Association of Body Length with Ocular Parameters in Mice.

PURPOSE: To determine the association between changes in body length with ocular refraction, corneal radii, axial length, and lens thickness in two different mouse strains. METHODS: Body length, ocular refraction, corneal radii, axial length, and lens thickness were measured for two inbred mouse strains: 129S1/SvJ (n = 7) and C57BL/6 J (n = 10) from 4 to 12 weeks of age. Body length, from tip of nose to base of tail, was obtained using a digital camera. Biometric parameters, corneal radii, and refractions were measured using spectral-domain optical coherence tomography, automated keratometry, and infrared photorefraction, respectively. A mixed-model ANOVA was performed to examine the changes in ocular parameters as a function of body length and strain in mice controlling for age, gender, and weight over time. RESULTS: C57BL/6J mice had significantly longer body length (average body length at 10 weeks, 8.60 ± 0.06 cm) compared to 129S1/SvJ mice (8.31 ± 0.05 cm) during development (P < .001). C57BL/6J mice had significantly hyperopic refractions compared to 129S1/SvJ mice across age (mean refraction at 10 weeks, 129S1/SvJ: +0.99 ± 0.44D vs. C57BL/6J: +6.24 ± 0.38D, P < .001). Corneal radius of curvature, axial length, and lens thickness (except 10 weeks lens thickness) were similar between the two strains throughout the measurement. In the mixed-model ANOVA, changes in body length showed an independent and significant association with the changes in refraction (P = .002) and corneal radii (P = .016) for each mouse strain. No significant association was found between the changes in axial length (P = .925) or lens thickness (P = .973) as a function of body length and strain. CONCLUSIONS: Changes in body length are significantly associated with the changes in ocular refraction and corneal radii in different mouse strains. Future studies are needed to determine if the association between body length and ocular refraction are related to changes in corneal curvature in mice.

Genome-Wide Scleral Micro- and Messenger-RNA Regulation During Myopia Development in the Mouse.

PURPOSE: MicroRNA (miRNAs) have been previously implicated in scleral remodeling in normal eye growth. They have the potential to be therapeutic targets for prevention/retardation of exaggerated eye growth in myopia by modulating scleral matrix remodeling. To explore this potential, genome-wide miRNA and messenger RNA (mRNA) scleral profiles in myopic and control eyes from mice were studied. METHODS: C57BL/6J mice (n = 7; P28) reared under a 12L:12D cycle were form-deprived (FD) unilaterally for 2 weeks. Refractive error and axial length changes were measured using photorefraction and 1310-nm spectral-domain optical coherence tomography, respectively. Scleral RNA samples from FD and fellow control eyes were processed for microarray assay. Statistical analyses were performed using National Institute of Aging array analysis tool; group comparisons were made using ANOVA, and gene ontologies were identified using software available on the Web. Findings were confirmed using quantitative PCR in a separate group of mice (n = 7). RESULTS: Form-deprived eyes showed myopic shifts in refractive error (-2.02 ± 0.47 D; P < 0.01). Comparison of the scleral RNA profiles of test eyes with those of control eyes revealed 54 differentially expressed miRNAs and 261 mRNAs fold-change >1.25 (maximum fold change = 1.63 and 2.7 for miRNAs and mRNAs, respectively) (P < 0.05; minimum, P = 0.0001). Significant ontologies showing gene over-representation (P < 0.05) included intermediate filament organization, scaffold protein binding, detection of stimuli, calcium ion, G protein, and phototransduction. Significant differential expression of Let-7a and miR-16-2, and Smok4a, Prph2, and Gnat1 were confirmed. CONCLUSIONS: Scleral mi- and mRNAs showed differential expression linked to myopia, supporting the involvement of miRNAs in eye growth regulation. The observed general trend of relatively small fold-changes suggests a tightly controlled, regulatory mechanism for scleral gene expression.

Comparison of refractive development and retinal dopamine in OFF pathway mutant and C57BL/6J wild-type mice.

