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Objective: Transcorneal electrical stimulation (TES) is a promising approach to delay retinal degeneration by inducing extracellular electric field-driven neuroprotective effects within photoreceptors. Although achieving precise electric field control is feasible in vitro, characterizing these fields becomes intricate and largely unexplored in vivo due to uneven distribution in the heterogeneous body. In this paper, we investigate and characterize electric fields within the retina during TES to assess the potential for therapeutic approaches Methods: We developed a computational model of a rat's head, enabling us to generate predictive simulations of the voltage and current density induced in the retina. Subsequently, an in vivo experimental setup involving Royal College of Surgeon (RCS) rats was implemented to measure the voltage across the retina using identical electrode configurations as employed in the simulations., Results: A stimulation amplitude of 0.2-0.3 mA may be necessary during TES in rats to induce a current density of at least 20 A/[Formula: see text] in the retina, which is the lower limit for triggering neuroprotective effects according to culture studies on neural cells. Measurement taken from cadaveric pigs' eyes revealed that a stimulation amplitude of 1 mA is necessary for achieving the same current density., Conclusion: The computational modeling approach presented in this study was validated with experimental data and can be leveraged for predictive simulations to optimize the electrode design and stimulation parameters of TES., Significance: Once validated, the flexibility and low research cost of computational models are valuable in optimization studies where testing on live subjects is not feasible.

The impact of plastic pollution on living organisms have gained significant research attention. However, the effects of nanoplastics (NPs) on retina remain unclear. This study aimed to investigate the effect of long-term polystyrene nanoparticles (PS-NPs) exposure on mouse retina. Eight weeks old C57BL/6 J mice were exposed to PS-NPs at the diameter of 100 nm and concentration of 10 mg/L in drinking water for 3 months. PS-NPs were able to penetrate the blood-retina barrier, accumulated at retinal tissue, caused increased oxidative stress level and reduced scotopic electroretinal responses without remarkable structural damage. PS-NPs exposure caused cytotoxicity and reactive oxygen species accumulation in cultured photoreceptor cell. PS-NPs exposure increased oxidative stress level in retinal pigment epithelial (RPE) cells, leading to changes of gene and protein expression indicative of compromised phagocytic activity and cell junction formation. Long-term PS-NPs exposure also aggravated light-induced photoreceptor cell degeneration and retinal inflammation. The transcriptomic profile of PS-NPs-exposed, light-challenged retinal tissue shared similar features with those of age-related macular degeneration (AMD) patients in the activation of complement-mediated phagocytic and proinflammatory responses. Collectively, these findings demonstrated the oxidative stress- and inflammation-mediated detrimental effect of PS-NPs on retinal function, suggested that long-term PS-NPs exposure could be an environmental risk factor contributing to retinal degeneration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Photobiomodulation (PBM) therapy uses light of different wavelengths to treat various retinal degeneration diseases, but the potential damage to the retina caused by long-term light irradiation is still unclear. This study were designed to detect the difference between long- and short-wavelength light (650-nm red light and 450-nm blue light, 2.55 mW/cm 2 , reference intensity in PBM)-induced injury. In addition, a comparative study was conducted to investigate the differences in retinal light damage induced by different irradiation protocols (short periods of repeated irradiation and a long period of constant irradiation). Furthermore, the protective role of PARP-1 inhibition on the molecular mechanism of blue light-induced injury was confirmed by a gene knockdown technique or a specific inhibitor through in vitro and in vivo experiments. The results showed that the susceptibility to retinal damage caused by irradiation with long- and short-wavelength light is different. Shorter wavelength lights, such as blue light, induce more severe retinal damage, while the retina exhibits better resistance to longer wavelength lights, such as red light. In addition, repeated irradiation for short periods induces less retinal damage than constant exposure over a long period. PARP-1 plays a critical role in the molecular mechanism of blue light-induced damage in photoreceptors and retina, and inhibiting PARP-1 can significantly protect the retina against blue light damage. This study lays an experimental foundation for assessing the safety of phototherapy products and for developing target drugs to protect the retina from light damage., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

To study the macroglia and microglia and the immune role in long-time light exposure in rat eyes, we performed glial cell characterization along the time-course of retinal degeneration induced by chronic exposure to low-intensity light. Animals were exposed to light for periods of 2, 4, 6, or 8 days, and the retinal glial response was evaluated by immunohistochemistry, western blot and real-time reverse transcription polymerase chain reaction. Retinal cells presented an increased expression of the macroglia marker GFAP, as well as increased mRNA levels of microglia markers Iba1 and CD68 after 6 days. Also, at this time-point, we found a higher number of Iba1-positive cells in the outer nuclear layer area; moreover, these cells showed the characteristic activated-microglia morphology. The expression levels of immune mediators TNF, IL-6, and chemokines CX3CR1 and CCL2 were also significantly increased after 6 days. All the events of glial activation occurred after 5-6 days of constant light exposure, when the number of photoreceptor cells has already decreased significantly. Herein, we demonstrated that glial and immune activation are secondary to neurodegeneration; in this scenario, our results suggest that photoreceptor death is an early event that occurs independently of glial-derived immune responses.

Purpose: Environmental light pollution due to artificial light may increase the rate and severity of retinal diseases, and plant-based nutritional interventions with antioxidant properties have the potential to reverse this phenomenon. We aimed to investigate the potential effects of allyl isothiocyanate (AITC) against white light-emitting diode (LED)-induced retinal degeneration (RD) in the rats., Methods: Twenty-eight male rats were allocated as: (i) Control, (ii) LED, (iii) LED + AITC (10 mg/kg BW), (iv) LED + AITC (20 mg/kg BW). Rats were administered with AITC for 28 days, followed by two days of intense environmental LED light (750 Lux ) exposure to the eyes. Animals were sacrificed immediately at the end of the study, then the blood and eyeballs were taken for the biochemical, western blotting, and histopathology examinations., Results: AITC lowered the serum and retina malondialdehyde (MDA) levels while significantly ( p <  0.05) improving the retinal antioxidant enzyme activities in a dose-dependent manner. AITC improved retinal and outer nuclear layer (ONL) thickness as compared to the LED group ( p <  0.05). AITC increased the levels of Bax, caspase-3, HO-1, GAP43, and VEGF, while decreasing IL-1β, IL-6, NF-κB, Bcl-2, GFAP, Grp78, activating ATF4 and ATF6 as compared to the LED group ( p <  0.05)., Conclusion: In conclusion, four weeks of AITC administration to the rats showed specific protective effects against two days of intense LED light-induced retinal damage; through antiinflammatory, antioxidant, anti-apoptotic, and modulating mitochondrial metabolic pathways.

Bright light exposure in animals results in the selective degeneration of the outer retina, known as "retinal photic injury" (RPI). The susceptibility to RPI differs among rat strains. WKY rats display susceptibility to RPI with extensive retinal degeneration observed in the sagittal eye specimen, whereas LEW strain rats are resistant to it, showing only slight or no degeneration. In the present study, we first established an ethological screening method using the Morris water maze to discern differential susceptibility among the living rats. WKY and LEW were crossed to produce the first filial generation (F 1 ) offspring. Maze-trained individuals were exposed to bright, white light. The screening test results demonstrated that the susceptibility to light-induced visual impairment in rats is a dominant Mendelian susceptibility trait, as F 1 rats were susceptible to visual impairment like WKY rats. Therefore, F 1 rats were backcrossed with recessive LEW to produce the first backcross offspring (BC 1 ). Subsequent recurrent backcrossing while selecting for the susceptibility, indicated a segregation ratio of ca. 24% in BC 1 and BC 2 generations, indicating the involvement of two or more genes in the susceptibility. Further, microsatellite analysis of BC 1 -to-BC 4 individuals using microsatellite markers mapped two susceptibility loci on chromosome segments 5q36 and 19q11-q12, named RPI susceptibility (Rpi)1 and Rpi2, respectively. This study provides an insight into mechanisms underlying differential susceptibility, which could help decipher the mechanism underlying the onset/progression of human age-related macular degeneration., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We developed a model of retinal degeneration in rabbits based on exposure to light with a wavelength of 405 nm. This model allows reproducing structural and functional disorders in the central parts of the retina, including primarily degeneration of the outer layers of the retina (retinal pigment epithelium and layer of photoreceptor cells), and is designed to study the mechanisms of formation, progression and effectiveness of new drugs and methods of treatment of degenerative diseases of the retina.

