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. 2004 Aug;45(8):2737-46.
doi: 10.1167/iovs.03-1047.

Temporal changes in gene expression after injury in the rat retina

Félix Vázquez-Chona  1 , Bong K Song, Eldon E Geisert Jr
Affiliations

Temporal changes in gene expression after injury in the rat retina

Félix Vázquez-Chona et al. Invest Ophthalmol Vis Sci. 2004 Aug.

Abstract

Purpose: The goal of this study was to define the temporal changes in gene expression after retinal injury and to relate these changes to the inflammatory and reactive response. A specific emphasis was placed on the tetraspanin family of proteins and their relationship with markers of reactive gliosis.

Methods: Retinal tears were induced in adult rats by scraping the retina with a needle. After different survival times (4 hours, and 1, 3, 7, and 30 days), the retinas were removed, and mRNA was isolated, prepared, and hybridized to the Affymatrix RG-U34A microarray (Santa Clara, CA). Microarray results were confirmed by using RT-PCR and correlation to protein levels was determined.

Results: Of the 8750 genes analyzed, approximately 393 (4.5%) were differentially expressed. Clustering analysis revealed three major profiles: (1) The early response was characterized by the upregulation of transcription factors; (2) the delayed response included a high percentage of genes related to cell cycle and cell death; and (3) the late, sustained profile clustered a significant number of genes involved in retinal gliosis. The late, sustained cluster also contained the upregulated crystallin genes. The tetraspanins Cd9, Cd81, and Cd82 were also associated with the late, sustained response.

Conclusions: The use of microarray technology enables definition of complex genetic changes underlying distinct phases of the cellular response to retinal injury. The early response clusters genes associate with the transcriptional regulation of the wound-healing process and cell death. Most of the genes in the late, sustained response appear to be associated with reactive gliosis.

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Figures

Figure 1
Figure 1
The responses of the rat retina to injury. Retinas labeled with anti-glial fibrillary acidic protein (GFAP; A, D, G, J), lectin from Phaseolus vulgaris (PHA-E; B, E, H, K), and Hoechst nuclear staining (C, F, I, L). Shown are four regions of retina: normal (A– C), 3 days after injury (D– F), and two different retinas 30 days after injury (G– I, J– L). In normal retinas, anti-GFAP labeled astrocytes and Müller cell end feet at the GCL (A). PHA-E lectin stained the normal RPE, choroid, and outer segments (OS, B). At 3 days after injury, cells invading the retinal tear (F, arrow) labeled with anti-GFAP (D, arrow) or PHA-E lectin (E, arrow); whereas, near the injury anti-GFAP labeled mainly Müller cells and astrocytes at the GCL (D). At 30 days after injury, two cellular responses were observed at the site of injury. In one response, the cells filling the tear (I, arrows) were mainly glia cells (H, arrowheads). In the second response, RPE cells mainly filled the tear (K and L, arrows). In some instances, epiretinal membranes with glia (G, arrow) and RPE cells (H, arrow) formed over the retinal surface. A– C are of the same magnification and (D– L) are of the same magnification. Scale bar: (A, L) 50 μm.
Figure 2
Figure 2
Differentially expressed genes (n = 194) were clustered into three major profiles: (A) an early response, (B) a delayed response, and (C) a late, sustained response. Genes were clustered using principal component analysis. Early-response genes displayed a transient surge in expression after injury. Genes fitting the delayed-response profile had a transient peak in expression at either 1 or 3 days after injury. Genes within the third expression profile had a late expression peak (i.e., at either 3 or 7 days) and sustained overexpression at 30 days after retinal injury. A complete list of upregulated genes organized by clusters is found online in Supplemental Table 3. The distribution of genes by functional category reveals that those within each expression profile are functionally related (D).
Figure 3
Figure 3
Temporal distribution of genes and functional categories after retinal injury. Location of genes describes relative gene expression peaks: early expression corresponds to a peak expression at 4 hours, delayed expression corresponds to a peak expression at 1 or 3 days, and late expression corresponds to a peak expression at 7 or 30 days. For specific expression profiles, see Supplemental Table 4.
Figure 4
Figure 4
Crystallin-α, -β, and -γ were dramatically upregulated after retinal injury. The microarray expression changes for Cryab and Crygd (A, formula image) were confirmed with RT-PCR (A, □). Immunoblots show increased levels of crystallin-α, -β, and -γ proteins (B). Crystallin-α, -β, and -γ expression in normal retinas (C, E, G, respectively) and injured retinas 7 days after injury (D, F, H, respectively) was localized by immunohisto-chemistry. In normal retina, crystallin immunoreactivity was present mainly in the GCL (C, E, G). Seven days after injury, there was a strong crystallin immunoreactivity throughout the scar spanning the retinal tear and protruded into the vitreous space (data not shown). High immunoreactivity levels for crystallin-α (D), -β (F), and -γ (H) were found immediately adjacent to the retina at GCL and outer segment layer (OS). No staining was seen when secondary antibody control was used (data not shown). (A) Significant changes from normal: t-test, *P < 0.05, **P < 0.01.
Figure 5
Figure 5
Cd81 clustered into the late-response profile along with tetraspanins and glial reactive markers. RT-PCR confirmed the sustained upregulation of Cd81 after injury (A). A second round of principal component analysis was used to define further the associations between Cd81 and the genes within the late-response profile. The subclusters were visualized by plotting the first two components (B). Cd81 clustered tightly with tetraspanins Cd9 and Cd82, as well as with the genes for associated proteins MHC class I (RT1Aw2 and RT1.Dau) and MHC class II (RT1.C/E). (A) Significant changes from normal: t-test, *P < 0.05, **P < 0.01.
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