Effect of FGF2 and sciatic nerve grafting on ChAT expression in dorsal root ganglia neurons of spinal cord transected rats

ContentslistsavailableatScienceDirect

Neuroscience

Letters

j ou rn a l h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / n e u l e t

Research

paper

Effect

of

FGF-2

and

sciatic

nerve

grafting

on

ChAT

expression

in

dorsal

root

ganglia

neurons

of

spinal

cord

transected

rats

Fausto

Pierdoná

Guzen

_,

_Dayane

_Pessoa

_de

_Araújo

_,

_Eudes

_Euler

_de

_Souza

_Lucena

_,

Hécio

Henrique

Araújo

de

Morais

_,

_José

_Rodolfo

_Lopes

_de

_Paiva

_Cavalcanti

_,

Expedito

Silva

do

Nascimento

Jr.

_,

_Miriam

_Stela

_Maris

_de

_Oliveira

_Costa

_,

Jeferson

Sousa

Cavalcante

a_{LaboratoryofExperimentalNeurology,HealthScienceCenter,StateUniversityofRioGrandedoNorte,Mossoró,RN,Brazil} b_{LaboratoryofNeuroanatomy,DepartmentofMorphology,FederalUniversityofRioGrandedoNorte,Natal,RN,Brazil} c_{LaboratoryofNeurochemicalStudies,DepartmentofPhysiology,FederalUniversityofRioGrandedoNorte,Natal,RN,Brazil}

h

i

g

h

l

i

g

h

t

s

•TransectedspinalcordasamodelforstudyofDRGregeneration.

•PeripheralnervegraftsasafavorableenvironmenttoDRGneuroprotection.

•FGF-2potentiatesneuroprotectiveeffectinDRGafterspinalcordinjury.

•FGF-2plussciaticnervefragmentimproveDRGplasticityofratssubmittedtocompletetransectionsofspinalcord.

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24June2015

Receivedinrevisedform17August2015 Accepted23August2015

Availableonline28August2015

Keywords:

Dorsalrootganglia Fibroblasticgrowthfactor-2 Neuroprotection

Sciaticnervegraft Spinalcord

a

b

s

t

r

a

c

t

NeurotrophicfactorsandperipheralnervesareknowntobegoodsubstratesforbridgingCNStrauma. Theinvolvementoffibroblastgrowthfactor-2(FGF-2)activationinthedorsalrootganglion(DRG)was examinedfollowingspinalcordinjuryintherat.WeevaluatedwhetherFGF-2increasestheabilityof asciaticnervegrafttoenhanceneuronalplasticity,inagappromotedbycompletetransectionofthe spinalcord.Theratsweresubjectedtoa4mm-longgapatlowthoraciclevelandwererepairedwith saline(Salineorcontrolgroup,n=10),orfragmentofthesciaticnerve(Nervegroup,n=10),orfragment ofthesciaticnervetowhichFGF-2(Nerve+FGF-2group,n=10)hadbeenaddedimmediatelyafterlesion. TheeffectsoftheFGF-2andfragmentofthesciaticnervegraftsonneuronalplasticitywereinvestigated usingcholineacetyltransferase(ChAT)-immunoreactivityofneuronsinthedorsalrootganglionafter8 weeks.Preservationoftheareaanddiameterofneuronalcellbodiesindorsalrootganglion(DRG)was seeninanimalstreatedwiththesciaticnerve,aneffectenhancedbytheadditionofFGF-2.Thus,the additionofexogenousFGF-2toasciaticnervefragmentgraftedinagapoftheratspinalcordsubmitted tocompletetransectionwasabletoimproveneuroprotectionintheDRG.Theresultsemphasizedthat themanipulationofthemicroenvironmentinthewoundmightamplifytheregenerativecapacityof peripheralneurons.

©2016PublishedbyElsevierIrelandLtd.

1. Introduction

Trophicfactors,suchasnervegrowthfactor(NGF),brainderived neurotrophicfactor(BDNF)andglia-derivedneurotrophicfactor

∗ Correspondingauthorat:DepartmentofBiomedicSciences,HealthSciences Faculty,StateUniversityofRioGrandedoNorte,Mossoró59607-360,RioGrande doNorte,Brazil.

E-mailaddress:faustoguzen@uern.br(F.P.Guzen).

