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Neuroprotective effect of bilberry extract in a murine model of photo-stressed retina

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Excessive exposure to light promotes degenerative and blinding retinal diseases such as age-related macular degeneration and retinitis pigmentosa. However, the underlying mechanisms of photo-induced retinal degeneration are not fully understood, and a generalizable preventive intervention has not been proposed. Bilberry extract is an antioxidant-rich supplement that ameliorates ocular symptoms. However, its effects on photo-stressed retinas have not been clarified. In this study, we examined the neuroprotective effects of bilberry extract against photo-stress in murine retinas. Light-induced visual function impairment recorded by scotopic and phototopic electroretinograms showing respective rod and cone photoreceptor function was attenuated by oral administration of bilberry extract through a stomach tube in Balb/c mice (750 mg/kg body weight). Bilberry extract also suppressed photo-induced apoptosis in the photoreceptor cell layer and shortening of the outer segments of rod and cone photoreceptors. Levels of photo-induced reactive oxygen species (ROS), oxidative and endoplasmic reticulum (ER) stress markers, as measured by real-time reverse transcriptase polymerase chain reaction, were reduced by bilberry extract treatment. Reduction of ROS by N-acetyl-L-cysteine, a well-known antioxidant also suppressed ER stress. Immunohistochemical analysis of activating transcription factor 4 expression showed the presence of ER stress in the retina, and at least in part, in Müller glial cells. The photo-induced disruption of tight junctions in the retinal pigment epithelium was also attenuated by bilberry extract, repressing an oxidative stress marker, although ER stress markers were not repressed. Our results suggest that bilberry extract attenuates photo-induced apoptosis and visual dysfunction most likely, and at least in part, through ROS reduction, and subsequent ER stress attenuation in the retina. This study can help understand the mechanisms of photo-stress and contribute to developing a new, potentially useful therapeutic approach using bilberry extract for preventing retinal photo-damage.
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RESEARCH ARTICLE
Neuroprotective effect of bilberry extract in a
murine model of photo-stressed retina
Hideto Osada
1
, Tomohiro Okamoto
1,2
, Hirohiko Kawashima
1,2
, Eriko Toda
1
,
Seiji Miyake
1,3
, Norihiro Nagai
1,2
, Saori Kobayashi
3
, Kazuo Tsubota
2
, Yoko Ozawa
1,2
*
1Laboratory of Retinal Cell Biology, Department of Ophthalmology, Keio University School of Medicine,
Tokyo, Japan, 2Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan,
3Wakasa Seikatsu Co., Ltd., Kyoto, Japan
*ozawa@a5.keio.jp
Abstract
Excessive exposure to light promotes degenerative and blinding retinal diseases such as
age-related macular degeneration and retinitis pigmentosa. However, the underlying mech-
anisms of photo-induced retinal degeneration are not fully understood, and a generalizable
preventive intervention has not been proposed. Bilberry extract is an antioxidant-rich sup-
plement that ameliorates ocular symptoms. However, its effects on photo-stressed retinas
have not been clarified. In this study, we examined the neuroprotective effects of bilberry
extract against photo-stress in murine retinas. Light-induced visual function impairment
recorded by scotopic and phototopic electroretinograms showing respective rod and cone
photoreceptor function was attenuated by oral administration of bilberry extract through a
stomach tube in Balb/c mice (750 mg/kg body weight). Bilberry extract also suppressed
photo-induced apoptosis in the photoreceptor cell layer and shortening of the outer seg-
ments of rod and cone photoreceptors. Levels of photo-induced reactive oxygen species
(ROS), oxidative and endoplasmic reticulum (ER) stress markers, as measured by real-time
reverse transcriptase polymerase chain reaction, were reduced by bilberry extract treat-
ment. Reduction of ROS by N-acetyl-L-cysteine, a well-known antioxidant also suppressed
ER stress. Immunohistochemical analysis of activating transcription factor 4 expression
showed the presence of ER stress in the retina, and at least in part, in Mu
¨ller glial cells. The
photo-induced disruption of tight junctions in the retinal pigment epithelium was also attenu-
ated by bilberry extract, repressing an oxidative stress marker, although ER stress markers
were not repressed. Our results suggest that bilberry extract attenuates photo-induced apo-
ptosis and visual dysfunction most likely, and at least in part, through ROS reduction, and
subsequent ER stress attenuation in the retina. This study can help understand the mecha-
nisms of photo-stress and contribute to developing a new, potentially useful therapeutic
approach using bilberry extract for preventing retinal photo-damage.
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 1 / 17
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OPEN ACCESS
Citation: Osada H, Okamoto T, Kawashima H, Toda
E, Miyake S, Nagai N, et al. (2017) Neuroprotective
effect of bilberry extract in a murine model of
photo-stressed retina. PLoS ONE 12(6): e0178627.
https://doi.org/10.1371/journal.pone.0178627
Editor: Alfred S Lewin, University of Florida,
UNITED STATES
Received: February 4, 2017
Accepted: May 16, 2017
Published: June 1, 2017
Copyright: ©2017 Osada et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: The study was supported by Wakasa
Seikatsu Co., Ltd. SK is an employee of Wakasa
Seikatsu Co., Ltd. and provided a resource. The
funder had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript. The funder provided support in the
form of salary to HO and in the grant to YO, but did
not have any additional role in the study design,
data collection and analysis, decision to publish, or
Introduction
Photons are indispensable for visual function; however, excessive exposure may overwhelm
the receptor capacity of the retina, resulting in stress and degeneration of the tissue. Retinal
damage can be caused by staring at the sun, termed solar retinopathy, as well as staring at com-
puter monitors while playing games [1]. Retinitis pigmentosa [2] and age-related macular
degeneration [3,4] are the leading causes of blindness and are characterized by retinal degen-
eration exacerbated by light exposure [511]. Thus, preventive interventions for reducing
photo-stress have been suggested in order to lower the risk of disease progression. However,
no specific treatment has been established except for blocking light using glasses, which may
not be helpful for patients with sub-average visual function who may require bright light to
recognize targets.
Previous reports have suggested that rhodopsin metabolism plays a role in photo-induced
retinal degeneration [12,13]. Although reactive oxygen species (ROS) are generated during
normal physiological metabolism, intrinsic mechanisms are involved in the maintenance of
homeostasis. However, when ROS levels exceed processing capacity, they cause abnormal
modifications of cellular components, such as proteins, and subsequently dysregulate multicel-
lular organelles and induce cellular death [14]. Similarly, excessive light exposure increases
ROS generation and causes retinal photoreceptor death [15,16].
When protein abnormalities are detected in the endoplasmic reticulum (ER), cells maintain
homeostasis by triggering an unfolded protein response (UPR) [17,18], which suppresses
overall protein production to prevent the accumulation of abnormally modified proteins.
However, when abnormalities exceed the homeostatic capacity of the UPR, cell death signaling
molecules such as the CCAAT-enhancer-binding protein (C/EBP) homologous protein
(CHOP) are induced to promote the transcription of pro-apoptotic molecules. Therefore, ER
stress can be involved in cell death, and the pathogenesis of blindness due to retinitis pigmen-
tosa, an inherited retinal degeneration, at least partly involves ER stress [1921]. Light expo-
sure-induced ER stress was previously reported in a photoreceptor cell line [22,23] and in the
retinal pigment epithelium (RPE), which maintains photoreceptors, of autophagy-deficient
mice [24,25], and in the neural retina [23]. However, the relationship between ROS accumula-
tion and ER stress in light-induced photoreceptor degeneration is not fully understood.
Bilberry extract is an antioxidant-rich supplement that contains various anthocyanins. It is
not only believed to ameliorate ocular symptoms but has also been used in clinical trials for
eye fatigue [26]. It can reduce ROS, as shown in in vitro experiments [27,28], and suppress the
pathogenesis of innate retinal inflammation [29] and diabetes [3032] in animal models. How-
ever, its effects on photo-stressed retinas have not been clarified. In the present study, we eval-
uated the preventive effects of bilberry extract on photo-induced retinal degeneration in Balb/
c mice. We focused particularly on the degeneration of rod photoreceptor cells, which form
the visual field, and cone photoreceptor cells, which determine visual acuity in humans, with
an emphasis on local ROS accumulation and ER stress. The influence of ROS on ER stress was
further analyzed in the retina using a well-known antioxidant, N-acetyl-L-cysteine (NAC),
which reduces retinal ROS [16].
Materials and methods
Animals
Seven- to eight-week-old male Balb/c mice (CLEA Japan, Tokyo, Japan) were housed in an
air-conditioned room maintained at 22 ±2˚C under a 12-h dark/light cycle, with free access to
a standard diet (CLEA Japan, Tokyo, Japan) and tap water. The mice were randomly divided
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 2 / 17
preparation of the manuscript. The specific roles of
these authors were articulated in the ‘author
contributions’ section.
Competing interests: HO, KT, and YO received
grants from Wakasa Seikatsu Co., Ltd. SM was
previously, and SK is currently, an employee of
Wakasa Seikatsu Co., Ltd. This does not alter our
adherence to PLOS ONE policies on sharing data
and materials.
into three groups (non-light exposed control and light-exposed mice treated with vehicle, as
well as light-exposed mice treated with either bilberry extract or NAC, as described below). All
animal experiments were conducted in accordance with the Association for Research in Vision
and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research,
and the guidelines of the Animal Care Committee of Keio University. The animal experimen-
tal protocols were approved by the Animal Care Committee of Keio University (Approval No.
08002).
Light exposure
Mice were exposed to light as previously described [15,3335]. Briefly, the mice were dark-
adapted by maintaining them in complete darkness for 12 h before light exposure. Their pupils
were then dilated with a mixture of 0.5% each of tropicamide and phenylephrine (Mydrin-P
1
,
Santen Pharmaceutical, Osaka, Japan) just before exposure to light. The mice were exposed to
a white fluorescent lamp (FHD100ECW, Panasonic, Osaka, Japan) at 3000 lux for 1 h (starting
at 0900) in a dedicated exposure box maintained at 22 ±2˚C containing stainless steel mirrors
on each wall and the floor (Tinker N, Kyoto, Japan). After light exposure, the mice were
returned to their cages and maintained under a dim cyclic light (5 lux, 12 h on/off) until they
were euthanized with sodium pentobarbital (70 mg/kg BW) at specific time points according
to the experimental protocol. The control mice without light exposure were also maintained
under dim cyclic lighting and were euthanized with sodium pentobarbital (70 mg/kg BW) at
corresponding times.
