Myopia or nearsightedness is a condition in which the axial length of the eye is too long relative to its optical focal length. This condition is reaching epidemic levels worldwide, and has become a tremendous public health burden. Myopia is one of the leading causes of vision loss and high myopia significantly increases the risk of permanent blindness. Consequently, myopia cannot be considered as a benign condition and early interventions aimed at slowing down or even stopping the progression of myopia rather than merely correcting the associated optical focusing error is of great importance.

The consistent evidence from animal model studies showing that imposed hyperopic defocus, if sustained, comprises an effective myopogenic stimulus, accelerating eye growth, while imposed myopic defocus slows ocular elongation, has motivated clinical studies using bifocal and progressive addition spectacles for myopia control. While these studies, reviewed in Chapter 2 of this dissertation, failed to provide convincing evidence for a clinically significant treatment effect, i.e., slowed myopia progression; smaller scale, non-randomized studies using multifocal soft contact lenses and orthokeratology lenses have shown much more promising effects in terms of slowing myopia progression. However, the mechanism(s) underlying the latter anti-myopia treatment effects are poorly understood, thereby limiting their further refinement. In addition to the aforementioned systematic review of relevant clinical evidence for optical interventions for myopia control, this dissertation described 3 other studies using chick model, representing efforts to further understand how multifocal optical environments affect normal ocular development, specifically the process of emmetropization. These manipulations have also been used as a tool to investigate the mechanism underlying the myopia controlling effect of the novel contact lens applications referred above.

Chapter 2 describes a systematic review and meta-analysis performed on randomized controlled trials investigating the effects of three traditional optical interventions, bifocal and progressive addition spectacles, and rigid contact lenses, all believed to control myopia progression, based on anecdotal evidence. The overall treatment effect was estimated to be only small and clinically insignificant. A number of reasons were proposed to explain the discrepancy between the results of related animal studies and these clinical trials - strong support for optical control of myopia from the former studies and inconclusive results from the latter studies. Poor compliance to the spectacle corrections in clinical studies, inaccurate measurement of adherence to the treatments, inadequate and potentially, ambiguous classification systems for clinical myopia, and potential flaws in the optical designs are likely to have contributed to the discouraging results from the clinical trials.

Chapter 3 describes the first of three studies in which the chick model was used to test the effects on ocular growth of a series of custom-designed 2-zone multifocal "spectacle" lenses. This and the two follow-up studies using this model have the following merits; they made use of standardized experimental paradigms and objective methods of measurement to track ocular changes. The first study applied the lenses in a simple experimental paradigm, in which monocular lenses were attached to the normal eyes of young chickens. The 2-zone lenses included a plano zone, either in the center or peripheral surround, with either positive or negative power (+5 or -5 D) incorporated into the other zone. The size of the central zone was also allowed to vary, to control the size of the unifocal central and peripheral retinal areas, and intermediate multifocal zone, as was the placement of the powered zone, i.e. in the center or periphery of the lenses. Single vision lenses were included as a control treatment. Two important observations from this study were that 1) peripheral optical defocus can influence both peripheral (off-axis) and central (on-axis) refractive error development and 2) the inhibitory effect on axial ocular growth of myopic defocus imposed using 2-zone lenses (positive zones) could exceed that induced by single vision lenses of the same power. These results suggested complex interactions between adjacent retinal regions, with the peripheral retina apparently able to decode optical defocus, as well as complex interactions between the eyes own optical aberrations and those of the 2-zone lenses, which introduced large amounts of spherical aberration.

Chapter 4 described a closely related study in which subsets of the same 2-zone lenses were tested on eyes that had undergone surgical manipulations (sectioning of the ciliary nerve, CNX or iridectomy, ID) to investigate the influence of the pupil size of the eye as well as accommodation on the effects of the 2-zone lenses. Both were uncontrolled in the first study; yet create a dynamic optical system on which the multifocal optical environment was imposed. The ID surgery produced a fixed dilated pupil without any effect on accommodation (confirmed in another study, reported in Appendix I), while the CNX surgery produced a similarly enlarged pupil while also eliminating accommodation. This study revealed pupil size to be a critical factor in the treatment effects of 2-zone lenses, likely to reflect at least in part, its influence on the optical experience of various retinal zones (center to periphery), and also suggested a significant role of accommodation in the decoding of imposed optical defocus stimuli, in this case, complex multifocal optical stimuli.

