycellinfiltrationinthecoreandpenumbra zoneofischemia[12,13].Ischemiaevokesstrongreactive astroglio-sisthatactivatesinducednitricoxidesynthase(iNOS)andNADPH oxidasetoproduceNOandsuperoxide,whichareinvolvedinlipid peroxidation,andthereleaseofcytokines,suchasTNF andIL-1 [12].Thesecytokines,dependingontheconcentrations,mayinhibit synaptictransmissionoractasneuromodulators[14].Afterbrain ischemia,astrogliosishasbothbeneficialanddeleteriouseffects andastrocyteactivationisanimportantproliferativecomponent duringsynaptogenesis[8,15].Studiesshowthatastrocytesrelease mediatorsthatinfluencefromthegenesisofsynapsesuntiltheir stabilization[16,17].Inadditiontoastrogliosis,neurotrophic fac-torssuchasBDNFplayapivotalroleinsynapticplasticity[18], neuronalsurvival, and growth[19].Studiesshowthat systemic administrationofBDNFpromotesrecoveryafterstroke[20].

Rosmarinic acid (RA) is a phenolic compound with potent antioxidantandanti-inflammatoryactivities[21].RAhas neuro-protectiveactioninanimalmodelsofneurodegenerativediseases suchasAlzheimer’sdisease[22–24]and,Parkinson’sdisease[25], aswellasinischemia/reperfusionmodels[26].However,currently, therearenoreportsregardingtheprotectiveeffectsofrosmarinic acidonmemorydeficitsfollowingfocalcerebralischemia.Thus,the aimofthepresentworkwastoinvestigatetheeffectofRAon mem-orydeficitsinducedbypermanentfocalcerebralischemia,thereby investigatingthemechanismofaction.

2. Methods

2.1. Subjects

MaleSwissmiceweighing25–30gobtainedfromtheCentral AnimalHouseofPhysiologyandPharmacologyDepartmentof Fed-eralUniversityofCearáwereused.Animalswerehousedundera

12-h-light,12-h-darkcycleand allowedfreeaccesstofoodand water.Allproceduresinthisstudywereinagreementwiththe GuidefortheCareandUseofLaboratoryAnimalsbytheInstitute forLaboratoryAnimalResearchoftheNationalResearchCouncil publishedbytheNationalAcademiesPress(Washington,District ofColumbia,USA)andwereapprovedbytheethicscommitteeon animalexperimentationoftheFederalUniversityofCeará.

2.2. Drugs

The following drugs were used: rosmarinic acid (SIGMA, USA);xylazine(2%,Kensol®

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

,König,Argentina).Allotherreagentswereof analyti-calgrade.

2.3. Inductionofpermanentmediacerebralarteryocclusion (pMCAO)

Permanentmiddlecerebralarteryocclusion(pMCAO)was pro-ducedbyelectrocoagulationoftheleftmiddlecerebralarteryas reportedpreviously[27].Briefly,animalswereanaesthetizedwith xylazine (10mg/kg, intraperitoneally) and ketamine (90mg/kg, intraperitoneally), an incision was made on the left temporo-parietalregion,andthetemporalismusclewaspartiallyremoved.A burrholewasdrilledintotheskulloverthemiddlecerebralartery andthevesselwasoccludeddirectly,proximaltothelateral lenticu-lostriatebranches,usingelectrocoagulationwithamicro-unipolar coagulator.Thecompleteinterruptionofthebloodflowwas con-firmedbyvisualinspection.Bodytemperaturewaskeptcloseto 37◦_C. _{Sham-operated}_animals_(SO_group)_were_subjected_to_the

sameprocedure,withtheexceptionofcauterizationofthe mid-dlecerebralartery.Thirtyminutesbeforeand 1hafterpMCAO, theanimalsreceivedeitherNaCl0.9%(pMCAOgroup)orRA(0.1,1 and20mg/kg/day,intraperitoneally)for5consecutivedays.There weretwogroupsofsham-operatedanimals:onegroupreceived NaCl0.9%andtheothergroupwastreatedwithRA(20mg/kg/day). Inthefirstsetofexperiments,animalsweretestedfor neurologi-calevaluationandeuthanizedforischemicdamageevaluation24h

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afterischemia(n=6/group).Inasecondset,animalswere evalu-atedforlocomotoractivityandworkingmemory(n=8/groups)at 72haftersurgery,andimmunohistochemistry(n=4/group)at96h aftersurgery.Inathirdset,animalswereevaluatedat4and5days for recognitionandspatial memories(n=8/group) and western blot(n=4/group).Immunohistochemistryandwesternblot anal-ysisandbehavioralinvestigationswereperformedattheindicated timepoints(Fig.1).

