Investigative Ophthalmology & Visual Science

PurposeAqueous humor outflow through the trabecular meshwork (TM) is segmental, demonstrating high flow (HF) and low flow (LF) regions. Here, we investigate transcriptomic differences between flow regions in naive mouse and non-glaucomatous human TM tissue to better understand intraocular pressure (IOP) regulation. MethodsHuman eyes (<6 hours postmortem) from two donors were perfused with a fluorescent tracer to identify HF and LF regions and fixed. In parallel, one pair of 8-month-old C57Bl/6J mouse eyes were similarly processed. Sagittal sections from HF and LF regions underwent whole-transcriptome spatial profiling. Differential expression and gene set variation analysis were conducted to identify transcript and pathway-level differences between HF vs. LF TM. Selected targets were validated by immunolabeling. ResultsGenes with relevance to TM outflow were identified as significantly differentially expressed in the human dataset, including ADAM metallopeptidase domain 15 (ADAM15), vimentin (VIM), chitinase 3-like 1 (CHI3L1), and several transcription factors (e.g., FOS, JUNB, ESR1). Pathways related to epigenetic modifications were also differentially enriched in human eyes. In both human and mouse eyes, myocilin was significantly upregulated in HF regions, despite greater protein labeling in LF regions of human eyes. In both species, rho-kinase signaling pathways showed increased enrichment in LF regions, while cell stress pathways, and TNF- signaling were increased in HF TM. ConclusionsHF regions maintain a more active stress response that facilitates greater outflow, whereas LF regions exhibit more matrix accumulation and contractility. This characterization of segmental flow regions can inform future studies to target trabecular outflow and lower IOP.

Fuchs endothelial corneal dystrophy (FECD) is a disease of the corneal endothelium (CE) characterized by the loss of corneal endothelial cells (CECs) and guttae formation, ultimately resulting in corneal edema and vision loss. FECD primarily affects the central CE while sparing the peripheral CE, however the underlying mechanism contributing to the spatial differences remain unknown. Oxidative stress has been increasingly recognized as a key contributor to the pathogenesis of FECD, with CECs being particularly susceptible to damage from reactive oxygen species (ROS), high metabolic activity and ultraviolet induced DNA damage. The non-proliferative nature of CECs, along with the accumulation of oxidative damage can ultimately lead to CEC loss, a key feature of FECD. In this study, we induced oxidative stress with hydrogen peroxide (H2O2) on ex-vivo corneal specimens and observe increased cell death in the central region compared to the peripheral CE. To investigate these underlying differences, we performed bulk RNA sequencing (RNA-seq) on the central and peripheral regions of CE from FECD and normal cadaveric donors. Pathway analysis identified an enrichment of genes involved in collagen and extracellular matrix between the central and peripheral regions of CE in both normal and FECD, as well as between normal and FECD CE. Intriguingly, we identified the long non-coding RNA (lncRNA), NEAT1 as a top differentially expressed gene, with reduced expression in the central CE compared to the peripheral CE and lower expression in FECD compared with normal CE. Using corneal endothelial cell lines and ex-vivo specimens from FECD patients and normal cadavers, we found decreased NEAT1 expression levels in FECD and increased susceptibility to H2O2-induced oxidative stress. We observed that NEAT1 knockdown in normal and FECD cells exacerbated H2O2-mediated oxidative stress, and that NEAT1 overexpression protected FECD cells. We report in this study, a novel insight in the spatial differences in gene expression in the CE and identify reduced expression of NEAT1 in the central CE as a potential contributor to oxidative stress-related cell death in FECD. These findings provide novel insight into FECD pathogenesis and why FECD pathology preferentially affects the central CE. Antioxidants targeting NEAT1 signaling could be developed into novel therapeutics aimed at preventing FECD pathogenesis.

