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Protective Effects of L-902,688, a Prostanoid EP4 Receptor Agonist, against Acute Blood-Brain Barrier Damage in Experimental Ischemic Stroke

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Title:
Protective Effects of L-902,688, a Prostanoid EP4 Receptor Agonist, against Acute Blood-Brain Barrier Damage in Experimental Ischemic Stroke
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Frontiers in Neuroscience 2018 Feb 20;12:89. doi: 10.3389/fnins.2018.00089.
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Candelario Jalil, Eduardo
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Frontiers Media
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Ischemic stroke occurs when a clot forms in the brain vasculature that starves downstream tissue of oxygen and nutrients resulting in cell death. The tissue immediately downstream of the blockage, the core, dies within minutes, but the surrounding tissue, the penumbra is potentially salvageable. Prostaglandin E2 binds to four different G-protein coupled membrane receptors EP1-EP4 mediating different and sometimes opposing responses. Pharmacological activation of the EP4 receptor has already been established as neuroprotective in stroke, but the mechanism(s) of protection are not well-characterized. In this study, we hypothesized that EP4 receptor activation reduces ischemic brain injury by reducing matrix metalloproteinase (MMP)-3/-9 production and blood-brain barrier (BBB) damage. Rats underwent transient ischemic stroke for 90 min. Animals received an intravenous injection of either the vehicle or L-902,688, a highly specific EP4 agonist, at the onset of reperfusion. Brain tissue was harvested at 24 h. We established a dose-response curve and used the optimal dose that resulted in the greatest infarct reduction to analyze BBB integrity compared to vehicle-treated rats. The presence of IgG, a blood protein, in the brain parenchyma is a marker of BBB damage, and L-902,688 (1 mg/kg; i.v.) dramatically reduced IgG extravasation (P < 0.05). Consistent with these data, we assessed zona occludens-1 and occludin, tight junction proteins integral to the maintenance of the BBB, and found reduced degradation with L-902,688 administration. With immunoblotting, qRT-PCR, and/or a fluorescence resonance energy transfer (FRET)-based activity assay, we next measured MMP-3/-9 since they are key effectors of BBB breakdown in stroke. In the cerebral cortex, not only was MMP-3 activity significantly decreased (P < 0.05), but L-902,688 treatment also reduced MMP-9 mRNA, protein, and enzymatic activity (P < 0.001). In addition, post-ischemic administration of the EP4 agonist significantly reduced pro-inflammatory cytokines IL-1β (P < 0.05) and IL-6 (P < 0.01) in the ischemic cerebral cortex. Most importantly, one injection of L-902,688 (1 mg/kg; i.v) at the onset of reperfusion significantly reduces neurological deficits up to 3 weeks later (P < 0.05). Our data show for the first time that pharmacological activation of EP4 with L-902,688 is neuroprotective in ischemic stroke by reducing MMP-3/-9 and BBB damage.
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Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Eduardo Candelario Jalil.

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ORIGINALRESEARCH published:20February2018 doi:10.3389/fnins.2018.00089 FrontiersinNeuroscience|www.frontiersin.org 1 February2018|Volume12|Article89 Editedby: MatildeOtero-Losada, InstitutodeInvestigaciones Cardiolgicas,Argentina Reviewedby: LuigiaTrabace, UniversityofFoggia,Italy KevinDonaldBroad, UniversityCollegeLondon, UnitedKingdom ShekherMohan, ManchesterUniversity,UnitedStates *Correspondence: EduardoCandelario-Jalil ecandelario@u.edu Specialtysection: Thisarticlewassubmittedto Neuropharmacology, asectionofthejournal FrontiersinNeuroscience Received: 05December2017 Accepted: 02February2018 Published: 20February2018 Citation: DeMarsKM,McCreaAO, SiwarskiDM,SanzBD,YangCand Candelario-JalilE(2018)Protective EffectsofL-902,688,aProstanoid EP4ReceptorAgonist,againstAcute Blood-BrainBarrierDamagein ExperimentalIschemicStroke. Front.Neurosci.12:89. doi:10.3389/fnins.2018.00089 ProtectiveEffectsofL-902,688,aProstanoidEP4ReceptorAgonist,againstAcuteBlood-BrainBarrierDamageinExperimentalIschemicStrokeKellyM.DeMars,AustinO.McCrea,DavidM.Siwarski,BrianD .Sanz,ChangjunYangand EduardoCandelario-Jalil DepartmentofNeuroscience,McKnightBrainInstitute,Univ ersityofFlorida,Gainesville,FL,UnitedStates Ischemicstrokeoccurswhenaclotformsinthebrainvascula turethatstarves downstreamtissueofoxygenandnutrientsresultingincell death.Thetissueimmediately downstreamoftheblockage,thecore,dieswithinminutes,b utthesurrounding tissue,thepenumbraispotentiallysalvageable.Prostagl andinE 2 bindstofourdifferent G-proteincoupledmembranereceptorsEP1–EP4mediatingdi fferentandsometimes opposingresponses.PharmacologicalactivationoftheEP4 receptorhasalreadybeen establishedasneuroprotectiveinstroke,butthemechanis m(s)ofprotectionarenot well-characterized.Inthisstudy,wehypothesizedthatEP 4receptoractivationreduces ischemicbraininjurybyreducingmatrixmetalloproteinas e(MMP)-3/-9productionand blood-brainbarrier(BBB)damage.Ratsunderwenttransien