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.

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: 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: To 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. Methods: This 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. Results: Five 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+/-372.6um, maximal corneal thickness was 1624.0+/-383.5um and maximal height and diameter of pre-Descemet layer/Descemet membrane (PDL/DM) detachment was 1014.6+/-366.4um and 4456.0+/-839.4um, 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. Conclusions: This 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.

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.

Purpose: To 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. Methods: 165 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. Results: The 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. Conclusions: This 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.

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.

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.

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.

Age-related macular degeneration is a common ocular disease that causes vision loss in the elderly, with a complex set of risk factors and proposed mechanisms of pathogenesis. A powerful method for investigating changes in disease is metabolomics, by which small molecules can be identified and quantified simultaneously. We report here the metabolic analysis of human RPE-choroid tissue in aging and macular degeneration (AMD), as well as comparisons of human macular and extramacular RPE-choroid and neural retina. Levels of 215 metabolites were determined in young donors, AMD donors (early/intermediate, geographic atrophy, and neovascularization) and age-matched controls. The largest number of metabolite differences were observed between young and healthy aged controls, as opposed to between aged controls and any stage of AMD. Two notable metabolites found to be increased in aging choroids are trimethylamine N-oxide and uric acid, both of which were significant after Bonferroni correction. A mouse endothelial cell line treated with a high concentration of uric acid exhibited reduced migration in a wound closure assay. This study provides initial insights into the metabolome of human choroids in varying states of age and macular degeneration, as well as functional implications of these changes in the aging choroid.

Objective: The study aims to evaluate the real-world effectiveness of Highly Aspherical Lenslets spectacle (HAL; Essilor(R) Stellest(R)) in slowing myopia progression among Indian children and adolescents aged between 4 and 16 years. Methods and analysis: This was a multicentre retrospective study conducted across 10 leading ophthalmic centers. The study participants comprised children aged between 4 and 16 years who were prescribed HAL spectacles (Essilor(R) Stellest(R)). Data were extracted from electronic medical records at three time points: T1: One year prior to intervention; T2: Baseline at HAL spectacle prescription; T3: 6 to 24 months after prescription. The primary endpoint was the myopia progression and axial elongation in the year following prescription, compared with the untreated year and with published meta-regression models. Only data from the right eye were analysed, with the expected physiological progression estimated based on the individual progression trajectory after adjusting for age-related slowing as reported in published meta-regression models. Results: A total of 372 myopic children were included in the study. The annual myopia progression was -0.72 {+/-} 0.47 D/year during the untreated period, reducing to -0.11 {+/-} 0.29 D/year with HAL spectacle wear. The expected progression without treatment was -0.65 D/year, based on trajectory-adjusted modelling, indicating a treatment effect of 0.54 D/years and an estimated 83% slowing in myopia progression compared to expected progression. The expected axial elongation under physiological conditions was 0.29 mm/year, estimated using age-adjusted meta-regression models; with HAL lens wear, axial elongation was 0.11 {+/-} 0.16 mm/year, corresponding to a [~]62% relative slowing of elongation. Conclusion: The present study demonstrates the real-world evidence validating the efficacy of HAL lenses as an effective myopia control intervention in Indian children and adolescents. The retrospective design and limited follow-up period warrant future prospective, long-term studies to validate these findings.

Purpose: To compare hypopyon detection using anterior segment optical coherence tomography (ASOCT) versus slit lamp examination (SLE) in microbial keratitis, and to evaluate intra-and inter-grader agreement for ASOCT hypopyon measurement. Methods: Two masked graders independently evaluated ASOCT images for hypopyon presence or absence in eyes with microbial keratitis, with disagreements resolved by consensus. A subset of hypopyon eyes underwent triplicate height measurement using two methods (endothelial length, vertical height). Cohen's kappa, intraclass correlation coefficients (ICC), sensitivity, and specificity were calculated comparing diagnostic performance of ASOCT versus SLE. Results: Inter-grader agreement for hypopyon detection on ASOCT was excellent (k=0.94; 95% CI 0.84-1.00) and intra-grader agreement was excellent (k=0.89-1.00). ASOCT detected hypopyon in 67.1% of eyes versus 57.0% by SLE (sensitivity 83.0%, specificity 96.2% using ASOCT as reference). Intra-grader reproducibility was excellent for both endothelial length and vertical height measurements (ICC 0.977-0.996). Inter-grader agreement was good for endothelial length (ICC 0.831) and vertical height (ICC 0.827), though a statistically significant inter-grader bias was identified for vertical height only (Wilcoxon p=0.008). Conclusions: ASOCT detected hypopyon with greater sensitivity than SLE and demonstrated excellent intra-grader and good inter-grader measurement reproducibility. Endothelial length showed slightly superior inter-grader concordance to vertical height measurement.