Microglia are immune cells of the central nervous system capable of distinct phenotypic changes and migration in response to injury. These changes most notably include the retraction of fine dendritic structures and adoption of a globular, phagocytic morphology. Due to their characteristic responses, microglia frequently act as histological indicators of injury progression. While algorithms seeking to automate microglia counts and morphological analysis are becoming increasingly popular, few exist that are adequate for use within the retina and manual analysis remains prevalent. To address this, we propose a novel segmentation routine, implemented within FIJI-ImageJ, to perform automated segmentation and cell counting of retinal microglia. We show that our routine could perform cell counts with accuracy similar to manual observers using the I307N Rho model. Tracking cell position relative to retinal vasculature, we observed population migration towards the photoreceptor layer beginning 12 h post light damage. Using feature selection with Chi 2 and principal component analysis, we resolved cells along a morphological gradient, demonstrating that extracted features were sufficiently descriptive to capture subtle morphological changes within cell populations in I307N Rho and Balb/c TLR2 -/- retinal degeneration models. Taken together, we introduce a novel automated routine capable of efficient image processing and segmentation. Using data retrieved following segmentation, we perform morphological analysis simultaneously on whole populations of cells, rather than individually. Our algorithm was built entirely with open-source software, for use on retinal microglia., (Published by Elsevier Ltd.)

Purpose: Retinitis pigmentosa (RP) is a blinding neurodegenerative disease of the retina that can be affected by many factors. The present study aimed to analyze the effect of different environmental light intensities in rd10 mice retina., Methods: C57BL/6J and rd10 mice were bred and housed under three different environmental light intensities: scotopic (5 lux), mesopic (50 lux), and photopic (300 lux). Visual function was studied using electroretinography and optomotor testing. The structural and morphological integrity of the retinas was evaluated by optical coherence tomography imaging and immunohistochemistry. Additionally, inflammatory processes and oxidative stress markers were analyzed by flow cytometry and western blotting., Results: When the environmental light intensity was higher, retinal function decreased in rd10 mice and was accompanied by light-dependent photoreceptor loss, followed by morphological alterations, and synaptic connectivity loss. Moreover, light-dependent retinal degeneration was accompanied by an increased number of inflammatory cells, which became more activated and phagocytic, and by an exacerbated reactive gliosis. Furthermore, light-dependent increment in oxidative stress markers in rd10 mice retina pointed to a possible mechanism for light-induced photoreceptor degeneration., Conclusions: An increase in rd10 mice housing light intensity accelerates retinal degeneration, activating cell death, oxidative stress pathways, and inflammatory cells. Lighting intensity is a key factor in the progression of retinal degeneration, and standardized lighting conditions are advisable for proper analysis and interpretation of experimental results from RP animal models, and specifically from rd10 mice. Also, it can be hypothesized that light protection could be an option to slow down retinal degeneration in some cases of RP.

Purpose: Maintaining levels of nicotinamide adenine dinucleotide (NAD+), a coenzyme critical for cellular energetics and biosynthetic pathways, may be therapeutic in retinal disease because retinal NAD+ levels decline during retinal damage and degeneration. The purpose of this study was to investigate whether systemic treatment with nicotinamide riboside (NR), a NAD+ precursor that is orally deliverable and well-tolerated by humans, is protective in a mouse model of light-induced retinal degeneration., Methods: Mice were injected intraperitoneally with vehicle or NR the day before and the morning of exposure to degeneration-inducing levels of light. Retinal function was assessed by electroretinography and in vivo retinal morphology and inflammation was assessed by optical coherence tomography. Post mortem retina sections were assessed for morphology, TUNEL, and inflammatory markers Iba1 and GFAP. Retinal NAD+ levels were enzymatically assayed., Results: Exposure to degeneration-inducing levels of light suppressed retinal NAD+ levels. Mice undergoing light-induced retinal degeneration exhibited significantly suppressed retinal function, severely disrupted photoreceptor cell layers, and increased apoptosis and inflammation in the outer retina. Treatment with NR increased levels of NAD+ in retina and prevented these deleterious outcomes., Conclusions: This study is the first to report the protective effects of NR treatment in a mouse model of retinal degeneration. The positive outcomes, coupled with human tolerance to NR dosing, suggest that maintaining retinal NAD+ via systemic NR treatment should be further explored for clinical relevance.

Photoreceptor cells are first-order retinal neurons that directly contribute to the formation of vision. Photoreceptor degeneration is the primary cause of vision impairment during the course of retinopathies such as retinitis pigmentosa and age-related macular degeneration, for which photoreceptor-targeted therapies are currently unavailable. Shihu Yeguang Pill (SYP), a classic formula in traditional Chinese medicine, has a long histology of clinical application for the treatment of a wide range of retinopathies in China. However, whether SYP is pharmacological effective at protecting photoreceptor cells is unclear. The current study thus directly addressed the pharmacological implications of SYP in photoreceptor degeneration in a mouse model characterized by bright light-induced retinal degeneration. Non-invasive full-retinal assessment was carried out to evaluate the effect of SYP on the retinal structure and function through optical coherence tomography and electroretinography, respectively. In addition, photoreceptor apoptosis, second-order neuron impairment and reactive changes in retinal microglial and müller cells, hallmark pathologies associated with photoreceptor degeneration, were assessed using immunohistochemistry and real-time PCR analyses. The results showed that SYP treatment attenuated bright light-induced impairment of the retinal structure and function. Moreover, SYP treatment suppressed photoreceptor apoptosis, alleviated the impairment of bipolar and horizontal cells and mitigated the reactive changes of müller and microglial cells in the bright light-exposed retinas. Real-time PCR analyses showed that dysregulated expression of pro-apoptotic c-fos and c-jun and anti-apoptotic bcl-2 as well as proinflammatory TNF-α in the bright light-exposed retinas was partially normalized as a result of SYP treatment. In summary, the work here demonstrates for the first time that SYP treatment protects the retinas from developing bright light-induced photoreceptor degeneration and associated alterations in second-order neurons and glial cells. The findings here thus provide experimental evidence to better support the mechanism-guided clinical application of SYP in the treatment of related retinal degenerative diseases., Competing Interests: Declaration of Competing Interest The authors declare no commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Purpose: To analyze the role of microglial and Müller cells in the formation of rings of photoreceptor degeneration caused by phototoxicity., Methods: Two-month-old Sprague-Dawley rats were exposed to light and processed 1, 2, or 3 months later. Retinas were dissected as whole-mounts, immunodetected for microglial cells, Müller cells, and S- and L/M-cones and analyzed using fluorescence, thunder imaging, and confocal microscopy. Cone populations were automatically counted and isodensity maps constructed to document cone topography., Results: Phototoxicity causes a significant progressive loss of S- and L/M-cones of up to 68% and 44%, respectively, at 3 months after light exposure (ALE). One month ALE, we observed rings of cone degeneration in the photosensitive area of the superior retina. Two and 3 months ALE, these rings had extended to the central and inferior retina. Within the rings of cone degeneration, there were degenerating cones, often activated microglial cells, and numerous radially oriented processes of Müller cells that showed increased expression of intermediate filaments. Between 1 and 3 months ALE, the rings coalesced, and at the same time the microglial cells resumed a mosaic-like distribution, and there was a decrease of Müller cell gliosis at the areas devoid of cones., Conclusions: Light-induced photoreceptor degeneration proceeds with rings of cone degeneration, as observed in inherited retinal degenerations in which cone death is secondary to rod degeneration. The spatiotemporal relationship of cone death microglial cell activation and Müller cell gliosis within the rings of cone degeneration suggests that, although both glial cells are involved in the formation of the rings, they may have coordinated actions and, while microglial cells may be more involved in photoreceptor phagocytosis, Müller cells may be more involved in cone and microglial cell migration, retinal remodeling and glial seal formation.

