Retinal Proteome Analysis Reveals a Region-Specific Change in the Rabbit Myopia Model

Abstract

Uncovering region-specific changes in the myopic retina can provide clues to the pathogenesis of myopia progression. After imposing form deprivation myopia in the right eye of 6-week-old rabbits, we investigated the proteome profile of each retinal region (central, mid-periphery, and far-periphery retina), using accurate high-resolution mass spectrometry. Protein expression was analyzed using gene ontology and network analysis compared with that of the control, the left eyes. Among 2065 proteins detected from whole retinal samples, 249 differentially expressed proteins (DEPs) were identified: 164 DEPs in the far-periphery, 39 in the mid-periphery, and 83 in the central retina. In network analysis, the far-periphery retina showed the most significant connectivity between DEPs. The regulation of coagulation was the most significant biological process in upregulated DEPs in the far-periphery retina. Proteasome was the most significant Kyoto Encyclopedia of Genes and Genomes pathway in downregulated DEPs in the central retina. Antithrombin-III, fibrinogen gamma chain, and fibrinogen beta chain were identified as hub proteins for myopia progression, which were upregulated in the far-periphery retina. Proteomic analysis in this study suggested that oxidative stress can be the primary pathogenesis of myopia progression and that the far-periphery retina plays a role as the key responder.

Keywords: coagulation; myopia; oxidative stress; proteomics; regional; retina.

Figure 1

Myopia induction and proteomic analysis of myopic retina in each retinal region: (A) to verify the induction of myopia, refraction and axial length were measured (* p < 0.01); (B) using LC-MS/MS with MaxQuant processing, 2065 proteins in total were identified from the whole retinal sample.

Figure 2

Hierarchical clustering of expressed proteins in each retinal region: (A) principal component analysis (PCA) demonstrated that far-periphery myopia retinas and far-periphery control retinas were distinguished; on the other hand, the mid-periphery myopia retinas and control retinas were not differentiated. Central-myopia retinas and control retinas were well distinguished on PCA; (B) unsupervised hierarchical clustering of the whole retinal proteome generated a heat map similar to PCA.

Figure 3

Differentially expressed proteins (DEPs) in each retinal region: (A) in total, 249 DEPs were identified from whole retinal regions; in total, 164 DEPs were identified in the far-periphery retina occupying the largest proportion of DEPs, followed by the central retina (83 DEPs) and the mid-periphery retina (39 DEPs); (B) heatmap of DEPs in each retinal region; the box color indicates log2 fold changes of DEPs from red (increasing) to blue (reducing), when comparing a myopic eye to a control eye.

Figure 4

Network modeling of differentially expressed proteins in each region of myopia versus control retina: (A) protein–protein interaction network of DEPs in each retinal region (node color: −log2 (fold changes); border color: –log10 (p-value); thickness of edge: connectivity between nodes); (B) gene ontology (GO) analysis of whole DEPs with biological process terms in the far-periphery and central retina; (C) GO analysis of upregulated and downregulated DEPs in the far-periphery retina with biological process terms.

Figure 5

Hub protein identification based on MCC values and its GO analysis: (A) identification of hub proteins using the CytoHubba plugin; the maximal clique centrality (MCC) score was calculated, and nodes with the top 10 MCC values were identified as hub proteins colored yellow to red. All nodes with MCC value ≥ 10 were included in the GO analysis. MCC values of blue nodes were equal to or more than 10, but not in the top 10 values; (B) GO analysis with hub proteins whose MCC value was ≥10.

Figure 6

Preparation of retinal tissue sample after myopia induction. The retina was divided into three regions: central, mid-periphery, and far-periphery retina. Before the excision of the cornea, marking was performed along the limbus at 1, 4, 7, and 11 o’clock. Then, the eyeball wall was excised along the line from the marking spot to the optic nerve into a petaloid shape. The central retina was defined as the rectangular area bordered by the point at a distance of 4 mm in width and 3 mm in length on both sides from the point, which was ventral 3 mm from the optic nerve head. After separating the central retina, the remaining retina was divided evenly into two parts. The inner portion was defined as the mid-periphery retina, and the outer portion was defined as the far-periphery retina.