The Journal of Nutrition
Nutrient Supplementation with n3
Polyunsaturated Fatty Acids, Lutein, and
Zeaxanthin Decrease A2E Accumulation and
VEGF Expression in the Retinas of Ccl2/Cx3cr1-
Deficient Mice on Crb1rd8Background1–3
Hema L. Ramkumar,4,7,8Jingsheng Tuo,4De F. Shen,4Jun Zhang,5Xiaoguang Cao,4,9Emily Y. Chew,6
and Chi-Chao Chan4,5*
4Laboratory of Immunology,5Histology Core, and6Epidemiology and Clinical Applications, National Eye Institute, National Institutes
of Health, Bethesda, MD;7Howard Hughes Medical Institute, Chevy Chase, MD;8Department of Ophthalmology, Shiley Eye Center,
University of California-San Diego, San Diego, CA;9Department of Ophthalmology, Peking University People?s Hospital, Beijing, China
The Age-Related Eye Diseases Study 2 (AREDS2) clinical trial is assessing the effects of higher dietary xanthophyll (lutein
and zeaxanthin)and long-chainn3 polyunsaturated fatty acid (LCPUFA)docosahexaenoic acid (DHA) and eicosapentaenoic
acid (EPA) intake on progression to advanced age-related macular degeneration (AMD). This study?s purpose was to
examine the retinal effects of the AREDS2 formulation on Chemokine (C-C motif) ligand 2 (Ccl22/2)/CX3C chemokine
receptor 1 (Cx3cr12/2) mice on Crumbs homolog 1 retinal degeneration phenotype 8 (Crb1rd8) background (DKO), which
develop focal retinal lesions with certain features similar to AMD. DKO and C57BL/6N rd8 background mice (WT) were
bred and randomized into 4 groups. Two groups, WT mice on AREDS2 diet (A-WT) and DKO mice on AREDS2 diet
(A-DKO), were supplemented daily with 1.76 mmol of lutein, 35.1 mmol of zeaxanthin, 215 mmol EPA, and 107 mmol of
DHA, and 2 control groups, WT mice on control diet (C-WT) and DKO mice on control diet (C-DKO), were fed an isocaloric
diet. All mice had monthly fundus photos and were killed after 3 mo for biochemical and histologic analyses. After 3 mo,
81% of A-DKO mice had lesion regression compared with 25% of C-DKO mice (P < 0.05). Toxic retinal 2-[2,6-dimethyl-8-
1-yl) 1E,3E,5E,7E-hexatrienyl]-pyridinium (A2E) concentrations were significantly lower in A-DKO compared with C-DKO
mice. The outer nuclear layer thickness in A-DKO mice was significantly greater than that in C-DKO mice. Retinal
expression of inducible nitric oxide synthase (iNos) tumor necrosis factor-a (Tnf-a), Cyclooxygenase-2 (Cox-2),
interleukin1beta (IL-1b), and vascular endothelial growth factor (Vegf) was significantly lower in A-DKO compared with
C-DKO mice. Xanthophylls and LCPUFAs have antiinflammatory, neuroprotective, and antiangiogenic properties. Our data
provide potential mechanisms by which the AREDS2 formula has a protective effectonretinal lesionsinDKOmice.J. Nutr.
143: 1129–1135, 2013.
Age-related macular degeneration (AMD)10is the leading cause
of irreversible central vision loss in the elderly population in the
United States and the world (1). Most patients with AMD
experience slowly progressive, high-resolution central vision loss
after age 60 y, making activities such as driving and reading
difficult. The pathogenesis of AMD is not well understood, but it
10Abbreviations used: AA, arachidonic acid; A-DKO, DKO mice on AREDS2 diet;
Eye Diseases Study 2; A-WT, WT mice on AREDS2 diet Ccl2, Chemokine (C-C
motif) ligand 2; C-DKO, DKO mice on control diet; COX-2, Cyclooxygenase-2;
Crb1, Crumbs homolog 1; C-WT, WT mice on control diet; Cx3cr1, CX3C chemokine
receptor 1; DKO, Ccl22/2/Cx3cr12/2; INL, inner nuclear layer; iNOS, inducible nitric
oxide synthase; IS, inner segment; long-chain PUFA, LCPUFA; OLM, outer limiting
membrane; ONL, outer nuclear layer; OS, outer segment; rd8, retinal degeneration
phenotype 8; RPE, retinal pigment epithelium; VEGF, vascular endothelial growth
factor; WT, C57BL/6N retinal degeneration phenotype 8 background.
