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Myopia, or short-sightedness, is a highly prevalent refractive disorder in which the eye’s focal length is too short for its axial dimension in its relaxed state. High myopia is associated with increased risks of blinding ocular complications and abnormal eye shape. In addition to consistent findings on posterior segment anomalies in high myopia (e.g., scleral remodeling), more recent biometric and biomechanical data in myopic humans and animal models also indicate anterior segment anomalies (e.g., corneal biomechanical properties). Because the cornea is the anterior-most ocular tissue, providing essential refractive power and physiological stability, it is important to understand the biochemical signaling pathway during myopia development. This study first aimed to establish the entire chicken corneal proteome. Then, using the classical form deprivation paradigm to induce high myopia in chicks, state-of-the-art bioinformatics technologies were applied to identify eight differentially expressed proteins in the highly myopic cornea. These results provide strong foundation for future corneal research, especially those using chicken as an animal model for myopia development.

The vitreous humor (VH) is a transparent gelatin-like substance that occupies two-thirds of the eyeball and undergoes the most significant changes during eye elongation. Quantitative proteomics on the normal growth period in the VH could provide new insights into understanding its progression mechanism in the early stages of myopia. In this study, a data-independent acquisition (SWATH-MS) was combined with targeted LC-ESI-MS/MS to identify and quantify the relative protein changes in the vitreous during the normal growth period (4, 7, 14, 21 and 28 days old) in the chick model. Chicks were raised under normal growing conditions (12/12 h Dark/light cycle) for 28 days, where ocular measurements, including refractive and biometric measurements, were performed on days 4 (baseline), 7, 14, 21 and 28 (n = 6 chicks at each time point). Extracted vitreous proteins from individual animals were digested and pooled into a left eye pool and a right pool at each time point for protein analysis. The vitreous proteome for chicks was generated using an information-dependent acquisition (IDA) method by combining injections from individual time points. Using individual pool samples, SWATH-MS was employed to quantify proteins between each time point. DEPs were subsequently confirmed in separate batches of animals individually on random eyes (n = 4) using MRMHR between day 7 and day 14. Refraction and vitreous chamber depth (VCD) were found to be significantly changed (p < 0.05, n = 6 at each time point) during the period. A comprehensive vitreous protein ion library was built with 1576 non-redundant proteins (22987 distinct peptides) identified at a 1% false discovery rate (FDR). A total of 12 up-regulated and 26 down-regulated proteins were found across all time points compared to day 7 using SWATH-MS. Several DEPs, such as alpha-fetoprotein, the cadherin family group, neurocan, and reelin, involved in structural and growth-related pathways, were validated for the first time using MRMHR under this experimental condition. This study provided the first comprehensive spectral library of the vitreous for chicks during normal growth as well as a list of potential growth-related protein biomarker candidates using SWATH-MS and MRMHR during the emmetropization period.

Most of the previous myopic animal studies employed a single-candidate approach and lower resolution proteomics approaches that were difficult to detect minor changes, and generated limited systems-wide biological information. Hence, a complete picture of molecular events in the retina involving myopic development is lacking. Here, to investigate comprehensive retinal protein alternations and underlying molecular events in the early myopic stage, we performed a data-independent Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) based proteomic analysis coupled with different bioinformatics tools in pigmented guinea pigs after 4-day lens-induced myopia (LIM). Myopic eyes compared to untreated contralateral control eyes caused significant changes in refractive error and choroid thickness (p < 0.05, n = 5). Relative elongation of axial length and the vitreous chamber depth were also observed. Using pooled samples from all individuals (n = 10) to build a species-specific retinal ion library for SWATH analysis, 3202 non-redundant proteins (with 24,616 peptides) were identified at 1% global FDR. For quantitative analysis, the 10 individual retinal samples (5 pairs) were analyzed using a high resolution Triple-TOF 6600 mass spectrometry (MS) with technical replicates. In total, 37 up-regulated and 21 down-regulated proteins were found significantly changed after LIM treatment (log2 ratio (T/C) > 0.26 or < −0.26; p ≤ 0.05). Data are accepted via ProteomeXchange with identifier PXD025003. Through Ingenuity Pathways Analysis (IPA), “lipid metabolism” was found as the top function associated with the differentially expressed proteins. Based on the protein abundance and peptide sequences, expression patterns of two regulated proteins (SLC6A6 and PTGES2) identified in this pathway were further successfully validated with high confidence (p < 0.05) using a novel Multiple Reaction Monitoring (MRM) assay on a QTRAP 6500+ MS. In summary, through an integrated discovery and targeted proteomic approach, this study serves as the first report to detect and confirm novel retinal protein changes and significant biological functions in the early LIM mammalian guinea pigs. The study provides new workflow and insights for further research to myopia control.

