The National Eye Institute J© Annual Report RscaJYear 1980 // 7-^/'S/ THE NATIONAL EYE INSTITUTE^ 6 -. 3 H M Retinal and Choroidal Diseases 15 10 49 $ 984* 52 $ 866 $1,850 40 Corneal Diseases 10 9 34 750 11 210 960 21 Cataract 3 8 98 7 122 220 5 Glaucoma 4 15 305 5 95 400 9 Sensory and Motor Disorders of Vision 11 19 33 678 24 485 1,163 25 TOTALS 43 38 139 $2 ,815 99 $1 ,778 $4,593 100 * $13,000 of this total represents NEI's co-funding of two T35s (short-term training program) under the auspices of the NIGMS for four predoctoral positions. 11 The FY 1980 appropriation for the NEI included $3,000,000 for research grants for construction of vision research facilities. The program was formally announced on September 19, 1980. Applications will be due in May 1981. The NEI has made several policy changes of interest to the vision research community. First, the NEI has adopted the NIH guidelines for the New Investigator Research Award (NIRA) program. Basically, this research grant program is intended to support young investigators in the very early stages of their careers. Because of the overall similarities between the NIRA guidelines and the NEI Academic Investigator Award program, we have discontinued use of the Academic Investigator Award mechanism. In addition, the guidelines for NEI Core Grants have been revised. A committee composed of members of the National Advisory Eye Council, a member of the Vision Research Program Committee, and NEI staff met over a six-month period to evaluate all aspects of the Core Grant program. The main concern was the potential for expansion of the Core Grant program at a time when funds for research grants were decreasing. The major policy changes are (1) the maximum amount the NEI will devote to the Core Grant program would be 5 percent of the extramural grant budget, (2) a dollar limit of $150,000 per year direct costs has been established, (3) the appropriateness of modules within a Core Grant has been greatly clarified, (4) review criteria have been better defined. Both of these policy changes will greatly enhance our ability to implement our research grant programs. Immunology Workshops The National Advisory Eye Council, in its 1977 report entitled Vision Research — A National Plan; 1978-1982 , identified the need for increased research effort on the immunological aspects of ocular diseases and for recent advances in the general field of immunology to be applied to the study of the visual system. To accomplish this objective, the Council believes that immunologists should be encouraged to become better acquainted with ocular tissue and systems in order that these researchers may exploit the eye as an immunological model. An attempt to stimulate discussion and collaboration among vision researchers and immunologists was initiated through the development of an immunology workshop series. These workshops were planned by the NEI staff with full assistance from members of the National Institute of Allergy and Infectious Diseases staff in the selection of appropriate conference partici- pants. The first workshop in this series, "Immunogenetics and Transplantation Immunity," was held on December 5-7, 1979, and identified potentially pro- ductive applications of recent advances in immunogenetics and transplantation immunology in eye research. The following specific recommendations were made by workshop participants: 1. The NEI should develop mechanisms for attracting immunologists to departments of ophthalmology and training them to carry out research on 12 ocular problems. Veterinary ophthalmologists should be encouraged to work with such immunologists to develop good animal models for basic investigations into ocular immunologic mechanisms. 2. Corneal transplantation studies: a. The antigens and cell types in each layer of the cornea which may participate in graft rejection should be defined. b. Organ specific antigens in the cornea should be studied. c. Investigations should be undertaken on the various mechanisms by which graft rejection can take place. d. Graft rejection studies should explore the effect of matching histocompatibility leukocyte antigens (HLA) at both the A and B locus. These antigens appear on the cellular surface of lymphocytes in the peripheral blood and are controlled by genes located at histocompatibility zones A and B on the two number 6 chromosomes in humans . 3. Autoimmunity and infections of the eye: Attempts should be made to define individual antigens responsible for ocular autoimmune diseases such as sympathetic ophthalmia and infectious diseases such as herpetic keratouveitis. i 4. True and false malignant lymphoid tumors of the eye: Investigators should determine whether lymphocytes (white blood cells) associated with these tumors are derived from the thymus, fetal liver and gut or lack markers from any of the above tissues; whether the lymphocytes arise from one clone of cells; and what function they perform. 5. Association of HLA antigens with ocular disease: a. The extent to which individual immune responses to defined ocular antigens vary should be studied. b. Epidemiological studies should investigate suspected associations of certain HLA antigens with ocular diseases. 6. Chemical and physical factors which lead to vascularization of corneal transplants and their resultant rejection should be delineated. Methods should then be sought to limit such vascularization. 7. The mouse should be adopted as a good immunogenet ic animal model for study of immunological insult to the eye. A second workshop entitled "Autoimmune Phenomena and Ocular Disorders" was subsequently convened on March 5-7, 1980, and provided promising avenues for— additional research in the immunological mechanisms which govern auto- immunity in relation to specific ocular diseases. 13 The research priorities developed by the workshop participants are listed below in rank order: 1. Much consideration should be given to the long-term training of both full-time immunologists involved in vision research and ophthalmologists engaged in research on the ocular immune response. A collaboration between these two classes of investigators should be encouraged. 2. Animal models should be recognized and developed, in particular those which represent spontaneous ocular autoimmune diseases. This may be achieved mainly through liaison with the veterinary community. In parallel, experimentally induced autoimmune diseases should be further developed, with the purified ocular antigens. 3. As many specific ocular antigens as possible should be isolated, characterized, and identified from experimental animals and, where feasible, from human subjects. 4. The possible associations between autimmune ocular diseases in man and histocompatibility (HLA) antigens should be further examined, along with population and family studies. In parallel, the immunogenet ics of ocular autoimmune diseases should be further analyzed in experimental animals with well-defined genetic makeup. 5. Longitudinal clinical studies should be conducted on patients identified as being afflicted with various ocular autoimmune diseases. Estab- lishment of the abnormal features in these patients through all phases of their diseases should provide helpful information concerning the respective pathogenic mechanisms. 6. The pathogenic mechanisms involved in autoimmune ocular diseases should be further investigated, both in man and in experimental animals. Better knowledge in this research area should bring about better approaches to therapy. 7. New approaches to immune modulation should be applied to ocular immune diseases such as the use of immunosuppressive agents, suppressor cells, and their products and specific ant i-idiotype immunity. The third and final workshop in this series, "Infection, Inflammation, and Allergy," was held on June 25-27, 1980, and represented the completion of the NEI initiatives in the field of immunology for FY 1980. This workshop provided a forum for discussion of potentially promising approaches to minimizing the destructive effects of inflammation associated with ocular disease while maximizing the protective effects of the inflammatory process. The following specific recommendations were formulated by workshop participants: 1. Early biochemical events in ocular inflammatory disease(s) requires investigation. Chemical mediators need to be identified which can regulate chemotaxis of inflammatory cells and function as causal agents 14 for neovascularization in both the cornea and the retina. The events in the inflammatory response should be differentiated so that components deleterious to the host may be avoided. 2. Development of naturally occurring and/or genetically defined animal models of human ocular infections, inflammatory and allergic diseases should be encouraged. Increased recognition and promotion of the use of such animals should be fostered. 3. Support should be provided for investigations of the primary defense mechanisms exhibited by ocular mucous surfaces to elucidate which cellular events in barrier disruption lead to inflammation and ultimate structural damage. 4. The effects of specialized nonlymphoid inflammatory cells including mast cells and eosinophils should be determined in human and animal model systems of inflammation. 5. Studies should be continued on the evaluation of the individual types of interferon alone, in mixtures, and in combination with antiviral drugs on such ocular diseases as recurrent stromal herpes keratitis, herpes ziysteir, and adenovirus infections. 6. The potential use of ant i- idiot op ic antibodies either directly or through 'the subsequent generation of appropriate suppressor cells for therapy (and possible prevention) of ocular infections, ocular tumors, and uveitis should be explored. These specific stimulators should also be utilized in development of diagnostic techniques for specific ocular inflammatory diseases. 7. The development of knowledge about the chemical nature of glucocorticoid receptors and the modification of such receptors through drug action should be pursued in ocular tissues. 8. An increased knowledge of nonsteroid drugs which are capable of inhibiting individual rate limiting steps in the inflammatory process in needed. 9. Drug delivery systems which either increase the penetration or perme- ability of drugs through topical application to the eye or through subconjunctival implantation are in urgent need of development. 10. New antiviral drugs should be developed, especially those compounds which can eradicate herpes virus at the ganglion level. 11. Evaluation of new antibiotics and antifungals for treatment of ocular disease should be carried out both as to increased ocular penetration and pharmokinetics as well as toxicity of such compounds. Upon completion of the revision and editing of the individual manuscripts in each workshop, the proceeding of that workshop will subsequently be published as a special supplement to the journal Immunology Abstracts in 1980 and 1981. Copies of the three-volume set of publications will be provided to each of the workshop participants and^:o interested peraona in -the vision and immunology communities through appropriate journal announcements. 