Miaozhen Pan

The Development of the Refractive Status and Ocular Growth in C57BL/6 Mice

PURPOSE To investigate refraction, corneal curvature, axial components, and the correlations between the refraction and ocular growth during the emmetropization in the C57BL/6 mouse. METHODS Ten groups of 10 mice underwent ocular measurements at 22 to 102 days after birth. Refraction was measured by photoretinoscopy and corneal radius of curvature (CRC) was measured by keratometry. Corneal thickness (CT), anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), retinal thickness (RT), and axial length (AL) were measured by optical coherence tomography (OCT) with focal plane advancement. RESULTS Refraction was -1.49 +/- 3.17 diopters (D; mean +/- SD) at day 22 and the highest myopia was at day 25 (-4.61 +/- 2.96 D). The refractive error then increased and reached a hyperopic peak (+9.43 +/- 3.33 D) on day 47. The overall change in refraction was significant from 22 to 102 days (P < 0.05). All measured ocular components changed significantly during the study period except for CT and RT (P > 0.05 for CT and RT; P < 0.05 for others). The CRC, ACD, LT, and AL increased from 22 to 47 days. The increase in ACD, LT, and AL continued after 47 days; however, the CRC increased slowly after this age. The ACD became stable around 67 days and LT and AL at 81 days. CONCLUSIONS In C57BL/6 mouse eyes, myopia developed early and then the refractive error increased rapidly in the hyperopic direction to reach a peak at around 47 days with the major contributing changes being in axial length and corneal curvature.

Spontaneous axial myopia and emmetropization in a strain of wild-type guinea pig (Cavia porcellus).

PURPOSE To describe a wild-type guinea pig strain with an incidence of spontaneous axial myopia, minimal pupil responses, lack of accommodation, and apparently normal spatial vision. Such a strain is of interest because it may permit the exploration of defective emmetropization and mapping of the underlying quantitative trait loci. METHODS Twenty-eight guinea pigs were selected from 220 animals based on binocular myopia (exceeding -1.50 diopter [D]) or anisometropia (difference between both eyes exceeding 10 D) at 4 weeks of age. Refractions and pupil responses were measured with eccentric infrared photoretinoscopy, corneal curvature by modified conventional keratometer, and axial lengths by A-scan ultrasonography once a week. Twenty-one guinea pigs were raised under a normal 12-hour light/12-hour dark cycle. From a sample of 18 anisometropic guinea pigs, 11 were raised under normal light cycle and 7 were raised in the dark to determine the extent to which visual input guides emmetropization. Spatial vision was tested in an automated optomotor drum. RESULTS In 10 guinea pigs with myopia in both eyes, refractive errors ranged from -15.67 D to -1.50 D at 3 weeks with a high interocular correlation (R = 0.82); axial length and corneal curvature grew almost linearly over time. Strikingly, two patterns of recovery were observed in anisometropic guinea pigs: in 12 (67%) anisometropia persisted, and in 6 (33%) it declined over time. These ratios remained similar in dark-reared guinea pigs. Unlike published strains, all guinea pigs of this strain showed weak pupil responses and no signs of accommodation but up to 3 cyc/deg of spatial resolution. CONCLUSIONS This strain of guinea pigs has spontaneous axial refractive errors that may be genetically or epigenetically determined. Interestingly, it differs from other published strains that show no refractive errors, vivid accommodation, or pupil responses.

cAMP Level Modulates Scleral Collagen Remodeling, a Critical Step in the Development of Myopia

The development of myopia is associated with decreased ocular scleral collagen synthesis in humans and animal models. Collagen synthesis is, in part, under the influence of cyclic adenosine monophosphate (cAMP). We investigated the associations between cAMP, myopia development in guinea pigs, and collagen synthesis by human scleral fibroblasts (HSFs). Form-deprived myopia (FDM) was induced by unilateral masking of guinea pig eyes. Scleral cAMP levels increased selectively in the FDM eyes and returned to normal levels after unmasking and recovery. Unilateral subconjunctival treatment with the adenylyl cyclase (AC) activator forskolin resulted in a myopic shift accompanied by reduced collagen mRNA levels, but it did not affect retinal electroretinograms. The AC inhibitor SQ22536 attenuated the progression of FDM. Moreover, forskolin inhibited collagen mRNA levels and collagen secretion by HSFs. The inhibition was reversed by SQ22536. These results demonstrate a critical role of cAMP in control of myopia development. Selective regulation of cAMP to control scleral collagen synthesis may be a novel therapeutic strategy for preventing and treating myopia.

