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ARVO 2016 Annual Meeting Abstracts 364 Novel signaling mechanisms in refractive development Tuesday, May 03, 2016 3:45 PM–5:30 PM 608 Paper Session Program #/Board # Range: 3785–3791 Organizing Section: Anatomy and Pathology/Oncology Program Number: 3785 Presentation Time: 3:45 PM–4:00 PM Crystalline lens thickness is modulated by spectacle lens defocus in chicks Sally A. McFadden1, Natalia Bilton2, 1, Sheree Harrison1, Marc H. Howlett3, 1. 1Psychology, University of Newcastle, Callaghan, NSW, Australia; 2School of Biomedical Sciences, Charles Sturt University, Port Macquarie, NSW, Australia; 3Netherlands Institute for Neuroscience, Amsterdam, Netherlands. Purpose: Eyes become myopic because they grow excessively. The signals underlying eye growth are studied using spectacle lens compensation (SLC), in which growth can be inhibited or enhanced after exposure to myopic or hyperopic defocus respectively. The underlying signals likely arise locally in the retina, and are rapidly translated to the choroid prior to subsequent eye growth in the posterior sclera. However, emmetropisation involves a balance between both posterior eye growth and the changing optics of the eye. Therefore, we looked for changes in the crystalline lens and the choroid during the early response to SLC in chicks. Methods: 63 chicks (White leghorn, Steggles, Amatil, NSW, Australian Poultry Ltd) were reared on a 12/12 h light/dark cycle, wearing a monocular spectacle lens of one of 8 different powers (-8D, -6D, -4D, -2D, +2D, +4D, +6D, +7.5D; n=6/group) or no lens (n=8) from 3 days of age for 24 h. The lens-wearing eye was repeatedly measured every 4 h over 24 h using a high frequency ultrasound (resolution 10 μm) in anaesthetised chicks. Results: Choroid thickness was bi-directionally modulated by the sign and the magnitude of imposed defocus (Fig 1A). The maximum change occurred in the middle of the day (Fig 2), and the gain was larger for hyperopic than myopic defocus. The thickness of the crystalline lens was also bi-directionally changed (Fig 1B), growing mostly during the day. These changes in the lens were not a passive consequence of eye size as they were in the opposite direction to that predicted by stretch, and after one day of lens-wear, there was no relationship between lens thickness and axial length (R2 = 0.003, p = 0.73). In contrast, choroid thickness was significantly correlated with crystalline lens thickness (R2 = 0.193; p = 0.004). Conclusions: The depth of the crystalline lens is sensitive to early signals which predict the direction of eye growth and is thicker in eyes that subsequently develop myopia and thinner in eyes developing hyperopia. The source of the signal is unknown, but since SLC is unaffected after the elimination of lenticular accommodation in chicks, it may reflect a sensitivity of the crystalline lens or ciliary muscle to factors released from the retina in response to defocus exposure. Fig 2. Choroidal rhythm after subtracting the increase in thickness over 24 h. Commercial Relationships: Sally A. McFadden, None; Natalia Bilton; Sheree Harrison, None; Marc H. Howlett, None Support: Australian Research Council ARC-SG G0177409; RMC-G0180055 University of Newcastle Program Number: 3786 Presentation Time: 4:00 PM–4:15 PM Investigation of the role of ZNF644 in emmetropization and refractive error using zebrafish Ross F. Collery1, Terri L. Young2, Brian Link1. 