ventilator manuscript

8/22/2019 Ventilator Manuscript

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AConstant-VolumeVentilatorandGas

RecaptureSystemforHyperpolarized

GasMRIofMouseandRatLungs

JohnNouls,ManuelFanardjian,Larry?,BastiaanDriehuys


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HyperpolarizedgasMRIhasintroducednewmeanstovisualizepulmonaryfunction

regionally,non-invasivelyandwithhighresolution.Thistechnologyhasseenwide-

rangingapplicationinclinicalresearch1-8,butonlymodestapplicationinanimal

studies9-12.ThisispartlybecausehyperpolarizedgasMRIismorestraight-forward

toconductinhumansubjects,whocaninhaleandholdtheirbreath,thanin

uncooperativeanimalswhichmustbeanesthetizedandventilated.Nonetheless,

manyapplicationsareemergingforhyperpolarizedgasMRIinsmallanimals,but

thishasbeenlimitedtoafewcentersthathavetheexpertiseneededtoprecisely

deliverhyperpolarizedgasestosmallanimals.Whilethemajorityofsmallanimal

hyperpolarizedgasMRItodatehasbeenconductedinrabbits13-15,guineapigs16,

andrats17-22,thereisalsoacompellingneedtoimagemicegiventheirprominent

roleinbiomedicalresearch.Thispresentsevengreaterchallengesfor

hyperpolarizedgasMRIbecausecomparedtorats,mouselungvolumesare10-fold

smaller,breathingrateis2-foldfaster,andhigherresolutionisrequiredtovisualize

theirairways.

Deliveryofhyperpolarizedgasesforsmallanimalimagingrequiresadedicated

ventilatorthatcanbeusedinproximitytoanMRmagnet.MR-compatible

ventilatorshavebeenavailablesincethemid80stofacilitatesmall-animalproton

imagingofthelungandintegratethecapabilitytoadministergaseousanesthesia,

controlbreathingandtriggerimaginginsynchronywiththerespiratoryandcardiac

cycles23-27.Specifically,itwasrecognizedthathigh-resolutionimagingofthesmall

animallungrequiredbuildingupimagesovermultiplebreaths,whichinturn


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requiredrepositioningthelungwithhighprecisionduringeachcycle.However,

theseventilatorswerenotdesignedtodeliverhyperpolarizedgases,whichmustbe

handledusingonlyspecificmaterialsthatpreservenuclearspinpolarization28,29.To

thisend,Hedlundetal,pioneeredthefirstventilatorsthatcoulddeliver

hyperpolarizedgasesinawell-controlledfashion,whileusingcomponentsthat

avoideddepolarization.Theseventilatorsenabledthefirstinvivo3Heimagesof

guineapiglungs16,andsubsequentimprovementsenabledthedeliveryofasmaller

tidalvolumeinrats30.Theseventilatorsreliedonacustom-made,pneumatically-

controlledvalvepositionedclosetotheanimal,containingseveraldelicateflexible

membranes31-33.Acommercialvalverecentlybecameavailable34.However,the

intricatedesign,rigorousfabricationrequirementsorphysicalsizeofthesevalves

limitedtheiradoptionatothercenters.

Recently,Chenetal.introducedadifferenthyperpolarizedgasventilator22that

eliminatedtheneedforacustomdeliveryvalve.Thischangesignificantlysimplified

thedesign,andwiththereductionofdeadvolumesandcontinuedevolution,

permittedhyperpolarizedgasMRItobecomepossibleinmice35.Thisventilator

designhasbeenusedbyourlaboratorytoconductstudiesusing3Heand129Xe,in

bothratsandmice36,37.Ourrecentstudiesusingbronchoconstrictivechallengeto

studyairwayshyperresponsivenessinmice38haveledustointroduceseveralnew

designelementsthatmaketheconstantvolumedeliveryofhyperpolarized3He

morerobustandpermitquantitativeanalysis39.Furthermore,thepaucityandcost

of3Heandthefrequentuseofexpensiveisotopicallyenriched129Xecreateastrong


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incentivetorecapturetheexhaledgasesafterimaging.Hence,itistheobjectiveof

thismanuscripttoclearlylayoutthekeydesignaspectsofthissimpleandrobust

ventilatorandrecapturesystem,toprovideanexperimentalvalidationofits

performance,andtoincludeacomprehensivelistofpartstoenableduplicationby

otherswishingtoconductpreclinicalhyperpolarizedgasMRimaging.

