Investigative ophthalmology & visual science最新文献

Purpose: To investigate how chronic psychological stress alters circadian, immune, and lipid regulatory networks in meibomian glands (MGs) and to assess the efficacy of β-adrenergic blockade in mitigating these effects.

Methods: Male C57BL/6J mice were exposed to daily 4-hour restraint stress for 14 days. Experimental groups included control, stress alone, stress with propranolol (a nonselective β-adrenergic receptor antagonist), and stress with metyrapone (a glucocorticoid synthesis inhibitor). MGs were collected at 3-hour intervals across a 24-hour period for bulk RNA sequencing. Additional analyses included single-cell RNA sequencing, untargeted lipidomics, and immunohistochemistry. Circulating levels of corticotropin-releasing hormone, adrenocorticotropic hormone, corticosterone, norepinephrine, and epinephrine were measured. Adrenal glands and superior cervical ganglia were examined to evaluate neuroendocrine activation.

Results: Chronic stress activated both the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis, leading to broad transcriptional reprogramming in MGs. Circadian gene expression became phase-dispersed, immune-related pathways were suppressed, and lipid profiles shifted toward elevated triglycerides and reduced phospholipid content. Propranolol, but not metyrapone, partially restored circadian rhythmicity, immune signaling, T-cell infiltration, and lipid composition. Increased proliferation of MG epithelial cells under stress was reduced by propranolol. β-Adrenergic receptors ADRB1 and ADRB2 were localized to MG epithelial subsets.

Conclusions: Chronic psychological stress disrupts MG homeostasis through sympathetic overactivation, affecting circadian regulation, immune responses, and lipid metabolism. β-Adrenergic blockade partially reverses these changes, highlighting a potential therapeutic approach for stress-related evaporative dry eye.

Purpose: To investigate whether the alternative pathway (AP) of complement inhibition restores the epithelial phenotype and ameliorates dry AMD biomarkers in AMD patient induced pluripotent stem cell-derived RPE (iPSC-RPE) monolayers.

Methods: iPSC-RPE monolayers were established from an AMD patient iPSC line; ARPE-19 monolayers were used for comparison. For AP inhibition, monolayers were treated with CR2-fH (fusion protein composed of the iC3b/C3d/C3dg-binding region of complement receptor type 2 [CR2] linked to the inhibitory domain of human factor H [fH]) either as a soluble protein or by adeno-associated virus transduction. Complement challenge used 5% complement competent human serum (CC-HS); 5% heat-inactivated serum served as a control. Membrane attack complex, apolipoprotein E, lipid deposits, and autophagy were analyzed by immunostaining. Morphology and integrity were evaluated in brightfield images, using zonula occludens-1 (ZO-1) immunostaining, followed by ImageJ analysis, and transepithelial electrical resistance measurements.

Results: iPSC-RPE monolayers expressed the RPE-specific markers zonula occludens-1 and RPE65 and exhibited a honeycomb pattern, as confirmed by nearest-neighbor analysis. Exposure to CC-HS significantly reduced transepithelial electrical resistance; increased membrane attack complex, apolipoprotein E, and lipid deposits; and altered levels of autophagy-related proteins. Preincubation with soluble CR2-fH or CR2-fH expression reversed these features. Quantitatively, similar changes were observed in ARPE-19 cell monolayers.

Conclusions: The AP inhibitor CR2-fH delivered either as a soluble protein or via gene therapy is efficacious in preventing serum-induced complement activation that led to monolayer disruption and AMD-like pathology in iPSC-RPE cells. These results contribute to existing data highlighting the importance of the AP of complement in dry AMD pathology and its potential role in AMD treatment.

Purpose: The purpose of this study was to investigate the morphological and hemodynamic characteristics of the ophthalmic artery (OA) in ocular ischemic syndrome (OIS) and identify features potentially associated with disease pathogenesis.

Methods: This retrospective case-control study included 33 patients with OIS, 22 patients with internal carotid artery stenosis (ICAS), and 29 healthy controls. Clinical data and ophthalmic examinations were collected. The morphology and hemodynamics of the OA were quantified using three-dimensional (3D) reconstruction and computational fluid dynamics (CFD). Particle image velocimetry provided a qualitative consistency check for the CFD results.