Administration of bilberry extract and NAC
Bilberry extract (containing about 39% anthocyanins) was provided by Wakasa Seikatsu
(Kyoto, Japan), and was dissolved in phosphate-buffered saline (PBS) and orally administered
through a stomach tube (750 mg/kg body weight, BW) at 12 h and 30 min before light expo-
sure. Mice receiving vehicle received PBS alone. Alternatively, intraperitoneal injection of
NAC (Nakalai Tesque, Kyoto, Japan, 250 mg/kg BW) diluted in PBS was administered at 12 h
and 30 min before light exposure. Again, mice receiving vehicle received PBS alone. The dose
and time points of bilberry extract and NAC administration were chosen based on optimiza-
tion studies performed previously by our group [16,29]. The control mice without light expo-
sure received either two oral administrations or intraperitoneal injections of vehicle at the
corresponding time points in the respective experiments.
Electroretinograms (ERG)
The mice were dark-adapted for at least 12 h and then placed under dim red illumination
before conducting ERGs. The mice were anesthetized with 60 mg/kg BW sodium pentobarbi-
tal (Dainippon Sumitomo Pharmaceutical Co., Osaka, Japan) and kept on a heating pad
throughout the experiment. Mouse pupils were dilated using a single drop of a mixture of
0.5% each of tropicamide and phenylephrine. The ground and reference electrodes were then
placed on the tail and in the mouth, respectively, while the active gold wire electrodes were
placed on the cornea.
The recordings were made with a PowerLab System 2/25 (AD Instruments, New South
Wales, Australia). Full-field scotopic ERGs were measured in response to a flash at intensities
ranging of 2.12 to 2.89 log cd s/m
2
. Photopic ERGs were measured after 10 min of light adap-
tation. Flash stimuli ranging from 0.48 and 1.48 log cd s/m
2
were used for recording with a
background of 30 cd s/m
2
(PowerLab System 2/25; AD Instruments; New South Wales, Aus-
tralia), and the results of 20 single-flash trace trials were averaged. The responses were
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 3 / 17
differentially amplified and filtered through a digital bandpass filter ranging from 0.3 to 1000
Hz. Each stimulus was delivered using a commercial stimulator (Ganzfeld System SG-2002;
LKC Technologies, Inc., Gaithersburg, MD) and the a-wave amplitude was measured from the
baseline to the trough while the b-wave amplitude was measured from the trough of the a-
wave to the peak of the b-wave. The implicit times of the a- and b-waves were measured from
the onset of the stimulus to the peak of each wave. The peak points were automatically indi-
cated by the system and confirmed by the examiner. The experiments were performed 4 days
after light exposure when the photo-stress-induced ERG change was obvious according to our
previous reports [15,3335].
Histological analyses
Mouse eyes were enucleated and fixed in 4% paraformaldehyde (PFA) overnight at 4˚C. After
fixation, the eyes were embedded in paraffin (Sakura Finetek Japan, Tokyo, Japan), and the
sections (6- to 8-μm thick), which included the optic nerve head to the most peripheral region
of the retina, were deparaffinized by passing them through the following treatment series
thrice for 5 min each: xylene, 1:1 xylene-alcohol, 100% ethanol, 90% ethanol, 70% ethanol, and
50% ethanol. The ethanol was then removed by rinsing with distilled water.
Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)
nick-end labeling (TUNEL) assay was performed using the ApopTag red apoptosis detection
kit (Millipore, Bedford, MA, USA) according to the manufacturer’s protocol. The nuclei were
stained with Cellstain-4’,6-diamidino-2-phenylindole (DAPI) solution (2 μg/mL, Dojindo
Molecular Technologies, Kumamoto, Japan). The TUNEL-positive cells in each section were
counted and averaged. All the groups were analyzed 2 days after light exposure, which was a
time point where a clear change in photoreceptor apoptosis was previously detected [15,33
35].
To measure the thickness and length of retinal components, sections were stained with
hematoxylin and eosin or incubated with either a rabbit anti-rhodopsin antibody (1:10,000;
Cosmo Bio Co., Ltd., Tokyo, Japan) or a rabbit anti-blue opsin antibody (1:500; Merck Milli-
pore, Darmstadt, Germany) overnight at 4˚C. Antibody-incubated sections were then incu-
bated with Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen Japan, Tokyo, Japan) with
subsequent counterstaining with DAPI solution (2 μg/mL) for 1 h at room temperature. Mea-
surements were performed using ImageJ software (developed by Wayne Rasband, National
Institutes of Health, Bethesda, MD, USA; available at http://rsb.info.nih.gov/ij/index.html),
and averaged as described previously [16,34,36,37]. All the groups were analyzed 4 days after
light exposure, which was a time point where clear changes in the photoreceptors were previ-
ously detected [15,3335].
For double immunostaining of activating transcription factor 4 (ATF4) and glutamine
synthetase, sections obtained 12 h after light exposure were immersed in preheated antigen-
retrieval Immunosaver solution (Wako, Tokyo, Japan) and boiled for 45 min at 90˚C. Next,
sections were stained sequentially with a rabbit anti-ATF4 antibody (1:100; CST JAPAN,
Tokyo, Japan) and a mouse anti-glutamine synthetase antibody (1:500; BD Transduction
Laboratories, San Jose, CA, USA) overnight at 4˚C. Signals were obtained using Alexa
488-conjugated goat anti-rabbit IgG and Alexa 555-conjugated goat anti-mouse IgG, respec-
tively, with subsequent counterstaining with DAPI solution (2 μg/mL), all for 1 h at room
temperature.
Sections were examined under a microscope equipped with a digital camera (Olympus Co.,
Tokyo, Japan), and fluorescent images were obtained using a confocal microscope (TCS-SP5;
Leica, Tokyo, Japan).
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 4 / 17
ROS measurement
The mouse eyes were enucleated and the retinas were immediately frozen and homogenized in
PBS using a Mixer Mill MM 300 homogenizer (Qiagen Inc., Chatsworth, CA, USA). The tissue
homogenates were incubated with 2’,7’-dichlorofluorescein-diacetate (DCFH-DA) (50 μM,
Sigma-Aldrich, St. Louis, MO, USA) at 37˚C in the dark for 1 h. Then, the samples were
centrifuged at 3000 rpm for 5 min at 4˚C. Finally, pellets were washed with cold PBS twice
and resuspended in cold PBS. DCFH-DA fluorescence was measured using Synergy4 (BioTek
Instruments, Inc., Winooski, VT, USA) at 1, 3, and 6 h after light exposure. The data at 6 h,
when significant differences between the groups were observed, are presented.
Real-time reverse-transcription polymerase chain reaction (RT-PCR)
Total RNA was isolated from mouse retinas or RPE-choroid complex with TRIzol reagent
(Life Technologies, Carlsbad, CA, USA). RNA concentration was measured using a NanoDrop
1000 (Thermo Fisher Scientific, Waltham, MA, USA) and 1 μg RNA was reverse-transcribed
using the SuperScript VILO master mix (Life Technologies, Carlsbad, CA, USA) according to
the manufacturer’s instructions. The following primer sequences were used: glyceraldehyde
3-phosphate dehydrogenase (GAPDH), forward 5'-AACTTCGGCCCCATCTTCA-3' and
reverse 5'-GATGACCCTTTTGGCTCCAC-3'; ho-1, forward 5'-ACGCATATACCCGCTACC
TG-3' and reverse 5'-CCAGAGTGTTCATTCGAGCA-3'; bip, forward 5'-TGCAGCAGGAC
ATCAAGTTC-3' and reverse 5'-TTTCTTCTGGGGCAAATGTC-3', chop, forward 5'-
CTGGAAGCCTGGTATGAGGA-3' and reverse 5'-GGACGCAGGGTCAAGAGTAG-3'; atf4,
forward 5'-GAAACCTCATGGGTTCTCCA-3' and reverse 5'-TCCATTTTCTCCAACATCC
A-3'; xbp1s, forward 5'-CTGAGTCCGCAGCAGGTG-3' and reverse 5'-TGCCCAAAAGGA
TATCAGACT-3'; and sec24d, forward 5'-CACCACAGCTCCAGAGGAAG-3' and reverse
5'-GCCCCTGGTATTGGTCGTAT-3'. Real-time PCR was performed using the StepOnePlus
PCR system (Applied Biosystems, Foster City, CA, USA) and gene expression was quantified
using the ΔΔCT method. All mRNA levels were normalized to those of GAPDH as described
previously [16,34,38,39]. The mRNA levels were measured at 6, 12, and 24 h after light expo-
sure, and the data at 12 h, when significant differences between the groups were observed, are
presented.
Flat mount immunohistochemistry
Mouse eyes were enucleated and the cornea, lens, vitreous, and retina were removed prior to
preparing flat-mount eyecups 24 h after light exposure. The eyecups were prefixed with 4%
PFA for 1 h at 4˚C and flattened by making four radial cuts before being returned to 4% PFA.
The samples were blocked with 5% BSA in PBS for 30 min at room temperature and then incu-
bated overnight with anti-ZO-1 antibody (1:500; Invitrogen) at 4˚C. Signals were obtained
using an Alexa Fluor 488 goat anti-rabbit IgG antibody and DAPI solution (2 μg/mL). Fluores-
cent images of the flat mounts were obtained using a confocal microscope. The number of
intact RPE cells visible under ZO-1 immunostaining of the intracellular face of an entire cell
membrane and the total RPE cells were counted in four quadrants of a 400-μm square in the
central part of the retina (superior, inferior, nasal, and temporal).
Statistical analyses
All results are expressed as mean ±standard deviation (SD). A one-way analysis of variance
(ANOVA) with the Tukey’s post hoc test was used to assess the statistical significance of the
differences, and results with P-values <0.05 were considered significant.
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 5 / 17
Results
Suppression of photo-induced visual function impairment by bilberry
extract
The effects of bilberry extract on visual function in the light-exposed mice were determined by
performing ERG 4 days after light exposure (Fig 1a–1h). In scotopic ERG (Fig 1a–1e), the
amplitudes of the a- and b-waves, which show rod photoreceptor function and subsequent ret-
inal neuronal activity, respectively, were lower in the vehicle-treated light-exposed mice than
in the vehicle-treated unexposed mice kept in dim cyclic light, indicating that these illumina-
tion conditions were stressful to the retina. However, the reduction in the amplitude was atten-
uated by treatment with the bilberry extract (Fig 1b and 1c). In addition, the b-wave amplitude
in photopic ERG, which represents cone photoreceptor system function [40,41], decreased
upon light exposure. However, this reduction was attenuated by the bilberry extract (Fig 1f
and 1g). No changes were observed in the implicit times of the a- and b-waves between the
groups (Fig 1d, 1e and 1h). These data suggest that the bilberry extract attenuated visual func-
tion impairment of both rod and cone photoreceptors in the photo-stressed mice.
