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InvivoimagingofthephotoreceptormosaicofarodmonochromatJosephCarroll,StaceyS.Choi,DavidR.WilliamsDepartmentofOphthalmology,MedicalCollegeofWisconsin,TheEyeInstitute,925North87thStreet,Milwaukee,WI53226,USADepartmentofOphthalmology&VisionScience,UniversityofCalifornia-Davis,Sacramento,CA95817,USACenterforVisualScience,UniversityofRochester,Rochester,NY14627,USA VisionResearch48(2008)2564…2568 ContentslistsavailableatScienceDirectVisionResearchjournalhomepage:www.else brought to you by CORE View metadata, citation and similar papers at core.ac.uk provided by Elsevier - Publisher Connector Recently,opticalcoherencetomography(OCT)imagesofpatientswithachromatopsiarevealedanabsenceofphotoreceptorre”ec-tivityinthefovea,andthiswasinterpretedasbeingduetoanab-senceofhealthyconephotoreceptors(Barthelmesetal.,2006;Varsányi,Somfai,Lesch,Vámos,&Farkas,2007),thoughthetwostudiesdifferontheabnormalityoffovealthicknessinachromats.Nishiguchietal.(2005)detectedanormallayerofpho-toreceptorsusingOCTinasubsetofachromatswithCNGB3muta-tions.Despitetheirwideclinicaluse,commercialOCTscansofferlimitedresolutionofthephotoreceptorlayer(thoughaveragingmultipleframescanenhancetheseimages;Sander,Larsen,Thrane,Hougaard,&Jørgensen,2005).Thus,itisdif“culttodraw“rmcon-clusionsaboutthephotoreceptorcomplementofrodmonochromats.Bycorrectingfortheeyesopticalaberrations,anadaptiveop-tics(AO)funduscameraenablesdirectvisualizationofthephoto-receptormosaicinvivoLiangetal.,1997).Thisimagingtoolhasbeenusedtostudyboththenormalretina(Miller,Williams,Mor-ris,&Liang,1996;Roorda,Metha,Lennie,&Williams,2001;Roordaetal.,2002)andthediseasedretina(Carroll,Neitz,Hofer,Neitz,&Williams,2004;Choietal.,2006;Duncanetal.,2007;Roorda,2000;Wol“ng,Chung,Carroll,Roorda,&Williams,2006).Here,weimagedtheretinaofanachromat(ofknowngenotype)inordertocharacterizethephotoreceptormosaic.Thisimagingapproachislikelytoyieldbene“tincharacterizingthephotoreceptorcomple-mentinotherretinaldegenerations,especiallyinthosewhererestorativetherapiesarebeingdeveloped.2.Methods2.1.SubjectsSubjectsprovidedinformedconsentafterthenatureandpossibleconsequencesofthestudywereexplained.AllresearchadheredtothetenetsoftheDeclarationofHelsinki,andstudyprotocolswereapprovedbytheinstitutionalresearchboardattheUniversityofRochester.Amale(ACH0016)diagnosedwithcompleteachroma-topsia(age28)andhismother(ACH0015),acarrier(age50),wererecruitedforthisstudy.Completeophthalmologicexaminationswereperformedonbothsubjectsjustpriortoretinalimaging,whichwasperformedovera2-dayperiod.Colorvisionwasassessedusingavarietyoftests,includingtheRayleighmatch,pseudoisochro-maticplates(AO-HRR,DvorineandIshihara)andtheNeitztestofcolorvision.DNAanalysiswasdonepreviously,revealingthatACH0016washomozygousforthecommon1basepairdeletion(1148delC)intheCNGB3subunitoftheconeCNGchannelandhismother(ACH0015)washeterozygousforthemutation.