The intrinsically photosensitive retinal ganglion cell (ipRGC) mediated pupil response in young adult humans with refractive errors

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This is the published version of a paper published in Journal of Optometry.

Citation for the original published paper (version of record):

Chakraborty, R., Collins, M J., Kricancic, H., Moderiano, D., Davis, B. et al. (2021) The intrinsically photosensitive retinal ganglion cell (ipRGC) mediated pupil response in young adult humans with refractive errors

Journal of Optometry

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www.journalofoptometry.org

ORIGINAL ARTICLE

The intrinsically photosensitive retinal ganglion cell (ipRGC) mediated pupil response in young adult humans with refractive errors

Ranjay Chakraborty

^a,b,^∗

, Michael J. Collins

^c

, Henry Kricancic

^c

, Daniel Moderiano

^a

, Brett Davis

^c

, David Alonso-Caneiro

^c

, Fan Yi

^c

, Karthikeyan Baskaran

^d

aCollegeofNursingandHealthSciences,OptometryandVisionScience,SturtNorth,FlindersUniversity,SturtRd,BedfordPark, SA5042,Australia

bCaringFuturesInstitute,FlindersUniversity,SturtRd,BedfordPark,SA5042,Australia

cContactLensandVisualOpticsLaboratory,SchoolofOptometryandVisionScience,QueenslandUniversityofTechnology, VictoriaParkRoad,KelvinGrove4059,Brisbane,QLD,Australia

dDepartmentofMedicineandOptometry,LinnaeusUniversity,Kalmar,Sweden

Received15September2020;accepted1December2020

KEYWORDS Intrinsically

photosensitiveretinal ganglioncells;

Pupil;

Melanopsin;

Refractiveerror;

Myopia

Abstract

Purpose:Theintrinsicallyphotosensitiveretinalganglioncells(ipRGCs)signalenvironmental light,withaxonsprojectedtothemidbrainthatcontrolpupilsizeandcircadianrhythms.Post- illuminationpupilresponse(PIPR),asustainedpupilconstrictionaftershort-wavelengthlight stimulation,isanindirect measureofipRGC activity.Here,wemeasuredthePIPR inyoung adultswithvariousrefractiveerrorsusingacustom-madeopticalsystem.

Methods:PIPRwasmeasuredonmyopic(−3.50±1.82D,n=20)andnon-myopic(+0.28±0.23 D,n=19)participants(meanage,23.36±3.06years).Therighteyewasdilatedandpresented withlong-wavelength(red,625nm,3.68×10¹⁴photons/cm²/s)andshort-wavelength(blue, 470nm,3.24×10¹⁴ photons/cm²/s)1sand5spulsesoflight,andtheconsensualresponse wasmeasuredinthelefteyefor60sfollowinglightoffset.The6sand30sPIPRandearlyand lateareaunderthecurve(AUC)for1and5sstimuliwerecalculated.

Results:Formostsubjects, the6sand 30s PIPRwere signiﬁcantlylower (p<0.001),and theearlyandlateAUCweresigniﬁcantlylargerfor1sbluelightcomparedtoredlight(p<

0.001),suggestingastrongipRGCresponse.The5sbluestimulationinducedaslightlystronger melanopsinresponse,comparedto1sstimulationwiththesamewavelength.However,noneof thePIPRmetricsweredifferentbetweenmyopesandnon-myopesforeitherstimulusduration (p>0.05).

∗Correspondingauthorat:CollegeofNursingandHealthSciences,OptometryandVisionScience,SturtNorth,FlindersUniversity,Sturt Rd,BedfordPark,SA5042,Australia.

E-mailaddress:ranjay.chakraborty@ﬂinders.edu.au(R.Chakraborty).

https://doi.org/10.1016/j.optom.2020.12.001

Pleasecitethisarticleinpressas:R.Chakraborty,M.J.Collins,H.Kricancicetal.,Theintrinsicallyphotosensitiveretinal ganglion cell (ipRGC) mediated pupil responsein young adult humans withrefractiveerrors, Journal of Optometry, https://doi.org/10.1016/j.optom.2020.12.001

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Conclusions: Weconﬁrmpreviousresearchthatthereisnoeffectofrefractiveerroronthe PIPR.