PurposeMitochondrial dysfunction contributes to major blinding diseases, including age-related macular degeneration and glaucoma. Although mitochondrial transplantation has shown therapeutic potential in multiple organ systems, translation to the eye remains limited, partly due to uncertainty regarding optimal delivery. We summarize the biologic rationale and preclinical evidence supporting ocular mitochondrial transplantation and present feasibility data evaluating clinically relevant delivery routes. MethodsWe conducted a focused narrative review of ocular mitochondrial transplantation. For feasibility experiments, mitochondria with an endogenous fluorescent dye were isolated from liver donor mice. Postnatal day 7 pups received subretinal injections, and adult CD1 mice received intravitreal injections, including optic nerve head directed delivery. Eyes were analyzed using fluorescence microscopy and immunohistochemistry. Mitochondrial uptake was assessed in cultured retinal pigmental epithelial (RPE) cells using co-incubation assays. Suprachoroidal delivery feasibility was evaluated in cadaveric human near-real surgical specimens using a novel dedicated suprachoroidal injector. ResultsThe literature on ocular mitochondrial transplantation remains limited and consists primarily of small preclinical studies using intravitreal delivery and imaging-based detection. In our experiments, intravitreal delivery produced donor signals predominantly within inner retinal layers, with enrichment along retinal nerve fiber bundles when directed toward the optic nerve head. Cultured RPE cells demonstrated dose-dependent uptake of exogenous mitochondria. Subretinal delivery localized donors signal to the RPE and adjacent outer retina. Suprachoroidal injections demonstrated procedural feasibility with reliable access to the suprachoroidal space and visible injectate distribution. ConclusionsOcular mitochondrial transplantation is in an early stage of investigation. Our feasibility data indicate that established posterior-segment delivery routes expose distinct retinal compartments and that route selection strongly influences anatomic distribution. Further studies are needed to verify intracellular uptake, define dosing and durability, and evaluate safety in disease-relevant models.

Background: Despite strong evidence from controlled trials, uncertainty remains about the real-world use of 0.05% atropine in patients with lighter irises due to tolerability concerns, and predictors of treatment response are poorly understood. Here, we evaluated the effectiveness, tolerability, and early biometric response to 0.05% atropine in clinical practice among patients with predominantly light irises. Methods: This prospective cohort study included 33 patients treated with 0.05% atropine (82% with light irises). Cycloplegic spherical equivalent refraction (SER) was measured at baseline and 3-month intervals. Axial length (AL), photopic pupil diameter, accommodation amplitude, and subjective side effects were monitored more frequently initially. Results: Median age at treatment initiation was 11.97 years, SER -5.38 D, and AL 25.42 mm. Over 12 months, SER changed by -0.078 {+/-} 0.349 D (mean {+/-} SD), and AL increased by 0.052 {+/-} 0.115 mm. Eighty-eight percent of participants had a SER change of <0.5 D, and 91% had axial elongation of <0.2 mm, indicating clinically limited myopia progression. Photopic pupil diameter was larger, and accommodation amplitude was reduced throughout follow-up. Early in treatment, side effects, including photophobia and near-work difficulties, were common but minimally disruptive. Their incidence decreased rapidly and rarely required treatment modification. In exploratory analyses, early AL changes predicted 12-month AL outcomes, with associations detectable as early as 1 week and strengthening over time. Conclusions: 0.05% atropine was well tolerated and effective in this population with light irises. Early AL changes may predict 12-month treatment response. These findings support the implementation of 0.05% atropine in routine clinical practice in populations with light irises and highlight the potential for early AL monitoring to guide timely treatment adjustments.

