Biochemistry of the Eye
Structural and biochemical changes in the sclera of experimentally myopic eyes Neville A. McBrien, Hadi 0. Moghaddam, Anne P. Reeder and Suzanne Moules Department of Optometry, University of Wales, College of Cardiff, Cardiff CF I 3XF, U.K.
Introduction The refraction of the eye is determined by four ocular components: corneal power, anterior chamber depth, lens power and vitreous chamber depth. From birth to adulthood the axial diameter of the human eye increases by approximately 7 mm. This change would result in significant refractive error ( > 15 D) if compensatory changes in the other ocular components did not occur. However, for the majority of individuals (approx. 65%) there is precise regulation of the development of the ocular components such that the image of distant objects is brought to a sharp focus on the photoreceptive layer of the retina. This process of coordinated development of the ocular components is known as emmetropization. In a significant minority of the population, however, there is a breakdown of this regulatory process such that hyperopia or more commonly myopia (shortsightedness) results. Myopia is a condition that affects approximately 25% of the population in both western European countries and the U SA. [ 1, 21, with an even higher incidence reported in Asian populations. In addition to the economic impact involved owing to the cost of spectacles, progressive or pathological myopia is a major cause of world blindness [3] as a result of degenerative changes that take place as a sequelae to the excessive axial elongation of the eye. In recent years research on experimental models of pathological myopia has concentrated more on the biochemical changes that may be involved. Several studies have demonstrated changes in retinalneurotransmitter levels of myopic eyes [4,51, and that altering neurotransmitter levels in the eye can reduce or eliminate the excessive axial elongation of the eye found in experimentally induced myopia [6-81. However, it is only very recently that work has begun in an attempt to elucidate the biochemical mechanisms responsible for the structural changes observed in the sclera of myopic eyes.
Structural and biomechanical changes in myopic sclera The major functions of the sclera are to provide a protective coat for the light-sensitive components of
Abbreviations used: IOP, intraocular pressure; MD, monocularly deprived; /?-APN, /?-aminopropionitrile.
the eye and to withstand the considerable expansive force generated by the intraocular pressure. Human sclera is fibrous in nature with collagen content reported to be 50-75% of the dry weight [9, 101. Collagen type is mainly type I (90-95%) with evidence of a small amount of type 111, particularly in young eyes [ 11, 121. Analysis of bovine sclera has shown that the proteoglycans of the extracellular matrix are exclusively proteodermatan sulphates which have been separated into two populations of differing molecular mass and proteiddermatan sulphate ratios [13, 141. In the highly myopic human eye, enlargement occurs in all diameters but principally the anteroposterior, the globe taking an ellipsoid or prolate spheroidal shape. This axial enlargement of the myopic eye is often associated with marked thinning of the posterior sclera [ 151. Microscopic evaluation of myopic sclera in human shows not only marked thinning at the posterior pole of the eye, but also a reduction in fibril diameter and a dissociation of collagen-fibre bundles [ 16- 191. Monocular deprivation of pattern vision has been found to produce high levels of axial myopia in chicks [20),tree shrews [21] and monkeys [22]. In mammalian models of experimentally induced myopia (monkey, tree shrew), similar microscopic changes in both scleral thickness and/or fibril diameter have been noted [23-251. The finding of a reduction in fibre diameter has led to the suggestion that defective fibrillogenesis might be a factor in the excessive axial elongation of the eye in myopia [ 161. Hiomechanical changes have also been recorded in myopic human sclera with reduction in the tensile strength and increase in elasticity, especially at the posterior pole [ 171. These findings have been interpreted by some to suggest that the thinner sclera in myopic eyes is a result of passive stretching. The mechanical forces of the extraocular muscles and/or accommodation causing marked cyclic increases in intraocular pressure (IOP), resulting in scleral stretch. Studies in vitro on rabbit eyes by Greene and colleagues [26, 271 have demonstrated that cyclic IOP increases, at or slightly above physiological temperatures, result in significant creep (stretch) of the posterior sclera. However, scleral creep also occurred at normal physiological temperature and pressure, which questions the validity of such in vitro studies.