Fig 1. Suppression of photo-induced visual function impairment by bilberry extract. Analysis of full-field scotopic (a-e) and photopic
(f-h) ERGs following light exposure. Representative waveforms of scotopic (a) and photopic (f) ERG from individual mice treated with vehicle
or bilberry extract in response to one flash 4 days after light exposure. The amplitude of a- and b-waves in scotopic ERG (b and c) and b-
wave in photopic ERG (g) was decreased by light exposure, and these changes were attenuated by treatment with bilberry extract. No
differences were observed in a- or b-wave implicit times in both scotopic and photopic ERG (d, e and h). n = 6/ group. ERG,
electroretinogram. **P<0.01 and *P<0.05.
https://doi.org/10.1371/journal.pone.0178627.g001
Bilberry extract protects retina from photo-stress
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Reduction in photo-induced apoptotic cells by bilberry extract
Next, we analyzed the effect of the bilberry extract on histological changes in the photo-
stressed retina by measuring the number of TUNEL-positive and apoptotic cells 2 days after
light exposure (Fig 2a). Apoptotic cells were extensively observed in the outer nuclear layer
(ONL), corresponding to the photoreceptor layer of the vehicle-treated light-exposed mice,
while the number was negligible in those not exposed to light. However, bilberry treatment
substantially reduced the number of apoptotic cells in the photo-stressed retinas.
Suppression of photoreceptor loss and damage by bilberry extract
The measurement of ONL thickness 4 days after light exposure revealed that it was signifi-
cantly thinner in the vehicle-treated light-exposed mice than in the non-light exposed mice
kept in dim cyclic light (Fig 2b). However, administration of the bilberry extract significantly
attenuated ONL thinning, indicating that it attenuated the photoreceptor loss due to photo-
stress. Moreover, the length of the rod outer segments (OSs), where rhodopsin was concen-
trated, decreased 4 days after light exposure in the vehicle-treated mice, while this phenome-
non was clearly suppressed in the bilberry extract-treated mice (Fig 2c). Moreover, the length
of blue cone OSs evaluated by blue opsin staining showed light-induced shortening, which was
suppressed by the bilberry extract (Fig 2d). These results indicated that photo-induced photo-
receptor degeneration was ameliorated by the bilberry extract.
Antioxidative effects of bilberry extract on the retina
To evaluate the degree of retinal oxidative stress, ROS levels were measured using a DCFH-DA
probe (Fig 3). The photo-induced increase in fluorescence intensity of retinas at 6 h after light
exposure was significantly attenuated by treatment with the bilberry extract compared to that
reported with vehicle treatment.
NAC, a well-known antioxidant that plays a role in the maintenance and metabolism of
glutathione, an intrinsic antioxidant [42], has been proven to reduce ROS levels in the retina
[16]. Therefore, we measured retinal ROS levels in light-exposed mice treated with NAC
and showed comparable levels of reduction to that in the bilberry extract-treated animals
(Fig 3). The administration protocol for NAC (via intraperitoneal injection) differed from
that for bilberry extract (oral), however, ROS levels did not differ between the retinas of
mice treated either with intraperitoneal or oral control PBS, both under light-exposed and
non-light-exposed conditions, respectively (S1 Fig). The results suggest that the bilberry
extract showed an antioxidant activity in a manner similar to that of NAC under these
conditions.
Photo-induced ER stress and the effect of bilberry extract
To determine whether ER stress is involved in photo-induced retinal degeneration, we ana-
lyzed markers of ER stress using real-time RT-PCR after verifying the mRNA level of an oxi-
dative stress marker, ho-1 [43] (Fig 4a). Interestingly, the retinal mRNA levels of bip, chop,
atf4, spliced-xbp1 (xbp1s), and sec24d were all upregulated 12 h after light exposure (Fig
4b–4f). However, these increases in mRNA expression were attenuated by bilberry extract
treatment, indicating that the bilberry extract attenuated ER stress in photo-stressed retinas.
NAC treatment also induced a similar effect to bilberry extract treatment (Fig 4b–4f,S2
Fig).
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 7 / 17
Fig 2. Suppression of photo-induced histological changes in the retina by bilberry extract. (a) TUNEL assay
performed 2 days after light exposure. TUNEL-positive cells (red) appeared only in the ONL following light exposure.
The number of apoptotic cells was significantly reduced by bilberry extract. DAPI staining of the control is shown as a
guide for retinal layers. (b) H&E staining of retinal sections 4 days after light exposure. ONL thickness was lower in
vehicle-treated, light-exposed mice than in vehicle-treated, non-light-exposed mice. Photo-induced ONL thinning was
significantly attenuated by bilberry extract treatment. (c and d) Rhodopsin (c) and blue opsin (d) immunostaining 4 days
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 8 / 17
Suppression of photo-induced translocation of ATF4 in Mu
¨ller glial cells
by bilberry extract
One pathway of ER stress induces the transcription factor ATF4 [44]. Interestingly, the trans-
location of ATF4 to the nuclei was observed in Mu¨ller glial cells, as determined by the expres-
sion of its marker, glutamine synthetase, 12 h after light exposure (Fig 5a and 5b). However,
translocation was suppressed by both bilberry extract (Fig 5c) and NAC (Fig 5d).
Suppression of photo-induced RPE damage by bilberry extract
Photo-induced oxidative stress disrupts the tight junctions of the RPE [45], which maintains
photoreceptor conditions. Immunostaining for a tight junction marker, ZO-1, in flat mount
samples 24 h after light exposure showed a decrease in RPE cells with an intact ZO-1 pattern at
all cell edges per total RPE cell (Fig 6a and 6b). During the study time-period, there was no
obvious nuclear condensation in the RPE cells of animals exposed to light and treated with
either vehicle or bilberry extract, suggesting a lack of RPE cell death. Under this condition, the
expression of an oxidative stress marker, ho-1, was increased by light exposure but suppressed
by bilberry extract (Fig 6c). There was no difference in the mRNA levels of ER stress markers
under this condition (data not shown).
after light exposure. The OS lengths of both rod (c) and cone (d) photoreceptors were lower in vehicle-treated light-
exposed mice than in vehicle-treated non-light exposed mice. Photo-induced OS shortening was significantly
attenuated by bilberry extract treatment (n = 4/ group). Scale bar, 25 μm. ONL, outer nuclear layer; DAPI,
4’,6-diamidino-2-phenylindole; H&E, hematoxylin and eosin; IS, inner segment; OS, outer segment. **P<0.01.
https://doi.org/10.1371/journal.pone.0178627.g002
Fig 3. Inhibition of photo-induced ROS accumulation in the retina by bilberry extract. Retinal ROS
levels were evaluated by DCFH-DA fluorescence. Photo-induced increases in retinal ROS levels were
suppressed by bilberry extract and NAC treatment 6 h after light exposure; n = 4/ group. ROS, reactive
oxygen species; DCFH-DA, 2’,7’-dichlorofluorescein-diacetate; NAC, N-acetyl-l-cysteine. *P<0.05.
https://doi.org/10.1371/journal.pone.0178627.g003
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 9 / 17
Discussion
In this study, we showed the suppression of light-induced visual impairment (Fig 1) and rod
and cone photoreceptor damage/apoptosis (Fig 2) by the administration of bilberry extract,
which reduced light-induced retinal ROS levels and ER stress (Figs 3and 4, respectively). A
standard antioxidant, NAC, also similarly attenuated ER stress caused by light exposure (Figs 3
and 4, respectively). One ER stress marker, ATF4, showed a photo-induced nuclear transloca-
tion in Mu¨ller glial cells, which was suppressed by both bilberry extract and NAC (Fig 5). The
photo-induced disruption of tight junctions in the RPE was also attenuated by bilberry extract
(Fig 6).
The scotopic ERG results showed that both the a- and b-wave amplitudes decreased after
light exposure, indicating that rod photoreceptor function was impaired. The a-wave abnor-
malities represented an original change in the photoreceptors, and b-wave abnormalities most
likely were responsive to the decrease in the input from the photoreceptors to downstream
retinal neurons, thus reflecting the visual response to damaged photoreceptors. Moreover, the
photopic ERG recorded after light adaption showed impairment of cone photoreceptor
function. In photopic ERG, the b-wave is commonly regarded as an indicator of cone system
function because the a-wave amplitude is too small to evaluate, and because only cone
Fig 4. Suppression of photo-induced retinal oxidative and ER stress by both bilberry extract or NAC. (a–f) mRNA expression of
markers of oxidative and ER stress was measured using real-time RT-PCR. Expression of (a) ho-1, (b) bip, (c) chop, (d) aft4, (e) xbp1s, and
(f) sec24d mRNA was upregulated at 12 h in photo-stressed retinas; however, treatment with bilberry extract attenuated these increases in
mRNA expression. n = 5/ group. ER, endoplasmic reticulum. **P<0.01 and *P<0.05.
https://doi.org/10.1371/journal.pone.0178627.g004
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 10 / 17
photoreceptors (and not rod photoreceptors) respond under photopic conditions [40,41].
This photoreceptor disorder was further evidenced by the TUNEL-positive cells in the photo-
receptor layer preceding visual function impairment and a clear decrease in ONL thickness
representing photoreceptor loss. The shortening of both rod and cone OSs demonstrated a
reduction in photon receptors in the remaining photoreceptors, and was likely involved in the
impaired photoreceptor responses observed in the ERG. These results showing photo-induced
rod photoreceptor degeneration, including visual function impairment and histological
Fig 5. Suppression of nuclei ATF4 in Mu¨ller glial cells by bilberry extract. (a-d) Co-immunostaining for
ATF4 (green) and glutamine synthetase (red), a marker for Mu¨ller glial cell, 12 h after light exposure. ATF4
staining was observed in the nuclei (DAPI, blue) of Mu¨ller glial cells after light exposure; however, this
translocation was suppressed by bilberry extract (c) or NAC (d). n = 5/ group. Scale bar, 10 μm.
https://doi.org/10.1371/journal.pone.0178627.g005
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 11 / 17
changes, were consistent with the results of previous studies [13,15,16,33,34]. Similar results
were obtained in the current study with respect to cone photoreceptors.
