The protective effects of plasma gelsolin on stroke outcome in rats

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Title:
The protective effects of plasma gelsolin on stroke outcome in rats
Series Title:
Experimental & Translational Stroke Medicine
Physical Description:
Mixed Material
Language:
English
Creator:
Le, Huong T.
Hirko, Aaron C.
Thinschmidt, Jeffrey S.
Grant, Maria
Li, Zhimin
Peris, Joanna
King, Michael A.
Hughes, Jeffrey A.
Song, Sihong
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BioMed Central
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Abstract:
Background: To date, recombinant tissue plasminogen activator (rtPA) is the only approved drug for ischemic stroke. It is intravenously administered functioning as a thrombolytic agent and is used to obtain reperfusion of the affected area of the brain. Excitotoxicity, inflammation and apoptosis are all involved in delayed neuronal death following stroke and offer multiple opportunities to intervene with neuroprotective agents. Gelsolin (GSN) is an actin- and calcium-binding protein mediating the disassembly of actin filaments and activity of calcium channels. It also functions as a regulator of apoptosis and inflammatory responses. This study tests the hypothesis that increasing the concentration of the form of GSN known as plasma GSN (pGSN) near an infarct will provide neuroprotection following ischemic stroke. Methods: We induced middle cerebral artery occlusion (MCAO) in male rats via intracranial injection of endothelin- 1 (ET-1), a potent vasoconstrictor, and then treated with local delivery of pGSN. Whole brain laser Doppler perfusion imaging was performed through the skull to assess MCAO effectiveness. Cylinder and vibrissae tests evaluated sensorimotor function before and 72 h after MCAO. Infarct volumes were examined 72 h after MCAO via 2, 3, 5-triphenyltetrazolium chloride (TTC) assay. Results: Estimates of relative cerebral perfusion were significantly decreased in all groups receiving MCAO with no differences detected between treatments. Despite equivalent initial strokes, the infarct volume of the pGSN treatment group was significantly reduced compared with the untreated MCAO rats at 72 h. ET-1 induced significant deficits in both cylinder and vibrissae tests while pGSN significantly limited these deficits. Conclusion: Gelsolin could be a promising drug for protection against neurodegeneration following ischemic stroke. Keywords: ischemic stroke, plasma gelsolin, protective effect, endothelin-1 induced MCAO
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Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/ufir) at the time of funding. The Institutional Repository at the University of Florida (IR@UF) is the digital archive for the intellectual output of the University of Florida community, with research, news, outreach and educational materials

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RESEARCH OpenAccessTheprotectiveeffectsofplasmagelsolinon strokeoutcomeinratsHuongTLe1,AaronCHirko2,JeffreySThinschmidt2,MariaGrant2,ZhiminLi3,JoannaPeris3,MichaelAKing2,4, JeffreyAHughes5andSihongSong1*AbstractBackground: Todate,recombinanttissueplasminogenactivator(rtPA)istheonlyapproveddrugforischemic stroke.Itisintravenouslyadministeredfunctioningasathrombolyticagentandisusedtoobtainreperfusionofthe affectedareaofthebrain.Excitotoxicity,inflammationandapoptosisareallinvolvedindelayedneuronaldeath followingstrokeandoffermultipleopportunitiestointervenewithneuroprotectiveagents.Gelsolin(GSN)isan