An Automated Device to Increase Screening Throughput of ...

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An Automated Device to Increase ScreeningThroughput of Zebrafish LarvaeFuoad Saliou-SulleyUniversity of Maine, [email protected]

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ANAUTOMATEDDEVICETOINCREASESCREENINGTHROUGHPUTOF

ZEBRAFISHLARVAE

By

FuoadSaliou-Sulley

B.S.UniversityofMaine,2014

ATHESIS

SubmittedinPartialFulfillmentofthe

RequirementsfortheDegreeof

MasterofScience

(inBiologicalEngineering)

TheGraduateSchool

TheUniversityofMaine

August2017

AdvisoryCommittee:

PaulJ.Millard,AssociateProfessorofChemicalandBiologicalEngineering,

Advisor

DouglasW.Bousfield,ProfessorofChemicalandBiologicalEngineering

SaraWalton,Lecturer,ChemicalandBiologicalEngineering

ii

©2017FuoadSaliou-Sulley

AllRightsReserved

ANAUTOMATEDDEVICETOINCREASESCREENINGTHROUGHPUTOF

ZEBRAFISHLARVAE

ByFuoadSaliou-Sulley

ThesisAdvisor:Dr.PaulMillard

AnAbstractoftheThesisPresented

inPartialFulfillmentofthe

Requirementsforthe

DegreeofMasterofScience

(inBiologicalEngineering)

August2017

Theuseofthezebrafishasananimalmodelalternativetomammalian

specieshasspawnedresearchadvancementsinseveralmedicalfields1.Sincethe

zebrafishsharesahighdegreeofsequenceandfunctionalhomologywithmammals,

studiesusingthisorganismcanprovidein-depthinsightintohostresponseto

diseaseandprovideaplatformfortestingarangeoftreatmentoptions.Theoptical

transparencyofzebrafishatearlystagesofdevelopmentpermitseasyassessmentof

theeffectsoftreatments,occurrenceoftumorsorotherabnormalgrowth,disease

progression,andimmuneresponse.Thesecharacteristicsmakeitidealforstudying

humandiseasessuchastheInfluenzaAVirus(IAV).Theconventionalmethodof

IAVtransmissionisbyaerosol,andsincethezebrafishdoesnotaccommodatethis

modeofentry,thevirusisinjectedintothespecimenunderstudy.

Thelarvaetypicallyrequiremanipulationduringpreparatoryprocedures

priortoassessment,aprocessthatcanbetimeconsumingandstressfulforthe

organism.

Inthisthesis,Idescribethedevelopmentofadevicedesignedtoeliminate

problemsassociatedwithmanipulatingzebrafishlarvaebyautomatically

conductingspecimenfromareservoirdirectlyintoanentrapmentdock,whereit

willbeimmobilizedforinjectionandrapidlyremovedpost-injection.Thiswillhelp

toreducethehandlingtimeoflargesamplesets,therebyincreasingthescreening

throughput.Zebrafishhavefastgrowthrates4hencepreparatoryproceduresfor

analysislikeinjectionshouldbeasquickandefficientaspossible.Thedeviceusesa

systemtoconduct48-72houroldzebrafishthroughaliquidmedium(eggwater)

usingasyringepump.Thecompletesystemconsistsofthreemainsubsystems,

namelythepump,opticaldetectionandentrapmentcomponents.A3Dprinted

housingenclosestheelectricalcomponentsoftheentiresystem.Thedeviceworks

byaspiratingindividualfishthroughatubeviaapressuregradientcreatedwitha

syringepump.Eachcycleofthedeviceinvolvesthefollowingsteps:(1)loading,(2)

sensing,(3)trapping,(4)injection,and(5)flushing.Duringloading,asinglelarvais

extractedfromthereservoirandconductedthroughatubepasttheoptical

detectionsubsystem.Atthesensingstage,theopticaldetectionsubsystem

composedofaphotodiodeandalaser,sensestransmittedlightfromthelaserand

discernstheentryoflarvafromairbubblesanddebriswithprecision.Uponlarva

recognition,thespecimenisthenconductedtotheentrapmentdock(step3)where

itwillbeimmobilizedforinjection(step4).Thefinalstep(5)involvesconducting

thelarvaoutoftheentrapmentdockandsubsequentlyoutofentiresystemfor

furtheranalysis.Thisdevicewillprimarilyservezebrafishresearcherswhointend

tointroducevaccines,pathogensandotherexperimentalmaterialsintomany

individualzebrafishlarvae.

iii

ACKNOWLEDGEMENTS

Iwouldliketousethisopportunitytothankmythesisadvisor,Dr.Paul

Millard,forwelcomingmeintohislabandforbeingmymentoroverthepastfew

years.Throughhissupport,mygraspforthesubjecthasimprovedvastlyandIam

verygratefulforhispatienceandguidance.IwouldalsoliketothankDr.Doug

BousfieldandDr.SaraWaltonforprovidingvaluablecontributionstothiswork.

AdditionallyIwouldliketothankSeanMichaelTaylorfromtheIMRCforproviding

criticaldesigncontributionsandforhelpingmetobecomefamiliarwiththe

prototypingequipment.IwouldalsoliketoacknowledgeMadelineMazjanisfor

helpingwiththecontrolsoftwareandforconductingdevicetests.

Withagreatdealofgratitude,Iwouldliketothankmyfamilyfortheir

continuoussupportinmyendeavors.Theyhavealwaysbelievedinmeand

encouragedmetobethebestversionofmyselfandtonevergiveuponmydreams,I

willforeverbegratefulfortheirsupport.

iv

TABLEOFCONTENTS

ACKNOWLEDGEMENTS………………………………………………………………………………………iii

LISTOFTABLES…………………………………………………………………………………………………vii

LISTOFFIGURES……………………………………………………….……………………………………...viii

CHAPTER1. LITERATUREREVIEW…………………………………………………………………….1

1.1 InfluenzaVirusOverview…………………………………………………………………………..1

1.2 InfluenzaVirusVariants…………………………………………………………………………….1

1.2.1 Epidemiology-IAV………………………………………………………………………….2

1.2.2 IAVTransmissionandViralPropagation………………………………………….3

1.2.3 ClinicalFeaturesofinfection…………………………………………………………...5

1.3 CurrentIAVResearch……………………………………………………………………….………..5

1.4 AnimalModels…………………………………………………………………………………………..6

1.4.1ZebrafishasAnInfluenzaAVirusModel………………………………………….7

1.4.2PathologicalPhenotypeObservedinIAVInfectedZebrafish……..……10

1.5 SystemicIAVInfection……………………………………………………………………………..11

1.5.1 α2,6-linkedSialicAcidsinZebrafish……………………………………………..14

1.6 ZebrafishAntiviralDefenses…………………………………………………………………….16

1.7 InjectionofZebrafishEmbryos………………………………………………………………...17

1.7.1 StandardTechniques…………………………………………………………………….21

1.7.2 RapidPrototypingandInfluenceofTechnologyonResearch………….21

1.7.3 NotableDevicesforZebrafishInjection………………………………………….22

v

CHAPTER2. METHODS…………………………………………………………………………………….27

2.1 Overview………………………………………………………………………………………………..27

2.2 PumpSubunitComponents……………………………………………………………………..29

2.3 OpticalDetectionSubunitComponents……………………...……………………...……..31

2.4 EntrapmentDock………………………………………………………...………………………….32

2.4.1 Biocompatibility…………………………………………………………………………...37

2.5 SpecificationsandParameters…………………………………………………………………38

2.6 Electronics……………………………………………………………………………………………...38

2.6.1 ControlMaps/SoftwareProgramming…………………………………………..42

CHAPTER3. EXPERIMENTALRESULTSANDDISCUSSION…………………………………46

3.1 InputReservoirEfficacyTest…………………………………………………………………...46

3.1.1 Procedure…………………………………………………………………………………….46

3.1.2 Results…...…………………………………………………………………………………….46

3.2 OpticaldetectionUnitEfficacyTest………………………………………………………….48

3.2.1 Procedure…………………………………………………………………………………….48

3.2.2 Results…………………………………………………………………………………………49

3.3 EntrapmentDockTests…………………………………………………………………………...50

3.3.1 Procedure…………………………………………………………………………………….50

3.4 AverageDurationofScreeningSteps(1Cycle)……………………….…………………51

3.4.1 Procedure…………………………………………………………………………………….51

3.5 QuantitativeAssessmentofAnimalHealth……………………………………................53

3.5.1 Procedure…………………………………………………………………………………….53

3.5.2 Results……………………..…………………………………………………………………..53

vi

CHAPTER4. SUMMARY,CONCLUSIONANDFUTUREWORK……………………………...56

4.1 Summary……………………..………………………………………………………………………….56

4.2 Conclusions……………………..………………………………………………………………………56

4.3 FutureWork……………………..……………………………………………………………………..57

BIBLIOGRAPHY………………………………………………………...………………………………………..60

APPENDIXA:ZEBRAFISHHUSBANDRY………………………………………………………………71

APPENDIXB:VALVE/SYRINGEPUMPCONTROLMAP…………………………………………73

APPENDIXC:MATERIALSLIST…………………………………………………………………………...75

BIOGRAPHYOFTHEAUTHOR…………………………………………………………………………….77

vii

LISTOFTABLES

Table1. Averagedurationofscreeningstepsfromthereservoirtothelight

detectoratdifferentflowrates.(0.08cminnerdiameterand88cm

length)………………………………………………………………………………………….52

Table2. Calculateddurationofscreeningstepsfromthephoto-detectortothe

entrapmentdockatdifferentflowrates.(0.08cminnerdiameterand

80cmlength)………………………………………………………………………………...52

Table3. Averagetimerequiredforinjection……………………………………………….52

Table4. Calculateddurationofscreeningstepsfromtheentrapmentdockto

theexit(oftheentiresystem)atdifferentflowrates.(0.08cminner

diameterand62cmlength)…………………………………………………………...52

viii

LISTOFFIGURES

Figure1. Processofinfluenzavirusinfection………………………………………………..13

Figure2. Atypicalmicromanipulatorformanualinjectionneedleoperationin

allthreeaxes(x,yandz)……………………………………………………………….20

Figure3. Deviceschematicanddesign…………………………………………………………28

Figure4. Animageoftheinputreservoir……………………………………………………..29

Figure5. AquaCulturesingleoutletaquariumairpump……………………………….30

Figure6. NewEraNE-1000xbidirectionalprogrammablesyringepump………31

Figure7. Animageoftheopticaldetectionunitcomponents………………………...32

Figure8. Imageoftheentrapmentdockwithhighlightedchannelsand

dimensionsinmillimeters……………………………………………………………..33

Figure9. Imagesoftheentrapmentdockanditssupportingcomponents

designedusingsolidedgecadsoftware………………………………………….36

Figure10. Electroniccomponents………………………………………………………………….39

Figure11. Cole-Parmertwo-waynormallyclosedsolenoidpinchvalve…………..40

Figure12. Imageofthefunctionalcircuitdiagramofthevoltagecomparator….42

Figure13. LabVIEWcontroldashboard………………………………………………………….43

Figure14. Valve/syringepumpcontrolmap…………………………………………………..44

Figure15. Abargraphdepictingthenumberofzebrafishlarvaecollectedafter

60secatdifferentflowrates…………………………………………………………48

Figure16. Awaveformchartofthephotodiodevoltagespikeswithrespectto

time……………………………………………………………………………………………...50

ix

Figure17. Imageoftheentrapmentdockillustratingtheimmobilizedzebrafish

larvaeinthetwopossibleorientations…………………..………………………51

Figure18. Comparisonofmorphologicalabnormalitiesofexperimental

zebrafish,atdifferentflowrates,withacontrolzebrafish……………....54

Figure19. Quantitativeassessmentofzebrafishhealthatdifferentflowrates

(n=100)………………………………………………………………………………………..55

1

CHAPTER1

LITERATUREREVIEW

1.1 InfluenzaVirusOverview

Thecontagious,acuterespiratoryillnessknownasthefluhasafflicted

humansforcenturies,yetitspreciseoriginremainsamystery.Theworking

hypothesisisthatwildaquaticbirdsweretheprimaryreservoirforbothbirdand

mammalianvirusspecies.Thereisextensiveevidenceforthedirectandindirect

transmissionofinfluenzafrombirdstootherspeciessuchaspigsandhorses5.

