Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the elderly population. Sodium iodate (NaIO3), a stable oxidizing agent, has been injected to establish a reproducible model of oxidative stress-induced RPE and photoreceptor death. The aim of our study was to evaluate the morphological and molecular changes of retina and retinal pigment epithelium (RPE)-choroid in NaIO3-treated mouse using multimodal fundus imaging and label-free quantitative proteomics analysis. Here, we found that following NaIO3 injection, retinal degeneration was evident. Fundus photographs showed numerous scattered yellow-white speckled deposits. Optical coherence tomography (OCT) images indicated disruption of the retinal layers, damage of the RPE layer and accumulation of hyper-reflective matter in multiple layers of the outer retina. Widespread foci of a high fundus autofluorescence (FAF) signal were noticed. Fundus fluorescein angiography (FFA) revealed diffuse intense transmitted fluorescence mixed with scattered spot-like blocked fluorescence. Indocyanine green angiography (ICGA) presented punctate hyperfluorescence. Due to the atrophy of the RPE and Bruch\'s membrane and choroidal capillary complex, the larger choroidal vessels become more prominent in ICGA and optical coherence tomography angiography (OCTA). Transmission electron microscope (TEM) illustrated abnormal material accumulation and damaged mitochondria. Bioinformatics analysis of proteomics revealed that the differentially expressed proteins participated in diverse biological processes, encompassing phototransduction, NOD-like receptor signaling pathway, phagosome, necroptosis, and cell adhesion molecules. In conclusion, by multimodal imaging, we described the phenotype of NaIO3-treated mouse model mimicking oxidative stress-induced RPE and photoreceptor death in detail. In addition, proteomics analysis identified differentially expressed proteins and significant enrichment pathways, providing insights for future research, although the exact mechanism of oxidative stress-induced RPE and photoreceptor death remains incompletely understood.

BACKGROUND: Cordyceps cicadae (C.cicadae), named \"Chan Hua\", an anamorph of Isaria cicadae Miquel, is an entomogenous complex formed by fungi parasitizing on the larvae of cicadas and belongs to the Claviciptaceae family and the genus Codyceps, which traditionally holds a significant place in Chinese ethnopharmacology, specifically for eye clarity and as a remedy for age-related ocular conditions. The underlying mechanisms contributing to its eyesight enhancement and potential effectiveness against Age-related macular degeneration (AMD) remain unexplored.
OBJECTIVE: This study aims to elucidate the protective role of C.cicadae and its active ingredient, Myriocin (Myr), against AMD.
METHODS: A chemical inducer was employed to make retinal pigment epithelium (RPE) damage in vitro and in vivo. The key ingredients of C.cicadae and their related mechanisms for anti-AMD were studied through bioinformatic analysis and molecular biological approaches.
RESULTS: Myr was identified through high-performance liquid chromatography (HPLC) as an active ingredient in C.cicadae, and demonstrated a protective effect on RPE cells, reducing the structural damage and cell death induced by sodium iodate (SI). Further, Myr reduced eyelid secretions in AMD mice and restored their retinal structure and function. The differentially expressed genes (DEGs) in Myr treatment are primarily associated with TNF and Necroptosis signaling pathways. Molecular docking indicated a strong affinity between TNF and Myr. Myr inhibited the TNF signaling pathway thereby reducing the expression of inflammatory factors in ARPE-19 cells. Additionally, Myr had consistent action with the necroptosis inhibitor Necrostatin-1 (Nec-1), inhibited the RIPK1/RIPK3/MLKL pathway thereby protecting ARPE-19 cells.
CONCLUSIONS: The findings present Myr, as a potent protector against SI-induced AMD, predominantly through modulation of the TNF-RIPK1/RIPK3/MLKL signaling pathway, offering the insights of therapeutic C.cicadae as viable candidates for AMD treatment.

