Modulation of microglial activation enhances neuroprotection and functional recovery derived from bone marrow mononuclear cell transplantation after cortical ischemia

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ContentslistsavailableatSciVerseScienceDirect

Neuroscience

Research

j our na l h o me p age : w w w . e l s e v i e r . c o m / l o c a t e / n e u r e s

Modulation

of

microglial

activation

enhances

neuroprotection

and

functional

recovery

derived

from

bone

marrow

mononuclear

cell

transplantation

after

cortical

ischemia

Edna

C.S.

Franco

a

_,

_Marcelo

_M.

_Cardoso

a

_,

_Amauri

_Gouvêia

b

_,

_Antonio

_Pereira

c

_,

_Walace

_Gomes-Leal

a,∗

a_Laboratory_of_Experimental_{Neuroprotection}_and_{Neuroregeneration,}_Institute_of_Biological_Sciences,_Federal_University_of_Pará,_Brazil

b_Laboratory_of_{Neurosciences}_and_Behavior,_Institute_of_Biological_Sciences,_Federal_University_of_Pará,_Brazil

c_Brain_Institute,_Federal_University_of_Rio_Grande_do_Norte,_Natal,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received8September2011

Receivedinrevisedform24February2012

Accepted13March2012

Availableonline23March2012

Keywords: Acutestroke Stemcells Microglia Inﬂammation Minocycline Neuroprotection

a

b

s

t

r

a

c

t

Activatedmicrogliamayexacerbatedamageinneuraldisorders;however,itisunknownhowthey affectstemcellstransplantedafterstroke.Focalischemiawasinducedbymicroinjectionsof40pmolof endothelin-1intothemotorcortexofadultrats.Ischemicanimalsweretreatedwithsterilesaline(n=5), bonemarrowmononuclearcells(BMMCs,n=8),minocycline(n=5)orconcomitantlywith minocy-clineandBMMCs(n=5).BMMC-treatedanimalsreceived5×106_BMMCs_through_the_caudal_vein₂₄_h

post-ischemia.Behavioraltestswereperformedtoevaluatefunctionalrecovery.Morphometricand histo-logicalanalyseswereperformedtoassessinfarctarea,neuronallossandmicroglia/macrophageactivation upto21dayspost-ischemia.Treatmentswithminocycline,BMMCsorminocycline-BMMCsreduced infarctarea,increasedneuronalsurvivalanddecreasedthenumberofcaspase-3+andED-1+cells,but theseeffectsweremoreprominentintheminocycline-BMMCgroup.Behavioralanalysesusingthe mod-ifiedsticky-tapeandopen-fieldtestsshowedthatischemicratsconcomitantlytreatedwithBMMCsand minocyclineshowedbettermotorperformancethanratstreatedwithBMMCsorminocyclineonly.The resultssuggestthatpropermodulationoftheinflammatoryresponsethroughtheblockageofmicroglia activationenhancesneuroprotectionandfunctionalrecoveryinducedbyintravenoustransplantationof BMMCsaftermotorcortexischemia.

1. Introduction

Acuteneuraldisorders,includingstrokeandbrainandspinal cordtrauma,are untreatableconditionsand commoncausesof death or functional deficits (Zhang and Chopp, 2009). In cen-tralnervoussystem(CNS)diseases, severalpathological events, suchasexcitotoxicity, free radical formationand inflammatory response,induceinexorabletissuedamageandsubsequent func-tionaldeficits(Woodruffetal.,2011).

Stroke,spinalcordinjury(SCI)andotheracuteneuraldisorders areaccompaniedbyanintenseinﬂammatoryresponse, character-izedbycytokinerelease(Tuttolomondoetal.,2008),recruitment ofneutrophils(Gomes-Lealetal.,2005;Schnelletal.,1999; Souza-Rodriguesetal.,2008)and lymphocytes(Schroeteretal.,1994; Thoredetal.,2009)andintensemicrogliaactivation(Gomes-Leal

∗ Correspondingauthorat:LaboratoryofExperimentalNeuroprotectionand

Neuroregeneration,InstituteofBiologicalSciences,FederalUniversityofPará,Rua

AugustoCorrêaS/N,CampusdoGuamá,CEP:66075-900,Belém,Pará,Brazil.

Tel.:+559132017741;fax:+559132017741.

E-mailaddresses:[email protected],[email protected](W.Gomes-Leal).

etal.,2004,2005;Moriokaetal.,1993;Thoredetal.,2009)inthe damagedCNSparenchyma.

Microglialactivation is animportant component of theCNS inflammatoryresponse(RansohoffandPerry,2009).Several stud-iessuggestthatmicrogliamaybeprotectiveafterstrokeandother CNSdiseases(Lalancette-Hebertetal.,2007;Neumannetal.,2008; Thoredet al., 2009) by modulating theinflammatory response throughthereleaseofanti-inflammatorycytokines(Battistaetal., 2006)andgrowthfactors(Lalancette-Hebertetal.,2007;Thored etal.,2009)andbyengulfingneutrophils(Neumannetal.,2008) orincreasingadultneurogenesisinvivo(Thoredetal.,2009)and invitro(Waltonetal.,2006).

Thereisalsosubstantialexperimentalevidencesuggestingthat anexacerbatedmicroglialresponsemaybedetrimentalafterstroke andotherCNS diseases(Blocket al.,2007;Hamby etal.,2007; Yrjanheikki et al., 1999). Recently, it has been suggested that minocyclinemaybebeneﬁcialinacuteischemicstrokeinhumans (Lampletal.,2007).Moreover,severalstudiescorroboratethe neu-roprotectiveactionsofminocyclineinotherCNSdiseases (Yong etal.,2004),indicatingthatanuncontrolledmicroglialresponse isacommonandimportantpathologicaleventunderlyingseveral acuteandchronicneuraldisorders(Blocketal.,2007).

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Transplantationofstemcellsisconsideredapromising ther-apeuticapproach tominimizetheburden ofneurodegenerative diseases(LindvallandKokaia,2006).Bothembryonic(Takahashi et al., 2008) and adult stem cells (Brenneman et al., 2010; de Vasconcelos Dos Santos et al., 2010; Keimpema et al., 2009; Sarnowska et al., 2009) induce neuroprotective and functional recoveryafterstroke.

It has been shown that bone marrow mononuclear cells (BMMCs),afractionofbonemarrowcontainingbothmesenchymal andhematopoieticstemcells(Weissmanetal.,2001),areas effec-tiveasmesenchymalstemcells(MSCs)ininducingneuroprotection and locomotor recovery after experimentalstroke (Brenneman etal.,2010;deVasconcelosDosSantosetal.,2010;Iihoshietal., 2004).BMMCisolationdoesnotinvolvelongculturingprocedures, whichdecreasestheriskofcontaminationandrendersthesecells potentiallysuitable fortransplantation a fewhoursafterstroke onset(Brennemanetal.,2010;deVasconcelosDosSantosetal., 2010;Iihoshietal.,2004),even inhumans(BarbosadaFonseca etal.,2010;Mendez-Oteroetal.,2007).