Introduction

The ipRGCsarea distinctsubtype of ganglioncells in the mammalianretina thatcontain ablue light-sensitivepho- topigment, melanopsin, with a peak sensitivity at ∼482 nm.^1---3 These cells constitute only about 1---2% of the entire ganglioncellpopulation, withwidespreaddendritic coverage across the entire retina, except the fovea.^4,5 The ipRGCs are considered to be environmental irradi- ancedetectorsthatcandirectlydetectlightlevelsthrough melanopsin, without any input from the rod and cone pathway.^2,6 The ipRGCaxonsprojecttoseveralbraincen- ters and primarily regulate non-image forming functions, such as photoentrainment of circadian rhythms and con- trollingthepupillarylightreﬂex(PLR).^5---8However,thereis evidencethatipRGCsarealsoinvolvedinimageformation, contributingtocontrastand colourdetectionand pattern vision.^9---11

The stimulation of ipRGCs signiﬁcantly inﬂuences retinal networks that are integral to the physiology of the retinaanddifferentvisualfunctions.¹²Inadditiontointrin- sic activation, the ipRGCs also receive extrinsic synaptic inputfromrodandconephotoreceptorsviabipolarcells.^5,6 Studieshaveshownsynapticconnectionsbetweendopamin- ergic amacrine cells and ipRGCs in the inner plexiform layerof theretina^5,13 andevidencethatthesemelanopsin cells may affect retinal dopamine release.^14,15 Increased releaseofretinaldopamine throughON-bipolarcellactiv- ity inhibits experimental myopia in chicks^16,17 and mice¹⁸ reared underhigh-intensity illumination.Furthermore, the protectiveeffectsofbrightlightingonexperimentalmyopia in primates^19,20and guinea pigs²¹are believed to be medi- atedbylight-inducedincreasesinretinaldopaminerelease.

Clinically,somecharacteristicsoflightinghavebeenhypoth- esisedtoinfluencehumanrefractivedevelopment.Several cross-sectional and longitudinal studies have shown that children whospendmore timeoutdoorshave significantly reduced odds of myopia, which is believed to mediated by increased dopamine release in the retina.^22---25 It is therefore possible that the ipRGCs and melanopsin may influenceeyegrowthandmyopiathroughchangesinretinal dopamine.

Previouslypublishedresearchhasfoundthatrods,cones and ipRGCs control different phases of the PLR.^1,26,27 With lightstimulation, initial pupil constriction is primarily regulated by rod and cone photoreceptors,²⁸ while the post-illuminationpupil response(PIPR) following light offset is attributed to the ipRGCs.^1,29 PIPR is charac- terised by a sustained constriction of the pupil following short-wavelengthstimulation.^1,29---31Thissustainedmiosisis attributed to prolonged ﬁring of melanopsin cells follow-

inglightoffset.¹ThePIPRhasbeen reliablyestablishedas arobust biomarker for estimating the intrinsicactivity of melanopsin cells.^29,30,32 Impaired ipRGC function and PIPR has been recognised in several ocular diseases, including glaucoma,³³ age-related macular degeneration,³⁴ retinitis pigmentosa³⁵ and diabetic retinopathy.³⁶ Despite circum- stantialevidencepointingtowardsapossiblelinkbetween melanopsinfunctionandrefractiveerrordevelopment,previous studies have found noeffect of refractiveerror on theipRGC-driven PIPRinyoung adultsand children.^30,37,38 Thegoalofthisstudywastoexaminetheseprevious ﬁnd- ings usinga custom-built opticalsystem and measurethe ipRGC-drivenPIPR in a cohort of young adult myopicand non-myopicparticipants.

Material and methods

Participants

Thirty-ninemyopic(n=20,male=8;female=12),hyperopic (n = 5, male = 3; female = 2) and emmetropic (n = 14, male= 5; female = 9) participants between the ages of 20and 35 years(mean age ± SD, 23.36 ± 3.06 years) were recruited to examine the association between the ipRGC-driven pupilresponse and refractiveerror. Prior to participation,allsubjectsunderwentacomprehensiveeye examination to assess their refractive status and ocular health.Themeansphericalequivalentrefraction(SER)was

−3.50±1.82,+0.58±0.23and−0.02±0.12DSforpar- ticipantsinthemyopic,hyperopicandemmetropicgroups, respectively.Noneofthepupilmetricsdataweresigniﬁcan- tly differentbetween the low hyperopic and emmetropic participants (p > 0.05, data not shown), and hence they were grouped together as one ‘‘non-myopic’’ group (n = 19; mean SER, +0.28 ± 0.23 DS). All subjects had nor- malvisualacuityof 0.00logMARor better,andastigmatic refractive error of ≤1.00 DC. No participants had ocular pathology or a history of any major eye or refractive surgery.