Bilberry extract treatment attenuated all the observed photo-induced photoreceptor degen-
eration, including the increased ROS levels in the retina of photo-stressed mice. Furthermore,
the ROS levels in the bilberry extract-treated retinas were almost equal to those observed in the
retinas of light-exposed mice treated with a dosage of NAC previously reported to suppress
photo-induced photoreceptor degeneration [16]. These data and previous reports showing in
vitro antioxidant effects support the idea that the neuroprotective effects of the bilberry extract
against photoreceptor apoptosis and degeneration observed in the current study may be attrib-
utable to its antioxidant properties.
Increases in retinal ROS were observed as early as 6 h after light exposure, which was sup-
pressed by the bilberry extract. The oral consumption of bilberry extract rapidly increased the
serum levels of the major component of bilberry extract, anthocyanins [46], which peaked 15–
Fig 6. Suppression of photo-induced tight junction disruption in the RPE by bilberry extract. (a) Immunostaining for ZO-1 in
flat mount samples 24 h after light exposure showed a decrease in RPE cells. (b) The number of RPE cells with an intact ZO-1
pattern at all cell edges per total RPE cells is graphically represented. (c) ho-1 mRNA expression was measured using real-time
RT-PCR. RPE, retinal pigment epithelium; n = 6/ group. **p<0.01. Scale bar, 20 μm.
https://doi.org/10.1371/journal.pone.0178627.g006
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 12 / 17
30 min after ingestion in mice [47]. The rapid systemic absorption of anthocyanins may have
contributed to the effective suppression of the early increase in retinal ROS levels after light
exposure, and therefore attenuated photoreceptor degeneration.
Although the increase in retinal ROS levels were as much as 20%, this amount may have
been sufficient to cause retinal degeneration. The retinal protective effects of bilberry extract
and NAC were most likely attributable to the removal of locally accumulated ROS. Alterna-
tively, Narimatsu et al. showed that angiotensin II type 1 receptor (AT1R) signaling occurs
upstream of photo-induced ROS generation in the retina [16]. Furthermore, AT1R blockade
efficiently suppressed light-induced photoreceptor apoptosis, OS shortening, and functional
impairment [16]. A subgroup of the anthocyanins, proanthocyanidins, has been reported to
bind to and inhibit AT1R [48,49]. It would be interesting to study the effects of the bilberry
extract on AT1R and ROS generation in future research.
Treatment with the bilberry extract, as well as NAC, reduced ROS accumulation in the
retina and attenuated ER stress, suggesting that photo-induced ER stress was activated by
ROS. This was consistent with a previous report on brain neuronal death showing that ER
stress in an ischemia/reperfusion model was suppressed in transgenic mice overexpressing
superoxide dismutase 1, an antioxidant enzyme [50]. The relationship between oxidative and
ER stress may be explained by the oxidation of both ER-resident and other proteins trans-
ported by the ER, which may activate the UPR [51,52], although the involvement of a ROS-
independent pathway in ER stress activation cannot be excluded after light exposure. There-
fore, reducing oxidative stress may be important for maintaining normal ER functions,
including UPR.
Retinitis pigmentosa is a photoreceptor degenerative disease caused by genetic mutations.
In a mouse model of rd10 retinitis pigmentosa, the suppression of ER stress by inhibiting aden-
osine triphosphatase (ATPase) activity using a valosin-containing protein attenuated photore-
ceptor loss [53]. Moreover, photoreceptors with a gene mutation for rhodopsin differentiated
from induced pluripotent stem (iPS) cells derived from an affected patient showed an increase
in ER stress and apoptosis, which were attenuated by rapamycin [54], suppressing ER stress
[55], suggesting the involvement of ER stress in retinitis pigmentosa-induced photoreceptor
death. In the current study, light exposure induced CHOP in the retina, suggesting that the
misfolded and unfolded proteins may have increased in the retina regardless of the presence of
mutation. One of the mechanisms underlying the progression of retinitis pigmentosa caused
by light exposure may involve a scenario where photo-induced ER stress may easily exceed the
capacity of UPR in situations where UPR is already in effect owing to mutation-coded abnor-
mal proteins.
In addition, Mu¨ller glial cells were involved in photo-induced ER stress in the current
study, suggesting the involvement of impaired photoreceptor-Mu¨ller glia interaction which
induced abnormality in photoreceptor maintenance.
The disruption of tight junctions in the RPE by photo-induced oxidative stress was also
suppressed by bilberry extract, although the pathway may not involve ER stress (data not
shown). Because RPE also maintains photoreceptor survival [56], bilberry extract may also
have exerted a protective effect on photoreceptors by protecting RPE conditions.
In this study, the reduction of oxidative stress subsequently reduced ER stress and apoptosis
in photo-stressed retinas. Moreover, the micronutrient-containing bilberry extract supplement
clearly acted as an antioxidant in the treatment of retinal oxidative stress, reducing ER stress
and apoptotic signaling (S3 Fig). The current study will facilitate the elucidation of photo-
stress pathogenesis and development of a therapeutic approach for preventing the progression
of retinal degeneration.
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 13 / 17
Supporting information
S1 Fig. Equivalent ROS levels after treatment with control vehicle by intraperitoneal or
oral administration. Retinal ROS levels evaluated by DCFH-DA fluorescence were similar fol-
lowing treatment with control vehicle by either the intraperitoneal (IP) or oral route, 6 h after
light exposure; n = 4/ group. ROS, reactive oxygen species; DCFH-DA, 2’,7’-dichlorofluores-
cein-diacetate. P<0.05.
(TIF)
S2 Fig. Equivalent levels of oxidative and ER stress markers after treatment with control
vehicle by intraperitoneal or oral administration. (a–f) mRNA expression of markers of oxi-
dative and ER stress was measured using real-time RT-PCR. Expression of (a) ho-1, (b) bip, (c)
chop, (d) aft4, (e) xbp1s, and (f) sec24d mRNA was similar under the control conditions of oral
or intraperitoneal (IP) PBS administration for 12 h in photo-stressed retinas. n = 5/ group. ER,
endoplasmic reticulum. P<0.01 and P<0.05.
(TIF)
S3 Fig. Model of the mechanisms of photo-induced visual function impairment and the
protective role of bilberry extract. Excessive light exposure causes ROS accumulation, which
induces ER stress to initiate photoreceptor apoptosis and degeneration and subsequent visual
function impairment. A pathway for ROS-independent ER stress could not be excluded. ROS-
dependent RPE changes may also cause photoreceptor disorder. Bilberry extract at least partly
reduced ROS and ER stress to protect photoreceptors and visual function.
(TIF)
Acknowledgments
We would like to thank Professor Masato Yasui (Department of Pharmacology, Keio Univer-
sity School of Medicine) and his laboratory members, as well as all the members of the Retinal
Cell Biology Laboratory, for their kind assistance during this study.
Author Contributions
Funding acquisition: YO.
Investigation: HO TO HK ET SM.
Project administration: YO.
Resources: SK.
Supervision: NN SK KT.
Writing – original draft: HO.
Writing – review & editing: YO.
References
1. Kishi S, Li D, Takahashi M, Hashimoto H. Photoreceptor damage after prolonged gazing at a computer
game display. Jpn J Ophthalmol. 2010; 54(5):514–6. https://doi.org/10.1007/s10384-010-0849-2
PMID: 21052924.
2. Reme CE, Grimm C, Hafezi F, Marti A, Wenzel A. Apoptotic cell death in retinal degenerations. Prog
Retin Eye Res. 1998; 17(4):443–64. PMID: 9777646.
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 14 / 17
3. Suzuki M, Tsujikawa M, Itabe H, Du ZJ, Xie P, Matsumura N, et al. Chronic photo-oxidative stress and
subsequent MCP-1 activation as causative factors for age-related macular degeneration. J Cell Sci.
2012; 125(Pt 10):2407–15. https://doi.org/10.1242/jcs.097683 PMID: 22357958.
4. McCarty CA, Mukesh BN, Fu CL, Mitchell P, Wang JJ, Taylor HR. Risk factors for age-related maculo-
pathy: the Visual Impairment Project. Arch Ophthalmol. 2001; 119(10):1455–62. PMID: 11594944.
5. Nag TC, Wadhwa S. Ultrastructure of the human retina in aging and various pathological states. Micron.
2012; 43(7):759–81. https://doi.org/10.1016/j.micron.2012.01.011 PMID: 22445096.
6. Paskowitz DM, LaVail MM, Duncan JL. Light and inherited retinal degeneration. Br J Ophthalmol. 2006;
90(8):1060–6. https://doi.org/10.1136/bjo.2006.097436 PMID: 16707518.
7. Fain GL. Why photoreceptors die (and why they don’t). Bioessays. 2006; 28(4):344–54. https://doi.org/
10.1002/bies.20382 PMID: 16547945.
8. Fahim AT, Daiger SP, Weleber RG. Retinitis Pigmentosa Overview. In: Pagon RA, Adam MP, Ardinger
HH, Wallace SE, Amemiya A, Bean LJH, et al., editors. GeneReviews(R). Seattle (WA) 1993.
9. Armstrong RA, Mousavi M. Overview of Risk Factors for Age-Related Macular Degeneration (AMD). J
Stem Cells. 2015; 10(3):171–91. PMID: 27125062.
10. Sui GY, Liu GC, Liu GY, Gao YY, Deng Y, Wang WY, et al. Is sunlight exposure a risk factor for age-
related macular degeneration? A systematic review and meta-analysis. Br J Ophthalmol. 2013; 97
(4):389–94. https://doi.org/10.1136/bjophthalmol-2012-302281 PMID: 23143904.
11. Marquioni-Ramella MD, Suburo AM. Photo-damage, photo-protection and age-related macular degen-
eration. Photochem Photobiol Sci. 2015; 14(9):1560–77. https://doi.org/10.1039/c5pp00188a PMID:
26198091.
12. Maeda A, Maeda T, Golczak M, Chou S, Desai A, Hoppel CL, et al. Involvement of all-trans-retinal in
acute light-induced retinopathy of mice. J Biol Chem. 2009; 284(22):15173–83. https://doi.org/10.1074/
jbc.M900322200 PMID: 19304658.
