November 2000
Volume 41, Issue 12
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Biochemistry and Molecular Biology  |   November 2000
Scleral Remodeling during the Development of and Recovery from Axial Myopia in the Tree Shrew
Author Affiliations
  • Neville A. McBrien
    From the Department of Optometry and Vision Sciences, The University of Melbourne, Victoria, Australia.
  • Patrick Lawlor
    From the Department of Optometry and Vision Sciences, The University of Melbourne, Victoria, Australia.
  • Alex Gentle
    From the Department of Optometry and Vision Sciences, The University of Melbourne, Victoria, Australia.
Investigative Ophthalmology & Visual Science November 2000, Vol.41, 3713-3719. doi:https://doi.org/
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      Neville A. McBrien, Patrick Lawlor, Alex Gentle; Scleral Remodeling during the Development of and Recovery from Axial Myopia in the Tree Shrew. Invest. Ophthalmol. Vis. Sci. 2000;41(12):3713-3719. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Recent investigations have suggested that scleral thinning in mammalian eyes with axial myopia is a consequence of the loss of scleral tissue, rather than the redistribution of existing tissue as the eye enlarges. The present study investigated whether further changes in the distribution and metabolism of scleral tissue occur during the process of recovery from axial myopia. Scleral glycosaminoglycan (GAG) synthesis and content as well as scleral dry weight changes were monitored as indicators of remodeling in myopic and recovering tree shrew sclerae.

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.

