Purpose:Pseudomonas aeruginosa (PA) is a Gram-negative opportunistic bacterial pathogen that can infect the cornea as a result of trauma or contact lens wear. Mitochondria, originally derived from bacteria, play important roles in host defense. This study investigated the effect of PA on mitochondrial structure and function during the early stages of infection in corneal epithelial cells. Methods:Telomerase-immortalized human corneal epithelial cells and primary cultured human corneal epithelial cells were infected with a standard invasive test strain of PA (strain PAO1). Changes in mitochondrial dynamics, mitophagy, and metabolic proteins were assessed by Western blot. Mitochondrial polarization was quantified using JC-1 labeling. Mitochondrial metabolism was measured in real time using Seahorse, and mass spectrometry was performed for metabolomic analysis. Mitochondrial morphology was assessed by transmission electron microscopy. Results:PA infection induced rapid mitochondrial fission, followed by PINK1-mediated mitophagy. PA infection also reduced NADH-linked respiration through a reduction in complex I and impaired dihydroorotate and glycerolipid metabolism. Other changes in the metabolite profile included an increase in arginine biosynthesis, purine metabolism, and the pentose phosphate pathway, all pathways that can be readily exploited by bacteria. Following treatment with gentamicin to kill all extracellular bacteria, metabolic flux analysis showed that corneal epithelial cells were able to restore mitochondrial function despite the continued presence of intracellular PA. Conclusions:Taken together, these data demonstrate that extracellular PA triggers mitochondrial dysfunction and metabolic rewiring in corneal epithelial cells. This may represent a potential mechanism whereby PA disables host cell mitochondria to facilitate invasion.
Purpose:Considerable ganglion cell (GC) loss occurs over the normal human lifespan and even more in ocular diseases such as glaucoma. Current clinical methods, however, lack sufficient resolution and sensitivity to directly detect GC loss. Here, we used adaptive optics optical coherence tomography (AO-OCT) to overcome these limitations, enabling longitudinal tracking of individual GC somas and direct measurement of their loss in healthy and glaucomatous eyes. Methods:Six healthy subjects and two patients with glaucoma were imaged at multiple retinal locations using AO-OCT. Healthy subjects were imaged annually for up to 3 years (4 total timepoints); patients with glaucoma were reimaged 2 to 3 years after baseline. Individual ganglion cell layer (GCL) somas were identified and tracked across sessions and soma densities were quantified. A flicker-based image comparison approach enabled sensitive detection of soma loss from which cumulative loss was determined by subject and location. Results:The GCL soma mosaic was highly stable, with 23,586 somas tracked in healthy subjects. Soma densities agreed with histologic values, including near the fovea, and correlated strongly with GCL thickness (R2 = 0.96). The mean soma loss rate in healthy subjects was -0.10%/year (95% confidence interval [CI] = -0.09 to -0.11%/year), with no significant differences across subjects or locations. By contrast, one glaucoma patient showed a loss rate within an arcuate defect over 100× greater (-31.6%/year; corresponding GCL thinning rate of -13%/year), whereas the other demonstrated a near-normal rate (-0.26%/year). Conclusions:In vivo measurement of individual GC soma loss is now feasible and provides a sensitive tool for longitudinal evaluation of GC health.
