Effect of muscarinic agents on sclera fibroblast and their role in myopia 1

Lens Induced Myopia LIM The other form of experimental myopia, lens induced myopia results from raising an animal with a negative lens over one eye, imposing hyperopic defocus image behi

I Introduction

1.1 Myopia

Myopia has reached epidemic proportions in Singapore (Rajan et al 1994, Saw et al 1996) Myopia is 1.5 to 2.5 times more prevalent in adult Chinese residing in Singapore than similarly aged European-derived populations in the United States and Australia where the sociodemographic associations are similar (Wong et al 2000) In the United States 25% of children become myopic and it affects 15-20% of the adult population (Sperduto et al

1983, Hirsch and Weymouth 1990) Optically, myopia is defined as a mismatch between the refractive optics and the length of the eye that causes an image to be focused in front of the retina leading to an out of focus image High myopia is an important cause of visual disability (Klein et al 1995) It has been noted as the cause of blindness due to the many associated complications, such as retinal break, retinal detachment and myopic retinopathy

Myopia also places a burden on society and on the individual The cost of glasses, contact lenses and refractive surgery has been estimated to be $13 billion annually (Sheedy 1996) Quality of life issues associated with myopia are also considerable Myopia limits career choices, social interaction and in underdeveloped countries, myopes may not have corrective choices Despite the high prevalence and associated social and economic costs

of correcting myopia, we know little about the etiology of myopia

The eye is approximately 17mm long at birth From birth to age 6, the eye grows by approximately 5 mm During this period there will be a loss of 4D of corneal power, and 20D of lens power Through the process of emmetropisation, the distribution of refractive error becomes narrow and the prevalence of myopia is only 2% at age 6 During the next 8 years, the average eye will grow approximately a millimeter The prevalence of myopia during this time will increase more than seven fold, to 15% by 15 years of age (Mutti et al 1996)

Gross and Wickman (1995) concluded that both emmetropisation and juvenile onset myopia are best explained by a retinal image-mediated biochemical mechanism that modulates eye growth However, the nature of this growth mechanism has not been elucidated

The emmetropisation mechanism in chickens and tree shrews appear to require visual signals to guide the elongation of the eye If the focal length is artificially lengthened by wearing a minus-power lens, the eye will elongate until the axial length approximately matches the amount of increase in imposed focal length (Irving et al 1991, Siegwart et al 1993) When the eyes are deprived of form vision with a translucent diffuser, there is no visual image to indicate that the appropriate axial length has been reached In this situation, elongation continues unchecked, moving the retina past the focal plane (Pickett-Seltner et al 1988, Wallman et al 1987)

If humans have an emmetropisation mechanism similar to that demonstrated in animals, juvenile-onset myopia may occur if a child inherits a dysfunctional emmetropisation mechanism It is not clear if dysfunction occurs in photoreceptors, in the communication of

an unknown signal to the sclera, or in some intrinsic control of sclera growth This suggests that there may be an emmetropisation feedback loop, and this becomes disrupted

in myopia Several studies have found pharmacological treatments that reduce axial elongation in animal models (Stone et al 1989, McBrien et al 1993, Rohrer et al 1993, Seltner et al 1993), and there are current clinical trials testing some of these in children

1.1.2 Genetic influences

Several lines of evidence point to a role of genetics in the development of myopia Monozygotic twins tend to resemble each other more closely in refractive error than do dizygotic twins Estimates of heritability (proportion of phenotypic variance explained by heredity) for myopia obtained from monozygotic twins were higher than dizygotic twins (Minkovitz et al 1993,Teikari et al 1988), suggesting a genetic influence

A family history of myopia is associated with the likelihood of developing myopia, although this could also be a result of visual habits such as amount of reading from parents A greater prevalence of myopia exists among the children of myopic parents than among the children of non-myopic parents According to the Orinda longitudinal study, prevalence of myopia in children with two myopic parents is 30-40% whereas it is reduced to 20-25% in children with one myopic parent and to <10% in children with non-myopic parents (Zadnik

et al 1994) Overall the parental history of myopia explains significantly more variance in children’s refractive error and ocular component values than children’s near work activity (reading, watching television, playing video games) It is unknown, however to what extent these familial patterns are due to genetic or environmental factors Children from ages 6 to 14 years at risk for the onset of myopia due to a positive family history, have longer eyes than children of similar age without myopic parents, even before the onset of juvenile myopia (Zadnik et al 1993)

The association between increased axial length and juvenile onset myopia is known largely from cross-sectional studies and from limited longitudinal investigations (Fledelius et al

1980, 1982, Sorsby et al 1970, Tokoro et al 1969) However, exactly how and why normal eye growth goes awry to produce myopia is still unknown

There is a much higher prevalence of myopia in Singapore This has been attributed to its rigorous education system and high near work demands (Au Eong et al 1993, Wong et al 2000) Other research points to the association of myopia and increased “near work” such

as reading during childhood (Angle et al 1980, Richler et al 1980) In addition, experiments using animal models of myopia have demonstrated that an environmental component has an important role in eye growth (Raviola et al 1985, Wallman et al 1987)

1.1.4 Experimental Myopia

Animal models of myopia

Animal models have been developed to determine the influence of accommodation and the visual environment on emmetropisation and refractive error Two different types of myopia have been developed They are Form Deprivation Myopia (FDM) and Lens Induced Myopia (LIM)

Animal studies on myopia began in the 1960’s (Young 1961, Lauber et al 1965), and several animal models have been developed (Wiesel and Raviola 1977, Sherman et al 1977, Wallman et al 1978, Somers 1978, Wilson and Sherman 1977) Several species have been examined and three models have emerged such as chick (Wallman et al 1978), tree shrew (Sherman et al 1977) and monkey (Wiesel and Raviola 1977)

Animal models have provided new insights into the understanding of myopia; they have allowed studies into the cellular mechanism, which can not be undertaken in humans Whether these mechanisms are exactly similar to those controlling axial growths in humans requires careful analysis as mentioned by Zadnik and Mutti (1995)

Animal experiments provide a basis for understanding the mechanisms that have evolved to control the refractive development of the mammalian eye and it is probable that some of them are common to all species

1.1.5 Methods of inducing experimental myopia

Form Deprivation Myopia (FDM)

This method for producing experimental myopia deprives a young eye of form-vision but not light by means of lid-suture or the wearing of translucent diffusers This form of myopia can be produced in animal models such as chicks (Hodos et al 1984, Sivak et al