tischemicstrokefor90min. Animalsreceivedanintravenousinjectionofeithertheveh icleorL-902,688,ahighly specicEP4agonist,attheonsetofreperfusion.Braintiss uewasharvestedat24h. Weestablishedadose-responsecurveandusedtheoptimaldo sethatresultedin thegreatestinfarctreductiontoanalyzeBBBintegritycom paredtovehicle-treated rats.ThepresenceofIgG,abloodprotein,inthebrainparen chymaisamarkerof BBBdamage,andL-902,688(1mg/kg;i.v.)dramaticallyredu cedIgGextravasation ( P < 0.05).Consistentwiththesedata,weassessedzonaocclude ns-1andoccludin, tightjunctionproteinsintegraltothemaintenanceoftheB BB,andfoundreduced degradationwithL-902,688administration.Withimmunobl otting,qRT-PCR,and/ora uorescenceresonanceenergytransfer(FRET)-basedactiv ityassay,wenextmeasured MMP-3/-9sincetheyarekeyeffectorsofBBBbreakdowninstr oke.Inthecerebral cortex,notonlywasMMP-3activitysignicantlydecreased ( P < 0.05),butL-902,688 treatmentalsoreducedMMP-9mRNA,protein,andenzymatica ctivity( P < 0.001). Inaddition,post-ischemicadministrationoftheEP4agoni stsignicantlyreduced

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DeMarsetal. EP4ActivationReducesStrokeDamage pro-inammatorycytokinesIL-1 b ( P < 0.05)andIL-6( P < 0.01)intheischemiccerebral cortex.Mostimportantly,oneinjectionofL-902,688(1mg/ kg;i.v)attheonsetof reperfusionsignicantlyreducesneurologicaldecitsup to3weekslater( P < 0.05). Ourdatashowforthersttimethatpharmacologicalactivat ionofEP4withL-902,688 isneuroprotectiveinischemicstrokebyreducingMMP-3/-9 andBBBdamage. Keywords:prostaglandinE 2 ,EP4receptor,ischemia,stroke,blood-brainbarrier,matri xmetalloproteinase-9, neuroinammation INTRODUCTIONStrokeislistedasthefthleadingcauseofdeathintheUSAandabout87%ofstrokesareischemic( Mozaarianetal.,2016 ). Recombinanttissueplasminogenactivator(rtPA)istheonlyFDA-approveddrugforischemicstroke.OnlyasmallproportionofstrokepatientsareeligibletoreceivertPAbecauseitcar ries ahighriskofbleeding/hemorrhagictransformationinaddi tion todirectneurotoxicity( Kauretal.,2004 )andhasashort eectivetimewindowofonly4.5hafterstrokeonset( DelZoppo etal.,2009 ).Itisthereforeessentialtosearchforalternative pharmaceuticalinterventionstoreachalargerpercentageofischemicstrokepatients. Anischemicstrokeoccurswhenamajorcerebralarteryis occluded,andcellsjustdownstreaminthecoreofthestrokenecroticallydiewithinminutes.Celldeathisperpetuatedin to thesurroundingpenumbraoverthecourseofhourstodayslater.Reactiveoxygen/nitrogenspecies(ROS)formationfurt her compromisestheintegrityofanalreadydegradedblood-brai n barrier(BBB)byactivatingmatrixmetalloproteinases(MMP)i.e.,MMP-3,MMP-9thatcleavethebasementmembraneoftheneurovascularunitandthetightjunctionproteins(TJPs)betweenendothelialcells( Roselletal.,2008;Soodetal.,2008; Candelario-Jaliletal.,2011;TurnerandSharp,2016;Hafez etal., 2018 ).Thistriggersaninammatoryresponseandinltration ofimmunecellswhichhavebeenassociatedwithincreasedce ll death,formationoffreeradicals/ROS,andsecondaryinjury( Yilmazetal.,2006;Jinetal.,2010;Benakisetal.,2014 ). Followinganischemicstroke,breakdownoftheBBB, vasogenicedema,andhemorrhagicconversionaremainlymediatedbyMMPs,inparticularMMP-3andMMP-9,whichhavebeenshowntobecriticalininammation-mediatedneurovasculardamage( Asahietal.,2000;Candelario-Jalil etal.,2009;StanimirovicandFriedman,2012;Lakhanetal. 2013 ).GeneticknockoutorinhibitionofMMP-3orMMP9dramaticallyreducesneurovascularinjuryfollowingfoca l cerebralischemiainrodents( Asahietal.,2000;Harrisetal., 2005;Suzukietal.,2007;Dejonckheereetal.,2011;Hafezetal.,2016,2018 ).Neuroinammation-mediatedBBBdisruption signicantlycontributestotheprogressionofbraininjury inthe penumbraafterstroke.Therefore,understandingmechanism sof Abbreviations: PGE 2 ,prostaglandinE 2 ;EP4,ProstaglandinE 2 receptor4;MMP, Matrixmetalloproteinase;tPA,Tissueplasminogenactivator;BBB,Blo od-brain barrier;ZO-1,Zonulaoccludens-1;MCAO,Middlecerebralarteryocclus ion; CCA,Commoncarotidartery;TBS,Tris-bueredsaline;TBST,Tris-bu ered salinewith0.1%Tween20. BBBdamagecouldleadtotheidenticationofnoveltargetsf or therapeuticintervention. Aspartoftheneuroinammatoryresponsetostroke,alarge quantityofarachidonicacidreleasedfromthemembranebyphospholipasesismetabolizedintoprostaglandinH 2 mainlyby cyclooxygenase-2(COX-2),andthenfurthermetabolizedint o severalprostanoids.ProstaglandinE 2 (PGE 2 )isoneofthemajor prostanoidsformedafterischemicstrokebyincreasedCOX-2activity( Nogawaetal.,1997;Manabeetal.,2004;Kawanoetal., 2006;Candelario-Jaliletal.,2007 ).Prostaglandinsareshort-lived, lipidmediatorsthatareessentialtoinammatorysignaling .PGE 2 canhaveparacrineorautocrineeectsandistheendogenousligandforfourG-proteincoupledreceptorsEP1-EP4.PGE 2 can haveopposingeectsdependingonwhichreceptorisactivated( SugimotoandNarumiya,2007 ). Inischemicstroke,theincreaseinCOX-2-derivedPGE 2 formationcorrelateswithBBBopeningandinltrationofperipheralimmunecells( Candelario-Jaliletal.,2007 ).Moreover, invivo datashowthatdirectinjectionofPGE 2 intotherat brainleadstoincreasedpermeabilityoftheBBB( Schmidley etal.,1992;Messripouretal.,2015 ).Inthecontextoffocal cerebralischemia,previousstudieshaveshownthatactivat ion ofEP1andEP3PGE 2 receptorssignicantlyexacerbatestroke injury( Manabeetal.,2004;Kawanoetal.,2006;Ahmadetal., 2007,2008;Abeetal.,2009;Fukumotoetal.,2010;Zhenetal ., 2012;Shimamuraetal.,2013 ).Werecentlyshowedthatgenetic deletionorpharmacologicalblockadeoftheEP1receptorresul ts inadramaticreductioninstrokeinjuryandBBBpermeability whichcorrelatedwithreducedlevelsofMMP-3andMMP-9( Frankowskietal.,2015 ).Stroke-inducedBBBdamageis signicantlyreducedinEP3decientmiceorinwild-typeanimalstreatedwithanEP3receptorantagonist( Ikeda-Matsuo etal.,2011 ). UnlikeEP1andEP3receptors,activationofEP2andEP4 receptorshaspreviouslybeenshowntobeneuroprotectiveinstroke( McCulloughetal.,2004;Ahmadetal.,2005;Liang etal.,2011;Akrametal.,2013 ).Althoughseveralstudies haveprovidedstrongevidenceofaprotectiveroleofEP4inneuroinammationandcerebralischemia,nothingisknownoftheeectsofEP4activationonBBBpermeabilityafterstroke.Inthisstudy,ourobjectivewastoinvestigatewhet her EP4receptoractivationwouldimpactBBBpermeabilityandneurobehavioraloutcomesinaclinicallyrelevantanimalmo del oftransientfocalcerebralischemia.WehypothesizedthatEP4receptoragonismwithL-902,688reducesinfarctsizeandneurologicaldecitsbyreducingMMP-3,MMP-9,andBBBdamage. FrontiersinNeuroscience|www.frontiersin.org 2 February2018|Volume12|Article89

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DeMarsetal. EP4ActivationReducesStrokeDamage MATERIALSANDMETHODSAnimalsAdultmalerats(10–12weeks, 280–320g,SpragueDawleyfrom CharlesRiverLaboratoriesInternational,Wilmington,MA,US)wereallowedtoacclimatizefor1weekbeforeexperimentsinhousingfacilitiesona12hlight/darkcyclewithfreeacces sto foodandwaterwithtworatspercage.AllanimalprocedureswereperformedinaccordancewithapprovedguidelinesoftheNationalInstitutesofHealthfortheCareandUseofLaboratoryAnimals,theARRIVEguidelines(https://www.nc3r s. org.uk/arrive-guidelines),andtheguidelinesapprovedbyt he InstitutionalAnimalCareandUseCommitteeattheUniversit y ofFlorida(protocol#201406503).Experimentswereplannedtoreducethetotalnumberofanimalsusedandtoreducepotentia l painandsuering.IntraluminalFilamentModelofTransientFocalIschemiaandDrugTreatmentTomimicischemicstroke,ratsweresubjectedto90minoftransientmiddlecerebralarteryocclusion(MCAO)usingth e intraluminallamentmodeloffocalischemia,describedindetailinourpreviouspublications( Candelario-Jaliletal.,2007; Hawkinsetal.,2013,2017 ).Ratsweredeeplyanesthetizedwith 2–2.5%isouraneinmedicalgradeoxygenandmaintainedataconstant37 Cthroughoutsurgeryonaheatedplatform (Cat#TP-700T/Pump;StrykerGlobalIndustries,Kalamazoo,MI,USA).Amidlineventralcutwasmade,andthecommoncarotidartery(CCA)wasseparatedfromthevagusnerveandligatedwitha4-0silksuture(Cat#SP116;HarvardApparatus,Holliston,MA,USA).Theexternalcarotidartery(ECA)andpterygopalatinearteriesweretemporarilyclippedwithamicrovascularcliptopreventincorrectplacementoftheoccludinglament.AnarteriotomywasperformedontheCCAafewmillimetersabovetheligationtoallowfora4-0silicone-coatedlament(Cat#403523PK10;DoccolCorporation,Sharon,MA,USA)insertionthroughtheinternalcarotidarteryupintothemiddlecerebralarteryuntildete ction ofaslightresistance.Aftertemporarilyclosingtheventra l incision,ratswereallowedtorecoverinatemperaturecontr olled heatedchamber(Cat#ICSDW-1Warmer,Thermo-Care,PasoRobles,CA,USA)forabout80mintopreventhypothermiabeforere-anesthetizingtheanimaltoremovethelament.At the onsetofreperfusion,animalsrandomlyreceivedanintraven ous injectionofvehicle(saline; n D 10),0.3mg/kgL-902,688( n D 8), or1.0mg/kgL-902,688( n D 8).L-902,688(5-[(1E,3R)-4,4diuoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-te trazol5R-yl)hexyl]-2-pyrrolidinone)wasobtainedfromCaymanChemical(AnnArbor,MI,USA;Cat#10007712).L-902,688isapotentEP4agonistwithaK i valueof0.38nMandanEC 50 valueof0.6nM.Itdisplays > 4,000-foldselectivityforEP4over otherprostanoidreceptorsandhasahalf-life invivo of 12h inrats( Youngetal.,2004 ).Treatmentschedulewasdetermined bysimplerandomizationusingacoiniptodeterminetheinitialtreatmentandthentreatmentwasalternated.Visua l conrmationofocclusionwasdemonstratedbycurlingandcirclingbehaviorduringthe90-minocclusionperiod.Inthi s strokemodel,thecoreofthestrokeisrepresentedbysubcorti cal celldeath,andthepotentiallyviablepenumbraisrepresentedb y thecortexinwhichcelldeathoccursmainlybyapoptosisatlat er timepoints.TissueCollectionandHomogenizationRatsweredeeplyanesthetizedwith150mg/kgi.p.pentobarbita l andperfusedwithice-coldphysiologicalsaline.Brainswereextractedandslicedat2mmintervalsinaratbrainmatrix(ZivicInstruments,Pittsburgh,PA,USA).Thefourthslice(anteriortoposterior),whichroughlycorrespondstobregma and representsthecoreofthestrokeinthismodel,wasdissectedintoipsilateralandcontralateralcortexandstriatum/sub cortex, andimmediatelyfrozenondryiceformolecularanalyses.Theremainingsliceswereusedforinfarctcalculation.Tiss ue wasweighedandhomogenizedwithaTissue-Tearorinradioimmunoprecipitationbuercontaining1%sodiumdodecylsulfate(SDS),1%sodiumdeoxycholate,150mMNaCl,50mMTris-HClpH7.6,and1%IGEPAL R r CA-630at10 m L/mgof tissueandHALTProteaseInhibitorCocktail,HALTPhosphata se InhibitorCocktailand0.5MEDTA(Cat.No.78430;Cat.No.78428;andCat.No.1860851,respectively;ThermoFisherScientic,Waltham,MA,USA)at10 m L/mLoftotalvolume. SamplesweresonicatedwithaVibra-Cell TM sonicator(Sonics& MaterialsInc.,Newtown,CT,USA)twicefor15sseparatedby15-minincubationsonicebeforecentrifugationat14,000 gfor 20minat4 