Investigative Opthalmology & Visual Science

PurposeEndophthalmitis is a serious complication of intraocular surgery due to the risk of irreversible retinal damage. Because retinal cell populations are highly heterogenous, single-cell resolution is required to uncover the detailed mechanisms of infection response. Using a mouse model of bacterial endophthalmitis, we investigated transcriptional changes across resident and infiltrating cell types in the retina. MethodsA methicillin-sensitive strain of Staphylococcus aureus was isolated from a patient with endophthalmitis. Adult C57Bl/6J mice received an intravitreal injection of phosphate-buffer solution (PBS) with or without 5000 CFU S. aureus (n=3 per group). 24 hours later, retinas were isolated and single-cell suspensions were sent to the Penn Genomic Core for sequencing with an Illumina NovaSeq 6000. After standard pre-processing of the data, differential genes and pathways were identified for each cell type (adjusted p < 0.01, log2FC > 1 or < -1). ResultsOur analysis identified all expected retinal cell types, including a population of infiltrating neutrophils in the infected retinas. We surveyed genes known to be upregulated at the bulk-retina level in this model (e.g. Tlr2, Nlrp3, Il1b), and found that infiltrating cells mainly drove this expression. Several genes were altered across nearly all retinal cell types, including upregulation of Hsph1 and Stat3. Muller glia downregulated Gpx4 while upregulating Acsl4 and iron importers Tfrc, Zip14, and Dmt1. Top pathways for macrophages/microglia included chemotaxis, cell-cell adhesion, and wound healing. Vascular cells upregulated angiogenesis-related genes. Cellular respiration was a commonly affected pathway across several neuronal populations, with most genes decreasing. ConclusionsThis study advances our understanding of the pathobiology of bacterial endophthalmitis. Muller glia appear to be undergoing ferroptosis, potentially while activating a program to sequester iron away from bacteria. Decreased cellular respiration may indicate hypoxia among neurons. Our results reveal several trends in the retinal response to infection, including iron dysregulation and hypoxia. Understanding these cell-type-specific responses to endophthalmitis may help design therapies to combine with antibiotics.

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.

Albino individuals are clinically recognized to exhibit heightened susceptibility to light-induced retinal injury, yet the cellular and metabolic mechanisms underlying this vulnerability remain poorly defined. Here, we investigated whether retinal pigment epithelium (RPE) pigmentation governs mitochondrial structure, metabolism, and inflammatory responses that ultimately determine retinal resilience to blue light stress. Using pigmented (C57BL/6J) and albino (Balb/c) mice, we demonstrate that albino animals exhibit markedly increased retinal phototoxicity following blue light exposure, manifested by fundus lesions, outer nuclear layer (ONL) disruption, and structural degeneration evident by OCT. Primary RPE cultures derived from albino mice exhibited profound difference in mitochondrial morphology, characterized by increased mitochondrial number, reduced size, and enhanced fragmentation, accompanied by elevated mitochondrial DNA copy number. These structural changes correlated with transcriptional skewing toward mitochondrial fission (increased Drp1) and suppression of mitochondrial fusion (Mfn1, Mfn2, OPA1). Functionally, albino and depigmented RPE displayed impaired oxidative phosphorylation, reduced ATP production, and diminished reliance on mitochondrial pyruvate carrier (MPC)-dependent metabolism. In parallel, albino RPE demonstrated cell-cycle accumulation at G2/M and heightened basal and blue light-induced secretion of pro-inflammatory cytokines, particularly IFN-{beta}1, IL-6, and TNF-. Importantly, exogenous melanin supplementation partially restored mitochondrial fusion gene expression, pyruvate-dependent respiration, and inflammatory restraint. Together, these findings identify melanin as a critical regulator of RPE mitochondrial architecture, metabolic substrate utilization, and inflammatory signaling, providing a mechanistic framework to explain enhanced photo-vulnerability in the albino retina. These insights establish pigmentation-dependent mitochondrial metabolism as a determinant of retinal resilience and suggest mitochondrial bioenergetics as a therapeutic target.

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.

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.

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.

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.