The major structural correlate to the development of myopia is axial elongation of the vitreous chamber. The relationship between myopia and vitreous chamber depth (VCD) has been demonstrated in the development of both juvenile-onset 1 and adult-onset myopia 2 and is found in both low and high degrees of myopia. In high degrees of axial myopia there is increased risk of pathologic changes to the retina and sclera, illustrated by the fact that the prevalence of retinal lesions in high myopia is reported to be 30% to 50%. 3 Such changes can result in irreversible loss of vision. Investigation of postmortem highly myopic human eyes has revealed marked thinning of the sclera, particularly at the posterior pole, 4 with changes in the scleral extracellular matrix. 5 Similar changes have been observed in mammalian models of axial myopia, with marked thinning of the sclera at the posterior pole detected in monkeys 6 and tree shrews. 7 8 Reductions in collagen fibril diameter have also been observed. 6 9 Such findings have led to the hypothesis that thinning of the sclera in highly myopic eyes of human and animal models is a result of changes in scleral extracellular matrix (ECM) metabolism. 10 11 12  
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 incorporation 16 17 and DNA synthesis 25 26 as well as in the activity of degradative enzymes. 22 However, the mechanisms of scleral remodeling 10 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 collagen 28 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. 
Materials and Methods
Animals
Tree shrew (Tupaia belangeri) pups were maternally reared in our breeding colony, under conditions identical with those previously reported by this laboratory, 29 and transferred from the breeding room 15 days after natural eye opening (20 days ± 3 SD), on the day that experimental procedures commenced. Animals were randomly allocated to one of eight experimental groups. Each group consisted of 5 animals and was as balanced as possible, for right/left eye treatments and gender. The eight experimental groups consisted of: 1 group of animals who had 5 days of normal binocular vision (5 day normal); 1 group of animals who were monocularly deprived (MD) of pattern vision with a translucent occluder for 5 days (5 days MD); 1 group of animals who had a negative lens (−10 D) placed over one eye for 5 days (5 days lens-defocus); 5 groups of animals who had 5 days MD followed by removal of the occluder and either 1 (5 + 1 Rec), 3 (5 + 3 Rec), 5 (5 + 5 Rec), 7 (5 + 7 Rec), or 9 (5 + 9 Rec) days of unoccluded vision. Previous data from this laboratory has shown that tree shrew eyes with myopia induced using either the form-deprivation or negative lens-defocus paradigm show similar changes in ocular component parameters. 26 Accordingly, in the present study the recovery process was only assessed after form-deprivation. 
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. 
Results
Development of Myopia
Refractive and Biometric Findings.
After 5 days of form-deprivation or lens-defocus, treated eyes had developed significant relative myopia compared with contralateral control eyes (P < 0.005; Fig. 1A ). When ocular refractive errors were corrected for the small eye artifact (∼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. 
Sulfate Incorporation into Scleral Glycosaminoglycans in Myopia.
Incorporation of radiolabeled sulfate (cpm/mg dry tissue weight) was reduced in the anterior (−20% ± 3%) and posterior (−36% ± 7%) sclera of the myopic eye in 5 day MD animals (P < 0.01 anterior and posterior sclera; Fig. 2A ) compared with contralateral control eyes (paired t-test) and the eyes of normal animals (ANOVA). Sulfate incorporation was also significantly reduced in the posterior (−42% ± 4%) sclera of the myopic eyes of lens-defocus animals (P < 0.001) when compared with contralateral control and normal eyes. There was no significant difference between sulfate incorporation into posterior scleral samples from myopic eyes of 5 day form-deprived animals and lens-defocus animals. If incorporation of radiolabeled sulfate was expressed per microgram of DNA (cpm/μg DNA), instead of per milligram of dry tissue weight, the findings were essentially identical (see Figs. 2A 2B ). There was found to be no significant difference in levels of sulfate incorporation (cpm/mg) between normal eyes (n = 10) and contralateral control eyes (n = 10) of 5 day MD and lens-defocus animals for either anterior or posterior sclera (P = 0.66 anterior; P = 0.89 posterior). There was no correlation between the interocular reduction in GAG synthesis and either the interocular difference in refractive error (P = 0.77) or VCD (P = 0.99), indicating that the level of scleral GAG synthesis was not a good predictor of the degree of refractive error or ocular elongation. 
Recovery from Induced Myopia
Refractive and Biometric Findings.
The amount of myopia induced in recovery groups after 5 days of form-deprivation was not significantly different from the amount of myopia induced in 5 days MD and lens-defocus groups, when compared by ANOVA (P = 0.18, Fig. 1A ). The degree of myopia, induced by 5 days of form-deprivation, had reduced by 16% after only 1 day of recovery (P = 0.14) and was significantly less than its starting value (P < 0.01) after 3 days of unoccluded visual experience. Almost complete recovery from induced myopia had been achieved after 9 days (Fig. 1A) . The recovery from induced myopia was found to be axial in origin, with significant reductions measured in the VCD differences between treated and contralateral control eyes in all but the 1 day recovery group (Fig. 1B) . Differences in equatorial diameter between treated and control eyes had returned to nonsignificant values by 5 days of recovery (8.49 ± 0.05 versus 8.47 ± 0.06 mm, P = 0.68). 
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 weight 11 and DNA content 26 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. 
Scleral Dry Tissue Weight, Glycosaminoglycan Content, and DNA Content
There was a significant reduction in total scleral dry weight (Fig. 3B) of treated eyes in the 5 days MD animals when compared with contralateral control eyes (P < 0.05), and this reduction was greatest in the posterior sclera (−4.5%). By 5 days of recovery there was a significant increase in scleral dry weight relative to contralateral control eyes (P < 0.05). After 7 and 9 days of recovery there was no significant difference between treated and control eyes in regional or total scleral dry weight, although small increases were still present in these groups. 
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. 
Discussion
The present study has established that the metabolic changes in the mammalian sclera during recovery from induced myopia occur very rapidly in response to a change in visual conditions and are specific to the direction and degree of change in eye size. These findings strongly support the hypothesis that changes in eye size during the recovery from axial myopia are facilitated by active remodeling of the sclera and not simply a redistribution of existing tissue. This is consistent with findings in eyes developing myopia, as reported in this and other studies of mammalian refractive error development. 11 12 26 37 38 The results of the present study also support the view that the scleral mechanisms of myopia development are common to both the form-deprivation and lens-defocus paradigms, the general mechanisms of which have been a matter of debate in the recent literature. 39 40 41  
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 remodeling 16 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 human 43 and tree shrew eyes. 8 The findings of the present study support those of previous workers 10 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 recovery 26 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 previous 26 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  
In summary, the present study has shown that scleral remodeling is intimately associated with both axial myopia development and recovery in this mammalian model of refractive error. The loss of scleral tissue supports the view, put forward in previous reports, that a thinned sclera in myopia is not simply due to a redistribution of existing tissue. The fact that changes in GAG content account for only a minor proportion of the change in scleral dry weight indicates a regulatory and/or biomechanical role for GAGs in ocular growth regulation and refractive error development. Furthermore, changes in scleral remodeling in recovery are observed less than 24 hours after removal of the occluder and, thereafter, a close relationship exists between the changes observed in vitreous chamber depth and the patterns of remodeling displayed in the sclera. 
 