Spontaneous high myopia in one eye will affect the development of form deprivation myopia in the fellow eye.

Purpose: Whether there is an interaction between eyes of individual subjects in refractive development is an important issue to guide experimental designs and help understand mechanisms involved in development of refractive errors. This study investigated whether spontaneous high myopia in one eye will affect refractive development of the fellow eye treated with form deprivation. Methods: Thirty-four guinea pigs were divided into four groups: MD (monocularly form-deprived animals with a pre-treatment anisometropia ≤ 2D, n = 8), anisometropic MD (monocular form deprivation on a relatively hyperopic eye in animals with a pre-treatment anisometropia ≥ 10D, n = 9), normal control (non-form deprivation in animals with a pre-treatment anisometropia ≤ 2D, n = 8), and anisometropic control (non-form deprivation in animals with a pre-treatment anisometropia ≥ 10D, n = 9). All eyes in different groups underwent biometric measurements on days 0, 12, 24, and 36 of the experiment. Results: High myopia in one eye reduced form deprivation myopia in the fellow treated eye. The change in refraction from 0 to 36 days in the deprived eyes was -3.07D for the MD group, but -1.22D for the anisometropic MD group (-3.07D vs. -1.22D: p = 0.009, independent sample t-test). The amount of vitreous chamber lengthening over the same period in the deprived eyes was 0.19 mm for the MD group, but 0.12 mm for the anisometropic MD group (0.19 mm vs. 0.12 mm: p = 0.038, independent sample t-test). Myopic development in the anisometropic animals is mainly inhibited within the first 12 days compared to normal MD animals. Conclusions: These results indicate that an interaction in refractive development may exist temporarily between two eyes of a highly anisometropic animal if the visual environment has been changed.

The Effect of Temporal and Spatial Stimuli on the Refractive Status of Guinea Pigs Following Natural Emmetropization

PURPOSE To understand the visual information essential for maintaining stable refraction after emmetropization, we investigated the effects of spatial and temporal stimuli on the refractive status of guinea pigs. METHODS Eighty-eight guinea pigs (4 weeks old) were randomly divided into 10 groups. Thirty animals were raised in backgrounds of gray (N = 13), square-wave (N = 9), or sine-wave grating (N = 8). Thirty-one animals were raised in gray backgrounds with three frequencies of flicker: gray-1-Hz (n = 10), gray-6-Hz (n = 12), and gray-20-Hz (n = 9). Eighteen animals were raised in regular cages with different frequencies of flicker (n = 6 respectively in 1-Hz-, 6-Hz-, and 20-Hz-flicker groups). Nine animals were raised in regular cages with no additional stimuli and used as normal controls. Ocular biometry was measured before and after 3 weeks of exposure to the test environments. RESULTS Guinea pigs raised in the gray background for 3 weeks developed myopia, -6.1 ± 2.1 diopters (D), whereas those exposed to either sine-wave or square-wave gratings, or raised in regular cages, retained stable refractions. Animals in the gray-6-Hz group developed lower myopia, -2.7 ± 2.7 D, than the gray group not exposed to flicker. Animals stimulated with a range of flickering frequencies in regular cages also developed myopia but to a lower degree, -3.1 to 0.2 D, than those in gray backgrounds, -5.0 to -2.7 D. CONCLUSIONS Guinea pigs require both spatial and temporal stimuli to maintain stable refractions. The influence of temporal stimuli on refraction varies with the type and amount of spatial information available in the visual environment.

The role of cGMP in ocular growth and the development of form-deprivation myopia in guinea pigs.

PURPOSE Development of myopia is associated with remodeling of the sclera, a tissue composed principally of collagen. Cyclic guanosine monophosphate (cGMP) regulates collagen synthesis in several organs; therefore, we investigated the effects of soluble guanylyl cyclase (sGC) stimulation and inhibition on refraction and ocular growth in guinea pigs under normal and form-deprived (FD) conditions. METHODS Retinal and scleral cGMP concentrations were measured in normal and monocularly FD guinea pigs at 2 days, 1 week, and 2 weeks of form deprivation and following 2 days recovery. Stimulation of sGC by BAY41-2272 and inhibition by NS-2028 were achieved by daily subconjunctival injection in normal and FD eyes. Refraction and axial parameters were measured at the commencement, middle, and cessation of the experiment. cGMP levels were also determined at the end of the experiment. RESULTS Retinal and scleral cGMP concentrations increased in FD eyes from 2 days to 2 weeks (P ≤ 0.029). Levels decreased after 2 days of recovery (P ≤ 0.003). Daily injections of BAY41-2272 induced a myopic shift (P ≤ 0.001) and ocular elongation (P ≤ 0.01) in normal animals, but did not alter myopia in FD eyes (P > 0.05). In contrast, daily injections of NS-2028 partially reduced myopic shifts (P ≤ 0.012) and ocular elongation (P ≤ 0.015) induced by form deprivation, but did not affect ocular growth and refraction in normal eyes (P > 0.05). Retinal and scleral cGMP levels were increased by BAY41-2272 in normal eyes and decreased by NS-2028 in FD eyes. CONCLUSIONS Changes in cGMP signaling contribute to myopic development. Thus, cGMP may be a potential therapeutic target for preventing/treating myopia.