Inmostofthestudiesavailable,stemcellsweretransplanted duringthe acutephase ofstroke in anenvironment of intense inflammatoryresponse(Brennemanetal.,2010;deVasconcelos DosSantosetal.,2010;Keimpemaetal.,2009).Thisraisesthe possi-bilitythattransplantedcellscouldbeharmedbytheinflammatory environment, which includes activatedmicroglia. This possibil-ityissupportedbyreportssuggestingthatbonemarrowstromal cells transplantedintotheintactadultbrainarerejectedbyan inflammatory response (Coyne et al., 2006) and that activated macrophages may impair both the survival and integration of embryonic stem cells transplantedat 3days afterbrain trauma (Molcanyietal.,2007).

Itisunknownwhetherpropercontrolofmicroglialactivation intheischemicenvironmentcouldfavorthetherapeuticactions of transplanted stem cells. We explored whetherthe modula-tionoftheinﬂammatoryresponsebyminocycline,aninhibitorof microglialactivation,wasbeneﬁcialforthetherapeuticactionsof BMMCstransplantedduringtheacutephaseofcorticalischemia.

2. Materialsandmethods

2.1. Experimentalanimals

MaleadultWistarrats(250–300g)wereobtainedfromthe Fed-eralUniversity ofParáCentralAnimalFacility.Allanimalswere housed under standard conditions with food and water avail-ableadlibitum.Allexperimentalprocedureswerecarriedoutin accordance withthePrinciples of LaboratoryAnimal Care(NIH publicationNo.86-23,revised1985) andEuropeanCommission Directive86/609/EECforanimalexperimentsunderlicenseofthe EthicsCommitteeonExperimentalAnimalsoftheFederal Univer-sityofPará.Allpossibleeffortsweremadetoavoidanimalsuffering anddistress.

2.2. Surgicalproceduresandexperimentalmodeloffocal ischemia

Thefocalischemiawasinducedbymicroinjectionsofthe vaso-constrictorpeptideendothelin-1(ET-1)(Sigma,SaintLouis,MO, USA)aspreviouslydescribedbySouza-Rodriguesetal.(2008). Ani-malswereanesthetizedwithketaminehydrochloride(72mg/kg, i.p.) and xylazine hydrochloride (9mg/kg, i.p.) and held in a stereotaxic frame after their corneal reﬂex was abolished. A homoeothermicblanketunitwasusedtomaintaintheanimal’s body temperature,as measuredbya rectal thermometer. After craniotomy,40pmolofET-1(Sigma,SaintLouis,MO,USA)in1␮l

ofsterilesalinewasinjectedintotheratmotorcortex(n=5–8per survivaltime/animalgroup)overaperiodof2minusingaﬁnely drawnglasscapillaryneedle.Thecapillaryneedlewasleftin posi-tionfor3minbeforebeingslowlywithdrawn.Controlanimalswere injectedwiththesamevolumeofsterilesaline(n=5persurvival time).Weusedthefollowingstereotaxiccoordinatesinrelationto thebregma:+2.3mmlateral;+1.2mmposteriorand0.50mmdeep fromthepialsurfaceinthedorsoventralaxis(PaxinosandWatson, 2007).Toidentifytheinjectionsite,asmallquantityofcolanylblue wasaddedtobothET-1andvehiclesolutions.Aftersurgery, ani-malswereallowedtorecoverwithfreeaccesstofoodandwater for7,14and21days.

2.3. Animalgroups

To investigate whether microglial activation inﬂuences the effectsofBMMCstransplantedduringtheacutephasefollowing motorcortexischemia,wedelineatedfourexperimentalgroups: animalsinjectedwithET-1andintravenouslytreatedwithsterile PBS(Group1,n=5);animalsinjectedwithET-1andintravenously transplantedwithBMMCs(Group2,n=8);animalsinjectedwith ET-1andtreated(i.p.)withminocycline(Group3,n=5)andanimals injectedwithET-1 andconcomitantlytreated withminocycline (i.p.)andintravenouslytransplantedwithBMMCs(Group4,n=5). 2.4. Bonemarrowharvestingandprocessing

TheharvestingofBMMCsfollowedtheprotocoldescribedby Giraldi-Guimaraesetal.(2009).Briefly,ratswereanesthetizedwith ketaminehydrochloride(72mg/kg,i.p.)and xylazine hydrochlo-ride(9mg/kg,i.p.).Thefemoralandtibialbonesweredissected, andtheirepiphyseswerecutundersterileconditionsina lami-narflowhood(VECO,CFLH-09).Bonemarrowwasextractedwith serum-freeDMEM-F12(Sigma,SaintLouis,MO,USA)andgathered inside steriletubes.Bonemarrowcells werethenmechanically dissociatedandcentrifugedfor5minandresuspendedin3mlof serum-freeDMEM-F12(Sigma,SaintLouis,MO,USA).Thisvolume wasgentlyaddedover3mlofHistopaque1083(Sigma,SaintLouis, MO,USA)andcentrifugedfor30min.BMMCswerecollectedfrom theboundarybetweentheDMEM-F12 (Sigma, SaintLouis,MO, USA)andtheHistopaque1083(Sigma, USA).Cellswerewashed withHankssolutionwithoutglucosein3consecutiveseriesof cen-trifugation(5mineach).Afterwashing,thecellswerere-suspended inDMEMF12(Sigma,SaintLouis,MO,USA),supplementedwith 10%fetalcalfserum(SBF)andcountedusingaNeubauer’schamber toafinalconcentration5×106_cells/ml.

TolabelthetransplantedBMMCs,bonemarrowdonoranimals receiveddailyinjections(50mg/kg,i.p.,twiceadayfor7days)of bromodeoxyuridine(BrdU,Sigma,SaintLouis,MO,USA).The exclu-siontestusingtrypanbluewasperformedtoassesscellviability.

Twenty-fourhoursafterET-1-inducedfocalischemia,Group2 animalswereintravenouslytransplantedwith5×106_BMMCs_by tailveininjection(n=8/survivaltime).Inthecontrolgroup,animals wereinjectedwithsterilePBS(n=5/survivaltime).

2.5. Minocyclinetreatment

Minocycline has beenshown tobe an effective inhibitor of microglialactivation(Yrjanheikkietal.,1999).Toinhibitmicroglial activationinthefirstweekfollowing corticalischemia,animals (Group3)receiveddailyinjections(twiceaday)ofminocycline (Sigma,SaintLouis,MO,50mg/kg,i.p.)duringthefirsttwodays.The firstdosewasadministered2haftertheET-1microinjections. Sub-sequentdosesofminocycline(25mg/kg,i.p.)wereadministered onceadayoverthefoursubsequentdays.Theminocyclinedose usediswidelyacceptedasaneffectivedoseinreducingmicroglia

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activationandinducingneuroprotection(Guimaraesetal.,2010; HewlettandCorbett,2006;Yrjanheikkietal.,1999).Aseparate groupofanimalswastransplantedwithBMMCs24hpost-ischemia andconcomitantlytreatedwithminocyclinefor6days(n=5,Group 4).Theseanimalswerekilledat7dayspost-ischemia.This sur-vivaltimewaschosenbecausemicroglialactivationismaximalin theﬁrstweekfollowingbothET-1-inducedfocalischemia( Souza-Rodriguesetal.,2008)andMCAO(Moriokaetal.,1993).

