A microarray analysis of retinal transcripts that are controlled by image contrast in mice

Abstract

Purpose: The development of myopia is controlled by still largely unknown retinal signals. The aim of this study was to investigate the changes in retinal mRNA expression after different periods of visual deprivation in mice, while controlling for retinal illuminance.

Methods: Each group consisted of three male C57BL/6 mice. Treatment periods were 30 min, 4 h, and 6+6 h. High spatial frequencies were filtered from the retinal image by frosted diffusers over one eye while the fellow eyes were covered by clear neutral density (ND) filters that exhibited similar light attenuating properties (0.1 log units) as the diffusers. For the final 30 min of the respective treatment period mice were individually placed in a clear Perspex cylinder that was positioned in the center of a rotating (60 degrees) large drum. The inside of the drum was covered with a 0.1 cyc/degree vertical square wave grating. This visual environment was chosen to standardize illuminances and contrasts seen by the mice. Labeled cRNA was prepared and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays. Alterations in mRNA expression levels of candidate genes with potential biological relevance were confirmed by semi-quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).

Results: In all groups, Egr-1 mRNA expression was reduced in diffuser-treated eyes. Furthermore, the degradation of the spatial frequency spectrum also changed the cFos mRNA level, with reduced expression after 4 h of diffuser treatment. Other interesting candidates were Akt2, which was up-regulated after 30 min of deprivation and Mapk8ip3, a neuron specific JNK binding and scaffolding protein that was temporally regulated in the diffuser-treated eyes only.

Conclusions: The microarray analysis demonstrated a pattern of differential transcriptional changes, even though differences in the retinal images were restricted to spatial features. The candidate genes may provide further insight into the biochemical short-term changes following retinal image degradation in mice. Because deprivation of spatial vision leads to increased eye growth and myopia in both animals and humans, it is believed some of the identified genes play a role in myopia development.

Figure 1

Detailed representation of the treatment protocol. A: shows the protocol for the control animals. A-D: An arrow above each time bar signals (1) the time when mice were brought into light, (2) when each mouse was put into the rotating drum, and (3) when mice were sacrificed. B-D: The arrows below the time bars mark the beginning of each treatment: 30 min (B), 4 h (C), and 6+6 h (D) diffuser and ND filter attachment. Three animals were treated per day, and were brought into light as follows: the first mouse at 8.00 a.m., the second at 8.30 a.m., and the third animal at 9.00 a.m. Accordingly, the entire treatment schedule was shifted 30 min or 1 h later (to the right) for the 2nd and 3rd animals, respectively.

Figure 2

Analysis of GeneChip® results by real-time reverse transcriptase polymerase chain reaction. Delta values were calculated between the normalized expression values of the diffuser- and neutral density (ND) filter-treated eyes and are plotted on the ordinate. Six mice were used for each experiment. Results were given as follows for each treatment group: (A) 30 min treatment group, (B) presents 4 h group and (C) 6+6 h group. Single asterisk represents p<0.05 *; while double asterisk indicates p<0.01 **. Error bars denote standard errors of the mean.

Figure 3

Expression of Egr-1 and cFos in control animals and as a function of time in treated animals. The mRNA expression of Egr-1 (A) and cFos (B) in diffuser- and neutral density (ND) filter-treated eyes is displayed as a function of time. Normalized expression values were plotted on the ordinate. Error bars denote standard errors of the mean (SEM). Statistical results represent the ANOVA followed by Dunnett's test (comparisons with a control). Single asterisk indicates p<0.05 *; while triple asterisk denotes p<0.001 ***. Three control animals and six treated animals were used for each experiment.

Figure 4

Retinal mRNA expression levels in untreated (control) and treated (diffuser/filter) animals. Normalized expression values were plotted on the ordinate. Error bars denote standard errors of the mean (SEM). Statistical results represent the ANOVA followed by Tukey-Kramer test. A single asterisk indicates p<0.05 *, while a double asterisk denotes p<0.01 **, and a triple asterisk marks p<0.001 ***. Three control animals and six treated animals were used in these experiments.

Figure 5

mRNA expression levels in the diffuser-treated eyes as a function of time. Normalized expression values for Fgf13 (A), Mapk8ip3 (B), Trak2 (C), Jag1 (D), Stk3 (E), and Klf9 (F) were plotted on the ordinate. Each black dot represents the approximate expression level changes as determined by the GeneChip® experiment. Error bars denote standard errors of the mean (SEM). Statistical results represent the ANOVA followed by Tukey-Kramer test. A single asterisk indicates p<0.05 *, a double asterisk equals p<0.01 **, and a triple asterisk representsp<0.001 ***. Three control animals and six treated animals were used for each experiment.