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Current Eye Research
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Compensation for spectacle lenses involves changes in
proteoglycan synthesis in both the sclera and choroid
Debora L. Nickla; Christine Wildsoet; Josh Wallman
First Published on: 01 April 1997
To cite this Article: Nickla, Debora L., Wildsoet, Christine and Wallman, Josh (1997)
'Compensation for spectacle lenses involves changes in proteoglycan synthesis in
both the sclera and choroid', Current Eye Research, 16:4, 320 - 326
To link to this article: DOI: 10.1076/ceyr.16.4.320.10697
URL: http://dx.doi.org/10.1076/ceyr.16.4.320.10697
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Current Eye Research
Compensation for spectacle lenses involves changes in
proteoglycan synthesis in both the sclera and choroid
Debora L. Nickla1, Christine Wildsoet2 and Josh Wallman1
1 Biology
Department, City College of CUNY, New York, USA and 2 School of Optometry, Queensland University of Technology,
Brisbane, Queensland 4001, Australia
Abstract
Purpose. It has been demonstrated that chick eye growth compensates for defocus imposed by spectacle lenses: the eye elongates in response to hyperopic defocus imposed by negative
lenses and slows its elongation in response to myopic defocus
imposed by positive lenses. We ask whether the synthesis of
scleral extracellular matrix, specifically glycosaminoglycans,
changes in parallel with the changes in ocular elongation. In
addition, there is a choroidal component to compensation for
spectacle lenses; the choroid thickens in response to myopic
defocus and thins in response to hyperopic defocus. We ask
whether choroidal glycosaminoglycan synthesis changes in
parallel with changes in choroidal thickness.
Methods. Chicks wore either a 115 diopter (D) or 215 D spectacle lens over one eye, or they wore one lens of each power
over each eye for 5 days. At the end of this period, we measured refractive errors and ocular dimensions by refractometry
and A-scan ultrasonography, respectively. Pieces of the scleras
and choroids from these eyes were put into culture and the synthesis of glycosaminoglycans was assessed by measuring the
incorporation of radioactive inorganic sulfur.
Results. We here report that the compensatory modulation of
the length of the eye involves changes in the synthesis of glycosaminoglycans in the sclera, with synthesis increasing in
eyes wearing 215 D spectacles lenses and decreasing in eyes
wearing 115 D lenses. In addition, changes in the synthesis of
glycosaminoglycans in the choroid are correlated with changes
in choroidal thickness: eyes wearing 115 D lenses develop
thicker choroids and these choroids synthesize more glycosaminoglycans than choroids from eyes wearing 215 D lenses.
Conclusions. Changes in scleral glycosaminoglycan synthesis
accompany lens-induced changes in the length of the eye. Furthermore, changes in the thickness of the choroid are also asso-
Correspondence: Current affiliation: Debora L. Nickla, New England College
of Optometry, 424 Beacon St., Boston, MA, 02115, USA
ciated with changes in the synthesis of glycosaminoglycans.
These results are consistent with the regulation of the growth
of the eye being bidirectional, and with the retina being able to
sense the sign of defocus. Curr. Eye Res. 16: 320–326, 1997.
Key words: chicks; emmetropization; eye growth; hyperopia;
myopia; proteoglycans
Introduction
During postnatal ocular development, the length of the eye
usually becomes matched to the optics of the cornea and lens
so that images of distant objects fall on the retina; that is, the
eye becomes emmetropic. The notion that this optical “tuning”
is visually guided has been extensively studied since the finding that depriving the eye of patterned input results in excessive growth and myopia: monkeys (1), chicks (2), tree shrews
(3, 4) and cats (5, 6). Stronger evidence for the visual regulation of eye growth comes from more recent studies showing
that the eyes of chicks, monkeys, tree shrews and guinea pigs
all show compensatory changes in length and refractive error
for defocus imposed by spectacle lenses: chicks (7–10), monkeys (11), tree shrews (12), guinea pigs (13). In the chick, at
least, the retina appears to be able to determine not only the
magnitude of imposed defocus, but also its direction, and to
initiate compensation over a wide range of myopic and hyperopic defocus, although the “bidirectionality” of this compensatory mechanism is not universally accepted. In this paper we
looked for biochemical correlates of the compensatory growth
responses of eyes wearing positive and negative lenses.