2.6. Analysisofsensorimotordysfunction

Sensorimotoroutcomewasassessedusingbehavioraltests per-formedbyblindedinvestigators.Animalsinallgroupsweretested onedaybeforeand1,3and7daysafterischemia.Thefollowing testswereused:

1.Theopen-fieldtest(Bresnahanetal.,1987):Thistestwas per-formedinanopenfieldwith60cm× 60cm× 50cmdimensions andcontaining16squaresubdivisionsofequalsize.Experiments consistedofthreetrials(5mineach)inwhichtheanimalwas removedfromitscageandplacedatthecenteroftheopenfield. Theanimals’motorperformancewasrecordedbyavideo cam-era(Sony,USA).Thebehavioralparametersrecordedincluded thenumberofoccurrencesoftheexploratorybehaviorof stand-inguponthehindlegs(rearing),bodyself-cleaning(grooming), latency(timetaken toleavethestarting point)and distance traveledintheopenfield.

2. The modified sticky-tape test: We used a protocol adapted from(Sughrueetal.,2006).Asmallnon-removabletapesleeve (3.0cm×1.0cm)wasplacedaroundtheanimal’sforepaw.The timespent attendingtothis stimuluswasrecorded.Animals werepre-trained24hbeforeischemia(twotimesadayfor30s) witha1-hintervalbetweeneachtest.Afterischemia,thetest wasperformedonceadayinthefirst,thirdandseventhdays afterischemia.Eachtest consistedoffive trialsin whichthe twobestperformanceswereconsideredforquantitative analy-sis.Theratiooflefttorightperformancewascalculatedtoassess sensorimotorperformance(Sughrueetal.,2006).

2.7. Perfusionandtissuepreparation

Aftersurvivaltimesof7,14and21days,animalsweredeeply anesthetized with ketamine hydrochloride (72mg/kg, i.p.) and xylazinehydrochloride(9mg/kg,i.p.)andtranscardiallyperfused withheparinized0.9%phosphate-bufferedsaline(PBS) followed by4% paraformaldehyde.Surgicalmanipulation wasperformed onlyafterboththecornealandthepawwithdrawreflexeswere abolished.Brainswerepost-fixedfor24hinthesamefixativeand cryoprotectedindifferentgradientsofsucrose-glycerolsolutions over 7days. Thetissue wasthen frozenin TissueTek, and 30-␮mcoronalsectionswerecutusingacryostat(CarlZeissMicron, Germany). Sections were mounted onto gelatinized slides and storedinafreezerat−20◦_C.

2.8. Grosshistopathology

Thelesionareawasvisualizedinsectionsstainedwithcresyl violet(Sigma,Brazil).ThesiteoftheET-1injectionwasrecognized bythepresenceofcolanylblue,tissuepallorandnecrosisinduced byfocalischemia(Souza-Rodriguesetal.,2008).

2.9. Antibodies

To evaluate the patterns of neuronal loss, microglial/ macrophage activation and apoptosis in the different experi-mentalgroups, weperformed aseries ofimmunohistochemical

procedures.Table1showsdetailsregardingtheantibodiesused forthispurpose.

2.10. Immunolabelingprotocol

Theimmunolabelingprotocolusedinthisstudywasdetailed elsewhere(Gomes-Leal etal.,2004).Brieﬂy, slide-mounted sec-tionswereremovedfromthefreezer,keptinaheatingovenat 37◦Cfor30minandrinsedoncein0.1MPBSfor5min.Toimprove labelingintensity,sectionswere then pretreated in 0.2Mboric acid(pH9.0)previouslyheatedto65◦Cfor25min.This temper-aturewas maintainedconstantlyover thepretreatmentperiod. Sectionswerefurtherallowedtocoolforabout20minandwere incubatedunder constantagitation in1% hydrogenperoxidein methanolfor20min.Sectionswererinsed3 times(5mineach) in0.05%PBS/Tween(Sigma,SaintLouis,MO,USA)andincubated withnormalserum(Table1)inPBSfor1h.Withoutfurtherrinsing, sectionswerethenincubatedwiththeprimaryantibodydilutedin PBSfor24h,rinsedinPBS/Tweensolutionfor5min(3times),and incubatedwithappropriatesecondaryantibody(Table1)for2h. Bothprimaryandsecondaryantibodieswereincubatedatroom temperature(20◦C).Asanegativecontrol,PBS,ratherthanthe pri-maryantibody,wasused.Sectionswererinsedagainfor5min(3 times)andincubatedinanavidin–biotin–peroxidasecomplex(ABC Kit,VectorLaboratories)for2h.Sectionswerethenrinsed4times (3mineachrinse)andDAB-reactedaccordingtoaprotocol pub-lishedelsewhere(Gomes-Lealetal.,2004).AftertheDABreaction, sectionswererinsed3times(3mineach)in0.1Mphosphatebuffer, dehydratedusingalcoholsandxylene,andcoverslipped.Some sec-tionswerealsocounterstainedwithcresylviolet.

2.11. Qualitativeanalysis

Allsectionsstainedwiththedifferenthistologicalmethodswere surveyedbylightmicroscopy(OlympusBX41).Illustrativeimages fromallexperimentalgroupswereobtainedusingadigitalcamera (OlympusEvoltE-330)attachedtothemicroscope.

2.12. Quantitativeanalysis

Themeasurementofthelesionarea(mm2₎_was_performed_using photomicrographs obtained from cresyl violet-stained sections forthedifferentexperimentalgroups(3sections/animal/survival time)usingtheNIH’sfreesoftware,ImageJ.

Weusedcoronalsectionscontainingthedamagedmotorcortex tocountthenumberofactivatedmicroglia/macrophages(ED1-1+ cells),apoptoticcells(activecaspase-3+cells)andmatureneuronal bodies(NeuN+cells)perfieldusingasquare0.25-mm-widegrid (objective40×)intheeyepieceofamicroscope.Inthe40× objec-tive,thisgridcorrespondstoanareaof0.0625mm2_._We_counted 3fieldspersectionand3sections/animal(n=5animals/survival time).Fieldswerecountedintheregionsofhighestcelldensity alongalinefromthelesionepicenter(centralfield)andin2 addi-tionalfieldsat1-mmintervals(1mmmediallyand1mmlaterally). CountswereaveragedandplottedinCartesiancoordinates.This protocolwasadaptedfrom(Schnelletal.,1999).

2.13. Statisticalanalysis

Averagesandstandarddeviationswerecalculatedforallcounts. Comparisonsbetweendifferentgroupswereassessedbyan analy-sisofvariance(ANOVA)withBonferroniposthoctest.Statistical signiﬁcance was accepted for P<0.05. All statistical analyses wereperformedusingtheBioEstat5.0software(SociedadeCivil Mamiraua’/CNPQ-Brazil).

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Table1

Antibodiesandnormalserumused.