15 RETINAL AND CHOROIDAL DISEASES Introduction Retinal and choroidal diseases are the leading causes of blindness in the United States. These diseases interfere with communication, seriously restrict employment opportunities and limit mobility for a large segment of our population. Further evidence of the devastating effects of retinal diseases is that ophthalmologists consider them to be the most difficult ocular disorders to treat, and the most in need of basic as well as clinical research. Diseases of the retina are diverse in origin and the research programs to attack them are complex. In previous years, the subprogram's activities, as outlined in Vision Research - A National Plan: 1978-1982 , were divided predominantly into fundamental and clinical studies. A more appropriate division which demonstrates the interrelationship between basic and applied research is under consideration by the Retina Program Planning Panelists. As proposed, the retina program would be divided into four comprehensive sub-programs to include: A. Vascular, Neoplastic and Inflammatory Disorders of the Retina and Choroid o Diabetic Retinopathy, Other Vascular and Circulatory Disorders and Retinopathies o Inflammatory Disorders o Tumors B. Degenerative Disorders of the Retina o Developmental and Hereditary Disorders o Macular Degeneration o Retinal Detachment, Vitreous Disorders and Trauma C. Fundamental Processes and Retinal Disorders o Photoreceptors, Visual Pigments, and Transduction o Interaction between Visual Cells and Pigment Epithelium o Retinal Organization, Neurotransmission, and Visual Function o Non-Invasive Techniques in the Study of Retinal Disorders D. Special Areas of Future Interest o Toxic, Nutritional and Environmental Disorders o Low Vision and Myopia o Retinal Regeneration and Glial Cells and the Retinal Environment o Tissue Acquisition and Distribution: Human Donor Eyes and Animal Models 17 The NEI's Retinal and Choroidal Diseases Program supports a cadre of research scientists from the basic and clinical areas such as biochemistry, biomedical engineering, cell biology, electrophysiology , epidemiology, molecular biology, genetics, immunology, molecular biology, neuropharmacology, ophthalmology, optometry, pathology and psychophysics . They are all striving to prevent, diagnose, and understand the mechanisms of visual disorders, and to improve the quality of life through treatment of these diseases. In FY 1980, the Extramural and Collaborative Program's Retinal and Choroidal Diseases Branch has been responsive to the objectives outlined in Vision Research - A National Plan: 1978-1982 . Although significant advances have been made in most subprograms, the staff has decided to highlight, in the annual report, progress in the following areas: diabetic retinopathy, allergic uveitis, hereditary and other retinal degenerations, ocular tumors, photoreceptor renewal, and visual transduction. Also included in this are the recommendations of the Ocular Melanoma Task Force which convened April 17-18, 1980 at the National Institutes of Health to evaluate recent literature on ocular melanoma and to recommend research initiatives aimed at the improved diagnosis and treatment of this disease. Diabetic Retinopathy Diabetic retinopathy, besides causing severe visual impairment, is one of the major leading causes of new cases of blindness in the United States. It is characterized pathologically by excessive leakage of fluid from retinal capillaries, microaneurysms, occlusion of retinal vessels, and proliferation of newly-formed blood vessels and fibrous tissue along the inner surface of the retina. Impaired vision results from: o Leakage from capillaries into the macula causing swelling and distortion with impaired reading vision, sometimes progressing to legal blindness. o Impairment of retinal blood supply leading to loss of part of the visual field, including reading vision when the macula is involved. o Bleeding from the newly formed blood vessels into the vitreous and detachment or distortion of the retina caused by contraction of the fibrous tissue and resulting in total or nearly total blindness. The NEI has utilized a comprehensive approach to reduce or eliminate the ocular complications from diabetes which involves testing the safety and efficacy of various therapies, determining risk and protective factors, and understanding how the observed pathological changes result in visual disorders. 18^ In 1973, 46 percent of ophthalmologists surveyed considered retinal research to be the most inadequately developed, especially research on heredi- tary retinal and macular degenerations and diabetic retinopathy. In just seven years, intensive research in the area of diabetic retinopathy has made the latter part of the previous statement obsolete. The use of randomized controlled clinical trial methodology to determine the safety and efficacy of photocoagulation in treating diabetic retinopathy is without doubt one of the major accomplishments in ophthalmic research of this decade. The Diabetic Retinopathy Study (IIR&) showed that photocoagulatioa treatment can reduce the risk of severe visual loss by 60 percent in people with moderate to severe diabetic retinopathy. It is estimated that 300-300 thousand known diabetics have retinopathy serious enough to warrant consider- ation for this treatment. Photocoagulation is now offered at-inany hospitals and most university medical centers throughout the United States. The safety and efficacy of this technique for use by practicing ophthalmologists is assured due to the identification of four risk factors which indicate when a patient is at high risk of developing severe visual loss. Thus, this information can be used to determine when to photocoagulate eyes of patients with diabetic retinopathy. Since the above mentioned studies were performed in people with moderate to severe diabetic retinopathy, some clinical researchers have postulated that earlier treatment of diabetic retinopathy by photocoagulation may slow or even prevent visual loss. Thus, a new multicenter trial, the Early Treatment Diabetic Retinopathy Study was begun in 1977 and if this study is as success- ful as the DRS, it may be possible to enhance the quality of life for a greater number of people with diabetes by maintaining their visual function. Restoring sight or decreasing the progression of visual impairment through the application of photocoagulation could not have been achieved without studying a large, well-defined population of .patients whose ophthal- mologists were convinced that the only ethically and scientifically feasible way to determine efficacy of this treatment was through a randomized con- trolled clinical trial. The ultimate success of photocoagulation therapy will depend on ophthalmologists and patients with diabetic retinopathy being aware of its usefulness and the safe application of this technique by all ophthalmologists. The potential impact of the DRS on the lives of many who would have otherwise been visually impaired or blind for a significant portion of their adult lives points to the importance of further clinical research on diabetic retinopathy and related diseases. Other randomized controlled clinical trials which currently receive NEI grant support are: Branch Vein Occlusion Study . The objective of this study is to determine whether argon laser photocoagulation can (1) prevent the development of neovascularization, (2) prevent vitreous hemorrhage in branch vein occlusion patients with neovascularization and (3) improve or maintain visual acuity in patients who have macular edema secondary to branch vein occlusion. 19 Macular Photocoagulation Study . The objective of this study is to determine whether the use of argon laser photocoagulation to obliterate extrafoveal choroidal neovascularization is of value in preventing perman- ent loss of central visual function in patients with senile macular degen- eration and in patients with presumed ocular histoplasmosis. Photocoagulation in Disciform Macular Disease . The objective of this study is to assess the therapeutic value of argon laser photocoagulation in the disciform process of senile macular degeneration. While research on the treatment of diabetic retinopathy will continue in the 1980s more attention will be directed toward prevention, early diag- nosis, and understanding the underlying mechanisms in this disorder. Epidemiological studies are extremely important in preventive medicine because they can help determine the magnitude of the problem, provide a pro- file of the susceptible person, and determine what risk or protective factors are associated with the disorder. Klein at the University of Wisconsin analyzed the medical records of over 5,000 diabetic individuals from southern Wisconsin. He recorded a bimodal distribution for age of onset of diabetes with peak frequencies in the 10-14 year age band and the 60-64 year age band; diabetes was diagnosed in approximately fifteen percent of the population before the age of twenty-five years. These results confirm previous studies on the prevalance of diabetes. At the University of Minnesota, Ramsey and his colleagues are testing the hypothesis that susceptibility to microangiopathy in diabetics is geneti- cally determined. The group is trying to determine whether one or more "microangiopathy genes" are linked to histocompatibility (HLA) genes. It has been well substantiated that diabetic patients with microangiopathy are more often positive for HLA B8, BIS and B8/15 and that normal volunteers are more often positive for HLA B7 and B12. When the HLA antigens from juvenile insulin dependent diabetics (IDDM) with non-proliferative diabetic retinopathy are compared with the HLA antigens from diabetics with proliferative diabetic retinopathy (PDR), patients with PDR are less often positive for HLA B7 and significantly more often positive for HLA B15. These observations suggest that HLA B15 may be a susceptibility marker for PDR. Since several autoimmune disorders in humans have been associated with HLA B8 and there is evidence for abnormal immune mechanisms in some cases of IDDM, it is important to identify the allele primarily associated with dia- betes mellitus in humans. Ramsey and his colleagues studied the interrelation between IDDM and the HLA B8 and the D locus in forty one unrelated patients. They demonstrated significant increases in the frequencies of HLA B8, BIB and Dw3 and significant decrements in the frequencies of HLA B7 , B12 and Dw2 in patients with IDDM. The investigators concluded that the HLA B8 excess seen in diabetics was secondary to the excess of HLA Dw3 , thus indicating that the pathogenesis of the disease is closely associated with the D locus of the major histocompatibility system. 