Investigative ophthalmology & visual science最新文献

Purpose: Calcium (Ca2+) is involved in regulating many cellular functions, including the intercellular barrier integrity. This study investigated the expression, localization, and permeability of TRP-Vanilloid (TRPV1-4) channels to Ca2+ in human corneal endothelial (hCE) cells.

Methods: Gene and protein level expression of the TRPV1 to 4 channels was determined by PCR, immunostaining, and Western blot analysis in hCEs from fresh human donor tissues and cultured primary cells. Colocalization of the channels with actin and junction proteins zonula occludens-1 (ZO-1) and N-cadherin was quantified by calculating Pearson's coefficient using ImageJ software. Changes to Ca2+ influx in response to channel-specific agonist and antagonist treatment were measured using Fluo-4 AM dye.

Results: TRPV1 to 4 expression was confirmed in cells by PCR and Western blot. Staining revealed distinct membrane and cytoplasmic localization of these four channels. All four channels showed high colocalization with N-cadherin (r ≥ 0.7), whereas TRPV4 colocalized with N-cadherin and ZO-1 equally (r ≥ 0.8). An increase in Ca2+ influx was noted in >80% of the cells when TRPV1, TRPV2, and TRPV4 channels were activated using channel-specific agonists. A similar response was evident in only 50% of cells following TRPV3 channel activation. Pretreatment of cells with antagonists reduced agonist-induced Ca2+ influx, as noted by a significant reduction in the area under the curve of the influx profile, relative to agonist treatment.

Conclusions: This study confirms TRPV1 to 4 expression, enumerates their distinct localization and colocalization with junction proteins, and their permeability to Ca2+ in hCEs. Taken together, these results suggest a potential role for these channels in maintaining Ca2+ homeostasis and barrier function in these cells.

Purpose: To cross-sectionally compare parameters from the quick contrast sensitivity function (qCSF)-area under the log CSF (AULCSF) and sensitivity at each spatial frequency-in older eyes with normal macular health and early, intermediate, and late AMD. Our purpose was to determine whether qCSF could be a promising functional outcome measure in interventions designed to slow AMD progression from aging to late AMD.

Methods: qCSF contrast sensitivity was measured at 1.0 to 18 cycles per degree (cyc/deg). Models evaluated the impact of diagnostic group on the AULCSF and individual spatial frequencies, adjusting for age and lens status. The AREDS nine-step classification system defined AMD presence and severity.

Results: Eight hundred twenty-three eyes were studied: 383 normal macular health, 203 early AMD, 208 intermediate AMD, and 29 late AMD (23 geographic atrophy, six neovascular AMD). AULCSF decreased with increasing AMD severity; this was mostly attributable to the late AMD eyes having lower AULCSF than other groups. Late AMD eyes had significantly worse sensitivity at 1 and 1.5 cyc/deg compared to all other groups, with other groups having similar sensitivity. At frequencies near or just beyond the CSF peak (3-12 cyc/deg), late AMD eyes again had the worst sensitivity, with intermediate AMD eyes having slightly worse sensitivity than early AMD eyes. There were no group differences at 18 cyc/deg.

Conclusions: The qCSF will be most useful in trials studying the transition from intermediate AMD to late-stage AMD, rather than in studying the transition from aging to early and intermediate AMD.

Purpose: Previously, we reported that the apicobasal polarity-determinant Pard3 regulates mouse corneal epithelial (CE) stratification and barrier function. Herein, we describe a complementary cell culture model to study the functions of PARD3 in greater detail.

Methods: PARD3-knockout (PARD3KO) cells were generated by CRISPR/Cas9-mutagenesis in hTCEpi, a telomerase-immortalized human CE cell line. We compared hTCEpi and PARD3KO cellular proliferation by CellTiter-Blue, crystal violet, xCELLigence Real Time Cell Analyzer, Ki67 staining and flow cytometry, extracellular matrix (ECM) affinity by adhesion assays and trypsinization time, actin polymerization by phalloidin staining, barrier function by Real-Time Cell Analyzer and FITC-dextran permeability assays, and relevant markers' expression by quantitative RT-PCR, immunoblots, and immunofluorescence.