Changes in the rate of growth of the chick eye are correlated
with changes in the rate of synthesis of scleral extracellular
matrix; specifically, scleras of eyes that are rapidly elongating
in response to form deprivation show an increase in synthesis
of matrix glycosaminoglycans, the sulfated side chains of proteoglycans, whereas scleras of eyes that have slowed their
elongation in response to removal of the diffusers show a
Received on June 19, 1996; revised on October 30 and accepted on October 31, 1996
© Oxford University Press
Downloaded By: [Mt Sinai School of Medicine, Levy Library] At: 17:18 14 April 2008
Spectacle lenses and proteoglycan synthesis in chick eyes
decrease in synthesis of glycosaminoglycans (14–16). Furthermore, scleras from form-deprived eyes contain significantly
more of the core protein of the cartilage proteoglycan, aggrecan, than do scleras from normal eyes (14), indicating that
changes in the rate of synthesis of aggrecan underlie the
changes in growth rate. We measured GAG synthesis in scleras
of eyes wearing positive and negative spectacle lenses to see
whether changes in the rate of synthesis might underlie the
defocus-induced changes in the length of the eye. Furthermore,
to make a start toward understanding the mechanism whereby
the choroid changes its thickness in response to myopic or
hyperopic defocus (i.e. positive or negative lenses), we also
measured GAG synthesis in the choroids from the same eyes.
We studied refractive error and 3 ocular dimensions: (1) The
axial length (cornea to sclera), which we expected to be most
closely related to scleral growth (2), the vitreous chamber
depth, the distance of the retina from the lens, which should be
well correlated with refractive error, and (3) the choroid 1 retinal thickness, because the choroid participates in compensation by becoming thicker in response to myopic defocus and
thinner in response to hyperopic defocus, thereby moving the
retina.
Predictably, we found that eyes wearing negative lenses
were longer than eyes wearing positive lenses. Our new findings are that the scleras of eyes wearing negative lenses synthesized more GAGs than the scleras of eyes wearing positive
lenses, and furthermore, that the thicker choroids from eyes
wearing positive lenses synthesized more GAGs than the thinner choroids of the negative lens treatment group. Part of this
work has been presented as an abstract (17) and the data in Figure 3a have been published in Wallman et al., 1995 (9).
Methods
Subjects
Subjects were White Leghorn chickens (Gallus gallus)
obtained as day-old hatchlings from Truslow Farms (Chestertown, MD). Birds were housed in wire cages with raised floors
within temperature controlled chambers. The light cycle was
14L/10D. Food was sifted to minimize dust accumulation on
the lenses. Lenses were cleaned every 3–4 hours over the
period during which the lights were on. All experiments conformed to the ARVO Resolution on the Use of Animals in
Research.
Lenses and experimental paradigms
Lenses were made from PMMA material with a back optic
radius of 7 mm. To attach the lenses to the chicks, lenses were
first glued to plastic rings that were affixed to Velcro rings. A
mating ring of Velcro was glued to the feathers around the eye.
This system allowed for their easy removal for frequent cleaning. Lenses were put onto the eyes on day 5 after hatching. On
the fifth day of lens wear, the refractive errors and ocular
dimensions were measured by refractometry and A-scan ultrasonography respectively. The animals were then sacrificed with
321
an overdose of sodium pentobarbitol and the eyes dissected
and put into culture (described below).
Two groups of birds comprise the data in this paper. In one
group (“binocular lenses”, n 5 8), one eye wore a 115 D lens
and the other eye wore a 215 D lens. (The +15 D and 215 D
lenses had effective powers at the cornea, of 116.5 D and
213.8 D respectively, based on an average vertex distance of 6
mm). A second group (“monocular lenses”, n 5 13) wore
either a 115 D or 215 D lens over one eye (115 D, n 5 7;
215 D, n 5 6), while the fellow control eye wore a plano (0 D)
lens. Ocular dimensions and refractive errors were measured in
all 21 birds. Proteoglycan synthesis was measured in the
scleras and choroids of the “binocular lenses” group, and in 4
birds of the “monocular lenses” group (n 5 2 for each lens
power and 4 plano eyes). Statistical comparisons between
groups used a two-tailed t-test, unless otherwise indicated.
