Experimental Eye Research

Purpose: To identify transcript-level variants in crystallin genes in paediatric patients with unilateral cataracts. Methods: Anterior capsulorhexis (n=12) from patients underwent surgical management of congenital unilateral cataracts was collected. Total RNA was isolated from lens epithelial cells, and complementary DNA (cDNA) was synthesized. Full-length RNA transcripts of 10 lens-specific crystallin genes were PCR-amplified and analysed via Sanger sequencing. Identified transcript variants were further validated using genomic DNA (gDNA) through Sanger sequencing. In addition, the full-length (~7,535 bp) CRYBA1 genomic region was sequenced using Oxford Nanopore Technology. Results: Aberrant low molecular weight (LMW) amplicons (~370 bp) of the CRYBA1 transcript were identified in three patients presented with unilateral cataract. Of 3 patients, 2 had persistent fetal vasculature (PFV) and 1 had pre-existing posterior capsular defect (PPCD). Sanger sequencing revealed a precise loss of exons 2 to 4 in the CRYBA1 RNA transcript. No coding, splice-site, or large deletion variants were detected in the genomic DNA of the patients or their parents. In silico analysis predicted two possible truncated proteins arising from these alternatively spliced transcripts: one comprising the first 11 amino acids of the N-terminal region with a loss of all Greek key motifs, and another comprising 90 amino acids encoded by exons 5 and 6, initiated from an alternative start codon in exon 5, and loss of Greek key motifs 1 & 2. Conclusion: The precise skipping of exons 2 to 4, consistent with canonical splicing signals (5-prime-GU...AG-3-prime), in the absence of genomic alterations, suggests the presence of alternatively spliced (AS) CRYBA1 transcripts in human lenses. This is the first report documenting AS-CRYBA1 transcripts in association with childhood cataracts with PFV and PPCD.

Early glaucoma detection and treatment are critical to prevent irreversible blindness. Glaucoma polygenic risk scores (PRS) offer an effective approach for stratifying disease risk and are increasingly available in clinical practice. However, the psychosocial impact of receiving glaucoma PRS results is currently unknown. As such, this study investigated short-term psychosocial outcomes of disclosing glaucoma PRS to individuals over 50 years from the general population. Individuals from the bottom 10%, middle 45 to 55%, and top 10% of PRS scores were invited to receive their results and complete surveys before and 2 weeks after receiving results to assess anxiety, test-related distress, decisional regret, recall and understanding. Of invited participants, 51.7% (136/263) enrolled with 133 completing both surveys. Two weeks after disclosure, PRS recall was high (78.2%), although PRS knowledge remained limited. Privacy concerns were moderate, not differing across PRS groups (X^2 = 4.17, p = .124). Small reductions in glaucoma-related anxiety (z = -2.93, p = .003), generalised anxiety (z = -3.75, p < .001) and stress (z = -2.49, p = .013) were observed following disclosure. While scores remained within normal ranges, higher glaucoma-related anxiety (t = -2.36, p = .020), higher negative emotions (X^2 = 20.80, p < .001), and lower positive experience (F = 5.70, p = .004) were seen for high-risk participants compared to lower-risk participants. Decisional regret was low and did not differ across PRS groups (X^2 = 0.28, p = .869). These findings support the psychosocial safety of glaucoma PRS testing while highlighting the need for improved education and longer-term follow-up to support clinical implementation.