1Supported by the Intramural Research Program of the National Eye Institute,
NIH, and Howard Hughes Medical Institute.
2Author disclosures: H. L. Ramkumar, J. Tuo, D. F. Shen, J. Zhang, X. Cao, E. Y.
Chew, and C.-C. Chan, no conflicts of interest
3Supplemental Figure 1 and Supplemental Tables 1 and 2 are available from the
‘‘Online Supporting Material’’ link in the online posting of the article and from the
same link in the online table of contents at http://jn.nutrition.org.
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
ã 2013 American Society for Nutrition.
Manuscript received September 17, 2012. Initial review completed October 16, 2012. Revision accepted April 1, 2013.
First published online May 15, 2013; doi:10.3945/jn.112.169649.
at NATIONAL INSTITUTES OF HEALTH (NIH) on June 25, 2013
Supplemental Material can be found at:
is thought that cumulative oxygen and light exposure over a
lifetime might lead to oxidative stress and inflammation, which
injure the retinal pigment epithelium (RPE), resulting in loss of
retinal outer layers and photoreceptor cells (2). Although vascular
endothelial growth factor (VEGF) has been a drug target for
patients with neovascular AMD, the majority of patients with
geographic atrophic AMD have no therapeutic options.
Free radical species expedite oxidative stress-induced damage
of RPE cells, and nutritional supplements that quench these
species have been a great source of interest in AMD research.
The Age-Related Eye Disease Study was a large-scale, random-
ized, controlled clinical trial that showed a beneficial effect of
supplementation with vitamins C and E, b-carotene, and zinc
with copper in reducing the risk of progression to advanced
AMD in patients with intermediate or advanced AMD (3).
Observational studies (4) demonstrated an inverse relationship
between macular xanthophylls (lutein and zeaxanthin) and n3
long-chain PUFA (LCPUFA) supplementation and advanced
AMD (5,6). The Age-Related Eye Disease Study 2 (AREDS2),
a large, multi-center, randomized clinical trial, is prospectively
evaluating the efficacy of lutein, zeaxanthin, and n3 LCPUFA
supplementation in participants who are at risk of developing
The purpose of our study is to evaluate the effects of a diet
supplemented with lutein, zeaxanthin, DHA, and EPA in
DKO mice on clinical and histopathological retinal lesions,
retinal inflammatory gene expression, VEGF expression,
cyclohexen-1-yl) 1E,3E,5E,7E-hexatrienyl]-pyridinium (A2E)
concentrations, and photoreceptor loss. We selected the Chemokine
(C-C motif) ligand 2 (Ccl2)
(Cx3cr1)2/2mouse on Crumbs homolog 1 retinal degeneration
phenotype 8 (Crb1rd8) background (DKO), which develops focal
retinal lesions that have clinical, biochemical, and pathological
features of AMD. In addition to the lesions of Crb1rd8(7,8), these
micehavefocalphotoreceptorand RPE degenerationand atrophy
and a few drusenoid deposits (9,10).
2/2/ CX3C chemokine receptor 1
Materials and Methods
Mice. DKOmice andC57BL/6Nrd8/rd8(WT)mice werebredin-house.
These mice were termed DKO in our previous publications (9,11), and
now DKO refers to DKO on an rd8 background (7). The study was
conductedin compliance with theAssociationfor Research in Vision and
Ophthalmology statement for the ethical use of animals. Two of 54 mice
died duringtheexperiment fromafightwitha mouseinthe samecage,so
data from these mice were excluded from analysis. All animal experiments
were performed under protocols approved by the National Eye Institute
(NEI)?s Institutional Animal Care and Use Committee.
