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The current study aimed to investigate the differential protein expression in guinea pig retinas in response to lens-induced myopia (LIM) before fully compensated eye growth. Four days old guinea pigs (n=5) were subjected to −4D LIM for 8 days. Refractive errors were measured before and at the end of the lens wear period. Ocular dimensions were also recorded using high-frequency A-scan ultrasonography. After the LIM treatment, retinas of both eyes were harvested and soluble proteins were extracted. Paired retinal protein expressions in each animal were profiled and compared using a sensitive fluorescence difference two-dimensional gel electrophoresis. The quantitative retinal proteomes of myopic and control eye were analysed using computerised DeCyder software. Those proteins that were consistently changed with at least 1.2-fold difference (P<0.05) in the same direction in all five animals were extracted, trypsin digested and identified by tandem mass spectrometry. Significant myopia was induced in guinea pigs after 8 days of lens wear. The vitreous chamber depth in lens-treated eyes was found to be significantly elongated. Typically, more than 1,000 protein spots could be detected from each retina. Thirty-two of them showed differential expression between myopic and untreated retina. Among these proteins, 21 spots were upregulated and 11 were downregulated. Eight protein spots could be successfully identified which included β-actin, enolase 1, cytosolic malate dehydrogenase, Ras-related protein Rab-11B, protein-L-isoaspartate (D-aspartate) O-methyltransferase, PKM2 protein, X-linked eukaryotic translation initiation factor 1A and ACP1 protein. The present study serves as the first report to uncover the retinal 2D proteome expressions in mammalian guinea pig myopia model using a top-down fluorescent dyes labelling gel approach. The results showed a downregulation in glycolytic enzymes that may suggest a significant alteration of glycolysis during myopia development. Other protein candidates also suggested multiple pathways which could provide new insights for further study of the myopic eye growth.

To examine the difference between primary and secondary retinal ganglion cell (RGC) degeneration, the protein expression at four regions of retina including superior, temporal, inferior and nasal quadrant in a rat model of partial optic nerve transection (pONT) using 2-D Fluorescence Difference Gel Electrophoresis (DIGE) were investigated. Unilateral pONT was performed on the temporal side of optic nerves of adult Wistar rats to separate primary and secondary RGC loss. Topographical quantification of RGCs labeled by Rbpms antibody and analysis of axonal injury by grading of optic nerve damage at 1 week (n=8) and 8 weeks (n=15) after pONT demonstrated early RGC loss at temporal region, which is considered as primary RGC degeneration and progressing RGC loss at nasal region, which is considered as secondary RGC degeneration. Early protein expression in each retinal quadrant (n=4) at 2 weeks after pONT was compared with the corresponding quadrant in the contralateral control eye by DIGE. For all comparisons, 24 differentially expressed proteins (>1.2-fold; P<0.05; ≥3 non-duplicated peptide matches) were identified by mass spectrometry (MS). Interestingly, in the nasal retina, serum albumin and members of crystallin family, including αA, αB, βA2, βA3, βB2 and γS indicating stress response were upregulated. By contrast, only αB and βA2 crystallin proteins were altered in temporal quadrant. In the superior and inferior quadrants, βB2 crystallin, keratin type I, S-arrestin and lamin-B1 were upregulated, while heat shock cognate 71 kDa protein and heterogeneous nuclear ribonucleoproteins A2/B1 were downregulated. In summary, the use of DIGE followed by MS is useful to detect early regional protein regulation in the retina after localized optic nerve injury.

The molecular pathophysiology of corticosteroid-induced ocular hypertension (CIH) is not well understood. To determine the biological mechanisms of CIH, this study investigated protein expression profiles of human trabecular meshwork (hTM) cells in response to dexamethasone and prednisolone treatment. Both discovery-based sequential windowed data independent acquisition of the total high-resolution mass spectra (SWATH-MS) and targeted based high resolution multiple reaction monitoring (MRM-HR) confirmation were applied using a hybrid quadrupole-time-of-flight mass spectrometer. A comprehensive list of 1759 proteins (1% FDR) was generated from the hTM. Quantitative proteomics revealed 20 differentially expressed proteins (p-value ≤ 0.05 and fold-change ≥ 1.5 or ≤ 0.67) commonly induced by prednisolone and dexamethasone, both at 300 nM. These included connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1), two proteins previously implicated in ocular hypertension, glaucoma, and the transforming growth factor-β pathway. Their gene expressions in response to corticosteroids were further confirmed using reverse-transcription polymerase chain reaction. Together with other novel proteins identified in the data sets, additional pathways implicated by these regulated proteins were the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, integrin cell surface interaction, extracellular matrix (ECM) proteoglycans, and ECM-receptor interaction. Our results indicated that an integrated platform of SWATH-MS and MRM-HR allows high throughput identification and confirmation of novel and known corticosteroid-regulated proteins in trabecular meshwork cells, demonstrating the power of this technique in extending the current understanding of the pathogenesis of CIH.