Axonal regeneration in hippocampal and spinal cord organotypic slice cultures
Inauguraldissertation
zur
Erlangung der Würde eines Doktors der Philosophie vorgelegt der
Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel
von
Brenda Bonnici
aus Malta
Basel, 2008
Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von
Prof. Josef Kapfhammer Prof. Markus Ruegg Prof. Cordula Nitsch
Basel, den 16 Oktober 2008
Dekan
Prof. E. Parlow
T ABLEOF C ONTENTS :
LISTOFABBREVIATIONS v
SUMMARY 1
1. GENERALINTRODUCTION 3
1.1 AxonaldegenerationintheCNS 3
1.1.1 TheimpactoftraumaticinjurytotheCNS 3 1.1.2 Theneurobiologyofaxonalregeneration 4
1.2 Factorsinvolvedinaxonregenerationfailure 6
1.2.1 Extrinsicfactors 6
a. Glialscar 6
b. Myelinassociatedinhibitors 7
c. Signallingmechanisms 9
Promotorsignals 9
Inhibitorysignals 10
1.2.2 Intrinsicfactors 12
1.3. Experimentalapproachestoimproveaxonalregeneration 13
1.3.1 Transplantationandcellreplacement 13
a. TransplantationofSchwanncellsorOECs 13
b. Transplantationofyoungcells 14
c. Transplantationofstemcells 14
1.3.2 Inhibitionoftheglialscar 15
1.3.3 Neutralisationofinhibitorymoleculesinmyelin 15 1.3.4 Modificationofsignaltransductionpathways 17
a. PKCinhibitors 17
b. RhoandROCKinhibitors 18
c. cAMPactivators 19
1.3.5 Summarytable 19
1.4 Hippocampus 21
1.4.1 Anatomicalstructure 21
1.4.2 Thehippocampalcircuits 22
1.5 Spinalcord 23
1.5.1 Majordescendingtracts 24
1.5.2 Ascendingtracts 25
a. Dorsalcolumn 25
b. Spinothalamictract 25
c. Spinocerebellartract 26
1.5.3 Propriospinalconnectionsinthespinalcord 26
1.6 Organotypicslicecultures 27
1.6.1 (Enterohinal)hippocampalorganotypicsliceculturemodel 28 1.6.2 Studiesthatpromoteregenerationoftheperforantpathway
inorganotypicslicecultures 29
1.6.3 Spinalcordorganotypicslicecultures 30
1.6.4 OrganotypiccoͲculturesystems 31
2. AIMSOFTHEPROJECT 32
2.1. Todevelopofanovelspinalcordorganotypicsliceculturemodel towhichallowsustobetterstudyspinalcordinjury 32 2.2. Toassessspontaneousregenerationofspinalcordfibresaftera
mechanicallesion 32
2.3. Toassesstheeffectivenessofvarioustreatments(interactingwith
varioussignaltransductionmechanisms)whichpromoteaxonal 33 regenerationinentorhinoͲhippocampalslicecultures
3. HIPPOCAMPUS 34
Modulators of signal transduction pathways can promote axonal regenerationinentorhinoͲhippocampalslicecultures
3.1. Abstract 35
3.2. Introduction 35
3.3. MaterialsandMethods 36
1. Organotypicslicecultures 36
2. Lesions 37
3. Immunohistochemistry 38
4. Analysisofslices 38
5. Semiquantitativeevaluationofregeneration 38
6. Statisticalanalysis 39
3.4 Results 39 1. Evaluationofaxonalgrowththroughamechanicallesionin
entorhinoͲhippocampalslicecultureslicesinvitro 39
2. Unlesionedanduntreatedcontrols 39
3. TreatmentwithproteinkinaseCinhibitors 40
4. TreatmentwithcAMPactivators 42
5. Othertreatments 42
3.5 Discussion 44
1. Regenerationofentorhinalfibresinorganotypicslicecultures 44 2. PromotionofregenerationbyPKCinhibitorsandcAMPactivators 45 3. Promotionofregenerationbyinhibitorsofothersignallingpathways 46
3.6 Conclusions 48
4. SPINALCORD 49
Spontaneousregenerationofintrinsicspinalcordaxonsinanovelspinalcord sliceculturemodel
4.1. Abstract 50
4.2. Introduction 50
4.3. MaterialsandMethods 51
1. Organotypicslicecultures 51
2. TreatmentwithRolipram 52
3. Immunohistochemistryandmicroscopy 53
4. Semiquantitativeevaluationofregeneration 53
5. Cryosections 54
4.4. Results 55
1. SagittalspinalcordsliceculturesmaintainventroͲdorsalpolarity 55 2. Neurofilamentstainingrevealslongitudinallyextendingfibersin
spinalcordcultures 56
3. Synapticcontactsandmyleinationinspinalcordcultures 58 4. Aftermechanicallesions,spinalcordfibresregeneratespontaneously 60 5. Comparablefibregrowthinculturesderivedfrommiceatpostnatal
agesP0–P6 62
6. Promotionofaxonalgrowththroughthelesionsitecanbe
inducedbypharmacologicaltreatment 63
4.5. Discussion 63
1. Spinalcordslicecultures 64
2. Synapticcontactsandmyleinationinspinalcordcultures 65 3. Spontaneousaxonalregenerationinspinalcordslicecultures 66
5. COͲCULTURES 68
AxonalgrowthinspinalcordorganotypicslicecoͲcultures
5.1 Introduction 69
5.2 Materialsandmethods 70
1. Spinalcord–cerebellarslicecultures 70 2. Cortexspinalcordslicecultures 70
3. Immunohistochemistryandmicroscopy 71
5.3 Results 72
1. Spinalcord–cerebellarslicecultures 72
a. Untreatedcontrols 73
b. TreatmentwithproteinkinaseCinhibitors 74
c. TreatmentwithcAMPactivators 74
2. Cortex–spinalcordcultures 74
5.4 Discussion 75
6. GENERALDISCUSSION 78
6.1 Useofvariousorganotypicsliceculturemodelstostudyaxonal
growthandregeneration 78
6.2 Modificationofsignaltransductionpathwaystopromoteaxonal
regeneration 79
6.3 OrganotypicsliceculturesversusinͲvivo 80
7. CONCLUSIONS&OUTLOOK 81
8. REFERENCES 82
9. ACKNOWLEDGEMENTS 92
10. CURRICULUMVITAE 93
L ISTOF A BBREVIATIONS :
Ab antibody
BDA biotindextranamine cAMP cyclicAMP
CNS centralnervoussystem
CSPG chondrotinsulphateproteoglycan CST corticospinaltract
DG dentategyrus
DIV daysinvitro
DRG dorsalrootganglion EC enterohinalcortex GAG glycosaminoglycan
GFX GF109203X
Gö Gö6976
GPI glycosylphosphatidylinositol MAG myelinassociatedglycoprotein MBP myelinbasicprotein
MOG myelinoligodendrocyteprotein
LY LY294002
NgR NogoReceptor
NGS normalgoatserum
OMgp oligodendrocyteͲmyelinglycoprotein OEC olfactoryensheathingcells
P0,P6 postnatalday0,postnatalday6 PBS phosphatebufferedsaline
PFA paraformaldehyde PI3ͲK phosphoinositide3Ͳkinase PKA proteinkinaseA
PKC proteinkinaseC
PNS peripheralnervoussystem PP perforantpath
PTX pertussistoxin
RGC retinalganglioniccells ROCK Rhokinase
RT roomtemperature
SC spinalcord
SCI spinalcordinjury TBI traumaticbraininjury
VAChT vesicularacetylcholinetransporter XSp xestospongin
S UMMARY :
Undernormalconditions,axonalregenerationafterlesionsisnotpossibleinmatureCNSbut canoccurinembryonicandearlypostnatalnervoussystems.Inrecentyears,anumberof possiblestrategiestoenhanceaxonalregenerationandeventuallytreatspinalcordandbrain injuries have been identified, some of which have been used successfully in animal experiments,buttillnowthereisstillnosuccessfultreatmentavailableforpatients.This problemispartlyduetothecomplexityoftheanimalexperimentswhichmakesitdifficultto comparedifferenttreatmentstrategies.
Inthisproject,wehaveusedorganotypicsliceculturemodelstotesttheeffectivenessof pharmacologicalcompoundsthatinterferewithvarioussignaltransductionmechanisms,to promoteaxonalregeneration.WeusedtheentorhinoͲhippocampalsliceculturestoassess regenerationofentorhinalfibersprojectingtothedentategyrusaftermechanicallesionsand treatment.Itwaspreviouslyshown(Pranget.al.,2001)thatthereisamarkeddecreasein regeneratingfiberswhenalesionismadeat6Ͳ7daysinvitroorlaterinslicesderivedfrom postnatalday5Ͳ6mice.Wetookthisasacontrolmodelwherethereislittlespontaneous axonalregeneration,addedtreatmentsonthedayoflesionandlatertracedforentorhinal axonswithbiotinylateddextranamine(BDA).Inthisstudyitwasshownthatcompounds actingonthecAMP,PKCandGͲproteinscanpromoteregeneration.Furthermore,wehave identifiedtheinhibitionofthePI3ͲkinasepathwayandtheIPͲ3receptoraspotentialdrug targetsthatpromoteaxonalregeneration.
Inordertostudyaxonalgrowthinaspinalcordenvironmentwehavedevelopedaspinalcord longitudinalorganotypicsliceculturemodelwhichallowedustofollowaxonsalongtherostroͲ caudalextensionofthespinalcord.Slicesofcervicalspinalcordwerecutinthesagittalplane fromearlypostnatalmiceandweremaintainedincultureforvarioustimeperiodsupto4 weeks.Histologicalandimmunohistochemicalstainingsofthecultureshaveshownthatthese sliceculturesmaintaintheventroͲdorsalpolarityofthespinalcordandthatanintrinsicfibre projectiondevelopswhichrunsalongtherostroͲcaudalextensionofthespinalcordslice culture.Aftermechanicallesion,thesefibreshavetheabilitytoregeneratespontaneously demonstratingtheintrinsicabilityofthespinalcordforrepair,butthisabilityisdecreased
expressedsynapticmarkers.Theseculturescouldthusservealsoasamodelformyelin formationandsynaptogenesis.