(GDNF)showsimportantandselectiveeffectsonsurvivaland phe-notypicexpressionofprimarysensoryneuronsinthedorsalroot ganglion (DRG)followingnervous systeminjury and peripheral inflammationTrophicfactors,suchasnervegrowthfactor(NGF), brainderived neurotrophic factor(BDNF) andglia-derived neu-rotrophicfactor(GDNF)showimportantandselectiveeffectson survivalandphenotypicexpressionofprimarysensoryneuronsin thedorsalrootganglion(DRG)followingnervoussysteminjuryand peripheralinflammation[1–4].

http://dx.doi.org/10.1016/j.neulet.2015.08.043

positivesubpopulationofsensoryneurons[6].

Evidencefromseveralinvitroandinvivostudieshasshown that FGF-2is not only present in the nervoussystem but also mediatessurvival-promotingeffectsandstimulatesthe transmit-termetabolismofseveralneuronsduringdevelopmentandafter injury[15,16].ApplicationofFGF-2totheproximalstumpofthe transectionedsciaticnervepreventsthelesion-induceddeathof sensoryneuronsofDRGL4-L6[12].

Animal studies have shown that if continuity is restored betweenthespinal cordand theventralroots ofnerves atthe lumbar[17] orcervical[18–20]levelsofthespinalcord, motor neuronaxonscanregrowintotheirrespectiveperipheralnerves withconcomitantrecoveryofmotorfunctions.Thereturnofmotor functionsafterimplantationofavulsedspinalnerverootsintothe spinalcordhasalsobeenreportedinoneclinicalcase[21],aswell asinsomeanimalstudies[22–24].

FGF-1and FGF-2treated cultures can promote a significant increaseinneuriteoutgrowthofventralspinalcordneuronsand stemcells,suggestingthatbothFGFscaninfluenceneuronal devel-opment[25,26].

InordertodeterminefunctionalrolesofFGF-2inthe periph-eralnervoussystemweanalyzedtheexpressionofcholineacetyl transferase(ChAT)inspinalgangliaafterspinalcordlesiontreated withFGF-2.

2. Materialsandmethods

2.1. Animaltreatment

AdultmaleWistarrats(n=30)fromtheFederalUniversityof Pernambuco,Brazil(bodyweight[b.w.]180–200g),wereusedin thepresentstudy.Thestudywasconductedaccordingto proto-colsapprovedbytheAnimalCareandUseEthicCommitteeatthe FederalUniversityof Rio GrandedoNorte usingtheSanPoiley outbreedingmethod[27].

2.2. Microneurosurgery

Ratswerepre-anaesthetizedwithanintramuscularinjectionof ketaminechloridrate10%(AgenerUnião,Brazil,0,1ml/100gb.w.) andxylazine2%(AgenerUnião,Brazil,0,01ml/100gb.w.)andthen anesthetized with isoflurane inhalation (Isoflorine®

) (Cristália, Brazil).Asmalllaminectomyatthetenth/elevenththoracic lev-elswasperformed.Completetransectionscreateda4 mm-long gapattheeleventh/twelfthspinalcordlevels.Agelfoamsoaked in10␮lof0,9%salinewasleftatthebottomofthegapcloseto

thevertebralbody in10rats(SalineorControlgroup).Another groupof 10ratsreceivedgelfoamfilledwithsciaticnerve frag-ment(Nervegroup).Additionally,afurther10ratsreceivedsciatic nervefragmentand10␮lofaFGF-2(SantaCruzBiotechnology,

Germany)dissolvedin0.1Mphosphatebufferedsaline(PBS),pH 7.4,for48h.

Agroupof6uninjuredanimalswerealsoperfusedandtheir DRGs fromthe same location in the thoraxwere taken out. A microtome(Leica,SM2000R,Germany)wasusedtoprepare20␮m

thickentransversalfrozensectionsfromtheDRG.

2.4. ImmunohistochemicalproceduresforChAT

Immunoreactivitywasdetectedusingtheavidin–biotin peroxi-dasetechnique[31–33].Seriesofsectionswerewashed2×10min in0.1MPBS,pH7.4andincubatedwith5%normalgoatserum (NGS,Sigma)for 30minatroomtemperature.Serieswerethen incubatedfor24hat4◦_{Cwithgoatmonoclonalantiseraagainst} ChAT(Millipore, diluted1:100). Theantibodieswere dilutedin PBScontaining0.3%TritonX-100(Sigma)and1%bovineserum albumin (Sigma). The series of sectionswere washed again in PBS (2×10min) and incubated with biotinylated donkey anti-goat(ChAT)immunoglobulinsdiluted1:1000(Jackson,USA)for1 hour.ThesectionswerewashedagaininPBSandincubatedwith anavidin–biotinperoxidasecomplex(bothdiluted1:100, Vectas-tain,Vector) for90min.Immunoreactivitywasvisualizedusing 3-3′_{-diaminobenzidinetetrahydrochloride(DAB,Sigma)asa} chro-mogenandH2O2asasubstrate.