2.4. QuantificationofinfarctareathroughTTCstaining

Six animals of each group were euthanizedby cervical dis-location 24h after ischemia. Brains were removed and 2-mm coronal sections were made from the prefrontal cortex to the midbrain. Slices were immersed in 1% solution of TTC (2,3,5-triphenyltetrazolium)innormalsalineat37±◦_C_for₁₅_min._This

saltacceptsaprotonfromsuccinatedehydrogenaseintheinner membraneofthemitochondriawhichreducesittoitsred, insol-ubleformknownasformazan[28].Thus,anareawithinactive enzymesandtheinfarctionisnotstainedandappearspale.The unstainedareasweremeasuredbytheOsirissoftware(University ofGeneva,Switzerland,Switzerland)andcalculatedasthepercent oftheentirecoronalsection.

2.5. Neurologicalevaluation

Neurologicalevaluationwasperformed24hafterischemiaby aninvestigatorwhowasblindedtotheexperimentalgroupdesign. Theneurologicfindingswerescoredusingascaledescribed previ-ouslybyGarciaetal.[29].Sixitemsweremeasuredandthetotal scorerangedfrom3to18;thehigherthescore,thebetterthemotor performance.Items1–4(spontaneousactivity,symmetryof move-ments,symmetryofforelimbs,andclimbingthewallofwirecage) measuredmotorperformance,items5–6(reactiontotouchonand responsetovibrissaetouch)measuredsensoryfunction(Table1).

2.6. Open-fieldtest

Theanimalsinthesubgroup2weretestedforlocomotor activ-ityusinganopen-fieldapparatus,whichconsistedofablackacrylic chamber(30×30cm)withthefloordividedintoninesquaresof equalareas[30].Seventy-twohoursaftersurgery,theanimalwas positionedinthecentreofthearenaandallowedtoexplorefreely. Thenumberofcrossings(horizontalexploration)andrearings (ver-ticalexploration)werescoredfor5min.Thearenawascleanedwith 20%ethylalcoholtoremoveanyodorsbeforethenexttest.

2.7. Y-mazetest

The third day after surgery, spatial working memory was assessed byrecordingspontaneousalternation (SA)behavior in theY-maze[31].Themazewasconstructedofwhitewoodwith threeidenticalarms(40×15×4.5cm)positionedatequalangles. Animalswereplacedattheendofonearmandallowedtomove freelythroughthemazeduring an8-minsession. Theseriesof armsentrieswasrecordedvisuallyandarmentrywasconsidered tobecompletewhenthehindpawsofthemicewerecompletely placedinthearm.Theabilitytoalternaterequiresthatthemice torememberwhicharmshavealreadybeenvisited.Each experi-mentwasscored,andthepercentageofspontaneousalternation wascalculatedusingthefollowingformula:

SAs(%)= Alternationbehavior

Maximumalternations×100

wherealternationbehaviorisdefinedasconsecutiveentriesinto eachofthethreearms,withoutrepetition,andmaximum alterna-tionsarethetotalnumberofarmentries,minustwo.

2.8. Objectrecognitiontest

Thefourthdayaftersurgery,recognitionmemorywasassessed by the object recognitiontest [32]. Theobject recognitiontest wasperformedin ablack acrylicchamberopen-fieldapparatus (30×30cm).Animalswereplaced intheexperimentalroomat least30minbeforetesting.Intheacquisitionsessionanimalswere allowedtofreelyexploretwoobjectsplacedinthebackleftand rightcornersoftheapparatusfloor-fixedfor10min.Theanimals were placed at the mid-point of the wallopposite thesample objects.Whenplaced,itsbodywasparalleltothesidewallsand itsnosepointingawayfromtheobjects.Aftertheplanned sample-objectexposuretime,theanimalwasremovedfromtheapparatus andreturnedtothecage.Theobjectswerecleanedbetweeneach trialusing20%ethylalcoholtoavoidodortrails.Inthetest ses-sion (1h after acquisition session), one of the familiar sample objectswasplaced inonebackcorneroftheapparatusand the novelobjectintheotherbackcorner.Theobjectsweredifferent in shapesandcolors. Recognitionmemorywasevaluatedusing arecognitionindex(RI)calculatedforeachanimalusingthe for-mula:(N−F/N+F)correspondingtothedifferencebetweenthe timeexploringthenovel(N)andthefamiliarobject(F),corrected fortotaltimeexploringbothobjects.

2.9. Watermazetest

Inthisversionofthewatermazetest,previouslydescribedby Morrisetal.[33],themicelearntoswim atashortestpossible

Table1

DefinitionoftheneurologicalscoreaccordingtoGarciaetal.[29].