Primaryantibodies Secondaryantibodies Normalserum(10%) Labelingpurpose

Anti-BrdU(1:100,Novocastra) Horseanti-mouse(1:100

VectorLaboratories)

Horse Transplantedcells(Lietal.,2001)

Anti-activecaspase-3(1:250,Promega) Goatanti-rabbit(1:100

VectorLaboratories)

Goat Apoptoticcells(Lalancette-Hebertetal.,2007)

Anti-NeuN(1:100,Chemicon) Horseanti-mouse(1:100

VectorLaboratories)

Horse Matureneurons(Mullenetal.,1992)

Anti-ED-1(1:200,Serotec) Horseanti-mouse(1:100

VectorLaboratories)

Horse Activatedmicroglia/macrophages(Robinsonetal.,1986)

3. Results

3.1. VisualizationoftransplantedBMMCs

Transplanted BrdU+ BMMCs were present in different CNS regions,includingtheischemiccorticalparenchyma,meningesand perivascularspace(Fig.1).FewBMMCswerepresentinthe perin-farctarea,whichisinagreementwithpreviousstudiesshowing that most of thetransplanted cells are localized tonon-neural organs(i.e.,thespleen)andthatmostcellsreachingtheCNShas atransientexistence(Brennemanetal.,2010;Keimpemaetal., 2009).

3.2. BMMCtreatmentdecreasesinfarctareaaftercortical ischemia

Microinjectionsof40pmolofET-1intothemotorcortexofadult ratsinducedconspicuoustissue lossaccompaniedbyanintense inﬂammatoryreaction(Fig.2),whichisinagreementwithprevious studiesusingtheET-1modeloffocalischemia(Souza-Rodrigues et al.,2008).BMMC treatmentdecreased the tissue lossfor all survivaltimes(Fig.2A–F),asconﬁrmedbyquantitativeanalysis

(P<0.05,ANOVA-Bonferroni)oftheinfarctarea(Fig.2G).The aver-ageinfarctareaswere1.82(±0.59),1.97(±0.12)and1.79(±0.32) mm2_at_7,₁₄_and₂₁_days,_{respectively,}_in_the_vehicle_group_and_1.15 (±0.59),0.80(±0.18)and0.82(±0.31)mm2_in_the_BMMC-treated animals(Fig.2G).

3.3. BMMCtreatmentreducesapoptosisandpromotesneuronal preservationintheischemiccortex

ET-1microinjectionsinducedconspicuouslossofneuronalcell bodies(NeuN+cells)forallsurvivaltimes(Fig.3).BMMCtreatment increasedneuronalpreservationforallsurvivaltimes(Fig.3A–F) asconfirmed(P<0.01,ANOVA-Bonferroni)byquantitativeanalysis (Fig.3G).TheaveragenumbersofNeuN+cellswere49(±17),35 (±17)and39(±14)cells/fieldat7,14and21days,respectively,in thevehiclegroupand75(±22),62(±22)and77(±28)cells/field intheBMMC-treatedanimals(Fig.3G).

ET-1microinjectionsincreasedthenumber ofapoptoticcells (activecaspase-3+cells)mainlyinthemotorcortexperinfarctarea (Fig.4A–F).BMMCtreatmentreducedthenumberofthesecells forallsurvivaltimes,asconﬁrmed(P<0.05,ANOVA-Bonferroni) byquantitativeanalysis(Fig.4H).Theaveragenumbersofactive

Fig.1.BrdU-labeledBMMCsfromdonorratsinjectedintothecaudalveinofischemicanimals.Labeledcellswerepresentintheischemiccortex(AandB),perivascularspace

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Fig.2.BMMCtreatmentreducestissuelossaftercorticalischemia.Grosshistopathologyincresylviolet-stainedcoronalsections.Ischemicanimalstreatedwithsterilesaline

(A,C,E)orBMMCs(B,D,F)at7(AandB),14(CandD)and21(EandF)dayspost-injury.Asterisksindicatetheischemicnecroticcenter.Therewasastatisticallysigniﬁcant

decreaseintheinfarctareaafterBMMCtreatmentforallsurvivaltimesascomparedtothevehiclecontrol(G,*P<0.05ANOVA-Bonferroni).Scalebar:250␮m.

caspase-3+cellswere38(±11),45(±11)and50(±22)cells/ﬁeldat 7,14and21days,respectively,inthevehiclegroupand30(±11), 31(±8)and31(±12)cells/ﬁeldinBMMC-treatedanimals(Fig.4H). 3.4. BMMCtreatmentreducesmicroglial/macrophageactivation atalatersurvivaltimeaftercorticalischemia

Microinjections of ET-1 induced an intense inflammatory responsecharacterizedbythepresenceofalargenumberof acti-vatedmicroglia/macrophages(ED1+cells) in both theischemic coreandperipheralregions(Fig.5A–F).ThenumberofED1+cells was similar (P>0.05, ANOVA-Bonferroni) in both vehicle- and BMMC-treatedanimalsat7(84_±24and81_±18cells/field)and14 (65±25and 64±28cells/field)dayspost-injury(Fig.5G). How-ever,there wasa decrease (P<0.05, ANOVA-Bonferroni)in the

numberofED1+cellsinBMMC-treatedanimals(63±25cells/ﬁeld) ascomparedtothevehiclegroup(77±25cells/ﬁeld)at 21days post-injury(Fig.5G).

3.5. InhibitionofmicroglialactivationimprovesBMMC-induced neuroprotectionandfunctionalrecovery

Totestthehypothesisthatthemodulationofmicroglial acti-vationin theischemicenvironment mightbebeneﬁcial forthe therapeutic actions of BMMCs transplanted during the acute phase of cortical ischemia, microglial activation was inhibited using minocycline(Yrjanheikkiet al., 1999).Minocycline treat-mentreduced thenumberof ED1+cells inthemotor cortexof ischemicanimalsat7dayspost-injury(Fig.6CandM).Concomitant

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Fig.3. BMMCtreatmentincreasesneuronalpreservationaftercorticalischemia.Anti-NeuNimmunohistochemistryincoronalsectionsfromischemicanimalstreatedwith

sterilesaline(A,C,E)orBMMCs(B,D,F)at7(AandB),14(CandD)and21(EandF)dayspost-injury.ThedensityofNeuN+cellswasstatisticallyincreasedintheischemic

cortexafterBMMCtreatmentforallsurvivaltimesascomparedtothevehiclecontrol(G,*P<0.05ANOVA-Bonferroni).ArrowsindicateNeuN+cells.Scalebar:100␮m.

treatment with minocycline and BMMCs induced a greater decrease(P<0.05)inthenumberofED1+cells(Fig.6DandM).

ConcomitantBMMCandminocyclinetreatmentreducedinfarct areacompared to controlanimals, but not in comparison with minocyclineorBMMCalone(notshown).Concomitanttreatment reducedthenumberofapoptoticcells comparedtocontroland BMMCtreatment,butnotincomparisontominocycline(Fig.6I–L). Neuronalpreservationwasenhancedby concomitanttreatment compared to control, but not in comparison to minocycline or BMMCtreatmentalone(Fig.6E–H)at7dayspost-injury(Fig.6E–H). Weinvestigated sensorimotorrecoveryinanimalsbelonging toallexperimentalgroups(Fig.7).Inamodiﬁedsticky-tapetest, animals treated with BMMCs plus minocycline performed bet-ter at 7days post-injury than animals treated with BMMCs or minocyclinealone (Fig.7A, P<0.05,ANOVA-Bonferroni,). Inthe open-ﬁeld test, animals treated with BMMCs plus minocycline

performedbetterinallanalyzedparameters(i.e.,meandistance traveled,rearing,groomingandlatencytime)thanvehicle-treated animals(Fig.7B–E,P<0.05,ANOVA-Bonferroni).

