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UnderstandingthePathologyofAlzheimer’s
DiseaseintheSubstantiaNigraoftheBrainstem
throughUnbiasedStereologyHimaRajana
LeaT.GrinbergLab
UCSFMemoryandAgingCenter
November2014
StructuredAbstract:
Background:Alzheimer’sDisease(AD)affectsmillionsofpeopleworldwide,and
withouragingpopulation,theprevalenceofADisonlyincreasing.Recentresearchshows
thatADpathologybeginsinthebrainstemevenbeforeclinicalsymptoms,suchasmemory
lossandimpairedcognition,appear.Thesubstantianigra(SN)isadopamineproducing
nucleusinthebrainstemcloselytiedtoothernucleithathaveshownchangesinearlyAD,
suchasthelocuscoeruleus.ThepresentstudyanalyzeschangesintheSNbywayoftau-
proteinaccumulation,ahallmarkofAD,inbrainsofearlyADpatientsusingunbiased
stereology.Methods:Humanbrainstemsfromsevensubjectsaged46-71withearlyAD
wereobtainedfromtheBrainBankoftheBrazilianAgingBrainStudyGroup,fixedin
celloidin,andprocessedusingimmunohistochemistryforunbiasedstereologicalanalysis
toquantitativelycharacterizethetau-proteinburden.Results:Althoughtherewasasmall
samplesizeofonlysevenbrains,thereappearstobeapositiverelationshipbetweentau
burdenandageofpatients,whichistrendingtowardsignificance.Therewasnocorrelation
betweenBraakstageandtauburden.ConclusionsandFurtherResearch:Thefindings
areinlinewithrecentlypublishedworkdescribinganincreaseintauburdenwithage,
independentofAD.However,thesmallsamplesizegivesuslittlepowerwhenmaking
conclusions,andforthisreason,animmediateexpansionofsamplesizeisnecessaryto
createanormativebaseforfurtherstudiesofearlyADintheSN.
I. Introduction
Asof2014,over5millionindividualsaged65andolderinAmericahavebeen
diagnosedwithAlzheimer’sdisease,alongwithanadditional200,000individualsunder65
diagnosedwithearly-onsetAlzheimer’sdisease(Alz.org).Currently,oneinninepeople,or
11%ofthepopulationaged65orolderhasorwillhaveAlzheimer’sdisease(Alz.org).By
2030,seniorcitizensareexpectedtomakeup19%oftheAmericanpopulation,compared
to12.4%intheyear2000,withthenumberofseniorsdoublingto72.1millionindividuals
(AdministrationonAging).Becauseoftheincreasingnumberofindividualsaged65and
olderintheUnitedStates,thenumberofnewcasesforAlzheimer’sandotherdementiasis
predictedtodoublebytheyear2050(Alz.org).
Aseveryindividualages,thebrainchanges,andtheybegintodevelopplaquesand
neurofibrillarytangles.Theprevalenceofplaquesandneurofibrillarytanglesin
Alzheimer’sdisease,however,ispurportedlymuchhigher(Hardy2002),andincreases
withtheprogressionofthedisease.Plaquesoccurwhenpiecesofthebeta-amyloidprotein,
whichisapartofthemembranesurroundingneurons,startstocomeoffthemembrane
andclumptogetheroutsidethecells.Sincethereisnogeneticchangeintheamyloidbeta
precursorprotein,weknowthatplaquesarecausedbytranscriptionalorposttranslational
changes(Selkoeet.all1988).Neurofibrillarytanglesarecausedbyhyperphosphorylated
tauproteinsinsidecells.ThetaugenecodesforRNA,whichinturn,codesforthetau
protein.Theproteinhasseveralepitopes,whichareactivatedandinactivatedby
phosphorylation.Thiscanbecomparedtothecockpitofaplane;eachofthemanybuttons
controlsaspecificfunction,andcanbeturnedonandoff.Theabnormallyfunctioningtau
protein,whichusuallysupportsthemicrotubules(MTs)inneurons,nolongerhasthesame
affinityfortheMTs(Geschwind2003).Subsequently,theMTsinneuronscollapse,
combinationofplaquesandtanglesimpedesnormalfunctionsincells,andeventually
precipitatescelldeath.