2.5. Morphometric/microdensitometricimageanalysis

TheChATimmunoreactivitywasmeasuredinonesectionper ratintheganglioncentralregion.Anopticalmicroscope (Olym-pusBX41)witha40×objectiveinbrightfieldwasusedtoobtain thedigitalimage.Thecountsandmeasurementsweremadeusing ImageJsoftware.Digitalimagesofrepresentativesectionswere obtainedusingadigitalvideocamera(NikonDXM1200).

2.6. Statisticalanalysis

Statisticalanalyseswereperformedusingtheanalysisof vari-ance(ANOVA)andsignificantinteractionswerefollowed-upwitha TukeyandBonferronipost-testcomparison.Allstatisticalanalyses wereperformedusingSPSS22,andsignificancewassetatp<0.05.

3. Results

PhotomicrographsinFig.1illustratestheNisslstainedDRG neu-ronsofanimalswithoutinjury(Fig.1).

Uninjured animals used as parameters had a mean area of neuronal cell body of 9.1␮m, compared to the saline group

(6.86␮m2), the nerve group (7.85␮m2) and the nerve+FGF-2

group(8.54␮m2).Consideringtheinjuredgroups,thegroupthat

Fig.1. PhotomicrographsillustrateNissl-stainedDRGneuronsofanimalswithoutinjury.

Fig.2. AreaofChAT-immunoreactivecellbodiesintheDRGofuninjuredratstreatedwithsaline,graftsoffragmentsofthesciaticnerve(nerve)orfragmentsofthesciatic nervewithaddedFGF-2(nerve+FGF-2).Means±S.E.M.⋆p<0.05,⋆⋆p<0.01and⋆⋆⋆p<0.001accordingtoANOVA–TukeyandBonferroni.

ofcellbodyareacomparedtothesalinegroup(⋆⋆⋆p=0.0001)and

comparedtothenervegroup(⋆⋆p=0.006)(Fig.2).

TheprofilesofChATimmunoreactiveneuronalcellbody diam-eterintheDRG,intheuninjuredcontrolanimals,hadanaverage diameterof2.96␮mneuronal cellbody.Intheinjuredanimals,

thesalinegrouphad1.75␮m, inthenervegroup2.55␮m, and

in the nerve+FGF-2 group had 2.81␮m. Thus, the group that

Fig.3.DiameterofChAT-immunoreactivecellbodiesintheDRGofanimalstreatedwithsaline,graftsoffragmentsofthesciaticnerveorfragmentsofthesciaticnervewith addedFGF-2.Means±S.E.M.⋆p<0.05,⋆⋆p<0.01and⋆⋆⋆p<0.001accordingtoANOVA–TukeyandBonferroni.

Fig.4.ChAT-immunoreactiveneurons.(A),salinegroup;(B),nervegroup;(CandD),nerve+FGF-2group.ArrowspointrelevantChAT-immunoreactivecellbody.Bars: 20mm2_(A–D).

Fig.4A–C shows profiles of ChAT immunoreactive neuronal cell bodies of DRG frominjured groups. Fig. 4A (saline group) showsthecellbodyflatteningattheperipheryofganglion(arrow). Greaterpreservationwasobservedinthegroupsnerve(Fig.4B) andnerve+FGF-2(Fig.4CandD).

Morphologicalanalysisshowsthattheplasticbehaviorsinthe treatedgroupswerehigherthanthecontrolgroup.Thenodesof treatedanimalsshowedamorepreservedarchitecture(Fig.4B–D) thanthecontrol.

4. Discussion

theeffectsofexogenousFGF-2aremediatedmainlybythetyrosine kinaseFGFR1,whichislocalizedintheplasmamembraneaswell asinthenucleiofneuronsintheperipheralnerve[7,38,39].

Somereports have shown that peripheral nerve lesionscan accelerateregenerationofthecentralprimarysensoryneuronsin thedorsalspinalroots[40–42].However,mostoftheaxonsarestill unabletoreenterthespinalcord,andthosethatsucceedpenetrate nofurtherthanthesuperficialdorsalhornofthespinalcord.These reportsalsosuggestthataddingsome“intrinsicorextrinsic fac-tors”[42–44]canleadtotheregenerationofthecentralprocesses oftheDRG,towardgainingentryintothespinalcord.

