Experimental Eye Research

PurposeAnti-vascular endothelial growth factor (anti-VEGF) therapy is the standard of care for neovascular age-related macular degeneration (AMD), yet many patients exhibit persistent retinal degeneration, fibrosis, and incomplete therapeutic response. The molecular pathways underlying this incomplete response remain poorly understood. We sought to identify VEGF-independent signaling pathways active in the vitreous of anti-VEGF-treated AMD patients. MethodsWe performed multiplex antibody-based proteomic profiling of 1,000 human proteins in vitreous samples from patients with neovascular AMD receiving anti-VEGF therapy (n=8) and comparative controls (n=6). Differential protein expression was assessed using one-way ANOVA, followed by gene ontology and pathway enrichment analyses. Drug-target relationships were evaluated to identify potential opportunities for therapeutic repositioning. ResultsWe identified 107 differentially expressed proteins (p<0.05), including key regulators of immune signaling, angiogenesis, and metabolism. Notably, multiple components of cytotoxic lymphocyte pathways were dysregulated, including IL-21R, SIGLEC-7, CTLA4, and IL-2-associated signaling. Enrichment analyses revealed significant activation of pathways related to T-cell activation, interleukin signaling, and leukocyte-mediated cytotoxicity. These immune signatures persisted despite suppression of VEGF signaling. Several clinically available immunomodulatory agents--including abatacept, sirolimus, and dupilumab--targeted pathways identified in this dataset. ConclusionsAnti-VEGF-treated neovascular AMD exhibits persistent cytotoxic immune signaling in the vitreous, suggesting that VEGF-independent immune mechanisms may contribute to ongoing retinal damage and incomplete therapeutic response. These findings provide a rationale for combination therapeutic strategies targeting both angiogenic and immune pathways in AMD.

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.

Aim: To evaluate the potential of a three-dimensional microscope combining Laser scanning confocal imaging and white-light interferometry for quantitative topographic characterisation of Descemet's membrane (DM) in Fuchs endothelial corneal dystrophy (FECD). Methods: Descemet's membranes were collected from 38 FECD patients undergoing endothelial keratoplasty and 4 healthy donors. After flat-mounting on glass slide and drying, specimens were analysed using the VK-X3000 system (KEYENCE). Entire samples were reconstructed by image stitching at low magnification (x10) in white-light interferometry mode (0.01nm axial resolution). Higher magnifications (x20-x150) in confocal mode (12nm axial resolution) enabled detailed structural analysis. Three-dimensional height maps were generated to calculate standardised surface roughness parameters. Guttae and other DM features were classified according to spatial organisation and elevation profiles. Results: White-light interferometry enabled full-field mapping of whole 8mm diameter DMs with nanometric vertical resolution (~2 hours/sample). Surface roughness (Sa) was higher in FECD than in controls (median{+/-}IQR: 0.571{+/-}0.259 m vs 0.239{+/-}0.161 m ; p = 0.0018). In FECD, three zones were identified: central (guttae buried in the posterior fibrillar layer; Sa 0.442 {+/-} 0.112 m), paracentral (large uncovered guttae; Sa 0.562{+/-}0.170 m ; p = 0.0423), and outer zone (no confluent guttae; Sa 0.261{+/-}0.143 m ; p < 0.0001). Confocal 3D imaging revealed radial striae, embossments and furrows in the DM, confluent central guttae, and fused or buried structures. Conclusions: Combining white-light interferometry and confocal microscopy enables label-free, high-resolution surface characterisation of DM in FECD, providing quantitative metrics to compare histological subtypes and supporting the predominance of radial structural organisation.

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: Exfoliation glaucoma (XFG) is the most common secondary glaucoma. Prior studies suggest a higher incidence in women and links to reproductive history, implying estrogen-related pathways. Metabolomic data also indicated inverse associations with steroid-related plasma metabolites, suggesting steroid involvement in XFG pathogenesis. Methods: We conducted a nested case-control study within the Nurses' Health Study (NHS) (1980-2018), NHSII (1989-2019), and Health Professionals Follow-up Study (1986-2018), with 217 XFG suspect (XFGS)/XFG cases and 217 matched controls (62 men and 372 women). We evaluated 18 endogenous steroids and five steroid classes using conditional logistic regression. Secondary analyses examined effect modifications by age and residential latitude, and heterogeneity by disease severity (XFGS vs. XFG). Metabolite set enrichment analysis (MSEA) was used for class-level associations. Multiple comparisons were addressed using the number of effective tests (NEF) for individual steroids and false discovery rate (FDR) for steroid classes. Results: No individual steroid or steroid class met NEF- or FDR-adjusted significance thresholds, overall or by sex. Nonetheless, across both sexes, MSEA demonstrated a non-significant inverse trend between androgen levels and XFG/XFGS risk (FDR=0.22), with 11-ketotestosterone showing a nominal inverse association (OR=0.54; 95%CI=0.31-0.93; P=0.03). Progestogens showed enrichment scores in the positive trend (FDR=0.31), with a borderline positive association between progesterone and XFG/XFGS (OR=2.21; 95%CI=1.00-4.87; P=0.05). Conclusions: Although we observed no statistically significant associations with steroids after correction for multiple testing, the suggestive patterns for androgens and progestogens support the possibility of steroid-related pathways in XFG etiology and support further evaluation in larger studies.