C.Theresultingsupernatantswerestoredat 80 C untiluse.InfarctCalculationTomeasuretheinfarctsize,brainslices1–3and5–6wereincubatedinthedarkin2%2,3,5-triphenyltetrazoliumchlo ride inphosphate-bueredsolution(PBS)for30minatroomtemperature,andplacedin4%paraformaldehyde.Livetissuestainsred,anddeadtissueremainswhite.Sectionsweresca nned withanHPScanjet8300(PaloAlto,CA,USA)at600dpirostralsidedownexceptforthe3rdslicewhichwasalsoscannedcaudalsidedowntorepresenttherostralsideofthe4thslice.Duetothesignicantedemaproducedbythisstrokemodel,infarctswerecalculatedindirectly( Swansonetal.,1990;Frankowskietal., 2015 ).UsingAdobePhotoshopCS5,theredtissuewasdelineated foreachsliceandthestrokesurfacearea(mm 2 )wascalculated bysubtractinglive,redtissueontheipsilateralsidefromth ered tissueonthecontralateralside.Tocalculatetotalinfarc tvolume, thesurfacearea(mm 2 )ofdeadtissuewassummedforeachslice andmultipliedbythethicknessoftheslice(2mm).ELISAandMMPActivityAssayTomeasureBBBpermeability,weperformedELISAanalysesforimmunoglobulinG(IgG).BloodproteinslikeIgGarenotpresen t inthebrainparenchymaunlesstheBBBwascompromised,providinganindirectmethodofBBBpermeability;wetherefor e measuredIgGin100 m gproteinfrombrainlysatesprepared fromthoroughlyperfusedratbrains.WeusedacommerciallyavailableratIgGELISAkit(Cat#E101,BethylLaboratories ,Inc., Montgomery,TX,USA). FrontiersinNeuroscience|www.frontiersin.org 3 February2018|Volume12|Article89

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DeMarsetal. EP4ActivationReducesStrokeDamage TwolargecontributorsofBBBdegradationinstrokeare MMP-3andMMP-9whichproteolyticallycleavetight-junctionproteinsbetweenendothelialcellsandcollagenIVinthebasementmembranealongtheendothelium( Candelario-Jalil etal.,2009 ).Usingauorometricimmunocaptureassaythat ourteamdeveloped( Hawkinsetal.,2013 ),wemeasuredMMP3andMMP-9activityin50 m gofbrainlysate.Briey,96wellplateswerecoatedwithProteinA/Gtostablyorientandimmunocaptureantibodies,coatedwitheitheranMMP-3antibody(Cat#SC-6839-R,SantaCruzBiotechnology,Dalla s, TX,USA)oranMMP-9antibody(Cat#SC-6841-R,SantaCruzBiotechnology),andwasincubatedwith50 m goftotalprotein, thenprobedwithaspecicFRETpeptidesubstrate(ForMMP3:SubstrateXIII,Cat#60580-01orMMP-9:SubstrateIII,Cat#60570-01).Thesubstrate(5-FAM/QXL TM 520)isboundto aquenchermoleculethatcanbecleavedbyeitherMMP-9orMMP-3toallowuorescence.Valueswerenormalizedto1ngofrecombinantratMMP-9orMMP-3.ImmunoblottingWeprobed40 m goftotalproteinforthetightjunctionproteins occludin(Cat#ab167161;AbCam)at1:1,000reducedin5%b -mercaptoethanolanddenaturedwith10minofboiling,and zonulaoccludens-1(Cat#61-7300;LifeTechnologies,Carls bad, CA,USA)(ZO-1)at1:500reducedin2% b -mercaptoethanol withoutboiling.Becauseitisknowntodegradetight-junct ion proteins,sowealsoprobed50 m goftotalproteinforMMP-9(Cat #ab76003,Abcam)at1:5,000reducedin5% b -mercaptoethanol anddenaturedwith10minofboiling.Toseparateproteins,weransamplesthrough4–20%Mini-PROTEANTGXgels(Bio-Rad,Hercules,CA,USA)at200Vfor45minin0.1%SDSTris-glycinebuer.GelswereequilibratedinTris-glyc ine buercontaining10%methanolfor10min,thentransferredat25Vfor30minontoeitheranitrocellulose(Cat#926-31092,Li-Cor,Lincoln,NE,USA)orPVDF(Immobilon-FL,Millipore,Billerica,MA,USA)membraneusingthesemi-dryTrans-BlotTurbotransferapparatus.Membraneswereblockedin5%milkinTBSfor1hatroomtemperature,thenincubatedovernightat4 Cwithprimaryantibodyin5%milkinTBST. Membraneswerewashed4timeswithTBST,andincubatedwithgoatanti-rabbitIRDye800CW(1:30,000;Li-Cor)in5%milkinTBSTcontaining0.01%SDSfor1hatroomtemperature.ExcessantibodywasremovedwithfourmoreTBSTwashes,andincubatedwithprimaryantibodyagainst b -actin(1:10,000,Cat #A1978,Sigma-Aldrich,SaintLouis,MO,USA)for1hatroom temperaturetoensureequalproteinloading.MembraneswerewashedfourtimeswithTBST,incubatedwithdonkeyanti-mou se IRDye680LT(1:40,000;Li-Cor)in5%milkinTBSTcontaining0.01%SDSfor1hatroomtemperature,andscannedwithanOdysseyinfraredscanningsystem(Li-Cor).Targetproteins ignal wasdividedbyactinsignaltoobtaindensitometricvalues,a nd normalizedacrossblotsbydividingbyacontrolsample.qRT-PCRTissue(3mm)correspondingtothecoreofthestrokenearbregmawasdissectedintoipsilateralandcontralateralhemispheresandfurtherdividedintostriatalandcorticals ections andplacedinRNAlaterRNAStabilizationReagent(Cat.No.76106,Qiagen,Germany)at10 m L/mgoftissue.RNAwas isolatedwiththeAurumTotalRNAFattyandFibrousTissuekit(CatNo.732-6830;Bio-Rad)accordingtothemanufactur er's instructions.OnemicrogramofRNAwasreverse-transcribedintocDNAwithiScript TM ReverseTranscriptionSupermix(Cat# 1708841,BioRad),anddilutedto10ng/ m LwithIDTEbuerpH 8.0(Cat#11-05-01-13;IntegratedDNATechnologies,Coral ville, IA,USA).TwentynanogramsofcDNAfromipsilateralandcontralateralcorticalandsubcorticaltissuewererunint riplicate, probedwithexon-exonspanningprimers(500nM,IntegratedDNATechnologies)for IL-1 b IL-6 Mmp-9 ,or Mmp-3, and normalizedtothehousekeepinggene Ywhaz ( Frankowskietal., 2015 ; Table1 forprimersequences)withPerfeCTa R r SYBR R r GreenFastmix R r (Cat#95072-012;QuantaBiosciences,Beverly, MA,USA)usingaBio-RadCFX96TouchReal-TimePCRDetectionSystemwiththefollowingprocedure:polymeraseactivation/DNAdenaturationphaseat95 Cfor30s,then40 cyclesofdenaturingat95 Cfor5sandannealingat60 C