Investigative Ophthalmology & Visual Science

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.

PurposePitx2-associated developmental glaucoma is characterized by anterior segment dysgenesis, ocular hypertension, and optic neuropathy. Its consequences for corneal sensory innervation remain poorly understood. We investigated whether this disease alters corneal nerve structure and sensory function in a sex-dependent manner. MethodsMale and female Pitx2egl1/egl1 and Pitx2+/+ mice were examined at 1 and 3 months. Ocular phenotyping included intraocular pressure, fundus imaging, visual evoked potentials, and optic nerve ultrastructure. RNA sequencing of corneas and trigeminal ganglia was performed at 3 months. Corneal innervation was assessed by {beta}III-tubulin immunofluorescence and volumetric quantification of nerve fibers. Corneal sensitivity was measured using Von Frey filaments. ResultsPitx2egl1/egl1 mice developed progressive ocular hypertension, fundus abnormalities, reduced visual evoked potential amplitudes, and optic nerve degeneration, supporting the model as early-onset glaucoma. Baseline sex-related transcriptional differences were limited in both cornea and trigeminal ganglia. In contrast, Pitx2 mutation induced sex-dependent molecular responses. Female corneas showed broader transcriptional changes enriched in inflammatory, stress-response, and tissue-remodeling pathways, whereas male corneas showed a more restricted response involving metabolic and homeostatic processes. Similar sex divergence was observed in trigeminal ganglia. Corneal nerve fiber volume was reduced in both sexes at 3 months but not at 1 month, whereas reduced sensitivity was detected only in mutant males. ConclusionsThis study identifies sexual dimorphism as a component of Pitx2-associated developmental glaucoma. Furthermore, our findings suggest that glaucoma affects the corneal sensory system beyond optic nerve pathology, highlighting a potentially overlooked dimension of disease relevant to ocular surface monitoring and patient management. HighlightsO_LIDisruption of the cornea-trigeminal ganglion axis with coordinated molecular and functional alterations in Pitx2-associated early-onset glaucoma C_LIO_LISex-dependent modifications in both cornea and trigeminal ganglion responses to early-onset glaucoma C_LIO_LIProgressive corneal neurodegeneration in early-onset glaucoma C_LI

Objective: To investigate the performance of anterior segment (AS) OCT quantitative imaging of anterior chamber inflammation in uveitis patients with diverse demographics. Design: Prospective cross-sectional study. Participants: 144 adult patients managed at a tertiary care service in the UK Methods: Repeated swept-source ASOCT imaging was performed pre- and post-pupil dilation (i.e. 4 scan sets). Inflammation was quantified using a validated human in the loop automated image analysis pipeline, Minuscule Cell Detection (MCD), which identified and counted putative inflammatory cells on AS-OCT. Main Outcome Measures: Test-retest variability of ASOCT and diagnostic accuracy of various ASOCT derived measurands (minimum, maximum, median counts per cross sectional image, and total counts across volume image sets per eye or MINCC, MAXCC, MEDCC and TOTCC) versus Standardization of Uveitis Nomenclature (SUN) grading system as assessed by a uveitis specialist. Results: A total of 281 eyes were included in the analysis. Median age was 48 years (IQR 36 to 64). Strong test-retest measurand reliability was demonstrated, with a 95% tolerance interval ratio 0.3 to 3.0. The best diagnostic performances for SUN activity were observed with the MINCC threshold of 3 particles (negative predictive value for clinical activity of 89.8%, 95% CI 83.0 to 94.1). Associations between ASOCT measurands and patient age (adjusted coefficient 7.5 additional particles, 95% CI 0.5 to 14.6, p<0.04 for age over 60 years versus under 44), and pigment load (52.8, 11.8 to 92.9, p<0.01 in eyes with AC pigment versus without) were noted. Conclusions: ASOCT assessment of anterior chamber inflammation in uveitis meets current recommendations for quantitative imaging biomarkers, demonstrating strong repeatability, linearity with clinical assessment scores and stability with pupil dilation and patient characteristics of ethnicity and lens status. The absence of variability in diagnostic indices across derived measurands suggests similar performance across different acquisition protocols. Further longitudinal cross-platform studies are needed to determine limitations of use.