Chapter 5 described a third study which attempted to develop a clinically more relevant scenario; specifically, 2-zone lenses that incorporated two different negative powers (-5 & -10 D) in two optical zones (center & periphery or vice versa), were tested on both normal eyes and eyes made myopic before being fitted with one of the 2-zone lenses. The latter combination was intended to simulate the ocular conditions created when concentric multifocal contact lenses, i.e. with a near addition, are prescribed to human myopes, one of the novel, myopia control treatments currently being explored. When the 2-zone lenses were fitted to normal eyes, they induced myopia, of a magnitude falling between the values expected, had single vision lenses of the same powers been used. However, on myopic eyes, the lenses had a strong myopia inhibiting effect with the already induced myopia undergoing substantial regression. This result supports the further investigation of appropriately designed concentric multifocal contact lenses for the control of myopia progression.

In summary, the studies reported in this dissertation indicate complex interactions between central and peripheral retinal regions in decoding and responding to complex defocus signals as well as critical influences of pupil size and accommodation on these processes. The strong and consistent inhibitory effects on ocular growth of concentric 2-zone lenses incorporating a zone of either positive power or a near addition, 2-zone designs lend plausibility to the notion of using custom-designed novel optical treatments for the control of myopia progression.

Myopia prevalence has steadily climbed worldwide in recent decades with the most dramatic impact in East Asian countries. Treatments such as eyeglasses, contact lenses, and laser surgery for the refractive error are widely available, but none cures the underlying cause. In progressive high myopia, invasive surgical procedures using a scleral buckle for mechanical support are performed since the patient is at risk of becoming blind. The treatment outcome is highly dependent on the surgeon's skills and the patient's myopia progression rate, with limited choices in buckling materials. This dissertation, in four main studies, represents efforts made to control high myopia progression through the exploration and development of biomaterials that influence scleral growth.

First, mRNA expression levels of the chick scleral matrix metalloproteinases, tissue-inhibitor of matrix metalloproteinases, and transforming growth factor-beta 2 were assessed for temporal and defocus power effects. The first study elucidated the roles that these factors play in scleral growth regulation and suggested potential motifs that can be incorporated in future biomaterials design. Second, poly(vinyl-pyrrolidone) as injectable gels and poly(2-hydroxyethyl methacrylate) as solid strips were implanted in chicks to demonstrate the concept of posterior pole scleral reinforcements. This second study found that placing appropriate biomaterials at the posterior pole of the eye could directly influence scleral remodeling by interacting with the host cells. Both studies advanced the idea that scleral tissue remodeling could be potentially controlled by well-designed biomaterials.

These findings led to the exploration of biomimetic hydrogels comprising enzymatically-degradable semi-interpenetrating polymer networks (edsIPNs) to determine their biocompatibility and effects on the chick posterior eye wall. This third study demonstrated the feasibility of stimulating scleral growth by applying biomimetic injectable materials. Fourth, the muscarinic antagonist drug, atropine, was encapsulated within the edsIPNs and delivered to the chick eye posterior pole to evaluate the local effect of atropine release. This fourth study offered an alternative method of ocular drug delivery for treatment of myopia, with the potential to elucidate the actual location of the inhibitive effect of atropine on myopia progression. In summary, this dissertation contributes to the design and use of biomaterials specific to myopia therapy and adds novel insights to scleral tissue engineering.

Myopia, or nearsightedness, is a common refractive error characterized by an abnormally large increase in eye elongation that leads to a mismatch between the eye's refracting power and the location of the retina. Besides causing blurry vision when left uncorrected, myopia carries a significant risk of vision-threatening complications such as glaucoma, retinal detachment, myopic

maculopathy, and choroidal neovascularization. Billions of dollars are spent worldwide on the correction of myopia and treatment of its associated complications. In addition to the threat it poses

to the ocular health of working-age populations, the steady climb in myopia prevalence reported in most countries adds urgency to the need for innovative and reliable myopia-control therapies.

Taking inspiration from the concept behind scleral buckling surgery - in which a strip of cadaver sclera is used to brace the weakened posterior pole of the eye - we sought to develop a hydrogel-based

therapy with the intended action of slowing or preventing subsequent excessive elongation, by way of rehabilitating the myopic sclera in the early stages of the disease.

Chapter 2 describes the development of a guinea pig myopia model, for use in testing our experimental myopia-control therapy. Guinea pigs have emerged as a popular mammalian myopia model because of their ease of housing and husbandry, relatively large eyes, and better visual acuity than other laboratory rodents (mice and rats). After establishing a breeding colony using breeders donated from a myopia lab at the University of Auckland, we successfully induced myopia in guinea pigs using a form deprivation model. In the course of our studies, we also tried to induce myopia using defocusing lens and form deprivation in guinea pigs from a commercial research supplier. To our surprise, those animals proved resistant to any kind of myopia-inducing stimuli. The research

implications of such potential strain-related variability in responses are discussed, including the need to exercise due caution in comparing results from different myopia research laboratories.

