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From the Department of Optometry and Vision Sciences, The University of Melbourne, Victoria, Australia.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. Myopia was induced in tree shrews by monocularly depriving them of pattern vision. Some animals then had the occluder removed and were allowed to recover from the induced myopia for periods of 1, 3, 5, 7, and 9 days. Newly synthesized GAGs were radiolabeled in vivo with [35S]sulfate. Sulfate incorporation and total GAG content in the sclera was measured through selective precipitation of GAGs from proteinase K digests with alcian blue dye. Dry weights of the sclerae were also determined. Changes in ocular refraction and eye size were monitored using retinoscopy, keratometry, and ultrasonography.
RESULTS. Eyes developing myopia showed a significant reduction in scleral GAG synthesis, particularly in the region of the posterior pole (-36% ± 7%) compared with contralateral control eyes. Scleral dry weight was also significantly reduced in these eyes (-3.7% ± 1.2%). In recovering eyes, significant changes in GAG synthesis were apparent after 24 hours of recovery. After 3 days of recovery, significantly elevated levels of GAG synthesis were found (+79% ± 15%), returning to contralateral control eye values after 9 days of recovery. Interocular differences in scleral dry weight were shown to follow a similar pattern to that observed for GAG synthesis.
CONCLUSIONS. Active remodeling, resulting in either the loss or replacement of scleral tissue and not passive redistribution of scleral tissue, is associated with changes in eye size during both myopia development and recovery. Regulatory changes in scleral metabolism can be rapidly evoked by a change in visual conditions and the direction of regulation is related to the direction of change in eye size.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mammalian sclera is a structural and protective connective tissue, predominantly composed of fibrillar collagens, proteoglycans, and small amounts of various glycoproteins.13 Proteoglycans, which consist of a central core protein and side chains of negatively charged disaccharide polymers known as glycosaminoglycans (GAGs), are complex molecules that regulate many of the properties of the ECM. They are involved in the control of collagen fibril separation, matrix hydration and biomechanics as well as in making fibrillar collagen particularly difficult to degrade. In addition, they provide binding sites for other regulatory molecules within the ECM.14
The measurement of sulfate incorporation into GAG polymers is a useful index of proteoglycan synthesis and has been used as a marker of scleral metabolism during the investigation of myopia development in both birds and mammals.15 16 Changes in GAG sulfate incorporation represent just one of a number of biochemical changes reported in the sclera during the development of myopia, however, the patterns of change have been found to be quite different between birds and mammals.10 11 12 15 17 Subsequent alterations to the hydrational and biomechanical properties of the sclera have also been demonstrated and represent a potential mechanism through which ocular elongation may occur.18 19 20 Changes in dry tissue mass,10 11 associated with ocular elongation, and findings of increased levels of degradative enzymes,19 21 22 known to be involved in the process of ECM remodeling, are also indicative of ocular growth through scleral remodeling.
Eyes with induced myopia have demonstrated the ability to recover from the imposed refractive error in both avian and mammalian models of myopia.23 24 This recovery from induced axial myopia has also been shown to be associated with changes in GAG sulfate incorporation16 17 and DNA synthesis25 26 as well as in the activity of degradative enzymes.22 However, the mechanisms of scleral remodeling10 11 and reduction in vitreous chamber depth,26 27 found in recovering eyes, have also been shown to occur through a somewhat different mechanism in birds and mammals.