Objective: To define CSC genetic architecture and identify implicated ocular tissues, cell types, genes, and circulating proteins. Data Sources: Genome-wide data were assembled from FinnGen, All of Us, Mass General Brigham Biobank, Million Veteran Program, and a Dutch chronic CSC cohort. Serum protein quantitative trait loci, human single-cell ocular atlases, and UK Biobank macular optical coherence tomography (OCT) imaging were used for downstream analyses. Study Selection: Five European-ancestry cohorts with genome-wide data and cohort-specific CSC case-control definitions were included, comprising 2,584 cases and 1,044,455 controls. Variants present in at least 2 cohorts were meta-analyzed. Data Extraction and Synthesis: Cohort-level GWASs were adjusted for age, age squared, sex, genotyping array or batch, and 10 genetic principal components, then combined using fixed-effects inverse-variance meta-analysis. Post-GWAS analyses included gene prioritization, colocalization, Mendelian randomization, single-cell disease-relevance scoring, and testing of a CSC genetic risk score in UK Biobank OCT images. Main Outcome(s) and Measure(s): Genome-wide significant CSC loci, effector genes and proteins, tissue and cell-type enrichment, and CSC-relevant OCT abnormalities. Results: Across 11,068,938 variants, 10 loci reached genome-wide significance (P < 5e-8), including 3 novel loci near TGFB1, LINC00551, and LOC105375630 and 7 replicated loci near CFH, CD46, NOTCH4, PREX1, PTPRB, GATA5, and TNFRSF10A. Integrative analyses prioritized 10 candidate effector genes. Colocalization and Mendelian randomization implicated circulating TNFRSF10A, TGFB1, and CASP10 levels. Single-cell analyses localized genetic risk to sclera (P = 2.0e-4) and vascular endothelial cells (P = 4.0e-4), with fibroblast enrichment. In UK Biobank, OCT abnormalities were more frequent in the top vs bottom 1% of CSC genetic risk (18 of 109 [16.5%] vs 8 of 134 [6.0%]; odds ratio, 4.05; 95% CI, 1.65-10.87; P = .002). Conclusions and Relevance: In this GWAS meta-analysis, CSC susceptibility localized predominantly to scleral and vascular biology rather than primary retinal pigment epithelial dysfunction. These findings support CSC as a sclerovascular disorder and nominate complement regulation, endothelial signaling, and extracellular matrix pathways for future study.

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

ObjectiveTo assess how whole genome sequencing and varying phenotype definitions influence genetic discovery for primary open-angle glaucoma (POAG) in a diverse population. DesignAncestry-stratified genome-wide association studies (GWASs) and cross-ancestry meta-analyses of POAG cases and controls using two phenotype definitions. ParticipantsCases (age>40) and controls (age>65) were identified in the National Institutes of Health All of Us Research Program v8 data release and sub-divided into genetically inferred ancestral groups. Using the relaxed phenotype (ICD codes only), case/control counts were: European (1,846/84,654), African (1,042/15,966), and Latino/Admixed American (305/10,167). Using the stringent phenotype (ICD codes and evidence of glaucoma treatment in the electronic health record), case/control counts were: European (1,528/79,276), African (862/14,076), and Latino/Admixed American (250/9,668). Cross-ancestry meta-analyses included 3,193 cases/110,787 controls for the relaxed phenotype and 2,640 cases/103,020 controls for the stringent phenotype. MethodsGWASs were conducted within European, African, and Latino/Admixed American ancestry groups individually using firth logistic regression with age, sex, and the top 10 genotype principal components included as covariates. The ancestry-stratified GWASs were then meta-analyzed using a fixed-effects, inverse variance-weighted approach. Main Outcome MeasuresIdentification of genome-wide significant loci (P < 5x10-8) for POAG using different phenotype definitions and ancestry groups. ResultsKnown POAG risk loci (e.g., TMCO1, CDKN2B-AS1, and GMDS) reached genome-wide significance in both the European GWASs and cross-ancestry meta-analyses (odds ratio (OR) range: 1.19-1.38). A novel risk locus near CYP2A7 (rs76935404[T], OR = 1.35) was identified in the African ancestry GWAS using the stringent phenotype definition. Effect sizes for known POAG risk loci from prior large-scale meta-analyses strongly correlated with effect sizes in this study (Pearson r = 0.75-0.84, P < 1 x 10- for all). The strength and consistency of these correlations support the robustness of the findings. ConclusionsThis study demonstrates the value of whole genome sequencing, diverse ancestry inclusion, and phenotypic refinement in uncovering novel POAG genetic risk loci. The findings underscore the need to prioritize both genetic diversity and refined case/control definitions to advance understanding of this complex ocular disease. PrecisThis study identifies a novel primary open-angle glaucoma risk locus in individuals of African ancestry using whole genome sequencing and varying phenotype definitions in the diverse All of Us Research Program dataset.