ConceptualOverview

Theventilatorisdesignedtodeliveramixtureofnitrogenandoxygenduring

normalbreathing(usually80%N2and20%O2)andtosubstitutehyperpolarized

gasinplaceofnitrogenduringhyperpolarizedgasMRI40.Asshowninfigure1,the

deliveryofnitrogenandoxygeniscontrolledbyfast-switchingsolenoidvalvesthat

resideinthe0.1Tfringefieldofthe2Teslamagnet.Afterpassingthroughthese

valves,thebreathinggasesaredeliveredthroughalong,butlow-deadvolume

polymertubetotheanimalinthemagnetbore.Asimilartubedirectsexpiredgasto

theexhalevalve,alsoresidinginthefringefield.Aswithmosthyperpolarizedgas

ventilatordesigns,thehyperpolarizedgasisdeliveredfromaTedlarbag,whichis

housedinsidearigid,pressurizedcylinder.

Akeydesignrequirementfortheventilatoristorepeatedlydeliverallnecessary

gasesinprecisevolumes,withoutneedingamixingvalvethatoperatesclosetothe

animalstrachea.Thisrequirementismetbyflowingthegasesfromapressure


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muchhigherthanphysiologiclungpressure(severalpsig),throughaprecisionhigh-

impedanceflowconstrictortoachieveawell-definedflowrate.Thetidalvolumeof

eachgasisthendeterminedbytheproductofthisflowrateandthedurationfor

whichthedeliveryvalveisopened(slightlyincreasedbymechanicaldeadspace).

Thisdesignensuresthatthegasflow,andthusthetidalvolume,aredominatedby

theimpedanceoftheflowrestrictorandarelargelyunaffectedbytheimpedanceof

theconnectingtubing,oranychangesinlungimpedanceduringimaging.

Theinspirationphaseendswhenthevalvescontrollingtheflowofoxygen,

nitrogenorhyperpolarizedgasareclosed,initiatingabriefbreath-holdduring

whichimagingdataistypicallyacquired.Afterthis,thesolenoidvalvecontrolling

exhalationisopenedtoinitiatepassiverelaxationofthelungsbacktofunctional

residualcapacity.Formice,typicaltimingoftheventilatorycycleisabreathingrate

of100breathsperminute,with150msforinspiration,a150msbreath-hold,and

300msforexpiration.

Theoperationofthehyperpolarizedgaslineisslightlydifferentfromthe

oxygenandnitrogenlinesbecausethehyperpolarizedgascannotpassthrougha

typicalmetallicsolenoidvalvewithoutsubstantialpolarizationloss.Hence,the

deliveryofthehyperpolarizedgasiscontrolledusingarapidlyswitchingpneumatic

valveconstructedentirelyfromTeflon.Thepneumaticvalveisopenedandclosed

bypressurechangescontrolledbyasolenoidvalve.Additionally,theflowrestrictor

usedonthehyperpolarizedgaslineismadeofsapphireembeddedinnylon;both


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materialsarenon-depolarizing.Infigure1,thepartsoftheventilatorthatmustbe

constructedfromnon-depolarizingmaterialsareindicatedbyaboldline.They

includethehyperpolarizedgasvalveandrestrictor,aswellastheinhaletubing.