Test Score

0 1 2 3

Spontaneousactivity(in cagefor5min)

Nomovement Barelymoves Movesbutdonotapproach atleastthreesidesofthe cage

Movesandapproachat leastthreesidesofthe cage

Symmetryofmovements (4limbs)

Contralateralside:no movement

Contralateralside: slighmovement

Contralateralside:moves slowly

Bothsides:move symmetrycally Symmetryofforelimbs

(outstretchingwhileheld bytail)

Contralateralside:no movement,no outreaching

Contralateralside: slighmovementto outreach

Contralateralside:moves andoutreacheslessthan therightside

Symmetricaloutreach

Climbingwallofwirecage ... Failstoclimb Contralateralsideisweak Normalclimbing Reactiontotouchoneither

sideoftrunk

... Noresponseon Contralateralside

Weakresponseonright side

Symmetricalresponse

Responsetovibrissaetouch ... Noresponseon contralateralside

Weakresponseon contralateralside

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distancefromtheedgesofawatertankforahiddenplatformbelow thesurface.Theylearnguidedbycluesinthewallsorothervisual stimuliexternaltothetank.Thistaskspaceisdependentonthe hippocampus.

Theanimalwasplacedrandomlyinacircularpool(90cm diam-eter×60cmdeep)containingturbidwater(whitenontoxicpaint) at26◦_C,_divided_spatially _into_four_quadrants,_and_should_find_a platform(7cmdiameter)submerged2cmbelowthewaterlevel. Theanimalhad60stofindtheplatform(whichremainedinthe sameplaceeverytrail)andremainedtherefor10s.Thistraining wasconductedfourtimesadayatintervalsof30sfortwodays (learning)and48hafterthelasttrainingsession,theanimalswere tested,nowwithouttheplatform,whichwasassessedmemory retention.On occasion,theanimalremainedinthepoolfor60s andwasrecordedatthetimethattheanimalremainsinthe quad-rantwheretheplatformshouldbe,thelatencytimetofindthe locationoftheplatformandthenumberoftimeshecrossedthe exactlocationtheplatform.

2.10. MyeloperoxidaseassayBradleyetal.[34]

Eightanimalsofeachgroupwereeuthanized24hafterpMCAO andcortex,hippocampusandstriatumwereremoved.Briefly,brain sampleswerehomogenized(50mg/mL)inasolutionofphosphate buffer(PBS)(50mM,pH6.0)containing0.5%of hexadecyltrimethy-lammoniumbromide(HTAB)(Sigma–Aldrich,USA).Homogenates werecentrifugedat 14,000g,4◦_C, _for₂_min,_and _then₃₀_␮_L _of

thesupernatant was mixed with 0.167mg/mL of o-dionisidine dihydrochloride(Sigma–Aldrich, USA)and 0.0005%ofhydrogen peroxide(CarloHerba, Italy)inafinal volumeof230␮L,plated ina96wellplate.Thentheabsorbancechangeat460nmwas mea-suredwithaplatespectrophotometerin0.1and3min.Oneunit ofactivitywasdefinedasthatdegrading1␮molofperoxide/min. ResultsarepresentedasMPOactivity(U/mgtissue).

2.11. Immunohistochemistry

Brainswerefixedwith4%paraformaldehydebytranscardiac perfusionandpost-fixedinthesamesolutionfor24hat4◦_C_and

thenstoredincryoprotectantsolutionofsucrose30%.Free-floating sectionswerefirstrinsedinphosphatebufferedsalinefor5min and then three times for 5min with Tris buffered saline(TBS, 0.05MTrizmabasebuffercontaining150mMofNaCl,pH7.2)at roomtemperature.Slicesweresimultaneouslypermeabilizedand blockedwithTBScontaining0.2%TritonX-100and10%ofnormal goatserum,for1hatroomtemperature.Afterwards,sliceswere incubated,free-floating,withthe primaryantibodyprepared in theblockingsolution(anti-GFAP,1:500dilution,rabbitpolyclonal, Sigma–Aldrich,USA)overnightat4◦_C._Sections_were_then_rinsed

3×10mineach,inTBSandsubsequentlyincubated2hatroom temperature,withgoatanti-rabbitsecondaryantibodyconjugated withafluorophore(AlexaFluor594,diluted1:200intheblocking solution,Invitrogen,Portugal).Afterrising3×for10mininTBS, thebrainsectionswereincubatedwithDAPI(VectorLaboratories, UK)andthenmountedongelatincoatedslidesusingFluoromount mountingmedium(Sigma–Aldrich,USA).Theslideswereallowed todry protected fromlight beforebeingstored at −20◦_C _until

analysis.Sectionswerevisualizedunderafluorescencemicroscope (ZeissImagerZ2)orwithalaserscanningconfocalmicroscope(LSM 510Meta,Zeiss).Theimmunoreactivitywasquantifiedinaseries of8coronalsections(50␮mthickand300␮mapart) representa-tiveofthestriatumandcortexin4animalsfromeachexperimental group.Themeanfluorescenceintensity(MFI)wasusedasa mea-sureoftheimmunoreactivityandwasquantifiedusingtheprogram ImageJ.ThevaluesofMFIobtainedforthecontrolgroup(SO)were

averagedandalloftheMFIvalueswerecalculatedasapercentage ofthatmean.