Ocular inflammation contributes to the pathogenesis of blind-causing retinal degenerative diseases, such as age-related macular degeneration (AMD) or photic maculopathy. Here, we report on inflammatory mechanisms that are associated with retinal degeneration induced by bright visible light, which were revealed while using a rabbit model. Histologically and electrophysiologically noticeable degeneration of the retina is preceded and accompanied by oxidative stress and inflammation, as evidenced by granulocyte infiltration and edema in this tissue, as well as the upregulation of total protein, pro-inflammatory cytokines, and oxidative stress markers in aqueous humor (AH). Consistently, quantitative lipidomic studies of AH elucidated increase in the concentration of arachidonic (AA) and docosahexaenoic (DHA) acids and lyso-platelet activating factor (lyso-PAF), together with pronounced oxidative and inflammatory alterations in content of lipid mediators oxylipins. These alterations include long-term elevation of prostaglandins, which are synthesized from AA via cyclooxygenase-dependent pathways, as well as a short burst of linoleic acid derivatives that can be produced by both enzymatic and non-enzymatic free radical-dependent mechanisms. The upregulation of all oxylipins is inhibited by the premedication of the eyes while using mitochondria-targeted antioxidant SkQ1, whereas the accumulation of prostaglandins and lyso-PAF can be specifically suppressed by topical treatment with cyclooxygenase inhibitor Nepafenac. Interestingly, the most prominent antioxidant and anti-inflammatory benefits and overall retinal protective effects are achieved by simultaneous administrating of both drugs indicating their synergistic action. Taken together, these findings provide a rationale for using a combination of mitochondria-targeted antioxidant and cyclooxygenase inhibitor for the treatment of inflammatory components of retinal degenerative diseases., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The use of nanomaterials is an emerging therapeutic approach for the treatment of several pathologies. Cerium oxide nanoparticles have been studied for biomedical application, including neurodegenerative disorders, such as age-related macular degeneration in several animal models. The light damage model is characterised by oxidative stress upregulation followed by photoreceptor death and microglia activation in the outer retina. For this reason, the light damage model mimics some aspects involved in human age-related macular degeneration pathogenesis. In this review, we focus on the neuroprotective effects on retinal function and microglia activation in the light damage model, considering the administration of the nanoparticles both before and after the injury. The electrical responses of the retina and the microglia number and morphology are clearly modulated by the treatment, supporting the beneficial effects of cerium oxide nanoparticles to counteract the degeneration processes in the retina., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Declining mitochondrial function drives ageing. With age, mitochondrial membrane potential declines, reducing ATP production and elevating pro-inflammatory reactive oxygen species production leading to cell death. In the retina mitochondrial density is high and there is a 30% photoreceptor loss with normal ageing. But aged mitochondrial membrane potential and ATP can be improved by long wavelengths absorbed in mitochondrial respiration. Hence, we ask if exposure to such wavelengths for 8 months in 12 month old mice can reduce aged photoreceptor loss. We expose aged mice daily for 10mins to 670 nm light then counted their photoreceptors. Exposure significantly retarded aged photoreceptor loss. Control mice suffered an approximate 30% decrease in photoreceptor outer segments and in the thickness of the retinal layer containing their nuclei compared to 2 month old mice. But in aged mice exposed to 670 nm over 8 month, reductions in outer segments were only <15% and reductions in their nuclear layer were only <10%. Hence, improving mitochndrial function reduces the impact of aged cell loss., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Sphingosine 1-phosphate (S1P) is a potent lipid mediator that modulates inflammation and angiogenesis. In this study, we investigated the possible involvement of S1P in the pathology of light-induced retinal degeneration in vivo and in vitro. The intracellular S1P and sphingosine kinase (SphK) activity in a photoreceptor cell line (661W cells) was significantly increased by exposure to light. The enhancement of SphK1 expression was dependent on illumination, and all-trans-retinal significantly promoted SphK1 expression. S1P treatment reduced protein kinase B (Akt) phosphorylation and increased the protein expression of cleaved caspase-3, and induced photoreceptor cell apoptosis. In vivo, light exposure enhanced the expression of SphK1 in the outer segments of photoreceptors. Intravitreal injection of a SphK inhibitor significantly suppressed the thinning of the outer nuclear layer and ameliorated the attenuation of the amplitudes of a-waves and b-waves of electroretinograms during light-induced retinal degeneration. These findings imply that light exposure induces the synthesis of S1P in photoreceptors by upregulating SphK1, which is facilitated by all-trans-retinal, causing retinal degeneration. Inhibition of this enhancement may be a therapeutic target of outer retinal degeneration, including age-related macular degeneration.

Purpose: To investigate structural changes in the retinal outer layers and choroid using adaptive optics (AO) and optical coherence tomography (OCT) in eyes with myopia, and to correlate the changes with decreased macular light sensitivity (MLS)., Methods: This prospective study included 27 subjects with emmetropia and low myopia (EM/LM), 25 with moderate myopia (MM), and 25 with high myopia (HM). Microperimetry was used to quantify MLS in each subject, while AO and OCT images of fundus were analyzed to quantify cone density and regularity and thickness of outer retinal sublayers and choroid. Differences of MLS, cone distribution, and chorioretinal thicknesses were compared among the three groups, and the associations among photoreceptor morphological alterations, MLS, and other parameters were analyzed., Results: In HM, the MLS, cone density and regularity, and thicknesses of the myoid and ellipsoid zone (MEZ), Henle fiber layer and outer nuclear layer, interdigitation zone and RPE/Bruch complex, and choroid were lower than in EM/LM. Decreased MLS was correlated with lower cone density and regularity, and thinner MEZ and choroid in the inner region, and with lower cone density, thinner MEZ and choroid, and longer axial length in the outer region. Multivariate regression showed that better MLS was correlated with thicker MEZ in the inner region and with higher cone density in the outer region., Conclusions: Altered cone distribution and outer retinal thickness, especially cone density and MEZ thickness, were significantly correlated with decline of MLS in HM, which may help to evaluate and monitor visual impairment in HM.

Purpose: Zeaxanthin protects the macula from ocular damage due to light or radiation by scavenging harmful reactive oxygen species. In the present study, zeaxanthin product (OmniXan®; OMX), derived from paprika pods (Capsicum annum; Family-Solanaceae), was tested for its efficacy in the rat retina against photooxidation., Methods: Forty-two male 8-week-old Wistar rats exposed to 12L/12D, 16L/8D and 24L/0D hours of intense light conditions were orally administrated either 0 or 100 mg/kg BW of zeaxanthin concentration. Retinal morphology was analyzed by histopathology, and target gene expressions were detected with real-time polymerase chain reaction methods., Results: OMX treatment significantly increased the serum zeaxanthin concentration (p < 0.001) and ameliorated oxidative damage by increasing the antioxidant enzyme activities in the retina induced by light (p < 0.001). OMX administration significantly upregulated the expression of genes, including Rhodopsin (Rho), Rod arrestin (SAG), Gα Transducin 1 (GNAT-1), neural cell adhesion molecule (NCAM), growth-associated protein 43 (GAP43), nuclear factor-(erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase (HO-1) and decreased the expression of nuclear factor-κB (NF- κB) and GFAP by OMX treatment rats. The histologic findings confirmed the antioxidant and gene expression data., Conclusions: This study suggests that OMX is a potent substance that can be used to protect photoreceptor cell degeneration in the retina exposed to intense light.

Purpose: To test the hypothesis that oxidative stress in the outer retina (OR = distance from external limiting membrane to the retinal pigment epithelium-choroid boundary) can be detected by using antioxidants (AOs) to correct an impaired light-evoked response as measured by optical coherence tomography (OCT)., Methods: C57BL/6J mice were maintained in the dark for ∼20 hours and studied by OCT before and after 1 hour of light exposure. OR thickness in dark or light was measured, and the light-dark difference (i.e., the photoresponse) was calculated. Subgroups of mice were given either saline or d-cis-diltiazem (an inducer of transient and nondamaging OR oxidative stress) ± methylene blue (24 hours before examination) and α-lipoic acid (1 hour before examination); one group was kept only in the dark and given only AOs., Results: In uninjected or saline-injected control mice, the OR showed a similar and reproducible light-induced expansion; dark-adapted mice given AOs did not increase dark-adapted OR thickness. The d-cis-diltiazem-treated mice had no photoresponse (P > 0.05). The d-cis-diltiazem-treated mice given AOs corrected (P < 0.05) the suppressed OR photoresponse, indicating the presence of oxidative stress., Conclusions: QUEnch-assiSTed (QUEST) OCT reproduced results from previous gold standard assays, showing that oxidative stress impairs the OR photoresponse and that d-cis-diltiazem produces OR oxidative stress. We envision future applications of QUEST OCT in a range of oxidative stress-based retinopathies.

Purpose: To determine if there is structural and functional recovery of the retina from light induced retinal degeneration, and to evaluate the role of the oxidative stress response elements Nrf2, SOD1, DJ-1, and Parkin in such a recovery process., Methods: Eyes from C57BL/6J (B6J) mice and from oxidative stress response-deficient strains of mice were treated with intense light using the fundus camera-delivered light-induced retinal degeneration (FCD-LIRD) model. Fundus photographs, optical coherence tomography (OCT) images, and electroretinography (ERG) responses were obtained before the injury, during the "maximal injury phase" (days 4-7) and during the "recovery phase" (days 14-16) post light exposure and were evaluated for retinal damage and assessed for evidence of recovery from the injury., Results: We demonstrate that mice treated with a sub-lethal FCD-LIRD protocol show an initial acute retina injury phase peaking between days 4 to 7 followed by a recovery phase in which the outer retinal thickness/volume and retinal function partially recover. These observations are reproduced in B6J mice and in mice lacking oxidative stress response enzymes (SOD1, DJ-1, and Parkin) or the oxidative stress response master regulator Nrf2., Conclusions: Our data indicate that retinal recovery from injury can proceed via pathways that are independent from the common oxidative stress response elements Nrf2, SOD1, DJ-1, and Parkin. Furthermore, the model of retinal recovery from injury that we describe here mimics changes seen in a variety of clinical entities and may provide an excellent platform for dissecting general pathways of retinal recovery from sub-lethal injury.