Dopamine Receptor Subtypes Mediate Opposing Effects on Form Deprivation Myopia in Pigmented Guinea Pigs

Purpose We reported previously that changes in dopamine receptor (DR) subtype activation modulate spontaneous myopia progression in albino guinea pigs. To determine if DR control of refractive error development is different than in its normal counterpart, we evaluated the contribution of dopaminergic pathways to emmetropization and form deprivation myopia (FDM) progression in pigmented guinea pigs. Methods Monocular myopia was induced by unilateral form-deprivation (FD). The effects of agonists of D1R (SKF38393) and D2R (quinpirole), the corresponding antagonists (SCH23390 and sulpiride), and vehicle were tested by peribulbar injection around FD or untreated control eyes. High-performance liquid chromatography with electrochemical detection quantified retinal and vitreous dopamine (DA) and 4-dihydroxyphenylacetic acid (DOPAC) levels. Ocular refraction and axial dimensions were measured using eccentric infrared photoretinoscopy (EIR) and A-scan ultrasonography, respectively, initially and after 2 or 4 weeks of treatment. Results After treatment with any of these four agents for 2 weeks, retinal and vitreal DA and DOPAC levels were not significantly different in drug- and vehicle-treated eyes. Neither agonism nor antagonism of D1R or D2R activity affected emmetropization. In contrast, D1R activation by SKF38393 inhibited FDM progression, while D2R activation by quinpirole augmented this response. On the other hand, D2R antagonism with sulpiride slowed FDM progression while D1R antagonism with SCH23390 had no effect. Conclusions In pigmented guinea pigs, D1R activation inhibited, whereas D2R activation enhanced, FDM. These results closely mirror previous findings in albino animals and offer further evidence that DA and its cognate receptors affect refractive error regulation in guinea pigs.

Prostaglandin F2α Receptor Modulation Affects Eye Development in Guinea Pigs

Retinal arachidonic acid (ARA) levels in form‐deprived eyes decline in guinea pigs. As prostaglandin F2α (PGF2α) is an ARA metabolite and endogenous agonist of prostaglandin F receptor (FP), we have been suggested that down‐regulation of PGF2α‐FP receptor signalling pathway contributes to myopia onset. To test this hypothesis, this study determines whether: (i) retinal PGF2α levels decline during the development of form deprivation myopia (FDM) in guinea pigs; (ii) FP receptor agonism and antagonism alter emmetropization and myopia development. Pigmented guinea pigs were randomly assigned to normal vision and form‐deprived groups. Ultraperformance liquid chromatography coupled with a mass spectrometer (UPLC‐MS) measured retinal PGF2α levels 2 weeks after form deprivation (FD). The selective FP agonist, latanoprost acid (LAT) and its corresponding antagonist, AL8810, were peribulbarly injected into each group. An eccentric infrared photorefractor (EIR) monitored refraction. A‐scan ultrasonography measured axial elongation (AL) and vitreous chamber depth (VCD). Tonometry measured the intraocular pressure (IOP). Retinal PGF2α levels declined in form‐deprived eyes compared to those in normal eyes. Neither LAT nor AL8810 affected IOP with or without FD. On the other hand, after 4 weeks of daily 0.5 μg AL8810 treatment, a myopia of −1.99 ± 0.34 dioptre (D) developed, but LAT had no effect on emmetropization in a normal visual environment. Nevertheless, daily 30 μg LAT treatment for 4 weeks inhibited FDM development by 41% (vehicle control: −8.39 ± 0.45 D; LAT: −4.95 ± 0.39 D; two‐way anova with repeated measures, p < 0.05). Down‐regulation of PGF2α‐FP receptor signalling pathway may contribute to myopia onset as retinal PGF2α declined in myopic eyes and antagonism of FP receptor by AL8810 induced a myopic shift in normal vision environment. Meanwhile, up‐regulation of this pathway by LAT inhibited FDM development. However, the mechanism underlying LAT‐induced FDM inhibition needs further clarification. This uncertainty exists because its inhibition of FDM suggests that LAT strengthens the scleral framework which reduces axial elongation. On the other hand, its IOP‐lowering effect is attributed to thinning and weakening the scleral framework in glaucoma treatment.