InfluenzavirusesbelongtotheOrthomyxoviridaefamilyofRNAviruseswhichaffect

theupperandlowerrespiratorytract.Theyaresphericalorfilamentousandrange

insizefrom80to120nmindiameter6,7.Generalsymptomsofinfluenzainclude

fever,malaise,headacheandcough8.Thediseaseaffectsallagegroupsbuthashigh

prevalenceinpeopleunder25yearsandhasmoredrasticandlifethreatening

effectsininfantsandtheelderly9,7,10,11.Fluoutbreaksappearsuddenlyandcan

persistforafewdaystomonths,ifuncontrolled.Theseverityofanoutbreakmay

varydependingonthevariantofinfluenzaviruscausingthedisease.

1.2 InfluenzaVirusVariants

TherearesixgenerawithintheOrthomyxoviridaevirusfamily,andthree,

InfluenzaA,InfluenzaBandInfluenzaC,containvirusesthatcancauseinfluenzain

humansandbirds,aswellasothermammals12,13.Thesevariantsarecharacterized

bysegmentednegativestrand-RNAgenomesthataretypicallyenclosedinalipid

envelope.Subtleclassificationsdistinguishthethreesubfamilies.Viralsurface

2

proteinsserveasuniqueidentificationmarkersforviruses,andtypicalsurface

proteinsinclude:ahemagglutinin(HA),whichagglutinateserythrocytesandplaysa

keyroleinviralpropagation,neuraminidase(NA),whichisanenzymeessentialfor

thereleaseofprogenyvirusparticlesfromthesurfaceofaninfectedcell,and

membrane(M)proteins,whichserveasionchannels.

1.2.1 Epidemiology-IAV

ThemostprevalentformofinfluenzaisinfluenzaA,whichprimarilyaffects

humans,othermammalsandbirds.InfluenzaAisadiseaseofimmensecomplexity.

Itsoccurrenceandoutcomedependoninteractionsbetweenthevirus,whichis

constantlychangingitsgeneticandantigeniccomposition,andtheimmunesystem

ofthehost,whichmaynotbeabletorespondadequatelywithinarestrictedtime

frame.Theprincipalfactordeterminingwhetherinfluenzaoutbreaksoccuristhe

degreeofcomplementarypairingbetweenthesurfaceantigens(HAandNA)ofthe

virusandtheantibodiesagainstthem,whichareinthecurrentpopulation7.

Thisclassofvirusesisthemostlethalofthethree(InfluenzaA,InfluenzaBand

InfluenzaC),withtheabilitytocauseseverediseasesonthepandemicscale.

PandemicsofinfluenzaAhaveoccurredaboutthreetimespercenturysince

1700andweremanifestedbyglobalspreadofthedisease,typicallywithhigh

morbidityandmortality14.Themostextremepandemicrecordedinthe20thcentury

wastheSpanishinfluenza,prevalentbetween1918and1919,withadeathtoll

estimatedtohavebeenbetween20and40millionpeopleacrosstheglobe7,14,.Other

recentnotablepandemicsincludethe2009avianflu(H1N1)pandemic; thisvirus

3

wasauniquecombinationofinfluenzavirusgenesneverpreviouslyidentifiedin

eitheranimalsorpeople,infectingmillionsandkillingthousandsworldwide15.

Forseveraldecades,theCentersforDiseaseControlandPrevention(CDC)

hasmadeannualestimatesofinfluenza-associateddeaths,whichhavebeenusedin

influenzaresearchtodevelopinfluenzacontrolandpreventionpolicy.Outofthe5-

20%ofpeoplethatcontractthefluintheUnitedStateseachyear,theCDCestimates

over20,000laboratory-confirmedhospitalizationsannually,duetoIAVandother

relatedcomplications16,17.Inaddition,theeconomyisburdenedbyabout87billion

USDduetoIAVrelatedissues,rangingfrommedicalexpenses,paidworkabsences,

lossesinproductivity,declineinretailspending,andeventourism18.

1.2.2 IAVTransmissionandViralPropagation

Thefluishighlycontagiousandinfectedpeoplecanspreaditviaaerosolto

othersuptoabout6feetaway.Thevirusspreadsmainlythroughtinyaerosol

dropletsproducedwheninfectedindividualscough,sneezeor,occasionally,just

talk.Theaerosoldropletscontainingthevirusenterthemouth,noseorrespiratory

tractofpeoplewithinproximity.Inaddition,theviruscanbecontractedwhen

peoplecomeintodirectcontactwithsurfacesandobjectscontaminatedwiththe

virusandthenlatertouchtheirmouth,nose,oreyes.Contaminatedsurfacesmay

includedoorhandles,tablesurfaces,banknotes,andelectronicinputdeviceslike

keyboards.ResearchhasproventhatIAVcansurviveonsuchsurfacesforupto

eighthours19,20,21.Theflucanbecontagiousevenbeforesymptomsbecome

apparent.Mostadultscaninfectothersbeginningonedaybeforetheirsymptomsof

4

thediseasearedisplayedandcanremaincontagiousforuptooneweekafter

becomingsick19.Youngchildrenwithweakenedimmunesystems,however,canbe

contagiouswellpastaweek.

Uponinfection,thevirusreplicatesintheupperandlowerrespiratorytract,

reachingitspeakat2-3daysafterinfection.Symptomsofthediseasestartto

becomeapparentatthisstage19.Thevirusinducespathologicchangesthroughout

therespiratorytract,withthemostsignificantpathologyoccurringinthelower

respiratorytract.Bronchoscopiesofpersonswiththeflushowadiffuse

involvementofthetrachea,larynxandthebronchi,withacutemucosal

inflammationandedema.Thevirushasanaffinityforcellsintheepithelialliningof

therespiratorymucosa.Forviralpropagationtoinitiate,infectionofthesecells

mustoccur.Infectionbeginsbyvirusbindingtothesurfaceofthehostcell,followed

bythefusionofviralandhostcellmembranes.Theattachmentandfusionis

mediatedbyvirushemagglutininglycoprotein(HA)interactingandbindingtosialic

acidcellreceptors.Bindingtosialicacidoccursviaashallowcavitynearthedistal

tipoftheHAglycoprotein.Subsequently,influenzavirusentersthecellbyreceptor-

mediatedendocytosis22.SincethevirushasnoneedforDNAcoding,transcription

occurswithinthenucleus,followedby:replicationofviralRNAandsecondary

transcription,translationofviralRNAtoproduceviralproteins,thenculminating

withtheassemblyofviralstructuralcomponentsandthereleaseofprogenyvirus

intotheextracellularmedium.

5

1.2.3 ClinicalFeaturesofInfection

Inhumans,influenzaischaracterizedbythesuddenonsetofanacute

respiratoryillnessaccompaniedbyheadaches,chillsandnonproductivecough.This

isusuallyfollowedbymuscleaches,highfever,generalizedweaknessandlossof

appetite.Thefeverusuallydeclinesbythethirddayandisgoneafteraboutaweek,

butthecoughingandsignsofweaknesscanpersistforabouttwomoreweeks10.Due

tothevirus’natureofcompromisingtheimmunesystem,thehostcanbeproneto

infectionbyotherpathogensthatcanleadtofurthercomplications.Complications

oftheflucanincludeearinfections,sinusinfections,dehydration,andworseningof

chronicmedicalconditionssuchascongestiveheartfailureordiabetes.Moresevere

illnessessuchasprimaryinfluenzapneumoniacandevelop,whichcanbelethal.

Secondarybacterialpneumonia,amorerareformofthedisease,canalsooccurif

theflugoesuntreated23.Peopleover65yearsofage,thosewithheartconditions,

asthma,emphysema,AIDSorpeoplereceivingchemotherapyareatanincreased

riskofcomplicationsfrominfluenza.

1.3 CurrentIAVResearch

InfluenzaAviruses,duetotheirgeneticmakeupandmodeofpropagation,

arecontinuouslychanginginseveralanimalhosts,includingbirds,pigs,horsesand

humans24.Asaresultoftherapidandconstantemergenceofnewviralstrains,IAV

ishighlylikelytocausehumanepidemics.Itis,however,necessarythatnaturally

occurringIAVshouldbemonitoredandcharacterizedtoimprovethecurrent

understandingofthehosttropismaswellasthevirulenceofIAV.Therehavebeen

6

numerousresearchadvancementsinIAVresearchinthepastfewdecades,someof

whichinclude,butarenotlimitedto:viral/humansurveillanceandcharacterization,

influenzagenomediversityofevolution,vaccinationtechnologies,tissuepathology

andinfluenzaanimalmodelmanipulationtechnologies24,25,26,27,28,29,30.TheNational

InstituteofAllergyandInfectiousDisease(NIAID)InfluenzaResearchProgram

supportsresearchtolearnmoreaboutthestructureandpathogenesisofinfluenza

viruses.Suchresearchhasproventobeinvaluabletothedevelopmentofnew

vaccines,therapeutics,anddiagnostics31.Currentresearchinvolvesunderstanding

viralreplication,theemergenceofnewviralstrainsandtheeffectthosehaveonthe

immunesystem.AmajorNIAIDactivityistheInfluenzaGenomeSequencingProject.

Thisisajointefforttoobtaincompletegeneticfootprintsofthousandsofhuman

andavianinfluenzastrains,withthegoalofprovidingvaluablegenomicknowledge

thatcanbeusedtocreateimprovedpublichealthcountermeasures.AsofMay2014,

morethan16,000humanandavianisolateshavebeencompletelysequencedand

madepubliclyavailable32.

NIAIDalsosupportstheresearchcommunitybydevelopingnewanimal

modelsforpreclinicalevaluationofvaccineandtherapeuticcandidates.Animal

modelsareusefultoolsthatserveasbiologicalresourcesformicroarrays,clones,

peptides,andreagents.

1.4 AnimalModels

Animalmodelshavebeenpivotalinresearchfordecades,leadingtomajor

drugdiscoveriesandmedicalbreakthroughs33,34.Suitablemodelsareusually

7

selectedbasedonphysiological,anatomicalandgeneticresemblancewithhumans.

Theunlimitedsupplyandeaseofmanipulationaresomeofthegeneraladvantages

ofanimalmodels.Scientificresearchcansometimesinvolvestatisticalanalysisof

testspecimens,mostlybymanipulatingonlyonevariablewhilekeepingothers

constant,andthenobservingtheconsequencesofthatchange.Anadequatenumber

oftestspecimensare,however,requiredtoaccomplishthistaskeffectively.The

relativeeaseofmanipulatinglargesamplesizesmakesanimalmodelsinvaluableto

medicalresearch.

Rodentsarethemostcommontypeofmammalusedinexperimentalstudies.

Extensiveresearchhasbeenconductedusingrats,mice,gerbils,guineapigs,and

hamsters35,36.Amongtheserodents,themajorityofgeneticstudies,especiallythose

involvingdisease,haveemployedmicebecausetheirgenomesaresimilartothatof

humansandmanyuniquestrainshavebeendeveloped.Othercommon

experimentalorganismsincludefruitflies,baker'syeastandzebrafish37.

