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colleagues were examining the retinoic acid system, some of which had a relationship to some aspects of modifying the visual input.
In 2008, using the Affymetrix GeneChip Chicken Genome with more than 28,000 entries, Schippert, Schaeffel, and Feldkaemper were able to find 16 candidate genes that were differentially expressed and were further assessed by real-time PCR.17 Of the 16 genes, six were found to respond similarly to either positive or negative lenses, and three genes responded differentially to the presence of positive or negative lenses. In this study, lenses were worn for only a short time,24 but the implication was that novel gene programs were set into motion by short defocus on the retina. It is not clear how these signals could be transmitted to the sclera or if there were other gene programs set into motion in the sclera. However, it was found that six genes mapped to regions that were already known to be associated with families of human myopes. Due to the short time period of the modification of light input, perhaps ZENK would not be expected to appear.
ZENK was also found to respond to pharmacological control by atropine (muscarinic antagonist) and a dopamine agonist. These were injected in small amounts intravitreally in the chick eye just prior to fitting diffusers over the eye. These agents reversed the down regulation of ZENK due to diffusers alone.18
Relating the chick results with ZENK to mammals has been shown by examining the development of the eye in an EGR-1 knockout mouse. Eye growth and refractive error were followed by measuring the corneal radius of curvature along with refractive state and ocular dimersions.19 A myopic shift was found in the eyes of the knockout animals, and although changes declined with age, the myopic shift remained.
Thus, the situation with animal models of myopia is not substantially better than with the many gene candidates found in human studies. A substantial benefit has been the development of arrays for the chick. This is valuable as the chick model shows rapid myopic changes in response to modification of the visual input, and the eye is larger than the mouse, easing the technical hurdles.
Scleral Gene Expression in a Mouse Model of Myopia
The emphasis in all these studies has been on the retina, despite the realization that growth processes producing the posterior chamber elongation
336 R.W. Beuerman, L.K. Goh and V.A. Barathi
and myopia are located in the sclera. We have shown that two models of experimental myopia can be developed in Balb/cJ mice20,21 and also from two other groups.22,23 We determined that all five muscarinic receptor subtypes expressed in mouse sclera and RPE similar to human.24,25 In another study, we showed that the M1, M4, and M5 muscarinic receptor knock-out (KO) mice eye grew 200 m longer than M2 and 220 m longer than M3 knock-out mice.26 These results provide initial evidence that M1, M4, and M5 receptors may contribute more than the M2 and M3 receptor in terms of scleral growth in experimental myopia.
We have chosen to examine the sclera despite some technical issues, such as the ability to extract sufficient target from a single mouse sclera so that direct experimental-contra-lateral control comparisons can be made, thus increasing our statistical power. Pregnant Balb/cJ mice (Mus musculus) were obtained from the animal holding unit of the National University of Singapore. Animals gave birth in our animal holding unit. Naive control animals were housed in groups of six, while experimental animals were housed individually in standard mouse cages after 28 days of age at 25°C on a schedule of 12:12 hours of light on and off with mouse pellets and water available ad libidum. Approval was obtained from the SingHealth IACUC and all procedures performed in this study complied with the Association of Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmology and Vision Research. Procedures for myopia induction and data analysis for biometry measurements were as previously published.21
RNA, Target cDNA and Microarray Chip Preparation
Total RNA was isolated from a single mouse sclera (n = 6 at each time point) using MELTTM Total Nucleic Acid Isolation System (Ambion Inc., Austin, TX) according to the manufacturer’s instructions. RNA concentration and quality were assessed by absorbance at 260 nm and the absorbance ratio of 260/280 respectively using Nanodrop® ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE). cDNAs were synthesized and labeled with biotin using Genechip® Whole Transcript Sense Target Labeling Assay (Affrymetrix, Inc., Santa Clara, CA). Biotin-labeled cDNA were then hybridized to a Mouse Gene 1.0 ST array for naive samples and MOE gene expression chips for −10 D eyeglass lens treated and control samples (Affrymetrix, Inc., Santa Clara, CA) using
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Genechip® Hybridzation kit (Affrymetrix, Inc., Santa Clara, CA). The microarray chips were washed and stained using Genechip® Hybridization, Wash and Stain kit (Affrymetrix, Inc., Santa Clara, CA). Subsequently, the microarray chips were scanned using Genechip® Scanner 3000 7G (Affrymetrix, Inc., Santa Clara, CA).
Microarray Data Analysis
The microarray data (cel.files) were imported into Partek Genomic Suite 6.5 beta (Partek. Inc., Louis, MO) and normalized using GC-RMA. The variability of all samples was assessed using PCA plot (Fig. 1), and a Venn diagram (Fig. 2) was generated to compare the differentially expressed
Figure 1. PCA plot shows clustering of gene expression in mouse sclera at one week (T0), two weeks (T1), and eight weeks (T2) after eyeglass lens induced myopia and control sclera.
338 R.W. Beuerman, L.K. Goh and V.A. Barathi
Figure 2. Venn diagram shows the differentially expressed genes found among the age groups.
genes found among the age groups. Furthermore, the gene profiles of all samples were hierarchically clustered based on gene expression (Fig. 3). ANOVA analysis (P < 0.05) was performed on the data and a set of genes was selected using a two-fold change threshold. The set of genes was then further grouped into a biological process, cellular component, and molecular function using Gene Ontology enrichment. The Gene Ontology enrichment score for each functional group is calculated using the Chisquare test, and a bar chart was generated. In addition, a forest plot showing the gene expression of each functional group was also generated.
Scleral Gene Expression in the Myopic Mouse
Three time points for the induction of myopia have been tested thus far: T0 (week 1), T1 (week 2), and T2 (week 8). At each time point, batch
339 Gene Analysis in Experimental Myopia
Figure 3. Gene profiles of all samples were hierarchically clustered based on gene expression.
Figure 4. Selected up and down regulated gene expression in sclera after six weeks of eyeglass lens myopic induction compared to sclera of the contra-lateral control eye.
removal was applied, and then gene expression from these time points were combined for analyses. Principal component analysis (Fig. 1) showed clustering of gene expression. T0 and the control were clustered together as might be expected. Sources of variation indicated gene expression was the major signal.