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Biochemical changes in myopic sclera of chicks 862
Reduced scleral thickness in myopic eyes does not necessarily rule out the possibility of increased scleral growth. In the most extensively utilized model of axial myopia, the chick, high levels of induced myopia caused by increased axial elongation are not associated with a thinning of the overall sclera. Chick sclera differs from mammalian sclera in that it has both a cartilaginous layer and a fibrous layer, the cartilaginous layer consisting of type-I1 collagen. Gottlieb et al. [2S] found that the cartilaginous sclera was thicker in myopic eyes, with a decrease in cell density and an increase in binucleate cells. The fibrous-scleral layer was found to be thinner, resulting in no significant difference in overall scleral thickness between highly myopic and normal control eyes. Our own laboratory has observed similar thickness changes (see Fig. 1) in the sclera of myopic chick eyes (I,. Foster, I,. R. Williams & N. A. McHrien, unpublished work). These findings strengthen the possibility that, in chick at least, depriving the retina of pattern vision causes the adjacent sclera to undergo active growth, either as a consequence of, or resulting in, axial elongation and myopia. Recent results from our laboratory are presented that provide evidence of increased synthesis of nucleic acids, total protein, collagen and proteoglycans in the sclera of chick eyes developing myopia. Using standard liquid-scintillation dualcounting methods we measured DNA synthesis and protein synthesis by uptake of radiolabelled thymidine and leucine respectively. Chicks were monocularly deprived (MD) of pattern vision (translucent occluder) from day 3 post-hatching for a period of six days ( n= lo), and the radiolabel applied 24 h before optical measures and sacrifice. A 25% increase in [3H]thymidine uptake (d.p.m.) and a 16% increase in [14C]leucine uptake (d.p.m.) were measured in the total sclera of the deprived eye compared with the fellow open control eye (see Fig. 2). In binocularly open-control animals ( n = 10) the difference in uptake of ["]thymidine and [14C]leucineof right eyes compared with left eyes was - 4% and -0.1% respectively (see Fig. 2). Although there is significant equatorial enlargement in the myopic chick eye the greatest increase in size is in the anterior-posterior axis. In a subpopulation of MD chicks ( n = 5) the same labelling protocol as described above was performed, except that the sclera was dissected into two halves to give anterior and posterior portions before solubilization and counting. It was found that the increased synthesis
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Fig. I Sections (2 p m ) of chick sclera from the open eye (top) and the myopic eye (bottom) of a 21 day MD chick The cartilaginous sclera is thicker and fibrous sclera is thinner in the myopic eye when compared with the sclera of the fellow open control eye. Abbreviations used: CS, cartilaginous sclera; FS, fibrous sclera. Sections were stained with Toluidine Blue. Final magnification X 600.
cs
I
FS
cs
'
FS
I
was confined to the posterior sclera with a 44% increase in ['Hlthymidine uptake (d.p.m./mg) and 21% increase in [ '4C]leucine uptake (d.p.m./mg). There was no significant difference in DNA or protein synthesis in the anterior sclera between deprived and fellow open control eyes (see Fig. 2).
Biochemistry of the Eye
Fig. 2
Fig. 3
Uptake of [)H]thymidine and ['4C]leucine in the sclera of M D and binocularly normal chicks
Uptake of 3 5 S 0 4 in the sclera of M D chicks (n = 10)
( a ) Uptake of rHlthymidine in total sclera of MD (n= 10) and
binocularly normal (n= 10) chicks (d.p.m.). For a subpopulation of MD chicks (n = 5) a comparison of uptake in deprived (myopic) versus control eye in the anterior and posterior sclera was made (d.p.rn./mg). ( b ) Uptake of ['4C]leucinein total sclera of MD (n= 10) and binocularly normal (n= 10) chicks (d.p.m.). For a subpopulation of MD chicks ( n = 5 ) a comparison of uptake in deprived (myopic) versus control eye in the anterior and posterior sclera was made (d.p.m./mg). Error bars I SE.M.
Comparison was made between the deprived myopic eye and the fellow control eye for the total sclera (d.p.m.), and also for anterior and posterior halves of the sclera (d.p.m./mg). Error bars I S E M
*70
O
r
T I Total sclera
I"
~
~
Total sclera
-Y
Anterior sclera
Posterior sclera
Pm M D animals 25
0 N o r m a l animals
T
.I-
Total sclera
Anterior sclera
Posterior sclera
Leucine is found in type-I and type-I1 collagen at a frequency of about 25 residues/1000 [29]. However, while collagen is by far the major constituent of sclera, in the interfibrillar matrix characteristic macromolecular glycoconjugates such as dermatan sulphate proteoglycans are present. Leucine has been found to be present in both bovine (fibrous) sclera [ 141, and also in cartilage
Anterior sclera
Posterior sclera
proteoglycans [30]. To get a separate and more specific measure of both collagen synthesis and proteoglycan synthesis, measures were made of hydroxyproline content and "SO, uptake in the sclera of myopic and normal chick eyes. Total hydroxyproline content was measured in the sclera of normal and fellow myopic eyes of chicks ( n = 8) after 14 days of monocular deprivation using Woessner's method [31]. Results show an increase of 20% [1.51 m g f 0 . 0 9 6 versus 1.25 mgf0.075 (mean f S.E.M.)] in the hydroxyproline content, indicative of a similar increase in collagen content in the sclera of myopic eyes when compared with their fellow control eyes. Consistent with this increase in collagen content was a 16% increase in scleral dry weight in myopic eyes (22.5k0.73 mg versus 19.4f0.68 mg). Proteoglycan synthesis was measured by incorporation of "SO4 in both the anterior and posterior portions of the sclera of both eyes of chicks ( n = 10) after 7 days monocular deprivation. Results showed a 48% increase in uptake in the total sclera (d.p.m.) of myopic eyes. Again it was found that this increase was confined almost exclusively to the posterior sclera with a 64% increase in uptake (d.p.m./mg) in the posterior sclera of myopic eyes compared with fellow control eyes. Only a 7% increase in %30,incorporation was found in the anterior sclera of myopic eyes compared with fellow control eyes (see Fig. 3).
