Experimental eye research最新文献

Diabetic Retinopathy (DR), a leading complication of diabetes mellitus, has long been considered as a microvascular disease of the retina. However, recent evidence suggests that DR is a neurovascular disease, characterized by the degeneration of retinal neural tissue and microvascular abnormalities encompassing ischemia, neovascularization, and blood-retinal barrier breakdown, ultimately leading to blindness. The intricate relationship between the retina and vascular cells constitutes a neurovascular unit, a multi-cellular framework of retinal neurons, glial cells, immune cells, and vascular cells, which facilitates neurovascular coupling, linking neuronal activity to blood flow. These interconnections between the neurovascular components get compromised due to hyperglycemia and are further associated with the progression of DR early on in the disease. As a result, therapeutic approaches are needed to avert the advancement of DR by acting at its initial stage to delay or prevent the pathogenesis. Non-coding RNAs (ncRNAs) such as microRNAs, piwi-interacting RNAs, and long non-coding RNAs regulate various cellular components in the neurovascular unit. These ncRNAs are key regulators of neurodegeneration, apoptosis, inflammation, and oxidative stress in DR. In this review, research related to alterations in the expression of ncRNAs and, correspondingly, their effect on the disintegration of the neurovascular coupling will be discussed briefly to understand the potential of ncRNAs as therapeutic targets for treating this debilitating disease.

Currently, research on optic nerve injury predominantly focuses on the retina and optic nerve, but emerging evidence suggests that optic nerve injury also affects advanced visual structures like the superior colliculus (SC) and primary visual cortex (V1 region). However, the exact mechanisms have not been fully explored. This study aims to investigate the characteristics and mechanisms of pathology in the SC and V1 region after optic nerve crush (ONC) to deepen our understanding of the central mechanism of visual injury. After unilateral ONC, visual acuity in the injured eye declined, along with thinning of the retinal nerve fiber layer, and the latency and amplitude of FVEPs decreased. Furthermore, neuronal loss and degeneration were observed in the contralateral SC and V1 region, accompanied by astrocytic activation. Additionally, protein markers C3, and Serping1 for A1 astrocytes, which had neurotoxic effects and S100A10, and PTX3 for A2 astrocytes, which promoted tissue repair, were increased in the two regions. A1 astrocytes were mainly present in the early stages of observation, while A2 astrocytes were mainly increased later. Notably, NLRC4, GSDMD-N, cleaved caspase-1 expression, and IL-1β, IL-18 secretion increased in the contralateral SC and V1 region. Collectively, our findings reveal that A1 (neurotoxic) and A2 astrocytes (neuroprotective), NLRC4-mediated neuronal pyroptosis are enhanced in SC and V1 region contralateral to the ONC eye. The primary visual cortex responds to injury later than the superior colliculus after ONC, with less pronounced damage changes. Reactive astrocytes and NLRC4 inflammasome may act as promising targets for the prevention and treatment of optic nerve injury.

Radiotherapy is one of the conventional treatments for head and neck malignancies. Despite the implementation of protective measures to minimize the detrimental impact on healthy tissues surrounding the radiation site, radiation keratopathy remains a prevalent complication. We aimed to establish a mouse model of radiation keratopathy to characterize the pathophysiology of the disease and enable future identification of potential treatments. Thirty-six mice were divided equally into six groups. One eye of each mouse was irradiated with 5, 10, 15, 20, 25, and 30 Gy and the other eye used as a control. The mice were clinically monitored for one year, at which time eyes were tested using anterior segment optical coherence tomography, then the mice were euthanized, and the corneas dissected. Corneal sections were stained with hematoxylin and eosin, β-galactosidase, and CK12. The results indicated that animals experiencing increased doses of radiation had increased corneal vascularization, fibrosis, and opacity and conjuctivalization and a higher number of positive results of beta-galactosidase staining, which indicates an increase in the tendency of senescence. The results of β-III tubulin staining indicated that the density of corneal stromal nerves and the subepithelial nerve plexus decreases as the dose increases. Also, as the irradiation dose increases, the central corneal thickness decreases as well.