13. Wenzel A, Reme CE, Williams TP, Hafezi F, Grimm C. The Rpe65 Leu450Met variation increases reti-
nal resistance against light-induced degeneration by slowing rhodopsin regeneration. J Neurosci. 2001;
21(1):53–8. PMID: 11150319.
14. Duprez L, Wirawan E, Vanden Berghe T, Vandenabeele P. Major cell death pathways at a glance.
Microbes Infect. 2009; 11(13):1050–62. https://doi.org/10.1016/j.micinf.2009.08.013 PMID: 19733681.
15. Sasaki M, Yuki K, Kurihara T, Miyake S, Noda K, Kobayashi S, et al. Biological role of lutein in the light-
induced retinal degeneration. J Nutr Biochem. 2012; 23(5):423–9. https://doi.org/10.1016/j.jnutbio.
2011.01.006 PMID: 21658930.
16. Narimatsu T, Ozawa Y, Miyake S, Nagai N, Tsubota K. Angiotensin II type 1 receptor blockade sup-
presses light-induced neural damage in the mouse retina. Free Radic Biol Med. 2014; 71:176–85.
https://doi.org/10.1016/j.freeradbiomed.2014.03.020 PMID: 24662196.
17. Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat
Rev Mol Cell Biol. 2012; 13(2):89–102. https://doi.org/10.1038/nrm3270 PMID: 22251901.
18. Kim I, Xu W, Reed JC. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic
opportunities. Nat Rev Drug Discov. 2008; 7(12):1013–30. https://doi.org/10.1038/nrd2755 PMID:
19043451.
19. Griciuc A, Aron L, Ueffing M. ER stress in retinal degeneration: a target for rational therapy? Trends Mol
Med. 2011; 17(8):442–51. https://doi.org/10.1016/j.molmed.2011.04.002 PMID: 21620769.
20. Hiramatsu N, Chiang WC, Kurt TD, Sigurdson CJ, Lin JH. Multiple Mechanisms of Unfolded Protein
Response-Induced Cell Death. Am J Pathol. 2015; 185(7):1800–8. https://doi.org/10.1016/j.ajpath.
2015.03.009 PMID: 25956028.
21. Nguyen AT, Campbell M, Kiang AS, Humphries MM, Humphries P. Current therapeutic strategies for
P23H RHO-linked RP. Adv Exp Med Biol. 2014; 801:471–6. https://doi.org/10.1007/978-1-4614-3209-
8_60 PMID: 24664733.
22. Li GY, Fan B, Jiao YY. Rapamycin attenuates visible light-induced injury in retinal photoreceptor cells
via inhibiting endoplasmic reticulum stress. Brain Res. 2014; 1563:1–12. https://doi.org/10.1016/j.
brainres.2014.02.020 PMID: 24607296.
23. Nakanishi T, Shimazawa M, Sugitani S, Kudo T, Imai S, Inokuchi Y, et al. Role of endoplasmic reticulum
stress in light-induced photoreceptor degeneration in mice. J Neurochem. 2013; 125(1):111–24. https://
doi.org/10.1111/jnc.12116 PMID: 23216380.
24. Chen Y, Perusek L, Maeda A. Autophagy in light-induced retinal damage. Exp Eye Res. 2016; 144:64–
72. https://doi.org/10.1016/j.exer.2015.08.021 PMID: 26325327.
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 15 / 17
25. Chen Y, Sawada O, Kohno H, Le YZ, Subauste C, Maeda T, et al. Autophagy protects the retina from
light-induced degeneration. J Biol Chem. 2013; 288(11):7506–18. https://doi.org/10.1074/jbc.M112.
439935 PMID: 23341467.
26. Ozawa Y, Kawashima M, Inoue S, Inagaki E, Suzuki A, Ooe E, et al. Bilberry extract supplementation
for preventing eye fatigue in video display terminal workers. J Nutr Health Aging. 2015; 19(5):548–54.
https://doi.org/10.1007/s12603-014-0573-6 PMID: 25923485.
27. Ogawa K, Kuse Y, Tsuruma K, Kobayashi S, Shimazawa M, Hara H. Protective effects of bilberry and
lingonberry extracts against blue light-emitting diode light-induced retinal photoreceptor cell damage in
vitro. BMC Complement Altern Med. 2014; 14:120. https://doi.org/10.1186/1472-6882-14-120 PMID:
24690313.
28. Milbury PE, Graf B, Curran-Celentano JM, Blumberg JB. Bilberry (Vaccinium myrtillus) anthocyanins
modulate heme oxygenase-1 and glutathione S-transferase-pi expression in ARPE-19 cells. Invest
Ophthalmol Vis Sci. 2007; 48(5):2343–9. https://doi.org/10.1167/iovs.06-0452 PMID: 17460300.
29. Miyake S, Takahashi N, Sasaki M, Kobayashi S, Tsubota K, Ozawa Y. Vision preservation during retinal
inflammation by anthocyanin-rich bilberry extract: cellular and molecular mechanism. Lab Invest. 2012;
92(1):102–9. https://doi.org/10.1038/labinvest.2011.132 WOS:000298743600010. PMID: 21894150
30. Brader L, Overgaard A, Christensen LP, Jeppesen PB, Hermansen K. Polyphenol-rich bilberry amelio-
rates total cholesterol and LDL-cholesterol when implemented in the diet of Zucker diabetic fatty rats.
Rev Diabet Stud. 2013; 10(4):270–82. https://doi.org/10.1900/RDS.2013.10.270 PMID: 24841880.
31. Hoggard N, Cruickshank M, Moar KM, Bestwick C, Holst JJ, Russell W, et al. A single supplement of a
standardised bilberry (Vaccinium myrtillus L.) extract (36% wet weight anthocyanins) modifies glycae-
mic response in individuals with type 2 diabetes controlled by diet and lifestyle. J Nutr Sci. 2013; 2:e22.
https://doi.org/10.1017/jns.2013.16 PMID: 25191571.
32. Kim J, Kim CS, Lee YM, Sohn E, Jo K, Kim JS. Vaccinium myrtillus extract prevents or delays the onset
of diabetes—induced blood-retinal barrier breakdown. Int J Food Sci Nutr. 2015; 66(2):236–42. https://
doi.org/10.3109/09637486.2014.979319 PMID: 25582181.
33. Kubota S, Kurihara T, Ebinuma M, Kubota M, Yuki K, Sasaki M, et al. Resveratrol prevents light-induced
retinal degeneration via suppressing activator protein-1 activation. Am J Pathol. 2010; 177(4):1725–31.
https://doi.org/10.2353/ajpath.2010.100098 PMID: 20709795.
34. Narimatsu T, Ozawa Y, Miyake S, Kubota S, Yuki K, Nagai N, et al. Biological effects of blocking blue
and other visible light on the mouse retina. Clin Experiment Ophthalmol. 2014; 42(6):555–63. https://
doi.org/10.1111/ceo.12253 PMID: 24304494.
35. Kurihara T, Omoto M, Noda K, Ebinuma M, Kubota S, Koizumi H, et al. Retinal phototoxicity in a novel
murine model of intraocular lens implantation. Mol Vis. 2009; 15:2751–61. PMID: 20019883.
36. Kamoshita M, Ozawa Y, Kubota S, Miyake S, Tsuda C, Nagai N, et al. AMPK-NF-kappaB axis in the
photoreceptor disorder during retinal inflammation. PLoS One. 2014; 9(7):e103013. https://doi.org/10.
1371/journal.pone.0103013 PMID: 25048039.
37. Okamoto T, Ozawa Y, Kamoshita M, Osada H, Toda E, Kurihara T, et al. The Neuroprotective Effect of
Rapamycin as a Modulator of the mTOR-NF-kappaB Axis during Retinal Inflammation. PLoS One.
2016; 11(1):e0146517. https://doi.org/10.1371/journal.pone.0146517 PMID: 26771918.
38. Kamoshita M, Toda E, Osada H, Narimatsu T, Kobayashi S, Tsubota K, et al. Lutein acts via multiple
antioxidant pathways in the photo-stressed retina. Sci Rep. 2016; 6:30226. https://doi.org/10.1038/
srep30226 PMID: 27444056.
39. Narimatsu T, Negishi K, Miyake S, Hirasawa M, Osada H, Kurihara T, et al. Blue light-induced inflam-
matory marker expression in the retinal pigment epithelium-choroid of mice and the protective effect of
a yellow intraocular lens material in vivo. Exp Eye Res. 2015; 132:48–51. PMID: 25576667.
40. Lei B, Yao G, Zhang K, Hofeldt KJ, Chang B. Study of rod- and cone-driven oscillatory potentials in
mice. Invest Ophthalmol Vis Sci. 2006; 47(6):2732–8. https://doi.org/10.1167/iovs.05-1461 PMID:
16723493.
41. Lei B. Rod-driven OFF pathway responses in the distal retina: dark-adapted flicker electroretinogram in
mouse. PLoS One. 2012; 7(8):e43856. https://doi.org/10.1371/journal.pone.0043856 PMID: 22937111.
42. Kerksick C, Willoughby D. The antioxidant role of glutathione and N-acetyl-cysteine supplements and
exercise-induced oxidative stress. J Int Soc Sports Nutr. 2005; 2:38–44. https://doi.org/10.1186/1550-
2783-2-2-38 PMID: 18500954.
43. Applegate LA, Luscher P, Tyrrell RM. Induction of heme oxygenase: a general response to oxidant
stress in cultured mammalian cells. Cancer Res. 1991; 51(3):974–8. PMID: 1988141.
44. Deldicque L, Bertrand L, Patton A, Francaux M, Baar K. ER stress induces anabolic resistance in mus-
cle cells through PKB-induced blockade of mTORC1. PLoS One. 2011; 6(6):e20993. https://doi.org/10.
1371/journal.pone.0020993 PMID: 21698202.
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 16 / 17
45. Narimatsu T, Ozawa Y, Miyake S, Kubota S, Hirasawa M, Nagai N, et al. Disruption of cell-cell junctions
and induction of pathological cytokines in the retinal pigment epithelium of light-exposed mice. Invest
Ophthalmol Vis Sci. 2013; 54(7):4555–62. https://doi.org/10.1167/iovs.12-11572 PMID: 23761083.
46. Burdulis D, Ivanauskas L, Dirse V, Kazlauskas S, Razukas A. Study of diversity of anthocyanin
composition in bilberry (Vaccinium myrtillus L.) fruits. Medicina (Kaunas). 2007; 43(12):971–7. PMID:
18182842.