Wehaveanalyzedthepotentialofaxonsfromlongitudinalspinalcordculturestogrowintoan adjacentsliceofcerebellartissue.Wecouldshowthatspinalcordaxonsdoenterthe cerebellarsliceinparticularwhenearlypostnatalspinalcordiscombinedwithpostnatal cerebellum.Pharmacologicaltreatmentswereusedtoenhanceaxonalgrowth.Similarlytoour findingsintheentorhinoͲhippocampalmodel,cAMPactivatorsandPKCinhibitorspromoted axonalgrowthfromthespinalcordtothecerebellum.Incoculturesoflongitudinalspinalcord sliceswithcorticalsliceswehaveshownthatfibersfromthecorticalslicesgrewextensively intothespinalcordsliceandextendedcaudallyforsubstantialdistances.
Ourresultsdemonstratethatorganotypicsliceculturescanbeausefultooltostudyaxonal growth andregeneration. Intrinsicspinal cordaxons havea considerablepotentialfor spontaneousregenerationintheearlypostnatalperiodandareabletogrowboththrougha mechanicallesionandintoanothertissue. Moreover,compoundsinterferingwithsignal transductionmechanisms,particularlycAMP,PKC,PI3ͲKinase,GͲproteinsandIP3receptors, wereabletopromoteaxonalgrowthandregenerationindiversesliceculturemodelsmaking theminterestingdrugcandidatesforthepromotionofaxonalregeneration.
1. G ENERAL I NTRODUCTION
1.1. AxonaldegenerationintheCNS
CNSdamagecanoccurinmanyways.Onemajorcauseisneurotraumawhichcanoccurafter headinjury,i.e.,traumaticbraininjury(TBI)orinthespinalcord,i.e.,spinalcordinjury(SCI).
OthermajorcausesarevariousneurodegenerativediseasessuchasAlzheimer’s,Parkinson’s, and Huntington’s disease; neuroinflammatory diseases such as multiple sclerosis; and neurovasculardiseasessuchasstroke.Alltheseconditionswillresultinthelossofneurons andinthedamagetoaxonsleadingtoaseveredysfunctionoftheCNS.IntheadultCNS,the lossofneuronsnormallycannotbecompensatedbythegenerationofnewcellsfrom immature precursors, and damagedaxonal projections fail to regenerate(Cajal,1928).
However,axonsintheembryonicandearlypostnatalCNSdoregenerateafterinjury,anditis intheperinatalperiodthattheregenerativepotentialofaxonsislost(MartinGF,2000).Inthis thesiswehaveusedtheinvitromodelsystemoforganotypicsliceculturesofdifferentCNS regionsinordertocharacterizethisdecreaseofregenerationandhavetriedtoinduceaxonal regenerationbyinterferingwithvarioussignaltransductionmechanisms.Becauseofthe extraordinaryimportanceofspinalcordinjuryandtheabsenceofasuitableinvitrosystemto studyaxonalgrowthandregenerationinthespinalcordwehavedevelopedanewspinalcord sliceculturemodel.
1.1.1 TheimpactoftraumaticinjurytotheCNS
Therearearound1.5millionpeopleeachyearwhosustainaTBIinUSAalone.Outofthese around230000arehospitalizedandsurvive,50000dieandaround85000sufferfromlongͲ termdisability.ThecostofTBIinUSin1985wasaroundUSD38billion(ThurmanDJ,1999).
Therearealso10000newSCinjuriesperyear,andaround250000peoplelivingwithSCIin USA.Thishasgreatimpactnotonlyonthelivesofthepatientsandtheirfamilies,butalsoon economy.ItisestimatedthattheannualcostofSCIisaroundUSD7.7billion(DeVivo,1997).
SCIisclassifiedbytheAmericanspinalcord injury association using the following ASIA impairment scale, as described in Fig. 1.1 (ThuretS,2006):ScaleA:completelesion,no motororsensoryfunctionispreservedinthe sacralsegmentsS4ͲS5;B:incompletelesion, sensorybutnotmotorfunctionispreserved belowtheneurologicallevelandincludesthe sacralsegmentsS4ͲS5;C:incompletelesion, motor function is preserved below the neurologicallevel,andmorethanhalfofkey musclesbelowtheneurologicallevelhavea musclegradelessthan3,i.e.activemovement withfullrangeofmotionagainstgravitybutno resistance; D: incomplete lesion, motor functionispreservedbelowtheneurological level,andatleasthalfofkeymusclesbelow theneurologicallevelhaveamusclegradeof3 ormore;andE:motorandsensoryfunction arenormal.
Fig.1.1ASIAImpairmentScale:Spinalcordinjury severityasclassifiedbytheAmericanSpinalCord InjuryAssociation(takenfromThuret,2006)
1.1.2 Theneurobiologyofaxonalregeneration
GroundͲbreakingworkbyRamónyCajalconfirmedthatafewdaysafterinjury,thereisaxonal sproutingatthelesionsite(Cajal,1928).Seventyyearslater,DavidandAguayoshowedthat retinalganglioniccells(RGCs)couldregeneratetheiraxonsthroughperipheralnervegrafts (DavidS,1981).CaroniandSchwabhavelatershownthatadultCNSisnonpermissivefor axonalregenerationtotakeplace,andthisisduetoaxongrowthinhibitorsassociatedwith myelin(CaroniP,1988a;CaroniP,1988b).
NowadaysweknowthatthereducedregenerationcapacityofCNSaxonsisduetoseveral factors,includingtheactivationofaxonalͲgrowthinhibitionrelatedtothelesion,thepresence ofinhibitorsintheadultCNSandothersignallingmechanismsinhibitinggrowth.Thesewillbe furtherdiscussedinthisthesis.Forregenerationtotakeplace,manyfactorsneedtobe targeted.Mostimportant,thedamagedneuronmustsurvive,andaxonkeepcontactorreͲ extenditsprocessesandmakesynapsestoitsoriginalneuronaltargets.Inaddition,these contactsshouldalsobefunctional,thustheaxonshouldberemyelinated.(seeFig.1.2;fora reviewrefertoHornerandGage,2000).
Fig.1.2 Stepsneededforaxonalregenerationtotakeplace(takenfromHornerandGage,2000)
In recent years, various ways to improve axonal regeneration have been identified.
Unfortunately,sofarwestillneedtohavebettertreatmentforSCIandTBI.Recently,there werevariouspromisingclinicalstudiesgoingonforSCI.Ofparticularinterestarethecell permeableRhoinhibitorCethrindevelopedbyBioAxoneonASIAcategoryApatients(Baptiste DC,2006;ThuretS,2006).AnotherclinicaltrialonASIAApatientsusingantiͲNogoͲAantibody, whichwasdevelopedbyM.Schwabandhiscolleagues(SchnellL,1990;FouadK,2004)is
1.2 Factorsinvolvedinaxonregenerationfailure
TherearevariousfactorsthataccountforaxonregenerationfailureinadultCNS(forareview, refertoGoldbergandBarres,2000).Thesecanbedividedintointrinsicandextrinsicfactors.
HerewewillhaveamoreinͲdepthoverviewofthesefactorsandthemechanismsbywhich theyeffectregeneration.Inalaterchapter(Chapter1.3)wewillthenhaveanoverviewofthe treatmentmethodstotargetthesemechanismsandinduceregenerativegrowth.
1.2.1 Extrinsicfactors:
a. Glialscar
Afterinjury,theglialscarisoneofthemajorobstaclesforaxonalregenerationtotakeplace (Fig.1.3,SilverJ,2004).Theglialscaractsbothasaphysicalbarrier,butalsoinhibitsaxonal growthduetoinhibitorymoleculeswithinthescaritself(DavidS,2003).
Oninjury,microglia,oligodendrocyteprecursors,meningealcellsandastrocytesareattracted tothelesionsite.Thisformsathickcellularbarrierwhichinhibitsaxonsfromcrossingthrough thescar.TheseaxonsweredescribedbyRamónyCajalashaving‘dystrophicendballs’which hethoughtareincapableofregeneration(Cajal,1928).Laterstudiesshowthattheseendings arehighlyactivestructures,whichdonotlosetheirabilitytoregenerate.Moreovertheglial scarrequiresacertaintimetomatureandblockaxonalgrowth(SilverJ,2004);inadultratsit can take around 2 weeks (Berry M, 1983). This gives a ‘window of opportunity’ for regenerationtotakeplace.Theglialscarasaphysicalbarrieralsohasabeneficialeffect,asit canisolatetheinjurysiteanddecreasethe areaofinflammationandcelldeath(YiuG, 2006).