Results: Deconvolution of sequencing data, immunoblots, and immunofluorescence confirmed PARD3 ablation in PARD3KO cells, which displayed 1.44- to 1.66-fold faster proliferation with a shorter doubling time of 19.06 hours compared with 21.84 hours for hTCEpi cells. The G1-S phase regulators CCND2, CCNE, and CDK2 were upregulated, while the mitosis-promoting factor components CCNB1 and CDK1 were downregulated in PARD3KO cells, consistent with their increased stalling at S phase revealed by flow cytometry. PARD3KO cells also displayed 0.5- to 0.8-fold weaker adhesion to different components of ECM, 1.37-fold faster trypsin-mediated detachment, 1.4-fold faster gap filling, disrupted actin cytoskeleton, nuclear β-catenin, upregulated epithelial-mesenchymal transition markers ZEB1, TWIST1, SLUG, and α-smooth muscle actin, and defective barrier function coupled with decreased expression of cell junctional components.

Conclusions: PARD3 regulates human CE cell proliferation, ECM-binding, migration, F-actin polymerization, and barrier function. Loss of PARD3 activity pushes CE cells toward epithelial-mesenchymal transition.

Purpose: To investigate effects of near work performed under short-wavelength "blue" and long-wavelength "red" light on choroidal thickness (CT) and axial length (AL).

Methods: Twenty-five participants (9 myopes, 16 nonmyopes), aged 20 to 36 years, completed four visits, consisting of a 30-minute near-work task (5 D demand) under broadband, blue (457 nm), and red (628 nm) light, and a control distance viewing condition under broadband light. Right eye CT and AL were measured before and after the task and after 30-minute recovery using optical coherence tomography (Spectralis) and biometry (Lenstar). Dynamic accommodative responses (n = 11) were recorded under each light condition (Grand Seiko).

Results: CT decreased and AL increased after near work in broadband (mean ± SEM CT: -11 ± 2 µm, AL: +10 ± 3 µm, P < 0.01) and red light (CT: -6 ± 1 µm, AL: +7 ± 2 µm, P < 0.01) but not under blue light or the control condition (P > 0.05 for all). After recovery, CT and AL returned to baseline, except in blue light, where AL decreased compared to baseline (AL: -4 ± 2 µm, P = 0.04). The accommodative response was significantly reduced under blue light (P < 0.01).

Conclusions: Near work under broadband and red light produced acute choroidal thinning and axial elongation. Blue light was associated with a posttask reduction in axial length and reduced accommodative response. Findings suggest that blue light may mitigate biomechanical stress on the posterior eye that is induced by sustained near work.

Purpose: To identify the basic genomic profile of Acanthamoeba, obtain information on Acanthamoeba endosymbionts, and analyze the correlation between these endosymbionts and the prognosis of Acanthamoeba keratitis (AK) patients.

Methods: Whole-genome sequencing was conducted on 30 cornea-derived Acanthamoeba strains. Pan-genome analysis was performed, and endosymbionts were identified by metagenomic analysis. Gimenez staining, fluorescence in situ hybridization, and transmission electron microscopy were used to prove the existence of endosymbionts. Linear discriminant analysis effect size was used to associate endosymbiont species with AK clinical prognosis. The correlation between the endosymbiont Ralstonia and pathogenicity was experimentally validated by assessing the biological characteristics of Acanthamoeba and by performing clinical and histopathological evaluations in AK mouse models.

Results: Whole genome sequencing revealed that the Acanthamoeba genome size was 37.1-105.0 Mb and GC content was 53.9%-60.5%. Pan-genomic analysis indicated an open state of the Acanthamoeba genome. Metagenomic analysis identified the presence of endosymbionts within Acanthamoeba, notably the endosymbiont Ralstonia, which was associated with poor prognosis at the genus level (P = 0.047). Acanthamoeba harboring the endosymbiont Ralstonia exhibited an increased migration area, enhanced adhesion, and had a more pronounced cytopathic effect. The size of clinical scores and corneal ulcers showed a significant increase in mouse models induced by Acanthamoeba with endosymbiont Ralstonia.