Figure 1.
 
(A) After 5 days, treated eyes of animals in all form-deprived and lens-defocus groups displayed significant amounts of myopia, relative to contralateral control eyes. Following removal of the occluder, a reduction was observed in the amount of myopia after 1 day of recovery. This reduction was highly significant by 3 days of recovery (as indicated by asterisks), and the induced myopia had completely recovered by 9 days. (B) The structural correlate to the recovery from induced myopia was a reduction in the VCD difference between eyes. Significant reductions in VCD differences were found by 3 days of recovery, and this difference was eliminated by 7 days of recovery. (C) Reductions in the VCD differences between treated and control eyes in recovery were predominantly due to reductions in the VCD of treated eyes. Significant reductions were found in the VCD of treated eyes, relative to the VCD after 5 days of form-deprivation, by 3 days of recovery (as indicated by asterisks). There was no significant difference between the VCD of treated eyes and control eyes after 7 days of recovery. No significant changes were found in the VCD of control eyes, relative to the depth after 5 days, throughout the recovery period. T, treated eye; C, control eye. n = 5 in all groups. Error bars, 1 SEM.** P < 0.01, *P < 0.05.
Figure 1.
 
(A) After 5 days, treated eyes of animals in all form-deprived and lens-defocus groups displayed significant amounts of myopia, relative to contralateral control eyes. Following removal of the occluder, a reduction was observed in the amount of myopia after 1 day of recovery. This reduction was highly significant by 3 days of recovery (as indicated by asterisks), and the induced myopia had completely recovered by 9 days. (B) The structural correlate to the recovery from induced myopia was a reduction in the VCD difference between eyes. Significant reductions in VCD differences were found by 3 days of recovery, and this difference was eliminated by 7 days of recovery. (C) Reductions in the VCD differences between treated and control eyes in recovery were predominantly due to reductions in the VCD of treated eyes. Significant reductions were found in the VCD of treated eyes, relative to the VCD after 5 days of form-deprivation, by 3 days of recovery (as indicated by asterisks). There was no significant difference between the VCD of treated eyes and control eyes after 7 days of recovery. No significant changes were found in the VCD of control eyes, relative to the depth after 5 days, throughout the recovery period. T, treated eye; C, control eye. n = 5 in all groups. Error bars, 1 SEM.** P < 0.01, *P < 0.05.
Figure 2.
 
(A) Significant reductions in sulfate incorporation, relative to dry tissue weight (cpm/mg), were found in the posterior sclera of eyes developing myopia (5 days MD and lens-defocus). Significant reductions in incorporation were also found in the anterior sclera of form-deprived, but not lens-defocused eyes. The reduced sulfate incorporation levels in myopic eyes of 5 day MD animals were no longer evident after 24 hours of recovery, with levels similar to control eye values. Sulfate incorporation was significantly elevated, relative to contralateral control eyes, in both the anterior and posterior sclera, after 3 days of recovery and values peaked in the posterior sclera after 5 days of recovery. Although still elevated, levels of scleral sulfate incorporation in treated eyes were returning to control eye values after 9 days of recovery. (B) The above patterns of scleral sulfate incorporation were found to be similar when expressed relative to scleral DNA content (cpm/μg DNA). n = 5 in all groups. Error bars, 1 SEM.** P < 0.01, *P < 0.05.
Figure 2.
 
