紫杉醇减少脊髓损伤后疤痕形成并促进轴突再生

Title MicrotubuleStabilizationReducesScarringandCausesAxonRegenerationAfterSpinalCordInjuryFaridaHellaletal Science331 928 2011 DOI 10 1126 science 1201148 Reporter Supervisor SecondarySupervisor Backgrounds Hypertrophicscarringandpoorintrinsicaxongrowthcapacityconstitutemajorobstaclesforspinalcordrepair Microtubuledynamicsregulatekeyprocessesduringscarring includingcellproliferation migration anddifferentiationaswellasintracellulartraffickingandsecretionofextracellularmatrix ECM molecules 1 2 Moreover moderatemicrotubulestabilizationpreventsaxonalretractionandswellingoftheaxontipaftercentralnervoussystem CNS injury 3 andstimulatesaxongrowthofculturedneurons 4 enablingthemtoovercomethegrowthinhibitoryeffectofCNSmyelin 3 Assumptions TheyhypothesizedthatmoderatemicrotubulestabilizationwithTaxol anapproveddrug wouldfacilitateaxonalregenerationafterspinalcordinjury SCI bydecreasingscarformationandenhancingintrinsicaxonalgrowth Methods TheyfirstexaminedwhetherTaxoltreatmentreducedscarringafterSCI Adultratsunderwentadorsalhemisectionattheeighththoracicspinalcordlevel Taxol 256ng day wascontinuouslydeliveredattheinjurysiteusinganintrathecalcatheterconnectedtoanosmoticminipump Whathappened7dayslater FaridaHellaletal Science2011 331 928 931 PublishedbyAAAS Fig 1Taxoldecreasesscarringinducedbyspinalcordinjury A Representationoflesionedspinalcord box BandC Midsagittalsectionsoflesionsitefromratstreatedwith B vehicleor C Taxol 256ng day 7daysafterinjury Scalebars 300 m D Taxolsignificantlydecreasesfibroticscarring expressedaspercentageofvehiclecontrol n 12ratspergroup P 0 002 two tailedttest withoutaffectingglialcompaction 28daysafterinjury E orinjurysize F n 7to10ratspergroup P 0 951 two tailedttest Dataexpressedasmean SEM GFAP glialfibrillaryacidicprotein FigureS1 Taxoldecreasesfibronectin NG2proteoglycanandcollagenIVaccumulationinducedbyspinalcordinjury A Representationoflesionedspinalcord box B D Mid saggitalsectionsoflesionsitefromrattreatedwithVehicleorTaxolat7dayspost injury TaxoldecreasesECMdepositinthecoreofthelesionasassessedbyfibronectin B collagenIV C andNG2proteoglycan D immunostainings Thedashedlinesoutlinethelesionsite Scalebar 150 m A Representationoflesionedspinalcord box B C Mid saggitalsectionsoflesionsitefromratstreatedwithVehicleorTaxol 7dayspost injury stainedwithDAPI B andphospho histoneH3 C Scalebar 150 m D Taxoldoesnotaffectcellproliferationinducedbyspinalcordinjury Resultsexpressedasmean SEM p 0 591 two tailedttest n 7animalspergroup FigureS2 Taxoldoesnotinterferewithproliferation FigureS3 Taxoldoesnotinduceapoptosisintheinjuredspinalcord A Representationoflesionedspinalcord box B C Mid saggitalsectionsoflesionsitefromratstreatedwithVehicleorTaxolstainedwithTUNELat3 B and7 C daysafterdorsalhemisectioninjury Scalebar 150 m D Taxoldoesnotaffectthecelldeathinducedbyspinalcordinjury Resultsexpressedasmean SEM 3dpi p 0 628 7dpi p 0 679 two tailedttest n 7animalspergroup Conclusion1 Atlowdoses Taxolreducedfibroticscarringbymechanismsindependentofcellproliferationorapoptosis 2 Then theyexaminedwhetherstabilizingthemicrotubulenetworkhindersTGF signalingandattenuatesfibrogenesis AkeyeventinfibroticscarringafterCNSinjuryistheactivationoftransforminggrowthfactor TGF signaling FollowingSCI TGF expressiondramaticallyincreases whichfavorsfibrosis 10 12 Integrityofthemicrotubulenetworkiscrucialforthetransductionofthissignal 13 Smad2 thedownstreameffectoroftheTGF pathway bindstomicrotubulesthroughconventionalkinesin 1 14 Fig 