for30s.Specicityofeachprimerwasconrmedusingnon-templatecontrolsandmeltcurves.Thenormalizedexpressionshowninthebargraphs( Figure4 )wascalculatedusingthe CFXManager TM software(Bio-Rad)andrepresenttherelative quantityofthetargetgenenormalizedtothereferencegene( Ywhaz ),andfurthernormalizedtothebiologicalcontrol (contralateralsampleofthevehicle-treatedgroup).AssessmentofLong-TermNeurologicalDecitsRatsweretrainedontasks24and48hbeforeinducingMCAOandtested48h,andat1,2,and3weekspost-ischemia.Tomeasurelong-termsensoryandnemotorcontroldecitswit h theadhesiveremovaltest,ratsweretrainedtoremoveasmal l TABLE1| PrimersequencesforqRT-PCRexperiments. GeneAccessionnumberForward Reverse IL-1B NM_0315125 0 -GTGCTGTCTGACCCATGT-3 0 5 0 -TTGTCGTTGCTTGTCTCTCC-3 0 IL-6 NM_0125895 0 -CAGAGCAATACTGAAACCCTAGT-3 0 5 0 -CCTTCTGTGACTCTAACTTCTCC-3 0 Mmp9 NM_0310555 0 -GAACTCACACAACGTCTTTCAC-3 0 5 0 -GGAGGTCATAGGTCACGTAGG-3 0 Mmp3 NM_1335235 0 -CTATTCCTGGTTGCTGCTCAT-3 0 5 0 -CTGTGGAGGACTTGTAGACTG 3 0 Ywhaz NM_0130115 0 -GAAGAGTCGTACAAAGACAGCA-3 0 5 0 -GCTTCTGCTTCGTCTCCTTG-3 0 IL,interleukin;Mmp9,matrixmetalloproteinase-9;Mmp3,matrixmetal loproteinase-3;Ywhaz,tyrosine3-monooxygenase/tryptophan5-monooxygenaseactiv ationprotein,zeta. FrontiersinNeuroscience|www.frontiersin.org 4 February2018|Volume12|Article89

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DeMarsetal. EP4ActivationReducesStrokeDamage FIGURE1| ReducedInfarctwithEP4AgonistL-902,688.Usingaone-wayA NOVAwithaDunnett'sMultipleComparisonposttest,wefoun dthat1.0mg/kg signicantlyreducedinfarctsizeinboththe (A) cortex( p D 0.0012)andthe (B) subcortex( p D 0.0018). (C) Totalinfarctvolumeisreducedwith1.0mg/kgL-902,688 (Students' t -test* P D 0.0123) (D) RepresentativeTTC-stainedslicesfromavehicle-andL-90 2,688-treatedbrainsafter24hofreperfusionfollowing90 minofMCAO. Vehicle( n D 10),0.3mg/kg( n D 8),and1.0mg/kg( n D 8)L-902,688.* P < 0.05and** P < 0.01comparedwithvehicle. FrontiersinNeuroscience|www.frontiersin.org 5 February2018|Volume12|Article89

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DeMarsetal. EP4ActivationReducesStrokeDamage stickerplacedonthefront,contralateralpaw,andthelatenc y toremovewasrecordedfor3trials,andtheaverageofthetwolowestvalueswaschosenforanalysis.Tomeasuremotordec its, ratsweretrainedtostayonarotarodthatacceleratedfrom4to40rpm.Latencytofallowasmeasuredfor5trialsandtheaverageofthetwohighestvalueswerenormalizedtoeachanimal'sbaselinevalues.Experimentaldetailsoftheadhes ive removalandacceleratingrotarodtestshavebeendescribed by ourgroupinrecentpublications( Hawkinsetal.,2017;Yangetal., 2017 ). StatisticsInfarctmeasurementwasperformedwithaone-wayANOVAwithaDunnett'smultiplecomparisonpost-test.PCRdatawasanalyzedwithaStudent's ttestcomparingvehicle-treated ipsilateraldatatoL-902,688-treatedipsilateraldata.Beh avioral performancewasanalyzedusinga ttestbetweenvehicle-treated ratsandL-902,688-treatedratsateachtimepoint.Statisti cswere analyzedwithGraphPadPrismversion6.0,anda p -valueof lessthan0.05wasconsideredstatisticallysignicant.Da taare reportedasmean SEM. RESULTSAfter24hofreperfusion,infarctvolumewassignicantlyreducedinthecortex( P < 0.05, P < 0.01, Figure1A )and subcortex( P < 0.05, P < 0.01, Figure1B )with0.3and1.0 mg/kgL-902,688,respectivelycomparedtothevehicle-treat ed group.Becauseonly1.0mg/kgL-902,688signicantlyreduce d totalinfarctvolume( P < 0.05, n D 8–10, Figure1C ),thisdose wasusedfortherestofthestudy.RepresentativeTTC-stainedbrainsectionsareshownin Figure1D forbothtreatmentgroups, whichhelptobetterappreciatethereductionininfarctsizei n strokedratsreceivingtheEP4agonist,L-902,688,attheon setof reperfusion(after1.5hofstrokeonset). Toelucidatethemechanismofprotectionwith1.0mg/kg L-902,688,IgGextravasationintothebrainparenchymawas measuredwithanELISA.BecauseIgGisabloodprotein,thereisminimalamountdetectedinathoroughlyperfusedbrainunlesstheBBBintegrityiscompromised.With1.0mg/kgL-902,688,wefoundsignicantreductionofIgGintheipsilater al cortex( P < 0.05, Figure2A )andtheipsilateralsubcortex ( P < 0.05, Figure2B )comparedtotheipsilateralvehiclecortex andsubcortex. BecauseMMP-3andMMP-9aremajorcontributorsto BBBdamageafterstroke,wewantedtoseeifthereducedBBBdamageevidencedbyreducedIgGextravasationwasassociatedwithreducedMMP-3/MMP-9activityandproteinlevels.Densitometricanalysisofimmunoblotsshowedreduc ed MMP-9levelsintheipsilateralcortex( P < 0.05, Figure3A ) andanon-signicantreductionintheipsilateralsubcortex( p D 0.0792, Figure3B )comparedtoipsilateralvehiclevalues. ThiseectwasmirroredinourMMP-9activityassaydatainthecortical( P < 0.05, Figure3C ),butnotsubcortical( Figure3D ) tissue.BecauseMMP-3canactivateMMP-9,wealsomeasuredMMP-3activityandfound1.0mg/kgL-902,688reducedMMP-3activityintheipsilateralcortex( P < 0.05, Figure3E ),butnot inthesubcortex( Figure3F ). Wealsomeasuredreducedexpressionofcorticaland subcorticalIL-1 b andIL-6expression( P < 0.05, P < 0.01, Figures4A–D ).Treatmentwith1.0mg/kgL-902,688also signicantlyreducedMMP-9( P < 0.001)andMMP-3 expression( P < 0.01)inthecortex( Figures4E,G )andnonsignicantlyreducedmRNAlevelsofMMP-9andMMP-3inthesubcortex( p D 0.2335, Figure4F; p D 0.5104, Figure4G ). ReducedIgGextravasationandreducedMMP-9and MMP-3activitysuggestedthat1.0mg/kgL-902,688reducedstroke-inducedBBBdamage.WethereforemeasuredlevelsofthetightjunctionproteinsZO-1andoccludinincorticaltissuewithimmunoblotting.Wefoundanon-signicantpreservationofZO-1intheipsilateralcortexinL-902,688-treatedratscomparedtovehicle-treatedrats( Figure5A ). Additionally,degradationofthe125-kDaoccludindimerwassignicantlyreducedintheipsilateralcortexwith FIGURE2| ReducedIgGinthebraininstrokedratstreatedwiththeEP4Ag onistL-902,688. (A) ThereissignicantlyreducedIgGafter24hofreperfusionint he1.0 mg/kgL-902,688CXIgroupcomparedtothevehicleCXIgroup.