Thehyperphosphorylated,andsubsequentlymisfolded,tauproteinspreadsthroughout
thebrainfollowingacharacteristicpattern.TheBraakstagingsystem,initiallypublishedin
1991,isaqualitativemethodofcategorizingthedegreeofADpathologyinpostmortem
brainsfromstagesItoVI(BraakandBraak1991).Forthisstudyofchangesinthebrainin
earlyAD,brainswithBraakstages0toIIwereused.In1991,itwasestablishedthatthere
islittletonotau-positiveneurofibrillarytangleburdenintheneocortex,theupperpartof
thecerebralcortexinearlystages.Becausethecerebralcortexhousesareasdealingwith
cognitivefunction,suchaslanguage,memory,motorcommands,andspatiallanguage,the
disease-definingsymptomsofADarenotmanifesteduntilthelaterstages.TheBraak
stagingsystemisstillusedtoday,butwasreviewedandmodifiedin2011toinclude
changesinthelocuscoeruleus,asubcorticalnucleus,intheearlystages(Grinberget.al
2011).Thisexplainshowdiseasepathologydevelopsevenbeforeclinicalsymptoms
appear,andfurtherresearchcouldleadtoaneffectivetreatmentthattargetsADbeforeit
spreadsintotheneocortex.
TheneuronallossinADoriginatesinanareaofthebrainstemknownasthe
isodendriticcore.Theisodendriticcoreismadeupoffourmainnuclei:thedorsalraphe,
locuscoeruleus,parabrachialnucleus,,andthesubstantianigra(SN).Thesubstantianigra
isaffectedinseveralneurodegenerativediseases,includingintheearlystagesof
Parkinson’sdisease(Braak2003),andproducestheneurotransmitterdopamine,whichis
knowntoplayaroleinhappinessandthebrain’srewardsystem.Mostdopamine-producer
neuronsoftheSNalsoharborneuromelanin,apigmentthatcausesthecharacteristicdark
coloroftheSN.
Asthediseaseprogresses,thedegreeofdementiaiscloselyrelatedtoneuronallossin
ADpatients.Whilewedoknowthatneurofibrillarytanglesandneuronallossarehallmarks
ofAD,wedonotknowif,orhow,theyarerelated.Thereisagapofstudiesdoneofwell-
characterizedindividualstounderstandthetwolesionsinthecontextofoneanother.The
overallgoalofthelabthatthisstudywasconductedat,theGrinbergLab(Memoryand
AgingCenter,UCSF),istoquantitativelystudythisrelationshipbetweenproteinbuildup
andneuronallossthroughoutthefourmajornucleioftheisodendriticcoreusing
innovativemethods.Myproject,specifically,focusesonanucleusoftheisodendriticcore
knownasthesubstantianigra,andthisisfurtherdiscussedbelow.
DespitealltheeffortsandmoneytoputintounderstandingandcuringADoverthelast
threedecades,includingallkindsofsophisticatedexperimentalmodelsdesignedtomimic
thedisease,excellenttreatmentresultsondrugstriedintheseanimalmodelsandhuge
effortsinhumanclinicaltrialsusingthesamedrugs,weareyettofindsomethingtocureor
evendelaytheprogressionofAD.Atthispoint,wemustgobacktothefundamentalsofAD
pathology,andlookathowthediseaseprogressesinhumans.Becausetherehasbeenlittle
successintranslatingpositiveresultsfromanimalmodelstohumanmodelsbeyondearly
stageclinicaltrials,itisimportantthatwegainathoroughunderstandingofthewayAD
functionsinthehumanbrainbeforeidentifyingtherapeutictargetsanddeveloping
treatments.
Currently,littledataisavailableasabaselineofnormativechangesintheSNthrough
aging,sothisstudyseekstocreatealibraryofdataforthepathologyofADintheSN.
Additionally,thechangesintheSNduringaging,suchasthenaturaloccurrenceoftangles
andplaques,aswellasneuronalloss,arestillcontroversial.SincetheSNisbilateral,itis
alsonotknownifthebuild-upoftanglesandplaquesdiffersontheleftandrightsides
(Alho2014)
InlookingattheSNthroughthelensofAD,weemployadoublestainingtechniqueto
identifyboththetau-negativeneurons,stainedwithgallocyanine,andthetau-positive
neurons,stainedwithCP13toanalyzechangesintheSNwithageandwithprogressionof
AD(Theofilas2014).WelackdataonwhatcharacterizesthepathologyofearlyADinthe
brainstem.Theresultscontributetoabaseofknowledgeforimaging,clinical,and
anatomicalstudiesoftheSNintheearlystagesofAD.MyhypothesisisthatbecausetheSN
isapartoftheisodendriticcoreandconnectedtoothernucleioftheisodentriticcore
vulnerabletoAD,therewillalsobechangesintheSNinearlyAD.However,becauseof
whatisknownofBraakstages,wherethereislittleproteinburdenintheearlystagesof
AD,therewillbecorrelationbetweenageandtauburden.