Cone photoreceptors are essential for daylight vision, and their degeneration has more profound visual consequences than rod loss in retinal degenerative diseases. Metabolic dysfunction is closely associated with cone degeneration, however the relatively small population of cones in mice and humans have limited our understanding of cone-specific metabolism. Here, we leveraged cone-dominant and cone-degeneration mouse models including Nrl-/-, Cnga3-/-, and high-dose Triiodothyronine (T3) treatment to investigate cone-specific metabolism and their metabolic impacts on the retinal pigment epithelium (RPE). Across models, cone-dominant retinas consistently showed lower pyruvate abundance alongside increases in glutathione, purines, pentose phosphate pathway intermediates, and one-carbon metabolites. Increases in several key amino acids were also associated with higher cone abundance, such as proline, arginine, alanine, valine, leucine, and hypotaurine. Strikingly, aminoadipate, an intermediate in lysine catabolism, was the most robustly changed metabolite in the retina, showing highly consistent increases across models. Relative cone increases were also associated with metabolic changes in the RPE/choroid. Like the retina, RPE/choroids showed consistent increases in aminoadipate, proline, and hypotaurine, as well as xanthosine and betaine, alongside decreases in uracil. Moreover, proteomic analysis of Nrl-/- mice showed decreases in many key metabolite transporters in the RPE/choroid, including carriers for glucose, lactate, aspartate, glutamate, serine, lysine, taurine, and proline. Collectively, these findings further our understanding of cone-specific metabolism and highlight potential cone-specific metabolic vulnerabilities in retinal degeneration.

Purpose: To characterize the biomechanical response of the optic nerve head (ONH) in myopic versus non-myopic individuals with normal-tension glaucoma (NTG), and to evaluate the association between ONH strains and the rate of visual field (VF) progression. Design: Retrospective clinical cohort study. Participants: A total of 118 NTG eyes from 77 patients with [&ge;] 2 years of follow-up and 5 VF tests were enrolled, including 66 eyes with myopia and 52 eyes without. Methods: Each participant underwent three-dimensional (3D) optical coherence tomography (OCT) imaging of the ONH under three mechanical loading conditions: 20{degrees} adduction, 30{degrees} abduction and acute intraocular pressure (IOP) elevation by applying a 0.80 N force to the lower eyelid. Effective strains across the global ONH complex and within specific segmented tissues (pre-lamina, lamina cribrosa, choroid, and sclera) were quantified using a validated 3D strain-mapping algorithm. Results: Myopic NTG eyes exhibited significantly higher ONH strains compared to non-myopic eyes under acute IOP elevation (10.1{+/-}2.7% vs. 9.3{+/-}1.8%, P = 0.044) and abduction (9.2{+/-}2.3% vs. 8.4{+/-}2.1%, P = 0.048). In the non-myopic group, ONH strains varied significantly across conditions (P < 0.001), with adduction (9.9{+/-}2.0%) and acute IOP elevation (9.3{+/-}1.8%) eliciting greater strains than abduction (8.4{+/-}2.1%). These condition-dependent variations were less pronounced in the myopic group (10.1{+/-}2.7% vs. 9.8{+/-}2.7% vs. 9.2{+/-}2.3%, P = 0.07). Multivariable linear mixed-effects analysis revealed that in myopic NTG, higher ONH strains induced by adduction were significantly associated with faster MD decline (estimate: -0.139 dB/year per 1% increase, P = 0.03, adjusted for age, spherical equivalent and baseline perimetric mean defect). Conversely, larger strains during acute IOP elevation were associated with slower progression (estimate: 0.184 dB/year per 1% increase, P = 0.004). Tissue-specific analysis confirmed that adduction-induced strains in the choroid and pre-lamina tissue as well as IOP-induced strains across all ONH tissues were significantly correlated with the rate of perimetric progression in myopic eyes (all P < 0.05). Conclusions: In this cohort, greater adduction-induced ONH strain was associated with faster perimetric deterioration in myopic NTG. These findings support further investigation of gaze-related optic nerve traction as a potential non-IOP-related biomechanical factor in myopic NTG. Keywords: Normal tension glaucoma, Myopia, Optic nerve head, Ocular biomechanics, Visual field Progression, adduction.