PurposeHyperosmolar stress (HOS) is a major contributor to corneal epithelial cell damage in dry eye disease. We have previously shown that HOS damages mitochondria and impairs cell metabolism in corneal epithelial cells. Small extracellular vesicles (sEVs) are cell-derived lipid envelopes that are present in all body fluids, including tears. Prior studies suggest that sEV release and composition may be linked with changes in cell metabolism. In this study, we tested the effects of HOS on sEV release and composition, and found that sEV cargo may reflect early, underlying changes in dry eye disease. MethodsTelomerase-immortalized human corneal epithelial (hTCEpi) cells were treated with 450 mOsm NaCl for five days to induce chronic HOS. sEVs were isolated using differential centrifugation followed by iodixanol density gradient flotation. Particle number was determined using Nanoparticle Tracking Analysis (NTA). Mass spectrometry was used to assess the sEV proteome, and selected proteins were validated by immunoblot. Proteome pathways were analyzed using KEGG and CORUM. ResultsPathway analysis revealed an increase in metabolic proteins and proteasome components in sEV cargo released from hTCEpi cells exposed to HOS. These proteins were increased more than fourfold in HOS-sEVs. Examination of proteins involved in the endosomal pathway and NTA further confirmed an increase in HOS-sEV release. ConclusionOur findings suggest a potential mechanism whereby corneal epithelial cells exposed to HOS retain proteins involved in maintaining tissue integrity, while simultaneously releasing unneeded proteins involved in cell metabolism. The presence of metabolic proteins in sEVs may serve as early indicators of dry eye disease.

Purpose Considering that myopia is associated with thinning of the ocular coats, this study investigated the inter-relationship of retinal, choroidal and scleral thickness in foveal regions in Indian high myopes. Methods A total of 23 high myopes (spherical equivalent refraction [&le;]-6.00D) aged 16 to 35 years underwent posterior segment imaging with swept-source optical coherence tomography. The retinal, choroidal and scleral thickness was determined using semi-automated custom-designed software at sub-foveal regions. Axial length was determined using Lenstar LS 900 non-contact biometer. Results The mean plus-or-minus sign SD axial length was 30.17 plus-or-minus sign 2.23 mm, sub-foveal retinal thickness was 245 plus-or-minus sign 28 lower case Greek mum, sub-foveal choroidal thickness was 82 plus-or-minus sign 46 lower case Greek mum, and sub-foveal scleral thickness was 254 plus-or-minus sign 68 lower case Greek mum. The choroid was significantly thinner compared to the retina and sclera (p<0.001). With a 1 mm increase in axial length, there was no significant variation in sub-foveal retinal (increased by 0.86 lower case Greek mum) and scleral thickness (decreased by 4.31 lower case Greek mum, p[&ge;]0.05), but sub-foveal choroidal thickness decreased by 10.35 lower case Greek mum (p=0.02). For a 1D decrease in spherical equivalent refraction, the choroidal thickness reduced significantly (decreased by 5.88 lower case Greek mum, p<0.001), while there was no significant variation in retinal (decreased by 0.68 lower case Greek mum, p=0.55) and scleral thickness (increased by 0.13 mum, p=0.98). The association of the sub-foveal retinal, choroidal, and scleral thickness was weak and was not significant in high myopes (p[&ge;]0.10). Conclusions With increasing axial length and severity of myopia in high myopes, compared to scleral and retinal thickness, the choroidal thickness alone decreased significantly. Our findings indicate that the changes in the choroid do not necessarily reflect the changes in retinal and scleral thickness and highlight the importance of the choroid as a marker for axial elongation even in high myopes.