4. Discussion

We tested thehypothesis that microglialactivationis detri-mentaltoBMMCstransplantedduringtheacutephase(24h)of corticalischemiaandthatinhibitionofmicroglialactivationwith minocyclinemightfacilitatethetherapeuticeffectsofBMMCsin theischemicenvironment.

ThepresentdatashowthatBMMCtreatmentinducesa consid-erablereduction oftheinfarctareaandapoptosisandincreases neuronalpreservationandfunctionalrecoveryat7,14and21days aftercorticalischemia.Inaddition,weshowthattransplantation of BMMCsconcomitantwithminocycline treatmentintheﬁrst

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Fig.4.BMMCtreatmentreducesapoptosisaftercorticalischemia.Stainingforactivatedcaspase-3incoronalsectionsfromischemicanimalstreatedwithsterilesaline(A,C,

E)orBMMC(B,D,F)at7(AandB),14(CandD)and21(EandF)dayspost-injury.Thedensityofactivecaspase-3+cellswasstatisticallydecreasedintheischemiccortexafter

BMMCtreatmentforallsurvivaltimesascomparedtothevehiclecontrol(H,*P<0.05ANOVA-Bonferroni).Negativecontrolinwhichtheprimaryantibodywasreplacedby

normalserum(G).SectionsinA,CandFwerecounterstainedwithcresylviolet.Arrowsindicateactivecaspase-3+cells.Scalebar:100␮m.

weekaftercorticalischemia improvesthe neuroprotection and functionalrecoveryaffordedbytreatmentwithBMMCsalone.

Theseresultsconﬁrmandextendpreviousreportssuggesting that bone marrow-derived stem cells are highly neuroprotec-tivefollowing CNSdisorders, includingstroke(Baoet al.,2011; Brennemanetal.,2010;deVasconcelosDosSantosetal.,2010; Keimpemaetal.,2009)andspinalcordtrauma(Sobanietal.,2010). Inthesestudies,isolatedMSCs(Baoetal.,2011;Keimpemaetal., 2009) or BMMCs(Brenneman et al.,2010; de VasconcelosDos Santosetal.,2010)wereusedfortransplantation.

After intravenous transplantation, BMMCs (BrdU+) were presentin severalCNSregions,including thecorticalperinfarct area, meninges and perivascular space, which is in agreement withpreviousreports(Brennemanetal.,2010;Choppetal.,2009; Keimpemaetal.,2009).FewBMMCswerepresentintheischemic cortexmostlyintheperinfarctarea,whichisalsoinagreementwith

previousstudies(Choppetal.,2009;Keimpemaetal.,2009).Ithas beenshownthatthesecellsarepreferentiallyfoundinnon-neural tissues(i.e.,thespleen)andhaveanearlyandtransientexistence intheischemicenvironment(Keimpemaetal.,2009).Keimpema etal.(2009)showedthatafterMCAO,around95%oftransplanted BMMCsaretrappedinthespleenshortlyafterarterialinjection andtheyarriveintheischemiccortexbetween6and12hafter MCAO.Similarﬁndingshavebeenobtainedusingtheintravenous route(Yangetal.,2011).Themigrationofthesecellstowardsthe lesionsiteseemstoinvolvethereleaseofstroma-derivedfactor-1␣ (SDF-1␣)byastrocytes,neuronsandendothelialcellsaswellasthe expressionoftheSDF-1␣receptorbyMSCs(Choppetal.,2009).

Despite their low numbers and transient existence, several studieshave shown that bone marrow-derived cells are highly neuroprotective after experimental stroke (Bao et al., 2011; Brennemanetal.,2010;deVasconcelosDosSantosetal.,2010;

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Fig.5. BMMCtreatmentreducesmicroglia/macrophageactivationatalatersurvivaltimeaftercorticalischemia.Anti-ED1immunohistochemistryincoronalsectionsfrom

ischemicanimalstreatedwithsterilesaline(A,C,E)orBMMCs(B,D,F)at7(AandB),14(CandD)and21(EandF)dayspost-injury.ThedensityofED1+cellswas

statisticallydecreasedintheischemiccortexafterBMMCtreatmentat21dayspost-injury(F)ascomparedtothevehiclecontrol(G,*P<0.05ANOVA-Bonferroni).Sections

werecounterstainedwithcresylviolet.ArrowsindicateED1+cells.Scalebar:100␮m.

Keimpemaetal.,2009).However,mostofthesestudiesevaluated onlyafewhistopathologicalparameters(mainlytheinfarctarea), andapoptosisandneuronaldensitywerenotcommonlyassessed (Brennemanetal.,2010;deVasconcelosDosSantosetal.,2010; Keimpemaetal.,2009;Schwartingetal.,2008).

In this study,intravenous transplantation of BMMCs at24h post-ischemia decreased the infarct area and apoptosis and increasedneuronal densityup to21days post-ischemia. Senso-rimotorperformance wasalsohigherinBMMC-treatedanimals duringthisperiod.Theseresultssuggestalong-lastingprotective effect of BMMCs after cortical ischemia. Although the mecha-nismsunderlyingthe BMMC-derivedprotective actionsare not fullyunderstood,theyprobablydonotinvolvetransdifferentiation withcellreplacement.Rather,theylikelyinvolvethemodulation oftheischemicenvironmentbyreleaseofgrowthfactorsand anti-inﬂammatorycytokines(Choppetal.,2009;Sarnowskaetal.,2009; Schwartingetal.,2008).

Severalsolublefactorsaredirectlyreleasedbybone marrow-derived cells, including ﬁbroblast growth factor-1 (FGF-2),

epidermalgrowthfactor(EGF),brain-derivedneurotrophicfactor (BDNF),nerve growthfactor (NGF),vascular endothelialgrowth factor(VEGF),brainnatriureticpeptide(BNP),erythropoietinand anti-inflammatorycytokines(Choppetal.,2009;Sarnowskaetal., 2009).Moreover,BMSCscanmodulatetheactivityofboth astro-cytesandmicroglia,inducingthesecellstoreleasegrowthfactors and/oranti-inflammatorycytokines(Choppetal.,2009;Sarnowska etal.,2009).Inaddition,ithasbeenshownthatMSCsmayexert therapeutic effects byenhancing endogenous neurogenesisand protectingnewlyborncellsafterMCAO(Yooetal.,2008).Inthe presentstudy,BMMCsmighthavereleasedsuchsoluble factors tocontributetoapoptosisreductionand neuronalpreservation. This is in agreement witha recent study showing that BMSCs mayreleaseNGF,FGFandIGFinhippocampalorganotypicslice cultures(Sarnowskaetal.,2009).Accordingtotheseauthors, dif-fuseneurotrophicfactorscanprovideneuroprotectionwithoutthe necessityof cell–cellcontact. Thesefindings canexplainhow a lownumberofBMMCsmayinducesignificantneuroprotectionin theischemicparenchyma.It hasbeenshownthatmostBMMCs

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Fig.6.Concomitantminocycline/BMMCtreatmentenhancesneuroprotectionanddecreasesapoptosisaftercorticalischemia.ImmunohistochemistryforED1,NeuNand

activecaspase-3incoronalsectionsfromischemicanimalstreatedwithsterilesaline(A,E,I),BMMCs(B,F,J),minocycline(C,G,K)andBMMC/minocycline(D,H,L)at

7dayspost-injury.BMMC/minocyclineconcomitanttreatmentenhancedtheneuroprotective,anti-inﬂammatoryandanti-apoptoticeffectsaffordedbyisolatedtherapies

(G,P<0.05ANOVA-Bonferroni,ascomparedtovehicle*orothergroups#_)._Sections_B,_C,_E_and_F_were_{counterstained}_with_cresyl_violet._Arrows_indicate_{immunolabeled}_cells.