Purpose:Defects in primary cilia and ciliary signaling are associated with an array of neurodegenerative diseases. Primary cilia are sensory organelles that transmit and regulate various cellular communication pathways, including the significant Sonic hedgehog and Wnt signal pathways. Extensive studies have shown that neuronal cilia in the central nervous system contribute to early patterning, neuronal maturation, and survival. The behavior of cilia on astrocytes in glaucoma and aging remains poorly understood. This study used rodent and human retinal astrocytes to examine primary cilia and their alteration during aging and disease. Methods:Retinal flatmounts from wild-type mice, one macaque, and two humans were immunostained for astrocyte (glial fibrillary acidic protein [GFAP]) and ciliary (Arl13b and gamma-tubulin) markers and analyzed by confocal microscopy. Ciliation was also assessed in models of ocular hypertension and optic nerve crush prepared in 3- and 20-month-old mice. Results:Primary cilia were found in most of the mouse retinal astrocytes (98.81% ± 4.03% in 3-month-old mice and 91.05% ± 9.95% in 20-month-old mice). Interestingly, astrocyte ciliation was significantly reduced in the peripheral retinas of the aged mice. Both human and nonhuman primate retinas expressed primary cilia in bundle-shaped and star-shaped GFAP-positive astrocytes. As a result of alterations in the primary cilia of retinal astrocytes, response processes varied in different glaucoma mouse models, including the ocular hypertension and optic nerve crush models. Conclusions:Aged and diseased mouse retinas demonstrated a novel alteration of their ciliary profile. These findings suggest the possibility that primary cilia contribute to age-associated retinal diseases.
Purpose:To evaluate association of retinal morphological alteration with retinal sensitivity (RS) based on a point-to-point correlation with optical coherence tomography (OCT) and microperimetry in a 10-year follow-up of patients with central serous chorioretinopathy (CSC). Methods:Macular OCT and microperimetry of patients with an acute CSC episode were acquired prospectively at disease onset and 12 weeks after fluid resorption. Ten years later patients were invited for a cross-sectional follow-up visit including Spectralis-OCT and microperimetry. Multivariable models evaluated point-to-point correlation of RS2 and OCT parameters. Results:Thirty patients were included in the acute setting and 23 patients were included for the 10-year follow-up. In the acute setting, RS2 is associated with subretinal fluid (SRF; estimate = 37.01; P = 0.014), SRF height (estimate = -32.95; P < 0.001), irregularities in the ellipsoid zone (estimate = -35.28; P < 0.001), and interdigitation zone (estimate = -14.86; P < 0.001), patient age (estimate = -1.26; P = 0.03), and prior photodynamic therapy (estimate = -33.14; P = 0.001). Baseline parameters affecting RS2 after fluid resolution were SRF height (estimate = -11.93; P < 0.001), retinal pigment epithelium (RPE)/Bruch's membrane thickening (estimate = -55.34; P < 0.001), and prior photodynamic therapy (estimate = -118.05; P < 0.001). For the 10 -year follow-up RPE detachment (estimate = -89.02; P < 0.001), RPE atrophy (estimate = -377.14; P < 0.001), female sex (estimate = -81.50; P = 0.014), and prior photodynamic therapy (estimate = -162.18; P < 0.001) were associated with worse RS2. Conclusions:Long-term functional outcome in CSC is mainly determined by chronic structural damage, especially RPE atrophy and persistent RPE detachment. The association with prior photodynamic therapy reflects disease chronicity rather than treatment-related harm. Early recognition of high-risk morphological features may reduce irreversible functional loss.
Purpose:To characterize the longitudinal changes of macular sensitivity in best vitelliform macular dystrophy (BVMD) and explore microperimetry (MP)-derived outcome measures for clinical trials. Methods:Natural history cohort study (IRDs-OSR, NCT07265895) on subjects with BVMD undergoing mesopic MP (68-point grid, central 10°; MAIA, CenterVue). Test points were classified relative to normative data as normal (P ≥ 5%), relative scotoma (P < 5%, sensitivity ≥ 1dB), or deep scotoma (sensitivity <1 dB). Per-eye annual rates of change (RoC) of mean sensitivity (MS) were computed globally and within point subsets, together with point counts. Results:Twenty-three eyes of 13 patients with clinical BVMD, with a median age at baseline of 29.4 years, completed an average of three tests over a median of 4.4 years. The median MS at baseline was 21.4 dB, with 44% of points being normal, 54% relative scotomas, and 2% deep scotomas. Globally MS showed no significant decline (mean RoC = -0.16 dB/year; P = 0.21). Restricting analysis to normal points at baseline revealed small but significant decline (mean RoC = -0.36 dB/year; P = 0.006), steepest within 5° (mean RoC = -0.56 dB/year; P = 0.028). Average increase of deep scotoma points was ∼1 every five years (mean RoC = 0.22 points/year; P = 0.015), with no significant change in relative scotoma or normal point counts (all P > 0.05). Conclusions:Macular sensitivity within the central 10° remained stable over four years in BVMD when averaged across several test points, reflecting the disease slow natural history. Restricting analysis to normal points unmasked measurable decline, which may serve as an MP-derived outcome measure for gene therapy trials.
