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l a b o r a t o r y s c i e n c e
Blue light-absorbing intraocular lens and retinalpigment epithelium protection in vitro
 Janet R. Sparrow, PhD, Ashley S. Miller, Jilin Zhou, MD
 Purpose:
To compare the Alcon AcrySof
Natural (SN60AT) and AcrySof(SA60AT), the AMO Sensar
(AR40e) and ClariFlex
, and the Pfizer CeeOn
Edge911A intraocular lenses (IOLs) as to their ability to protect retinal pigment epithe-lial (RPE) cells from light damage mediated by the lipofuscin fluorophore A2E.
Setting:
Department of Ophthalmology, Columbia University, New York, NewYork, USA.
 Methods:
Cultured human RPE cells (ARPE-19 cell line) that had accumulated A2E were exposed to blue (430 nm
30), green (550
10 nm), or white (390 to750 nm) light with and without an IOL in the light path.
 Results:
The blue light-absorbing AcrySof Natural IOL was associated with sig-nificant reduction (78% to 82%;
P
.01) in the death of A2E-laden RPE that wereexposed to blue, white, and green light. The decrease in the incidence of celldeath was greater in magnitude than would be expected from the amount of lightthat was absorbed by the IOL. The considerably smaller declines in cell death ob-served with the AcrySof, Sensar, ClariFlex, and CeeOn Edge IOLs were likely dueto nonspecific reductions in light transmittance.
Conclusions:
By absorbing blue light, the AcrySof Natural IOL shields RPE cellsthat have accumulated the aging lipofuscin fluorophore A2E from the damaging ef-fects of light. A long-term population-based clinical trial would determine whethera blue light-absorbing IOL can reduce the risk for or progression of age-relatedmacular degeneration.
 J Cataract Refract Surg 2004; 30:873–878
2004 ASCRS and ESCRS
T
he design of intraocular lenses (IOLs) should be humanlens, sincethelatter yellowswith agewhileIOLsbased on the properties of the human ocular lens,in current use are colorless.
2,9–13
The color change in theespecially in the transmission properties of the IOL.naturallensislikelyattributabletooxidationproductsof Notably, the human crystalline lens absorbs most ultra-tryptophan (n-formyl-kynurenine) and to glycosylationviolet light between 300 nm and 400 nm.
1
Thus, toof lens proteins.
14
It results in a progressive increaseprovide the same protection to the retina afforded by in absorbance within the blue range of the visiblethe natural lens, the use of ultraviolet light-absorbingspectrum.
2,9
IOLs became the standard of practice once these lensesFilteringthe shorterwavelengthsofthe visiblespec- weredeveloped.
2–8
However,thetransmissionpropertiestrum is particularly significant because it is also thisof most IOLs are not comparable to those of the naturalportion of the spectrum that produces photochemicaldamage to the retinal pigment epithelium (RPE).
15–17
Itisnowgenerallyacceptedthatatleast1oftheintracel-
 Accepted for publication January 27, 2004.
lular chromophores responsible for the blue light sensi-
ReprintrequeststoJanetR.Sparrow,PhD,DepartmentofOphthalmol-
tivity of RPE cells
18,19
is the lipofuscin constituent
ogy, Columbia University, 630 West 128th Street, New York, New York 10032, USA. email: jrs88@columbia.edu.
A2E.
20–22
This fluorophore is unique to RPE cells and
2004 ASCRS and ESCRS 0886-3350/04/$–see front matterPublished by Elsevier Inc. doi:10.1016/j.jcrs.2004.01.031
 
LABORATORY SCIENCE: BLUE LIGHT-ABSORBING IOL
CeeOn
Edge (911A) (silicone, 20.0 D, 6.0 mm optic
has been shown to accumulate in human RPE cells
diameter).
throughout the lifetime of an individual, with levelsbeing highest in the aged eye.
21
Thus, replacement of 
 A2E Accumulation in Culture 
a senile cataractous crystalline lens with a colorless IOL
Human RPE cells (ARPE-19, American Type Culture
mayleavetheRPE vulnerableatanagewhenitscontent
Collection), which are devoid of endogenous A2E,
35
 were
of blue light sensitive A2E is already high.
grown in 8-well, plastic chamber slides (Laboratory-Tek,
The RPE fluorophore A2E is maximally excited
Nunc), as described.
