Matthew LaVail - US grants

Our proposed research deals with environmental light damage to the retina of normal mice and rats and of those undergoing various forms of inherited retinal degeneration. Our goals are to clarify the cytopathologic steps in light damage due to fluorescent lighting, with particular emphasis on the involvement of the retinal pigment epithelium and eye pigmentation. We hope to define a genetic factor in susceptibility to light damage in otherwise normal retinas and to explore the role of the interphotoreceptor matrix in light damage. Our analysis of the influence of environmental lighting on several inherited retinal degenerations should offer useful guidelines on some aspects of the management of patients with retinitis pigmentosa.

Our overall objective is to gain an understanding of the mechanism(s) by which the pigment epithelium (PE) supports the normal functioning of the neural retina. This ultimate goal can be reformulated into the following immediate goals: to obtain a greater understanding of PE ion and metabolite transport; to assess the contribution of potassium to electroretinogram slow waves and to long-term changes in the standing potential; to determine if the PE buffers extracellular potassium; to assess PE involvement in spreading depression of the inner retina; to determine if the PE interacts with the cones in the same way that it interacts with the rods; to obtain an understanding of the passive and active ionic mechanisms of mammalian PE; to describe hormonal control of disc shedding and phagocytosis in Xenopus. It is proposed to study these PE mechanisms and PE retina interactions with a combined electrophysiological, epithelial-transport and anatomical approach.

The most common inherited disorder causing blindness in man is retinitis pigmentosa, a family of diseases in which the photoreceptor cells of the retina degenerate disappear slowly and progressively over a period of years. Similar inherited diseases are known in several laboratory animals. In the past several decades the most widely studied mutant has been the Royal College of Surgeons (RCS) rat with inherited retinal dystrophy. Four congenic strains of RCS rats are also available that either serve as genetic control animals or have different types of eye pigmentation and different rates of retinal degeneration. The goals of this proposal are 1) to maintain a breeding colony of the RCS rats and four congenic strains, 2) to maintain the congenic status of the congenic strains, and 3) to distribute animals and eye tissues to other investigators who request them. It is anticipated that increased availability of these animals will stimulate research by attracting new and established investigators to the field of inherited retinal degenerations.

Our proposed research deals with environmental light damage to the retina of normal mice and rats and of those undergoing various forms of inherited retinal degeneration. We will study the damaging effects of fluorescent lighting at illuminance levels often found in office and industrial work environments. Our research objectives are three-fold. First, we shall clarify the sequence of cytopathologic steps in light damage and explore the possible role of the retinal pigment epithelium and the interphotoreceptor matrix in the degenerative process. To do this we will define the subcellular changes by quantitative electron microscopy. In addition, we will carry out studies using histochemistry, immunocytochemistry, and autoradiography of protein metabolism and transport or diffusion of various sugars. Second, we will study two determinants of the severity of light damage: eye pigmentation and genetic factors. To assess the protective role of eye pigmentation, we will use quantitative morphlogical methods and coisogenic and congenic strains of animals. To characterize the recently described genetic factors that regulate the susceptibility of the retina to light damage, we will use classical genetic crosses and recombinant inbred strains of mice to determine a) whether resistance to light damage is imparted by a single-gene or polygenic factor, b) the genetic mode of inheritance, and c) the chromosomal localization of the gene(s). In addition, we will probe the cellular and molecular mechanisms of resistance to light damage by the use of experimental mouse chimeras and collaborative biochemical studies. Third, we will examine the potentially damaging effects of light on the rate and character of the inherited retinal degenerations in the nervous and Purkinje cell degeneration mutant mice and in the RCS rat with inherited retinal dystrophy.

The most common inherited disorder causing blindness in man is retinitis pigmentosa, a family of diseases in which the photoreceptor cells of the retina degenerate disappear slowly and progressively over a period of years. Similar inherited diseases are known in several laboratory animals. In the past several decades the most widely studied mutant has been the Royal College of Surgeons (RCS) rat with inherited retinal dystrophy. Four congenic strains of RCS rats are also available that either serve as genetic control animals or have different types of eye pigmentation and different rates of retinal degeneration. The goals of this proposal are 1) to maintain a breeding colony of the RCS rats and four congenic strains, 2) to maintain the congenic status of the congenic strains, and 3) to distribute animals and eye tissues to other investigators who request them. It is anticipated that increased availability of these animals will stimulate research by attracting new and established investigators to the field of inherited retinal degenerations.