Conclusions: Whole-genome sequencing highlighted the symbiotic relationship between Acanthamoeba and associated microorganisms. The presence of the endosymbiont Ralstonia influenced the biological characteristics of Acanthamoeba and was correlated with clinical poor prognosis in AK, suggesting its potential as a target for clinical intervention.

Purpose: Mitochondria are highly dynamic organelles that continuously undergo fission and fusion, and their dysfunction is associated with various age-related disorders. This study aimed to elucidate the role of mitochondrial fission in the development of choroidal neovascularization (CNV), a hallmark of neovascular age-related macular degeneration (AMD), and to evaluate the therapeutic potential of its pharmacological inhibition.

Methods: The murine CNV model was created by laser photocoagulation using C57BL/6J mice. Expression changes of mitochondrial fission-related protein during CNV development were examined using western blotting and immunofluorescence. To assess the effectiveness of pharmacological inhibition of mitochondrial fission, the effects of mitochondrial division inhibitor-1 (Mdivi-1) and mitochondrial fusion promoter (M1) were evaluated by CNV area measurement, fluorescein angiography, and western blot analysis. The pro-angiogenic mechanisms associated with mitochondrial fission were further investigated in RPE cells cultured under hypoxic condition.

Results: In a murine laser-induced CNV model, mitochondrial fission-related proteins increased in the retinal pigment epithelium (RPE)-choroid complex, and the high expression of phosphorylated dynamin-related protein 1 (DRP1) was observed in RPE cells surrounding the CNV lesion. Additionally, intravitreal injection of Mdivi-1 or M1 suppressed CNV formation, vascular leakage, and pro-angiogenic factor production. In RPE cells exposed to hypoxia, DRP1-mediated mitochondrial fission was rapidly activated, accompanied by increased mitochondrial reactive oxygen species production. Moreover, inhibition of mitochondrial fission suppressed mitochondrial bioenergetic dysfunction and the upregulation of vascular endothelial growth factor.

Conclusions: These findings support that pharmacological inhibition of activated mitochondrial fission could serve as a potential therapeutic approach for neovascular AMD.

Purpose: The purpose of this study was to characterize the early transcriptomic and pathologic changes of Fuchs endothelial corneal dystrophy (FECD) using the Col8a2Q455K/Q455K mutant mouse model.

Methods: The Col8a2Q455K/Q455K mutant mice were divided into the early-stage (≤2-month-old) group and the late-stage (≥8-month-old) group, based on corneal endothelial changes evaluated by slit-lamp microscopy, optical coherence tomography (OCT), and confocal microscopy. The corneal endothelial cells from early-stage mutant and age-matched wild-type (WT) mice were collected for transcriptomic analysis and validated by quantitative PCR and immunofluorescence staining.

Results: The Col8a2Q455K/Q455K mutant mice showed no observable corneal abnormality before 2 months of age. Morphological changes of the corneal endothelium appeared at 4 months and aggravated continuously with apparent corneal edema. However, when analyzing transcriptomic changes of the corneal endothelium, we found that the early-stage mutant mice exhibited 221 upregulated and 55 downregulated genes compared with age-matched WT mice; these differences were even more pronounced in the late-stage mutant mice. The upregulated genes predominantly enriched three signaling processes, including extracellular matrix (ECM) remodeling (e.g. Lgals3, Timp1, and Mmp3), endoplasmic reticulum (ER) stress (e.g. Hspa5, Dnajb9, and Atf3), and early activation of immune-related pathways (e.g. Icam1, Bpifb1, and C1q). Moreover, the qPCR and immunofluorescence staining further validated changes in gene and protein expressions prior to the morphological abnormalities in the mutant mice.

Conclusions: The Col8a2Q455K/Q455K mutant mice exhibit aberrant activation of ECM remodeling, ER stress. and immune responses in the corneal endothelium prior to observable pathogenic changes, providing the first in vivo evidence of potential early biomarkers and therapeutic treatments for FECD.