35
Once confluent, the cells were allowed
by light in the blue region of the spectrum.
18
 When
toaccumulatesynthesizedA2E
21
froma20
Mconcentration
irradiated,A2Egeneratessingletoxygenthatproceedsto
added to the medium. With this protocol, A2E accumulates
add to carbon–carbon double bonds of A2E to generate
in the lysosomal compartment of the cells to levels that are
highly reactive epoxides (A2E-epoxides) along the side-
comparable to amounts present in vivo.
18,35
armsof themolecule.
23,24
The cellularinjuryinduced by the illumination of A2E-laden RPE includes oxidative
Illumination and Placement of the IOL
DNA base changes,
25,26
and it is likely that as electro-
Immediately before illumination, the culture medium
philes that can readily react with many cellular mole-
was replaced with phosphate-buffered saline containing cal-cium, magnesium, and glucose. For quantitative measure-
cules, A2E-epoxides may account for much of the
ments of light-induced cell death, the cells were exposed to
cellular damage accrued.
25
The photochemical events
blue light (430 nm
30 [SD], 8 mW/cm
2
), green light
provoked by the irradiation of A2E-laden RPE ulti-
(550
10 nm, 8 mW/cm
2
), or white light (246 mW/cm
2
)
mately result in the initiation of a cell death program.
over a 0.8 mm
8.5 mm field. The light was delivered
The sensitivity to blue light conferred by the lipofuscin
from a tungsten halogen source for 20 minutes, and power
fluorophore A2E may explain why atrophic age-related
 was measured with a Newport optical power meter (model
macular degeneration (AMD) has been linked to both
840). The spectral range of the lamp was 390 to 750 nm,
RPE lipofuscin
27–33
and cumulative light exposure.
34
 with a lower output at the shorter wavelengths.
Moreover, the loss of RPE cells in atrophic AMD is a
The IOL to be tested was applied to the undersurfaceof the culture well, where it remained attached, unaided. It
criticaleventasitleadstophotoreceptorcelldegeneration.
 was centered over the light path.
Given that the attenuation of blue light afforded
To obtain a visual representation of IOL protection,
by the yellowed senescent lens may defend lipofuscin-
1.0 mm diameter disks were cut from the center of the IOL
filled RPE cells against blue light damage, we are in-
using a trephine blade (Katena Products, Inc.). The IOL
terested in efforts being made to develop IOLs that
disks were positioned on the undersurface of the well, which
replicate the transmission characteristics of the aging
 was thenirradiated (430
30 nm, 16 mW/cm
2
)from below.
crystalline lens. In this study, we constructed a cellculture system that allowed us to compare several IOLs
Detection of Nonviable Cells 
as to their ability to protect A2E-laden RPE from blue
The nuclei of dead RPE cells were labeled with a mem-
light damage. In this cell culture system, A2E accumu-
brane impermeant dye (Dead Red, Molecular Probes), and
lates in the lysosomal compartment of a human RPE
the nuclei of all cells with 4
,6
-diamino-2-phenylindole
cell line, as it does in RPE of the eye.
35
Moreover, the
(DAPI), as reported.
25,26
Blue light-illuminated A2E-con-
levels of A2E are comparable to the level occurring in
taining RPE labeled in this way are undergoing an apoptoticform of cell death.
36
Digital images (5 fields per illumination
vivo.
18,35
This model also allows us to study RPE cells
zone) were obtained using a Zeiss Axioplan II microscope
 with and without intracellular deposits of A2E.
 with Axiocam camera and KS400 image processing software.Subsequently, Dead Red and DAPI-stained nuclei were
Materials and Methods
counted. Dead cells were quantified as a percentage of thetotal number of cells in a field. Means are based on 3The IOLs studied were the Alcon AcrySof 
Naturalexperiments.(SN60AT) (acrylic, 20.0 diopters [D], 6.0 mm optic diame-Statisticalanalysiswasbyananalysisofvariancefollowedter) and AcrySof (SA60AT) (acrylic, 20.0 D, 6.0 mm opticby the Newman-Keul multiple comparison test (Prism,diameter); the AMO ClariFlex
(CLRFLXB) (silicone,GraphPad Software). A 
P 
value of 0.05 or less was consid-20.0 D, 6.0 mm optic diameter) and Sensar
(AR40e)(acrylic, 20.0 D, 6.0 mm optic diameter); and the Pharmacia ered significant.
