Gene expression signatures in tree shrew sclera during recovery from minus-lens wear and during plus-lens wear.

ABSTRACT

Purpose: In juvenile tree shrews that have developed minus lens-induced myopia, if lens treatment is discontinued, refractive recovery (REC) occurs. However, in age-matched juvenile animals, plus-lens wear (PLW) produces little refractive change, although the visual stimulus (myopia) is similar (an "IGNORE" response). Because the sclera controls axial elongation and refractive error, we examined gene expression in the sclera produced by PLW and compared it with the gene expression signature produced by REC to learn whether these similar refractive conditions produce similar, or differing, scleral responses. Methods: Eight groups of tree shrews (n = 7 per group) were examined. Four groups wore a monocular -5 D lens for 11 days until 35 days of visual experience (DVE). Lens wear was then discontinued, and the animals recovered for 0 h (REC-0), 2 h (REC-2h), 1 day (REC-1d), or 4 days (REC-4d). Starting at 35 DVE, three groups wore a monocular +5 D lens for 2 h (PLW-2h), 1 day (PLW-1d), or 4 days (PLW-4d). A normal group (PLW-0) was examined at 38 DVE to provide baseline measures. Using quantitative real-time PCR (qPCR), we examined scleral mRNA levels in recovering, plus-lens treated, and untreated control eyes for 55 candidate genes whose protein products included signaling molecules, metallopeptidases (MPs) and their inhibitors (tissue inhibitors of metallopeptidases [TIMPs]), and extracellular matrix proteins. Results: No refractive recovery was measured in the REC-2h group. The scleral mRNA expression pattern for recovering versus untreated control eyes after 2 h of recovery was similar to that found for the group (REC-0) that had no recovery time. Many genes in both groups still had downregulated expression in the treated eyes versus the control eyes. The REC-1d group showed little refractive recovery (0.1 ± 0.1 D, mean ± standard error of the mean [SEM]), and the mRNA expression pattern was similar to that of the REC-2h group, but had fewer statistically significantly downregulated genes in the recovering eyes. The REC-4d group recovered refractively by 2.6 ± 0.4 D, and displayed a "STOP" gene expression signature of mostly upregulated mRNA expression in the recovering eyes compared with the untreated control eyes. The PLW-0 (normal) group and the PLW-2h group showed no statistically significant differential gene expression. The PLW-1d group showed a small hyperopic shift (0.1 ± 0.2 D). Two genes were differentially expressed NPR3 was upregulated in the plus lens-wearing eyes, and IGF1 was downregulated. The PLW-4d group showed a similar hyperopic shift (0.3 ± 0.4 D), confirming that the plus lens-induced 5 D of myopia produced little refractive change. In the sclera, there was an IGNORE pattern of general differential upregulation of genes in the treated eyes (22 upregulated, one downregulated) that was distinct from the STOP signature found in recovery. Ten genes were upregulated in the REC-4d group and the PLW-4d group. However, ten other genes were differentially expressed in recovery, but not in plus-lens wear, while 12 genes were differentially expressed in plus-lens wear but not in recovery. Conclusions: One day of recovery is not long enough for the emmetropization mechanism to produce significant gene expression changes in the sclera or refractive recovery. After 4 days, recovery and plus-lens wear produced altered scleral gene expression, but the patterns ("signatures") differed as to which genes showed altered expression, and whether the gene expression was up- or downregulated. Thus, myopia produced altered scleral mRNA expression in recovery and plus-lens wear, confirming that signals initiated by the retina reached the sclera, but the sclera in the elongated recovering eye responded differently from a normal sclera. This might have occurred because the recovering-eye sclera had remodeled during minus-lens compensation, making the sclera respond differently to the signals initiated by the retina. However, the myopia-produced retinal signals in plus lens-wearing animals also may have differed from those in the recovering eyes by the time the signals passed through the RPE and choroid to reach the sclera.