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Year: 2019

Vascular density and distribution in neocortex

Schmid, Franca ; Barrett, Matthew J P ; Jenny, Patrick ; Weber, Bruno

Abstract: An amazingly wide range of complex behavior emerges from the cerebral cortex. Much ofthe information processing that leads to these behaviors is performed in neocortical circuits that spanthroughout the six layers of the cortex. Maintaining this circuit activity requires substantial quantities ofoxygen and energy substrates, which are delivered by the complex yet well-organized and tightly-regulatedvascular system. In this review, we provide a detailed characterization of the most relevant anatomicaland functional features of the cortical vasculature. This includes a compilation of the available data onlaminar variation of vascular density and the topological aspects of the microvascular system. We alsoreview the spatio-temporal dynamics of cortical blood flow regulation and oxygenation, many aspectsof which remain poorly understood. Finally, we discuss some of the important implications of vasculardensity, distribution, oxygenation and blood flow regulation for (laminar) fMRI.

DOI: https://doi.org/10.1016/j.neuroimage.2017.06.046

Posted at the Zurich Open Repository and Archive, University of ZurichZORA URL: https://doi.org/10.5167/uzh-146003Journal ArticleAccepted Version

The following work is licensed under a Creative Commons: Attribution-NonCommercial-NoDerivatives4.0 International (CC BY-NC-ND 4.0) License.

Originally published at:Schmid, Franca; Barrett, Matthew J P; Jenny, Patrick; Weber, Bruno (2019). Vascular density anddistribution in neocortex. NeuroImage, 197:792-805.DOI: https://doi.org/10.1016/j.neuroimage.2017.06.046

1

Vasculardensityanddistributioninneocortex

FrancaSchmid1,MatthewJ.P.Barrett2,3,PatrickJenny1,BrunoWeber2,3

1. InstituteofFluidDynamics,ETHZurich,Sonneggstrasse3,8092Zurich,Switzerland

2. InstituteofPharmacologyandToxicology,UniversityofZurich,Winterthurerstrasse

190,CH-8057Zurich,Switzerland

3. NeuroscienceCenter,UniversityandETHZurich,Winterthurerstrasse190,CH-8057

Zurich,Switzerland

Correspondingauthor:FrancaSchmid,[email protected]

2

Abstract

Anamazinglywiderangeofcomplexbehavioremergesfromthecerebralcortex.Muchof

theinformationprocessingthatleadstothesebehaviorsisperformedinneocorticalcircuits

thatspanthroughoutthesixlayersofthecortex.Maintainingthiscircuitactivityrequires

substantialquantitiesofoxygenandenergysubstrates,whicharedeliveredbythecomplex

yetwell-organizedandtightly-regulatedvascularsystem.Inthisreview,weprovidea

detailedcharacterizationofthemostrelevantanatomicalandfunctionalfeaturesofthe

corticalvasculature.Thisincludesacompilationoftheavailabledataonlaminarvariationof

vasculardensityandthetopologicalaspectsofthemicrovascularsystem.Wealsoreview

thespatio-temporaldynamicsofcorticalbloodflowregulationandoxygenation,many

aspectsofwhichremainpoorlyunderstood.Finally,wediscusssomeoftheimportant

implicationsofvasculardensity,distribution,oxygenationandbloodflowregulationfor

(laminar)fMRI.

Keywords:corticalmicrovasculature,vasculardensity,neurovascularcoupling,

hemodynamicresponse,cerebraloxygenation,laminarcharacteristics

1 Introduction

Thebrainconsumesapproximatelyaquarterofthebody’stotalglucoseandafifthofthe

oxygen,anorderofmagnitudemorethanwhatwouldbeexpectedonaweightbasis.This

remarkableenergydemandrequiresarobustenergysupplyviathebloodstream,anda

complexcerebrovascularsystemhasevolvedtomeetthisdemand.Adetailedknowledgeof

thecerebralvasculatureiscrucialtounderstandthebasicprinciplesofcerebralbloodflow

(CBF),itscouplingtoneuralprocessing,andalsotounderstandnon-invasivehumanbrain

imaging.Giventheextraordinaryimportanceofstructuralandfunctionalmagnetic

resonanceimaging(fMRI),itisquitesurprisingthatquantitativedataregardingcerebral

bloodvesselsaresparse.Inparticular,themicrovascularsystemissignificantly

understudiedbothintermsofitsstructuralandfunctionalcharacteristics.Theaimofthe

presentreviewistoprovideadetailedoverviewofthecerebralmicrovascularsystem,with

afocusontheneocortexanditsimportanceforlaminarMRI.

Thehumanbrainreceivesapproximately15-20%ofthetotalcardiacoutputandthisblood

istransportedfromthetrunkviafourlargevessels,theleftandrightinternalcarotid

arteriesandtheleftandrightvertebralarteries.Beforeramifyingintothelargefeeding

arteriesofthebrain,thevertebralarteriesjoinintothebasalarteryandtogetherwiththe

carotidvesselsformaring-likestructurecalledthecircleofWillis.Thisstructureintroduces

redundancyandservesasaprecautionarymeasure.Themiddlecerebralartery’scirculation

territoryisthelargestandcompriseslargepartsofthefrontal,temporalandparietalcortex.

Withoutbeingtoodetailed,theposteriorcerebralarteryfeedstheinferiorandmedial

surfaceoftheoccipitalandtemporalcortices,whereastheanteriorcerebralarteryis

responsiblefortheperfusionofmedialaspectsofthefrontalandparietalcortex.Whereas

thefeedingterritoriesofthelargecerebralarteriesplayanimportantroleinthediagnosis

ofneurologicaldisorders,theyhaveonlyaminorimportanceforcorticalMRI.Ofmuch

greaterimportanceisthecerebralmicrovasculatureandhencethisreviewwillbefocused

3

onthemammaliancorticalmicrovasculature(forarecentreview,pleasealsoseeHirschet

al.,2012).

Themaindutyofthecerebrovascularsystemistodeliveroxygenandglucosetothetissue.

Thehumanbrainmetabolizesabout31mmolglucoseper100gramsoftissueperminuteto

produceadenosine-triphosphate(ATP),whichistoalargeextentformedbyoxidative

phosphorylationinthetricarboxylicacidcycle.Thismetabolicpathwayrequiresalarge

amountofoxygen,whichdiffusesfrommicrovesselstomitochondriaofthebraincells.

Withinthebloodvessels,chemicallyboundoxygenmoleculesdissociatefromhemoglobin

anddissolvedoxygendiffusesthroughtheredbloodcellmembrane,bloodplasma,the

capillarywall,andinterstitialfluiduntilitenterstheneuronsandglialcells,whereitis

metabolized.Blood-borneglucosetravelsalongthesameroute;however,incontrastto

oxygen,whichcanfreelydiffuse,glucosemoleculesaretransportedacrossmembraneswith

thehelpofglucosetransporters.AmajorwasteproductofbrainenergymetabolismisCO2,

whichrapidlydiffusesoutofthenervoustissueandisclearedfromthebrainalsoviathe

vascularsystem.Thebrainvascularsystemisnotonlyresponsibleforsupplyandwaste

collection,butalsoactsasabarrierthatrestrictsthepassageofmoleculesfrombloodto

brain.Theendothelialcellsofthebrainvesselsarecoupledbytightjunctionsandformthe

blood-brainbarrier.

Itwasrecognizedasearlyasthe19thcenturythatthebrain’sbloodsupplyisdynamic,and

thatchangesinneuralactivityaremirroredbypreciselycontrolledchangesin

hemodynamics(Mosso,1881;RoyandSherrington,1890).Thisspatialandtemporal

neurovascularcouplinghasbeensystematicallyusedtogeneratedetailedmapsof

hemodynamicchangesthatareassumedtobesurrogatesoftheactualregionalneural

activation.Themostimportantapplicationofthisrelationshipistheso-calledblood

oxygenationlevel-dependent(BOLD)contrast(Kwongetal.,1992;Ogawaetal.,1990)used

infMRI.

Theneocortexisoneofthemostintriguingstructuresofthebrain.Itshowsan

extraordinaryflexibility,giventhelargerangeofbehaviorthatemergesdespiteonlylimited

variationinitsanatomicalorganizationacrossthedifferentcorticalareas(Douglasand

Martin,2004).Thelaminarorganizationofthecortexanditsimpactonstructuraland

functionalMRIisthetopicofthisspecialissue.Thesix-layeredstructure,withwell-defined

microcircuits(DouglasandMartin,2004),islargelypreservedacrossspecies.Similarly,the

vascularsystemoftheneocortexalsoshowsahighdegreeoforganizationthroughoutthe

corticaldepth,anorganizationthatisanalogousinallthestudiedanimalmodelsaswellas

inthehumancorticalmicrovasculature.

Thepresentreviewintroducesthevascularsystemoftheneocortexandisstructuredinthe

followingway.Afterashortexplanationofthemostimportantmethodsusedtoinvestigate

brainbloodvessels,weintroducethegrossorganizationfollowedbyadetailed

characterizationoflaminardensityvariation,topologicalaspects,andflowregulationofthe

microvascularsystem.Weendthereviewwithadiscussionoftherelevanceofthe

vasculaturesystemfor(laminar)fMRI.

2 Methodstostudybrainvasculature

ClassicalstudiesofthemicrovascularnetworkarebasedonIndianinkfillingsandscanning

electronmicroscopyofvascularcorrosioncasts(Duvernoyetal.,1981;Reina-DeLaTorreet

4

al.,1998;Weberetal.,2008).Thecombinationofimmunohistochemistryandstereology

alsoallowsestimatingvesseldiametersandvasculardensity(Weberetal.,2008).

However,thethree-dimensionalvascularnetworktopologyoftheneocortexhasnotbeen

studiedindetailuntilrecently,whennewtechnologiesbecameavailable.Inorderto

describethenetworkinthreedimensions,methodsarerequiredthathaveaspatial

resolutiontoresolvecapillariesinasufficientlylargefieldofviewtocoveranentirecortical

column.Inordertoestimatethediameterofcapillaries,submicronspatialresolutionis

required.DavidKleinfeldandcolleagueshavedevelopedtheso-calledall-opticalhistology

technique(Tsaietal.,2003;Tsaietal.,2009),wherebytwo-photonmicroscopyisusedto

imagethefluorescentlylabeledvasculature,andlaserablationisusedtoincreasethefield

ofview,whichwouldnormallybelimitedtoafewhundredmicrometers.Anotherpossible

approachistheuseofsynchrotronradiation-basedX-raymicroscopy,whereahighphoton

fluxisexploitedtoacquireabsorption-basedtomographicimagesofthevasculature

(Guibertetal.,2010;Heinzeretal.,2006;Plouraboueetal.,2004;Reicholdetal.,2009).