2.12. Westernblot

Cortical tissue lysates were prepared fifthday after pMCAO andRAtreatment.Briefly,brainsampleswerehomogenizedin5 volumesofice-cold buffer(0.32Msucrose, 1mMEDTA, 10mM Hepes and 1% BSA, pH7.4) usinga ground-glass homogenizer. Homogenateswerecentrifugedat3000×gfor10min,thenthe supernatantwascollectedandcentrifugedat25,000×gfor1h.The pelletwasresuspendedin5%SDSwithproteaseinhibitorcocktail andproteinconcentrationwasdeterminedbytheLowrymethod. Samples were heated at 95◦_C _for ₅_min, _and _proteins ₍₂₅_␮_g)

wereseparatedbySDS-PAGE(12%gels)andelectricallytransferred ontoa PVDFmembrane (BIORAD,USA).Theblots wereblocked with5%nonfatdrymilkinTris-bufferedsalinein0.1%Tween20 (TBS-T)at4◦_C_and_incubated_with_rabbit_{anti-synaptophysin}

anti-body(1:2,000;SantaCruzBiotechnology,CA)andmouseanti-␣

tubulinaantibody(1:4,000;Sigma,St.Louis,MO).Afterwashing withbufferTBS-Tthemembranewasincubatedwith horseradish-peroxidase-conjugated goatanti-mouse IgG (1:2,000; Millipore, Bedford, MA) or horseradish-peroxidase-conjugated goat anti-rabbit IgG (1:3,300; Bio-Rad; USA) for 1 hour. Immunoreactive bandswerevisualizedwith3,3′_{-diaminobenzidine.}_The_density_of

synaptophysinimmunoreactivebandswasmeasuredusingImageJ software.

2.13. BDNFassay

Sixanimalsofeachgroupwereeuthanized120hafterpMCAO and hippocampus, and striatum were removed. Briefly, brain samples were homogenized (10% v/v) in a solution of phos-phatebuffer(PBS)(50mM,pH6.0)containingproteaseinhibitor (Sigma–Aldrich,USA).Homogenateswerecentrifugedat14.000g, 4◦_C,_for₃₀_min,_and_the_{concentrations}_of_BDNF_in_the_supernatans

weremeasuredwithELISAkit(Quantikine®_Elisa_R&D_SYSTEMS),

accordingtothemanufacturesdirections.

2.14. Statisticalanalysis

DataareexpressedasmeanSEMorasmedian(interquartile range)and statistical differenceswereestimated usingeither a one-way analysis of variance, followed by a Newman-Keuls or Bonferronipost-hoc test,or Kruskal–WallisandMann–Whitney U-tests,withasignificancelevelof95%.

3. Results

3.1. Rosmarinicacidtreatmentreducesinfarctarea24hafter pMCAO

Infarctvolumeswereevaluatedat24hafterpMCAO.The rep-resentative photomicrographs shown in Fig. 2A indicated that the infarctareas were located mainly in the ipsilateral cortex, andtoalesserextentinstriatumandhippocampus.Theanimals treated withrosmarinicacidat dosesof 1 and 20mg/kg had a significantlowerischemicneuronaldamagecomparedtopMCAO group(pMCAO+RA0.1:6.39±1.77%,pMCAO+RA1:3.43±0.7%5, pMCAO+RA20:2.65±0.85%)(Fig.2B).

3.2. RAtreatmentpreventedneurologicaldeficit24hafter pMCAO

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com-Fig.2. Rosmarinicacid(RA)treatmentreducestheischemia-inducedinfarctedarea.SwissmalemicereceivedNaCl0.9%andRA(0.1,1or20mg/kg,i.p.).(A)Representative coronalsectionsstainedwith1%triphenyltetrazoliumchloride.Thepale-coloredregionshowstheischemicregionwhereasthedarkcoloredregionshowstheviabletissue. (B)Bargraphshowingthepercentageofinfarctarea24hafterpMCAO.a_vs_SO,b_vs_pMCAO,_p_<_0.05._Data_are_expressed_as_mean

±S.E.M.,n=6pergroup,Kruskall–Wallis andMann–Whitneytest.

pared to sham-operated animals (SO: 18 (17–18); pMCAO:16 (14–17).ThepMCAOanimalspresentedreducedmotorabilityand alsodecreasedabilitytorespondtostimulionthesideofthebody contralateral toischemia.Animals treatedwithrosmarinic acid showedasignificantimprovementinneurologicaldeficitsinduced by pMCAO at doses of 1 and 20mg/kg (pMCAO+RA 0.1:15.5 (15–16),pMCAO+RA1:17.5(16–18),pMCAO+RA20:17(17–18), p<0.05)(Fig.3).

3.3. RAhasnoeffectinlocomotoractivityinpMCAOmice

Intheevaluationoflocomotoractivity72hafterischemiawere notobserved significantchanges inthe numberof crossingsor rearingsafterpMCAOand/orRAtreatment(Fig.4AandB).

