MUSCARINIC MECHANISMS IN A MOUSE MODEL OF MYOPIA VELUCHAMY A BARATHI (B.Vet. Sc.,) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF OPHTHALMOLOGY, FACULTY OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2005 Acknowledgements I would like to express my sincere gratitude to Professor Roger W. Beuerman for his supervision and guidance throughout this project. I am grateful for his comments and advice. I would like to thank all the staff of SERI especially Dr. Chi Luu, Jaime Chew, Jaimie Hoh, Sung Rhan and Dr. Li Jing, who assisted me in many ways. I would like to thank my husband for his love and support throughout my endeavours. ii TABLE OF CONTENTS I. Introduction……………………………………………………………… 1.1 Background……………………………………………………………… 1.1.1 Definition of Myopia…………………………………………………… . 1.1.2 Human Refractive Development…………………………………………. 1.1.2.1 Development of emmetropisation………………………………………… 1.1.2.2 Age of one to six………………………………………………………… 1.1.2.3 Age of six to adulthood…………………………………………………… 1.1.3 Epidemiology of Myopia……………………………………………… . 1.2 Experimental Myopia Research: Literature Review……………………… 1.2.1 Animal models of myopia………………………………………………… 1.2.2 Species difference………………………………………………………… 10 1.2.3 Induction of myopia………………………………………………………. 12 1.2.4 The differences between FDM and LIM in experimental myopia……… 14 1.2.5 Similarities between FDM and LIM…………………………………. 14 1.2.6 Clinical models of myopia……………………………………………… . 16 1.3 Sclera and its Role in Experimental Myopia…………………………… . 17 1.3.1 Retinal control of sclera growth………………………………………… 20 1.3.2 Scleral fibroblast control…………………………………………………. 21 1.3.2.1 Tyrosine kinases…………………………………………………………. 22 1.3.2.2 Muscarinic receptors……………………………………………………… 22 1.3.2.3 Muscarinic sites of action in the eye……………………………………… 26 1.4 Pharmacological Intervention…………………………………………… 30 1.4.1 Effect of atropine…………………………………………………………. 31 iii 1.4.2 Use of atropine and anti-muscarinic therapy for myopia retardation…… 32 1.4.3 Molecular basis of atropine and anti-muscarinic action………………… 33 1.4.4 Prospect for new therapeutic strategies…………………………………… 34 1.5 Summary and Aims………………………………………………………. II. Materials and Methods………………………………………………… 37 2.1 Experimental Myopia…………………………………………………… 37 2.1.1 Animals…………………………………………………………………… 37 2.1.2 Form-deprivation myopia (figure 2.2.1 A)……………………………… 37 2.1.3 Lens-induced myopia (figure 2.2.1 B)………………………………… . 37 2.2 Treatment Protocols…………………………………………………… . 38 2.3 Refraction and Axial length…………………………………………… . 41 2.4 Histological Studies………………………………………………………. 43 2.4.1 Paraffin embedding procedure………………………………………… . 43 2.4.2 Plastic embedding procedure…………………………………………… . 44 2.5 Immunohistochemistry………………………………………………… 45 2.5.1 Procedures on frozen section…………………………………………… . 45 2.6 Immunocytochemistry……………………………………………………. 45 2.6.1 Procedure…………………………………………………………………. 45 2.7 Total RNA Extraction…………………………………………………… 46 2.7.1 Procedure…………………………………………………………………. 46 2.7.2 RNA concentration measured by spectrophotometer…………………… 47 2.7.3 Agarose gel electrophoresis…………………………………………… . 48 2.8 First strand cDNA synthesis prior to PCR……………………………… 48 2.8.1 Procedure…………………………………………………………………. 48 2.8.2 cDNA concentration measured by spectrophotometer………………… 49 35 iv 2.9 Reverse Transcription Polymerase Chain Reaction (RT-PCR)………… . 49 2.9.1 Procedure…………………………………………………………………. 49 2.9.2 Agarose gel electrophoresis and photograph…………………………… 51 2.10 QIAEX II Gel Extraction……………………………………………… 52 2.10.1 Principle………………………………………………………………… 52 2.10.2 Procedure…………………………………………………………………. 52 2.11 TOPO TA Cloning……………………………………………………… 54 2.11.1 Purpose………………………………………………………………… 54 2.11.2 Procedure…………………………………………………………………. 54 2.11.3 One shot ® chemical transformation…………………………………… . 55 2.12.4 Colony selection and bacterial culture………………………………… 55 2.12 Plasmid Prep using QIAGEN kits………………………………………… 55 2.12.1 Procedure…………………………………………………………………. 55 2.13 Checking transformants………………………………………………… . 