PhysicalImplementation

Theventilatorisconstructedusinginexpensive,off-the-shelf,fast-switching

solenoid-actuatedvalves(modelEC-2-12-H,Clippard,Cincinnati,OH),whichcontrol

theflowofoxygen,nitrogen,exhalation,andtheactuationofthepneumaticvalve

deliveringhyperpolarizedgas.Foreachgasline,thepressureiscontrolledusing

compactpneumaticregulators(modelR7010,AirLogic,Racine,WI).Oxygenand

nitrogenflowthroughbrassflowrestrictors(modelMLP-1-BRtoMLP-5-BR,andL-

6-BRtoL-30-BR,O'KeefeControls,Trumbull,CT).Hyperpolarizedgasflowsthrough

afast-switching,pneumaticallyactuatedvalvewithall-Teflonconstruction(model

PV-1-1134,PartekDivision,ParkerHannifin,Tucson,AZ)andanon-depolarizing

sapphireconstrictor(modelF-3-NYtoF-30-NY,O'KeefeControls,Trumbull,CT).

Thesizeoftherestrictordependsonthetidalvolumerequiredbytheanimaland

thetypeofgasadministered(Table1).Asisdiscussedlater,itisimportantto

minimizethemechanicaldeadspacebetweentherestrictorsandthevalves,which

isdonebyintegratingtheconstrictordirectlyintothehosebarb,asshowninfigure

2.

Thenitrogen,oxygen,andhyperpolarizedgaslinesarecombined

immediatelyuponexitingtheirrespectivecontrolvalveintoasingle1-m-long,


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polyetheretherketone(PEEK)inhaleline(modelTPK130,ViciValcoinstruments,

Houston,TX),whichlinkstheventilatortotheanimalinthecenteroftheMRI

magnet.Becausethislinecarrieshyperpolarizedgas,itmustbeconstructedfrom

non-depolarizingmaterials,inthiscase,PEEK.Furthermore,hyperpolarizedgasand

oxygentraveltogetherthroughtheinhaleline,andoxygenisapotentsourceof

depolarization,itiscriticaltolimittheirinteractiontimebyminimizingthedead-

volume.Inoursystem,thedeadvolumeoftheinhalelineis0.4ml(inner-diameter

1/32in).Duringtheadministrationofhyperpolarizedgastoamouse,theusualtidal

volumeof0.25mlresultsinaninteractionbetweenoxygenandhyperpolarizedgas

lasting1.6breaths(960ms),whichresultsinlessthan10%polarizationlossduring

transit.

Exhalationproceedsthroughpolyurethanetubingwithaslightlylargerinner

diameter(1/16in)toprovidelowimpedanceandtoensurethatfullexhalation

occursaftereachbreath(modelURH1-0402,Clippard,Cincinnati,OH).Throughout

thebreathingcycle,theairwaypressureismonitoredusingacompactpressure

transducer(modelXFGM-6065KPGSR,FujikuraAmericaInc.,SantaClara,CA)

mountedatthejunctionoftheinhaleandexhalelines.Thissolid-statetransducer

wasselectedbecauseitprovidesrobustpressurereadingsuptoafieldstrengthof7

Tesla,whichisimportantbecauseitresidesintheboreoftheMRImagnetduring

imaging.


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Allsolenoidvalvesinthesystemareactuatedbysolid-staterelays,whichare

computer-controlledbyadatainput/outputboard(modelPCI-6602,National

Instruments,Austin,TX)drivenbyreal-timeinstrumentationsoftware(Labview

8.0,NationalInstruments,Austin,TX).Thissoftwareisusedtosetthedurationof

theinspiration,breath-hold,andexpiration,aswellasthebreathingrate.Italso

providesa5Vtriggersignalthatcanbepositionedanywhereintherespiratorycycle

(usuallyatend-inspiration)toinitiateMRacquisition.Thissoftwarealsocontrols

theanimal'sbodytemperatureandmonitorsheartrate.Althoughitisnotdiscussed

indetailinthismanuscript,thesoftwareisavailableasanon-linesupplementto

thismanuscript,alongwithdetailedplumbingandwiringdiagrams,schematicsof

thepowerdistributionelectronicsandanextensivelistofcomponentsand

suppliers,athttp://www.civm.duhs.duke.edu/VentilatorMouseRat.