( P D 0.0413). (B) Inanimalsreceiving1.0mg/kgL-902,688,theipsilateralsub corticalIgGlevelsare alsosignicantlyreduced.(Student's ttest* P D 0.0254).CXI,ipsilateralcortex;CXC,contralateralcorte x;STI,ipsilateralsubcortex;STC,contralateralsubcorte x. Vehicle( n D 10)and1.0mg/kgL-902,688( n D 8). FrontiersinNeuroscience|www.frontiersin.org 6 February2018|Volume12|Article89

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DeMarsetal. EP4ActivationReducesStrokeDamage FIGURE3| ReducedMMP-9andMMP-3ActivityinischemicratsgiventheE P4AgonistL-902,688.WemeasuredreducedMMP-9proteinwit himmunoblottingafter 24hofreperfusioninthe (A) cortex(* P < 0.05)and (B) thesubcortex( P D 0.0792).TherewasreducedMMP-9activitywithL-902,688tr eatmentinthecortex (C) ,butnotinthesubcortex (D).(E) Inthecerebralcortex,1.0mg/kgL-902,688signicantlyreduc esMMP-3activityintheipsilateralsideofthetreatedgrou pvs.the vehiclegroup.* P D 0.0215. (F) Inthesubcortex,theeffectofL-902,688intheipsilateralh emisphereislesspronounced( P D 0.1304).CXI,ipsilateralcortex;CXC, contralateralcortex;STI,ipsilateralsubcortex;STC,con tralateralsubcortex.Vehicle( n D 10)and1.0mg/kgL-902,688( n D 8). EP4receptoractivation.Thisisassociatedwithreducedinjury-inducedlowmolecularweight65-kDaoccludin( Figure5B ). Finally,wewantedtoconrmthatthereductionininfarctsi ze wasassociatedwithreducedneurologicaldecitslong-ter m.EP4 activationwithoneintravenousinjectionof1.0mg/kgL-90 2,688 attheonsetofreperfusionshowedsustainedimprovementinneurologicalfunction.Animalsreceiving1.0mg/kgL-902, 688 performedbetterattheadhesiveremovaltestat1,2,and3wee ks afterstroke( P < 0.05, P < 0.01, Figure6A )andwerealso abletostayontherotarodlongerthanvehicle-treatedratsa t1,2, and3weeksafterstroke( P < 0.05, Figure6B ). FrontiersinNeuroscience|www.frontiersin.org 7 February2018|Volume12|Article89

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DeMarsetal. EP4ActivationReducesStrokeDamage FIGURE4| ReducedIL-1 b ,IL-6,MMP-9,andMMP-3expressionwithL-902,688treatment (A )IL-1 b isdecreasedintheipsilateralcortex(** P D 0.0082)inrats givenL-902,688. (B) Therewasanon-signicant( P D 0.0888),butsubstantialdecreaseinIL-1 b intheipsilateralsubcortex. (C) IL-6issignicantlydownregulatedin theipsilateralcortex(** P D 0.0089)andslightlyreducedinthesubcortex (D) ( P D 0.1186) .(E) L-902,688decreasedMMP-9intheipsilateralcortex(*** P < 0.001) (F) TherewasatrendtowardMMP-9downregulationwithtreatmen tintheipsilateralsubcortex,butitdidnotreachsignican ce( P D 0.2335). (G) EP4receptor activationwithL-902,688potentlyreducesMMP-3mRNAexpr essionintheischemiccerebralcortex(** P D 0.0083). (H) NosignicantreductioninMMP-3 expressionwasfoundintheischemicsubcorticalregionbet weentreatmentgroups( P D 0.5104).Student's t -testcomparingvehicleandL-902,688ipsilateralgroups. CXI,ipsilateralcortex;CXC,contralateralcortex;STI,ips ilateralsubcortex;STC,contralateralsubcortex.Vehicl e( n D 10)and1.0mg/kgL-902,688( n D 8). FrontiersinNeuroscience|www.frontiersin.org 8 February2018|Volume12|Article89

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DeMarsetal. EP4ActivationReducesStrokeDamage FIGURE5| EffectofL-902,688onTightJunctionProteins.Weperforme dimmunoblotstomeasure (A) ZO-1invehicle( n D 10)andL-902,688( n D 8)groupsand foundanon-signicanttrendtowardZO-1preservationinthe ipsilateralhemisphereofthetreatedgroupcomparedtothe vehicleincorticalsamples. (B) Degradation ofthedimericformofoccludin( 125kDa)wasreducedintheipsilateralcortexofL-902,688a ndthiswasassociatedwithdecreasedinductionofthelowmo lecular weightoccludin( 65kDa).Dataarereportedasipsilateraldividedbycontral ateralduetothewidevariabilityintheoccludincontentin boththecontralateralandthe ipsilateralhemispheresofvehicle-treatedrats.CXI,ipsi lateralcortex;CXC,contralateralcortex;STI,ipsilatera lsubcortex;STC,contralateralsubcortex.* P < 0.05 comparedwithvehicle-treatedanimals. DISCUSSIONHere,weshowforthersttimethatactivationoftheEP4rece ptor withL-902,688givenattheonsetofreperfusionsignicantl y reducesinfarctsize,blood-brainbarrier(BBB)breakdown MMP-3andMMP-9levels,degradationoftightjunctionproteins,andstroke-inducedincreaseintheexpressionofth e pro-inammatorycytokinesIL-1 b andIL-6.Moreimportantly, post-ischemictreatmentwithL-902,688resultedinanimprov ed long-termneurologicalrecoveryasassessedusingtheadhe sive removalandacceleratingrotarodtests. Expressedincardiovascular,neuronal,andimmunecells ( Sandoetal.,1994;HataandBreyer,2004;Yokoyamaetal.,20 13 ) theEP4receptorisuniquelysuitedtoinuenceinfarctoutco me inischemicstroke.TheBBBcanbeconceptuallydissectedint o neurovascularunitscomprisedofneurons,astrocyticendfeet pericytes,andendothelialcells.Typically,theneurovascula r unitsynergisticallymaintainshomeostaticlevelsofperme ability forvasculaturetoparenchymasubstanceexchange.Duringanischemicevent,theBBBundergoesbiphasicopeningat3and48hafterreperfusionwhichiscorrelatedwithMMPlevelsinthi s strokemodel( Rosenbergetal.,1998 ).Thisisrelevantbecause MMP-9-inducedBBBopeningisdetrimentalintheacutephaseofstroke( Soodetal.,2008;Yangetal.,2010 ). Becauseendothelialcellsareonthefrontline,theyarethe rstcelltypetobeaectedbyhypoxia.OnemechanismofneuroprotectionmaybeviaEP4receptor-inducedvasodilatio n, alteringcerebralbloodowinresponsetoischemia( Taniguchi FrontiersinNeuroscience|www.frontiersin.org 9 February2018|Volume12|Article89