1990, Wallman et al 1987,1978), monkeys (Tigges et al 1990, Wiesel et al 1977), marmosets (Troilo et al 1993), tree shrews (Marsh-Tootle et al 1989, McBrien et al 1992), guinea pigs (Lodge et al 1994), squirrels (McBrien et al 1993) and rabbits (Beuerman et al 1993)

Lid suture was used in young monkeys to produce myopia (Wiesel and Raviola, 1977) After suturing the lids for periods up to 26 months the eyes were found to be myopic by retinoscopy (-1 to -13.5D) Both axial length and equatorial diameter were increased The posterior sclera was thinner than the fellow control eye while the anterior segment of the eye was similar on the control and experimental two sides Wiesel and Raviola carried out further experiments and found that lid-suture in dark reared animals with lid-suture did not alter the refraction or axial length (Raviola et al 1978) They also reported in 1979 that opacification of cornea by injection of latex particles produced an increase in axial length in primates (Wiesel et al 1979) This result can also be explained as the effect of a reduction

in pattern vision Raviola and Wiesel (1985) went on to examine the effects of optic nerve section on lid suture myopia In one stump tailed macaque myopia was prevented by optic nerve section, while in three rhesus macaques it was not This suggested that in the retina might release regulatory molecules whose production is influenced by the pattern of light stimulation

In newly hatched chicks myopia can be produced either rapidly by lid suture or wearing simple occluders The effects of lid-suture on the morphology and refractive state of the chick eye have been extensively described (Yinon 1980,1983) The morphological changes included increase in axial length and equatorial diameter It has also been reported that dark rearing results in the enlargement of the posterior segment, accompanied by corneal flattening resulting in a net hyperopia when combined with lid suture and light diffusers (Yinon 1986, Gottleib 1987) It should be noted that dark rearing does not only remove the visual input but it changes the circadian rhythms

Lens Induced Myopia (LIM)

The other form of experimental myopia, lens induced myopia results from raising an animal with a negative lens over one eye, imposing hyperopic defocus (image behind retina) leading to axial elongation In a similar manner, hyperopia can be induced by raising animals with positive spectacle lens over the eye to impose myopic defocus (image focused

in front of the retina)

Lens induced myopia and hyperopia were developed first in chickens and more recently in rhesus monkeys, tree shrews, and marmosets (Schaeffel et al 1988, Irving et al 1991,1995, Siegwart and Norton 1993, Hung et al 1995, Graham and Judge 1995,1999)

This important observation suggests that the eye can actively control growth to optimise visual acuity This finding could be a clinical problem because negative lenses are fitted to children with growing eyes to correct refractive error It is therefore important to understand this growth control mechanism, and whether it occurs in humans

To follow up on the finding that the application of a simple lens to the eye of the young chick can produce an eye enlargement, experiments have been designed to occlude the whole visual field as well as a portion of the visual field Domes, arches and rings, were applied to the eye of three-day old chicks (Hayes et al 1986) The domes degraded the image over the entire visual field of one eye The arches degraded only the lateral visual field, leaving unobstructed vision in the frontal and posterior visual field The rings did not occlude the visual field, but served as a control Chicks were refracted in the lateral visual field at the age of 3 to 7 weeks The application of the dome device in particular produced

a large shift to myopic refraction The rings did not affect eye growth while the arch significantly increased the dorsoventral equatorial diameter of the eye The dome device had the most dramatic effect on eye morphology and resulted in an increase in both axial and equatorial dimensions Eyes fitted with the dome device had a steepened cornea, increased anterior chamber depth, more open angle and a greater corneal diameter than control eyes The axial length of the posterior segment was also increased These results suggested scleral growth be topographically related to the sector of the retina undergoing visual deprivation (Hodos et al 1984, 1985, Hayes et al 1986)

In the chick, experimental myopia developed even after optic nerve section Thus, the overgrowth is produced by systems confined to the eye and need not involve visual feedback from the brain to the eye (via accommodation or some other efferent system) An explanation could be that retinal cells sensitive to image quality could produce substances, which locally modulate sclera growth However, there is no mechanism to explain that effect

1.1.6 Recovery from experimental myopia

Wallman and Adams (1987) investigated the susceptibility to and recovery from axial myopia in the chick produced by occluders Recovery occurred when the occluders were removed within the first six weeks and amounted to 20 D in two weeks Troilo and Wallman (1988) have reported that chick eyes made hypermetropic by dark rearing or myopic by occluders can recover toward emmetropia when reverting to normal visual conditions Recovery was also found to proceed when the Edinger Westphal nucleus was lesioned (eliminating accommodation) and when the optic nerve is cut

Chicks were found to compensate for a large range of imposed refractive errors There was nearly complete compensation within a week for lenses from -10 to + 20 D (Irving et

al 1992), with greater compensation for positive lenses than negative lenses Whether this bi-directional compensation occurs in humans is uncertain

1.1.7 Clinical models of experimental myopia

Clinical observations have suggested that a condition of image deprivation myopia occur in humans that are similar to FDM in animal models

Birth injuries of the cornea, often unilateral have been associated with subsequent myopia (Lloyd 1938) Myopia has been observed in patients with corneal opacities (Muramatsu 1982) Premature infants with or without retinopathy of prematurity (ROP) may develop abnormal eyes with impaired vision, refractive errors and strabismus Numerous studies on refraction in pre-term infants have shown a predisposition to childhood myopia ( Birge

1956, Kalina 1969, Fledelius 1976, Shapiro et al 1980, Kushner 1982, Koole et al 1990, Gallo et al 1991, Fledelius 1993)

Johnson and colleagues (1982) have described axial myopia in one eye in one sibling of a pair of identical twins, which had a posterior subcapsular cataract

Von Noorden and Lewis (1987) examined 10 young patients who had unilateral cataracts and in seven out of ten cases the involved eye revealed increased axial length

Robb (1977) described an association between hemangiomas of the eye and high myopia in young children Hoyt and colleagues (1981) described eight infants with lid closure caused

by nerve palsy, obstetric trauma, and hemangioma, all were found to have increased axial length and myopia

1.1.8 Species differences

Few experiments have been carried out on compensational myopia in mammals A main difference between birds and mammals is the time course of the response to visual deprivation and the structure of the sclera

In chicks, when deprivation is removed, the rate of ocular elongation returns to normal within days In contrast, monkey eyes continue to elongate for months after visual deprivation is removed If marmosets are visually deprived during the period of normal emmetropisation and form deprivation ends, the eye continues to elongate and become increasingly myopic for several months (Troilo et al 1993)