2 TaxoldampensTGF signaling A Taxoltreatmentincreasestotaltubulinanddecreasestyrosinatedtubulininthelesionsite enablingkinesin 1tobindtightlytomicrotubules 15 B Kinesin 1enrichmentinmicrotubulefractionofTaxol treatedlesionsite C His Smad2bindstokinesin 1andendogenousSmad2coimmunoprecipitateswithkinesin 1 D ofbrainandspinalcordextracts Fig 2 TaxoldampensTGF signaling EandF Taxolaltersmicrotubule basedcargotransport E Overlayofcolorcodedtimeseriesofredfluorescentprotein RFP labeledperoxisomesboundtothekinesin 1 KIF5 motordomainuponRapalogaddition Bluemarkstheinitialdistribution theredgradientshowsthedistributionovertime 30min Scalebars 10mm F Timetracesofradiusofcircleenclosing90 oftotalfluorescenceintensityforKIF5 ordyneinadaptor BICDN linkedperoxisomes MeanTSEMof5to8COS 7cellspercondition P 0 004 two tailedttestforbothKIF5andBICDN ThissuggestingthatTaxolwouldhinderSmad2trafficking Indeed inTGF 1 stimulatedastrocytes TaxolcausedSmad2 3tolocalizepersistentlytomicrotubules fig S4 andinhibited70 ofitstranslocationtothenucleus Fig 2 GandH FigureS4 TaxolsequestersSmad2ontomicrotubulesAstrocytestreatedwithDMSOorTaxol 100nM 30minutespriortoTGF 1 2ng ml stimulation CellswerepermeabilisedduringfixationtoextractsolublecomponentsofthecytoplasmandstainedwithSmad2 3antibody TaxolinducesSmad2 3co localizationwithmicrotubules Scalebar 10 m Indeed inTGF 1 stimulatedastrocytes TaxolcausedSmad2 3tolocalizepersistentlytomicrotubules fig S4 andinhibited70 ofitstranslocationtothenucleus Fig 2 GandH Fig 2 TaxoldampensTGF signaling GandH Inculturedastrocytes TaxolcounteractstheTGF 1 inducednucleartranslocationofSmad2 3 arrowheads causingcytoplasmicSmad2 3accumulation arrow Resultsin H aremeanTSD threeindependentexperiments P 0 041 one wayanalysisofvariance ANOVA Intime lapsemicroscopy overexpressedSmad2fusedtoPAGFP photoactivatablegreenfluorescentprotein movedintothenucleuswithinminutesafterTGF b1stimulation whereasTaxoltreatmentabolishedthismovement moviesS1andS2 Fig 2 TaxoldampensTGF bsignaling I Invivo 7daysafterSCI phosphorylatedSmad2 3translocatedintothenucleusin95 ofvehicletreatedanimals comparedwithonly30 oftheTaxol treatedanimals n 13ratspergroup Scalebar 20mm BSA bovineserumalbumin DAPI 4 6 diamidino 2 phenylindole DMSO dimethylsulfoxide Indeed inculturedmeningealcells TaxolreducedtheTGF 1 stimulatedproductionoffibronectin Fig 3A andimpairedTGF 1 stimulatedmigration Fig 3 BandC Fig 3 Taxoldecreasesmeningealcellmigrationandglycosaminoglycanreleaseinvitroandinvivo A TaxoldecreasesfibronectindepositsinducedbyTGF b1inmeningealcells Scalebar 300mm BandC Threedaysinvitro Taxol 1and10nM decreasesmeningealcellmigrationinducedbyTGF b1 Arrowsindicatetheinitialgapsize Scalebar 300mm resultsin C aremeansTSDfromthreeindependentexperiments P 0 003 one wayANOVA Conclusion2 LowdosesofTaxolpreventfibroticscarringafterSCIbyinterferingwithSmaddependentTGF signalingandreducingextracellularmatrixsecretionandcellmigration 3 The3rdquestioniswhetherTaxoldecreasesCSPGsafterSCI TGF signalingalsoregulatestheproductionoftheaxongrowthinhibitorychondroitinsulfateproteoglycans CSPGs 10 FigureS1 Taxoldecreasesfibronectin NG2proteoglycanandcollagenIVaccumulationinducedbyspinalcordinjury A Representationoflesionedspinalcord box B D Mid saggitalsectionsoflesionsitefromrattreatedwithVehicleorTaxolat7dayspost injury TaxoldecreasesECMdepositinthecoreofthelesionasassessedbyfibronectin B collagenIV C andNG2proteoglycan D immunostainings Thedashedlinesoutlinethelesionsite Scalebar 150 m At7daysafterinjury