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DeMarsetal. EP4ActivationReducesStrokeDamage FIGURE6| Post-ischemictreatmentwithL-902,688Reduces Stroke-InducedNeurologicalDecits. (A) L-902,688administrationreduces thelatencytocontralateraladhesiveremovalinratssubje ctedtostroke. (** P < 0.01,* P < 0.05). (B) L-902,688increasesthetimespentontherotarod comparedtovehicle-treatedrats(* P < 0.05).Dataanalyzedwithmultiple t -tests; n D 10ineachtreatmentgroup. etal.,2014 ).ThismakessenseinlightofthefactthatEP4 receptoractivationincreaseseNOSandphospho-Ser 1177 eNOS, increasinglocalNOlevels.Directprotectionmayalsoshiel d neuronsfromagainststrokeinsult,aswellbecauseneurona lEP4 receptoractivationreducescelldeath invitro and exvivo after excitotoxicchallengeand invitro afterhypoxic/hypoglycemic challenge( Liangetal.,2011 ).Theseeectslikelytranslate intoreducedinfarctsizewithasinglebolusofEP4receptoragonistL-902,688attheonsetofreperfusioninourstrokemodeloftransientfocalischemia.Thisreductioniscorrel ated withdecreasesinseveralmeasuresofBBBpermeability.EP4receptoractivationreducesIgGextravasationinboththeco re ofthestrokerepresentedbytheipsilateralsubcortexandinthepenumbrarepresentedbytheipsilateralcortex.EP4receptoractivationsimilarlyreducedstroke-inducedMMP-9andMMP-3activity,particularlyinthecortex,andstroke-inducedMMP-9mRNAlevels.MMP-9andMMP-3arekeycontributorstoBBBdisruptioninthecontextofischemicstrokesincetheseproteasesdegradethebasallaminaand tightjunctionproteinsessentialtothebarrierfunctionof the neurovascularunit( Rosenbergetal.,1998;Asahietal.,2001; Roselletal.,2008;Candelario-Jaliletal.,2009;Turneran dSharp, 2016 ). Notsurprisingly,EP4receptoragonismreducedproinammatorygenetranscriptionalongwithreducedinfarcta nd BBBdamage.IL-1 b activatesmicroglia/macrophages,stimulating moreIL-1 b releaseandtriggeringimmunecellinltrationthat contributetoincreasedBBBpermeabilityandapoptoticdeathi n penumbralneurons( Yamasakietal.,1995;HawkinsandDavis, 2005;McColletal.,2007;Clausenetal.,2008;SandovalandW itt, 2008 ).WefoundrobustreductionsinIL-1 b geneexpression inthecortexandthesubcortexwithEP4receptoractivation.L-902,688furtherreducedacutephaseIL-6expression,andincreasedIL-6iscorrelatedwithlargerstrokevolumeandworseoutcome( Waje-Andreassenetal.,2005 ).Thesedataare interestingbecauseEP4activationhasshowntoupregulateI L-6 expressioninsomecelltypes( HataandBreyer,2004;Zhouetal., 2016 ).L-902,688-dependentIL-6reductionsarelikelyreective ofreducedcelldamage/infarction( Tarkowskietal.,1995;Suzuki etal.,1999,2009;Smithetal.,2004 ).Inamodelofsubarachnoid hemorrhageinrats,averyrecentstudyfoundthatAE1-329,a n EP4receptoragonist,signicantlyreducedBBBdamage,edem a, andexpressionofIL-1 b ,IL-6,andTNFa ( Xuetal.,2017 ). EP4agonismprotectsthetightjunctionproteinsbetween endothelialcellsthatarevitaldeterminantsofBBBpermeab ility. Inthecortex,wefoundanon-signicanttrendtowardZO-1preservationwithEP4receptoractivation,andsignicantpreservationofdimericoccludin(125kDa)intheipsilateralcortexwhichwasassociatedwithreducedstroke-induced65 -kDa occludin,alikelyphosphorylatedformofoccludin.Increasedlevelsofthe65-kDabandofoccludinhavebeendetectedinmodelsofischemicstrokeandareassociatedwithBBBdisruptio n ( Kagoetal.,2006;Takenagaetal.,2009;Fukumotoetal.,201 0; Muthusamyetal.,2014;Frankowskietal.,2015 ).Nochangein thelowermolecularweightoccludinband(50kDa)wasobserve d betweenipsilateralandcontralateralhemispheresirrespecti ve ofthetreatmentgroup.Ourmodeloffocalischemiatypicallyinducessuchoccludinalterations( Frankowskietal.,2015 ). OxidativestressisakeymechanismofBBBdisruptionand neuronaldeathinischemicstroke( Chan,2001;Lietal.,2017 ). Freeradicals/ROSdirectlydamageendothelialcellscomposin g theBBBandindirectlyactivateMMPs,whichleadtoproteolyticbreakdownofbasallaminaproteinsandTJPsresultingininjur y totheneurovascularunit( Grsoy-Ozdemiretal.,2004;Kahles etal.,2007;Hafezetal.,2018 ).Onepotentialmechanismthrough whichEP4receptoractivationcouldreducestroke-inducedBB B openingisreductionofoxidativedamageduringthereperfusi on phase.ThisnotionissupportedbypreviousstudiesshowingthatEP4agonistsreducefreeradicalformationinneuronsa nd microgliaexposedtoamyloid b ( Echeverriaetal.,2005 )or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin(MPTP)( Pradhan etal.,2017 ).Itremainstobedeterminedwhethertreatmentwith L-902,688orotherEP4agonistsreducesoxidativestress invivo afterstroke. TheEP4receptorisexpressedinmanycelltypesincluding endothelium,neurons,microglia,astrocytes,andperiphera l FrontiersinNeuroscience|www.frontiersin.org 10 February2018|Volume12|Article89