Mammalian models of myopia such as tree shrews and monkeys have an advantage over avian models as the structure and biochemistry of their eyes is more similar to humans Sherman, Norton and Casagrande (1977) introduced tree shrews as a model for myopia Visually induced changes in eye growth of tree shrew and monkey are smaller than that of chickens and continuous treatment with diffuser spectacle or lid suture is more difficult but has been successful (Wiesel and Raviola 1977,1985, Smith and Hung 1999,2002, Smith et

al 2000)

The major obstacle in using monkeys are the longer time required for the effect and additional level of care There are many results from chickens that have not yet been obtained from monkeys but are essential for the understanding of human myopia

The chicken is currently the most frequently studied animal model due to its easy availability and advantages of rapid eye growth, good optical quality and sensitivity to moderate modifications of vision The chick can develop up to 20D of axial myopia in a week and can compensate for imposed defocus of 4D in 3 days The chicken model was introduced by Wallman, Trachtman and Turkel (1978)

However the results from chickens are difficult to apply to mammals The pharmacology and morphology of pupillary and accommodation responses are different in chicks and mammals Fortunately, there are similarities with regard to the roles of neurotransmitters and neuropeptides, such as acetylcholine, dopamine and vasoactive intestinal polypeptide (VIP) in the control of eye growth (Schaeffel et al 1995, Stone et al 1988)

1.1.9 Pharmacology of myopia

Several studies have reported the modulation of the effect of pharmacological agents on experimental myopia Raviola and Wiesel (1985) found that atropine had no effect in the rhesus macaque, but prevented axial elongation in the stump-tailed macaque Wildsoet and Pettigrew (1988) have shown that intravitreal injections of neurotoxin kainic acid induced axial elongation in chickens Kainic acid is a glutamate analogue, while another glutamate analogue amino-phosphonobutyric acid also (APB) has been reported to reduce axial elongation (Smith, Fox and Duncan, 1985)

It was observed that retinal concentrations of neurochemicals were altered in experimental myopia, specifically VIP was increased in monkey and dopamine was decreased in both chick and monkey (Iuvone et al 1989, Stone et al 1988,1989)

A recent study found that glucagon-containing amacrine cells respond differentially to the sign of defocus and may mediate lens-induced changes in ocular growth refraction (Fischer

et al, 1999) Local application of the dopamine agonist, apomorphin blocked the axial elongation that ordinarily follows visual deprivation in chick and in rhesus monkey (Stone

et al 1989,1991, Iuvone 1991) In chicks, normalisation of retinal dopamine correlated with recovery from myopia (Pendrak et al 1997)

Use of atropine

Historically atropine was used because it is a cycloplegic agent in human antagonising the mAChRs of the cilliary muscle It also reduces axial elongation in deprivation models of myopia (McBrien et al 1993, Stone et al 1991)

In the case of chicks, the ciliary muscle is striated and therefore is innervated via nicotinic rather than muscarinic cholinergic receptors (McBrien et al 1993) Therefore the effect of

atropine must have been exerted on the retina or directly on the sclera Indeed, there is evidence that atropine inhibits cellular proliferation of sclera chondrocytes and extra cellular matrix production (Marzani et al 1994, Lind et al 1997)

Atropine has been advocated as a myopia therapy for some time, based on the assumption that it prevents the accommodative response Several studies have shown that daily topical ocular instillation of 1% or 0.5% atropine markedly reduces the progression rate of myopia (Sampson 1979, Bedrossian 1971, Brodstein et al 1984, Dyer 1979, Goss 1982, Brenner

1985, Kao et al 1988, Kelly et al 1975, Yen et al 1989, Shih et al 1999) Side effects, systemic and ocular such as blurred vision, photophobia, allergic dermatitis, possible UV related retinal damage and direct effect of the heart rate make long term use of atropine impractical (Brodstein 1984, Chou et al 1997)

Muscarinic receptors (mAChRs)

Muscarinic receptors are members of a superfamily of G protein coupled receptors Molecular cloning studies identified five subtypes of mAchR genes (m1-m5) widely expressed in various tissues (Kubo et al 1986, Peralta et al 1987, Hulme et al 1990, Receptor Nomenclature suppl, 1993) All these receptors share a similar three-dimensional structure consisting of a tightly packed bundle of seven transmembrane helices linked by three extracellular and three intracellular loops (Wess et al 1995) Pharmacological studies have revealed four different subtypes of mAChR (m1-m4) based on their ability to bind specific ligands and activate second transduction pathways Intracellular signals are transmitted by coupling of receptors to cytoplasmic guanine nucleotide-binding regulatory

proteins (G proteins) (Hulme et al 1990, Lanbert et al 1992) with subsequent generation of second messengers

The molecular weight of the subtypes predicted from amino acid sequences range between

51 kd and 66 kd (Kerlavage et al 1987, Ashkenazi et al 1988)

A mechanical hypothesis has been suggested to explain excessive axial elongation It has been suggested that myopic eyes may experience higher externally applied forces, such as from blinking or rubbing of the eye (Greene 1979, Ku and Green 1981), higher intraocular pressure (Arciniesgas 1980, Maurice and Mushin 1966, Tokoro 1970, Pruett 1988) or higher extra ocular muscle tensions (Greene 1980) It has been suggested that these higher forces may result in sclera creep

Another possible cause would be the elastic stretching of the sclera shell due to lower elastic modulus in the myopic eye Tree shrew sclera from myopic eyes does appear to

stretch more easily than control eyes Philips and McBrien (1995) studied this in tree shrews by measuring the modulus of elasticity and sclera thickness of tree shrew eyes that were monocularly form-deprived They simulated the elastic deformations of the control and form-deprived eyes with a finite element model and found that both the measured sclera thickness and elastic modulus were lower in the myopic eyes Weakening of the sclera in high myopia may be the result of the structural change (Curtin et al,1958)

Biochemical and structural studies of myopic eyes support the idea that sclera growth and remodelling both contribute to the development of myopia

For example, lower levels of extracellular matrix proteins and collagen content have been observed in the sclera of experimentally myopic tree shrew (Norton et al 1995) This could

be due to increase activity of matrix metalloproteinases (MMPs), decreased activity of tissue inhibitors of metalloproteinases (TIMPs) or a fundamental lack of protein synthesis Similarly, light and electron microscopic analyses of myopic monkey and human eyes have demonstrated changes in collagen fibre diameter and bundle spacing (Funata 1990, Liu 1986)

All these studies suggest that scleral elongation in experimental myopia involves more than just passive stretching of the sclera and biochemical or molecular changes are probably associated with growth and remodelling

