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191
Advances in Anatomy
Embryology
and Cell Biology
Editors
F.F.Beck,Melbourne·F.Clascá,Madrid
M.Frotscher,Freiburg·D.E.Haines,Jackson
H.-W.Korf,Frankfurt·E.Marani,Enschede
R.Putz,München·Y.Sano,Kyoto
T.H.Schiebler,Würzburg
A.B. Tonchev · T. Yamashima
G.N. Chaldakov
Distribution and Phenotype
of Proliferating Cells
in the Forebrain of Adult
Macaque Monkeys
after Transient Global
Cerebral Ischemia
With65Figuresand11Tables
123
AntonB.Tonchev,Dr.
GeorgeN.Chaldakov,Dr.
DivisionofCellBiology
DepartmentofForensicMedicine
VarnaUniversityofMedicine
55MarinDrinovstr.
9002Varna
Bulgaria
e-mail:[email protected]
[email protected]
TetsumoriYamashima,Dr.
DepartmentofRestorativeNeurosurgery
DivisionofNeuroscience
KanazawaUniversityGraduateSchoolofMedicalScience
Takara-machi13-1
Kanazawa920-8641
Japan
e-mail:[email protected]
ISSN 0301-5556
ISBN 978-3-540-39613-0 SpringerBerlinHeidelbergNewYork
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ListofContents
1 Introduction.............................................. 1
1.1 StudiesonCellProliferationinAdultPrimateBrain .................. 1
1.2 MethodologicalConsiderationsinDetectingCellProliferation........... 2
1.3 CellProliferationinRodentBrainAfterIschemia .................... 3
1.4 GlobalCerebralIschemiainPrimates ............................ 6
2 MaterialsandMethods ...................................... 8
2.1 AnimalSubjects ........................................... 8
2.2 BromodeoxyuridineInfusionProtocol............................ 8
2.3 TissueProcessing .......................................... 9
2.4 Immunohistochemistry ...................................... 10
2.5 DetectionofDNADamageandDegeneratingCells ................... 10
2.6 ElectronMicroscopy ........................................ 14
2.7 ImageAcquisitionandAnalysis ................................ 15
2.8 StatisticalAnalysis.......................................... 17
3 Results.................................................. 17
3.1 HippocampalFormation ..................................... 21
3.1.1 DentateGyrus............................................. 21
3.1.2 CornuAmmonis ........................................... 35
3.1.3 Subiculum ............................................... 42
3.2 SubventricularZoneoftheInferiorHornoftheLateralVentricle ......... 42
3.3 TemporalLobe ............................................ 47
3.3.1 ParahippocampalRegion ..................................... 47
3.3.2 TemporalNeocortex ........................................ 49
3.4 SubventricularZoneoftheAnteriorHornoftheLateralVentricle ........ 56
3.5 RostralMigratoryStreamandOlfactoryBulb....................... 68
3.6 FrontalCortexandStriatum................................... 76
4 Discussion ............................................... 83
4.1 BrdUasaProliferationMarker ................................. 83
4.2 EffectsofIschemiaonCellProliferationandDifferentiation ............ 84
4.3 SustainedProgenitorCellExistenceinGerminativeZones.............. 91
4.4 ImplicationsofMonkeyFindingsforTherapiesinHumans ............. 93
5 Summary ................................................ 95
References..................................................... 97
SubjectIndex................................................... 107
Preface
Mostoftheinvestigationsdescribedinthepresentpaperwereperformedatthe
Department of Restorative Neurosurgery, Graduate School of Medical Sciences,
KanazawaUniversity,Kanazawa,Japan,andsomewerecarriedoutintheDivision
ofCellBiology,VarnaUniversityofMedicine,Varna,Bulgaria.Theadultmonkey
cellproliferationprojectwasinitiatedtoprovideinformationinnon-humanpri-
mates relevant to clinical conditions in humans. The proliferation patterns seen
inadultmonkeybrainsafterglobalischemiaarejustasteptowardadeeperun-
derstanding of the molecular and cellular mechanisms that specify cell fate in
postischemicprimatetelencephalon.Additionalstudiesinmonkeysarenecessary
toidentifyprimate-specificbrainrepairmechanismsoccurringafterischemicin-
sultsandotherinjuries,focusingonfocalischemicmodelsasfocalstrokeismore
commonthanglobalischemiainhumans.