Because there were no statistically significant differences
between the plano lens-wearing eyes paired with negative
lenses versus those paired with positive lenses, these data were
combined for all analyses.
Ocular measurements
For measurement of refractive error and ocular dimensions,
birds were anaesthetized with chloropent, a mixture of chloral
hydrate and sodium pentobarbitol. Mydriasis (and presumably
cycloplegia) was obtained using 6 drops/eye of a mixture of
vecuronium bromide (Norcuron, Organon, West Orange, NJ)
and benzalkonium chloride (10 mg/ml and 0.26 mg/ml, respectively) in saline.
Refractive error was measured using a Hartinger refractometer (Jena Coincidence Refractometer). The pupillary axis of the
eye was aligned with the refractometer by centering in the
pupil the corneal reflection of a ring of light mounted coaxially
with the instrument. Two pairs of measurements along orthogonal meridians were made and the data averaged. The eye was
realigned twice more, and two more pairs of measurements for
each orthogonal axis were taken each time. The median of
these 6 average spherical equivalent refractive errors per eye (2
measurements per 3 realignments) yielded the refractive data
presented here. Refraction data were not corrected for the artifact of retinoscopy.
We measured ocular dimensions by A-scan ultrasound. A 7.5
MHz transducer with an attached water-filled standoff aligned
along the optic axis of the eye, and with an intervening layer of
gel between the cornea and the standoff. The signals were
recorded on diskettes by a Nicolet digital oscilloscope; four
sweeps per eye were subsequently analyzed and averaged to get
the data for each eye. Because this measuring system could not
resolve the retinal/choroidal interface, the thickness of the retina
and choroid combined was measured and is referred to in the
text and graphs as “retina 1 choroid” thickness. Nonetheless,
because we know that the thickness of the retina does not
change over the 5 days of lens wear (based on unpublished
observations using a high frequency ultrasound system which
resolves the two layers), we can confidently state that the thickness changes observed are due solely to changes in the choroidal
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D. L. Nickla et al.
layer. Vitreous chamber depth is defined as the distance between
the posterior lens surface and the anterior retinal surface. Axial
length is defined as the distance between the anterior cornea and
the anterior sclera.
Measurement of GAG synthesis
To dissect out the choroid and sclera, eyes were placed in cold
medium and the extraocular muscles were cleared from the
globe. The eyes were then bisected along the ora serrata using
a razor blade, and a 7 mm punch of tissue from the posterior
pole of the eye was obtained using a trephine. The retina/retinal pigment epithelium (RPE) was separated from the underlying choroid and sclera and discarded. The choroid was then
gently dissected away from the sclera, and any adhering RPE
was removed using a small sable paint brush. Pieces of sclera
and choroid were incubated in separate wells in medium labeled with Na235SO4 (30 mCi/ml) for 24 hours. The medium
used for all experiments was the chemically defined medium (N2) of Bottenstein (18). This medium is a 1:1 ratio of Dulbecco’s Modified Eagle’s Medium and Ham’s mixture (F-12)
with added sodium bicarbonate, sodium selenite, progesterone,
putrescine, pyruvate, glutamine, transferrin, catalase and insulin. Antibiotics added were streptomycin, garamycin and
fungizone.
To assay newly synthesized glycosaminoglycans, tissue was
digested in 0.05% proteinase-K (protease type XXVIII, Sigma,
in 10 mM EDTA, 0.1M sodium phosphate, pH 6.5) overnight
at 578C. This treatment results in complete digestion of the cartilaginous and fibrous sclera, and of the choroid. Glycosaminoglycans were precipitated by the addition of 0.5% cetylpyridinium chloride (CPC) in 2 mM Na2SO4 in the presence of
unlabeled chondroitin sulfate (1 mg/ml in distilled H2O). Samples were incubated for 1 hour at 378C and the precipitated glycosaminoglycans were collected on Whatman filters (GF/A)
using a Millipore 12-port manifold. Filters were rinsed 5 times
with 0.1% CPC containing 0.05 M NaCl. Radioactivity in the
filters was measured by liquid scintillation counting in 10 ml
scintillation fluid (CytoScint, Fischer). Further details of this
procedure are in Rada et al., 1992 (15).
