Dissection of Human Vitreous Body Elements for Proteomic Analysis.

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by  Skeie, Jessica M.

JournalofVisualizedExperiments www.jove.com
VideoArticle
Dissection of Human Vitreous Body Elements for Proteomic Analysis
JessicaM. Skeie,VinitB. Mahajan
DepartmentofOphthalmologyandVisualSciences,OmicsLaboratory,UniversityofIowa
Correspondenceto:[email protected]
URL:http://www.jove.com/details.php?id=2455
DOI:10.3791/2455
Citation:Skeie J.M.,Mahajan V.B.(2011).DissectionofHumanVitreousBodyElementsforProteomicAnalysis.JoVE.47.http://www.jove.com/details.php?id=2455,doi:10.3791/2455
Abstract
Thevitreousisanopticallyclear,collagenousextracellularmatrixthatfillstheinsideoftheeyeandoverliestheretina.1,2Abnormalinteractions
betweenvitreoussubstructuresandtheretinaunderlieseveralvitreoretinaldiseases,includingretinaltearanddetachment,macularpucker,
macularhole,age-relatedmaculardegeneration,vitreomaculartraction,proliferativevitreoretinopathy,proliferativediabeticretinopathy,and
inheritedvitreoretinopathies.1,2Themolecularcompositionofthevitreoussubstructuresisnotknown.Sincethevitreousbodyistransparentwith
limitedsurgicalaccess,ithasbeendifficulttostudyitssubstructuresatthemolecularlevel.Wedevelopedamethodtoseparateandpreserve
thesetissuesforproteomicandbiochemicalanalysis.Thedissectiontechniqueinthisexperimentalvideoshowshowtoisolatevitreousbase,
anteriorhyaloid,vitreouscore,andvitreouscortexfrompostmortemhumaneyes.One-dimensionalSDS-PAGEanalysesofeachvitreous
componentshowedthatourdissectiontechniqueresultedinfouruniqueproteinprofilescorrespondingtoeachsubstructureofthehuman
vitreousbody.Identificationofdifferentiallycompartmentalizedproteinswillrevealcandidatemoleculesunderlyingvariousvitreoretinaldiseases.
Protocol
1.Anterior Segment Dissection.
1. Thecorneaisremovedbyfirstmakinganincisionatthelimbusintotheanteriorchamberusinga15°blade(Figure1).Thenonebladeof
curvedcornea-scleralscissorsisinsertedintotheanteriorchamber.Circumferentialcutsaremadeinthecornea,justanteriortothelimbus.
2. 0.12ColibriforcepsandWestcottscissorsareusedtocuttheiriscircumferentiallyanteriortotheciliarybody.
3. Thelensnucleusisremovedwith0.12Colibriforcepsora15°blade(supersharp)andthecortexandcapsulewithforceps.
2. Vitreous CoreAspiration.
1. A23-gaugeneedleona5-ccsyringeisinsertedintothemid-vitreous(Figure1).
2. Approximately1mLormoreofvitreouscoreisgentlyaspirated.
3. Thesampleisthenplacedinamicrofugetubeandthenintoliquidnitrogen.
3.Anterior Hyaloid Dissection.
1. Theanteriorhyaloidisseenasasemi-transparentringbyadjustingtheincidentlightfromagooseneckmicroscopelight.Previousaspiration
ofthevitreouscoreseparatestheanteriorhyaloidfromotherstructuresforeasieridentification.
2. ThesheetofelastictissueiscarefullypulledawayfromtheciliarybodywithColibriordressingforcepsandcutwithVannasscissors.This
maneuverisrepeated.
3. Thesampleisthenplacedinamicrofugetubeandthenintoliquidnitrogen.
4. Vitreous Base Dissection.
1. Using0.12forcepstostabilizetheeyecup,Westcottscissorsareusedto"flower"theeyebymakingfourequallyspacedstress-relievingcuts
fromtheciliarybodytowardstheopticnervehead.
2. Theparsplicataoftheciliarybody(Figure2)isremovedfromeachofthequadrantsusingWestcottscissors.
3. Thevitreousbaseisthengraspedoneithersideoftheoraserratawithforcepsovertheparsplanaandtheretina(Figure2).
4. Continuoustractiononthevitreousbaseisappliedwiththeforceps.SequentialcuttingwithWestcottscissorsextractsasemi-transparent
tissuewitha"stringofpearls"appearanceasitisexcised.
5. Thesampleisthenplacedinamicrofugetubeandthenintoliquidnitrogen.
5. Vitreous Cortex Removal.
1. TheparsplanaisexcisedfromeachofthequadrantswithWestcottscissorsbycuttingtheleafletapproximately3-mmposteriortotheora
serrata(Figure2).