Fig.1.3 Glialscarformationthroughalargestab lesionAstrocytesalignatthelesionsiteforminga barrierwhichinhibits growth. Later,astrocytes produceCSPGswhichalsoinhibitgrowth(taken fromSilverandMiller,2004)
Reactiveastrocytesintheglialscararerichinproteoglycans,particularlyCSPGs(including aggrecan,brevican,neurocan,versican,phosphacanandNG2)whichinhibitaxonalgrowth (McKeonRJ,1991).CSPGsecretionstartswithin24hafterinjuryandcanlastforseveral months(JonesLL,2003).CSPGshaveagrowthinhibitingeffect,althoughinsomecasesthey canalsohavegrowthpromotingeffect,andduringdevelopmentplayaroleinguidingaxonsto theirappropriatedestinations(SnowDM,1990).CSPGsaremoleculescharacterizedaslarge, highlysulphatedglycosaminoglycan(GAG)chainsattachedtoaproteincore.Theinhibitory effectofproteoglycansisduetoGAGandcanbeneutralizedwithchondrotinaseABC,an enzymethatremovesGAGchainsfromtheproteincore(SilverJ,2004).Itisthoughtthatthe inhibitoryeffectofCSPGsismediatedthroughasignallingpathwayinvolvingRhoA(Dergham P,2002).InadditiontoCSPGs,othermoleculesarepresentattheglialscarwhichmediate growthinhibitionandareupregulatedatthelesionsitesuchastenascines,Semaphorin3, ephrinͲB2andslitproteins(SilverJ,2004).
b. Myelinassociatedinhibitors
Inadditiontotheeffectsoftheglialscar,myelinassociatedinhibitorsplayamajorrolein hinderingaxonalregeneration.Infact,beforethescarmatures,themainhindrancefor regenerationcomesfrominhibitormoleculesassociatedwithmyelin(Filbin,2003).These includeNogo,MAG,OMgP(Fig.1.4),andothers(suchassemaphorin4DandephrinB3which willnotbediscussedinthisthesis).Around20yearsago,Schwabandhiscolleaguesshowed thatCNSmyelincontainsmoleculeswhichinhibitaxonalgrowthandregeneration(CaroniP, 1988b).AmonoclonalantibodyagainstmyelinproteinnamedINͲ1(latercalledNogo)was thangeneratedwhichwasabletoatleastpartiallyneutralizetheinhibitoryeffectsofCNS myelin(CaroniP,1988a;SchnellL,1990).NOGOisprobablythebestknownmyelinassociated inhibitor.Itexistsin3formsNogo–A,–Band–C,whichoriginfromasinglegeneandhavea commonCͲterminaldomainof188aminoacidswhich‘anchors’theNogo;apropertyshared bythereticulonproteinfamily.Itwasindependentlystudiedby3differentlabs(ChenMS, 2000;GrandPréT,2000;PrinjhaR,2000)usingdifferentcloningapproaches;whichalso yieldeddifferentresultsrelatedtoaxonalregeneration.NogoͲAisthelargest,andishighly expressedonthesurfaceofoligodendrocytes(ChenMS,2000;GrandPréT,2000),andonthe innermostloopofmyelin(HuberAB,2002),whereitcancontactaxons.NogoͲAcontains2
receptor(NgR)aGPIanchoredprotein,hasbeenclonedbasedontheidentificationofNogoͲ66 (FournierAE,2001).NgRformsacomplexwithp75(WangKC,2002b),p75beingoneofthe coͲreceptorsforNogo,MAGandOMgp(WangKC,2002a).
MyelinͲassociatedglycoprotein(MAG)isanothermyelinassociatedneuritegrowthinhibitor (MukhopadhyayG,1994;McKerracherL,1994).Itisamemberoftheimmunoglobulin superfamily,containingfiveimmunoglobulinͲlikedomainsinitsextracellularregion.Itis expressedonbothCNSoligodendrocytesandPNSSchwanncellsandisthoughttobeinvolved intheformationandmaintenanceofmyelin.Inculture,MAGinducesaxonal(butnot dendritic)growthconecollapse(ShibataA,1998).Itsinhibitoryeffectisrestrictedtoadult neurons,whileinyoungneuronsithasagrowthͲpromotingeffect.Thistransitionoccurswith neuronalmaturationandisthoughttobecAMPdependant(CaiD,2002).
Another myelin associated inhibitor identified more recently is oligodendrocytes myelin glycoprotein OMgp(WangKC,2002b).OMgpisaGPI (glycosylphosphatidylinositol) –linked protein,andislocalizedatthesurfaceof myelin(MikolDD,1990).Itisnotonly highly expressed in oligodendrocytes, butalsoinseveralneurons(HabibAA, 1998)andinthePNS(MikolDD,1990).
Fig.1.4 MyelinAssociatedInhibitorsinhibit axonal regeneration through various receptors.(modified fromHannila et. al., 2007)
c. Signallingmechanisms
Varioussignallingmechanismscontributetoeitherthepromotionortheinhibitionofaxonal regeneration. Pharmacologicaltreatments targeting thesemechanismsshould thushelp improvegrowth.Thischapterattemptstogiveageneraloverviewofsuchmechanisms.In Chapter1.3.4aͲd,therelevantcompoundsaffectingthesemechanismswillbediscussedin furtherdetail.
Promotersignals
AwellknownpromoteriscAMP(seealsoFig.1.5).NeurotrophinsactivatecAMP(CaiD,1999) (byactivatingTrkandErkreceptors,whichinturnproducesatransientinhibitionofPDE4 activity).Inturn,cAMPactivatesPKA,whichthanactivatesCREB.Thiswillallowneuronsto overcomeinhibitorsofmyelinandinduceaxonalgrowth(HannilaSS,2008).Anincreasein cAMPalsoincreasesthelevelsofCa2+
within the neuron, which activates calcium – calmodulin – dependant protein kinase II (CaMKII) and also promotes axonal growth (Wen Z, 2004).Filbinandhercolleagueshave doneseveralstudieswhichshowthat on increasing cAMP, axonal regeneration is promoted. Of particularinterestispharmacological activity of the cAMP promoter Rolipram,whichcanbeapromising drugtoinduceaxonalregeneration.
Fig.1.5cAMPpathwayanditsagonistspromoteaxonalgrowth
Itiswellknownthatphosphoinositide3Ͳkinase(PI3ͲK)signallingpathwayaffectsgrowth, survival,andmovementofcells.Moreover,thispathwayplaysanimportantroleinaxonaland dendriticmorphogenesisduringnervoussystemdevelopment,particularlyaxonalelongation and guidance (RodgersEE, 2002).In addition, thePI3ͲK signallingisessential forthe maintenanceoftheneuronalconnectivitywithintheadultbrain(KwonCH,2006).Thereareat
totheaxonalgrowthcone;thisinhibitsdownstreamRhoactivation(discussedabove),thus promotingaxonalgrowth.PI3ͲKsignallingalsopromotesaxonalelongationbyactivatingAkt.
Inturn,AktinhibitsGSKͲ3ɴ,leadingtoincreasedmicrotubulestability.LY294002(LY)isa specificinhibitorofPI3ͲKactivity(VlahosCJ,1994).Whileitwasshownthatacutetreatmentof sensorygrowthconeswithLY294002hadacollapsingeffectongrowthcones,thesegrowth conesrecoveredrapidlyandresumedoutgrowthinthecontinuedpresenceofLY294002.
ChronicLY294002treatmentasappliedinourculturesmightdesensitizethePI3K/Aktpathway andmakeneuritesunresponsivetosignalsnormallyinducinginhibitionofaxonalgrowth throughinhibitionofthePI3K/Aktpathway.
InhibitorySignals
TheProteinKinaseC(PKC)pathway(Fig.1.6)isknowntoregulateaxonalgrowthbyactingon cytoskeletonregulatorslikeGAPͲ43(LauxT,2000).ProteinKinaseC(PKC)belongstothe familyofkinases,whichhavekeyrolesinregulatingmultiplecellularactivities.Activationof PKCmightbeinvolvedindifferentiationandinitiationofneuriteoutgrowthinPC12cells (KorshunovaI,2007);itisalsoinvolvedinthetransmissionofinhibitorysignalsleadingto growthconecollapse(ConradS,2007).MyelininhibitorsandCSPGsareshowntoinducePKC activation.Inturn,PKCactivatesRho(discussedlater)whichisinvolvedinsignaltransduction mechanismsinhibitingaxonalgrowth(SivasankaranR,2004).Blockingofthispathwayshould leadtoaxonalgrowth.Pranget.al.haveshownforthefirsttimethatpharmacological inhibitionofPKCactivitydoesindeedpromoteregenerativeaxonalgrowth.Sivasankaranet.al.
havetakenthisastepfurtherbyshowingthatdeliveryofthePKCinhibitorGö6967intothe lesionsitepromotesregenerationofdorsalcolumnaxonsinthespinalcordinͲvivo.Thismay alsoprovetobeapromisingtreatmentinthefuture.
Anotherwellknownintracellularsignallingmoleculewhichinhibitsaxonalregenerationis RhoA(Fig.1.6).TheRhofamilyofsmallGTPasesisknowntotransduceextracellularsignalsto theactincytoskeletontomodulategrowthconemotility.Whilesomeofthesemembers inducegrowth,RhoAinhibitsgrowthbyinducinggrowthconerepulsionandcollapse.Various groupshavedemonstratedthatRhoAisactivatedbymyelinassociatedinhibitors(YamashitaT, 2002,FournierAE,2003).MyelininhibitorsactivateRhoAbyrecruitingRhoͲGDI,which interactswithp75(NTR)toallowthereleaseofRhoͲGDPfromitsboundstateleadingtothe
releaseofRho(YamashitaT,2003).OneofthedownstreameffectorsofRhoisROCK(Rho kinase).TheactivationofROCKinducesgrowthconecollapsethroughitsmultipledownstream effectors.InanimalmodelsofSCI,deliveryoftheROCKantagonist,YͲ27632wasshownto promoteregenerationandfunctionalrecovery.
Fig.1.6 Signalsinhibitingaxonalgrowthandtheirantagonistdrugs
Pertussistoxin(PTX)inhibitssignaltransductionthroughthePI3K/Aktpathway(Fig.1.6).It actsbybindingtoGiͲandGoͲproteinstoinactivatethem(PostGR,1996).GͲproteinsareknown toinhibitcAMPandpromotePKCproduction;thusbyinhibitingGͲproteins,thereisan increasedcAMPlevelandimprovedaxonalregeneration.ItwaspreviouslyshownthatPTX allowsneurotrophinͲtreatedneuronstoovercomeinhibitionbymyelin(CaiD,1999;Igarashi M,1993).Inaddition,Pertussisinducedextensivegrowthinlesionedorganotypicslicecultures (PrangP,2001).Followingtheseresults,althoughinͲvivostudiesarecurrentlynotavailable, PTXandotherGͲproteininhibitorsshouldbefurtherinvestigatedasapotentialtreatmentof CNSinjury.