56 2.13.1 Restriction digests to check for insert (target) DNA……………………… 56 2.13.2 Sequencing of the insert DNA………………………………………… . 57 2.13.2.1 DNA sequencing method……………………………………………… . 57 2.14 Northern Blot…………………………………………………………… . 58 2.14.1 Principle………………………………………………………………… . 58 2.14.2 Agarose gel electrophoresis…………………………………………… . 58 2.14.3 Transfer of RNA to membrane………………………………………… 59 2.14.4 Preparation of labelled probe…………………………………………… 61 2.14.5 Monitoring incorporation using the rapid labelling assay……………… . 62 2.14.6 Hybridization and stringency washes for northern blots…………………. 63 2.14.7 Reprobing blots ………………………………………………………… 65 v 2.15 Real-Time Comparative PCR ……………………………………………. 66 2.15.1 Procedure…………………………………………………………………. 66 2.15.2 Data analysis by comparative CT (

CT) method …………………… . 67 2.16 Data Analysis …………………………………………………………… 67 2.17 Reagents………………………………………………………………… . 68 2.17.1 Components………………………………………………………………. 68 2.17.2 Buffers organize into northern blots etc………………………………… 70 III. Experimental Results… ……………………………………………… 72 3.1 Experimental Myopia ……………………………………………………. 73 3.1.1 Axial elongation …………………………………………………………. 73 3.1.2 Refractive error ………………………………………………………… . 83 3.1.3 Scleral thickness …………………………………………………………. 87 3.2 Effect of atropine sulphate (muscarinic antagonist) treatment ………… . 92 3.2.1 Atropine treatment reduced axial elongation ……………………………. 92 3.2.2 Atropine treatment increased scleral thickness ………………………… 96 3.3 Localization of Muscarinic Receptors (mAChR )………………… . 98 3.4 Gene Expression of Muscarinic Receptor Subtypes …………………… . 103 3.4.1 Expression of mAchR subtypes; M1-M5 on naive and sclera from mice with induced myopia……………………………………………………… 103 3.4.2 Expression of mAchR subtypes in atropine treated and normal saline treated sclera of mice that have undergone experimental myopia………. 103 3.5 Sequencing of Plasmid DNA with insert RT-PCR products ……………. 116 3.5.1 mAChR insert DNA for the five receptor subtypes were sequenced…… 116 3.6 Northern Blot Analysis ………………………………………………… . 119 3.7 Quantification of Real-Time PCR .………………………………………. 130 vi 3.7.1 Amplification plot………………………………………………………… 130 3.7.2 Comparative analysis by real-time PCR………………………………… 136 3.7.2.1 Gene expression of myopic and atropine treated myopic sclera compared with normal saline treated sclera … …………………………………… 136 3.7.2.2 Gene expression of Experimental Sclera compared with their contralateral control sclera ……………………………………………………… 136 3.7.2.3 Gene expression of all scleral samples compared with mouse brain…… 137 3.7.2.4 Gene expression of experimental and control sclera compared with naive sclera……………………………………………………………………… 138 IV. Discussion………………………………………………………………… 176 4.1 Mouse model of experimental myopia ………………………………… 176 4.1.1 Changes of axial length and experimental myopia……………………… 176 4.1.2 Effects of refractive development and ocular growth…………………… 177 4.1.3 Myopia development …………………………………………………… 178 4.2 Atropine and Experimental Myopia … …………………………………. 179 4.2.1 Atropine reduced axial length elongation ……….……………………… 180 4.2.2 Effect of atropine treatment on sclera ………………… 181 4.3 Scleral remodelling in myopic mouse eye …………………………… 183 4.3.1 Histological findings …………………………………………………… . 183 4.4 Muscarinic receptors in growth control………………………………… 184 4.4.1 Atropine treatment and expression of mAChR subtypes on mouse sclera…………………………………………………………………… 185 4.5 Clinical implication …………………………………………………… 187 4.6 Conclusions…………………………………….………………… . 189 4.6.1 Atropine effect and clinical trials ………………………………………… 189 vii 4.6.2 Clinical usefulness of atropine to retard myopia progression in humans 189 4.6.3 Muscarinic receptors effect, interaction and signal transduction……… . 190 4.7 Prospect for future studies … ………………………………………… . 