II. Methods:
Priortomyanalysisofthetissueusingunbiasedstereology,thebrainstemwascutinto60
micrometersectionsandprocessedusingimmunohistochemistryandspecificbrainareas
weresampledaccordingtoBBBABSGprotocol(Grinbergetal.2007).Ashortsummaryof
thismethodfollows.
a. Participants:
Ageoftimeofdeath Case BraakStage Gender
44 7020.12 0 Female
46 9379.13 2 Female
47 7678.13 0 Female
56 9526.12 1 Male
70 6664.12 1 Female
71 6366.13 0 Male
71 6931.12 2 Female
Brainstemtissuefrom7individualsobtainedfromtheBBBABSGwasused.Grinbergand
colleagueshavedescribedtheBBBABSGprotocolindetail.
b.EmbeddingandSectioning
Thebrainstemwasembeddedincelloidintoreducetissuedistortion,byfirst
dehydratingitwithprogressivelystrongerethanolsolutionsandthengoingthrougha
dessicationprocesstostronglysecurethetissue.Whenenoughliquidhasbeenremoved
fromthecelloidintoreachanIndiarubberconsistency,theblocksweresectionedusinga
slidingmicrotome.Brainstemsweresectionedalternatelyintoone300micrometerand
five60micrometersections.Thethicksectionsaretheodd-numberedsections,usedto
calculateanunbiasedestimatefortheneuronalpopulationofthesixnucleiinthe
isodendriticcore.Theeven60micrometersections,usedtoevaluatethetauprotein
burdenineachofthenuclei.AcomparisonoftheburdenintheearlyoflatestagesofAD,as
wellaswithcontrols,isdoneinthe60micrometersections.Duringthecuttingprocess,
eachsectionwasphotographedusinganEOS5DMarkII.
b. StainingandImmunohistochemistry
`Thethin60micrometersectionswerefirststainedusingamonoclonalCP13
antibody,whichstainedforbothcytoplasmicboundandextracellularphosphorylatedtau.
Beyondthis,theywerecounterstainedovernightin2.0pHgallocyaninetostainthe
nucleoliandcreatefurthercontrastbetweentaupositiveneuronsandsurroundingcells.
Then,thesectionsweremountedtoslides,coverslipped,andlabeledbynumber.
c. StereologyintheSubstantiaNigra
Intermsofphysicalorientation,theSNisabilateralnucleuslocatedinthemidbrain,
adjacenttothecerebralpeduncles.Itisdividedintothreeparts:theparcompacta,parts
diffusa,andparsreticulata.Theparscompactahasthehighestneurondensityandismade
upofthelargest,mostpigmentedneurons.Theparsdiffusaisnotasdense,andhassmaller,
lesspigmentedneurons,althoughtherearesomeclustersofneuronsresemblingthose
foundintheparscompacta.Finally,theparsreticulatacontainsthickdendritesofthe
neuronsintheparscompactaregion,aswellasafewspread-outneuronslackingthe
neuromelaninpigment.
StereologicalanalyseswereperformedusingtheStereoInvestigatorprogram(MBF
StereoInvestigatorv.10,MBFBioscience,Williston,VT,USA).Thisset-upwascomprisedof
amotorizedstagesystem,whichallowedmovementofthestagetoviewadifferentsection
oftheslideifnecessary,abrightfieldmicroscope(AxioA2,ZeissMicroscopy,Thornwood,
NY,USA),andacolorcameratofacilitateuseofthemicroscopeonanexternalmonitor.
Figure1:Thestereologyset-uprequiresabrightfieldmicroscopewithamotorizedstageanda
motorizedstagecontrollertoadjustviewingframe.
Basedonpreviousstereologicalanalyses,itwasdeterminedthatcountingevery
otherevensection,asopposedtoeveryevensection,producedaccurateresults,sothis
processhasbeenimplementedinordertoincreaseefficiency.Priortobeginning
stereologicalanalysis,itisimperativetofindtheoptimalparameterstoestimateneuronal
numbers.Indoingso,thesubstantianigrawasfirstdelineatedusingtwoseparatecontours
forthetwoseparatesidesusingthe5x/0.16objective,asoutlinedintheoptical
fractionatorworkflowofStereoInvestigator.Theboundariesoftheregionofinterestwere
decidedbasedonvisualcuessuchasincreasinglyspareneuronsandtheAtlasofthe
CytoarchitectureoftheHumanBrainstem(OlszewskiandBaxter1982)CP13positivecells
weremarkedinthe40x/1.30oilobjective.BothobjectivesarefromZeissMicroscopy.The
cellswerecountedusingtheStereoInvestigatorOpticalFractionatorworkflow,as
describedinpreviousworks(SchmitzandHof2007).
d. Resample-OversampleProbe
Followingtheinitialcountingofcellsinthepilotstudy,theStereoInvestigator
Resample-Oversampleprobeisruntohelpdeterminetheoptimalparameters.Thegoalof
thepilotstudyistodeterminetheminimumcountingandsamplingrequiredtoensure
accuracyofsampling.Inotherwords,thegoalistocountenoughcellswithminimalwork.