PurposeFuchs endothelial corneal dystrophy (FECD) is a progressive disease, causing premature death of corneal endothelial cells (CECs). Iron-dependent lipid peroxidation and ferroptosis mediate cell death in FECD. We aimed to determine whether FECD progression is mediated by derangements in ferritinophagy - a form of autophagy that degrades ferritin to release labile ferrous iron - and whether ultraviolet A (UVA) exposure drives FECD progression by activating ferritinophagy. MethodsEndothelium-Descemet membrane (EDM) tissues were collected from patients with end-stage FECD undergoing endothelial keratoplasty and from healthy age-matched donor corneas. Separately, immortalized FECD and healthy control CEC lines were cultured. Cellular levels of NCOA4 production and LC3 activation, both markers of ferritinophagy, were quantified using western blotting and PCR. UVA-exposed immortalized cells were plated on coverslips, stained for immunohistochemistry (IHC), and analyzed using confocal microscopy. Corneal endothelial peels were stained and analyzed using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). ResultsSurgically explanted FECD CECs showed significantly increased levels of NCOA4 compared to healthy controls. LC3 activation was increased in FECD immortalized CECs; UV exposure further increased LC3 activation. Additionally, UVA exposure showed trends of increased expression of NCOA4 in immortalized FECD and healthy CECs. On IHC of FECD surgical explant tissue, ferritin was decreased markedly, NCOA4 localized in a dramatic punctate pattern, and both ferritin and LC3 localized within cell nuclei. Spectrometry images showed higher iron levels correlating with areas of higher FECD disease burden. ConclusionsOur results demonstrate ferritinophagy in FECD indicated by the increase of NCOA4 and LC3 ferritinophagy markers in FECD patient and cell culture models. Our finding that UVA activates ferritinophagy implicates this mechanism in UVA-mediated FECD progression. Altogether, aberrant iron dysregulation associated with FECD and ferroptosis may be mediated by ferritinophagy, providing a biomarker to assess disease severity as well as a potential target for future medical therapeutics.

Fungal keratitis (FK) is a major cause of visual impairment worldwide, yet the development and evaluation of new therapeutic approaches are limited by experimental models that inadequately reproduce deep stromal infection or permit longitudinal quantification of fungal burden. Here, we developed an ex vivo porcine corneal model of Aspergillus fumigatus keratitis that supports reproducible fungal invasion into the deeper corneal stroma and enables non-destructive monitoring of infection over time. Optimisation of the culture conditions demonstrated that reducing nutrient availability markedly promoted stromal penetration compared with nutrient rich conditions. Using a YFP expressing A. fumigatus strain combined with confocal microscopy and three-dimensional image analysis, fungal burden could be quantified repeatedly within individual corneas, allowing changes in established infection to be monitored longitudinally. We applied the model to evaluate natamycin and photoactivated chromophore for keratitis corneal cross-linking (PACK-CXL), alone and in combination. Both monotherapies significantly restricted the increase in fungal burden observed in untreated corneas, but neither consistently reduced pre-existing fungal burden. In contrast, combined natamycin and PACK-CXL treatment reduced fungal burden in all treated corneas and was significantly more effective than either treatment alone. Together, these findings establish a quantitative ex vivo platform for investigating invasive A. fumigatus corneal infection and demonstrate its ability to distinguish between treatments that restrict fungal expansion and those that reduce established fungal burden. The model provides a useful approach for the longitudinal evaluation and optimisation of therapeutic interventions for fungal keratitis.

Mustard keratopathy, caused by exposure of the cornea to sulfur or nitrogen mustard vesicants, chemical warfare agents, can lead to severe and often irreversible vision loss. Despite considerable efforts to develop medical countermeasures, including anti-inflammatory, antioxidant, anti-fibrotic, and anti-angiogenic therapies, no treatment effectively targets the underlying mechanisms responsible for mustard-induced tissue injury or prevents long-term disease progression. In the present study, we comprehensively define mitochondrial mechanisms underlying nitrogen mustard-induced corneal injury in both our cell culture model in vitro and a mouse model in vivo. DNM1L (aka Drp1) is a mitochondria-localized dynamin-related GTPase that executes mitochondrial fission and facilitates the autophagic elimination of damaged mitochondrial components. Using complementary in vitro and in vivo models, we demonstrate that nitrogen mustard rapidly induces excessive mitochondrial fragmentation, bioenergetic collapse, membrane depolarization, oxidative stress, intracellular acidification, mitophagy, and apoptotic cell death. Pharmacological inhibition of DNM1L with Mdivi-1 preserves mitochondrial structure and function, restores cellular metabolism, reduces oxidative damage, and markedly improves corneal epithelial integrity, and tissue repair following nitrogen mustard exposure. Collectively, these findings establish mitochondrial dysfunction as a central pathological mechanism in mustard keratopathy and identify DNM1L-mediated mitochondrial remodeling as a therapeutically actionable target. Our work provides strong preclinical evidence supporting mitochondrial-directed therapy as a promising strategy for treating mustard keratopathy.