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma that results from impeded fluid drainage. The increase in outflow resistance is caused by trabecular meshwork (TM) cell dysfunction and excessive extracellular matrix (ECM) deposition. Baicalein (Ba) is a natural flavonoid and has been shown to regulate cell contraction, fluid secretion, and ECM remodeling in various cell types, suggesting the potential significance of regulating outflow resistance and IOP. We demonstrated that Ba significantly lowered the IOP by about 5 mmHg in living mice. Consistent with that, Ba increased the outflow facility by up to 90% in enucleated mouse eyes. The effects of Ba on cell volume regulation and contractility were examined in primary human TM (hTM) cells. We found that Ba (1–100 µM) had no effect on cell volume under iso-osmotic conditions but inhibited the regulatory volume decrease (RVD) by up to 70% under hypotonic challenge. In addition, Ba relaxed hTM cells via reduced myosin light chain (MLC) phosphorylation. Using iTRAQ-based quantitative proteomics, 47 proteins were significantly regulated in hTM cells after a 3-h Ba treatment. Ba significantly increased the expression of cathepsin B by 1.51-fold and downregulated the expression of D-dopachrome decarboxylase and pre-B-cell leukemia transcription factor-interacting protein 1 with a fold-change of 0.58 and 0.40, respectively. We suggest that a Ba-mediated increase in outflow facility is triggered by cell relaxation via MLC phosphorylation along with inhibiting RVD in hTM cells. The Ba-mediated changes in protein expression support the notion of altered ECM homeostasis, potentially contributing to a reduction of outflow resistance and thereby IOP.

Background In the past decade and during the COVID pandemic, the prevalence of myopia has reached epidemic proportions. To address this issue and reduce the prevalence of myopia and its complications, it is necessary to develop more effective interventions for controlling myopia. In this study, we investigated the combined effects of narrowband lights and competing defocus on eye growth and refraction in chicks, an important step in understanding the potential for these interventions to control myopia. This is the first time these effects have been characterized. Methods Three groups of five-day-old chicks (n = 8 per group) were raised in three different lighting conditions: white, red, and blue for 13 days in a 12/12-h light/dark diurnal cycle. One eye was randomly selected for applications of a dual-power optical lens (− 10 D/ + 10 D, 50∶50), while another eye was left untreated as control. Vitreous chamber depth (VCD), axial length (AL), choroidal thickness (CT) and refractive errors were measured at pre-exposure (D0) and following 3 (D3), 7 (D7), 10 (D10), and 13 days (D13) of light exposure. Results Under white light, the dual-power lens induced a hyperopic shift [at D13, mean spherical equivalent refraction (SER), treated vs. control: 4.81 ± 0.43 D vs. 1.77 ± 0.21 D, P  < 0.001] and significantly reduced the progression of axial elongation (at D13, change in AL, treated vs. control: 1.25 ± 0.04 mm vs. 1.45 ± 0.05 mm, P  < 0.01). Compared to white light alone, blue light alone induced a hyperopic shift (at D13, mean SER, blue vs. white: 2.75 ± 0.21 D vs. 1.77 ± 0.21 D, P  < 0.01) and significantly reduced axial elongation (at D13, change in AL, blue vs. white: 1.17 ± 0.06 mm vs. 1.45 ± 0.05 mm, P  < 0.01) in control eyes. When comparing all conditions, eyes exposed to blue light plus dual-power lens had the least axial elongation (at D13, change in AL, 0.99 ± 0.05 mm) and were the most hyperopic (at D13, mean SER, 6.36 ± 0.39 D). Conclusions Both narrowband blue light and dual-power lens interventions were effective in inducing a hyperopic shift in chicks, and provided protection against myopia development. The combination of these interventions had additive effects, making them potentially even more effective. These findings support the use of optical defocus interventions in combination with wavelength filters in clinical studies testing their effectiveness in treating myopia in children.