3.4. RAreducesmemorydeficitsinducedbypMCAO

Theanimalssubjectedtoischemiashoweddeficitson work-ingmemory(SA-SO:70.2±2.5%;SO+RA20:69.2±6.0%;pMCAO: 60.9±2.6%)andontherecognitionmemory(recognition index-SO: 0.21±0.1; SO+RA 20: 0.39±0.13; pMCAO: −0.09±0.07) (Figs. 5 and 6). In assessing the retention of spatial memory, 120h after ischemia, pMCAO group had significant deficits in theparameterslatencytoreachtheplatform,number of cross-ings and time spent in the quadrant (latency: SO: 5.8±1.1s; SO+RA20:9.0±2.1s;pMCAO:14.4±2.5s, numberofcrossings: SO:6.5±0.6;SO+RA20:4.2±0.5;pMCAO:4.0±0.3,timespent inthequadrant:SO:22.3±1.6s;SO+RA20:20.0±1.2s;pMCAO: 17.2±1.0s)(Fig.7B–D).

Workingandrecognitionmemorydeficitswereprevented sig-nificantly byRA (SA:pMCAO+RA0.1: 62.9±4.3%; pMCAO+RA

Fig.3.Rosmarinicacid(RA)treatmentreducesischemia-inducedneuronaldeficits. SwissmalemicereceivedNaCl0.9%andRA(0.1,1or20mg/kg,i.p.).Bargraph showingthetotalscoresrangedfrom3to18ofsixitemsmeasured;thehigher thescore,thebetterthemotorperformance.a_vs_SO,b_vs_pMCAO,_p_<_0.05._Data areexpressedasmedian(interquartilerange),n=6pergroup,Kruskall–Wallisand Mann–Whitneytest.

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Fig.4. Rosmarinicacid(RA)treatmentpromotednoalterationsonmotoractivity. SwissmalemicereceivedNaCl0.9%andRA(0.1,1or20mg/kg,i.p.).(A)Bargraph showingthenumberofcrossings.(B)Bargraphshowingthenumberofrearings. Dataareexpressedasmean±S.E.M.

3.5. RAtreatmentpreventtheincreasedlevelsofMPOinducedby pMCAO

Ischemicmice showedincreased levelsof MPOin temporal cortex and striatum 24h after ischemia (Temporal cortex: SO: 0.85±0.42 U/mg tissue; SO+RA 20: 1.25±0.71U/mg tissue; pMCAO:7.4±1.67U/mgtissue,striatum:SO:0.39±0.27U/mg tis-sue;SO+RA20:0.75±0.36U/mgtissue;pMCAO:1.76±0.38U/mg tissue), but not in hipoccampus (SO: 0.50±0.27U/mg tissue; SO+RA 20: 0.60±0.39U/mg tissue; pMCAO: 0.68±0.20U/mg tissue).RA(20mg/kg/day)preventedsignificantlytheincreaseof MPOlevelsintemporalcortex(pMCAO+RA20:3.50±0.87U/mg tissue) but not in striatum (Striatum: pMCAO+RA 20: 1.29±0.23U/mgtissue)(Fig.8A–C).

Fig.5. Rosmarinicacid(RA)treatmentpreventsischemia-inducedworking mem-orydeficits.SwissmalemicereceivedNaCl0.9%andRA(0.1,1or20mg/kg,i.p.). Bargraphshowingthepercentageofspontaneousalterations72hafterpMCAO.a vsSO,b_vs_pMCAO,_p_<_0.05._Data_are_expressed_as_mean

±S.E.M.,n=8pergroup, Kruskall–WallisandMann–Whitneytest.

Fig.6.Rosmarinic acid(RA)treatment preventsischemia-inducedrecognition memorydeficits.SwissmalemicereceivedNaCl0.9%andRA(0.1,1or20mg/kg, i.p.).Bargraphshowingtherecognitionindex.a_vs_SO,b_vs_pMCAO,_p_<_0.05._Data areexpressedasmedian(interquartilerange),n=6pergroup,Kruskall–Wallisand Mann–Whitneytest.

3.6. RAtreatmentpreventtheastrogliosis

GFAPexpressionwasfoundtobehigherinischemichemisphere ofpMCAOgroupcomparedtoSOgroup(SO:109.2±4.3%ofmean control;pMCAO:152.9±8.8%ofmeancontrol).Asignificant reduc-tioninGFAPexpressionwasobservedin ischemicmicetreated withRA(20mg)ascomparedtopMCAOgroup(pMCAO+RA20: 124.4±6.2%ofmeancontrol)(Fig.9A–G).

3.7. RAtreatmentpreventthedecreaseofSYPinducedbypMCAO

Ischemicanimalsshowedlowerexpressionofsynaptophysinin thestriatum5daysaftertheischemia,RAtreatmentinhibitedthe reductionofsynaptophysininischemicanimals(SO:100.0±1.9%; pMCAO:73.6±6.5%;pMCAO+RA20:88.6±4.0%)(Fig.AandC). No alterationwasfoundin thehipoccampus (SO: 105.3±9.5%; pMCAO:90.7±17.6%;pMCAO+RA20:96.2±13.1%)(Fig.10Aand B).