Experimental protocol. DKO and WT mice were separated from their
mothers at 3 wk of age, randomly assigned to 2 groups, and separately
fed the standard diet. Feeding habits were observed and we calculated
that themean consumption ofboth WTand DKOmicewas 4.06 0.2g/d
diet and the mean body weight was 16 6 0.3 g. The doses of these
ingredients in the experimental formula were determined using the
human AREDS2 clinical trial dose (12) and converting this to the
mouse dose using allometry formulas (A diet). The 2 pelleted, purified
animal diets used (provided by Dyets) were based on the AIN-93G
formulation (13,14) with several modifications. The isocaloric control
diet is (C diet) identical to the AIN-93G diet with the exception of an
increased amount of soybean oil (117 vs. 70 g/kg diet) and the experimental
diet is identical to the AIN-93G diet with the following modifications: no
soybean oil, 1.76 mmol zeaxanthin/kg diet, 17.6 mmol lutein/kg diet, 54.9
mmol EPA/kg diet, and 25.2 mmol DHA/kg diet (Supplemental Table 1).
The effective daily dose is: 1.76 mmol lutein, 35.1 mmol zeaxanthin, 215
mmol EPA, and 107 mmol DHA. The ratio of EPA:DHA is 2:1. The animal
diets were also quantitatively tested for the presence of baseline concentra-
tions of lutein, zeaxanthin, and DHA+EPA at 0, 4, and 9 wk at Covance
(Supplemental Table 2). DKO and WT mice were fed for 3 mo then killed
for further studies. Of the 4 experimental groups, 2 groups (A-WT and A-
DKO) were supplemented daily with 1.76mmol of lutein, 35.1mmol of
zeaxanthin, 215mmol EPA, and 107mmol of DHA (AREDS2 formula).
C-WT and C-DKO were fed an isocaloric diet (control formula). Only
one eye per mouse was assayed for each analysis. The experiment was
repeated 3 times. In total, each group had a mean of 25 mice.
Fundus photography. After pupil dilation, sequential funduscopic
examinations and photography were performed every month from 1 mo
of age until 4 mo of age using methods previously described (14). Retinal
lesion change was evaluated by comparing the sequential photos taken
over time in the same fundus area. The lesions were quantitatively
evaluated using a previously described scale (15) in which a negative
integer indicates lesion regression and a positive integer represents lesion
progression. Grading of the pictures was conducted by a masked
measurements. Eyes were harvested following killing of the mice at
4 mo of age. The eyes were processed using previously described
methods (14). The photoreceptor outer nuclear layer (ONL) thickness
was evaluated on photomicrographs of retinal sections with a 203
objective of a photomicroscope (E800; Nikon) and a digital camera
(DXM1200; Nikon). Histopathological examination and measurement of
ONL thicknesses in 10 different areas were performed on 4 A-DKO eyes, 3
were made across the inferior and superior retinas every 100 mm from the
optic nerve head.
Transmission electron microscopy. One eye from 2 mice in each diet
group was used for transmission electron microscopic study. Eye cups
were fixed in 2% paraformaldehyde in 0.1 mmol/L phosphate buffer.
embedded in Epon-812 resin. Six 1-mm-thick sections stained with
toluidine blue were examined under light microscopy. The lesions shown
on thick sections guided the selection of ultrathin sections, which were
stained with uranyl acetate and lead citrate for examination under a
JEOL1010 electron microscope.
A2E extraction and quantification. A fluorophore found in lipofuscin
and RPE phagolysosomes, A2E, is generated during the visual cycle.
Quantitative A2E measurement was performed on 10 A-DKO eyes, 10
CT-DKO eyes, 10 A-WTeyes, and 10 C-WTeyes. The mice were kept in
the dark for >12 h before being killed. Whole eyes were removed in a
dark room under dim red light and homogenized. A2E was extracted
with chloroform/methanol as previously described (16). Detection and
quantification of A2E was performed as we previously described (14).
ELISA. Because n3LCPUFAsupplementationcaninfluencethemetabolism
of arachidonic acids (AAs), we measured PGE2, an inflammatory, biolog-
ically active metabolite of AAs. Serum was collected from all experimental
groups. PGE2levels were determined by monoclonal antibody-based
ELISA. Assays were performed using the EIA kit (Cayman Chemical)
following the manufacturer?s instructions.