Abstract Purpose: To expand the clinical and mechanistic understanding of the +57C>T seed-region mutation in miR-184 causing EDICT (endothelial dystrophy, iris hypoplasia, congenital cataract, and stromal thinning) syndrome. Design: Cross-sectional analysis and laboratory confirmation Participants: 18 members of a four-generation family with known +57C>T miR-184 status Methods: We used optical biometry, corneal topography, and medical history to characterize the clinical phenotype. Carrier effects on ocular biometric measurements were estimated using polygenic linear mixed models incorporating a pedigree-derived kinship matrix, adjusted for age and sex. Patient-derived and control induced pluripotent stem cells (iPSCs) were generated and differentiated into corneal endothelial cells (CECs). Main Outcome Measures: Axial length, keratometry (in diopters), white-to-white corneal diameter, topography mapping, central and peripheral corneal thickness, and history of retinal detachment or corneal transplant were compared between mutation carriers and noncarriers, adjusting for age and sex. Cellular analysis was conducted with immunostaining (ZO-1, ATP1A1), morphometric quantification, qRT-PCR of endothelial markers, and transendothelial electrical resistance (TEER). Results: 10 of 18 family members were heterozygous for +57C>T, with retinal detachment occurring in 5/10 affected individuals compared to 0/8 unaffected individuals (p=0.04). Affected eyes had 2.2 mm shorter axial length (p=0.02), 9.3 D steeper mean keratometry (p=0.004), 1.6 mm smaller horizontal corneal diameter (p=0.0001), and 139-micrometer thinner central corneas (p=0.003). Mutant iPSC-derived CECs were associated with irregular borders, increased cell and nucleus area, widened intercellular gaps, disrupted ATP1A1 membrane localization, and reduced barrier function on TEER (all p<0.05). Gene expression analysis showed downregulation of COL4A1, COL4A3, and AQP1 with upregulation of COL8A1. Conclusions: The miR-184 +57C>T mutation produces a broad ocular phenotype that includes smaller, thinner corneas and microphthalmia. Mechanistically, it disrupts CEC junctional integrity, extracellular matrix and pump-related genes, supporting a role for miR-184 in coordinated anterior-posterior eye morphogenesis.

Purpose: Photoreceptor Kv2.1/Kv8.2 voltage-gated potassium channels carry an outward potassium current, helping to set the resting membrane potential and to shape dim light responses. Because potassium flux in the outer retina influences extracellular osmolarity and fluid distribution, we hypothesized that Kv2.1/Kv8.2 channels also contribute to fluid homeostasis in this region of the retina. Methods: OCT imaging was performed in Kv8.2 heterozygous (Het) and knockout (KO) mice aged 4-7 weeks under dark- and light-adapted conditions. Light-dark differences in the distance between the external limiting membrane (ELM) and retinal pigment epithelium (RPE) ({Delta}ELM-RPE) were calculated to quantify light-evoked expansion of the subretinal space (SRS). As a secondary outcome, outer nuclear layer (ONL) thickness was also measured under both lighting conditions. Retinal gene expression was assessed by RNA-seq and droplet digital RT-PCR. Retinal protein expression was determined by western blotting and immunolabeling. Results: {Delta}ELM-RPE was significantly reduced in Kv8.2 KO mice compared with Het controls, indicating reduced SRS hydration. ONL thickness exhibited a small but significant light-dark change that was different between genotypes. Transcriptomic analyses revealed upregulation of osmosensitive genes, including osmolyte transporters and aquaporins. AQP1 protein expression in photoreceptors increased. Conclusions: These findings reveal a previously unrecognized role for Kv2.1/Kv8.2 channels in outer retinal fluid homeostasis and support a model in which photoreceptor potassium efflux contributes to osmotic water movement into the subretinal space.

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.

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.

New approach methodologies (NAMs), including induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs) and retinal pigment epithelium (iRPE), are increasingly applied to study retinal disease mechanisms and therapeutic strategies. However, these models often remain relatively immature. Given the high lipid content and complex metabolism of the retina, it is unclear to what extent iPSC-derived systems recapitulate the human retinal lipidome. Here, we compared the lipidomic profiles of ROs and iRPE, collected at several differentiation stages, with those of post-mortem adult human macular, non-macular and RPE plus choroid (pmRPE). The lipidome of iRPE differed markedly from pmRPE, whereas prolonged differentiation of ROs resulted in a lipidomic profile increasingly resembling that of the post-mortem retina. Moreover, ROs showed similarities to both macular and non-macular lipidome. These findings show that iPSC-derived models can become valuable NAMs to study lipid-related retinal disorders and provide a framework to optimize differentiation protocols.