PurposeThe inner wall of Schlemms canal (SC) is a mechanosensitive endothelial monolayer that provides resistance to conventional aqueous humor drainage, a process dependent on pore formation. This study examined how microtubule (MT) stability affects SC cell mechanobiology, transcellular pore formation, and aqueous humor outflow dynamics. MethodsMT stability in cultured SC cells from normal and glaucomatous human donors was manipulated pharmacologically. Changes in MT acetylation, phosphorylated myosin light chain, and F-actin were assessed by immunofluorescence and immunoblotting. GEF-H1 was knocked down using siRNA. Cellular stiffness was measured by atomic force microscopy. Transcellular pore formation was quantified using an established pore formation assay. Outflow facility was measured in enucleated mouse eyes using the iPerfusion system. ResultsMT stabilization in normal SC cells decreased actomyosin contractility and cellular stiffness, whereas MT destabilization increased contractility and stiffness; these effects involved the MT-associated Rho guanine nucleotide exchange factor GEF-H1. MT stability was also mechano-responsive to substrate stiffness. Furthermore, SC cells derived from glaucomatous donors exhibited reduced MT stability compared with normal SC cells. MT stabilization increased transcellular pore formation in both normal and glaucomatous SC cells. In ex vivo mouse eyes, paclitaxel perfusion to stabilize MTs significantly increased outflow facility relative to contralateral control eyes. ConclusionsOur data suggest that MT stability influences SC cell contractility, stiffness, and transcellular pore formation and can alter aqueous humor outflow facility. These findings identify MT-dependent cytoskeletal remodeling as an important contributor to the biomechanics of the conventional outflow pathway and suggest that MT-associated pathways may represent potential targets for improving outflow function in glaucoma.

ObjectiveTo systematically review automated nerve morphometry tools and independently benchmark their performance on independent optic nerve datasets. DesignSystematic review and comparative benchmarking study. ControlsBenchmarking was performed using paraphenylenediamine-stained mouse (n = 85) and rat (n = 44) optic nerve images with manually annotated axon counts as ground truth. MethodsPublished studies describing automated or semi-automated neural tissue morphometry tools were identified through systematic searches of PubMed, Embase, and Scopus through January 2026 following PRISMA guidelines. Data extraction covered 70 fields across tool capabilities, imaging modality, species, automation level, and validation approach. Eighteen eligible tools (8 deep learning [DL], 10 classical computer vision [CV]) were benchmarked on both mouse and rat independent datasets. Main Outcome MeasuresPerformance was assessed by mean absolute percentage error (MAPE), Pearson correlation, and median predicted-to-ground-truth ratio. Tools were ranked per image and compared using Friedman tests with Nemenyi post-hoc analysis. ResultsSeventy-one studies met inclusion criteria, spanning from 1999 to 2026. Deep learning methods represented 38% (27/71) of studies, increasing from 0% before 2017 to over 55% of publications after 2020. Axon counting was the most common output (73%, 52/71), while only 35% (25/71) reported g-ratio. Among benchmarked tools, Marina (CV, 2010) achieved the lowest average MAPE (32.9%). The top five tools (MAPE ranging from 32.9 to 44.8%) included both CV and DL methods and were statistically indistinguishable by Friedman-Nemenyi analysis (p > 0.05). Performance varied substantially across datasets: AxonJ (CV) achieved the second best MAPE on rat images (27.7%) but the worst on mouse images (438.6%). ConclusionsNo single tool demonstrated consistently superior performance across both datasets. Classical and deep learning approaches achieved comparable accuracy for axon counting. Tool selection should be guided by target species, tissue preparation protocol, and desired morphometric outputs. This systematic review and independent benchmarking study provide an evidence base for tool selection in optic nerve research.

BackgroundThe developmental pattern of the crystalline lens provides a unique model to study biological aging and its effects on the posttranslational modification of long-lived proteins. The orderly differentiation of lens fiber cells leads to a spatiotemporal gradient where mature, organelle-free fiber cells are packed in the lens nucleus surrounded by con-centric rings of successively younger fiber cells in the cortex. MethodsPig lenses were separated into six layers by dissolution in a hypotonic buffer. The changes in protein abundance, oxidation, and phosphorylation that occur across the spatiotemporal gradient of the lens were assessed quantitatively by using data independent acquisition label-free proteomic analysis of these six fractions. ResultsExpected changes in protein abundance of major lens protein which reflect the maturation process of lens fiber cells across the spatiotemporal gradient were found. Significant differences were noted in phosphorylation sites on crystallins, phakinin, and actin. Significant changes in oxidation of residues on essential lens proteins, as well as several glycolytic enzymes, were found across the spatiotemporal gradient. ConclusionDissolution of the lens followed by high resolution data-independent acquisition proteomics is a powerful technique for spatial mapping of protein abundance and posttranslational modification changes in the lens. The oxidation and phosphorylation sites noted in this study may play important roles in both lens development and cataractogenesis.

