Investigative ophthalmology & visual science最新文献

Purpose: Mitochondria regulate cellular activity in a tissue-selective manner. The role of mitochondria in corneal fibrosis is elusive. This study investigated changes in mitochondrial DNA (mtDNA) and mitochondrial transcription factor A (TFAM) in human corneal stromal fibroblasts (CSFs) and corneal myofibroblast (CMFs) and effects on corneal fibrosis in vitro and ex vivo.

Methods: Healthy donor human corneas were used to generate CSFs and ex vivo culture. CMF formation was induced by transforming growth factor beta-1 (TGFβ1) in vitro and human cornea by nitrogen mustard (NM) ex vivo. mtTFA/TFAM CRISPR/Cas9 KO plasmid, Lipofectamine CRISPRMAX, and TrueCut Cas9 Protein v2 were used for gene editing. Long-range PCR and quantitative reverse-transcription PCR (qRT-PCR) measured mtDNA transcription, mtDNA quantity, and ratios of mtDNA to nuclear DNA (nDNA). Immunofluorescence and immunoblotting quantified protein expression. The MitoSOX assay was used to analyze mitochondrial reactive oxygen species (mtROS).

Results: Human CMFs showed significantly reduced mtDNA copies (P < 0.01) and mtDNA-to-nDNA ratios (P < 0.05) compared to CSFs. Significant time-dependent increases in mRNA levels of α-smooth muscle actin (αSMA) and nDNA-transcribed genes and decreases in TFAM and mtDNA-transcribed genes were noted during CSF transdifferentiation to CMFs (P < 0.05, P < 0.001, or P < 0.0001). Correspondingly, time-dependent decreases in TFAM and increases in Rieske iron-sulfur (Fe-S) and αSMA protein (P < 0.0001) and mtROS and ROS levels (P < 0.0001) were observed. TFAM silencing arrested fibrotic events and exhibited reduced αSMA and enhanced mtDNA (P < 0.001). The NM-induced fibrotic human cornea showed decreased TFAM and increased αSMA compared to naïve corneas (P < 0.01).

Conclusions: We observed that mtDNA plays an important role in corneal fibroblast transdifferentiation to myofibroblast and that TFAM has the potential to modulate this process in an injured cornea. Additional studies are warranted.

Purpose: Optic nerve head (ONH) swelling, a critical feature in idiopathic intracranial hypertension (IIH) and non-arteritic anterior ischemic optic neuropathy (NAION), can present diagnostic challenges. We explored a multimodal deep learning (DL) approach integrating optical coherence tomography (OCT) scans and fundus photographs to enhance diagnostic accuracy for differentiating IIH, NAION, and healthy eyes.

Methods: We developed two separate models using 7019 OCT scans (3D-ResNet-18) and 17,657 fundus photos (ResNet-50) to classify eyes with papilledema (2315 OCT, 6349 fundus), NAION (841 OCT, 1814 fundus), and healthy eyes (3863 OCT, 9494 fundus). We arranged the dataset so that the test set consisted entirely of same-day OCT scans and fundus photos, with each modality (OCT and fundus) contributing at least 15% of the data for each class. We combined output probabilities from both models using two methods: an F1-weighted sum by class (F1WS), as well as an XGBoost model. Performance of each was evaluated with AUC-ROC, accuracy, precision, recall, and F1 scores.

Results: The OCT model alone achieved a test accuracy of 93.5%, with the fundus photo model reaching 93.9%. The multimodal F1WS and XGBoost models achieved an accuracy of 97.5% and 98.3%, respectively.

Conclusions: Combining OCT and fundus photographs improves the classification of IIH, NAION, and healthy eyes, showing the value of using complementary imaging modalities. This approach supports the use of DL to aid diagnosis and clinical management of optic nerve head swelling. It may also be extended to leverage DL from additional data sources, such as macular scans or visual field tests.

Purpose: To characterize proteomic profiles and underlying biological processes in vitreous humor and plasma of patients with proliferative diabetic retinopathy (PDR), diabetic patients without retinopathy (Non_DR), and non-diabetes mellitus patients (Non_DM), and to identify the molecular endotypes within PDR patients.