The present study sought to determine whether the scleral changes in a mammalian model of refractive error development are representative of a controlled remodeling process or whether they simply reflect the redistribution of existing tissue. The experimental model used in this study was the tree shrew, which possesses a fibrous sclera composed of predominantly type I collagen28 and undergoes similar scleral changes during the development of axial myopia to those observed in highly myopic humans.8 Scleral changes were monitored through a time course investigation of scleral GAG synthesis and dry tissue weight changes during the process of recovery from induced myopia.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Vivo Experimental Procedures
All experimental procedures were performed in accordance with the
ARVO Statement for the Use of Animals in Ophthalmic and Vision
Research. Axial myopia was induced by monocular deprivation (MD) with
translucent occluders or through hyperopic defocus with negative lenses
fitted to a head mounted goggle, as previously
described.26
Ocular biometric measures consisted of
corneal curvature (keratometry), ocular refraction (retinoscopy) and
axial ocular dimensions (A-scan ultrasonography) and were collected
with the animal under anesthesia (ketamine 90 mg/kg, xylazine 10 mg/kg)
using instruments and procedures previously described.30
The sympathomimetic drug phenylephrine (2%) was used to dilate the
pupil during data collection from all animals, since the use of a
muscarinic antagonist, to produce mydriasis and cycloplegia, was
considered inappropriate in the light of a recent report that such
drugs affect scleral production of GAGs.31
Pilot data
demonstrated that interocular axial dimensions were not significantly
altered by the topical administration of phenylephrine (up to 10%),
thus any effects on ocular vasculature did not translate to changes in
choroidal dimensions.
After 5 days of lens or occluder treatment, ocular biometric and refractive data were gathered from all groups of animals. Animals in the normal, 5 days MD, and lens-defocus groups underwent biometric and refractive measurement and were then administered terminal anesthesia before the ocular tissues were collected. Animals in recovery groups also had their occluder removed and ocular measures taken, allowing subsequent individual ocular component changes to be monitored, before being allowed the specified time periods of recovery without the occluder in place. A final set of optical and biometric measures were taken after the specified period for each group of recovering animals, before tissue was collected under terminal anesthesia.
On the morning of the final measurement day (5th experimental day for normal, 5 day MD, and lens-defocus groups and the 6th, 8th, 10th, 12th, and 14th experimental day for recovery groups), 115 µCi of 35S-labeled aqueous sulfate (1000 Ci/mmol; Amersham International, Arlington Heights, IL) was injected intraperitoneally, and the animals were returned to their cages. Final structural measures were taken 6 hours after administration of the radiolabeled sulfate.
Materials
Proteinase K was obtained from Stratagene (La Jolla, CA); alcian
blue 8GX from Fluka (Ronkonkoma, NY); calf thymus DNA, dermatan
sulfate, and Hoechst 33258 from Sigma (St. Louis, MO); Cytoscint liquid
scintillant from ICN (Irvine, CA); and all other chemicals were
supplied by either Fisher Scientific (Pittsburgh, PA) or Sigma.
Multiwell Durapore (GAG synthesis assay) and mixed cellulose ester
filter (total GAG assay) plates (pore size 0.45 µm), with disposable
punch tips, were obtained from Millipore (Bedford, MA).
Tissue Preparation and Digestion
Eyes were enucleated under deep anesthesia (120 mg/kg, sodium
pentobarbital), and equatorial diameters measured with a digital
calliper. Corneas were dissected out with a circumferential cut around
the limbus, and the iris, crystalline lens, and vitreous were removed.
A 5-mm trephine, centered on the posterior pole, was used to separate
the eye cup into an anterior/equatorial sample and a posterior sample,
and then the retina and choroid were carefully removed from the sclera
before the cornea and anterior and posterior scleral samples were
snap-frozen in liquid nitrogen. Frozen tissue samples were vacuum-dried
overnight (Micro Modulyo; Edwards, Wilmington, MA), and dry weights
were measured to the nearest 10 µg, before homogenization. Samples
were homogenized by digestion with proteinase K as previously
described.16
Biochemical Analysis of Tissue Homogenates
Incorporation of the radiolabel into scleral GAGs was assessed by
selective precipitation with alcian blue, using a previously reported
modification of the dye-binding method of Masuda et
al.16
32
Total GAG content was also assessed, using a
different modification of this method,33
in selected
groups of myopic and recovering animals, to give a general picture of