Quantification of gene expression by RT-PCR. Approximately 100
retinal cells (RPE and neuronal cells) were microdissected from a frozen
section of an ocular slide. The primers for TNFa, IL-6, and VEGF were
synthesized by SuperArray and supplied as the RT2Real-Time Gene
ExpressionAssay kit. RT-PCR was performed usingpreviously described
methods (14). The levels of the target mRNAs were quantified, using
masked procedures, relative to the level of the housekeeping gene,
b-actin, by the comparative DDCT method. The formula is the fold of =
10^DDCt/standard curve slope, DDCt = [Ct (target gene of the tissue) 2
Ct (b-actin of the tissue)] – [Ct (target gene of the reference) 2 Ct
1130Ramkumar et al.
at NATIONAL INSTITUTES OF HEALTH (NIH) on June 25, 2013
(b-actin of the reference)]. In the event that an individual mRNA level
was more than 2 SDs above the group when not included, it was
considered an outlier and excluded. The results were calculated by
using universal total RNA as the reference (SABiosciences). Each
sample was analyzed twice.
Retina fatty acid analyses. Fiveeyesineachtreatmentgroupwereused
for retinal fatty acid analyses. Total cellular lipids were extracted from
the retina samples as previously described (17). Briefly, retinas were
manually homogenized in a small volume of ice-cold buffer (50 mmol/L
Trizma, 1 mmol/L EDTA, pH 7.4). After acidification with 0.1 nmol/L
HCL, total lipids were extracted with chloroform-methanol (2:1). The
organic phase was concentrated under a stream of nitrogen gas. FAMEs
were preparedbybase-catalyzedmethylation (0.5mol/Lsodiummethoxide
in methanol) and analyzed using an Agilent Technologies gas chromato-
graph equipped with 60- 3 0.25-mm i.d.-fused silica capillary column with
a 0.15-mm film thickness (DB-23 column). FAMEs were identified by
comparing retention times with those from commercial standards. Data are
expressed as mol/100 mol of the total FAMEs identified.
Statistical analysis. Median fundus lesion scores were compared
unpaired Mann-Whitney U test. Rates of progression and regression
and 3 mo of treatment. Data are presented with chi-squared statistic
(df) (P value). A2E concentrations (C-DKO vs. A-DKO and C-WT vs. A-
WT), gene expression level (A-DKO vs. C-DKO), retinal outer layer
thickness (C-WT vs. A-WT and C-DKO vs. A-DKO), serum PGE2
concentration (A-DKO vs. C-DKO), and retinal fatty acid concentrations
(A-WT vs. C-WTand A-DKO vs. C-DKO) were compared after 3 mo of
treatment using a 2-tailed unpaired Mann-Whitney U test. Values in the
text are presented as median (minimum value, maximum value) (U = Mann-
for chi-squared and Mann-Whitney U analyses. XLSTAT software version
2011.4.02 and Microsoft Excel were used for statistical analysis.
Three independent experiments were performed on DKO and
WT mice fed either the control diet (C-DKO and C-WT) or the
AREDS2-supplemented diet (A-DKO and A-WT) for 3 mo. The
results of all 3 experiments were comparable. Data (fundoscopic
photography, histopathology, fatty acid analysis, etc.) were
pooled and are presented below. Dietary analysis performed and
confirmed that these nutrients were present at the expected
levels in the diets and did not change over time (Supplemental
Table 2). Analyses of the diets revealed that the amounts of the
starting ingredients were 100-140% of the expected values.
Clinical ocular features. Fundus photographs were scored
with comparison to baseline photographs using the afore-
mentioned standardized scale. We measured lesion progres-
sion as a positive score and regression as a negative score.
C-WT and A-WT mice did not develop retinal lesions and
therefore had a score of 0 (Supplemental Fig. 1). As previously
described, by 4 wk of age, all DKO mice developed a retinal
phenotype of progressive focal photoreceptor loss and RPE
mottling (Fig. 1A).
The fundus scores were 0.75 (22, 2) in C-DKO mice
and 20.25 (22, 2) in A-DKO mice at 1 mo (U = 152; P = 0.09);
1 (20.5, 2) in C-DKO mice and 20.5 (23, 0) in A-DKO mice
(U = 118; P = 0.001) at 2 mo; and 1 (20.5, 2) in C-DKO mice
and 21 (23.5, 1) in A-DKO mice at 3 mo (U = 121; P = 0.001).
The contingency table of fundus scores of 54 mice during 3 mo
(one eye per mouse from 3 separate experiments) is shown
(Fig. 1B). At the end of 3 mo of treatment, more A-DKO than
C-DKO mice had lesion regression [x2= 20.6 (8); P = 0.008].