20 Bresnick at the University of Wisconsin is employing a non-invasive electrophysiologic technique to identify eyes that are at high risk of developing PDR. Preliminary data from diabetic patients with retinopathy ranging from mild non-proliferative to proliferative showed a reduction in ERG oscillatory potentials with advancing retinopathy. This recently initiated study also has the potential for helping identify candidates for vitreous surgery from among diabetic patients with vitreous hemorrhage. For the surgeon, evaluating patients with cloudy media by existing methods is difficult, and such a diagnostic technique would be extremely useful. The blood-retinal barriers (BRB) appear to be located at the endothelium of the retinal capillary and at the retinal pigment epithelium; in diabetic retinopathy the BRB is disrupted. Neither the basic mechanism of disruption nor the factors responsible for its repair have been determined. However, researchers supported by NEt are actively seeking answers to this problem. One approach is to compare the morphology of normal retinal vessels with that of abnormal retinal vessels. Frank at Wayne State University is using-the scanning electron microscope to study small retinal blood vessels. Initial observations of retinal vessels from a patient with proliferative retinopathy revealed a bulbous crenulated microaneurysm with several attached acellular capillaries whose basement membrane showed multiple ridges and furrows, rather than a smooth surface. This technique will be useful in determining the architecture of retinal blood vessels and in correlating morphology with biochemical and physiological changes in micro- angiopathy. Wallow at the University of Wisconsin is examining the morphology of the BRB by electron microscopy. He found that in the new vessels, the blood-retinal barrier was altered by the formation of fenestrae bridged by diaphragms. Occasionally, tight junctions between endothelial cells appeared widened. Thus, the characteristic "leakiness" observed clinically by fluorescein angiography may be caused by fenestrae and incompetent tight junctions. 9 Some evidence is emerging from the works of Peczon and Mukai in Boston that the alterations in basement membranes in diabetic retinal vessels may include not only a thickening of the basement membrane but that the cellular metabolism may also be different from the retinal vessels of non- diabetics. Autoradiographic studies of the basement membrane of normal and streptozotocin-induced diabetic rats showed that the basement membrane had increased perivascular collagen fibrils. How the BRB in diabetic eyes recovers after chronic photocoagulation was examined by Wallow and Davis. In donor eyes from diabetics treated with photocoagulation, they found that burns of 0.5 seconds or longer resulted in complete destruction of the neurosensory retina with only the retinal pigment epithelium providing the resource of repair to bridge the retinal defect created within the center of the burn; the pigment epithelium cells were often devoid of pigment. Following less severe burns from argon photocoagulation of 0.1 second or less, retinal glial cells participated in the reparative process 21 together with the pigment epithelium cells and the pigment-laden macrophages. Such information is extremely important since it can be utilized to correlate safe levels of laser energy with efficacy of treatment. Scientists are just beginning to probe into the mechanisms responsible for description of the blood-retinal barrier in diabetic patients. A thorough analysis of the problem requires expertise in such disciplines as cell biology, morphology, physiology, biochemistry and endocrinology. To facil- itate the process, the RCDB is planning to convene a multi-disciplinary task force in the Spring 1981 to discuss research needs in the area of diabetic retinopathy. This activity will complement the NEl's third program planning exercise currently underway. Hereditary and Other Retinal Degenerations Hereditary and developmental disorders are responsible for an estimated thirty-three percent of all blindness among school children in the United States. Presently, little can be done to prevent the onset and progression of most or these disorders, however, jeveral researchers within the past year have made significant contributions in early detection of the disorder, identification of genetic carriers and evaluation of nutritional therapies. Retinitis Pigmentosa . Light-evoked electrical responses from the human eye recorded at the cornea have provided criteria for evaluating the func- tional integrity of specific layers of the human retina. The a-wave and b-wave depend on both rod and cone systems. The c-wave depends on the rod system and a healthy pigment epithelium. Thus, electrophysiological tests can be utilized to determine cell layer interaction and normalcy of cells. Approximately 35 percent of patients with retinitis pigmentosa (RP) are males with no family history of RP, or males with one or more affected male relatives and no affected female relatives. Berson and his colleagues at Harvard University were able to show that the full field electroretino- grams (ERGs) of female carriers were either reduced in amplitude to white light under dark adapted conditions or delayed in cone b-wave implicit time, or both. Daughters of carriers had either normal ERGs or abnormal ERGs similar to those recorded for carriers. Thus, ERG testing of female relatives of males with retinitis pigmentosa can establish for a given family whether the mode of inheritance is X-chromosome linked or autosomal recessive. Preliminary data from Corwin's laboratory at the University of Rochester suggest that some psychophysical tests can be used to discriminate between normal volunteers and patients with RP. A procedure for measuring two- flash reso).ution was developed and implemented using a microcomputer-con- trolled interactive tracking procedure. The mean threshold inter-flash interval for a normal population was 60.0 msec compared with 79.2 msec for one confirmed RP patient. This test yielded reliable results when administered to inexperienced subjects and, it may have potential use for large-scale visual screening of a population at risk. 22. Berson and his group are attempting„to identify abnormal structures in the retinal cell layers of RP patients. The postmortem histological analysis of an eye from a patient with a slowly progressing form of KP (visual acuity of 20/300) revealed a reduced number of cones with shortened outer segments in the fovea and some rods on the fovea slope, the absence of photoreceptors in the mid periphery, and preserved rods and cones anterior to the zone of bone spicule pigmentation. The pigment epithelium cells adjacent to the photoreceptors in the central retina contained decreased melanin, and autophagic vacuoles in the cone photoreceptors. It has been shown that patients with some types of mucopolysaccharidoses have RP. The levels and kinds of glycosaminoglycans (GAGs) were measured in a pigment epithelium cell line derived from a patient with X-chromosome-linked RP. The GAGs secreted by these cells were similar to that secreted by normal cell lines. Thus, a defect in GAG metabolism appears not to be associated with X-chromosome-linked RP. Gyrate Atrophy . Gyrate atrophy of the choroid and retina (GA) is an inherited progressive chorioretinal degeneration. The vi sual ab normalities in GA are characterized by the onset of myopia and decreasing peripheral and night vision in late childhood or early adolescence. Progressive destruction of the visual fields results in tunnel vision and eventual blindness by the age of fifty. Metabolic studies have shown that GA patients have a deficiency in the enzyme, omithine-amino-transf erase (OAT). Since this is an inheri- table disease, researchers have been developing sensitive and reliable assays for detecting GA and determining whether a particular form of GA can respond to therapy. O'Donnell at the University of California in San Francisco and Valle at Johns Hopkins University have demonstrated that cultured cells from GA patients have decreased levels of OAT. In addition, the former investigator was able to assay cells obtained by amniocentesis for OAT activity. Thus, this assay can potentially be used in genetic counseling. Detecting the different mutations leading to OAT deficiency is essential in understanding variability among the clinical manifestations of the disease. Valle and his colleagues have developed a genetic complementation assay to test for different mutations which involves hybridizing fibroblasts from different patients with GA and measuring the recovery of OAT activity. Fourteen cell lines have been tested so far and none were shown to be comple- mentary. Thus, all tested cell lines appeared to have allelic mutant genes. Valle and Kaiser of the National Eye Institute's Clinical Branch have treated a small number of GA patients by diet modification. Eight patients have been on arginine-deficient diets from 3-28 months and have had a 2-6 fold reduction in plasma ornithine. One patient who had been on the diet for over 24 months has shown improvement in dark adaptation, ERG and visual fields. In another GA patient who is vitamin B^-responsive, daily doses of vitamin B, were effective in lowering. serum ornithine levels with a con- comitant improvement in ERG response. 