PurposeTo evaluate associations between green autofluorescence (GAF) and structural and functional measures relevant to retinal and optic neuropathies, and to establish normative GAF values across the optic nerve head (ONH), macula, and papillofoveal bundle (PFB) in rhesus macaques. MethodsEighty-two macaques with normal ONH morphology by spectral-domain optical coherence tomography (SD-OCT) were included with a mean {+/-} SD age of 12.76 {+/-} 7.18 (range 0.11-29.39) years. The GAF images were acquired in the ONH, macula and PFB with the OcuMet Beacon. In a subset of macaques (n=19), pattern electroretinogram (PERG) and photopic full-field ERG including the photopic negative response (PhNR) were recorded. ResultsThe GAF significantly increased with age in the ONH, macula and PFB. After adjusting by age, there were no sex differences, but IOP showed a positive association with macular GAF. At the ONH, higher GAF correlated with thinner retinal nerve fiber layer, inner and outer segment complex, and total retinal thickness. In the macula, inner nuclear layer thickness was positively associated with GAF, whereas outer plexiform layer and inner and outer segment complex were inversely associated. The PERG amplitudes inversely tracked ONH GAF. ConclusionsGAF rises with age and IOP, couples to retinal structure, and at the ONH, aligns with inner-retinal functional indices. This study provides a regional reference for GAF in rhesus macaques. Translational RelevanceNormative GAF data in healthy rhesus macaques provide a framework for interpreting this noninvasive signal in translational studies of retinal and optic nerve disease.

Iris biomechanics plays a key role in primary angle-closure glaucoma (PACG). Soft tissue biomechanics is heavily influenced by tissue water content; therefore, accurately assessing water content is needed to describe iris biomechanics in a physiologically relevant manner. Yet, accurate experimental measurements of iris water content are rare in the literature. Therefore, our objective was to experimentally assess iris water content and potential sex differences in New Zealand White rabbit iris--a suitable model for studying iris biomechanics. We also examined the effect of exposure to phosphate-buffered saline (PBS) on iris water content, given its common use to maintain hydration during soft-tissue biomechanical testing. Our results showed slightly higher water content in males (89.2% {+/-} 2.3%) than in females (87.7% {+/-} 2.2%) (p = 0.022), while we found no significant differences in gross iris morphology between the sexes, indicating subtle compositional differences. Furthermore, PBS incubation had a minimal effect on water content only in female irides. Overall, our results indicated high iris water content in both sexes compared with anatomically adjacent ocular tissues and other soft connective tissues. These results improve our understanding of iris composition and physiology and can serve as a baseline for constructing biomechanical models to assess its role in PACG.

The aim of this study was to characterize the BXD51 mouse strain as a reproducible model of chronic progressive glaucoma. Unlike the highly susceptible DBA/2J (D2) mutant strain, BXD51 is a genetically stable recombinant inbred line derived from C57BL/6J (B6) and D2 parental lines. Longitudinal assessments of intraocular pressure (IOP), visual acuity (VA), contrast sensitivity (CS), and pattern electroretinogram (pERG) demonstrated that BXD51 mice undergo a delayed decline in visual and retinal ganglion cell (RGC) function. Their decline is biphasic, with a period of initial ocular stress followed by a late-onset, accelerated structural and functional deterioration of RGCs. Anterior segment structural analysis by optical coherence tomography (OCT) and histology demonstrated increasing pigment dispersion and subsequent iridocorneal angle closure. Immunofluorescence analysis of structural neuronal markers (TUBB3 and MAP1A/2) exhibited thinning of the ganglion cell layer (GCL) and inner plexiform layer (IPL) together with axonal degeneration, mirroring the laminar degeneration seen in human glaucoma patients. BXD51 also revealed marked spatial heterogeneity between peripheral and central retina. Multivariate analysis confirmed that BXD51 follows a distinct clinical trajectory that separates it from both wild-type (B6) and a severe glaucoma model (D2). By spanning the range between resistance and extreme susceptibility to glaucomatous neurodegeneration, this study establishes the BXD51 mouse as a translational platform for mechanistic studies and for evaluating long-term neuroprotective strategies.