Iron-induced oxidative stress was thought to be the reason why the a-wave amplitude of the electroretinogram (ERG) dropped when iron ions were present. It is assumed that reactive oxygen species (ROS) are generated in the presence of iron ions, and this leads to a decrease in hyperpolarization of the photoreceptor. It is known that in age-related macular degeneration (AMD), sodium iodate can induce oxidative stress, apoptosis, and retinal damage, which mimic the effects of clinical AMD. Here, the reduction of the a-wave amplitude in mice with sodium iodate-induced age-related macular degeneration is explained.
The leading edge of the a-wave is divided into voltages developed by cones and rods. The same oxidative stress model is applied here since sodium iodate causes the creation of ROS in a manner similar to that caused by iron ions, with the exception that the retina is treated as a circuit of various resistances when computing the photoresponse. Moreover, sodium iodate also leads to apoptosis and, hence, may cause misalignment in cones (not in rods) during the initial stage of apoptosis in AMD. To include the effects of apoptosis and shortening in cones and rods, we have used a factor representing the fraction of total cones and rods that are alive. To include the effect of misalignment of cones on the reduction of the a-wave amplitude, we have used the Stiles-Crawford function to calculate the number of photoisomerizations occurring in a photoreceptor misaligned at an angle θ. The results are compared with experimental data.
In sodium iodate-treated eyes, the ROS produced can attract calcium ions in the photoreceptor, which increases the calcium influx. In the case of the cones, the inclusion of the misalignment angle in the phototransduction process helps in determining the voltage and slope of the voltage vs. time graph.The smaller the fraction of active photoreceptors, the smaller the amplitude of the a-wave. The calcium influx, misaligned photoreceptors, and total photoreceptor loss all cause the amplitude of the a-wave to decrease, and at any time from the beginning of phototransduction cascade, the calcium influx causes the slope of the a-wave to increase.
The reduction in the a-wave amplitude in the eyes of sodium iodate-treated mice is attributed to oxidative stress in both cones and rods and cone misalignment, which ultimately lead to apoptosis and vision loss in AMD.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of blindness in elderly people in the developed world, and the number of people affected is expected to almost double by 2040. The retina presents one of the highest metabolic demands in our bodies that is partially or fully fulfilled by mitochondria in the neuroretina and retinal pigment epithelium (RPE), respectively. Together with its post-mitotic status and constant photooxidative damage from incoming light, the retina requires a tightly-regulated housekeeping system that involves autophagy. The natural polyphenol Urolithin A (UA) has shown neuroprotective benefits in several models of aging and age-associated disorders, mostly attributed to its ability to induce mitophagy and mitochondrial biogenesis. Sodium iodate (SI) administration recapitulates the late stages of AMD, including geographic atrophy and photoreceptor cell death.
METHODS: A combination of in vitro, ex vivo and in vivo models were used to test the neuroprotective potential of UA in the SI model. Functional assays (OCT, ERGs), cellular analysis (flow cytometry, qPCR) and fine confocal microscopy (immunohistochemistry, tandem selective autophagy reporters) helped address this question.
RESULTS: UA alleviated neurodegeneration and preserved visual function in SI-treated mice. Simultaneously, we observed severe proteostasis defects upon SI damage induction, including autophagosome accumulation, that were resolved in animals that received UA. Treatment with UA restored autophagic flux and triggered PINK1/Parkin-dependent mitophagy, as previously reported in the literature. Autophagy blockage caused by SI was caused by severe lysosomal membrane permeabilization. While UA did not induce lysosomal biogenesis, it did restore upcycling of permeabilized lysosomes through lysophagy. Knockdown of the lysophagy adaptor SQSTM1/p62 abrogated viability rescue by UA in SI-treated cells, exacerbated lysosomal defects and inhibited lysophagy.
CONCLUSIONS: Collectively, these data highlight a novel putative application of UA in the treatment of AMD whereby it bypasses lysosomal defects by promoting p62-dependent lysophagy to sustain proteostasis.

Purpose: Previously, we identified increased retinal degeneration and cytokine response in a mouse model of dry age-related macular degeneration (AMD) in the presence of systemic inflammation from rheumatoid arthritis (RA). Histone deacetylases (HDACs) regulate cytokine production by reducing acetylation and are found to be dysregulated in inflammatory diseases, including RA and AMD. Therefore, this current study investigates the effect of HDAC inhibition on AMD progression in the presence of systemic inflammation. Methods: Collagen induced arthritis (CIA) was induced in C57BL6J mice, followed by sodium iodate (NaIO3)-induced retinal degeneration. Mice were treated with a selective HDAC class I inhibitor, MS-275, and retinal structure [optical coherence tomography (OCT)], function (electroretinography), and molecular changes quantitative real-time polymerase chain reaction (RT-qPCR, Western Blot) were assessed. Results: NaIO3 retinal damage was diminished in CIA mice treated with MS-275 (P ≤ 0.05). While no significant difference was observed in retinal pigment epithelium (RPE) function, a trend in increased c-wave amplitude was detected in CIA + NaIO3 mice treated with MS-275. Finally, we identified decreased Hdac1, Hdac3, and Cxcl9 expression in CIA + NaIO3 mouse RPE/choroid when treated with MS-275 (P ≤ 0.05). Conclusions: Our data demonstrate that HDAC inhibition can reduce the additive effect of NaIO3-induced retinal degeneration in the presence of systemic inflammation by CIA as measured by OCT analysis. In addition, HDAC inhibition in CIA + NaIO3 treated mice resulted in reduced cytokine production. These findings are highly innovative and provide additional support to the therapeutic potential of HDAC inhibitors for dry AMD treatment.