Methods: Proteomic profiling of paired vitreous humor and plasma samples from 47 PDR patients, 26 Non_DR patients, and 48 Non_DM patients were conducted with Olink platform. The Olink platform includes 13 panels targeting 1161 proteins. Gene set enrichment analysis was applied for enriched pathways, and the K-means clustering method was used to identify different PDR clusters based on vitreous proteomic profiles.

Results: Proteomic analysis revealed significant differences in the vitreous humor of PDR patients compared to those in the Non_DR or Non_DM groups, with elevation of carbonic anhydrase (CA) family members as potential contributors to PDR pathophysiology. Plasma samples from PDR group exhibited less profound differences in proteomic profiles compared to the other two groups. Clustering analysis of PDR vitreous samples identified three distinct clusters as molecular endotypes of PDR patients. Of those, Cluster 3 was characterized by enrichment in CCL/CXCL/IL6/IL18 chemokines and pro-inflammatory signaling pathways, which may contribute to more severe PDR phenotypes.

Conclusions: Significant differences in proteomic profiles were observed in PDR patients, especially in vitreous samples, with CA family members identified as potential therapeutic targets for PDR. Endotyping analysis of vitreous samples uncovered unique patient population with enriched CCL/CXCL/IL6/IL18 inflammatory pathways, highlighting the significance of local protein signature changes in PDR disease heterogeneity and its potential applications in patient stratification and therapeutic treatment.

Purpose: Aniridia-associated keratopathy (AAK) leads to loss of corneal transparency because of epithelial, inflammatory, and pathological vascular changes. Here, we sought to understand this process at the transcriptomic level while evaluating an experimental pharmacotherapy for potential modulatory effects.

Method: 17 Pax6+/- Small-eye (Sey) heterozygous mice with p.Gly208* Pax6 mutation and 10 wild-type 129S1/SvImJ mice at four months of age were examined to identify dysregulated genes and pathways in established AAK. We next evaluated the potential efficacy of 10 µM duloxetine administered as eye drops twice daily for 90 days, assessing outcomes at the transcriptomic level via microarray and protein level with Western blot and immunostaining.

Results: Transcriptomic analysis of the cornea revealed enrichment of Ccl21 gene family members associated with lymphangiogenesis, along with upregulation of genes involved in inflammation, cell adhesion, differentiation, motility, and keratinization, and downregulation of drug metabolism with significantly dysregulated genes emerging as potential therapeutic targets, including Gpha2, Chrnb3, Epgn, Cnfn, kallikreins and inflammation mediators Il18r1 and classical complement factors. Duloxetine therapy failed to regress AAK in adult corneas; however, transcriptomic profiling indicated duloxetine suppressed inflammatory genes and promoted anti-inflammatory and protective activity while modulating drug metabolism, suggesting potential beneficial effects in the cornea.

Conclusions: Transcriptomics reveals multiple unexplored pathways and genes altered in the AAK mouse model. Although clinical results with duloxetine are promising, our current regimen and delivery method did not improve established disease. Duloxetine's therapeutic potential requires further study.

Purpose: To evaluate the therapeutic efficacy of an adeno-associated virus serotype DJ (AAV-DJ) vector delivering MYOC-targeting short-hairpin RNA (shMYOC) in a MYOCP370L transgenic glaucoma mouse model (Tg-MYOCP370L) for the treatment of open-angle glaucoma (OAG) associated with MYOC mutations.

Methods: An AAV-DJ vector, selected for its high transduction efficiency and tropism for trabecular meshwork (TM), was used to deliver shMYOC via a transpupillary intravitreal approach in Tg-MYOCP370L mice. Post-treatment evaluations included myocilin accumulation, ER stress marker expression, intraocular pressure (IOP), aqueous humor outflow facility, retinal ganglion cell (RGC) survival, and visual function.

Results: AAV-DJ-shMYOC markedly reduced myocilin accumulation and ER stress markers in TM cells in vivo, effectively preventing age-dependent IOP elevation, preserving aqueous humor outflow facility, and maintaining RGC survival and visual function in young Tg-MYOCP370L mice. In aged Tg-MYOCP370L mice, AAV-DJ-mediated MYOC silencing similarly lowered IOP and improved outflow facility.

Conclusions: AAV-DJ-mediated MYOC silencing effectively alleviated glaucomatous pathology in Tg-MYOCP370L mice, highlighting its potential as a gene therapy strategy for myocilin-associated glaucoma.