Results
Efficacy of spectacle lens treatment
Refractive compensation was observed in response to both the
positive and negative spectacle lenses worn (albeit only partially for negative lenses, probably due to the relatively short
duration of lens wear compared to other studies) (Fig. 1). Furthermore, the degree of compensation to the lenses did not differ depending on whether the bird wore two lenses of opposite
sign, or one plano lens: Eyes wearing 115 D lenses became
hyperopic (binocular lenses: mean 5 116 D; monocular lenses:
mean 5 112 D), whereas those wearing 215 D lenses became
myopic (mean 5 26 D for both groups (Fig. 1a); all comparisons between eyes wearing lenses of different powers are significantly different, p , 0.001). Corresponding to these refractive changes, the now hyperopic eyes that had worn 115 D
lenses had significantly shorter vitreous chambers (back of lens
to retina) than either the myopic eyes that had worn 215 D
lenses, or the eyes that had worn plano lenses (Fig. 1b, all comparisons between lenses significantly different). For vitreous
chamber depth, too, the compensation for the lenses was similar, regardless of the power of the lens worn on the fellow eye
(compare bars in the 115 D and 215 D groups, Fig. 1b). The
refractive error was highly correlated with the vitreous chamber depth (Fig. 1c, r = 20.71, p , 0.001), and linear regression
analysis yielded a slope of 19 D/mm, implying that changes in
the length of the vitreous chamber are largely responsible for
the changes in refractive error. (The average length of these
eyes was approximately 9mm. We estimated the refractive
change per mm change in axial length in these eyes to be
17.5D/mm, based on the formula in Wallman et al., 1995 [9]).
Scleral GAG synthesis and eye length
We find that GAG synthesis in scleras from eyes wearing spectacle lenses is changed in the same direction as the eye length
(Fig. 2). Specifically, the scleras of eyes wearing 115 D lenses,
which are shortest, synthesize significantly fewer GAGs than
the scleras of eyes wearing 215 D lenses, which are longest
(means: 215 vs 456 mmoles sulfur per punch; p , 0.01, Fig.
2a, compare to Fig. 2b). The scleral GAG synthesis in control
eyes wearing plano lenses is intermediate between the two
(mean: 370 mmoles sulfur per punch) although not significantly different from either, probably because of the small
number of eyes in this group. The facts that the axial length
(cornea to sclera) predominantly reflects scleral growth, and
that the eyes wearing negative lenses are significantly longer
than eyes wearing positive lenses (binocular lenses: p , 0.01;
monocular lenses: one-tailed t-test, p , 0.05) are consistent
with the hypothesis that changes in the synthesis of scleral
GAGs induced by spectacle lenses are causally related to
changes in eye length.
Choroidal GAG synthesis and choroid thickness
We find that the choroid, too, shows changes in synthesis of
GAGs in the direction appropriate for the changes in its thickness (Fig. 3). Specifically, the choroid 1 retinas in the eyes that
are compensating for the myopic defocus are thicker than those
from eyes compensating for hyperopic defocus (115 D vs 215
D lenses: binocular lenses: mean 5 0.85 vs 0.42 mm, p ,
0.0005; monocular lenses: mean 5 0.68 vs 0.44 mm, p , 0.05,
Fig. 3b) and thicker than in eyes wearing plano lenses (mean:
0.68 vs 0.45 mm, p , 0.05). These thicker choroids show significantly greater GAG synthesis than the choroids from eyes
wearing negative lenses (mean: 19 vs 12 mmoles sulfur/punch,
p , 0.001, Fig. 3a). In addition, although we (unlike Wildsoet
and Wallman, 1995 [10]) did not detect a difference in the
thickness of the choroid + retinas from eyes wearing plano
lenses versus those wearing negative lenses (Fig. 3b), the choroids from eyes wearing negative lenses synthesized significantly fewer GAGs than those from control eyes (mean: 12 vs
17 mmoles sulfur/punch, p , 0.005, Fig. 3a). These results
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Spectacle lenses and proteoglycan synthesis in chick eyes
323
Figure 1.
suggest that changes in glycosaminoglycan synthesis may be
related to changes in choroidal thickness.