Purpose:The purpose of this study was to noninvasively test the hypothesis that dysregulated photoreceptor metabolism occurs with early retinal degeneration in dark-reared Pde6brd10 mice. Methods:Postnatal day 23 (P23) phosphodiesterase (PDE) mutant (Pde6brd10) mice, P23 wild-type (WT) mice were continuously dark-reared, and 2-month-old dark-adapted (DA) C57BL/6J WT mice were studied following acute injection of the carbonic anhydrase inhibitor acetazolamide (ACZ) or saline control. Two optical coherence tomography (OCT) metabolic biomarkers were measured: (i) external limiting membrane-retinal pigment epithelium (ELM-RPE) thickness, a proxy for subretinal space acidosis, and (ii) the profile shape aspect ratio of the hyper-reflective band just distal to the ELM (band 2), a proxy for mitochondrial configuration within photoreceptor inner segments. Retinal laminar thicknesses were also assessed. Results:P23 Pde6brd10 mice responded to ACZ with ELM-RPE contraction, but no change in band 2 profile shape aspect ratio; outer nuclear layer thickness was subnormal. In contrast, P23 WT mice showed no change in either OCT metabolic biomarker following ACZ administration. DA 2-month-old WT mice showed ELM-RPE contraction with ACZ; band 2 profile shape aspect ratio increased in male mice, with no change in female mice. Conclusions:P23 Pde6brd10 mice with limited rod atrophy had biomarker values suggestive of dysregulation of RPE fluid/ion transport but with stable rod mitochondrial configuration following ACZ. DA 2-month-old male WT mice biomarkers were responsive to ACZ in a manner similar to those in light-adapted 2-month-old male WT mice; the difference in biomarker response pattern in female WT mice suggests a possible hormonal influence.
Purpose:Visual acuity (VA) is a key outcome measure in clinical practice, epidemiological studies, and in clinical trials. However, letter-based charts may disadvantage individuals who are illiterate or are unfamiliar with Latin text, thereby limiting subject participation and complicating comparisons across studies. We compared VA using Early Treatment Diabetic Retinopathy Study (ETDRS) charts for Latin letters, Landolt-C, or Tumbling-E optotypes to determine their association and measurement variability. Methods:Seventy-eight patients from an eye clinic underwent VA testing using the various optotypes on an ETDRS logMAR chart. VA was measured with a pinhole (PH), uncorrected, and corrected for the three optotypes, and compared with results in severe VA loss. Regression slopes, coefficients of determination, and limits of agreement (LoA) were determined. Results:All three optotype VAs showed a similar response to PH use, with an average improvement of approximately three logMAR lines. For uncorrected VA, Tumbling-E and Landolt-C showed slopes near 1.0 relative to letter VA across a broad range with R2 > 0.93. For corrected VA, slopes were 0.73 (Tumbling-E) and 0.58 (Landolt-C), with R2 ≈0.70. Combining our data with prior studies extended the range to 20/2000 for Landolt C and letter VA, yielding slopes near 1.0 and R2 > 0.94. LoA across comparisons ranged from logMAR ±0.12 to ±0.23. Conclusions:Non-letter optotypes provide clinically useful and broadly comparable measures of VA at the population level supporting their use in clinical practice or population/clinical studies. However, the wide variability means they cannot be considered interchangeable for individual subjects.