Immunohistochemistry (IHC) is widely used to assess protein expression in corneal tissue, yet staining outcomes are strongly influenced by tissue preparation methods and regional differences within the cornea. This study aimed to systematically compare three preparation techniques including paraffin (wax) embedding, wax embedding with antigen retrieval (wax AR), and cryosectioning for IHC analysis in embryonic day 18 chicken corneal tissue. Markers representing key biological functions were evaluated, including progenitor activity (PAX6, P40), tissue architecture (actin), and immune surveillance (TAP1, CD68), across central and limbal regions. Cryosectioning consistently produced the most specific staining for nuclear and antigen-sensitive markers. PAX6 and P40 exhibited strong, nuclear-localized expression in the corneal epithelium only under cryo conditions, whereas wax-based methods resulted in reduced specificity and irregular signal distribution. TAP1-positive immune cells were detectable in the limbal stroma exclusively in cryosections, highlighting improved antigen preservation. In contrast, actin staining, was best preserved with wax AR, and provided superior structural clarity and expected expression patterns across corneal layers. CD68 showed minimal or inconsistent staining in corneal tissue across all methods despite positive control validation. These findings demonstrate that optimal IHC outcomes in corneal tissue are marker-dependent and influenced by preparation methods and regional tissue context. Cryosectioning is recommended for detecting nuclear and immune-related antigens, while wax AR is preferable for preserving tissue architecture. This study provides a practical framework for improving reproducibility and interpretation of corneal immunostaining in avian models.

ObjectiveTo determine whether uveitis shares genetic similarity with extraocular immune-mediated inflammatory diseases (IMIDs), we performed network analysis of putative causal genes associated with ocular inflammatory disease, IMIDs and eye-specific diseases, including age-related macular degeneration and monogenic disorders. MethodsWe identified putative causal genes for genome-wide significance variants from uveitis, IMIDs and ocular diseases using OpenTargets and published studies. To assess the gene-level pleiotropy between disease groups, we quantified the causal gene overlap between groups, and the Jaccard Similarity Indices for individual disease pairs. We then used a network approach to assess the molecular genetic similarity between diseases at a biological pathway level and comparative statistics to identify diseases with greater network similarity to uveitis. ResultsSeventy-five percent of the putative causal genes for uveitis are also causal for IMIDs, while no uveitis genes are shared with primary ocular disorders. Network analysis revealed that 1) uveitis genes are more closely networked with systemic IMIDs disease genes than with ocular-specific disease genes; and 2) significant network similarity links uveitis and specific IMIDs, such as ankylosing spondylitis and sarcoidosis. ConclusionsOverlapping causal genes and network similarity indicate that uveitis is predominantly an inflammatory disease, sharing genetic architecture with other IMIDs. Future studies aimed at dissecting genetic heterogeneity within uveitis may determine whether subgroups share common immune pathways that could nominate endotype-specific therapeutic approaches.

Purpose: To develop and evaluate deep learning models for automated detection of corneal perforation in microbial keratitis using anterior segment optical coherence tomography (ASOCT) images. Methods: We enrolled 150 patients with microbiologically confirmed keratitis. Contralateral healthy eyes served as controls. Four convolutional neural network models using ResNet architecture were trained and evaluated using ASOCT images to classify the presence or absence of corneal perforation at the eye level. Ground truth labels for perforation were established following consensus grading by two masked ophthalmologist graders. Models differed in inclusion of healthy controls and masking of non-corneal anterior segment anatomy. Results: The best-performing model (Model 1), which included healthy controls and randomly applied masking of the inferior image portion during training, achieved an AUC of 0.965 (95% CI, 0.911-0.995), sensitivity of 84.0% (95% CI, 70.0%-97.1%), and specificity of 97.8% (95% CI, 96.1%-99.3%) for detection of corneal perforation. Models including healthy controls outperformed those without, and lens masking improved discrimination. Conclusions: Deep learning models achieved high diagnostic accuracy for detecting corneal perforation on ASOCT imaging in eyes with microbial keratitis. These findings support the potential role of automated ASOCT analysis as a clinical decision support tool for identifying this vision-threatening complication.