Glaucoma is an optic neuropathy characterized by progressive retinal degeneration. Population genetic analyses describe an association of variants in angiopoietin-like protein 7 (ANGPTL7) with lower intraocular pressure (IOP) and decreased glaucoma susceptibility. In addition to its reported role in the trabecular meshwork (TM), we demonstrate that ANGPTL7 is also expressed within disease-relevant cell types of the human posterior eye, specifically prelaminar optic nerve head astrocytes (ONHA). Contrasting with previously published findings, we observe no difference in IOP between Angptl7-/- mice and wild-type littermates. Despite absence of an IOP phenotype, loss of ANGPTL7 protects from IOPinduced retinal ganglion cell (RGC) damage in a rodent glaucoma neurodegeneration model. ANGPTL7 may signal through LAIR1-positive immune cells, potentially contributing to pro-inflammatory signaling and phenotypic modulation of resident immune cells, with evidence suggesting that oligomerization of ANGPTL7 is necessary for its activity. Comprehensively, our data highlight an unappreciated role of ANGPTL7 in modulating neuroinflammation in the posterior eye, contributing to glaucoma progression.

A cardinal feature of nonexudative age-related macular degeneration (AMD) is the accumulation of drusen between the retinal pigment epithelium (RPE) and Bruch's membrane, yet the mechanisms that initiate and promote drusen formation remain to be fully understood. Hydroxyapatite (HAP), a calcium-phosphate mineral identified within human drusen, has been proposed to contribute to drusen nucleation and is associated with increased likelihood of RPE atrophy. However, mechanistic studies providing insight into RPE-associated mineralization have been limited by a lack of easy-to-use and manipulatable culture models. Here, we established a rapid, inducible ARPE-19-based model of HAP mineralization. We found that ARPE-19 cells cultured in minimum essential media (MEM) supplemented with nicotinamide (NIC), sodium phosphate, and ascorbic acid (NaPAA) reproducibly formed HAP positive deposits, whereas mineralization was not observed while using standard DMEM/F12 medium, DMEM/F12 with NaPAA, MEM-NIC medium, or MEM-NIC medium with alternative osteogenic supplements (such as beta-glycerophosphate, ascorbic acid, and dexamethasone). Scanning electron microscopy with energy dispersive X ray spectroscopy demonstrated that the formed deposits were enriched in calcium (Ca) and phosphorus (P) with Ca/P ratios comparable to drusen-associated HAP deposits from AMD donor tissue. ARPE-19-based HAP deposits were also closely associated with neutral lipid-rich material, staining positive for BODIPY around their core structure. Bulk RNA sequencing of mineralized ARPE-19 cells revealed extracellular matrix, angiogenic, inflammatory, and metabolic transcriptional changes in the absence of strong enrichment of canonical osteogenic pathways. Co-treatment of mineralizing cells with either pyrophosphate, EDTA, or sodium citrate significantly reduced mineral deposition, but these approaches were not able to reverse it. Together, these findings establish a tractable RPE mineralization model that recapitulates key features of AMD-associated HAP and provides a platform for investigating mechanisms of drusen-associated mineral formation.