PurposeOculocutaneous albinism type 1 (OCA1) is an inherited disorder caused by tyrosinase (TYR) gene mutations. Affected individuals experience visual impairment and severe photosensitivity from ocular hypomelanosis, with no current treatments. We evaluated the safety and efficacy of a TYR-encoding adeno-associated virus (AAV) vector in healthy rhesus macaques as a potential OCA1 treatment. MethodsA novel AAV2-based capsid (ATX002) was packaged with the human VMD2 promoter and TYR (hTYR) fused with mGreenLantern (mGL). Two adult rhesus macaques were injected with ATX002-hVMD2-hTYR-mGL subretinally (OD) and intravitreally (OS). Safety and efficacy were assessed via comprehensive ophthalmic examination, fundus photography, spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinography at baseline and defined timepoints up to 12 weeks post-injection, followed by post-mortem immunohistochemistry (IHC). ResultsBoth subretinal doses induced localized hypermelanosis by 3 weeks post-injection, which persisted through the study endpoint and was accompanied by measurable thickening of the retinal pigment epithelium (RPE) on SD-OCT. Histological IHC confirmed successful RPE transduction via robust mGL fluorescence, corroborating in vivo findings by revealing localized RPE hyperplasia and transgene-expressing cells adjacent to regions of de novo hypermelanosis. Intravitreal delivery did not induce any changes to the RPE. Transient uveitis was observed but successfully managed with anti-inflammatory treatment. ConclusionsSubretinal AAV-TYR delivery is a safe and effective approach with the potential to induce RPE pigmentation. These findings support the use of AAV-TYR gene therapy for OCA1, demonstrating efficacy and a manageable safety profile in a large-animal model, and provide a critical bridge toward human clinical translation.

Purpose: Lymphatic system plays a critical role in fluid regulation and immune response. The conjunctiva of the ocular surface is endowed with a rich lymphatic network, and it provides an ideal site to study lymphatic structure and function. The purpose of this study is to investigate potential morphological and functional changes of conjunctival lymphatics with aging, a time-dependent biological deterioration process. Methods: Young and aged fluorescently labeled transgenic mice of Prox-1, the master control gene for lymphatic determination, were used in the study. For morphological assessment, conjunctival lymphatic vessels were examined in vivo by our advanced live imaging system. For functional analysis, lymphatic drainage efficiencies were measured by fluorescently labeled tracer injection. Results: Compared to young mice, both vascular branching points and intraluminal valves were significantly reduced in conjunctival lymphatic vessels of aged mice. Moreover, lymphatic functional deterioration and drainage deficiencies, such as fluid leakage and reflux, were also detected in the aged condition. Conclusions: Conjunctival lymphatic system undergoes morphological as well as functional changes with aging. Further investigation into this phenomenon may provide novel insights into lymphatic and age-related diseases inside and outside the eye.

Col8a2Q455K/Q455K (Q455K) mice exhibit features of early-onset Fuchs endothelial corneal dystrophy (FECD), including decreased endothelial cell density (ECD) and guttae formation. Within the context of these clinical features, this study longitudinally evaluates ferroptosis in Q455K and wild-type (WT) mice using in vivo imaging, PCR and immunohistochemistry. Fifty-six Q455K and 56 WT mice were evaluated from 3 to 24 months of age with in vivo confocal microscopy; ECD and guttae were measured. Ferroptosis marker expression was determined with PCR and immunohistochemistry (IHC). Data were analyzed using two-way ANOVA with Tukeys post hoc test, Chi-square test and a paired t-test. The ECD significantly decreased in both groups from 3 to 24 months of age, but more markedly in Q455K (2285-/+317 to 1012-/+58 cells/mmSquare) versus WT mice (2714-/+139 to 2057-/+149 cells/mmSquare, P<0.0001). Guttae were observed exclusively in Q455K mice beginning at 3 months of age and increased over time (P=0.0003). The Q455K mice demonstrate guttae at the vertices of corneal endothelial cells rather than their centers (74.3% vs. 25.7%P<0.001). Expression of ferroptosis-related genes (Tfrc, Slc40a1, Ftl1, Gpx4) were significantly increased in the Q455K versus WT mice (P<0.05). Furthermore, corresponding protein expression (transferrin receptor 1, ferroportin, ferritin and glutathione peroxidase 4) was significantly elevated adjacent to guttae in Q455K versus WT mice (P<0.05). These findings implicate guttae in the initiation of ferroptosis as it relates to the pathophysiology of FECD and provide an optimal window for testing novel FECD therapies using this murine model, particularly those that target ferroptosis.