PURPOSE: Proper visual transmission depends on the retinal ON and OFF pathways. We used Vsx1-/- mice with a retinal OFF visual pathway defect to determine the role of OFF pathway signaling in refractive development (RD) of the eye. METHODS: Refractive development was measured every 2 weeks in Vsx1-/-, Vsx1+/+ (both on 129S1/Sv background), and commonly used C57BL/6J mice from 4 to 12 weeks of age. Form deprivation (FD) was induced monocularly from 4 weeks of age using head-mounted diffuser goggles. Refractive state, corneal curvature, and ocular biometry were obtained weekly using photorefraction, keratometry, and 1310 nm spectral-domain optical coherence tomography. Retinal dopamine and its metabolite, 3,4-dihydroxyphenylacetate (DOPAC), were measured using high-performance liquid chromatography (HPLC). RESULTS: During normal development, the Vsx1-/- and Vsx1+/+ mice showed similar myopic refractions at younger ages (4 weeks, Vsx1-/-: -5.28±0.75 diopter (D); WT: -4.73±0.98 D) and became significantly hyperopic by 12 weeks of age (Vsx1-/-: 3.28±0.82 D; WT: 5.33±0.81 D). However, the C57BL/6J mice were relatively hyperopic at younger ages (mean refraction at 4 weeks, 3.40±0.43 D), and developed more hyperopic refractions until about 7 weeks of age (8.07±0.55 D) before stabilizing. Eight weeks of FD did not induce a myopic shift in the 129S1/Sv animals (0.16±0.85 D), as opposed to a significant shift of -4.29±0.42 D in the C57BL/6J mice. At 4 weeks of visual development, dopamine turnover (the DOPAC/dopamine ratio) was significantly greater in the 129S1/Sv mice compared to the C57BL/6J mice. FD did not alter the levels of dopamine between the goggled and opposite eyes for any genotype or strain. CONCLUSIONS: OFF pathway signaling may not be critically important for normal refractive development in mice. Elevated retinal dopamine turnover in early refractive development may prevent FD myopia in 129S1/Sv mice compared to C57BL/6J mice.

Visually-driven ocular growth in mice requires functional rod photoreceptors.

PURPOSE: Proper refractive eye growth depends on several features of the visual image and requisite retinal pathways. In this study, we determined the contribution of rod pathways to normal refractive development and form deprivation (FD) myopia by testing Gnat1(-/-) mice, which lack functional rods due to a mutation in rod transducin-α. METHODS: Refractive development was measured in Gnat1(-/-) (n = 30-36) and wild-type (WT) mice (n = 5-9) from 4 to 12 weeks of age. FD was induced monocularly from 4 weeks of age using head-mounted diffuser goggles (Gnat1(-/-), n = 9-10; WT, n = 7-8). Refractive state and ocular biometry were obtained weekly using a photorefractor, 1310 nm optical coherence tomography, and partial coherence interferometry. We measured retinal dopamine and its metabolite, DOPAC, using HPLC. RESULTS: During normal development, the refractions of WT mice started at 5.36 ± 0.68 diopters (D) and became more hyperopic before plateauing at 7.78 ± 0.64 D. In contrast, refractions in Gnat1(-/-) mice were stable at 7.39 ± 1.22 D across all ages. Three weeks of FD induced a 2.54 ± 0.77 D myopic shift in WT mice, while Gnat1(-/-) mice did not respond to FD at any age. Axial lengths of Gnat1(-/-) and WT mice increased with age, but differences between genotypes or with goggling did not reach statistical significance and fell within the precision of the instruments. The DOPAC levels were significantly lower in Gnat1(-/-) mice from 2 to 12 weeks of age with DOPAC/dopamine ratio peaking earlier in Gnat1(-/-) compared to WT mice. No differences in dopamine were seen in response to FD or between genotypes. CONCLUSIONS: Functional rod photoreceptors are critical to normal refractive development and the response to FD in mice. Dopamine levels may not directly modulate the refractive state of the mouse eye, but tonic levels of dopamine during development may determine susceptibility to myopia.

Refractive index measurement of the mouse crystalline lens using optical coherence tomography.