Recently,theclassicalIndianinkfillingmethodhasbeencombinedwithaserialsection

microscopictechnique,calledMicro-OpticalSectioningTomography(MOST),andwasused

toacquirethevascularsystemoftheentiremousebrain(Xueetal.,2014).

Withtheadventofadvancedtissueclearingmethods,selectiveplaneillumination

microscopy(SPIM)orultramicroscopy(Erturketal.,2012)isbecominganincreasingly

powerfulapproachtoacquireandreconstructfluorescentlylabeledvesselsinlargesamples.

Todate,thespatialresolutionandavaryingpointspreadfunctionacrossthefieldofview

aretechnicalchallengesthatneedtobeovercomeforthemethodtobecomeanovel

standard.

Allthehistologicaltechniquessharetheproblemoftissuedeformation,eitherdueto

fixationand/orclearingofthetissue.Therefore,measurementsofthemicrovascular

networkneedtobecorrectedforthesealterations.Onewayofdoingthisistomeasure

vasculardiametersinvivousingtwo-photonmicroscopyandtousethediameter

distributiontotransformtheexvivomeasurements(Tsaietal.,2009).Moreover,future

studiescoulddeploydeepinvivotwo-photonmicroscopytoacquirethecorticalvascular

systeminitsentiredepth.

3 Grossstructureofthecorticalvascularsystem

Thecerebralarteriesrunalongthesurfaceofthecortexandramifyintoacomplexnetwork

ofso-calledpialarteries.Thesearteriesaresituatedinthesubarachnoidspaceandare

surroundedbyapialcelllining.Thetopologyofthispialarterialnetworkensuresarobust

deliveryofbloodtothecerebralcortex(Section5.1).

Onceanarteryleavesthepialnetwork,itpenetratesperpendicularlyintothecortex(Figure

1).Thepialcellensheathmentofthearteriescontinuesintheformofaperivascularchannel

(Zhangetal.,1990).Thispialcelllayerisperforatedatthearteriolarlevelandiscompletely

absentoncapillariesandveins.Aperivascularspace,termedtheVirchow-Robinspace,

surroundsthecorticalarteriesandveinsinafunnel-shapedmanner.Aroundthelargepial

anddivingvessels,astrocytesdefinetheVirchow-Robinspace.Thisperivascularspaceisa

clearingrouteforinterstitialsolutes,muchasthelymphaticsystemisfortherestofthe

body(IliffandNedergaard,2013;Iliffetal.,2012;Iliffetal.,2013;Xieetal.,2013).Thisso-

calledglymphaticsystemconsistsofapara-arterialinfluxroute;apara-venousclearance

5

route;andatrans-parenchymalpathwaydependingontheaquaporin-4waterchannelin

astrocytes.

Thecorticalarteriessendoffcollateralsatdifferentcorticaldepths.Thisfactledtothe

introductionofaclassificationschemebyDuvernoyetal.(1981),inwhichavesseloftype1

wouldfeed/drainsuperficialcorticallayers,whereashigherordervesseltypeswould

feed/draindeeperlayers.Somecorticalarteriesevenpenetratetheentirecortexwithout

anycollateraluntilreachingwhitematter(Section5.2).Itisimportanttonotethatthese

classesarenotcompletelyseparable,butappearratherasacontinuum.Corticalarteries

divergeintoarterioles(smallarteries)andeventuallyendinthecapillarynetwork(Section

5.3).Thecapillarynetwork,wheremostoftheexchangeofenergysubstratesandoxygen

occursbetweenbloodandtissue,convergesintothevenoussystem.Again,principal

corticalveinsareorientedperpendicularlytothecorticalsurface(Section5.2).Thebasic

buildingprincipleofthecorticalvasculatureis,therefore,deliveryofbloodfromthecortical

surface,penetrationintothecortexinaperpendicularmannerand,afterthecapillary

passage,drainagebacktothesurface,wherethebloodiseventuallytransportedawayvia

thevenoussystemthatconvergesintolargesinuses.

Therearedistinctdifferencesintheultrastructurebetweenthedifferenttypesofcerebral

bloodvessels.Commontoarteries,capillariesandveinsaretheendothelialcelllayerand

thethinbasalmembrane.Endothelialcellsarecoupledbytightjunctionsandformthe

blood-brainbarrierthatrestrictsthepassageofmoleculesfrombloodtobrain.Whereas

capillariesonlyconsistofthesetwoelements,arteriesandtoamuchlesserextentalso

veinsarecoveredwithasmoothmusclesheath.Thesesmoothmusclesareresponsiblefor

regulatingthevascularresistancebychangingthevesseldiameter(Section6).Pericytesare

averyheterogeneouscelltypethathaveaclaw-likeappearanceandarelocatedonthe

abluminalsideofendothelialcells(Armuliketal.,2011).Theyalsohaveacontractile

capacity,buttheirroleasactiveregulatorsofvascularresistanceisdebated(Section6,

(Fernandez-Klettetal.,2010;Halletal.,2014;Hamiltonetal.,2010;Peppiattetal.,2006)).

Furthermore,allmicrovesselsarealmostcompletelycoveredbyastrocyticendfeet

(Mathiisenetal.,2010).Thisperivascularastrocyticsheathisthoughttoplayapivotalrole

inthetranscellulartraffickingofmetabolitesaswellaswaterandionexchangeatthe

blood-braininterface.

1 mm

6

Figure1Grossstructureofthecorticalvasculature.Left:Scanningelectronmicrographofavascularcorrosioncastfrom

themonkeyvisualcortex(primaryvisualcortex).Arteriesareshadedinredandveinsareblue.FigurefromHirschetal.

(2012).Right:Schematicrepresentationofthecorticalvasculatureanditskeycomponents.

4 Densityofthecorticalmicrovascularsystem

Thediametersofcorticalvesselsrangefromaround4µm(capillaries)toafewtensof

microns(arteries/veins)(Blinderetal.,2013;Duvernoyetal.,1981;Gutiérrez-Jiménezetal.,

2016;Halletal.,2014;Tsaietal.,2009;Weberetal.,2008).Whenlookingatthe

distributionofthecalibers,itbecomesobviousthatthemostfrequentvesseltypeisthe

capillary,irrespectiveofthespecies(Weberetal.,2008).Thecapillarynetworkcanbe

regardedasaredundantnetworkwithameshwidthofapproximately50µm(Section5.3).

Thismeshwidthisprobablyadjustedtothediffusionconstantofoxygeninbraintissue.

Theoverallvascularvolumefractionrangesbetween1and3%ofthetotalbrainvolume,

dependingonthespeciesandtheappliedmethod(Blinderetal.,2013;Lauwersetal.,2008;

Risseretal.,2009;Tsaietal.,2009;Weberetal.,2008).Althoughsimilarvascularnetwork

characteristicscanbefoundthroughoutthecortex,therearevariations,bothacrossthe

corticallayersandbetweendifferentcorticalareas(Figure2).Intheprimarysensoryareas,

thehighestvasculardensitycanbefoundinlayerIV.Thisismostobviousintheprimate

primaryvisualcortex,wherelayerIVcβ displaysanincreaseinvasculardensitythatiseasily

detectable(FontaandImbert,2002;Weberetal.,2008).

Severalauthorshaveinvestigatedtherelationshipbetweenvascularandneuronaldensity,

buttheevidencesuggeststhattherelationshipisweak(Tsaietal.,2009;Weberetal.,

2008).Inthemacaquecortex,thiscorrelationwasparticularlyweakintheupperlayersof

cortex(Weberetal.,2008).However,amuchstrongercorrelationbetweenthepatternof

oxidativemetabolismandmicrovasculardensitywasobserved(Kelleretal.,2011;Weberet

al.,2008).

TheKleinfeldgrouphasstudiedthemousebarrelcortexandfoundthatthevariationin

cellulardensityacrossthecorticaldepthwasmorepronouncedthanthatofthevascular

density(Tsaietal.,2009).Moreover,thevascularradiusdistributionwasverysimilaracross

thecorticaldepth.Ifwelookatthemicrovasculardensityalongthecorticallayersinthe

primaryvisualcortex,thereisamarkedgradientwiththehighestdensityinlayerIVc-

β (Weberetal.,2008;Zhengetal.,1991).

Fromadevelopmentalperspective,FontaandImbert(2002)wereabletodemonstratethat

therelativevasculardensitydevelopedinparalleltocytochromeoxidaseactivityandwas

highestinlayerIVc-α inthefirstpostnatalmonth.(Cytochromeoxidaseactivityisa

histochemicalproceduretoassesstheoxidativemetabolicdemandofagivenbrainregion.)

Inthenextdevelopmentalphase,thevasculardensityandcytochromeoxidaseactivityin

thetwolayersaresimilar,beforelayerIVc-βeventuallybecomesthemostdensely

vascularizedlayer.Inthecat,asimilarchangeinsteady-statemetabolicdemandand

vasculardensitywasobserved.ItwasfoundthatlayerIVinthecatstriatearea17showed

thehighestrelativevasculardensityandrelativeglucoseutilization.However,thislaminar

differencecouldonlybefoundintheadultbutnotin5-week-oldkittens(Tiemanetal.,

2004).Changesinvascularorganizationcanalsobeobservedintheadultanimal.Whereas

acutehypoxiaiscompensatedwithanincreaseinCBF,prolongedhypoxialeadstoamarked

7

increaseincapillarydensity(DiemerandHenn,1965;Hariketal.,1994;LaMannaetal.,

1992;MillerandHale,1970;Opitz,1951).

Weberandcolleaguesstereologicallydeterminedvasculardensityvaluesonthebasisof

anti-collagenimmunohistochemistryfromalargenumberofsamplesfromthemacaque

visualcorticesV1,V2,V3andV4(Weberetal.,2008).Theauthorsfoundthattheoverall

vascularlengthdensityinvisualgraymatterlayaround478mm/mm3,whereasthevolume

fractionwasapproximately2.1%.Aselaboratedabove,thevascularnetworkofthestriate

cortexalsodisplaysaspeciallaminarorganization.Thevolumefractionandlengthdensityin

layerIVc-βwere2.70%and627.83mm/mm3respectively.Thelowestvascularlength

densitywasfoundinlayerI(408.44mm/mm3),whereaslayerIIshowedthelowestvolume

fraction(1.93%).Thesesuperficialdensitydatacoincidewiththerelativelylowcellbody

densityandhighproportionofmyelinatedandunmyelinatedfibersinlayerIandII.The

vasculardatafromV2,V3andV4wereverysimilar,withlayerIVshowingthehighest

density(volumefraction2.18%)andlayerVIthelowest(volumefraction2.01%).Itis

importanttonote,thatdifferentvesselcaliberscontributedifferentiallytothedifferent

vasculardensitymetrics.Whenfocusingonthemerelengthasinlengthdensity,itis

obviousthatbyfarthelargestcontributionisprovidedbythecapillaries.However,forother

quantitiesthecontributionofthenon-capillariesmaybehigher,accordingtotheir

respectivedependenceonthevesselradius.(Thelengthdensity’sdependenceonthevessel

diameterisd0,thatofthesurfacedensityisd1andthatofthevolumefractionisd2.)Dueto

thevolumefraction’squadraticdependenceonthediameter,thecontributionoflarge

vesselssurpassesthatofthecapillaries,despitetheirmuchlowerfrequency(Figure2).