Genetherapywithadenoviralvectorencodinganeurotrophinis capableofenhancinganddirectingtheregenerationofa subpopu-lationofdorsalrootaxonsthroughtotheCNSenvironmentinrats. However,theregeneratingaxons,inthestudycited,didnotspread widelythroughoutthegraymatterorawayfromtheinjectionsite, andnosensoryrecoverywasmentioned[45].

AnalysisofnervoustissueinratsshowedFGF-2 immunoreactiv-ityinmanydiversesizeneurons.Manyresearchershaveproposed bothneurotrophicandregenerativefunctionsforFGF-2.This sug-geststhatFGF-2mayperformsimilarfunctionsinDRGneurons [46,47].

OurfindingsshowChATimmunoreactivityintheDRGadjacent tothespinalcordinjury.Theanimalsthatreceivedtreatmentwith sciaticnervehadneuroprotectionwhentheareaanddiameterof cellbodieswereanalyzed,aneffectpotentiatedbyFGF-2.

Spinalcordtreatmentwithsciaticnerveandsciaticnerveplus FGF-2 allowedrecovery of hind limb movements compared to control,manifestedbysignificantlyhigherbehavioralscoresand higheramountsofMAP-2andGAP-43immunoreactive.Thus FGF-2addedtothenervegraftfavoredthemotorrecoveryandfiber regrowth[30].

FGF-1enhancesneuriteoutgrowthandstimulatesexpressionof GAP-43andT␣-tubulininculturedneuronsfromadultratdorsal

rootganglia.Aftertransectionandrepair,theanimalsthatreceived treatmentwithNGFshowedrecoveryinbothmotorandsensory nervefunctions[48,20].Duringperipheralnerveregeneration, FGF-2is up-regulatedinboth thecrushednerve andtherespective spinalganglia,suggestingapossiblephysiologicalfunctionofFGF-2 duringtheregenerationprocess[11].Treatmentwithacupuncture hasbeenseentopromoteeffectsontheexpressionofGDNFand FGF-2intheleftsixthlumbarDRGfollowingremovalofadjacent dorsalrootganglia[49].

Thesefindingsindicatethatourtreatmentstrategyusingnerve graftsalongwithFGF-2canenhanceaxonoutgrowthfromtheDRG, protectingthespinothalamicandspinocerebellartractsresulting inimprovementsinsensorystimulation,tone,postureand move-ment.

5. Conclusion

TheadditionofexogenousFGF-2toasciaticnerve fragment graftedintoagapontheratspinalcordwhichhadbeensubmitted tocompletetransectionisabletoimproveneuroprotectioninthe DRG.Theresultsemphasizedthatthemanipulationofthe microen-vironmentatthewoundisabletoamplifytheregenerativecapacity ofperipheralneurons.

Acknowledgments

ThisstudywassupportedbyfundingfromtheNationalCounsel ofTechnologicalandScientificDevelopment(CNPq)andthe Coor-dinationforImprovementofHighLevelStaff(CAPES).TheEnglish versionofthistextwasrevisedbySidneyPratt,Canadian,MAT(The JohnsHopkinsUniversity),RSAdip(TEFL,UniversityofCambridge).

References

[1]R.R.Ji,T.A.Samad,S.X.Jin,R.Schmoll,C.J.Woolf,P38MapkactivationbyNgf inprimarysensoryneuronsafterinflammationincreasesTrpv1levelsand maintainsheathyperalgesia,Neuron36(2002)57–68.

[2]F.Amaya,G.Shimosato,M.Nagano,M.Ueda,S.Hashimoto,Y.Tanaka,H. Suzuki,M.Tanaka,NgfandGdnfdifferentiallyregulatetrpv1expressionthat contributestodevelopmentofinflammatorythermalhyperalgesia,Eur.J. Neurosci.20(2004)2303–2310.

[3]S.Pezet,S.B.McMahon,Neurotrophins:mediatorsandmodulatorsofpain, Annu.Rev.Neurosci.29(2006)507–538.

[4]F.P.Guzen,R.J.deAlmeidaLeme,M.S.deAndrade,B.A.deLuca,G.Chadi,Glial cellline-derivedneurotrophicfactoraddedtoasciaticnervefragmentgrafted inaspinalcordgapamelioratesmotorimpairmentsinratsandincreaseslocal axonalgrowth’,Restor.Neurol.Neurosci.27(2009)1–16.