Asshowninfigure1b,theventilatorassemblyiscompactandresidesona

portablecart.Duringimaging,theventilatorcartispositionedattheedgeofthe

magnetbore,connectedtotheanimalbytheinspirationandexpirationlines.The

ventilatorcartfacilitatesshuttlingtheanimalbetweentheanimalpreparationarea

andthemagnet,andcanbeusedondiverseimagingsystems.Itisconnectedtoan

externalsupplyofnitrogenandoxygenbyextensiblepneumaticlines,andtothe

computerandpower-distributionelectronicscontrollingvalveactuationbya25-pin

cable(D-subminiatureDB-25).


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GasRecapture

Duringhyperpolarizedgasimaging,exhalationiscontrolledbyadifferent

expirationvalvefromtheoneusedduringnormalnitrogen/oxygenbreathing.From

thisvalve,theexhaled3Heor129Xemixtureisdirectedtoacaptureballoonmadeout

ofmetalizedmylar.Byusingthisseparateexhalevalve,theballoonfillsonlywith

exhaled3Heor129Xe(plusresidualbreathinggases),andminimizesdilutionwithair

thatwouldoccurifcapturealsocontinuedduringnormalbreathing.Afterthe

imagingsession,themylarballoonisattachedtoaseparatestationthatfirst

withdrawsthegasfromtheballoonandthencompressesitintoagascylinderfor

storage(figure3).Thisisaccomplishedusingapneumatically-actuatedpiston(part

number6498k424,McMaster,Aurora,OH).Oncethestoragecylinderhasreached

capacity,itcanbesenttoareprocessingfacilitysothat3Heor129Xeisseparated

fromnitrogen,oxygen,carbondioxideandothergasesandrenderedsuitableforre-

useinsubsequentnon-clinicalorpre-clinicalhyperpolarizedgasstudies.

PerformanceMetricsandConsiderations

Areasonableruleofthumbforsmallanimalventilationistouseatidalvolumeofair

of1mlperminute,pergramofbodymass41,approximately.Atypical25gmouse

canbeventilatedat100breathsperminute,withatidalvolumeof0.25ml.Weuse

thisexampletofurtherdiscusstheperformancemetricsandlimitationsofthe

ventilatordesign.


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Foranyofthegaslines,theflowrateQisgivenby

Qsupplied =P

Rrestrict +Rtubing +Rlung

P

Rrestrict (1)

wherePisthepressuredropacrosstheentireflowpath,andtherelevant

resistancesRarethoseoftheflowrestrictor,thetubing,andthelungs.Bydesign,

wesetRrestrict>>Rtubing+Rlung,tomaketheapproximationvalid.Forreference,we

commonlyemployflowrestrictorswithanimpedanceof1250cmH2Os/ml,

whereasthetubingimpedancewasmeasuredtobe85cmH2Os/ml.Thelung

impedancerangesfrom0.5cmH2Os/mlnormallyto2cmH2Os/mlduring

bronchoconstriction42.

Takingtheoxygenlineasanexample,andassumingventilationofa25gmouse,the

inhalationandexhalationproceedsasfollows.Theoxygenpressureismaintainedat

2psigattheentranceofaflowrestrictorwithanimpedanceof0.26psimin/ml,

resultinginaflowrateof7.7ml/minwhenthevalveisopen.Duringthe150-ms-

longinspiration,0.019mlisdispensedtowardsthemouse.Attheendofinspiration,

thevalveclosesandoxygennolongerflowsthroughthevalve.However,oxygen

continuesflowingthroughtherestrictor:thepressureinthemechanicaldeadspace

betweentherestrictorandtheclosedvalverisesuntilitequilibrateswiththe

drivingpressureof2psig.Atthebeginningofthenextinspirationcycle,thevalve

opensandthisvolumeofcompressedgasisimmediatelyreleasedandaddstothe

nextinspiration.Specifically,thedeadspacewasmeasuredtobe0.030ml.That


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volumeofoxygen,originallycompressedto2psig,expandswhentheoxygenvalve

openstoavolumeof0.034mlclosetoatmosphericpressure.Hence,thetotal

volumeflowingthroughtheoxygenvalve,perinspiration,is0.019+0.034=0.053ml.