Theprimaryvisualcortexseemstobeuniquewithrespecttothemicrovascularsystemas

well,sinceoverallvasculardensityisclearlyhigherinthestriatecortexthanintheextra-

striatecortices(FontaandImbert,2002;Tiemanetal.,2004;Zhengetal.,1991).However,

differencesbetweentheextra-striateareasaresmallorevennegligible(Weberetal.,2008).

Thevasculardensitydifferencesbetweentheprimaryandnon-primaryareaswerealso

foundinthesomatosensoryandauditorycortex,andthevasculardensitiesofthe

secondaryauditoryandsomatosensoryareaswerecomparabletothoseoftheextrastriate

cortex(ownunpublishedobservations).Takentogether,itseemsthatnon-primarycortical

areasshareasimilarmicrovasculararchitecture.Therefore,basedonthevascularstructure

alone,itappearsthatlargedifferencesinthehemodynamicresponseandlaminarMRIare

nottobeexpectedwithintheseareas.However,cautionisadvisedwhenhemodynamic

responsepatternsaredirectlycomparedbetweenprimaryandnon-primaryareas(Section

8).

Itissurprisingthatwedonotseesignificantdifferencesinthevasculardensityoflarge

vesselsovercorticaldepth,despitethefactthatDuvernoyetal.(1981)statesthatDAsand

AVsofgroup4(Table1)arethemostfrequentgroupofpenetratingtrees.Firstofall,it

shouldbenotedthatDuvernoy’sclassificationhasbeenderivedforthevasculatureofthe

humanbrainandthusspeciesdependentdifferencesmightexist.Furthermore,differences

invesseldiameterfordifferentgroupsofpenetratingtreesmightblurthosecharacteristics.

Inadditiontotheoverallvasculardensity,therelativevolumefractionoccupiedbyblood

vesselsofdifferentcategories,i.e.arteries,capillariesandveins,isrelevantformany

applications.Forexample,thefractionofbloodfoundinthedifferentvesseltypesisan

importantparameterincompartmentalbiophysicalmodels.

Themeasurementsfromthemacaquevisualcortexdiscussedaboveestimatedthat

capillaries(definedasvesselswithdiameter<8µm)madeupapproximately41%ofthe

8

totalvascularvolumefraction(Weberetal.,2008).Thisissimilartoanestimateof~48%

obtainedfromIndianinkinjectedsectionsofhumancortex,whichweretakenfromthe

fusiformandparahippocampalgyri(Lauwersetal.,2008).Whilethereisarangeof

microvasculardatacomparingtherelativenumberofdescendingarteries(DAs)and

ascendingveins(AVs,Section5.2),toourknowledgenosuchdataexistdescribingthe

distributionofbloodvolumebetweenarterialandvenouscompartments.However,itis

possibletousedatafromarangeoflowerresolutiontechniquessuchMRIandPET,and

basedonthisapproachBarrettetal.(2012)estimatedarteriesmakeup~29%oftotalblood

volume,andveinscontribute~27%,usingmostlyhumanandprimatedata.

Figure2Vasculardensityofthecorticaldepth.Vasculardensityovercorticaldepthfordifferentspecies,differentareas

anddifferentvesseltypeswithsimilar(A)anddifferent(B)x-axesscales.Datafrom:Lauwersetal.(2008)(Human);Weber

etal.(2008)(Macaque);Risseretal.(2009)(Marmoset);Tsaietal.(2009)(Mouse).Thecut-offdiametertodifferentiate

betweencapillariesandlargevesselsdiffersfordifferentspecies.Here,thefollowingvalueswereused:Human:10µm,

Macaque:6µm,Marmoset:11.2µm,Mouse6µm.

9

5 Topologicalaspectsofthecorticalvascularsystem

InSection3weintroducedthegrossanatomicalstructureofthecorticalvasculature

consistingofpialandpenetratingvesselsandthecapillarybed.Here,wefocusonspecific

topologicalcharacteristicsandontheroleofthethreevesseltypesinthedistributionof

blood.Aprofoundknowledgeofthevasculartopologyisrelevantnotonlyfor

understandingneurovascularcouplingbutalsotocommentontheseverityofvessel

occlusionatdifferentlocations.Unlessstatedotherwisethedescribedcharacteristicsare

validacrossspecies.

5.1 Thepialnetwork

Thepialnetworkisa2-D-planarnetworklocatedatthecorticalsurface.Thepialarteries

distributebloodfromthelargecerebralarteriestotheintracorticalvesselsandthepial

veinscollectit.

Asthepialarteriesareatthebeginningofthepathwayofbloodthroughthecortex,a

robustnetworktopology,whichguaranteesaconstantsupplyofblood,iscrucial.Thisis

achievedbyalargenumberofarterialanastomoses,whichonaveragecontainfouredges

(Blinderetal.,2010;Duvernoyetal.,1981;Schafferetal.,2006).Overall,thestructureof

thepialarterialnetworkiscomparabletothatofahoneycomb(Blinderetal.,2010).

Forathoroughanalysisoftheredundanciesatthepiallevel,Blinderetal.(2010)introduce

theconceptofthebackboneofthepialnetwork(Figure3A).Thebackbonespansthewhole

territoryofthepialnetwork,eventhoughitismadeupofonly~11%ofthepialarteries.In

addition,nearly75%ofalldescendingarteries(DAs)startatabackboneedge.Thisproperty

furtherincreasestherobustness,becausetwodifferentpialvesselscanfeedthosearteries.

Occlusionexperimentshavedemonstratedtherobustnessofthepialnetwork(Blinderet

al.,2010;Schafferetal.,2006).Schafferetal.(2006)showedthatsinglevesselocclusion

inducesaredistributionofflowwiththepositiveeffectthatallvesselsstayperfused(Figure

3F).Nonetheless,theflowratesinindividualvesselsaresignificantlyaffectedandflow

reversals,reductionsandevenincreasesareobserved(Schafferetal.,2006).Blinderetal.

(2010)notedthatinlowfluxDAstheflowispreservedinresponsetopialarteryocclusion,

whileitdecreasesinhighfluxDAs.ThissuggeststhathighfluxDAscontainbloodreserves

thatcanberedistributedincaseofocclusion.

Thenetworktopologyofthepialveinsissignificantlylessstudied.Theprevailingviewisthat

ithasfeweranastomosesthanitsarterialcounterpartandisrathera“drainagesystemlike

ariverwatershed”(Adamsetal.,2014).Generally,pialveinsarelargerindiameterthanpial

arterioles.Duvernoyetal.(1981)observethatinthehumanbrainlargepialveinstendto

surpassthesulciandremainatthecorticalsurface.Thisisincontrasttopialarterieswhere

themaintrunkisoftenlocatedwithinthesulci.Furthermore,pialarteriesnormallyrun

abovepialveins(Duvernoyetal.,1981).

5.2 Thepenetratingvessels

TheDAsdeliverbloodtothecapillarybedovertheentiredepthofthecortex.Afterthe

bloodpassesthecapillarynetworkitiscollectedintheascendingveins(AVs)andreturns

towardsthecorticalsurface.ItiswellestablishedthatDAs,aswellasAVs,haveatree-like

10

structureanddiffersignificantlyintheirpenetrationdepth(Blinderetal.,2013;Cassotet

al.,2009;Duvernoyetal.,1981;Guibertetal.,2012;Hirschetal.,2012;Lauwersetal.,

2008;Reina-DeLaTorreetal.,1998).Themostwidespreadclassificationforpenetrating

vesselsisbyDuvernoyetal.(1981),andissummarizedinTable1.Theauthorsproposed

thatdifferentgroupsofDAsareresponsibleforfeedingdifferentcorticallayers.Guibertet

al.(2012)providedsomeevidenceforthishypothesisintheirnumericalwork,andshowed

thatthedepthofthefeedingregionstronglycorrelateswiththepenetrationdepthofthe

DA.

VesselGroup 1 2 3 4 5 6

Penetration

depth(cortical

layer)

I-II IIIa IIIc-Va VI downto

WM

downtoWM

without

branching

Table1ClassificationofpenetratingvesselsbasedontheirpenetrationdepthbyDuvernoyetal.(1981).Theclassification

isvalidforDAsaswellasAVs(withtheexceptionofgroup6whichonlyexistsforDAs).Allpenetratingtrees,with

exceptionofgroup6,haveoffshootsalongtheirdepth.WM:whitematter.

TheDAsbelongingtogroup4arethemostnumerous(Duvernoyetal.,1981),andthe

numberofbranchesfeedingthecapillarybedpeaksatcorticallayerIV(Figure3E)(Blinder

etal.,2013;Schmidetal.,2017).WhilethisevidencesuggeststhattheDAtopologyis

designedwiththemajorpurposeofsupplyingbloodtolayerIV,topologicalcharacteristics

alonedonotpredictflow.Indeed,Schmidetal.(2017)showedthat,incontrasttothe

numberofcapillarystartingpoints,theRBCinfluxismaximalclosetothecorticalsurface

(Figure3E).

OffurtherinterestisthedistributionofDAswithrespecttoeachotherandtotheAVs.The

ratioofDAstoAVsishighlyspeciesdependentandwhileforprimatestherearemoreDAs

thanAVs,theoppositetrendisobservedforrodents(Table2).Theevolutionarybasisfor

thesedifferencesremainsunclear.

Species Human Monkey Rat Mouse

RatioDA:AV 2.2:1 2.1:1 1:1.8 1:3.0

DAspermm2 1.0 7.9 8.3 3.9

AVspermm2 0.5 3.6 10.3 -

Table2AverageratiosofDAstoAVs,averagenumberofDAsandAVs2fordifferentspecies.References:Human:(Cassot

etal.,2009;Lauwersetal.,2008);Monkey:(Adamsetal.,2014;Guibertetal.,2010;Risseretal.,2009;Weberetal.,

2008);Rat:(Blinderetal.,2010;Nguyenetal.,2011);Mouse:(Blinderetal.,2010;Blinderetal.,2013).