190 V. References ………………… 192 Appendices ………………… 233 viii SUMMARY Myopia is an important cause of visual disability throughout the world (Takashima et al 2001, Midelfart et al 2002, Saw et al 2002, Saw et al 2005) both the prevalence and the incidence of myopia appears to be increasing. The demonstration that myopia can be induced in macaque monkeys by form deprivation (Wiesel 1977) was a milestone in our understanding of this complex disorder. Many other animal models have been developed since then. In this present study, we developed a new experimental model of form deprivation and lens-induced myopia model in the Balb/cJ mouse and it is suggested that this model can be used to develop a cell-molecular understanding of myopia in order to develop targeted therapies, an effort aided by the great assortment of cellmolecular tools available for mouse. In the case of chick, rabbit, marmoset and treeshrew only a few cell-molecular tools are available. We have determined the effect of the treatment of atropine during the development of mouse myopia and also investigated the expression of muscarinic receptor sub-types from the normal and experimentally induced myopic mouse scleral fibroblasts. It is my hypothesis that atropine, a general muscarinic antagonist for all five known subtypes of the muscarinic acetylcholine receptors, effectively reduced axial elongation (3.58 ± 0.05 mm) in the contact lens covered eye compared with nonatropine treated lens-covered eye (3.81 ± 0.06 mm). Axial length of lens treated eyes was 111% of their controls and 103% of controls following atropine treatment. We have positively confirmed that atropine an antagonist of muscarinic receptor effectively reduced axial elongation in lens wearing eyes. Atropine did not have any ix significant effect on axial length of control eye, which received clear visual input. The major morphological changes in myopic sclera are decreased thickness at the equator as well as at the posterior pole when compared to control sclera. Genes for muscarinic receptor subtypes; M1-M5 were found in naive, control and experimental mouse sclera fibroblasts by immuno-histochemistry, RT-PCR and northern blot analysis. All specific M1-M5 primers were sequenced and blasted with NCBI gene data bank. DNA sequencing was matched with mouse muscarinic receptor genome data for all M1-M5. Real-time PCR showed that message levels for M1, M3 and M4 were up regulated in atropine treated myopic sclera, but M2 and M5 showed little change. This finding is helpful for our understanding of the specific roles of muscarinic receptor subtypes in the mouse sclera during the myopia development. This study confirmed that the muscarinic receptor sub types; M1-M5 are present in mouse sclera and that long term application of atropine could lead to new, differential levels of expression of message. The results suggest that atropine may act on one or more mAChRs to differentially regulate expression levels of specific receptors and that modulation of the scleral matrix may be controlled by scleral fibroblast involvement of mAChRs. 87 3.1.3 Scleral Thickness: Scleral thickness increased from anterior to posterior in naive and control eyes. In contrast, the suture (Figure 9A, 9B, 9C) and lens induced myopic eyes (Figure 10A, 10B) sclera was thinner at equator and posterior sites. In naive eyes, scleral thickness increased from anterior (16.9 ± 0.10
m, mean ± s.d.), equator (41.8 ± 0.12
m) to posterior (91.7 ± 0.16
m). In eyes that underwent lid closure, the sclera was similar in thickness anteriorly (16.7 ± 0.05
m); however, the sclera was thinner at the equator (33.3 ± 0.10
m) and posterior sites (58.33 ± 0.08
m) when compared to control eyes and also similar in lens induced eyes (all at a level of p < 0.001, n = 24 eyes in lid-sutured group, n = 10 eyes in lens-induced group and n = eyes in naive group, Figure 11 and Table 4). The anterior and posterior segment was measured with histology photos using microscope eye piece calibrated with stage micrometer as well as by magnified video image using trans-illuminated globes, accuracy ±