23 Animal Models . Animal models which mimic human disorders are valuable resources to the investigator. Three models of hereditary retinal degenera- tions have been studied extensively: the dog model (specifically Irish setters, Norwegian elkhound and miniature poodle), the Royal College of Surgeon (RCS) dystrophic rats, and several mutant mouse genotypes. A mutant mouse, pcd / pcd , in which photoreceptor cells degenerate slowly over the course of a year is bging studied by La Vail at the University of California, San Francisco. Rods were found to degenerate faster than cones. In the two month old mutant mouse, the rate of rod outer segment disc synthesis and the disc shedding values after light onset were approximately one-half that of the values obtained in control mice. 19 Using the dog model, Aguirre at the University of Pennsylvania is studying the kinetics of rod outer segment renewal in Irish setters with rod-cone dysplasia. He has demonstrated a lack of synchronous production of new rod outer segment discs at the time normal photoreceptors are elongating. In addition, no defect in phagocytosis by the pigment epithelium was detected. The abnormally high levels of cGMP in the retinas of dogs with rod-cone dysplasia results from the lack of photoreceptor-specif ic phosphodiesterase (cGMP-PDE). The activity of this enzyme can be regulated by calmodulin, a calcium-dependent protein activator. In examining the relationship between calmodulin and cGMP metabolism during photoreceptor differentiation, Aguirre and his colleagues found that in the normal retina, cGMP-PDE changes from calmodulin-dependent to calmodulin-independent , whereas in affected dogs, cGMP-PDE fails to become calmodulin-independent with aging. These observations suggest that the disease may be modulated or corrected by delivering calmodulin to the visual cells. Ultrastructural and biochemical studies have been performed on the retinas of RCS rats to define the cellular and molecular events of retinal degeneration. La Vail, demonstrated that changes occur in the distribu- tion and quantity of mucopolysaccharides of the interphotoreceptor matrix in RCS rats before cell death; such changes may be involved in the mechanism of photoreceptor cell death in these animals. A decline in opsin synthesis was the only detectable change in protein synthesis observed in RCS retinas. . Renewal Processes in Photoreceptor Outer Segments The shedding of photoreceptor outer segments and subsequent phagocytosis by the retinal pigment epithelium have in recent years been established as normal events in the vertebrate retina. A reasonable hypothesis is that disturbances in the delicate balance between outer segment degradation and reassembly may underlie several forms of retinal degenerative disease. Investigators have increasingly directed their research towards better understanding those processes which maintain or disrupt this balance. NEI intramural scientists and grantees have been the leading investigators in this promising area of research. 2it> 22 23 The classic studies of Young ' established that rod photoreceptor outer segment membranes are not static structures, but structures which are continually renewed. Despite structural differences between rod and cone outer segments, it is now clear that renewal is a universal feature of vertebrate photoreceptors. ' La Vail discovered that rod outer segment shedding was not a con- tinuous, random process, but was a rhythmic event synchronized by the daily light cycle. This unexpected finding galvanized new research efforts into all aspects of this phenomenon. Studies by Young, Bunt, and Anderson and Fisher later demonstrated the cyclic pattern of shedding in cone outer segments. The timing of outer segment membrane shedding by rods and cones, however, is 12 hours out of phase. Rod shedding occurs near the beginning of the light cycle; cone shedding occurs at^ the^ beginning of the dark cyde^ Regular cycles of disc shedding continue in rats for at least 12 days in constant darkness. Thus, the light/dark cycle in mammals may entrain an endogenous circadian rhythm which more directly controls the sheddine process. Constant light dramatically reduces rod outer segment disc shedding ' ' indicating that a dark-dependent control process is operative as well. The circadian nature of the outer segment shedding process has prompted a search for extraocular control mechanisms. The pineal gland was implicated in early experiments by La Vail ' who discovered that reserpine (a drug known to block other circadian rhythms by depleting pineal norepinephrine) abolished shedding. However, shedding was quickly shown to continue after surgical removal of the pineal gland, the superior cervical ganglion,, the pituitary and parathyroid gland and after the cutting of the optic nerves. In both frogs and rats it has been demonstrated- in eye-patching experiments that shedding may be initiated monocularly. ' Finally, in perhaps the defin- itive experiment, Besharse et al . have established culture conditions for open frog eye cups in which it is possible to demonstrate light-evoked disc shedding. Taken together, these observations clearly indicate that the major controlling processes of the shedding phenomenon are intrinsic to the eye. This discussion has focussed on the degradative aspects of the photo- receptor renewal process. But, it is now evident that photoreceptor membrane assembly, and RNA^ R^°£f^2* ^^'^ glycoprotein synthesis are also influenced by the photoperiod. ' ' ' Photoreceptor renewal emerges from these studies as the end product of a series of complex, highly-orchestrated meta- bolic processes. Promising new approaches to the study of retinal degenera- tive diseases have been made possible. Visual Transduction in Vertebrate Rod Photoreceptors A central problem in visual photoreceptor physiology, and in the physi- ology of all excitable tissues, remains unanswered: What mechanisms bring about the generation, amplification, and modulation of transmembrane ionic currents? Specifically, vrtiat process links the absorption of light by rhodopsin molecules in the rod disc membrane with the conductance mechanism of the physically separate plasma membrane? Recent research, much of it 25 conducted by NEI grantees, has led to important findings which bear on this problem. Two salient points are generally agreed upon. A transmitter (unknown) must link the site o£ photon absorption and the plasma membrane. Secondly, many sodium channels are inactivated by a single photon absorption, that is, an amplification process takes place. 44 Hubbell and Bownds have recently reviewed the two general schemes which researchers have used to account for these phenomena. "The calcium hypothesis" and similar models depend upon compartmentalization of the transmitter by the disc membrane and the participation of rhodopsin in trans- membrane processes. Alternatively, enzymatic schemes have been proposed which do not depend on transmembrane phenomena and involve only events at the membrane surface. The calcium hypothesis holds that calcium accumulates within the discs in the dark-adapted state and is released into the cytoplasm upon rhodopsin activation. The released calcium is translocated in such a way as to inhibit sodium conductance. Indirect evidence in favor of the calcium hypothesis is strong. Fung and Hubbell by comparing the lactoperoxidase catalyzed iodination and papain proteolysis of rhodopsin in native and reconstituted disc membranes have unequivocally demonstrated the transmembrane orientation of rhodopsin. Hargrave and his associates have provided other evidence, which relates to the transmembrane organization of the rhodopsin molecule. ' The results of photochemical labeling experiments with membrane impermeable probes indicate that the rhodopsin carboxy-terminal region is located at the cytoplasmic (external) surface of the disc membrane while the amino terminus is located at the intradiscal membrane surface. Thus, rhodopsin is physically situated within the disc membrane in such a way that gated calcium channel functions or other transmembrane functions may be mediated. While several experiments have indicated that calcium participates in the excitatory process, direct evidence of an amplified release of calcium is lacking. Brown and coworkers have shown that an intracellular injection of calcium hyperpolarizes the photoreceptor, mimicking the effects of light. Several groups have ^Ru^d that calcium chelating agents reduce photoresponses of rod receptors. ' Many investigators have demonstrated a light activated release of calcium from native and reconstituted disc membranes. The estimated amounts of calcium released, however, do not exceed 1.0 calcium ion per activated rhodopsin molecule. Amplification of the calcium response in these systems has yet to be demonstrated, and may in vivo, depend on intact rod structure. An alternative explanation holds that calcium release sets in motion a cascade of events which result in the amplified attenuation of sodium conductance. 26 The second type of scheme which has been proposed to account for trans- mission and amplification involves enzymatic processes. Here cyclic 3' , 5' guanosine monophosphate (cGMP), present in unusually high levels in photo- receptor outer segments, has been proposed to play a central role in trans- duction. A number of investigators have shown that light brings about a dramatic reduction in the intracellular cGMP concentration and that the reduction is largely mediated by phosphodiesterase. Woodruff et al. have established a number of correlations between cGMP levels and permeability changes in rod outer segments. ' Bleaching of, a single rhodopsin molecule was found to bring about the disappearance of 10 -10 molecules of cGMP. The half-time of the decrease in cGMP was about 125 milliseconds (msec.) and the latency was less that 50 msec. A steady-state level of cGMP was reached within seconds of illumination and varied as a function of the light intensity. Changes in cGMP levels and the suppression of plasma membrane permeability occurred over the same range of light intensity, and were similarly affected by pharmacological agents. Changes in cGMP levels occur rapidly enough and with sufficient amplifi- cation to be involved in the rod phototransduction process. Miller and Nicol have recently reported, in support of this possibility, that intra- cellular injection of cGMP increased both the latency and amplitude of the rod light response. It is also entirely possible that cGMP may subserve some other function, possibly visual adaptation. Whether cGMP is the transmitter which links photon absorption with perme- ability changes of the rod plasma membrane or whether, as now seems likely, other transmitters (6*2. , calcium) or steps are involved remains to be proven. Yau, Lamb, and Baylor have introduced a novel "suction" electrode tech- nique, making possible the study of the photoresponses of single photoreceptor outer segments. It should soon be possible to compare the biochemical measure- ments here reviewed with rod outer segment conductance measurements — an excit- ing experimental opportunity for investigators in the field. Allergic Uveitis The importance of immunology in ocular diseases has been emphasized by NEI in its sponsorship of three immunology workshops this fiscal year. The purpose of these workshops was to bring together eminent immunologists and ophthalmologists to discuss the state-of-the-art in immunology. NEI grantees within RCDB participated in these very important workshops. Currently, the NEI supports eighteen grants in its Retinal and Choroidal Diseases program related to the immunology of the eye for a total of $1.1 million dollars. Allergic uveitis is a non-infectious inflammatory disease of the uvea and is thought to arise from an immunologic insult. The principal causes of blindness from inflammatory diseases of the retina and the choroid are destruction of the photoreceptors in the macula, damage to the nerve fibers that transmit visual impulses to the optic nerve, and opacification of the vitreous overlying the inflammatory lesion. 