Atthesametime,themodelofglobalcerebralischemiaprovidesanexcellent
opportunitytostudytheregenerativecapabilitiesofdistantfromoneanotherte-
lencephalic regions after acommon forall insult.Thus, we initially investigated
globalcerebralischemia,andwearenowanalyzingin-depththemoleculardeter-
minants involved in modulating cellular responses. We hope the data described
inthispapermaytriggerfurtherinterestinnon-humanprimateneurogenesisre-
search.Wearegratefultothosewhosupportedusduringthesestudies.Hideyuki
Okano,KazunobuSawamoto,MasahikoWatanabe,NobuyukiTakakura,LuigiAloe
andMarcoFioreprovidedstimulatingandcriticaldiscussions.MasaoYukie,Hi-
roshi Yamamoto, Ivan Stankulov and Peter Ghenev were always enthusiastic in
theirsupport.WeexpressourgratitudetoLiangZhaoandXiangdiWangfortheir
excellent technical assistance. We are also indebted to Kiyoko Wada, Eiko Sak-
aguchiandPenkaFilipovaforsecretarialassistance.WethankRonMathisonfor
critically reading parts of the manuscript. Finally, our special thanks go to our
familiesfortheirunwaveringsupport.
Abbreviations
BDNF Brain-derivedneurotropicfactor
bFGF Basicfibroblastgrowthfactor
BrdU Bromodeoxyuridine
CA CornuAmmonis
(cid:1) (cid:1) (cid:1)
CNP 2,3-Cyclicnucleotide3-phosphodiesterase
CNS Centralnervoussystem
DG Dentategyrus
DGL Dentategranulecelllayer
EGF Epidermalgrowthfactor
GAD Glutamicaciddecarboxylase
Gadd45 GrowtharrestandDNAdamageinduciblegene45
G-CSF Granulocytecolony-stimulatingfactor
GDNF Glialcellline-derivedneurotrophicfactor
GFAP Glialfibrillaryacidicprotein
Ham56 Humanalveolarmacrophage56antigen
HB-EGF Heparin-bindingEGF-likegrowthfactor
Iba1 Ionizedcalciumbindingadaptermolecule1
IGF-1 Insulin-likegrowthfactor-1
IT Inferiortemporalcortex
ITG Inferiortemporalgyrus
MTG Middletemporalgyrus
NeuN Neuronalnuclei
PHG Parahippocampalgyrus
PHR Parahippocampalregion
PSA-NCAM Polysialylatedneuralcelladhesionmolecule
RMS Rostralmigratorystream
SCF Stemcellfactor
α α
SDF-1 Stromalcell-derivedfactor-1
SGZ Subgranularzone
STG Superiortemporalgyrus
SVZ Subventricularzone
VEGF Vascularendothelialgrowthfactor
TUC4 TOAD/Ulip/CRMP4
TUNEL Terminaldeoxynucleotidyltransferase(TdT)-mediatedUTPnick
endlabeling
Introduction 1
1
Introduction
1.1
StudiesonCellProliferationinAdultPrimateBrain
Until a few decades ago, a central postulate in neuroscience had been that the
adult mammalian brain was unable to regenerate its neurons (Ramon y Cajal
1928). Although early studies reporting mitoses in postnatal (Hamilton 1901)
and adult (Allen 1912) rat brain suggested the existence of postnatal progenitor
cellsintheadultmammaliancentralnervoussystem(CNS),itwasnotuntilthe
demonstration ofdenovo-generated cells(e.g.,SchultzeandOehlert 1960)with
tritiated(H3)-thymidinewasthepotentialoftheadultCNStoreplacesomeofits
neuronsconfirmed.Whileatfirstmainlynonneuronalcellswereinvestigatedfor
H3-thymidinelabeling(Messieretal.1958;Altman1962a),aseriesofstudiesin
the 1960s by Joseph Altman and co-workers was the first to show that de novo
generation ofneuronsoccursinthehippocampusandpossiblyinotherregions
oftheadultmammalianbrain(Altman1962b,1963;AltmanandDas1965,1966).
Altman’sresultsinrodentswereconfirmedinthenextdecadesbyMichelKaplan
andhiscollaborators(KaplanandHinds1977;Kaplan1981;KaplanandBell,1983;
1984)aswellasinbirdsbyFernandoNottebohmandcoworkers(Goldmanand
Nottebohm1983;PatonandNottebohm1984;reviewedbyNottebohm2002).