Scalebar:100␮m.

dieshortlyafterenteringtheischemictissue,buttheymodulate theischemicenvironmenteven duringtheirtransientexistence (Keimpemaetal.,2009).

Themaingoalofthisstudywastoinvestigatewhether mod-ulationof microglialactivation would facilitate thetherapeutic effectsofBMMCsintheischemicenvironment.Minocycline treat-mentreduced thenumber of ED1+cells inthe firstweekafter corticalischemiaand clearlyimprovedtheneuroprotection and functional recovery afforded by treatment of ischemic animals withBMMCsonly.Theseresultssuggestthatexacerbatedmicroglia activation impairsBMMC actions in the ischemicenvironment andthatmicrogliamodulationwithminocyclinecreatesamore permissiveenvironment for thetransplanted BMMCs.It iswell establishedthat overactivatedmicrogliaaredetrimental follow-ingfocalischemiaandthatthemicroglialinhibitor,minocycline, reducesinfarctareaandcontributestofunctionalrecoveryafter stroke(Lampl et al., 2007; Yrjanheikki et al., 1999). Neverthe-less,nopreviousstudiesinvestigatedwhetheractivatedmicroglia influencetransplanted stem cells in theischemic environment. Thepresentresultsaresupportedbyreportsshowingthatbone marrow stromal cells transplanted into the intact adult brain are rejectedby an inflammatory response (Coyne et al., 2006)

andthat activatedmacrophages impairsurvivaland integration of embryonic stemcells transplanted 3days afterbrain trauma (Molcanyi et al., 2007). In addition, a recent paper has shown thatminocyclinetreatmentincreasesthesurvivalofporcinefetal neurons intrastriatally transplanted into the rat brain ( Michel-Monigadon et al., 2010). In this study, inhibition of microglia activation and lymphocyte inﬁltration in a later survival time wasclearly associated withsurvival of transplanted stem cells (Michel-Monigadonetal.,2010).

Weinvestigatedsensorimotorrecoveryinanimalsbelongingto allexperimentalgroups.AnimalstreatedwithBMMCplus minocy-clineanimalsshowedbetterperformance inallbehavioraltests administeredmainlyat7dayspost-injury.Theresultssuggestthat concomitanttreatmentusingminocyclineandBMMCsenhances theeffectofBMMCsonfunctionalrecoveryaftercorticalischemia. OtherstudieshaveshownthattreatmentwithisolatedMSCs(Bao etal.,2011;Schwartingetal.,2008)orBMMCs(Brennemanetal., 2010;deVasconcelosDosSantosetal.,2010)improves sensori-motorrecoveryafterstroke.Histologicalpreservationinducedby thetransplantedBMSCsthroughthemechanismsdiscussedabove likelyunderliesthebehavioralimprovementshowninthese stud-ies.Inhibitionofmicroglialikelyimprovestheseeffectsbycreating

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Fig.7.Concomitantminocycline/BMMCtreatmentenhancesthefunctionalrecoveryprovidedbyBMMCsaftercorticalischemia.Performanceofanimalsinmodiﬁed

sticky-tape(A)andopen-ﬁeldtests(CandE)at1,3and7dayspost-injury.BMMC-minocycline-treatedanimalsshowedbetterperformanceinthemodiﬁedsticky-tapetest

thananimalstreatedwithBMMCorminocyclinealoneat7dayspost-injury(P<0.05,ANOVA-Bonferroni,ascomparedtovehiclecontrolanimals).Intheopen-ﬁeldtest,

BMMC-minocycline-treatedanimalsshowedbetterperformanceinallanalyzedparametersat7dayspost-injuryascomparedtothevehicle*orsham#_groups_(B–E,_P_<_0.05,

ANOVA-Bonferroni).

amorepermissiveenvironmentforneuralrepair(Keimpemaetal., 2009;Lalancette-Hebertetal.,2007;Schwartingetal.,2008).

Although the data suggest a deleterious effect of activated microgliaontherapeuticactionsofBMMCs,therearereports sug-gestingthatBMSCsmayexertneuroprotectiveactionsbychanging the ischemic environment through interactions with microglia (Keimpemaetal.,2009;Schwartingetal.,2008).Thephenotypeof theseglialcellsislikelymodulatedbyanti-inflammatorycytokines andgrowthfactorsreleasedbyBMSCs,renderingthembeneficial afterischemia(Keimpemaetal.,2009;Schwartingetal.,2008).It hasbeenshownthatmicrogliamayprovidebenefitsafterischemia byreleasingIGF-1andanti-inflammatorycytokines( Lalancette-Hebertetal.,2007;Thoredetal.,2009).Itfollowsthatacomplete inhibitionofmicroglialactivationisnotanidealapproach;rather, modulationofmicroglial activitymaybea bettersolution.The experimentaldataavailablesuggestthatminocyclinedoes mod-ulatemicroglialactivity.Itispossiblethatminocyclineinhibitsthe activationofdeleteriouspopulationsofmicrogliawithoutaffecting thebeneficialones.Thisshouldbeinvestigatedinfurther stud-ies using double immunofluorescence with specific phenotypic

markersformicroglia.Moreover,minocyclinehasotherpleiotropic effectsbeyondmicrogliainhibition(Yongetal.,2004),whichcan bealsopartiallyresponsibleforitsneuroprotectiveeffects.

5. Conclusion

Our data support the notion that uncontrolled microglial activation is detrimental to the therapeutic actions of BMMCs transplantedduringtheacutephaseofcorticalischemia.Aproper modulationofthemicroglialresponseintheischemicenvironment providesbetterneuroprotectionandfunctionalrecoverythanthe transplantationofBMMCsonly.Thisanti-inﬂammatoryapproach mayrepresentanimportantadjuvanttherapytoimprove thera-peuticactionsofstemcellstransplantedduringtheacutephaseof CNSdiseases.

Conﬂictofintereststatement

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Acknowledgements

ThisworkwassupportedbytheBrazilianNationalCouncilfor ScientiﬁcandTechnologicalDevelopment(CNPq)andFundac¸ãode AmparoAPesquisadoEstadodoPará(FAPESPA).WGomes-Lealis aprincipalinvestigatoringrantnumber573872/2008-2fromthe MinistryofScienceandTechnology(MCT),MinistryofHealth(MS) and CNPq (Edital CT-Biotecnologia/MCT/CNPq/MS/SCTIE/DECIT no.17/2008)and FAPESPA(PRONEX-FAPESPA-CNPQ-Edital 012-2009).

