Christine Wildsoet - US grants

The many studies reporting myopia in response to form deprivation (either with diffusers or lid suture) provide convincing evidence for an effect of visual experience on ocular development. More significantly, there is compelling evidence based on studies involving spectacle lenses that vision actively directs eye growth; specifically, negative lenses which impose hyperopic defocus cause eyes to grow toward myopia, whereas eyes with myopic defocus imposed by positive lenses grow toward hyperopia. These growth changes include choroidal and scleral components, both of which serve to reduce the imposed defocus, a process known as emmetropization. In addition, there appears to be another process that keeps the two eyes yoked (coupled) together such that compensatory growth changes in one eye cause the other eye to change its growth in the same direction, although to a lesser extent. We understand very little about these two mechanisms, yet both are potentially of great clinical significance. Understanding emmetropization may provide the key to preventing myopia in humans. Understanding interocular yoking may help us to understand anisometropia (i.e. the two eyes have different refractions), a common condition in infants. In relation to emetropization, this proposal asks whether the brain is involved. The brain seems not to be required in the case of form deprivation myopia as optic nerve section does not prevent it. However, this is unclear in the case of the lens-induced changes. By interfering with eye-brain communication, I propose to resolve this important issue for the choroidal and scleral components of emmetropization. If preliminary results are confirmed indicating that the brain does play a role, I will try to identify the pathways involved using selective lesioning of the most likely efferent pathways. In relation to interocular yoking in eye growth, this proposal seeks to clarify the conditions under which these interactions occur. I will also assess how the refractive state of each of the eyes (natural or altered experimentally), influences their interactions. If the existence of yoking is confirmed, I propose to identify the pathways involved, again using a selective lesioning approach.

DESCRIPTION (provided by applicant): The retinal pigment epithelium (RPE) lies in the back of the eye between the neural retina and its choroidal blood supply. This layer of epithelial cells serves to protect the health and integrity of the outer retina. In the choroid, abnormal blood vessel growth or choroidal neovascularization (CNV) occurs in diseases such as age related macular degeneration (AMD) - the major cause of blindness for people over the age of 60. Vascular endothelial growth factor (VEGF) and other pro- or anti-angiogenic factors are constitutively secreted by the RPE into the extracellular space on both sides of the epithelium. The effects of these molecules on RPE physiology are not known. Evidence from human pathologic specimens and from rodent models suggests that increased VEGF production by the RPE is the source of CNV. Research in this area is severely hampered by the lack of a small animal model in which VEGF secretion by the RPE induces CNV. In preliminary experiments, we have used gene transfer techniques to develop a rat model for stimulating blood vessel growth in the back of the eye. This model will be used to test the hypothesis that secretion of VEGF by RPE is critically important in generating choroidal neovascularization (CNV). Gene transfer techniques will be also used to inhibit or reduce this VEGF-induced abnormal blood vessel growth. These experiments should provide a deeper understanding of CNV and the basis for a potential therapy for diseases like AMD. Normally, there is a very close anatomical relationship between the RPE and the retina. Separation of these two tissues occurs in a whole host of pathologies that lead to the abnormal accumulation of fluid in the extracellular or subretinal space. This separation or retinal detachment can lead to a loss of vision. One of our goals is to develop a rat model of retinal detachment. This model will allow us evaluate putatively therapeutic molecules that work directly on the RPE to remove fluid from the subretinal space. This removal will restore the normal anatomical relationship between RPE and retina and should improve vision.

DESCRIPTION (Provided by applicant): Emmetropization is the regulatory process that normally matches the optical power of the eye to its length, so that objects can be focused on the retina. Abnormalities can cause myopia (shortsightedness), in which the eye grows too long for its optical power, leading to sight-threatening complications. Refractive surgery that overcorrects myopia may lead to renewed eye growth, as may certain lighting conditions. Near-epidemic prevalence of myopia in several populations makes it critical and timely to understand the determinants of emmetropization, without which effective treatments are unlikely. Studies: The chick eye has proven to be an effective experimental model for emmetropization: many optical, surgical and pharmacological manipulations can be performed, with time and cost effectiveness, and results generalize well to mammals and primates. Our recent studies in the chick used optic nerve section (ONS) to dissociate retina and brain, and suggested that emmetropization is largely controlled locally, by the retina. Emmetropization with ONS is abnormal. We ask whether this is due to cell loss and retinal rewiring, which are side effects of ONS. Aim 1 (Role of inner retinal cells in emmetropization): We will (a) dissociate retina and brain with a ganglion cell toxin that spares certain fibers destroyed by ONS, (b) use molecular markers to test if ONS causes loss of glucagonergic amacrine cells, and (c) use growth factors to protect ganglion cells in ONS eyes. Visual stimuli are well known to affect posterior segment growth. What determines corneal growth? Aim 2 (Role of the retina in anterior segment growth): We will (a) measure effects of continuous light on corneal flattening, and consequent posterior chamber growth, (b) use radial keratotomy to control for effects of non-visually induced corneal flattening, and (c) measure intersegmental fluid exchange as a potential conduit for retinal factors that control corneal growth. It is still not known how the retina distinguishes the sign of a defocused image. Aim 3 (Decoding defocus in emmetropization): We will manipulate defocus cues with (a) reverse chromatic aberration lenses, (b) blue and red monochromatic rearing, and (c) optical control of object distance, spatial frequency, contrast, and monochromatic aberrations. Reduced retinal acuity may explain the high refractive errors in congenital outer retinal pathologies. Aim 4 (Retinal acuity & emmetropization): We will study in 3 low-acuity preparations: (a) albino chicks, (b) normal chicks reared in UV light, and (c) normal chicks reared in dim light.