Tomitigatethepotentialinjurycausedbytheinstantaneousreleaseofcompressed

gastothelungsandavoidbarotrauma,themechanicalspaceinwhichthis

compressedgasaccumulatesmustbeminimized(figure2).

Thetidalvolumeofoxygenthatactuallyreachesthelungissmallerthancalculated

above:

TVlung=TVsupplied-Vdead

(2)

whereTVlungisthetidalvolumeofoxygendeliveredtothelungs,TVsuppliedisthe

tidalvolumeflowingthroughtheoxygenvalve,andVdeadisthedeadvolume

betweentheanimal'stracheaandthejunctionteeoftheinhaleandexhaleline.This

deadvolumemustbeminimizedtoensuresufficientoxygenationoftheanimal,as

illustratedbyfigure1and2c.Ingeneral,wealsoemployamixtureslightlyricherin

oxygen,consistingof25%O2ratherthan20%.

Theoperationofthenitrogenlineissimilartothatdescribedforoxygen;fora25g

mouse,itdelivers0.2mlofnitrogenperinspiration.Sincebothgasesemergefrom

high-impedanceflowrestrictors,theirvolumessimplyaddlinearlytoprovidethe

totaltidalvolumedeliveredof0.25mlofbreathingmixtureperinspiration.Asis

apparentfromthediscussionabove,theapproximatetidalvolumeofagivengascan


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bedeliveredbyidentifyinganadequateflowrestrictor,andthenmakingsmall

adjustmentsinthedrivingpressuretosetthevolumeexactly.Becauseofthis

operatingprinciple,theventilatorcanbesimplychangedtoventilatemiceandrats,

oradministerdifferenttypesofgas,byswappingtherestrictorsandtubingsizeas

outlinedintable1.

MeasurementofTidalVolume

Thetidalvolumeemergingfromtheendoftheendotrachealtubewasdetermined

byfeedingitsoutputintoaninvertedgraduatedcylinderinawaterbath,and

measuringthedisplacedwatervolumeduringaknownnumberofinspirations,as

illustratedinfigure4a.Thismethodwasusedtoshowthelinearincreasesintidal

volumesofoxygen,nitrogen,xenonandheliumthatareachievedbyincreasingthe

drivingpressure.Inaddition,animportantconsiderationistherobustnessoftidal

volumeagainstincreasinglungresistance.Thiseffectwassimulatedbyplacingthe

outputoftheendotrachealtubesuccessivelydeeperunderwater,therebyincreasing

theback-pressureagainstwhichtheventilatorwasworking.Asshowninfigure4d,

thetidalvolumedeliveredremainsconstantoveraphysiologicallyrelevantrangeof

pressures.

AnimalPreparationandInVivoImaging

Protocolsdescribinganimal-relatedworkandguaranteeinganimalwelfarewere

approvedbytheInstitutionalAnimalCareandUseCommitteeatDukeUniversity.


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Two6-to-8week-oldmaleBalb/C,20-to-25gmicewereanesthesized,underwent

tracheotomyandreceivedtail-veininjectionsofmethacholinefor

bronchoconstrictorchallengeasdescribedbyThomasetal38.

Theanimal'sendotrachealcatheterwasconnectedtotheventilatorprovidinga150

msinspiration,a150msbreathhold,anda300msexpiration,atabreathingrateof

100breathsperminute.Thetidalvolumewascomposedof0.07mloxygen,and

0.15mlnitrogenreplacedbyheliumorxenonduringhyperpolarizedgasimaging.

Heartrateandairwaypressureweremonitoredinreal-time.

Theanimalwasplacedintoadual-frequencyquadraturebirdcagecoil,providing

both1Hand3He,or1Hand129Xesignals(m2mimagingcorp.,Cleveland,OH).The

coilcontainingthemouse,linkedtotheventilatoronarollingcart,wasmovedfrom

theanimalpreparationtabletothemagnet,andthecoilwasinsertedintothebore.

Therodent'sbodytemperaturewasmeasuredbyarectalthermistorandfedback

intoasystemcirculatingwarmairinthemagnetbore,maintainingthebody

temperaturebetween35and37C.