PurposeOculocutaneous albinism type 1 (OCA1) is an inherited disorder caused by tyrosinase (TYR) gene mutations. Affected individuals experience visual impairment and severe photosensitivity from ocular hypomelanosis, with no current treatments. We evaluated the safety and efficacy of a TYR-encoding adeno-associated virus (AAV) vector in healthy rhesus macaques as a potential OCA1 treatment. MethodsA novel AAV2-based capsid (ATX002) was packaged with the human VMD2 promoter and TYR (hTYR) fused with mGreenLantern (mGL). Two adult rhesus macaques were injected with ATX002-hVMD2-hTYR-mGL subretinally (OD) and intravitreally (OS). Safety and efficacy were assessed via comprehensive ophthalmic examination, fundus photography, spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinography at baseline and defined timepoints up to 12 weeks post-injection, followed by post-mortem immunohistochemistry (IHC). ResultsBoth subretinal doses induced localized hypermelanosis by 3 weeks post-injection, which persisted through the study endpoint and was accompanied by measurable thickening of the retinal pigment epithelium (RPE) on SD-OCT. Histological IHC confirmed successful RPE transduction via robust mGL fluorescence, corroborating in vivo findings by revealing localized RPE hyperplasia and transgene-expressing cells adjacent to regions of de novo hypermelanosis. Intravitreal delivery did not induce any changes to the RPE. Transient uveitis was observed but successfully managed with anti-inflammatory treatment. ConclusionsSubretinal AAV-TYR delivery is a safe and effective approach with the potential to induce RPE pigmentation. These findings support the use of AAV-TYR gene therapy for OCA1, demonstrating efficacy and a manageable safety profile in a large-animal model, and provide a critical bridge toward human clinical translation.

Purpose: To predict retinal nerve fiber layer thickness (RNFLT) norms from fundus images. Methods: We selected 18,000 OCT scans and visual fields (VF) from the Massachusetts Eye and Ear Glaucoma Service. A U-Net-based deep learning model was developed to predict RNFLT norms from OCT en face fundus images. A total of 10,000 OCT scans with normal VFs (mean deviation [MD] [&ge;] -1 dB, glaucoma hemifield test within normal limits, and pattern standard deviation probability > 5%) tested within 30 days were used for training, while the remaining 8,000 OCT scans (mean VF MD: 3.3 +/- 4.9 dB), including 2,419 scans with normal VFs, were used for evaluation. Structure-function correlations between RNFLT maps and VFs were assessed using linear regression and VGG-16 across original RNFLT maps, deviation maps, and their combination. Performance was evaluated using correlation coefficients, mean absolute error (MAE), and R-squared. Results: Predicted RNFLT norm maps showed agreement with baseline RNFLT maps in eyes with normal VFs (R-squared = 0.81 +/- 0.13). RNFLT deviation maps correlated more strongly with VF MD than original RNFLT maps (R = 0.42 vs. 0.19, p < 0.01). In deep learning-based VF prediction, combining original and deviation maps achieved the best performance (MAE = 3.31 dB, R-squared = 0.39), outperforming the model (p < 0.05) using original RNFLT maps alone (MAE = 3.36 dB, R-squared = 0.35). Conclusions: Deep learning can estimate individualized RNFLT norms and improve structure-function assessment in glaucoma. Translational Relevance: Personalized RNFLT norm prediction may improve detection of glaucomatous damage.