3.8. RAtreatmentincreasedBDNFlevelsinpMCAOmice

IschemicanimalsshowedincreasedlevelsofBDNFin hippocam-pusanddecreasedlevelsinstriatum,andRAtreatmentincreased BDNF levels in hippocampus of sham-operated and ischemic miceafter5 daysofpMCAOcomparedtopMCAOgroupandto SO group (Hippocampus: SO: 316.1±38.64pg/mL; SO+RA 20: 1148.0±92.0pg/mL; pMCAO: 516.8±49.51pg/mL; pMCAO+RA 20: 868.2±64.30pg/mL). RA treatment increased BDNF levels in striatumof ischemicmiceafter 5days of pMCAOcompared topMCAOgroup(Striatum:SO:511.2±65.8pg/mL;SO+RA20: 336.1±27.17pg/mL; pMCAO: 239.9±55.80pg/mL; pMCAO+RA 20:492.7±42.42pg/mL)(Fig.11AandB).

4. Discussion

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cat-Fig.7.Rosmarinicacid(RA)treatmentpreventsischemia-inducedspatialmemorydeficits.SwissmalemicereceivedNaCl0.9%andRA(0.1,1or20mg/kg,i.p.).(A)Acquisition trailinthewatermaze,(B)latencytoreachthepreviouslocationoftheplatform,(C)numberofcrossings,(D)timespentinthequadrant.a_vs_SO,b_vs_pMCAO,_p_<_0.05._Data arepresentedasmean±SEM,n=8pergroup.Kruskall–WallisandMann–Whitneytest.

echin, a polyphenol, in thedrinking water for 2 weeks. In the presentwork,infarctionareaandneurologicalevaluationshowed thatRAhadaneuroprotectiveeffectconsistentwiththeworkby Luanetal.[26]thatdemonstratedreducedcerebralinfarctvolume andneurologicaldeficitsinischemicdiabeticratsaftertreatment withRA(50mg/kg).Although,theliteraturehasdemonstratedthe neuroprotectiveeffectofRAonischemicdamage,nostudieshave demonstratedtheRAeffectonmemorydeficitinducedbycerebral ischemia.OurstudyfoundthatRAsignificantlyimprovedworking, recognitionandspatialmemoryincerebralischemicmice.

Y-mazeandobjectrecognitiontestsarerelatedtothe frontal-subcortical circuits rather than to thehippocampus [37]. Zhou et al. [38] demonstrated that transient focal cerebral ischemia lesionsofthestriatumandhippocampuscausedworkingmemory deficitsinratsassessedbytheY-mazetest.Recognitionmemory impairmentisrelatedtowhitemattersub-corticalstrokelesions inmice[39]andparietalcortexinC57B/6miceafterpMCAO[40]. Thesefindingsareconsistentwithourresultsthatshowedthatthe striatumandthecortexwerelargelyaffectedbyischemia,which inducedworkingandrecognitionmemorydeficits.Furthermore, poorperformanceintheMorriswater-mazetestwascorrelated withCA1regiondamage[41],althoughtheimpairmentinthe spa-tialmemoryinducedbypMCAOismorelikelyrelatedtocortical andstriatumdamage[42].Theneuralsubstratesmostfrequently implicatedinvariousformsofspatiallearningarethehippocampus andthedorsalstriatum,withtheformerbeinginvolvedin learn-ingwithreferencetoanarrayofdistalcues.Thisprocessisoften referredtoastheformationofa“cognitivemap”[43,33].Thedorsal striatumisinvolvedintheformationofanassociationbetweena proximalcueintheenvironmentandtheanimal’sownkinesthetic response,typicallyovermanyrepeatedtrials[44].Inourstudy,we observedthatmicewithpMCAOshowedspatialmemory

impair-ments,andRAsignificantlyattenuatedthesedeficits.RAprotects againstworkingandrecognitionmemoryimpairmentinducedby A␤(25–35)inmice[23]andenhancescognitiveperformancesinthe

Morriswater-mazetaskinrats[45].Previousstudieshaveshown that RAmetabolites, suchasCaffeic acid, have neuroprotective actions[46].Caffeicacidisreportedtoreducebraindamageand preventmemorydeficitsafterpermanentfocalcerebralischemia inmiceduetoanti-inflammatoryactions[47].