actin-andcalcium-bindingproteinmediatingthedisassemblyofactinfilamentsandactivityofcalciumchannels.It alsofunctionsasaregulatorofapoptosisandinflammatoryresponses.Thisstudyteststhehypothesisthat increasingtheconcentrationoftheformofGSNknownasplasmaGSN(pGSN)nearaninfarctwillprovide neuroprotectionfollowingischemicstroke. Methods: Weinducedmiddlecerebralarteryocclusion(MCAO)inmaleratsviaintracranialinjectionofendothelin1(ET-1),apotentvasoconstrictor,andthentreatedwithlocaldeliveryofpGSN.WholebrainlaserDoppler perfusionimagingwasperformedthroughtheskulltoassessMCAOeffectiveness.Cylinderandvibrissaetests evaluatedsensorimotorfunctionbeforeand72hafterMCAO.Infarctvolumeswereexamined72hafterMCAOvia 2,3,5-triphenyltetrazoliumchloride(TTC)assay. Results: EstimatesofrelativecerebralperfusionweresignificantlydecreasedinallgroupsreceivingMCAOwithno differencesdetectedbetweentreatments.Despiteequivalentinitialstrokes,theinfarctvolumeofthepGSN treatmentgroupwassignificantlyreducedcomparedwiththeuntreatedMCAOratsat72h.ET-1induced significantdeficitsinbothcylinderandvibrissaetestswhilepGSNsignificantlylimitedthesedeficits. Conclusion: Gelsolincouldbeapromisingdrugforprotectionagainstneurodegenerationfollowingischemic stroke. Keywords: ischemicstroke,plasmagelsolin,protectiveeffect,endothelin-1inducedMCAOIntroductionStrokeorbrainattackoccurswhenthebloodsupplyto thebrainisinterrupted,usuallybecauseabloodvessel isblockedbyaclotorlosesstructuralintegritypermittinghemorrhage.Thediseaseisnotsubjecttoaparticularraceorethnicgroup[1].In2009,795,000strokes occurredintheUnitedStates,i.e.astrokeoccursonce every40secondsandadeathoccursevery4minutes [2].AccordingtotheCentersforDiseaseControland Prevention(CDC),thetotalcostofstrokewas$68.9 billionandthenumberisexpectedtorise.Ofall strokes,87%areischemic[2 ].Currently,recombinant tissueplasminogenactivator(rtPA)istheonlyFDAapprovedtherapeuticagentforischemicstroke.rtPAis effectiveonlyifintravenouslyadministeredwithin3to 4.5hofstrokeonset,andcanhaveadverseneurotoxic effectsevenwithproperuse[3].Thedrugcanonlybe usedwithinanarrowtimewindowafterastrokebegins andonlyabouttwopercentofstrokepatientsareable toaccessrtPAtherapy.Therefore,developmentofnew agentsforstrokeisessential. Themechanismsinvolvedinstrokeinjuryandrepair areextremelycomplex,involvingexcitotoxicityand necroticcelldeathoccurrin gwithinminutesofstroke *Correspondence:shsong@ufl.edu1DepartmentofPharmaceutics,UniversityofFloridaCollegeofPharmacy, Gainesville,FL32610,USA FulllistofauthorinformationisavailableattheendofthearticleLe etal Experimental&TranslationalStrokeMedicine 2011, 3 :13 http://www.etsmjournal.com/content/3/1/13 2011Leetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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onset[4].Aswell,thereisincreasingevidenceshowing thatgeneticallyprogrammedcelldeathduringpostischemictissueinflammation(thatcanlastdaysto weeks)hasadetrimentaleffect[5,6].Therefore,therapeuticstrategiestargetingthatdelayordampening inflammatoryresponsescouldinhibittheprogressionof thetissuedamageandimprovetheoveralloutcomeof stroke. Gelsolin(GSN)isaubiquitous[7,8]actinfilamentsevering,cappingandactinnucleationproteinofeukaryotes.Originallydescribedasanactin-bindingprotein, GSNexistsinbothintracellular(cytoplasmicprotein, cGSN)andextracellular(asecretedproteinorplasma gelsolin,pGSN)forms.pGSN,alsoknownasbrevinand actin-depolymerizingfactor,consistsofasingle755aminoacidpolypeptidechain(84kDa)includinga25aminoacidN-terminalexten sion[9]thatdistinguishes