DESCRIPTION (provided by applicant): Emmetropization is the regulatory process that matches the eye's length to its optical power, bringing distant objects to focus on the retina with accommodation relaxed. Myopia, or shortsightedness, is the failure of emmetropization in which the eye grows too long for its optical power. Myopia's association with sight-threatening complications, and near-epidemic prevalence in several populations, make it critical to understand the determinants of emmetropization, without which effective treatments are unlikely to be developed. The chick eye has provided a very fruitful model of emmetropization, clarifying the optical conditions that lead to myopia, providing evidence for local retinal control of eye growth, and identifying optical manipulations that inhibit eye elongation, which are therefore potential myopia treatments. Aims and Studies: (1) We will study local molecular signaling across the retinal pigment epithelium (RPE), which separates the image-processing retina from the choroid and eye shape-determining sclera, using molecular techniques to delineate patterns of gene expression in eyes undergoing experimentally altered growth through exposure to myopic and hyperopic defocus, and in cultured RPE treated with presumptive retinal growth modulators. We will then conduct in vivo tests of growth factors identified in gene expression studies. (2) We will use these genetic signatures, in combination with drugs known from previous work to modulate responses to imposed myopia and hyperopia, to study the mechanisms of eye growth induced by simultaneous, competing myopic and hyperopic defocus produced by several types of multifocal and astigmatic lenses. We will evaluate hypotheses regarding retinal processing, optical aberrations, and peripheral retinal growth to explain lens effects. (3) We will develop a guinea pig model to extend our findings on local ocular growth regulation to mammalian eyes, and to compare mammalian and avian responses to simple and competing defocus stimuli. PUBLIC HEALTH RELEVANCE Emmetropization is the regulatory process that matches the eye's length to its optical power, bringing distant objects to focus on the retina with accommodation relaxed. Myopia, or shortsightedness, represents the failure of emmetropization in which the eye grows too long for its optical power, in near-epidemic prevalence in several populations, with associated sight-threatening complications, making it critical to understand the determinants of emmetropization, without which effective treatments are unlikely to be developed. This project will apply modern molecular biology and optical techniques to investigate the mechanisms underlying emmetropization, with a view to developing novel treatments for myopia.

DESCRIPTION (provided by applicant): Biopolymer Application for Myopia Control Abstract Significance: This proposal describes a novel approach to the prevention and treatment of myopia (near- sightedness), which results from increased scleral remodeling, leading to excessive eye elongation. Recent years have witnessed a rapid rise in the prevalence of myopia, especially within Asian communities where it has reached epidemic (>90%) levels in some young adult student populations. Trends show a decreasing age of onset, and higher average amounts of myopia. High myopia (>-6D), once considered rare, is increasingly common, and so represents a serious public health concern due to potentially blinding retinal complications. Topical ophthalmic atropine is currently the only drug treatment for myopia although its use is mostly limited to "high-risk" Asian communities, because of associated significant ocular side-effects and compliance problems. We propose to use a synthetic and environmentally responsive biomimetic hydrogel (sIPNs) that can regulate the behavior of scleral cells and be used as a slow release drug delivery device. A nanoparticle formulation of atropine will be made for use with sIPNs. Our treatment goals for these products are to stabilize the weakened scleras of high myopes and to slow elongation in eyes showing myopia progression, with minimal side-effects. Planned experiments in this pilot project will allow synthesis, characterization and biocompatibility testing of the products as well as limited i vivo testing. There are no generally accepted treatments for myopia, a significant cause of visual impairment and blindness around the world. Novel, tissue-engineering-based treatments for myopia are likely outcomes of this study. PUBLIC HEALTH RELEVANCE: Myopia (shortsightedness) involves excessive elongation of the eye due to changes in the sclera, the outer supporting wall of the eye, and is in near-epidemic prevalence in several populations, with associated with sight-threatening complications making it critical that effective treatments be developed. Currently topical ophthalmic atropine is only drug treatment and has many side-effects. This project will apply modern tissue engineering principles to develop novel treatment for myopia that target the sclera.