(A) Significant reductions in sulfate incorporation, relative to dry tissue weight (cpm/mg), were found in the posterior sclera of eyes developing myopia (5 days MD and lens-defocus). Significant reductions in incorporation were also found in the anterior sclera of form-deprived, but not lens-defocused eyes. The reduced sulfate incorporation levels in myopic eyes of 5 day MD animals were no longer evident after 24 hours of recovery, with levels similar to control eye values. Sulfate incorporation was significantly elevated, relative to contralateral control eyes, in both the anterior and posterior sclera, after 3 days of recovery and values peaked in the posterior sclera after 5 days of recovery. Although still elevated, levels of scleral sulfate incorporation in treated eyes were returning to control eye values after 9 days of recovery. (B) The above patterns of scleral sulfate incorporation were found to be similar when expressed relative to scleral DNA content (cpm/μg DNA). n = 5 in all groups. Error bars, 1 SEM.** P < 0.01, *P < 0.05.
Figure 3.
 
(A) Levels of GAG synthesis were the major contributing factor to the patterns of scleral sulfate incorporation detailed in this study. The figure shows that when normalizing parameters (scleral dry weight or DNA content), both of which also change during myopia development and recovery, are removed and raw data (cpm) from whole sclera is presented, patterns are essentially the same as shown in Figures 2A and 2B . (B) Total scleral dry weight was reduced in myopic eyes, relative to contralateral control eyes, after 5 days of form-deprivation or lens-defocus. Scleral dry weight was found to increase gradually in recovering eyes, when compared with the reductions observed after 5 days of MD. After 5 days of recovery, scleral dry weights of recovering eyes were significantly elevated relative to contralateral control eyes. This pattern was remarkably similar to the pattern seen in total scleral sulfate incorporation, although the elevation in scleral weight appeared to occur slightly later than was found for sulfate incorporation. (C) Changes in sulfate incorporation did not translate to significant differences in scleral GAG content, although there was a trend to reduced GAG content in myopia development and a replacement of GAGs in recovery. R and L, right and left eyes of normal animals, respectively; T, treated eye; C, control eye. n = 5 in all groups. Error bars, 1 SEM.** P < 0.01, *P < 0.05.
Figure 3.
 
(A) Levels of GAG synthesis were the major contributing factor to the patterns of scleral sulfate incorporation detailed in this study. The figure shows that when normalizing parameters (scleral dry weight or DNA content), both of which also change during myopia development and recovery, are removed and raw data (cpm) from whole sclera is presented, patterns are essentially the same as shown in Figures 2A and 2B . (B) Total scleral dry weight was reduced in myopic eyes, relative to contralateral control eyes, after 5 days of form-deprivation or lens-defocus. Scleral dry weight was found to increase gradually in recovering eyes, when compared with the reductions observed after 5 days of MD. After 5 days of recovery, scleral dry weights of recovering eyes were significantly elevated relative to contralateral control eyes. This pattern was remarkably similar to the pattern seen in total scleral sulfate incorporation, although the elevation in scleral weight appeared to occur slightly later than was found for sulfate incorporation. (C) Changes in sulfate incorporation did not translate to significant differences in scleral GAG content, although there was a trend to reduced GAG content in myopia development and a replacement of GAGs in recovery. R and L, right and left eyes of normal animals, respectively; T, treated eye; C, control eye. n = 5 in all groups. Error bars, 1 SEM.** P < 0.01, *P < 0.05.
Figure 4.
 
Percentage change in GAG synthesis and vitreous chamber depth change during the recovery period. The amount of recovery during each time period was estimated as a percentage of full recovery (100%) by extrapolation of data across the time points. It was found that the greatest change in GAG synthesis preceded the greatest change in vitreous chamber depth. 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. n = 5 in all groups. Error bars, ± 1 SEM.
Figure 4.
 