The most common form of inherited human blindness is retinitis pigmentosa, a family of disorders in which the photoreceptor cells of the retina progressively degenerate and disappear over a period of years. The paucity of human donor tissues at early stages of these diseases and others, such as age-related macular degeneration, has led scientists worldwide to turn to similar retinal degenerations in laboratory animals. These animal models of the human diseases have played a prominent role in vision research in the past several decades, and much has been learned from them about the cellular mechanisms of photoreceptor degenerations and potential therapeutic measures for these diseases. Among the various species with retinal degenerations, mice and rats have been used most extensively, primarily because of the experimental advantages of short gestation time; small size; powerful genetic control in the form of several readily available retinal degeneration mutants, multiple inbred and congenic strains with genetic controls and different eye pigmentation types; and the potential to carry out certain embryological and genetic procedures such as the production of experimental chimeras and transgenic animals. The rapidly escalating costs to maintain animal colonies on individual grant budgets are beyond the means of most vision scientists, and most investigators do not have the time or expertise in mammalian genetics to develop or maintain various inbred, congenic, and transgenic rodent strains. Thus, the goals of this proposal are 1) to maintain breeding colonies of such rats that are appropriate for studies on various forms of inherited and age- related retinal degenerations, and 2) to distribute these animals and eye tissues to investigators who request them. The lines that will be developed and/or maintained and distributed are 1) Royal College of Surgeons (RCS) rats with inherited retinal dystrophy; 2) three rat strains congenic with RCS that serve as genetic controls and with different eye pigmentation and rates of retinal degeneration; 3) P23H mutant rhodopsin transgenic rat lines with 3 different rates of degeneration; and 4) S334ter mutant rhodopsin transgenic rat lines with 5 different rates of degeneration. Based on past performance, this colony and the distribution of animals will benefit scores of vision scientists and will have a major impact on research leading to the causes, prevention and treatment of retinal degenerative diseases.

[unreadable] DESCRIPTION (provided by applicant): The broad objective of this application is to facilitate study of the structure, development and function of the visual system in health and in blinding diseases, with the aim of preventing, mitigating or curing such diseases through the application of the most sophisticated available techniques, including the methods used in molecular biology and molecular genetics. Five support and service modules will help achieve the broad objective, as follows: [unreadable] [unreadable] I. Imaging Support Module (computer-aided image analysis; production of graphics for data analysis, presentation and publication, including poster printing); [unreadable] [unreadable] ll. Molecular Bioloqy and Genetic Analysis Service Module (DNA sequencing, PCR and Southern blot genotyping of transgenic animals and in situ hybridization); [unreadable] [unreadable] lll. Confocal and Digital Microscopy Support Module (digital data acquisition using confocal microscopy and optical brightfield, darkfield, phase contrast or fluorescence microscopy) [unreadable] [unreadable] IV. Computer/IT Support Module (assistance in computer and information technology hardware and software selection, installation, instruction in use, maintenance and minor repairs, networking, and programming for custom research needs); [unreadable] [unreadable] V. Electrical and Machine Shop Service Module (design, manufacture, maintenance and repair of specialized research instruments and devices). [unreadable] [unreadable] This application is a competing renewal of a Core Vision Research Grant submitted by the Principal Investigator and 28 other funded vision scientists. Sixteen are NEI-funded with 20 separate research projects, two are NIH- but not NEI-funded, and 11 others are funded by non-NIH sources. Of the latter 11, two have just completed NEI K08 grants and have submitted NEI R01 grant renewals or new grant applications, two others have submitted NEI R01 grant renewals or new grant applications, and others plan to submit in the future. [unreadable] [unreadable]