Histological and ultrastructural features. Histopathological
examination was conducted on 8 A-DKO, 4 C-DKO, 8 A-WT,
and 6 C-WT eyes. C-WT and A-WT eyes had a similar histopa-
thology regardless of diet, with normal morphology of the outer
and inner segments (OSs/ISs), inner nuclear layer (INL) and ONL,
and RPE cell layer (Fig. 2A,B). C-DKO mice had focal RPE
hypertrophy and hypopigmentation, loss of the OS and IS layers,
and focal disorganization of the ONLs and INLs with abundant
photoreceptor loss (Fig. 2C). In contrast, an A-DKO eye had
preserved organization of the OSs/ISs and normal RPE mor-
phology. Although there were some areas of retinal dystrophy
(photoreceptor nuclear migration to the outer plexiform layer)
characteristic for mice with the rd8 background (18), no
pseudorosettes or retinal folds were detected in these mice.
Photoreceptor architecture and separation of the INL and ONL
was preserved in A-DKO eyes (Fig. 2D).
At theultrastructural level, A-WTandC-WTeyes had normal
RPE morphology and pigmentation, clear demarcation of the
OS/IS, and an outer limiting membrane (OLM) (Fig. 3A,B). In
C-DKO eyes, liposomes and lipofuscin accumulated in the RPE,
with pigment extravasation, a shorter OS/IS, and complete loss
of the OLM. The photoreceptor IS appeared vacuolated,
collapsed, and disorganized (Fig. 3C). In contrast, the RPE cell
in the A-DKO eye showed normal melanosomes and intact
structure without excess lipofuscin granules. The OS/IS of the
treatment with a control or AREDS2 diet. C-DKO mice (A) and A-DKO
mice (B) are shown at 1 mo of age prior to treatment (left) and after 3
mo of treatment (right). C-DKO mice had an increase in the number
and size of lesions (arrows), whereas A-DKO mice had lesion
regression (arrows). (C) A 3D contingency table of the fundus lesion
scores of A-DKO and C-DKO mice showing a significant difference in
the rate of lesion progression after 3 mo of treatment, n = 16 (A-DKO),
12 (C-DKO), P = 0.008. A-DKO, DKO on AREDS2 diet; AREDS2, Age-
Related Eye Diseases Study 2; C-DKO, DKO on control diet; Cx3cr1,
CX3C chemokine receptor 1; DKO, Ccl22/2/Cx3cr12/2.
Periodic monitoring of fundus lesions in DKO mice after
Nutrients for an age-related macular degeneration model 1131
at NATIONAL INSTITUTES OF HEALTH (NIH) on June 25, 2013
photoreceptors were more organized, appear healthy without
vacuolization, and had preserved length. Additionally, the OLM
was easily identified (Fig. 3D, arrow).
Retinal outer layer thicknesses. The results of the histopath-
ological examination and measurement of ONL thicknesses
were summarized with mean thickness and SD. The retinal ONL
thicknesses (mm) in C-WT eyes [33.4 (30.0, 39.0)] were similar
to A-WT eyes [32.7 (20.7, 101)]. The ONL thickness (mm) in a
C-DKO eye was 0.75 (0, 11.2) and<18.3 (0, 29.5) in the A-DKO
eye (U = 33.5; P < 0.0001). Overall, ONL thickness was
significantly greater in A-DKO eyes than in C-DKO eyes.
Quantification of a biochemical component in RPE
phagolysosomes. Quantitative A2E measurement was per-
formed on 10 each of A-DKO, CT-DKO, A-WT, and C-WTeyes.
A2E concentrations were significantly higher in C-DKO eyes
than in C-WT eyes, as previously published (10,11,19). The
retinal A2E concentration in C-WTeyes did not differ from that
in A-WT eyes. A2E (pmol/eye) in C-DKO eyes [62.3 (53.8,
77.6)] was greater than in A-DKO eyes [38.0 (25.9, 51.7)] (U =
90; P = 0.0003) and did not differ from C-WT concentrations.