47. Sakakibara H, Ogawa T, Koyanagi A, Kobayashi S, Goda T, Kumazawa S, et al. Distribution and excre-
tion of bilberry anthocyanins [corrected] in mice. J Agric Food Chem. 2009; 57(17):7681–6. https://doi.
org/10.1021/jf901341b PMID: 19663426.
48. Caballero-George C, Vanderheyden PM, De Bruyne T, Shahat AA, Van den Heuvel H, Solis PN, et al.
In vitro inhibition of [3H]-angiotensin II binding on the human AT1 receptor by proanthocyanidins from
Guazuma ulmifolia bark. Planta Med. 2002; 68(12):1066–71. https://doi.org/10.1055/s-2002-36344
PMID: 12494331.
49. Jaakola L, Maatta K, Pirttila AM, Torronen R, Karenlampi S, Hohtola A. Expression of genes involved in
anthocyanin biosynthesis in relation to anthocyanin, proanthocyanidin, and flavonol levels during bil-
berry fruit development. Plant Physiol. 2002; 130(2):729–39. https://doi.org/10.1104/pp.006957 PMID:
12376640.
50. Hayashi T, Saito A, Okuno S, Ferrand-Drake M, Dodd RL, Chan PH. Damage to the endoplasmic retic-
ulum and activation of apoptotic machinery by oxidative stress in ischemic neurons. J Cereb Blood Flow
Metab. 2005; 25(1):41–53. https://doi.org/10.1038/sj.jcbfm.9600005 PMID: 15678111.
51. Sozen E, Karademir B, Ozer NK. Basic mechanisms in endoplasmic reticulum stressand relation to car-
diovascular diseases. Free Radic Biol Med. 2015; 78:30–41. https://doi.org/10.1016/j.freeradbiomed.
2014.09.031 PMID: 25452144.
52. Naidoo N. ER and aging-Protein folding and the ER stress response. Ageing Res Rev. 2009; 8(3):150–
9. https://doi.org/10.1016/j.arr.2009.03.001 PMID: 19491040.
53. Ikeda HO, Sasaoka N, Koike M, Nakano N, Muraoka Y, Toda Y, et al. Novel VCP modulators mitigate
major pathologies of rd10, a mouse model of retinitis pigmentosa. Sci Rep. 2014; 4:5970. https://doi.
org/10.1038/srep05970 PMID: 25096051.
54. Yoshida T, Ozawa Y, Suzuki K, Yuki K, Ohyama M, Akamatsu W, et al. The use of induced pluripotent
stem cells to reveal pathogenic gene mutations and explore treatments for retinitis pigmentosa. Mol
Brain. 2014; 7:45. https://doi.org/10.1186/1756-6606-7-45 PMID: 24935155.
55. Appenzeller-Herzog C, Hall MN. Bidirectional crosstalk between endoplasmic reticulum stress and
mTOR signaling. Trends Cell Biol. 2012; 22(5):274–82. https://doi.org/10.1016/j.tcb.2012.02.006
PMID: 22444729.
56. D’Cruz PM, Yasumura D, Weir J, Matthes MT, Abderrahim H, LaVail MM, et al. Mutation of the receptor
tyrosine kinase gene Mertk in the retinal dystrophic RCS rat. Hum Mol Genet. 2000; 9(4):645–51.
PMID: 10699188.
Bilberry extract protects retina from photo-stress
PLOS ONE | https://doi.org/10.1371/journal.pone.0178627 June 1, 2017 17 / 17
... Here, we focused on endoplasmic reticulum (ER) stress, which is induced after light exposure (LE) [12][13][14], as well as oxidative stress [8,12,15,16]. The ER is an interconnected network of branching tubules and flattened sacs and plays a major role in biosynthesis, posttranslational modification, folding, and assembly of newly synthesized proteins [17][18][19]. ...
... Here, we focused on endoplasmic reticulum (ER) stress, which is induced after light exposure (LE) [12][13][14], as well as oxidative stress [8,12,15,16]. The ER is an interconnected network of branching tubules and flattened sacs and plays a major role in biosynthesis, posttranslational modification, folding, and assembly of newly synthesized proteins [17][18][19]. ...
... ER stress is involved in various human diseases, such as diabetes, cancer, and neurodegeneration [17][18][19][20][21][22][23]. ER stress has a close relationship with oxidative stress and inflammation in neurodegeneration and retinal diseases [12][13][14][17][18][19][20][21][22]. However, whether ER stress has a critical role and could be a therapeutic target against the influences of photo-stress have not been determined, and no methods or pharmaceuticals have been developed for its management. ...
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Exposure to excessive visible light causes retinal degeneration and may influence the progression of retinal blinding diseases. However, there are currently no applied treatments. Here, we focused on endoplasmic reticulum (ER) stress, which can cause cellular degeneration and apoptosis in response to stress. We analyzed functional, histological, and molecular changes in the light-exposed retina and the effects of administering an ER-stress inhibitor, 4-phenylbutyric acid (4-PBA), in mice. We found that light-induced visual function impairment related to photoreceptor cell loss and outer segment degeneration were substantially suppressed by 4-PBA administration, following attenuated photoreceptor apoptosis. Induction of retinal ER stress soon after light exposure, represented by upregulation of the immunoglobulin heavy chain binding protein (BiP) and C/EBP-Homologous Protein (CHOP), were suppressed by 4-PBA. Concurrently, light-induced oxidative stress markers, Nuclear factor erythroid 2–related factor 2 (Nrf2) and Heme Oxygenase 1 (HO-1), and mitochondrial apoptotic markers, B-cell lymphoma 2 apoptosis regulator (Bcl-2)-associated death promoter (Bad), and Bcl-2-associated X protein (Bax), were suppressed by 4-PBA administration. Increased expression of glial fibrillary acidic protein denoted retinal neuroinflammation, and inflammatory cytokines were induced after light exposure; however, 4-PBA acted as an anti-inflammatory. Suppression of ER stress by 4-PBA may be a new therapeutic approach to suppress the progression of retinal neurodegeneration and protect visual function against photo-stress.
... Rd10 mice (RP) [375] Prph2/rds mouse [376] Ganglion cell damage models: Glaucoma model in rats [377] Mouse, ischemia/reperfusion [378] (rasagiline + idebenone) Others: Retinal detachment: NCT02068625 (Macula off-retinal detachment) [379] Norgestrel/ Progesterone IRD and retinal damage models: Pde6b Rd10 mouse [380][381][382] Rd1 mice (L + Z + lipoic acid + glutathione + Lycium barbarum) Rd1 mouse [383] Rd10 mice [384] Acute light-induced degeneration model in mice [382,385] Ganglion cell damage models: Rat models of ocular ischemia [386] Review: [387] IRD and retinal damage models: Light-induced photoreceptor degeneration in rats (+L) [390] Light damage in rabbits [391] MNU-induced damage in rats [392] Other retinal disease models: Oxygen-induced retinopathy in mice [393] IRD and retinal damage models: Photo-stressed murine model [394] Light damage in rabbits [395] AMD models: OXYS rats [396] DR models: STZ rats [397] Other retinal disease models: Oxygen-induced retinopathy in mice [398] Ganglion cell damage models: Optic nerve crush in mice [399] Uveitis models: Endotoxin-induced uveitis in mice [400] Metabolism and Clearance of Cyanidin ...
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Inherited retinal dystrophies (IRDs) are a large group of genetically and clinically heterogeneous diseases characterized by the progressive degeneration of the retina, ultimately leading to loss of visual function. Oxidative stress and inflammation play fundamental roles in the physiopathology of these diseases. Photoreceptor cell death induces an inflammatory state in the retina. The activation of several molecular pathways triggers different cellular responses to injury, including the activation of microglia to eliminate debris and recruit inflammatory cells from circulation. Therapeutical options for IRDs are currently limited, although a small number of patients have been successfully treated by gene therapy. Many other therapeutic strategies are being pursued to mitigate the deleterious effects of IRDs associated with oxidative metabolism and/or inflammation, including inhibiting reactive oxygen species’ accumulation and inflammatory responses, and blocking autophagy. Several compounds are being tested in clinical trials, generating great expectations for their implementation. The present review discusses the main death mechanisms that occur in IRDs and the latest therapies that are under investigation.
... The number of cells in the outer nuclear layer (ONL), the photoreceptor layer, and quantification of inner segment (IS) areas in a 50 µm length of the retina was evaluated at each point using ImageJ software (National Institutes of Health, Bethesda, MD, USA; available at http://rsb.info.nih.gov/ij/index.html (accessed 27 September 2021) and averaged as described previously [21][22][23][24]; data at 200 µm distance from the optic nerve in the superior retina were shown. ...
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Retinitis pigmentosa (RP) is a hereditary blinding disease characterized by gradual photoreceptor death, which lacks a definitive treatment. Here, we demonstrated the effect of 4-phenylbutyric acid (PBA), a chemical chaperon that can suppress endoplasmic reticulum (ER) stress, in P23H mutant rhodopsin knock-in RP models. In the RP models, constant PBA treatment led to the retention of a greater number of photoreceptors, preserving the inner segment (IS), a mitochondrial- and ER-rich part of the photoreceptors. Electroretinography showed that PBA treatment preserved photoreceptor function. At the early point, ER-associated degradation markers, xbp1s, vcp, and derl1, mitochondrial kinetic-related markers, fis1, lc3, and mfn1 and mfn2, as well as key mitochondrial regulators, pgc-1α and tfam, were upregulated in the retina of the models treated with PBA. In vitro analyses showed that PBA upregulated pgc-1α and tfam transcription, leading to an increase in the mitochondrial membrane potential, cytochrome c oxidase activity, and ATP levels. Histone acetylation of the PGC-1α promoter was increased by PBA, indicating that PBA affected the mitochondrial condition through epigenetic changes. Our findings constituted proof of concept for the treatment of ER stress-related RP using PBA and revealed PBA’s neuroprotective effects, paving the way for its future clinical application.
... Substantial evidence from animal studies also indicated that prolonged light stimulation to the retina can cause accumulation of oxidative damage [52][53][54]. Exposure to ultraviolet (UV) radiation initiates oxidative DNA damage and inflammatory response in RPE. Taken together, these events cause overproduction and accumulation of lipofuscin and formation of toxic aggregates of amyloid-β (Aβ) peptides. ...