Becauseoxidativedamageandinflammationareimplicatedin theetiology of neurologicalcomplications incerebralischemia, floodingthesystemwithantioxidantsandanti-inflammatory com-poundsmaybeasensible step.Phenoliccompoundsareknown bytheirantioxidantandanti-inflammatoryeffects,whichinturn haveneuroprotectiveeffectsduringcerebralischemia[48]. Ros-marinicacidisaphenoliccompoundwithpotentantioxidantand anti-inflammatoryactionsandiscontainedinvariousLaminaceae herbsusedcommonlyasculinaryherbs,suchasRosmarinus offici-nalis[49,21].RAshowedpotentantioxidantactivitybymodulating H2O2-inducedcelldeathinhumandopaminergicneuronsthrough

reactiveoxygenspecies(ROS)scavenging[50].RAwasalsofoundto suppresslipidperoxidation[51].Additionally,ithasbeenreported thatRAhastheabilitytoinhibitcomplementfixationand lipoxy-genaseandcyclooxygenaseactivity[52–54].Furthermore,RAwas alsoabletoinhibitseasonalallergicrhinoconjunctivitisinhumans bysuppressingtheinflammatoryresponse[55].Inrats,RAisrapidly absorbedinthedigestivetract,andthemajormetabolitesfound inurinearecaffeicacid, ferulicacidandcoumaricacid[56].RA isalsoabsorbedinhumansafteroraladministrationandspecific metabolitescanbedetectedinurine[57].

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Fig.8.Effectofrosmarinicacid(RA)20mg/kgonthemyeloperoxidase(MPO)activityinthecortex(A),inthestriatum(B)andinthehipoccampus(C)ofmicesubmittedto pMCAO.a_vs_SO,b_vs_pMCAO,_p_<_0.05._Data_are_presented_as_mean_±_S.E.M.,_n₌₈_per_group._One-way_ANOVA_followed_by_{Newman–Keuls}_post_hoc-test.

impairmentinducedbycerebralischemia.Ithasbeenshownthat RApossesses anti-inflammatoryactionsbydiminishingIL-1␤in anototoxicitymodelinducedbycisplatininmice[58],decreased COX-2positivecellsinamodelofcollageninducedarthritis[59], anddecreasedpro-inflammatorycytokinesIL-6,IL-1␤andTNF-␣

inaratmodelofsystemicinflammation[60].Furthermore,caffeic acid,amajorRAmetabolite,attenuateshippocampal inflamma-tionbydiminishing neutrophilinfiltrationin micesubjectedto pMCAO [47], RA diminished MPO activity induced by a renal ischemia/reperfusionmodelin rats[61],andotherpolyphenols, suchaspinocembrin, diminishedneutrophilinfiltrationina rat modelofglobalcerebralischemia/reperfusion[62].Forghaniand colleagues[63]demonstratedthatMPO-knockoutmicesubjected totransientMCAOshowedreducedlesionvolumeandimproved neurobehavioraleffects,suggestingthattheMPOactivityblockade is animportant neuroprotective strategy to attenuateischemic damage after 24h of stroke onset. In accordance with these findings,wealsofoundthatRA(20mg/kg)abolishedtheincrease MPOactivityafterischemicinjuryincerebralischemicmice.

BDNFis widelydistributed in nerve terminals and synapses throughout the brain, particularly in the hippocampus [64]. IncreasedBDNFlevelspromoteneuronalsurvivalandinfarct tol-erance[65].StudiesshowthatBDNFlevelsinischemicmiceare elevated,anditisprobablyduetothecleavageoftheexisting pro-BDNFinthecoreofthelesion[66,67].Inthelesionedhemisphere,

matureBDNFiselevated,althoughinthecontralateralhemisphere, bothpro-andmatureBDNFareelevated,indicatingthatinthe non-lesionedhemisphere,theoverexpressionofbothformscouldbe theresultofgeneinduction[67].TheoverexpressionofBDNFin ischemicconditionsmaydescribetheself-protectingpropertiesof neuronsafterischemicinjury[68].Inthepresentstudy,wefound anincreasinglevelofBDNFinthehippocampusofpMCAOmice 5daysafterstrokeonset.RAincreasesBDNFlevels,causing sig-nificantlyincreasedBDNFlevelsinthehippocampuscomparedto sham-operatedandischemicmice.Itoandcolleagues[69] demon-stratedthatRAinducedcellproliferationofnewborncellsinthe dentategyrusofthehippocampusofmice.Thesestudies corrobo-rateourresults,whichshowsthatRAhastheabilitytoself-induce cellproliferationthroughtheBDNFincreaseinthedentategyrus inthehippocampus;thismayexplaintheneuroprotectiveeffectof RAonmemorydeficits.Thestriatumisaregionofthebrainthat lacksBDNFmRNA[64],althoughtheBDNFproteincanbe trans-ferredbyananterogradetransport fromneuronalcellbodiesto theirterminals[70].BDNFwasfoundtobedecreasedinthecortex 7daysaftertransientMCAO,anareathatisthecoreofthesetypes ofischemiclesions[71].Thisfindingisconsistentwithourresults, whichshoweddecreasedBDNFlevelsinthestriatumofpMCAO mice5daysafterpMCAO.