References

Bao,X.,Wei,J.,Feng,M.,Lu,S.,Li,G.,Dou,W.,Ma,W.,Ma,S.,An,Y.,Qin,C.,Zhao,R.C.,

Wang,R.,2011.Transplantationofhumanbonemarrow-derivedmesenchymal

stemcellspromotesbehavioralrecoveryandendogenousneurogenesisafter

cerebralischemiainrats.BrainRes.1367,103–113.

BarbosadaFonseca,L.M.,Gutﬁlen,B.,RosadodeCastro,P.H.,Battistella,V.,

Golden-berg,R.C.,Kasai-Brunswick,T.,Chagas,C.L.,Wajnberg,E.,Maiolino,A.,Salles

Xavier,S.,Andre,C.,Mendez-Otero,R.,deFreitas,G.R.,2010.Migrationand

homingofbone-marrowmononuclearcellsinchronicischemicstrokeafter

intra-arterialinjection.Exp.Neurol.221,122–128.

Battista,D.,Ferrari,C.C.,Gage,F.H.,Pitossi,F.J.,2006.Neurogenicnichemodulation

byactivatedmicroglia:transforminggrowthfactorbetaincreasesneurogenesis

intheadultdentategyrus.Eur.J.Neurosci.23,83–93.

Block,M.L.,Zecca,L.,Hong,J.S.,2007.Microglia-mediatedneurotoxicity:uncovering

themolecularmechanisms.Nat.Rev.Neurosci.8,57–69.

Brenneman,M.,Sharma,S.,Harting,M.,Strong,R.,CoxJr.,C.S.,Aronowski,J.,Grotta,

J.C.,Savitz,S.I.,2010.Autologousbonemarrowmononuclearcellsenhance

recoveryafteracuteischemicstrokeinyoungandmiddle-agedrats.J.Cereb.

BloodFlowMetab.30,140–149.

Bresnahan,J.C.,Beattie,M.S.,Todd,F.D.,Noyes3rd,D.H.,1987.Abehavioraland

anatomicalanalysisofspinalcordinjuryproducedbyafeedback-controlled

impactiondevice.ExpNeurol95,548–570.

Chopp,M.,Li,Y.,Zhang,Z.G.,2009.Mechanismsunderlyingimprovedrecovery

ofneurologicalfunctionafterstrokeintherodentaftertreatmentwith

neu-rorestorativecell-basedtherapies.Stroke40,S143–S145.

Coyne, T.M., Marcus, A.J., Woodbury, D., Black, I.B., 2006. Marrow stromal

cells transplanted to the adult brain are rejected by an inﬂammatory

responseandtransferdonorlabelstohostneuronsandglia.StemCells24,

2483–2492.

deVasconcelosDosSantos,A.,daCostaReis,J.,DiazParedes,B.,Moraes,L.,Jasmin

Giraldi-Guimaraes,A.,Mendez-Otero,R.,2010.Therapeuticwindowfor

treat-mentofcorticalischemiawithbonemarrow-derivedcellsinrats.BrainRes.

1306,149–158.

Giraldi-Guimaraes,A.,Rezende-Lima,M.,Bruno,F.P.,Mendez-Otero,R.,2009.

Treat-mentwithbonemarrowmononuclearcellsinducesfunctionalrecoveryand

decreasesneurodegenerationaftersensorimotorcorticalischemiainrats.Brain

Res.9,108–120.

Gomes-Leal, W., Corkill, D.J., Freire, M.A., Picanco-Diniz, C.W., Perry, V.H.,

2004. Astrocytosis, microglia activation, oligodendrocyte

degenera-tion,and pyknosis following acute spinal cord injury. Exp. Neurol.190,

456–467.

Gomes-Leal,W.,Corkill,D.J.,Picanco-Diniz,C.W.,2005.Systematicanalysisofaxonal

damageandinﬂammatoryresponseindifferentwhitemattertractsofacutely

injuredratspinalcord.BrainRes.1066,57–70.

Guimaraes,J.S.,Freire,M.A.,Lima,R.R.,Picanco-Diniz,C.W.,Pereira,A.,Gomes-Leal,

W.,2010.Minocyclinetreatmentreduceswhitematterdamageafterexcitotoxic

striatalinjury.BrainRes.

Hamby,A.M.,Suh,S.W.,Kauppinen,T.M.,Swanson,R.A.,2007.Useofa

poly(ADP-ribose)polymeraseinhibitortosuppressinﬂammationandneuronaldeathafter

cerebralischemia-reperfusion.Stroke38,632–636.

Hewlett,K.A.,Corbett,D.,2006.Delayedminocyclinetreatmentreduceslong-term

functionaldeﬁcitsandhistologicalinjuryinarodentmodeloffocalischemia.

Neuroscience141,27–33.

Iihoshi,S.,Honmou,O.,Houkin,K.,Hashi,K.,Kocsis,J.D.,2004.Atherapeutic

win-dowforintravenousadministrationofautologousbonemarrowaftercerebral

ischemiainadultrats.BrainRes.1007,1–9.

Keimpema,E.,Fokkens,M.R.,Nagy,Z.,Agoston,V.,Luiten,P.G.,Nyakas,C.,Boddeke,

H.W.,Copray,J.C.,2009.Earlytransientpresenceofimplantedbonemarrow

stemcellsreduceslesionsizeaftercerebralischaemiainadultrats.Neuropathol.

Appl.Neurobiol.35,89–102.

Lalancette-Hebert,M.,Gowing,G.,Simard,A.,Weng,Y.C.,Kriz,J.,2007.Selective

ablationofproliferatingmicroglialcellsexacerbatesischemicinjuryinthebrain.

J.Neurosci.27,2596–2605.

Lampl,Y.,Boaz,M.,Gilad, R.,Lorberboym, M.,Dabby,R.,Rapoport,A.,

Anca-Hershkowitz,M.,Sadeh,M.,2007.Minocyclinetreatmentinacutestroke:an

open-label,evaluator-blindedstudy.Neurology69,1404–1410.

Li,Y.,Chen,J.,Chopp,M.,2001.Adultbonemarrowtransplantationafterstrokein

adultrats.CellTransplant10,31–40.

Lindvall,O.,Kokaia,Z.,2006.Stemcellsforthetreatmentofneurologicaldisorders.

Nature441,1094–1096.

Mendez-Otero,R.,deFreitas,G.R.,Andre,C.,deMendonca,M.L.,Friedrich,M.,

Oliveira-Filho,J.,2007.Potentialrolesofbonemarrowstemcellsinstroke

therapy.Regen.Med.2,417–423.

Michel-Monigadon,D.,Nerriere-Daguin,V.,Leveque,X.,Plat,M.,Venturi,E.,Brachet,

P.,Naveilhan,P.,Neveu,I.,2010.Minocyclinepromoteslong-termsurvivalof

neuronaltransplantinthebrainbyinhibitinglatemicroglialactivationand

T-cellrecruitment.Transplantation89,816–823.