Noparticipantsweretakinganyprescriptionmedication knownto affect the pupil size or sleep patterns(such as melatonin).Inaddition,participantswereaskedtorefrain fromalcohol, caffeine, andnicotine for 12 h priortothe pupilmeasurements.Allparticipantsweretestedbetween 9:00amand12:00pmtominimizetheeffectsofcircadian variationonipRGCfunctionandthePIPR.³⁹ The studywas approvedby the SouthernAdelaide LocalHealth Network (SALHN,ID:156.17)ethicscommittee,andallparticipants provided written informed consent prior to their participation. All subjects were treated in accordance withthe DeclarationofHelsinki.

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Figure1 Overviewoftheopticalsystemusedinthestudy;sketch(A)andrealimage(B).TwoFresnellenses,F1andF2(both 10.16cmfocallengthanddiameter),wereplacedattwicetheirfocallengthapart.RedandblueLEDswereplacedatoneendof theopticalsystem.Thesubject’sdilatedrighteyewasalignedattheotherend,whiletheundilatedlefteyewasrecordedbythe infraredcamera(IRcamera)attachedtothecomputer.Thediffuserhada5degdiffusingangle.

Opticalsystemforpupilmeasurements

ThePIPRwasmeasuredusingacustom-builtopticalsystem, similartotheonedescribedbyKankipatietal.,³¹asshown inFig.1AandB.Theilluminationsystemconsistedofaset ofredandbluelight-emittingdiodes(LEDs).Thelightfrom theredandblueLEDswastransmittedtotherighteyevia twoFresnellenses;F1andF2,each 10.16cmindiameter andwitha10.16cmfocallength(EdmundOptics,Barring- ton,NJ).The blue(470nm, 3mm diameter,fullwidthat half maximum [FWHM] 22 nm) and red LEDs (625 nm, 3 mmdiameter,FWHM20nm)(JaycarElectronics,Rydalmere, Australia) were positionedat the focallength of the ﬁrst Fresnellens,F1.ThetwoFresnellenseswerekept20.32cm apart(i.e.separatedbytwice theirfocallength).A20.32

×20.32cmholographicdiffuserof5-degreediffusingangle (EdmundOptics,Barrington,NJ)wasplacedinfrontofthe secondFresnellens(F2),andtheparticipant’srighteyewas positionedatthefocalpointofF2.DuringthePIPRmeasure- ment,theright eyewaspresentedwiththelightstimulus, andtheeffectoflightstimulationwasmeasuredinthecon- tralaterallefteye.AmodiﬁedLogitechC920HDProwebcam (Logitech,Newark,CA)withilluminatinginfraredLEDs(940 nm,5mmdiameter,CoreElectronics,NSW,Australia)was usedtorecordthepupilresponsesfromthelefteyeatarate of15frames/s.Thepresentationoflightstimulusandthe durationofPIPRrecordingwerecontrolledviaasmallsingle- boardcomputer,RaspberryPi3ModelB(CoreElectronics, NSW, Australia). During the experiment, the participants werepositionedinachinrestandinstructedtolookstraight aheadatasmallredlaserspotonthewallatadistanceof 4m.

Pupillometry

TheredandblueLEDsilluminatingtheeyeﬂickeredat 10 Hzwithadutycycleof80%.Thetwostimulusdurationsof 1sand5susedinthisstudywerewithintherangeofprevi- ouslypublishedstudies.^29,37 The cornealirradiancelevels, measured using an optical power meter (Newport Corpo- ration, Irvine, CA), were 3.24 × 10¹⁴ photons/cm²/s for thebluestimulus(470nm)and3.68×10¹⁴ photons/cm²/s for the red stimulus (625 nm). These corneal irradiances were close to previously used irradiance levels in young, healthyparticipants.^37,38,40Table1showsexcitationforeach

Table1 Individualphotoreceptorexcitation(␣-opticlux) with470nm3.24×10¹⁴photons/cm²/sand625nm3.68× 10¹⁴photons/cm²/slightstimuli(basedonLucasetal.⁴¹).

Photoreceptor

class Preﬁx ␣-opticlux

470nm 625nm

Scone Cyanopic 939.26 0.00

Melanopsin Melanopic 981.08 0.61

Rod Rhodopic 674.45 4.42

Mcone Chloropic 324.37 87.44

Lcone Erythropic 160.49 328.50

photoreceptor classestimated using the toolbox provided by Lucas et al.⁴¹ The L cones have higher sensitivity to the625-nm light, whereasmelanopsin, rods, and S cones havehigher excitationtothe470-nmlight comparedtoL cones.