PurposeMacrophage migration inhibitory factor (MIF) is a pleiotropic cytokine implicated in many inflammatory and fibrotic diseases; however, its role in primary open-angle glaucoma (POAG) and trabecular meshwork (TM) dysfunction remains unknown. In this study, we investigated whether MIF-CD74 signaling regulates TM pathobiology through modulation of the transcription factor, Blimp-1, and downstream cytoskeletal reorganization and extracellular matrix (ECM) remodeling. MethodPrimary human TM cells (HTMC) were exposed to glaucomatous stressors, including TGF-{beta}2, rMIF, or a pro-inflammatory milieu. Expression of MIF, its receptor CD74, and Blimp-1 was measured by qPCR and immunoblotting. ECM proteins and phosphorylated myosin-light chain (pMLC) were evaluated by immunofluorescence staining. In vivo, MIF-CD74 and Blimp-1 expression were examined in the TM/anterior segment (AS) tissue of Tg.CreMYOCY437H and lentiviral (LV)-TGF-{beta}2-induced ocular hypertension (OHT) mouse models. Functional involvement of MIF signaling in TM pathobiology was examined using the irreversible MIF inhibitor 4-IPP and the immunomodulatory metabolites agmatine and thiamine. ResultsGlaucomatous stressors significantly upregulated MIF and CD74 expression with concomitant suppression of Blimp-1 in HTMC. Similarly, TM/AS tissue from both OHT models (Tg.CreMYOCY437H and LV-TGF-{beta}2) demonstrated increased MIF-CD74 expression accompanied by reduced Blimp-1 levels. Activation of MIF-CD74 signaling triggered pro-inflammatory and cell death pathways and promoted ECM remodeling, characterized by increased fibrotic protein expression and enhanced RhoA/ROCK-mediated MLC phosphorylation, indicating modulation of TM contractility. Pharmacological inhibition of MIF attenuated inflammatory signaling, reduced ECM deposition and cytoskeletal remodeling, and suppressed RhoA/ROCK/MLC activation, restoring a protective TM phenotype. ConclusionOur findings identify MIF-CD74 signaling as a previously unrecognized regulator of TM dysfunction in POAG. MIF-mediated suppression of Blimp-1 mechanistically links inflammatory signaling to cytoskeletal contractility and fibrotic ECM remodeling, key determinants of aqueous humor outflow resistance. Targeting the MIF-CD74/Blimp-1 axis may represent a novel therapeutic strategy to restore TM homeostasis and reduce intraocular pressure in glaucoma.

Pentosan polysulfate (PPS) is a semisynthetic sulfated polysaccharide that was approved by the United States Food and Drug Administration (FDA) for treatment of interstitial cystitis (IC). A 2018 study by our group described a vision-threatening macular toxicity associated with long-term use of PPS. However, given the relatively recent characterization of PPS maculopathy, we have limited knowledge of its pathophysiology. The present study therefore investigated the pathophysiology of PPS maculopathy in a cell culture model, assessing impacts of PPS exposure on morphology and mitochondrial function. We treated ARPE-19 cells with increasing doses of PPS and investigated both mitoprotective and cytoprotective mechanisms, mitochondrial reactive oxygen species production (ROS) and respiration, cellular structure, and retinal pigment epithelium (RPE) dysfunction through phagocytosis assays. We found that PPS increased mitochondrial superoxide accumulation and that increased doses of PPS impaired basal and maximal respiration in a Seahorse assay without the expected response of increases in the cellular energy sensor pAMPK. PPS exposure disrupted mitochondrial and cell protective mechanisms against ROS accumulation as assessed through examination of mitochondrial biogenesis markers PGC-1 and SIRT1 and autophagy markers LC3 and p62. PINK1 expression increased with increasing duration of exposure to PPS. Further, we found that PPS led to functional and structural changes to RPE cells, which exhibited an increase in cell aspect ratio and impaired phagocytosis with higher doses of PPS. Lastly, we found an increase in cell death in response to higher doses of PPS, evident through ethidium homodimer cell viability assays. Taken together, our study shows PPS exposure has profound effects on RPE viability and function through impairment of mitochondrial respiration and mito- and cyto-protective mechanisms and highlights mitochondrial insult as a potential focus of future PPS research.