Age-related macular degeneration (AMD) shows substantial clinical heterogeneity that remains unexplained despite extensive genetic and clinical characterization. We evaluated whether proteomic stratification could provide insight beyond clinical phenotype and genetic risk. We performed 384-plex plasma proteomics in a cohort of 215 individuals, including patients with early and late neovascular AMD, other complement-associated retinal diseases, and age-matched controls. Proteome-based reclassification identified four disease-overarching clusters. Neovascular AMD cases were partitioned almost exclusively between two clusters (30/36). Early AMD cases were predominantly assigned to one of these clusters (10/18), whereas only two localized to the other (2/18). Both AMD-associated clusters shared elevated levels of a protein module enriched for lipoprotein-related functions compared to the other clusters. However, the cluster containing both early and neovascular AMD cases showed higher levels of additional protein modules enriched for complement pathways and cellular stress-response pathways compared with the other AMD-associated cluster. Importantly, this molecular divergence in neovascular AMD could not be explained by genetic predisposition (i.e., 52-variant AMD genetic risk score), signatures of biological ageing, nor by other clinical features. Together, these findings support two proteomic endotypes of neovascular AMD with distinct involvement of cellular stress pathways.

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.

The Microphthalmia-associated transcription factor (MITF) plays a critical role in retinal pigment epithelium (RPE) development and function. Dysfunctional autophagy and lysosomal degradation in the RPE have been implicated in age-related retinal degeneration, yet the contribution of MITF to these pathways remains incompletely understood. Here, we show that reduced Mitf expression impairs autophagy in mouse and human RPE cells. Primary RPE cells from Mitfmi-vga9/+ heterozygotes mice displayed altered autophagic flux characterized by accumulation of LC3B-II and p62, while MITF knockdown in human ARPE-19 cells promoted autophagosome accumulation. Ultrastructural analysis further revealed age-dependent accumulation of autolysosomes and lipofuscin-like granules in mutant RPE cells. In addition, expression of autophagy-related genes was altered in mutant RPE tissue, supporting disrupted lysosomal-autophagic homeostasis. Together, our findings identify MITF as an important regulator of autophagy in the RPE and suggest that impaired MITF-dependent homeostasis may contribute to retinal degeneration.

Rhodopsin (RHO) P23H is one of the most common mutations causing autosomal dominant retinitis pigmentosa (adRP), yet the relationship between retinal electrophysiology, structure and visually guided behaviour in rodent models remains unclear. We characterised changes in heterozygous P23H (Sakami line) mice and P23H line 3 (P23H-3) rats using full-field electroretinography (ERG), optomotor response (OMR) assays and, in rats, optical coherence tomography (OCT). ERG assessed rod- and cone-mediated responses relative to wild-type controls, whereas OMR under scotopic and photopic conditions quantified contrast sensitivity and visual acuity. In P23H mice, scotopic ERG responses were significantly reduced from postnatal day 16 and declined further from 4 months. Scotopic OMR contrast sensitivity remained largely preserved until 2 months, and photopic acuity was comparable to wild-type up to 6 months. In 13-week-old P23H-3 rats, ERG amplitudes were significantly reduced, and OCT revealed retinal thinning. OMR showed a decline in contrast sensitivity at 7 and 15 weeks, whereas photopic acuity was maintained. Thus, in both models, electrophysiological and structural abnormalities precede detectable OMR deficits, with implications for the selection of outcome measures in preclinical studies. Summary StatementThis study compares electrical and behavioural measures of vision in rodent models of inherited blindness, revealing that retinal dysfunction appears well before measurable vision loss.

The choroid is critical for maintaining vision and implicated in several ocular diseases, being the sole source of nutrients and waste removal for the outer retina. Genetic discovery can help elucidate the pathways through which choroidal features influence disease risk. Our meta-analysis of genome-wide association studies (n= 78,682 participants) identified 30 genomic regions, including 20 novel loci, associated with choroidal thickness. Findings suggest inflammatory and vascular processes drive choroidal thickness, with overlapping mechanisms shared with refractive error. Genome-wide independently significant SNPs accounted for 18.7% of the genetic variance in choroidal thickness. Mendelian randomisation analyses showed a causal effect of age-related macular degeneration on choroidal thickness, and suggest a bidirectional causal effect between choroidal thickness and primary angle-closure glaucoma. These findings provide insight into the shared genetic architecture and biological pathways linking choroidal thickness and related diseases.