On the day of experiment, the participant’s right eye was dilated with 1% Mydriacyl (tropicamide, Alcon, Fort Worth,TX)toensureconsistentretinalilluminationwithin andbetweensubjectsduringthePIPRmeasurement.After 20-minutes,allsubjectsweredarkadaptedfor5min(∼2---5 lux)beforecommencingthepupilmeasurements.Afterdark adaptation, the right eye was presented with 1 and 5 s long-wavelength(red)andshort-wavelength(blue)narrow- bandlightstimuliwhile theconsensualpupilresponsewas measured in the undilated left eye as a measure of the ipRGC-inducedPIPR,asshowninFig.2.Theorderofstim- ulus presentation was: 1 s red, 1 s blue, 5 s red, and 5 s blue. Testing with 1 s light pulses always preceded the 5 s pulses. Red and blue stimuli were alternated in all sessions,similartopreviously publishedexperiments,³⁰ to controlfor theeffectofmelanopsin bistability.⁴² Aslight- inducedmelanopsinresponsecouldpersistforupto3---5min afterlightoffset,^29,43the5-minutedarkadaptationperiod between 1s and5 s trials wasnecessary to avoid poten- tiation of the response from previous light stimulation.⁴⁴ Tworepeats for eachstimulus (470nmand625 nm)were recordedforeachstimulusdurationandwereaveragedfor furtheranalysis.

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Figure2 Pupilstimulationprotocolsfortheexperiment.Darkadaptation(5min)wasfollowedbya10sbaselineand1sred stimuluswith60spupilmeasurementsafterstimulusoffset.Afteranother10sbaseline,thesameprotocolwasrepeatedfor1s bluestimulus.Following1smeasurements,adarkadaptationperiodof5minwasobservedbeforerepeatingthesameprotocolfor 5sredandbluestimuli.Tworepeatsforeachstimulus(470nmand625nm)wererecordedforeachstimulusdurationandwere averaged.

Table2 Pupilmetrics usedtoquantifyphotoreceptorcontributionstothepost-illuminationpupilresponse.Metricsinclude baselinepupildiameter(%),peakconstriction(%ofbaseline),6sand30spost-illuminationpupilresponse(PIPR,%ofbaseline) andearlyandlateareaunderthecurve(AUC,unitless).

Metric Deﬁnition Unit Expectedchange Photoreceptor

contribution Baselinepupildiameter 10spre-stimulusperiod

before

long-and-short-wavelength stimulation

Percent(%)

Peakconstriction Maximumpupilconstriction %oftheaverage baselinepupil diameter

Smallervalue indicatesgreater constriction

Combinationof rod/coneandinner retinalactivity 6sPIPR Meanpupildiameter6---7s

afterstimulusoffset

%oftheaverage baselinepupil diameter

Smallervalue indicatesgreater ipRGCactivity

ipRGCactivity

30sPIPR Meanpupildiameter30---31 safterstimulusoffset

%oftheaverage baselinepupil diameter

Smallervalue indicatesgreater ipRGCactivity

ipRGCactivity

EarlyAUC Logoftrapezoidal approximationofthe integralof100%baseline minustheinterpolated% pupildiameter,0---10safter stimulusoffset

Unitless Largervalueindicates greateripRGCactivity

ipRGCactivity

LateAUC Logoftrapezoidal approximationofthe integralof100%baseline minustheinterpolated% pupildiameter,10---30s afterstimulusoffset

Unitless Largervalueindicates greateripRGCactivity

ipRGCactivity

Dataanalysis

The change inpupil diameterinresponsetoredand blue stimuli was measured from the pupil camera recordings usingacustomMatlabprogram(Matlab2017b,version9.3, MathWorks,Natick,MA).Forboth1and5strials,theMat- labprogramanalysed thechange in pupilarearelativeto the averagebaseline pupilareafor each wavelength (i.e.

theaverageof10spre-stimulusperiodbeforeredandblue stimulation).Tocalculate thepupilarea,theMatlab algo- rithmcycledthrougheachframeoftherecording,andthe

