Limbal stromal cells constitute a heterogeneous cellular compartment that supports tissue homeostasis and may contribute to regional progenitor niches within the corneoscleral limbus. Although stromal cells have been described in both anterior and posterior limbal regions, their in vivo distribution and relationship to α-smooth muscle actin (αSMA) lineage activity remain poorly understood. This study investigated the distribution of αSMA-lineage (αSMA-Lin) cells in the cornea and limbus of αSMA-CreER[T2]/Ai9 mice. Intraperitoneal administration of tamoxifen was used to induce tdTomato fluorescence in cells with an active αSMA promoter at the time. Tissues were analysed at 2, 14, and 28 days after induction using wholemount imaging, three-dimensional reconstruction, and immunofluorescence staining. αSMA-Lin (+) cells were expanded by day 28 compared to earlier time points. They were more abundant in the limbus compared to the cornea and sclera, and in the posterior limbus (positive in 69.9 ± 3.5% of cells) compared to the anterior limbus (7.6 ± 2.2%, n = 3, P < 0.01). The superior (435 ± 20 μm) and temporal (422 ± 23 μm) limbal quadrants exhibited wider αSMA-Lin (+) areas compared to the nasal quadrant (345 ± 15 μm) (n = 6, P < 0.05). There was some colocalisation with αSMA antibody labelling on day 2. The αSMA-Lin (+) cells co-expressed vimentin and TRA-1-60. Some αSMA-Lin (+) cells colocalised with pericyte marker NG2. There was no colocalisation with the fibroblast marker FSP1. These findings provide an anatomical map of an αSMA-lineage stromal cell compartment that is preferentially enriched in the posterior limbus. Our findings provide new insights into the structure of the corneoscleral limbus.

Body weight is a potential modulator of vascular homeostasis. However, its effects on the development and pathological progression of fundus blood vessels in C57 mice remain unclear. This study aimed to investigate the impact of body weight on fundus vascular development and pathological processes. Wild-type C57 mice were selected, with the three lightest and three heaviest mice from each litter chosen to evaluate retinal vascular development. Choroidal tissues were isolated for culture, and three pathological models (oxygen-induced retinopathy, choroidal neovascularization, and STZ-induced diabetic retinopathy) were established. Body weight significantly affected retinal vascular development and oxygen-induced pathological indices, but had no obvious effect on choroidal neovascularization and diabetic retinopathy. Disease course was the key factor determining the severity of adult fundus lesions. These findings indicate that body weight is crucial for juvenile retinal vascular development, while disease course dominates adult fundus pathological changes, laying a foundation for standardized research on fundus blood vessels.

This study investigated the role of Integrin β1 subunit in conjunctivochalasis (CCH) using clinical specimens, shRNA-mediated in vitro models, and an MMP-3-induced in vivo rabbit model. Clinical and rabbit CCH tissues exhibited severe stromal edema along with collagen and elastic fiber degradation. In CCH-derived human conjunctival fibroblasts, Integrin β1 subunit and Collagen I were significantly downregulated, while MMP-2 and MMP-9 were upregulated. Mechanistically, shRNA-mediated knockdown of Integrin β1 subunit directly triggered a sharp increase in MMP-2 and MMP-9 expression, suppressed Collagen I synthesis, and severely impaired fibroblast proliferation and migration. Furthermore, the MMP-3-induced rabbit model successfully recapitulated hallmark clinical features of CCH, including loose conjunctival folds, tissue thinning on anterior segment OCT, all of which strongly correlated with the localized downregulation of Integrin β1 subunit in rabbit conjunctival stroma. In conclusion, Integrin β1 subunit deficiency is a pivotal driver of CCH pathogenesis. Its loss accelerates extracellular matrix degradation via aberrant MMP-2/9 activation and induces fibroblast dysfunction, leading to the tissue thinning and laxity characteristic of CCH.

PAX6 is a key regulator of corneal epithelial differentiation, expressed as multiple variants that result either from alternative splicing or by the use of an alternative internal promoter. These variants include the canonical PAX6, PAX6(5a), and the paired-less PAX6ΔPD variant. This study evaluated the functional roles of these variants in rabbit RCE1(5T5) corneal epithelial cells. Endogenous PAX6 and PAX6(5a) proteins were detected in a 2:1 ratio, consistent with previously reported mRNA expression, while PAX6ΔPD was not clearly observed. Overexpression of each variant revealed distinct biological effects. PAX6 and PAX6(5a) did not enhance proliferation and led to reduced growth upon serial passage; in contrast, PAX6ΔPD significantly increased cell proliferation and sustained growth capacity. Consistently, PAX6 and PAX6(5a) promoted differentiation, as evidenced by increased expression of the terminal differentiation marker KRT3, while PAX6ΔPD inhibited differentiation and led to an increase in ΔNp63α expression, a marker associated with proliferative and stem/progenitor-like states. Mechanistically, bimolecular fluorescence complementation (BiFC) assays demonstrated that all variants could form homo- and heterodimers, with PAX6ΔPD exhibiting enhanced dimerization potential, particularly with PAX6(5a). Mutation of a conserved motif in PAX6ΔPD reduced these interactions, suggesting that protein-protein interactions underlie its functional effects. Overall, these findings suggest that PAX6 and PAX6(5a) promote corneal epithelial differentiation, whereas PAX6ΔPD antagonizes this process by sustaining proliferation, likely through competitive interactions with other PAX6 variants.