Retinal transcript expression profile. We investigated the
retinal mRNA expression of several pathologic proinflammatory
genes, including tumor necrosis factor-a (Tnf-a), cyclooxygenase-
2 (Cox-2), interleukin-1b (Il-1b), and inducible nitric oxide
synthase (iNOS), in our treatment groups (Fig. 4). The expression
ofTnf-a (U = 18;P =0.028),Cox-2 (U= 15;P = 0.036),Il-1b (U =
15; P = 0.036), and iNOS (U = 21; P = 0.017) in A-DKO micewas
lower than in C-DKO mice. Vegf levels were also significantly
higher in C-DKO mice compared with A-DKO mice (U = 22; P =
0.04). A-DKO mice had lower expression of inflammatory and
proangiogenic genes linked to advanced AMD.
Retinal accumulation of fatty acids and reduction in AA
concentrations. To ascertain that dietary consumption of fatty
acids results in retinal accumulation, analyses of fatty acid
concentrations were performed using 5 eyes from each treatment
group. AA concentrations (nmol/mg retina) were lower in A-WT
eyes [1.7 (1.5, 2.5)] compared with C-WT eyes [3.0 (2.8, 3.2)]
(U = 25; P = 0.008) and in A-DKO eyes [1.2 (1.2, 1.6)] compared
with C-DKO eyes [2.6 (2.5, 3.1)](U = 25; P = 0.008). There were
higher concentrations of EPA (nmol/mg of retina) in A-WT mice
[0.40 (0.38, 0.44)] compared with C-WT mice [0.067 (0, 0.15)]
treatment with a control or AREDS2 diet. Representative photomi-
crographs taken after 3 mo of treatment. (A,B) In C-WT and A-WT
eyes, there is a clear INL, OPL, ONL, IS, OS, and RPE. The RPE
appears homogenous in size and character. (C) C-DKO mice have focal
areas of photoreceptor loss (arrow), loss of the IS/OS and OPL, and
collapse of the ONL and INL. RPE shows vacuole accumulation,
atrophy, and hypertrophy. (D) In contrast, A-DKO mice have preser-
vation of retinal architecture and a relatively normal RPE, OS, IS, and
OLM with only scattered areas of nuclear photoreceptor migration
(arrows) in the OPL (hematoxylin and eosin, original magnification
3200). AREDS2, Age-Related Eye Diseases Study 2; A-DKO, DKO on
AREDS2 diet; A-WT, WT on AREDS2 diet; C-DKO, DKO on control
diet; C-WT, WT on control diet; INL, inner nuclear layer; IS, inner
segment; OLM, outer limiting membrane; ONL, outer nuclear layer;
OPL, outer plexiform layer; OS, outer segment; RPE, retinal pigment
epithelium; WT, C57BL/6N retinal degeneration phenotype 8 back-
Ocular photomicrographs of all treatment groups after
1132 Ramkumar et al.
at NATIONAL INSTITUTES OF HEALTH (NIH) on June 25, 2013
(U = 0; P = 0.011) and in A-DKO mice [0.53 (0.50, 0.57)]
compared with C-DKO mice [0.11 (0, 0.14)] (U = 0; P = 0.012).
Retinal DHA concentrations (nmol/mg retina) were 9.4 (8.8,
9.9) in C-WTeyes, <11.0 (10.3, 11.4) in A-WTeyes (U = 0; P =
0.008), and 9.1 (8.6, 9.4) in C-DKO mice, significantly <10 (9.9,
11) in A-DKO mice (U = 0; P = 0.008).
Profile of AA derivatives. The concentration of PGE2, a pro-
inflammatory metabolite of AA, was measured in all treatment
groups (56 retinas). The median PGE2 serum concentration
(nmol/L) of C-DKO mice [5.49 (0.32, 22.7)] did not significantly
differ from that of A-DKO mice [9.78 (0.32, 11.4)].
In this study, DKO and WT mice were fed either a diet
supplemented with lutein, zeaxanthin, and n3 LCPUFAs or
an isocaloric diet. The results demonstrate a benefit of the
AREDS2 diet on retinal AMD-like lesions, RPE lipofuscin and A2E
accumulation, pathologic gene expression, and preservation of
out a chemokine (Ccl2) and a chemokine receptor (Cx3cr1) on a
Crb1rd8background. Although nonprimate models do not have a
macula, this model mimics the A2E accumulation and focal
photoreceptor and RPE degeneration found in AMD. The rd8
background may contribute to focal retinal dystrophic lesions in
both WT and DKO mice. DKO mice develop RPE pathology
(lipofuscin accumulation, hypertrophy, and hypotrophy) and
photoreceptor and synaptic degeneration that is more charac-
teristic in AMD (20).