Background: The ciliary muscle, a critical intraocular smooth muscle, plays a potent role in ocular accommodation. Investigating the potential detrimental effects on the ciliary muscle during prolonged contraction and precise mechanism underlying the cell damage hold significance in treating ciliary muscle dysfunction. The effect and mechanism of vitamin E (VitE), an antioxidant, in mitigating these adverse effects of prolonged contraction remains to be thoroughly elucidated.

Methods: A guinea pig model of prolonged contraction of the ciliary muscle was established through topical administration of carbachol, and primary ciliary muscle cells isolated from guinea pigs were used for in vitro experiments.

Results: The ophthalmic examination results demonstrated that prolonged contraction of the ciliary muscle impaired accommodative function in guinea pigs. This condition may lead to disrupted cellular migration, intracellular adenosine triphosphate depletion, decreased mitochondrial membrane potential, and reactive oxygen species accumulation. Further examination revealed that carbachol induced darker-stained mitochondrial membranes and diminished cristae density, consistent with morphological features of ferroptosis. Moreover, sustained cell contraction modulated specific contraction-related proteins (α-smooth muscle actin), concurrently decreased the expression of antioxidant proteins (glutathione peroxidase, superoxide dismutase, catalase, and GSH) in both tissue specimens and cells, culminating in ferroptosis in vivo and in vitro experiments. Our findings demonstrated that pretreatment with VitE alleviated oxidative injury and mitigated ferroptosis. Additionally, knocking down acetyl-coenzyme A thiosterase 7 attenuated the beneficial effect of VitE.

Conclusions: These findings collectively indicated that VitE presented a viable approach to ameliorate oxidative stress and ferroptosis induced by prolonged contraction of the ciliary muscle via activating acetyl-coenzyme A thiosterase 7.

Purpose: The pathological effects of pre-mRNA processing factor 8 (PRPF8) mutations on the retinal pigment epithelium (RPE) are not fully understood. We aimed to identify disease-specific cellular and molecular phenotypes in PRPF8 retinitis pigmentosa (RP) patient-derived induced pluripotent stem cell (iPSC)-RPE and to test whether adenine base editing (ABE), which corrects the PRPF8 mutation in iPSCs, can reverse abnormal RPE phenotypes.

Methods: We obtained patient-derived iPSCs with the heterozygous PRPF8 (c.5792C>T) mutation and created an induced mutation iPSC line by introducing the same mutation into wild-type iPSCs using CRISPR/Cas9. These cells were differentiated into RPE cells. We measured PRPF8 expression, barrier integrity, and apicobasal polarity. Electron microscopy examined apical microvilli and pigment granules. RNA sequencing quantified splicing events and affected pathways. ABE corrected the PRPF8 mutation in patient iPSCs, and the corrected clones were re-differentiated into RPE cells for evaluation.

Results: PRPF8-mutant RPE cells exhibited decreased PRPF8 mRNA and protein levels, weakened barrier function, and disrupted cell polarity. Ultrastructural analysis showed loss of apical microvilli and pigment granules. Transcriptomic analysis identified abnormal splicing events, with enrichment in cilium assembly and melanosome pathways. ABE correction restored PRPF8 expression, normalized barrier integrity, apicobasal polarity, and rescued the defects in apical microvilli and pigment granules.

Conclusions: PRPF8 mutations in patient-derived iPSC RPE cause functional and ultrastructural defects driven by splicing abnormalities. ABE correction of the PRPF8 mutation in iPSCs can restore PRPF8 expression and alleviate cellular and molecular defects in RPE and highlights the therapeutic potential of precise gene editing correction strategies for RP.

Purpose: Levodopa (L-DOPA), a precursor for melanin and dopamine, has been linked to reduced intravitreal injection burden and delayed onset of neovascular age-related macular degeneration (AMD). Further, L-DOPA and dopamine receptor D2 (DRD2) agonists inhibit laser-induced choroidal neovascularization (CNV). However, the contributions of endogenous versus exogenous L-DOPA signaling, their effects in alternative CNV models, and the contributions of DRD2 versus GPR143, the receptor for L-DOPA, signaling remain unresolved.

Methods: Choroidal sprouting assays (CSA) were performed using wild-type (WT) and tyrosinase-mutant (Tyr-/-) mice with and without dopamine pathway agonists and antagonists. CNV number and area were measured in pigmented Vldlr-/- and albino Vldlr-/-Tyr-/- mice with and without L-DOPA or the DRD2 agonist quinpirole.