itfromcGSN(82kDa).MostcellssecretepGSN,howeversmooth,skeletalandcardiacmusclecellsproduce largeramountsofpGSN[7].Theplasmaconcentration ofpGSNis200-300mg/L[10-12]andisolatedhuman andrabbitpGSNhaveaplasmahalf-lifeof2.3days [13].BecausepGSNderivesfrommuscletissue,itmust passthroughinterstitialfluidoftheextracellularmatrix tolocalizeintheblood.pGSNalsoexistsinhumancerebrospinalfluid(CSF)[14].Althoughcertainfunctions fortheintracellularisoformshavebeendescribed,the function(s)oftheplasmaisoformsremainunclear.The highaffinityofpGSNforfilamentousactin(F-actin)(Ka>109/mol/L)[15]suggeststhatitsphysiologicalfunction islikelyrelatedtoitsactin-bindingproperties.pGSN mayscavengeactinleakedfrominjuredtissueandlimit subsequentdamageinstigatedbyextracellularfilamentousactin[16].Studieshaveshownthatlargeamounts ofF-actincouldpotentiallyincreasethebloodviscosity andperturbbloodflowthroughthemicrovasculature [17]. ItisalsowellestablishedthatpGSNlevelsdecreasein bloodinacuteinflammationconditionsthatinvolvetissuedamage[18-21].ConsistentwiththeideathatpGSN isnotonlyabiomarkerforinflammationbutalsoan importantprotectivefactor,repletionofpGSNina mousemodelofendotoxemicsepsisledtosolubilization ofcirculatingactinaggregatesandsignificantlyreduced mortalityinmice[22].GSNknockoutmiceneuronsare vulnerabletoglucose/oxygendeprivation,andpharmacologicalbrainactindepolymerizationrestoredresistancetoischemicstrokei nknockoutmice[23].The knockoutmiceresultscouldnotdeterminewhichendogenousformofgelsolinisresponsible,orwhethergelsolininorneartheinfarctmediatesneuroprotective effects.Gelsolin-overexpressingtransgenicmicedemonstrateneuroprotectionagainstexperimentalstroke[23], butitisnotknownwhethertheseeffectsaremediated bypGSNorcGSN,orwhetheritisGSNnearthe infarctthatmediatestheprotection. Totestthehypothesisthatproximaladministrationof pGSNcanantagonizestrokepathology,weinduced transientmiddlecerebralarteryocclusion(tMCAO)in maleratsviaintracranialinjectionofET-1,apotent vasoconstrictor,andpost-treatedwithdiscretebrain injectionofpGSN.Cylinderandvibrissaetestswere usedtoexaminesensorimotorfunctionbeforeand72h afterMCAOtoassessfunctionaldeficits.Wholebrain laserDopplerperfusionimagingwasperformedthrough theskulltoverifyMCAOeffectiveness.Infarctvolumes wereexamined72hafterMCAOusing2,3,5-triphenyltetrazoliumchloride(TTC)assay.MaterialsandmethodsMaterialsEndothelin-1(1-39)rat(ET-1),purchasedfromAmericanPeptideCompany,Inc.(CA,U.S.A.),wasdissolved insterilephosphatebufferedsaline(PBS)tomakea stockconcentrationof80 M.Thestocksolutionwas storedat-20C.ET-1wasthawed,centrifugedand placedoniceuntilreadytoinject.Humanplasmagelsolin(pGSN)wasagenerousgiftfromCriticalBiologics Corporation(MA,U.S.A.)Arti ficialcerebrospinalfluid (aCSF)wasobtainedfromFisherScientific,Inc.(PA,U. S.A.)AnimalmodelExperimentswereconductedon19maleSprague-Dawleyratsweighingfrom210-270g.Animalswererandomlydividedintofivegroups:twogroupsofthreefor laserDopplermeasurement,twogroupsoffivefor experimentalischemicstrokebyET-1pluspGSNor controlinjections,withevaluationofbehaviorandbrain damage,agroupofthreeforartificialcerebrospinalfluid (aCSF)injection.Twoanimalsinthegroupoffive injectedwithET-1alonewereeliminatedfromthe study:onediedonday2andonedidnotshowany movementinbehavioralstudies.OneanimalwaseliminatedfromtheET-1pluspGSNgroupsincewecould notdefinetheinfarctionarea.Theexclusionratewas3/ 13forthetreatmentstudyand0/6forthelaserDoppler study.Allprocedureswereimplementedaccordingtoa protocolapprovedbytheUniversityofFlorida InstitutionalAnimalCareandUseCommittee ( IACUC ). AmodelofreversiblefocalischemiausingET-1was usedaspreviouslydescribed[24,25].WhenET-1is injectedproximaltothemid dlecerebralartery(MCA), itactsonvascularsmoothmusclecellreceptorsETAandETB[26],causingarterialconstrictionandreduced bloodflowtoareasinthebrainsuppliedbytheipsilateralMCA(anterolateralneocortex,corticalareas,and caudatenucleus).AnesthesiawasinitiatedwithLe etal Experimental&TranslationalStrokeMedicine 2011, 3 :13 http://www.etsmjournal.com/content/3/1/13 Page2of9