Purpose: Animal models of myopia typically induce monocular refractive shifts via form deprivation (FD) or lens-induced myopia (LIM), modeling susceptibility to myopia, but with potentially limited applicability to childhood myopia. Here we describe a novel, genetically diverse mouse model of naturally occurring refractive error (NORE) with three distinct refractive phenotypes: hyperopic, myopic, and intermediate. Methods: C57BL/6J mice were mated to 129S2/SvPasCrl mice to create F1 or F2 offspring. Refractive errors in male and female F1 (N=21) and F2 (N=101) mice were assessed on postnatal days (P) 28 and 42 using photorefractometry. In a subset of mice (N=30 - 40), corneal radius of curvature, axial ocular dimensions, retinal and visual function were assessed. Results: F2 mice were classified as NORE with either hyperopic (RE [&ge;] 0 diopters (D) at P28 and P42), myopic (RE<0D at P28 and P42) or intermediate (RE<0D at P28 and RE [&ge;] 0D at P42) refractions based on individual trajectories. All ocular parameters changed with age, with significantly slower growth in axial length and vitreous chamber depth in the intermediate versus myopic mice (p<0.05). Lens thickness was smaller in the myopic group at P28. Differences in refraction were not attributed to variances in retinal function or dopamine signaling. Conclusions: NORE mice represent a novel, genetically diverse wild-type mouse model that, unlike traditional models, does not require interventions such as FD or LIM to induce myopia. NORE mice provide a valuable tool for future investigations of genetic and environmental mechanisms and targeted therapeutic strategies for refractive errors.

Col8a2Q455K/Q455K (Q455K) mice exhibit features of early-onset Fuchs endothelial corneal dystrophy (FECD), including decreased endothelial cell density (ECD) and guttae formation. Within the context of these clinical features, this study longitudinally evaluates ferroptosis in Q455K and wild-type (WT) mice using in vivo imaging, PCR and immunohistochemistry. Fifty-six Q455K and 56 WT mice were evaluated from 3 to 24 months of age with in vivo confocal microscopy; ECD and guttae were measured. Ferroptosis marker expression was determined with PCR and immunohistochemistry (IHC). Data were analyzed using two-way ANOVA with Tukeys post hoc test, Chi-square test and a paired t-test. The ECD significantly decreased in both groups from 3 to 24 months of age, but more markedly in Q455K (2285-/+317 to 1012-/+58 cells/mmSquare) versus WT mice (2714-/+139 to 2057-/+149 cells/mmSquare, P<0.0001). Guttae were observed exclusively in Q455K mice beginning at 3 months of age and increased over time (P=0.0003). The Q455K mice demonstrate guttae at the vertices of corneal endothelial cells rather than their centers (74.3% vs. 25.7%P<0.001). Expression of ferroptosis-related genes (Tfrc, Slc40a1, Ftl1, Gpx4) were significantly increased in the Q455K versus WT mice (P<0.05). Furthermore, corresponding protein expression (transferrin receptor 1, ferroportin, ferritin and glutathione peroxidase 4) was significantly elevated adjacent to guttae in Q455K versus WT mice (P<0.05). These findings implicate guttae in the initiation of ferroptosis as it relates to the pathophysiology of FECD and provide an optimal window for testing novel FECD therapies using this murine model, particularly those that target ferroptosis.

The retinal nerve fiber layer, composed of axon bundles converging toward the optic nerve, is a key biomarker for diagnosing and monitoring glaucoma and other neurodegenerative diseases. High-resolution en face imaging of individual nerve fiber bundles offers morphological information beyond what conventional optical coherence tomography provides, yet clinical integration remains limited by the lack of automated analysis tools and normative data. Here, we imaged 14 healthy volunteers using time-domain full-field optical coherence tomography and adaptive optics scanning laser ophthalmoscopy, and developed automated pipelines to quantify bundle width, trajectory, tortuosity, and orientation. Bundles were on average 25% wider at shallower retinal depths, width measurements were consistent across imaging modalities, and estimated axon count per bundle decreased significantly with age. Global trajectory analysis revealed systematic deviations of high resolution data from existing mathematical models, particularly in the temporal sector, leading us to propose two refined trajectory models. These normative results provide a foundation for high resolution biomarkers for use in investigations of retinal neurodegeneration.