Duringhyperpolarizedheliumimagingofthefirstmouse,onethree-dimensional

imagewasacquiredinfiveminutes(respiratorygatedradialacquisition,FOV

202032mm,TE0.9ms,TR5ms,BW31.25kHz,10,001projections,matrix

128128128,resolution156156250m3).Then,aseriesoftwo-dimensional

3Heimageswereacquiredconsecutively,before,duringandaftermethacholine


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challenge.Thesedatawereacquiredoneimageevery12seconds,foratotaloffour

minutes(respiratorygatedradialacquisition,FOV20x20mm,TE0.9ms,TR5ms,

BW31.25kHz,400projections,matrix128128,resolution156156m2).

Specifically,afteracquiringtworeferenceimages,a250g/kgbolusof

methacholinewasinjectedtoinduceseverebroncho-constrictionandimaging

continuedfor18moreimagestocapturetheresponse.Throughouttheimaging,the

peakinspirationpressurewasrecorded.Aftercompletionofthe3Heimaging,a3D

1Himagewasacquiredtoprovideananatomicalreference.Thisacquisitiontook10

min(respiratorygatedradialacquisition,FOV404040mm,TE0.9ms,TR5ms,

BW31.25kHz,20,001projections,matrix128128128,resolution313313313

m3).

Hyperpolarized129Xeimagesofthesecondmousewereacquiredover20min

(respiratorygatedradialacquisition,FOV303032mm,TE0.9ms,TR5ms,BW

31.25kHz,10,001projections,matrix12812832,resolution2342341000

m3).Thiswasagainfollowedbyan1Himageoftheanatomyasdescribedabove.

DiscussionofVentilatorPerformance

Theventilatordescribedhereisusedroutinelyatourlaboratorytoventilatemice

andrats,sometimesforperiodsoftimeupto6hrs.Inaddition,itenablesthe

acquisitionofHPgasimagesaccumulatedoverhundredsofbreaths,whichrequirea

highly-reproducibletidalvolumeandpositioningofthediaphragm.Itsperformance

forimaging1H,3Heand129Xeinamouseisillustratedbyfigure5.Notethegood


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airwaydelineationinboththe3Heand129Xemaximumintensityprojections.

Similarly,thesharplowerlungborderonthecentralslicefrom3D1Himages

indicatesreproduciblepositioningofthediaphragmoverthe4000breath-holds

requiredtoacquiretheimage.

Figure6illustratestheimportanceofconstanttidalvolumedeliveryovertheentire

durationoftheimageacquisition.Theleft-hand-sideimagewasacquiredwithalow

drivingpressureandlargediameterrestrictor.Duringtheprogressionofimage

acquisition,thetidalvolumedecreasesbecausethebagholdinghyperpolarizedgas

becomeslessandlesscompliantasitempties.Consequently,theimageisseverely

degradedbythegradualdecreaseintidalvolume.Conversely,theright-hand-side

imagewasacquiredwithahighdrivingpressureandasmallconstrictordiameter.

Thedrivingpressureissufficienttoovercomethechangesincomplianceofthebag.

Itenablesthedeliveryofaconstanttidalvolumeresultinginaimagedevoidof

motionartifacts.

Amorestringenttestoftheventilatoristheabilitytodeliveraconstanttidal

volumewhenthelungimpedancechanges,asisthecaseduringseverebroncho-

constriction.Theconsistencyof3Hetidalvolumecanbeassessedfromthe

magnitudeofthefirstdatapointofeachradialviewofk-spaceduringimaging.This

exploitsanadvantageofradialimaging:thecenterofk-spaceisacquiredoneach

viewanditsmagnitudeisdirectlyproportionaltotheamountofmagnetization

inhaled.AplotofthissignalintensityisshowninFigure7foralltheviewsacquired


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duringmethacholinechallenge,alongwiththecorrespondingpeakinspiration

pressure.Notethatthepeakinspirationpressureincreasesduringmethacholine

challengebecausetheventilatedlungvolumeisreducedwhiletheamountofgas

deliveredremainsfixed.The3Hesignalexhibitsaslowexponentialdecayovertime,

whichisattributableto3Herelaxationwithinthebagholdinghyperpolarizedgas.It

showsonlyaslightdecrease(10%)duringbroncho-constriction,indicatingthatthe

ventilatorcontinuestodeliverarelativelyconstanttidalvolumeof3He.