IthasbeenhypothesizedthatthedistributionofDAscorrelateswiththelocationof

functionalneuronalunits,suchasbarrels(somatosensorycortex)orblobs(visualcortex).

Forthebarrelcortexnosuchcorrelationcouldbedetected(Figure3B;Blinderetal.(2013)).

Fortheblobsthematterremainscontroversial.Kelleretal.(2011)observedanincreased

DAdensitybetweentheblobs.However,thisisincontrasttoresultsfromAdamsetal.

(2014),whodidnotobserveanydifferencesintheDAdensity.

Althoughthereisnoclearevidencesupportingastrongcorrelationbetweenfunctional

unitsandthedistributionofDAs,itseemslikelythateachDAisresponsibleforfeedinga

11

specifictissuevolume.Shihetal.(2013)occludedDAsintheratcortextoestimatethe

feedingregionofindividualDAs.Theystatethattheinfarctvolumeisproportionaltothe

baselinefluxintheoccludedDAandonaverageaffectsacylindricalvolumewitharadiusof

460µmandadepthof1.17mm.

Guibertetal.(2012)determinedthefeedingvolumebasedonnumericalbloodflow

simulationsinthemarmosetcortex.Theyobtainedafeedingvolumewitharadiusof386

µmandadepthof2mm.Consideringthelargesizeofthebrainthisvaluemightseem

comparablysmall.However,itshouldbekeptinmindthattheDA:AVratiois>1for

primateswhileitis<1forrodents.Furthermore,thevolumemeasurementpresentedby

Guibertetal.(2012)isaconservativeestimatebecauseitisdefinedasthevolumewhichis

exclusivelyfedbyoneDA,andnotthewholevolumeaffectedbyoneDA.Accurate

comparisonsarealsocomplexbecausetheDAdensityincreaseswiththedistancetothe

originofthemiddlecerebralartery(MCA),andconsequentlythefeedingvolumeislikelyto

decrease(Blinderetal.,2010).Additionally,thedensityofpenetratingvesselsdiffers

dependingoncorticalarea(Risseretal.,2009).

ThestudiesbyNishimuraetal.(2007)andBlinderetal.(2013)furtherunderlinethecrucial

roleofDAsinthesupplyofblood(Figure3F).TheeffectofaDAocclusionisapparentupto

tensegmentsfromthesiteofocclusion(Blinderetal.,2013).Thereisstillincreased

variabilityinRBCvelocitiesevenafteradistanceof350µm,wherethemeanRBCspeed

maintainsitsbaselinevalue(Nishimuraetal.,2007).

EventhoughevidencesuggeststhatanAVocclusionisascriticalasaDAocclusion(Shihet

al.,2013),theAVshavereceivedsignificantlylessattentionthantheDAs.Nguyenetal.

(2011)hypothesizedthatiftheratioofDAstoAVsislargerthan1anAVocclusionismore

severeandviceversa.Forthemarmoset,macaqueandhuman,wheretheratioofDA:AVis

greaterthan1,thedrainingregionofoneAVissignificantlylargerthanthefeedingregionof

oneDA(Guibertetal.,2012;Lorthoisetal.,2011,Weberetal.,2008).Furthermore,

Lorthoisetal.(2011)showedthatthedrainagevolumeincreaseswiththediameterofthe

AV.

Thediameterofthepenetratingvesselsisstronglyspeciesdependent.Blinderetal.(2013)

statethatinthemousethemeandiameteris11µmand9µmforDAsandAVs,

respectively.Opposingtrendshavebeenrecordedinthehumanbrain,herethevessel

diameterofAVsislargerthantheoneofDAs(65µmvs.35µmforpenetratingvesselsof

group4)(Duvernoyetal.,1981).Moreover,themeanvesseldiameterincreaseswiththe

penetrationdepthoftheDA/AV.Interestingly,incontrasttotheDAs,thenumberofAV

offshootsdecreaseswithcorticaldepth(Blinderetal.,2013).

RelativelylittleisknownabouttherelativeplacementofDAsandAVs.Basedontheanalysis

ofmicrovascularnetworkfromthemousecortexBlinderetal.(2013)suggestedarhombic

latticewhereoneDAisencircledbysixAVs.Acomparableconceptwasproposedby

Duvernoyetal.(1981)whostatethata“vascularunit”inthehumanbrainconsistsofanAV

surroundedbyseveralDAs(PleasebearinmindthatinthehumanbraintheDA:AVratio

approximately2).FromasolefluiddynamicalpointofviewitisplausiblethattheAVs/DAs

arefedequallybythecentralDAs/AVs.However,opposingtrendshavebeenobservedin

numericalworksfromGuibertetal.(2012)andSchmidetal.(2017).Guibertetal.(2012)

statethat61.5%oftheflowofoneDAisdrainedintooneAV.AdditionallySchmidetal.

(2017)observethat72%ofRBCsdrainedbyanAVoriginatefromoneDA.Those

observationscanonlybeexplainedbyaspecificmicrovasculaturetopologyorflowpattern

thatfavorsthedirectionalflowtowardsoneAV.Nonetheless,ithastobekeptinmindthat

12

althoughthereseemstobeapreferentialAVforeveryDA,theDAsandAVsarehighly

interconnected.Indeed,Guibertetal.(2012)showedthatinthemarmosetoneDAis

connectedto22AVs.

Allinall,multipleexperimentalaswellasnumericalstudiesdemonstratethatthe

penetratingvesselsarecrucialtomaintainasufficientsupplyofbloodtothetissue.

However,manyopenquestionsconcerningtheirdistributionandlayer-specificfeeding

remaintobeanswered.

5.3 Thecapillarybed

Thecapillarybedhasamesh-likestructure,generallydescribedashomogeneousandhighly

interconnected(Blinderetal.,2013),andhenceitstopologyissignificantlymoredifficultto

analyzethanthepialvessels’orthepenetratingtrees’(Blinderetal.,2013;Cassotetal.,

2009;Hirschetal.,2012;Lauwersetal.,2008).Nonetheless,theflowfieldinthecapillary

bedisveryheterogeneouswithalargerangeofRBCvelocitiesandahighcapillarytransit

timeheterogeneity(JespersenandØstergaard,2012).

Thisisalsoreflectedinthenumberofavailablepathwaysthroughthecapillarybedandthe

frequencieswithwhichthesearechosen.FiveexemplaryRBCpathwaysthroughthecortical

microvasculatureareillustratedinFigure3D.Werecentlyshowedthatforeachcapillary

startingpointthereareonaverage8differentRBCpathwaysleadingfromDAtoAV.

However,formorethan50%ofallcapillarystartingpointsthereisapreferentialpathwhich

ischosenwithafrequency>50%(Schmidetal.,2017).Furthermore,ourresultsrevealed

thatthecapillarystartingpointandthecapillaryendpointarestronglycorrelated(Figure

3C)andhence,theRBCtendtomove“in-plane”throughthecapillarybed(Schmidetal.,

2017).Itseemslikelythatthecapillarybedisdesignedtoencouragethein-plane(i.e.

paralleltothesurface)motionofRBCs.

Allinall,eventhoughthestructureofthecapillarybediscommonlydescribedasbeing

homogeneousitsflowfieldishighlyheterogeneous.Furtherinvestigationsarenecessaryto

thoroughlyexplaintheobservedcharacteristics.

Similarlytothepialandpenetratingvessels,occlusionexperimentshavebeenperformedto

assesstheoverallrobustnessofthecapillarybedinthedistributionofflow(Figure3F;

(Nishimuraetal.,2007;Shihetal.,2013)).Nishimuraetal.(2007)andShihetal.(2013)

showedthattheimpactofamicrovesselocclusionisminimalandthattheRBCflux

recoveredto45%ofitsbaselinevaluebythreebranchesdownstreamoftheocclusion.The

robustnessofthecapillarybedcanbeexplainedbyitsmesh-likestructure,whichis

beneficialforanefficientredistributionofflow.Furthermore,Guibertetal.(2012)show

that63%ofallcapillariesarefedbymorethanoneDAandhencearedundancytowardsDA

occlusionpersistsaswell.Interestingly,therobustnessofthecapillarybedtowardsDA

occlusionincreaseswithdepth(Figure3G;Guibertetal.(2012)).Ithasalsobeensuggested

thatcapillarybeddensitymightcorrelatewithneuronalfunctionalunits.Forthebarrel

cortexnosuchcorrelationwasobserved(Blinderetal.,2013).Similarlytothepenetrating

vessels,theresultsofKelleretal.(2011)andAdamsetal.(2014)divergefortheblobsinthe

visualcortex,andonlyKelleretal.(2011)notedaslightlyincreasedvesseldensitywithinthe

blobs.

13

Figure3Topologicalcharacteristicsofthecerebralvasculature.(A)Exemplarypialnetwork.Thebackboneofthepial

networkishighlightedinblack(FigurefromBlinderetal.(2010)).(B)Feedingregionsofdescendingarteries(DAs)andtheir

distributionwithrespecttowhiskerbarrels(“goldenbands”).ThecolorsindicatefeedingregionsfordifferentDAs.(Figure

fromBlinderetal.(2013).ReprintedbypermissionfromMacmillanPublishersLtd:NatureNeuroscience,2013).(C)Cortical

depth(CD)ofcapillarystartandcapillaryendpointstoillustratethein-planemotionofRBCsinthecapillarybed(CB)

(FigurefromSchmidetal.(2017)).(D)ExemplaryRBCtrajectoriesfordifferentdepthsofcapillarystartingpoints(Figure

fromSchmidetal.(2017)).(E)Layer-specificfeedingcharacteristicsofthecapillarybed(CB)(FigurefromSchmidetal.

(2017)).(F)Impactofocclusionfordifferentvesseltypes(DA:descendingarteries,C:capillaries,PA:pialarteries).The

impactismeasuredbasedonthefractionalRBCspeedinvesselsegmentsdownstreamthesiteofocclusion(D1:one

segmentdownstream,D2:twosegmentsdownstream,D3&D4:threeandfoursegmentsdownstream)(Figureadapted

from:Nishimuraetal.(2007)).(G)Robustnessindexofthecapillarybed(CB)fordifferentcorticaldepths.Therobustness

indexisdefinedasthenumberofdescendingarteriesfeedingacapillarysegment(Figureadaptedfrom:Guibertetal.

(2012)).

6 Flowregulation

Intheprevioussectionwedescribedstaticcharacteristicsofthecerebralvasculature,yet

thevasculatureisconstantlyadaptingtothemetabolicneedsoftheparenchyma.The

resultinghemodynamicchangesarethebasisofamultitudeofimagingtechniques.