Results: The OA diameter in the OIS group was significantly smaller than that in the control and ICAS groups. Compared with the control group, the OIS group showed lower blood flow velocity and wall shear stress (8.25 ± 5.34 Pa vs. 13.50 ± 6.24 Pa; P = 0.004) at the OA origin. A smaller OA diameter and lower wall shear stress at the OA origin were significantly associated with the presence of OIS. A low wall shear stress was observed at the OA origin regardless of the flow direction in the OIS. Patients with high-velocity retrograde OA flow present severe ocular ischemic manifestations.

Conclusions: Low wall shear stress may be a consistent hemodynamic characteristic of eyes with OIS. This hemodynamic feature may induce inward remodeling of the OA, leading to further luminal narrowing and exacerbation of ocular ischemia. The presence of high-velocity retrograde OA flow may indicate a severe degree of ocular ischemia, highlighting the potential of OA hemodynamics for improving risk stratification and guiding management.

Purpose: Mitochondrial dysfunction is increasingly recognized as a pivotal factor in cancer pathogenesis. We thus explored the causal role of mitochondrial-related genes (MRGs) in uveal melanoma (UM) and the underlying mechanisms.

Methods: We performed Mendelian randomization (MR) analysis using 1693 cis-expression quantitative trait loci (cis-eQTLs) of MRGs as instrumental variables and genome-wide association data (GWAS) of UM. Colocalization analysis assessed whether gene expression and UM risk shared a common causal variant. Further, mediation MR and in vitro functional assays were used to validate key findings and explore their biological relevance. A molecular docking-based virtual screening strategy, followed by experimental validation, was used to identify potential therapeutic compounds.

Results: MR analysis identified 57 MRGs significantly associated with UM risk after Bonferroni correction (P < 0.05/1693). Among them, MTG2 or GTPBP5, encoding a mitochondrial ribosome-associated GTPase, showed strong colocalization with UM susceptibility (PP.H4 = 75%). Silencing MTG2 in UM cell lines induced PARP cleavage and markedly suppressed cell proliferation and colony formation. Mediation analysis revealed that mitochondrial DNA (mtDNA) heteroplasmy at chrM:567 (A:ACCCCCC) partially mediates MTG2's effect on UM (4%). Moreover, both genetically driven analysis and qPCR confirmed a positive association between MTG2 expression and mtDNA copy number. Notably, dimercaptosuccinic acid (DMSA), identified as a potential MTG2 inhibitor, reduced MTG2 levels and promoted apoptosis in UM cells.

Conclusions: Our integrative genomic and experimental approach uncovers MTG2 as a causal contributor to UM pathogenesis, potentially through modulation of mtDNA heteroplasmy and copy number. DMSA emerges as a promising therapeutic agent targeting mitochondrial dysregulation in UM.

Purpose: The purposes of this study were to assess whether optic nerve head (ONH) biomechanics, quantified by tissue strain, improves classification of progressive visual field (VF) loss patterns in glaucoma beyond morphology, and to use saliency maps to identify ONH regions associated with the predictions.

Methods: We recruited 249 patients with glaucoma (mean age 69 ± 5 years, 54% female patients). One eye per subject was imaged under (1) primary gaze and (2) primary gaze with IOP elevated to approximately 35 millimeters of mercury (mm Hg) via ophthalmo-dynamometry. Twelve subjects were excluded due to poor scan quality/limited lamina cribrosa (LC) visibility. Experts classified subjects into four categories based on the presence of specific visual field defects (VFDs): (1) superior nasal step (N = 26), (2) superior partial arcuate (N = 62), (3) full superior hemifield defect (N = 25), and (4) other/non-specific defects (N = 124). Automatic segmentation and digital volume correlation computed neural tissue and LC strains. Biomechanical and structural features were input to a PointNet model. Three classification tasks were performed to detect: (1) superior nasal step, (2) superior partial arcuate, and (3) full superior hemifield defect. Data were split 80/20 (train/test). Area under the curve (AUC) assessed performance. Saliency maps (an explainable artificial intelligence [XAI] technique) highlighted ONH regions most critical to classification.

Results: Models achieved AUCs of 0.77 to 0.88 across VFD classifications. The structure-only model reached an AUC of 0.83 ± 0.02 for superior arcuate defects, which significantly improved to 0.87 ± 0.02 (P < 0.05) with the addition of strain information, demonstrating that ONH biomechanics enhance prediction beyond morphology. Strain-sensitive regions were localized to the inferior and inferotemporal rim, expanding with increasing severity of VF loss.