Because the selective estrogen receptor modulator tamoxifen was shown to be retina-protective in the light damage and rd10 models of retinal degeneration, the purpose of this study was to test whether tamoxifen is retina-protective in a model where retinal pigment epithelium (RPE) toxicity appears to be the primary insult: the sodium iodate (NaIO3) model. C57Bl/6J mice were given oral tamoxifen (in the diet) or the same diet lacking tamoxifen, then given an intraperitoneal injection of NaIO3 at 25 mg/kg. The mice were imaged a week later using optical coherence tomography (OCT). ImageJ with a custom macro was utilized to measure retinal thicknesses in OCT images. Electroretinography (ERG) was used to measure retinal function one week post-injection. After euthanasia, quantitative real-time PCR (qRT-PCR) was performed. Tamoxifen administration partially protected photoreceptors. There was less photoreceptor layer thinning in OCT images of tamoxifen-treated mice. qRT-PCR revealed, in the tamoxifen-treated group, less upregulation of antioxidant and complement factor 3 mRNAs, and less reduction in the rhodopsin and short-wave cone opsin mRNAs. Furthermore, ERG results demonstrated preservation of photoreceptor function for the tamoxifen-treated group. Cone function was better protected than rods. These results indicate that tamoxifen provided structural and functional protection to photoreceptors against NaIO3. RPE cells were not protected. These neuroprotective effects suggest that estrogen-receptor modulation may be retina-protective. The fact that cones are particularly protected is intriguing given their importance for human visual function and their survival until the late stages of retinitis pigmentosa. Further investigation of this protective pathway could lead to new photoreceptor-protective therapeutics.

UNASSIGNED: Age-related macular degeneration (AMD) is one of the eye diseases that can affect a person\'s central vision. Retinal pigment epithelium (RPE) cells are damaged in this medical condition and some pigments are presented in these cells. Here, we aimed to investigate melanin and lipofuscin granules of RPE cells as a precursor of AMD.
UNASSIGNED: Hooded rats (n=18) were divided into two groups and received 100 μl of sodium iodate (SI) into the retro-orbital sinus of their eyes at 40 and 60 mg/kg doses. The total number of melanin and lipofuscin granules, different types of granules, cytoplasmic dispersion of granules as well as morphological changes in the shape and number of nuclei of RPE cells were evaluated over the course of 1-30 days.
UNASSIGNED: The total number of melanin pigments increases over time at a dose of 40 mg/kg and decreases at a dose of 60 mg/kg. Also, the total number of lipofuscin granules in 40 mg/kg increases over time and decreases in 60 mg/kg. Autofluorescent intensity (AF) is also increased at 40 mg/kg, but at 60 mg/kg, the highest intensity is on day 7. Also, the highest number of multinucleated giant cells was on day 7 at 60 mg/kg and the most changes in cell appearance due to sodium iodate injection were seen on the first day after injection.
UNASSIGNED: We demonstrated that granules and autofluorescent intensity appear to decrease at high doses of sodium iodate, which is similar to the advanced stage of the AMD disease, where the number of granules and AF intensity increase in the middle and even early stages of the disease.

Sodium iodate (NaIO3) is a commonly used model for age-related macular degeneration (AMD), but its rapid and severe induction of retinal pigment epithelial (RPE) and photoreceptor degeneration can lead to the premature dismissal of potentially effective therapeutics. Additionally, little is known about how sex and age affect the retinal response to NaIO3. This study aims to establish a less severe yet reproducible regimen by testing low doses of NaIO3 while considering age- and sex-related effects, enabling a broader range of therapeutic evaluations. In this study, young (3-5 months) and old (18-24 months) male and female C57Bl/6J mice were given an intraperitoneal (IP) injection of 15, 20, or 25 mg/kg NaIO3. Damage assessment one week post-injection included in vivo imaging, histological examination, and qRT-PCR analysis. The results revealed that young mice showed no damage at 15 mg/kg IP NaIO3, with varying degrees of damage observed at 20 mg/kg. At 25 mg/kg, most young mice displayed widespread retinal damage, with females exhibiting less retinal thinning than males. In contrast, older mice at 20 and 25 mg/kg displayed a more patchy degeneration pattern, outer retinal undulations, and greater variability in degeneration than the young mice. The most effective model for minimizing damage while maintaining consistency utilizes young female mice injected with 25 mg/kg NaIO3. The observed sex- and age-related differences underscore the importance of considering these variables in research, aligning with the National Institutes of Health\'s guidance. While the model does not fully replicate the complexity of AMD, these findings enhance its utility as a valuable tool for testing RPE/photoreceptor protective or replacement therapies.