Understandingthevasculartopologygoeshandinhandwithunderstandingthevascular

responsetoneuronalactivation.Hence,inthefollowingwesummarizethecurrent

knowledgeonvascularregulationmechanisms.Webeginbydescribingthecandidate

locationsforregulationandthecontractilecellsintheirsurroundings.Subsequently,we

commentonspatio-temporalandlayer-specificdynamicsofregulation.Theneurovascular

signalingpathwaysarenotaddressedinthismanuscript.Agoodoverviewisgiveninthe

reviewsbyIadecolaandNedergaard(2007),Attwelletal.(2010)andHillman(2014).

6.1 Contractilecellsofthecerebralvasculature

(A) Structure of the pial network (B) Distribution of DAs with respect

to barrels

(C) In-plane motion of RBCs

through the CB

(D) Exemplary RBC trajectories

Analy

sis

Layer

1

2

3

4

5

(E) Layer-specific

feeding of CB

(F) Vessel specific

occlusion effects

(G) Robustness index of

CB over depth

14

Twocelltypeshavebeenreportedtomodulatevasculardiameterinvivo:smoothmuscle

cellsandpericytes.Whiletheinvolvementofsmoothmusclecellsinneurovascularcoupling

iswellestablished,thecontractilityofpericytesinvivohasbeenshownonlyrecentlyand

theirroleinneurovascularcouplingisamatterofongoingdebate(Attwelletal.,2010;

Attwelletal.,2015;Fernandez-Klettetal.,2010;Fernández-KlettandPriller,2015;Hallet

al.,2014;Hilletal.,2015;Hillman,2014;ItohandSuzuki,2012).

Arterialsmoothmusclecells(SMCs)aretightlywrappedaroundarteriesandhenceperfectly

positionedtocontrolvesseldiameters(Figure4A).Pericytesdiffersignificantlyintheir

shapeandintheexpressionof𝛼smoothmuscleactin(𝛼-SMA).Whileinthesmooth

musclecellsaroundDAs𝛼-SMAisomnipresentinpericytesitonlypersistsclosetotheDA

(Figure4A;(Attwelletal.,2015;Hartmannetal.,2015;Hilletal.,2015)).However,it

remainsunclearif𝛼-SMAisaprerequisiteforthecontractilityofpericytesorifother,asyet

unknownmechanismsforalteringthediameterofcapillariesexist(Fernández-Klettand

Priller,2015).

6.2 Vascularresponseofdifferentvesseltypestoneuronalactivation

Theoverallhemodynamicresponsetoneuronalactivationresultsfromaninterplayof

differentvascularresponses(Hillman,2014).Inthissubsection,wediscusstheresponseand

spatio-temporaldynamicsofindividualvesseltypes,namely:(1)pialanddescending

arteries,(2)pre-capillaryarteriesandcapillaries,and(3)venulesandveins.Table3

summarizesthekeycharacteristicsofthevascularresponseofdifferentvesseltypestobrief

stimulation(<5s).

Ingeneralthelocationwherethevascularresponseisinitiateddependsonthecorticalarea

underinvestigationandthetypeofstimulusapplied.Fortheratprimarysomatosensory

cortexandthestimulationoftheforepawtheprevailingviewisthattheresponsestartsas

deepas0.6-0.9mminthecortexandspreadstowardsthesurfacewithapropagationspeed

of~0.9mm/s(Tianetal.,2010;Uhlirovaetal.,2016b).Thevasculatureitselfmaybe

responsibleforthepropagationofthedilatorysignal(Chenetal.,2011;Chenetal.,2014;

Hillman,2014;Iadecolaetal.,1997;Uhlirovaetal.,2016b),butitremainsunclearif

vasodilationistriggeredataspecificvesseltypeonly.

Lindvereetal.(2013)werethefirsttomonitordiameterchangessimultaneouslyina

microvascularnetworkspanning0.5x0.5x0.6mm.Theyshowthatevenalongindividual

vesselstheresponseisheterogeneous,whichaddstothecomplexityofthevascular

response.Inordertoanalyzechangesinthewholenetwork,theyintroducetheconceptof

earlyandlaterespondingvessels.Whiletheyobserveanupwardpropagationofearly

dilationsandlateconstrictions,theearlyconstrictionsandlatedilationsspreadwith

increasingdepth.

Thestimuluslengthaswellastheusedanesthesiacanplayaroleinthevascularresponse.

Drewetal.(2011)showedthatthelongerthestimulusthelargerthevasodilationuntila

plateaudilationisreached.TheeffectsofanesthesiahavebeeninvestigatedbyLyonsetal.

(2016)andMasamotoetal.(2009).Theirworksshowthatthelevelofanesthesiaaffectsthe

increaseincerebralbloodflowaswellasthetissueoxygenpartialpressure.Furthermore,

onehastobearinmindthatbaselinefluctuationsindiameterpersist,whichforthepial

vesselshavethesameorderofmagnitudeasfunctionaldiameterchanges(Drewetal.,

15

2011).Allinall,thespatio-temporaldynamicsofneurovascularcouplingarehighlycomplex

anddifficulttoelucidate,partlyduetotheirmutualinterdependence.

Vesseltype Maximum

dilation

Averagepeak

dilation

Onsettime Timetopeak

Pialarteries 31.6±4.1% 11.0±3.5% 1.0±0.1s 2.9±1.1s

Descending

arteries

31.0±5.0% 9.7±2.2% 0.8±0.2s 2.4±0.4s

Capillaries* 40% 13.3±2.0% 1.7±1.0s 2.8s

Table3:Keycharacteristicsofthevascularresponseofdifferentvesseltypes.Thegivenvaluesaretheaverage

andthestandarddeviationofallvaluesfoundinliteratureforshortsensorystimulation(<5s).Capillaries*:

Dataoninvivocapillarydilationisverysparse.Thus,wealsoconsideredthemeasurementsbyHalletal.

(2014)althoughthestimulusdurationis15s.Furthermore,forcapillariesitwasnotalwaysclearifthegiven

valuesareforactiveorpassivedilation.Maximumdilation:maximumdilationthathasbeenmeasuredin

individualvessels.Averagepeakdilation:averageoverallmeasureddilations.Onsettime:timeafterstartof

stimulusuntildilationisinitiated.Timetopeak:timeafterbeginofthestimulusuntilthemaximumdilationis

reached.Referenceliterature:Pialarteries:(Devoretal.,2008;Devoretal.,2007;Drewetal.,2011;Hillmanet

al.,2007;Sekiguchietal.,2014;Tianetal.,2010;Uhlirovaetal.,2016b).Descendingarteries:(Sekiguchietal.,

2014;Tianetal.,2010;Uhlirovaetal.,2016b).Capillaries:(Halletal.,2014;Tianetal.,2010).

Pialanddescendingarteries:

AsmentionedinSection5.1thepialnetworkisresponsibleforarobustsupplyofbloodto

differenttissueregions.Furthermore,ithasbeenhypothesizedthatitredistributesblood

duringneuronalactivation(Devoretal.,2007;Shihetal.,2015).

Asthepialvasculatureiscomparablyeasytoaccess,manyworkshaverecordedvascular

changesatthepiallevel(Chenetal.,2014;Devoretal.,2008;Devoretal.,2007;Drewet

al.,2011;Hillmanetal.,2007;Ngaietal.,1988;NgaiandWinn,2002;Sekiguchietal.,2014;

Tianetal.,2010;Uhlirovaetal.,2016b).Despitedifferencesinmethodologyand/orspecies,

allstudiesagreethatmultiplepialarteriesaltertheirdiameterinresponsetoneuronal

activation.

Thelargestchangesarelocatedclosetothecenterofactivation(CoA),andtheamplitudeof

therelativediameterchangedecreaseswiththedistancefromtheCoA(Figure4B).Positive

diameterchangeshavebeenobservedupto3mmawayfromtheCoA;however,for

distances>2mmthevascularresponseispredominantlynegative(i.e.moreconstrictions

thandilations(Devoretal.,2008;Devoretal.,2007)).

Formanypialarteriesthereisaperiodofconstrictionafterstimuluscessation,forreasons

notcurrentlyunderstood(Figure4B).However,theamplitudeofconstrictionissignificantly

smallerthanthatfordilation(Devoretal.,2007;Drewetal.,2011;Hillmanetal.,2007;

Uhlirovaetal.,2016b).

TheDAsfeedthecapillarybedwithbloodfromthecorticalsurface.Astheyposethe

“bottleneckofperfusion”(Nishimuraetal.,2007)itseemslikelythattheyarealsoideally

placedforalocalizedincreaseinbloodflow,andthereisalargebodyofevidenceshowing

thedilationofDAsduringactivation(Attwelletal.,2010;Halletal.,2014;Hillman,2014;

IadecolaandNedergaard,2007;Lindvereetal.,2013;Tianetal.,2010;Uhlirovaetal.,

2016b).BasedontheresultsofHalletal.(2014)~50%ofthemonitoredDAsrespondedto

16

neuronalactivation.However,itremainsunknownhowthosearedistributedwithrespect

totheCoA.

RecentstudieshavereportedthatthedilationofDAsisinitiateddeepinthecortex

(measurementsupto0.9mm)andpropagatestowardsthecorticalsurface(Figure4C,

(Lindvereetal.,2013;Tianetal.,2010;Uhlirovaetal.,2016b)).

Uhlirovaetal.(2016b)showedthatapproximately50%ofallrespondingDAsexperiencea

post-stimulusconstrictionphase.Asforthepialvessels,theamplitudeofconstrictionis

significantlylowerthanfordilation.

Itisstilldebatediftheamplitudeofdilationisafunctionofcorticaldepth,andresults

concerningthismatterhavebeendiverging(Figure4C)(Lindvereetal.,2013;Sekiguchiet

al.,2014;Tianetal.,2010;Uhlirovaetal.,2016b).EventhoughitiswellknownthatDAs

dilateduringactivationthedetailedpatternsoftheirresponsestillhavetobeelucidated.

Althoughlackingtheresolutiontodistinguishbetweenchangesoriginatingfrompialand

descendingarteries,arangeofMRItechniqueshavealsoreportedsubstantialincreasesin

arterialbloodvolumeuponstimulation,generallyconsistentwiththediameter

measurementsdiscussedabove(Hoetal.,2011;Kimetal.,2007;KimandKim,2010).

Afurtherinterestingaspectofthevascularresponseisthereturntobaseline.However,this

aspectislessstudiedthantheonsetofdilationandstronglydependsonthedurationof

stimuli.Ithasbeenhypothesizedthatthereisadistinctregulationmechanism,whichis

responsibleforthedecayphase(Chenetal.,2011).Approximately4safterstimulus

cessation,thepialvesselsreachtheirbaselinediameteratwhichtheyremainorcontinueto

constrict(Devoretal.,2008;Sekiguchietal.,2014;Tianetal.,2010).Thereturntobaseline

ofpialvesselsdoesnotdependonthedistancetotheneuronalCoA(Devoretal.,2007).