DESCRIPTION (provided by applicant): Short term training is requested for 12 optometry students during the summers after their first and second year of optometry school. The training will be conducted by established vision science and clinical science researchers, primarily mentors in the Group in Vision Science who are also faculty members in the UC Berkeley School of Optometry. Each trainee will be engaged in a maximum of two separate short term training periods (summers) following completion of their first and second year of optometry school. The training program is designed to attract talented students to clinical research by developing their interest, awareness and enthusiasm for a career in biomedical or behavioral research. Training will be provided within the laboratories of 20 mentors in the School of Optometry, where currently 43 pre-doctoral students and approximately 20 postdoctoral fellows are involved in the Graduate Program in Vision Science. The mentors also train graduate students in other disciplines (e.g. neuroscience, computer science, molecular and cell biology, psychology and infectious disease). The Group Program in Vision Science has been in existence for over 60 years on the Berkeley campus and has graduated more than 178 trainees with advanced degrees - the majority now actively engaged in vision research. In the past 15 years, the program has attracted many clinicians to its Ph.D. training program. The same mentors that train PhD students will also mentor optometry students on this short-term training grant. A wide range of basic science as well as clinical research projects are on-going in the laboratories of the mentors offering numerous opportunities for involvement by the short-term trainees. Optometry schools from across the country will be encouraged to support our effort to attract talented health professional students to our short term training program. The long term goal is to encourage clinicians to continue research training/involvement after completion of their professional training.

DESCRIPTION (provided by applicant): This grant will support the 5th in a series of annual translational conferences at UC Berkeley, to support the NEI-funded K12 training program for clinician scientists. The themes for these conferences represent cutting edge, clinically applicable research, and change annually. The theme for the 2010 conference is innovative solutions involving optical manipulations or targeting optical problems. The following topics will be covered: 1) optical solutions to presbyopia, an age-related loss in ones ability to focus (accommodate) on nearby objects, 2) surgical solutions to optical problems of the eye, including presbyopia, 3) novel optical treatments for ocular diseases including myopia (near-sightedness) and keratoconus, and 4) technological advances in 3D simulations of the natural environment and their applications. The final session of the conference will be a panel discussion, involving all speakers, who will be challenged to think into the future. Targeted speakers include international leaders in their respective research fields, with a poster session providing opportunity for K12 trainees to show case their research. Trainees will also be involved as chairs, speakers and discussants of the paper sessions. In addition to K12 trainees, the conference targets local scientists, clinicians, graduate students and members from industry R&D. Travel grants will be offered to encourage involvement of trainees from interstate K12 programs and interested minority undergraduate students from the SETT program. The conference will serve as forum for sharing ideas, and establishing new collaborations, including industry links. PUBLIC HEALTH RELEVANCE: This grant will support the 5th in a series of annual translational conferences at UC Berkeley covering innovative solutions involving optical manipulations or targeting optical problems. The following topics will be covered: 1) optical solutions to presbyopia, an age-related loss in ones ability to focus (accommodate) on nearby objects, 2) surgical solutions to optical problems of the eye, including presbyopia, 3) novel optical treatments for ocular diseases including myopia (near-sightedness) and keratoconus, and 4) technological advances in 3D simulations of the natural environment and their applications.

DESCRIPTION (provided by applicant): Myopia (short-sightedness) results from a failure of emmetropization in which eyes grow too long for their optical power. Myopia's association with sight-threatening complications and near-epidemic prevalence in several populations make it critical that effective treatments for slowing myopia progression be developed. The chick eye has provided a very fruitful model, yielding evidence for local retinal control of eye growth, identifying multifocal (MF) optical manipulations that inhibit ocular elongation as potential myopia treatments, and providing initial insights into the mechanism underlying atropine's anti-myopia action. The guinea pig, a mammalian model with a fibrous sclera more like human sclera, has yielded valuable translational data. Our proposal relied on recent findings from many labs, including our own, to develop a multi-pronged approach to myopia control. It exploits novel tools and advanced technologies to pursue 4 aims: (1) We will use our novel optical imaging (cone) devices and MF spectacle lenses combined with neurotoxins to further investigate the mechanisms underlying MF lens effects on eye growth, and the variables influencing treatment efficacy. (2) We will investigate the anti-myopia actions of atropine, to better understand the roles of retinal and nonretinal mechanisms and to determine effective dosing regimens. (3) We will investigate whether treatment efficacy can be improved by combining MF lenses designed to slow myopic growth with either or both atropine and very bright light, which is also known to slow myopia progression. Experiments under Aim 3 will make use of results from earlier experiments (Aims 1 & 2). We will apply molecular biology tools to study retinal/retinal pigment epithelium (RPE) signal pathways. RPE gene expression signatures of myopic and hyperopic growth developed in the last grant period will be exploited to obtain insights into nature of treatment mechanisms and interactions. (4) We will test MF contact lenses in guinea pig to translate our recent related MF spectacle lens studies and look for parallels with chick retinal and RPE molecular signatures that may be applied to understand ocular growth signals.