Theresample-oversampleprocessbeginswithanexhaustiveanalysisofeachandevery
siteinthetissueinpredeterminedblocks.Afterthis,theprogramusesaformulato
determinetheparameterssuchthatthecounterisgettingthemostaccurateresultswith
theleastamountofworkpossible.
Intheoversampleparameters,thegridsizewasautomaticallygenerated,andthe
opticalfractionatortopguardzonewassetas5micrometersanddissectorheightat25
micrometers,basedontheminimumsectionthickness.Sectionthicknesswasmeasured
manuallyusingamotortogothroughthez-axiseachtimeaneuronwascounted,andthe
measureusedincalculationswasanaverageofallthicknessmeasurements.Newresample
parameterswereestablisheduponrunningtheprobeandevaluatingthedataplottedinthe
resampleoversamplegraph.
Figure2:Thisgraph,generatedusingtheResampleOversampleprobein
StereoInvestigator,wasgeneratedinMicrosoftExcel.Itshowshowclosethe
neuronalestimatesareincountingeveryintervalofdissectorsites,fromevery
sitetoevery20thsite.Accordingtothisgraph,theoptimalintervalisevery3rd
dissectorsite,becausetheestimatesusingone,two,andthreecountersare
closesttoeachother.Therefore,countingeverythirddissectorsitewillallowus
tomaintainaccuracywithminimalwork.
Thepilotstudyisrecountedaccordingtotheparametersgivenbytheresample
oversamplegraph,andthedataiscomparedtodeterminehowclosetheresampleand
oversampleestimatesofneuronalpopulationsare.Theoretically,theyshouldbethesame,
becausetheparametersarecalculatedsuchthatthesamelevelofaccuracyismaintained,
evenwithfewerdissectorsites.Iftheestimatesaresignificantlydifferent,theoversample-
resampleprocessisrepeateduntiltheestimatesarereliableandcanbeusedtocount
additionalcasesusingthesameparameters.
ACP13-negativeneuronisstainedlightbrownbecauseoftheneuromelanintheSN,
whilethenucleusandnucleolusarestainedbluebecauseofthegallocyaninestain.Only
neuronswherethenucleusandnucleolusareclearlyvisiblearecounted,becausethe
sectioningprocesscancutoffneuronsalongthez-axis,creatingfragmentsthatcanskew
neuronalestimatesifcountedasneurons.Additionally,usingthenucleolusandnucleusas
markersinstereologyisinlinewiththestainingtechniquesdescribedinthestaining
section.IncomparisontotheCP13-negativeneurons,CP13-postiveneuronsarecoveredin
adarkbrownblanketofCP13stain.Thestainingisapproximatelyuniformacrossthecell
Figure2:Thesubstantianigraistracedat5x.Thisimageisthecompleteright
sideofthenucleus.Thecharacteristicwavystructure,wherethecontourgoes
inandout,followingthelinesoftheSN,isvisiblehere.
body,andthenucleolusmaynotbevisible.Inmostcases,granulesarevisible.TheCP13
stainalsopenetratesintothecellprocessesastheneuronsinteractwithoneanother,
makingthedendritesoftheneuronclearlyvisibleastheyreachbeyondthecellbody.
Figure4:CP13-stainedneuron,whereCP13blanketsthecellevenlyand
granulesarevisible.ThepresenceofCP13stainincellprocessesdelineatesthe
dendritesoftheneuron.
Inordertoensurethateachneuroniscountedamaximumofonetimewhenthe
programplacesthedissectorrandomly,anyneuronsincontactwiththered-edgedpartof
thesquare(seebelowfigureforreference)werenotcounted,eveniftheymeetthecriteria
accordingtothepreviousparagraph.Anyneuronstouchingthegreen-edgedpartofthe
square,whethercompletelyorpartiallyinsidethesquare,arecounted.Additionally,
severaldissectorsintersectedwiththeborderofthecontour,asshownintheimagebelow.
Figure5:Thesubstantianigracontourisbrokenintomanycountingframes,andthe
dissectorsite,wheretheusercountsneurons,isconsistentlyinthebottomleft
corner.Thesmallerthecountingframe,themoredissectorsitestocount.