(Dr.IreneMaumenee,personalcommunication).Thismutationresultsinaframe-shiftdown-streamofaminoacidThreonine383andintroducesaprematurestopcodon,whichiseffectivelyanullallele(Peng,Rich,&Varnum,20032.2.AdaptiveopticsimagingImagesofthephotoreceptormosaicwereobtainedusinganAO-funduscameraHoferetal.,2001;Putnametal.,2005,foraschematicandsystemdetails).Thesubjectsrighteyewasdilatedandaccommodationparalyzedthroughuseofacom-binationofPhenylephrineHydrochloride(2.5%)andTropicamide(1%).Theheadwasstabilizedusingadentalimpressiononabitebar.Inacontinuousclosedloop,wemeasuredtheeyesmonochromaticaberrationsovera6.8-mmpupilwithaShack-Hartmannwavefrontsensorandcorrectedforthemuntiltheroot-mean-squarewavefronterrorfellbelow0.1morafter800mselapsed,whicheverhap-pened“rst.Additionaldetailsonwavefrontmeasurementandcompensationhavebeenpreviouslypublished.Onceawavefrontcorrectionwasobtained,aretinalim-agewasacquiredbyilluminatingtheretinawitha1diameter,4-ms”ash(630,25nmbandwidth;fullwidthathalfmaximum)fromakryptonarc”ashlamp.Theshortdurationofthe”ashhelpedtominimizetheeffectsofmotionblurontheaccompanyingretinalimage.Acircular“xationtarget(front-illuminatedwithadimlong-wavelengthlight)wasusedtorecordtherelativeretinallocationofeach2.3.ImageanalysisImagesfromthesameretinallocationwereregisteredwithsubpixelaccuracy(accountingfortranslationandrotation)andaveragedusingacustomMatLab(MathWorks,Natick,MA)imageregistrationprogram(Putnametal.,2005).Wesummatedbetween3and12individualf
ramestocreateeachoftheimagesusedforanalysis.Conedensitywascalculatedusingpreviouslypublishedmethods(rolletal.,2004)aswellasamodi“edversionofasoftwareprogramthathasbeenusedtoautomaticallyidentifyphotoreceptorsinAOretinalimages(Li&Roorda,).ImageJ(NIH,Bethesda,MD)wasusedtomeasurephotoreceptordiametersAbramoff,Magelhaes,&Ram,2004).Thiswasdonebyexaminingtheplotpro“leof30pseudo-randomlyselectedcellsfromeachimage(seeFig.2c).AnaxiallengthmeasurementfromtheZeissIOLMaster(Dublin,CA)wasusedtoderivearetinalmagni“cationfortheimagesasdescribedbyBennett,Rudnicka,andEdgar(1994)3.Results3.1.ClinicalphenotypeDilatedfundusexaminationofACH0016revealedmildRPEatrophyOUandablondfundusconsistentwiththepatientscom-plexion.Therewasaloculatedareaofsubretinal”uid(notinvolv-ingthefovealcenter„inferonasalmacula,OS),consistentwithcentralserousretinopathy.Best-correctedvisualacuitywas20/150.DilatedfundusexaminationofACH0015showedmultiplesmalldrusenOU,notuncommonforherage.ColorvisiontestswerenormalforACH0015,whereasACH0016showedacompletelackofcolordiscrimination.Bothrod-andcone-basedERGswerenormalforACH0015,whiletherewasnodetectableconeresponsefromACH0016(althoughrodamplitudeswerenormal).3.2.AdaptiveopticsimagingImagesforACH0016wereobtainedat1.25,2.5,and10temporalfrom“xation(OD).Numerousphotoreceptorsarevisibleinallfourimages,thoughthephotoreceptormosaicisseverelydis-ruptedcomparedtoimagesfromanormalretina(Fig.1).Theden-sityofthephotoreceptorswasconsistentwithnormalrod,notcone,densities(seeTable1).Normalconedensitypeaksinthefo-veaanddeclinesrapidlymovingawayfromthefovea,whereasrodphotoreceptorsareabsentinthecentralfoveaandreachamaxi-mumdensitynear10eccentricity(Curcio,Sloan,Kalina,&Hend-rickson,1990).Thephotoreceptordensityfortheachromatexaminedinthisstudyremainedrelativelyconstantbetweentheand4images,thoughtherewasaslightelevationat10Imagesfromtheheterozygouscarriermother(at1.25and2.5OD)revealednoobviousdisruption.Ananalysisofherretinalimagesrevealedacontinuousmosaicinwhichthephotoreceptorswereofadensitycharacteristicofcones(Table1Fig.2ashowsanimagefrom10temporalfrom“xationforACH0016.Remarkably,despiteonlytwoframesbeingusedtocre-atethisimage,