1Medical College of Wisconsin, Milwaukee, WI; 2Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, WI. Purpose: Multiple pedigree analyses have shown that mutations in human ZNF644 are associated with high-grade myopia. ZNF644 is widely expressed in many tissues throughout the body, including the eye. ZNF644 has recently been shown to regulate gene repression through its interaction and recruitment of the G9/GLP histone lysinespecific methyltransferase complex. It is thought that ZNF644, via its zinc finger domains, binds DNA at defined recognition sites and provides locus specificity for H3K9 methylation. To better understand the role of ZNF644 in myopia and emmetropia, we have inactivated zebrafish orthologs znf644 a and b. We also overexpressed wild-type and myopia-associated mutants of human ZNF644 in zebrafish to assess the effect on eye size and refractive state. Methods: Zebrafish ZNF644 orthologs were inactivated using CRISPR/Cas9 methods. Transgenic zebrafish expressing human ZN644 were generated using Tol2 methods. Eye size and refractive state were measured using spectral-domain optical coherence tomography. Results: znf644a and b are expressed throughout the developing embryo, with enrichment within the nervous system and eye. Inactivation of znf644b in zebrafish increased eye size (n=8 eyes; p=0.0005, Mann-Whitney test). Human ZNF644-eGFP can be ubiquitously expressed in zebrafish without affecting survival. Consistent with its known role in gene regulation, overexpressed ZNF644 protein is enriched within the nucleus. Conclusions: Inactivating ZNF644 orthologs in zebrafish provide insight into the function of the human gene and its effects on eye size and refractive state in wild-type and mutant contexts. In a complementary fashion, zebrafish engineered to overexpress human normal and disease-associated variants of ZNF644, may shed light on the precise links and mechanisms underlying ZNF644 alteration and myopia. Fig 1. Mean choroid thickness (A) and crystalline lens thickness (B) in the lens-treated eye. These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts A, B. in situ hybridization showing znf644a and znf644b mRNA expression at 3 dpf. C, D, D’. Human ZNF644-eGFP is enriched in the nucleus in zebrafish epithelial and muscle cells. E, F. inactivation of zebrafish znf644b causes enlargement of the eye axial length and lens diamter with respect to body length. Commercial Relationships: Ross F. Collery, None; Terri L. Young, None; Brian Link, None Support: NIH R01EY016060; NIH P30EY001931; NIH R01EY014685-11A1; Research to Prevent Blindness; University of Wisconsin Centennial Scholar Funds Program Number: 3787 Presentation Time: 4:15 PM–4:30 PM Ascorbic acid, and not L-DOPA, protects against formdeprivation myopia in retinal degeneration mouse models Erica Landis1, 2, Hanna Park1, Ranjay Chakraborty1, Curran Sidhu1, P. Michael Iuvone1, 5, Machelle T. Pardue3, 4. 1Ophthalmology, Emory University, Atlanta, GA; 2Neuroscience, Emory University, Atlanta, GA; 3Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA; 4Center for Visual and Neurocognitive Rehabilitation, Veterans Affairs Medical Center, Atlanta, GA; 5Pharmacology, Emory University, Atlanta, GA. Purpose: Mouse models of retinal degeneration have shown increased susceptibility to form deprivation myopia, potentially due to decreased dopamine (DA) turnover (Park et al., 2013) or loss of dopaminergic amacrine cells (DACs; Ivanova et al., 2015). The purpose of this work is to test whether systemic injections of the DA precursor, L-3,4-dihydroxyphenylalanine (L-DOPA) will protect against form deprivation (FD) myopia in wild-type and retinal degeneration mice. Methods: Susceptibility to FD myopia was measured beginning at post-natal day 28 (P28) in Pde6brd10/rd10 (rd10) mice and age-matched C57BL/6J wild-type (WT) mice. A subset of animals was given monocular FD lenses following baseline measurements. At P28, mice received daily systemic injections of L-DOPA only (n=17), L-DOPA + the vehicle, ascorbic acid (AA) (n=18-25), or AA only (n=18-21). Weekly measurements of refractive error, corneal curvature, and ocular biometry were performed until P42, using photorefraction, keratometry, and spectral-domain optical coherence tomography. At P44 retinas were collected to measure dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) with HPLC. Results: WT mice exposed to FD developed a significant myopic shift (right-left eye) with AA only treatment (-3.54 ± 1.14D); that was significantly decreased by L-DOPA + AA (0.283 ± 0.588D, p<0.01). Rd10 mice showed the opposite response such that AA only treatment