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andcentrifugingless thanat !00010% each.exposedgroupwere inance.RetinalPhototoxicitygroupsall data fromcontrolof variancedifferencesfor multiplecomparisonsdifferenceswerewerean LED-cohort.LED-exposed(Kruskal-WaUiswhereof variationgroup,the CWF.exposed,SignificantperformedInstitutionalin this study wereStatet3minimized14phototoxicityt5Sixteen16this experimentand were housedt7(CWF-exposedrats) and two experimental18housed19Cleveland,20arrays.2tin a cylindrical22uniformany ups werefurther analyzedusing thewith an experiment-wisein a chamberDawleyLED-exposedof experimentalwere housed underrats (300-350g,and testedfitted with two14-watttogetherlight exposureto the animaland approvedto assurealpha of 0.05in groupsCWFbulbs (catalogin a separateand 8 inchesor CWF lighting.Gilroy,rats).# GE FI4TI2,chamberin height).animalsCWF-exposedrats wereGE Lighting,fitted with two LEDfitted with aluminumas well as an unobstructedCA) were used inof four: two control(LED-exposedcagesIn the retinalLED lightingLaboratories.by the San Josethat experimentsanimals.animalsrats were housedin diametereitherSimonsenrats were kept in shoebox-typering (8 inchesreviewedCare and Use Committeepain and discomfortthe animalsmale SpragueIndividualan unexposedfrom one light illuminancewere unequal),12OH).coefficientsStudyAll experimentsUniversityincludedby analysisMethodfor eachIIassaygroupifp 0.05.Student-Newrnan-Keulsi0The intra- and interassaystudy (each illuminance),animalsbecauseEach melatoninand a CWF-exposedFor eachcontrolx g for 30 min.sheet metal shapedThis design helpedlight path.provideThe light sources7

Iwere2exposed3was produced4between5adjusting6study was 100 lux (corresponding7tW/cm:8fluorescentlight and the other exposing9maintainedso as to keep externalmountedfrom the ceilingsdirectlyfrom overheadby maskingwith one CWFthe CWFthe distancebetweenwith lights12important13changes14measured15and maintenance16Rochester,17Electroretinographyon at 0700factorsin 1photoreceptorchambers,light sourcethe extentand morphologywas done underirradiancewas establishedbyused in thisfrom the LED panelone exposingbethe distanceThe illuminanceand 30.1two rats to CWFfollowingtoxic12:12 LD cyclesand the cyclicof light-induceddim red lightingretinaland(KodakdamageSafelight(14).for 14 days,lightinglight exposurelight exposureschedule(13).Quantitativeare mostreliablyAll animal#2 - EastmanarehandlingKodak,of 0.4 - 1.5 lux.day of the retinal phototoxicityovernightusingof exposurethe start of toxichad an illuminancedark adaptationCWFtwo rats to LED light, were both constructedthe durationon the 14 m day or beyond19LED irradiancewouldlight from entering.when determiningOn the fifteenth18The two exposureThe desiredfoil as well as adjustingwere 28.5 tW/cm'to their respectivehours.NY) whichThe desiredirradiancesthat each animalbulb or LED panel.the LED panel and the rat cages.from the CWF source).IIto ensurebulbs v,ith aluminumthe CWF bulbs and the rat cages.Rats were exposedIOof each of the chambers,study, rats were movedto a darkroom(the last dark cycle of the 14 m day) for scotopic(ERG) assessment.cells that are best studiedNinetyfive percentin dark adaptationof the rat retina(15).is comprisedERG was performedof rodon the rats