Photoreceptor outer segments are sensory cilia whose maintenance depends on a balance between basal disc renewal and tip shedding, controlled by intraflagellar transport and axonemal microtubule organization. Microtubule plus-end proteins regulate microtubule dynamics and are strong candidates for roles in this process. In this study, mCherry-tagged EB1, EB3, and DCX were overexpressed in zebrafish (Danio rerio) cone photoreceptors under a cone-specific promoter. Eyes were examined at 5 and 10 dpf, and eyecup depth, diameter, and cone photoreceptor area were quantified relative to uninjected controls. At 5 dpf, all three constructs produced eyes indistinguishable from those of controls. By 10 dpf, all three constructs significantly increased eye cup depth and cone photoreceptor area. EB1 and DCX also significantly increased eye cup diameter. EB1 and, more severely, EB3 also caused retinal holes, mainly in the retinal pigment epithelium and at the outer nuclear/outer plexiform layer, along with misshapen cells near the inner plexiform layer. DXC did not cause retinal holes, but, like EB1 and EB3, produced enlarged, bulbous cone outer segments. The results show that overexpression of any of the three +TIPs results in a similar eye and photoreceptor overgrowth phenotype, while also producing construct-specific defects: EB1 and EB3 disrupt the broader retinal architecture, whereas DCX produces enlarged eyes. The shared outer segment hypertrophy suggests an imbalance between cargo delivery at the basal end and shedding of the distal tips. The organomegaly may reflect altered progenitor signaling in the ciliary marginal zone.

Retinopathy of prematurity (ROP) is caused by hyperoxic exposure of prematurely born infants. The mouse model of oxygen-induced retinopathy (OIR) recapitulates pathological features of both phase I and phase II ROP. We here looked at the retinal proteins that change in response to hyperoxia in phase I of the mouse model of OIR. Using tandem mass tag labeled proteomics, we found several differentially expressed proteins (DEPs) in phase I of OIR. Of all the DEPs, we investigated the role of previously unknown protein NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2). NDUFA4L2 protein and its paralog NDUFA4 are both mitochondrial complex I proteins; however, here we demonstrate that NDUFA4L2 changes in both phases of OIR, with no changes in its paralog NDUFA4, implying its unique function in pathophysiology of the disease. We demonstrate that NDUFA4L2 is an oxygen-sensitive protein and regulates retinal endothelial cell migration by rescuing isocitrate dehydrogenase flux impaired by hyperoxia in phase I of OIR.

New approach methodologies (NAMs), including induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs) and retinal pigment epithelium (iRPE), are increasingly applied to study retinal disease mechanisms and therapeutic strategies. However, these models often remain relatively immature. Given the high lipid content and complex metabolism of the retina, it is unclear to what extent iPSC-derived systems recapitulate the human retinal lipidome. Here, we compared the lipidomic profiles of ROs and iRPE, collected at several differentiation stages, with those of post-mortem adult human macular, non-macular and RPE plus choroid (pmRPE). The lipidome of iRPE differed markedly from pmRPE, whereas prolonged differentiation of ROs resulted in a lipidomic profile increasingly resembling that of the post-mortem retina. Moreover, ROs showed similarities to both macular and non-macular lipidome. These findings show that iPSC-derived models can become valuable NAMs to study lipid-related retinal disorders and provide a framework to optimize differentiation protocols.

BackgroundKeratoconus (KC) is a multifactorial disorder with unclear etiology, characterized by localized thinning and a cone-like protrusion of the cornea. The complex etiology of KC exacerbates the lack of an animal model. Previous studies by Tachibana et al. (2002) described an inbred mouse strain (SKC) with a spontaneous, androgen-dependent, cone-like corneal morphology. This study aimed to investigate the corneal phenotypes of SKC mice through an in-depth ophthalmic examination. MethodsMice (n=53) were examined via slit lamp biomicroscopy with fluorescein staining. Spectral-domain optical coherence tomography (SD-OCT) enabled central corneal thickness (CCT) measurement in selected mice (n=26 eyes), and OCT-based pachymetry mapping (n=16 eyes). In vivo corneal confocal microscopy was conducted on eyes to assess cellular morphology (n= 9 eyes). Eyes were collected for histology analysis (n=22). ResultsLesions and epithelial breaks were present in [~]95% of eyes (n=101). Neovascularization, perforation, scarring, and hydrops were seen primarily in males. An opaque, unilateral cone-like morphology was exclusive to males (n=11). Male and female corneas showed no significant difference in CCT, though pachymetry mapping revealed regional thinning patterns in both sexes. Loosened epithelial tight junctions, stromal fibrosis, vascularization, and inflammation of variable severity were identified in both sexes. ConclusionThis study identified previously unreported corneal phenotypes in SKC mice through ophthalmic examination. Unlike previous studies, gross and histological abnormalities were observed in female SKC mice. Our findings suggest a lower penetrance of the cone-like phenotype ([~]20%) than previously reported ([~]33%) and support that the conical phenotype in male mice may be secondary to keratitis.