Percentage change in GAG synthesis and vitreous chamber depth change during the recovery period. The amount of recovery during each time period was estimated as a percentage of full recovery (100%) by extrapolation of data across the time points. It was found that the greatest change in GAG synthesis preceded the greatest change in vitreous chamber depth. 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. n = 5 in all groups. Error bars, ± 1 SEM.
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Figure 1.
 
(A) After 5 days, treated eyes of animals in all form-deprived and lens-defocus groups displayed significant amounts of myopia, relative to contralateral control eyes. Following removal of the occluder, a reduction was observed in the amount of myopia after 1 day of recovery. This reduction was highly significant by 3 days of recovery (as indicated by asterisks), and the induced myopia had completely recovered by 9 days. (B) The structural correlate to the recovery from induced myopia was a reduction in the VCD difference between eyes. Significant reductions in VCD differences were found by 3 days of recovery, and this difference was eliminated by 7 days of recovery. (C) Reductions in the VCD differences between treated and control eyes in recovery were predominantly due to reductions in the VCD of treated eyes. Significant reductions were found in the VCD of treated eyes, relative to the VCD after 5 days of form-deprivation, by 3 days of recovery (as indicated by asterisks). There was no significant difference between the VCD of treated eyes and control eyes after 7 days of recovery. No significant changes were found in the VCD of control eyes, relative to the depth after 5 days, throughout the recovery period. T, treated eye; C, control eye. n = 5 in all groups. Error bars, 1 SEM.** P < 0.01, *P < 0.05.
Figure 1.
 
(A) After 5 days, treated eyes of animals in all form-deprived and lens-defocus groups displayed significant amounts of myopia, relative to contralateral control eyes. Following removal of the occluder, a reduction was observed in the amount of myopia after 1 day of recovery. This reduction was highly significant by 3 days of recovery (as indicated by asterisks), and the induced myopia had completely recovered by 9 days. (B) The structural correlate to the recovery from induced myopia was a reduction in the VCD difference between eyes. Significant reductions in VCD differences were found by 3 days of recovery, and this difference was eliminated by 7 days of recovery. (C) Reductions in the VCD differences between treated and control eyes in recovery were predominantly due to reductions in the VCD of treated eyes. Significant reductions were found in the VCD of treated eyes, relative to the VCD after 5 days of form-deprivation, by 3 days of recovery (as indicated by asterisks). There was no significant difference between the VCD of treated eyes and control eyes after 7 days of recovery. No significant changes were found in the VCD of control eyes, relative to the depth after 5 days, throughout the recovery period. T, treated eye; C, control eye. n = 5 in all groups. Error bars, 1 SEM.** P < 0.01, *P < 0.05.
Figure 2.
 
(A) Significant reductions in sulfate incorporation, relative to dry tissue weight (cpm/mg), were found in the posterior sclera of eyes developing myopia (5 days MD and lens-defocus). Significant reductions in incorporation were also found in the anterior sclera of form-deprived, but not lens-defocused eyes. The reduced sulfate incorporation levels in myopic eyes of 5 day MD animals were no longer evident after 24 hours of recovery, with levels similar to control eye values. Sulfate incorporation was significantly elevated, relative to contralateral control eyes, in both the anterior and posterior sclera, after 3 days of recovery and values peaked in the posterior sclera after 5 days of recovery. Although still elevated, levels of scleral sulfate incorporation in treated eyes were returning to control eye values after 9 days of recovery. (B) The above patterns of scleral sulfate incorporation were found to be similar when expressed relative to scleral DNA content (cpm/μg DNA). n = 5 in all groups. Error bars, 1 SEM.** P < 0.01, *P < 0.05.
Figure 2.
 