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isoflurane(5%inO2andmaintainedwith1.5-2.5%in O2).Bodytemperaturewasmaintainedbetween36.537.5Cthroughoutsurgeryusingawater-jacketedheatingblanket.Theanimalswereplacedinastereotaxic frame(BraintreeScientific,Inc.,MA,U.S.A.)and securedintheflatskullposition.Amidlineincisionwas madespanningthebregmaandlambdalandmarks,and asmallhole(3mmi.d)wasdrilledinthecraniumadjacenttotheleftMCA(coordinates:0.2mmanterior,5.2 mmlateral,and1mmdorsaltothebottomofthe skull).A27-gaugeneedlewasusedtoinject3 LofET1(240pmol)at1 L/min.AfterET-1injection,theneedlewasleftinplaceforanadditional3minbeforebeing slowlywithdrawn.ThesystemwasflushedbyDIwater andpGSNwasloadedintheneedle.Approximately,510minafterET-1injection,3 LofpGSN(3 gor 35.71pmol)wasinjected, afterwhichtheneedlewas againleftinplacefor3min,thenslowlyremoved.The incisionwasclosedandanimalswerekeptwarmat37C untiltotallyrecoveredfromanesthesia.CerebralPerfusionMeasurementIneachanimal,skinonthescalpwasremovedandtwo holes(3mmi.d)weremadeaboveineachhemisphere. ET-1orET-1pluspGSNwereinjectedintheleftside ofthebrainandtheothersidewasusedasaninternal control.Relativebloodflux/perfusionmeasurements weremadeusingLaserDoppler(MOORLDI,Moor InstrumentLtd,UK)atfourdifferenttimepoints:preinjection,10-20min,30-35min,and55-65minfrom theET-1injectiontime.Themeasurementswerebased onthemovingbloodinthemicrovasculaturethat causesaDopplerfrequencyshiftofthescatteredlaser light,whichisphotodetectedandthenprocessedto buildacolorcodedmapofrelativebloodflow.Adigital camerarecordsacolorclini calphotographatthesame time,correspondingcloselywiththebloodflowimage insizeandaspect.Thepseudocoloringisrelativeflux (numberofredbloodcellsmultipliedbyspeed)where warm colorsindicaterelativehighfluxand cool colorsrepresentrelativelowflux.The16-bitcolorscans weremadewitharbitrarilyassignedunitfrom0(lower limit)to1,000ormore(upperlimit).Thescanspeed was10ms/pixelandthetotalscan(scanareawasabout 1.8cm2.3cm)durationperanimalwasapproximately10min.ActualbloodflowisimpossibletomeasurewithLaser-Dopplerflowmetry(LDF),butishighly correlatedtofluxexceptatsupranormalpressures[27]. Indeed,percentagereductionsofcerebralperfusionare actuallyrelativepercentagereductionsofflowandwere calculatedusingthefollowingformula: Pi/Pc 100 % (1) wherePiisflowvaluesatipsilateralside(injectedside) andPcisflowvaluesatcontralateralside(unaffected side);botharemeasuredinperfusionunit(PU)BehavioraltestsBehavioraltestsusedincludethelimb-useasymmetry (cylinder)andvibrissae-st imulatedforelimbplacing (vibrissae)forsomatosensoryasymmetry.Animalswere testedbeforeand72hpost-surgery.Thecylindertest examinesthelevelofpreferenceforusingthenonimpairedforelimbforweightshiftingmovementsduring spontaneousverticalexploration.Non-infarctedanimals typicallyusebothlimbsequallyforuprightsupport,but afterdamagetothemotorsystem,animalsshowan asymmetricrelianceontheless-affected(ipsilateral) limb[28].Ratswereplacedinaspeciallydesignedtransparentcylinder(20cmi.d,40cmheight)for3min.The cylinderwashighenoughthattheanimalcouldnot