Pre-capillariesandcapillaries:

Overthepastyearsmoreevidencefortherelevanceofcapillariesforneurovascular

couplinghasemerged(Halletal.,2014;Tianetal.,2010).Ascapillariesarethevesselsmost

proximaltothelargestpartoftissueitseemsplausiblethattheymayplayaroleintheup-

regulationofflow,oxygenandenergysubstratedelivery.However,thereisrelativelylittle

dataavailable,anditischallengingtodifferentiatebetweeneffectsresultingfromarteriole

andcapillarydilationinvivo.Inthefollowing,wecommentondirectandindirectevidence

thatsupportsthehypothesisofanactiveinvolvementofcapillariesinneurovascular

coupling.

AcrucialargumentforactivecapillarydilationhasbeenprovidedbyHalletal.(2014).They

demonstratethatcapillariesdilateonaverage1.4spriortoarterioles,whicheliminatesthe

possibilityofapurelypassiveresponseofcapillaries.Additionally,theyreportthat

capillariesaremorelikelytorespondinthevicinityofpericytes(50%vs.22%response

frequency)andthatthefrequencyofrespondingcapillariesdecreaseswithbranchingorder.

Thisobservationisinlinewithadecreasein𝛼-SMAforpericyteslocatedathigher

branchingorders(Attwelletal.,2015;Hartmannetal.,2015;Hilletal.,2015).Similarlyto

DAs,capillarieslocateddeepinthecortexdilateearlierthantheonesclosetothecortical

surface(Tianetal.,2010).Whetherornottheresistanceatthelevelofsmallvesselsis

regulatedbypericytesorsmoothmusclecellsremainsamatterofongoingdebate(Attwell

etal.,2015;Hartmannetal.,2015;Hilletal.,2015),whichmaybecausedinpartby

differentdefinitionsofpericytesand/orcapillaries.

However,theobservationsbyHalletal.(2014)areincontrasttotheresultsofTianetal.

(2010)whostatethatthedilationspreadsfromtheDAstothecapillarybed.Theworkby

17

Chenetal.(2011)supportsanonsetofvasodilationatthecapillarylevel,becausethey

reportedanincreaseintotalhemoglobinintheparenchymapriortotheincreaseinthe

arterioles.Theoriginofthosedifferencesisnotyetclear.

Anumberofstudiesprovidefurtherindirectevidenceforanactiveregulationmechanismat

thecapillarylevelleadingtoahomogenizationofflow(Gutiérrez-Jiménezetal.,2016;Lee

etal.,2014;Leeetal.,2015;Stefanovicetal.,2008).Leeetal.(2015)measuredtheRBCflux

in~200capillariessimultaneously,duringbaselineandactivation,andshowedthatthe

standarddeviationofRBCfluxdecreased2spriortotheincreaseinthemean.Thisagrees

withtheobservationsofStefanovicetal.(2008)andGutiérrez-Jiménezetal.(2016),who

reportedthatlowbaselinefluxcapillariesexperiencealargerresponsetostimulation.

Furthermoreduringactivation,thecapillarytransittimeheterogeneityisreduced

(Gutiérrez-Jiménezetal.,2016).ThealteredRBCvelocitiesandthehomogenizationofflow

followingneuronalactivationmayalsopartlyresultfromapO2-dependentincreaseinRBC

flexibility(Weietal.,2016).

Itshouldalsobekeptinmindthatcapillarydilationcanbeaneffectivemeanstoalterthe

distributionofRBCs(Schmidetal.,2015),andthatflowhomogenizationduringneural

activationmayplayanimportantroleinoxygendelivery(BarrettandSuresh,2013;

JespersenandØstergaard,2012;Vazquezetal.,2008).

VenulesandVeins

Noactivedilationofvenulesandveinshasbeenreported;however,thereareconflicting

reportsaboutthepresenceand/orsignificanceofpassivedilationofvenulesandveins.For

example,directopticalmeasurementsofvenousdiameterhaveshowneithernoincrease

(Hillmanetal.,2007)orverysmallincreases(Drewetal.,2011),whereasMRI-based

approacheshavereportedconsiderableincreasesinvenousbloodvolume(ChenandPike,

2009;ChenandPike,2010).However,usingabiophysicalmodel,Barrettetal.(2012)

demonstratedthatthesediscrepanciescouldbeexplainedbydifferencesinstimulation

length(typicallyshortinopticalimagingexperiments,butlonginMRIexperiments)andthe

factthatevenrelativelysmallchangesincapillaryand/orvenousdiameterscanleadtolarge

changesinvenousCBV.Takenalongsidetheexperimentalevidence,thisresultsuggests

that,forbriefstimulation,dilationofarteriesandarteriolescontributesthemajorityof

bloodvolumeincreases;however,dilationofpost-arteriolarvesselsisrelevantduring

longerstimulation(>10s).

6.3 Layer-specificregulation

Inthecortexthedistributionofneuronsandconsequentlythemetabolicneedsvaryover

depth.Wehavealreadybrieflycommentedonthepropagationofvasodilationoverdepth.

Yetthequestionremainswhetherthosedifferencesarealsoanindicationforlayer-specific

regulationmechanisms.Here,wesummarizetheavailableevidencefortheexistenceof

layer-specificregulation,andwediscusswhylayer-specificregulationseemsplausiblewith

respecttotheflowfieldfromafluiddynamicspointofview.

Directevidenceforlayer-specificregulationmechanismsisstillrelativelysparse.To

investigatewhethertheresponsetostimulationvariesoverdepth,Gutiérrez-Jiménezetal.

(2016)measuredRBCvelocity,RBCfluxandcapillarytransittimewithtwo-photon

18

microscopy.ArangeofhighresolutionMRIstudieshavealsoobservedlayer-dependent

changesinCBFand/orCBV(Goenseetal.,2012;Hiranoetal.,2011;Hoetal.,2011;Huber

etal.,2015;Huberetal.,2016;KimandKim,2010,2011;Zhaoetal.,2006).Both

approachesagreethatthehemodynamicresponsevariesoverdepth,whichpointstowards

layer-specificregulationmechanisms.However,thesedifferencescouldalsoresultpassively

fromlaminarvariationsinvasculartopology.

Inthecerebralmicrovasculaturedepth-dependentflowandpressurecharacteristicspersist

(Figure4D)andprovidefurtherevidencewhylayer-specificregulationmightbebeneficial

(Gutiérrez-Jiménezetal.,2016;Kleinfeldetal.,1998;Leeetal.,2014;Schmidetal.,2017).

WerecentlyanalyzedthepressuredropalongthepathwayofindividualRBCsandshowed

thatthedeepertheRBCentersthecapillarybedthelargerthepressuredropintheDAand

thesmallerinthecapillarybed(Figure4E,Schmidetal.(2017)).Thepressuredrophasa

strongimpactontheincreaseinflowrateresultingfromdilation.Hence,ourresultssupport

thehypothesisthatlayerspecificregulationcouldbeadvantageous.

Additionally,variousexperimentalaswellasnumericalworksobservethattheRBCvelocity

decreasesoverdepthandconsequentlythecapillarytransittimeincreases(Figure4D)

(Gutiérrez-Jiménezetal.,2016;Kleinfeldetal.,1998;Leeetal.,2014;Schmidetal.,2017).

ThesefactorsstronglyimpacttheamountofoxygendischargedfromRBCsandthereforeit

seemslikelythattheoxygendischargealsovariesoverdepth(Section7.3).

6.4 Dimensionoftheareaaffectedbyneuronalactivation

Inordertodiscussthedimensionsoftheareaaffectedbyneuronalactivationitisimportant

todistinguishbetween(1)theareainwhichthevasculaturerespondstostimulationand(2)

theareathatisaffectedbythealteredvesseldiameters.

Relativelylittleisknownabouttheprecisespatialpatternofthevascularresponse.Atthe

pialleveldiameterchangeshavebeenrecordedupto3mmapartfromtheCoA(Devoret

al.,2008;Devoretal.,2007).Basedontheconceptofretrogradepropagationitseemslikely

thatthosevesselsarelocatedupstreamoftheneuronalCoA(ErinjeriandWoolsey,2002;

Iadecolaetal.,1997).However,theresultsfromChenetal.(2011)suggestthatthe

vasodilationspreads“spatiallyoutwards”andisindependentoftheflowdirection.Evenif

theprecisepatternofvesselrecruitmentisnotyetfullyunderstood,aselectiverecruitment

ofvesselsseemslikely(ErinjeriandWoolsey,2002;Hillman,2014).Abetterknowledgeof

thesignalingpathwaysandmeasurementsofdiameterchangeswithhightemporaland

spatialresolutionarenecessarytoadvanceourunderstandingofthevascularresponse

patterns.

Thedimensionoftheareathatisaffectedbythealteredvesseldiameterstronglydepends

onthelocationofthevesselalongthevascularpathway:thefurtherupstreamthelarger

theareaaffected.IllustrativeproofisgiveninthenumericalworkbyReicholdetal.(2009).

TheyshowedthatifthesiteofdilationalongtheDAisclosetothecorticalsurfaceitsarea

ofinfluenceislargerthanifitislocatedfurtherdownstream.

Toinvestigatethisinvivoisdifficult,becausechangesinalargevolumehavetobe

monitoredsimultaneously.Frequently,techniqueswhichmeasure2Dprojectionsofthe

flowfieldareapplied.Forexample,Dunnetal.(2005)usemulti-wavelengthreflectance

imagingtoestimatethespatialextentofhemodynamicchangesduringfunctional

19

activation.Theyshowthatthesurfaceareawhereachangeintotalhemoglobinisnoted

differsforforepawandwhiskerstimulation:~2.0mm2and~1.6mm2,respectively.

Figure4Vascularresponsetoneuronalactivationandflowcharacteristics.(A)Schematicrepresentationofsmoothmuscle

cellandpericytesinthecerebralvasculature(Figurefrom:Hartmannetal.(2015)).(B)B.1:Responseofpialarteriesto

stimulationasafunctionoftimefordifferentdistancestotheneuronalcenterofactivation(CoA).Distances:red:0-0.5

mm,darkred:0.5-1.5mm,orange:1.5-2.5mm.B.2:Maximumdilation/constrictionforpialarteriesasafunctionofthe

distancetotheCoA(Figureadaptedfrom:Devoretal.(2007).Pialvasculatureininsertadaptedfrom:Schafferetal.

