Investigative Ophthalmology & Visual Science

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.

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.

PurposeTo uncover the molecular mechanisms of corneal sensory nerves (CSN)s involvement in the initiation of Pseudomonas aeruginosa (PA) keratitis and the roles of the miR-183/96/182 cluster (miR-183C) in this process. MethodsmiR-183C conventional knockout (KO) or sensory neuron-specific (SNS) conditional (C)KO mice and their age- and sex-matched wild type (WT) controls were used. TG SN were isolated. Neurite growth and branching were analyzed by neurite tracing. Custom-made microfluidic chambers (MFC) were used to separate the neuronal cell bodies in the soma chamber and their neurites/nerve endings in the axon chamber. TG SNs response to lipopolysaccharide (LPS) or PA infection of the neurites/nerve endings was studied by ELISA assays of CX3CL1 and substance P (sP) in the axon chamber. Target luciferase reporter assays were performed to validate key downstream target genes of miR-183C. ResultsThe total neurite length and number of branches per TG SN were decreased in the CKO vs WT mice, and in the male vs female WT mice. PA infection, but not LPS alone, induced the production and secretion of CX3CL1 and sP in WT mice; while TG SN of miR-183C KO mice responded to both LPS and PA and were significantly enhanced when compared to WT mice. Antagonists to TLR4 and/or FPR1 inhibited PA-induced responses. Target luciferase reporter assays confirmed that genes encoding NRP1, TAC1-the precursor gene of sP, CX3CL1 and ADAM10, a metalloproteinase involved in the production of soluble CX3CL1, were direct targets of miR-183C. ConclusionsPA directly activates TG SN and induces chemokine and neuropeptide production/secretion through TLR4 and FPR1 receptors, which may contribute to the initiation of PA keratitis. miR-183C regulates TG SN neurite growth, chemokine and neuropeptide production/secretion and the response to PA infection by targeting a collection of key genes involved in axon guidance/projection-, chemokine and neuropeptide biogenesis- and receptors mediating PA-induced activation.

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.

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.

ObjectiveTo determine whether non-axon optic nerve morphometric features correlate with clinical visual function as strongly as the traditional axon count gold standard. DesignCross-sectional histological analysis with longitudinal clinical correlation. SubjectsEighteen mice from three strains: C57BL/6J (n=6), BXD51 (n=6), and DBA/2J (n=6). MethodsLeft eye (OS) optic nerves from mice euthanized at 12 months of age were resin-embedded and stained with p-phenylenediamine. Bright-field cross-sectional images were segmented using an AxonDeepSeg-based workflow to generate axon, myelin, whole nerve, and glial coverage masks for morphometric quantification. Seven morphometrics were extracted: axon count (nAx), axon density (AxDen), glial coverage area ratio (GliaR), mean solidity (Sol), mean axon diameter (AxDiam), mean myelin area (MyArea), and mean axon-myelin area (AxMyArea). Morphometrics were correlated with longitudinal clinical data collected at 1, 3, 6, 9, and 12 months, including visual acuity (VA), contrast threshold, intraocular pressure (IOP), and pattern electroretinography P50 and N95 amplitudes (PERG P50 and N95). Main Outcome MeasuresPearson correlation coefficients were used to assess associations between morphometric features and clinical measures, and Fisher z-transformed meta-analytic correlations were used to aggregate these associations across ages. ResultsVA and contrast threshold demonstrated strong correlations with GliaR that matched or exceeded nAx. Meta-analysis across ages revealed GliaR correlated with VA (r = -0.84, p = 4.49 x 10-21) and contrast threshold (r = 0.86, p=7.55 x 10-23), comparable to nAx correlations with VA (r = 0.80, p=8.13x10-17) and contrast threshold (r = -0.80, p= 1.74x10-16). Structure-function relationships shifted with age: at 6 months, GliaR had the strongest correlation with contrast threshold (r = 0.96), while at 12 months, AxDiam became the dominant correlate of both VA (r = 0.77) and contrast threshhold (r = -0.74). IOP, PERG P50, and PERG N95 exhibited weak correlations with all morphometrics (|r| < 0.27). ConclusionsNon-axon morphometrics, particularly glial coverage area ratio, correlate with visual function as strongly as traditional axon count. Automated optic nerve assessment should incorporate glial and other non-axon features. Further, stage-aware biomarker selection may better capture structure-function relationships in glaucoma.