PurposeAssessing visual function in patients with ultralow vision (ULV), particularly those with retinitis pigmentosa (RP), remains a significant challenge in therapeutic development. Full-field stimulus test (FST) provides a quantitative measure of retinal light sensitivity and may serve as a valuable clinical endpoint. We investigated FST in ULV RP by examining its associations with functional measures and daily activity-based tasks. DesignObservational, cross-sectional study. ParticipantsPatients with RP and visual acuity in the worse-seeing eye below counting fingers (CF) were enrolled. MethodsAfter dilation and 45-minute dark adaptation, FST was performed monocularly with brief full-field white-light flashes across three visits. Visual acuity was classified into four groups: no light perception (NLP), light perception (LP), hand motion (HM), and CF or better. We assessed functional vision using two tabletop object-recognition and exploration tasks, two mobility tasks, and three vision-related questionnaires. FST thresholds were compared across visual acuity groups, and associations with functional outcomes were analyzed. Main Outcome MeasuresFST thresholds and their associations with functional vision outcomes. ResultsThirty-five patients (70 eyes; median age, 62 years, range 39-84) were included. Median FST thresholds (log cd*s/m{superscript 2}) by visual acuity group were as follows: NLP, 1.13 (-0.63-2.54); LP, -0.27 (-2.70-2.91); HM, -1.13 (-6.24-0.51); CF or better, -2.82 (-5.67- -1.73) (p < 0.001). Measurable FST thresholds were obtained in 9 of 14 NLP eyes (64.3%). FST thresholds showed significant correlations with tabletop performance (r = -0.70 to - 0.46) and mobility performance (r = -0.65), whereas no significant association was observed with questionnaire scores. Test-retest variability across three visits showed no systematic bias, with a coefficient of repeatability of {+/-}0.66 to {+/-}0.82 log cd{middle dot}s/m{superscript 2}. ROC analyses identified FST cutoffs of -1.75 to -0.87 log cd{middle dot}s/m{superscript 2} at which patients first achieved nonzero functional task performance. ConclusionsFST quantifies residual visual function in ULV RP and correlates strongly with performance-based measures of functional vision in daily life. These findings support FST as a clinically meaningful endpoint for therapeutic trials in advanced RP and other severe visual impairments and highlight the value of anchoring FST thresholds to functional task performance.

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.

Thyroid eye disease (TED) is a debilitating condition characterized by orbital fibroblast (OF) activation and excessive hyaluronic acid (HA) accumulation within the retro-ocular space. While IGF-1R blockade with teprotumumab has significantly advanced TED management, incomplete clinical responses and disease relapse underscore the need to identify alternative targets. In this study, we used high-throughput RNA sequencing to map the transcriptomic landscape in TED OFs compared with non-TED OF controls. Our analysis identified robust enrichment of pathways critical to the TED phenotype, including PI3K-AKT signaling, the platelet-derived growth factor (PDGF) pathway, and extracellular matrix remodeling. We validated several key upregulated mediators that may contribute to orbital remodeling, including FOXC2, HGF, MET, and HMGA2, alongside the downregulation of the Wnt antagonist SFRP2. By employing a computational drug-repositioning approach, we identified the multi-kinase inhibitor sorafenib, which targets VEGFR, PDGFR, and RAF, as a potent candidate to neutralize the TED-specific gene signature. Functional assays demonstrated that sorafenib dose-dependently inhibited PDGF-induced AKT phosphorylation and significantly attenuated HA synthesis in primary TED OFs. These results define a persistent, receptor tyrosine kinase-driven program in the TED orbit and suggest that multi-kinase inhibition represents a viable therapeutic strategy for refractory TED. HighlightsO_LIThyroid eye disease (TED) orbital fibroblasts exhibit a transcriptomic signature characterized by elevated PI3K/AKT, angiogenic, and growth factor signaling. C_LIO_LIComputational drug prediction identifies sorafenib as a candidate to reverse the TED gene signature. C_LIO_LISorafenib dose-dependently inhibits AKT activation and hyaluronic acid production in TED orbital fibroblasts. C_LI

The retinal nerve fiber layer, composed of axon bundles converging toward the optic nerve, is a key biomarker for diagnosing and monitoring glaucoma and other neurodegenerative diseases. High-resolution en face imaging of individual nerve fiber bundles offers morphological information beyond what conventional optical coherence tomography provides, yet clinical integration remains limited by the lack of automated analysis tools and normative data. Here, we imaged 14 healthy volunteers using time-domain full-field optical coherence tomography and adaptive optics scanning laser ophthalmoscopy, and developed automated pipelines to quantify bundle width, trajectory, tortuosity, and orientation. Bundles were on average 25% wider at shallower retinal depths, width measurements were consistent across imaging modalities, and estimated axon count per bundle decreased significantly with age. Global trajectory analysis revealed systematic deviations of high resolution data from existing mathematical models, particularly in the temporal sector, leading us to propose two refined trajectory models. These normative results provide a foundation for high resolution biomarkers for use in investigations of retinal neurodegeneration.