InadditiontoProteinkinaseC,theIP3signallingpathway(Fig.1.6)isalsoactivatedbymyelin inhibitorsthroughactivationofphospholipaseC(HasegawaY,2004)whichactsonbothPKC andIP3(seeFig1.6).PhospholipaseCactivationleadstothehydrolysisofPIP2toproduce diacylglycerol(DAG)andIP3(Berridge,1998).IP3thenbindstotheIP3receptortoinduce releaseofCa2+fromtheintracellularstore.DAGtogetherwithanelevationofCa2+directly activatesPKCtoinhibitgrowth.ActivationoftheIP3receptorthusalsomightbebeneficialfor inducingaxonalregenerationinaninhibitoryenvironment,althoughontheotherhandIP3 receptoractivationhasalsobeenimplicatedinthestimulationofneuriteoutgrowth(reviewed inHasegawaY,2004).AlthoughthegrowthpromotingeffectsofXestosponginhavenotyet beenstudied,itmightpromoteaxonalregenerationinaninhibitoryenvironment.
1.2.2 Intrinsicgrowthpotentialoflesionedaxons
Intrinsic factors are thought to influence axonal growth capability. The growth cone cytoskeleton modulators GAP43 and CAP23 (Skene JH, 1981; Aigner L, 1995), are downregulatedintheCNSduringdevelopment(LauxT,2000).Theseproteinscaninduceaxon elongationinadultDRGneuronsinvitro,andtriggeranincreaseinregenerationofDRGaxons inadultmiceafterspinalcordinjuryinvivo(BomzeHM,2001).ThedownregulationofGAP43 isinverselyrelatedtomyelination.Inadultratsmanipulatedtolackmyelinationinthespinal cord,GAP43expressionisstronglyincreased(KapfhammerJP,1994a;KapfhammerJP,1994b).
AnotherpotentialintrinsicstimulatorofaxonalgrowthistheantiͲapoptoticgene,BclͲ2.Itwas shown that BclͲ2 promotes regeneration of retinal axons in vivo (Chen DF, 1997).
Unfortunately,theseresultscouldnotbereproduced,andotherstudiesshowthatBclͲ2 overexpressionincreasescellsurvivalbutthereisnoregenerationbothinvivo(LodovichiC, 2001;InoueT,2002),andinvitro(SoléM,2004).
cAMPisalsoessentialinregulatingaxonalgrowth.ThereishighexpressionofcAMPlevels duringdevelopment.AxonalgrowthispromotedbyinhibitingPKA,adownstreameffectorof cAMP;thuspreventinginhibitionbyMAG.IntheadultCNS,cAMPlevelsarelowandare inhibitedby MAG(CaiD,2002).After experimental elevation of cAMPlevels (e.g.by conditioninglesionNeumannS,1999),thereisasignificantincreaseinregenerationinadult CNSafterlesion(SpencerT,2004).
1.3
Experimentalapproachestoimproveaxonregeneration
Variousapproachestopromoteaxonalregenerationandfunctionalrecoveryhavebeen studiedinmanylabs.Someofthesestudieshaveevenbeenextendedtouseinpatientsin clinicaltrialsandmightonedaybeusedtotreatTBIandSCI.Inthissectionwewillgeta generaloverviewofthevarioustreatmentmethodsandmechanismsusedtopromoteaxonal growth.
1.3.1 TransplantationandCellReplacement a.TransplantationofSchwanncellsandOECs
Aswehavediscussedearlier,axonalregenerationcanoccurinthePNSbutnotinthe CNS.Inthe1980s,Aguayoandhiscolleaguesusedthisideatograftpermissive SchwanncellsinCNStissue(Fig.1.7)(DavidS,1981;Bray,1987).Thisresultedinaxons growingwellintothegraftedPNS,butveryfewmanagedtoleavethegraftbacktothe CNS.Thustherewaslimitedfunctionalrecovery.
Fig.1.7 Grafting of PNS tissue in CNS done in the 1980s by Aguayo and colleagues(adaptedfromBray,1987)
Anewerapproachisthetransplantationofolfactoryensheathingcells(OECs).OECsare producedfromstemcellsintheolfactorymucosaandcanbeculturedfromtheadultolfactory bulbormucosa(RaismanG,2007).UnlikeSchwanncells,OECsaresupposedto‘crossback’
fromthegraftedtissuetotheCNS(LiY,1997)andmigrateoverlongdistanceswithinthehost tissue(RamónͲCuetoA,1998).Moreover,OECgraftspromotemyleination,growthfactor secretionandenhancementofsprouting,leadingtofunctionalrecoveryevenincompletely transactedSC(RamónͲCuetoetal.,2000).TheseoptimalresultsusingOECtransplantsledto theideathattheycouldprovidethebasisofanewtherapyinhumans.Aclinicalstudyinspinal
cordinjuredpatientswasrecentlydoneinChina.Unfortunately,therewerevariousmedical complicationsandnoclinicallysignificantfunctionalrecoverywasobserved(DobkinBH,2006).
b. Transplantationofyoungcells
Transplantationofyoungpermissivetissuecanalsopromoteaxonalgrowth.InastudybyLiet.
al.,‘old’hippocampalcultureswerecoͲculturedwith‘young’ECfromearlypostnatalrats.In theseculturestherewasarobustaxonalprojectionfromtheECtothehippocampus(LiD, 1995).ThesamegroupalsotransplantedembryonicECinadulthippocampusinͲvivo,and showedthattheamountofprojectionmadebythistissuedependsonthetimingofthe transplant(ZhouCF,1989).Inanotherstudy,humanembryonicneuraltissuetransplantedin ratsyieldedlongaxonalprojectionsalongthenigrostriatalandthecorticospinaltract(Wictorin K,1990).Inanadultratmodeloftemporallobeepilepsy,CA3wasgraftedfromyoungratsasa waytorestrainmossyfibersprouting.Intheserats,astrongaxonalprojectionformed, reducingtheamountofsproutingandseizures(ShettyAK,2005).
c. TransplantationofStemCells
Stemcelltransplantationisanotheralternativetohelpimproveaxonalrepair.Stemcellscan comefrombothfetalandadulttissue;butusingautologousadultprogenitorcellshasthe advantagesthatthecelltransplantsarenotrejectedandalsoreducesvariousethicalconcerns.
Adultstemcellscanbeobtainedfromvarioustissues,includingbrain,spinalcord,olfactory system,bonemarrowandblood.Moreover,nowadaysstemcellscanbeobtainedfromthe umbilicalcordandstoreduntilneedforuse.Varioussuccessfulstemcellstudieshavebeen performedinrodents(Schultz,2005).TransplantationofHSC(hematopoieticstemcells)and BMSC(bonemarrowstromalcells)fromadultbonemarrow(KodaM,2005);andNPC(neural progenitorcells)frombrain(KarimiͲAbdolrezaeeS,2006),promotedvariousdegreesof functionalrecoveryinmice.Someyearsago,BMSCwerealsotransplantedinSCIpatientsina smallclinicaltrial(Reier,2004).Althoughimprovementswereobserved,insuchatrialitwas difficulttohavecontrolsandthusassessproperlytheoutcomeofthetherapy.
1.3.2 Inhibitionoftheglialscar
Asdiscussedearlier,CSPGsarethemaininhibitorsofaxonalregenerationintheglialscar(Fig.
1.3).TheinhibitoryeffectofproteoglycansisduetoGAG(glycosaminoglycan)chains(SilverJ, 2004).SeveralstudieshaveshownthattheseGAGchainscanbedegradedbychondrotinase ABC,thusenhancingregeneration.Inratspinalcord,thistreatmentwasshowntoimprove regenerationofthenigrostriatalpathway(MoonLD,2001),andthedorsalcolumnsandthe CST,withimprovedfunctionalrecoveryinvivo(BradburyEJ,2002).Inadifferentstudy,this treatmentwascombinedwithSchwanncellgraftsinaspinalcordinjurymodel,andwas showntoimproveregeneration(ChauCH,2004).Itwasalsoshowninvitrothatanotherway ofovercominginhibitionbyCSPGsisbyinactivatingRho(DerghamP,2002).
Otherstudiestargetedotherpotentialgrowthinhibitorsintheglialscar,includingGFAPand EphA4. SpinalcordinjuredGFAP(MenetV,2003)andEphA4(GoldshmitY,2004)knockout miceshowimprovedregenerationandfunctionalrecovery.
1.3.3 Neutralisationofinhibitorymoleculesinmyelin
Inafirstattempttoneutralisemyelinassociatedinhibitors,INͲ1monoclonalantibodywas produced(CaroniP,1988a).INͲ1Abwasfoundtopromotelongdistanceaxonalregeneration andfunctionalrecovery(SchnellL,1990;BregmanBS,1995;ThallmairM,1998). Thiseffect wasobservedinvariousCNSlesionmodels.Moreover,itwasnotedtheINͲ1Ableadsto sproutingofbothlesionedandunlesionedaxons(SchweglerG,1995;ThallmairM,1998;
Z'GraggenWJ,1998).RecentlyanantiͲNogoͲAspecificantibodywasalsousedsuccessfullyto treatSCIinmonkey;whichyieldedpromisingresults,ie,axonalsproutingandfunctional recovery(FreundP,2006). ThisbringsusastepclosertothispromisingtherapyforCNS, particularlySCinjuredhumans.