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The above findings of biochemical changes in the sclera of myopic chick eyes are qualitatively and quantitatively similar to other recent abstracts [32-351. The above results clearly support the hypothesis of increased scleral growth in myopic chick eyes. They indicate that deprivation of form vision causes a large increase in scleral proteoglycan production with a smaller increase in collagen production and also a hyperplasia of scleral cells. In the chick, deprivation of a localized area of the retina results in elongation and scleral changes in just that part of the sclera adjacent to the deprived retina [36]. This suggests that in some way the retina signals to the underlying sclera to increase its metabolic activity. Apart from the likelihood that this involves growth factors, how the signal crosses the choroidal blood barrier is unclear. This problem is actively under investigation in several laboratories at present.
Biochemical changes in myopic sclera of mammals While the above findings clearly indicate increased growth in the sclera of myopic chick eyes the critical question is whether these findings are indicative of the situation in human myopia. Biochemical studies of sclera in human myopia are rare owing to the difficulty of obtaining suitable tissue. Avetisov and colleagues have investigated post-mortem eyes and found a reduction in both scleral thickness and collagen content (hydroxyproline content) in the posterior sclera of myopic eyes compared with agematched emmetropic eyes [17]. In cases of degenerative myopia abnormal levels of both testosterone and 17P-oestradiol have been reported in male and female patients respectively [ 371. Increased urine levels of mucopolysaccharides have also been reported in high myopia in humans [38,391. In the family of connective-tissue disorders known as Ehlers-Danlos syndrome, defective organization of collagen-fibril bundles is commonly observed. This results in an unusual biomechanical hyperextensibility of the affected skin and is sometimes associated with myopia [40]. A somewhat similar defect of collagen-bundle organization has been cited as the cause of scleral extensibility in human high myopia [ 151. In Ehlers-Danlos type V there is known to be a deficiency of lysyl oxidase [41]. T o investigate the importance of collagen cross-linking in high myopia McRrien & Norton [42] gave MD tree shrews systemic injections of P-aminoproprionitrile (P-APN), a lathyritic agent that blocks the cross-linking of newly formed collagen. P-APN works by irreversibly blocking the
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enzyme lysyl oxidase which is involved in the initial step in the formation of collagen cross-links by converting specific lysine and hydroxylysine residues, giving rise to aldehydes. Animals were monocularly deprived before natural eye opening. Intraperitoneal injections of P-APN were given during the susceptible period for development of experimental myopia in tree shrew (i.e. after 20 days of MI) from natural eye opening). Owing to the reported short-term activity of P-APN (4.11. injections were given every day for 21 days. Visual deprivation was continued beyond the end of the series of injections, with measurements taken of the animals eyes after 75 days of MD. Animals injected systemically with P-APN showed a marked increase in experimental myopia in the deprived eye ( - 23.6 k 3.3 D, n = 5) compared with saline-injected 75-day-MD control animals ( - 11.0 k 0.8 D, n = 5). The axial elongation of the vitreous chamber was nearly double that found in saline-control-MD animals (0.85 _+ 0.09 mm versus 0.44k0.03 mm). The non-deprived eye of animals systemically injected with P-APN showed no significant differences in refraction or vitreouschamber depth from normal eyes. Animals treated with a different lathyrogen, i)-penicillamine, which works by preventing condensation of aldehydes, formation of Schiff bases and also by chelation of Cuz+,showed qualitatively similar results although of a lesser degree. Interestingly, when the experiment was repeated in chicks [42], the MD eye did not show increased vitreous-chamber elongation or myopia despite animals developing clear systemic effects. Why the lathyrogens have such a selective effect on only the deprived eye of mammals is uncertain. Histological analysis of the lathyritic sclera showed marked scleral thinning at the posterior pole of the deprived eye compared with the fellow control eye [ 25 1. Also obvious tessellation of the fundus, a sign of thinning of the retinal-pigment epithelium, was observed in lathyritic-deprived eyes. These results are consistent with the suggestion that in mammalian high myopia the sclera is stretched to cover the expanded eye, although it has been shown that this thinning is greater than would be expected from simple stretching [24]. It could also be argued that as lathyritic agents only prevent cross-linking of newly formed collagen, such a selective effect on the deprived eye might suggest increased synthesis of collagen. However, the marked thinning of the sclera at the posterior pole in mammals may be indicative of an increased rate of degradation of scleral collagen in myopic eyes,
Biochemistry of the Eye
which might be accentuated by the lathyritic agents. Studies are underway to further elucidate the mechanism of scleral changes in experimentally induced mammalian myopia. W e thank Joanne Loades for technical assistance. This work was supported in part by a grant from the K.N.I.H. 1. Fledelius, H. C. (1983) Acta Ophthalmol. 61,
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Received 22 July 1991
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