Purpose:To evaluate changes in visual acuity (VA) and stereoacuity in children with amblyopia following monocular or stereopsis training delivered alongside standard clinical care and to examine factors associated with training-related improvements in visual outcomes. Methods:Forty-three children with amblyopia (7.3-14.8 years; 37 anisometropic, five strabismic, and one mixed) completed approximately 29 sessions of either monocular training (grating acuity or motion discrimination targeting the amblyopic eye, n = 23) or binocular stereopsis training (dichoptic disparity discrimination, n = 20). All participants continued standard clinical management, including optical correction and prescribed patching. Pre- and post-training assessments included amblyopic-eye VA, interocular acuity difference (IOD), and Randot stereoacuity. Results:Both training protocols produced significant task-specific learning. Amblyopic-eye VA improved significantly in both groups, with no evidence of differential effects between training approaches. Stereoacuity also improved in both groups, with significantly greater gains following stereopsis training than monocular training. Exploratory analyses indicated that poorer baseline amblyopic-eye VA was associated with larger VA gains; however, this association did not survive correction for multiple comparisons. Stereoacuity improvement was not significantly associated with baseline VA, IOD, age, or changes in VA. Conclusions:Visual training combined with standard clinical care improved amblyopic-eye VA and stereoacuity in children with amblyopia. Although both training approaches yielded comparable gains in VA, stereopsis training produced larger improvements in stereoacuity, supporting its potential role in addressing residual binocular deficits after conventional treatment.
Purpose:To quantitatively map the spatial architecture of mechanical stress and pressure in the retinal pigment epithelium (RPE) and to test the central hypothesis that the mechanical state of normal mouse RPE is spatially patterned from center to periphery and that this spatial pattern is maintained during adult aging. Methods:Whole-mount flat images of C57BL/6J mouse RPE spanning multiple age groups were analyzed using a tissue-scale stress inference framework under the assumption of mechanical equilibrium. Individual RPE cells were segmented, intercellular junctions were skeletonized, and the resulting geometric networks were used to estimate local intracellular pressure and junctional tension. Radial profiles of these mechanical fields were computed from the tissue center to the periphery and systematically compared across age groups. Results:Inferred intracellular pressure exhibited a robust and monotonic decrease from the central to peripheral RPE across all age groups, whereas junctional tension displayed no consistent radial dependence. We did not detect substantial age-associated changes in the inferred pressure and tension profiles among the usable flatmounts analyzed, consistent with apparent preservation of the large-scale mechanical organization across the ages examined. Conclusions:Tissue stress inference revealed robust spatial patterning in inferred RPE mechanics, characterized by a pronounced decline in inferred intracellular pressure toward the periphery and relatively uniform junctional tension across the tissue. No strong age-associated changes were detected in the inferred mechanical profiles, suggesting that the spatial mechanical signatures of the RPE are largely preserved across the ages studied. Together, these data establish a quantitative biomechanical baseline against which AMD- or genotype-associated remodeling of RPE mechanics can be evaluated.
Purpose:The molecular pathway for increased outflow resistance in the trabecular meshwork (TM) remains unknown. Secreted protein, acidic and rich in cysteine (SPARC) has been shown to regulate intraocular pressure (IOP) correlating to extracellular matrix (ECM) alterations. In other tissues, SPARC binds integrin-linked kinase (ILK) and regulates ECM organization through signaling cascades. We hypothesized SPARC regulates ECM proteins partly through ILK in the TM. Methods:Adenovirus carrying cDNA of human SPARC (Ad.SPARC) was used to overexpress SPARC and a lentivirus carrying short-hairpin RNA (shRNA) targeting human ILK (shILK) was used to inhibit ILK in live mice, primary human TM cells, and perfused human cadaveric anterior segments. IOP was measured in mice and human anterior segments. Selected ECM proteins were analyzed by immunoblotting and immunostaining. Results:SPARC overexpression elevated IOP 1.87 ± 0.50 millimeters of mercury (mm Hg; P = 0.009, n = 15) in mice. Coinfection with ILK inhibition reduced IOP by 3.64 ± 0.57 mm Hg (P < 0.001, n = 15) and histologically reduced ECM proteins compared to SPARC overexpression. In human anterior segments, SPARC overexpression elevated IOP 2.10 ± 0.25-fold (P = 0.021, n = 4), whereas addition of shILK attenuated SPARC's effect and decreased IOP 0.61 ± 0.31-fold (P = 0.015, n = 4). In human TM cells, Ad.SPARC infection elevated levels of SPARC and laminin. Coinfection with shILK reduced levels of ILK, collagen I, collagen VI, and laminin. Conclusions:ILK inhibition attenuated the effects of SPARC overexpression on IOP and ECM proteins, suggesting ILK signaling may contribute to the pathway of SPARC-mediated regulation of ECM homeostasis in TM. Future studies evaluating the downstream transcription factors may provide an elucidated pathway for POAG pathogenesis.