Purpose: To quantify the binocular integrated visual field (IVF) loss patterns with archetypal (AT) analysis and their associations with patients' Quality of Life (QoL). Design: Retrospective study. Participants: Over 125,000 patients from three datasets from Massachusetts Eye and Ear and Glaucoma Research Network Consortium. Methods: We used: (1) the Glaucoma Research Network excluding the Massachusetts Eye and Ear subset for the binocular archetypal model training (77, 270 IVFs from 77 270 patients), (2) Massachusetts Eye and Ear dataset for demographic correlation analysis (47,965 IVFs from 47,965 patients), and (3) the MEE Quality of Life Survey dataset for QoL correlation analysis (75 IVFs from 75 patients). The whole study was restricted to the most recent VF measurements from each subject and binocular VFs were constructed by the integrated visual field method, which was taking the higher sensitivity at each test location. We first applied archetypal analysis to cluster 24-2 binocular VFs into archetypal patterns. The total number of patterns was determined by the Bayes factor. Pearson's correlations analyzed the associations between patients demographic information, binocular VF patterns and QoL scores, and the coefficients were set to 0 if p-values corrected by multiple comparisons < 0.05. Main Outcome Measures: A binocular VF archetypal patterns and its relationships with demographic divergences and QoL. Results: We identified 17 binocular VF loss patterns. Patterns with major vision impairment (AT10, AT12, AT13, AT14, and AT17) were more common in older patients, while Black or African Americans exhibited a broader spectrum of visual loss, notably AT5 and AT12, compared to Asian and White counterparts. 81 MEE patients with QoL survey data was analyzed to investigate the impact of demographic and vision-related variables on QoL. Older age and female gender were significantly associated with lower QoL. Binocular central vision loss (AT 5) and total vision loss (AT 12) had a significantly greater impact on QoL than binocular peripheral vision loss (AT 2, AT 5, AT 16). Conclusions: Individuals with central or total vision loss, as well as certain demographic groups, experience a significantly greater impact on quality of life. The quantifications of binocular VF loss patterns by archetypal analysis may help better understand glaucoma's impact on patients' quality of life.

Secondary degeneration following optic nerve crush (ONC) is driven in part by mitochondrial dysfunction and microglial activation. Inspired by hibernation, where reduced succinate oxidation limits reactive oxygen species (ROS) production, we tested whether pharmacological inhibition of this pathway confers neuroprotection. Using in vivo ONC models and in vitro microglial assays, we evaluated the effects of dimethyl malonate (DMM), an inhibitor of succinate dehydrogenase, and a cell-permeable succinate analog (succinate-NV). Succinate-NV increased pro-inflammatory cytokine expression (IL-1{beta}) and reduced anti-inflammatory IL-10, whereas non-permeable succinate had no effect, indicating that intracellular succinate can drive microglial activation. In hibernating animals, succinate-NV disrupted neuroprotection and reduced retinal ganglion cell (RGC) survival following optic nerve injury. Although DMM partially reduced select inflammatory cytokines, it failed to normalize IL-1{beta} or IL-10 and suppressed microglial phagocytosis while exhibiting cytotoxic effects. In vivo, DMM-treated animals showed reduced IBA1{square} microglia but increased CD68{square} activation and accumulation of DAPI{square} cells at 7 days post-injury at the crush site. RGC somas persisted but were Caspase3+ consistent with impaired clearance. Astrocyte reactivity increased at lesion borders, while reduced and fragmented GFAP at the lesion site indicated localized astrocyte loss. Collectively, these findings demonstrate that inhibition of succinate oxidation alone is insufficient for neuroprotection and underscore the need for coordinated metabolic and immune regulation that cannot be achieved through single-pathway pharmacological intervention.