Background and Objective As optical coherence tomography (OCT) has enabled the identification of an expanding set of age related macular degeneration (AMD) risk biomarkers and become central to routine clinical practice, there remains a need for a simplified grading scheme that allows physicians to communicate and synchronize AMD grading directly from standard OCT imaging rather than relying on traditional color fundus imaging. This study aims to establish a standardized OCT based AMD classification that balances diagnostic accuracy with practicality for use across clinical and research settings. Patients and Methods Spectral domain optical coherence tomography scans were independently graded by two retinal specialists following the newly proposed Stanford OCT Based AMD Classification (SOAC). Discrepancies were adjudicated by a third independent retinal specialist. Intergrader agreement was assessed using weighted kappa coefficients. Results Among the 109 eyes from 108 patients, AMD staging based on SOAC was distributed as follows: normal aging in 9 patients (8.3%), early AMD in 16 (14.7%), intermediate AMD in 32 (29.4%), neovascular AMD (nAMD) in 18 (16.5%), geographic atrophy (GA) in 20 (18.3%), and combined nAMD and GA in 14 (12.8%). The overall intergrader agreement demonstrated robust consistency, with a weighted kappa value of 0.95 (95% CI: 0.92 to 0.98), signifying excellent intergrader reliability and reinforcing the validity of SOAC. Conclusion SOAC provides a standardized, OCT based framework for AMD grading that demonstrates high intergrader agreement. By enabling consistent classification from commonly acquired OCT scans, SOAC supports reliable disease staging and facilitates integration across clinical studies and translational research. As imaging and molecular data continue to expand, SOAC can serve as a common OCT based reference for phenotype refinement and longitudinal AMD studies.

PurposeMachine learning approaches for automated quantification of optic nerve histology have emerged as potential tools for objective assessment of axonal injury in experimental glaucoma models. However, the generalizability of these models to independent datasets remains unclear. Guided by a scoping review of the literature, this study performed independent validation testing of publicly available models on a novel rat optic nerve dataset to assess their generalizability. MethodsWe conducted a scoping review following PRISMA-ScR guidelines. PubMed, EMBASE, Scopus, and Cochrane CENTRAL were searched from 2000 through 2025. Two reviewers independently screened records and extracted data on model characteristics and performance metrics. Additionally, we performed independent validation of three models (AxoNet, AxonDeepSeg, AxoNet 2.0) on a novel rat optic nerve dataset comprising 57 images with 9,514 manually annotated axons. Because AxonDeep is not publicly available, we instead evaluated AxonDeepSeg, a separate publicly available deep learning-based tool that, while not previously applied to optic nerve tissue, is widely used for nerve fiber segmentation. ResultsFrom 2,036 records, four manuscripts describing three deep learning models met inclusion criteria. Published correlation coefficients between model predictions and reference counts ranged from 0.959 to 0.99. On independent validation, performance was reduced: AxoNet 2.0 achieved the highest correlation (r = 0.89), followed by AxonDeepSeg (r = 0.86) and AxoNet (r = 0.79). Segmentation quality metrics revealed high precision (>0.94) but low recall (0.18 to 0.27), with Dice coefficients of 0.29 to 0.40, substantially below published benchmarks of 0.81. ConclusionsDeep learning models for optic nerve histology demonstrate strong within-study performance but show meaningful performance decrements when applied to independent datasets. The observed generalizability gap (correlations 0.07 to 0.182 points below published values) demonstrates the need for standardized validation datasets and multi-center testing before widespread adoption of these tools.

PurposeQuantitative metrics obtained from retinal fundus images (such as vessel length, tortuosity and other scale-dependent measures) are increasingly used as potential biomarkers for systemic diseases, including cardio- and neurovascular conditions. However, with the increasing prevalence of myopia and related axial growth, this study aims to evaluate if axial length scaling significantly alters the overall distributions of the inferred biomarkers when compared to biomarker data obtained without axial length scaling and if these effects can be corrected. Methods2,309 clinic visits from patients aged [&le;]21 years were analysed and extracted for axial-length scaling analysis (range) 20 to 28 mm). The retinal fundus photographs were automatically segmented using Automorph to extract biometric data, including vascular metrics. The parameters were further corrected for axial length using correction factors based on the Bennett-Littmann formula and true axial length. ResultsAxial length significantly influenced biometric parameters (vessel metrics) derived from fundus photography. The magnitude of error in diameter and length of blood vessels was approximately 4-5% for each 1 mm deviation from the reference axial length of 24 mm, whereas the error in vessel area was approximately 9-10% per 1 mm, consistent with the geometric expectation that area scales with the square of linear dimensions. The scaling corrections for different axial lengths are presented. ConclusionsAxial-length-related magnification introduces systematic bias into retinal vascular metrics from fundus photographs. Bennett-Littmann correction using true axial length reduces these errors and should be adopted in quantitative fundus imaging and Al biomarker development.