Discussion
This paper reports two main findings: First, scleral GAG synthesis changes in concert with eye length, both increasing with
compensation for the hyperopic defocus imposed by negative
lenses and both decreasing with compensation for the myopic
defocus imposed by positive lenses. Second, the thickened
choroids in eyes wearing positive lenses synthesize more
GAGs than choroids in control eyes, and the choroids from
eyes wearing negative lenses synthesize fewer GAGs.
Much recent evidence indicates that the regulation of eye
growth in the chick is at the level of the synthesis of scleral
extracellular matrix molecules. Form deprivation results in an
increase in scleral dry weight, as well as in the synthesis of
protein, DNA and glycosaminoglycans relative to normal eyes
(dry weight, protein and DNA: [19]; glycosaminoglycans
[14]). Conversely, scleras of eyes that had normal vision
restored and were recovering from form deprivation myopia
showed decreased GAG synthesis (15, 16). Our finding of increased scleral GAG synthesis in eyes that were elongating
more rapidly in response to negative lenses, and decreased synthesis in eyes that were elongating more slowly in response to
positive lenses lends further support to the hypothesis that the
regulation of eye length occurs via the modulation of scleral
extracellular matrix synthesis.
The chick sclera consists of two layers, an inner cartilaginous one and an outer fibrous one (20). The cartilaginous layer
is composed of chondrocytes embedded in a matrix of collagen
fibers and proteoglycans, predominantly aggrecan. The fibrous
layer is composed of fibroblasts, collagen and predominantly
small proteoglycans, like decorin. These two layers show
opposite responses to both form-deprivation and negative spectacle lens wear in the rate of synthesis of GAGs: specifically,
the cartilaginous layer shows increases in synthesis while the
fibrous layer shows decreases in synthesis (21). Because the
uptake of sulfur into proteoglycans in the cartilaginous layer is
approximately 100 times greater than that in the fibrous layer,
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324
D. L. Nickla et al.
Figure 2.
analyses done using whole pieces of sclera predominantly
reflect the response of the cartilaginous layer (21). Indeed, in
whole scleral pieces, most of the incorporation of sulfur is into
a proteoglycan of approximately the same size as the cartilage
proteoglycan aggrecan (14, 22). Furthermore, the newlysulfated GAGs on the proteoglycans are cleaved by the enzymes chondroitinase and keratanase (14, 22) leading us to
conclude that the molecules we are measuring are mostly
aggrecan, and that the cells responsible for the production of
these molecules are in large measure the chondrocytes in the
cartilaginous layer of the sclera. However, because we are
measuring sulfur incorporation into the GAG side chains, and
because newly sulfated GAGs can be added to pre-existing
core proteins, we cannot assert that changes in levels of newly
synthesized molecules reflect changes in the rate of synthesis
of intact proteoglycans, only that they reflect changes in the
synthesis rate of the glycosaminoglycans.
It should be noted that, in analyzing the biochemical data,
we did not normalize the data to either DNA or protein, raising
the possibility that the differences in synthesis between groups
are an epiphenomenon of a change in cell number. There is evidence, however, that the density of the chondrocytes in the cartilage of scleras from form-deprived myopic eyes is lower than
that in normal eyes (23), and that these scleras too show
increased GAG synthesis (14). Furthermore, given the evidence that the GAG synthesis measured in scleral tissue predominantly reflects synthesis in the cartilaginous layer, we can
conclude with some confidence that the greater synthesis in the
scleras of eyes wearing negative lenses is not the result of an
increase in the number of cells, but of an increase in the rate of
synthesis of GAGs.