in the morningsheddingno earlierthan 0930hoursto avoidvariabilityassociatedwith photoreceptordisc(16).3The rats were injected4Pupils were dilatedwith5held open and the e10% phenylephrinewas protectedproducingMA)i.p. with urethanewhiteon the cornea,10cheek.The ityrepresented19maximal130 lux to 0.1 lux.2orecording21neutraland displayedCT).distanceof 9 cm individuals,a 0.5-logan ERGERG recordingERGand steppingInstruments,in the rats.Monocularwire as the recordingand l-kHzon the(highand amplitudesystem(RetinoGraphics,of light iatensitywhich adjusts the powerof the stimulusfilters in the path of the light.Inc.,was a blue LED light placed aThis methodologyconfirmeddown to the lowestThe range of illuminancewasAwaswith the Grass PS22to a series of brief flashes varying in intensityintensityThe rangeeyelidand a ground electrodethe entire retina.waveformssolution).Grass Medicalfor cursor placementused in the ERG recordingswith eliciteddrops.response(low frequency)the eye to illuminateunit.Stimulator,on the foreheadBPM-100ERG responsesat the highestthe rat'sloop of platinum-iridiumon a PC monitorThe photic stimulusPhotowith 0.l-kHzusing the computerizedAider anesthesia,to confirmelectrodewere AC-amplifiedcutoffs20% aqueousfrom drying with methylcelluloseusing a 3ram diametera referencebody weight;eye drops.light (Grass PS22was used as a stimuluswere obtained(0.15g/kgintensity.wereEach step inof the brief flashes was from awas controlledelectricallyby the BPM-100ratherERGthan by placementof

The averageintervalof eight responsesof 20 seconds.determineLater the ERGthe a- and b-wavethe stimulusintensityway-ANOVAsubjectsconfirmedwhereby directexperiment.Uponresultsenergyand rement,an interstimulusplacementtovalueswere plottedagainstStatisticalevaluationof the dataused ofactorand light sourcesignificantdata collection,in modifiedgithusing cursorTheseifp 0.05.at the beginningof ERGand placedmeasuredtimes.as a within-subjectswerecompletionat each intensity,and implicit(intensity-responseright eyes were removed10waveformsamplitudesv,ith ERG stimulusfactor,was obtainedas a between-Stimulusof the experimentintensityand at the end of theeach rat was sacrificedKarnovsky'sfixativewaswith CO,- gas.for histologicalThepreparationHistologyTo determinewhether13rats of the retinal14total retinal15LED-housed163 control17to the experimental18(250 lux, lights on at 0700)19modified2Oat least 72 hours.21on the verticallight 'sto the ERG procedure.in an animalfixativebut maintainedvivarium(1% paraformalyde,and embeddedrod outer segments,histological7 of the CWF-housedenvironments,Follov, ing fixation,meridianThe quantitativestudy,rats that were not subjectedin the retinas of the LED and CWF.exposedthe retinal epithelium,were inspected.rats from the ERGoccurredanalysiswas carried out on 7rats from the ERG study,The 3 controlEyes were fixed1.5% glutaraldehydeeach eye was postfixedof CWFlightby immersionin cacodylatein 2% osmiumas well asrats were not exposedon a 12:12 LD cyclefor 14 days.in epon-aralditerod nuclei, andtetroxide,buffer)inforbisectedplastic.10

The retinalInstruments).examinedSemi-thinon a Nikonmeasurementsoptictissue was sectionedweresectionsresearchwith a Sorvalwere stainedmicroscopeJB-4ultramicrotomewith toluidineblue (!% in 1% borax)with an ocular micrometermade under 100x oil immersion(Dupontat 270 micronsattachment.and wereRetinaland 450 micronsfrom thenerve head.The was inspectedepithelium,vacuolationand vesicles.and observedwas countedfor any signsperpendicularof damagewas measuredperpendicularI!used one-wa ANOVA,120.05.13RESULTSto the reas oflengthThe numberLastly.epithelium.betweenof missingThe rod outer segmentand disorganization.to the retinal pigmentedIOfor the presencewasof rod nucleithe thicknessof the retinaStatisticalevaluationof the datawere consideredsignificantif p 1415MelatoninSuppression2 showsStudy16Figurethe pineal17their respective18differentdays (often19groups.In both experimental2O(lO0,21control22induced23the samelight nsBecauseby weeks),animalslight conditions40, 10. l, and 0.1 lux), the rats showedvs. CWFand controlby an LED illuminanceilluminanceof CWFvs. LED;Tablesignificantlylight (p 0.05for each of the 3 groupseach light exposurewere compared(CWFand LED).significantmelatoninexperimenttook place onto their respectiveat all illuminancessuppression1). In no case was the melatonindifferentof rats atfrom the melatonincontrolexamined(p 0.05 forsuppressionsuppressionelicitedbyfor LED vs. CWF at all illuminances).II