‘starburst’algorithmwasusedtodetect thepupiloutline andﬁtanellipticalshapetotheboundary.Foreachframe, theareaoftheellipse wasdeterminedusingtheformula, A = a*b*pi (where a and b arethe two semi-axes of the ellipse).Frameswhereanellipsecouldnotbedetecteddue toblinksorpoorﬁxationwereautomaticallyremovedfrom

theanalysis.Datawassmoothedusingthemovingaverage ﬁlterwithawindowoflength1s. Finally,atime-stamped seriesofrelativepupilresponseswasgeneratedforfurther analysis.AsoutlinedinTable2,thePIPRwasdescribedby 5metrics,thepeakconstriction, the6and30s PIPR,and theearly andlateareaunderthe curve(AUC).These are welldeﬁned,robustandreliablemetricsthathavebeenfre- quentlyusedbypreviousstudies.29,37,38,45---47Allpupilmetrics areshown as‘‘normalized change’’ to the average baselinepupildiameter(expressedasapercentage).Whilstpeak pupilconstrictionrepresentsbothrod/coneandinnerreti- nalactivity,the other fourmetricsarecommonly usedto describetheipRGCactivity.^29,30

Statistical analyses were performed using commercial software(SigmaStat3.5,AspireSoftwareInternational,Ash- burn, VA). For both 1 and 5 s stimulus durations, the differenceinpupilmetricsbetweenmyopesandnon-myopes 4

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withredandbluestimuliwereanalysedwithtwo-wayanal- ysis of variance (ANOVA) and Holm-Sidak post-hoc tests forstatistical significance,using‘‘refractiveerror’’asthe between-subjectsfactorand‘‘wavelength’’asthewithin- subjectsfactor.Todeterminethewithin-subjectvariability ofthePIPRmetrics,theintrasessioncoefficientofvariation (CVorSD/mean)wascalculated.TheCVprovidesareliable measurementofvariabilitybecauseitisdimensionlessand is not affectedby the changesin measurement units.⁴⁸ A p-valueoflessthan0.05wasconsideredtobestatistically significant.Alldataareexpressedasmean±standarderror ofmean(SEM).

Results

Effectsof1and5sbluestimulationonthe ipRGC-drivenPIPR

Thechangeinpupilmetricswith1and5slong-wavelength (red) and short-wavelength(blue) stimuli for myopes and non-myopes is shown in Table 3. Following 5 min of dark adaptation, baselinepupil areaof theundilated lefteyes was not signiﬁcantly different between 1 s red (mean ± SEM for the two refractive groups, 100.96 ± 0.94%) and bluestimuli(100.85±0.53%,two-wayANOVAmaineffectof wavelengthF(1,75)=0.017,p=0.895).For36participants (92%),pupilsre-dilatedrapidlyafter lightoffsetfollowing red stimulation; whereas, with blue stimulation the rate ofre-dilationtothebaselinepupildiameterwasconsider- ablyslower(Fig.3).Exposuretothebluestimuluscauseda greaterconstrictionofthepupilthantheredstimulus(red stimulus, 36.65± 1.60%;blue stimulus, 29.42± 1.82%,two- wayANOVAmaineffectofwavelength,p<0.001,Table3).

Compared to the 1 s red stimulus, the 6 s (red stimulus, 81.02± 1.51%;bluestimulus,56.93±4.20%)and30sPIPR (redstimulus, 98.18± 0.77%;blue stimulus86.11± 2.83%) weresigniﬁcantlysmallerforthebluestimulusacrossboth refractive groups (two-way ANOVA main effect of wavelength, p < 0.001, Fig. 3A and B). In addition, the early (redstimulus,1.06±0.03,bluestimulus1.28±0.04)and lateAUC(redstimulus,0.60 ±0.07,bluestimulus1.15± 0.09)weresigniﬁcantlylongerforthebluelightthanthered light(two-wayANOVAmaineffectofwavelength,p<0.001, Fig.3CandD).TheseresultsindicateastrongipRGC-induced PIPRfollowingshort-wavelengthstimulation.

As illustrated in Fig.4, 5 s blue stimulationinduced a slightlystrongermelanopsinresponsecomparedto1sstimu- lationwiththesamewavelength.Allpupilmetrics,including thepeakconstriction,the6sand30sPIPR,andtheearly and lateAUC indicated a strong PIPR in responseto blue stimulationforbothgroups(two-wayANOVAmaineffectof wavelength,p<0.001forall,Table3).

AsshowninFigs.3and4,noneofthepupilmetricswere signiﬁcantlydifferentbetweenmyopicandnon-myopicparticipants for either 1 s stimulus or 5s stimulus (two-way ANOVAmaineffectofrefractiveerror,p>0.05forall).