The n3 LCPUFA supplementation fed during gestation
ameliorated retinal lesions, reduced retinal A2E levels, and
decreased concentrations of serum AA metabolites in DKO mice
and influence retinal cell gene expression, cellular differentiation,
and survival (21). DHA acts as a transcription factor for PPARa,
which prevents endothelial cell dysfunction, photoreceptor death,
and vascular remodeling (22–24). Humans lack the capacity for de
novo synthesis of the precursors of EPA and DHA, necessi-
tating dietary supplementation. AMD patients who reported
the highest intake of n3 LCPUFAs were less likely to have
neovascular AMD at baseline or to progress to advanced AMD at
the end of 12 y (25,26).
In this study, LCPUFAs, lutein, and zeaxanthin were admin-
istered in combination to mice after 1 mo of age, and clinical and
histopathological improvement was observed after only 4 wk of
nutritional supplementation. The addition of omega-3 LCPUFA
to oral supplementation of lutein/zeaxanthin for 6 months did
not change the serum levels of lutein and zeaxanthin in persons
with or without AMD (27). There may be a greater protective
effect of the combination of xanthophylls and LCPUFAs on the
retina compared with LCPUFAs alone in AREDS2, a long-term
clinical trial, but one limitation of this study is that the nutrients
were not studied individually. It has been shown in humans that
dietary lutein supplementation increases serum and macular retinal
concentrations of lutein (28). Because of the extremely low serum
and tissue concentrations of lutein and zeaxanthin in rodents, these
carotenoids were not measured in the experimental mice in this
study. It therefore cannot be determined if the effects of these
carotenoids on the retina are direct or indirect. Lutein supple-
mentation was shown to be beneficial in the LAST trial (29) and
there is an inverse relationship between dietary consumption and
advanced AMD (30–32). Lutein suppresses STAT3 activation by
inflammatory cytokines and extracellular signal-regulated kinase
(ERK) activation, slowing DNA damage and preserving a-wave
ERG amplitude in mouse models (33). Lutein attenuates hypoxia
inducible factor 1a expression, inhibits reactive oxidative species
production, and decreases VEGF expression (34).
Incomplete degradation of the photoreceptor OS in lysosomes
of RPE cells creates lipofuscin granules (35) and produces A2E,
which correlates with poor RPE longevity and photoreceptor
degeneration in the central retina. Retinal A2E is directly related
to complement activation, destabilization of cell membranes,
and choroidal neovascularization in vivo (36–38). A2E accumu-
lation is an appropriate biomarker of AMD pathology in mice,
because classic AMD Bruch?s membrane changes and drusen are
after treatment with a control or AREDS2 diet. In a normal RPE, a
distinct OLM, IS, and OS are seen clearly in C-WT (A) and A-WT (B)
mice. (C) In C-DKO mice, the IS and OS are shortened and
photoreceptors have vacuoles (asterisk). RPE cells have abundant
lysosomes, lipofuscin granules, loss of infoldings, and pigment
extravasation (dashed arrow). (D) In A-DKO mice, the IS and OS
appear normal and intact with fewer photoreceptors vacuoles. The
OLM is preserved (arrow) and RPE cells are normal (magnification
32500). A-DKO, DKO on AREDS2 diet; A-WT, WT on AREDS2 diet;
AREDS2, Age-Related Eye Diseases Study 2; C-DKO, DKO on control
diet; C-WT, WT on control diet; IS, inner segment; OLM, outer limiting
membrane; OS, outer segment; RPE, retinal pigment epithelium.
Retinal ultrastructural images of all treatment groups
after treatment. Retinal gene expression of Tnf-a, Vegf, Cox2-2, Il-1b,
and iNos in A-WT and A-DKO mice are plotted on a scatter plot. The
median values are noted with a dash, n = 7 (A-DKO), 4 (C-DKO). The
asterisk indicates that medians differ, P , 0.05. A-DKO, DKO on
AREDS2 diet C-DKO, Ccl22/2/Cx3cr12/2Crb1rd8mice fed isocaloric
control; Cox-2, Cyclooxygenase-2; Cx3cr1, CX3C chemokine receptor
1; DKO, Ccl2/2/Cx3cr12/2; A-WT, WT on AREDS2 diet; iNos, inducible
nitric oxide synthase; Vegf, vascular endothelial growth factor.