The most common form of inherited human blindness is retinitis pigmentosa. a family of disorders in which photoreceptor cells of the retina progressively degenerate and disappear over a period of years. The paucity of human donor tissues at early stages of these diseases and others, such as age-related macular degeneration, has led scientists worldwide to turn to similar retinal degenerations in laboratory animals. These animal models of the human diseases have and continue to play a pivotal role in vision research on the cellular mechanisms of photoreceptor degenerations and the development of potential therapeutic measures for these diseases. Among the various species with retinal degenerations, mice and rats have been used most extensively, primarily because of the experimental advantages of short gestation time;small size;powerful genetic control in the form of several readily available retinal degeneration mutants, multiple inbred and congenic strains with genetic controls: and the potential to carry out certain embryological and genetic procedures such as the production of experimental chimeras and transgenic animals. For most therapeutic studies in the areas of neuroprotection, drug delivery, transplantation and visual prostheses, the rat is the species of choice among rodents because an intermediate-sized eye, larger than that of a mouse, is required, yet large numbers of genetically controlled animals are needed. The high costs to maintain animal colonies on individual grant budgets are beyond the means of most vision scientists, and most investigators do not have the time or expertise in mammalian genetics to develop or maintain various inbred, congenic and transgenic rat strains. Thus, the specific aims of this proposal are 1) to maintain breeding colonies of mutant rats that are appropriate for studies on various forms of inherited retinal degeneration, and 2) to distribute these animals and eye tissues to investigators who request them. These lines are 1) highly inbred Royal College of Surgeons (RCS) rats with inherited retinal dystrophy;2) three rat strains congenic with RCS that serve as genetic controls and with different eye pigmentation and rates of retinal degeneration;3) P23H mutant rhodopsin transgenic rat lines with 2 different rates of degeneration;and 4) S334ter mutant rhodopsin transgenic rat lines with 4 different rates of degeneration. The overall goal of this project is to facilitate research on retinal degenerations by producing, maintaining and distributing rat models necessary for this research. Based on past performance and the increasing use and demand for these animals (almost doubling in the past 5 years), this colony and the distribution of animals will benefit scores of vision scientists and will have a major impact on research leading to the causes, prevention and treatment of retinal degenerative diseases. More than 70 current and pending grant projects are dependent upon this Retinal Degeneration Rat Model Resource established by the National Eye Institute in 1986.

DESCRIPTION (provided by applicant): The broad objective of this Core Vision Research Grant application is to facilitate study of the structure, development and function of the visual system in health and in blinding diseases, with the aim of preventing, mitigating or curing such diseases through the application of the most sophisticated available techniques, including the methods used in molecular biology and molecular genetics. Five support and service modules will help achieve the broad objective, as follows: I. Imaging Support Module (computer-aided image analysis; production of graphics for data analysis, presentation and publication, including poster printing); II. Molecular Biology and Genetic Analysis Service Module (PCR and Southern blot genotyping of transgenic animals and DMA sequencing); III. Confocal and Digital Microscopy Support Module (digital data acquisition using confocal microscopy and optical brightfield, darkfield, phase contrast or fluorescence microscopy); IV. Computer/IT Support Module (assistance in computer and information technology hardware and software selection, installation, instruction in use, maintenance and minor repairs, networking, and programming for custom research needs); V. Electrical and Machine Shop Service Module (design, manufacture, maintenance and repair of specialized research instruments and devices). This application is a competing renewal of a Core Vision Research Grant submitted by the Principal Investigator and 32 other funded vision scientists. Twenty are NEI-funded with 23 separate research projects (R01, K08 and K23), 5 are funded by other NEI mechanisms, one is NIH- but not NEI-funded, and 8 others are funded by private, non-NIH sources. Of the latter 8, two are newly recruited vision scientists who plan to submit new NEI R01 grant applications, and two others are more senior scientists who plan to resubmit productive R01 applications in the near future. Overall, Core investigators are involved in 38 different, active research projects. The Core Vision Research Grant has been highly successful in enhancing the productivity of vision research and facilitating collaborative studies on the visual system at UCSF.