Conclusions: ONH strain enhances classification of glaucomatous VF loss patterns. The neuroretinal rim, rather than the LC, was most critical, suggesting rim strain may play a dominant role in axonal injury and functional loss.

Purpose: Fuchs' endothelial corneal dystrophy (FECD) is the leading cause of corneal endothelial dystrophy. This study aimed to investigate the protective effects and mechanism of taurochenodeoxycholic acid (TCDCA) in FECD.

Methods: TCDCA levels were quantified in aqueous humor from patients with FECD and ultraviolet A (UVA)-induced FECD mice. Corneal endothelial cell (CEC) morphology and function were evaluated by optical coherence tomography (OCT) and ZO-1 staining following TCDCA treatment. In vitro, UVA-induced human corneal endothelial cells (HCECs) were treated with TCDCA, and cell viability, mitochondrial membrane potential, ATP, and reactive oxygen species (ROS) levels were measured. RNA sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) were used to explore molecular mechanisms, and the role of Ca²⁺ signaling was validated using the inhibitor 2-APB in vivo.

Results: Analysis of FECD aqueous humor revealed significantly elevated TCDCA levels. In UVA-induced mouse model, TCDCA administration ameliorated corneal endothelial dysfunction, as evidenced by reduced corneal thickness, increased endothelial cell density, and a lower percentage of abnormal cells. Further, in vitro studies revealed a concentration-dependent effect of TCDCA, with 100 µM TCDCA significantly enhancing cell viability, reducing ROS production, restoring mitochondrial membrane potential, and promoting ATP synthesis. RNA-seq and functional studies identified that TCDCA exerts its beneficial effects on corneal endothelial function by activating the calcium signaling pathway.

Conclusions: TCDCA demonstrated a protective effect on corneal endothelial cells during the pathogenesis of FECD. Therefore, TCDCA may be a promising novel therapeutic target for attenuating the progression of FECD.

Purpose: Recent evidence suggests that simultaneous inhibition of multiple diabetes-induced molecular abnormalities is a valuable approach toward inhibiting the development of early stages of diabetic retinopathy (DR). The methylxanthine, theophylline acts by multiple mechanisms in different diseases, and we investigated its effect on the development of lesions of early DR.

Methods: Wild-type C57Bl/6J male mice were made diabetic with streptozotocin at two to three months of age, and some were given theophylline or structurally related analogs for one to two months to assess effects on visual function and biochemical and physiological abnormalities in the retina, or for eight months to assess retinal vascular histopathology. Effects of theophylline on leukocytes were examined ex vivo, because leukocytes contribute to the retinal vascular pathology of diabetes.

Results: Retinal superoxide generation, expression of inflammatory proteins, leukocyte-mediated cytotoxicity against retinal endothelial cells, and degeneration of retinal capillaries were significantly increased in retinas of control diabetic mice, and theophylline inhibited each of these abnormalities. Of recognized actions of theophylline (adenosine receptor antagonist, antioxidant and anti-inflammatory agent, inducer of histone deacetylase activity, inhibitor of phosphodiesterases), the inhibition of phosphodiesterases, oxidative stress and inflammation seem most likely to account for the beneficial effects of the drug in DR. In contrast to the beneficial effect of theophylline on capillary degeneration, it did not inhibit the diabetes-induced dysfunction of the neural retina.

Conclusions: Theophylline significantly inhibited the degeneration of retinal capillaries in early DR and can help investigate mechanistic differences in the pathogenesis of retinal capillary degeneration and neural dysfunction in diabetes.

Purpose: Nystagmus involves abnormal head positions (AHPs) toward the null point for improving visual acuity. While surgical interventions are the standard therapy, the use of yoked prisms is a possibility not often used. Our team used low-value prisms in children to mitigate AHP.

Methods: Yoked prisms were tailored to the direction of head turn. Thirty-one children were reviewed. Outcomes were binocular best-corrected visual acuity (BBCVA) and AHP measures with and without prisms.