1.4.1 ZebrafishasAnInfluenzaAVirusModel

Animalmodelsofinfluenzaareessentialtoresearchthatfocusesongaininga

betterunderstandingofthevirusanditseffectonhumans.Therearecurrently

variousresearchapplicationsofanimalmodelsrangingfromthepathogenesisand

hostresponsetoIAVtoantiviraldrugscreeningsandpre-clinicaltestingofantiviral

drugsandvaccines.IAVanimalmodelsarealsousedtoinvestigatethecompetency

ofvaccinesanddrugstocombatthevirus,preventfuturecomplicationsand

commonly,toreducethesymptomsofinfluenza.Inselectingthemodelsfor

8

research,acoupleoffactorshavetobeconsidered.Themostimportantcriterionis

thesusceptibilityoftheanimaltoinfluenzavirusinfectionwhilesupportingviral

replication,andexhibitinghumanhistopathologicalandmorphologicalsymptomsof

aninfluenzavirusinfection38.

Zebrafish(Daniorerio)modelsforinfectiousdiseasesarewellestablishedfor

characterizationofbacterial,fungalandviralinfections39,40,41.TheNational

InstitutesofHealth(NIH)alongwithotherresearchbodieshasapprovedthe

zebrafishanimalmodelforuseintheadvancementofbiomedicalstudies.Someof

itsinherentadvantages,suchasopticaltransparency,minimalhusbandry,large

clutchsizeandfastreproductivecyclemakethezebrafishanidealspecimenfor

high-throughputdrugtesting.Anotherimportantfactortoconsideristhatzebrafish

arepropagatedattheoptimalincubationtemperatureforvirusesduringinfection

studies.Zebrafishcanbegrowninatemperaturerangewithinthatofthehuman

respiratorytract(from25to33°C).

Asaresult,thezebrafishmightbewellsuitedtomodelinghumanrespiratoryviral

infections,giventhesimilaritiesbetweenthezebrafishswimbladderandthehuman

lung42,43,44,45.Therehavealsobeenrecentstudiesofzebrafishswimbladder

infectionswithhumanfungalpathogens.Furthermore,zebrafishattheearlystage

ofdevelopmentarehighlyamenabletogeneticmanipulationandtransgenesis2.

Amongallofthebenefitsofthezebrafishanimalmodelsystem,oneofthe

mostimportantcharacteristicsthatmakethissystemidealforIAVresearchisthe

zebrafish’simmunesystem.Duringtheonsetofdevelopment,zebrafishareknown

torelysolelyontheinnateimmuneresponseforthefirst4to6weeks46,47,48.

9

Hematopoiesisinzebrafishproduceslargelythesamedifferentiatedcelltypesas

observedinmammals.Monocytes,neutrophils,macrophagesandeosinophilshave

allbeendescribedwithinthezebrafishmyeloidlineage47.Inaddition,othercells

withmammaliancytotoxicproperties,suchasmastcellsandnaturalkillercells,

havealsobeenclassifiedwithinzebrafish49.Theinnateimmunefunctionsofthese

cellsprovideaneffectivedefensesystemduringtheearlystagesofdevelopment50.

Themajorityoftheresearchconductedusingthezebrafishmodelinvolves

fish-specificviralpathogensthathavetheabilitytoinfectandcausedisease;

however,fewhumanviruseshavebeendemonstratedtoinfectthezebrafish51.

Somerecentstudieshaveproventhezebrafishtobesusceptibletoviralinfectionby

mammalianviruses,ascharacterizedbyBurgosetal,wherethezebrafishwasused

asamodelofherpessimplexvirustype1(HSV-1)infectionofthenervoussystem52.

Gaboretal,attheUniversityofMainedescribedazebrafishmodelforhuman

influenzaAVirus(IAV)infectionandshowedthatzebrafishembryosaresusceptible

toinfectionwithbothinfluenzaAstrainsAPR8andX-31(Aichi).Influenza-infected

zebrafishshowedanincreaseinviralburdenandmortalityovertime.The

expressionofinnateantiviralgenes,thegrosspathologyandthehistopathologyin

infectedzebrafishrecapitulateclinicalsymptomsofinfluenzainfectionsinhumans2.

Thestudywasconductedbasedonthepremisethatanantiviralstatesimilartothat

inhumanswasinducedininfectedspecimens,leadingtothedeductionthat

zebrafishcanbeasuitablealternativetovertebrateanimalmodelsusedforIAV

studies.RecombinantinfluenzavirusescarryingaGFPreportergeneintheNS

segment(NS1-GFP)wereusedtocharacterizeinfectioninthezebrafish.Although

10

attenuatedcomparedtothewildtypevirus,itistypicalforthesemodifiedviral

strainstoreplicateefficiently53.Todeterminewhetherthemodifiedhumanisolates

ofIAVcouldcausediseaseinzebrafishembryos,differentstrainsofIAV[APR8

(H1N1)andX-31(H3N2)]wereinjectedintravenouslyinto48hpf(hourspost

fertilization)zebrafishthroughtheDuctofCuviertomimicviralinfectioninthe

humanbloodstream.Aninertphosphatebufferedsalinesolutionwasalsoinjected

intoanumberofzebrafishembryosinacontrolsetup2.

DatafromthestudydemonstratedthatIAVreplicateswithinzebrafishand

suggeststhatthedeathofIAV-infectedzebrafishislikelytobecausedbyIAV

infection.Toinvestigatetheeffectoftreatmentonthezebrafishimmunesystem,an

antiviralcompoundusedforthetreatmentofIAVinhumanswasintroduced,which

reducedthemortalityoftheinfectedfishaswellasthevirulenceofIAV.

1.4.2 PathologicalPhenotypeObservedinIAVInfectedZebrafish

Inanefforttocharacterizetheinnateimmuneresponseofzebrafishtothe

presenceofeitherstrainofIAV(APR8 or X-31)inthebloodstream,infected

embryosweremonitoredovertimeforbehavioralandphenotypicchanges2.A

controlledsetofembryoswereinjectedwithPBS(phosphate-bufferedsaline)and

monitoredunderidenticalconditions.Whilefishinthecontrolsetupdisplayedno

abnormalbehaviororsignsofviremia,theIAV-injectedzebrafishexhibited

symptomsoflethargy,withsignsofedemaobservedinthepericardiumandyolk

sac.Furthermore,theedemaworsenedovertimeandadditionalpathological

phenotypes,includingvaryingdegreesoflordoticcurvatureofthespine,were

11

observed.Otherobservationsincludedpigmentationdefects,cranialirregularities

andeyedeformities.Gaboretalextendedtheinvestigationtofurthercharacterizetheinnate

immuneresponseofzebrafishtoIAVinfectionbyconductingpathologicalanalysis

onthecellularlevel.Influenzavirustypicallyreplicatesintheepithelialcells

throughouttherespiratorytractofhumansand,theviruscanberecoveredfrom

boththeupperandlowerrespiratorytractofindividualswhohavebeennaturally

orexperimentallyinfected.IAVviremiainitsacutestagecanbecharacterizedby

multifocaldestructionanddesquamationofthepseudostratifiedcolumnar

epitheliumofthetracheaandbronchi29.Itisalsotypicalforthebasallayerofthe

epitheliumtobetheonlystructureremaining.Edemaandcongestionofthe

submucosacanalsobeobserved.

Inthecaseofthezebrafish,Gaboretal.preparedtissuesamplesfromIAVinfected

zebrafish48afterinjection.Analysisofthetissuesconfirmedtheclinicalsymptoms

ofinfluenza,includingnecrotictissuesandedema.

1.5 SystemicIAVInfection

Thevascularsystemallowstheflowofbloodfromthehearttoallpartsofthe

body,supplyingoxygentothemostvitalorganssuchasthekidneys,thebrainand

theliver.Sincebloodwithinthissystemisdistributedthroughoutthebodyfrom

theaorta,introductionofabloodbornepathogensuchasIAVintothevasculature

expeditesviralinfectionthroughoutthebody.IAVischaracterizedbyhemagglutinin

(HA),anantigenicglycoproteinusuallyfoundontheirsurfaces.Theseglycoproteins

12

areresponsibleforbindingthevirustothecellthatisbeinginfected54.Theviral

hemagglutininproteinsbindtosialicacidgroupsofcellularsurfaceglycoproteinsto

achieveviralattachmentandentry.ThespecificityoftheHA-Sialicbindingcomplex

isoneofthemajordeterminantsforcontrollingviraltropismandhostspecificity

Thisexplainswhysomeorganismsaresusceptibletocertaindisease-causing

pathogens,whileothersarenot.

Typically,humaninfluenzaviruseshaveabindingpreferenceforα2,6-linked

sialicacid.Avianinfluenzavirus,ontheotherhand,bindstotheα2,3-linkedsialic

acidofthehostbird56.Besidestheconventionalviralinfectionmechanism,thevirus

cansometimestraveltoacidicendosomesformembranefusionviaclathrinor

caveolinmediatedendocytosis57.Alternatepathwaysintothecellbyviruseshave

alsobeenstudied,demonstratingthatinfluenzaviruseshavetheabilitytoemploy

differentcellentrymechanismstoachieveviralinfection.Itis,however,notknown

whetherallinfluenzaviruseshavetheabilitytousetheothervariouspathwayswith

identicalpreferences56,58,59,60.FollowingtheinteractionbetweentheviralHAand

theα2,6-linkedsialicacid,endocytosisoftheviralparticleistriggered.Figure1

demonstratesatypicalprocessofcellentryoftwodifferentviruses:Influenzaand

HIV.

13

Schematic diagrams of HIV-1 and influenza virus fusion and entry processes. MajorconformationalchangesintheviralenvelopeglycoproteinsoccurforfusionofbothHIV-1andinfluenzavirus,buttherequirementsforfusiondiffer,asdotheviraltargettypes(notshown).ForHIV-1,gp120binds sequentially to its primary receptor CD4 and, after an initial conformational change, to co-receptors(chemokinereceptor)CCR5orCXCR4(notshown)(step1a).Co-receptorinteractiontriggersfusionoftheviralandcellularmembranes,initiatedbytheHIV-1fusionpeptidethatislocatedingp41(step2a).FusionandentryoftheHIV-1genomicRNAandaccompanyingviralproteinsintothetargetcelloccursatthecellsurfaceatneutralpH.Followingentry,theHIV-1RNAgenomesaretranscribedbyreverse transcriptase intoDNA(step3a)andtheHIV-1pre-integrationcomplex is transportedto thenucleusforintegrationintotargetcellgenomicDNAtoinitiatechronicinfection.OndifferentcelltypesfromthoseaffectedbyHIV-1,influenzavirusbindsviahaemagglutinin1(HA1)toterminalsialicacidspresentonglycoproteinsoronglycolipids(step1b).Thevirusissubsequentlyinternalizedbyreceptor-mediatedendocytosisintoalowpHcompartment(endosome),triggeringconformationalchangesthatexpose the viral fusion peptide that is located in HA2 (step 2b). Subsequently, the genomicribonucleoproteincomplexistransportedtothenucleustoinitiatetranscriptionandreplicationoftheviralgenome(step3b).

Figure1:Processofinfluenzavirusinfection.

Karlssonetal.SchematicdiagramsofHIV-1andinfluenzavirusfusionandentryprocesses.Diagram.ThechallengesofelicitingneutralizingantibodiestoHIV-1andtoinfluenzavirus.Fall2008;6(2):148.AvailablefromNatureReviewsMicrobiology.AccessedMay62017.

14

Influenzavirusbindstoterminalsialicacidspresentonglycoproteinsor

glycolipidsviaHA1.Thevirusissubsequentlyinternalizedbyreceptor-mediated

endocytosisintoanendosome.Thisinitiatesconformationalchangesthatexpose

theviralfusionpeptidelocatedonHA2.Theentireviralproteincomplexisthen

transportedtothenucleus,wheretranscriptionandreplicationoftheviralgenome

willoccur

ReplicationandtranslationoftheviralRNAproducesnewviral

macromolecules,whichareultimatelyassembledintocompleteviralparticlesfor

releaseandinfectionofnewcells62.