The cholesterol content in the membranes of the fiber cells of the human eye lens is significantly higher than in any other cell of the body. This review examines the existing literature on the origin and function of this unique feature as one of multiple factors that may help protect against age-related cataract formation throughout a person's life. Three independent sets of experimental data are highly suggestive that high cholesterol content in the fiber cell membranes may protect against cataract formation during aging: (1) saturating cholesterol content preserves the physical properties of the lipid bilayer of the lens cell membranes when the lipid composition of the bilayer changes; (2) high cholesterol content hinders the binding of cytoplasmic α-crystallin to the lipid membrane, which reduces light scattering; and (3) genetic upregulation of cholesterol biogenesis in zebrafish lenses protects against cataract formation in predisposed mutants, whereas administration of cholesterol-lowering statins cause cataracts to reappear.Understanding why the lens contains such high levels of cholesterol is essential for describing its fundamental biology, determining how environmental and genetic factors impact its transparency, and developing treatments for lens opacities. As humans age and are repeatedly exposed to oxidative stress and environmental damage, it is crucial for both researchers and clinicians to comprehend the mechanisms that protect against cataract formation.

Purpose: Aniridia, driven by PAX6 mutations, causes aniridia-associated keratopathy (AAK), a progressive condition linked to limbal stem cell deficiency. A major hurdle to developing targeted therapies for AAK is the incomplete understanding of the molecular abnormalities in affected corneas. To address this, we leveraged Pax6± (Pax6 het) mice, a model of AAK, and applied single-cell RNA sequencing (scRNA-seq) to profile the transcriptomic changes at a single-cell resolution.

Methods: ScRNA-seq of corneal/limbal tissues of wild type (WT) and Pax6 het mice were conducted. Immunostaining was performed to examine the expression of specific markers for stem cells.

Results: ScRNA-seq identified a quiescent limbal epithelial stem cell (LESC)-like cell cluster and an early transient amplifying cell (eTAC)-like cluster. An increase in the cell numbers in these two clusters in the Pax6 het mouse corneas was observed. Immunostaining detected a marked increase in markers for these two clusters including Tmem176b, Apoe, and Krt15 in the corneal epithelium of Pax6 het mice, suggesting an increase of these LESC/eTA-like cells into the corneal epithelium. The Pax6 deficiency inhibited the expression of genes involved in cell proliferation in the eTAC-like cluster as well as the expression of genes related to corneal epithelial cell fate and differentiation compared with WT mice.

Conclusions: Our single cell transcriptome of the limbus and cornea of Pax6 het mice indicates that AAK may be due to the increase of dysfunctional stem/eTACs with defects in committing to a corneal epithelial cell fate and differentiation.

Purpose: This study aimed to investigate the role of sensory neuropeptide calcitonin gene-related peptide (CGRP) in postrefractive surgery dry eye caused by corneal nerve severing in mice.

Methods: A mouse model was established in adult C57BL/6J mice by surgical corneal severing. Additional validation was performed using a photorefractive keratectomy (PRK) model. Postoperative evaluations included corneal sensitivity, corneal nerve density, tear secretion, corneal fluorescein staining, immunofluorescence staining, and corneal RNA sequencing. Transient receptor potential melastatin 8 (TRPM8) of the trigeminal ganglia (TG), c-FOS, and in vivo electrophysiologic recording of the brain superior salivatory nucleus (SSN) were determined. The therapeutic efficacy of CGRP and an engineered CGRP (eCGRP) was assessed in treating postrefractive surgery dry eye.

Results: Corneal nerve severing led to a significant reduction of corneal CGRP contents, accompanied by the development of dry eye symptoms, including tear secretion reduction, epithelial barrier dysfunction, and subbasal nerve degeneration. These pathologic alterations were effectively rescued by prophylactic CGRP and significantly attenuated by therapeutic CGRP via enhancing the intact neural circuit of tear secretion. Compared with native CGRP, topical application of eCGRP exhibited superior efficacy of increasing tear secretion, restoring epithelial homeostasis, and reducing corneal inflammation, without pain-related side effects. In the laser-based PRK model, eCGRP similarly alleviated dry eye, confirming its therapeutic efficacy in a more clinically relevant surgery.

Conclusions: Our data demonstrate that CGRP expression in the cornea is downregulated in postrefractive surgery dry eye through decreasing TRPM8-SSN activity. eCGRP may provide an effective therapeutic approach for its prevention and treatment.