reachthetopedgeorescape,butencouragesvertical explorationoftheglasswallswiththeforepawsaswell aslanding(returntofloor)activity.Forepawuseduring explorationoftheratswasscoredbyanalyzingslowmotionplaybackofdigitalvideorecordedfromacamera installedoverthecylinder,byanexperimenterblindto thetreatmentcondition.Thefractionofeventsinwhich theanimalusedtheipsilateral(affected),contralateral (un/lessaffected),orbothforelimbswascalculatedasa dependentmeasureofasymmetry. Vibrissae-stimulatedfore pawandextinctionplacing testsevaluatesensorimotor/proprioceptivedeficits.To determinewhetherananimalhasasymmetricalsensorimotorperception,theanimalisheldbythetorsowith itsforelimbshangingfreely,andthenslowlymovedlaterallytowardtheedgeofthetableorcountertopuntil thevibrissaeofonesidem akescontactwiththeedge. Intactratsquicklyplaceth eipsilateralforepawonthe edgeorthesurfaceofthetablewhentheipsilateral vibrissaebrushthetableed ge.Incontrast,ratswith damagetothemotorsystemoftendonotrespondto vibrissaestimulationonthea ffectedside(contralateral side).UsingWindowsMovieMaker2007(Microsoft),a treatment-blindedviewers coreddigitalvideorecords foreachside,usingtheframerateandthenumberof framesbetweenvibrissaestimulationandforepawcontacttodetermineplacingresponsedelayintervals.HistologyAnimalsweresacrificedat72hforTTCstaining.The brainswerequicklyisolated,placedincoldPBS(0-4C) for30minandthensectionedinto2-mmthickcoronal slicesusingabrainslicematrix(LeicaMicrosystems,IL, U.S.A.)ThetissuesectionswereheldincoldPBSfor3 minbeforetheywereincubatedinTTCsolution(0.05%Le etal Experimental&TranslationalStrokeMedicine 2011, 3 :13 http://www.etsmjournal.com/content/3/1/13 Page3of9

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TTCinPBS)for30minat37C.Thesectionswere washedthreetimeswithPBS(oneminuteeach)and fixedin0.1Mphosphatebufferedformaldehyde(PBF). Thesectionsthenwereplacedonaflatbedscanner (EPSON1680)toobtainimages.InfarctionAreaAnalysisCalibrateddigitalimagesoftissuesectionsweremadeat 600-dpiscannerresolutionwith48-bitcolorandsaved asTIFFfiles.Theinfarctionareaswerequantifiedby visualthresholdingofTTC-labeled(normal)andunlabeled(infarct)tissue,andmeasurementofeacharea, usingImageJversion5.0(NIH).Infarctvolumewas estimatedastheproductofareaoneachsectionand thenumberof2mmsectionsexhibitinginfarct.The ratioofinfarcttonormalvolumewasusedasdependent measuresforevaluatingpGSNeffects.StatisticalanalysisAlldataareexpressedasarithmeticmeanss.e.m. Two-wayANOVA(timeandtreatment)wasfollowed byBonferroni posthoc testtocomparebehavioral dependentvariablesbetweengroups.Comparisonsof infarctareaweremadebytwo-tailedStudent st-test; andp-valueslessthan0.05wereconsideredtobe significant.ResultsTreatmentofpGSNdidnotinterruptET-1inducedartery contractionInordertotesttheeffectofpGSNandET-1on MCAO,cohorts(n=3)ofratswereinjectedwithET1toinducetransientmiddlecerebralarteryocclusion (MCAO).Approximately5-10minafterET-1injection,pGSNorsalinewasinjectedatthesamelocation. ThetimepointsofscanningwerebasedonET-1injectiontime.Therelativeperfusionunit(PU)orblood flowvaluesofanimalsbeforeinjectionwereinthe rangeof800-1,600PUandthedifferencebetween twohemispheresofthebrainwasnotstatisticallysignificant.After20minfollowingET-1injection,the fluxvaluesontheinjectionsidedroppedtotherange of300-600PU.Thecalculationsweremadeusingformula1describedinthemetho ds.Relativeflowvalues showedarapiddecreaseto~50%ofbaselineinall animalsandflowremainedmaximallydecreasedfor 20-30minafterET-1injection(Figure1).ET-1injectionresultedinadropinperfusionimmediatelyafter