Molcanyi,M.,Riess,P.,Bentz,K.,Maegele,M.,Hescheler,J.,Schafke,B.,Trapp,T.,

Neugebauer,E.,Klug,N.,Schafer,U.,2007.Trauma-associatedinﬂammatory

responseimpairsembryonicstemcellsurvivalandintegrationafter

implan-tationintoinjuredratbrain.J.Neurotrauma24,625–637.

Morioka,T.,Kalehua,A.N.,Streit,W.J.,1993.Characterizationofmicroglialreaction

aftermiddlecerebralarteryocclusioninratbrain.J.Comp.Neurol.327,123–132.

Mullen,R.J.,Buck,C.R.,Smith,A.M.,1992.NeuN,aneuronalspeciﬁcnuclearprotein

invertebrates.Development116,201–211.

Neumann,J.,Sauerzweig,S.,Ronicke,R.,Gunzer,F.,Dinkel,K.,Ullrich,O.,Gunzer,

M.,Reymann,K.G.,2008.Microgliacellsprotectneuronsbydirectengulfmentof

invadingneutrophilgranulocytes:anewmechanismofCNSimmuneprivilege.

J.Neurosci.28,5965–5975.

Paxinos,G.,Watson,C.,2007.Theratbraininstereotaxiccoordinates,6thEdition.

AcademicPress,NewYork,pp.456.

Ransohoff,R.M.,Perry,V.H.,2009.Microglialphysiology:uniquestimuli,specialized

responses.Annu.Rev.Immunol.27,119–145.

Robinson,A.P.,White,T.M.,Mason,D.W.,1986.Macrophageheterogeneityintherat

asdelineatedbytwomonoclonalantibodiesMRCOX-41andMRCOX-42,the

latterrecognizingcomplementreceptortype3.Immunology57,239–247.

Sarnowska,A.,Braun,H.,Sauerzweig,S.,Reymann,K.G.,2009.Theneuroprotective

effectofbonemarrowstemcellsisnotdependentondirectcellcontactwith

hypoxicinjuredtissue.Exp.Neurol.215,317–327.

Schnell,L.,Fearn,S.,Klassen,H.,Schwab,M.E.,Perry,V.H.,1999.Acute

inﬂamma-toryresponsestomechanicallesionsintheCNS:differencesbetweenbrainand

spinalcord.Eur.J.Neurosci.11,3648–3658.

Schroeter,M.,Jander,S.,Witte,O.W.,Stoll,G.,1994.Localimmuneresponsesinthe

ratcerebralcortexaftermiddlecerebralarteryocclusion.J.Neuroimmunol.55,

195–203.

Schwarting,S.,Litwak,S.,Hao,W.,Bahr,M.,Weise,J.,Neumann,H.,2008.

Hematopoi-eticstemcellsreducepostischemicinﬂammationandameliorateischemicbrain

injury.Stroke39,2867–2875.

Sobani,Z.A.,Quadri,S.A.,Enam,S.A.,2010.Stemcellsforspinalcordregeneration:

currentstatus.Surg.Neurol.Int.1,93.

Souza-Rodrigues,R.D.,Lima,R.R.,Guimaraes-Silva,J.,Costa,A.M.,DosSantos,C.D.,

Picanc¸o-Diniz,C.W.,Gomes-Leal,W.,2008.Inﬂammatoryresponseandwhite

matterdamageaftermicroinjectionsofendothelin-1intotheratstriatum.Brain

Res.1200C,78–88.

Sughrue,M.E.,Mocco,J.,Komotar,R.J.,Mehra,A.,D’Ambrosio,A.L.,Grobelny,B.T.,

Penn,D.L.,ConnollyJr.,E.S.,2006.Animprovedtestofneurological

dysfunc-tionfollowingtransientfocalcerebralischemiainrats.JNeurosciMethods151,

83–89.

Takahashi,K.,Yasuhara,T.,Shingo,T.,Muraoka,K.,Kameda,M.,Takeuchi,A.,Yano,

A.,Kurozumi,K.,Agari,T.,Miyoshi,Y.,Kinugasa,K.,Date,I.,2008.Embryonic

neuralstemcellstransplantedinmiddlecerebralarteryocclusionmodelofrats

demonstratedpotenttherapeuticeffects,comparedtoadultneuralstemcells.

BrainRes.1234,172–182.

Thored,P., Heldmann, U.,Gomes-Leal, W., Gisler, R.,Darsalia, V., Taneera,J.,

Nygren,J.M.,Jacobsen,S.E.,Ekdahl,C.T.,Kokaia,Z.,Lindvall,O.,2009.

Long-termaccumulationofmicrogliawithproneurogenicphenotypeconcomitant

withpersistentneurogenesisinadultsubventricularzoneafterstroke.Glia57,

835–849.

Tuttolomondo,A.,DiRaimondo,D.,diSciacca,R.,Pinto,A.,Licata,G.,2008.

Inﬂam-matorycytokinesinacuteischemicstroke.Curr.Pharm.Des.14,3574–3589.

Walton,N.M.,Sutter,B.M.,Laywell,E.D.,Levkoff,L.H.,Kearns,S.M.,Marshall2nd,

G.P.,Schefﬂer,B.,Steindler,D.A.,2006.Microgliainstructsubventricularzone

neurogenesis.Glia54,815–825.

Weissman,I.L.,Anderson,D.J.,Gage,F.,2001.Stemandprogenitorcells:origins,

phenotypes,lineagecommitments,andtransdifferentiations.Annu.Rev.Cell

Dev.Biol.17,387–403.

Woodruff,T.M.,Thundyil,J.,Tang,S.C.,Sobey,C.G.,Taylor,S.M.,Arumugam,T.V.,

2011.Pathophysiology,treatment,andanimalandcellularmodelsofhuman

ischemicstroke.Mol.Neurodegener.6,11.

Yang,B.,Strong,R.,Sharma,S.,Brenneman,M.,Mallikarjunarao,K.,Xi,X.,Grotta,J.C.,

Aronowski,J.,Savitz,S.I.,2011.Therapeutictimewindowanddoseresponseof

autologousbonemarrowmononuclearcellsforischemicstroke.J.Neurosci.Res.

89,833–839.

Yong,V.W.,Wells,J.,Giuliani,F.,Casha,S.,Power,C.,Metz,L.M.,2004.Thepromise

ofminocyclineinneurology.LancetNeurol.3,744–751.

Yoo,S.W.,Kim,S.S.,Lee,S.Y.,Lee,H.S.,Kim,H.S.,Lee,Y.D.,Suh-Kim,H.,2008.

Mesenchymalstemcellspromoteproliferationofendogenousneural stem

cellsandsurvivalofnewborncellsinaratstrokemodel.Exp.Mol.Med.40,

387–397.

Yrjanheikki,J.,Tikka,T.,Keinanen,R.,Goldsteins,G.,Chan,P.H.,Koistinaho,J.,1999.A

tetracyclinederivative,minocycline,reducesinﬂammationandprotectsagainst

focalcerebralischemiawithawidetherapeuticwindow.Proc.Natl.Acad.Sci.

U.S.A.96,13496–13500.

Zhang,Z.G.,Chopp,M.,2009.Neurorestorativetherapiesforstroke:underlying