Figure5:Thegallocyanine-stainedneuronsaresmaller,andadarkerareaof
thenucleusandnucleolus,wherethestainisdeeper,isvisible.
A
At40x,itisquitedifficulttotellwherethedissectorisinrelationtothecontourasawhole
andrelativetothepositionoftheslide,sohavinganoverallmapofthecontoursindicating
wherethedissectoristellsushowthedissectorfallsonthecontour,andconsequently
whichneuronscanbecountedandwhichonesfalloutsidethepreviouslydelineated
contourisnecessary.TheMacroviewfeatureonStereologyinvestigatorhelpsovercome
thisissue,asshownintheimagebelow.
e. StatisticalAnalysis
Theresampleoversampleprobeusedaformulaembeddedintheprogramtocalculate
theappropriateintervalfortheresample.Thecoefficientsoferrorforthetau-positive
neuronalestimateswerecalculatedusingthepredictionmethodsfromthe
StereoInvestigator,theGundersenandSchmitz-Hof’scoefficientoferrormeasurements.
Figure6:TheMacroviewfeature,locatedintheupperlefthandcorner,shows
theentirecontour,withasmallerboxindicatingwherethedissectorsite,
displayedontherightsideat40x,isinrelationtothewholecontour.Becauseof
this,weknowtocountonlytheneuronsontheleftsideoftheorangecontour
line,sincetheyaretheonlyonesinsidethecontour.
Thesemeasurementsareusefulfordeterminingtheprecisionofneuronalestimates,and
havebeendescribedpreviously(Gundersenetal.1999;Schmitz,1998).Plannedstatistical
analysisforfurtherstudiesfollowsinthefurtherresearchsection.
III. Results
The60micometergallocyanine-stainedsectionsfulfilledthebasicrequirementsof
unbiaseddesign-basedstereology,whichhasproventobetheoptimalmethodforcell
countingtimeandagainforitsabilitiestodetectthemostminutecellgroupdifferences
(SchmitzandHof2005).Everycellinsidetheregionofinteresthadequalopportunitytobe
selectedforcountingfortwomainreasons.First,theserialsectioningofthebrainstem
allowedustoevaluatethefullthicknessoftheSNalongthez-axis,sowedidnotmissany
neurons.Second,theboundariesoftheSNwereeasilydetectable,andwewereableto
drawcontoursaroundallofthem.Althoughthesectionsweresupposedlyall60
micrometersthick,thicknesswasmeasuredtoaccountforanyshrinkageordiscrepancies
incutting.Thesectionthicknessofeachcasewasanaverageofthemeasuredsection
thicknessesoreachdissectorsites.Thishelpedaccountforanyfolding,waviness,or
Table1:Thistableshowsallofthedta
warpingofthetissue.Themeansectionthicknessacrossallcaseswas48.7micrometers
withastandarddeviationof0.28micrometers.
Throughtheresampleoversampleprobe,IdeterminedthatIcouldcounteverythird
dissectorsitewithoutlosingaccuracy.Duetotheexperimentalnatureofstereology,Ihad
toconducttheoversample-resamplefourtimesbeforereceivingsatisfactoryresults.Iwas
thenabletousetheseparameterstocountsixothercases.Originally,IcountednotCP13-
negativecells,inadditiontothedataofCP13positiveneuronspresentedinthispaper.
Unfortunately,therewereproblemswiththecounterstainintheimmunohistochemistry
process,sotheseresultshavebeendeemedunreliable.Boththeresample-oversampleand
theCP13-negativecellissueswillbeexplainedfurtherinthediscussionsection.
Ofthesevencasescounted,fourappearedtohavenotauburden,andtheremaining
threehadsimilarburdens.Afterblindcounting,theBraakstagesandageofpatientwere
matchedwiththedata,andwetestedforcorrelation.Basedonthisdata,thereisno
correlationbetweentauburdenandBraakstage,asallofthecasesthathadtauburdens
wereBraakstage0or1.Whilethesamplesizeistoosmalltoclaimacorrelationbetween
tauburdenandage,thereisapositivetrendbetweenageandtauburden.Outofthree
patientsolderthan65attimeofdeath,eachhadadifferentBraakstageof0,1,and2,but
twoofthethreeshowedsimilarpresenceofneurofibrillarytangles.Outofthe4patients
underage65attimeofdeath,only1patientshowedanytauburden,andthelevelwas
similartothetwopatientsover65.Thus,thepositivecorrelationbetweenageandtau
burdenistrendingtowardssignificance,andtheimmediatecourseofactionwouldbeto
expandthesamplesizeofthisstudy.Thiswouldallowustodetermineifthecorrelationis
simplytheresultofasmallsamplesizeorcanbesupportedwithfurtherdata.