Intrasessionvariability

To quantifythewithin-subjectvariabilityinthePIPRmet- rics,wecalculatedtheintrasessionCVforeachofthepupil

metricsforboth1and5sstimuli(Table4andSupplementary FigureA.1).Forboththe1and5sstimuli,theintrasession CVfor thepeakconstrictionandthe6and30sPIPRwere generallygreaterforthebluestimuluscomparedtothered stimulus,buttheywereall<20%, whichis consideredlow andacceptablefor PIPRmeasurements.²⁹ Theintrasession CVforboth 1and5sstimuliweresigniﬁcantlygreaterfor AUCparameters,particularlyforthelateAUCwithCV>20%

forbothwavelengths(SupplementaryFigureA.1).

Discussion

Thisstudyconﬁrmsprevious ﬁndingsthatstimulationwith 1sand5spulsesofshort-wavelengthbluelightgenerates astrongmelanopsin-drivenPIPRinyoung, healthyparticipants.Usingacustom-builtopticalsystem,wehavefurther validatedthatthereisnoeffectofrefractiveerroronthe PIPRinyoungadults.³⁰

In the current study, the optical system based on the design by Kankipati et al.³¹ effectively induced the melanopsin-driven PIPR in our participants. A number of previous studies using narrowband short-wavelength blue light (wavelength used across different studies, 448---470 nm) and similar irradiance levels to our study have shown a strong melanopsin response in young healthy subjects.29---31,37,38,40,45,49Inacomprehensivestudy,Adhikari et al. showed that the PIPR amplitude was largest with 1 s short-wavelength pulses (465 nm) of ≥12.8 log quanta.cm⁻².s⁻¹.²⁹ Consistent with this observation, we were able to generate a strong PIPR using 1 s short- wavelengthstimulus(470nm)of3.24×10¹⁴photons/cm²/s (or14.5logquanta.cm⁻².s⁻¹estimatedusingthetoolboxby Lucasetal).⁴¹ Forboth1sstimulusand5sstimulus,pupil re-dilationwasslower afterblue stimulationcomparedto redstimulation.This is indicated bythe smaller6 and30 sPIPRandlargerearlyandlateAUCvaluesfollowingblue lightstimulation(Figs.3and4).Forbothredandbluestim- uli,allmetricswerelargerfor5sstimulationcomparedto 1sstimulation(i.e.,lowerPIPRvaluesandhigherAUCval- uesasshowninTable3),suggestingastrongermelanopsin responsewiththe5sstimulus.³⁷ Similarly,previousstudies inhumans²⁹ andmice⁵⁰ have alsoreportedan increase in thePIPRdurationwithincreasingstimulusduration,possi- blyduetoincreasedlightadaptationofmelanopsinsignaling overtime.

Given the evidence of synaptic connections between the ipRGCsand dopaminergic amacrine cells in the inner retina,^5,13 and the fact that dopamine agonists inhibit experimentalandspontaneousmyopiainchicksandguinea pigs,^51,52ithasbeenhypothesisedthattheipRGCscanmod- ulate eye growth and myopia through changes in retinal dopaminelevels.However,inagreementwithearlierobser- vationsinchildrenandadults,^30,37,38thisstudyalsofoundno effectofrefractiveerrorontheipRGC-drivenpupilresponse inthiscohortofyoungadults.Futurestudiesshouldexamine theeffectsofotheropticalstimuli(suchasopticaldefocus andlightexposure)onthePIPRtofurtherexplorethepoten- tialassociationbetweentheipRGCpathwaysandrefractive error.

Pupilsizeisinﬂuencedbyseveralfactors,includingage, accommodation, psychological state, lighting, drugs and 5

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Figure3 Changeinpupilmetricswith1sredandbluestimulationformyopes(n=20)andnon-myopes(n=19).The6s(A)and 30s(B)post-illuminationpupilresponses(PIPR)weresigniﬁcantlylowerwiththebluelightcomparedtotheredlight(two-way ANOVAmaineffectofwavelength,p<0.001).Theearly(C)andlate(D)areaunderthecurve(AUC)weresigniﬁcantlygreater followingbluelightstimulationcomparedtoredlightstimulation(two-wayANOVAmaineffectofwavelength,p<0.001).ThePIPR valuesareshownasnormalizedchangerelativetothebaselinepupildiameter;whereastheAUCvaluesareshowninlogunits.