Purpose: MicroRNA-182 (miR-182) is abundantly expressed in mammalian retinas; however, the association between miR-182 and retinal function remains unclear. In this study, we explored whether miR-182 contributes to functional decline in retinas using a miR-182 depleted mouse., Methods: Electroretinogram (ERG) amplitudes at different ages were measured in miR-182 knockout (KO) mice. The thickness and lamination of retinas were assessed using a color fundus camera and high-resolution optical coherence tomography. Expression levels of key photoreceptor-specific genes and the miR-183/96/182 cluster (miR-183C) were quantified using quantitative real-time PCR. RNA sequencing and light-induced damage were carried out to observe the changes in the retinal transcriptome and sensitivity to light damage in the miR-182 KO mice., Results: The ERG recording reveals that the ERG response amplitude decreased both at early and later ages when compared with control littermates. The expression of some key photoreceptor-specific genes was down-regulated with deletion of miR-182 in retina. RNA sequencing indicated that some biological processes of visual system were affected, and the numbers of potential target genes of miR-182 were presented in the mouse retina using bioinformatics analysis. The miR-182 KO mice were characterized by progressively losing the outer segment after being treated with light-damage exposure. The thickness and lamination of retina as well as compensatory expression of miR-183C showed no apparent changes in retina of miR-182 KO mice under normal laboratory lighting condition., Conclusions: Our findings provided new insights into the relationship between the miR-182 and retinal development and revealed that miR-182 may play a critical role in maintaining retinal function.

Purpose: We determine if monomethyl fumarate (MMF) can protect the retina in mice subjected to light-induced retinopathy (LIR)., Methods: Albino BALB/c mice were intraperitoneally injected with 50 to 100 mg/kg MMF before or after exposure to bright white light (10,000 lux) for 1 hour. Seven days after light exposure, retinal structure and function were evaluated by optical coherence tomography (OCT) and electroretinography (ERG), respectively. Retinal histology also was performed to evaluate photoreceptor loss. Expression levels of Hcar2 and markers of microglia activation were measured by quantitative PCR (qPCR) in the neural retina with and without microglia depletion. At 24 hours after light exposure, retinal sections and whole mount retinas were stained with Iba1 to evaluate microglia status. The effect of MMF on the nuclear factor kB subunit 1 (NF-kB) and Nrf2 pathways was measured by qPCR and Western blot., Results: MMF administered before light exposure mediated dose-dependent neuroprotection in a mouse model of LIR. A single dose of 100 mg/kg MMF fully protected retinal structure and function without side effects. Expression of the Hcar2 receptor and the microglia marker Cd14 were upregulated by LIR, but suppressed by MMF. Depleting microglia reduced Hcar2 expression and its upregulation by LIR. Microglial activation, upregulation of proinflammatory genes (Nlrp3, Caspase1, Il-1β, Tnf-α), and upregulation of antioxidative stress genes (Hmox1) associated with LIR were mitigated by MMF treatment., Conclusions: MMF can completely protect the retina from LIR in BALB/c mice. Expression of Hcar2, the receptor of MMF, is microglia-dependent in the neural retina. MMF-mediated neuroprotection was associated with attenuation of microglia activation, inflammation and oxidative stress in the retina.

There have been a number of studies on the negative effects of blue light exposure in various species; however, little information is available on the impacts of blue light intensity and duration on fish. We investigated the effects of blue light spectra on stress in the retinas of goldfish, using a blue (460 nm) light-emitting diode (LED) at three intensities (0.5, 1.0, and 1.5 W/m 2 ). The experiment was conducted for 4 weeks, and sampling was performed at intervals of 1 week. We measured changes in the expression of cortisol, and the concentrations of hydrogen peroxide (H 2 O 2 ), melanin-concentrating hormone receptor (MCH-R), and caspase-3 in the retinas of goldfish. In addition, we measured histological changes in the retina. We used a transferase dUTP nick end labeling (TUNEL) assay to evaluate the apoptotic response to blue LED spectra. Levels of cortisol, H 2 O 2 , MCH-R, and caspase-3 increased with exposure time and light intensity. Histological analysis revealed that the thickness of melanin granules increased with exposure time and light intensity. The progressive TUNEL assay revealed many apoptotic cells after exposure to blue LED light, increasing with exposure time and light intensity. Irradiation with blue light for longer than 1 week induced increased retinal stress and may induce apoptosis in the retinas of goldfish, even at a low intensity.

The aim of this study was to explore the role of autophagy in response to blue light damage in aged mice and in human retinal pigmented epithelium (hRPE) cells. Blue light damage to the retina was induced in 10-month-old (10 mo) C57 mice and hRPE cells. Flash electroretinography was used to assess retinal function. Retinal structure changes were observed by electron microscopy. Western blot was conducted to determine the expression levels of the following proteins: cleaved caspase-3, p38 mitogen-activated protein kinases, protein kinase R-like endoplasmic reticulum kinase (PERK), autophagy marker light chain 3 (LC3), P62, and Beclin-1. On day 1 after light damage to the 10 mo mice, retinal function was changed. The latent periods of a-wave and b-wave were delayed, and amplitude was reduced. The electron microscopy results revealed mitochondria damage in the retinal pigmented epithelium and a disorganized photoreceptor outer segment (OS). PERK, LC3, and Beclin-1 were upregulated, whereas P62 was not. On day 5 after the blue light damage, restoration of electroretinography and OS was observed. PERK, LC3, and Beclin-1 were downregulated, whereas P62 was not. Protein changes in vitro were consistent with in vivo. The present study provided structural and functional evidence that autophagy plays an important role in the response to blue lightinduced retinal damage.

Purpose: Lipocalin 2 (LCN2) is reported to be one of the key regulators of cell survival and death; however, its effect on retinal degeneration is unclear. Therefore, we aimed to investigate the role of LCN2 and its underlying mechanisms in light-induced retinal degeneration., Methods: A recombinant lentivirus expressing a short hairpin RNA targeting LCN2 mRNA and a recombinant lentivirus overexpressing LCN2 were used to downregulate and upregulate retinal LCN2, respectively. Seven days after intravitreal injection of the lentiviruses, rats were exposed to blue light (2500 lux) for 24 hours. Retinal function and morphology were evaluated with ERG and hematoxylin-eosin staining, respectively. TUNEL staining was used to detect apoptotic cells. The levels of reactive oxygen species (ROS) were evaluated with dihydroethidium labeling. Western blotting and real-time PCR were used to examine protein and mRNA expression levels, respectively., Results: Retinal LCN2 expression was significantly upregulated after light exposure. Light exposure reduced the amplitudes of a- and b-waves on the ERG and the thickness of the outer nuclear layer and promoted photoreceptor apoptosis. These phenomena were clearly attenuated by LCN2 knockdown, whereas LCN2 overexpression had the opposite effects. The overexpression of LCN2 facilitated photoreceptor apoptosis by increasing ROS generation and Bim expression. On the opposite, LCN2 knockdown mitigated the generation of light-exposure-induced ROS and the activation of the Bim-mediated mitochondrial apoptotic pathway., Conclusions: Light-induced LCN2 is a proapoptotic factor in the retina, and LCN2 knockdown protects photoreceptors from apoptosis by inhibiting ROS production and Bim expression. LCN2 is a potential therapeutic target for light-induced retinal degeneration.

To determine the characteristics of the damages of the retinal pigment epithelium (RPE) and photoreceptors of pigmented mice induced by exposure to blue light emitting diode (LED) light, and to determine the mechanisms causing the damages. Exposure to blue LED light for 3 days induced retinal damage, and the characteristics of the damage differed from that induced by white fluorescent light exposure. Ophthalmoscopy showed that blue LED exposure for 3 days induced white spots on the retina, and histological examinations showed materials accumulated at the IS/OS junction of the photoreceptors. The accumulated materials were stained by ionized calcium binding adapter molecule-1 (Iba-1), a marker for macrophages. The debris was also positive for periodic acid-Schiff (PAS). An enlarging the area of RPE was detected just after the blue LED exposure especially around the optic nerve, and this led to a secondary degeneration of the photoreceptors. Exposure of pigmented mice to 3 consecutive days of blue LED light will cause RPE and photoreceptor damage. The damage led to an accumulation of macrophages and drusen-like materials around the outer segments of the photoreceptors. This blue light exposed model may be useful for investigating the pathogenesis of non-exudative age-related macular degeneration., (Copyright © 2018. Published by Elsevier Ltd.)