ADMINISTRATIVE CORE Principal Investigator Matthew M. LaVail, Ph.D. is an internationally known senior vision scientist who has used many different types of research approaches, including anatomical, biochemical, classical genetic, molecular genetic and electrophysiological methods, as well as animal model development and neurotrophic factor and gene therapy studies for retinal degenerative diseases. His discoveries of circadian rod outer- segment disk shedding and the pharmaceutical rescue of degenerating photoreceptors have led to the establishment of new fields of vision research. He is highly respected at UCSF and in the vision research community. Dr. LaVail has been funded continuously by the NEI for the past 35 or more years with 6 different co-operative agreements, contracts and long-running research grants, so he is fully aware of both the research needs and regulations involved with NIH grants. Also, he has been an active member of the Core Grant Core Directors' Advisory Committee (see below), serving as the Imaging Core's first Director beginning in 1998. Through his leadership and effort in designing and constructing the Core, the Imaging Core has been the most heavily used of our Cores. Dr. LaVail distinguished himself, as well, in establishing procedures that would assure equitable access to the Core, even with very high usage at several times during the year just prior to several research meetings. One year before the retirement of the former Principal Investigator, Dr. Steven Kramer, Dr. LaVail was approved by the NE I to become the new Principal Investigator of the Core Grant effective July 1, 2002. We will use this process in case a Core Director or PI moves, retires or becomes unable to carry out the duties of the position, in which case the person will be identified by the Advisory Committee (see below), and the name will be submitted to the NEI for approval.

III. COMPUTER/IT RESOURCE CORE Research Plan of Computer/IT Resource Core Computer technology now permeates every facet of vision research. Computers are used by all scientists for preparing manuscripts and lectures, communicating with colleagues, ordering supplies, searching the Internet, and reading the literature. In addition to these routine tasks, they are also vital for acquisition, storage, and analysis of scientific data. In some laboratories, computers are required to actually carry out experiments, especially those that require presentation of visual stimuli and recording of neuronal responses from the retina or brain. In recent years, experimental rigs that once relied on complex electronic circuitry have evolved to carry out the same hardware functions using computers equipped with specialized software. This shift in instrumentation means that a computer specialist has taken over many tasks that once were the job of an electronics shop. Every investigator has some degree of familiarity with computers, often gained through painful trial and error experience. However, the field of computer technology continues to advance rapidly, and most scientists cannot keep up. In fact, it is difficult simply to maintain adequate laboratory computer systems, as equipment becomes obsolete and new software is introduced. Many scientists find themselves unsure what new computer equipment they need, and how they can design and apply computer systems to meet their needs most efficiently. Sometimes the solution to a computer problem is quite straightforward, but documentation is inadequate, and hours can be wasted dealing with equipment incompatibility, software bugs, or missing components. The worst scenario is a laboratory that does not realize that it needs a new computer backup system until a critical collection of data is lost. Most scientists prefer to spend their time and energy on science, not dealing with computer problems, but few laboratories can afford the luxury of hiring a computer specialist, nor require one full-time. The Computer/IT Core has been immensely successful over the past decade because it has provided all vision laboratories with a highly skilled computer programmer for every aspect of basic computer services. Indeed, since the Core's inception, its work log has documented that over 100 different users ~ from graduate students to post-doctoral fellows to principal investigators ~ have come to the Computer/IT Core for help. The Computer/IT Core is available for the simplest of problems (my printer won't work) to the most sophisticated challenges, such as writing custom programs to run vision experiments. It is built around a single, highly experienced computer specialist who functions as a jack of all trades, while at the same time providing sophisticated support for specialized applications. This includes a spectrum of services, from software and hardware installation, troubleshooting, automatic data backup, creation of web-based lab databases, to writing of custom programs for data collection and analysis. The vision science community at UCSF shares a strong consensus that dedicated IT support constitutes an exam pie of crucial core support.