Results: Endogenous L-DOPA deficiency (Tyr-/-) did not affect CSA sprouting or CNV in Vldlr-/- mice. Exogenous L-DOPA suppressed angiogenesis ex vivo in both WT and Tyr-/- choroidal explants in a dose-dependent manner. In vivo, L-DOPA reduced CNV lesion number, lesion area, and macrophage infiltration in albino but not pigmented Vldlr-/- mice. Dopamine and quinpirole produced modest anti-angiogenic effects, and eticlopride partially reversed L-DOPA inhibition in choroidal explants. Quinpirole suppressed CNV lesion number, lesion area, and macrophage infiltration in pigmented Vldlr-/- mice.

Conclusions: Our findings show that L-DOPA's anti-angiogenic effects are exogenous, more effective in tyrosinase-mutant mice, and mediated by both the DRD2 and non-DRD2 pathways, potentially GPR143. The saturation of GPR143 signaling in pigmented eyes provides a mechanistic basis for reduced responsiveness, highlighting the importance of pigmentation biology in the development of L-DOPA-based therapeutics.

Purpose: Macular degeneration impairs central vision, compelling patients to use their peripheral vision for reading, which is difficult due to reduced spatial resolution and crowding. Although perceptual learning improves reading, single-session anodal transcranial direct current stimulation (a-tDCS) over the visual cortex has shown inconsistent outcomes, with transient improvements observed in English reading but no benefit for Chinese reading in macular degeneration patients. This randomized controlled trial investigated whether combining multi-session a-tDCS with perceptual learning enhances Chinese reading performance in these patients compared to sham stimulation.

Methods: Twenty Chinese-reading patients with macular degeneration (39-90 years old) were randomized to receive either active (n = 10) or sham (n = 10) a-tDCS during six sessions of rapid serial visual presentation (RSVP) reading training. Trained outcomes (RSVP reading) and untrained functions (sentence reading, crowding, contrast sensitivity, and visual acuity) were compared at baseline, 1 day, and 1 month post-training.

Results: Perceptual learning significantly improved RSVP reading speed (P < 0.001) in both groups, with effects lasting at least a month. No additive effect of active versus sham a-tDCS was observed (group × time P = 0.99). Transfer effects to untrained functions were limited to visual acuity and critical print size for sentence reading.

Conclusions: Perceptual learning enhances Chinese reading performance in individuals with macular degeneration, but a-tDCS confers no additional benefit. This contrasts with previous results where non-invasive brain stimulation enhanced English reading in macular degeneration. The results emphasize the need for more refined neuromodulation strategies for improving logographic reading.

Purpose: Micropulse laser trabeculoplasty (MLT) is an established treatment for primary open-angle glaucoma; however, the mechanisms underlying its IOP-lowering effect remain incompletely understood, particularly in vivo. We investigated the mechanisms of MLT in vivo, with a focus on the trabecular meshwork outflow pathway.

Methods: Pigmented rabbits received MLT at 0 to 1500 mW, and IOP was monitored for up to 7 days posttreatment. Outflow facility was assessed, and aqueous outflow was visualized using fluorescent dextran tracer imaging. Angle tissues were collected at 1 and 7 days for quantitative RT-PCR analysis of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and extracellular matrix (ECM) components. Aqueous humor samples were analyzed for MMP enzymatic activity.

Results: At 1000 mW, IOP was significantly reduced on day 7, and at 1500 mW, it was significantly reduced on days 3 and 7. Aqueous outflow facility was significantly increased in the 1000- and 1500-mW irradiated eyes. Tracer imaging consistently demonstrated stronger signal intensity near the angle in the 1500-mW irradiated eyes. Gene expression analysis exhibited significant upregulation of MMP-1, -3, and -9 at day 1, with sustained elevation of MMP-9 at day 7 after the 1500-mW treatment. TIMP-1 was transiently elevated, whereas TIMP-2 remained unchanged. Aqueous humor MMP activity was significantly increased at 1 day after 1500-mW irradiation.

Conclusions: MLT induced transient MMP activation, enhanced aqueous outflow, and reduced IOP. These findings imply that MLT lowers IOP in pigmented rabbits by promoting ECM remodeling through MMP upregulation.