IV. Discussion
Asourworldwidepopulationages,theprevalenceofneurodegenerativediseases,
especiallydementiaandAlzheimer’s,isincreasing.Atthesametime,thescientific
community’seffortstoworktowardsacure,oreventreatmentofAlzheimer’sdisease,have
beenstalledbythedifficulttransitionfromanimalmodelstohumanmodels.Something
abouthumanbrainsisdifferentenoughthatwhateversemblanceofprogressisachievedin
animalmodelsdoesnottranslatetohumanmodels.Forthisreason,itisnecessarytostudy
thewayADprogressesinhumansspecifically,becausethereisagapofknowledgethatcan
onlybefilledbyusingpostmortembraintissuehelpcharacterizeearlystagesofAD.
Currently,theseisalackofunbiaseddataonthesubstantianigra,akeynuceusofthe
isodendriticcore,knownchieflyforthedarkpigmentasaresultofdopamineproduction.
Althoughweknowofsomechangesintheothernucleioftheisodendriticcore,including
thelocuscoeruleus,littleisknownabouttheSNinthecontextofAD.Becauseoneofthe
hallmarksofADistauproteinbuildup,weanalyzedproteinbuildupintheSNusing
unbiasedstereologyinapost-mortemsampleof7adultsaged46-71attimeofdeath.
Quantitativeneuropathologicalmethodsaretimeconsuming,tediousandusuallybiased
duetotheenourmousamontofneuronsinthebrain.Design-basedstereologyis
transformingthewayquantitativeneuropathologyisperformed,allowingustomake
predictionsfortheneuronnumberanddensityofatissuewithoutcountingeachandevery
neuronthroughanestablishmentofparametersusingaresample-oversampleprocess.In
addition,therandomnessofstereologyallowsustomakeunbiasedmeasurementsandbe
abletotrustinthevalidityofresults.
Althoughthereseemedtobenodiscernablerelationshipbetweentauburdenand
Braakstage,theredoesappeartobeapositiverelationshipbetweenageandtauburden,
trendingtowardsignificance.Thisisinlinewithaveryrecentlypublishedpaperontau
buildupasrelatedtoageinthebrainstem.Craryandcolleaguessuggesttheuseofanew
term:primaryage-relatedtauopathy(PART)todescribetaubuildupinthebrainthat
seemstobetiedonlytoage(Craryet.al.2014).Pathologically,thiscanbedistinguished
fromAD,frontotemporaldementia,orotherneurodegenerativediseasesbythelackofthe
beta-amyloidplaques.Althoughthelackofbeta-amyloidplaquespointsoadiagnosesis
earlyAD,therearesomeclinicaldifferences,sincePARTusuallyhasalessercognitive
impact.LookingatPARTthroughthelensofAD,Craryandcolleaguesfoundthatmany
patientswithmild-to-moderateneurofibrillaryburdensimilartoearlystageADlackedthe
betaamyloidplaquescharacteristicofAD.Thus,theneurofibrillarytanglesmaybe
involvedinanonADagingrelatedprocess.TheincidenceofPARTwasmuchhigherin
olderpatients,whichsupportsthepossiblepositiverelationshipbetweentauburdenand
age.
EventhoughmyfindingsaresupportedbyCrary’swork,itisdifficulttoconfirm
anythingworkingwithsuchasmallsamplesize,especiallyinhumans.Humansaresuch
diverse,varied,anduniquebeingsthatvariationbecomesnottheexceptionbutthenorm.
Forthisreason,manycasesarenecessarytodrawageneralconclusionthatcanbeapplied
totheentirepopulation,andthisstudydoesnothavethehighsamplesize.Becauseofthe
gapofknowledgeconsideringthesubstantianigraandAD,weareunabletocalculatethe
exactnumberofcasesneededtohavepowerintermsofstatistics.Thebiggestreasonfor
thesmallsamplesizeinthisstudyisthedifficultyofprocuringhumanbrainswiththeearly
ADdiagnosis.Thiskindofanalysiscanonlybedonepostmortem,andprocessingthe
brainsforstereologyinhumanmodelstakesaverylongtimecomparedtoratbrains,
whicharemuchsmaller.Itisstillimportanttostudythisinhumansbecausethedisease
manifestsitselfinhumans.Therefore,themaingoalofthisstudyisnotnecessarilytomake
sweepingconclusionaboutthenatureofADintheSN,butrathertobuildaknowledgebase
tocharacterizetheSNinearlyAD.AsIwritethispaper,Iamintheprocessofincreasing
samplesize,andbytheendofthiswinter,Ishouldhavearound20cases,whichgivesmuch
morepowerintermsofbeingrepresentativeofearlyADbrains,thanseven.