Abstract Myopia (near-sightedness) refers to the ocular condition in which there is a mismatch between the eye's optical power and its length, typically the product of increased scleral remodeling and eye elongation during childhood and/or adolescence, and leading to blurred distance vision. Myopia is associated with increased risk of potentially blinding pathologies, including glaucoma, maculopathies and retinal detachments, with risks increasing with the level of myopia. Myopia is now one of the leading causes of acquired blindness worldwide. Interestingly, a number of epidemiological studies have reported myopia to be more common among females who also show faster progression of the disease, while realizing poorer clinical outcomes. Traditionally, these apparent sex-specific differences have been attributed to behavioral differences, with little supportive scientific evidence. However, associations between myopia and growth spurts and early menarche as well as accelerated progression around puberty have also been reported. The project described here is based on the hypothesis that the observed sex-related bias in myopia reflects, at least in part, sex-specific, endocrine-related influences on the sclera, as has been observed in other connective tissues in the body. The over-riding goal of proposed research is understand the role and related mechanisms for endocrine regulation of scleral and thus eye growth. Unpublished pilot data has confirmed the expression of key class III sex hormone receptors in the sclera of young chicks, which will be used as our myopia model. Under Aim 1, we will profile scleral sex hormone receptors and related enzymes during development in young normal chicks, age-matched chicks induced to undergo precocious puberty and chicks reaching puberty naturally (validation step). Age-related changes in circulating levels of key sex hormones will also be measured. Experiments will investigate how expression of scleral sex hormone receptors and related enzymes vary, at both the gene and protein levels, with age and the onset of puberty, with sex being an over- riding variable. Local scleral and systemic levels of key ligands and enzymes will also be assessed. Precocious puberty will be induced using systemic tamoxifen. This approach takes into account practical and time constraints of an R21 grant. Under Aim 2, we will determine how transcriptional and translational levels of sex hormone receptors and enzymes, as well as local scleral sex hormone levels, change during myopia development in tamoxifen-treated compared to normal chicks. In experiments under both Aims, RT-PCR, western blotting and immunohistochemical analyses will be undertaken to examine gene and protein expression and LC-MS/MS used to assess local scleral and systemic levels of key ligands. An improved understanding of the link between sex and myopia prevalence and severity has potentially broad implications related to environmental endocrine disruptors and the timing of school vision screening programs, as well as for the individual patient, customization of clinical intervention strategies for myopia control.

PROJECT SUMMARY Support is requested for 15 profession optometry graduate students to participate in short-term training in vision research upon successful completion of their first year of optometry school. The training will be conducted by established vision researchers and clinical scientists in the School of Optometry at UC Berkeley, and who are also members of its Graduate Program in Vision Science. Trainees will each complete one short-term training program, during the summer months following completion of their first year of optometry school. The long-term goal of this program is to encourage clinicians to pursue advanced research training and/or continue involvement in research after completion of their professional training. Thus the training program is designed to attract talented students to clinical and/or translational research by developing their interest, awareness and enthusiasm for a career in biomedical or behavioral research. Optometry schools from across the US, including Puerto Rico, are targets of our recruitment efforts aimed at attracting a diverse pool of talented students to our program. Training will be provided within the laboratories of 23 mentors in the School of Optometry, where currently 39 pre-doctoral students and approximately 70 postdoctoral fellows are involved in vision and eye-related research, through the Graduate Program in Vision Science in the former case. The multidisciplinary nature of this program is also reflected in engagement of mentors in the training of graduate students from other disciplines among which are molecular and cell biology, immunology, infectious diseases, neuroscience, psychology and bioengineering. Over the past 23 years, the Short Term Training for Optometry Students training program has attracted many clinicians to the PhD program in Vision Science, supported by an NIH/NEI T32 Training grant (Program in Vision Research). This interdisciplinary research program in Vision Science has been in existence for over 70 years on the Berkeley campus and over this period has trained and graduated 241 trainees mostly with PhD degrees. The majority of these graduates are now actively engaged in vision research on a professional level. The same mentors that train PhD students have also mentored and will continue to mentor optometry students undertaking short- term training via the NIH/NEI T35 grant for which renewal is being sought in this application. A wide range of basic science as well as clinical research is on-going in the laboratories of these potential mentors, offering numerous, equally wide-ranging research opportunities for the short- term trainees.