m. The values (Table 5) represented that there was axial growth in the posterior segment and not in anterior. In lid sutured, there was slight change in the anterior chamber; this could have been due to slight pressure by the suture on the eyelid. 88 Table 4. Scleral Thickness in Lid-Suture and Lens Induced Model (
m) Dimensions (
m) Anterior Sclera LidSutured 16.7 ± 0.05 Control Control Naive 16.7 ± 0.02 LensInduced 16.7 ± 0.05 16.9 ± 0.04 16.9 ± 0.10 Age (weeks) Equator Sclera 33.3 ± 0.10 41.7 ± 0.07 30.8 ± 0.11 42.5 ± 0.08 41.8 ± 0.12 Posterior Sclera 58.3 ±0.08 54.6 ±0.06 91.6 ± 0.12 91.7 ± 0.16 91.2 ± 0.09 Values are represented as mean ± s.e, significance at p < 0.05, n = at each time point Table 5. Anterior and Posterior Segment Measurements in Both Models (mm) Dimensions (mm) Anterior Segment LidSutured 0.27 ± 0.98 Posterior Segment Control Control Naive 0.36 ± 1.03 LensInduced 0.34 ± 0.54 0.37 ± 0.95 0.35 ± 0.50 2.57 ± 0.92 2.14 ± 1.01 2.62 ± 0.85 2.17±0.93 2.01±1.03 Values are represented as mean ± s.e, significance at p < 0.05, n = 50 at each time point 89 9A 100
m 9B 100
m 9C 100
m Figure 9. Photomicrograph of anterior, equator and posterior scleral thickness of Suture (A), control (B) and normal sclera (C), (n=6), 63X original magnification. The sutured eye equator and posterior sclera was thinner as compared with contra-lateral control and normal sclera. 90 10A . 100
m 10B 100
m Figure 10. Photomicrograph of anterior, equator and posterior scleral thickness of lens induced (10A) and control eyes (10B, n=6), 63X original magnification. The experimental eye equator and posterior sclera was thinner as compared with contralateral control sclera. 91 100 Ante rior Scle 90 Equa tor Scle Thickness (mm) 80 Poste rior Scle 70 60 50 40 30 20 10 Normal LI Cont (LI) LS Cont (LS) Figure 11. Scleral thickness of anterior, equator and posterior was plotted for suture (LS, n = 12), control (cont LS, n = 12), lens induced (LI, n = 5), control (cont LI, n = 5) and normal eyes (n = 6). Scleral thickness increased from anterior to posterior in normal and control eyes. In sutured and lens-induced eyes the sclera was thinner at equator and posterior sites. Data was represented as mean ± s.e. 92 3.2 Effect of Atropine sulphate (Muscarinic Antagonist) Treatment 3.2.1 Atropine treatment reduced axial elongation Mice at four weeks of age, were randomly allocated to one of two groups, an Atropine treatment group (ATG, n=50) received sub-conjunctival injections of 10
l, 100
M atropine and the other group (n=50) received similar injections of 10
l, 0.9% sterile normal saline (NSG). Contact lenses were attached unilaterally as before and the atropine treatment began at the same time and continued for four weeks. To avoid complications of the systemic effects of atropine, both eyes of each mouse were given the same injections in this experiment. Following four weeks of treatment, refractive errors for all mice were measured by streak retinoscopy. It was demonstrated in this experiment that the effect of sub-conjunctival atropine injection treatment reduced axial elongation and refractive error. Axial length of enucleated globes was measured by calibrated digital calliper as well as by magnified video imaging using trans illumination. Four weeks of atropine sub-conjunctival injection treatment significantly reduced myopia in lens wearing eyes. Following atropine treatment (ATG) the axial length of myopic eyes was 3.58 ± 0.05 mm, for the control without lenses the axial length was 3.44 ± 0.05 mm (p < 0.05 t-test for independent samples, n = 50). In contrast, animals with induced myopia that received normal saline (NSG) axial length was 3.81 ± 0.06 mm and axial length of control eyes was 3.47 ± 0.07 mm (p < 0.05 t-test for independent samples, n = 50, Figure 12 and Table 6). The refractive errors of atropine treated myopic eyes were 1.5D ± 0.06, and for control eyes it was 6.25D ± 0.07, p < 0.05; whereas normal saline receiving eyes refractive error were -2.75D ± 0.05 and for control eyes it was 6D ± 0.08, p < 0.05 (Figure 13). 