PurposeRetinal ganglion cells (RGCs) are essential for visual signal transmission, yet they are vulnerable to injury and degeneration. Gene modulation in RGCs using adeno-associated virus (AAV) offers a promising avenue for neuroprotection and regeneration, but promoters lack sufficient RGC specificity, limiting precision needed for preclinical studies. This study aims to identify novel promoter-enhancer combinations (PECs) to achieve gene expression preferentially in RGCs. MethodsWe evaluated existing transcriptomic data to identify Neuritin 1(Nrn1) as a gene with highly restricted RGC expression in the retina. Synthetic PECs derived from human and mouse Nrn1 loci were incorporated into AAV2 vectors driving expression of a nuclear-targeted reporter GreenLantern. AAVs were delivered via intravitreal injection into C57BL6/J mice, and transduction efficiency and RGC specificity were evaluated in both young and aged retinas and those subjected to intraorbital optic nerve crush (ONC), using immunohistochemistry and quantitative analysis of RBPMS+ cells. ResultsWe found that AAV2 with a human Nrn1 PEC drives gene expression in RGCs. Quantitative analysis revealed that over 83% of transduced cells were RBPMS-positive, indicating robust RGC selectivity and significantly outperforming ubiquitous promoters. Notably, the Nrn1 PEC retained strong and selective transgene expression in RGCs in aged mice and following ONC, demonstrating its resilience under aged and injury conditions. ConclusionThe Nrn1 PEC enables efficient and injury-resilient gene expression in RGCs, addressing a key limitation in cell-specific targeting. This AAV-incorporated PEC offers a robust platform for evaluating neuroprotective interventions and accelerates translational development of gene therapies for glaucoma and other optic neuropathies.

Open-angle glaucoma (OAG) affects approximately 57.5 million individuals worldwide and is characterized by the progressive loss of retinal ganglion cells (RGC) and irreversible optic nerve damage resulting from chronic ocular hypertension. Intraocular pressure (IOP) is the only major modifiable risk factor in OAG and clinical treatments necessarily aim to lower IOP in order to preserve RGCs and prevent vison loss. Pharmacological therapies, such as prostaglandin analog containing eye drops, are known to be effective at reducing IOP, but are critically undermined by poor patient compliance and are unable to control for potentially damaging diurnal fluctuations in IOP, leading to vision loss even in patients diagnosed early. Herein we evaluate the effectiveness of a long-acting, single use, prostaglandin-based recombinant adeno-associated virus (rAAV)-mediated IOP-lowering gene therapy treatment in glaucomatous DBA/2J mice and demonstrate that sustained IOP reduction leads to preservation of both optic nerve anatomy and function in end-stage glaucomatous disease. One Sentence SummaryIOP-lowering gene therapy provides partial anatomical and functional rescue in glaucomatous mouse model following single dose treatment

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.

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.

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.

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.

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.

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.

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.

Purpose: To determine whether spatial decomposition of longitudinal retinal nerve fiber layer (RNFL) change maps reveals distinct modes of glaucomatous progression masked by conventional averaging, and to validate these modes through structure function mapping and genetic association analysis. Methods: Pixel wise RNFL rates of change were computed from longitudinal optic disc OCT scans of 15,242 eyes (8,419 adults with primary open angle glaucoma [POAG]; Massachusetts Eye and Ear, 1998 to 2023). A loss only constraint zeroed all thickening values, reflecting the biological prior that adult RNFL does not regenerate. Nonnegative matrix factorization decomposed these maps into spatial progression components (80% training set). Components were evaluated in a heldout set (20%) for retinotopic structure function concordance, visual field (VF) progressor classification against global and quadrant RNFL rates, and enrichment of genetic association signals at established POAG loci. Results: Six anatomically distinct progression patterns emerged, including diffuse circumferential loss, focal peripapillary defects, and arcuate bundle degeneration. Pattern based models significantly outperformed global RNFL rate for classifying VF progressors (area under the curve, 0.750 [95% CI, 0.709 to 0.790] vs. 0.702; P = .0096) and explained additional variance in functional decline (Nagelkerke pseudoR2, 0.301 vs. 0.198; P = .0011). Structure function mapping confirmed retinotopic coherence. Spatial phenotypes recovered stronger genetic signals than global rates at 85.3% of established POAG loci, suggesting they capture more biologically homogeneous endophenotypes of progression. Conclusions: Glaucomatous structural progression occurs through spatially distinct modes with independent structure function and genetic signatures that conventional RNFL averaging obscures.

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.

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 determine whether circadian timing defines critical molecular windows in myopia development and to assess the transferability of circadian gene programs across ocular tissues, disease stages, and species. MethodsPublicly available retinal and choroidal RNA-seq datasets from chick models of form-deprivation myopia were analyzed using unsupervised transcriptomic profiling and multistage machine-learning classification. Circadian windows were defined based on Zeitgeber time, and samples were grouped accordingly for downstream analyses. Classification model robustness was evaluated through cross-tissue and cross-stage validation and further assessed using external validation in an independent dataset. Functional translation to humans was examined using ortholog-based Gene Ontology enrichment analysis to identify conserved biological processes and higher-order regulatory pathways. ResultsA circadian critical window at ZT8-ZT12 exhibited the strongest transcriptional divergence during both myopia onset and progression. Gene signatures derived from this window generalized across retina and choroid and remained predictive across disease stages, supporting coordinated molecular regulation between ocular tissues. External validation confirmed the reproducibility of these signatures despite differences in experimental design and gene coverage. Functional mapping revealed that conserved molecular components in chicks are reorganized into more complex neuroendocrine and regulatory networks in humans, indicating cross-species conservation with increased functional complexity. ConclusionsCircadian timing strongly shapes myopia-related gene expression and underlies coordinated retina-choroid signaling. These findings highlight circadian biology as a key factor of refractive development and suggest that time-dependent mechanisms may influence myopia susceptibility, progression, and response to treatment.

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.