Purpose: To investigate the association between mitochondrial DNA (mtDNA) copy number (CN) and the rate of visual field progression in patients with primary open-angle glaucoma. Design: Retrospective cohort study Participants: 200 patients with primary open-angle glaucoma (POAG) with a mean of 11 years of longitudinal clinical data and DNA extracted from peripheral blood. Methods: DNA extraction was performed on peripheral blood leukocytes collected from 200 POAG patients who had an average follow-up of 11 years. Mitochondrial CN was quantified by multiplex quantitative TaqMan PCR using custom primers and probes against cytochrome b and beta-2 microglobulin as measures of mitochondrial and nuclear genes, respectively. Univariable and multivariable linear regression models were used to evaluate the association between mtDNA CN and rates of visual field progression, quantified by the slope of standard automated perimetry (SAP) mean deviation (MD) over time. Main Outcome Measures: Association of mtDNA CN with MD slope. Results: Mean mtDNA CN was 107.6 copies per cell (interquartile range, 70.5 to 126.7). Higher ln(mtDNA CN) was associated with slower rates of MD loss in both univariable (beta = 0.29, p < 0.001) and multivariable analyses (beta = 0.25, p = 0.003). Higher peak IOP was independently associated with faster visual field progression in both analyses. A significant interaction was observed between mtDNA CN and peak IOP (beta = -0.054, p < 0.001), with the association between lower mtDNA CN and faster SAP progression being more pronounced at lower levels of peak IOP. Conclusion: Lower mtDNA CN was associated with faster SAP progression, with the association being most pronounced at lower levels of peak IOP. These findings support mtDNA CN as a potential systemic biomarker of mitochondrial genomic reserve and susceptibility to visual field loss.

Purpose: Oxidative stress is thought to contribute to the development of age-related cataracts (ARCs), but the mechanisms by which oxidative damage leads to the opacification of the lens remain unclear. Previous studies suggest that oxidative stress can disrupt lens proteostasis. Therefore, it was hypothesized that ARCs arise from proteomic changes driven by an age-associated decline in oxidative stress defenses that interact with the lens proteostatic state. To test this hypothesis, proteomic analyses of lenses exposed to oxidative stress were performed to examine oxidative and proteostatic stress responses in vivo. Methods: Cataract formation was induced by injecting hydrogen peroxide into the aqueous humor of adult zebrafish. nrf2fh318/fh318 zebrafish were used to model the reduced oxidative stress protection observed in aged human lenses, while cryaba-/- zebrafish were used to model impaired lens proteostasis. Resulting opacities in WT, cryaba-/-, nrf2fh318/fh318, and cryaba-/-; nrf2fh318/fh318 lenses were quantified and proteomic changes in the cortex were analyzed using data independent acquisition Parallel Accumulation Serial Fragmentation mass spectrometry. Results: Hydrogen peroxide treatment induced the formation of cortical cataracts. Proteomic results showed that, dependent on genotype and day, oxidative stress activates the unfolded and mitochondrial unfolded protein responses. Additional changes were also observed in energy metabolism, Ca2+ homeostasis, protein degradation, and cytoskeletal and extracellular matrix remodeling pathways. Conclusions: Treated zebrafish lenses successfully model ARC and mass spectrometry proteomics identified the unfolded and mitochondrial unfolded protein responses as potential therapeutic targets for ARC.

Purpose. To determine which proposed keratoconus (KC) mechanisms carry its genetic susceptibility and how it relates to corneal structure, and which describe its corneal tissue. Methods. Six mechanism modules and two attribution sets (transforming growth factor-{beta} [TGF-{beta}] and canonical type-2 atopy) were tested for enrichment in three independent European KC genome-wide association studies (GWAS) and in GWAS of central corneal thickness (CCT) and corneal resistance factor (CRF), with colocalization at matrix loci. Tissue expression was pooled across nine arms from eight studies with control-source meta-regression; activator protein-1 (AP-1)/immediate-early regulatory activity was scored in 107 donors, spatial expression in 21, and serum responses in human and rabbit stromal cells. Results. Extracellular matrix (ECM) was the only module enriched in all three KC sources (P [&le;] 5.46 x 10-) and for CCT, CRF, and thickness-independent CRF (P [&le;] 2.07 x 10-). COL5A1 shared causal variants with CCT in all three sources (posterior probability 0.985-0.991), and FNDC3B and COL1A1 with CRF or its thickness-independent component (0.961-0.986). Pooled, KC corneas had lower general and corneal-local inflammatory and stress/immediate-early expression (log2 fold-change -0.97, -0.98, -0.82; P [&le;] 0.049). In whole-cornea and stromal tissue, ECM expression reversed between postmortem-donor and pathological controls (log2 fold-change +0.36, -2.34), whereas inflammatory and TGF-{beta} programs were lower against both. AP-1/immediate-early regulatory activity was lower in four donor cohorts (Hedges' g -1.50) and stress/immediate-early expression in two spatial cohorts; serum raised immediate-early readouts in human and rabbit cells. Conclusions. KC combines matrix-mechanical genetic susceptibility with a low-inflammatory, low-immediate-early corneal state whose matrix expression direction depends on the control source. Because this susceptibility is genetically shared with corneal thickness and resistance, interventions on the inherited disease are judged by corneal structure and mechanics.