TheseH3-thymidinemarkerstudiesinlowermammalsraisedthequestionasto
whetherneuronalreplacementbyimmature(progenitor)cellsalsooccursinadult
primatebrain.Studiesreportingthepresenceofmitoticcellsinthesubependymal
layer(alsoreferredtoassubventricularzone,SVZ)ofthelateralventricleofadult
monkeybrain(Lewis1968;Kaplan1983)suggestedthepresenceofimmaturecells
inadultprimateCNSwhoseprogenycouldpotentiallybeglialorneuronalcells,
andthuspromptedforafurtherinvestigation.Subsequentexperimentsperformed
inthelaboratoryofPaskoRakicdemonstratedrenewalofoligodendrocytes,astro-
cytes,microglia,andvascularcellsinadozenofpostpubertalmonkeysinjected
withH3-thymidine,butfailedtodetectevidenceforneuronalreplacementinany
ofthemajorbrainsubdivisionsstudied:neocortex,hippocampus,olfactorybulb,
basal ganglia, thalamus, retina, cerebellum, brain stem, and spinal cord (Rakic
1985a,b).H3-thymidineincorporationwasdetectedinthenucleiofprogenitor-
like cells or astrocytes in the hippocampus, but not in neurons (Eckenhoff and
Rakic1988).Thus,adultneurogenesiswasassumednottooccurinprimateCNS,
althoughwiththeintroductionofnewinvestigativetoolsthisconclusionhasbeen
reversed.
The first advance came with the visualization of DNA synthesis immunohis-
tochemically using bromodeoxyuridine (BrdU) (Miller and Nowakowski 1988).
Thesecondsignificantadvancewastheidentificationofnewmarkersthatallowed
neurons to be distinguished from glia and that more precisely determined the
developmentalstageofcellpopulations(reviewedbyPevnyandRao2003).These
2 Introduction
techniques,nowusedincombination,permittedthedeterminationofthe“birth-
day”ofaselectivecellphenotype,suchthatthegenerationofnewneuronsinat
leasttworegionsoftheadultrodentbrain—thehippocampaldentategyrus(DG)
andSVZ—wasconvincinglydemonstrated(reviewedbyGageetal.1998;Garcia-
Verdugoetal.1998;Gage2000).Inprimates,BrdUstaininginadultmonkeySVZ
(McDermottandLantos1991)supportedpreviousworkwithH3-thymidine(Ka-
plan 1983). Subsequently, double-labeling experiments using BrdU and various
cellmarkersprovidedevidencefortheadditionofnewneuronstoadultmonkey
DG(Gouldetal.1998;Gouldetal.1999a;KornackandRakic1999)andolfactory
bulb(KornackandRakic2001a;Bedardetal.2002a).Importantly,incorporation
ofBrdUinDGneuronswasdetectedinthehippocampusofadulthumansupto
781daysafterBrdUinjection(Erikssonetal.1998).Asaconsequence,thedogma
thatnonewneuronsareaddedtotheadultbrainneededtobereconsidered(Gross
2000).
WhilethepresenceofneurogenesisinadultmonkeyDGandolfactorybulbis
nowgenerallyaccepted,itsexistenceoutsidetheseregionsremainscontroversial.
Reports of neuronal renewal in normal monkey neocortex (Gould et al. 1999b,
2001; Bernier et al. 2002) were challenged (Kornack and Rakic 1999b; Koketsu
et al. 2003), and a similar controversy exists for rodents, with both the absence
(Magavi et al. 2000)and presence (Dayer et al. 2005)of neurogenesis in normal
neocortexbeingclaimed.Adultneurogenesisinthestriatum(Bedardetal.2002b)
andamygdala(Bernieretal.2002)ofmonkeyshasbeenproposed.Whilestudiesin
monkeysmostlyinvolvenormalanimals,onereportshowedalterationsofmonkey
DGprogenitorproliferationafterstress(Gouldetal.1998),thusdemonstratingthe
abilityofprimateprecursorstorespondtochangesintheenvironment.Inaddition,
severalpapershavereportedinvitroneurogenesisinhumanhippocampus(Roy
etal.2000),olfactorybulb(Paganoetal.2000),andcortex(Nunesetal.2003).
1.2
MethodologicalConsiderationsinDetectingCellProliferation
Forpracticalpurposesthemethodsusedtostudycellproliferationcanbedivided
into two groups: (1) those that allow labeling of both dividing cells and their
postmitoticdaughtercells;and(2)thosethatselectivelyidentifydividingcellsbut
cannotidentifytheirpostmitoticprogeny.
The H3-thymidine and BrdU methods label both dividing cells and their
progeny.ThesechemicalsbecomeincorporatedintoDNAofdividingcellsduring
theSphaseofthecellcycle,andarecarriedintothedaughtercells.However,strictly
speakingH3-thymidineandBrdUindicateDNAsynthesis,butdonotclearlyprove
that cell division has occurred (Nowakowski and Hayes 2001). This distinction
is important as there are several instances when BrdU (or H3-thymidine) can
be incorporated into DNA by nonmitotic processes such as DNA repair (Rakic
2002a,b),apoptosis(Katchanovetal.2001;Kuanetal.2004),orthedevelopment
of polyploidy (Yanget al. 2001).As cell proliferation is frequently studiedusing