The chick eye shows good compensation, in both refractive
error and vitreous chamber depth, to lenses of different powers
and opposite sign (7, 10). We here show that scleral GAG synthesis is higher in eyes wearing negative lenses and lower in
eyes wearing positive lenses compared to eyes wearing plano
lenses. One interpretation of these results is that the eye can
either increase or decrease its growth in response to the sign of
the defocus; that is, that there are two opposite signals arising
from the retina, depending on whether the image plane is in
front of or behind it, with the plano lens (or no lens) being the
neutral point. An alternative interpretation is that there is only
one signal, perhaps the same signal involved in form-deprivation myopia, the level of which depends on the amount of blur
experienced over the day. Because the chick is presumed to
spend much of its time viewing close objects, a positive lens
may reduce the total amount of blur experienced, whereas negative lenses may increase the amount of blur. The weakness of
this second interpretation is that it implicitly makes strong presumptions about the operation of accommodation. Either
accommodation must not be used (as might be the case if the
lens is applied monocularly in species with unyoked accommodation), or the defocus remaining in the presence of accommodation in eyes wearing negative lenses is adequate to cause
myopia. Despite this weakness, this hypothesis is favored by
some. One reason for its prevalence is that in mammals, the
response to positive lenses is limited to low powers (11, 12),
suggesting that the action of the lens is to correct the infantile
hyperopia, rather than to produce a myopic refractive error to
which the eye shows a compensatory response. However,
recent findings suggest that rhesus monkeys, at least, can be
induced to become very hyperopic in compensation for positive lenses, if the lens power is increased gradually (E. Smith
of the University of Houston at the 1996 International Congress of Eye Research, Yokohama), suggesting that the eye can
respond to positive lenses but only within a certain range of
defocus.
Although the evidence arguing for one or two mechanisms
of lens compensation is equivocal at present, we would expect
that, if the compensation for positive and negative lenses used
the same mechanism as the response to diffusers, manipulations that affect the response to one would affect the responses
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Spectacle lenses and proteoglycan synthesis in chick eyes
325
Figure 3.
to the others. Recent work describes five manipulations that
reduce form-deprivation myopia: flickering light, constant
light, treatment with atropine (a muscarinic acetylcholine
antagonist), reserpine (a depleter of retinal serotonin and
dopamine) or 6-hydroxydopamine; all of these also reduce the
compensatory myopia resulting from negative lens wear, but
have little effect on the compensatory hyperopia resulting from
positive lens wear: flicker (24), atropine (25), reserpine (26),
constant light (27), 6-hydroxydopamine (28); see also Wallman, 1995 (29). Furthermore, the response to negative lenses
differs from the response both to diffusers and to positive
lenses in that optic-nerve-section attenuates the former but not
the latter two (10). We are inclined to interpret this set of findings as suggesting that two different mechanisms are involved
in the compensation to positive and negative lenses, although
the mechanism underlying compensation to negative lenses
appears similar in most cases to that underlying form-deprivation induced myopia.
The choroidal thickening response is the major component
of the early phase of compensation to myopic defocus in the
chick eye, serving to push the retina toward the image plane
(9). This response occurs within hours (unpublished results),
and in the absence of a connection to the brain (10, 30). In this
paper, we report that the thicker choroids from eyes wearing
positive lenses synthesize more glycosaminoglycans than the
thinner choroids from both control eyes and eyes wearing negative lenses. We have also previously reported that the choroid
thins significantly in response to form deprivation (22) and to
negative lenses (9, 10), although in the present study we did
not observe this thinning in eyes wearing negative lenses, presumably because of the lower resolution of the ultrasonography system used. Nonetheless, we found that the synthesis of
GAGs by the choroids from eyes wearing negative lenses was
significantly lower than in choroids from eyes wearing plano
lenses. We raise the possibility that changes in the rate of synthesis of proteoglycans underlie the changes in choroidal thickness. Proteoglycans are large, highly sulfated, anionic macro-
molecules that are abundant in the extracellular matrices of
connective tissue (review: [31]). Because they are extremely
hydrophilic, increased synthesis could cause tissue expansion
by increasing movement of water into the tissue. Conversely,
choroidal thinning could be affected by a decreased synthesis
(and/or increased degradation) of proteoglycans. Nonetheless,
our data do not preclude the alternative possibilities either that
other mechanisms are involved, or that proteoglycans subserve
some other function in the choroid.
In summary, the compensatory responses of the eye to positive and negative lenses involve changes in glycosaminoglycan
synthesis in both the sclera and the choroid. In the case of the
sclera, the observed changes in synthesis of GAGs are consistent with, and may underlie, the lens-induced changes in ocular
growth and thus indirectly, the compensatory changes in
refraction. The changes in the choroidal GAG synthesis may
play a role in regulating choroidal thickness in order to move
the retina forward and back, thereby providing a fast component of the emmetropization mechanism.
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