Background: Mitochondrial dysfunction is an emerging metabolic hallmark of age-related diseases, yet tools to directly profile mitochondrial pathways and test metabolic interventions in the living human eye remain limited. Multi-omics ocular liquid biopsy enables real-time proteomic and metabolomic profiling of the intraocular microenvironment, complementing systemic biomarkers and imaging surrogates. Here, we used this approach to define mitochondrial and tricarboxylic acid (TCA) cycle dysregulation in geographic atrophy (GA) and to assess whether oral -ketoglutarate (-KG) supplementation can modulate mitochondrial metabolites within the eye. Methods: Mitochondrial and TCA cycle-related proteins were profiled in aqueous humor (AH) samples from patients with GA using DNA-aptamer-based proteomics. In a phase 0 study, a second cohort undergoing sequential cataract surgery provided paired AH samples collected at first-eye surgery and at second-eye surgery after interim -KG supplementation. These samples underwent targeted metabolomic profiling using hydrophilic interaction liquid chromatography coupled with mass spectrometry. Results: In GA, 64 mitochondrial proteins were differentially expressed, including coordinated TCA-cycle deficiencies marked by reduced expression of enzymes regulating TCA entry and flux, including PDHB and DLST. In the phase 0 cohort, oral -KG supplementation significantly increased intraocular -KG levels and the -KG-to-succinate ratio (P < 0.05), with coordinated shifts across TCA intermediates consistent with enhanced TCA cycle flux. Conclusions: AH proteomics demonstrated mitochondrial pathway depletion in GA, consistent with reduced oxidative bioenergetic capacity. AH metabolomics provided first-in-human in vivo evidence that systemic -KG supplementation can modify intraocular metabolites and may enhance intraocular energy metabolism. These findings support ocular liquid biopsy as a precision-health framework for per-patient biomarker-guided metabolic trials in GA.

PurposeTo highlight the roles of intraoperative optical coherence tomography (iOCT) in managing acute corneal hydrops (ACH) and outcomes of iOCT-guided pneumodescemetopexy and corneal compression sutures. MethodsThis was a retrospective, consecutive, interventional case series of patients with keratoconus who presented with significant ACH and underwent iOCT-guided pneumodescemetopexy (18% sulfur hexafluoride gas) and compression sutures at Birmingham and Midland Eye Centre, UK, between Aug 2023 and May 2025. ResultsFive patients were included; mean age was 32.3{+/-}6.6 years old and 3 (60%) were male. The mean follow-up duration was 16.3{+/-}5.6 months. At presentation, the mean corrected-distance-visual-acuity (CDVA) was 1.90{+/-}0.67 logMAR, central corneal thickness (CCT) was 1187.6{+/-}372.6m, maximal corneal thickness was 1624.0{+/-}383.5m and maximal height and diameter of pre-Descemet layer/Descemet membrane (PDL/DM) detachment was 1014.6{+/-}366.4m and 4456.0{+/-}839.4m, respectively. The surgery successfully achieved complete PDL/DM attachment in all cases, with a mean time from surgery to ACH resolution of 17.8{+/-}8.0 days. iOCT successfully visualized the area of PDL/DM break/detachment, revealed the involvement of PDL (evidenced by a persistent taut type 1 DM detachment after gas tamponade), and guided the placement of compression sutures. Compared to preoperative, there was a significant improvement in the mean CDVA (0.52{+/-}0.32 logMAR; p=0.014) at last follow-up. One patient required a repeat procedure to fully attach the PDL/DM. ConclusionsThis study demonstrated favorable outcomes of iOCT-guided pneumodescemetopexy and corneal compression sutures. iOCT revealed the involvement of PDL in ACH and provided plausible explanations why pneumodescemetopexy alone may not be able to resolve significant ACH rapidly in certain cases.