2^ The etiology of allergic uveitis is still unknown. However, an inmune response to ocular antigen has been detected in some patients with uveitis. Wacker and his co-investigators at the University of Louisville in Kentucky, along with Nussenblatt, Gery and Ballintine from the NEI's intramural program, have shown that a soluble retinal antigen (S-antigen) purified from human and bovine retinas can stimulate the lymphocytes of some patients to divide. This phenomenon was observed in patients who had both active and inactive retinal lesions. Some patients with posterior uveitis responded to crude retinal extracts but not to the purified S-antigen, indicating that other retinal antigens may also be involved in posterior uveitis. Neither control subjects nor patients with anterior uveitis manifested a positive response to the S-antigen. These responses may play some role in the pathogenesis of the disease. The question of whether the immune response is the cause of or the result of inflammation remains to be answered. Other researchers are using animal models of experimental allergic uveitis (EAU) to investigate the possible immunologic etiology of allergic uveitis. Three animal models for posterior uveitis are presently being studied: (1) a rhodopsin-induced guinea pig model, (2) an S-antigen-induced EAU rabbit, and (3) guinea pig models. Wacker was able to show that the dose of S-antigen used to induce uveitus in the rabbit was associated with the severity of the pathologic response. High doses of S-antigen (50 micro- grams) produced an acute and severe inflammation including fibrinoid necrosis of the retinal vessels whereas low doses (10 micrograms) produced a less severe inflammation with mononuclear infiltrates. Meyers-Elliot at the University of California, Los Angeles, has induced EAU in guinea pigs by injecting them with 500 micrograms of purified bovine rhodopsin in complete Freunds' adjuvant. The retinal pathology of EAU was character- ized by destruction of the photoreceptor cell layer. At no time was a substantial cellular infiltrate noted in the retina. Wacker and his co-workers showed that guinea pigs sensitized with disc membranes developed clinical symptoms of EAU between 3 and 6 weeks. Histologically, these animals exhibited a mild non-granulomatous choroid- itis with little or no involvement of the anterior uvea segments. Silverstein and co-workers at Johns Hopkins University are investiga- ting the immune response in inflammatory ocular disorders. They have recently described an intraocular immune response accompanying a Ijnnphokine-induced [Sea Star Factor, (SSF)] inflammatory response in rabbits. The SSF produced earlier and more devastating changes in the retina and the uveal tract than the control antigen, ovalbumin. The time of appearance of these abnormal changes suggest that SSF produced far more inflammation than could be due simply to its immunogenic properties alone. A series of lethally x-irradiated rabbits developed neither retinitis nor vitritis during the early post-inoculation period. Thus, the damage seen by SSF may be attributed to its ability to attract and to activate macrophages with resultant destruction of ocular tissue. 29 Tumors Ocular tumors are the only eye disorder which can result in death. Tumors of the eye arise principally in the retina and the uvea, although some originate from other areas such as the lids, conjunctiva, and orbit. Retinoblastoma is the most common ocular tumor among children; it rivals neuroblastoma as the most common congenital tumor. According to various studies in the United States and in Western Europe, retinoblastoma occurs at the rate of one per 23,000 live births. The most alarming feature of retino- blastoma is that it appears to be increasing in frequency. Although these tumors can be treated with up to ninety percent effectiveness by enucleation, blindness and a reduced quality of life are inescapable. There is some evidence that hereditary fragile chromosomal regions (the long arm of chromosome 13) may be related to the development of retino- blastoma. Murphree at Children's Hospital in Los Angeles is attempting to define the linkage between a marker enzyme, esterase D chromosome 13, and the hereditary forms of retinoblastoma. The clinical application of linkage analyses can be used for genetic counseling and for early identification of persons who are at risk of developing the disease. Albert and his colleagues at the Massachusetts Eye and Ear Infirmary in Boston have demonstrated that fibroblasts from patients with hereditary retinoblastoma are more sensitive to killing by x-ray than those from patients with sporadic retinoblastoma or normal volunteers. These studies offer preliminary evidence that x-ray sensitivity may be related to a deletion observed in a specific region of chromosome 13. The role of the immune system in preventing tumors or containing their growth is poorly understood. Albert is studying a lymphocyte-mediated cytotoxic response to human retinoblastoma cells. The assay system in- volves the use of human retinoblastoma derived cells (cell line Y-79) in a 14-hour radioactive chromium release assay. Lymphocytes from healthy donors showed greater cytotoxic activity against the Y-79 cells than did lymphocytes from patients with retinoblastoma; these results would suggest some type of immune surveillance in healthy donors. The treatment of ocular tumors is an area of interest to several vision researchers because of the tumor's potential to cause vision loss and death if metastasis occurs. The application, of drug delivery systems for chemotherapy is being explored by Liu in Boston. He is experimenting with a silicone balloon injected with 1,3-bis (2-chloroethyl)-l nitrosourea (BCNU) and implanted in the episclera of rabbits who have developed Greene's malignant melanoma. In ten eyes so treated, tumor growth was delayed, and eight of the ten eyes retained normal size and shape thirty days after treatment. Sery at Wills Eye Hospital in Philadelphia has employed a novel form of therapy, photodynamic inactivation, to treat rabbits whose eyes have^successful implants of retinoblastoma or Greene's malignant melanoma. This approach 29 is based on the theory that in the presence of visible light, some porphyrins release molecular oxygen causing photoxidative damage to a biological system. Since a variety of neoplasms preferentially take up hematoporphyrin and hematoporphyrin derivatives, the feasibility of such a technique being useful appears good. Data exist in the literature which support the effectiveness of photoinactivation in treating tumors in both animals and humans. Using the rabbit model, Sery had shown that tumor death occurs in animals sensitized with hematoporphyrin for three days prior to irradiation with a cold red beam at 620-640 nm from a dye laser. He and his clinical associates have received approval from the Food and Drug Administration to apply this therapeutic procedure to human tumors. Malignant melanomas of the uvea are the most frequent type of ocular tumor and comprise eighty percent of ocular malignancies in adults. Differ- ential diagnosis is difficult and the appropriate management of these tumors is controversial. In addition, little is known about the epidemiology, etiology, natural history or biology of the tumor. One of the major activ- ities of the RCDB this year was to assemble a task force to recommend research initiatives in this area following evaluation of the literature. A summary of its recommendations follows. Epidemiology of Ocular Tumors . o Conduct demographic studies to examine risk factors, such as socio- economic group, occupation, geographic locale, latitude, eye, skin and hair color, exposure to ultraviolet light, and presence of cutan- eous melanomas. o Collect complete family history data. o Use appropriate control groups for data analysis. Biology of Ocular Tumors . o Investigate the etiology of ocular tumors, including the role of environment, chemical, genetic and viruses. o Establish ocular melanomas cell lines, study the growth prop- erties of tumor cells in culture, and investigate factors which influence the growth rate of tumor cells. o Determine the oncogenic potential of various cell types and lesions. o Characterize tumor specific antigens and determine markers of oncogenity. Diagnosis of Ocular Melanomas . o Develop improved non-invasive methods for sizing tumors and determining growth rate in vivo. o Develop non-invasive methods to differentiate in vivo between malignant melanomas and nevi . 30 Pathology of Ocular Melanomas. o Standardize clinical and pathological terms. o Standardize terminology for classifying lesions. o Define methods to predict the prognosis from tumor pathology such as staging of tumors, o Determine which cell types are associated with malignancy. Natural History Studies . Set up regional centers to collect data on patients with melanomas using a standardized form. The type of data collected would include: o Time when tumor was initially diagnosed. o Tumor size. o Growth rate of tumor. o Description and location of lesion. o Survey of all cutaneous surfaces for the presence of melanomas. o Complete metastatic work-up including spleen and liver scans, liver function tests, general physical examination and chest x-ray. o Type of intervention, if any. Prospective Studies . The task force suggested that a randomized controlled clinical trial be conducted using as the study group ocular melanoma patients who present with extrascleral extensions. The suggested type of intervention was surgery with newer forms of adjunctive therapy such as thymosin, interferon or transfer factors. Chemotherapy and other forms of immunotherapy such as BCG were not highly recommended. The protocol for such a trial should include a minimum of treatment modalities and should be written in cooperation with the partici- pating research clinicians. In addition, a determination should be made with respect to the sample size required for a meaningful study and the number of ocular melanoma patients eligible for the study. Underlying the recommendations was the task force's belief in a need to share resources, a need for greater cooperation among ophthalmologists, and a need for increased communication among ophthalmologists, epidemiologists, and medical oncologists. 3p References Retinal and Choroidal Diseases 1. Hammond EC, Spalter HF: Survey of needs in ophthalmic research and development. Am J Ophthalmol 76:389-394, 1973. 2. Four risk factors for severe visual loss in diabetic retinopathy. The Third Report from the Diabetic Retinopathy Study. Arch Ophthalmol 97:654-655, 1979. 3. Klein R: Annual Progress Report, EY 03083, July 1979. 4. Barbosa J, Ramsay R, Knobloch W, Cantrill H, Noreen M, Chern M, King R, de Liva A, and Yunis E: Genetic contributions to diabetic microangio- pathy. The HLA and retinopathy. Am J Ophthalmol (in press). 5. Barbosa J, Chern MM, Reinsmoen N, Noreen H, Ramsey R, and Greenberg L: HLA-Dw antigens in unrelated juvenile, insulin-dependent diabetes. Tissue Antigens 14:426-436, 1979. 6. Bresnick GH: Annual Report, EY 03084, March 1980. 