For the 100-1ux intensity ,CWF,and LED groups 71. andshowed(mean(meanCWF, SEM),(meanand LED groups158 z 12 pg/gland(mean SEM),showedand LED groups164 12 pg/glandthe control.of 464 85.239 SEM),CW'F, and LED groupsthe control.experiment,showed SEM),showed SEM),StudyNo evidence of retinal phototoxicity13CWF,the control,of 650 124, 218 42. andFor the 0. l-lux intensitymelatoninthe control,of 353 34, 257 13. and 231 6 pg/glandFor the 1.0-1ux iment,(meanof 1569 126, 365 34. and 462 50 pg/glandFor the 10-1ux intensityrespectively.pinealof 1166 136, 393 41. and 439 25 pg/glandFor the 40-1uxmelatoninthe control. CWF. and LED groups showed pinealExamples of individualwas found in the ERG responses from rats housed14in LED or CWF light.ERG waveforrnsare shown in Figure 3. ERG15response-intensity16Figures 4 and 5 plot the a-wave implicit times and amplitudesfor the LED and CWF groups.17Figuresfor the LED and CWF18ERG a-waves were rarely observed in the lower photic stimulus19below the -1.5 log intensity in both LED and CWF animals.20amplitudes21treatment/exposure22b-wave implicit times and amplitudescurves are shown (mean 1 S.D.) for both exposure groups in Figures 4 - 7.6 and 7 plot the b-waveimplicittimesand amplitudesof the ERG a- and b-waves were not significantlygroups (Table 2: Light Source).as expectedgroups.intensities and were undetectableThe mean implicit times anddifferent for the twoERG stimulusintensity affected a-wave and(Table 2: ERG Stimulus).Lastly, there was no12

interactionbetween2: LightsourceRetinalHistology4light sourcex ERGNo evidenceand ERGofphototoxicityresultsof the quantitative6retinalpigmented7their thicknesswas normal.scontrolgroupsin the measurements9nuclei,or overallIoDISCUSSIONThis study showsequivalent13we have ationalstandards19reportedhere in terms20(laW/era:)or retinalNo significantthat a novelLED light to maintainsuch as grossfor greaterare no f photometry.utilitylighting.numberLED,of outer(Tablepinealglandat similarintensitydifferencesin retinalfunctionfor animalsIn addition,drinkingour parallelas assessedand clearlayerANOVA).light sourceWe have reportedto the researchThe retinas,betweencan suppressentrainmentin animalfoundlength,by light microscopy)to CWFfi r specifyingwere3, one-wayCWF3 showsof all the eyes were in gooddifferences(TableTableon the rat retinas.LED light source(determinedexposedmorphology.of rod outer segmentof the retinathat thereto animalsperformedto a conventionalstructurein retinaland rod outer segments,thicknessconcentrationswas foundmeasurementsepithelium12for any of the four ERG inlevels.Also,(determinedexposedto LEDsstudyas reportedthroughbehavioralTo compare(9). we haveour light intensitiesour resultsframedbyin (5),tothe studiesin radiometrictermscommunity.13

!Light suppressionof2it is a gnals8rapidsuppression9TheThe sensitiveinitiallyglandof the effectsnaturedemonstratedto light duringinducesof pinealmetabolismrhythmsin mammals,11(20).12(21) and gy.This responseknownthe rh thmic(22) of the light.LED light and CWFlight.in melatoninsuppressionto LED light at illuminancesat the atonin.significantlythat it can be equallyFurtherrangingilluminanceeffectivetests on otherThe exposurelaboratorynucleiof pinealof the hypothalamusgland whichglandpinealand secretionsynthesisfromindicaterats exposedlowerintensitiesin regulatingparameterscomparableother aspectsare requiredthat thereis noThe significantof CWFof LED light to suppressat low illuminances,comparisonto CWF light and rats100 lux to 0.1 lux.that evento suppressa meaningfulfrom these experimentsbetweenof melatoninon the intensityof each light sourceused provided(19).for circadianaxis to light is dependentthe abilityleads tomelatoninpacemakersglandratpathway,to be the endogenousThe abilitycircadianand otherof the mammalianto the pinealloweringintensitiesThe dataof rodentsaxis in the whiteare transmittedTherefore,at each of the lightin this study becausein the retinohypothalamicof the gnalsand subsequenthave beenincludingmelatoninas a bioassayin the suprachiasmaticnucleienzymesnucleiand colleaguesneuronalfrom the suprachiasmatic10was usedof light on the circadianby Mirmemanglucosesuprachiasmaticmelatoninof the retinal-h,,9othalamic-pinealthe nightin turn elevatesglandpinealand LED lightpinealglandto that of the CWF light,of circadianto confirmsystemthis.14