Age-related macular degeneration (AMD) is a common, complex disease affecting older individuals that can lead to severe vision loss. It is characterized by early anatomical changes in the retina, retinal pigment epithelium (RPE), and choroid, especially in the central (macular) region. AMD can progress to severe atrophy and/or pathologic angiogenesis that leads to visual decline. Over 30 genetic loci have been identified as contributing to AMD risk; however, the mechanisms by which genetic variants affect pathology has not been thoroughly explored. In this report we examined single-nucleus gene expression in the retina, RPE and choroid of 88 individuals categorized by AMD stage, as well as 37 previously published samples. Genotyping was performed on 1.8 million SNPs, with additional SNPs imputed, on each donor to identify expression quantitative trait loci (eQTLs). We found that two AMD-risk loci (PILRB and ARMS2/HTRA1) affected the expression of PILRB and HTRA1, respectively. The risk allele of PILRB was associated with increased PILRB RNA in cones, fibroblasts, choroidal macrophages, and RPE, whereas the HTRA1 risk locus was associated with decreased HTRA1 RNA in the RPE. We also identified an age-related decrease in complement inhibitors in the choriocapillaris, a tissue susceptible to complement mediated damage in AMD.

PurposeTo identify the ocular biometric parameters associated with refractive outcomes in Chinese Primary angle closure glaucoma (PACG) patients receiving phacoemulsification and intraocular lens (IOL) implantation (PEI) surgery. Methods165 Chinese PACG patients receiving PEI and goniosynechialysis (GSL) and 53 cataract patients as controls only receiving PEI surgery were recruited. The prediction accuracy of IOL power calculation was assessed by the prediction error (PE), mean absolute error (MAE), median absolute error (MedAE), and proportions of eyes with a PE within {+/-} 0.25 diopters (D), {+/-} 0.50 D, {+/-} 0.75 D, and {+/-} 1.00 D. The association of different ocular biometric parameters with the PE of IOL calculation were evaluated. ResultsThe PACG patients had significantly higher absolute of PE as compared to the control subjects, especially the acute PACG patients. The axial length (AL), changes in aqueous depth pre- and post-surgery ({bigtriangleup}AD), and the ratio of {bigtriangleup}AD/AL were significantly associated with the PE in acute PACG patients. The association of {bigtriangleup}AD with the PE of IOL power calculation was found in PACG patients with AL [&ge;] 22 mm. ConclusionsThis study revealed the association of AL and {bigtriangleup}AD with the PE of IOL calculation in Chinese PACG patients. Precisely predict the {bigtriangleup}AD is necessary for acute PACG patients, especially for those with AL [&ge;] 22 mm, to improve the refractive outcomes.

PurposeThe aim of this clinical investigation was to evaluate the safety and effectiveness of iota-carrageenan (I-C) eye drops in the treatment of mild-to-moderate dry eye disease (DED). MethodsIn this prospective, single arm, open label clinical investigation, thirty adult participants with mild-to-moderate DED applied I-C eye drops three times daily for four weeks. Before start and after end of treatment, participants rated DED symptoms (foreign body sensation, burning/stinging, itching, pain, sticky feeling, blurred vision and photophobia), both after exposure to a normal controlled environment (NCE) and to an adverse controlled environment (ACE). Additional endpoints were changes in ocular surface disease index (OSDI), Change in Dry Eye Symptoms Questionnaire (CDES-Q), corneal and conjunctival surface damage, tear film break-up time, tear evaporation and production. Tolerability was assessed by participants at start and end of treatment. Safety, including visual acuity and intraocular pressure, was monitored throughout the investigation. ResultsAfter four weeks of treatment with I-C eye drops, the mean total DED score after ACE was significantly reduced by -11.89 points (95% CI: -15.11, -8.67), p<0.001. The mean score reduction between baseline and final visit after NCE was slightly less pronounced, with -8.07 points (95% CI: -10.71; -5.43), p<0.001. The vast majority of participants (93% after ACE and 89% after NCE exposure) recorded a reduction in total DED score between baseline and final visit. Mean OSDI score significantly decreased by -7.75 points (95% CI: -10.85, -4.63), p<0.001. ACE-induced deterioration of tear film stability as well as corneal and conjunctival damage were reduced following treatment. All adverse events were mild and transient in nature. 93% of the patients described I-C eye drops as well or very well tolerated. Treatment did not negatively impact any of the safety parameters. ConclusionI-C eye drops are effective, safe and well tolerated. Treatment with I-C eye drops alleviates DED symptoms, stabilizes tear film and protects the ocular surface in patients with mild-to-moderate DED even under adverse environmental conditions. Trial RegistrationNCT06262100.