(2006)).(C)C.1:Responseofdescendingarteriestostimulationasafunctionoftimefordifferentdepths.C.2:Onsettime

andtimetopeakfordescendingarteriesasafunctionofcorticaldepth(Figureadaptedfrom:Uhlirovaetal.(2016b).

Schematicdrawingofadescendingarteryadaptedfrom:Duvernoyetal.(1981)).(D)RBCvelocityinthecapillarybedover

depth(Figurefrom:Schmidetal.(2017)).(E)PressuredropalongRBCtrajectoriesforanalysislayer1(AL1:0–200µm

corticaldepth)andanalysislayer5(AL5:800–1000µmcorticaldepth)fordifferentvesseltypes.Theanalysislayersare

200µmthickslicesforwhichdifferentflowcharacteristicsareanalyzed.(Figurefrom:Schmidetal.(2017)).

7 Oxygenation

Impairmentstocerebralbloodflowontheorderofafewminutesaresufficienttocause

irreversiblehypoxicischemicinjury(Jonesetal.,1981;Moskowitzetal.,2010).Therefore

supplyingoxygen,alongwithmetabolicsubstrates,isthemostcriticalroleofthebrain

vasculature.Inthissectionweprovideabriefoverviewofcerebraloxygenation,especially

asitrelatestolaminarfMRI.

7.1 Baselineoxygenation

Inthetraditionalviewofoxygenationinthebrain,arteriesprovideconstantsupplyof

oxygenatedblood,capillariesarethesiteofoxygendelivery,andveinsdraindeoxygenated

blood.However,evidencehasemergedthatchallengesthisconcept.Forexample,several

studiesreportedthatbloodincorticalarteriesandparticularlyarteriolesisnotcompletely

(A) Contractile cells

(B) Response of pial arterioles

20

oxygenated(Devoretal.,2011;Lyonsetal.,2016;Sakadžićetal.,2014;Vazquezetal.,

2010;Vovenko,1999;Yaseenetal.,2011).Inaddition,directmeasurements(Sakadžićetal.,

2014)andcombinedmorphologicalandfunctionaldata(Kasischkeetal.,2011)suggestthat

arteriolessupplyasignificantfractionofthetotaloxygentothetissueunderbaseline

conditions.Furthermore,thereisconflictingevidenceaboutthepresenceand/or

significanceofoxygengradientssurroundingvenulesandveins(Devoretal.,2011;Vazquez

etal.,2010;Vovenko,1999),andoxygenshuntsfromarterialtovenousvesselswerealso

directlyobservedinvivo(Lecoqetal.,2011),consistentwithanumberofstudiesthathave

reportedincreasesinvenouspO2(Sakadžićetal.,2014;Vazquezetal.,2010;Vovenko,

1999;Yaseenetal.,2011).Nonetheless,inrodents,pO2valuesinarteriesandarterioles

typicallyfallbetween60and110mmHg,valuesincapillariesvaryfrom20-60mmHg,and

valuesinveinsandvenulesrangefrom30to60mmHg(Lyonsetal.,2016;Parpaleixetal.,

2013;Sakadžićetal.,2014;Vazquezetal.,2010;Vovenko,1999;Yaseenetal.,2011).

Itisimportanttonotethatdifferencesinoxygenationbetweenawakeandanaesthetized

animalshavebeenreported,andalthoughoverallcapillarypO2appearedsimilarinthe

mouseolfactorybulbandsomatosensorycortex,thismaynotbetrueforallregions,

particularlysincemeasurementsofRBCfluxandlineardensitydiddifferbetweenthetwo

regions(Lyonsetal.,2016).Inaddition,sinceexperimentalandmodelingevidencesuggests

theexistenceoferythrocyte-associatedtransients(EATs;(GolubandPittman,2005;

Hellums,1977;Lecoqetal.,2011;Lückeretal.,2015;Parpaleixetal.,2013)),theremaybe

substantialdiscrepanciesbetweentruehemoglobinsaturationandthatestimatedbasedon

averagebloodpO2(Lyonsetal.,2016).Theoppositecaseisalsorelevant,sinceoxygen

saturationisoftenmeasuredusingtechniqueslikeintrinsicimagingofopticalsignals(Dunn

etal.,2003)orphotoacoustictomography(PAM;(Yaoetal.,2015))andthemeasured

saturationmaythereforeoverestimatemeanbloodpO2.

7.2 Activation-inducedchangesinoxygenation

Inresponsetoactivation,dynamicpO2increaseshavebeenreportedinallvesseltypes

(Lecoqetal.,2011;Parpaleixetal.,2013;Vazquezetal.,2010;Yaseenetal.,2011).Priorto

themainpositiveresponse,abrief,relativelysmalldecreaseinpO2hasalsobeenobserved

incapillariesandparenchymalregionsclosetocapillaries(Lecoqetal.,2011;Parpaleixet

al.,2013;Weietal.,2016).Thisearlyresponse,oftencalledthe‘initialdip’,hasbeen

controversialintheopticalimagingandfMRIcommunitiesduetoconflictingreports

(Buxton,2001;HuandYacoub,2012).Somereportssuggestthattheinitialdipmaybemore

spatiallyspecificthanthesubsequentincreaseinsignal(Vazquezetal.,2010).

ThereisalsosomeuncertaintyregardingthemagnitudeandimportanceofpO2increasesin

thetissuefollowingactivation.ManystudieshaveobservedrobustincreasesintissuepO2

(Lecoqetal.,2011;Parpaleixetal.,2013;Thompsonetal.,2003;Vazquezetal.,2010)and,

usingadrugthatpre-dilatedarteriestopreventfurtheractivation-inducedCBFincreases,

Masamotoetal.(2008)showedthatthesetissuepO2increasesundernormalconditions

occurredinspiteofincreasedoxygendemandinthetissue.Incontrast,Devoretal.(2011)

reportedthat,duringsustainedstimulation,therewasnopO2increaseinregionsofthe

tissuewithlowbaselinepO2.Theauthorsproposedthatactivation-inducedincreasesinCBF

mayoccurtopreventdangerouslevelsofhypoxiaintheseregions;however,itisunclear

21

whetherchangesinCBFwithoutanyincreaseinCMRO2wouldleadtopO2increasesin

theseregions,oriftheyweresimplytoofarfromthevasculaturetobenoticeablyaffected.

7.3 Laminaroxygenation

Tothebestofourknowledge,thereareonlyveryfewstudiesthathavereporteddirect

laminarmeasurementsofoxygenationinthebrain,partlybecauseofthetechnical

difficultiesinobtainingsuchdata.Forexample,electrodemeasurementsareinvasive,

especiallyatdepth,andcurrentopticalapproacheshavelimiteddepthpenetration(Lecoq

etal.,2011;Sakadžićetal.,2010).Furthermore,thestudiesthatdoexistaresomewhat

contradictory.Usingtwo-photonphosphorescencelifetimemicroscopy(2PLM)inalpha-

chloraloseanesthetizedrats,Devoretal.(2011)reportedasubstantialdecreasein

estimatesofarterialoxygenationfromthesurfaceto~200µmintothecortex;however,

Lyonsetal.(2016),usingthesametechniqueinawakemice,didnotobserveanysignificant

depth-dependenceinarterialorvenouspO2intheupper400µmofthecortex.Devoretal.

(2011)alsoobservednoticeabledecreasesintissuepO2intheupper~300µmofthecortex,

whichisconsistentwithpreviousmeasurementsusingoxygensensitivemicroelectrodes

(Masamotoetal.,2003).Masamotoetal.(2003)wereabletomeasurethroughoutthe

corticalgreymatter,deeperintothecortex.Allthreesomatosensoryregionsconsidered

showedasubstantialpO2decreasefromthesurfacetolayerII,buttheforelimband

hindlimbareasshowedfurthervariation,peakingaroundlayerV,whilethetrunkregion

remainedfairlyconstant.Notethatinallofthesestudies,pO2intheuppercorticallayers

mayhavebeeninfluencedbythesurgicalpreparation,includingremovaloftheskull.

Althoughtheirmeasurementtechnique(constantpotentialamperometry)wasnotableto

generatemeasurementsofbaselinepO2,Lietal.(2011)reportedsimultaneous,high

temporalresolutionmeasurementsofchangesoftissuepO2atmultiplecorticaldepthsin

responsetoelectricalstimulationoftheratwhiskerpad.Theauthorsobservedagenerally

biphasicresponse,wherebythepO2initiallydecreased,moststronglyinlayerIV,and

subsequentlyincreased,withthelargestincreasesoccurringintheuppercorticallayers.

Applyinganitricoxidesynthaseinhibitortoreduceactivation-inducedCBFincreasesmade

thepO2responsesmorenegativeatalldepths.Whilethisstudyoffersaninterestinginitial

viewintolaminardifferencesinoxygenation,furtherstudies,ideallyusingcomplementary

techniquessuchas2PLMorPAM,areneededtoinvestigatethesecomplexchanges.

8 Relevanceofvasculaturefor(laminar)fMRI

ThevasculatureisrelevantforanumberofMRItechniques,includingthediffusionand

perfusionweightedimagingapproachescommonlyusedinclinicaldiagnosisandtreatment

ofneuropathologiessuchastumors,stroke,andtransientischemicattack(Finketal.,2015;

Souillard-Scemamaetal.,2015).However,inthisreviewwefocusontherelevanceofthe

vasculatureforBOLD-fMRI.

AlthoughthedetailsoftheunderlyingphysicsofthefMRIsignalarebeyondthescopeof

thisreview,theequationfromBuxton(2013)listedbelow(Equation1)servestosummarize

thewaysinwhichthevasculaturecaninfluencetheBOLDsignal.(Readersinterestedinthe

derivationofthisequationoramorerigorousintroductiontotherelevantphysicsarehighly

recommendedtoconsultBuxton(2013)).Wefocushereonthemorecommonlyused

22

gradientecho(GE)technique,butnotethatthereareimportantdifferencesbetween

gradientechoandspinecho(SE)sequences.Forexample,GEsequencesaretypicallymore

sensitivetolargervessels,particularlyveins,thanSE(Boxermanetal.,1995b;Menon,

2012).