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Age-related macular degeneration (AMD) is a multifactorial disease associated with anatomical changes in the inner retina. Despite tremendous advances in clinical care, there is currently no cure for AMD. This review aims to evaluate the published literature on the therapeutic roles of natural antioxidants in AMD. A literature search of PubMed, Web of Science and Google Scholar for peer-reviewed articles published between 1 January 2011 and 31 October 2021 was undertaken. A total of 82 preclinical and 18 clinical studies were eligible for inclusion in this review. We identified active compounds, carotenoids, extracts and polysaccharides, flavonoids, formulations, vitamins and whole foods with potential therapeutic roles in AMD. We evaluated the integral cellular signaling pathways including the activation of antioxidant pathways and angiogenesis pathways orchestrating their mode of action. In conclusion, we examined the therapeutic roles of natural antioxidants in AMD which warrant further study for application in clinical practice. Our current understanding is that natural antioxidants have the potential to improve or halt the progression of AMD, and tailoring therapeutics to the specific disease stages may be the key to preventing irreversible vision loss.
... It has been reported that TNFa can induce Müller cells to transform from non-proliferative gliosis to the retinal regeneration responses [27]. However, when Müller cells are overactivated, the overactive gliosis is harmful, forming glial scars and promoting retinal remodeling [28]. Retinal microglia may affect the behaviors of Müller cells induced by laser exposure [29]. ...
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... Blueberry is considered as one of the richest sources of anthocyanins among common fruits [80,83], which are responsible for a variety of health promoting values [42,77]. Several studies have shown that blueberry can be used in treatment of many diseases such as diabetes [13,16,20,26], preventing diabetic retinopathy and neuro protection [29,34,62,79], malignant tumors [7,11,23,67], chronic diseases [46], prevention of cardiovascular diseases [15,41,84], having antimicrobial activity [70,73,74], improving memory and protection against Alzheimer's disease [34,40,78]. All these properties of blueberry and its unique set of anthocyanins content, make it especially attractive for food and pharmaceutical preparations. ...
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... ERG recording was performed as previously described [20][21][22] . Briefly, mice were dark-adapted for 12 h and placed under dim-red illumination until they were anesthetized. ...
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Lipid metabolism-related gene mutations can cause retinitis pigmentosa, a currently untreatable blinding disease resulting from progressive neurodegeneration of the retina. Here, we demonstrated the influence of adiponectin receptor 1 (ADIPOR1) deficiency in retinal neurodegeneration using Adipor1 knockout (KO) mice. Adipor1 mRNA was observed to be expressed in photoreceptors, predominately within the photoreceptor inner segment (PIS), and increased after birth during the development of the photoreceptor outer segments (POSs) where photons are received by the visual pigment, rhodopsin. At 3 weeks of age, visual function impairment, specifically photoreceptor dysfunction, as recorded by electroretinography (ERG), was evident in homozygous, but not heterozygous, Adipor1 KO mice. However, although photoreceptor loss was evident at 3 weeks of age and progressed until 10 weeks, the level of visual dysfunction was already substantial by 3 weeks, after which it was retained until 10 weeks of age. The rhodopsin mRNA levels had already decreased at 3 weeks, suggesting that reduced rhodopsin may have contributed to early visual loss. Moreover, inflammation and oxidative stress were induced in homozygous KO retinas. Prior to observation of photoreceptor loss via optical microscopy, electron microscopy revealed that POSs were present; however, they were misaligned and their lipid composition, including docosahexaenoic acid (DHA), which is critical in forming POSs, was impaired in the retina. Importantly, the expression of Elovl2 , an elongase of very long chain fatty acids expressed in the PIS, was significantly reduced, and lipogenic genes, which are induced under conditions of reduced endogenous DHA synthesis, were increased in homozygous KO mice. The causal relationship between ADIPOR1 deficiency and Elovl2 repression, together with upregulation of lipogenic genes, was confirmed in vitro. Therefore, ADIPOR1 in the retina appears to be indispensable for ELOVL2 induction, which is likely required to supply sufficient DHA for appropriate photoreceptor function and survival.
... Retinal pigmented cells and retinal Müller cells, which support the interactions between retinal neurons for homeostasis, nutrition, and metabolism, play key roles in the blood-retinal-barrier (BRB) function. In particular, Müller gliosis indicated by GFAP staining reflects the severity of the oxidative damage of retinal neurons [35,36], which leads to photoreceptor degeneration, intraretinal vascular leak, and microvasculopathy. Additionally, the mislocalization of M opsins is the consequence of a defect in the chromophore 11-cis retinal provided by the retinal pigmented cells and retinal Müller cells involved in visual cycles. ...
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Danshensu, a traditional herb-based active component (Salvia miltiorrhiza Bunge), has garnered attention, due to its safety, nutritional value, and antioxidant effects, along with cardiovascular-protective and neuroprotective abilities; however, its effect on the retinal tissues and functional vision has not been fully studied. The objective of this study was to analyze the protective effect of danshensu on retinal tissues and functional vision in vivo in a mouse model of light-induced retinal degeneration. High energy light-evoked visual damage was confirmed by the loss in structural tissue integrity in the retina accompanied by a decline in visual acuity and visual contrast sensitivity function (VCSF), whereas the retina tissue exhibited severe Müller cell gliosis. Although danshensu treatment did not particularly reduce light-evoked damage to the photoreceptors, it significantly prevented Müller cell gliosis. Danshensu exerted protective effects against light-evoked deterioration on low spatial frequency-based VCSF as determined by the behavioral optomotor reflex method. Additionally, the protective effect of danshensu on VCSF can be reversed and blocked by the injection of a dopamine D1 receptor antagonist (SCH 23390). This study demonstrated that the major functional vision promotional effect of danshensu in vivo was through the dopamine D1 receptors enhancement pathway, rather than the structural protection of the retinas.
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Bilberry (Vaccinusm myrtillus L.) is one among the most extravagant characteristic wellsprings of anthocyanins. These polyphenolic components offer bilberry its blue/dark shading and high inhibitor content, and that they are accepted to be the key bioactives liable for a few announced wellbeing points of interest of bilberry. in spite of the fact that bilberry is advanced most normally for up vision, it's been accounted for to bring down glucose, to possess restorative medication and lipid-bringing down impacts, and to advertise inhibitor protection and lower aerophilous pressure. Accordingly, bilberry is of potential worth inside the treatment or impedance of conditions identified with irritation, dyslipidemia, hyperglycemia or enlarged aerophilous pressure, disorder (CVD), malignancy, diabetes, and madness and distinctive age related sicknesses. There are reports that bilberry has contradicted microorganism action. Berries are an upscale supply of a vast sort of unhealthful, healthy, and fictive mixes like a pigments, resin, artificial addtives resin sap bitter, aromatic hydrocarbon, and phenolic substances further as nutritious compounds like sugars, basic grease, carotenes, nutrients, and mine. Bioactivity mixes with berries have intense inhibitor, anticancer, anticarcinogenic, antibiotices, therapeutic medication, and anti-neuroprogressive effects, each in test tube and in living body.
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Introduction Age-related macular degeneration (AMD) is the leading cause of blindness in individuals over age 50 in developed countries. Current therapy for nonexudative AMD (neAMD) is aimed at modifying risk factors and vitamin supplementation to slow progression while intravitreal anti-vascular endothelial factor (VEGF) injections are the mainstay for treatment of choroidal neovascularization in exudative AMD (eAMD). Areas covered Over the past decade, promising therapies have emerged that aim to improve the current standard of care for both diseases. Clinical trials for neAMD are investigating targets in the complement cascade, Vitamin A metabolism, metformin, and tetracycline whereas clinical trials for eAMD are aiming to decrease treatment burden through novel port delivery systems, increasing drug half-life, and targeting new sites of the VEGF cascade. Stem cell and gene therapy are also being evaluated for treatment of neAMD and eAMD. Expert opinion With an aging population, the need for effective, long term, low burden treatment options for AMD will be in increasingly high demand. Current investigations aim to address the shortcomings of current treatment options with breakthrough treatment approaches. Therapeutics in the pipeline hold promise for improving the treatment of AMD, and are on track for widespread use within the next decade.
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Purpose: The determination of the molecular mechanism underlying retinal pathogenesis and visual dysfunction during innate inflammation, and the treatment effect of rapamycin thereon. Methods: The endotoxin-induced uveitis and retinitis mouse model was established by injecting lipopolysaccharide. The mice were subsequently treated with rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. The rhodopsin mRNA and protein expression level in the retina and the photoreceptor outer segment (OS) length in immunohistochemical stainings were measured, and visual function was recorded by electroretinography. Inflammatory cytokines, their related molecules, mTOR, and LC3 levels were measured by real-time PCR and/or immunoblotting. Leukocyte adhesion during inflammation was analyzed using concanavalin A lectin. Results: The post-transcriptional reduction in the visual pigment of rod photoreceptor cells, rhodopsin, OS shortening, and rod photoreceptor cell dysfunction during inflammation were suppressed by rapamycin. Activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induction of inflammatory cytokines, such as interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), and the activation of the downstream signaling protein, signal transducer and activator of transcription 3 (STAT3), which reduces rhodopsin in the retina during inflammation, were attenuated by rapamycin. Increased leukocyte adhesion was also attenuated by rapamycin. Interestingly, although mTOR activation was observed after NF-κB activation, mTOR inhibition suppressed NF-κB activation at the early phase, indicating that the basal level of activated mTOR was sufficient to activate NF-κB in the retina. In addition, the inhibition of NF-κB suppressed mTOR activation, suggesting a positive feedback loop of mTOR and NF-κB during inflammation. The ratio of LC3II to LC3I, which reflects autophagy induction, was not changed by inflammation but was increased by rapamycin. Conclusions: Our results propose the potential use of rapamycin as a neuroprotective therapy to suppress local activated mTOR levels, related inflammatory molecules, and the subsequent visual dysfunction during retinal inflammation.
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Oxidative stress in the retinal pigment epithelium (RPE) is a well-accepted pathogenic change in vision-threatening diseases such as age-related macular degeneration. One source of oxidative stress is excessive light exposure, which causes excessive activation of the visual cycle. Because short wavelength light (blue light) has more energy, it is reported to be more harmful to photoreceptor cells than the other wavelengths of light. However, the biological effect of blue light in the RPE of living animals and the protective effect of a yellow intraocular lens (IOL) material that blocks blue light is still obscure. Therefore, we compared the pathogenic effect in the RPE-choroid complexes of mice exposed to light in a box made of a clear or a yellow IOL material. We measured the level of reactive oxygen species (ROS) using 2', 7'-dichlorodihydrofluorescein diacetate, the mRNA levels of inflammatory cytokines and a macrophage marker by real-time polymerase chain reaction, and the protein levels of monocyte chemotactic protein-1 (MCP-1) and macrophage markers by ELISA. The ROS level after light exposure was suppressed in the RPE-choroids of light-exposed mice in the yellow IOL material box. In parallel, all the inflammatory cytokines that we measured and a macrophage marker were also suppressed in the RPE-choroids of light-exposed mice in the yellow IOL material box. Therefore, a yellow IOL material suppressed, and thus blue light exacerbated, the increase in the ROS level and inflammatory cytokine expression as well as macrophage recruitment in the RPE-choroid in vivo after light exposure. Copyright © 2015. Published by Elsevier Ltd.