I. IMAGING RESOURCE CORE Research Plan of Imaging Core We have capitalized on the revolution in research-oriented imaging that has occurred over the last decade. The newer innovations, available to Core vision scientists, includes advances in confocal microscopy, sophisticated image analysis software, much more powerful and faster computer hardware, very sensitive CCD cameras, easily utilized computer graphics, and high quality printing. Thus, virtually all vision scientists no longer view graphics imaging and analysis as a novelty technique, but instead, require the use of graphic image equipment and support for their research. Thus, it is not surprising that our Imaging Core has been very heavily used since it was first established. More than 100 individuals have used the Imaging Core for their research work in the past 5 years, and there are currently 66 users with 24-hour access codes (see Resources, next page). Moving all image analysis from the Morphology to the Imaging Core will further increase usage of the Imaging Core (see INTRODUCTION to Resubmission and below). A key attribute of the Imaging Core is the availability of the Core Technician, Suling Wang. Many of the Core investigators have some experience with imaging software. Core investigators often can use the basics of Photoshop, but knowledge of the full power of the application is usually beyond their capabilities. Ms. Wang is an expert in this and the 19 other graphics and image analysis applications, and large numbers of people seek her help for instruction and help with analysis of digital data and preparing digital images for publication and presentation. Moreover, many Core investigators are unfamiliar with the software and image analysis applications. Ms. Wang is available to do some of the routine outputting of images, where interaction by the investigator is not required. More important, though, she is available to instruct Core investigators and their junior colleagues in the best use of the software applications. With Ms. Wang's extensive computer graphic arts background (see BUDGET JUSTIFICATION), this both improves the output images and saves time for the research groups in learning the software applications. Another important reason the Imaging Core is so heavily used is that most Core investigators cannot afford to purchase the expensive software applications and their upgrades, and we are restricted from copying them for individuals by U.S. copyright laws and by our University. These are all available and kept up to date in the heavily used Imaging Core.

IV. MACHINE SHOP SERVICE CORE Research Plan of Machine Shop Service Core The Machine Shop in the Department of Physiology has a long track record of excellent machine shop service, and it has been part of the Core Grant for the past 15 years. The Machine Shop Core provides dedicated space and technical expertise to support the design and manufacture of specialized research instruments that are required by (3ore investigators but are not available commercially. Vision research, especially that done on intact animals, involves a complex set of mechanical devices that are custom-designed by each laboratory. It is common to find that the complex demands of vision research simply cannot be met by commercially available devices, making the shop core an essential component of vision research at UCSF. The Machine Shop, for example, manufactures custom implants that are both superior to and more suitable than those available from companies. The shop provides essential repair services for equipment that was manufactured in the shop, as well as equipment that was purchased from outside. In many instances, the equipment is no longer being made, but is essential to the vision research of one or more laboratories, making this repair service of utmost importance. Further, the shop has the capacity to provide immediate service when things break during an ongoing experiment, and to make lab-calls to repair equipment that is difficult to bring to the shop. This capacity, especially, underscores the value of this service Core to the vision research community. The Machine Shop produces professional quality equipment that is customized to the exact needs of the investigators. Evidence of the high level of quality of their products comes from the large number of requests for its customized products from young scientists who are leaving UCSF and moving to their own faculty jobs. The shop is both efficient and productive. The business model for the shop is that investigators have access to shop services for free if (and only if) their research support pays part of the salary of the shop personnel. Ten years ago, 50% of the NEI Vision Core Grant investigators had access to the shops because they were members of the Department of Physiology. That percentage has dropped considerably, and only a few of the Core investigators support the shops with non-Core Grant funding sources. Therefore, their only means of access to the shops is through the Core Grant support for the shop personnel. Moreover, this support is the only means of access to the shops for vision scientists who are not in the Department of Physiology. Because of this demographic change, the business model of the Machine Shop changed from that proposed at the last Core Grant renewal in 2007, at the end of the first year of this past period (2008). From that point on. Vision Core Investigators have received service from the Machine Shop free of charge. Since the services provided by this machine shop are crucial to the research needs of many vision scientists, we propose to continue to provide salary support of the shop personnel to allow free access to all vision scientists at UCSF.