significantly reduced the myopic shift in response to FD (-0.769 ± 0.464D) compared to L-DOPA + AA (-3.206 ± 0.734) or L-DOPA only treatments (-5.752 ± 0.761D, p<0.05). No significant changes were seen in corneal curvature or ocular parameters. Conclusions: Similar to findings in other animals, L-DOPA treatment protects WT mice from FD myopia. However, L-DOPA treatments did not halt or slow FD myopia in rd10 mice, while AA only treatments completely eliminated the myopic shift. We hypothesize that L-DOPA is not protective effect in rd10 mice due to dysfunctional DACs that aren’t able to convert L-DOPA to DA. Furthermore, the anti-oxidant effects of AA on extracellular DA (Neal et al., 1999) may preserve DA in rd10 retinas that is not rapidly degraded by defective DACs. We will confirm this hypothesis with the measurement of dopamine and DOPAC levels with HPLC in each treatment group. Commercial Relationships: Erica Landis, None; Hanna Park, None; Ranjay Chakraborty, None; Curran Sidhu, None; P. Michael Iuvone, None; Machelle T. Pardue, None Support: NIH NEI R01 EY016435, R01 EY004864, P30 EY006360, T32 EY007092-29, Research to Prevent Blindness, and Dept. of Veterans Affairs Rehabilitation R&D Service Research Career Scientist Award C9257S Program Number: 3788 Presentation Time: 4:30 PM–4:45 PM Intrinsic Ocular Mechanisms Underlie Lens-Induced Astigmatism in Chicks William K. Stell1, Vanessa Popa1, Chea-Su Kee2. 1Cumming School of Medicine, University of Calgary, Calgary, AB, Canada; 2School of Optometry, The Hong Kong Polytechnic University, Kowloon, Hong Kong. Purpose: Ocular astigmatism is a refractive error due to differential meridional powers of ocular components, causing blurred vision at all viewing distances. The cause(s) remain poorly understood. Here we used a novel animal model of lens-induced astigmatism to test the hypothesis that processing of astigmatic images in retinal circuits causes the optical abnormality Methods: We induced astigmatism by mounting +4.00/-8.00D crossed-cylinder lenses over the right (treated) eyes of 7-dayold chicks (P7), in groups of n=12, with the -8.00D axis oriented vertically (at 90°) or horizontally (180°); the left (fellow) eyes wore no lens. Net refractive errors of both eyes were measured by streak retinoscopy, before and after 1 week of lens-wear; in selected cases the corneal component was measured by keratometry. To test whether neuronal pathways between retina and brain are required, we injected tetrodotoxin (TTX; 7µL of 10-4M) or PBS (7µL) into the vitreous of the treated eyes on P7, P9 and P11; we assessed the efficacy and duration of action of TTX by the pupillary light reflex and optokinetic response (n=6 each). To confirm that retinal circuitry is required, we injected mixed excitotoxins (2µmol N-methyl-D-aspartate, 0.2µmol quisqualic acid, 0.2µmol kainic acid; in 20µL water) into the treated eyes of n=12 chicks at P7. Fellow eyes always received vehicle alone. Interocular differences (treated - fellow) were assessed by 2-tailed unpaired t-test, or 2-way ANOVA + Tukey’s post-test. Results: Crossed-cylinder goggles reliably induced refractive astigmatism. Maximum astigmatic error was induced at 90°, by -8.00DC axis oriented vertically; this compensated for the imposed defocus. Treated eyes developed astigmatism after injecting TTX or PBS, but not after excitotoxins. Keratometry confirmed that the cornea itself was astigmatic. Conclusions: In our chicks, crossed-cylinder lenses reliably induced compensatory astigmatism. Prevention of astigmatism by excitotoxins showed that the mechanism requires the retina, and not some other light-sensitive ocular tissue (e.g., iris); furthermore, the failure of TTX to affect astigmatism showed that extraretinal neural These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts pathways are not required. We suggest that lens-induced astigmatism is due to local mechanisms of scleral growth-regulation by image defocus, plus mechanical deformation of the cornea by the distorted sclera. Commercial Relationships: William