Pathogenic variants in IFT140 are associated with a spectrum of syndromic and non-syndromic ciliopathies, with retinal degeneration as a common feature. Despite advances in understanding IFT140 function across various tissues, human retina-specific models are lacking. Here, we show that knock-in mice homozygous for the IFT140 patient variant c.932A>G (p.Y311C) did not develop retinal degeneration, while mice with the homozygous variant c.1451C>T (p.T484M), associated with non-syndromic retinal dystrophy, were embryonic lethal. Therefore, to understand the effect of these variants on retinal homeostasis, we generated novel human in vitro models of IFT140-associated retinal dystrophy, including CRISPR/Cas9 IFT140 knock-out (IFT140KO) induced pluripotent stem cells (iPSC) and patient-derived iPSC retinal pigment epithelium (iPSC-RPE) and retinal organoids (iPSC-ROs). IFT140KO iPSC-RPE cells display stubby cilia compared to isogenic controls, while IFT140T484M/T484Mpatient-derived iPSC-RPE cells exhibit slightly shorter cilia and cilia tip protein accumulation. Both IFT140KO and IFT140T484M/T484M iPSC-ROs show accumulation of cilia proteins at the connecting cilium and outer segment of photoreceptors, and mislocalization of rhodopsin to the inner segments and outer nuclear layer. Pharmacological screening of compounds previously reported to improve cilia structure identified the flavonoid eupatilin as the most effective molecule. Treatment with eupatilin improved cilium length and IFT traffic in iPSC-RPE, and IFT traffic and rhodopsin localization in iPSC-ROs. These findings emphasize the importance of human stem cell derived models to investigate tissue specific disease mechanisms and highlight the therapeutic potential of eupatilin to ameliorate cilia defects in retinal tissue.

The mammalian retina lacks meaningful regenerative capacity, and degeneration usually leads to irreversible vision loss. Although lower vertebrates regenerate retinal neurons through Muller glia, this capacity has generally been considered absent in humans. Using long-term organotypic retinal cultures from 39 adult donors, we show that defined humoral cues alone are sufficient to unlock a latent neurogenic program in human Muller cells. FGF-2 treatment and GSK-3 inhibition induced robust proliferation across both peripheral and central retina, with 79.09 {+/-} 6.32% of dividing cells identified as Muller glia, some completing multiple cell cycles. Single-cell transcriptomics revealed activation of progenitor-like and neuronal differentiation pathways, whereas immunohistochemistry demonstrated expression of early and late neuronal markers spanning all major retinal lineages. Newly generated cells expressed markers of cone, rod, bipolar, horizontal, amacrine, and ganglion cell identities, together with evidence of early synaptogenesis. These findings reveal an intrinsic regenerative potential in adult human Muller glia, with implications for future vision-restoration strategies in degenerative retinal disease. SummaryAdult human Muller glia retain an intrinsic capacity for proliferation and neural lineage commitment independent of donor age or gender. In long-term organotypic cultures of human donor retina, defined humoral cues, without genetic manipulation, induce Muller glia proliferation and the onset of neuronal differentiation. These findings reveal intrinsic neurogenic potential in human Muller glia and provide a human-relevant platform for retinal regeneration studies.