In Chapter 3, we detail the optimization and in vitro characterization of a hyaluronic acid (HyA) hydrogel investigated as a myopia-control therapy. Using parallel plate rheology we determined the modulus of hydrogels of two different monomer contents, 2.0 and 3.3 weight percent by volume. Both were much softer than the native sclera, i.e. 200 and 800 Pa compared to ~2 MPa. Through

cytotoxicity and proliferation assays we found that both softer and stiffer hydrogels were biocompatible with guinea pig scleral fibroblasts, although proliferation rates were higher for cells

cultured on the tissue culture polystyrene control surface. Interestingly, cell proliferation proved to be independent of the concentration of the bsp-RGD(15) cell-binding peptide included in the hydrogel. This suggests that scleral fibroblasts were able to engage with other binding sites on the scaffold, perhaps hyaluronan receptor CD44. Likewise, cells were observed to migrate through hydrogels with no bsp-RGD(15) peptide in a novel migration assay in which cells were allowed to first form a stable monolayer before being exposed to the hydrogels.

Chapter 4 presents the results of in vivo testing of 200 Pa HyA hydrogel containing 380 μM of cell binding

peptide in our established guinea pig myopia model. After a 7-day period of myopia induction through form deprivation, guinea pigs received a posterior sub-Tenon's capsule injection of either HyA hydrogels or the triethanolamine buffer used to prepare the hydrogel (sham treatment). After an additional three weeks of form deprivation, both groups of treated guinea pigs (hydrogel and sham) exhibited a significant amount of myopia control, as indicated by normalization of the axial lengths of treated eyes relative to their contralateral (fellow) eyes. In fact, the treatment effects were not significantly different from each other. The myopia control effect is tentatively attributed to thickening of Tenon's capsule at the surgical site, with the secondary effect of enhancing the mechanical stability of the posterior sclera. The changes in Tenon's capsule could be

the product of a wound healing response triggered by the surgical manipulation. Functional measurements (flash electroretinograms and visual acuity) revealed no evidence of adverse effects of

the treatments. Intraocular pressure was also unaffected by them. In discussing the findings of this study, two possible novel myopia-control therapies are proposed for further investigation: an HyA

hydrogel-based implant customized to also deliver an anti-myopia drug for additional control, and a sub-Tenon's capsule surgical manipulation, of the type used for sham injections of buffer.

The eye is an incredibly complex organ, composed of a wide spectrum of tissues. This ranges from the vascular (e.g. choroid) to the extracellular matrix (ECM) rich (e.g. cornea) to the neural (e.g. retina). Working together in concert, these tissues coordinate a wide variety of disparate activities, with exquisite temporal, spatial and functional precision, in order to facilitate vision. Given this high degree of complexity and variability across its components, the eye in many ways approximates the body in miniature. And just like the body at large, ocular tissues utilize the full complement of steroid hormones to facilitate these activities; both in the normal and under pathological conditions.

The impact of steroid hormones has been particularly well studied in the context of (1) sexually dimorphic processes and structures and (2) tissue growth and remodeling of ECM rich tissues (e.g. solid tumors and skin), as they appear exquisitely sensitive to steroid hormone signaling to drive their growth. Interestingly, myopia, an ocular pathology that is one the leading drivers of blindness worldwide, is characterized by (1) excessive ECM tissue remodeling of the sclera, leading to scleral thinning and ocular elongation (often, with greatest effect, during childhood and/or adolescence), and (2) its sexually dimorphic character; on the average it presents both more commonly and with greater phenotypic severity in females. Despite these characteristics, the affect and regulation of steroid hormones and their receptors on myopia progression, and with particular emphasis on the sclera, has received sparingly little study.

In this dissertation we work to remedy this. Here we explore the changes that occur in steroid hormones and their cognate receptors, both systemically and locally (i.e. at the level of the sclera), as myopia progresses, in a well validated form-deprivation, chicken based model of the pathology.

Leveraging a host of biometric (i.e. high frequency a-scan ultrasonography and retinoscopy), biochemical (i.e. western blotting), analytical chemistry (tandem mass spectrometry) and molecular biology (i.e. quantitative real-time polymerase chain reaction) tools we discover a strong associations between glucocorticoid metabolism in the sclera and myopia progression. More explicitly we observe lower scleral levels of the major glucocorticoid species in chicken,

corticosterone, concomitant with a drop in transcripts of its biosynthetic enzyme (HSD11β1) and myopia progression, irrespective of sex (chapter 3).

We also find several sex-specific disparities including: higher degrees of myopia in female subjects (Chapter 2), differences in the transcriptional expression levels of several sex hormone receptors (Chapter 4) and elevated progesterone levels in female form deprived sclera (Chapter 3). In aggregate our findings argue for a possible role of steroid hormones regulating ocular growth and tissue remodeling during myopia progression, both in manners that are independent from and dependent upon subject’s sex.