In recent years, there has been a growing interest for using mouse models in refractive development and myopia research. The crystalline lens is a critical optical component of the mouse eye that occupies greater than 50% of the ocular space, and significant increases in thickness with age. However, changes in refractive index of the mouse crystalline lens are less known. In this study, we examined the changes in thickness and refractive index of the mouse crystalline lens for two different strains, wild-type (WT) and a nyx mutant (nob) over the course of normal visual development or after form deprivation. Refractive index and lens thickness measurements were made on ex vivo lenses using spectral domain optical coherence tomography (SD-OCT). Comparison of refractive index measurements on 5 standard ball lenses using the SD-OCT and their known refractive indices (manufacturer provided) indicated good precision (intra-class correlation coefficient, 0.998 and Bland-Altman coefficient of repeatability, 0.116) of the SD-OCT to calculate mouse lens refractive index ex vivo. During normal visual development, lens thickness increased significantly with age for three different cohorts of mice, aged 4 (average thickness from both eyes; WT: 1.78 ± 0.03, nob: 1.79 ± 0.08 mm), 10 (WT: 2.02 ± 0.05, nob: 2.01 ± 0.04 mm) and 16 weeks (WT: 2.12 ± 0.06, nob: 2.09 ± 0.06 mm, p < 0.001). Lens thickness was not significantly different between the two strains at any age (p = 0.557). For mice with normal vision, refractive index for isolated crystalline lenses in nob mice was significantly greater than WT mice (mean for all ages; WT: 1.42 ± 0.01, nob: 1.44 ± 0.001, p < 0.001). After 4 weeks of form deprivation to the right eye using a skull-mounted goggling apparatus, a thinning of the crystalline lens was observed in both right and left eyes of goggled animals compared to their naïve controls (average from both the right and the left eye) for both strains (p = 0.052). In form deprived mice, lens refractive index was significantly different between the goggled animals and non-goggled naïve controls in nob mice, but not in WT mice (p = 0.009). Both eyes of goggled nob mice had significantly greater lens refractive index (goggled, 1.49 ± 0.01; opposite, 1.47 ± 0.03) compared to their naïve controls (1.45 ± 0.02, p < 0.05). The results presented here suggest that there are genetic differences in the crystalline lens refractive index of the mouse eye, and that the lens refractive index in mice significantly increase with form deprivation. Research applications requiring precise optical measurements of the mouse eye should take these lens refractive indices into account when interpreting SD-OCT data.

Retinal degeneration increases susceptibility to myopia in mice.

PURPOSE: Retinal diseases are often associated with refractive errors, suggesting the importance of normal retinal signaling during emmetropization. For instance, retinitis pigmentosa, a disease characterized by severe photoreceptor degeneration, is associated with myopia; however, the underlying link between these conditions is not known. This study examines the influence of photoreceptor degeneration on refractive development by testing two mouse models of retinitis pigmentosa under normal and form deprivation visual conditions. Dopamine, a potential stop signal for refractive eye growth, was assessed as a potential underlying mechanism. METHODS: Refractive eye growth in mice that were homozygous for a mutation in Pde6b, Pde6b(rd1/rd1) (rd1), or Pde6b(rd10/rd10) (rd10) was measured weekly from 4 to 12 weeks of age and compared to age-matched wild-type (WT) mice. Refractive error was measured using an eccentric infrared photorefractor, and axial length was measured with partial coherence interferometry or spectral domain ocular coherence tomography. A cohort of mice received head-mounted diffuser goggles to induce form deprivation from 4 to 6 weeks of age. Dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels were measured with high-performance liquid chromatography in each strain after exposure to normal or form deprivation conditions. RESULTS: The rd1 and rd10 mice had significantly greater hyperopia relative to the WT controls throughout normal development; however, axial length became significantly longer only in WT mice starting at 7 weeks of age. After 2 weeks of form deprivation, the rd1 and rd10 mice demonstrated a faster and larger myopic shift (-6.14±0.62 and -7.38±1.46 diopter, respectively) compared to the WT mice (-2.41±0.47 diopter). Under normal visual conditions, the DOPAC levels and DOPAC/dopamine ratios, a measure of dopamine turnover, were significantly lower in the rd1 and rd10 mice compared to the WT mice, while the dopamine levels were similar or higher than WT in the rd10 mice. Lower basal levels of DOPAC were highly correlated with increasing myopic shifts. CONCLUSIONS: Refractive development under normal visual conditions was disrupted toward greater hyperopia from 4 to 12 weeks of age in these photoreceptor degeneration models, despite significantly lower DOPAC levels. However, the retinal degeneration models with low basal levels of DOPAC had increased susceptibility to form deprivation myopia. These results indicate that photoreceptor degeneration may alter dopamine metabolism, leading to increased susceptibility to myopia with an environmental visual challenge.