Constitutional variants in the RB1 gene predispose individuals to the development of Retinoblastoma (RB) and the occurrence of second tumors in adulthood. Detection of causal RB1 gene variants is essential to establish the genetic diagnosis and to performing familial studies and counseling. In our cohort of 579 Spanish RB patients, 15% of cases suspected to have a genetic origin remained negative after traditional Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) of RB1 gene, likely due to the possibility of mosaicism or non-coding variants. A specific next-generation sequencing (NGS) gene panel was designed to analyze the complete sequence of the RB1 gene. While many familial RB cases showed variants through Sanger and MLPA, the analysis of 65 available sporadic RB patients using the NGS gene panel identified a causative variant in an additional 6 of 26 (23%) bilateral cases and 6 of 39 (15.4%) unilateral cases. Seven of these cases exhibited different degrees of mosaicism (26%, 20%, 15.8%, 8%, 6%, 5.9% and 3%) while 5 cases had heterozygous deep intronic variants, all of them previously described in RB patients. Additional cases with suspected variants, not detected in blood but present in tumor tissue, were also analyzed using NGS PCR amplicons, and mosaicism was confirmed in other 10 sporadic cases. Altogether, the use of NGS increased the diagnostic yield, particularly for patients with sporadic RB in 10 bilateral cases and in 12 unilateral cases.

Senescent retinal pigment epithelial cells play a key role in neovascular age-related macular degeneration (nAMD); however, the mechanisms underlying the angiogenic ability of these cells remain unclear. Herein, we investigated the effects of the senescent adult retinal pigment epithelial cell line-19 (ARPE-19) on wound healing, cell migration and survival, and tube formation abilities of human umbilical vein endothelial cells (HUVECs). Additionally, we used Brown Norway rats to establish a laser-induced choroidal neovascularization (CNV) model for further nAMD-related studies. We found that the wound healing, cell migration, and tube formation abilities of HUVECs were significantly enhanced following culture in conditioned media from senescent ARPE-19 cells; this was attributed to the activation of the transforming growth factor β-activated kinase 1 (TAK1)/p38 MAPK pathway. Consistently, we found that the TAK1 inhibitors 5Z-7-oxozeaenol and takinib reversed the effects of conditioned media from senescent ARPE-19 cells on the wound healing, migration, survival, and tube formation abilities of HUVECs. We further investigated the therapeutic effects of 5Z-7-oxozeaenol on the laser-induced CNV rat model. We found that TAK1 was activated in IB4+ areas in laser-induced CNV lesions; inhibiting the activity of TAK1 using 5Z-7-oxozeaenol significantly alleviated CNV lesion formation and fluorescein leakage in fundus fluorescein angiography and greatly improved a-waves, b-waves, and OP values, as recorded by electroretinography. Thus, senescent RPE cells may promote angiogenesis via the TAK1/p38 MAPK pathway. Further, inhibiting TAK1 expression alleviates pathological neovascularization and improves retinal function in a laser-induced CNV rat model, highlighting the therapeutic potential of this approach for treating nAMD.

Purpose: Keratin contamination is a common problem in mass spectrometry proteomic analyses, particularly in bottom-up mass spectrometry. The purpose of this study was to determine the protein contaminants introduced during the proteomic analysis of tear fluid.

Methods: Human tear fluid samples were collected using Schirmer strips. Proteomic analyses were performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on blank Schirmer strips and tear fluid samples, with empty vials serving as controls for assessing environmental contaminant proteins.

Results: We detected 26 contaminant proteins (18 keratins and 8 non-keratins). 98.2% of the total protein contamination can be attributed to the 9 keratins, including KRT10 (23.6%), KRT1 (23.5%), KRT2 (15.7%), KRT14 (7.6%), KRT16 (7.0%), KRT5 (6.1%), KRT9 (5.9%), KRT6B (4.6%), and KRT6A (4.3%). A comparison to the proteomic profile of blank Schirmer strips and controls (empty vials) found a strong correlation (R² = 0.9753), indicating that these proteins were not from the blank Schirmer strips but are environmental contaminants. On the other hand, several keratins including KRT19, KRT13, KRT4, KRT7, KRT15, KRT8 and KRT18 were present in tear fluid, but either not detected or were negligible in blank strips. Another set of keratins, including KRT5, KRT6A, KRT14, KRT16, and KRT17, were identified as components of tear fluid as well as environmental contaminants.