Myopia (near-sightedness) is one of the most common ocular disorders in humans. Due to the dramatic increases in prevalence of myopia worldwide, especially in children and young adults, myopia has also become a significant public health problem, both socially and economically. While the prevalence and severity of myopia continue to increase, effective therapeutic interventions for myopia remain limited. Currently, management of myopia is largely limited to traditional optical corrections - spectacles, contact lenses, and refractive surgery - which correct distance vision but have no effect on myopia progression. While slowed myopia progression has been reported in clinical studies using the contact lens-based, corneal reshaping therapy and atropine, a pharmaceutical agent, these approaches come with limitations and in the latter case, significant ocular side-effects. Uncontrolled progression may lead to high "degenerative" myopia, for which posterior scleral reinforcement surgery remains the only treatment option and a last resort directed at preserving vision. Thus there is a clear need for new myopia control treatments. Understanding more about the molecular and cellular mechanisms underlying myopic eye growth has the potential to uncover novel treatment options.

This dissertation presents results from three investigations into the role of the retinal pigment epithelium (RPE) in eye growth regulation, focusing on molecular and cellular mechanisms, and using both in vivo animal models and in vitro cell culture models. In the first in vivo study (Chapter 2), we investigated expression of candidate genes in chick RPE of imposing short-term optical defocus. Specifically, gene expression levels of the three bone morphogenetic proteins (BMP-2, 4 & 7) were examined after 2 and 48 h of treatment, with negative and positive lenses used to impose defocus of opposite sign. These growth factors were observed to be differentially and bidirectionally expressed in RPE, expression generally increasing with imposed myopia, which is associated with ocular growth inhibition. Because eyes had little chance to change their dimensions with such short-term lens treatments, these genes are assumed to play important roles in the onset and early phase of defocus-induced ocular growth changes. For this reason, these genes represent potential targets for molecular-based myopia treatments. In the second study (Chapter 3), high-through gene expression profiling was employed to examine changes in gene expression in chick RPE with long-term imposed hyperopic defocus, which resulted in eyes being longer than normal and highly myopic. This DNA microarray screening revealed changes in the expression of many genes, including BMPs, noggin (NOG), dopamine receptor D4 (DRD4), retinoic acid receptor, beta (RARB), and retinal pigment epithelium-derived rhodopsin homolog (RRH). Some of these genes showed increased expression while others showed decreased expression. It is plausible that some may be linked the ocular pathological complications seen in myopia, while others may be linked to ocular growth regulation, the imposed visual conditions resulting in sustained, increased ocular growth. The third and final study (Chapter 4), addressed the possibility of an RPE site for the anti-myopia action of apomorphine (APO), a dopamine receptor agonist, observed in animal studies. We further investigated the possibility that TGF-â secretion from RPE mediates this inhibitory growth effect. APO applied to cultured human fetal RPE cells was found to alter the secretion of both TGFâ1 and TGFâ2, which was biased towards the basal (choroidal) side. These growth factors also exhibited constitutive polarized secretion, albeit biased in the opposite direction to APO-induced paracrine secretion. The results for APO are consistent with its observed inhibitory (anti-myopia) effects in vivo and offer the RPE as a possible site of action.

In summary, the research reported in this dissertation provides evidence that RPE plays an important role in postnatal eye growth regulation, (including myopic growth), as a conduit for relaying growth modulatory retinal signals to choroid/sclera. Genes and molecules identified in these studies offer potential directions for novel anti-myopia treatments, with the RPE being a potential target for the same.

The research described in this dissertation investigates the relationship between electronic device use and myopia progression and focus on how screen settings influence choroidal thickness as a potential biomarker for myopia. The study is ifluences by the global rise in myopia prevalence, especially among students exposed to digital screens for long periods. The research examines the effects of blue light exposure and contrast polarity of elctronic devicess screen settings on ocular parameters, including choroidal thickness, and their potential role in myopia progression.

Chapter 1's literature review discusses the increasing prevalence of myopia, and explores choroidal thickness as a potential biomarker for myopia progression. It also reviews studies on blue light and contrast polarity, highlighting inconsistencies in their effects on choroidal thickness and myopia and the need for further research.

The first of three studies surveyed UC Berkeley students, showing a high prevalence of myopia (63%) with different screen settings usage. Chapters 3 and 4 present findings from four-week studies on the effects of blue light filters and contrast polarity screen settings on choroidal thickness, axial length, and refractive error. While the results did not fully align with previous studies, they suggest baseline differences in choroidal thickness related to habitual screen settings, affirming further investigation.

Overall, the studies introduce new factors related to electronic device use in myopia prevention, emphasizing the need to understand screen-setting habits among heavy digital device users in the student population.

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