(A) Significant reductions in sulfate incorporation, relative to dry tissue weight (cpm/mg), were found in the posterior sclera of eyes developing myopia (5 days MD and lens-defocus). Significant reductions in incorporation were also found in the anterior sclera of form-deprived, but not lens-defocused eyes. The reduced sulfate incorporation levels in myopic eyes of 5 day MD animals were no longer evident after 24 hours of recovery, with levels similar to control eye values. Sulfate incorporation was significantly elevated, relative to contralateral control eyes, in both the anterior and posterior sclera, after 3 days of recovery and values peaked in the posterior sclera after 5 days of recovery. Although still elevated, levels of scleral sulfate incorporation in treated eyes were returning to control eye values after 9 days of recovery. (B) The above patterns of scleral sulfate incorporation were found to be similar when expressed relative to scleral DNA content (cpm/μg DNA). n = 5 in all groups. Error bars, 1 SEM.** P < 0.01, *P < 0.05.
Figure 3.
 
(A) Levels of GAG synthesis were the major contributing factor to the patterns of scleral sulfate incorporation detailed in this study. The figure shows that when normalizing parameters (scleral dry weight or DNA content), both of which also change during myopia development and recovery, are removed and raw data (cpm) from whole sclera is presented, patterns are essentially the same as shown in Figures 2A and 2B . (B) Total scleral dry weight was reduced in myopic eyes, relative to contralateral control eyes, after 5 days of form-deprivation or lens-defocus. Scleral dry weight was found to increase gradually in recovering eyes, when compared with the reductions observed after 5 days of MD. After 5 days of recovery, scleral dry weights of recovering eyes were significantly elevated relative to contralateral control eyes. This pattern was remarkably similar to the pattern seen in total scleral sulfate incorporation, although the elevation in scleral weight appeared to occur slightly later than was found for sulfate incorporation. (C) Changes in sulfate incorporation did not translate to significant differences in scleral GAG content, although there was a trend to reduced GAG content in myopia development and a replacement of GAGs in recovery. R and L, right and left eyes of normal animals, respectively; T, treated eye; C, control eye. n = 5 in all groups. Error bars, 1 SEM.** P < 0.01, *P < 0.05.
Figure 3.
 
(A) Levels of GAG synthesis were the major contributing factor to the patterns of scleral sulfate incorporation detailed in this study. The figure shows that when normalizing parameters (scleral dry weight or DNA content), both of which also change during myopia development and recovery, are removed and raw data (cpm) from whole sclera is presented, patterns are essentially the same as shown in Figures 2A and 2B . (B) Total scleral dry weight was reduced in myopic eyes, relative to contralateral control eyes, after 5 days of form-deprivation or lens-defocus. Scleral dry weight was found to increase gradually in recovering eyes, when compared with the reductions observed after 5 days of MD. After 5 days of recovery, scleral dry weights of recovering eyes were significantly elevated relative to contralateral control eyes. This pattern was remarkably similar to the pattern seen in total scleral sulfate incorporation, although the elevation in scleral weight appeared to occur slightly later than was found for sulfate incorporation. (C) Changes in sulfate incorporation did not translate to significant differences in scleral GAG content, although there was a trend to reduced GAG content in myopia development and a replacement of GAGs in recovery. R and L, right and left eyes of normal animals, respectively; T, treated eye; C, control eye. n = 5 in all groups. Error bars, 1 SEM.** P < 0.01, *P < 0.05.
Figure 4.
 
Percentage change in GAG synthesis and vitreous chamber depth change during the recovery period. The amount of recovery during each time period was estimated as a percentage of full recovery (100%) by extrapolation of data across the time points. It was found that the greatest change in GAG synthesis preceded the greatest change in vitreous chamber depth. 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. n = 5 in all groups. Error bars, ± 1 SEM.
Figure 4.
 
Percentage change in GAG synthesis and vitreous chamber depth change during the recovery period. The amount of recovery during each time period was estimated as a percentage of full recovery (100%) by extrapolation of data across the time points. It was found that the greatest change in GAG synthesis preceded the greatest change in vitreous chamber depth. 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. n = 5 in all groups. Error bars, ± 1 SEM.
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