The cardinal characteristic of myopia is axial elongation of the posterior part of the eye All animal models have developed excessive vitreous chamber elongation (Wallman et al

1978, Norton et al 1990) Active sclera growth is a primary event in the axial elongation therefore control of the sclera fibroblast may lead to a possible therapy for myopia prevention or decreasing the development of myopia

1.2.1 Matrix Metalloproteinases (MMPs)

Evidence has been obtained that the sclera extracellular matrix (ECM) remodelling of the sclera is part of myopic process Increased levels of gelatinase A was observed in the sclera of experimentally myopic chick eyes (Rada et al 1995,1999)

MMPs are a family of related zinc metalloproteinases that are involved in normal development, wound healing, cancer, arthritis, and angiogenesis MMPs are secreted as inactive pro-enzymes Activation of these zymogens is the critical step that leads to tissue catabolism MMP pro-enzymes are activated by proteinases, thiol modifying reagents (mercurials, oxidised glutathion, HOCl ) and heat The endogenous protein inhibitors (TIMPs) inhibit activated MMPs There are at least 17 different MMPs and 4 TIMPs expressed in human tissue (Woessner 1998) The balance of between MMP and TIMP expression determines ECM homeostasis

Growth factors and hormones can regulate MMP and TIMP expression Various cell types including fibroblasts produce TIMPs Their expression is regulated by various cytokines such as Interlukin-1,6,11 (IL-1, IL-6, IL-11), transforming growth factor (TGF-β) and epidermal growth factor (EGF) (Roeb et al 1993, Leco et al 1994,1993) Selective stimulation of MMP is under control of growth factors such as fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and tumour necrosis factor (TNF-α) (Moses 1997, Wojtowicz-Praga et al 1997)

1.2.2 Sclera fibroblast control

Sclera cells are the final effectors in a complex signal cascade leading to axial elongation Several studies have established the effectiveness of atropine in decreasing the progression

of axial length in humans and in experimental myopia, however the mechanism is still elusive Atropine may act directly on sclera fibroblasts through the G protein coupled muscarinic receptor to modulate the development of myopia

It has been observed in myopic eyes that some growth factor expression levels were altered FGF-2 level was significantly lower in the sclera of myopic eyes than the control and TGF-β level was significantly higher in myopic eyes (Seko et al 1994) Atropine may have an effect on growth factors, which then control sclera fibroblast activity through tyrosine kinases, MMPs and TIMPs

1.3 Growth factors in myopia

Intravitreal and subconjunctival injections of FGF-2 have prevented axial elongation induced by form deprivation (Rohrer et al 1993,1994) FGF-2, which is reduced in myopic eye, may provide a “stop” signal for the growing eye although it is generally regarded as fibroblast proliferative factor The effect could be mimicked by FGF-1 but with potency approximately 160 times less than that of FGF-1

TGF-β was not found to induce myopia or to increase myopia however it acted as a potent

inhibitor of FGF-1 when injected together with FGF-1 (Rohrer et al 1994)

Phosphorylation of tyrosine kinases receptor

Tyrosine kinases couple cell surface events to the regulation of many intracellular activities such as gene expression, proliferation and ion channel modulation Studies show that growth factors, cytokines, integrins, antigens and G protein coupled receptors also utilise tyrosine kinase pathways to transduce intracellular signals (Zachary and Rozengurt 1992, Aoki et al 1994, August et al 1994, Chen et al 1994, Ihle et al 1994)

1.3.1 Fibroblast Growth Factor (FGF)

The FGF family has at least 18 different isoforms, which display a remarkable affinity to heparin and are also called heparin binding growth factors (HBGF)

FGF-2 is an 18Kd protein consisting of 155 amino acids, and an isoelectric point of 9.6, without disulfide bonds or glycosylation Some higher weight forms of FGF-2 (22-24Kd) have been also described The smallest form (18Kd) occurs predominantly in the cytosol while the higher molecular weight forms are associated with the nucleus and ribosomes

An isoform of FGF-2 associated with the endoplasmic reticulum has been designated altFGF-2 (Burgess and Maciag 1989, Baird and Klagsburn 1991, Goldfarb 1990,

Gospodarowicz et al 1987)

FGF-2 is found in almost all tissues of mesodermal and neuroectodermal origin and also in tumors derived from these tissues Endothelial cells produce large amounts of FGF Some FGF-2 is associated with the extracellular matrix of epithelial and endothelial cells (Abraham et al 1986) Many cells express FGF-2 only transiently and store it within the cytoplasm in a biologically inactive form (Bugler et al 1991)

FGF-2 is released after tissue injury (Fiddes et al 1991, Hebda et al 1990), during inflammatory processes and also during the proliferation of tumor cells (Chodak et al 1988, Fujimoto et al 1991)

At the sequence level, FGF-1 and FGF-2 display 55 percent homology FGF-1, like FGF-2 does not possess a signal sequence that would allow secretion of the factor by a classical secretion pathway The mechanism underlying the release of FGF-2 is unknown (Baird and Klagsbrun 1991, Burgess and Mariag 1989, Goldfarb 1990)

A truncated variant of FGF-1 generated by alternative splicing and removal of the entire second coding exon is only 60 amino acids long (6.7 Kd) elicits only minimal fibroblast proliferation and antagonises the effect of full length FGF-1 when added exogenously or when co-expressed with FGF-1 in 3T3 fibroblasts (Yu et al 1992) FGF-1 binds to the same receptor as FGF-2 FGF-1 displays similar activities as FGF-2 however, it is 50-100 fold less active than FGF-2

Fibroblast growth factor receptor (FGF-R)

FGF receptors are encoded by a gene family consisting of at least four receptor tyrosine kinases (FGF-R1,2,3,4) that transduce signals for cell growth and differentiation (Armstrong et al 1992, Avivi et al 1993, Eisemann et al 1991, Keegan et al 1991) Binding

of FGF-2 to one of its receptors requires the interaction with heparan sulfate and heparan sulfate proteoglycans (syndecan) of the extracellular matrix (Keifer et al 1991, Ornitz et al

1992, Raprager et al 1991) Heparan has been shown to protect FGF-2 from inactivation

by proteases, acids and heat Heparan also increases the biological activity of FGF-2

Binding of FGF-2 (also FGF-1) to receptor can be inhibited by suramin and also by protamin (Yayon et al 1991)

It has been observed that binding of FGF-R leads to phosphorylation by an intracellular protein kinase, which may additionally alter FGF activity and bioavailability (Partanen et al

