Microarray analysis of murine retinal light damage reveals changes in iron regulatory, complement, and antioxidant genes in the neurosensory retina and isolated RPE

Abstract

Purpose: The purpose of this study was to investigate light damage-induced transcript changes within neurosensory retina (NSR) and isolated retinal pigment epithelium (RPE). Similar studies have been conducted previously, but were usually limited to the NSR and only a portion of the transcriptome. Herein most of the transcriptome, not just in the NSR but also in isolated RPE, was queried.

Methods: Mice were exposed to 10,000 lux cool white fluorescent light for 18 hours and euthanized 4 hours after photic injury. NSR and isolated RPE were collected, and RNA was isolated. DNA microarray hybridization was conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Microarray analysis was performed using probe intensity data derived from the Mouse Gene 1.0 ST Array. For the genes of interest, confirmation of gene expression was done using quantitative real-time PCR. Immunofluorescence assessed protein levels and localization.

Results: Numerous iron regulatory genes were significantly changed in the light-exposed NSR and RPE. Several of these gene expression changes favored an iron-overloaded state. For example, the transferrin receptor was upregulated in both light-exposed NSR and RPE. Consistent with this, there was stronger transferrin receptor immunoreactivity in the light-exposed retinas. Significant changes in gene expression following light damage were also observed in oxidative stress and complement system genes.

Conclusions: The concept of a photooxidative stress-induced vicious cycle of increased iron uptake leading to further oxidative stress was introduced.

Figure 1.

Purity of NSR and isolated RPE. Graphs showing Rpe65 (A) Rho (B) mRNA levels within NSR and RPE and Pecam1 (C) within RPE and RPE/choroid. Isolated RPE has significantly higher expression of Rpe65 relative to NSR (NSR was normalized to 1), whereas NSR has significantly higher expression of Rho (RPE was normalized to 1). Pecam1 mRNA is significantly higher within the RPE/choroid relative to RPE. *P < 0.05.

Figure 2.

Light-induced changes in visual cycle genes. Graphs showing significant reduction in expression of Rho within NSR (A) and Rpe65 within RPE (B) following light damage, measured by qPCR. *P < 0.05.

Figure 3.

PCA of the processed microarray gene expression data. PCA analysis using all genes where NSR is represented in blue and RPE in red, and light exposure and no light exposure as triangles and squares, respectively. Tissue type (NSR and RPE) is the strongest determinant of global gene expression variability and is captured by the first principal component (x-axis). Light exposure is captured by the second principal component (y-axis).

Figure 4.

Light-induced changes in iron regulatory genes expression measured by qPCR. Graphs showing significant changes in mRNA levels for iron regulatory genes in NSR following light damage (LD) (A, C, E, G). The right column shows changes of the same genes within the isolated RPE (B, D, F, H). *P < 0.05.

Figure 5.

Light damaged retinas have increased TfR1 immunoreactivity. Fluorescence photomicrographs of light-damaged retinas (n = 3; A, bottom panels) show increase in TfR1 immunoreactivity relative to untreated controls (n = 3 each, top panels; scale bar: 25 μm). Relative pixel density for TfR1 within the RPE (B) shows significantly higher immunoreactivity in LD samples relative to untreated. Double-labeling of PECAM-1 (C, top) and TfR1 (C, bottom) shows colocalization with endothelial cells. *P < 0.05.

Figure 6.

Light-induced changes in oxidative stress–related genes measured by qPCR. Graphs showing significant upregulation in Hmox1, Cas-1, Gpx, and Sod1 mRNA levels in NSR following LD (A, C, E, G). The right column shows oxidative stress–related genes in RPE following LD, with only Hmox1 and Gpx being significantly upregulated (B, D, F, H). *P < 0.05.

Figure 7.

Light-induced changes in complement system–related genes measured by qPCR. Graphs showing significant upregulation of C3, C3ra1 mRNA levels in NSR following LD (A, C, E). The right column shows complement system–related genes in RPE following LD (B, D, F). *P < 0.05.