Purpose: To determine the neuroprotective effect of estrogen on the structure and function of the retina exposed to an oxidative stress., Methods: Male Sprague-Dawley rat pups were exposed to either hyperoxia (O 2 E: from P8 to P14) or bright light (LE: from P14 to P28) with or without 17 β-estradiol (βE 2 ) treatment. Retinal structure (histology) and function (ERG) were assessed at selected time points., Results: In the O 2 E model, βE 2 injections caused a significant reduction of the ERG and a significantly thinner OPL compared to untreated oxygen-exposed group (O 2 -exposed) rats. In contrast, in the LE model βE 2 , treatment was beneficial to the retinal structure (thicker ONL) and function (better preserved ERG amplitudes) compared to untreated light-exposed group (light-exposed rats)., Conclusion: Our results show that in conditions where the primary target of the oxidative stress is the outer retina (i.e., the photoreceptors) estrogen can protect the retina, while in situations where the inner retina (or retinal vasculature) is the main site of oxidative damage, estrogen may potentiate the detrimental effect of oxidative stress on the retina.

The transepithelial potential difference (TEP) across the retinal pigment epithelial (RPE) is dependent on ionic pumps and tight junction "seals" between epithelial cells. RPE cells release neurotrophic growth factors such as pigment epithelial derived factor (PEDF), which is reduced in age-related macular degeneration (AMD). The mechanisms that control the secretion of PEDF from RPE cells are not well understood. Using the CCL2/CX3CR1 double knockout mouse model (DKO), which demonstrates RPE damage and retinal degeneration, we uncovered an interaction between PEDF and the TEP which is likely to play an important role in retinal ageing and in the pathogenesis of AMD. We found that: (a) the expression of ATP1B1 (the Na + /K + -ATPase β1 subunit) was reduced significantly in RPE from aged mice, in patients with CNV (Choroidal Neovascularization) and in DKO mice; (b) the expression of PEDF also was decreased in aged persons and in DKO mice; (c) the TEP across RPE was reduced markedly in RPE cells from DKO mice and (d) an applied electric field (EF) of 50-100 mV/mm, used to mimic the natural TEP, increased the expression and secretion of PEDF in primary RPE cells. In conclusion, the TEP across the RPE depends on the expression of ATP1B1 and this regulates the secretion of PEDF by RPE cells and so may regulate the onset of retinal disease. Increasing the expression of PEDF using an applied EF to replenish a disease or age-reduced TEP may offer a new way of preventing or reversing retinal dysfunction., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Juvenile neuronal ceroid lipofuscinosis (jNCL) is a rare but fatal inherited lysosomal storage disorder mainly affecting children. The disease is caused by mutations in the CLN3 gene that lead to the accumulation of storage material in many tissues, prominent immune responses and neuronal degeneration. One of the first symptoms is vision loss followed by motor dysfunction and mental decline. The established Cln3 Δex7/8 mouse model mimics many pathological features of the human disease except the retinal phenotype, which is very mild and occurs only very late in these mice. Here, we first carefully analyzed the retinal structure and microglia responses in these animals. While prominent autofluorescent spots were present in the fundus, only a moderate reduction of retinal thickness and no prominent microgliosis was seen in young CLN3-deficient mice. We next genetically introduced a light-sensitive RPE65 variant and established a light-damage paradigm that showed a high susceptibility of young Cln3 Δex7/8 mice after exposure to 10,000 lux bright light for 30 min. Under these 'low light' conditions, CLN3-deficient mice showed a strong retinal degeneration, microglial activation, deposition of autofluorescent material and transcriptomic changes compared to wild-type animals. Finally, we treated the light-exposed Cln3 Δex7/8 animals with the immunomodulatory compound minocycline, and thereby rescued the retinal phenotype and diminished microgliosis. Our findings indicate that exposure to specific light conditions accelerates CLN3-dependent retinal degeneration, and that immunomodulation by minocycline could be a possible treatment option to delay vision loss in jNCL patients.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

NMNAT1 (nicotinamide mononucleotide adenylyltransferase 1) encodes a rate-limiting enzyme that catalyzes the biosynthesis of NAD + and plays a role in neuroprotection. Mutations in NMNAT1 have been identified to cause a recessive, non-syndromic early form of blindness genetically defined as Leber Congenital Amaurosis 9 (LCA9). One of the most common alleles reported so far in NMNAT1 is the c.769G > A (E257K) missense mutation, which occurs in 70% of all LCA9 cases. However, given its relatively high population frequency and the observation of individuals with homozygous E257K variant without phenotype, the pathogenicity of this allele has been questioned. To address this issue, we have studied the pathogenic effects of this allele by generating a knock-in mouse model. Interestingly, no obvious morphological or functional defects are observed in Nmnat1 E257K homozygous mice up to one year old, even after light-damage. Together with the previous clinical reports, we propose that the E257K allele is a weak hypomorphic allele that has significantly reduced penetrance in the homozygous state. In contrast, compound heterozygous Nmnat1 E257K/- mice exhibit photoreceptor defects which are exacerbated upon exposure to light. Furthermore, retina tissue- specific Nmnat1 conditional knockout mice exhibit photoreceptor degeneration before the retina has terminally differentiated. These findings suggest that NMNAT1 plays an important role in photoreceptors and is likely involved in both retinal development and maintenance of photoreceptor integrity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Light induced retinal degeneration (LIRD) is a useful model that resembles human retinal degenerative diseases. The modulation of adenosine A1 receptor is neuroprotective in different models of retinal injury. The aim of this work was to evaluate the potential neuroprotective effect of the modulation of A1 receptor in LIRD. The eyes of rats intravitreally injected with N6-cyclopentyladenosine (CPA), an A1 agonist, which were later subjected to continuous illumination (CI) for 24 h, showed retinas with a lower number of apoptotic nuclei and a decrease of Glial Fibrillary Acidic Protein (GFAP) immunoreactive area than controls. Lower levels of activated Caspase 3 and GFAP were demonstrated by Western Blot (WB) in treated animals. Also a decrease of iNOS, TNFα and GFAP mRNA was demonstrated by RT-PCR. A decrease of Iba 1+/MHC-II+ reactive microglial cells was shown by immunohistochemistry. Electroretinograms (ERG) showed higher amplitudes of a-wave, b-wave and oscillatory potentials after CI compared to controls. Conversely, the eyes of rats intravitreally injected with dipropylcyclopentylxanthine (DPCPX), an A1 antagonist, and subjected to CI for 24 h, showed retinas with a higher number of apoptotic nuclei and an increase of GFAP immunoreactive area compared to controls. Also, higher levels of activated Caspase 3 and GFAP were demonstrated by Western Blot. The mRNA levels of iNOS, nNOS and inflammatory cytokines (IL-1β and TNFα) were not modified by DPCPX treatment. An increase of Iba 1+/MHC-II+ reactive microglial cells was shown by immunohistochemistry. ERG showed that the amplitudes of a-wave, b-wave, and oscillatory potentials after CI were similar to control values. A single pharmacological intervention prior illumination stress was able to swing retinal fate in opposite directions: CPA was neuroprotective, while DPCPX worsened retinal damage. In summary, A1 receptor agonism is a plausible neuroprotective strategy in LIRD., Competing Interests: The authors have declared that no competing interests exist.

Photobiomodulation (PBM) with 670 nm light has been shown to accelerate wound healing in soft tissue injuries, and also to protect neuronal tissues. However, little data exist on its effects on the non-neuronal components of the retina, such as Müller cells (MCs), which are the principal macroglia of the retina that play a role in maintaining retinal homeostasis. The aim of this study was to explore the effects of 670 nm light on activated MCs using in vivo and in vitro stress models. Adult Sprague-Dawley rats were exposed to photo-oxidative damage (PD) for 24 h and treated with 670 nm light at 0, 3 and 14 days after PD. Tissue was collected at 30 days post-PD for analysis. Using the in vitro scratch model with a human MC line (MIO-M1), area coverage and cellular stress were analysed following treatment with 670 nm light. We showed that early treatment with 670 nm light after PD reduced MC activation, lowering the retinal expression of GFAP and FGF-2. 670 nm light treatment mitigated the production of MC-related pro-inflammatory cytokines (including IL-1β), and reduced microglia/macrophage (MG/MΦ) recruitment into the outer retina following PD. This subsequently decreased photoreceptor loss, slowing the progression of retinal degeneration. In vitro, we showed that 670 nm light directly modulated MC activation, reducing rates of area coverage by suppressing cellular proliferation and spreading. This study indicates that 670 nm light treatment post-injury may have therapeutic benefit when administered shortly after retinal damage, and could be useful for retinal degenerations where MC gliosis is a feature of disease progression., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Light causes damage to the retina (phototoxicity) and decreases photoreceptor responses to light. The most harmful component of visible light is the blue wavelength (400-500 nm). Different filters have been tested, but so far all of them allow passing a lot of this wavelength (70%). The aim of this work has been to prove that a filter that removes 94% of the blue component may protect the function and morphology of the retina significantly. Three experimental groups were designed. The first group was unexposed to light, the second one was exposed and the third one was exposed and protected by a blue-blocking filter. Light damage was induced in young albino mice (p30) by exposing them to white light of high intensity (5,000 lux) continuously for 7 days. Short wavelength light filters were used for light protection. The blue component was removed (94%) from the light source by our filter. Electroretinographical recordings were performed before and after light damage. Changes in retinal structure were studied using immunohistochemistry, and TUNEL labeling. Also, cells in the outer nuclear layer were counted and compared among the three different groups. Functional visual responses were significantly more conserved in protected animals (with the blue-blocking filter) than in unprotected animals. Also, retinal structure was better kept and photoreceptor survival was greater in protected animals, these differences were significant in central areas of the retina. Still, functional and morphological responses were significantly lower in protected than in unexposed groups. In conclusion, this blue-blocking filter decreases significantly photoreceptor damage after exposure to high intensity light. Actually, our eyes are exposed for a very long time to high levels of blue light (screens, artificial light LED, neons…). The potential damage caused by blue light can be palliated.