1.5.1 α2,6-linkedSialicAcidsinZebrafish

Thepresenceofcompatiblesialicacidmodificationsonhostglycolipidsor

glycoproteinsisoneofthemaindeterminantsofviralinfectioninmostanimal

species.Thepresenceofα2,6-linkedsialicacidinthezebrafishmakesthisa

valuablemodelsystemforthestudyoftheinfluenzaAvirus.Inspiteofthis,there

arenoknownreportednaturallyoccurringviralinfectionsofthezebrafish63.Inan

efforttodeterminewhetherzebrafishembryoscouldserveasahostforinfection

withmammalian/humanisolatesofIAV,Gaborconductedastudytoanalyzethe

sialicacidlinkagetypespresentonzebrafishglycoproteinsorglycolipids2.Analysis

usingHighPerformanceAnionExchangeChromatographywithPulsed

AmperometricDetection(HPAEC-PAD)demonstratedthatα2,6-linkedsialicacids

werepresentinzebrafishembryostwodaysafterfertilization.Therewere,

however,noα2,3-linkedsialicacidsdetectedinthetwoday-oldembryos2.There

15

areanumberofchemicalvariantsofsialicacids,andinfluenzastrainsvaryintheir

affinityforthem.Terminallylinkedsialicacidscanoccurinα-2,3,α-2,6,orα-2,8

linkages.Eventhoughthestudywasnotintendedtoidentifyα2,8-linkedsialic

acids,othershavepreviouslydemonstratedthatzebrafishhavetheabilityto

synthesizeavarietyofsialylatedglycoconjugatesduringtheonsetoftheir

development64,65,66.Theseuniquecharacteristicsofthezebrafishmodelsystem

makeitanattractivecandidateforstudyinginfectiousviraldiseases.Withfurther

studiesintosialylatedglycoconjugatespresentatvariousstagesofdevelopmentof

thezebrafish,additionalhumanviraldiseasescanfurtherbecharacterized.Someof

theknownhumanviralpathogensthathaveanaffinityforα2,6-linkedsialicacids

includecertainmembersofthefollowingvirusfamilies:

coronaviridae,paramyxoviridae,caliciviridae,picornaviridae,reoviridae,polyomavi

ridae,adenoviridaeandparvoviridae.Mostofthesegroupscontainmajorpathogens

forhumansthataffectallpartsofthebody,rangingfromsmallorganslikethe

salivarygland(inthecaseofmumps)tocellularnetworksasintricateasthe

immunesystem2,67,68.

Avianinfluenzavirusstrainshavethetendencytobindtosialicacids

attachedtogalactoseviaanα2,3-linkage,whichisthemajorsialicacidonepithelial

cellsoftheduckgut. Ciliatedepithelialcellsinthehumantrachealack α2,3linked

sialicacids,suggestingthatthelackofasuitablereceptoraccountsfortheinefficient

replicationofcertainhumanvirusesinduckintestineandofcertainavianvirusesin

humans55,69.

16

1.6 ZebrafishAntiviralDefenses

Althoughthezebrafishanimalmodelhasbeenproveneffectivefortesting

medicaltreatmentsandvirologicalassays,itcanserveasanidealtoolfor

understandinghostbiologicalresponsesafterviralinfections,particularlythe

InfluenzaAvirus.Aclearandthoroughunderstandingofahost’simmunity(both

innateandadaptive)canprovideinsightintonewtreatmentsandeffective

therapies.Thezebrafish,likemanyorganismspossessesinnateandadaptive

immunity,howeverthezebrafishreliessolelyonitsinnateimmuneresponse

systemforthefist4-6weeksofdevelopment2.Withinthisperiodofgrowth,

zebrafishdevelopfromtheembryotothelarvaestage.Thistemporalseparationof

innateandadaptiveimmunityinzebrafishallowsforthestudyoftheinnate

immuneresponsetoviralinfectionindependentofadaptiveimmunity.Uponviral

attack,oneoftheprimaryprocessesinitiatedistheinductionofhostantiviralstate,

whichslowsdowntherateofprogressionofviremia2.Themajormolecule

responsiblefortheantiviralstateisinterferon(IFN).Cellsthatarealreadyinfected

withavirussecreteinterferonstopreventfurthervirusexposureofhealthycells.

ResponsetoIFNinvolvesarapidanddirectsignaltransductionmechanismthat

quicklyreportsthepresenceofextracellularcytokinestothecellnucleus.This

preservesthespecificityinherentincytokine-receptorinteractionstoinduce

expressionofasetofgenesviatranscription,leadingtoencodingofimportant

antiviralproteins.Establishmentoftheresultingantiviralstateprovidesacrucial

initiallineofdefenseagainstviralinfection.StudiesofIFN-deficientcellsand

animalsderivedbygenetargetinghavedemonstratedtheessentialnatureofIFN-

17

mediatedinnateimmunity70,71,72.

Allbloodcellsinhumansbeginashematopoieticstemcells,andthendifferentiate

intomyeloidcells(erythrocytes,megakaryocytes,monocytes,neutrophils,

basophils,oreosinophils)orlymphoidcells(T-lymphocytesandB-lymphocytes).

Theseimmunecellstypicallyperceiveandrespondtomicrobialstimuli.Zebrafish

arelargelysimilartohumanswithrespecttocelldifferentiation.Theyhavedistinct

stagesofhematopoiesisthatoccurindiscreteyetfunctionallyanalogoussites.

Zebrafishmyeloidlineages,includingmonocytes,macrophages,neutrophilsand

eosinophils,havebeenreviewedandhavebeencharacterizedassimilartothosein

humans47.Theinnateimmunerolesofthesecellsofferanefficientdefense

mechanismduringtheonsetofdevelopment.Inthecaseofaviralinfectionsuchas

influenza,theimmunecellsaretriggeredandfunctionaccordinglytoquarantinethe

virusbyinducingtheantiviralstate.Macrophagesandneutrophils(large,mobile

phagocyticcells)canalsoberecruitedtothesiteofinfectiontoengulfinvading

species.Macrophageshavebeenknowntorespondmorequicklytoinfectionthan

neutrophilsatabout30hpf73.Therelativelyslowerneutrophilsrespondatabout

52hpftocomplementactivitiesofthemacrophages,partlybecausethephagocytic

capabilityofneutrophilsisrelativelylowcomparedtothatofmacrophages74.

Precursorsofthelymphoidcells(T-cellandB-cells)begintheprocessof

rearrangementandrecombinationofthegenesforimmunoglobulinandTcell

receptormoleculesbyday4.Thisistheonsetofadaptiveimmunesystem

development.Thethymus,whichisalsoalymphoidorgan,producesTcellsandB

cellsforthezebrafishimmunesystem,thepancreashasalsobeenreportedto

18

produceBcells75,76.Eventhoughtheadaptiveimmunesystembeginsformingearly

inzebrafishdevelopment,studieshaveshownthatthezebrafishasincapableof

mountingafullantibodyresponseuntilearlyadulthood,whichisabout4weeks

postfertilization.Themodeofpathogenicdefenseupuntilthispointisexclusively

dependentontheinnateimmunesystem77.

Inanefforttounderstandthedifferentmaturationstatesoftheimmune

systemduringzebrafishdevelopment,S.H.Lametalinvestigatedtheexpressionof

siximmune-relatedgenesofthezebrafishnamely:Ikaros,thatencodesa

transcriptionfactorwhichisusedasanearlylymphoidmarker;Rag-1

(Recombinationactivatinggene-1),whichencodesaproteininvolvedingenomic

rearrangementoftheT-cellreceptor,immunoglobulinloci,andisasuitablemarker

formaturinglymphocytes;T-cellReceptorAlphaConstantregiongenes(TCRAC)

whichencodeportionsoftheantigenreceptorsofmatureT-lymphocytes;three

immunoglobulinlightchainconstantregiongenes(IgLC)whichencodeportionsof

theantigenreceptorsofmatureBlymphocytes.Althoughtherehavebeencloning

andcharacterizationstudiesonimmune-relatedgenespriortothisstudy78,79,there

wasverylittleknownaboutthematurationofthezebrafishimmunesystemwith

regardtoitsformandfunction.Byemployingstandardprofilingtechniquesto

assessgeneexpression(quantitativereal-timepolymerasechainreaction,insitu

hybridization(ISH),immuno-affinitypurificationandWesternblotting),Ikaroswas

detectedfirst,at1dpf.Theexpressionthereafterincreasedgraduallytomorethan

two-foldbetween28and42dpfbeforedecreasingtolessthantheinitial1dpf

expressionlevelinadultfish(aged105dpf).Rag-1expressionlevelsincreased

19

rapidly(by10-fold)between3and17dpf,reachingamaximumbetween21and28

dpfbeforedecreasinggradually.Inadultfishaged105dpfhowever,theexpression

levelofRag-1droppedsignificantly,andwasequivalenttotheexpressionlevelat3

dpf.T-cellreceptoralphaconstantregiongenesandmRNAsofimmunoglobulin

lightchainconstantregion(IgLC)isotype-1,2and3weredetectedatlowlevelsby3

dpfandtheirexpressionlevelsincreasedsteadilytotheadultrangebetween4and

6weekspost-fertilization(wpf).Usingtissue-sectionISH,Rag-1expressionwas

detectedinheadkidneyby2wpfwhileIgLC-1,2and3weredetectedinthehead

kidneyandthethymusby3wpfonwards.SecretedIgwasonlydetectableusing

immuno-affinitypurificationandWesternblottingby4wpf.Ahumoralresponseto

T-independentantigen(formalin-killedAeromonashydrophila)andT-dependent

antigen(humangammaglobulin)wasobservedinzebrafishthatwereimmunizedat

4and6wpf,respectively,indicatinganormalimmuneresponse.Thefindings

revealedthatthezebrafishimmunesystemismorphologicallyandfunctionally

matureby4–6wpf77.

1.7 InjectionofZebrafishEmbryos

Oneofthemostcommontechniquesusedtointroducematerialsinto

zebrafishembryosandlarvaeisinjection.Althoughinvasive,itallowsprecise

targetingoftissuesandorgans.Whenconductedunderappropriateconditions,such

asmildsedation,injectionsposeminimalrisktozebrafishlarvae.Duringinjection,

certainsubstancessuchasnucleicacids,proteins,ordrugsareintroducedintothe

zebrafish,usuallyattheearlystagesofdevelopment80.Injectionsaretypically

20

conductedwithglassneedlesfilledwiththematerialtobedelivered.Theglass

needlescanbefabricatedeasilybycarefullydrawingoutaheatedcapillarytube

withtheappropriateinnerandouterdiameter81.Theglassneedlesarethenheldby

amicromanipulatorthatcontrolsandchangesthepositionoftheneedlerelativeto

theembryowithhighprecision.Pressurefromcompressedairisusedtoreleasethe

contentintotheembryoorlarva.

Figure2:Atypicalmicromanipulatorformanualinjectionneedleoperationinallthreeaxes(x,yandz).Itallowsforcoarseadjustmentreadingswithanaccuracyof100micrometers.WarnerInstruments,ModelMM-33,HamdenCT.

Injectionallowsrapiddeliveryofmaterialintothezebrafish.Aftermanipulationof

embryosorlarvaeintoasuitablearray,anexperiencedresearchercaninjectseveral

hundredembryosinonehour,althoughtherelativelyhighchancesoferror

associatedwiththistechnique,cannegativelyaffecttheconsistencyofinjection.A

detailedinjectionprotocolconsistingofzebrafishloadingandpreparationhasbeen

characterized82.