Scleral hypoxia is a target for myopia control

Significance Myopia is the leading cause of visual impairment. Myopic eyes are characterized by scleral extracellular matrix (ECM) remodeling, but the initiators and signaling pathways underlying scleral ECM remodeling in myopia are unknown. In the present study, we found that hypoxia-inducible factor-1α (HIF-1α) signaling promoted myopia through myofibroblast transdifferentiation. Furthermore, antihypoxic treatments prevented the HIF-1α–associated molecular changes, thus suppressing myopia progression. Our findings defined the importance of hypoxia in scleral ECM remodeling and myopia development. The identification of the scleral hypoxia in myopia not only provides a concept for understanding the mechanisms of myopia development but also suggests viable therapeutic approach to control myopia progression in humans. Worldwide, myopia is the leading cause of visual impairment. It results from inappropriate extension of the ocular axis and concomitant declines in scleral strength and thickness caused by extracellular matrix (ECM) remodeling. However, the identities of the initiators and signaling pathways that induce scleral ECM remodeling in myopia are unknown. Here, we used single-cell RNA-sequencing to identify pathways activated in the sclera during myopia development. We found that the hypoxia-signaling, the eIF2-signaling, and mTOR-signaling pathways were activated in murine myopic sclera. Consistent with the role of hypoxic pathways in mouse model of myopia, nearly one third of human myopia risk genes from the genome-wide association study and linkage analyses interact with genes in the hypoxia-inducible factor-1α (HIF-1α)–signaling pathway. Furthermore, experimental myopia selectively induced HIF-1α up-regulation in the myopic sclera of both mice and guinea pigs. Additionally, hypoxia exposure (5% O2) promoted myofibroblast transdifferentiation with down-regulation of type I collagen in human scleral fibroblasts. Importantly, the antihypoxia drugs salidroside and formononetin down-regulated HIF-1α expression as well as the phosphorylation levels of eIF2α and mTOR, slowing experimental myopia progression without affecting normal ocular growth in guinea pigs. Furthermore, eIF2α phosphorylation inhibition suppressed experimental myopia, whereas mTOR phosphorylation induced myopia in normal mice. Collectively, these findings defined an essential role of hypoxia in scleral ECM remodeling and myopia development, suggesting a therapeutic approach to control myopia by ameliorating hypoxia.

Role of Cyclic Adenosine Monophosphate in Myopic Scleral Remodeling in Guinea Pigs: A Microarray Analysis

Purpose Myopia induction accompanies increased scleral cyclic adenosine phosphate (cAMP) levels and collagen degradation in mammalian models. We compared the scleral gene expression changes following monocular form deprivation (FD) with those induced by adenylate cyclase activation with forskolin (FSK) in guinea pigs. Methods Guinea pigs were assigned to FD, FSK-treated, and age-matched (AM) control groups. FSK was injected monocularly into the inferior palpebral subconjunctiva daily for 4 days. After scleral RNA extraction, a gene microarray scanner and software were used to evaluate the gene expression patterns, followed by pathway analysis using Gene Ontology tools. Quantitative PCR (qPCR) was used to analyze the expression of 10 candidate genes in separate sets of form-deprived, vehicle-injected, and AM animals. Results FSK injections differentially regulated 13 collagen subtypes compared to AM and FD groups. FSK also downregulated Acta2 and Tgf-β2 compared to the AM eyes. Collagen subtypes and Acta2 underwent larger downregulation in the FSK group than during FD. FSK differentially regulated Rarb, Rxrg, Fzd5, Ctnnd2, Dkk2, and Dkk3, which have been linked to ocular growth. Only a few genes were differentially expressed between the FD and AM groups. There was 80% agreement in the direction of gene regulation between microarray and qPCR results. No significant differences were identified between vehicle-injected and AM eyes. Conclusions Collagen, a major scleral extracellular matrix component, is degraded during myopia. Given that FSK and FD both promote myopia through increased collagen degradation, targeting cAMP signaling pathway genes could suppress myopia development.