ILight exposurepartialcan causeor total blindnessretinal2causing3the LED arrays causedphototoxicity4LEDs,is produced5determine6on the retina,7lighting8it would9oftheas light damagewhether(2,3,4,23,24).morewe comparedfunctionbe observedeffectsliretinal12by the photoreceptor13of the intact retina, is a result of c-pinealigof physiologicaland behavioral19does not reducephotoreceptorepithelium(25).can be relatedThe a-wavecells (25).as a result of on-bipolarin amplitudeloss.order22shownin animalexcitationwhichto loss of e presencedegenerationand CWF animalfunctionalfunction,retinalof signalsThe resultsmass responsecomponentsfunctionof thein albinoproducedof the ERGin Mullerwould be indicateda reducedsensitivityphototoxicityassessmentby theto light ormay also affect thefrom the eye to the centralof the ERGfollowingof any photoreceptorto occurto the retina,ion concentrationsor disrupt retinal functionwas performeddamageand is the componentpotassiumshowingeffectlayers of the neural retina and theERGLoss of retinalTohabitatis the most salient fast potentialin extracellular(25).light (2).have any deleteriousof the individualcellularwhetherband in componentThe ERG is the evokedis the first part oftheaxis due to the transmissionto determinelightingcells,to determineof visiblewouldIf there ,,'asto specificThe b-wave,importantwavelengthshabitatshabitatof the ERG a- and b-waves,In additionMorphological21by someThe amplitudescompositeloss of photoreceptorwavelengthin the electroretinogram.I02oreadilyusing ERG assessment.by changeswaveformof the narrowof LED animalto a flash of light (25).ERGand irreversibleIt was especiallybecausethe use of LED lightingon retinalretinadamageshowpacemakerthat LED(Figures 4 - 7).the ERG functionalassessmentcell damageor death.Past findingsrats exposedto light intensitiesinhavegreaterthan15

60 lux for 13 weeksdegenerationdata obtaineddays using5a 12:12in rats exposedhistological3usinga nderstoodin our studydoesthe effectsthe interactionis the studyrelativein termsiIof generalcommunication,12categoriesof light meter17whileIsduringthe daytime.19which"shape"2ohumanvisual21photometryis a special22serviceablenomenclatureon the selectivemeasuresa photometerthe radiantmeasurespowerA photometerthe detectorresponsesensitivityof the "standardbranchsystem,as a descriptorLight,herehowever,in photobiology.of the humanimportanta radiometerof the(29). Radiometrysystemin photobiologicalover a definedthat stimulatesthat has filtersas determinedby the CIE (30).the photometricfor describing(29,30).Athe humaneyeadded to the detectorobserver"techniquewhereasrange of wavelengths,(brightness)and measurementisresearch.the luminanceAlthoughitfor purposesand wavelength,visualon a surfacediscussThere are two broadto resembleof radiometry.is oftenof light is serviceableand photometricofSpecifically,most individualssuch as its energyflux fallingthe disciplineportions(27,28).This descriptionof light,for 14(,Table 3).and ultravioletradiometricTheLED lightingvisibleof a light sourceis simply100-1uxorganisms.is particularlythe luminousthatand livingresponsivenessof stimuli14:10 LD cycle (4).radiationvisualpropertiesno signs of retinalinvolvesto the humantechniques:showedphysiologyprocessesbut is less usefulquantificationdamagebiologicalcolor and brightness.on the physicalis basedthe conclusionretinalopticalstudyusingof light on mammalianbetweenl0exclusivelynot causeinfluenceand describedof its apparentsupportof how the infrared,spectrumAnotherto 194 lux for 4 - 6 monthsLD cycleInvestigatingLD cycle (26).responseof theThus,system providesthe light stimuliaused in this16

Imanuscript,it does not imply that the investigators2relevant3physiolog2,.4is very unlikely5the input6terms7animal8regulation9theseto

3 fluorescent or incandescent lamps. We evaluated a novel light-emitting diode (LED) light source 4 for use in animal habitat lighting by comparing its effectiveness to cool white fluorescent light 5 (CWF) in suppressing pineal gland melatonin and maintaini