21

1.7.1 StandardTechniques

Injectionofzebrafishhasbeencommonplacefordecadesandnumerous

procedureshavebeendevelopedtoinjectalmosteverypartofthelivefish,notably

thebrainventricle,thekidney,theswimbladderandtheDuctofCuvier(DC).The

DCisawidebloodcirculationvalleyontheyolksacconnectingthehearttothe

trunkvasculature.Injectionthroughthisductallowsquickandefficientsystemic

infectionofzebrafishandisparticularlyusefulforintroductionofbacteriainto

embryosbetween1and3dpf.Fortheseinjections,theneedleisinsertedintothe

startingpointoftheductofCuvierjustdorsaltothelocationwheretheductstarts

broadeningovertheyolksac.Thislocationisthedeepestsectionoftheductand

thereforeprovidesthelowestriskofpuncturingtheyolksac.Theinjectedmaterial

willfollowthebloodflowthroughtheductofCuvier,overtheyolksactowardthe

heart,andcanbemonitoreddirectlypost-injectionbymonitoringtheexpanding

volumeoftheduct83.

1.7.2 RapidPrototypingandInfluenceofTechnologyonResearch

Rangesoftechnologicaladvancementshavepavedthewayfornewand

innovativedevicesandtechniquesthathelpmakelaboratoryworkmoreefficient

andreliable.Therateoftechnologicaladvancementhasbeenhighwithinthispast

decade,whichmaybeattributableinparttotheincreaseincomputationalpower84.

Anothermajorinfluenceontechnologicaladvancementistheemergenceof3D

printingtechnologiesforrapidprototyping,whichispoisedtorevolutionizedevice

developmentinmanyindustries,includinghealthcare.Sinceitsinitialuse,additive

22

prototypingtechnologyhasadvancedtouseinawiderangeofapplications

includingtissueengineering,dentistry,construction,automotiveandaerospace

85,84,86.Thetechnologyisalreadychangingthewayobjectsaremade,withresulting

productsrangingfromtoolsandtoystoclothingandevenbodyparts.3Dprintingis

partofaprocessknownasadditivemanufacturingwhereanobjectiscreatedby

addingmateriallayerbylayer,usuallyatafractionofthecostandtimeofstandard

meanssuchasforging,moldingandsculpting.Inthemedicalworld,physiciansare

testingbiomaterialsforregenerativemedicine.Usingmedicalimagingand3D-

modelingtechnology,surgicalteamscannowalsouse3Dprinterstocreate

temporarytoolsthatareaffixedtotheskeletalstructureofthepatienttoprovidea

precise"blueprint"forreshapingbonestructuretoperfectlyaccommodate

standard-sizeimplants.Custom-printeddrillingguidespermitscrewstobeplaced

preciselytoensurethebestfitwithapatient'sbody87,88.

Withinthedynamicbranchofzebrafishresearch,prototypingtechnologies

aregraduallybeingintroducedtoimprovethespeedandqualityoflabwork30,92,97.

Thesetechnologiesmayhavethepotentialtomakecomplexandtedious

procedures,suchasinjectionandimagingofchorionatedembryos,relativelysimple.

Thefollowingparagraphhighlightsdevicesthatimplementrapidprototyping

techniquestomakeworkingwithzebrafishmoreefficient.

1.7.3 NotableDevicesforZebrafishInjection

Pardo-Martinetal.atMassachusettsInstituteofTechnologyandHarvard

Universitydevelopedahigh-throughputplatformforcellular-resolutioninvivo

23

pharmaceuticalandgeneticscreensinzebrafishlarvae30.Theautomatedsystemis

capableofsamplingzebrafishlarvaeandthenpositioningthemforimaging.The

systemisalsocapableofmanipulatingbothsuperficialanddeeporgans(via

femtosecondlasermicrosurgery).Eachcycleofthesystemincludesthefollowing

steps:loading,sensing,trapping,injectionandflushing.Duringloading,thesystem

extractslarvaefromareservoirfilledwithdistilledwater.Ahigh-speedoptical

detectionsystemcomposedofaphotodiodeandtwoLEDsdetectstheentryof

larvaeintotheloadingtube.Thephotodiodesensestransmittedandscatteredlight

fromthelaserlightsource.Bymonitoringthelightsignals,thesystemcan

distinguishalarvafromairbubblesanddebriswith99.99%reliability(n=100).

Afterloadinganddetection,thelarvamovesintoacapillarypositionedwithinthe

angleofviewofanopticalimagingandmanipulationsystem.Usingafastcamera

andanautomatedimage-processingalgorithm,thelarvaiscoarselypositionedby

thesyringepumpwithinfocusoftheopticalimagingsystem.Attheendofone

completecycle,animalscanbedispensedbackintoeitherindividualwellsorlarger

containersbyexecutingtheloadingprocessinreverse.Whilethisdeviceboastsof

effortlesszebrafishmanipulationforimaging,itfallsshortofaddressingthetedious

processofmicroinjection:afrequentandnecessarysteppriortoimaging.Designof

anentrapmentmechanismaspartofasamplingdevicetoaccommodateaninjection

needlewouldaddanimportantmanipulationcapability.

RohdeetalatMassachusettsInstituteofTechnologydevelopedahigh-speed

microfluidicsorterwiththeabilitytoisolateandimmobilizesmallliveanimalsina

well-definedgeometry(withinachip)forscreeningphenotypicfeaturesin

24

physiologicallyactiveanimals.Theintegratedchipcontainsindividually

addressablechambersthatcanbeusedforincubationandexposureofindividual

animalstobiologicalcompoundsandhighresolutionimagingwithouttheneedfor

anesthesia.Besidesitssortingandimmobilizingfunction,thechannelswithinthe

microfluidicchipcanbecombinedinvariousconfigurationstopermitamultitudeof

complexfunctionssuchassmallanimalincubation(eg.C.elegans),immobilization

andindependentsubcellularresolutionimaging89.Thissystemisversatilebut

sharesasimilarlimitationwiththesystemmadebyPardo-Martin30andhisteam:it

doesnotaddresstheupstreamprocedureofanimalinjection.Furthermore,thehigh

multifunctionalcapabilitiesofthissystemmightmakeitsusceptibletocross-

contaminationduringuse.Boththemechanicalandelectricalcomplexityofthe

systemalsoraisesdurabilityconcerns.

BischeletalatUniversityofWisconsin-Madisondesignedazebrafish

entrapmentdevicethatcanbeusedtoquicklyandrepeatedlypositionzebrafish

embryosinapredictablearraywiththehelpofapipette.TheZebrafishEntrapment

byRestrictionArray(ZEBRA)iswellsuitedforusewithautomatedmicroscope

stages,therebyreducingtheamountofimagingtimeandfurtherincreasing

throughputcomparedtotraditionalmethods. ZEBRAwasdesignedandoptimized

toimmobilize3–5dpfzebrafishembryosinapredictablearraywithouttheuseof

agarose.Thedeviceconsistsofanenclosedchannelwitharestrictionwidththatis

accessedthroughaninputandanoutputport90.Fornormalfunction,ZEBRAmust

befilledwithwater(thisprocessrequiresoxygen-plasmatreatmenttorenderthe

insideofthechannelshydrophilic).Afterinitialfilling,apipettecanbeusedtoadda

25

dropletofwatercontainingaselectedzebrafishembryototheinputportofthe

device.Themainfunctionofthedeviceisgovernedbypassivepumping:a

phenomenonbywhichsurfacetensioninducespressuredifferencestodrivefluid

movementinclosedchannels91.Asaresult,waterisdrivenfromthesmallerinput

port(withhighersurfacetension)tothelargeroutputport(withlowersurface

tension).Thezebrafishisthentrappedinplaceuponreachingthechannel

restriction.Userscanchoosewhethertoloadthezebrafishintothedeviceheadfirst

ortail-firstdependingonthedesiredapplication.Passivepumpingworks

reproduciblyaslongasthereisaclosedchannelbetweenaninletandanoutlet91.

Thismakesthedevicemorepracticalandportablesinceitisnotdependentona

powersource.Despiteitsinherentadvantages,thedevicedoesnotaccommodate

zebrafishinjection.Theflowrateandaspirationconditionswithinthechannels

appeartobedependentonthesizeofthedropletplacedattheinletofthedevice.As

thedropletcollapses,theaspirationratedecreasesthuscreatingadeviceconstraint

thatcanpotentiallylimitthelengthofthechannelbetweentheinletandtheoutlet

ports91.Althoughlessenergy-efficient,employingactivepumpinginthisscenario

canpotentiallyeliminatethisconstrainttopermitlongerchannelsthatcan

accommodatezebrafish,aswellasotheranimals,suchasC.elegans.

WesthoffetalatUniversityofHeidelbergformedapieceofbrasstoserveas

anegativemoldtocastagarosetoyieldaseriesofwellscapableofholdingzebrafish

forimagingandmicromanipulation92.Thebrassmoldcastsindentationsintoan

agarosegelbyinsertingthetipsofthemoldintounsetagarsolutionandallowing

thesolutiontogel.Themoldisremoved,leavingvoidsthatarecontouredtohold

26

zebrafishlarvaeforimaging.Whilethisdevicerequiressignificantmanual

interfacing,itminimizestheissueofhavingtoinsertandorientspecimensinan

agarmedium.Userssimplypipettetheirspecimenintothewellspriortoimaging.

Evenlyspacedwellsallowscreeningoflargenumbersofspecimensandcanbe

automatedwithatranslationstage.Whilethedeviceoffersasimplewayforusersto

probeandorganizelargequantitiesofspecimens,itleaveslittleroomfor

automationoftrans-locatingfishintothedevice.Asaresult,moreuserinteractionis

neededtomoveandorganizethespecimens.Automatingthisprocesswouldallow

forminimaluserinteractionbetweentheuserandthedevice.

Thedevicesdiscussedabovealldemonstrateinnovativeapproachesto

eliminatingcommonproblemsofworkingwithzebrafish,suchasmanipulation;

however,noneofthemisapplicabletotheprincipalfunctionofzebrafishinjection.

UsingthezebrafishasananimalmodelforIAVstudiesinvolvessystematicallyand

reliablyintroducingthevirusintoindividuallarvaeviainjection.Adeviceto

conductandimmobilizezebrafishspecimensforinjectionwillbeinvaluabletoIAV

studies,particularlywheresamplesizesareverylarge.Thisprojecthasbeen

directedtowarddesignandfabricationofanautomateddevicethatwillhelpsolve

thisproblem.Themaingoaloftheproposeddeviceistoprovideameansfor

storingandgentlyconductingzebrafishfromareservoirtoachamberfor

immobilizationandinjection.Thiswillminimizedirectinteractionbetweentheuser

andspecimen,withthegoalofreducinginjection/handlingtimeinexperimentsand

tomaximizereliabilityandlaboratoryefficiency,aswellastominimizelong-term

costs.

27

CHAPTER2

METHODS

2.1 Overview

Thedeviceemploysasystemtoconduct48-72houroldzebrafishthrougha

liquidmedium(eggwater)usingasyringepump.Thecompletesystemconsistsof

threemainsubsystemsnamelythepump,opticaldetectionandentrapment

components.Thedeviceworksbyaspiratingindividualfishthroughatubeviaa

pressuregradientcreatedwithasyringepump.Eachcycleofthedeviceinvolvesthe

followingsteps:(1)loading,(2)sensing,(3)trapping,(4)injectionand(5)flushing.

Duringloading,asinglelarvaisextractedfromthereservoirandconductedthrough

atubepasttheopticaldetectionsubsystem.Atthesensingstage,theoptical

detectionsubsystemcomposedofaphotodiodeandalaser,sensestransmittedlight

fromthelaseranddiscernstheentryoflarvafromairbubblesanddebriswith

precision.Uponlarvarecognition,thespecimenisthenconductedtothe

entrapmentdock(step3)whereitwillbeimmobilizedforinjection(step4).The

finalstep(5)involvesconductingthelarvaoutoftheentrapmentdockand

subsequentlyoutofentiresystemforfurtheranalysis.Thesyringepumpispre-

programmedandmanipulatedwithLabView,thecontrolsoftware.Thisdevicewill

primarilyserveIAVresearcherswhointendtointroducevaccines,pathogensand

otherexperimentalmaterialsintomanyindividualzebrafishlarvae.

28

Figure3:Deviceschematicanddesign.Thedeviceoffersminimalinteractionandseamlessaspirationofzebrafishspecimen,ultimatelytrappingandimmobilizingitformicroinjectionofbiomaterials.