K. Stell, None; Vanessa Popa, None; Chea-Su Kee, None Support: NSERC Grant RGPIN 131-2013; UGC-GRF PolyU 151011/14M Program Number: 3789 Presentation Time: 4:45 PM–5:00 PM Origin of different responses to myopia-inducing stimuli in two guinea pig strains Liqin Jiang, Sarah Kochik, Yang Shen, Christine F. Wildsoet. Ophthalmology and Visual Sciences, School of Optometry, UC Berkeley, Berkeley, CA. Purpose: We previously reported two strains of guinea pigs to vary significantly in their sensitivity to myopia-inducing stimuli, including negative lenses and form deprivation (M Garcia et al. 2015 ARVO E-abstract 2167). To further explore the mechanism underlying this difference, this study focuses on the choroid, whose thickness is known to be modulated optical defocus stimuli. Methods: For both guinea pig strains (EH & NZ), choroidal thickness was evaluated longitudinally in normal animals by posterior segment SD-OCT (Bioptigen), with data from left eyes reported here. Additional animals (NZ, n=3; EH, n=4; 10 months old), wore -5 and +5 D lenses over the right and left eyes respectively for 8 h, with choroidal thickness changes evaluated by A-scan ultrasongraphy. Results: The two strains of guinea pigs showed consistent differences in normal choroidal thickness, at both young and older ages (OCT data: 5.5 months, NZ vs EH: 118±11 vs 138±12 mm, p=0.02; 10 months, NZ vs EH: 110±11 vs 127±21 mm, p<0.05). Interestingly, the NZ animals appeared more sensitive to minus lenses, shown by choroidal thinning while the EH animals were more sensitive to plus lens-induced thickening (-5D treating: NZ vs. EH, -28±26 vs. -8±13 μm; +5D treating: NE vs. EH, -0.7±15 vs. 9±20 μm; ns.). Before the lens treatments, both strains showed minimal interocular difference in choroidal thickness (R vs. L: NZ, 108±9 vs. 102±7 μm; EH, 125±21 vs. 127±18 μm; ns.). Conclusions: Together with the past observation of differences in the sensitivity of the two strains of guinea pigs to myopia-inducing stimuli, the naturally occurring difference of choroid thickness and differences in responsiveness of their choroids to optical defocus reported here raises the question of whether similar differences in humans might contribute to apparent differences in susceptibility to myopia. Commercial Relationships: Liqin Jiang, None; Sarah Kochik, None; Yang Shen, None; Christine F. Wildsoet, None Support: NIH/NEI R01EY12392; National Natural Science Foundation of China 81570878 Program Number: 3790 Presentation Time: 5:00 PM–5:15 PM Visually Regulated Gene Expression of Apolipoprotein A-1 in Chick Eyes Jody A. Summers Rada, Angelica Harper, John Moore. Cell Biology, University of Oklahoma Health Science Center, Oklahoma City, OK. Purpose: Apolipoprotein A-1 (ApoA1) has recently been identified as an extracellular retinoic acid binding protein secreted by the chick choroid (Summers JA, et al. IOVS 2015; ARVO E-Abstract 2162). The current study was carried out to identify ApoA1 gene expression changes in chick ocular tissues during periods of visually guided eye growth. Methods: Form deprivation myopia was induced in two day old white leghorn chicks by application of translucent occluders for 10 days after which time occluders were removed and chicks were allowed to experience unrestricted vision for 0 – 20 days (recovery). Retina, choroids and sclera were isolated from control and treated eyes of chicks and snap frozen in liquid nitrogen. Tissues were homogenized and total RNA was purified, DNAase-treated, and used for the synthesis of cDNA with reverse transcriptase and random hexamers. Steady state ApoA1 mRNA concentrations were quantified from cDNA samples by Taqman real time PCR and normalized using the housekeeping gene, GAPDH. Cycle-threshold data was converted to relative gene expression using CFX-Manager (Bio-rad) and analyzed using paired Student’s t-tests and ANOVA. Results: No significant differences in ApoA1 gene expression were detected in retinas and scleras of control and treated chick eyes (p >0.05). In the choroid, ApoA1 mRNA levels