ConclusionandFutureDevelopment

Wepresentaconstant-volumeventilatorthatiseasytoreplicateandimplement.

Itssimplicitystemsfromcombiningoxygen,nitrogenandhyperpolarizedgas

distallyfromtheanimal.Longlinesdelivergastotheanimal'slungs.Byselecting

asufficientdrivingpressureandminimizingdeadvolume,theventilatordelivers

ofaconstanttidalvolumeofgas.Onlyoff-the-shelf,inexpensivecomponentsare

required,allowingrobustoperationandaneasyscalingoftheventilatorto

deliverothertidalvolumes.

Wedemonstratethattheventilatorachievesconstantvolumedelivery,even

duringdifferentstagesofbroncho-constrictionandenablesquantitative

hyperpolarizedgasquantitativeMRIofmiceandrats.Futuretechnical


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developmentsincludetheadditionofgaseousanesthesiaandanebulizerto

deliverdrugsbyinhalation.

Acknowledgements

TheauthorsgratefullyacknowledgeBomaFubaraandYiQiforanimalsupport,

MichaelFosterandErinPottsforusefuldiscussions.WorkperformedattheCenter

forInVivoMicroscopy,aNIH/NCRRNationalBiomedicalTechnologyResearch(P41

RR005959)andsupportedinpartbyR01-CA-142842.


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Figure1

a)Schematicoftheventilator.Fromtheupperrightcorneroftheschematics:

oxygenflowsthrougharegulator,apressuregauge,thenaflowrestrictor.Its

deliverytotheanimaliscontrolledbytheoxygenvalve.Thenitrogenlineoperates

similarly.HyperpolarizedgasisdeliveredfromaTedlarbagwhichresidesinarigid

chamberthatispressurizedbyN2.Thevalveandrestrictor,andtubingthathandle

HPgasarebuiltfrompolarization-preservingmaterials,asindicatedbythebold

line.Allgasesarecombinedatthevalveoutputsanddirectedthroughtheinhaleline

totheendotrachealcatheter.Afterinspirationandabriefbreath-holdforimaging,

gasesareexpelledbyopeningtheventingvalve(normalbreathing)orcapturevalve

(HPgasbreathing).Note:tubingrepresentedbyatriplelineindicatesthatthedead


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volumemustbeminimized.b)theventilatorandallthevalvesarepositionedinthe

fringefieldofthe2Teslamagnetduringimaging.One-meter-longlinesdirectthe

breathingmixturetowardstheanimalatthecenterofthemagnet,orrecollectthe

exhalemixture.


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Figure2

a)Tominimizethemechanicaldeadspacebetweentheconstrictorandthevalve,

thesolenoid-actuatedvalvesoftheoxygenandnitrogenlinesuseaconstrictor

integratedintothebrasshosebarb,highlightedbytheblackovalbackground.b)

Similarly,theflowrestrictorforhyperpolarizedgas(emphasizedbytheblackoval

background)islocatedascloseaspossibletotheTeflon-constructed,

pneumatically-actuatedvalve.c)Thevolumeoftubingfromthejunctiontee

betweentheinhaleandexhalelinetothetipoftheendotrachealcatheteris

subtractedfromthetidalvolumeoffreshmixtureinhaledbytheanimal.Thevolume

waslimitedto0.02ml(blackbackground).


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Figure3

Apparatustransferringtheexhaledmixturecontainedintheballoontoa

pressurizedcylinder.a)Aftertheballoonisconnectedtotherecapturesystem,the

pistonismovedup,withdrawingthecontentoftheballoonthroughacheckvalve

intothechamberofthepiston.Then,whenthepistonispushedin,anothercheck

valveforcesthecontentsofthepistontoflowtowardthecylinderwherethe

recapturedgasisstored.Thegasalsocontainsairandcarbondioxide.b)Mechanical

modelwherethecasingistransparenttoshowthemaincomponents,andc)the

deviceinuseatourlab.