Gene transcripts in the retina of A-WT and A-DKO mice
Nutrients for an age-related macular degeneration model 1133
at NATIONAL INSTITUTES OF HEALTH (NIH) on June 25, 2013
not usually seen (10). It is of great importance that the A-DKO
mice had lower A2E levels.
We measured the retinal expression of genes that are im-
portant in AMD pathology. NO mediates the proangiogenic
responses of VEGF (39), and NO inhibition attenuates VEGF-
induced angiogenesis (40). Expression of iNOS produces large
amounts of NO for a prolonged period, leading to free radical
production and posterior retinal degeneration (41,42). Sub-
macular choroidal blood flow control is also mediated in part
by iNOS (43). Our study shows that supplementation with
the AREDS2 diet significantly reduces retinal iNOS expression
TNFa, an inflammatory cytokine expressed by a subset of
microglia associated with retinal vessels (44), is directly respon-
sible for photoreceptor death (45) and choroidal neovascular-
ization (CNV). TNF inhibitors reduce the size and leakiness of
laser-induced CNV lesions in many animal models (46). We
reported lower ocular TNF-a transcripts in DKO mice treated
with naloxone, an inhibitor of microglia activation (47), and
improved retinal lesions in DKO mice treated with a TNF-
inducible gene 6 protein (48). These same findings with the
AREDS2 treatment suggest that lower ocular TNF-a may be
important in healing retinal lesions in DKO mice.
AA is the substrate for the synthesis of many mediators of
leukocyte chemotaxis and inflammatory cytokine production,
including prostaglandins, thromboxanes, and leukotrienes. EPA
supplementation results in incorporation into phospholipid
membranes at the expense of prostaglandins (49), decreasing
AA concentrations. COX-2 is the major enzyme that converts
AA to PGs in central nervous system inflammatory cells. AA
and COX-2 were both significantly reduced in the A-DKO
mice. Whereas n3 LCPUFA supplementation alone signifi-
cantly reduced PGE2concentrations (14), a significant differ-
ence was not found in our study.
IL-1b is produced in RPE cells and CNV membranes. It is a
diverse proinflammatory cytokine whose upregulation promotes
angiogenesis and induces and perpetuates neuroinflammation
(50). The inhibition of IL-1b by intravitreal injections of
recombinant IL-1 receptor antagonists has also been shown to
prevent CNV in a mouse model (51). Retinal IL-1b was sig-
nificantly reduced in A-DKO mice compared with C-DKO mice.
VEGF has been widely related to advanced AMD, especially
CNV (52). We reported that local delivery of AAV-5 mediated
sFLT01 gene therapy can stabilize retinal lesions in DKO mice
(15). Our group has shown that supplementation with n3
LCPUFAs alone did not alter expression of VEGF (14). In this
study, A-DKO mice had significantly less retinal VEGF expression
after 3 mo compared with C-DKO mice.
In addition, deranged RPE and photoreceptor metabolism
likely improved with the supplementation of DHA and lutein
based on the lower A2E levels, fewer retinal lesions, and preserva-
tion of the ONL. Retinal lesion regression in A-DKO mice may
indicate that oxidative damage was counteracted early enough to
halt RPE damage and photoreceptor loss. Overall, the results of our
study are promising for this murine model. These findings in a
homogenous mouse population may not necessarily translate to
findings in humans. We look forward to the AREDS2 controlled
clinical trial results to assess the potential benefit of these nutrients
in AMD patients.
The authors thank Dr. Ginger Tansey at the National Eye
Institute for helping with the experimental protocol and animal
care, Mark Milbank from DSM Nutritional Products for
providing the nutrients used in this study, Technical Mar-
keting Analytical Services for performing stability analyses,
Dr. Kevin Fritsche at the University of Missouri for perform-
ing the fatty acid analyses, and Dr. Ronald Bush at the
National Eye Institute for facilitating the retinal thickness mea-
surements. C.-C.C. and E.Y.C. designed research; H.L.R., J.T.,
D.F.S., J.Z., and X.C. conducted research; H.L.R., C.-C.C., J.T.,
J.Z., and D.F.S. analyzed data; and H.L.R. wrote the paper with
C.-C.C. and both parties have final responsibility for the final
content. All authors read and approved the final manuscript.
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