93 Table 6. Axial length and Refractive error measurements from atropine treated and normal saline treated mice Dimensions Ex (Atr) Con (Atr) Ex (NS) Con (NS) Age (weeks) Axial Length 3.58 ± 0.05 3.44 ± 0.05 3.81 ± 0.06 3.47 ± 0.07 (mm) Refractive Error ± 0.08 1.5 ± 0.06 6.25 ± 0.07 -2.75 ± 0.05 (diopters) Values are represented as mean ± s.e, n=50 at each time point, Ex (atr) means atropine treated myopic eye, Con (Atr) means atropine treated control eye, Ex (NS) means normal saline treated myopic eye, Con (NS) means normal saline treated control eye, significance at p < 0.05 94 4.1 3.8 Axial Length (mm) 3.5 3.2 2.9 2.6 2.3 2.0 Atropine treated Experimental Atropine treated Control Normal Saline treated Experimental Normal Saline treated Control Figure 12. This graph represents the axial length (mm) of the atropine treated group and normal saline treated group. The axial length of the globes was determined at the conclusion of the experiment using calibrated digital calliper (accuracy ± 0.01 mm) and by video imaging using trans-illumination (accuracy ± microns). Atropine (muscarinic antagonist) treated myopic eye axial length was almost close when compared to control axial length. There was no effect on sub-conjunctival injection of normal saline treated myopic eye. Data was represented as mean ± s.e. 95 8.0 Refractive Error (Diopters) 6.0 4.0 2.0 0.0 Atropine treated Experimental Atropine treated Control -2.0 Normal Saline treated Experimental Normal Saline treated Control -4.0 Figure 13. Refraction was determined two months after lid wearing. Lens-induced myopia caused elongation of the globe and reduced hyperopia. In this experiment, one group was treated with sub-conjunctival injection of atropine sulphate and another group was treated with normal saline for four weeks. At 8th week, refractive error was measured by streak retinoscopy without cycloplegia, see text for more detail. The myopic eye was treated with atropine sulphate and it was shifted from myopic to hyperopic (1.5D ± 0.06) when compared to saline treated myopic eye was till at myopic shift (-2.75D ± 0.05). There was no significant difference seen in the control eyes. Data was represented as mean ± s.e. 96 3.2.2 Atropine treatment increased scleral thickness As shown in Table 7, scleral thickness increased from anterior to posterior in both atropine treated myopic (Figure 14A) and atropine treated control eyes (Figure 14B). In the normal saline treated myopic eyes, the sclera was thinner at equator and posteriorly (Figure 15A); however, this was reversed in the contra-lateral control eyes (Figure 15B). Table 7. Atropine and saline treated group mice scleral thickness Dimensions (
m ) Anterior Sclera Ex (Atr) 16.7 ± 0.01 Con (Atr) 16.7 ± 0.02 Ex (NS) 16.4 ± 0.02 Con (NS) 16.9 ± 0.01 Equator Sclera 42.3 ± 0.01 41.7 ± 0.01 32.6 ± 0.04 43.5 ± 0.03 Posterior Sclera 90.5 ±0.05 91.2 ± 0.03 55.2 ±0.05 90.9 ± 0.01 Age (weeks) Ex (Atr) means atropine treated myopic eye, Con (Atr) means atropine treated control eye, Ex (NS) means normal saline treated myopic eye, Con (NS) means normal saline treated control eye Values are represented as mean ± s.e, significance at p < 0.05, n=6 at each time point 97 14 A 14 B 100
m 100
m Figure 14. Photomicrograph of anterior, equator and posterior scleral thickness of atropine treated myopic (A) and atropine control eyes (B, n=6), 40X original magnification. Atropine sulphate was increased the scleral thickness. 15A 100
m 15 B 100
m Figure 15. Photomicrograph of anterior, equator and posterior scleral thickness of normal saline treated myopic (A) and control eyes (B, n=6), 40X original magnification. There was no effect of normal saline on the scleral thickness of myopic sclera. 