7. Franks RN: Renewal Grant Application, EY 01857. 8. Wallow, IH: Annual Progress Report, EY 01634, April 1980. 9. Mukai N: Annual Report, EY 02062, December 1980. 10. Wallow IHL, and Davis MD: Clinicopathologic correlation of xenon arc and argon arc photocoagulation procedure in human diabetic eyes. Arch Ophthalmol 97: 2308-2315, 1979. 11. Berson EL, Rosen JB and Simonoff EA: Electroretinographic testing as an aid in detection of carriers of X-chromosome-1 inked retinitis pigmentosa. Am J Ophthalmol 84(4)460-468, 1979. 12. Corwin TR: Annual Progress Report, EY 02366-02, August 1980. 13. Reilly P, Szamier RB, Berson EL: Histopathological findings in an unusual form of retinitis pigmentosa. Invest Ophthal 19: 191(1980). 14. Berson EL: Annual Progress Report, EY 02014-04, June 1980. 15. O'Donnell JJ, Annual Progress Report, EY 02706, November 1979. 32 16. Valle DL: Annual Progress Report, EY 02948, March 1980. 17. Weleber RG: Annual Progress Report, EY 02527, May 1980. 18. La Vail MM: Annual Progress Report, EY 01919-04, June 1980. 19. Aguirre GD, Annual Progress Report, EY 01280, EY 01244-08, February 1980. 20. Buyukmkci N, Aguirre G, and Marshall J: Retinal degeneration in the dog II. Development of the retina in rod-cone dysplasia. Exg^ Eye Res (in press) . 21. Battelle BA, La Vail MM: Protein synthesis in retinas of rats with inherited retinal dystrophy. Exp Eye Res (in press). 22. Young RW and Droz B: The renewal of photoreceptor outer segments J Cell Biol 39:169-184, 1968. 23. Young RW and Droz B: The renewal of protein in retinal rods and cones. J Cell Biol 39:169-184, 1968. 24. Anderson DH, Risher SK, and Steinberg R: Mammalian cones: disc shedding, phagocytosis and renewal. Invest Ophthalmol Visual Sci 17:117-133, 1978. 25. Hogan MJ, Wood I, and Steinberg R: Cones of human retina: phagocytosis by pigment epithelium. Nature , Lond, 252-305-307, 1974. 26. La Vail MM: Rod outer segment disk shedding in rat retina: relationship to cyclic lighting. Science 194:1071-1074. 27. Young RW: The daily rhythm of shedding and degradation of rod and cone outer segment membranes in the chick retina. Invest Ophthalmol Visual Sci 17:105-116, 1978. 28. Bunt AH: Fine structure and radioautography of rabbit photoreceptor cells. Invest Ophthalmol Visual Sci 17:90-98, 1978. 29. Anderson DH and Fisher SK: The photoreceptors of diurnal squirrels: outer segment structure, disc shedding, and protein renewal. J Ultrastruct Res 55:119-141, 1976. 30. La Vail MM: Circadian nature of rod outer segment disc shedding in the rat. Invest Ophthalmol Visual Sci 19:407-411, 1980. 31. Besharse JC, Hollyfield JG, and Rayborn ME: Turnover of rod photoreceptor outer segments. II. Membrane addition and loss in relationship to light, J Cell Biol 75:507-527, 1977. 33 32. Teirsten PS, O'Brien PJ, and Goldman AI : Nonsystemic regula- tion of rat rod outer segment disc shedding. ARVO abstracts. Suppl. Invest Ophthalmol 17: Suppl. 1: 134(1978) . 33. Besharse JC: Light and membrane biogenesis in rod photoreceptors of vertebrates in: The effects of constant light on visual pro- cesses. TP Williams and BN Baker, Eds, Plenum Press, New York, pp. 409-431, 1980. 34. La Vail MM: Rod outer segment disc shedding in relation to cyclic lighting. Exp Eye Res 23:277-280, 1976. 35. La Vail MM and Wand PA: Studies on the hormonal control of cir- cadian outer segment disc shedding in the rat retina. Invest Ophthalmol Visual Sci 17:1189-1193, 1978. 36. Tamai MP, Tiersten A, Goldman A, O'Brien and Chader G: The pineal gland does not control rod outer segment shedding and phago- cytosis in the rat retina and pigment epithelium. Invest Ophthalmol Visual Sci 17:558-562, 1978. 37. Currie JR, Hollyfield JG, and Rayborn ME. Rod outer segments elongate an constant light: darkness is required for normal shed- ding. Visual Res 18:995-1003, 1978. 38. Teirsten PS, Goldman AI, and O'Brien PJ: Multiple circadian oscillators regulate rat ROS disc shedding. ARVO abstracts, Suppl Invest Ophthalmol Visual Sci , pp. 226-227, 1979. 39. Hollyfield JG and Basinger SF: Photoreceptor shedding can be initiated within the eye. Nature 274:794-796, 1978. 40. Besharse JC, Terrill RO, and Dunis DAL Light evoked disc shedding by rod photoreceptors in vitro. Nature , 1980 (in press). 41. Hollyfield JG and Basinger SF: Cyclic metabolism of photoreceptors and retinal pigment epithelium in the frog. Neurochemistry 1:103- 112, 1980. 42. Hollyfield JG and Basinger SF: RNA metabolism in the retina in relation to cyclic lighting. Vis Res Symposium Issue, 1980 (in press). 43. Cone RA: The internal transmitter model for visual excitation: some quantitative implications. In: Biochemistry and Physiology of Visual Pigments , ed. Langer H, pp. 275-282. New York, Springer, 1973. 44. Hubbell WL, and Bownds MD: Visual transduction in vertebrate photoreceptors. Ann Rev Neurosci 2:17-34, 1979. 34 45. Hagins WA: The visual process: excitatory mechanisms in the primary photoreceptor cells. Ann Rev Biophys Bioeng 1:131-158, 1972. 46. Fung BKK and Hubbell WA: Organization of rhodopsin in photo- receptor membranes. 2. Transmembrane organization of bovine rhodopsin: Evidence from proteolysis and lactoperoxidase - catalyzed iodination of native and reconstituted membranes. Biochemistry 17:4403-4410, 1978. 47. Mas MT, Wang JK, and Hargrave PA: Topography of rhodopsin in rod outer segment disk membranes. Photochemical labeling with N-(4-azido-2-nitrophenyl)-2-aminoethanesulf onate . Biochemistry 19:684-692, 1980. 48. Hargrave PA, Fong SL, McDowell JH, Mas MT, Curtis DR, Wang JK, Juszczak E, and Smith DP: The partial primary structure of bovine rhodopsin and its topography in the retinal rod cell disc membrane. Neurochemistry 1:231-244, 1980. 49. Brown JE, Coles JA, and Pinto LH: Effect of injection of calcium and EGTA into the outer segments of retinal rods of Bufo Marinus. J Physiol , London, 269:707-722, 1977. 50. Hagins W and Yoshikami S: Intracellular transmission of visual excitation in photoreceptors; electrical effects of chelating agents introduced into rods by vesicle fusion. In: Vertebrate Photoreceptors : ed. , HB Barlow, P Fatt, pp 97-139. London, Academic, 1977. 51. Baylor DA, Hodgkin AL, and Lamb TD: The electrical response of turtle cones to flashes and steps of light. J Physiol London 242:685-727, 1974. 52. Woodruff ML and Bownds MD: Amplitude, kinetics, and reversibility of a light-induced decrease in guanosine 3', 5 '-cyclic monophos- phate in frog photoreceptor membranes. J Gen Physiol 73:629-653, 1979. 53. Woodruff ML, Bownds MD, Green SH, Morrisey JL, and Shedlovsky A: Guanosine 3', 5'-cyclic mophosphate and the in vitro physiology of frog photoreceptor membranes. J Gen Physiol 69:667-679, 1977. 54. Miller HW and Nicol GD: Cyclic GMP injected into retinal rod outer segments increases latency and amplitude of response to illumination. Proc Nat Acad Sci 75:5217-5220, 1978. 55. Yau KW, Lamb TD, and Baylor DA: Light-induced fluctuations in membrane current of single toad rod outer segments. Nature 269:78-80, 1977. 35 56. Nussenblatt RB, Gery I, Ballintine EJ, and Wacker WB: Cellular innnune responsiveness of uveitis patients to retinal- 8-antigen. Am J Ophthalmol 89:173-179, 1980. 57. Wacker WB: Annual Progress Report, EY 00254, March 1980. 58. Meyers-Elliot RH, Sumner HL, and Shimizer I: Immunopatho- genesis of rhodopsin-induced experimental allergic retinitis. Cell Immunol (submitted for publication). 59. Marak GE, Jr., Schichi H, Rao NA and Wacker WB: Patterns of experimental allergic uveitis induced by rhodopsin and retinal rod outer segments. Ophthalmic Research (in press). 60. Silverstein AH: Annual Progress Report, EY 00279-17, May 1980. 61. Ehrenberg M, and Prendergast RA: Uveitis and retinitis induced by sea star factor (ssf), in the immunology and immunopathology of the eye, chapter 10, pp. 46-54. 62. Murphree L: Annual Progress Report, EY 02715, December 1979. 63. Little JB, Weichselbaum KR, Nove J, and Albert DM: X-ray sensitivity of fibroblasts from patients with retinoblastoma and with abnormalities of chromosome 13. In: Friedberg EL, and Hanawalt PC (eds): DNA repair mechanisms. New York, Academic Press, 1978. 64. Albert DM: Annual Progress Report, EY 01917-05, July 1980. 65. Liu HS, Refojo MF, Perry HD, and Albert DM: Sustained release of BCNU for the treatment of intraocular malignancies in animal models. Invest Ophthalmol Visual Sci (in press). 66. Sery TD: Annual Progress Report, EY 02131, April 1980. 36 CORNEAL DISEASES ■ Introduction The Corneal Diseases program of the National Eye Institute provides the focus within the federal government for research support for the investi- gation of disorders of the cornea, lids, conjunctiva, the lacrimal gland, diseases of the orbit and external eye, and refractive errors and injuries of the cornea. Although lesions of the cornea account for only approximately 6 percent of all cases of blindness in the United States, diseases and disorders which affect this tissue constitute 62 percent of the total incidence of acute and chronic disorders, diseases and injuries which affect the eyes. Ocular infections, conjunctival allergies and traumatic and/or foreign body injuries to the cornea constitute the bulk of these eye problems. In this report, only a few of the research areas of greatest programatic activity will be considered in relation to the research priorities for the Corneal Diseases program as outlined in the 1977 report of the National Advisory Eye Council, Vision Research — A National Plan; 1978-1982 . These areas are: ocular herpetic infections, trophic influences on corneal epi- thelium, control of corneal hydration and transparency and collagen type distribution in the cornea. Ocular Herpetic Infections Viral keratitis and other keratopathies represent debilitating diseases of major importance in the United States and throughout the world. Keratitis, which results from infection by herpes simplex virus (HSV) has an incidence of between 300 and 500,000 cases a year with a high level of morbidity associated with such attacks. After one ocular infection of ocular den- dritic keratitis, between 25 percent and 50 percent of patients will have another attack within two years with the risk increased if the patient has had two or more attacks. Current treatment of recurrent herpes simplex virus infections of the cornea is through use of antiviral drugs which are administered locally during periods of active infections. The application of such drugs as preventives of the disease is contraindicated, however, except when re- currences are frequent or there is a high risk of recurrent disease. Topical use of the antiviral protein interferon has been proposed as a ther- apeutic agent, but Kaufman and collaborators have failed to note any differences in the frequency of recurrence between placebo-treated patients and those patients treated with interferon in a double masked clinical trial of herpetic keratitis. The titer of human leukocyte