PurposeAtoh7 is a transiently expressed developmental transcription factor that gives rise to the seven major retinal cell types. Despite this broad lineage, Atoh7 is only required for retinal ganglion cell (RGC) genesis and survival, even though a significant portion of RGCs are Atoh7 negative based on lineage tracing in mice, suggesting a cell nonautonomous role for Atoh7 in the genesis and survival of all RGCs. Atoh7 function is conserved in zebrafish, yet the full retinal lineage, including the RGC population, has remained unidentified. Therefore, we sought to determine the atoh7 retinal lineage in wild type and atoh7 mutant zebrafish retinas. MethodsWe generated atoh7:iCre transgenic zebrafish and in combination with the established ubi:Switch lineage trace permanently labeled cells that represent the atoh7 lineage. A combination of in vivo live imaging and immunohistochemical techniques were used to validate atoh7:iCre transgene expression and the atoh7 lineage in embryonic, larval, and adult retinas as well as the adult brain. ResultsThe atoh7:iCre;ubi:Switch transgene combination successfully recapitulated the onset of endogenous atoh7 expression and transgene fluorophores persisted into adulthood labeling the atoh7 lineage. Most notably, we determined 79% of total RGCs in the wild type retina come from atoh7+ progenitor cells, a greater number than reported in the mouse retina. In atoh7 mutant retinas, we confirmed a complete loss of RGCs and observed a statistically significant increase in the proportion of atoh7+/Pax6+ amacrine cells, as well as an increase in the total number of Prox1+ bipolar cells. Interestingly, we discovered atoh7+ cells located outside the eye in other areas of the central nervous system. ConclusionsThese data demonstrate the presence of atoh7 positive and negative retinal cell types in the zebrafish retina, including RGCs, highlighting the potential to study survival mechanisms of atoh7 negative RGCs and fate switch paradigms using zebrafish retinal development models.

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.

In recent years, researchers have paid increasing attention on potential associations between respiratory and ocular diseases. To examine whether asthma is independently associated with macular degeneration (MD) and whether asthma can serve as a predictor of MD using data from the English Longitudinal Study of Ageing (ELSA). Data from the 2020-2021 wave of ELSA were analyzed. Statistical tests were performed on participants baseline characteristics. Multivariable logistic regression, stratified analysis, ROC curve analysis, smoothing curves and sensitivity analysis were conducted to assess the association, stability, predictive performance, dose-response relationship and robustness. A total of 6,703 participants were included. After adjusting for covariates, age and asthma were significantly associated with MD (p < 0.05), while diet and sex were not. Asthma was consistently linked to increased MD risk across three regression models (OR > 1, p < 0.05), with the association persisting in stratified analyses. ROC analysis showed moderate predictive performance (AUC = 0.757), and a positive dose-response relationship was observed. Sensitivity analyses confirmed the robustness of the association. Asthma may independently increase MD risk, providing novel insights into their relationship and implications for clinical risk stratification and preventive strategies.

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 safety and exploratory outcomes of a single intravitreal injection of OGX110, a peptide agonist of CXCR3, in eyes with persistent fluid secondary to neovascular age-related macular degeneration (nAMD) despite ongoing anti-vascular endothelial growth factor (anti-VEGF) therapy. Methods: This prospective, open-label, sequential dose-escalation phase I study (NCT05904691) enrolled subjects receiving standard-of-care intravitreal anti-VEGF therapy. Subjects received a single intravitreal injection of OGX110 at 0.5 mg, 1.0 mg, or 2.0 mg (n=3 per cohort), 7 to 14 days after the anti-VEGF injection. Results: All nine enrolled subjects completed follow-up through day 56. Two subjects (22%) experienced at least 1 adverse event (AE); all were mild and unrelated to study treatment. Exploratory analyses showed a BCVA change of +1.4 letters following anti-VEGF injection and +4.4 letters from OGX110 baseline to 4 weeks (P < 0.05). Six of 9 subjects gained at least 3 ETDRS letters after OGX110. Anatomic responses were heterogeneous. Four eyes showed a reduction in CRT after anti-VEGF injection that was maintained after OGX110 administration. One additional eye demonstrated a substantial reduction in CRT after OGX110 despite minimal response to anti-VEGF treatment. Conclusions: A single intravitreal injection of OGX110 was well tolerated. Exploratory functional and anatomic findings suggest biologic activity; interpretation is limited by small sample size, open-label design, absence of a concurrent control group, and inter-subject heterogeneity. These results support further study in a controlled trial. Translational Relevance: OGX110 represents a mechanistically distinct investigational approach for nAMD that may warrant further evaluation in eyes with persistent.