TaxoldecreasedtheamountofNG2 oneofthemostabundantCSPGs 17 fig S1 Fig 3 Taxoldecreasesmeningealcellmigrationandglycosaminoglycanreleaseinvitroandinvivo Fig 3D LesionsiteextractsfromTaxol treatedanimalsshowedasignificantreductionofGAGscomparedwithcontrols Fig 3 EandF Theconditionedmediumofculturedmeningealcellsandastrocytestreatedwith10nMTaxolshoweda35 and32 decreaseofGAGlevels respectively Fig 3G Moreover theCSPGsexpressedintheTaxoltreatedanimalslocalizedtotheintracellularspaceinsteadofscaffoldingthecellsasobservedinvehicle treatedanimals AdministrationoflowdosesofTaxoldecreasesCSPGsatthelesionsiteafterSCI Conclusion3 4 Further theyaskedwhethertheTaxol treatedlesionsitebecomespermissiveforregeneratingaxonsinvivobyevaluatingtheregenerativeresponseofdorsalrootganglion DRG neurons Theprocedure Theseneuronsaresetintoagrowth competentstatebyinjuringtheirperipheralaxon conditioning thatallowsthemtoregeneratetheirCNSaxon butonlyinascar freeenvironment 21 TheyassessedwhetherthereductionofthescarinducedbyTaxolispermissiveforconditionedaxonstogrow Taxolwasdeliveredatthelesionfor4weeks 2weeksaftercentralinjury theyconditionedthelumbarL4 6DRGneuronsbytransectingthesciaticnerve Whatabouttheresults Fig 4 Taxolpromotesaxonalregenerationandfunctionalrecovery A SpinalcordhorizontalsectionsofL4 6DRGaxonslabeledwithcholeratoxinB 6weeksafterinjury Taxoltreatmentpromotesregenerationofgrowthcompetentneurons arrowheads Scalebars 200mm B Longestregeneratingaxonperanimal SEM P 0 002 two tailedttest Thelongestaxonsperanimalgrew1199 250mmintheTaxol treatedgroupversus176 225mminthevehicle treatedanimals n 13animalspergroup P 0 002 twotailedttest TheTaxol treatedlesionsitethusbecomesfavorableforregenerationofgrowth competentaxons Conclusion4 Question5 BecauseTaxolalsoenhancesintrinsicaxongrowth 4 andtheelongationofculturedneuronsplatedonCSPGsorCNSmyelincomponents 3 fig S5 theyassessedwhetherTaxoltreatmentalonecouldpromotegrowthofinjuredCNSaxons FigureS5 Taxolpromotesaxonaloutgrowthofprimaryneuronsplatedoninhibitorysubstrates A Cerebellargranuleneurons CGNs platedonlamininorCSPGsat2DIV B TaxolincreasesneuritegrowthofCGNsplatedonlamininandCSPGs Resultsaremeans SDfrom3independentexperiments n 150 200cellspercondition p 0 016 p 0 038respectively two tailedttest C TaxolincreasesneuriteoutgrowthofprimarycorticalneuronsplatedonMAG NogoA Semaphorin3AorCSPGs Resultsaremeans SDfrom3independentexperiments n 150 200cellspercondition p 0 005 p 0 009 p 0 007 p 0 002andp 0 001 respectively onewayANOVA Fig 4 Taxolpromotesaxonalregenerationandfunctionalrecovery C Spinalcordsagittalsectionsstainedwithantibodyto5 HT4weeksafterinjury AfterTaxoltreatment thecaudalpartofthecordisenrichedinserotonergicfibers C arrowheads Scalebars 75mm D Quantificationof5 HT positivefiberscaudaltothelesion SEMafterdorsalhemisection n 16ratspergroup P 0 0001 twotailedttest D FigureS6 Taxolpromotesformationofgrowthcone likestructuresatthetipofthe5HT positiveaxons Tworepresentativeexamplesofaxonaltipsof5HT positiveaxonsfromVehicleandTaxoltreatedanimals4weeksafterinjury Scalebar 10 m Taxolinducesgrowthof5 HTaxonsafterdorsalhemisection Conclusion5 6 TheylastexaminedwhetherTaxoltreatmentleadstofunctionalrecoveryaftermoderatespinalcordcontusion 24 Fig 4 Taxolpromotesaxonalregenerationandfunctionalrecovery E Quantificationof5 HT positivefiberscaudaltothelesion SEMaftercontusioninjury n 10ratspergroup P 0 002 two tailedttest E F Taxoltreatmentimproveslocomotorperformanceovertime P 0 004 P 0 0001 two wayANOVA n s notsi