Stereologyisanexperimentalprocessinandofitself,becausetheResample
Oversampleprobeallowstheusertotailortheprogramtothetypeoftissuebeingcounted.
Priortoanystereologicalanalysisofmultiplecases,itisnecessarytorunacomplexpilot
studytoensurethatresultsarenotonlyreliable,butcanalsobereplicatedinotherlabs.As
mentionedintheresultssections,Ihadtoconducttheoversampleprobefourtimestoget
satisfactoryresults.ThealgorithminStereoInvestigatorrecommendedthatIcountevery
thirddissectorsitethreetimesinallfouroccurrences.Theoretically,theoverallneuronal
estimatesfortheoversampleandtheresampleshouldbethesame,butthatwasn’tthecase
intheinitialoversample.Throughouttheprocess,issueswithinconsistentneuron
estimates,inconsistentthicknessmeasurements,andinaccuratelydrawncontours
promptedmetoconducttheanalysisagain.Finally,onthefourthround,Ihadreliable
parameters,whichIthenusedtocountsixadditionalcases.
IoriginallycountedbothCP13positiveandCP13negativeneurons.Uponreviewofthe
resultsandcomparisonstootherstereologicalestimatesofsubstantianigraneuron
population,mynumberswereonly10-20%ofothers’results.Weevaluatedmycriteriafor
countingneuronsandthecountingparametersandeventuallyconcludedthatthe
inaccurateresultswerearesultofafaultycounterstain.AftertheCP13stainwasappliedto
thesections,theywerecounterstainedinagallocyaninewashtoilluminatetheCP13
negativeneurons.The60micrometersectionsmayhavebeenslightlytoothinforthe
harshchemicals,ormayhavereactednegativelytothetestedconcentrationofdetergentin
thesolution.Inordertoaccountforthisinfurtherstudies,neuronalestimateswillnowbe
measuringfromonlythethick300micrometersectionsofeachcase.Asofnow,the
stainingandstereologyprotocolhasbeenoptimizedforthe300micrometersections,with
consistentlyreliableresults.Aswehavelearnedfromthisstudy,theCP13staindone
throughimmunohistochemistryworkswellonthethin60micrometersections.Asperthe
cuttingprotocolfollowedbytheGrinberglab,300micrometersectionsand60micrometer
sectionsarecutalternately,sothe300micrometersectionsareanaccuraterepresentation
ofthe60micrometersections.Inordertounderstandtherelationshipanddynamic
betweenneuronallossandtauproteinaccumulation,wewillnowutilizeacombinationof
datafromthe300micrometersections,forneuronalestimates,and60micrometer
sections,forproteinburdenestimates.
V. ConclusionsandFurtherResearch
Thisstudywasaninvestigationofthehumansubstantianigraintheearlystagesof
AD.Thereisapositivetrendbetweenageandtauproteinaccumulation,andasofnow,no
relationshipbetweenBraakstageandtauburden.However,thesmallsamplesizegivesus
verylittlepowerinmakingapplicableconclusions.Theimmediatenextstepistoincrease
thesamplesizeinordertorunappropriatestatisticalanalysis.Theanalysiswilltestthe
individualcorrelationsbycomparinganatomicalchanges,suchasinclusionburden,with
ageandBraakstageusinglinearregressionanalysis.Theregressionmodelswillinclude
indicatorsforADgroupaswellasageatdeath.
Additionally,inordertoinvestigatetheroleofthesubstantianigrainAD,and
perhapslookfurtherintothestillundiscoveredrelationshipbetweenSNproteinburden
andBraakstage,wecanuseneuronalestimatesfromthe300micrometersections.
Comparingtheoverallneuronalestimatestothetau-positiveneuronestimateswould
enableustocalculateafractionoftheneuronsintheSNaretaupositiveatdifferentstages
ofearlyAD.ThisfractioncouldserveasametricusedtocharacterizeeachstageofADand
understandthepathwaythatADtakesthroughtheSN.Beyondthis,comparingthedata
fromtheSNtoconclusionsdrawninothernucleioftheisodendriticcorecouldshedlight
onADthroughoutthebrainstem,leadingtopotentialtherapeutictargetsfordrug
development.
Bibliography
"AdministrationonAging(AoA)."AgingStatistics.AdministrationforCommunityLiving,31
Dec.2000.Web.09Nov.2014.