98 3.3 Localization of Muscarinic Receptor (mAChR) sub-types Immunocytochemistry is the demonstration of specific antigens in tissue section, smears or cells by the use of antibodies (antibody-antigen) interactions, which are visualized by the attachment of a marker that can be seen under the microscope to the antigen. The visual marker may be a fluorescent dye, colloidal metal, hapten, radioactive marker or more commonly for light microscopy an enzyme. Ideally, maximal signal strength along with minimal background or non-specific staining is required to give optimal antigen localization. Fluorescein (mistakenly abbreviated by its commonly-used reactive isothiocyanate form, FITC) is a small organic molecule, and is typically conjugated to proteins via primary amines (i.e.lysines). Usually, between and FITC molecules are conjugated to each antibody; higher conjugations can result in solubility problems as well as internal quenching (and reduced brightness). Thus, an antibody will usually be conjugated in several parallel reactions to different amounts of FITC, and the resulting reagents will be compared for brightness (and background stickiness) to choose the optimal conjugation ratio. Fluorescein is typically excited by the 488 nm line of an argon laser, and emission is collected at 530 nm. To determine the presence of mAChR subtypes in the mouse scleral fibroblasts, mouse scleral fibroblasts were cultured on slides. The scleral fibroblasts and scleral tissues were stained using anti mouse m1-m5 receptor rabbit IgG-fluorescein conjugates. Positive immunostaining of mAChR subtypes were identified in the mouse scleral fibroblasts and scleral tissues. With immunoreactivity to M1, M2, M3, M4 and M5 mAChRs were seen in the control (Figure 16B-F respectively) and experimental (Figure 17B-F respectively) scleral fibroblast cells and in naive scleral tissues (Figure 18B-F respectively). Immunostaining was often associated with the 99 plasma membrane. Muscarinic receptors are members of the G-protein coupled super family of receptors, with an expected presence on the cell membrane. No immunostaining was observed when primary antibodies were omitted in control (Figure16A), experimental (Figure17A) cells and naive tissues (Figure18A) or when cells were incubated with only secondary antibody (negative control). 100 Figure 16. Immunocytochemistry of mAChR subtypes in control group mouse SF in culture. Subtype selective antibodies bound to cultured SF demonstrates the presence of M1 (B), M2 (C), M3 (D), M4 (E) and M5 (F) receptors (shown in green). When secondary FITC labelled antibody was used without the primary antibody no significant binding was observed (A, negative control). All experiments were done in triplicates. 101 P2-3 Scleral Fibroblasts from Experimental Eye A B C D E F Figure 17. Immunocytochemistry of mAChR subtypes in experimental group mouse SF in culture. Subtype selective antibodies bound to cultured SF demonstrates the presence of M1 (B), M2 (C), M3 (D), M4 (E) and M5 (F) receptors (shown in green). When secondary FITC labelled antibody was used without the primary antibody no significant binding was observed (A, negative control). All experiments were done in triplicates. 102 Figure 18. Immunohistochemistry of mAChR subtypes from naive group mouse scleral tissue. Subtype selective antibodies bound to cultured SF demonstrates the presence of M1 (B), M2 (C), M3 (D), M4 (E) and M5 (F) receptors (shown in green and nucleus shows in blue; stains with DAPI). When secondary FITC labelled antibody was used without the primary antibody no significant binding was observed (A, negative control). All experiments were done in triplicates. [...]... (Hodos and Kuenzel 19 84, 13 Wallman and Adams 19 87, Wallman et al 19 78) Naturally occurring FDM has been found in cases of unilateral haemangioma of one eye (Robb 19 77), ptosis (O’Leary and Millodot 19 79), uniocular anomalies affecting vision (Rabin et al 19 81) and unilateral congenital cataract (Johnson et al 19 82) Meyer et al (19 99) reported that patients with an early onset of phlyctenular keratitis... of animal models of myopia; this dramatic finding stimulated a large number of studies using animal models for myopia Many different experimental animal models [tree shrew (Sherman et al 19 77, Mckanna and Casagrande 19 78, Seigwart and Norton 19 93, McBrien and Norton 19 92), chick (Wallman et al 19 78, Osol et al 19 86, Wallman and Adams 19 87), grey squirrel (McBrien et al 19 93b), marmoset (Troilo et al... 49 years of age (Saw et al 2004) The most comprehensive data come from Singaporean where young males conscripted into the military have shown increases in the prevalence of myopia from 20–30% in the 19 60s to 80–90% in 19 90s (Seet et al 20 01) There is a general pattern of earlier onset, increasing prevalence and increasing severity of myopia, making myopia a major health problem in much of East Asia The... clinical observations have indicated that a condition of image deprivation myopia occurring in human is similar to FDM in animal models Birth injuries of the cornea, often unilateral have been associated with subsequent myopia (Lloyd 19 38) Myopia has been observed in patients with corneal opacities (Muramatsu 19 82) Premature infants with or without retinopathy of prematurity (ROP) may develop abnormal... prevalence of myopia and high myopia in the rural populations of India were 26.99% and 3. 71% , respectively (Raju et al 2004) In Australia, the Victoria Visual Impairment Project found that the prevalence of under corrected refractive error in 4735 participants age 40 years and older was 10 % (Liou et al 19 99) Data from Proyecto VER study in Arizona indicated that Mexican Americans aged 40 years and... incidence of myopia after 40 years (Wang et al 19 94) Newborns are usually 3 hyperopic Axial length increases along with thinning of the lens and flattening of the cornea, leading to emmetropia in children by ages 8 -10 years (Hosaka 19 88) The eye enlarges during this time and the image of a distant object continues to be in focus on the retina Myopia may be a breakdown in this regulatory mechanism Both... 19 99) decreased incorporation of precursors into glycosaminoglycans (GAGs McBrien and Lawlor 19 95), decreased GAG content (Norton and Rada 19 95), and increased levels of active gelatinase A (an enzyme involved in collagen degradation Guggenheim and McBrien 19 96) These findings imply that in the tree shrew, induced myopia is associated with remodelling of the sclera and a net loss of tissue at the posterior... (Takashima et al 20 01, Midelfart et al 2002, Tan 2002, Saw et al 2002, Saw et al 2005) both the prevalence (the number of cases that exists at a particular time in a defined population) and the incidence (frequency of occurrence) of myopia, as well as the severity, appears to be increasing (Lin et al 20 01, 2004, Saw et al 2002) Even a low degree of myopia has a significant deleterious effect on visual... During the next 8 years, an average eye will grow only an additional 1mm; however, in the western population the prevalence of myopia will increase more than seven fold to 15 % by age of 15 years (Mutti et al 19 96) Myopia develops between 6 and 14 years Thereafter, the prevalence of myopia remains relatively constant between the ages of 12 and 54 (Sperduto et al 19 83) There is also a decreasing incidence... impaired vision, refractive errors and strabismus Numerous studies on refraction in preterm infants have shown a predisposition to childhood myopia (Birge 19 56, Kalina 19 69, Fledelius 19 76, Shapiro et al 19 80, Kushner 19 82, Gallo et al 19 91, Fledelius 19 93) Johnson et al (19 82) have described an axial myopia in one eye, which had a posterior sub-capsular cataract in one sibling of a pair of identical . 30 1. 4 .1 Effect of atropine…………………………………………………………. 31 iii 1. 4.2 Use of atropine and anti -muscarinic therapy for myopia retardation…… 32 1. 4.3 Molecular basis of atropine and anti -muscarinic. number of cases that exists at a particular time in a defined population) and the incidence (frequency of occurrence) of myopia, as well as the severity, appears to be increasing (Lin et al 20 01, . values…………………………………………………… 15 0 11 C Comparative quantification of S, MS, AMS, AMCS, NSMS and NSMCS M3 to mouse brain C T values…………………………………………………… 15 1 11 D Comparative quantification of S, MS, AMS, AMCS,