II. MORPHOLOGY RESOURCE CORE Research Plan of Morphology Core Much of vision science requires morphological research either as a means to accomplish experiments or as the definitive product of the research. Microscopic images of many kinds must be studied, acquired and quantified before publication or presentation of the studies. This new Morphology Core has been established to provide morphological resources of most kinds that are needed by vision scientists at UCSF, and it incorporates the former Confocal and Digital Microscopy Support Core. The establishment of the Morphology Core is in response to needs of vision scientists at UCSF, and based on a survey of needs, this Core will be extremely heavily used. The facilities and services provided include the following: slit lamp examination and photography, fundus photography and fluorescein angiography using a Micron III unit, optical coherence tomography (OCT), paraffin and plastic embedding, sectioning and staining, cryosectioning for immunohistochemistry, light microscopy (brightfield, darkfield, phase contrast, DIC and fluorescence) and photomicrography, electron microscopy (EM) and confocal microscopy. Of the 16 respondents to the survey of vision scientists, each of these functions had a minimum of 5 needing them, and 13/16 wanted standard H&E-stained paraffin sectioning (this included 6/8 respondents holding ROI grants). The need for morphological methods in vision science is great, so not surprisingly >50% of NEI Core Grants in the U.S. offer some or all of these services. [Note: All image analysis has been moved to the Imaging Core, so there is now a clear distinction between the Morphology (image and tissue acquisition) and Imaging (image analysis) Cores.] As noted below and in the Resources, the Core Director, Assistant Core Director and staff head of the Core (with a To Be Hired, experienced technician) are/will be fully capable of carrying out the various histological services provided by the Core, as well as instructing members of Core Investigators' labs in the methods and procedures required for those working with the Core equipment. A majority of the equipment will be in Dr. Matthew LaVail's laboratory (see Resources for sharing description) or in the confocal microscopy room, both of which are located adjacent to one another and immediately across the hallway from the Imaging Core. There will be close interaction between the Morphology and Imaging Cores for Core Investigators. In addition, there are several special image-acquisition resources at UCSF, described at the end of Resources. All of the instruction, equipment use and services provided by the Core will be free of charge to Core Investigators, except for the optional, special UCSF resources described at the end of the Resources section and, if desired, some EM services. For the past 19 years. Dr. LaVail and his staff have successfully used the San Francisco VA Hospital Pathology Cell Imaging Lab for some EM services, and we propose to use this model for the Core Investigators as follows: the Morphology Core will take fixed tissues from Investigators, postfix in osmium tetroxide, embed in epoxy resin, thick section (1pm) and stain tissue blocks. In consultation with investigators, the Core staff will trim the blocks to nearly the desired area for EM analysis, make drawings with camera lucida or take light micrographs of the area for final trimming, and then contact the EM staff at the VA Hospital. The blocks and drawings/photographs will be sent to the VA unit on the UCSF-VA Shuttle, where the VA staff will take them, do final trimming and ultrathin sectioning. The key personnel at the VA unit are familiar with retina and eye structures, so we have them take survey EMs of different magnifications, which are returned to us electronically. When we need different areas photographed or higher magnifications, we communicate by telephone or Email. Alternatively, we sometimes go to the VA unit and use the EM, ourselves. The VA facility can also do EM immunostaining and EM negative staining. All of these services are recharged to our individual grant accounts and are very cost-effective and time-saving..

DESCRIPTION (provided by applicant): The broad objective of this NEI Center Core Grant for Vision Research application is to facilitate study of the structure, development and function of the visual system in health and in blinding diseases, with the aim of preventing, mitigating or curing such diseases, or the restoration of lost vision, through the application of the most sophisticated available techniques. Four resource and service Cores will help achieve the broad objective, as follows: I. Imaging Resource Core (morphometric analysis; computer-aided image analysis; production of graphics for data analysis, presentation and publication); II. Morphology Resource Core (ocular imaging, including slit lamp photography, fundus examination and fluorescein angiography with Micron III, and optical coherence tomography; paraffin and plastic embedding, sectioning and staining; cryosectioning for immunohistochemistry; light microscopy and photomicrography, including brightfield, darkfield, phase contrast, DIC and florescence; electron microscopy; and confocal microscopy); III. Computer/IT Resource Core (programming for custom research needs; assistance in computer and information technology hardware and software selection, installation, instruction in use, maintenance and minor repairs); IV. Machine Shop Service Core (design, manufacture, maintenance and repair of specialized research instruments and devices using state of the art computer numerically controlled machines). This is a resubmission application of a NEI Center Core Grant for Vision Research competing renewal submitted by the Principal Investigator and 13 other vision scientists who hold 17 active NEI ROI research grants. In addition, the UCSF vision research community supported by the NEI Vision Core Grant comprises 6 NEI-supported scientists with grant mechanisms other than ROI, 2 with other NIH funding, 1 with FDA R01 funding and 7 with private funding. There are 30 Core Investigators with 33 active research programs, overall, each using at least one Core at a moderate or extensive level. Using traditional and innovative approaches, this Core Vision Research Grant has been highly successful and instrumental in enhancing the productivity and impact of vision research, attracting scientists to vision research and facilitating collaborative studies on the visual syste at UCSF.