1992, Vainikka et al 1992, Werner et al 1992, Moscatelli 1991)

Biological activity

A multifunctional role of FGF-2 is suggested by the many different receptor phenotypes and their cell specificity (Alarid et al 1991, Gallicchio et al 1991) FGF-2 stimulates the growth of fibroblasts, myoblasts, osteoblasts, neuronal cells, endothelial cells, keratinocytes, chondrocytes and many other cell types (Araujo and Cotman 1992, Benezra

et al 1992, Davidson and Broadley 1991, Ray et al 1993, Sato et al 1991) FGF-2 has also been shown to be a promoting or inhibitory modulator of cellular differentiation In early embryos FGF-2 functions as a differentiation factor that induces tissue destined to produce ectodermal structure to differentiate into mesodermal tissues FGF-2 retards senescence of cells thus allowing maintenance of cells in vitro which would normally lose their differentiated phenotype in long-term cultures An FGF-2 influence the differentiation processes is probably the result of a complex interaction with other factors

Transgenic animal studies show that FGF-2 is essential for the morphogenesis of suprabasal keratinocytes and for the establishment of the normal program of keratinocyte differentiation (Werner et al 1993)

Treatment of various human tumor cell lines (melanomas, glioblastomas) with antisense RNA directed against FGF-2 have been shown to inhibit the proliferation of these cells and their ability to form colonies (Becker et al 1989, Morrison 1991)

1.3.2 Transforming growth factor (TGF-β)

TGF-βs are secreted from cells as latent complexes They are synthesised by macrophages, lymphocytes, endothelial cells, keratinocytes, granulosacells, chondrocytes, glioblastoma cells and leukaemia cells (Barnard et al 1990, Bonewald and Mundy 1989, Sporn and Roberts 1992) TGF-β can be induced by steroids, retinoids, epidermal growth factor (EGF), NGF, vitamin D3 and Interleukin (IL-1) The synthesis of TGF-β can be inhibited

by EGF, FGF, retinoids, calcium and follicle stimulating hormone (Border and Ruoslahti et

al 1992, Burt 1992, Hooper 1991) TGF-β is stored in an inactive form as associated with ECM and complexed with betaglycan and decorin The mechanisms underlying its release from these reservoirs is unknown TGF-β exists in at least 5 isoforms The amino acid sequence of the various isoform display 70-80 percent homology TGF-β1 is the prevalent form TGF-β is the most potent known growth inhibitor for normal and transformed epithelial cells, endothelial cells, fibroblasts, neuronal cells, lymphoid cells and other hematopoietic cell types, hepatocytes and keratinocytes (Miyazono and Heldin 1992, Pfeilschifter 1990, Robers and Sporn 1990, 1992,1993)

In many cell types TGF-β antagonises the biological activities of growth factors which have receptors of the tyrosine kinase group At extremely low concentrations, TGF-β is a growth inhibitor for smooth muscle cells, fibroblasts and chondrocytes At higher

concentration TGF-β stimulates proliferation of these cells This bimodal activity is mediated in part by platelet derived growth factor (PDGF) The synthesis and secretion of PDGF is stimulated by low concentrations of TGF-β, while higher concentrations lower the expression of PDGF receptors and hence diminish the biological effects of PDGF TGF-βstimulates the synthesis of ECM including collagen, proteoglycan, fibronectin and integrin (Roberts et al 1990, Ignotz and Massague 1986)

EGF-R

Epidermal growth factor receptor (EGF-R) is a 170kd membrane bound glycoprotein expressed on the surface of epithelial cells EGF-R is a receptor tyrosine kinase known to regulate cell cycle (Carpenter and Cohen 1987, Yarden and Ullrich 1988, Cadena and Gill 1992) The receptor is activated when its ligand (EGF, TGF-α) binds to the extracellular domain, resulting in autophosphorylation of the receptor’s intracellular tyrosine kinase domain (Cohen et al 1980, Schreiber et al 1983) Dimerisation and internalisation of EGF-

R molecules transduce proliferative signals Several reports have indicated that EGF-R is over-expressed in various cancers (Gullick et al 1986, Ozanne et al 1986, McKenzie et al 1991) There are also studies, which suggest a relationship between levels of EGF-R and levels of estrogens and progesterone receptors (Klijin et al 1992, Nicholson et al 1991, Sainsbury et al 1987, Koenders et al 1991)

1.4 Aims of Project

Atropine has been shown to prevent the development of myopia Evidence is available to show that atropine and more specific m1 muscarinic receptor antagonists, such as pirenzepine, may slow axial elongation in both experimental and clinical myopia (Cottrial et

al 1996) Pharmacological treatment of myopia and its potential widespread application raise a number of questions

Experimental myopia leads to an increase in ECM production and accumulation of proteoglycans within the cartilaginous sclera while fibrous sclera was opposite as similarly observed in monkey and tree shrew It has been suggested that increased growth of the cartilaginous sclera is associated with interaction between cartilaginous sclera and fibrous sclera (Marzani and Wallman 1997)

It is the hypothesis of this study that sclera fibroblasts may play a major role in axial elongation and atropine has a direct effect on this cell to decrease myopic progression The general goal of this study has been to determine the cellular response of sclera fibroblasts to atropine A culture system for sclera fibroblasts will be developed and used to test the effect of atropine on proliferation, receptor activity and intracellular signal transduction pathways

To test the overall hypothesis, the following specific phenomena will be studied

1 The presence of muscarinic receptors in sclera fibroblasts (SF)

2 The response of SF to muscarinic agents

3 The response of SF to growth factors

4 The changes in growth factors and matrix metalloproteinases in response to atropine

5 The changes in matrix metalloproteinases in response to growth factors

II Materials and Methods

2.1 Materials

2.1.1 Animals

Female Lohman Brown chicks were purchased from a local hatchery (Chew’s agriculture, Singapore) on the first day of hatching They were reared in our animal facility on a 12:12 hour light:dark cycle (average 110 lux)

New Zealand White rabbits (6 weeks old) and Balb/c mouse (8 weeks old) were purchased from the Animal Center of the National University of Singapore