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Lutein slows the progression of age-related macular degeneration (AMD), a leading cause of blindness in ageing societies. However, the underlying mechanisms remain elusive. Here, we evaluated lutein’s effects on light-induced AMD-related pathological events. Balb/c mice exposed to light (2000 lux, 3 h) showed tight junction disruption in the retinal pigment epithelium (RPE) at 12 h, as detected by zona occludens-1 immunostaining. Substantial disruption remained 48 h after light exposure in the vehicle-treated group; however, this was ameliorated in the mice treated with intraperitoneal lutein at 12 h, suggesting that lutein promoted tight junction repair. In the photo-stressed RPE and the neighbouring choroid tissue, lutein suppressed reactive oxygen species and increased superoxide dismutase (SOD) activity at 24 h, and produced sustained increases in sod1 and sod2 mRNA levels at 48 h. SOD activity was induced by lutein in an RPE cell line, ARPE19. We also found that lutein suppressed upregulation of macrophage-related markers, f4/80 and mcp-1, in the RPE-choroid tissue at 18 h. In ARPE19, lutein reduced mcp-1 mRNA levels. These findings indicated that lutein promoted tight junction repair and suppressed inflammation in photo-stressed mice, reducing local oxidative stress by direct scavenging and most likely by induction of endogenous antioxidant enzymes.
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Age related macular degeneration (AMD) is the leading cause of blindness in individuals older than 65 years of age. It is a multifactorial disorder and identification of risk factors enables individuals to make life style choices that may reduce the risk of disease. This review discusses the role of genetics, sunlight, diet, cardiovascular factors, smoking, and alcohol as possible risk factors for AMD. Genetics plays a more significant role in AMD than previously thought, especially in younger patients, histocompatibility locus antigen (HLA) and complement system genes being the most significant. Whether the risk of AMD is increased by exposure to sunlight, cardiovascular risk factors, and diet is more controversial. Smoking is the risk factor most consistently associated with AMD. Current smokers are exposed to a two to three times higher risk of AMD than non-smokers and the risk increases with intensity of smoking. Moderate alcohol consumption is unlikely to increase the risk of AMD. Optometrists as front-line informers and educators of ocular health play a significant role in increasing public awareness of the risks of AMD. Cessation of smoking, the use of eye protection in high light conditions, dietary changes, and regular use of dietary supplements should all be considered to reduce the lifetime risk of AMD.
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Vision is reliant upon converting photon signals to electrical information which is interpreted by the brain and therefore allowing us to receive information about our surroundings. However, when exposed to excessive light, photoreceptors and other types of cells in the retina can undergo light-induced cell death, termed light-induced retinal damage. In this review, we summarize our current knowledge regarding molecular events in the retina after excessive light exposure and mechanisms of light-induced retinal damage. We also introduce works which investigate potential roles of autophagy, an essential cellular mechanism required for maintaining homeostasis under stress conditions, in the illuminated retina and animal models of light-induced retinal damage. Copyright © 2015. Published by Elsevier Ltd.
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Age-related macular degeneration (AMD) is a degenerative retinal disease that causes blindness in people 60-65 years and older, with the highest prevalence appearing in people 90 years-old or more. Epidemiological estimates indicate that the number of cases is increasing, and will almost double in the next 20 years. Preventive measures require precise etiological knowledge. This is quite difficult, since AMD is a multifactorial condition with intricate relationships between causes and risk factors. In this review, we describe the impact of light on the structure and physiology of the retina and the pigment epithelium, taking into account the continuous exposure to natural and artificial light sources along the life of an individual. A large body of experimental evidence demonstrates the toxic effects of some lighting conditions on the retina and the pigment epithelium, and consensus exists about the importance of photo-oxidation phenomena in the causality chain between light and retinal damage. Here, we analyzed the transmission of light to the retina, and compared the aging human macula in healthy and diseased retinas, as shown by histology and non-invasive imaging systems. Finally, we have compared the putative retinal photosensitive molecular structures that might be involved in the genesis of AMD. The relationship between these compounds and retinal damage supports the hypothesis of light as an important initiating cause of AMD.
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Eukaryotic cells fold and assemble membrane and secreted proteins in the endoplasmic reticulum (ER), before delivery to other cellular compartments or the extracellular environment. Correctly folded proteins are released from the ER, and poorly folded proteins are retained until they achieve stable conformations; irreparably misfolded proteins are targeted for degradation. Diverse pathological insults, such as amino acid mutations, hypoxia, or infection, can overwhelm ER protein quality control, leading to misfolded protein buildup, causing ER stress. To cope with ER stress, eukaryotic cells activate the unfolded protein response (UPR) by increasing levels of ER protein-folding enzymes and chaperones, enhancing the degradation of misfolded proteins, and reducing protein translation. In mammalian cells, three ER transmembrane proteins, inositol-requiring enzyme-1 (IRE1; official name ERN1), PKR-like ER kinase (PERK; official name EIF2AK3), and activating transcription factor-6, control the UPR. The UPR signaling triggers a set of prodeath programs when the cells fail to successfully adapt to ER stress or restore homeostasis. ER stress and UPR signaling are implicated in the pathogenesis of diverse diseases, including neurodegeneration, cancer, diabetes, and inflammation. This review discusses the current understanding in both adaptive and apoptotic responses as well as the molecular mechanisms instigating apoptosis via IRE1 and PERK signaling. We also examine how IRE1 and PERK signaling may be differentially used during neurodegeneration arising in retinitis pigmentosa and prion infection. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
Article
Objective To describe the risk factors and associated population attributable risk for age-related maculopathy (ARM) and age-related macular degeneration(AMD) in Australians aged 40 years and older. Methods Residents were recruited from 9 randomly selected urban clusters and 4 randomly selected rural clusters in Victoria, Australia. At locally established test sites, the following information was collected: visual acuity, medical and health history, lifetime sunlight exposure, dietary intake, and fundus photographs. Age-related maculopathy and AMD were graded from the fundus photographs using an international classification and grading system. Backwards logistic regression was used to identify the independent risk factors for ARM and AMD. Results The participation rate was 83% (n = 3271) among the urban residents and 92% (n = 1473) among the rural residents. Gradable fundus photographs of either eye were available for 4345 (92%) of the 4744 participants. There were 656 cases of ARM, giving a weighted prevalence of 15.1% (95% confidence limit [CL], 13.8, 16.4); and there were 30 cases of AMD, giving a weighted prevalence of 0.69% (95% CL, 0.33, 1.03). In multiple logistic regression, the risk factors for AMD were as follows: age (odds ratio [OR], 1.23; 95% CL, 1.17, 1.29), smoked cigarettes for longer than 40 years (OR, 2.39; 95% CL, 1.02, 5.57), and ever taken angiotensin-converting enzyme inhibitors (OR, 3.26; 95% CL, 1.33, 8.01). The magnitude of all of these risk factors was slightly less for ARM, and having ever taken blood cholesterol–lowering medications was also significant (OR, 1.67; 95% CL, 1.12, 2.47; P = .001). Conclusion Smoking is the only modifiable risk factor for ARM and AMD, among the many environmental and systemic factors that were assessed.
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Abstract Many dietary supplements have been sold through advertising their large number of beneficial effects. The aim of this study was to determine whether bilberries (Vaccinium myrtillus) help to prevent diabetes-induced retinal vascular dysfunction in vivo. V. myrtillus extract (VME; 100 mg/kg) was orally administered to streptozotocin-induced diabetic rats for 6 weeks. All diabetic rats exhibited hyperglycemia, and VME did not affect the blood glucose levels and body weight during the experiments. In the fluorescein-dextran angiography, the fluorescein leakage was significantly reduced in diabetic rats treated with VME. VME treatment also decreased markers of diabetic retinopathy, such as retinal vascular endothelial growth factor (VEGF) expression and degradation of zonula occludens-1, occludin and claudin-5 in diabetic rats. In conclusion, VME may prevent or delay the onset of early diabetic retinopathy. These findings have important implications for prevention of diabetic retinopathy using a dietary bilberry supplement.
Article
To examine the effect of a dietary supplement containing bilberry extract (BE) on eye fatigue induced by acute video display terminal (VDT) loads. A prospective, randomized, double-blind, placebo-controlled study was performed from August 2012 to February 2013 in the Medical Corporation Jico-kai Yagi Hospital, and the Shinyokohama Shinoharaguchi Orthopedic Surgery and Dermatology Clinic, in Japan. Two hundred eighty-one office workers aged 20-40 years that used VDTs were screened by critical flicker fusion (CFF) and near point accommodation (NPA). The participants were randomized to either a BE (480 mg/day) or placebo (vehicle) group, and took allocated capsule, daily, for 8 weeks. The CFF, NPA, contrast visual acuity, functional visual acuity, keratoconjunctival epithelial damage, and fluorescein tear film break-up time were examined, and 18 subjective symptoms of eye fatigue were evaluated by questionnaire. Adverse events were reported via medical interviews. Data were collected both before and after VDT load at baseline, and 4, and 8 weeks after daily supplementation with either BE or placebo. Of 281 participants screened, 88 having relatively lower levels of CFF and NPA were enrolled in the study. Of these, 37 control and 43 BE group subjects completed the study. The VDT load-induced reduction in CFF was alleviated after 8 weeks of BE supplementation (95% confidence interval, 0.10-1.60; p=0.023), in contrast to placebo supplementation, while NPA variation was not. Of the subjective symptoms of eye fatigue, VDT load-induced ocular fatigue sensation, ocular pain, eye heaviness, uncomfortable sensation, and foreign body sensation were mitigated more in the BE group than in the control group, at week 8 (p<0.05). There were no severe adverse events in either group. BE supplementation improved some of the objective and subjective parameters of eye fatigue induced by VDT loads.