Noneofthepupilmetricsweresigniﬁcantlydifferentbetweenmyopicandnon-myopicparticipants(two-wayANOVAmaineffect ofrefractiveerror,p>0.05).Errorbarsrepresentstandarderrorofthemean.(E)Normalizedchangeinpupilsizefor1sredand bluepulsesacrossthetworefractivegroups.Pupilmetricsincludebaseline,peakconstriction,6sPIPR,30sPIPR,earlyAUC,late AUC.Shadedregionsrepresent95%conﬁdenceintervals.Stimulusisshowninyellow.

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Figure4 Changeinpupilmetricswith5sredandbluestimulationformyopes(n=20)andnon-myopes(n=19).The6s(A)and 30s(B)post-illuminationpupilresponses(PIPR)weresigniﬁcantlylowerwiththebluelightcomparedtotheredlight(two-way ANOVAmaineffectofwavelength,p<0.001).Theearly(C)andlate(D)areaunderthecurve(AUC)weresigniﬁcantlygreater followingbluelightstimulationcomparedtoredlightstimulation(two-wayANOVAmaineffectofwavelength,p<0.001).ThePIPR valuesareshownasnormalizedchangerelativetothebaselinepupildiameter;whereastheAUCvaluesareshowninlogunits.

Noneofthepupilmetricsweresigniﬁcantlydifferentbetweenmyopicandnon-myopicparticipants(two-wayANOVAmaineffect ofrefractiveerror,p>0.05).Errorbarsrepresentstandarderrorofthemean.(E)Normalizedchangeinpupilsizefor5sredand bluepulsesacrossthetworefractivegroups.Pupilmetricsincludebaseline,peakconstriction,6sPIPR,30sPIPR,earlyAUC,late AUC.Shadedregionsrepresent95%conﬁdenceintervals.Stimulusisshowninyellow.

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Table3 Summaryofpupilmetricsfor1sand5sredandbluestimuliformyopesandnon-myopes,alongwithp-valuesfrom thetwo-wayANOVAillustratingthemaineffectofwavelength,refractiveerrorandwavelengthbyrefractiveerrorinteraction.

Metricsincludebaselinepupildiameter(%),peakconstriction(%ofbaseline),6sand30spost-illuminationpupilresponse(PIPR,

%ofbaseline)andearlyandlateareaunderthecurve(AUC,unitless).Signiﬁcantpvalues(p<0.05)arehighlightedinbold.

Stimulusduration Pupilmetrics Wavelength Refractiveerror p-values

Myope Non-myope Wavelength Refractive Error

Wavelength

*refractive error

1s

Baseline Red 100.61±0.61% 101.31±1.26%

0.895 0.855 0.491

Blue 101.05±0.58% 100.65±0.49%

Peakconstriction Red 38.10±1.35% 35.20±1.85%

<0.001 0.172 0.767

Blue 30.35±1.62% 28.48±2.02%

6s PIPR

Red 81.02±1.43% 81.01±1.58%

<0.001 0.521 0.522

Blue 58.97±3.87% 54.89±4.54%

30s PIPR

Red 97.37±0.82% 98.98±0.72%

<0.001 0.844 0.346

Blue 87.34±2.06% 84.88±3.60%

Early AUC

Red 1.07±0.02 1.05±0.04

<0.001 0.615 0.977

Blue 1.29±0.03 1.27±0.06 Late

AUC

Red 0.61±0.08 0.59±0.06

<0.001 0.835 0.627

Blue 1.12±0.08 1.18±0.11

5s

Baseline Red 101.10±0.26% 100.85±0.24%

0.905 0.548 0.928

Blue 101.11±0.43% 100.92±0.44%

Peakconstriction Red 19.08±0.95% 20.51±1.71%

<0.001 0.188 0.844

Blue 13.32±0.67% 15.24±1.52%

6s PIPR

Red 74.03±1.40% 75.09±2.22%

<0.001 0.965 0.744

Blue 52.17±3.20% 51.37±3.95%

30s PIPR

Red 96.36±0.70% 97.47±0.78%

<0.001 0.401 0.226

Blue 79.30±3.58% 73.21±4.60%

Early AUC

Red 1.18±0.02 1.14±0.03

<0.001 0.286 0.809

Blue 1.35±0.02 1.32±0.04 Late

AUC

Red 0.72±0.05 0.72±0.07

<0.001 0.665 0.686

Blue 1.28±0.08 1.33±0.08

Table4 Summaryoftheintrasessioncoefﬁcientofvariation(CV) foreachofthePIPRmetrics forboth1and5sstimuli.