2.1.2 Reagents

Coomassie plus protein assay reagent (Pierce), Bovine serum albumin;BSA standard (Pierce), M-Per mammalian protein extraction reagent (Pierce), Atropine sulfate( Sigma), Pirenzepine (Sigma), Carbacol (Sigma), cyclic AMP (Sigma), ATP (Sigma), FGF-2 recombinant human protein (Oncogene), Anti muscarinic receptor (m1,m2,m3,m4,m5) antibody (Biogenesis), Anti FGF-2 mouse IgG peroxidase conjugate, Human Proenzyme MMP-2 proteins(Oncogene), Polyclonal anti MMP-2 (Calbiochem), Mouse monoclonal anti MMP-2 antibody (Neomarkers), Anti-Sheep IgG peroxidase conjugate (Pierce), Anti-mouse IgG peroxidase conjugate (Pierce), Anti rabbit IgG peroxidase conjugate (Pierce), Bromodeoxyuridine;BrdU (Calbiochem), Anti BRDU antibody (Phrarmingen), DEVD-pNA substrate (Promega), Anti FGF-1 rabbit IgG (Sigma), FGF-1 standard (Sigma), Anti human TIMP mouse IgG (Calbiochem), DMEM (Sigma), Antibiotic-antimycotic (Sigma), FBS (Hyclone), Sample buffer (0.5M Tris-HCl, Glycerol, 10% SDS, β-mercaptoethanol,

1% bromophenol blue), Non-reducing sample buffer (0.5M Tris HCl, Glycerol,10%SDS,1% bromophenol blue), Coomassie staining solution (0.1% Coomassie blue R-250), Destaining solution (40% methanol, 10% acetic acid), Renaturation buffer (2.5%Triton-X), Development buffer (50mmTris, 200mmNaCl, 5mm CaCl2, 0.02% Brij-35), Stop solution (Sulfuric acid), Substrate solution (Tetra methyl benzidine;TMB), X-ray film (Pierce), Super signal chemiluminescent detection reagent (Pierce), Blocking buffer (0.5% non-fat milk/PBST), Phosphate Buffered Saline, Tween-20 (Sigma)

2.2 Methods

2.2.1 Experimental Myopia

Myopia was induced in 80 Lohman Brown chicks by covering their right eyes with a negative (-15D) plastic lens (12mm in diameter, large optic zone 10.5-11.5mm, 8mm posterior surface radium, blue tint, Lenspec Technology, Singapore) for 2 weeks The lenses were attached to Velcro and mounted on a matching piece of Velcro, which had been cemented to the feathers around the chick eye using collodion (Fisher Chemical) The lenses were kept clean by maintaining chicks on raised floors, sieving food to remove small particles and cleaning the lenses every 4 hours from 9 AM to 5PM

2.2.2 Refraction

Refractions of the chick eyes were measured by streak retinoscopy (Neitz, Japan) after applying one drop of topical anaesthesia (Alcaine 0.5%, Alcon) A specially made retractor was used to move the lids away during refraction at a working distance of 33cm Streak retinoscopy was performed by two independent ophthalmologist (Blinded)

2.2.3 Measurement of eye size

Chicks were sacrificed after 14 days of lens wearing with an overdose of sodium pentobarbitol Eyes were enucleated and the extra-ocular tissue was removed, the axial (anterior-posterior) and equatorial dimensions were measured with a digital calliper (Starret, No.721AX Basic Series Electronic Digital Caliper, accuracy +/-0.03mm and reliability 0.01mm) Axial elongation was measured by two independent persons (Blinded)

2.2.4 In vivo atropine treatment

Chicks were randomly divided into two groups: one group received atropine while the other received saline injections in the lens wearing eyes 50 µl of freshly made 1% atropine sulfate in sterile normal saline or 50µl of 0.9% sterile saline was given as subconjunctival injection at the same time each day for 14 days The treated eyes were checked every day for infections Animals showing any sign of infection were excluded from the experiment Chicks were sacrificed by an overdose of sodium pentobarbitol Eyes were enucleated and scleras separated

2.2.5 Measurement of tissue protein

Scleras from five eyes were pooled and frozen in liquid nitrogen They were homogenised

in T-per (tissue protein extraction reagent, Pierce) Five scleras from each group were pooled as one single sample for statistical purpose Insoluble material was removed by centrifugation at 2000g for 5 min at 4°C The soluble protein content was measured using Coomasie plus protein assay reagent (Pierce) based on the Bradford method

2.2.6 Sclera fibroblast culture

Chick Fibroblast culture:

Chicks were sacrificed after 2weeks of lens wearing The fibrous and cartilaginous scleral layers were separated by peeling away the fibrous sclera from cartilaginous sclera Fibrous sclera was placed in 60mm culture dish in 3ml of Dubelcco’s Modified Eagle’s Medium (DMEM, Sigma) supplemented with penicillin, streptomycin and amphotericin B and 10% Fetal Bovine Serum (FBS, Hyclone) Tissue culture were incubated at 37°C, 5% CO2 and allowed to reach 80% confluence Cells were passaged sequentially by exposing cells to 0.25% Trypsin/0.5mM EDTA at 37°C for 5 to 10min All cells used in experiments are between passages 2 to 4 ( less than 30 days old)

Rabbit, mouse and human sclera fibroblast culture:

Sclera was cut into small pieces (5x5mm) and placed choroid side down on 60mm culture dish (5pc/dish) in 5ml of Dubelcco’s Modified Eagle’s Medium (DMEM, Sigma) supplemented with penicillin, streptomycin and amphotericin B and 10% Fetal Bovine Serum (FBS, Hyclone) Tissue culture were incubated at 37°C, 5% CO2 After reaching

80% confluence, cells were passaged sequentially by exposing them to 0.25% Trypsin/0.5mM EDTA at 37°C for 5 to 10min All cells were used in experiments between passages 2 to 4 ( less than 30 days)

2.2.7 Culture conditions

Fibroblasts were passaged from whole are of fibrous layer Passaged cells were plated at a concentration of 1x105 /well into 24 well plates containing DMEM with 10% FBS and allowed to attach The cells were then washed with serum free medium and serum starved overnight Serum-free medium was replaced with fresh DMEM containing different concentrations of atropine (0.1uM-100uM) and incubated for 24hours at 37°C/5%CO2 The conditioned medium (CM) and cell lysate (CL) were collected for further experiments For cell proliferation assays 100 µl of passaged sclera fibroblasts (1x105 cells/ml) were seeded into 96 well plates containing DMEM with 10% FBS Cells were allowed to attach overnight The cells were then washed and serum starved overnight Serum free medium was replaced with fresh DMEM containing 1µM bromodeoxy uridin (BRDU, Calbiochem), different concentrations of FGF-2, TGF-β, IL-1 (1-100 ng/ml) or atropine, pirenzepine (0.1-100 µM) They were incubated for 24hours at 37°C/5%CO2