Purpose: Photoreceptor Kv2.1/Kv8.2 voltage-gated potassium channels carry an outward potassium current, helping to set the resting membrane potential and to shape dim light responses. Because potassium flux in the outer retina influences extracellular osmolarity and fluid distribution, we hypothesized that Kv2.1/Kv8.2 channels also contribute to fluid homeostasis in this region of the retina. Methods: OCT imaging was performed in Kv8.2 heterozygous (Het) and knockout (KO) mice aged 4-7 weeks under dark- and light-adapted conditions. Light-dark differences in the distance between the external limiting membrane (ELM) and retinal pigment epithelium (RPE) ({Delta}ELM-RPE) were calculated to quantify light-evoked expansion of the subretinal space (SRS). As a secondary outcome, outer nuclear layer (ONL) thickness was also measured under both lighting conditions. Retinal gene expression was assessed by RNA-seq and droplet digital RT-PCR. Retinal protein expression was determined by western blotting and immunolabeling. Results: {Delta}ELM-RPE was significantly reduced in Kv8.2 KO mice compared with Het controls, indicating reduced SRS hydration. ONL thickness exhibited a small but significant light-dark change that was different between genotypes. Transcriptomic analyses revealed upregulation of osmosensitive genes, including osmolyte transporters and aquaporins. AQP1 protein expression in photoreceptors increased. Conclusions: These findings reveal a previously unrecognized role for Kv2.1/Kv8.2 channels in outer retinal fluid homeostasis and support a model in which photoreceptor potassium efflux contributes to osmotic water movement into the subretinal space.

Purpose: To assess the feasibility of quantitative fundus autofluorescence (FAF) measurement in early age-related macular degeneration (AMD) using the freely available ImageJ software, to characterize signal intensity across FAF patterns, and to evaluate interobserver reproducibility in pattern classification. Methods: Single-center, non-blinded, retrospective, consecutive-case analytical study. FAF images acquired with Spectralis OCT+HRA (Heidelberg Engineering) from patients with early dry AMD seen at a tertiary referral center between January 2010 and September 2016 were analyzed. A standardized 300x300-pixel region of interest (ROI) centered on the fovea was evaluated in ImageJ v2.0.0-rc54/1.51h (Fiji distribution). Mean, minimum, and maximum autofluorescence (AF) pixel intensity were recorded. Each image was independently classified according to the Bindewald classification system by two graders; a third senior grader adjudicated discordances. Cohen's kappa (k) was used to assess interobserver agreement. Results: Of 423 patients with available FAF studies, 107 had dry AMD; 45 met quality and diagnostic criteria for early AMD and were included in the quantitative analysis. Mean age was 73.47 +/- 8.1 years; 62.2% were female. Mean FAF intensity was 120.26 (range 74.76-160.79); mean minimum was 32.07 (range 3-63) and mean maximum was 205.80 (range 125-255). Seven of eight Bindewald patterns were identified; the stippled pattern was absent. The most frequent pattern was minimal changes (31.1%), followed by increased focal (24.4%) and patchy (15.6%). Reticular pattern showed the highest mean AF (143.8), while lace pattern showed the lowest (88.4). Interobserver agreement for Bindewald pattern classification was almost perfect (k = 0.969; 95% CI, 0.908-1.000; p < 0.001). Agreement for lesion extent was moderate (k = 0.531) and for foveal involvement was substantial (k = 0.622). Conclusions: Quantitative FAF evaluation of early AMD using ImageJ is feasible and reproducible. ImageJ represents a cost-free alternative for multimodal retinal image analysis, with potential for automated screening applications in resource-limited settings. Keywords: age-related macular degeneration; fundus autofluorescence; ImageJ; quantitative autofluorescence; image analysis; Bindewald classification; interobserver agreement