Conclusions: This study revealed nine major contaminant keratins in the mass spectrometry analysis. Several other keratins were identified as constituents of tear fluid. Background subtraction is necessary for the accurate analysis of tear fluid using mass spectrometry.

The morphology and thickness of the retinal layers are valuable biomarkers for retinal health and development. The retinal layers in mice are similar to those in humans; thus, a mouse is appropriate for studying the retina. The objectives of this systematic review were: (1) to describe normal retinal morphology quantitatively using retinal layer thickness measured from birth to age 6 months in healthy mice; and (2) to describe morphological changes in physiological retinal development over time using the longitudinal (in vivo) and cross-sectional (ex vivo) data from the included studies. A PubMed search was conducted for articles published from to 1980-2024 that included quantitative data. Prior to sexual maturity, an increase in the total retinal and inner plexiform layer thicknesses were observed, with a decrease in the inner nuclear layer thickness. After sexual maturity, an asymptotic decrease in thickness was observed up to age 6 months in all layers; during this period, no significant changes were observed in the outer nuclear layer or nerve fiber layer/ganglion cell layer complex. Potential sources of variability and inconsistency among the studies included differences in imaging modality, animal strain, measurement timing, and retinal segmentation/assignment techniques. These findings highlight the importance of including a control group in experimental designs and providing comparative data for further investigations.

Retinal damage accounts for irreversible vision loss following ocular alkali burn (OAB), but the underlying mechanisms remain largely unexplored. Herein, using an OAB mouse model, we examined the impact of oxidative stress (OS) in retinal damage and its molecular mechanism. Results revealed that OS in the retina was enhanced soon after alkali injury. Antioxidant therapy with N-acetylcysteine (NAC) preserved the retinal structure, suppressed cell apoptosis and decreased retinal inflammation, confirming the role of OS. Moreover, enhanced OS was linked to mitochondrial dysfunction, mtDNA leakage and initiation of the cytosolic DNA-sensing signaling. The activation of the major DNA sensors cyclic GMP-AMP Synthase (cGas) and cGAS-Stimulator of Interferon Genes (cGAS/STING) pathway was then identified. Notably, inhibiting cGAS/STING signaling with C-176 markedly reduced inflammation and cell apoptosis and ultimately protected the retina against OAB. Overall, our study reveals the vital function of OS in the occurrence of OAB-induced retinal damage and the involvement of cGAS/STING activation. Furthermore, our provides preclinical validation of the use of an antioxidant or a STING inhibitor as a potential therapeutic approach to protect the retina after OAB.

Choroid neovascularization (CNV) is a distinct type of age-related macular degeneration (AMD) with a poor prognosis and responsible for the majority of vision loss in the elderly population. The laser-induced CNV model is a well-established animal model frequently used to study CNV. In this study, we performed an integrated analysis of metabolomic and transcriptomic data from CNV samples, utilizing multiple approaches including single-sample gene set enrichment analysis (ssGSEA), correlation analysis, and weighted gene co-expression network analysis (WGCNA), alongside various bioinformatics platforms, to identify key metabolic and immune signatures and to investigate their interplay during angiogenesis. Dominant infiltration of macrophages and monocytes was detected and a positive correlation between dysregulated riboflavin metabolism and angiogenesis pathways was characterized. Hub genes such as ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) and acid phosphatase 5, tartrate resistant (ACP5) emerged as potential central regulators of immune-metabolic crosstalk in CNV. The classification of the immune and metabolic landscape and their critical interactions in CNV models will enhance the understanding of the pathogenesis of neovascular AMD and other neovascular eye diseases, contributing to the development of multi-targeted therapeutic strategies with better efficacy.