TostudyNogofurther(Fig.1.8),independentstudiesfrom3differentlabsmadedifferent micedeficientinNogoͲA–onelackingNogoͲAand ͲB(KimJE,2004),onelackingNogoͲA (SimonenM,2003),whileinanotherstudybothknockoutmodelswerestudied(ZhengB, 2003). Although all these mice were phenotypically normal, myelin growth inhibitory propertiesduetoNogowereexpectedtobereduced.Surprisinglyconflictingresultswere
obtained.WhereastheStrittmattergroupreportedastrongimprovementinregenerationand functionalrecoveryinyoungmice(KimJE,2004);inthemodelfromtheSchwabgroupthe regenerationwasmoremodest,whichcouldpartlybeexplainedbytheupregulationofNogoͲ B(SimonenM,2003);ontheotherhandthemodelsfromtheTessierͲLavignegroupfoundno significantregenerationofcorticospinaltractfibers(ZhengB,2003).Itwasrecentlyshownthat thesevariationsinregenerationareinpartduetodifferencesinthegeneticbackgroundofthe usedmousestrains(DimouL,2006).
Fig.1.8 3 isoforms of NOGO. NOGOͲA is foundpredominantlyinCNSandcontains2 inhibitorydomains:a 66ͲaminoͲacid domain extracellularily (green), and aminoͲ NOGO intracellularily (taken from Goldberg and Barres,2000)
Inanattempttoaddressproblemsrelatedtoantibodydelivery,vaccinationagainstmyelin inhibitorswasperformedandrobustregenerationandfunctionalrecoverywereobserved afterlesioninthevaccinatedanimals(HuangDW,1999).LaterexperimentstargetingNogoͲA werealsoperformedsuccessfully,and,moreimportantly,regenerationwasalsoobtained whenusingthevaccineafterinjury(HaubenE,2001).
AnotherwaytopromoteaxonalregenerationistotargettheNgR.SomeyearsagoNogoͲ66 wasidentified(FournierAE,2001)asthereceptortowhichNogo,MAGandOMgparebinding toinhibitaxonalgrowth(WangKC,2002a).ItwassubsequentlyshownthattheNogoͲ66 receptorantagonistpeptideNEP1Ͳ40(blocksNogoonly)(GrandPreT,2002;LiS,2004)and NgR(310)(blocksNogo,MAGandOMgp)promoteregenerationandfunctionalrecovery(LiS, 2004).Inasimilarstudyp75,whichisacoͲreceptorofNgRwastargeted,buttherewasno regenerationpresentinthiscase(SongXY,2004).
SimilartotheNogomolecules,alsothemyelinproteinMAGhasbeenimplicatedinthe inhibitionofaxonalregeneration.UsinganantibodyagainstMAGithasbeensuggestedthat inhibitionofMAGpromotesaxonaloutgrowthandregeneration(MukhopadhyayG,1994).To verifytheinhibitoryeffectofMAGonregeneration,atransgenicmouse(MAGͲ/Ͳ)mousewas used.Afterinjuryinvivo,noincreaseinregenerationwasfound(BartschU,1995).Inanin vitrostudyusingmyelinfromMAGͲdeficientmiceanincreaseinnumberandlengthof
regeneratedfibreswasfoundwhencomparedtowildtypemicebutthepresenceoffurther importantinhibitorsinCNSmyelinofMAGͲdeficientmicewasacknowledgedinthisstudy(Li M,1996).AlthoughinhibitionofMAGalonedoesnotappeartobesufficienttoinduce regeneration,itmightcontributesignificantlytotheinhibitoryactionofCNSmyelin.
1.3.4 Modificationofsignaltransductionpathways
Inthischapterwewilldiscussfurthertheuseofsomeofthecompoundsinvolvedinsignal transductionandtheireffectonaxonalregeneration.Themechanismofactionforthese treatmentsisfoundinChapter1.2.1c.Inthisprojectmanyofthesecompoundswillbeusedin anattempttoimproveaxonalregenerationinorganotypicslicecultures.
a. PKCinhibitors
PKCisactivatedbyinhibitorspresentinmyelinandtheglialscar,andinhibitsaxonalgrowth.
Moreover, PKC activates its downstream effector Rho, which in turn inhibits growth (SivasankaranR,2004)(formechanismandantagonistdrugactionrefertoFig.1.6). Thus blocking PKC activity pharmacologically or genetically, should help improve axonal regeneration.Variousexperimentshavebeenpreformed,mostlyshowingthatblockingPKC inducesaxonalgrowth.ItwasshownthatmyelinassociatedinhibitorsandCSPGscouldinhibit neuriteextensionbyinhibitingPKC(SivasankaranR,2004),(HasegawaY,2004).Intheearly 1990s,thePKCinhibitorsGö6076(MartinyͲBaronG,1993)andGF109203X(ToullecD,1991), (MartinyͲBaronG,1993)wereshowntobeselectiveinhibitorsofPKC.WhileGö6076acts specificallyontheCa2+ͲdependentclassicalisoformsofPKC,GF109203Xwasshowntoinhibit mostisoformsofPKC(MartinyͲBaronG,1993).GF109203Xwasshowntostimulateaxonal growthinhippocampalorganotypicslicecultures(PrangP,2001).Inanotherattempttostudy otherPKCinhibitors(chelerythrineandstaurosporine)inorganotypicsliceculturesitwas foundthatregenerationofSchaffercollateralsisinhibitedbystaurosporine(byamechanism thoughtobeindependentfromPKC),butnotbythemorespecificPKCinhibitorchelerythrine (ToniN,1997).
Ontheotherhand,aninͲvivostudyinratsshowsthatintrathecaldeliveryofthePKCinhibitor Gö6976stimulatesdorsalcolumnaxonalregeneration(SivasankaranR,2004). Although furtherinvestigationsarerequiredtoshowhowPKCactivatesRho,PKCinhibitorshaveshown toplayanimportantroleinthetreatmentofaxonregenerationfailure. Inthefuture, combinationtherapiesusingdrugsactingonthesepathwaysmayshowtohavesynergistic effects.
b. Rho&ROCKinhibitors
RhoA, together with its downstream effector ROCK, are also known to inhibit axonal regeneration by inducing growth cone collapse (Yamashita T, 2003). Pharmacological strategies to block Rho could thus promote axonal regeneration (for mechanism and antagonistdrugactionrefertoFig.1.6).TheenzymeC3transferasecaninactivateRho,and treatmentwiththisenzymecanstimulateneuritegrowthbothinͲvitroandinͲvivo.Instudies withspinalcordlesionedanimals,deliveryofC3transferasepromotedCSTregenerationand improvedfunctionalrecoveryinmouse(DerghamP,2002),butnotinrat(FournierAE,2003).
InanotherstudyC3transferasetreatmentpromotedregenerationinadultratopticnerve afterinjury(LehmannM,1999).ThemainproblemofC3transferasetreatmentisthatitisnot membranepermeable,thusmakingitdifficulttodelivertothelesionsite.Anothercell permeableRhoinhibitor,Cethriniscurrentlybeinginvestigatedinaclinicaltrialonspinalcord injuredpatients(BaptisteDC,2006).
AnotherwayofinhibitingtheeffectsofRhoistotargetitsdownstreameffector,ROCK.Y– 27632,anATPcompetitiveantagonistthatblocksRhoactivationwasshowntopromote axonalregenerationandfunctionalrecoveryinspinalcordinjuredmice(DerghamP,2002)and rats(FournierAE,2003).
c. cAMPactivators
VariousstudieshavepreviouslyshownthatanincreaseincAMPlevelsimprovesurvivalofCNS neuronsinresponsetoneurotrophicfactors(GoldbergJL,2000).Asdiscussedpreviously,on elevatingcAMPlevels,thereisasignificantincreaseintheregenerativepotentialofaxonsin adultCNS(SpencerT,2004)(formechanismandantagonistdrugactionrefertoFig.1.5).cAMP regulatesseveralprocessesincludingaxonalresponsetoguidancecues,neurotrophicfactors andmyelinassociatedinhibitors(SongH,2001).Neurotrophintreatment(BDNFandGDNF), can overcomethe inhibitory effectofMAGand myelin(Song HJ, 1997; CaiD,1999).
Moreover,itisthoughtthatneurotrophinsareabletoelevateneuronalcAMPlevelsby inhibitingphosphodiesterase4(PDE4),theenzymewhichdegradescAMP(GaoY,2003;QiuJ, 2002;NeumannS,2002).cAMPinturnleadstoPKAactivationwhichconsecutivelyleadsto activationofCREB,whichinturnactivatesArginaseIexpression,anenzymerequiredfor polyaminesynthesis(CaiD,2002).BothPKAelevationandpolyaminesarethoughttoblockthe inhibitoryeffectofmyelin(Filbin,2003).
TherearesomepharmacologicaltreatmentswhichelevatecAMPlevels,whichincludethe cAMP analogues, spͲcAMP and dbͲcAMP; neurotrophins (BDNF, GDNF); Forskolin and Rolipram.ThePDE4inhibitor,Rolipramisoneofthetreatmentsusedinthisproject.Itreadily crossesthebloodbrainbarrieranditsdosehasanestablished‘optimal’therapeuticwindow.
Rolipramwasshowntopromoteregenerationandimprovefunctionalrecoveryonlesioned adultratSC(NikulinaE,2004).SofarRolipramwastestedinpatientsasanantidepressantina clinicaltrial,whichwasstoppedduetothedrug’ssideeffects(nausea)(WachtelH,1986).
Sincefortreatingspinalcordinjury,Rolipramneedsonlytobedeliveredforashorterperiodof time,thebenefitsmayoutweighthesideeffectsandthusitmightstillbeapromisingdrugfor thetreatmentofCNSinjury.
1.3.5 Summary
Table1.1isasummaryofthevariousapproachesusedtoimproveaxonalregenerationand functionalrecoverydiscussedabove.