Purpose:Human eye growth is reported to follow a seasonal pattern, with accelerated eye growth leading to myopia being linked to limited exposure to daylight. To investigate how environmental lighting may contribute to eye growth and refractive errors in emmetropic children, we analyzed real-world seasonal variations in daylight intensity, duration, and spectrum and their association with healthy regulated human eye growth. Methods:Downwelling irradiance was collected around the 2 solstice and the 2 equinox dates to assess variations across 4 seasons at 60°N latitude location over 4 years (2022-2025). To assess seasonal changes in the relative levels of short- versus long-wavelength parts of daylight, we calculated ratios of alpha-opic equivalent daylight illuminance (EDI) for the photoreceptors. Gold-standard ocular measurements were obtained from emmetropic 7 to 11-year-old children who spend 1 to 2 hours outdoors every day and were used to explore the associations between healthy eye growth rates and seasonal variations in these ratios. Results:Daylight intensities were significantly lower during autumn and winter mornings, winter midday, as well as autumn and spring afternoons. The spectra were significantly short-wavelength enriched during winter mornings and midday, and spring afternoons. The seasonal cycle of daylight and human eye growth in 7 to 11-year-old children was in anti-phase, with peak growth delayed relative to the shortest winter days when daylight was short-wavelength enriched. Conclusions:The short-wavelength enrichment encompasses the sensitivities of both S opsin and melanopsin and invites speculation that the eye may have evolved to exploit seasonal variations in intensity and spectrum as cues that may contribute to the regulation of eye growth via photoreceptor opsin activation.
Background:To evaluate the associations of the triglyceride-glucose (TyG) index and related obesity-combined indexes with the incidence of major vision-threatening ocular diseases. Methods:This prospective population-based cohort study included 400,237 participants from the UK Biobank. The TyG index and derived indexes (combined with waist circumference, waist-to-height ratio, and body mass index) were calculated using baseline laboratory and anthropometric data. The outcomes were incident cases of cataract, POAG, AMD, and diabetic retinopathy (DR), ascertained using self-reported data, hospital inpatient records, and primary care records. Cox proportional hazards models were used, with restricted cubic splines fitted to characterize dose-response relationships. Secondary analyses assessed the associations with retinal thicknesses and vascular features. Results:Over a median follow-up of 13.0 years, 46,151 cataract, 6317 POAG, 8276 AMD, and 5220 DR incident cases were identified. Distinct nonlinear associations were observed: the TyG index exhibited a reverse L-shaped relationship with cataract and DR, a J-shaped association with AMD, and a U-shaped relationship with POAG (all P total < 0.01, all P nonlinear < 0.05), suggesting an estimated optimal range from 8.6 to 9.1. Furthermore, elevated TyG-obesity indexes exhibited monotonic positive associations with the risk of cataract, AMD, and DR (all P total < 0.001), whereas moderate elevation was associated with a reduced risk of POAG (all P total < 0.01, all P nonlinear < 0.05). Elevated TyG-obesity indexes were also associated with retinal thinning and vascular abnormalities, providing supportive discrimination and risk-stratification information for incident DR. Conclusions:The TyG index and related obesity-combined indexes show disease-specific associations with major sight-threatening conditions, accompanied by retinal structural alterations. Maintaining these parameters within optimal ranges could help mitigate the risk of vision impairment.