scleral GAG content relative to scleral GAG synthesis. Samples were
diluted in proteinase K (0.5 mg/ml), and dermatan sulfate standards
were prepared, also in proteinase K. Blanks consisted of proteinase K
(0.5 mg/ml). Samples, standards, and blanks were then mixed,
centrifuged, and loaded onto a plate (cellulose ester filters) as
described previously. Plates were incubated and drained then filters
were washed and allowed to air dry before being punched into glass
vials. Filters and precipitate were dissolved in dimethyl sulfoxide
with 0.5% sulfuric acid overnight. Sample absorbance was measured at
678 nm. Homogenates were analyzed for DNA content using the Hoechst dye
based fluorometric assay previously described.26
34
Data Analysis
Data are presented as the group mean and SEM of either absolute
values, or of the difference (T - C) or percentage difference
[(T - C)/C] between treated and control eyes. Normative data
are presented identically, but on the basis of the difference between
right and left eyes [R - L or (R - L)/L]. One-way
analysis of variance (ANOVA) with Tukey’s post hoc test or Student’s
t-test were used to assess differences between groups,
whereas the paired t-test was used to assess the
significance of differences between treated and control eyes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5 D in tree shrews of this age),35
on
average, treated eyes displayed an absolute myopic refractive error of
around -2.5 D, whereas contralateral control eyes exhibited +4.5 D of
hyperopia. Significant axial enlargement of the vitreous chamber depth
(P < 0.02), when compared with contralateral control
eyes (paired t-test) or normal eyes (P <
0.01; ANOVA; Fig. 1B
), was also apparent in myopic eyes. Equatorial
diameter was significantly greater in 5 day form-deprived (8.47 ±
0.03 versus 8.39 ± 0.04 mm, P < 0.05) and
lens-defocus (8.40 ± 0.05 versus 8.31 ± 0.04 mm,
P < 0.01) eyes when compared with contralateral
control eyes. No significant changes were found in any other
intraocular parameter or in the corneal curvature.
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Analysis of axial intraocular dimensions from individual treated and control eyes of recovering animals revealed that the reduction in VCD differences in recovery was almost entirely due to a reduction in the vitreous chamber depth of the treated eye (up to 0.12 mm), rather than an increase in control eye VCD (Fig. 1C) . Changes in VCD of recovering eyes were not a result of thickening of the choroid in the treated eye because ultrasound data revealed no significant changes in thickness of the retina + choroid complex. Because the variation of repeated ultrasound readings was ±0.01 mm, any choroidal changes sufficient to account for the shortening of the recovering eye (0.12 mm) would easily have been identified.
Sulfate Incorporation into Scleral Glycosaminoglycans in Recovery.
Compared with a difference of -36% ± 7% in sulfate incorporation
between the posterior sclera of treated and control eyes after 5 days
of MD, there was only a -7% ± 7% difference after only 24 hours of
recovery from myopia (Fig. 2A)
, which represented a significant change
(P < 0.05). After 3 days of recovery there was a
significant increase in sulfate incorporation in both the anterior and,
particularly, the posterior sclera of treated eyes (anterior,
P = 0.01; posterior, P < 0.01). The
increased sulfate incorporation in the posterior sclera was found to
reach a peak in the 5 day recovery group (+88% ± 14%,
P < 0.01). By 7 and 9 days of recovery differences in
sulfate levels were rapidly returning to control eye values (Fig. 2A)
.
The patterns reported were similar whether sulfate incorporation was
expressed with respect to dry scleral weight or scleral DNA content
(Figs. 2A
2B)
.
As both dry tissue weight11 and DNA content26 have been found to change in the sclera of tree shrews with induced myopia, it was important to determine whether the change in sulfate incorporation into scleral GAGs was preserved if the confound of normalization to a parameter that was also changing was removed. Figure 3A demonstrates that sulfate incorporation in terms of just cpm in the whole sclera gave a similar pattern to normalized values. The similarity of raw cpm data to the data expressed relative to scleral dry weight or DNA content indicates that the major change underlying these results was an alteration in GAG synthesis.
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The overall GAG content of tree shrew sclera (Fig. 3C) accounted for approximately 1% of dry tissue weight in individual eyes, which is consistent with levels found in human sclera.36 No significant differences in scleral GAG content were observed between right and left eyes of normals or between treated and control eyes of 5 days MD or recovery animals, and the small differences found accounted for no more than 2% of the differences in total dry weight between treated and control eyes.