2.2.8 Determination of protein concentration

Total protein concentration was determined using Coomassie plus protein assay reagent (Pierce) Briefly, when Coomassie binds to protein in an acidic medium, an immediate absorbance shift occurs from 465nm to 595nm with simultaneous color change of the reagent from green/brown (or red/brown) to blue

2.2.9 Conditioned medium(CM)

Culture medium (1ml) was carefully removed from the wells after 24hr incubation Total protein concentration was determined and if not used immediately for growth factor and matrix metalloproteinase analysis, they were stored at –70°C for less than 4 weeks

2.2.10 Cell lysate (CL)

Culture medium was removed from the adherent cells and 100-200 µl of M-Per mammalian protein extraction reagent (Pierce) was added to each well The plate was shaken gently for 5minutes and cell debris was removed by centrifugation at 13,000 rpm for 5-10min Clear supernatant was used to determine the protein concentration and if not used immediately for growth factor and matrix metalloproteinase analysis, they were stored at –

70°C for less than 4 weeks

2.2.11 BrdU ELISA; Cell proliferation assay

Scleral fibroblast cell proliferation was measured with the aid of BrdU SF were serum starved overnight before they were treated with growth factors or muscarinic agents BrdU was added to the fibroblast culture at the same time as the test agent, to be incorporated into newly synthesized DNA After 24 hours of incubation, culture medium was removed, cells were washed, fixed, permeabilised and the DNA was denatured to enable antibody binding to the incorporated BrdU A monoclonal antibody to BrdU was added into the wells (100µl) and incubated for 1hour at RT Unbound antibodies were washed away and horseradish peroxidase conjugated goat-anti mouse (1:2000) was added

A substrate solution (TMB) was added to each well, resulting in color change proportional

to the amount of incorporated BrdU in the cells The color reaction was stopped by stop solution and the optical density was determined using Spectrafluor Plus microplate reader (TECAN), set to 450nm/620nm

2.2.12 Zymography

Gelatinase activity was investigated using gelatin as a substrate Both CM and CL samples collected from scleral fibroblast cell culture were diluted with non-reducing sample buffer (0.5M Tris-HCl, Glycerol, 10% SDS, β-mercaptoethanol, 1% bromophenol blue), and loaded onto 10% tricine acrylamide gels containing gelatin Proteins were separated by electrophoresis (100 volt, 1hour) After electrophoresis, the gel was soaked in renaturation buffer (2.5%Triton-X) for 30 minutes with agitation The gel was rinsed and incubated in development buffer (50mmTris, 200mmNaCl, 5mm CaCl2, 0.02% Brij-35), at 37°C overnight Gels were stained (0.1% Coomassie blue R-250), and destained (40% methanol, 10% acetic acid), and photographed

Densitometry was performed on negative images of the zymogram using image analysis software (Labworks, UVP)

2.2.13 Immunoblot

Equal protein concentrations of samples were diluted in a sample buffer and heated (5min

in boiling water) and loaded onto 10% tris-HCl acrylamide gel (BioRad) Proteins were separated by electrophoresis (200 volt, 30min) and transferred to Immobilon-P (PVDF, Millipore) membrane using Trans Blot electrophoretic transfer cell After transfer, non-specific sites were blocked by blocking buffer (0.5% non-fat milk/PBST) The blot was

then incubated with primary antibody in blocking buffer for 1 hour with agitation at RT Blot was washed 2 times with PBS-T Secondary antibody conjugated with HRP (1:1000-1:5000) was added and incubated with agitation for 30 minutes Blot was washed and chemiluminiscent detection solution (Pierce) was added Membrane was wrapped in plastic and placed against X-ray film (Pierce) and exposed for an appropriate length of time (30 seconds-5 minutes) The film was developed according to the manufacturer’s recommendations

37°C for 60min inside a humidified chamber to allow the end-labelling reaction to occur The reaction was stopped and slides washed with PBS to remove unincorporated biotinylated nucleotides Endogenous peroxidase was blocked with 0.3% hydrogen peroxide (3min at RT) Streptavidin HRP (0.5mg/ml) solution was diluted 1:500 in PBS and added to each slide and incubated for 30min at RT Slides were washed with PBS DAB (diaminobenzidine) chromogen was added to each slide and allow to develop color (10min) Slides were rinsed several times in deionised water and mounted with aqueous medium (50% glycerol, 0.1% phenylendiamine dihydrochloride) and observed under light microscope

2.2.15 Protein Kinase Assay

Equal amounts (10 µg) of CL were added to 96 well coated with a pseudosubstrate (Calbiochem) and allowed the PKA (with cAMP) or PKC (with calcium and phosphatidyl serine) present in the sample to catalyse the phosphorylation of the pseudosubstrate Wells were washed with wash buffer and biotinylated antibody conjugate (Calbiochem), which binds to the phosphorylated substrate, was added and incubated for 60min at 25°C Wells are washed five times and a substrate solution added to each well Colors were allowed to develop for 5 min and stopped by adding stop solution The optical density was determined using Spectrafluor Plus microplate reader (TECAN) set to 450nm/620nm

2.2.16 MMP-2 activity assay

Equal amounts (200 µl) of CM samples were incubated with biotinylated gelatinase substrates for 30min at 37°C, allowing the MMP-2 present in the sample to cleave biotinylated substrates Uncleaved (remaining) substrates were then added to a biotin binding 96 well and detected with streptavidin enzyme complex Substrate solution was added, which results in color change The optical density was determined using a Spectrafluor Plus microplate reader (TECAN), set to 450nm/620nm Relative MMP-2 activity was calculated using MMP-2 positive and negative control

2.2.17 Immuocytochemistry

SF were grown on chamber slides Cells were washed with PBS and fixed with 3.7% paraformaldehyde/PBS for 10min at RT Cells were permeabilised with 0.5% Triton-X/PBS for briefly 2min at RT) Non-specific sites were blocked with 1% BSA/PBS for 15 min at RT Blocking buffer was removed and primary antibody (1µg/ml) was added and incubated 45min at RT Cells were washed three times with PBS and incubated with fluorescein labeled second antibody for 30min at RT Cells were washed with PBS Slides were covered with aqueous mounting medium and observed under fluorescence microscope (Olympus, BH2, Mercury burner, Fluorescent radiation 420 and above)

2.2.18 MMP-2 ELISA

Equal protein concentrations of CL were loaded to each well coated with anti-MMP-2 antibody Samples and standards were incubated for 2hr at RT and the wells washed Anti MMP-2 HRP-peroxidase was added to each wells and incubated for 1hr at RT The wells were washed and a substrate solution (TMB) added Colors were allowed to develop for 20min at RT The reaction was stopped by adding stop solution The optical density was determined using Spectrafluor Plus microplate reader (TECAN), set to 450nm/620nm