 J CATARACT REFRACT SURG—VOL 30, APRIL 2004
874
 
LABORATORY SCIENCE: BLUE LIGHT-ABSORBING IOL
that did not contain A2E (no A2E, no IOL) remainedviable, while blue light-illuminated (430 nm peak witha bandwidth of 60 nm, 8 mW/cm
2
) RPE that hadpreviously accumulated A2E underwent marked celldeath. In the present experiments, 41.1%
4.1% (3experiments) of the A2E-laden cells in a field of illumi-nation became nonviable after blue light exposure inthe absence of an IOL (Figure 1). When the yellow-tinted AcrySof Natural IOL was placed in the centerof the light path, transmission of the 430 nm light was reduced by approximately 50% and cell death wasreduced by 80% (
P 
.001) compared to irradiation inthe absence of an IOL (Figure 1). When the AcrySof,Sensar, ClariFlex, or CeeOn Edge IOLs were in place,the frequency of nonviable cells was not different
Figure 1.
(Sparrow) Quantitation of nonviable RPE cells after A2Eaccumulation and blue light illumination (430 nm) with and without
(
P 
.05) than cell death in the absence of an IOL. The
an IOL placed in the light path. The percentage of nonviable cells
modest but consistent declines in cell death that were
was determined by labeling all nuclei with DAPI and the nuclei of 
detected with these IOLs (Figure 1) were undoubtedly 
nonviable cells with a membrane-impermeable dye. The data were
duetothesmallreductionsinlighttransmission(
5%)
normalized to the percentage of nonviable cells in the absence of anIOL. Values are mean
SEM.
that were measured.TheabilityoftheAcrySofNaturalIOLtoattenuateblue light-mediated cell death was evident when fieldsofirradiatedcellswereimaged.IntheabsenceofanIOL,the nuclei of nonviable cells were uniformly distributedacross the illuminated field (Figure 2). Conversely,placement of the blue light-absorbing AcrySof NaturalIOL (a 1.0 mm disk) within the light path spared acircularzoneofcells,thediameterofthisareacorrespond-ing to the diameter of the IOL disc. Within this sector,all nuclei were labeled with DAPI but labeled nucleiof nonviable cells were only occasionally observed. With the use of wide-band white light (390 to750 nm; 246 mW/cm
2
) that included all wavelengthstoward which A2E exhibits sensitivity, the death of  A2E-laden RPE reached a frequency of 35% (35.4%
2.7% of the cells in an illumination zone; 3 experi-
Figure 2.
(Sparrow) A blue light-absorbing IOL protects A2E-
ments).Inthepresenceofthebluelight-absorbingAcry-
laden RPE from cell death due to 430 nm irradiation. The nuclei of nonviable RPE were labeled with a membrane impermeable dye (
 left
 ),
Sof Natural IOL, cell death from white light was
and all nuclei were stained with DAPI (
 right
 ). The left and right panels
reduced by 82% (
P 
.001) compared to that without
arecorrespondingfieldsofilluminatedA2E-ladenRPE.Intheabsence
an IOL (Figure 3). In comparison, the AcrySof IOL
ofanIOLinthelightpath(
top
 ),theentirefieldiscoveredwithnonviable
diminished the incidence of nonviable cells by 22%
cells. Placement of a blue light-absorbing IOL (1.0 mm diameter disk; AcrySof Natural) in the light path reduced the number of nonviable
(
P 
.05); with the Sensar, ClariFlex, and CeeOn Edge
cells in a circular zone corresponding to the area covered by the IOL
IOLs, cell death was decreased by 37% to 39% (
P 
(
 bottom
, dotted circle).
.01). Again, the decreases observed with the colorlessIOLs were likely related to the reductions in spectral
Results
transmittance associated with all IOLs.Using a fluorescence assay that labels the nuclei of When compared with blue and white light, greenlight (550
20 nm) delivered at an intensity that wasnonviable cells, it was observed that illuminated RPE
 J CATARACT REFRACT SURG—VOL 30, APRIL 2004
875
of 00

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