[5]P.Bohlen.Fibroblasticgrowthfactor.InMacrophage-derivedcellregulatory factors,Ed.Sorg.C,1(1989),204–228.

[6]B.Weise,K.Unsicker,C.Grothe,Localizationofbasicfibroblastgrowthfactor inasubpopulationofratsensoryneurons,CellTissueRes.267(1992) 125–130.

[7]C.Oellig,U.Pirvola,L.Taylor,R.Elde,T.Hokfelt,R.F.Pettersson,AcidicFgfand Fgfreceptorsarespecificallyexpressedinneuronsofdevelopingandadultrat dorsalrootganglia,Eur.J.Neurosci.7(1995)863–874.

[8]C.Grothe,C.Meisinger,P.Claus,Invivoexpressionandlocalizationofthe fibroblastgrowthfactorsystemintheintactandlesionedratperipheralnerve andspinalganglia,J.Comp.Neurol.434(2001)342–357.

[9]H.Liu,Q.F.Wu,J.Y.Li,X.J.Liu,K.C.Li,Y.Q.Zhong,D.Wu,Q.Wang,Y.J.Lu,L. Bao,X.Zhang,Fibroblastgrowthfactor7isanociceptivemodulatorsecreted vialargedense-corevesicles,J.Mol.Biol.Cell.(2015).

[10]R.R.Ji,Q.Zhang,X.Zhang,F.Piehl,T.Reilly,R.F.Pettersson,T.Hokfelt, ProminentexpressionofBfgfindorsalrootgangliaafteraxotomy,Eur.J. Neurosci.7(1995)2458–2468.

[11]C.Grothe,C.Meisinger,A.Hertenstein,H.Kurz,K.Wewetzer,Expressionof fibroblastgrowthfactor-2andfibroblastgrowthfactorreceptor1messenger Rnasinspinalgangliaandsciaticnerve:regulationafterperipheralnerve lesion,Neuroscience76(1997)123–135.

[12]D.Otto,K.Unsicker,C.Grothe,Pharmacologicaleffectsofnervegrowthfactor andfibroblastgrowthfactorappliedtothetransectionedsciaticnerveon neurondeathinadultratdorsalrootganglia,Neurosci.Lett.83(1987) 156–160.

[13]R.Elde,Y.H.Cao,A.Cintra,T.C.Brelje,M.Pelto-Huikko,T.Junttila,K.Fuxe,R.F. Pettersson,T.Hokfelt,Prominentexpressionofacidicfibroblastgrowthfactor inmotorandsensoryneurons,Neuron7(1991)349–364.

[14]B.Weise,T.Janet,C.Grothe,LocalizationofBfgfandFgf-receptorinthe developingnervoussystemoftheembryonicandnewbornrat,J.Neurosci. Res.34(1993)442–453.

[15]C.Unsicker,G.L ¨udecke,D.Otto,R.Westermann,Fibroblastgrowthfactors: theirrolesinthecentralandperipheralnervoussystem,in:S.E.Loughlin,J.H. Fallon(Eds.),NeurotrophicFactors,AcademicPress,NewYork,London,1993, pp.313–338.

[16]C.Grothe,K.Wewetzer,Fibroblastgrowthfactoranditsimplicationsfor developingandregeneratingneurons,Int.J.Dev.Biol.40(1996)403–410.

[17]T.P.Carlstedt,H.Linda,S.Culheim,Reinnervationofhindlimbmusclesafter ventralrootavulsionandimplantationinthelumbarspinalcordoftheadult rat,ActaPhysiol.Scand.128(1989)645–646.

[18]S.Cullheim,T.Carlstedt,H.Linda,M.Risling,B.Ulfhake,Motoneurons reinnervateskeletalmuscleafterventralrootimplantationintothespinal cordofthecat,Neuroscience29(1989)725–733.

[19]T.P.Carlstedt,R.G.Hallin,K.G.Hedstrom,I.A.Nilsson-Remahl,Functional recoveryinprimateswithbrachialplexusinjuryafterspinalcord

implantationofavulsedventralroots,J.Neurol.Neurosurg.Psychiatry(1993) 649–654.

[20]F.Huang,Z.Liu,H.Liu,L.Wang,H.Wang,Z.Li,Gm1andNgfModulateCa2+ homeostasisandGap43Mrnaexpressionincultureddorsalrootganglion neuronswithexcitotoxicityinducedbyglutamate,Nutr.Neurosci.10(2007) 105–111.