To explore the temporal profile of retinal proteomes specific to primary and secondary retinal ganglion cell (RGC) loss. Unilateral partial optic nerve transection (pONT) was performed on the temporal side of the rat optic nerve. Temporal and nasal retinal samples were collected at 1, 4 and 8 weeks after pONT (n = 4 each) for non-biased profiling with a high-resolution hybrid quadrupole time-of-flight mass spectrometry running on label-free SWATHTM acquisition (SCIEX). An information-dependent acquisition ion library was generated using ProteinPilot 5.0 and OneOmics cloud bioinformatics. Combined proteome analysis detected 2531 proteins with a false discovery rate of <1%. Compared to the nasal retina, 10, 25 and 61 significantly regulated proteins were found in the temporal retina at 1, 4, and 8 weeks, respectively (p < 0.05, FC ≥ 1.4 or ≤0.7). Eight proteins (ALDH1A1, TRY10, GFAP, HBB-B1, ALB, CDC42, SNCG, NEFL) were differentially expressed for at least two time points. The expressions of ALDH1A1 and SNCG at nerve fibers were decreased along with axonal loss. Increased ALDH1A1 localization in the inner nuclear layer suggested stress response. Increased GFAP expression demonstrated regional reactivity of astrocytes and Muller cells. Meta-analysis of gene ontology showed a pronounced difference in endopeptidase and peptidase inhibitor activity. Temporal proteomic profiling demonstrates established and novel protein targets associated with RGC damage.

This study used isotope-coded protein label (ICPL) quantitative proteomics and bioinformatics analysis to examine changes in vitreous protein content and associated pathways during lens-induced eye growth. First, the vitreous protein profile of normal 7-day old chicks was characterized by nano-liquid chromatography electrospray ionization tandem mass spectrometry. A total of 341 unique proteins were identified. Next, myopia and hyperopia were induced in the same chick by attaching −10D lenses to the right eye and +10D lenses to the left eye, for 3 and 7 days. Protein expression in lens-induced ametropic eyes was analyzed using the ICPL approach coupled to LCMS. Four proteins (cystatin, apolipoprotein A1, ovotransferrin, and purpurin) were significantly up-regulated in the vitreous after 3 days of wearing −10D lenses relative to +10D lens contralateral eyes. The differences in protein expression were less pronounced after 7 days when the eyes approached full compensation. In a different group of chicks, western blot confirmed the up-regulation of apolipoprotein A1 and ovotransferrin in the myopic vitreous relative to both contralateral lens-free eyes and hyperopic eyes in separate animals wearing +10D lenses. Bioinformatics analysis suggested oxidative stress and lipid metabolism as pathways involved in compensated ocular elongation.