29

Thefollowingsectionsdescribeindetailthedesignandconstructionofthe

devicecomponents.

2.2 PumpSubunitComponents

Figure4:Animageoftheinputreservoir.TheassembledreservoirisshowninFigure4.1;Figure4.2andFigure4.3showthedesignrenderingsofthetopandbottomreservoircaps,respectively.

30

Thedevicepicturedaboveservesastheentrypointofthezebrafish

specimenintothedevice.Withamaximuminternalvolumeof60mL,thereservoiris

capableofaccommodatingabout50individual48-72hpflarvae.Whenfilledwith

eggwater(Figure4.1),theuniqueshapeanddesignallowsenoughroomforthefish

toswimandeventuallyberemovedbysuctionthroughtheoutlettubepositioned

alongthewallofthereservoir’sbottomcap.Duringtesting,itwasobservedthatthe

zebrafishlarvaehadthetendencytosinkdownward,towardthedeepestpartofthe

bottomcap.Thismadeitdifficultforspecimentoberandomlyremovedfromthe

reservoirthroughthetube,thusdramaticallyreducingthelarvaeoutputrate.To

remedytheproblem,abubblerwasintroduced.Anarrowholewasdrilledthrough

thedeepestpartofthebottomcaptoallowforasiliconetubetobethreaded

throughandconnectedtothebubbler.AnAquaCulturesingleoutletaquariumair

pumpwasusedastheairsourceFigure5.Itincludesacheckvalvethatprevents

thesiphoningofreservoirwaterintotheairpumpintheeventofapowerloss.

Figure5:AquaCulturesingleoutletaquariumairpump.Thepumpisoriginallyanairpumpdesignedfora5-to15-gallonaquarium.Itincludesacheckvalve(bluevalvepictured)thatcanhelppreventthesiphoningofreservoirwaterintotheairpumpintheeventofapowerloss.

31

PressureisprovidedtothesystemwithaNewEraNE-1000XBidirectional

SyringePump.Thepumpsuppliesnegativepressure(withdrawmode)forabrief

periodtocausespecimentobewithdrawnfromthereservoirandintothesystem

whereitwillbesensedbythelaserdiode/photodiodephotodetectionsystem.The

pumpthenswitchestopositivepressure(infusemode)toallowthespecimento

advancethroughthesystem.

Figure6:NewEraNE-1000Xbidirectionalprogrammablesyringepump.Withfullyprogrammableandstand-alonecapabilities,thepumpcanbeprogrammedforupto41pumpingphasesthatchangepumpingrates,setdispensingvolumes,insertpauses,controlandrespondtoexternalsignals.Ithasadispensingaccuracyof±1%.

2.3 OpticalDetectionSubunitComponents

Theopticaldetectionunithelpswiththedirectionoftravelofspecimen

withinthesystembysensingthepresenceofzebrafish,whichpromptsthesyringe

pumptoswitchbetweensupplyingpositiveandnegativepressure.Thetwomain

elementsusedforsensingare:a5mmtransparentcylinderheadphotodiode(PD)

32

1

1 22

(LLSO5-A,SenbaOpticalElectricalCo.,Ltd)anda4.5V650nmredlaserdiode

module(#01444878,LightInTheBoxCo.,Ltd).Duringnormaloperation,thered

laserbeamisfocusedontothephotodiodeandabase(unobstructed)voltage

readingisobservedandrecorded(voltagecomparatorcircuityieldsaHIGHsignal).

Themovementofspecimenpastthelightbeamreducesthevoltagereading,

resultinginthevoltagecomparatorcircuitproducingaLOWsignal,indicatingthe

presenceofalarvaasdepictedinthecartoon(Figure7.1).

Figure7:Animageoftheopticaldetectionunitcomponents.Figure7.1depictsacartoonthathighlightsazebrafishlarvamovingpasttheredlaserbeamtoindicatethepresenceofaspecimen;theblueselectionwithinFigure7.2showstheactualsetupofthedetectionsystemmountedona3Dprintedharness.2.4 EntrapmentDock

Acriticalcomponentoftheinjectionsystemistheentrapmentdock,adevice

thatimmobilizesthespecimenforinjection.ThedockwasdesignedusingSolidEdge

rapid-prototypingsoftwareand3Dprintedusingatransparentphotopolymerthat

PEEK Tubing

Photodiode

Laser

33

providesasuitablelevelofsurfacesmoothness.Thepolymer(VeroClear-RGD810)

isarigid,nearlycolorlessmaterialwithdimensionalstabilityforgeneralpurpose,

fine-detailmodelbuildingandvisualsimulationoftransparentthermoplastics.The

entrapmentdockdimensionsarerepresentedinFigure8.

Figure8:Imageoftheentrapmentdockwithhighlightedchannelsanddimensionsinmillimeters.

Thechannelswithinthedockaredesignedtoaccommodatetheshapeofa2-

dayoldzebrafishlarva.Beginningfromoneendofthedock,thechannelbeginswith

a1mmdiameteropeningtoaccommodatethecapillarytubefromtheautomated

deliverysystem.Itthennarrowstoamicro-constrictionthatissmallenoughto

allowsomeexcesswatertoflowpastthespecimentotheotherendofthedock

whilesimultaneouslytrappingandimmobilizingthespecimenforinjection.An

34

openingperpendiculartothetrappedspecimenservesasanentryfortheinjection

needle.

Despitetheconvenienceandpracticalityof3Dprinting,theabilityto

fabricatesmall,intricatedesignssuchasmicrochannelscansometimesbelimited.

Althoughprintersandtechniquesexistformicrofabrication,thecostofsecuringa

high-resolutionprintingunitishigh,withpricesstartingat20,000USD.Less

expensiveunitscanperformsimilarfunctionstothemoreexpensivemachines,but

withlessprecision.Thelabinwhichtheentrapmentdockwasprinted(Innovative

MediaResearch&CommercializationCenter,UniversityofMaine)wasequipped

withanintermediateresolution3Dprinter(StratasysObjet30,SUP705support

material),andsoseveralchallengesinfluencedthefinaldesignoftheport.Most

notableamongstthesewastheproblemoflodgedsupportmaterialwithinthe

microchannels.Most3DprintersemploytheAdditiveManufacturing(AM)design

techniquewheresuccessivelayersofmaterialareformedundercomputercontrol

tocreateanobject;itisanalogoustobuildingabrickwalllayerbylayer.Ifa

particulardesignissupposedtohaveanenclosedopeningorchannel,support

materialisdepositedintothecavitiestoholdupthestructureastherestofthe

objectisbeingbuilt.Asaresultofthisprocess,alloftheentrapmentdockswere

filledwithsupportmaterialandpost-printingprocedurestoremoveitprovedfutile.

Someoftheproceduresusedwere:applyingmildheat,usingbiodegradable

solventsandmanualexcavationofthematerialusingathinsyringeneedle.These

problemsspawnedtheideaofbisectingtheoriginaldesignoftheporthorizontally

andprintingbothpiecesindependently,asdisplayedinFigure9.2.Thisexposesthe

35

channelsandallowsforeasyremovalofthesupportmaterialwithoutdamagingthe

integrityofthechannels.Asupportingharnesswaslaterdesignedand3Dprintedto

rejoinandclamptogetherthepiecesofentrapmentdockFigure9.3.

36

1 2

3 4

5

Figure9:ImagesoftheentrapmentdockanditssupportingcomponentsdesignedusingSolidEdgeCADsoftware.The3Dprintedsolidisatransparentmaterial(VeroClear-RGD810)thatprovidesdimensionalstabilityandarigidfinish.Figure9.1isanimageoftheassembledentrapmentdock;Figure9.2depictsthetwosplit-piecesoftheentrapmentdock;Figure9.3showstherenderingofthesupportingharnessthatclampsandkeepsthesplit-piecesinplace;Figure9.4showstherendingofthecompletelyassembleddevicewiththeentrapmentdockhighlightedingreen;Figure9.5isanimageofthecompletelyassembleddevice.

37

2.4.1 Biocompatibility

3Dprintingandphotopolymercompositionhaveimprovedtremendously

withinthepastfewyears,andproblemswithbiocompatibilityhavemostlybeen

overcomebypost-processingthe3Dprinteddeviceandoptimizingthechemistryof

theresinsusedtocreatemicro-andmilli-fluidicplatforms.Thepolymerusedto

printtheentrapmentdock(VeroClear-RGD810)isdimensionallystableandis

approvedformedicalapplications93.Infuturepursuitstoperfecttheentrapment

dockandtoaccommodatedifferentorganisms,anadvancedpolymer(MED610,

madebyStratasys)couldbeused.Thisplastichasawiderbiocompatibilityrange

andmightserveasabetteralternative94.

Macdonaldet.alattheUniversityofGlasgowassessedthebiocompatibilityof

fourcommerciallyavailable3Dprintingpolymers(VisiJetCrystalEX200,Watershed

11122XC,FototecSLA7150ClearandABSplusP-430)withzebrafishandobserved

thekeydevelopmentalmarkersinthedevelopingembryos.Theirresultsshowed

thatthefourphotopolymerswereverytoxictotheembryos,resultinginfatalityin

mostcases95.Theseresultsre-emphasizetheimportanceofusingbiocompatible

materialstoachievereliableresults,especiallywhenconductingexperimentsin

whichmorphologicalchangesareanalyzed.Itisbecomingwidelyacknowledged

thatmoredetailedbiocompatibilitytestingmustbeprovidedbymanufacturers

before3Dprintedobjectsareusedindifferentbiosystems96,97.

38

2.5 SpecificationsandParameters

1. The48-72hpfzebrafishlarvaecanbeaspirateduptoamaximumflowrate

of330mm3s-1,giventubingdimensionsof0.8mminnerdiameterx2.4mm

outerdiameter.

2. Themaximumspeedthelarvaecanattaininthistubingwithoutdeformation

is656mms-1

3. Inthissystem,Re=528at20◦C.

4. Themaximumaspirationrateatthedeliverytube/capillaryjunctionshould

bebelow83μLs-1toavoiddamagingthelarvae.

5. Theinnerdiameteroftheinputcapillaryis800μm

2.6 Electronics

Toautomatemovementofthelarvaeinthepresentsystem,electronic

sensors(e.g.photodiode)andactuators(e.g.syringepump),wereusedtocontrol

fishmovementthroughaLabVIEWinterface.ThemyDAQ(NationalInstruments,

Austin,TX)wasusedtodetecteventsandtocontroldevices,suchastheSC5

solenoidcontroller(RWAutomations,Figure10)usedtocontrolthesolenoidvalves.

39

Figure10:ElectronicComponents.RWAutomationSC5SolenoidControllerboardequippedwithpower,communication,andstatusLEDs(Left);NationalInstrumentsmyDAQ(Right)

ThemyDAQfromNationalInstruments(NI)isaDataAcquisition(DAQ)unit

thatinterfaceswiththegraphicalprogrammingsoftwarepackage,LabView,and

allowsforsimpleexternalcontrolofconnecteddevices.NImyDAQisasimpleand

intuitiveDAQdevicewithanaloginputs,analogoutputs,digitalinputsanddigital

outputs.ThedigitaloutputchannelsareusedtocontroltheTransistor-Transistor

Logiccommands(TTL)thattriggerthesolenoidpinchvalves.ThemyDAQwasalso

beusedasapowersupply.

TheRWAutomationSC5solenoidcontrollerpermittedprogrammable

controlofthesolenoidpinchvalvedepictedinFigure11.Theboardcontrolsfive

independentvalvesbyutilizingapeak-and-holdalgorithm.Thisallowstheuserto

40

energizeeachsolenoidvalveatitsfullratedvoltageforfastactuationandstrong

pull.TheSC5thenautomaticallyreducestheaveragevoltageappliedtothesolenoid

toconservepowerandpreventoverheatingofthesolenoidintheopen/onposition.