Crystalline lens aging reflects parallel processes: intrinsic chemical modification of exceptionally long-lived fiber-cell proteins, deterioration of lens-wide redox and transport homeostasis, and declining maintenance in metabolically active cellular compartments. Lens epithelial cells (LECs) support antioxidant defense, ion and water balance, stress responses, and the quality of newly formed fibers. Current lens and LEC evidence shows age-associated reductions in mitochondrial oxygen consumption in primary human LECs obtained during cataract surgery; SIRT1-related signaling is linked to epithelial stress tolerance; PINK1/Parkin-dependent mitophagy and autophagy-lysosomal function protect LECs in lens-relevant stress models; and transport systems shape the mature fiber-cell environment. Together, these findings identify NAD[+] homeostasis as a testable link among established maintenance components, although age-dependent NAD[+] change in normal human LECs and a causal epithelial-to-tissue pathway remain unproven. Direct lens evidence strongly supports crystallin modification, aggregation, insolubilization, redox disruption, and age-related material change. We therefore propose three falsifiable levels of validation: age-stratified measurement of NAD[+] homeostasis in ethically sourced human LECs; pathway-dependent testing of whether NAD[+] restoration improves sirtuin-dependent mitochondrial quality control (MQC); and determination of whether verified LEC MQC rescue alters intact-lens homeostasis, crystallin damage, material properties, transparency, or optical quality. Presbyopia and age-related cataract are considered distinct outcomes with partially overlapping determinants. This evidence-stratified synthesis positions the LEC NAD[+]-sirtuin-MQC network as a candidate modulator of crystalline lens aging whose contribution can be supported, restricted, or rejected experimentally.

Classical accounts of retinal aging emphasize oxidative, mitochondrial, inflammatory, vascular, proteostatic, and retinal pigment epithelium (RPE)-centered mechanisms. Omics studies now confirm and refine this non-uniformity, showing that aging-associated signals vary by cell type, anatomical niche, molecular layer, and functional reserve. Direct evidence from single-cell and single-nucleus transcriptomics is strong for lineage- and subtype-specific aging programs in neural retina, RPE, glia, vascular cells, and choroidal immune populations. Spatial omics can generate and test localization hypotheses across anatomically defined foveolar/foveal, parafoveal, perifoveal, peripheral, vascular, glial, optic nerve head, and RPE-Bruch's membrane-choroid niches, but direct spatial-omics evidence for normal human retinal aging remains sparse. Proteomic, metabolic, lipidomic, epigenomic, and imaging mass spectrometry data help determine whether transcriptional aging states translate into altered protein turnover, metabolic reserve, lipid handling, chromatin state, and inflammatory memory. AI-derived retinal age clocks provide population-scale imaging phenotypes, but color fundus photography alone is poorly depth-resolved; biological interpretation requires optical coherence tomography (OCT), visual function, biomarkers, destructive cell-type-resolved or spatially resolved tissue data, and longitudinal imaging or functional data. We integrate these findings into a multi-scale model in which normal retinal aging may lower resilience thresholds in defined cells and niches, thereby contributing to susceptibility to age-related macular degeneration, glaucoma, diabetic retinopathy, and inherited retinal degeneration. We also outline minimum standards for retinal aging omics, including anatomically specified sampling, donor metadata, evidence-source class interpretation, spatial validation, perturbation or rescue testing, and measurable endpoints of retinal resilience and visual function.

Although vision is one of the primary senses in humans, direct mechanistic research in patients is challenging both technically and ethically because of its multiscale complexity-spanning phototransduction, neural processing, and perception. The foundation of visual science is animal models, which offer controlled, systematic assess to every stage of the visual pathway. Animal models' visual phenotyping methods are highly significant for vision-related research. However, the selection of various experimental methods and critical differences between them are frequently disregarded in study design. To address this, we present a systematic paradigm for in vivo visual phenotyping built upon three complementary pillars: imaging, electrophysiology, and behavioral testing. This triad reveals the integrity of the visual pathway from structural, functional, and perceptual perspectives, respectively. This review details the methodological principles of each visual phenotyping approach and advocates for integrative analytical strategies that connect macroscopic visual deficiencies to microcircuit dysfunction. By setting clear guidelines for multimodal data fusion and translational benchmarking, this paradigm aims to accelerate mechanism-based pathological investigation and therapeutic development. Ultimately, it will help advance the field from descriptive phenomenology toward a predictive, integrative phenomics. These convergent approaches are essential for strengthening the bridge between animal studies and clinical applications, thereby accelerating the delivery of novel treatments to patients with visual impairments.