injectionregardlessofpGSNinjectionatthesame location.Reperfusionwasobservedinbothgroups after~60min.TheseresultsindicatethatpGSNinjectiondidnotinterrupttheinductionofarterycontractionbyET-1.TreatmentofpGSNsignificantlyreducedET-1induced behavioraldeficitsTotesttheprotectiveeffectofpGSN,behavioralstudies wereconducted.Initially,fiveratswererandomly assignedintoET-1injectionaloneorET-1pluspGSN group.IntheET-1onlygroup,oneratdiedandonerat wasparalyzedafterET-1injectionandwaseuthanized immediately.InET-1pluspGSNtreatedgroup,onerat didnotshowanyinfarctionbyTTCstainingandwas excluded.Therefore,threeanimalsinET-1groupand fouranimalsinET-1pluspGSNgroupwereexamined forbehavioraldeficitsandbraindamage.CylindertestAsshowninFigure2,ET-1inducedMCAOresultedin profoundimpairmentofcontralateralforepawfunction threedaysfollowinginjection.ET-1-treatedanimals showedsignificantreductionofsymmetricalforepawuse duringwallexploration(fromapproximately80%to 20%),whileartificialcerebrospinalfluid(aCSF)injection hadnoeffect.InthepGSNtreatmentgroup,thepercentageofsymmetricalforepawusewassignificantlyhigher thanthecontrolgroup(20%vs.70%,p-value<0.01). Thenumberofattemptstoexplorethewallwasalso lowerthanbeforesurgery(dataisnotshown).These resultsindicatethatpGSNtreatmentsignificantlypreventedMCAO-induceddamagetothemotorsystem.VibrissaetestTotesttheeffectofpGSNonsensorimotorsystem,we alsoperformedvibrissaetest ing.Asexpected,vibrissaestimulatedplacingoftheipsilateralforepawwasnot affected3daysafterexperimentalischemicstroke(Figure3A).Contralateralforepa wplacingwassignificantly slowed(from1sto18s)inthecontrol(ET-1alone) group(Figure3B).Intriguingly,pGSNtreatmentsignificantlyreducedthetimeofcontralateralforepawplacing comparedtocontrolgroup(18svs.9s,p-value<0.01). TheseresultsindicatedthatpGSNtreatmentsignificantlypreventedthelossofsensorimotorfunction inducedbyMCAO.TreatmentofpGSNreducedMCAOinducedbrain damageInthecontrolrats,ET-1producedlargeandreproducibleunilateralinfarctsthatinvolvedtherostro-central dorsolateralcortexandbas alganglia,correspondingto thefullextentoftheMCAterritory.Theinfarction volumeinthepGSN-treatmentgroupwasreducedby 49%comparedtothecontrolgroup(Figure4).Sparing wasobservedinbothcorticalandsubcorticalstructures.DiscussionThecurrentstudyreportsthefirsttimeuseofpGSNas aproteindrugtoreduceinjuryaftertransientlocalLe etal Experimental&TranslationalStrokeMedicine 2011, 3 :13 http://www.etsmjournal.com/content/3/1/13 Page4of9

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Figure1 Perfusionmeasurements (A) Colorphotographsofananimalbrainrecordedatdifferenttimepoints(pre-injection,10-20min,30-35 min,and60-65minfromET-1injectiontime)byadigitalcameraoflaserDopplersystem,correspondingcloselywiththebloodflowimage. Look-uptableshowsarbitrarilyassignedperfusionunit(PU)fromthelowerlimit0toupperlimit1,000andabove.Thescalpsdemonstratethe holesandinjectionsites. (B) Percentageperfusionreductionasafunctionoftimecalculatedusingformula1.AllratswereinjectedwithET-1 (240pmolin3 LPBS)proximaltotheleftmiddlecerebralartery.About5-10minafterET-1injection,pGSN(35.71pmolin3 Lsaline)was intracraniallyinjected(i.c)atthesamesiteinagroupandnoinjectionintheothergroup.ThedottedlinerepresentstheaveragelevelsinpGSN treatedgroup(N=3).Thesolidlinerepresentlevelsincontrolgroup(N=3).Thedifferencesatall-timepointswerenotstaticallysignificant. Le etal Experimental&TranslationalStrokeMedicine 2011, 3 :13 http://www.etsmjournal.com/content/3/1/13 Page5of9