Figure11:Cole-Parmertwo-waynormallyclosedsolenoidpinchvalve.12VDC,1/16"IDx1/8"ODtubing.Thesolenoidcontrolledpinchvalveclampswithaforceof15PSIwitha25msecresponsetime.

Whentriggered,thesolenoidcontrollerapplies12Vtothesolenoidvalvesfor

0.5sec,thenreducesthevoltagetoaholdinglevelof~4.5V.Thisminimizes

overheatingofthesolenoid.TheSC5controllerisunderTTLcontrolandopensor

closesthesolenoidpinchvalveswithhighorlowvoltagesignalsfromtheDIO

41

channelsofthemyDAQcontroller.LowtohighvoltageTTLinputtransitionstrigger

thesolenoidvalveopening,whilehightolowTTLvoltagetransitionsturnoffthe

solenoidvoltagetodisablethesolenoidandclosethevalve(NCstate).TTLinputsof

thesolenoidcontrollerboardareinterfacedwithDIOports0-4onthemyDAQand

controlledbyaLabViewVIwithBooleancontrols.

Toenhancetheclarityofdataandtopermithigherdataacquisitionrates

fromtheopticaldetectionsystem,aprecisionhigh-speedvoltagecomparatorwas

used(TexasInstrumentsLM339N)toconvertthephotodiodevoltageoutputtoa

TTLsignal.Thevoltagecomparatorfunctionsbycomparingthedifferentialvoltage

betweenthepositivepin(sporadicvoltagefromPD)andnegativepin(stable

predeterminedvoltagefrommyDAQ),thengeneratingaTTLsignalbasedonthe

inputdifferentialpolarityFigure12.1.Thiscircuitrendersthesystemlesssensitive

topotentialfalsereadingsfromthePD.

42

Figure12:Imageofthefunctionalcircuitdiagram.Voltagecomparator(left);wiringofcomparatoronabreadboard(Right)

TheLM339NseriesICconsistsoffourindependentprecisionvoltage

comparatorswithanoffsetvoltagespecificationaslowas2mVmaximumforall

fourcomparators.Thecomparatorisdevisedtooperatefromasinglepowersupply

overawiderangeofvoltagesandtodirectlyinterfacewithTTL(Transistor-

TransistorLogic)andCMOS(ComplementaryMetal–Oxide–Semiconductor).

2.6.1 ControlMaps/SoftwareProgramming

Mostofthesystemismanagedmanuallythroughavirtualinstrument

dashboarddesignedinLabView,Figure13.Theplatformallowsautomationofthe

solenoidvalvesandthesyringepumptoaspiratezebrafishlarvaeintothe

entrapmentdock.

43

Figure13:LabVIEWcontroldashboard.Itcontrolstheautomationparameterssuchasthedurationofinjection;solenoidon/offcommandsandsyringepumpfunctions(pump/withdraw).

44

LabViewTestSetupRoutine

ThefollowingisthedataflowdiagramfortheLabViewroutine,which

controlsthecoordinatedfunctionsofthesyringepump,thepinchvalvesandthe

opticaldetectionsystem.

Figure14:Valve/syringepumpcontrolmap

Allthesolenoidvalvesarenormallyclosed.Acyclebeginswiththeopening

ofvalve1andvalve3.Thesyringepumpstartswithdrawingatagivenflowrate

untilalarvaispickedupfromreservoir1,conductedthroughthetubeanddetected

bytheopticaldetectionsystem.Thesyringepumpimmediatelystopswithdrawing

andvalve1andvalve3close.Next,valve2andvalve3open,followedbythe

syringepump,ininfusemode,aspiratingthelarvaatthesetflowratetothe

entrapmentdock.Valve2closestopreventbackflow.Injectiontakesplace.Valve4

45

andvalve5openafterinjection.Syringepump2,ininfusemode,flushesthelarva

outoftheentiresystem.

Thedetailedsetupandprogramcommandsarehighlightedintheappendix.

46

CHAPTER3

EXPERIMENTALRESULTSANDDISCUSSION

3.1 InputReservoirEfficacyTest

Toevaluatethespecimenoutputrateofthereservoir,asystemtestwas

conducted.

3.1.1 Procedure

Thereservoirwasfilledtocapacity(60mL)witheggwater(60µg/mL)at

roomtemperature(21°C),Figure4.1.Thebubblerwasthenturnedontoprevent

settling,therebyincreasingthelikelihoodoflarvauptakeintothesystem.50

individualzebrafishlarvaewerethenintroducedintothereservoir.Thezebrafish

wereanesthetizedwitha200µg/mLsolutionoftricainebeforeuse.Withthelarvae

floatingfreelywithinthereservoir,theaspirationtubewasloweredintothe

chamberandthesyringepumpwassettowithdrawmodeatthefollowingflow

ratesfor60seconds:10mL/min,8mL/min,6mL/minand4mL/min.Thenumberof

larvaeaspiratedfromthereservoirwasthenrecordedandtheentireprocedurewas

repeated10times,consecutively.

3.1.2 Results

Fortheentrapmentsystemtobemoreefficientthanthetraditionalinjection

method,thereservoirshouldexpelthelarvaefromthesystematabaserateof3fish

perminute.Thetestofthereservoiratthehighestflowrate(10mL/min)generated

anaverageoutputofabout6larvaeperminuteFigure15.Thistranslatesto1larva

47

outputevery10seconds,whichisarelativelyshorterdurationthantheremaining

timeittakesalarvatocompletetherestofthecycle.Thisimpliesthatthereservoir

musthaveanoutputratehighenoughtosupporttheentiresystem.Inspiteofthe

highoutputrate,onlyonelarvacanbeprocessedbythesystemforanygivencycle.

Thehighoutputrateofthereservoirsuggeststhatithasthecapacitytofeed

multipleentrapmentsystemssimultaneously.

48

Figure15:Abargraphdepictingthenumberofzebrafishlarvaecollectedafter60secatdifferentflowrates.3.2 OpticalDetectionUnitEfficacyTest

Toevaluatethesensitivityoftheopticaldetectionunit,asystemtestwas

conductedroutinely.

3.2.1 Procedure

First,the650nmlasermodulewaspoweredon,withmajorityofthelight

beamcenteredonthephotodiode(PD).AtthispointtheLabViewsoftware,

registeredabasalhighTTLsignal(1,TRUE).Tenindividualzebrafishlarvae

(~48hpf)werethenfedthroughthetube,pastthelaser/PD,at10mL/min.The

49

decreaseinlightdetectedbythePDresultedinalowTTLsignal(0,FALSE)fromthe

comparatorasthelarvapassedbetweenthelaserandthephotodiodeandwas

monitoredbythecontrolsoftware.

3.2.2 Results

Theresultsfromsensortestsprovedthatthelightdetectionsystem

effectivelydetectedlarvae.Thewaveformgraph,Figure16,displays10individual

peaksthatcorrespondtothe10-zebrafishspecimensusedinthistest.Theinitial

voltagereading(withoutacomparatorcircuit)from7-14secondsindicatesthe

observedvoltagebeforewaterorzebrafishlarvaewereintroducedintothetube.

Thebackgroundnoiseapparentinseveralpositionsonthechart(ex.14-25

seconds)canbeattributedtoairbubbles.Thecontrolsoftwaretracestherecorded

voltagereadingsthatregisteredbelowthebasalreading(tube+water+specimen),

thusallowingustodiscernbackgroundnoisefromusefuldata.

50

1 234 1095 86

7

Figure16:Awaveformchartofthephotodiodevoltagespikeswithrespecttotime.ThebasereadingfromthePDis1V.Therestofthelabeledpeaksregisteringsub-basalvoltagesindicatethepresenceofazebrafishspecimen.Thecomparatorcircuitwasnotusedtoconditiontheserawvoltagesignals.

3.3 EntrapmentDockTests

Theentrapmentdockisthemainpartofthesystemwiththecorefunctionof

immobilizing48-72hpfzebrafishtoallowtailinjection.Aseriesoftesttrialswere

conductedtoassessitsabilitytoentrapandimmobilize48-72hpfzebrafish.

3.3.1 Procedure

Totestitsabilitytoaccomplishtrapping,individual72hpfzebrafishwere

introducedintothedockviatheinletchannelandsubsequentlyflushedout.For

everytrialthatwasconducted(n=100),thelarvawassuccessfullyimmobilized

regardlessoforientation(head/tailentry)asillustratedinFigure17.

51

Figure17:Imageoftheentrapmentdockillustratingtheimmobilizedzebrafishlarvaeinthetwopossibleorientations.Head-onarrival(leftimage)andtailarrival(rightimage)3.4 AverageDurationofScreeningSteps(1Cycle)

Theoveralldurationforonecyclethroughthesystemwasdeterminedinthistest.

3.4.1 Procedure

Bytimingeachautomatedstageoftheprocesses(loading,trapping,injection

andflushing)withinonecycle,theaveragecycletimewascalculated.Sincetheflow

rateisvariedatcertainstageswithinagivencycle,timingeachstepprovidesamore

52

accurateprojectionoftheoveralldurationofonecycle.

Table1:Averagedurationofscreeningstepsfromthereservoirtothelightdetectoratdifferentflowrates.(0.08cminnerdiameterand88cmlength)

Table2:Calculateddurationofscreeningstepsfromthephoto-detectortotheentrapmentdockatdifferentflowrates.(0.08cminnerdiameterand80cmlength)

Table3:AveragetimerequiredforinjectionStep 5mL/min 4mL/min 3mL/min 2mL/minEntrapmentDock→Exit 4 5 6 9Table4:Calculateddurationofscreeningstepsfromtheentrapmentdocktotheexit(oftheentiresystem)atdifferentflowrates.(0.08cminnerdiameterand62cmlength)

Step FlowRate(mL/min) Time(sec)

Reservoir→PD 10mL/min 9±6.5

Reservoir→PD 8.0mL/min 10±10.4



Step FlowRate(mL/min) Time(sec)

PD→EntrapmentDock 2mL/min 12

PD→EntrapmentDock 1.75mL/min 14



Step Time(sec)Injectionprocedure Userdependent:10±3

53

3.5 QuantitativeAssessmentofAnimalHealth

Theconditionofeachindividualzebrafishwasassessedafteritpassed

throughthesystem.Thisassessmentwasbasedonbothfunctionaland

morphologicalcriteria.Atallflowrates(2mL/min,1.75mL/min,1.5mL/min,

1.2mL/min),heartbeat,touch-response,structuralintegrityoftheyolksacand

melanocytes,wereevaluatedandcomparedtoacontrolsample.Thelarvaeinthe

controlsetup(sameage)wereaspiratedinanunobstructedtubespanningthe

lengthoftravelencounteredbylarvaeintheexperimentalsetup.

3.5.1 Procedure

Thezebrafishfromthetestsdescribedinsection3.4werecollectedand

viewedunderamicroscopetoevaluatethefollowingparameters:survival

(heartbeat)andmorphology(structuralintegrityoftheyolksacandmelanocytes).

ThefollowingdatawererecordedinFigure18.

3.5.2 Results

Atalloftheflowratesused,100%ofthelarvaesurvived(n=100)foratleast.

Tearingoftheyolksacwasneverobserved.Atthehighestinitialflowrateof

2mL/min,48%ofthelarvaeexhibitedmorphologicalabnormalities,specifically

slightdistortionofmelanocytesalongthetailandoftheyolkextension,Figure18.

Withtheslightlyslowerinitialaspirationratesof1.75mL/min,allhealthcriteria

matchedthoseofthecontrols.

54

Figure18:Comparisonofmorphologicalabnormalitiesofexperimentalzebrafish,atdifferentflowrates,withacontrolzebrafish.Theredarrowsshowdistortionsofthemelanocytesandthehighlightedregionshowsdistortionsoftheeyeandtheyolk.