Target Treatment Results&References TransplantandCellReplacement
SC
GraftsinCNS Robustregenerationinto,butnotoutofthe graftDavid,1981;Bray,1987
OEC GraftsinCNS RobustregenerationintoandoutofthegrafttothehosttissueLi,1997;
RamonͲCueto,1998;RamonͲCueto,2000
‘Young’
cells
1.YoungEC
2.YoungCA3 3.Neuroblasts
1.RobustaxonalprojectionfromtheECtothehippocampusinͲvitro
Li,1995
andinͲvivoZhou,1989
2.StrongaxonalprojectionpreventingsproutinginͲvivoShetty,2005
3.RobustaxonalprojectionsalongnigroͲstratalandcorticospinaltract
Wictorin,1990
Stem
cells
Variouse.g.HSC, BMSC,NPC
Various degrees of functional recovery Schultz,2005; Koda,2005; KarimiͲ Abdolrezaee,2006
GlialScar
CSPGs Intrathecal deliveryof chondrotinase ABC
ImprovedregenerationofthenigrostriatalpathwayMoon,2001,dorsal columnandCST,withimprovedfunctionalrecoveryBradbury,2002
inrat SCI
GFAP&
EphA4
Geneticdeletion Improved regeneration and functional recovery after SCI in GFAPMenet,2003andEphA4Goldshmit,2004
knockoutmice MyleinͲAssociatedInhibitors
MAG Geneticdeletion NoimprovementinregenerationinMAGͲ/ͲmiceBratsch,1995
NOGO 1.Genetic
manipulation
1. Results vary from regeneration & functional recoveryKim,2004;
Simonen,2003tonoregenerationZheng,2003
2.Antibody 2. Regeneration with both INͲ1 Ab in miceThallmir,1998; Bregman,1995;
Z’Graggen,1998
andantiͲnogoͲAspecificAbinmonkeysFreund,2006
3.Vaccination 3.RobustregenerationinexperimentstargetingmyelinHuang,1999and NogoͲAHauben,2001
NgR Intrathecal delivery
Robust regeneration and functional recovery with bothNEP1Ͳ 40GrandPre,2002
&NgR(310)Li,2004 p75 Geneticdeletion NoregenerationSong,2004 ModificationofSignalTransductionPathways
PKC inhibitor
1.GF109203X 2.Gö6976
1.Strongregenerationinhippocampalorganotypicslicecultureswith GFXPrang,2001
2.DorsalcolumnregenerationbutnoCSTregenerationwithGöͲ treatedSCIratsSivasankaran,2004
RhoA&
ROCK
1.C3transferase 2.YͲ27632
1. CST regeneration and improved functionalrecoveryin SCI in mouseDergham,2002
butnotinratFournier,2003
2. CST regeneration and improved functional recovers in SCI in mouseDergham,2002
andratFournier,2003
cAMP activator
cAMP,Rolipram
&Foskolin
AxonalregenerationandfunctionalrecoverywithRolipramNikulina,2004
GͲprot.
inhibitor
Pertussistoxin RegenerationinhippocampalorganotypicsliceculturesPrang,2001;
Igarashi,1993
Table1.1. Summaryofaxonalapproachestoimproveaxonalregeneration
1.4 Hippocampus
Thehippocampusislocatedinthetemporallobeofthebrainandisimportantforthe formationoflearningandmemory.
1.4.1 Anatomicalstructure
TheentorhinalͲhippocampalformation(heresometimesreferredtoashippocampus)hasa laminarorganisationandconsistsoftheentorhinalcortex(EC),dentategyrus(DG)and hippocampusproper(i.e.theCA1–CA3,cornusammonisregions).TheECisamajorrelay providingawiderangeofcorticalinputstothehippocampusandtransferringtheinformation processedinthehippocampusbacktothecortex.TheECisdividedinclearlydistinguished layers(layersItolayerVI).Thecellsthatgiverisetotheperforantpathwayaremainlypresent inlayersIIandIII.IntheDGthereiscontinuousneurogenesisgoingonevenintheadult;it alsoplaysaroleintheformationofnewmemories.TheDGisalsosubdividedinlayers,mainly thestratummoleculareandthestratumgranulare.TheaxonsofDGgranulecells,calledmossy fibres,extendtheiraxonstotheCA3pyramidalcells.Thesubiculumislocatedinthetransition areabetweentheentorhinalcortexandthehippocampus;andistheregionwhereaxonsare exitingthehippocampustotheentorhinalcortex.
ThehippocampusproperissubdividedintoCA1–CA3regionsandhasalaminarorganisation whichispreservedinthevariousregions.Theprincipalcellsarepyramidalcells(formingthe pyramidalcelllayer),whichareinterconnectedbyinterneurons.Theinnermostlayeristhe stratumorienswhichcontainsthebasaldendritesofthepyramidalneurons,whosecellbodies arefoundinthestratumpyramidale.NextintheCA1isthestratumradiatumandamore superficialstratumlacunosumͲmoleculare.Themossyfiberprojectionfromthedentategyrus whichcanbefoundinthestratumradiatum,andsynapseinthestratumlucidumendinthe CA3(AmaralDG,1989).
1.4.2 ‘Thehippocampalcircuits’
ThehippocampusformsauniͲdirectionalnetwork,andtraditionallyitisdescribedasa
`trisynapticcircuit`(AndersonP,1971;Fig.1.9).Basically,inputstartingfromtheECformsa projectiontotheDG(“perforantpath”,synapse1),whichisfollowedbyaprojectiontothe CA3pyramidalcells(“mossyfiberprojection”,synapse2),withthethirdprojectionfromthe CA3totheCA1pyramidalcells(“Schaffercollaterals”,synapse3).
Fig.1.9 Thehippocampaltrisynapticcircuit:inputfromtheECconnectswith DGinthePP,DGtoCA3asthemossyfiberprojectionandCA3toCA1asthe Shaffercollaterals(modifiedfromNeveset.al.,2008)
Amaincharacteristicofthehippocampusisitslaminatedorganization,inwhichdifferent afferentsinnervatedifferentlayersinahighlyorganizedfashion.Theperforantpathway originatesfromlayersIIandIIIoftheentorhinalcortex(Amaral,1993);withpyramidaland stellatecells(fromlayerII)projectingaxonsmainlytotheDG,whilesmallpyramidalneurons (fromlayerIII)projecttheiraxonstotheCA1ofthehippocampus.Theperforantpathfibers terminateintheoutermolecularlayerofthehippocampus,whiletheinnermolecularlayeris occupiedbyterminalsfromcommissuralaxons.ThemossyfiberpathwayextendsfromtheDG totheCA3pyramidalcells.MultiplegranulecellscansynapseontoasingleCA3pyramidalcell.
TheSchaffercollateralpathwayisderivedfromaxonsthatprojectfromtheCA3totheCA1 region.IpsilateralaxonscomefromCA3neuronsinthesamehippocampus,whilecontralateral axons(betterknownascommissuralfibres)comefromtheCA3oftheoppositehemisphere.
AxonsleavethehippocampusfromtheCA1throughthesubiculumandbacktotheentorhinal cortex.
1.5 Spinalcord
Thespinalcordprovidesa‘relay’forneuronsfrombraintotheperipheryandback.
Thespinalcordislocatedinsidethevertebralcanal,andrunsfromtheforamenmagnumto thefirstandsecondlumbarvertebrae.Itiscoveredbythespinalmeninges,whichfurther supportsandprotectstheSCwithin.TheSCismadeof31segments:8cervical(C1–C8),12 thoracic(T1–T12),5lumbar(L1–L5),5sacral(S1–S5),and1coccygeal.Inthecervicalpart:
C1participatesintheinnervationofneckmuscles,C2carriessensationfromthebackofthe headandscalpandmotorinnervationtomusclesintheneck,C3ͲC5innervatethediaphragm, andC5ͲT1providemotorcontrolfortheupperextremitiesandrelatedmuscles.Thespinal nervesenterandexitateachsegment.Sensorynerveswhichcontaintheircellbodiesinthe dorsalrootgangliacarryinformationtotheSCviathedorsalroots.Eachdorsalrootcontains theinputfromallthestructureswithinthedistributionofitscorrespondingbodysegment.
Dermatomalmapsportraysensorydistributionsforeachlevel.Thesemapsdiffersomewhat accordingtothemethodsusedintheirconstruction.Motorinformationiscarriedawayfrom theSCviatheventralroots.Thecellbodiesoriginatefromthegreymatteroftheventralspinal cord.
InacrossͲsectionofthespinalcordtheareaisdividedinthesoͲcalledwhitematterandgrey matter(Fig1.10).Thegreymatterandiscalledsobecauseitcontainscellbodies(thusmaking itlookdarker).Itcanbefurthersubdividedinto10layersofRexed(RexedI–X);layersI–VI constitutethedorsalhorn(sensorypart),layerVIIistheintermediatezone,VIIIandIX constitutetheventralhorn,andlayerXsurroundsthecentralcanal.Thewhitematterisonthe externalpartoftheSCandissubdividedinto3columns:thedorsalcolumnthatrelay somatosensoryinformationtothemedullaandinthemousecontainingthecorticospinaltract;
thelateralcolumncontainingaxonsfromsensory,motorandautonomiccontrol;theventral columncontainingmostlyaxonsdescendingfromthebrain.Thewhitemattercontainsthe descendingandascendingtracts.