Oxidative stress is proposed to be a driver of age-related diseases. Age-related macular degeneration is one such disease, where the retinal pigment epithelium (RPE) is affected early in the disease. Vasculature damage also occurs, sometimes preceding RPE damage. To model some aspects of dry AMD, we used the NaIO3 mouse model of oxidative damage. Disruption of the deep retinal vascular plexus, disorganization and death of capillaries within the choriocapillaris, and marked electroretinographic decline were observed. AAV overexpressing the transcription factor, NRF2, which induces anti-oxidation enzymes and represses inflammation, was tested for protection of damage. The BEST1 promoter limited expression to the RPE. The RPE, photoreceptors, and vascular architecture in both retinal and choroidal compartments were protected. Conditioned medium from RPE-choroid explants, infected by AAV8/BEST1-NRF2, was sufficient to transfer partial protection in vivo, indicating that NRF2 induces a protective secreted factor(s). Analysis of RNA-seq data identified growth differentiation factor 15 (GDF15) as a candidate downstream mediator. Injection of recombinant GDF15 reproduced key protective phenotypes in vivo, whereas Gdf15-deficiency attenuated NRF2-mediated rescue. Pharmacologic inhibition of TGF-{beta} receptor signaling diminished NRF2 associated protection, supporting involvement of this signaling pathway. In a laser-induced choroidal neovascularization model, intravitreal GDF15 injection reduced fluorescein leakage and lesion size. These findings support a model in which NRF2 activation in the RPE induces expression of GDF15, which is capable of protecting the RPE, photoreceptors, and the retinal and choroidal vasculature. NRF2 and GDF15 have therapeutic potential for ocular diseases, as well as for other diseases with vascular pathology.

Purpose: To evaluate the incidence and baseline predictors of intraocular pressure (IOP)-lowering treatment following detection of referable glaucoma by teleretinal screening. Design: Retrospective cohort study. Methods: Participants were derived from a safety-net teleretinal diabetic retinopathy screening program (2013-2024). Participants included individuals who screened positive for referable glaucoma (cup-to-disc ratio [CDR] [&ge;]0.6 or CDR asymmetry [&ge;]0.2) and completed in-office diagnostic evaluation. The primary outcome was initiation of IOP-lowering treatment (medication, laser, or surgery) and the secondary outcome was intervention with surgery. Cumulative incidence functions were estimated, accounting for loss to follow-up. Fine-Gray models were used to identify baseline screening predictors to risk stratify each outcome. Glaucoma diagnosis was approximated using diagnostic codes and chart review. Results: 2,367 participants were included. The cumulative incidence of treatment was 19.6% (95% CI: 18.0-21.2) at Year 1 and 45.1% (42.1-48.1) at Year 8. Early treatment occurred primarily in glaucoma cases, whereas treatment accumulated longitudinally in glaucoma suspects, reaching 36.5% (31.6-41.5) by Year 8. Surgery was less common (8-year incidence: 5.3%). Baseline screening data predicted treatment and surgery, enabling risk stratification. At Year 8, cumulative incidence differed substantially between high- and low-risk groups (treatment: 59.9% vs. 31.2%; surgery: 9.7% vs. 1.0%). Older age (sub-distribution hazard ratio [SHR] 1.03 per year, p<0.001), Black race (SHR 1.50, p<0.001), and personal history of glaucoma (SHR 1.90, p<0.001) were associated with treatment; Asian race was protective (0.71, p=0.03). Older age (SHR 1.06, p<0.001), worse visual acuity (SHR 5.11 per logMAR unit, p<0.001), and screening at a hospital-based site (SHR 2.46, p=0.003) were associated with surgical treatment. Conclusion: Nearly half of safety-net diabetic patients screening positive for referable glaucoma initiated IOP-lowering treatment over 8 years, while few received surgery. Baseline screening characteristics enabled risk stratification of treatment and surgery. These findings address an evidence gap about longitudinal consequences of screening and suggest that its impact extends beyond detection of prevalent glaucoma to include identification of high-risk glaucoma suspects who warrant ongoing surveillance.