The DNA content of whole sclera was elevated in the myopic eyes of 5 days MD animals; however, this elevation was found to be significant only when expressed relative to scleral dry weight (+7.4% ± 2.4%, P < 0.05), as was found in a previous study from this laboratory.26 No significant differences were found in scleral DNA content between treated and contralateral control eyes of either the lens-defocus or any of the recovery groups in the present study.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The findings clearly demonstrate a decrease in GAG synthesis in axial myopia development and an increase in GAG synthesis in recovery from axial myopia. Of particular note is the finding that the increases in GAG synthesis in recovery from axial myopia are three to four times greater than the decrease observed during myopia development. The increased magnitude of this signal in recovery suggests that the positive feedback in visual information during recovery induces a more substantive response in scleral metabolism. The rapid response (24 hours) of altered scleral metabolism to a change in visual signal supports earlier findings in demonstrating visual control of scleral remodeling16 and ocular growth.16 42
The loss of scleral tissue during axial myopia development was predominantly from the region surrounding the posterior pole of the sclera, although there was a smaller, but consistent, reduction of tissue in the remaining equatorial/anterior sclera, as previously reported.16 This selective regional loss of scleral tissue in myopia development in the present study is consistent with reports of reduced scleral thickness in myopic human43 and tree shrew eyes.8 The findings of the present study support those of previous workers10 11 12 16 in forcing a reinterpretation of the mechanisms responsible for the thinner sclera that is found in highly myopic human eyes. Contrary to previous views that the thinner sclera in myopia was due to a redistribution of the sclera over the enlarged globe due to stretching,44 the most recent findings clearly demonstrate that an actual loss of scleral tissue occurs in myopic mammalian eyes.
The potential effects of changes in GAG synthesis in the scleral matrix can broadly be considered as either structural or biomechanical, although the two are likely to be interdependent. Given that the overall contribution of the scleral GAGs to the changes in scleral dry weight was approximately 2%, during myopia development and recovery, this suggests that the role of altered GAG synthesis during eye size changes is more likely to be regulatory or biomechanical in nature, possibly as a result of changes in hydration of the scleral ECM. Recent findings have also demonstrated that DNA synthesis in the tree shrew sclera is downregulated in myopia development and markedly upregulated in recovery26 in a similar pattern to GAG synthesis. The similarity in the direction and magnitude of change indicates a common signaling factor. It seems likely that the resultant reduced cell proliferation in myopia contributes to reduced cellular production of proteoglycans and to the lower proteoglycan content of scleral tissue in myopia which has also been reported in a previous study.12
The present study has shown that full recovery is achieved almost exclusively through a reduction of the vitreous chamber depth of the treated eye. Biometric data from the present and previous26 studies in the tree shrew suggests that the joint contribution of choroidal and retinal thickness changes to the reduction in VCD of the treated eye, represents a relatively minor contribution. This finding is in contrast to the myopic avian eye, which demonstrates marked thickening of the choroid during the early stages of recovery.27
Analysis of percentage change in vitreous chamber depth over the recovery period compared with changes in GAG synthesis over the same period revealed that the most rapid change in GAG synthesis preceded the most rapid change in vitreous chamber depth (Fig. 4) . Furthermore, it appears that changes in VCD only started to occur after levels of GAG synthesis in the treated eye began to exceed levels in the control eye. Although the exact role of scleral GAG synthesis in the shortening process is unclear, given the intrinsic role of GAGs in ECM biomechanics, it is not unreasonable to hypothesize that increases in GAG synthesis contribute to a decreased scleral elasticity, resulting in a shorter eye. Such a view would certainly be consistent with biomechanical findings in the recovering sclera.20
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Footnotes |
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Submitted for publication January 7, 2000; revised March 23, and June 6, 2000; accepted June 23, 2000.
Commercial relationships policy: N.
Corresponding author: Neville A. McBrien, Department of Optometry and Vision Sciences, The University of Melbourne, Victoria 3010, Australia. n.mcbrien{at}optometry.unimelb.edu.au
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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