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Figure4

Measurementoftidalvolume.a)Asmallgraduatedcylinderisfilledwithwaterand

placedupsidedowninawaterbath.Theoutputoftheendotrachealcatheter

displaceswaterfromthegraduatedcylinderandallowsthetidalvolumetobe

determinedoverafixednumberofbreaths.Totestrobustnessagainstimpedance

changes,thegasoutputcanbeplacedincreasinglydeeperinthewaterbathto

increasethebackpressure.b)Nitrogenandoxygentidalvolumesincreaseinlinear

proportiontotidalvolume.Theverticalbarwithineachmarkershowsthestandard


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deviationofthedatafittedlinearly.Inthisexample,thetidalvolumeofnitrogen

increaseswithaslopeof0.0440.011ml/psi,indicatinghowpreciselythetidal

volumecanbeadjusted.c)Thetidalvolumedispensedremainslargelyconstant

regardlessofbackpressure.Theverticalbarwithineachmarkershowsthe

standarddeviationofthedatafittedtoastraightline.Thedecreaseintidalvolume

versusbackpressureisnegligible,at0.72.5l/cmH2O.


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Figure5

a)maximumintensityprojection(MIP)ofavolumeimagesetacquiredonaBalb/C

mouseventilatedwithoxygenandhyperpolarized3He.b)MIPfromadifferent

Balb/Cmouse,ventilatedwithoxygenandhyperpolarized129Xe.Becauseof

differentdiffusionproperties,xenonrevealsairwaysmoreprominentlythanhelium.

c)313-micrometerthick1Hslicethroughthelungs.Thesharpedgeatthebaseof

thelungssuggeststhattheventilatoraccuratelyrepositionedthediaphragm4000

consecutivetimes,thenumberofbreathingcyclesrequiredtoacquiretheimage.d)

Therecordedairwaypressureincreasesduringthe150-msinhalation,followedbya

150ms-longplateau,indicatingbreathhold.Theairwaypressuredecreasesduring

the300msexpiration.Eachbreathingcycleisidenticalandlasts600ms.The


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imagingtriggers,located160-msintothebreathingcycle,indicatethestartofthe

partialacquisitionoftheimage,whichlasts100ms(shadedrectangle).


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Figure6

Effectofavariabletidalvolumeonimagequality.a)Athree-dimensional3Heimage

arrayofmouselungswasacquiredusingarespiratorygatedradialacquisitionover

500breaths;thecenter-sliceisdepicted.Alowdrivingpressureof8.5cmH2Owas

usedtomovehyperpolarizedgasthroughalargediameterconstrictor,which

resistanceiscomparabletothatofthelungs.Thiscausedthetidalvolumetovary

andgraduallydiminishoverthecourseof500breaths,asthe3Hesupplybag

depletedandbecamelesscompliant.Asaconsequence,theimagequalityis

degraded.b)Astandardhigh-impedanceconstrictorandadrivingpressureof4.5

psigwereused,allowingthetidalvolumetoremainconstant,thereforerepeatedly

positioningthebaseofthelungsatthesamelocation,andgivingrisetoahigh-

qualityimage.


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Figure7

a)Duringa4-minute-longchallengeinducedby250g/kgdoseofthebroncho-

constrictormethacholineinamouse,aseriesof20two-dimensionalimageswere

acquiredconsecutively,oneimageevery12second.Themagnitudeofthe3Hesignal

(k=0)remainslargelyconstant(fullline),althoughanexponentialdecayisapparent

duetorelaxationwithintheTedlarbag.Thedashedlineshowsthepeakinspiration

pressure,revealingaclearincreaseinairwayresistanceaftertheinjectionof

methacholine,24safterthestartoftheexperiment.Animageofthemouselungsis

shownbeforemethacholineinjection(left),duringbroncho-constriction(middle)

wherethelargerairwayshavedecreasedindiameter(arrows),andafterrecovery

(right).

ventilator manuscript

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