Purpose:Resistance to adjuvant chemotherapy following enucleation continues to drive recurrence and metastasis in high-risk advanced retinoblastoma (RB), posing a major clinical challenge. To identify candidate genes, a previous transcriptomic analysis revealed markedly reduced expression of Rho GTPase-activating protein 9 (ARHGAP9) in the etoposide-resistant Y79/EDR cell line. This study aims to further investigate the effect of ARHGAP9 on chemoresistance in high-risk advanced RB. Methods:Whole-exome sequencing and reduced-representation bisulfite sequencing profiles of eight peripheral blood samples from patients with high-risk advanced RB were analyzed to identify genes with germline genetic or epigenetic alterations associated with RB prognosis. In vitro and in vivo studies on drug responsiveness and molecular mechanisms were conducted in stable ARHGAP9-silenced and ARHGAP9-overexpressing cell lines. Results:ARHGAP9 was identified through peripheral blood-based genomic and epigenomic analyses and further validated via functional study. Functional assays demonstrated that ARHGAP9 knockdown considerably decreased sensitivity to carboplatin and etoposide, whereas its overexpression restored drug responsiveness in vitro and in vivo. Mechanistic analysis indicated that ARHGAP9 downregulation markedly enhanced Ras-related C3 botulinum toxin substrate 1 (Rac1) activity as its catalytic substrate, promoted Rac1-β-catenin interaction, and facilitated their nuclear accumulation. This process induced the formation of lymphoid enhancer-binding factor 1 (LEF1)-β-catenin complex and increased expression of downstream proteins, such as c-Myc, cyclin D1, and Bcl-2, which contribute to chemotherapy resistance in RB. Conclusions:These findings provide mechanistic insights into how ARHGAP9 modulates drug responses via the Rac1/β-catenin/LEF1 signaling axis in RB and suggest that ARHGAP9 warrants further investigation as a candidate prognostic indicator or therapeutic target for advanced high-risk RB.
Purpose:Amplification of MYCN is a primary driver of aggressive retinoblastoma (RB), yet it remains a challenging therapeutic target. This study aimed to evaluate the therapeutic potential of the PROTAC molecule HLB-0532259 in degrading MYCN and suppressing RB growth. Methods:Y79 and WERI-Rb1 RB cell lines were treated with HLB-0532259, and MYCN protein degradation, cell viability, and time-dependent transcriptional changes were assessed by Western blotting, cell viability assays, RNA-seq, and proteomics. The therapeutic potential was further validated in an orthotopic Y79 xenograft mouse model through intravitreal administration, with tumor burden evaluated by histology and immunohistochemistry. Results:HLB-0532259 induced dose-dependent degradation of MYCN in both cell lines, with Y79 cells showing higher sensitivity. Transcriptomic and proteomic analyses revealed rapid activation of the p53-p21 pathway, downregulation of DNA repair and epigenetic regulators, and accumulation of DNA damage. Proteomic profiling confirmed upregulation of senescence-associated secretory phenotype factors and downregulation of MYCN-dependent effectors, including HMGA1 and TRAF6. In orthotopic xenografts, intravitreal administration of HLB-0532259 reduced intraocular tumor burden, decreased Ki-67 and MYCN-positive cells, and activated p53, p21, and NF-κB signaling. Conclusions:Targeted MYCN degradation by HLB-0532259 effectively inhibits RB growth and induces a DNA damage-associated senescence program, highlighting targeted MYCN degradation as a promising therapeutic strategy for MYCN-driven RB.
