Download ARVO 2016 Annual Meeting Abstracts 364 Novel signaling

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.
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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
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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.
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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.