were not significantly different between control and treated eyes following 10 days of form deprivation (0 days of recovery) and 1 day of recovery (p>0.05). In contrast, ApoA1 mRNA levels were significantly elevated in choroids following 4 – 20 days of recovery as compared with untreated contralateral control eyes (↑52% - ↑129%, p< 0.05, paired t-test). Conclusions: The results of the present study indicate that ApoA1 gene expression is modulated in the choroid during recovery from induced myopia. Considering that ApoA1 can function as a retinoic acid binding protein, our results also suggest that transcriptional control of ApoA1 gene expression in the choroid may provide an additional level of regulation of retinoic acid transport and activity in the eye. Commercial Relationships: Jody A. Summers Rada, None; Angelica Harper, None; John Moore, None Support: NIH Grant EY09391 Program Number: 3791 Presentation Time: 5:15 PM–5:30 PM Non-visual factors influencing emmetropization in chicks Xiaoying Zhu2, 1, Josh Wallman3, Sally A. McFadden2. 1Biology and Vision Sciences, SUNY College of Optometry, New York, NY; 2 School of Psychology, University of Newcastle, Callaghan, NSW, Australia; 3Biology, City College of New York, New York, NY. Purpose: Visually based treatments for myopia progression are largely based on animal models and human studies which show that eye growth is influenced by post-natal visual experience. Treatment options for school-aged myopia aim to change visual experience but none appear to completely halt or reverse the progression pattern. Since the size of all of our internal organs are closely maintained during development and adulthood, we hypothesize that, beside visual experience (“defocus-factor”), eye growth may also be influenced by an internal, homeostatic mechanism (“size-factor”) that prevents the eye from deviating from its normal size. We studied the role of the size factor in the chick lens-induced myopia model. Methods: (1) To study if the hypothesized size-factor alone can guide the recovery from eye growth perturbations, chicks wore a +7 D or –7 D lens over one eye for 4 to 7 days, then the lens was removed and the chicks were kept in either normal light or darkness for 2-3 days. (2) To study the interaction of the size- and defocusfactors, chicks first wore a –5 D or –7 D lens on one eye for 7 days (to generate longer eyes), then a –10 D and –15 D lens, respectively, for 4 more days. After the step up in lens power, the size- and defocus-factors would be working in the opposite directions, with the size-factor acting to decrease eye growth (since the eyes were already longer than normal) and the defocus-factor acting to further increase eye growth to compensate for the hyperopic defocus. As a control, another group of chicks wore a –10 D or –15 D lens from the beginning for 11 days. These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record. ARVO 2016 Annual Meeting Abstracts Results: (1) While kept in darkness (in the absence of the defocusfactor), all chicks eyes showed robust recovery from prior hyperopia or myopia similar to those that recovered in normal light, suggesting that the size-factor is involved in eye growth. (2) After compensating for –5 D or –7 D lenses, chicks eyes did not further elongate to compensate for –10 D and –15 D lenses, respectively; whereas they fully compensated for these higher-powered negative lenses if worn from the beginning, suggesting that the size-factor prevents the eye from further elongating. Conclusions: Non-visual factors relating to prior growth state or possibly binocular innate yoked growth mechanisms contribute to the control of eye growth in chicks and may provide an opportunity and/ or be necessary to target in order to maximize the effects of myopia treatments in humans. Commercial Relationships: Xiaoying Zhu, None; Josh Wallman, None; Sally A. McFadden Support: National Institute of Health Grants EY-02727 and RR03060 These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/ to access the versions of record.