This study aims to investigate the protective effects of trimethylamine N-oxide (TMAO), a natural occurring chaperone, on delaying diabetic cataract (DC) progression. Rats were divided into 4 experimental groups: untreated controls, TMAO treatment only, streptozotocin-induced diabetic mellitus (DM), and DM with TMAO therapy. After 10 weeks, aqueous humor TMAO levels and lens opacity were assessed. Immunofluorescence analyses and Western blotting were utilized to evaluate epithelial-mesenchymal transition (EMT)-related genes in lens epithelial cells (LECs). In vitro, HLE-B3 cells were treated with or without TMAO under high glucose (HG) environment (50 mmol/L), then collected for RNA-seq. Apoptosis and reactive oxygen species (ROS) were evaluated by flow cytometry while endoplasmic reticulum stress (ERS)-related genes were observed by western blotting. Elevated aqueous humor TMAO levels were detected in DC rats, compared to controls. In vivo and ex vivo, TMAO reduced HG-induced lens opacity and EMT in LECs. In vitro experiments demonstrated that HG resulted in enhanced apoptosis, migration, ROS and EMT-related genes in HLE-B3 cells while TMAO addition diminished these effects. RNA-seq revealed that the inhibition of ERS was the key factor to reverse the impacts of HG on LECs by TMAO and mechanistic investigations suggested that ERS-related pathway were significantly inhibited in TMAO treated LECs under HG or tunicamycin-induced ERS. Moreover, TMAO improved the solubility of lens protein in DC rats. Our study pioneered the confirmation of the protective role of TMAO in DC through inhibiting lens protein unfolding, suppressing ERS, and alleviating ERS-induced apoptosis, ROS and EMT in LECs.

Tricaine methanesulfonate (MS-222) is a commonly used amphibian and fish anesthetic that blocks voltage-gated sodium channels. There are documented deleterious effects of MS-222 on visual response properties in newts, frogs and humans. Yet, the impact of MS-222 on the amplitudes and kinetics of visual responses in the zebrafish (Danio rerio) retina remains unresolved. Here, we use ex vivo electroretinogram (ERG) recordings from zebrafish retinae to investigate the effects of brief exposure and prolonged exposure to MS-222 on photoreceptor and bipolar cell response properties. Zebrafish were anesthetized in either an ice-cooling bath or MS-222 until surgical-grade anesthesia was achieved (30 seconds - 2 minutes), before isolating the retinae for ex vivo recordings. This brief exposure to MS-222 did not significantly alter response amplitudes or kinetics, however, synaptic transfer analysis suggested a non-significant trend toward reduced retinal gain. Prolonged bath application of MS-222 to isolated retinae altered ERG a-wave amplitudes, b-wave recovery dynamics, and response latencies. Recovery from bath application of MS-222 was also hindered, as we observed small a-wave responses even after a 30-min washout recovery process, although response kinetics were observed to return to similar values as control during recovery. These findings demonstrate that prolonged direct MS-222 exposure alters retinal function in zebrafish, and suggests that anesthesia should be considered as an experimental variable in zebrafish visual physiology studies where prolonged anesthetic exposure may confound visual response measurements.

The lamina cribrosa (LC) and peripapillary sclera (PPS) form a biomechanical structure whose response to intraocular pressure (IOP) is central to glaucomatous optic nerve damage. Computational models predict strong biomechanical interactions between PPS and LC, yet experimental validation in human eyes has remained limited, particularly for full-thickness characterization. In this study, we applied 3D high-frequency ultrasound elastography technique to quantify full-thickness deformation of PPS and LC and to characterize their biomechanical interplay under controlled IOP elevation. Inflation testing from 15 to 30 mmHg was performed in 22 human donor whole globes. At each pressure step, 3D radiofrequency ultrasound data was acquired and analyzed using a validated speckle-tracking algorithm to estimate full-field tissue displacements and calculate the spherical Green-Lagrange strain tensor. PPS and LC boundaries were manually segmented on radial reconstructions to enable region-specific strain and morphometric analyses. Our results showed that IOP-induced shear strains were significantly correlated between the PPS and LC (all r > 0.6, P < 0.01). Among the morphometric parameters, PPS radius of curvature was correlated with all PPS and LC shear strains (r: 0.54 to 0.71). PPS thickness was negatively correlated with PPS out-of-plane shear strains (r: -0.44 to -0.48). LC depth was correlated with LC shear strains (r: 0.42 to 0.46), and LC thickness was correlated with PPS shear strains (r: 0.54 to 0.56). These findings provide experimental evidence that PPS/LC biomechanical coupling under IOP elevation is associated with shear deformation. Quantifying PPS and LC shear behavior may improve understanding of optic nerve head biomechanics and facilitate discovery of new biomechanical biomarkers relevant to glaucoma susceptibility.