Earlier studies showed that the expressions of the agonists of the cannabinoid receptors are reduced in the vitreous humor of patients with age-related macular degeneration (AMD), and the cannabinoid type 2 receptor is present in the retinas of rats and monkeys. The purpose of this study was to determine whether the cannabinoid type 2 receptor is involved in the light-induced death of cultured 661W cells, an immortalized murine retinal cell line, and in the light-induced retinal degeneration in mice. Time-dependent changes in the expression and location of retinal cannabinoid type 2 receptor were determined by Western blot and immunostaining. The cannabinoid type 2 receptor was down-regulated in murine retinae and cone cells. In the in vitro studies, HU-308, a cannabinoid type 2 receptor agonist, had a protective effect on the light-induced death of 661W cells, and this effect was attenuated by SR144528, a cannabinoid type 2 receptor antagonist. Because the cannabinoid type 2 receptor is a G-protein coupled receptor and is coupled with G i/o protein, we investigated the effects of the cAMP-dependent protein kinase (PKA). HU-308 and H89, a PKA inhibitor, deactivated PKA in retinal cone cells, and H89 also suppressed light-induced cell death. For the in vivo studies, a cannabinoid type 2 receptor agonist, HU-308, or an antagonist, SR144528, was injected intravitreally into mouse eyes before the light exposure. Electroretinography was used to determine the physiological status of the retinas. Injection of HU-308 improved the a- and b-waves of the ERGs and also the thickness of the outer nuclear layer of the murine retina after light exposure. These findings indicate that the cannabinoid type 2 receptor is involved in the light-induced retinal damage through PKA signaling. Thus, activation of cannabinoid type 2 receptor may be a therapeutic approach for light-associated retinal diseases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: To characterize the mediators of 5-HT2A serotonin receptor-driven retinal neuroprotection., Methods: Albino mice were treated intraperitoneally with saline or sarpogrelate, a 5-HT2A antagonist, immediately before light exposure (LE). Following LE, retinas were harvested for a high-throughput phosphorylation microarray to quantify activated phosphorylated proteins in G protein-coupled receptor (GPCR) signaling. To confirm microarray results and define temporal changes, Western blots of select GPCR signaling proteins were performed. Since both methodologies implicated MAPK/ERK activation, the functional significance of sarpogrelate-mediated ERK1/2 activation was examined by inhibition of ERK1/2 phosphorylation via pretreatment with the MEK inhibitor (MEKi) PD0325901. The degree of neuroprotection was evaluated with spectral-domain optical coherence tomography (SD-OCT) and electroretinography (ERG). To determine the effects of sarpogrelate on gene expression, a qPCR array measuring the expression of 84 genes involved in oxidative stress and cell death was performed 48 hours post LE., Results: Sarpogrelate led to an activation of the MAPK/ERK pathway. Temporal analysis further demonstrated a transient activation of ERK1/2, starting with an early inhibition 20 minutes into LE, a maximum activation at 3 hours post LE, and a return to baseline at 7 hours post LE. Inhibition of ERK1/2 with MEKi pretreatment led to attenuation of sarpogrelate-mediated neuroprotection. LE caused significant changes in the expression of genes involved in iron metabolism, oxidative stress, and apoptosis. These changes were prevented by sarpogrelate treatment., Conclusions: Sarpogrelate-mediated retinal protection involves a transient activation of the MAPK/ERK pathway, although this pathway alone does not account for the full effect of neuroprotection.

Purpose: We used acoustic radiation force optical coherence elastography (ARF-OCE) to map out the elasticity of retinal layers in healthy and diseased in vivo rabbit models for the first time., Methods: A healthy rabbit eye was proptosed and imaged using ARF-OCE, by measuring the tissue deformation after an acoustic force is applied. A diseased retinal inflammation model was used to observe the contrast before and after disease formation. Retinal histologic analysis was performed to identify layers of the retina corresponding with the optical images., Results: The general trend of the retinal layer elasticity is increasing stiffness from the ganglion side to the photoreceptor side, with the stiffest layer being the sclera. In a healthy rabbit model, the mechanical properties varied from 3 to 16 kPa for the five layers that were identified via optical imaging and histology (3.09 ± 0.46, 3.82 ± 0.88, 4.53 ± 0.74, 6.59 ± 2.27, 16.11 ± 5.13 kPa). In the diseased model, we have induced optical damage in a live rabbit and observed a change in the stiffness trend in its retina., Conclusions: High sensitivity elasticity maps can be obtained using the ARF-OCE system to differentiate different retinal layers. Subtle changes in the mechanical properties during the onset of diseases, such as retinal degeneration, can be measured and aid in early clinical diagnosis. This study validates our imaging system for the characterization of retinal elasticity for the detection of retinal diseases in vivo.

Purpose: Light-induced photoreceptor cell degeneration and disease progression in age-related macular degeneration (AMD) involve oxidative stress and visual cell loss, which can be prevented, or slowed, by antioxidants. Our goal was to test the protective efficacy of a traditional Age-related Eye Disease Study antioxidant formulation (AREDS) and AREDS combined with non-traditional antioxidants in a preclinical animal model of photooxidative retinal damage., Methods: Male Sprague-Dawley rats were reared in a low-intensity (20 lux) or high-intensity (200 lux) cyclic light environment for 6 weeks. Some animals received a daily dietary supplement consisting of a small cracker infused with an AREDS antioxidant mineral mixture, AREDS antioxidants minus zinc, or zinc oxide alone. Other rats received AREDS combined with a detergent extract of the common herb rosemary, AREDS plus carnosic acid, zinc oxide plus rosemary, or rosemary alone. Antioxidant efficacy was determined by measuring retinal DNA levels 2 weeks after 6 h of intense exposure to white light (9,000 lux). Western blotting was used to determine visual cell opsin and arrestin levels following intense light treatment. Rhodopsin regeneration was determined after 1 h of exposure to light. Gene array analysis was used to determine changes in the expression of retinal genes resulting from light rearing environment or from antioxidant supplementation., Results: Chronic high-intensity cyclic light rearing resulted in lower levels of rod and cone opsins, retinal S-antigen (S-ag), and medium wavelength cone arrestin (mCAR) than found for rats maintained in low cyclic light. However, as determined by retinal DNA, and by residual opsin and arrestin levels, 2 weeks after acute photooxidative damage, visual cell loss was greater in rats reared in low cyclic light. Retinal damage decreased with AREDS plus rosemary, or with zinc oxide plus rosemary whereas AREDS alone and zinc oxide alone (at their daily recommended levels) were both ineffective. One week of supplemental AREDS plus carnosic acid resulted in higher levels of rod and cone cell proteins, and higher levels of retinal DNA than for AREDS alone. Rhodopsin regeneration was unaffected by the rosemary treatment. Retinal gene array analysis showed reduced expression of medium- wavelength opsin 1 and arrestin C in the high-light reared rats versus the low-light rats. The transition of rats from low cyclic light to a high cyclic light environment resulted in the differential expression of 280 gene markers, enriched for genes related to inflammation, apoptosis, cytokine, innate immune response, and receptors. Rosemary, zinc oxide plus rosemary, and AREDS plus rosemary suppressed 131, 241, and 266 of these genes (respectively) in high-light versus low-light animals and induced a small subset of changes in gene expression that were independent of light rearing conditions., Conclusions: Long-term environmental light intensity is a major determinant of retinal gene and protein expression, and of visual cell survival following acute photooxidative insult. Rats preconditioned by high-light rearing exhibit lower levels of cone opsin mRNA and protein, and lower mCAR protein, than low-light reared animals, but greater retention of retinal DNA and proteins following photooxidative damage. Rosemary enhanced the protective efficacy of AREDS and led to the greatest effect on the retinal genome in animals reared in high environmental light. Chronic administration of rosemary antioxidants may be a useful adjunct to the therapeutic benefit of AREDS in slowing disease progression in AMD.