Purpose:Transcriptional heterogeneity observed in retinoblastoma (RB) is unexplained by the limited genetic alterations identified. Epifactors, which can modulate transcriptional activity and oncogenic pathways, may contribute to this heterogeneity. However, knowledge regarding the role of epigenetic machinery in RB remains limited. Here, we analyze epifactors' expression to uncover dysregulations and their contribution to RB tumor heterogeneity. Methods:Transcriptomic datasets of primary RB tumors and normal fetal retina were obtained from the NCBI Gene Expression Omnibus (NCBI-GEO). Differentially expressed epifactors were analyzed using edgeR, and the identified epifactors were functionally annotated. Non-negative matrix factorization (NMF) was used for clustering based on epifactor's expression. Results:We found 133 epifactors differentially expressed in RB compared with the healthy retina. In particular, epifactors involved in histone phosphorylation and RNA degradation were predominantly upregulated, whereas those involved in RNA methylation regulation were largely downregulated. Based on epifactor expression, NMF-based clustering stratified patients with RB into two clusters with distinct biological signatures. Cluster 1 patients showed enrichment for cell-cycle and MYC programs, and cluster 2 showed enrichment for interferon- and inflammatory-related pathways. Clusters were found to be associated with specific epigenetic factors that formed cluster-specific functional complexes, such as centromere/chromosomal passenger complexes in the proliferative cluster 1 and nuclear receptor/interferon-associated complexes in the immunologically active cluster 2. Conclusions:Transcriptomic heterogeneity in RB appears to be shaped by epifactor expression, suggesting that RB heterogeneity may be encoded within its epigenetic machinery. Epifactor profiling, clustering, and complex-level analysis identified key epigenetic factors that could serve as potential biomarkers and therapeutic interventions.
Purpose:To explore whether amniotic membrane stromal cells (AMSCs) can promote the expansion of limbal stem cells (LSCs) during corneal epithelial tissue engineering. Methods:Rabbit corneal limbal epithelium was cultured on amniotic membrane containing live stromal cells (L-dAM) or amniotic membrane with dead stromal cells (D-dAM). Morphological and microstructural characteristics of in vitro-expanded rabbit limbal epithelial sheets were evaluated using light microscopy and scanning electron microscopy. Stemness properties and differentiation status were assessed through colony forming efficiency (CFE) assays and immunofluorescence staining of ΔNp63α, PAX6, Ki67, and K14. Gene expression in the limbal epithelial sheets was analyzed by quantitative real-time polymerase chain reaction and western blotting. Transcriptomic alterations in LSCs and AMSCs were decoded through RNA sequencing. Intracellular NAD+ dynamics were quantified via liquid chromatography-tandem mass spectrometry/metabolomics and enzymatic NAD+/NADH ratio assays. Results:LSCs cultured on L-dAM exhibited superior tight junction integrity and colony-forming capacity compared with those on D-dAM, with upregulated expression of ΔNp63α, PAX6, Ki67, and K14, alongside downregulated expression of K12. Nicotinamide supplementation in D-dAM cultures restored NAD+-dependent stem cell homeostasis, achieving functional equivalence to L-dAM through complete recovery of CFE and coordinated molecular restitution of stem/progenitor markers with sustained K12 inhibition. Mechanistically, co-cultured AMSCs transferred NAD+ via exosomes to maintain LSC homeostasis, as evidenced by vesicle inhibition causing NAD+ depletion, stemness exhaustion, and accelerated differentiation. Conclusions:AMSCs could promote limbal epithelial stem cell expansion, offering clinical implication for corneal epithelial tissue engineering.
Purpose:To investigate the effects of acute radiation exposure on retinal microvascular health using the New Zealand White Rabbit (NZWR) model, aiming to enhance understanding of vascular response to total body irradiation (TBI). Methods:This research involved two phases: a repeatability assessment of imaging metrics and evaluating changes after TBI. Advanced imaging modalities, including fluorescein angiography, laser speckle contrast imaging (LSCI), and optical coherence tomography, were used for characterizing the static and dynamic attributes of retinal blood flow. Results:In phase I, six NZWRs were imaged over 2 years to validate repeatability and reproducibility. Vessel density metrics derived from fluorescein angiography exhibited moderate to high reliability, with intraclass correlation coefficients of 0.69 to 0.71. Dynamic LSCI metrics, including blood flow velocity index and related parameters, demonstrated varying levels of repeatability, with the Resistivity Index showing the greatest consistency. In phase II, 11 NZWRs underwent imaging at baseline and at 7 longitudinal time points after TBI. There were significant changes in heart rate-adjusted LSCI metrics at various postradiation days compared with baseline measurements in different vascular areas. Conclusions:This study validates retinal imaging tools in the NZWR model and longitudinally assesses radiation-induced vascular changes. Our findings support the candidacy of the NZWR retina as a robust model for studying the short- and long-term effects of diseases, medical interventions, and harmful stimuli such as radiation on microvascular status, with potential applications in understanding systemic vascular conditions and neurodegenerative diseases.