Briefly,theBOLDsignalchangenormalizedtobaseline,∆𝑆 𝑆!,canbedescribedsuchthat

∆𝑺 𝑺𝟎 = 𝒌 ∙ 𝑻𝑬 ∙ 𝑽𝟎 ∙ 𝑫𝟎

𝜷∙ 𝟏 − 𝒗 ∙ 𝒅

𝜷 , (1)

where𝑉!isthebaselinebloodvolume,𝐷!isthebaselineconcentrationof

deoxyhemoglobin,𝑣and𝑑arethedynamicbloodvolumeanddeoxyhemoglobin

concentrationnormalizedtotheirrespectivebaselines,𝑘isaconstantrelatedtotheMRI

fieldstrength,𝑇𝐸istheechotime,and𝛽isaconstantdescribingtheeffectof

deoxyhemoglobinontherelaxationrate.Whilethebloodvolumetermsarerelativelyself-

explanatory,itisworthemphasizingthatlocaldeoxyhemoglobinconcentrationreflectsthe

balanceoftwocompetingprocesses:oxygendelivery(viaCBF),andoxygenconsumption

(CMRO2).SinceCBFgenerallyincreasesmorethanCMRO2duringactivity(FoxandRaichle,

1986),theconcentrationofdeoxyhemoglobindecreases,andtheBOLDsignalincreases

(Ogawaetal.,1990).

AlthoughfMRIhasprimarilybeenusedtolocalizeneuronalactivity,thereisanincreasing

desiretouseitasaquantitativetooltomeasureCMRO2changes(Buxton,2013).Earlier

studiessuggestedthatCMRO2changesprimarilyreflectedenergyuseassociatedwith

neuronalsignaling(AttwellandLaughlin,2001).Assuch,alongstandingaimofthe

neuroimagingcommunityhasbeento‘unmix’or‘deconvolve’thevascularcomponentfrom

themeasuredfMRIresponse,inordertoisolatethemetaboliccontributiontothesignal.

However,amorerecenthypothesisproposesthatdifferenttypesofneuronalactivity,e.g.

excitatoryvsinhibitorysignaling,mayhavedistincteffectsonchangesinCBF,CMRO2,and

electricalactivity(Buxtonetal.,2014;Uhlirovaetal.,2016a).Inthefollowingsections,we

provideanoverviewofthewaysinwhichthevasculatureinfluencestheBOLDresponse,

includingthoseaspectsparticularlyrelevanttolaminarfMRI.

8.1 Baselinebloodvolumeanddeoxyhemoglobinconcentration

AsshowninEquation(1),thebaselinevaluesofCBVanddHbconcentrationacttoscalethe

magnitudeoftheBOLDsignalchangeforagivenchangeinCBVanddHb.Thismeansthat,

dependingonthebaselinevaluesofCBVanddHb,differentchangesinCBVanddHbcould

resultinthesameBOLDsignalchangeand,inversely,thatdifferentBOLDsignalchanges

couldresultfromthesamechangesinCBVanddHb.GiventhatCBVandpO2(likely

reflectingdifferencesindHb)varybetweendifferentcorticalregions,andindeedlayers

(Section4andSection7),thiseffectisimportanttoconsiderwhencomparingBOLD

responsesfromdifferentlocations.

ThebaselinevalueofdHbconcentrationalsoimposesatheoreticallimitonthemaximum

achievableBOLDincrease,sincedHbconcentrationcanonlydecreasefromitsbaseline

valuetozero,andnotbelow.Inaddition,itisalsoimportanttonotethat,despiteprevious

assumptionstothecontrary(Davisetal.,1998;Dunnetal.,2005;Hogeetal.,1999;

Mayhewetal.,2000),capillariesandevenarteriolescontainanon-negligibleamountof

deoxyhemoglobin(Section7;(Lyonsetal.,2016;Sakadžićetal.,2014;Yaseenetal.,2011)).

Evidencefromarecentmodelingstudysuggeststhatassumingarterialhemoglobinis

23

completelysaturatedcanleadtoerrorswhenestimatingCMRO2(BarrettandSuresh,

2015).

8.2 Activation-inducedchangesinbloodvolumeanddeoxyhemoglobin

DeoxyhemoglobinconcentrationreflectsthedynamicbalanceofchangesinCBFand

CMRO2,soitisimportanttoconsiderthespatiotemporalinterplayofthesetwoprocesses.

IntermsofCBF,someauthorssuggestthatthespacingbetweenpenetratingvessels

representsalimitontheminimalachievablepointspreadfunction(PSF)ofBOLD-fMRI

measurements(Turner,2016;UludağandBlinder,2017).Thiswouldbeconcerningforthe

utilityofhighresolutionandlaminarfMRI,particularlyconsideringthatcombined

anatomicalandfunctionaldatafromthemousebarrelcortexshowedthatthelocationof

descendingarteriesandascendingveinsdidnotcorrelatewithwhiskerbarrelregions

(Blinderetal.,2013).Inaddition,diameterchangesindescendingarteriesinthecatvisual

cortexwererecentlyshowntobelessselectiveforstimulusorientationthancalciumsignals

intheneuronssurroundingthem(O’Herronetal.,2016).However,theconclusionthat

penetratingvesselgeometrylimitsthePSFofBOLD-fMRImaybepremature,fortwo

reasons.

First,asdiscussedinSection6,flowregulationmayalsooccuratsitesdownstreamof

descendingarteries,suchaspre-capillaryarterioles,capillaries,andperhapsevenviared

bloodcellsthemselves(Weietal.,2016).ThiswouldallowCBFchangestoberegulatedover

amuchfinerspatialscalethantheterritoryofasingledescendingartery.

Secondly,changesinCBFrepresentonlyoneoftheeffectsleadingtothechangesindHb

thatdrivetheBOLDsignal;changesinCMRO2alsoplayavitalrole.Therefore,evenifCBF

increasesoccuroveralargerordifferentareathantheregionofneuronalactivity,orwith

differentkinetics,modelingapproachesthatmakeuseofmultimodaldatamaybeableto

decoupletheeffectsoflayer-dependentchangesinCBFandCMRO2ondHb(Gagnonetal.,

2015;Heinzleetal.,2016;Markuerkiagaetal.,2016).AnydifferencesbetweentheCBFand

CMRO2responsemayalsohelptoinferthenatureoftheunderlyingneuralactivity

(Uhlirovaetal.,2016a).

TheinfluenceofCBVchangesontheBOLDsignalarecomplex.Partofthismayberelated

todifficultiesindistinguishingbetweendirecteffectsofvolumechangesonthesignal,and

indirecteffectsthatrelatetovolumechanges.AsperEquation(1),earlysimulationsand

experimentsshowedthatpureincreasesinCBVtendtoreducetheBOLDsignalchange

(Boxermanetal.,1995a;Ogawaetal.,1993;YablonskiyandHaacke,1994).However,in

practice,anincreaseinarterialbloodvolumemaysomewhatincreasethesignal,by

exchangingvolumewiththeextravascularfluid,whichhasaweakersignal(Buxton,2013).

Furthermore,increasesinvenousbloodvolumewouldalsotypicallyincreasethe

concentrationofdHbinavoxel,leadingtoreducedBOLDsignal.

Severalrecentstudies,usingelegantapproachestoproducespatiallyconfinedresponses,

haveobservedthatactivation-inducedchangesinCBVappearedmorespecificandlocalized

toneuronalactivitythanBOLDresponses(Moonetal.,2013;Poplawskyetal.,2015).While

theseareinterestingresults,itisimportanttonotethatchangesinCBVareapurely

vascularresponse,andsodonotcontainanyinformationaboutthechangesinCMRO2

thoughttoderivemoredirectlyfromneuronalactivity.Nonetheless,combininghigh

resolutionCBVimagingwithCBFandBOLDmeasurementsintoadetailedbiophysicalmodel

24

wouldbeaparticularlypowerfulapproachforprobingneuronalactivitynon-invasively

(Uhlirovaetal.,2016a).

8.3 DirectMReffects

ThevascularstructureitselfcanalsohaveadirectinfluenceofontheBOLDsignal.Using

detailedreconstructionsofthemicrovasculaturecoupledwithhighresolutionfunctional

measurements,Gagnonetal.(2015)recentlydevelopeda‘bottomup’modeloftheBOLD

signalwhichpredictedthatactivation-inducedsignalchangeswouldvarybyupto40%,

dependingontheorientationofthecortextothescanner’sprimarymagneticfield.The

effectwaspresentonlywhenusinggradientecho,ratherthanspinecho,pulsesequences,

andderivesfromthefactthattheorientationoflargerveinsisnotisotropic,sincetheyare

predominantlyalignedeitherinparallelwithorperpendiculartothecorticalsurface

(Gagnonetal.,2015).Thisorientationdependenceisparticularlyrelevantfordatafrom

humanandprimatebrains,sincethefoldingpatternofgyriandsulciproducesasignificant

variationinlocalsurfaceorientation(Cohen-Adadetal.,2012),whichisnotpresentin

lissencephalicanimals.AlthoughthemodeldevelopedbyGagnonetal.(2015)usedthe

vascularstructurefrommice,ratherthanprimates,andfunctionaldatafromrats,

predictionsfromtheirmodelagreedverywellwithhumandata.However,giventhatthe

imagingvoxelsizewas3.4x3.4x6mm,itremainstobeseenwhethersuchorientation

dependenceexiststhroughthecortex,atthehigherspatialresolutiontypicalforlaminar

fMRI.

9 Outlook

Wehavetriedtocompilethecurrentstatusofresearchonthecorticalvascularsystemin

thepresentreview.Asmentioned,thevascularsystemisatthebasisformanyimportant

topicsinneuroscience,bothinhealthanddisease.Manyaspectsremaininsufficiently

understood.Firstandforemost,thehumancorticalvascularsystemhasnotbeenstudied

quantitatively,andmanyofourcurrentconceptsrelyonrodentdata.Itistherefore

importantthatnoveltechnicalapproachesemergethatallowvascularlabellingandimaging

ofhumanpost-mortemtissue.Thiswilldirectlyleadustoaddressingrelevantquestions

regardingtheinvolvementof(micro-)vascularnetworkalterationsinneurodegenerative

diseases.Anotherimportantfutureresearchdirectionconcernsthesizeofthe

reconstructednetworks.Muchofwhatweknowtodayreliesonrelativelysmall(fewcubic

millimeters)networks.Itisdesirablethatwholebrainvascularsystemreconstructions

becomeavailable,whichseemsfeasibleatleastinthemouse.Thiswouldsignificantly

reducetheproblemoftheboundaryconditionsformodelingbloodflowdynamics.

Furthermore,bloodflowdynamicsmusteventuallybestudiedinvivo,andmethodsare

neededthatcancoverentiremicrovascularnetworkswithsufficientspatialandtemporal

resolutiontocapturedynamicsatthesingleredbloodcelllevel.

10 Acknowledgements

25

FundingforthisworkwasprovidedbytheUniversityandETHZurichandtheSwissNational

ScienceFoundationGrantNo.140660.BWisamemberoftheClinicalResearchPriority

ProgramoftheUniversityofZurichonMolecularImaging.MBissupportedbythe

ForschungskreditoftheUniversityofZurich.

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