In this work we study the effects of an acute light-induced retinal degeneration on the population of melanopsin positive retinal ganglion cells (m + RGCs) and the expression of the melanopsin protein in the retina. The m + RGCs may be more resistant than other RGCs to lesion, but the effects of an acute light exposure in this population are unknown. Albino rats were exposed to white light (3000 lux) continuously for 48 h and processed 0, 3, 7 or 30 days after light exposure (ALE). Whole-mounted retinas were immunodetected with antibodies against melanopsin, Brn3a, and rhodopsin to study the populations of m + RGC, Brn3a + RGC and rods (which are the most abundant photoreceptors in the rat retina). Three days ALE there was substantial rod loss in an arciform area of the superior retina and with time this loss expanded in the form of rings all throughout the retina. Light exposure did not affect the number of Brn3a + RGCs but diminished the numbers of m + RGCs. Immediately ALE there was a significant decrease in the mean number of immunodetected m + RGCs that was more marked in the superior retina. Later, the number of m + RGCs increased progressively and reached normal values one month ALE. Western blot analysis showed that melanopsin expression down-regulates shortly ALE and recovers thereafter, in accordance with the anatomical data. This study demonstrates that there is a transient downregulation of melanopsin expression in the RGCs during the first month ALE. Further studies would be needed to clarify the long-term effect of light exposure on the m + RGC population., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Transgenic mice overexpressing serum retinol-binding protein (RBP4-Tg) develop progressive retinal degeneration, characterized by microglia activation, yet the precise mechanisms underlying retinal degeneration are unclear. Previous studies showed RBP4-Tg mice have normal ocular retinoid levels, suggesting that degeneration is independent of the retinoid visual cycle or light exposure. The present study addresses whether retinal degeneration is light-dependent and RBP4-dependent by testing the effects of dark-rearing and pharmacological lowering of serum RBP4 levels, respectively., Methods: RBP4-Tg mice reared on normal mouse chow in normal cyclic light conditions were directly compared to RBP4-Tg mice exposed to chow supplemented with the RBP4-lowering compound A1120 or dark-rearing conditions. Quantitative retinal histological analysis was conducted to assess retinal degeneration, and electroretinography (ERG) and optokinetic tracking (OKT) tests were performed to assess retinal and visual function. Ocular retinoids and bis-retinoid A2E were quantified., Results: Dark-rearing RBP4-Tg mice effectively reduced ocular bis-retinoid A2E levels, but had no significant effect on retinal degeneration or dysfunction in RBP4-Tg mice, demonstrating that retinal degeneration is light-independent. A1120 treatment lowered serum RBP4 levels similar to wild-type mice, and prevented structural retinal degeneration. However, A1120 treatment did not prevent retinal dysfunction in RBP4-Tg mice. Moreover, RBP4-Tg mice on A1120 diet had significant worsening of OKT response and loss of cone photoreceptors compared to RBP4-Tg mice on normal chow. This may be related to the very significant reduction in retinyl ester levels in the retina of mice on A1120-supplemented diet., Conclusions: Retinal degeneration in RBP4-Tg mice is RBP4-dependent and light-independent.

Purpose: The aim of the present study was to investigate the neuroprotective effects of glucocorticoid-induced leucine zipper (GILZ) in a light-induced retinal degeneration model and to explore the underlying mechanisms., Methods: Intravitreal injection of recombinant GILZ-overexpressing lentivirus (OE-GILZ-rLV) and short hairpin RNA targeting GILZ recombinant lentivirus (shRNA-GILZ-rLV) was performed to up- and downregulate retinal GILZ, respectively. Three days after stable transduction, rats were exposed to continuous bright light (5000 lux) for 2 days. Retinal function was assessed by full-field electroretinography (ERG), and the retinal structure was examined for photoreceptor survival and death in rats kept under a 12-hour light:2-hour dark cycle following light exposure. The expression levels of retinal Bcl-xL, caspase-9, and caspase-3 were examined by Western blotting or real-time PCR at 1, 3, 5, and 7 days after light exposure., Results: Exposure to bright light downregulated retinal GILZ in parallel with the downregulation of Bcl-xL and the upregulation of active caspase-3. Overexpression of retinal GILZ attenuated the decrease of Bcl-xL and the activation of caspase-9 and caspase-3 at 1, 3, 5, and 7 days after bright light exposure, respectively. GILZ silencing aggravated the downregulation of Bcl-xL induced by bright light exposure. Bright light exposure reduced the amplitude of ERG, increased the number of apoptotic photoreceptor cells, and decreased retinal thickness; and GILZ overexpression could attenuate all these effects., Conclusions: Overexpression of GILZ by OE-GILZ-rLV transduction protected the retina from light-induced cellular damage by activating antiapoptotic pathways.

Purpose: The complement system is involved in the pathogenesis of age-related macular degeneration (AMD). Because activated microglia are also associated with AMD, we studied the relationship between complement anaphylatoxin receptors and microglial recruitment., Methods: We assessed the effect of anaphylatoxin C3a receptor (C3aR) and C5a receptor (C5aR) knockout (KO) on light damage-induced migration of microglia/macrophages into the mouse outer retina via immunofluorescence and real-time quantitative PCR., Results: We found that the mRNA levels of C3, C5, C3aR, C5aR, and two activators of the complement alternative pathway, Cfb and Cfd, were all upregulated after light exposure. Retinal Iba1-positive microglia/macrophages express receptors for C3a and C5a. Light damage increased the number of retinal Iba1-positive cells and the mRNA levels of Iba1. Compared with the wild-type (WT) mice, these increases were attenuated in the C5aR KO mice but not in the C3aR KO mice., Conclusions: C5aR but not C3aR promoted the recruitment of microglia/macrophages. These divergent properties of complement anaphylatoxins in the light damage model provide a rationale for testing the differential effects of these receptors in additional retinal and neurodegeneration models.

Purpose: To characterize a light damage paradigm and establish structural and immunocytochemical measures of acute and protracted light-induced retinal degeneration in the rhodopsin (RHO) T4R dog model of RHO-autosomal dominant retinitis pigmentosa (ADRP)., Methods: Retinal light damage was induced in mutant dogs with a 1-minute exposure to various light intensities (0.1-1.0 mW/cm2) delivered with a Ganzfeld stimulator, or by fundus photography. Photoreceptor cell death was assessed by TUNEL assay, and alterations in retinal layers were examined by histology and immunohistochemistry 24 hours and 2 weeks after light exposure. Detailed topographic maps were made to document changes in the outer retinal layers of all four retinal quadrants 2 weeks post exposure., Results: Twenty-four hours post light exposure, the severity of photoreceptor cell death was dose dependent. Immunohistochemical analysis revealed disruption of rod outer segments, focal loss of the RPE integrity, and an increase in expression of endothelin receptor B in Müller cells with the two highest doses of light and fundus photography. Two weeks after light exposure, persistence of photoreceptor death, thinning of the outer nuclear layer, and induction of Müller cell gliosis occurred with the highest doses of light., Conclusions: We have characterized outcome measures of acute and continuing retinal degeneration in the RHO T4R dog following light exposure. These will be used to assess the molecular mechanisms of light-induced damage and rescue strategies in this large animal model of RHO-ADRP.

The aim of study was to establish a mouse model of blue light emitting diode (LED) light-induced retinal damage and to evaluate the effects of the antioxidant N-acetylcysteine (NAC). Mice were exposed to 400 or 800 lx blue LED light for 2 h, and were evaluated for retinal damage 5 d later by electroretinogram amplitude and outer nuclear layer (ONL) thickness. Additionally, we investigated the effect of blue LED light exposure on shorts-wave-sensitive opsin (S-opsin), and rhodopsin expression by immunohistochemistry. Blue LED light induced light intensity dependent retinal damage and led to collapse of S-opsin and altered rhodopsin localization from inner and outer segments to ONL. Conversely, NAC administered at 100 or 250 mg/kg intraperitoneally twice a day, before dark adaptation and before light exposure. NAC protected the blue LED light-induced retinal damage in a dose-dependent manner. Further, blue LED light-induced decreasing of S-opsin levels and altered rhodopsin localization, which were suppressed by NAC. We established a mouse model of blue LED light-induced retinal damage and these findings indicated that oxidative stress was partially involved in blue LED light-induced retinal damage.