IntrasessionCV(expressedin%)wascalculatedasstandarddeviation/meanofthetwolong-wavelength(red)andtwoshort- wavelength(blue) trialsforeachstimulus duration.Metricsincludebaselinepupil diameter,peak constriction,6sand30s post-illuminationpupilresponse(PIPR),andearlyandlateareaunderthecurve(AUC).

Pupilmetrics IntrasessionCVfor1secstimulus(%) IntrasessionCVfor5secstimulus(%)

Red(625nm) Blue(470nm) Red(625nm) Blue(470nm)

Baseline 0.57 2.13 0.75 1.76

Peakconstriction 10.14 12.67 11.65 13.27

6sPIPR 3.85 8.96 4.33 9.92

30sPIPR 2.46 8.01 2.98 8.19

EarlyAUC 13.75 15.90 9.16 11.70

LateAUC 43.84 27.93 44.90 22.97

autonomic input.⁵³ Several measures were taken in our experimentalprotocoltoavoidanyundueinﬂuenceofthese externalfactorsonpupilmeasurements.Thisincludedusing aquiet,darkroomformeasurements,presentingadistant ﬁxation targettoinduceminimal accommodation,exclud- ingsubjectsonprescriptionmedicationthatmayaffectthe pupilsize,performingmeasurementsatthesametimeofthe day,andrecruitingyoungparticipants(<40years)toavoid

theinﬂuenceofage-relatedlenticularlight scatteronthe PIPR.⁴⁵

We foundthat intrasessionCVfor both 1and5 s stimuli weregenerally lower for the 6 and30 s PIPR(≤10%), and higher for the AUC parameters,particularly the late AUC(SupplementaryFigureA.1).Previousstudieshavealso reporteddifferences in intrasessionCV for different PIPR metrics.^29,30,54 Importantly, other studies have also shown

8

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thattheintrasessionCVwasfoundtobelowerforthe6s PIPR(≤20%),andhigherfortheearlyandlateAUC(≥20%).³⁰ Althoughtheintrasessionvariabilityforthepeakconstric- tion,andthe6and30sPIPRwereslightlyhigherfortheblue stimulus comparedthe red stimulus, theywere all below 20%,whichisconsideredacceptableforthePIPRmetrics.²⁹ ThevariabilityinthePIPRresponsecanvarydependingon thestimulusirradianceandsize²⁹;therefore,futureexper- iments should consider these factors to control for the intrasessionvariability.

Similar to previous reports,^29,37 we found that the L cones hadhigher sensitivitytothe 625-nmlight, whereas melanopsin,rods,andSconeshadhigherexcitationtothe 470-nmlight (Table1).This happensbecauseall photore- ceptorshave distinctbutoverlapping spectraltuning,and evenamonochromaticlight matchedtothepeakspectral sensitivityofagivenphotoreceptorwillstimulateotherpho- toreceptorswithsimilarspectraltuning.⁵⁵ However,based ontherelativedifferencesintheindividualphotoreceptor excitationstoredandbluestimuli,wecandeduceasignif- icantcontributionofmelanopsincellstothePIPRfollowing short-wavelengthstimulation.Somestudies haveusedthe methodofsilentsubstitutionthatstimulateaspecificpho- toreceptor class in the living human retina while leaving otherclassesunstimulatedtoexaminethespecificcontribu- tionofthemelanopsincellsinthepupillarylightresponse.⁵⁵ In conclusion, the results of this study confirmed previous findings that stimulation with1 s and 5 s pulses of short-wavelengthbluelightgeneratesastrongPIPRinyoung adult participants.Similartoprevious research,wefound noeffectofrefractiveerror onany ofthe measuredPIPR metrics.

Funding

This work was supported by the Flinders University Col- lege of Nursing and Health Sciences Establishment Grant [01.529.41820]; and the Contact Lens and Visual Optics Laboratory,QueenslandUniversityofTechnology,Brisbane, Australia.

Declarations of interest

None.

Note

Aspectsofthearticlehavebeen presentedattheInterna- tionalMyopiaConference(IMC),September2019inTokyo, Japan.

Acknowledgements

WewouldliketoacknowledgeProf.NicolaAnstice,Flinders University,forcarefuleditingofthemanuscript.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.

1016/j.optom.2020.12.001.

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