PurposeTo characterize corneal surface temperature changes induced by different eye-rubbing techniques in healthy individuals and to investigate the factors influencing temperature change. SettingEskisehir Osmangazi University DesignCross-sectional experimental study MethodsThis study included 93 healthy volunteers aged 19-29 years with no ocular pathology. Participants performed three eye-rubbing techniques-fingertip, knuckle, and fingernail rubbing-while corneal temperatures were recorded with a high-resolution thermal camera (FLIR A8200sc, Teledyne FLIR Systems Inc., Boston MA, USA). Subjects rubbed their eyes for 20 seconds with their dominant hand. Linear mixed-effects models were used to compare corneal temperature before and after eye rubbing and to examine the effect of covariates. ResultsAll eye rubbing techniques significantly increased corneal temperature (fingertip: 1.02 +/-0.58 degrees Celsius, knuckle: 1.03 +/-0.54 degrees Celsius, fingernail: 1.12 +/-0.52 degrees Celsius; all p<0.001), with no significant differences between techniques (p>0.05). Age showed a negative correlation with corneal temperature increase across all rubbing methods (all unadjusted p<0.05), remaining significant only for the fingertip technique after FDR correction (p<0.001). IHA correlated positively with temperature increase for fingertip and knuckle rubbing after FDR adjustment (p= 0.003 and <0.001, respectively). The subgroup analysis indicated that approximately 0.6 degrees Celsius of every 1 degrees Celsius rise in corneal temperature could be attributed to eye closure alone, while the remainder was likely due to mechanical effects of eye rubbing. ConclusionFingertip, knuckle, and fingernail rubbing each produced a transient but significant rise of approximately 1 degree Celsius in corneal temperature. Greater temperature elevation was associated with younger age and higher corneal asymmetry.

PurposeAlanine transaminases (ALT), encoded by the GPT gene, catalyzes the reversible conversion of pyruvate and glutamate to alanine and alpha-ketoglutarate, thereby correlating carbohydrate and amino acid metabolism. However, its role in the human neural retina remains unclear. This study aimed to explore the expression, localization, and metabolic function of ALT in the human neural retina and its potential involvement in retinal diseases. MethodsALT1 and ALT2 expression and localization were examined in the retinas of healthy and diabetic retinopathy (DR) donors via immunoblotting and immunofluorescence. ALT function was assessed in ex vivo human retinal explants using pharmacological inhibition with beta-chloro-L-alanine (BCLA), followed by the analyses of enzyme activity, tissue injury, and transcriptomic responses. Stable-isotope tracing with 13C-and 15N-labelled substrates combined with GC-MS was used to define ALT-dependent carbon and nitrogen fluxes in macular and peripheral retinas. Redox level (NADPH/NADP+) was also evaluated under tert-butyl hydroperoxide-induced oxidative stress. ResultsALT1 and ALT2 were both expressed in the human neural retina, with prominent localization in Muller glia and photoreceptor inner segments. ALT1 displayed a diffuse cytoplasmic distribution, whereas ALT2 demonstrated a punctate pattern consistent with mitochondrial localization. In DR retinas, ALT1 expression was spatially disorganized and heterogeneous, while ALT2 remained comparatively preserved. Inhibition of ALT with BCLA markedly reduced ALT activity without causing overt cytotoxicity or major transcriptional changes. Isotope tracing demonstrated that retinal ALT predominantly channels pyruvate-derived carbon into alanine, whereas alanine was minimally contributed to pyruvate production under basal conditions. ALT inhibition suppressed alanine synthesis and release, redirected nitrogen flux towards glutamate, glutamine, and aspartate, and uncovered distinct metabolic adaptations in macular but not peripheral retinas. Under oxidative stress, ALT inhibition induced the decrease of NADP+/NADPH ratio and LDH release, indicating improved redox balance and reduced tissue injury. ConclusionsALT is previously unrecognized as a regulator of carbon and nitrogen partitioner in the human neural retina, contributing to redox homeostasis under stress. The altered distribution of ALT1 in DR retina and the protective metabolic effects of ALT inhibition suggest ALT as a potential contributor to retinal metabolic vulnerability and a candidate therapeutic target in retinal diseases.

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.

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.