Prolonged use of dexamethasone (DEX) elevates intraocular pressure (IOP) and increases the risk of developing glaucoma. In a previous study, we demonstrate that DEX stimulates the expression of thrombospondin-1 (THBS1) in primary human trabecular meshwork (hTM) cells, and that inhibiting THBS1 expression prevents DEX-induced elevation of IOP in mice. Therefore, we investigate the mechanism by which DEX regulates THBS1 expression. Treatment with the DNA methylation inhibitors, 5-azacytosine (5-AC) or 5-aza-2’-deoxycytidine (5-aza-dC), upregulates THBS1 protein levels in vitro and in vivo, reduces outflow facility in perfused mouse eyes, and elevates IOP in mice. In primary hTM cells, 7-day DEX treatment results in hypomethylation of the THBS1 promoter region and reduces transcript levels of 2 DNA methyltransferases (DNMTs), DNMT1 and DNMT3A. Taken together, we show that DEX reduces expression of DNMTs and DNA methylation of the THBS1 promoter region, supporting a critical role for THBS1 in DEX-induced outflow reduction. Epigenetic alternations in trabecular meshwork induced by dexamethasone are associated with hypomethylation of the thombospondin-1 promoter region and dysregulation of the fluid flow of aqueous humor and intraocular pressure.

The retina is a key sensory tissue composed of multiple layers of cell populations that work coherently to process and decode visual information. Mass spectrometry-based proteomics approach has allowed high-throughput, untargeted protein identification, demonstrating the presence of these proteins in the retina and their involvement in biological signalling cascades. The comprehensive wild-type mouse retina proteome was prepared using a novel sample preparation approach, the suspension trapping (S-Trap) filter, and further fractionated with high-pH reversed phase chromatography involving a total of 28 injections. This data-dependent acquisition (DDA) approach using a Sciex TripleTOF 6600 mass spectrometer identified a total of 7,122 unique proteins (1% FDR), and generated a spectral library of 5,950 proteins in the normal C57BL/6 mouse retina. Data-independent acquisition (DIA) approach relies on a large and high-quality spectral library to analyse chromatograms, this spectral library would enable access to SWATH-MS acquisition to provide unbiased, multiplexed, and quantification of proteins in the mouse retina, acting as the most extensive reference library to investigate retinal diseases using the C57BL/6 mouse model. Measurement(s) retina Technology Type(s) mass spectrometry Sample Characteristic - Organism Mus musculus Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.13128044

Background Mice are an emerging model for experimental myopia. Due to their small eye size, non-invasive optical coherence tomography is essential for evaluating ocular biometrics. There is currently no universally accepted protocol for those measurements. This study aims to compare ocular biometric measurements using two methods: Purkinje image-based alignment and optic nerve head alignment, utilizing spectral domain optical coherence tomography. Gaining an understanding of the implications of these methods in determining axial elongation in the normal growing eyes of wild-type C57BL/6J mice would offer valuable insight into their relevance for the experimental myopia model. Methods Ocular dimensions and refractive development were measured on postnatal days P21 (n = 10), P28 (n = 15), and P35 (n = 8). The Purkinje image-based alignment (P1) was determined using a photorefractor and aligned perpendicular to the corneal apex using SD-OCT. In comparison, due to the absence of a fovea in the mouse retina, the optic nerve head (ONH) alignment was used. Variance analysis, regression analysis, and Bland–Altman analysis were performed to compare the differences between alignment methods as well as the replication by another operator. Results Mice developed hyperopic ametropia under normal visual conditions. The photorefractor measured a technical variation of 3.9 D (95% CI, n = 170, triplicates). Bland–Altman analysis revealed a shorter (mean ± SD) axial length (− 26.4 ± 18.1 μm) and vitreous chamber depth (− 39.9 ± 25.4 μm) in the Purkinje image-based alignment. There was a significant difference in the relative growth trend in VCD (linear regression, p = 0.02), which was relatively stable and showed shortening when measured with ONH alignment from postnatal age 21 to 35 days. Conclusions SD-OCT allowed precise in-vivo measurement and segmentation of ocular dimensions, regardless of the methods adopted. P1 alignment consistently resulted in significantly shorter VCD and AL compared to ONH alignment at most time points. When considering temporal changes from P21 to P35, both methods showed similar results, with significant elongation of ACD, LT, and AL as expected. However, our findings revealed a significant shortening of VCD over time with the adoption of ONH alignment, while the change in P1 alignment was relatively stable. Therefore, AL provides a better measure for evaluating ocular growth in mice using optical coherence tomography than VCD for myopia research.