To save energy, the European directives from the Eco-design of Energy Using Products (2005/32/CE) have recommended the replacement of incandescent lamps by more economic devices such as Light Emitting Diodes (LEDs). However, the emission spectrum of these devices is enriched in blue radiations, known to be potentially dangerous to the retina. Recent studies showed that light exposure contributes to the onset of early stages of age-related macular degeneration (AMD). Here, we investigate, in albinos and pigmented rats, the effects of different exposure protocols. Twenty-four hours exposure at high luminance was compared to a cyclic (dark/light) exposure at domestic levels for 1week and 1month, using different LEDs (Cold-white, blue and green), as well as fluorocompact bulbs and fluorescent tubes. The data suggest that the blue component of the white-LED may cause retinal toxicity at occupational domestic illuminance and not only in extreme experimental conditions, as previously reported. It is important to note that the current regulations and standards have been established on the basis of acute light exposure and do not take into account the effects of repeated exposure., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Experimental studies demonstrated that saffron (Crocus sativus) given as a dietary supplement counteracts the effects of bright continuous light (BCL) exposure in the albino rat retina, preserving both morphology and function and probably acting as a regulator of programmed cell death [1]. The purpose of this study was to ascertain whether the neuroprotective effect of saffron on rat retina exposed to BCL is associated with a modulation of the endocannabinoid system (ECS). To this aim, we used eight experimental groups of Sprague-Dawley rats, of which six were exposed to BCL for 24 hours. Following retinal function evaluation, retinas were quickly removed for biochemical and morphological analyses. Rats were either saffron-prefed or intravitreally injected with selective type-1 (CB1) or type-2 (CB2) cannabinoid receptor antagonists before BCL. Prefeeding and intravitreally injections were combined in two experimental groups before BCL. BCL exposure led to enhanced gene and protein expression of retinal CB1 and CB2 without affecting the other ECS elements. This effect of BCL on CB1 and CB2 was reversed by saffron treatment. Selective CB1 and CB2 antagonists reduced photoreceptor death, preserved morphology and visual function of retina, and mitigated the outer nuclear layer (ONL) damage due to BCL. Of interest, CB2-dependent neuroprotection was more pronounced than that conferred by CB1. These data suggest that BCL modulates only distinct ECS elements like CB1 and CB2, and that saffron and cannabinoid receptors could share the same mechanism in order to afford retinal protection., Competing Interests: Competing Interests: This investigation was partially supported by the commercial companies Essse Caffè S.p.A. and Hortus Novus s.r.l. We confirm that this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Scope: Cyanidin-3-glucoside (C3G) is a major anthocyanin in berries and a potential nutritional supplement for preventing retinal degeneration. However, the protective mechanism of C3G and its metabolites, protocatechuic acid (PCA) and ferulic acid (FA), remain unclear. The molecular mechanisms of C3G and its metabolites against retinal photooxidative damage in vivo are investigated., Methods and Results: Pigmented rabbits were orally administered C3G, PCA, and FA (0.11 mmol/kg/day) for 3 weeks. Electroretinography, histological analysis, and TUNEL assay showed that C3G and its metabolites attenuated retinal cell apoptosis. The expression of oxidative stress markers were upregulated after light exposure but attenuated by C3G and FA, which may be attributed to the elevated secretion and expression of heme oxygenase (HO-1) and nuclear factor erythroid-2 related factor 2 (Nrf2). C3G, PCA, and FA attenuated the secretion or expression of inflammation-related genes; FA suppressed nuclear factor kappa B (NF-κB) activation. The treatments attenuated the light-induced changes on certain apoptotic proteins and angiogenesis-related cytokines., Conclusion: C3G and FA reduced light-induced retinal oxidative stress by activating the Nrf2/HO-1 antioxidant pathway. FA attenuated the light-induced retinal inflammation by suppressing NF-κB activation. C3G and its metabolites attenuated the photooxidation-induced apoptosis and angiogenesis in the retina., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Light-induced degeneration in rodent retinas is an established model for of retinal degeneration, including the roles of oxidative stress and neuroinflammatory activity. In these models, photoreceptor death is elicited via photo-oxidative stress, and is exacerbated by recruitment of subretinal macrophages and activation of immune pathways including complement propagation. Existing light damage models have relied heavily on albino rodents, and mostly using acute light stimuli. These albino models have proven valuable in uncovering the pathogenic mechanisms of such pathways in the context of retinal disease. However, their inherent albinism hinders comparability to normal retinal physiology, and also makes gene technology analysis time-consuming due to the predominance of the pigmented mouse strains in these applications. In this study, we characterise a new light damage model utilising C57BL/6J mice over a 7 day period of chronic light exposure. We use high-efficiency LED technology to deliver a sustained intensity of 100 k lux with negligible modulation of ambient temperature. We show that in the C57BL/6J mouse, chronic light exposure elicits the cardinal features of light damage including photoreceptor degeneration, atrophy of the choriocapillaris, decreased retinal function and increases in oxidative stress markers 4-HNE and 8-OHG, which emerge progressively over the 7 day period of exposure. These changes are accompanied by robust recruitment of IBA1+ and F4/80 + microglia/macrophages to the ONL and subretinal space, followed the strong up-regulation of monocyte-chemoattractants Ccl2, Ccl3, and Ccl12, as well as increases in expression of complement component C3. These findings are in agreement with prior damage models conducted in albino rodents such as Balb/c mice, and support the use of this new model in further investigating the causative features of oxidative stress and inflammation in retinal disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Optogenetics methods are rapidly being developed as therapeutic tools for treating neurological diseases, in particular, retinal degenerative diseases. A critical component of the development is testing the safety of the light stimulation used to activate the optogenetic proteins. While the stimulation needs to be sufficient to produce neural responses in the targeted retinal cell class, it also needs to be below photochemical and photothermal limits known to cause ocular damage. The maximal permissible exposure is determined by a variety of factors, including wavelength, exposure duration, visual angle, pupil size, pulse width, pulse pattern, and repetition frequency. In this paper, we develop utilities to systematically and efficiently assess the contributions of these parameters in relation to the limits, following directly from the 2014 American National Standards Institute (ANSI). We also provide an array of stimulus protocols that fall within the bounds of both safety and effectiveness. Additional verification of safety is provided with a case study in rats using one of these protocols., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

To compare patterns of gene expression following preconditioning cyclic light rearing versus preconditioning aerobic exercise. BALB/C mice were preconditioned either by rearing in 800 lx 12:12 h cyclic light for 8 days or by running on treadmills for 9 days, exposed to toxic levels of light to cause light-induced retinal degeneration (LIRD), then sacrificed and retinal tissue harvested. Subsets of mice were maintained for an additional 2 weeks and for assessment of retinal function by electroretinogram (ERG). Both preconditioning protocols partially but significantly preserved retinal function and morphology and induced similar leukemia inhibitory factor (LIF) gene expression pattern. The data demonstrate that exercise preconditioning and cyclic light preconditioning protect photoreceptors against LIRD and evoke a similar pattern of retinal LIF gene expression. It may be that similar stress response pathways mediate the protection provided by the two preconditioning modalities.

Purpose: To determine if sarpogrelate, a selective 5-HT2A receptor antagonist, is protective against light-induced retinopathy in BALB/c mice., Methods: BALB/c mice were dosed intraperitoneally with 5, 15, 30, 40, or 50 mg/kg sarpogrelate 48, 24, and 0 hours prior to bright light exposure (10,000 lux) as well as 24 and 48 hours after exposure. Additionally, a single injection regimen was evaluated by injecting mice with 50 mg/kg sarpogrelate once immediately prior to light exposure. To investigate the potential for additive effects of serotonin receptor agents, a combination therapy consisting of sarpogrelate (15 mg/kg) and 8-OH-DPAT (1 mg/kg) was evaluated with the 5-day treatment regimen. Neuroprotection was characterized by the preservation of retinal thickness and function, measured by spectral-domain optical coherence tomography (SD-OCT) and electroretinography (ERG), respectively., Results: Mice that were light damaged and injected with saline had significantly reduced outer retinal thickness, total retinal thickness, and ERG amplitudes compared with naïve mice. A 5-day administration of 15, 30, or 40 mg/kg of sarpogrelate was able to partially protect retinal morphology and full protection of retinal morphology was achieved with a 50 mg/kg dose. Both 15 and 30 mg/kg doses of sarpogrelate partially preserved retinal function measured by ERG, whereas 40 and 50 mg/kg doses fully preserved retinal function. Additionally, a single administration of 50 mg/kg sarpogrelate was able to fully preserve both retinal morphology and function. Administration of 15 mg/kg of sarpogrelate and 1 mg/kg of 8-OH-DPAT together demonstrated an additive effect and fully preserved retinal morphology., Conclusions: A 5- or 1-day treatment with 50 mg/kg sarpogrelate can completely protect the retina of BALB/c mice from light-induced retinopathy. Partial protection can be achieved with lower doses starting at 15 mg/kg and protection increases in a dose-dependent manner. Treatment with low doses of sarpogrelate and 8-OH-DPAT elicits an additive effect that results in full protection of retinal morphology.