interferon used in these studies may have been too low (6.4X10 units/ml) to be effective and such clinical research investigations should be repeated with a higher potency interferon from white blood cells and/or the newer immune interferon isolated from human lymphocytes after stimulation with staphylococcal entertoxin A. It has been previously shown by Nesburn and associates that the herpes simplex virus, after infection of the cornea, can reside in the 2,1 trigeminal ganglion of experimental animals in a latent phase between active episodes of the ocular disease. Haschke and coworkers have examined an alternative approach to treating ocular herpes during the latent phase through the use of an activiral drug 5-iodo-5' -amino -2' , 5' -dideoxyuridine (AIDU) , which was chemically synthesized with I and then coupled to horseradish peroxidase. The radioactive drug-protein conjugate was shown to be capable of being trans- ported in a retrograde manner in the axons of the trigeminal ganglion neurons by use of radioautography after corneal injection. These preliminary drug studies offer the hope of attacking the latent virus at the level of the ganglion during periods of remission of the active disease and thus preventing possible recurrences. The above drug strategy will probably require modifi- cation, however, if the strain of the herpes simplex virus, which resides in the ganglion, is determined to be different from the strain of the virus which produced the primary ocular infection. Another form of ocular herpetic infection, which has consistently proved to be a problem for drug therapy, is disciform stromal keratitis. Corticosteroids have often been employed for their anti-inflammatory and immunosuppressive effects in cases which are recalcitrant to therapy. Recently Smolin and coworkers provided hope for successful treatment of stromal herpetic infections through their studies with vitamin A. These workers have established a chronic herpes simplex virus model in rabbits by subconjunctival injections of low doses of corticosteroids to scarified corneas inoculated with the PH-strain of the virus. Animals treated with large doses of vitamin A (100,000 lU) by intraperitoneal injection were shown to develop milder, more rapidly healing epithelial lesions and either mild or no stromal disease, when compared to untreated rabbits. Vitamin A, thus, appears to be capable either of directly causing induction of this effect or acts by counteracting the immunosuppressive effect of cortico- steroids. Additional research will have to be conducted to determine whether this amelioration of symptoms can be obtained through topical treatment with the vitamin. In the 1977 report of the National Advisory Eye Council, Vision Research — A National Plan , the Corneal Diseases planning panel said that the search for better treatment of herpes infections of the cornea represented one of the major research objectives of the Program. Particular attention in that report was directed toward encouraging development of effective therapy for deep-seated, stromal and recurrent herpetic infections. Continuous attention must be given to encouraging further development of new strategies for treat- ment of the various forms of this debilitating viral disease. Trophic Influences on Corneal Epithelium The ability to manipulate specific metabolic pathways which govern the maintenance and repair of the corneal epithelium provides a possible approach to the management of certain diseases which affect the ocular surface. Because catecholamines and acetylcholine can physiologically modulate the activities of cells and tissues in many systems, recent basic research in vision has been directed toward demonstrating and clarifying such influences 3'8 on the epithelial cells of the cornea. The corneal epithelium has been shown experimentally to respond to catecholamines in several measurable ways. The first observations of ad- renergic influences on this tissue were made several decades ago by Friedenwald and Buschke who demonstrated that the mitotic rate of the corneal epithelial cells of the rat is suppressed by sympathetic stimulation (e.g., fright, systemic epinephrine) and by superior cervical ganglionectomy after a delay of several hours. Recent work by Butterfield and Neufeld have updated these observations by the demonstration that the decrease in mitotic rate following ganglionectomy is associated with beta-adrenergic stimulation of the corneal epithelium which probably results from an intraocular release of nonepinephrine from degenerating nerve terminals in the corneal stroma and iris. The beta-adrenergic stimulation of the epithelial layer is accompanied by an increase in cyclic AMP levels in the cornea. Although adrenergic stimulation has been suggested as influencing the diurnal rhythm of corneal epithelial cell division in some species, decentrali- zation of the adrenergic nerve supply to the eye of the rabbit failed to alter this rhythm. The implication from such studies is that the central nervous system does not provide the pacemaker input for the diurnal rhythm via an adrenergic innervation which stimulates corneal beta-adrenergic receptors. In vitro, catecholamines have been shown to stimulate active chloride ion transport by the corneal epithelium of several animal species. This influence is mediated by cyclic AMP which causes an enhanced permeability to anions. It has been^ recently observed by Neufeld and his collaborators in a series of studies ' ' that repeated topical application of epi- nephrine to the rabbit eye results in a decrease in the density of beta adrenergic receptors on the membranes of corneal epithelial cells which results in a decreased ability of the tissue layer to generate cyclic AMP and the failure of adrenergic agonists (e.g., epinephrine) to stimulate chloride ion transport. Thus, topical epinephrine is capable of depressing the entire adrenergic pathway from the cellular receptor site to the physio- logical response. On the other hand, an increase in beta-adrenergic receptor density has been noted to occur on such cells ffter repeated topical treatment with timolol, a beta-adrenergic antagonist. The chloride ion transport system, however, remains unresponsive to stimulation by catecholamines long after timolol has been cleared from the cornea. Recent experimental studies have also been conducted to gather evidence for a possible cholinergic pathway in the cornea. The neurotransmitter acetylcholine and its synthesizing enzyme choline acetyltransferase have been found by Mindel and Mittag to be in the corneas of all mammalian species except the cat. These workers have also demonstrated that surgical closure of the lids of adult rabbits for 24^hours caused a decrease in the corneal choline acetyltransferase activity. The degradative enzyme 3^ acetylcholinesterase has also^been located in both the epithelial and stromal layers of the cornea; the epithelial enzyme has been identified as specific for acetylcholine whereas the stromal activity is shown to be primarily pseudocholinesterase. Although the corneal epithelial cell layer contains both epithelial and neural elements, localization of cholinergic components have not been made to specific cell types in this layer. Denervation of the cornea failed to demonstrate a decrease in acetylcholine levels even though the substrate and its related enz)nnes have been demonstrated to be primarily in the epithelium. A transmitter function has been ascribed to acetylcholine in the cornea but neither cholinergic receptors in the tissue of the muscarinic or nicotinic type have been detected by specific radioligand assays. Thus, though there is evidence of adrenergic and cholinergic components in mammalian corneal epithelium, each pathway lacks certain components which are presently perceived as being required. The adrenergic component encompasses receptors, an intracellular messenger (cyclic AMP) and several partially understood cellular responses that presumably may be activated by stress. Missing, however, is the source to provide either the neural or humoral stimulus. The cholinergic component contains a substance which acts as a neurotransmitter with the related enzymes for its maintenance. Cholinergic receptors and evidence of cellular responses, however, are found to be lacking in corneal tissue. More research is required to complete our understanding of these possible pathways in the corneal epithelium and to correlate these observations with the recent observation that when human corneal epithelium receives thermal stimulation, only the sensation of pain is perceived in the eye of the subject. Control of Corneal Hydration and Transparency The maintenance of transparency in vertebrate corneas represents a process which expends metabolic energy and is primarily under the control of a pump mechanism which resides in the endothelial cell layer. This energy is utilized to keep the hydrophilic stroma at a minimum of physio- logical hydration. If the endothelial layer is damaged, an increase in corneal thickness is observed due to an imbition of water by the stromal tissue. The resultant swelling of the transparent stromal tissue layer ultimately results in corneal opacification. Although the importance of the endothelium in maintaining corneal transparency has been the concern of numerous studies, the exact mechanisms of how dehydration (deturgescence) can be effected are incompletely understood. A variety of compounds naturally present either in the endothelium or in the aqueous humor which bathes this layer have been recently investigated as to their respective roles in this process. 23 24 It has been observed by Riley and coworkers ' , that the tripeptide glutathione in the reduced state (GSH) at 24uM or in the oxidized state (GSSG) at 8uM can maintain perfused corneas at a normal state of hydration 40 for up to 5 hours. The intracellular levels of glutathione were noted to be higher in endothelia of such corneas than in those corneas swelled as a result of perfusion without glutathione. These findings are similar in effect to those results previously reported by Whikehart and Edelhauser using higher concentrations of the compound in the perfusing medium and point to the existence of some relationship between the tripeptide levels in the medium and its intracellular level. An alternative role, however, has been suggested for glutathione which differs from its reported effects on corneal hydration. Hull and coworkers have recently reported that catalase, the enzjrme which degrades hydrogen peroxidase can prevent loss of endothelial function which results from light exposure of photosensitized corneas. A redox buffer system such as GSH:GSSG could be important in the elimination of free radicals which are generated in the aqueous or cell membranes of the corneal endothelium. 