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ischemicstroke.WedemonstratethatpGSNcan improvesensorimotorrecoveryinconjunctionwithsubstantialreductionininfarctvolumepresentthreedays afterstroke. Transientmiddlecerebralarteryocclusion(tMCAO) inducedbyET-1,apotentvasoconstrictionpeptide, decreasedrelativecerebralbloodflowinbraintissue servedbytheMCAby50%inallgroups.Inprevious studies,injectionofET-1adjacenttoMCAhasbeen showntoreducebloodflow30-75%intheregionsuppliedbythearteryincludingcortexandbasalganglia, andtoproducesubsequentischemicneuropathologyin theseregionsofthebrain[24,25,29]. Ourresultsdemonstratethatafter10-20minfollowingET-1intracranialinjection,theipsilateralcerebral relativebloodflowdecreasedapproximately50%inall groupsofanimals(Figure1),indicatingthatallanimals initiallyhavecomparable ischemicstrokesandthat pGSNdidnotreducethemagnitudeoftheoriginal ischemicevent,butlimitedthesubsequentdegeneration andassociatedlossoffunction.ThissuggeststhatpGSN doesnotinterferewithET-1actiononitsreceptors,and correspondstostudiesinwhichGSNknockoutmice hadlargerinfarctvolumeat22heventhoughreductionsinCBFduringMCAOwerenotaltered[23]. AlthoughthisstudyshowsthatpGSNinornearan infarctcanreduceneuropathologyandfunctionalloss duetoischemicstroke,themechanismbywhichpGSN mediatesprotectionarenotyetclear.Onelikely mechanisminvolvesactindepolymerization.Upontissue Figure2 Cylindertest .Ratswereplacedinatransparentcylinder for3min.Animalforepawsusedduringexplorationwerescored. Eachbarrepresentstheaveragefractionofusingbothforepawsin theindicatedgroup.ET-1group,N=3,ET-1+pGSN,N=4,*:pvalue<0.05;**,p-value<0.01,***,p-value<0.001. Figure3 Vibrissaetest (A) Timeinsecondstoipsilateralforepaw placementonthecountertop. (B) Timeinsecondstocontralateral forepawplacementonthecountertop,ET-1group,N=3,ET-1+ pGSN,N=4,**:p-value<0.01. Figure4 Infarctionarealabeledformitochondrialactivity (triphenyltetrazoliumchloride,TTC) .ET-1-alonegroupN=3, pGSNtreatmentgroup,N=4.*,p-value<0.05.FourTTC-staining slicesfromtwoanimals,oneforeachgroup,attheareareceived mostofbloodfromMCA.Off-whitecolorareasshowdeathtissues whileredareasindicatevitaltissues. Le etal Experimental&TranslationalStrokeMedicine 2011, 3 :13 http://www.etsmjournal.com/content/3/1/13 Page6of9

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injuryduetoglucose/oxygendeprivation(CBFsignificantlyreduced),largeamountsofactincanbereleased fromdamagedcellsintotheextracellularspace.Since theionicconditionsintheextracellularfluidfavoractin polymerization,highamountsofF-actincouldbe releasedtopotentiallyincreasetheviscosityofblood andperturbbloodflowthroughthemicrovasculature. Theactinseveringproteingelsolinhasasecreted plasmaisoform(calledplasmagelsolin),whichisconstitutivelyactiveinthehighextracellularcalciumconcentrationsofplasma.Plasmagelsolinseversextracellular F-actintoshortfilaments,andbycappingbarbedends, preventspolymerizationandfavorsmonomerrelease. Therefore,pGSNactsas debriscleaner limiting inflammationandpossiblydecreasebloodclogging[22]. Anotherpossiblemechanismisthroughanti-apoptotic activityofpGNS.InJurkatcells,overexpressionofgelsolininhibitescytokineinducedapoptosis[30].Ithas beenreportedthatgelsolincanformcomplexwith phosphatidylinositol4,5-bisphosphateandinhibit capase-3and-9activities[31].Inaddition,pGSNmay alsoplayanimportantrolei nregulatinginflammation. FuturestudieswillfocusonthemechanismsofpGSN protection. Histonedeacetylaseinhibitor-mediatedneuroprotectionagainstMCAOhasbeenassociatedwithGSNupregulationandreductionsinfilamentousactin,neitherof whichwasshowntooccurinGSN-knockoutmicein