[21]T.Carlstedt,P.Grane,R.G.Hallin,G.Noren,Returnoffunctionafterspinalcord implantationofavulsedspinalnerveroots,Lancet346(1995)1323–1325.

[22]T.Y.Chuang,M.C.Huang,K.C.Chen,Y.C.Chang,Y.S.Yen,L.S.Lee,H.Cheng, Forelimbmuscleactivityfollowingnervegraftrepairofventralrootsinthe ratcervicalspinalcord,LifeSci.71(2002)487–496.

[23]C.F.Hoffmann,E.Marani,J.G.vanDijk,W.vdKamp,R.T.Thomeer, Reinnervationofavulsedandreimplantedventralrootletsinthecervical spinalcordofthecat,J.Neurosurg.84(1996)234–243.

[24]J.J.Katims,Electrodiagnosticfunctionalsensoryevalutaionofthepatientwith pain:areviewoftheneuroselectivecurrentperceptionthersholdandpain tolerancethershold,PainDig.8(1998)219–230.

[25]Y.Iwasaki,T.Shiojima,K.Ikeda,N.Tagaya,T.Kobayashi,M.Kinoshita,Acidic andbasicfibroblastgrowthfactorsenhanceneuriteoutgrowthinculturedrat spinalcordneurons,Neurol.Res.17(1995)70–72.

Neurochem.Int.25(1994)39–45.

[33]K.Fuxe,B.Tinner,G.Chadi,A.Harfstrand,L.F.Agnati,Evidenceforaregional distributionofhyaluronicacidintheratbrainusingahighlyspecific hyaluronicacidrecognizingprotein,Neurosci.Lett.169(1994)25–30.

[34]C.Basilico,D.Moscatelli,TheFgffamilyofgrowthfactorsandoncogenes,Adv. CancerRes.59(1992)115–165.

[35]A.J.Furusho,R.J.Franklin,R.Bansal,Fibroblastgrowthfactorsignalingin oligodendrocyte-lineagecellsfacilitatesrecoveryofchronicallydemyelinated lesionsbutisredundantinacutelesions,Glia(2015).

[36]C.Meisinger,C.Grothe,Differentialregulationoffibroblastgrowthfactor (Fgf)-2andFgfreceptor1MrnasandFgf-2isoformsinspinalgangliaand sciaticnerveafterperipheralnervelesion,J.Neurochem.6(1997)1150–1158.

[37]D.E.Johnson,L.T.Williams,Structuralandfunctionaldiversityinthefgf receptormultigenefamily,Adv.CancerRes.60(1993)1–41.

[38]L.Klimaschewski,C.Meisinger,C.Grothe,Localizationandregulationofbasic fibroblastgrowthfactor(Fgf-2)andFgfreceptor-1inratsuperiorcervical ganglionafteraxotomy,J.Neurobiol.38(1999)499–506.

[39]W.Nindl,P.Kavakebi,P.Claus,C.Grothe,K.Pfaller,L.Klimaschewski, Expressionofbasicfibroblastgrowthfactorisoformsinpostmitotic sympatheticneurons:synthesis,intracellularlocalizationandinvolvementin karyokinesis,Neuroscience124(2004)561–572.

[46]B.Malgrange,P.Delree,J.M.Rigo,H.Baron,G.Moonen,Imageanalysisof neuriticregenerationbyadultratdorsalrootganglionneuronsinculture: quantificationoftheneurotoxicityofanticanceragentsandofitsprevention bynervegrowthfactororbasicfibroblastgrowthfactorbutnotbrain-derived neurotrophicfactororneurotrophin-3’,J.Neurosci.Methods53(1994) 111–122.

[47]C.G.Acosta,A.R.Fabrega,D.H.Masco,H.S.Lopez,Asensoryneuron subpopulationwithuniquesequentialsurvivaldependenceonnervegrowth factorandbasicfibroblastgrowthfactorduringdevelopment,J.Neurosci.21 (2001)8873–8885.

[48]L.Mohiuddin,P.Fernyhough,D.R.Tomlinson,Acidicfibroblastgrowthfactor enhancesneuriteoutgrowthandstimulatesexpressionofGAP-43andTalpha 1alpha-tubulininculturedneuronesfromadultratdorsalrootganglia, Neurosci.Lett.215(1996)111–114.