Alho,AnaTerezaDiLorenzo."Three-dimensionalandStereologicalCharacterizationofthe
HumanSubstantiaNigraduringAging."(2014):n.pag.Web.2Nov.2014.
Association,Alzheimer's."2014Alzheimer’sDiseaseFactsandFigures."Alzheimer’s&
Dementia10.2(2014):n.pag.Alz.org.Alzheimer'sAssociation.Web.21Sept.
2014.
Braak,H.,andE.Braak."NeuropathologicalStageingofAlzheimer-relatedChanges."Acta
Neuropathologica82.4(1991):239-59.Web.21Oct.2014.
Braak,Heiko,K.DelTredi,U.Rub,andRADeVos."StagingofBrainPathologyRelatedto
SporadicParkinson'sDisease."TheNeurobiologyofAging24.2(2003):197-211.
Web.7Oct.2014.
Buttner-Ennever,JeanA.,A.K.E.Horn,andJerzyOlszewski.OlszewskiandBaxter's
CytoarchitectureoftheHumanBrainstem.N.p.:Karger,n.d.Print.
Crary,JohnF.,JohnQ.Trojanowski,JulieA.Scheider,andJoseF.Abisambra."PrimaryAge-
relatedTauopathy(PART):ACommonPathologyAssociatedwithHumanAging."
ActaNeuropathologica(2014):n.pag.Web.1Nov.2014.
Geschwind,DanielH."TauPhosphorylation,Tangles,andNeurodegeneration:TheChicken
ortheEgg?"Neuron40(2003):457-60.Web.15Sept.2014.
Grinberg,LeaTenenholz,RenataEloahLucenaFerretti,JoséMarceloFarfel,RenataLeite,
CarlosAugustoPasqualucci,SérgioRosemberg,RicardoNitrini,PauloHilário
NascimentoSaldiva,andWilsonJacobFilho."BrainBankoftheBrazilianAging
BrainStudyGroup—aMilestoneReachedandMorethan1,600CollectedBrains."
CellandTissueBanking8.2(2007):151-62.Web.18Sept.2014.
Grinberg,LeaTenenholz,UdoRueb,andHelmutHeinsen."Brainstem:NeglectedLocusin
NeurodegenerativeDiseases."FrontiersinNeurology2(2011):n.pag.Web.3Oct.
2014.
Gundersen,H.J.G.,E.B.V.Jensen,K.Kieu,andJ.Nielsen."TheEfficiencyofSystematic
SamplinginStereology-Reconsidered."JournalofMicroscopy193.3(1999):199-
211.Web.1Nov.2014.
Hardy,J."TheAmyloidHypothesisofAlzheimer'sDisease:ProgressandProblemsonthe
RoadtoTherapeutics."Science297.5580(2002):353-56.Web.10Sept.2014.
Kazee,AnnMarie,ChristopherCox,andEricK.Richfield."SubstantiaNigraLesionsin
AlzheimerDiseaseandNormalAging."AlzheimerDisease&AssociatedDisorders
9.2(1995):61-67.Web.19Oct.2014.
"MedicalNeurosciences."MedicalNeurosciences.UniversityofWisconsinMadison,n.d.
Web.13Oct.2014.
Schmitz,C.,andP.r.Hof."Design-basedStereologyinNeuroscience."Neuroscience130.4
(2005):813-31.Web.29Oct.2014.
Schmitz,Christoph."VariationofFractionatorEstimatesandItsPrediction."Anatomyand
Embryology198.5(1998):371-97.Web.1Nov.2014.
Selkoe,DennisJ."F3-AmyloidPrecursorProteinofAlzheimerDiseaseOccursas110-to
135-kilodaltonMembrane-associatedProteinsinNeuralandNonneuralTissues."
MedicalSciences85(1988):7341-345.Web.14Oct.2014.
Theofilas,Panos,LiviaPolichiso,XuehuaWang,LuziaC.Lima,AnaT.l.Alho,RenataE.p.
Leite,ClaudiaK.Suemoto,CarlosA.Pasqualucci,WilsonJacob-Filho,Helmut
Heinsen,andLeaT.Grinberg."ANovelApproachforIntegrativeStudieson
NeurodegenerativeDiseasesinHumanBrains."JournalofNeuroscienceMethods
226(2014):171-83.Web.27Sept.2014.
Yoshiyama,Yasumasa,BinZhang,andJenniferBruce."ReductionofDetyrosinated
MicrotubulesandGolgiFragmentationAreLinkedtoTau-inducedDegeneration
inAstrocytes."JournalofNeuroscience23.33(2003):10662-0671.Web.16Aug.
2014.