whichthetreatmentwasineffective[32].Also,GSNcan modulatetheactincytoskeletonregulationofnumerous ionchannelsresponsibleforelevatedcytotoxicintracellularcalciumandglutamateexcitotoxicity[33-35]. Gelsolinisregulatedbyphosphatidylinositol4,5bisphosphate(PIP2),andcontainsalipidsignalingbindingdomain.Thisdomainhasbeenshowntobindtoa numberofbioactivelipidsincludinglysophosphatidic acid(LPA),lipoteichoicaci d(LTA),andlipopolysaccharide(LPS)[36-39].LPAlevelshavebeenshownto beincreasedinpatientssufferingischemicstroke[40]. LPAsignalinghasalsobeenshowntoregulateanumberofpro-inflammatorygene s[41].Increasinggelsolin levelsduringstrokemayservetomodulatetheinflammatoryresponsetherebyofferingprotectionagainstthe inflammationrelatedneuro degenerationfollowing stroke. FurtheremphasisofthepotentialimportanceofGSN instrokecomesfromrecentreportsthatcirculating pGSNlevelsarereducedinischemicstrokesuffersand ishighlypredictiveforfirst-yearmortalityfromischemic stroke[37].Matrixmetalloproteinases(MMPs),zinccontainingendopeptidasesthatparticipateinbothnormalandpathologicalprocesses,areupregulatedduring inflammatoryconditions[42],includingstroke[43]. pGSNiscleaved invitro byMMP-3,MMP-2,MMP-1, MMP-14andMMP-9[44]whichmaybethecauseof theseveredepletionofpGSNobservedinpatientswho sufferischemicstroke.ReplacinglostpGSNmayinterruptpro-inflammatorycascadeandresultindecreased braindamage.ConclusionThecurrentstudyoffersaproofofprinciplethat deliveryofpGSNfollowingischemicstrokeresultsin neuroprotectionandcanreducebothsensoryand motordeficitsthatarisefollowingstroke.Future researchaimedatcharacterizingimproveddelivery, doseresponse,temporal,safety,pharmacokinetic issues,andphysiologicalme chanismsforfurtherpreclinicaldevelopmentofthispromisingstrategyare calledfor.Listofabbreviations ANOVA:Analysisofvariancebetweengroups;aCSF:Artificialcerebrospinal fluid;CBF:Cerebralbloodflow;CDC:CenterforDiseaseControland Prevention;ET-1:Endothelin-1;FDA:TheU.SFoodandDrugAdministration; GSN:Gelsolin;pGSN:Plasmagelsolin;LDF:Laser-Dopplerflowmetry;LPA: Lysophosphatidicacid;LTP:Lipoteichoicacid;LPS:Lipopolysaccharide;MCA: Middlecerebralartery;MCAO:Middlecerebralarteryocclusion;tMCAO: Transientmiddlecerebralarteryocclusion;MMPs:Matrixmetalloproteinases; PBF:Phosphatebufferedformaldehyde;PBS:Phosphatebufferedsaline;PU: Perfusionunit;rtPA:Recombinanttissueplasminogenactivator;TTC:2,3,5triphenyltetrazoliumchloride. Acknowledgements ThisworkwassupportedinpartbyNIHGrant#P01AG0485andtheU.S. DepartmentofVeteransAffairs.Publicationofthisarticlewasfundedinpart bytheUniversityofFloridaOpen-AccessPublishingFund. Authordetails1DepartmentofPharmaceutics,UniversityofFloridaCollegeofPharmacy, Gainesville,FL32610,USA.2DepartmentofPharmacology&Therapeutics, UniversityofFloridaCollegeofMedicine,Gainesville,FL32610,USA.3DepartmentofPharmacodynamics,UniversityofFloridaCollegeof Pharmacy,Gainesville,FL32610,USA.4DepartmentofVeteransAffairs MedicalCenter,Gainesville,FL32602,USA.5MedcoSchoolofPharmacy, FairleighDickinsonUniversity,Madison,NJ07940,USA. Authors contributions HTLandACHcontributedsubstantiallytoexperimentaldesign,experimental implementation,dataanalysis,andmanuscriptpreparation.JSTcontributed tothecerebralperfusionmeasurementinMG sLab,JLandJPcontributed tothebehavioraltests.MAK,JAHandSSconceivedofthestudy, contributedtoexperimentaldesign,dataanalysisandrevisedthe manuscript.Allauthorshavereadandapprovedthefinalversionofthe manuscript. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. 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