Results: Of the 31 patients aged 10.3 ± 3.6 years (16 male), 19 were diagnosed with idiopathic infantile nystagmus syndrome, 6 with oculocutaneous albinism, and 6 with other secondary nystagmus. Prism's average prescription was 3.9 ± 1.25 diopters. BBCVA for distance averaged 0.44 ± 0.22 logMAR, improving to 0.41 ± 0.23 logMAR (P = 0.001, small size effect). AHP reduced from 16.9° ± 9.9° to 4.6° ± 5.8° for distance vision (P < 0.0001) and 12.7° ± 12.9° to 3.8° ± 6.2° for near vision (P < 0.0001), both with large size effects. The angle of the prisms most frequently prescribed was 180° (26 cases), as well as 2 cases at 135°, 1 case at 30°, and 2 cases at 90°.

Discussion: Our study found an improvement in AHP using low-value prisms in a pediatric population with nystagmus. Allowing the use of primary gaze position, prisms may be a therapeutic option to reduce the physical and social difficulties from AHP in these cases.

Conclusions: This study provides the first quantitative assessment of the minimal prism values required to significantly reduce abnormal head posture in nystagmus, offering a valuable adjunctive treatment option for these patients.

Purpose: To quantitatively investigate disease progression and genotype-phenotype correlations in Stargardt disease patients harboring the common ABCA4 variant c.5882G>A p.(Gly1961Glu), aiming to evaluate the contribution of second ABCA4 variants to disease severity.

Methods: This multicenter study included 77 Stargardt disease patients uniformly carrying the ABCA4 c.5882G>A p.(Gly1961Glu) variant from Nijmegen, Bonn, and Basel. Ellipsoid zone (EZ) loss was measured using automated segmentation of optical coherence tomography images. Progression was assessed with linear mixed-effects models. Disease severity was quantified by the age-at-criterion EZ loss, defined as the estimated age when EZ atrophy reached 6.25 mm², allowing a comparison of the effects of different second alleles.

Results: A total of 52 patients were included in the study. The median age at first observation was 41 years (interquartile range, 27-52 years). The median age at criterion EZ loss across all patients was 43 years (interquartile range, 31-51 years). Substantial variability in the progression of EZ loss was observed among patients despite the shared c.5882G>A p.(Gly1961Glu) genotype. Differences in specific second ABCA4 variants were strongly associated with variation in disease progression, with some variants linked to markedly earlier progression (e.g., c.1957C>T, -50.7 years) and others associated with delayed progression (e.g., c.1648G>A, +19.8 years).

Conclusions: The ABCA4 c.5882G>A p.(Gly1961Glu) variant is associated with substantial interpatient variability in disease severity, heavily influenced by the nature of the second ABCA4 allele. These findings highlight the importance of precise genotypic characterization in prognostic counseling, clinical trial stratification, and the design of targeted gene therapies.

Purpose: Mutations in RPGRorf15 and PDE6B cause early-onset retinitis pigmentosa, yet the molecular factors driving photoreceptor degeneration remain poorly understood. Here, we characterize the pathological accumulation of RNA:DNA hybrids (R-loops) in two canine disease models-xlpra2/RPGRorf15 and rcd1/PDE6B-to provide new insights into disease pathogenesis.

Methods: Archival canine retinal tissues were used for immunohistochemistry, TUNEL, Western blotting, and PCR. Outer nuclear layer regions were isolated via laser-capture microdissection to assess gene expression changes.

Results: R-loop dynamics paralleled progression of apoptosis in both models. Most TUNEL-positive photoreceptor nuclei were also positive for R-loops and m⁶A RNA modification, suggesting potential crosstalk. Near peak apoptosis, colabeled nuclei accounted for ∼68% to 79% in xlpra2 (7-8 weeks old) and 59% to 78% in rcd1 (3-6 weeks old), although TUNEL-only nuclei were also present. At later stages (16 weeks), colabeling ranged from 76% to 100% in xlpra2 and 52% to 53% in rcd1. By 16 weeks, R-loop accumulation declined in both models, whereas γH2AX, a marker of early DNA double-strand breaks, increased. Downregulation of genes controlling R-loop metabolism was evident at 6 to 7 weeks, including CPSF6 and XRN2 (both models), as well as BRD4 and SF3B1 in xlpra2 and DHX9 in rcd1. These alterations may partly underlie the disease-associated splicing defects observed in RPGRorf15 pre-mRNA, encoded by the R-loop-prone ORF15 locus.

Conclusions: Photoreceptor degeneration in xlpra2 and rcd1 is accompanied by overlapping molecular features, including early R-loop- and m⁶A-associated apoptosis with concurrent transcriptional alterations in R-loop-regulatory genes, followed by a progressively R-loop-independent DNA damage response.