PurposeTo investigate the correlation of retinoblastoma (RB) aqueous humor (AH)-derived extracellular vesicle (EV) surface markers, particularly CD133, with clinical features of RB. MethodsAH samples (n=22) collected at diagnosis (n=17) or primary enucleation (n=5) from RB eyes were profiled using magnetic bead-based flow cytometry. Median fluorescence intensities (MFI) of tetraspanins (CD9, CD63, CD81) and CD133 were quantified and correlated with International Intraocular RB Classification (IIRC) stage, seeding location, seeding morphology, and enucleation. ResultsCD133+ EVs were detected in 16 of 22 (73%) samples. While CD133 levels showed a non-significant increasing trend with advanced IIRC stages, CD133 expression was significantly elevated in eyes with vitreous seeding vs. subretinal only (P = 0.010). Cuzicks trend analysis revealed a significant association between rising CD133 % MFI and complex seeding (P < 0.001). Conversely, CD63 displayed a significant inverse trend, with decreasing % MFI associated with complex seeding (P < 0.001). CD133high eyes (MFI>10,000, n=9) exhibited increased enucleation (P = 0.007) and predominantly sphere/cloud seeding patterns (P = 0.001). CD133high eyes demonstrated a coordinated CD63/CD81 co-expression pattern (r = 0.80), absent in the CD133low group. ConclusionsElevated CD133 level and percentage were associated with vitreous involvement and complex seeding morphology, suggesting potential as a minimally invasive indicator of disease burden. The inverse relationship between CD63 and seeding severity, combined with CD63/CD81 remodeling in CD133high eyes, suggests an EV profile indicative of aggressive disease. These findings support the association of CD133+ EVs with advanced RB seeding and warrant further prospective validation.

The multiligand endocytic receptor, megalin (LRP2), is expressed in the retinal pigment epithelium (RPE) and patients lacking the receptor develop high myopia. Despite its established role in retinal development, the contribution of megalin to retinal homeostasis in the normally developed/mature eye remains poorly understood. Here, we investigated megalin function using an inducible knockout mouse (KO) model and human iPSC-derived RPE with megalin knockdown (KD) to distinguish post-developmental homeostatic functions from developmental effects. In vivo, megalin ablation caused progressive retinal degeneration and visual impairment, with morphological abnormalities in the RPE but no changes in myopia-associated ocular phenotypes including axial length and intraocular pressure. Proteomic profiling of megalin-KO RPE revealed reduction of autophagy-related proteins. In line with this, megalin deficiency was associated with accumulation of pro-cathepsin D, and perturbed rhodopsin turnover. This was supported in vitro, where trafficking of photoreceptor outer segment (POS) containing phagosomes to lysosomes was reduced, suggesting disturbed phagosome maturation. Megalin KD did not measurably impair initial uptake of POS discs, but delayed rhodopsin degradation, indicating defective post-ingestion processing. Together, these findings establish megalin as a key regulator of retinal homeostasis in the mature eye by controlling phagosome-lysosome fusion in the RPE and suggest that megalin dysfunction contributes to slowly progressive retinal degeneration. This positions megalin as a potential therapeutic target in lysosomal degenerative diseases in the retina.

BackgroundDural defects, either from trauma, tumor resection, surgical approaches, genetics, or spontaneously represent a significant clinical challenge in neurosurgery. Given the established efficacy of riboflavin-sensitized ultraviolet-A (UVA) photo-crosslinking in ophthalmology, this study investigated its feasibility and dose-response characteristics as a novel strategy to biochemically augment the mechanical integrity and strength of ex-vivo bovine dura mater. MethodsForty bovine dura mater specimens were treated ex vivo using riboflavin concentrations of 2, 4, or 8 mM combined with UVA irradiation at 0.3 or 3 mW/cm{superscript 2}. PBS-treated specimens exposed to UVA served as controls. Atomic force microscopy nanoindentation was used to measure the local elastic modulus in matched regions before and after treatment, enabling paired assessment of treatment-induced mechanical changes while minimizing inter-sample variability. Post-treatment stiffness, fold-change from baseline, and riboflavin dose-response relationships were analyzed statistically. ResultsBaseline elastic moduli were equivalent across all groups (mean approximately 52 kPa, p=0.92). While UVA alone caused a modest approximately 2- to 3-fold stiffness increase, riboflavin-UVA treatment produced a dramatic, concentration-dependent effect. The highest treatment (8 mM RF, 3 mW/cm{superscript 2} UVA) increased the elastic modulus 150-fold, from approximately 53 kPa to approximately 8,000 kPa. Post-UV stiffness exhibited a strong linear relationship with riboflavin concentration (R{superscript 2} = 0.994), indicating a precisely titratable crosslinking effect. All treatment conditions were statistically distinguishable (p < 0.001). ConclusionRiboflavin-sensitized UVA crosslinking substantially increases the nanomechanical strength of ex vivo bovine dura mater in a controllable, dose-dependent manner. These findings establish a proof of concept for biochemical reinforcement of dural tissue that might be used clinically. As a next step evaluation using human dura, macroscopic biomechanical testing, penetration-depth analysis, and safety assessment is warranted.