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DeMarsetal. EP4ActivationReducesStrokeDamage immunecells( Sandoetal.,1994;HataandBreyer,2004; Yokoyamaetal.,2013;Bonll-Teixidoretal.,2017 ).Permeability oftheBBBafterstrokecanbealteredbycomplexcellularandmolecularinteractionsbetweencellsoftheneurovascular unit andtheperipheralimmunesystem.WefoundthatEP4receptoractivationwithL-902,688potentlyreduceslevelsofsomeofthekeymediatorsofstroke-inducedBBBdamageincludingIL 1 b ,IL-6,MMP-3,andMMP-9.Themaincellularsourcesof thesepro-inammatorymediatorsafterstrokeincludeactiv ated microgliaandastrocytes,aswellasinltratingneutrophil sand macrophages( Benakisetal.,2014;Amanteaetal.,2015 ).Ithas beenshownthatEP4signalingdecreasestheactivationofnu clear factor-kB(NF-kB),amasterregulatorofpro-inammatoryge ne transcription,inactivatedmicroglia( Shietal.,2010;Woodling etal.,2014 ),aswellasinperipheralimmunecells( Takayama etal.,2002,2006;Minamietal.,2008 ).Basedonourdata andpreviousreports,suppressionofimmunecellactivationandproductionofpro-inammatorymediatorsaresuggestedasmechanismsbywhichEP4agonismconfersneurovascularprotectioninischemicstroke. Mostimportantly,wefoundthatasingleadministrationofL902,688attheonsetofreperfusionreducedsensorimotorde cits intheadhesiveremovaltestandtherotarodassessmentupto 3 weeksafterstroke.Althoughinfarctsizeisnotalwaysacon sistent indicatorofstrokeoutcome,sensitivebehavioralassessm entslike theadhesiveremovalandrotarodtestsarerelativelyrelia ble indicatorsoffunctionalneurologicaldecitsthatcandet ect changesatleastupto3weeksafterinjury( Yangetal.,2017 ). Limitationsofourstudyincludetheutilizationofonetype oftransientstrokemodelthatincludesreperfusioninjury, and thesedatawouldbestrengthenedbyconrmingneuroprotecti on inotherstrokemodels.Weonlyusedyounghealthymalerats,whichisanotherlimitationofourstudy.Sinceage,diabete s, hypertension,andhypercholesterolemiaareamongthemostimportantriskfactorsforstroke,futurestudiesshouldinv estigate theeectsofEP4agonisminanimalsofbothsexeswiththesecomorbidconditions.Furthermore,becausewewerelimited to pharmacologicalinterventioninrats,itwouldbeofinteres t tosubjecttransgenicconditionalknockoutmicelackingEP 4 specicallyinmyeloidcells,endothelialcells,orneuronstostroketodeterminetherelativecontributionofEP4activa tion fromdierentcellsoftheneurovascularunit.Futurestudie s willdeterminewhetherdelayedadministrationofL-902,688(severalhoursafterstrokeonset)alsoconferssustained,lo ngtermneuroprotectionaswasfoundwithadierentEP4receptoragonistinmice( Liangetal.,2011 ).Thiswillprovidefurther supportthatEP4receptoractivationhasclinicalrelevanceifitprovestobeeectiveupto4.5hafterstroke,thecurrenttherapeuticwindowfortPA. Toourknowledge,wearetherstgrouptoestablishthatEP4 agonismwithasingleadministrationofL-902,688attheonsetofreperfusionisneuroprotectiveinatransientMCAOstrokemodelinratsupto3weeksafterischemia.Thisneuroprotecti on isduetothedynamiccrosstalkbetweeninammationandBBBdegradation.EP4activationreducespro-inammatoryIL 1 b genetranscriptionandmatrixmetalloproteinasesMMP-3 andMMP-9,majorcontributorsofBBBdamage.Theseeectsculminateinreducedtightjunctionproteindegradationtha t maintaintheintegrityoftheBBBandreducedlong-termneurologicaldecits.AUTHORCONTRIBUTIONSKD,AM,DS,BS,CY,andEC-J:Performedexperimentalprocedures;KDandEC-J:Designedresearchandplannedalltheexperiments;KD,AM,andEC-J:Analyzedthedataandpreparedthegures;KDandEC-J:Wrotethearticle;EC-J:Conceivedandledtheproject;Alltheauthorsreadandapprovedthenalversionofthemanuscript.FUNDINGFundingforthisprojectwasprovidedbytheNationalInstitute of Health(NIH),grantnumberR01-NS065849toEC-J.PublicationofthisarticlewasfundedinpartbytheUniversityofFlorida,OpenAccessPublishingFund. 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DeMarsetal. EP4ActivationReducesStrokeDamage Bonll-Teixidor,E.,Otxoa-de-Amezaga,A.,Font-Nieves,M., Sans-Fons,M.G., andPlanas,A.M.(2017).DierentialexpressionofE-typeprostano idreceptors 2and4inmicrogliastimulatedwithlipopolysaccharide. J.Neuroinammation 14:3.doi:10.1186/s12974-016-0780-7 Candelario-Jalil,E.,Gonzalez-Falcon,A.,Garcia-Cabrera,M.,Le on,O.S.,and Fiebich,B.L.(2007).Post-ischaemictreatmentwiththecycloo xygenase-2 inhibitornimesulidereducesblood-brainbarrierdisruptionandleuk ocyte inltrationfollowingtransientfocalcerebralischaemiainrats. J.Neurochem. 100,1108–1120.doi:10.1111/j.1471-4159.2006.04280.x Candelario-Jalil,E.,Thompson,J.,Taheri,S.,Grossetete,M.,Ada ir,J.C.,Edmonds, E.,etal.(2011).Matrixmetalloproteinasesareassociatedwithinc reasedbloodbrainbarrieropeninginvascularcognitiveimpairment. 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Neuroinammation 13:141.doi:10.1186/s12974-016-0607-6 ConictofInterestStatement: Theauthorsdeclarethattheresearchwas conductedintheabsenceofanycommercialornancialrelations hipsthatcould beconstruedasapotentialconictofinterest.Copyright2018DeMars,McCrea,Siwarski,Sanz,YangandCandela rio-Jalil. Thisisanopen-accessarticledistributedunderthetermso ftheCreativeCommons AttributionLicense(CCBY).Theuse,distributionorrepro ductioninotherforums ispermitted,providedtheoriginalauthor(s)andthecopyri ghtownerarecredited andthattheoriginalpublicationinthisjournaliscited,i naccordancewithaccepted academicpractice.Nouse,distributionorreproductionis permittedwhichdoesnot complywiththeseterms. FrontiersinNeuroscience|www.frontiersin.org 13 February2018|Volume12|Article89