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Fig.10.Effectofrosmarinicacid(RA)20mg/kgontheexpressionofsynaptophysin(A)inthestriatumand(B)inthehipoccampusofmicesubmittedtopMCAO.Theresults arepresentedasmean±S.E.M.,n=4pergroup,a_vs_SO,b_vs_pMCAO,_p_<_0.05._One-way_ANOVA_and_{Newman–Keuls}_test.

daysafterstrokeonset.Otherpolyphenols,suchasginsenosides, haveneuroprotectiveeffects, astheyincreasedneurogenesisby increasingBDNFlevelsinanoxygen-andglucose-deprivedmodel inPC12cells[72].OroxylinA, aflavonoidcompound,increased BDNFexpressioninthehippocampusandhasbeenshownto ame-liorate memory impairment and neuronal damage after global cerebralischemiainrats[68].Madinieretal.[67]demonstrated thatBDNFimmunoreactivity wascolocalizedinneuronsand in astrocytesafterpermanentfocalcerebralischemiaby photothrom-boticcorticalocclusioninrats.BDNFwasfoundtoreducecortical celldeathaftertransientMCAOinrats[73]andhasbeenshownto beessentialforsynaptogenesisandsynapticstructural composi-tionintheadultbrain[74,75].TherelationshipbetweenBDNFand synaptogenesisiswidelystudied.Chenetal.[76]haveshownthat statin,aBDNFupregulationinductor,isrelatedtoanincreaseof synaptophysinexpressionandtofunctionalrecoveryafterstroke. Pozzo-Milleretal.[77]haveshownthatBDNFknockoutmicehave areducedlevelofsynaptophysininhippocampalsynaptosomes.Jin etal.[78]havedemonstratedthatRA(10mg/kg)exertsa

neuropro-tectiveeffectbyincreasingastrocyticBDNFinthehippocampusvia modulationofERKphosphorylationinchronicunpredictablestress. Takentogether,synaptogenesisandneurogenesisaremechanisms involvedinlearningandmemoryrecoveryafterischemicevents [79,80].Thesemechanisms may,inpart,beresponsible forthe neuroprotectiveeffectofRAonmemorydeficitsinpMCAOinjured mice.

Synaptophysinhasarole inthebiogenesisofsecretory vesi-cles,stabilizationandmodificationofthefunctionofothersynaptic proteinsandprotectionduringischemicbraininjury.Itisusually overexpressed during neuronal remodeling [81]. Increased lev-elsof synaptophysinare interpretedtoindicate synaptogenesis [82].Ischemicpatientsshowmotorandcognitiverecoverysome time afterthestroke event[83],and themechanisms involved aresynaptogenesis,axongrowthandgenerationofnewcellsand bloodvessels[84,85].Studieshaveshownthatastrocytesplayan importantroleinsynaptogenesisthroughthereleaseofmediators thatmaintainthebrainmicroenvironmentevenunderconditions inwhichneuronslosetheirfunctionality,which occursin

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bralischemia.Thewayinwhichtheactivationofastrocytesafter ischemiaoccursdiffersdependingonthetimeandthebrainarea thatisobserved[86,87].Inpathologicalconditions,astrocytes sup-portneuronswithantioxidantsandenergeticmetabolites,suchas lactateandATP[88,89].Theprotectivefunctionofreactive astro-cytes results from their ability of glutamate re-uptake [90,91], production of antioxidants suchas glutathione and superoxide dismutase [88], and stimulation of neurogenesis [92–94]. Dur-ingastrogliosisastrocytichypertrophy,GFAPup-regulation,gliotic scarformation,proliferation,andde-differentiation,whicharethe mostimportantfeaturesoftheastrocyticresponse,occur[95–97]. The mostcommonsignaling agents influencing these reactions includeneurotransmittersand gliotransmitters(ATP,glutamate, noradrenalin),reactiveoxygenspecies,cytokinesandinterleukins [88,98].Thus, thereactive astrogliosisrevealsboth detrimental andneuroprotectiverolesinthecourseofpathologicalprocesses. RA prevented the decrease in the synaptophysin levels in the striatumanddecreasedastrogliosis,whichmeansthatRAmight preventlearningandmemoryimpairmentbyinhibitingthelossof synapsesorenhancingsynaptogenesisbydecreasingastrogliosis andincreasingneurotrophicfactors,suchasBDNF,afterischemic injury.

In conclusion, the present study is the first report that RA preventsmemorydeficitsinducedbycerebralischemia,probably byanti-inflammatoryandsynaptogenicmechanismsthroughthe BDNFpathway,althoughothermechanisms,suchasantioxidant andantiapoptoticactions,cannotbeexcluded.

Conflictofinterest

Therearenoconflictsofinterest.

Acknowledgements

TheauthorsthanktheBrazilianNationalResarchand Develop-mentCouncil(CNPq),INCT-IBISAB-Capes,andtheResearchSupport FoundationofCeará(FUNCAP)for financialsupportin theform ofgrantsandfellowshipawards.TheauthorsalsothankJuliana FernandesPereirafortechnicalassistance.

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