55

Figure19:Quantitativeassessmentofzebrafishhealthatdifferentflowrates(n=100)

56

CHAPTER4

SUMMARY,CONCLUSIONSANDFUTUREWORK

4.1 Summary

Adevicetoaccelerateexperimentsinvolvingmicroinjectionoflargenumbers

oflarvaewasdesignedandfabricatedusinginexpensiverapidprototyping

techniques.Chapter1introducestheinfluenzaAvirusanddescribesimportant

aspectsofthevirus,includingitsgenetics,distribution,transmissionandclinical

symptomsofthediseasethatitcauses.Thischapteralsoplacesemphasisoncurrent

IAVresearchadvancementsandreviewstheuseofanimalmodelsforinfectious

diseaseresearch.Chapter2describesthedevicecomponentsseparatelyand

reviewsprototypingmethods,modesofdevicefunctionandsupportingcontrol

software.Thedevicespecificationsandparametersarealsohighlightedinthis

chapter.Chapter3presentsefficacytestsofthemaincomponentsofthesystem.

4.2 Conclusions

Afullyfunctionalzebrafishimmobilizationdevicewasdesigned,fabricated,

andtested.48-72hpfzebrafishlarvaeweresuccessfullyimmobilizedwithina3D

printeddock.Theexperimentaltestsetupallowedmanipulationofthevolumetric

flowratewithinthetubes,pressurefromtheaircompressorusedtocreateair

bubblesinreservoir1,voltagewithintheopticaldetectionsystemandtheoverall

deviceoutputfrequency.Theloadingstagewasimprovedwiththeadditionofthe

bubblertoreservoir1.Thisplayedamajorroleindistributinglarvaeevenlywithin

thechamberduringoperation.

57

Automationofthesystemenhancedtestreproducibilitybyreducingthe

chancesforhumanerror.Theopticaldetectionsystemprovedeffective(see

waveformchart,Figure16.Individuallarvaeweresuccessfullyidentifiedbythe

detectionsystemwith100%precision(nofalsepositives).Thecontrolsoftware

tracedtherecordedvoltagereadingsthatregisteredbelowthebasalreading;

moreover,thecomparatorincreasedthedataacquisitionratetwo-fold,from62Hz

to124Hz.

Oneofthemostchallengingaspectsoftheprojectwasthefabricationofthe

immobilizationdock.The3Dprinterthatwasusedforthisprojectdeposited

supportmaterialwithinthechannelsandnomethodusedtoremovethematerial

wassuccessful.Bydevelopingthesplitdesigntechnique,Ieliminatedthisproblem

byexposingthechannelsforsimplifiedpost-printprocessing.Thetestsofthe

entrapmentdockweresuccessful,i.e.afterrunning100cyclesthroughthedockat

variousflowrates:48%oflarvae(n=25)wereimmobilizedwithoutany

deformationat2mL/min,76%oflarvae(n=25)wereimmobilizedwithoutany

deformationat1.75mL/min,100%oflarvae(n=25)wereimmobilizedwithoutany

deformationat1.50mL/min,100%oflarvae(n=25)wereimmobilizedwithoutany

deformationat1.25mL/min.

4.3 FutureWork

Atthisstageofdevicedevelopment,adjustingtheflowratewithinthesystem

duringcertainpartsofagivencyclecanalterthefrequencyofthedevice’soutput.In

spiteoftheautomationincorporatedintothedevice,theusermustmanuallytrigger

58

thecontrolprogramtoexecutecertaincommandssuchaswhentoinjectthe

specimen.Byaddingaminimalnumberofstepstotheexistingcontrolprogram,an

automaticinjectorequippedwithatranslationstagecouldbeincorporatedintothe

designtoreducetheneedformanualinput.Furthermore,arecoverysystemcould

easilyintegratedtowardtheendofthedevicetoreceivetheinjectedlarvaeafter

ejectionfromthesystem.Ultimately,thedevicewillprovideaseamlessuser

interfacethatrequiresminimaluserintervention.Bydepositingaknownnumberof

zebrafishlarvaeintotheinputreservoir,andinputtingthedosageofthecompound

orchemicalunderstudy,thedevicewillbeabletoinjectanddispenselarvaefor

analysis.

Microsystemsdesignedforcell-basedstudiesorapplicationssuchasthisone

requirefluidhandling.Flowwithinthesesystemsinevitablygeneratesfluidshear

stressthatmayadverselyaffectthehealthoftheorganismsunderstudy.Simple

assaysofspecimenviabilityandmorphologywereusedinthisprojecttodetectany

grossdisturbancestolarvalanatomyandphysiology.However,itwillbebeneficial

tospecificallyevaluatephysiologicalimplicationsoffluidshearstresswithinthe

tubesandtheentrapmentdock.Varmaetal.atMassachusettsInstituteof

Technologyexploredausefultechniquethatcanassistuswithunderstandingthe

fluiddynamicswithinthisdevice.Itinvolvesusingageneticallyencodedcellsensor

thatfluorescesinaquantitativefashionuponencounteringfluidshearstress

(FSS)98.Varmaetal.choseawidelyusedcellline(NIH3T3s)andcreateda

transcriptionalcell-sensorthatfluoresceswhentranscriptionofarelevantFSS-

inducedproteinisinitiated.Thesensorpathwayspecificityandfunctionalitywere

59

verifiedbynotinginducedfluorescenceinresponsetochemicalinductionoftheFSS

pathway,seenboththroughmicroscopyandflowcytometry.Inaddition,these

novelcellsensorscanbeinducedwitharangeofFSSintensitiesanddurations,with

alimitofdetectionof2dynes/cm2whenappliedfor30minutes,makingitmore

versatileinsimulatingseveralmicrofluidicflowconditions.Undertakingthe

improvementsdiscussedabovewillenableforamoreseamlessoperationofthis

deviceandpossiblypavethewayforsubsequentengineeringsystemswithwide

applicationsinthemicrosystemscommunity.

60

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APPENDIXA

ZEBRAFISHHUSBANDRY

ZebrafishwerehousedattheUniversityofMaineZebrafishFacilityin

recirculatingsystemswherethewatertemperaturewasmaintainedat28°Cwitha

totalsystemflowrateof150liters/min.Zebrafishweremaintainedinaccordance

withtheInstitutionalAnimalCareandUseCommitteestandardsoftheUniversityof

Maine.Fertilizedzebrafisheggsarecollectedaccordingtomethodsproposedby

PhennecieattheUniversityofMaineZebrafishFacility99.Collectedeggsareraised

for24hoursat28.0̊Cin100mm-diameterpetridisheshalffullofeggwater,60µg/L

InstantOceanSaltsinpurifiedreverseosmosiswater.Nomorethan100eggswere

raisedinone100mm-diameterpetridishatatime.

At24hourspostfertilization,eggwaterisreplacedineachpetridish.Chorions

areremovedfromtheembryosbyhandanddiscardedasbiologicalwaste.Live

embryosarestoredat28.0̊Cuntilthefollowingday.

At48hpf,embryosaremotile.Eggwaterisreplacedineachpetridishanddead

embryosareremovedfromthecontainer.Embryosarestoredat28.0̊Covernight.

At72hpf,specimensareconsideredlarvae.Zebrafisheggwaterisreplacedand

specimensareusedasneededinfurtherlaboratoryprocedures.

Zebrafishwereeuthanizedina600µg/LconcentratedstockofTricanefor

30minandfixedtominimizetheamountoflarvaeneededforrepeatedtesting.Fish

areremovedfromanestheticandplacedinfixativesolutionof1.5%gluteraldehyde,

0.5%para-formaldehyde,50mMPBS(phosphatebufferedsaline)forlongterm

storage100.Larvaewerefixedforatleast12hourspriortouse.Forflowtesting,

72

chemicallyfixedfishwereremovedfromfixativesolutionandplacedineggwater

withnomorethan0.25%gelatintominimizelarvaladhesiontotubing.

Forproceduresinvolvinglivespecimens,larvaeareanaesthetizedina

200µg/mLsolutionoftricanewithnomorethan0.25%gelatinaddedtominimize

specimenadhesiontotubing.Fishwereaspiratedthroughthedeviceinthe

anestheticgelatinsolution.

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APPENDIXB

VALVE/SYRINGEPUMPCONTROLMAP

SETUP:

MyDAQDIOchannels(0-5)

• Input–laserdetector(detectedfish=1,nothingdetected=0)

• Output–valve1(open=1,close=0)





• Output–syringepumpdirection(inject=1,withdraw=0)

• Output–syringepumpstart/stop(start=1,stop=0)

ProgramCommands:

• Allvalvesclosed,pumpstopped

• Setpumptowithdrawmode

• Closevalve2,Closevalve4,Closevalve5(shouldbeclosedalready)

• Openvalve1andvalve3

• Startpump1

• Monitordetector

� Ifpositivedetect→stoppump,closevalve1,closevalve3,openvalve2,

openvalve3setpumptoinject,startpump

• Pumpinjects.Duration:(inputvalue)seconds→stoppump,valve2close

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• Inject.Duration:(inputvalue)seconds

• Openvalve4,openvalve5

• Startpump2(infuse)

• Waitforfishtoflushout.Duration:(inputvalue)seconds

• Stoppump

• Closevalve5

• GotoStep1

75

APPENDIXC

MATERIALSLIST

PumpSystemMaterials:

� TygonMicroBoreTubing,Part#:TGY-030.ComponentSupplyCo.

� SiliconeTubing,Part#:SCT-063A.ComponentSupplyCo.

� BD60mLSyringe,Part#:309654.Becton,DickinsonandCompany.

� NewEraNE-1000XProgrammableSyringePump

� 2-waynormallyclosedsolenoidpinchvalve,Part#98302.ColePalmer

InstrumentCompany,LLC.

� AquaCultureSingleOutletAquariumAirPump,Part#0079285405132

OpticalDetectionMaterials

� 5mmtransparentcylinderheadphotodiode,Part#LLSO5-A.SenbaOptical

ElectricalCo.,Ltd

� 4.5V650nmredlaserdiodemodule,Part#01444878.LightInTheBoxCo.,

Ltd

� PROCELL1.5Vdrycellbatteries,Part#PC1500.DuracellInc.

EntrapmentDockMaterials

� Objet30RapidPrototypingSystem,SUP705SupportMaterial(WaterJet

Removable).StratasysLtd.

� CustomStageMount

� MakerBotReplicatorDesktop3DPrinter,PLAFilament.MakerBotIndustries,

LLC.

76

Electronics

� NationalInstrumentsmyDAQUnit

� SC5Five-ChannelSolenoidController/Driver.RWAutomations,LLC.

� SolidEdgeST7.SiemensProductLifecycleManagementSoftwareInc.

� NationalInstrumentsLabVIEW2016.NationalInstruments.

77

BIOGRAPHYOFTHEAUTHOR

FuoadSaliou-SulleywasborninAccra,Ghanaon16thJuly1991.Helived

withhisfamilyandattendedschoolinAccratillhewas17,whenhecametothe

UnitedStatesasanexchangestudent.Fuoadlivedwithahostfamilyin

Kennebunkport,MEandgraduatedfromKennebunkHighSchoolin2009.InMay

2014,hegraduatedMagnaCumLaudefromtheUniversityofMaineinOronowitha

degreeinBiologyandaminorinChemistry.Inthefallof2014,heenrolledinthe

schoolofChemicalandBiologicalEngineeringattheUniversityofMainedoing

researchinthelaboratoryofDr.PaulJ.Millard.

Fuoadintendstocontinuepursuingscientificresearchstudies,particularlyin

abiomedicalorpharmaceuticalinstitution.Wheneverhehasachanceto,Fuoad

likestospendtimewithfriends.Heisalsoveryfondofoutdoorsportsandactivities

likehiking,mountainbiking,soccerandbasketball.HeisacandidatefortheMaster

ofSciencedegreeinBiologicalEngineeringfromtheUniversityofMaineinAugust

2017.

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