2. Betweenthetissueleaflets,thevitreouscortexisvisualizedasasemitransparentfilm.
3. Theelasticfilmiseithergraspedwith0.12forcepsorheldbyadherencetoaWeck-Celsurgicalsponge.Thevitreouscortexisthenpulled
awayfromtheretinaandexcisedwithWestcottscissors(Figure1).
4. Thesampleisplacedinamicrofugetubeandthenintoliquidnitrogen.Allsamplesarestoredat-80°Cuntilutilizedforexperimentation.
6. Representative Results
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Tissuesamplescanbeprocessedbyavarietyofmethodsforspecificexperiments.Inourcase,samplesweresubmittedforproteinanalysisby
SDS-PAGE(Figure3).
Figure1.Cross-sectionalviewofthehumaneyedepictingdifferentsubstructuresofthevitreousbody.Themostanteriorvitreousisa
thincollagenouslayercalledtheanteriorhyaloid.Thevitreouscorecomprisestheentirecentralregionofthevitreousbody.Thisportionofthe
vitreousismoreaqueousincontrasttothevitreousbase,whichisviscousenoughtobegraspedbyforcepsandisfirmlyattachedtothe
underlyingciliarybodyandretina.Encompassingthevitreouscoreisaverythincollagenousshellcalledthevitreouscortex.
Figure2.Vitreousbaseanatomy.Thevitreousbaseisasemi-transparentsubstructureofthevitreousbodylocatedalongtheoraserrata(white
arrows),whichisthedividinglineseparatingtheciliarybodyandretina.Theanteriorborderofthevitreousbaseextendsovertheparsplana
(whiteline)oftheciliarybody.Theposteriorborderofthevitreousbaseextends2-3-mmposteriortotheoraserrata(whitedash).Toexcisethe
vitreousbase,forcepsareusedtograspthetissueandpullitawayfromtheunderlyingciliarybodyandretina.Onceelevated,Westcottscissors
areusedtocutalongthebase.
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Figure3.One-dimensionalSDS-PAGEofVitreousBodyElements.Totalproteinconcentrationsfortheanteriorhyaloid,vitreousbase,
vitreouscore,andvitreouscortexwere11.24,20.1,16.61,14.24mg/mL,respectively.Gelelectrophoresiswasperformedat200kVfor45
minutes,stainedwithFlamingo(Bio-Rad),andvisualizedusingaVersaDocImagingsystem(Bio-Rad).Theprofilesforthedifferenttissuesshow
severalsimilarbands,indicatingeitherconservedorcross-contaminatingproteins,aswellasuniquebands(asterisk),indicatingdifferentially
localizedproteins.
Discussion
Thevitreousbodyisasemi-transparentgelwhosemolecularcompositionispoorlyunderstood,especiallyatthelevelofitssubstructures:the
vitreousbase,core,cortex,andanteriorhyaloid.ThevitreouscorecontainscollagensII,V,IX,andXI,alongwithchondroitinsulphate
proteoglycans,heparansulphateproteoglycans,andhyaluronan.1,2Proteinbiomarkersinthevitreouscorehavebeenassociatedwithdiseases
suchasdiabeticretinopathy.3-5Howtheseproteinsaredifferentiallyexpressedineachofthesubstructures,andinmanycasesthespecific
proteinidentities,arenotknown.Thesedetailsmaygiveinsighttotheoriginofproteinsassociatedwithspecificvitreoretinaldiseasesandhelp
targetfuturetherapies.Althoughtheoptimumpostmortemintervalfortissuedissectionhasnotbeendetermined,proteindegradationmayaffect
downstreamexperiments.Forexample,immunohistochemistryisaffectedin12-hourpostmortemeyesandsomespecificenzymeactivitiesmay
bereducedwithinafewhours(unpublishedobservation).Alltissuesinthisstudywerecollectedbetween2and8hoursofdeathwithout
significantchangesinproteinexpressionorsuitabilityforproteomicsanalsyis.Theliquidnitrogenfreezingmethodofpreservationischosenover
fixationinordertopreventsmallchangesinproteinstructurecausedbyfixativecrosslinking,whichhasbeendemonstratedinothertissuesby
LC-MS/MS.6Proteomicstudieswilldependontheabilitytoaccuratelydissectthedifferentcompartmentsofthevitreous,asdemonstratedinthis
videoexperiment.Wehavevalidatedthedissectiontechniqueusing1-dimensionalSDS-PAGE.Asourresultssuggest,therearedifferentially
expressedproteinsinthevariousvitreousbodysubstructures.Identifyingtheseproteinswillprovideamoredetailedunderstandingofvitreous
compartmentalization.
Disclosures
Noconflictsofinterestdeclared.
Acknowledgements
FundingwasprovidedbyFightforSight.TissueswereobtainedfromtheIowaLionsEyeBank.
References
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