photoreceptorstructureisclearlyresolved.More-over,atalocationwhererodsoutnumberconesinthenormalretinabynearly10:1,weobserveahighdensityofcells.Inordertofurtherprobetheiridentity,wemeasuredthecelldiameterinthisimageaswellastheotherthreelocationsimaged.ShowninFig.2bisaplotofcelldiameterasafunctionofretinaleccentricity.At10retinaleccentricity,typicalroddiameteris2m,whileconesarenearly8mindiameter(Samy&Hirsch,1989).Theaver-age(±SEM)diameterofthestructuresintheimageinFig.2ais2.99±0.092m.Thisislargerthanthecalculatedfull-widthhalf-height(FWHH)ofthepointspreadfunctionoftheopticalsystem(6.8mmpupil,630nm),thereforewearecon“dentthatthesestructuresarenotsmallerthantheactualmeasurement.Fig.2shows2Dplotpro“lesfromthreedifferentlocationsintheimageFig.2a.Theindividualpro“leshavebeentranslatedverticallyforvisibility,butitillustratesthateveninourworstimageitisquiteeasytoobtainrobustestimatesofcelldiameter.Achromatstypicallyhavenystagmusandpoor“xation(Collewijn,&Nordby,1986),thoughACH0016hadminimalnystag-musandfairlyreliable“xation(thiscanbeassessedbythedis-placementofsuccessiveretinalimagesfromagivenreferenceframe).Moreover,wewereabletoreturntoimagethesameretinalJ.Carrolletal./VisionResearch48(2008)2564…2568 locationonsubsequentdays,consistentwithreliable“xation.However,eventhough“xationwassuf“cientlyreliableforimageregistration,anotherpotentialsourceoferrorcomesfromtheobservationthatachromatsoftenadoptpreferred“xationloca-tionsawayfromthefovea.Thus,theabsoluteretinallocationoftheimagesfromourachromatmightbedisplacedbyupto2Baseleretal.,2002).Nevertheless,thepositionofourimaginglocationsseemsreliable,andthustheinterpretationthatthephotoreceptorsintheimagesfromthemonochromatarerodsremainsthemostparsimonious.4.DiscussionHere,wepresentthe“rstinvivoimagesofthephotoreceptormosaicofarodmonochromat.Atfourdifferentretinallocationsalongthetemporalmeridian,imagesfromthemonochromatre-vealedaseverelydisruptedphotoreceptormosaic.Moreover,thesizeanddensityofthevisiblecellsweretypicalforrod,notcone,photoreceptors.Theseresultssuggestthat,atleastinthisachro-mat,theretinaislargelydevoidofhealthyconephotoreceptors.Theintermittentgapsinthemosai
ccouldbetheresultofeitherabsenceofconedevelopmentorconedegeneration.Itseemslikely,giventheavailablehistologicaldata(andthesmallpatchesofret-inaimagedhere),thattherecouldberesidualconestructureelse-whereinthispatientsretina.Inthenormalretina,conesareeasilyimagedwithadaptiveopticsasaresultoftheirstrongwaveguidenature(Roorda&Williams,2002).Thus,ifconespresentatthelocationsweimaged,theyaredamagedtothepointthattheyarenotfunctioning(giventheERGdatainthispatient)andnotvis-ibleintheretinalimages(likelyduetoimpairedwaveguiding„i.e.,lackofanoutersegment).Whileunlikely,analternateinterpretationofourdataisofcoursethatthephotoreceptorsintheseimagesaremorphologicallyandtopographicallyabnormalcones.Suchconescouldnotbefunction-ing,giventhepatientsphenotypeasacompleteachromat(non-detectableconeERG,nocolordiscrimination,photophobia,nystag-mus,andhishomozygousCNGB3nullmutation).Asmentionedabove,aninterpretationofthecurrentdatamightsuggestthatach-romatswithdifferentcausativemutationscanhavevaryinglevelsofresidualconefunction.WesuspectthatimagingsuchindividualswithAOophthalmoscopywillrevealsigni“cantdifferencesintheirphotoreceptormosaicsthatcorrelatewiththedegreeofresidualconefunction.Furthermore,examinationofgeneticallyclassi“edpatientswithhigh-resolutionretinalimagingislikelytoprovidevaluableinsightintopathogenesisofthediseaseaswellasidentifythoseindividualswhomightbemostreceptivetonewlydevelopedgenetherapies(Alexanderetal.,2007;Komaromyetal.,2007Ifaswebelievethesephotoreceptorsarerods,onewonderswhytheyarenotaseasilyvisualizedinthenormalretinaundersimilarimagingconditions.Toourknowledge,thereisonlyonere-portofinvivoimagesofrodsfromthenormalhumanretina.Inhigh-resolutionretinalimagestaken15…20from“xationinthenormalretina,Choiandcolleaguesobservedacontinuouscone Fig.1.Imagesoftheinvivophotoreceptormosaic.(aandc)Imagesfroma28-year-oldnormalmale,whohadbeenimagedaspartofanumberofunrelatedstudiesoverthecourseof3months.(bandd)Imagesfroma28-year-oldrodmonochromat.Imagesarefrom2.5(aandb)and4(candd)temporalretina.Scalebaris20 Table1Coneandroddensityinnormalsandarodmonochromat55,46649,30052,32282,99049,32326,539NDNDNormalcone(Curcioetal.,1990)46,08626,21520,6998559NormalconeAO44,33023,36215,6757656Normalrod(Curcioetal.,1990)22,03254,54278,011129,920Allvaluesareincells/mmJ.Carrolletal./VisionResearch48(2008)2564…2568 mosaicwithnumerousrodsintermingledthroughouttheimageChoietal.,2004).Thedif“cultyinimagingthem,andtherelativesparsenessatretinallocationswheretheyshouldoutnumbertheconesnearly10-fold,isconsistentwithpreviousdatathatrodsarelesseffectivewaveguidesthancones(Alpern,Ching,&Kithara,1983;vanLoo&Enoch,1975).Itmaybethatthisisonlyalimitingfactorwhentryingtoimagetheretina,wherethelightfromtheconesmaybereducingthecontrastoftheinterleavedrods.An-otherpossibilityisthatwhileinthenormalretinaconesarethoughttohavemoremitochondriathanrods(Hoang,Linsenme-ier,Chung,&Curcio,2002),therodsinthemonochromatretinamightbemoreactiveduetoachangeinlifestyleandasaresult,therodswouldhaveanincreasedmetabolicdemand.Thismightresultinanincreaseintherelativedifferenceinrefractiveindex,renderingthemmoreef“cientwaveguidesandeasiertovisualizewiththisparticularimagingtechnique.Alternatively,theabsenceofconephotoreceptorsinthisretinamayhaveallowedtheremainingrodstoswell,ashasbeenreportedtooccurwhenrodsarelostinnormalaging(Curcio,2001).Indeed,whilethephotore-ceptorsimagedaremoreconsistentwithrod,ratherthancone,diameters,theyareslightlylargerthanvaluesproposedinthelit-erature(Samy&Hirsch,1989).EvenaslightincreaseinsizewouldgreatlyimprovetheabilitytoresolvethemintheAOimages,sofurtherworkremainsindeterminingwhatmechanismsareactingtoincreaserodcontrastinthisretina.Finally,thephenotypicvariabilityingeneticallyidenticalachro-matsindicatesthatthereareothermitigatingfactorsthatin”uencethedegreeofresidualconestructureandfunctioninagivenrodmonochromatwillhave.Thisunderscorestheimportanceofimag-ingtheretinaeofindividualsubjectsratherthanmakinggeneralassumptionsabouttheachromatretina.Moreover,Jacobsonetal.propheticallystatedthat...identifyingandthentargetingretinallocationswithretainedphotoreceptorswillbeaprerequi-siteforsuccessfulgenetherapyinhumansŽ.Thisisworthreiter-ating,withthecautionarynotethatthestoryofconesurvivalinthemouseanddogmodelsofachromatopsiamaynotholdforallhumanachromats.Fortunately,boththetherapeut
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