Age-related macular degeneration (AMD) is a global health challenge. AMD causes visual impairment and blindness, particularly in older individuals. This multifaceted disease progresses through various stages, from asymptomatic dry to advanced wet AMD, driven by various factors including inflammation and oxidative stress. Current treatments are effective mainly for wet AMD; the therapeutic options for dry AMD are limited. Photobiomodulation (PBM) using low-energy light in the red-to-near-infrared range is a promising treatment for retinal diseases. This study investigated the effects of multi-wavelength PBM (680, 780, and 830 nm) on sodium iodate-induced oxidatively damaged retinal tissue. In an in vivo rat model of AMD induced by sodium iodate, multi-wavelength PBM effectively protected the retinal layers, reduced retinal apoptosis, and prevented rod bipolar cell depletion. Furthermore, PBM inhibited photoreceptor degeneration and reduced retinal pigment epithelium toxicity. These results suggest that multi-wavelength PBM may be a useful therapeutic strategy for AMD, mitigating oxidative stress, preserving retinal integrity, and preventing apoptosis.

A variety of techniques exist to investigate retinal and choroidal vascular changes in experimental mouse models of human ocular diseases. While all have specific advantages, a method for evaluating the choroidal vasculature in pigmented mouse eyes has been more challenging especially for whole mount visualization and morphometric analysis. Here we report a simple, reliable technique involving bleaching pigment prior to immunostaining the vasculature in whole mounts of pigmented mouse choroids. Eyes from healthy adult pigmented C57BL/6J mice were used to establish the methodology. The retina and anterior segment were separated from the choroid. The choroid with retinal pigment epithelial cells (RPE) and sclera was soaked in 1% ethylenediaminetetraacetic acid (EDTA) to remove the RPE. Tissues were fixed in 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Choroids were subjected to melanin bleaching with 10% hydrogen peroxide (H2O2) at 55 °C for 90 min, washed in PBS and then immunostained with anti-podocalyxin antibody to label vascular endothelium followed by Cy3-AffiniPure donkey anti-goat IgG at 4 °C overnight. Images of immunostained bleached choroids were captured using a Zeiss 710 confocal microscope. In addition to control eyes, this method was used to analyze the choroids from subretinal sodium iodate (NaIO3) RPE atrophy and laser-induced choroidal neovascularization (CNV) mouse models. The H2O2 pretreatment effectively bleached the melanin, resulting in a transparent choroid. Immunolabeling with podocalyxin antibody following bleaching provided excellent visualization of choroidal vasculature in the flat perspective. In control choroids, the choriocapillaris (CC) displayed different anatomical patterns in peripapillary (PP), mid peripheral (MP) and far peripheral (FP) choroid. Morphometric analysis of the vascular area (VA) revealed that the CC was most dense in the PP region (87.4 ± 4.3% VA) and least dense in FP (79.9 ± 6.7% VA). CC diameters also varied depending on location from 11.4 ± 1.97 mm in PP to 15.1 ± 3.15 mm in FP. In the NaIO3-injected eyes, CC density was significantly reduced in the RPE atrophic regions (50.7 ± 5.8% VA in PP and 45.8 ± 6.17% VA in MP) compared to the far peripheral non-atrophic regions (82.8 ± 3.8% VA). CC diameters were significantly reduced in atrophic regions (6.35 ± 1.02 mm in PP and 6.5 ± 1.2 mm in MP) compared to non-atrophic regions (14.16 ± 2.12 mm). In the laser-induced CNV model, CNV area was 0.26 ± 0.09 mm2 and luminal diameters of CNV vessels were 4.7 ± 0.9 mm. Immunostaining on bleached choroids with anti-podocalyxin antibody provides a simple and reliable tool for visualizing normal and pathologic choroidal vasculature in pigmented mouse eyes for quantitative morphometric analysis. This method will be beneficial for examining and evaluating the effects of various treatment modalities on the choroidal vasculature in mouse models of ocular diseases such as age-related macular degeneration, and degenerative genetic diseases.