Fig.1.10Transversesectionofthespinal cord showing grey and white matter (modifiedfromThuret,2006)
AscendingandDescendingtracts
Fig.1.11Spinalcord:Neuronsinvolvedinascendinganddescendingpathways (takenfromThuretet.al.,2006)
1.5.1 MajorDescendingtracts
Themaindescendingtractisthecorticospinaltract(CST),whichmainlyoriginatesinthe primarymotorcortex.CSTaxonscollecttoformalumpinthemedulla(calledpyramid).Inthe medullamostaxonscross(decussate)andcollectinthelateralcolumnofthespinalcord(in thehuman)andinthedorsalcolumns(intherodent).AxonsfromthelateralCSTsynapsewith alphamotorneuronsandinterneuronsmainlyfromRexedlaminaeIV,V,VI,andVII.TheCST decreasesinsizeatthemorecaudalspinalcordlevels,wherethefibersreachthedorsal surfaceofthespinalcord.CSTisrequiredforthecontrolofmovements.
1.5.2 Ascendingtracts
a. Dorsalcolumn
Thedorsalcolumnconsistsofsensoryaxonsascendingtothebrain.Thedorsalcolumnfibers haveafastervelocityandaccuracythanthoseofthespinothalamictractsandareresponsible forproprioceptionandsensationoflighttouch.Thedorsalcolumnfibersoriginatefromcell bodieswhichareagglomeratedinthedorsalrootganglionandhaveaperipheralbranch innervatingreceptorslocatedintheskinandacentralbranchenteringthespinalcord.The dorsalcolumnscanbefurthersubdivided:thefasciculusgraciliscontainsfibersfromsacral, lumbar,andthelowersixthoracicsegmentsandisresponsibleforlegsensation;whilethe fasciculuscuneatuscontainsfibersfromthecervicaltoT6oftheSCandisresponsibleforarm sensation.Theaxonsfromthedorsalcolumnterminateinthedorsalcolumnnucleiinthe medullaoblongata.Fromthemedulla,neuronsformanascendingbundlewhichsynapsesin thethalamus,andfromtheretheprojectioncontinuestoendintheprimarysomatosensory cortexlocatedinthepostcentralgyrus.
b. Spinothalamictract
Theconductionvelocityinthespinothalamictractisslowerandtheprojectionlessaccurate comparedtothedorsalcolumns,thistracttransmitscrudetypesofsensationstothebrain.
Thespinothalamictractconsistsoftwoparts:thelateralpartconcernedwithpainand temperaturesensationsandtheventralpartconcernedwithpressureandstrongtouch.The lateralspinothalamictractcarriesinformationfrompainandtemperaturereceptorsinthe skin,theventralspinothalamictractfromreceptorsofpressureandstrongtouch.Thesensory axonsenterthespinalcordthroughthedorsalrootandendinthedorsalhornofthegray matter.Eachaxonbifurcatesintoascendinganddescendingbranches,whichextendfor1–2 segmentsandthenenterthegreymatterofthedorsalhorntoterminateinRexedlaminaeI–
VI.TheaxonsfromthesecellscrossthemidlineandascendonthecontrolateralsideoftheSC toprojectviatheventralposteriornucleusofthethalamus,tothesomatosensorycerebral
c. Spinocerebellartract
Thespinocerebellartractprovidesinformationaboutthepositionofthebodyrelatedtoits surroundings;i.e.proprioception.Itcarriesinformationfromperipheralsensoryreceptors(in musclesandjoints)whichsendfastconductingmyelinatedaxonstotheClarke’scolumn,acell columninthelowerthoracicspinalcordgreymatter.Fromtherefiberscrossthemidlineand runtothecerebellumasthedorsalspinocerebellartract.Additionalspinocerebellartractsare the ventral spinocerebellar tract, the rostral spinocerebellar tract and the spinocuneocerebellartract.
1.5.3 PropriospinalconnectionsintheSC
PropriospinalneuronsoriginateandterminateintheSC andparticipate ininformation exchangeamongthespinalsegments.Propriospinalneuronsarepresentinconsiderable numbersintheSC,andaremostlymyelinated,thushavingslightlylargeraxonsthanother neuronspresentintheSC(ChungK,1983).TheirconnectionsascendanddescendtheSCand projecttovarioussitesincludinginterneuronsandmotorneurons.Theseneuronsarerequired forseveralbehaviouralandphysiologicalresponses,includingthecontrolofmovement.The axonlengthofpropriospinalneuronscanvary:someareshortascendanddescendonlyoneor two segments andsome long extending over several spinal cord segments. The long propriospinalneuronshaveaxonsthatascendanddescendintheanteriorfasciculusproprius toalllevelsoftheSC.Theseneuronshaveabilateralinfluenceonthemoremedialmotor neuronsandcoordinatemovementoftheneckandpelvis.Theaxonsoftheintermediate propriospinalneuronsextendforshorterdistancesintheventralpartofthelateralfasciculus propriusandinfluencethemotorneuronsthatinnervatethemoreproximalmusclesofthe limbs.Axonsfromthe shortpropriospinal neuronsareonlyfoundin thecervical and lumbosacralsegmentsandtravelinthelateralfasciculusproprius.Theseneuronsinfluence motorneuronsthatinnervatethemoredistalmusclesofthelimbs.
1.6 Organotypicslicecultures
Slicecultureshavebeenextensivelyusedinneurobiologyresearchparticularlyforstudies involvinggrowthandregeneration,butalsoforelectrophysiologystudies,synapseformation, and studies on several receptors.Variousmodels are available, including hippocampal, cerebellarandspinalcordmodels.
Therearedifferentwaysofpreparingorganotypicslicecultures(asexplainedbelowandinFig.
1.12).Thechoiceoftechniquedependsonthefinalthicknessoftheslicesandthesurvivaltime inculture(asreviewedbyGahwileretal.,1997).BasicallyCNStissueisdissectedandsliced into100–400ʅmsections.Theslicesarewashedtoremoveanydebris,andthenincubatedin anappropriategrowthmediumat37°Cwithsufficientoxygenation.Sterileconditionsneedto bekeptthroughouttheprocess.
Fig.1.12 Differenttechniquesusedforthepreparationoforganotypicslicecultures A.Rollertubemethod,B.MembranemethodandC.CultureDishMethod
(takenfromGähwiler,1987)
Inrollertubecultures,thetissueisembeddedinafibrinclotonacoverslipandthenplacedin aflatͲsidedplasticculturetubecontainingasmallamountofmedium.Thetubeisrotated slowly(10revs/h)toensurecontinuousalterationoffeeingandaeration.Theseculturescan surviveforseveralweeksandaresupposedtobecomethinnerthanthecorrespondingstatic membranecultures(Fig.1.12A;Gähwiler,1987).Inmembranecultures,slicesareplacedona
semiͲporousmembrane(millicellorTranswell),withmediumaddedatthebottomofthe membrane,whileoxygenisaccessedfromthesurfaceoftheculture,i.e.,theculturesare stationaryatanairͲmediuminterface(StoppiniL,1991).Theseculturescanalsosurvivefor severalweeks.Thiswastheculturemethodofchoiceforthisproject(Fig.1.12B;Gähwiler, 1987).Slicesculturedinpetridishesareplacedeitherdirectlyoncollagencoatedorembedded incollagengelsandaretotallycoveredbymedium.Theseculturesonlysurviveafewdaysand aremainlyusedforelectrophysiology(Fig.1.12C;Gähwiler,1987).
1.6.1 EntorhinoͲhippocampalorganotypicslicecultures
Hippocampalorganotypicslicecultureshavebeenusedextensivelynotonlytostudyaxonal growthandregeneration,butalsoforelectrophysiologystudies,andotherexperimentson various receptors. The entorhinoͲhippocampal perforant pathway develops normally in organotypicslicecultures(FrotscherM,1993),witharobustaxonalprojectionfromtheECto thehippocampus(LiD,1993).Thisprojectioncanbelesionedinvitrobycuttingthroughthe culturewithascalpelblade,andregenerationoftheentorhinalfiberscanbeassessed.
TovisualizefibersfromtheECtothehippocampusafterlesion,onecanuseeitherentorhinal cortexfromanimalswithdifferentgeneticbackgrounds,e.g.CB6micewithM6ratortauͲGFP mice;oraxonaltracingmethods.HippocampalsliceshavebeencoͲculturedwithECfromM6 (LiD,1993)andtauͲGFP(RadojevicV,2004)mice.Inbothcases,axonsfromtheEConlycould befollowedgrowingintothehippocampalpart.Fortracingthefibers,tracersinvarious coloursareavailable(e.g.biotin,BDA,DiI).Dependingonthetracerused,tracingcanbedone bothinͲvitroandinͲvivo(herewewillfocusonitsuseinͲvitro),andcanbeusedtotracefrom thecellbodytotheaxonterminal(anterograde)orviceversa(retrograde).Atracercanbe usedbothinthecrystalform,i.e.,byplacingthecrystalontopofthepartoftheculturetobe traced;andintheliquidform,i.e.,byusingamicrosyringetoinjectitintothetargetneuronal region.Tracers(withdifferentcolours)canbecombinedtovisualizefibersfromdifferent regionsofthebrain.OnecommontracerwhichisalsousedinthisprojectisBDA.BDAcanbe usedbothasananterogradeandasaretrogradetracer.Afterfixingthetissue,BDAcanbe developedwithavidincoupledeithertoDABortoafluorescentlabeltobeviewedeitherwith
lightorfluorescencemicroscopy(ReinerA,2000).InthisprojectwewillbeusingBDAinthe crystalformasananterogradetracer.
Itwaspreviouslyshownthatthereisamarkeddecreaseinfibersgrowingintoahippocampal slicewithincreasingtimeinculture(WoodhamsPL,1993).Whenalesionismadeat6–7days invitroorlaterinpostnatalday5Ͳ6mice,thereareveryfewregeneratingfibers(Fig.1.13;
PrangP,2001).Inthesecondpartofthisproject(Chapter3),wetakethisasacontrolmodel wherethereislittlespontaneousaxonalregeneration,andcheckwhethervarioustreatments (described previously and in Chapter 1.3.4) applied on the day of lesion can induce regeneration. A main advantage of
hippocampalsliceculturesisthatthey canbeeasilyculturedandmaintained for long periods of time, thus giving enoughtimeforaxonalregenerationto takeplace.Inaddition,biochemicaland pharmacologicaltreatmentsareeasyto performandcontrol.