Clinical relevance: Validated virtual control arms may provide population-level estimates of treatment effect and reduce reliance on untreated control allocations in myopia trials. Background: Untreated single-vision control arms in paediatric myopia efficacy trials are increasingly difficult to justify and retain. Several published models predict untreated childhood axial elongation by region or ethnicity. Here they are implemented unchanged in an open-source tool and validated against an untreated multi-ethnic cohort. Methods: Five published models predicted untreated elongation from baseline age, cycloplegic spherical equivalent, sex, and ethnicity, anchored at baseline axial length (AL) and evaluated at actual follow-up. Predictions were compared with 242 untreated myopic children (Chinese, Vietnamese, Indian) with AL measured at approximately 6 and 12 months, assessing bias, root-mean-square error, and prediction-interval coverage against pre-specified thresholds (bias <0.03 mm; coverage greater than or equal to 0.90). Results: The regional generalised estimating equation (GEE) and meta-regression models reproduced mean East Asian elongation without meaningful bias at 6 months (GEE bias -0.013 mm; equivalence to plus-or-minus 0.03 mm, p = 0.014) and at 12 months (-0.004 mm), although equivalence was not established at 12 months in an underpowered subgroup (n = 71, all Vietnamese; p = 0.068). Older age-only models under-predicted by 0.07 to 0.12 mm. Published individual prediction intervals were too narrow (coverage 0.77): the means were accurate, the individual uncertainty was not. Indian elongation fell between strata and was matched by no existing model. Conclusions: The models reproduce mean untreated East Asian elongation at 6 months, conditional on cohort independence; South Asian children remain unserved by any existing stratum. The tool is a group-level instrument, not an individual predictor, and a transparent unification of published models in open-source code. Its value for estimating treatment effect awaits back-testing against a trial with a known untreated arm, ideally over 24 to 36 months.

Purpose: To develop an interpretable automated pipeline for dome-shaped macula (DSM) detection and measurement from optical coherence tomography (OCT) B-scans. Methods: We analyzed one eye from each of 300 children aged 4-18 years in the Shanghai Child and Adolescent Large-scale Eye Study High Myopia (SCALE-HM) cohort (50 DSM-positive; 100 test eyes). A U-Net pretrained on OCTA-500 and fine-tuned on the development set segmented the retinal pigment epithelium (RPE) boundary. A geometric module located the apex, fitted a tangent-line baseline, and computed perpendicular height in physical coordinates. An eye was DSM-positive if any gradable B-scan had height >= 50 um. Two masked graders evaluated the test set; a third adjudicated disagreements. Results: The area under the receiver operating characteristic curve was 0.927 (95% confidence interval [CI], 0.850-0.986), sensitivity was 82.1% (95% CI, 64.4%-92.1%), and specificity was 100% (95% CI, 94.9%-100%) against the adjudicated reference. In concordant-positive eyes, automated height agreed with manual measurements (intraclass correlation coefficient, 0.949; mean absolute error, 6.9 um; bias, -5.7 um, versus Doctor A). Mean RPE-boundary error on pediatric test scans was 1.54 um. Conclusions: The pipeline may be considered a reliable and reproducible tool for automated DSM detection and height measurement on radial OCT, with each value traceable to landmarks on the source B-scan. External validation across populations, devices, and acquisition protocols is needed. Translational Relevance: The pipeline provides an objective and reproducible method to detect DSM and quantify dome height, width, and orientation across OCT meridians. Because each measurement can be verified on the source B-scan, it enables standardized DSM assessment.