Purpose:Ascl1 is a transcription factor (TF) that plays key roles in regulating retinal development. Recent studies have further uncovered its intriguing potential to drive retinal neuron regeneration by reprogramming Müller glial cells. However, the efficiency and specificity of Ascl1-mediated regeneration outcomes remain far from optimal, and the molecular mechanisms underlying its dual roles in retinal development and adult Müller reprogramming are not fully elucidated. Given the close developmental relationship between late-stage retinal progenitor cells (RPCs) and Müller glial cells, this study aimed to investigate the functional role and underlying molecular mechanisms of Ascl1 in governing retinal fate specification, using late RPCs as a model system. Methods:In vivo electroporation (IVE) of the mouse retinas was used to deliver Ascl1-overexpressing (Ascl1-OE) plasmids into late RPCs. Immunofluorescence (IF) staining was employed to recognize retinal cells and assess cell proliferation status. RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) were conducted to profile the transcriptomes of RPCs, and the assay for transposase-accessible chromatin using sequencing (ATAC-seq) was conducted to characterize the chromatin accessibility landscapes of RPCs. Results:Ascl1-OE caused late PRCs to exit the cell cycle prematurely and produce more rods but fewer bipolar and Müller glial cells. Integrative RNA-seq, scRNA-seq, ATAC-seq and chromatin immunoprecipitation (ChIP)-seq analyses of Ascl1-OE late RPCs revealed that Ascl1 promoted the expression of Notch pathway inhibitors and neurogenic genes by binding to and opening up relevant genomic regions. Conclusions:Ascl1 suppresses Müller glial and bipolar fates and promotes rod photoreceptor fate in late RPCs. Mechanistically, Ascl1 achieves the effect by repressing the Notch signaling and activating neurogenic genes through reprogramming the epigenetic landscape of the cells. The findings of this study may help elucidate the molecular mechanisms of Ascl1-driven Müller reprogramming in adult retinas and facilitate the development of novel methods to improve the efficiency and specificity of retinal neuron regeneration outcomes.
Purpose:The purpose of this study was to investigate the association between entropy values derived from the retinal pigment epithelium (RPE) layer using polarization-sensitive optical coherence tomography (PS-OCT) and RPE cellular metrics obtained by adaptive optics transscleral flood illumination (AO-TFI), including cell density, spacing, regularity, and dispersion. We further aimed to determine which cellular structural alterations in retinitis pigmentosa (RP) are reflected by entropy. Methods:Healthy controls and patients with RP who underwent both PS-OCT and AO-TFI imaging were included. Spearman correlation coefficients were calculated to assess the relationships between polarimetric entropy in PS-OCT and each AO-TFI-derived parameter. To account for repeated measurements within the same eye and the effects of group and eccentricity, linear mixed-effects models were constructed to evaluate the independent associations between entropy and AO-TFI metrics after adjustment for these factors. Results:Polarimetric entropy showed the strongest positive correlation with RPE cell density among the AO-TFI parameters and was negatively correlated with spacing of RPE cells. No clear associations were observed with regularity or dispersion. In linear mixed-effects models, the association between entropy and RPE cell density differed between groups, with a significant association observed in the RP group but not in healthy controls. Conclusions:PS-OCT-derived polarimetric entropy was associated with RPE cell density measured by AO-TFI, with the association primarily observed in RP rather than in healthy controls. These findings suggest that correspondence between the two imaging modalities may contribute to quantitative assessment of outer retinal-RPE complex alterations and has potential utility as a biomarker in retinal degenerative diseases.