Fig.1.13 Histogram of regenerating fibers. At DIV6, there is a sharp decrease in number of regeneratingfibers.(takenfromPrang,2001)
1.6.2 StudiesassessingaxonalregenerationofthePPinorganotypicslicecultures Inthisproject,westudytheeffectofcompoundsinterferingwithvarioussignaltransduction mechanismsonaxonalregenerationoftheperforantpathwayinorganotypiccultures.Similar studiesusingvariousapproacheshavebeendonebefore.GF109203X,aPKCinhibitor,and Pertussis,aGiproteininhibitor,treatmentshavebeenpreviouslystudied(PrangP,2001).
Thereisastrongregenerationinthesecultures(aswasalsoobservedinthisstudy).Inthis projectweextendthistreatmentstudy(ascanbeseeninTable3.1).
Studiesoftreatmentwithneurotrophicfactorsandgrowthfactorshavebeenperformed (PrangP,2001),(WoodhamsPL,1996).Withsomeofthesetreatmentsthereisnosignificant regeneration,whileGDNFandNT4(Prang),andaFGFandSchwanncellconditionedmedium
TheeffectofChABC(blockstheeffectofCSPG)andNEP1Ͳ40(blocksNogoͲ66bindingtoNgR) wasalsostudied(MingoranceA,2006).Withtreatmentsmadeimmediatelyafterlesion,both treatmentspromotedaxonalregeneration,althoughthecombinedtreatmentdidnothavea synergisticeffect.Interestingly,whenNEP1Ͳ40treatmentwasdelayed(atleast5daysafter lesion), axonal regeneration still occurred with a similar efficiency as with immediate treatment,thusshowingapromisingtimewindowwhenefficienttreatmentcouldstilloccur.
InastudybydelRioet.al.(delRioJA,2002),robust,layerspecificregenerationwasobserved wheninmatureenterohinohippocampalcultures,thehippocampalpartwasreplacedwith youngpermissivehippocampusenrichedinCajalͲRetziuscells,whilethematureECstayedin culture.Similartowhatisobservedinthedevelopinghippocampus,CajalͲRetziuscellsguide enterohinalaxonstotheirtargetlayers,i.e.,thereisformationofsynapticcontactswiththe correcttargetneurons.ThusCRderivedsignalscouldbeusedforrepair.
Inanotherapproach,BclͲ2transgenicmicewereusedtopreventapoptosisandpromote axonal outgrowth (Solé M, 2004). In these cultures, there was no improvement in regeneration,althoughcellsurvivalisimproved.
Muchmoreworkcouldbedonewiththismodele.g.combinationsoftreatments/grafting/
transplantations.
1.6.3 Spinalcordorganotypicslicecultures
Transverse spinal cord organotypic slice cultures have been very much studied both morphologically(DelfsJ,1989)andelectrophysiologically(RosatoͲSiriMD,2004).Inthese cultures the intersegmental connections of the spinal cordare lost. Developmentand distributionofvariousspinalcordneurons,includingsomaticandautonomicmotoneurons (BarberRP,1993)andinterneurons(PhelpsPE,1996)hasbeenextensivelystudied.
AtransverseorganotypicslicecultureofspinalcordcoͲculturedwithDRGandskeletalmuscle wasdevelopedtobeusedforelectrophysiologicalrecordings(BraschlerUF,1989).Later,the samelabusedthismodeltostudythestructureandfunctionofcentralsynapses(SpengerC, 1991),andtheelectricalpropertiesofmotoneurons,musclefibresanddorsalrootganglion
neurons(StreitJ,1991).Inanotherstudy,electrophysiologicalmeasurementsontransverse sectionsoforganotypicslicesweremadetomeasureexcitabilityofthespinaldorsalhorn, whichshouldreflectchronicpain(LuVB,2006).Themolecularmechanismforpathfindingand synapseformationofcorticospinalfiberswasfollowedusingtransverseSCcoͲculturedwith cortex(TakumaH,2002).LongitudinalspinalcordcultureswereusedbeforeinaspinalͲcord corticalcoͲculturesystem(KameiN,2004),butthemorphologicalpropertiesofthesecultures andtheregenerativeabilityoftheaxonswithinwerenotknown.
1.6.4 OrganotypiccoͲculturesystems
Inalaterpartofthisproject,differentcoͲculturemodelstostudyaxonalgrowthinspinalcord environmentwillbeintroduced.VariousorganotypiccoͲculturemodelshavebeenusedto assessaxonalgrowthandrepairinneuroscienceresearch.InentorhinoͲhippocampalcoͲ culturesECfromyoungrathasbeenshowntogrowintohippocampusfromoldmouse(LiD, 1995).Wehaveusedthisculturemodelfortheevaluationofpharmacologicalmodulationof signaltransductionpathwaysinordertostimulateaxonalregrowth(Chapter3). InacoͲ culturemodeloftheauditorysystem,themedialnucleusofthetrapezoidbodywasshownto projecttothelateralsuperiorolive(LohmannC,1999).CoͲculturesofcorticalandthalamic sliceshavebeenimportantforstudyingmechanismsofthalamocorticalprojections(BolzJ, 1992).Theconnectionsrelatedtothereflexarcwerealsostudiedusingaspinalcord–dorsal rootganglion–skeletalmusclecoͲculturesystem(SpengerC,1991);whiletheinnervations fromthedorsalrootgangliontothedorsalhornwasfollowedusingfoetalDRGscoͲcultures withspinalcordexplants(SmalheiserNR,1981).Asageneralfindingitisremarkablethatinall oftheseverydifferentpreparationsaxonalprojectionsmaintainahighdegreeoforderand specificity which makes such coͲculture models very suitable for the study of axonal projections.
2. A IMSOFTHE P ROJECT
Themain aimsofthis projectwere toestablish newandimprove currentlyavailable organotypic slice culture models to facilitate the testing of various pharmacological compoundswhicharethoughttoimproveaxonalregeneration.
2.1 Developmentofanovelspinalcordorganotypicsliceculturemodeltostudy spinalcordinjury
Invitromodelstostudyaxonalprocessesandregenerationhavesignificantadvantages:they couldbeusedtotestvariousapproachesforrepairofspinalcordinjury,withoutinitially needingtouseanimalstudies.
Organotypic slicecultures have previously beenused to assess axonal regeneration in entorhinoͲhippocampalandcerebellarcultures.Transversesliceculturesofspinalcordwere notsuitedforthistypeofstudy.Whilethecytoarchitectonicorganizationofthespinalcordis bestpreservedinthesecultures,thefiberprojectionsextendinginthelongitudinaldirection arelost.Inthefirstpartoftheproject,anovelspinalcordlongitudinalorganotypicslice culturemodelwasdevelopedallowingtofollowaxonsalongtherostroͲcaudalextensionofthe spinalcord.Slicesofcervicalspinalcordwerecutinthesagittalplanefromearlypostnatal miceandthepropertiesofsuchcultureswerethoroughlystudied.
ThemorphologicalpropertiesofthesecultureswerestudiedandcomparedwiththeinͲvivo situation.AnimportantaspectwaswhetherthesecultureswouldreallymaintaintheventroͲ dorsalpolarityofthespinalcord.
2.2 Evaluation of spontaneous regeneration of spinal cord fibres after a
mechanicallesion
Inthispartoftheproject,weinvestigatedtheabilityofintrinsicspinalcordaxonsto regenerateacrossalesion.Wetooktheculturedescribedabove(2.1)andintroduceda mechanicallesionbyatransversecutthroughtheculturewithascalpelblade.
Inaddition,weaskedwhethertherewouldbeadifferenceingrowthacrossalesionwith increasing age and increasing days in vitro. It was previously observed that in the hippocampus,withincreasingtimeincultureorwiththeuseofslicesfromolderpostnatal
animalstheregenerativepotentialoftheaxonsdecreased(Prangetal.).Eventuallywedida proofofprinciplestudyinvestigatingwhetherregenerationofintrinsicspinalcordfiberscould bestimulatedbyapharmacologicaltreatment.
2.3 Evaluation of the effectiveness of treatments which promote axonal
regenerationinhippocampalorganotypicsliceculturemodel
Axonalregenerationafterlesionisusuallynotpossibleintheadultcentralnervoussystembut canoccurintheembryonicandearlypostnatalnervoussystem.Hereweusedconditions wherethereisverylittleregenerationinthecultures,andtestedcompoundsfortheir effectivenesstoincreasefibregrowthacrossalesion.
InthisstudyweusedthemodelsystemofmouseentorhinoͲhippocampalsliceculturesto assessregenerationofentorhinalfibersprojectingtothedentategyrusaftermechanical lesionsandtreatmentwithpharmacologicalcompoundsinvitro.Thismodelhasbeenusedby others to study both axonal regeneration (using various methods discussed in the introduction)andfunctionalrecovery(usingelectrophysiology).Previousresultsfromourlab (Prangetal.2001)showthatinentorhinoͲhippocampalculturesthereisamarkeddecreasein regeneratingfiberswhenalesionismadeat6Ͳ7daysinvitroorlaterinpostnatalday5Ͳ6mice.
This DIV will be taken as a control model where there is little spontaneous axonal regeneration,inordertobeabletoassessthepotentialofvarioustreatmentstopromote regeneration.Byusingascoresystem,theamountofaxonalregenerationbothinthetreated culturesandtheuntreatedcontrolswasquantified.Wehaveusedcompoundsactingas modulatorsofsignaltransductionpathways,mainlycAMPactivatorsandPKCinhibitors,but alsoothercompoundsactingontheIP3andRhopathways.Wehaveonlyused1compound perculture,ie,nocombinationtreatments.