Purpose: To understand the functional impact of macular atrophy (MA) and fibrosis in eyes with treated neovascular age-related macular degeneration (nAMD) based on deep visual sensitivity losses. Design: Cross-sectional study. Participants: One-hundred and nine eyes from 97 individuals with nAMD that had been treated for >=12 months without significant retinal fluid. Methods: All participants underwent defect-mapping microperimetry (DMP) testing - a strategy optimized to quantify the spatial extent of deep visual sensitivity losses through single 10 decibel stimuli presentations - where 208 locations were sampled in the central 8 degrees radius region. Participants also underwent color fundus photography, fundus autofluorescence and OCT imaging, which were co-registered and manually annotated for MA and fibrosis to determine their two-dimensional pointwise extent at individual test locations, and global extent in the corresponding region where DMP was performed. Main Outcome Measures: Pointwise association between the presence of MA and fibrosis with missing a stimulus on DMP and global association between the extent of MA and fibrosis with the proportion of locations missed (PLM) on DMP. Results: At a pointwise level, the presence of MA and fibrosis were independently associated with an increased likelihood of missing a stimulus on DMP (odds ratio = 42.5 and 2.5 respectively; P < 0.001 for both). However, at a global level, only an increasing extent of MA was associated with a significantly higher PLM on DMP (P < 0.001), but not fibrosis (P <= 0.227). MA extent explained a substantial proportion of the variance in the PLM (R2 = 0.97) and there was a very strong correlation between the two parameters ({rho} = 0.93). Conclusions: This study confirmed the expected functional impact of MA and fibrosis on DMP testing at a pointwise level. However, it showed at the global level that only the extent of MA, and not fibrosis, was independently associated with functional loss on DMP, and that there was a very strong structure-function correlation based on MA. These findings thus suggest that evaluating MA alone may be sufficient for capturing structural changes strongly associated with the global extent of deep visual sensitivity losses in those with treated nAMD.

Published artificial intelligence for keratoconus is unproven in fellow eyes that topography and tomography call normal, the eyes where surgical decisions are made, although it reports near-perfect accuracy for early disease. In a registered systematic review of 573 studies (PROSPERO CRD420261441197), none of 2,035 claim-by-stratum units from 212 adjudicated studies established discrimination in these eyes. Most units (90.0%) answered a different question, never constructed the target population or certified its normality; external validation did not test label generation, and five systematic reviews did not assess it. In a public cohort, a frozen score yielded an area under the receiver operating characteristic curve of 0.996 against a same-eye index label and 0.549 against a contralateral proxy; its inputs reconstructed the label-generating index (r = 0.991), so accuracy there measured agreement with the index. Anchor-gap evaluation measures accuracy in these eyes against the contralateral clinical diagnosis.

Purpose: To evaluate the associations of the numerical AI-CO center index and visually classified central dark-area extent with Humphrey 10-2 mean deviation (MD). Methods: This single-center retrospective study included 320 eyes of 169 patients with AI-CO imaging, broad-field 24-2F or 30-2 testing, and 10-2 testing. Both visual field examinations were performed within 365 days before or after AI-CO imaging. Two ophthalmologists independently classified central dark-area extent into five Levels (0-4). Analyses assessed Spearman correlations, areas under the receiver operating characteristic curve (AUCs), distributions across final central dark-area Levels, and interrater reliability. Patient-cluster bootstrap confidence intervals and a one-eye-per-patient sensitivity analysis addressed inclusion of both eyes. Results: AI-CO center correlated with 10-2 MD (rho=0.829; 95% CI, 0.773-0.873). AUCs for 10-2 MD below -2, -6, and -12 dB were 0.902, 0.939, and 0.961, respectively. The numbers of eyes in Levels 0-4 were 66, 57, 94, 53, and 50; median 10-2 MD values were -0.59, -1.48, -3.54, -6.24, and -18.15 dB, respectively. Quadratic-weighted kappa was 0.970 (95% CI, 0.958-0.981). Sensitivity analyses yielded broadly similar findings. Conclusions: The numerical AI-CO center index and visually classified central dark-area extent were associated with central visual field damage. Assessing the extent of dark regions in the central image alongside the numerical index may provide useful information for interpreting central visual field status from the AI-CO display.