2.2.19 FGF-2 ELISA

FGF-2 content was determination by an ELISA Equal amount of Samples or standards were added to each well (anti-FGF-2 antibody precoated) and incubated for 2 hours at RT The wells were aspirated and washed with PBST Anti-FGF-2 mouse IgG conjugated with horse-radish peroxidase was added to the wells and incubated for 2 hours at RT Substrate solution was added to each well, resulting in color change proportional to the amount of FGF-2 present in the samples Samples were allowed to develop color for 20minutes at RT The reaction was stopped by adding stop solution and the optical density was determined within 30 minutes using Spectrafluor Plus microplate reader (TECAN), at dual wavelengths of 450nm/620nm

2.2.20 FGF-1 ELISA

FGF-1 was measured by an ELISA Equal amount of samples or standards were added to each well precoated with mouse monoclonal anti-FGF-1 antibody and incubated for 2 hours at RT The wells were aspirated and washed with PBST Anti FGF-1 rabbit IgG was added and incubated with 2 hour Unbound antibodies were washed off HRP conjugate that binds to rabbit IgG was added and incubated for 1 hour The wells were washed and substrate solution was added to each well, resulting in colour change proportional to the amount of FGF-2 present in the samples Samples were allowed to develop color for 20minutes at RT The reaction was stopped by adding Stop Solution and the optical density was determined within 30 minutes using Spectrafluor Plus microplate reader (TECAN), at dual wavelengths of 450nm/620nm

2.2 21 TGF-β1 ELISA

TGF-β1 ELISA kit (Amersham-Pharmacia) was used The kit detects the active form of TGF-β1 Therefore the samples were activated in acidic pH before ELISA was performed according to manufacturer’s instructions

Optical density was determined using Spectrafluor Plus microplate reader (TECAN), at dual wavelengths of 450nm/620nm

2.2.23 Sclera thickness

To measure sclera thickness, the posterior seclera was removed and dissected into three pieces per globe Tissue samples were fixed in 10% buffered formalin (ph7.4) at RT for 24 hrs Tissues were then processed through a series of ascending concentrations of ethanol and embedded in paraffin mould Tissues were sectioned at 20µm on a MT-910 microtome (Sowall), placed on clean glass slides and put into 30-40% ethanol to flatten Sections were transferred into a water bath at 60 °C for 10min and then into an oven at 60°C, for 30min

Paraffin was removed with xylen and samples were hydrated in a series of decreasing concentration of ethanol Slides were then stained with haematoxylin & eosin

Slides were mounted with Depex mounting medium (Fisher)

The thickness of the fibrous and carilaginous layer was measured using a calibrated eyepiece on the microscope with stage micrometer The average of three measurements was used to determine the thickness of the two layers for each globe

Images of samples and micrometers were taken at an equal magnification were taken and images were aligned

• Carbachol (muscarinic agonist) promoted SF cell growth

• Muscarinic receptor subtypes were found on human SF

• SF from myopic eyes demonstrate higher proliferation than SF from non myopic eyes

• Atropine increased FGF-2 release by SF

• Atropine decreased TGF-β1 release by SF

• SF from myopic eyes showed enhanced expression of MMP-2

• Atropine inhibited expression and activation of MMP-2

• FGF-2 decreased MMP-2 expression by SF

• TGF-β1 increased MMP-2 expression by SF

• SF from myopic eyes showed higher expression of EGF-R

• Atropine down regulated EGF-R expression

3.1 Atropine treatment reduced axial elongation

Experimental myopia was induced in chicks using hyperopic defocus by attaching a plastic lens (-15D) over the right eyes and left eyes were used as control Comparable degrees of myopia and globe enlargement were developed by imposed hyperopic defocus image All experimental (lens wearing) eyes examined in this study became myopic Fourteen days of lens wearing caused a significant increase in axial length, the difference in refractive error was significant (experimental: -11.5D±0.51, control: 4.40D±0.29, mean ±s.e, p<0.01, t-test for dependent samples, n=40, Fig1A) Increase in axial length between lens wearing eyes and control eyes was also significant (experimental: 9.78mm±0.08, control: 9.00mm±0.06, mean ±s.e, p<0.01, t-test for dependent samples, n=40, Fig1B)

In a separate study we corroborated the effect of subconjunctival atropine injection treatment to reduce axial elongation and refractive error Chicks were randomly allotted to

a group receiving atropine and another group receiving normal saline injection After two weeks of treatment, refractive errors of all chick eyes were measured by streak retinoscopy Axial length of enucleated globes was measured by a digital caliper Fourteen day atropine subconjunctival injection treatment significantly reduced myopia in lens wearing eyes Atropine was effective in reducing axial length (atropine treatment: 9.39mm± 0.11, myopic control: 9.69mm±0.07, p<0.01 t-test for independent samples, n=40 Fig2A) and refractive

error in the eye with experimental myopia (atropine treatment: -4.19D±1.5, myopic control: -12.03D±0.53, p<0.01, t-test for independent samples, n=40 Fig2B)

Atropine effect on structure

The chick sclera can be divided into two layers: a fibrous outer layer (FS) and a cartilaginous layer (CS) (Fig3.)

In light microscopic observations, the two layers showed opposite response to hyperopic defocus (Table 1) The posterior sclera was removed and dissected into three pieces and thickness of the two layer was measured The average three measurements was used to determine the thickness of sclera for each globe The cartilaginous layer becomes thicker (myopic: 68.3±1.78 µm, control: 54.3±2.83µm) during experimental myopia while the fibrous layer becomes thinner (myopic: 13.4±1.95 µm, control: 27.6±0.68 µm) When the thickness of the fibrous layer and cartilaginous layer were measured at the 14th day of atropine treatment It was found that atropine treatment increased the thickness of the fibrous layer of experimental (lens wearing) eye significantly when compared to control treatment (atropine: 24.2±1.28 µm, saline: 13.4±1.95 µm, p<0.01, t-test for independent samples, n=4) The increase was toward the non myopic control eye (atropine: 24.2±1.28

µm, control: 27.6±0.68 µm) In contrast, atropine decreased the thickness of cartilaginous layer when compared to control treatment (atropine: 58.7±0.42 µm, saline:68.3±1.78 µm, p<0.01, t-test for independent samples, n=4) The decrease was toward the non myopic control (atropine: 58.7±0.42 µm, control: 54.3 ±2.83 µm)