27 Anderson and Wright have studied the role of the thiol group in fluid transport in rabbit corneas by use of S-methyl derivatives of GSH and cysteine in their perfusion medium. These investigators determined that thiol-disulfide exchanges with GSSG were not necessary to achieve deturgescence of the stroma in these preswollen corneas. It was suggested that either potential Krebs cycle intermediates derivable from either the glutamyl group or gamma-aminobutyric acid (GABA) arising |Kom glutamate are obligatory in supporting the endothelial pump mechanism- It is interesting to note in this regard that GABA at a concentration of lOuM has been found to be effective in the deturgescence of preswollen rabbit„corneas and the maintenance of such corneas for periods of up to six hours. ' Metabolic events which take place in the cornea exclusive of the endo- thelial cell layer also have potential control of transparency in the total tissue. Preliminary studies conducted by Conrad and Woo indicate that the increase in transparency observed in developing chick cornea may be casually related to the degree of glycosaminoglycan sulfation which has occurred in the corneal stroma. The degree of sulfation of the principal glycosaminoglycan of the cornea, keratan sulfate, has been previously shown by Hart to^ be regulated by tl^e intracellular concentration of the sulfate donor, 3 - phos- phoadenosine" - phosphosulfate (PAPS), rather than by changes in the specific activity of the enzyme keratan sulfate sulfotransferase. Conrad and Woo tested corneal homogenates derived from chick embryos of different ages for their ability to synthesize PAPS and noted a 2.5-fold increase in such enzymatic activity between day 8 and day 16 of chick development which reached a peak on day 16. This sequence of events correlated well with the onset of the process of corneal transparency in the chick which begins on day 14 of development and reaches half completion by day 16. More research is needed into identification of natural chemical factors which may affect the functions of the corneal endothelium, both as a fluid barrier and as a metabolic pump. In humans, endothelial damage which may occur during surgery, e.g., cataract extraction or corneal 4J. transplantation may not become clinically evident as corneal edema with loss of vision for a period of several years. Knowledge of the metabolic processes which control corneal transparency may permit the design of pharmaceutical agents which could be employed to prevent the onset of corneal edema. Collagen Type Distribution in Cornea During the past decade, the study of the chemistry and biology of collagen in the eye and other tissues has been influenced by the discovery of the biosynthetic precursor, procollagen, and by the realization that collagens in tissues are heterogenous. Evidence has recenty accumulated that the heterogeneity in collagens results from the fact that the individual polypeptide chains observed in this group of proteins are produced at different genetic loci which are nonallelic. Structural and functional homologous proteins within the collagen family have thus become known as types . 33 Conrad and coworkers have used the developing chick cornea as a model for studying the factors that normally regulate the ratio and number of types of collagen in a fibrous connective tissue. Chick cornea appears to be somewhat unique when compared to corneal stromata of other animal species since it contains no type III collagen (composed of three type III and alpha-1 chains) post day 7 of embryonic development as determined by specific immunof luorescent staining. Prior to day 7 of development research by other investigators has shown that antibodies to type III collagen were found to bind to corneal epithelial cells but not to the primary chick stroma. After day 7, the primary and secondary stroma of chick cornea was found primarily to contain an abundance of type I collagen (composed'^-of two type I alpha-1 chains and one type I alpha-2 chain) . 33 Conrad and his associates have found, however, in older embryonic chick corneas, if the corneal epithelium and endothelium are gently removed after brief collagenase treatment of the stroma, the stromal fibroblasts which remain begin manufacturing type III collagen in addition to continuing to synthesize type I collagen. A similar ability to synthesize type III collagen also appeared within a few hours in corneal fibroblasts which were released from stroma and inoculated in vitro. These observations represent the first example of a switch in type of collagen synthesized by a fibroblast population although such shifts in collagen biosynthesis have been observed in cartilage cells. The region of the corneal stroma post day 7 of development that is devoid of type III collagen has been shown to be identical with the region that stains metachromatically for sulfated glycosaminoglycans (i.e., keratan sulfate). Indirect evidence suggests that type III collagen in the in vivo chick embryo cornea is repressed by production of keratan sulfate proteoglycan in this tissue. This polysaccharide reaches half of its maximal production in the stroma by day 7 when the cornea stops synthesizing type III collagen (see review of corneal hydration). 42 Type II collagen (composed of three type II alpha-1 chains) has also been reported by Linsenmayer and coworkers to be present in the anterior parts of developing chick, stroma. The protein is probably synthesized by the corneal epithelium although this activity in the latter tissue layer has so far only been shown for day 6 of development. Yue and Baum have examined in detail the collagens synthesized by cultures of corneal stroma cells derived from both normal individuals and patients with keratoconus. Type I collagen was found by SDS-gel electro- phoresis to be present in cell layer and medium fractions of both types of cultures. oT'^is observation confirmed the earlier catture~work^afStoesser and associates with human stromal fibroblasts. In addition, Yue and Baum found the A and B chains of "type VI" collagen associated mainly with the cell layer and small amounts (approximately 10 percent of total) of type III collagen present in the medium of their cultures. However, in comparison to normal control cultures, the relative amounts of "type VI" collagen synthesized by keratoconus cultures was found to be significantly increased. Studies on collagen synthesis in vivo generally agree with those studies performed in vitro on confluent cultures of corneal stromal cells. Freeman has determined that type I is_the main collagenous product in rabbit stroma and Davison and collaborators have isolated "type VI" segments from calf corneas. • Endothelial cell cultures have also been examined for ability to form collagen. A single basement membrane collagen classified as type IV has been isolated by Sundar Raj et al from rabbit corneal endothelium. Baum and coworkers have found the collagen synthesized from human endo- thelial cultures to be extremely heterogenous. Among all the collagenous proteins, type I collagen has been identified in both the cell layer and medium fractions of such cultures. This is in agreement with a previous ,„ study of the synthesis of Descemet 's membrane in vivo by Davison and Cannon. These latter workers have shown that Descemet 's membrane contains not only basement membrane collagen but also several other types of collagen, in addition to type I. The ultimate clarification of the factors which modulate the production and establish the ratio between the various type collagens in the individual layers of cornea will be important in the future for the design of therapies for achieving diverse effects within the anterior segment of the eye such as an acceleration of wound healing or alleviation of an inherited corneal dystrophy. Better understanding of the chemical structure of the individual chains of each collagen type will aid in development of a unified hypothesis for interaction of the protein with other macromolecules within corneal tissue. Radial Keratotomy At its May 1980 meeting, the National Advisory Eye Council expressed grave concern that the procedure, radial keratotomy, was being adopted widely, although recent reports from foreign countries and the United States 4? did not provide an adequate basis on which to assure the general public of its safety and efficacy. For this reason, the Council called for research on radial keratotomy and urged restraint on the part of patients and eye surgeons until the results of such research can be reviewed and evaluated by the ophthalmological community. The resolution on the surgical procedure as developed by the National Advisory Eye Council at the May meeting is presented below: 1. As the principal advisory body to the National Eye Institute, the Federal Government's chief source of support for vision research, the National Advisory Eye Council would like to express grave concern about potential widespread adoption of an operation intended to correct near- sightedness (myopia), a common condition that can be easily and safely corrected by the use of eyeglasses or contact lenses. The operation, called radial keratotomy, has received widespread publicity during the last year. It involves cutting the cornea with a series of deep incisions that extend from the sclera (the white tissue surrounding the cornea) toward, but not into, the center of the cornea. The incisions are intended to be deep enough to weaken the tissue so that internal eye pressure causes the edge of the cornea to bulge slightly, thereby flattening the central portion of the cornea which improves focusing. The incisions result in permanent corneal scars. 2. The Council considers radial keratotomy to be an experimental procedure because it has not been subjected to adequate scientific evaluation in animals and humans. Recent reports on radial keratotomy from foreign countries and the United States provide an inadequate basis on which to assure the procedure's safety. 3. The Council calls for carefully controlled research on radial keratotomy to determine the procedure's effectiveness, safety, short-term and long-term side effects, and the best surgical technique. The need for research on radial keratotomy and other forms of surgical correction of refractive errors (myopia, presbyopia, astigmatism) was recognized by the Council in a statement published in the journal Investigative Ophthalmology and Visual Science in August 1979. Until the ophthalmological .community has an opportunity to review and evaluate the results of such research, the Council urges restraint on the part of both patients and eye surgeons. 4. The Council therefore urges the National Eye Institute to take whatever measures are necessary to encourage research on radial keratotomy in animals, and also in humans provided patients are enrolled in scientif- ically designed clinical trials conducted by qualified investigators. 5. The Council strongly urges that its views on the subject of radial keratotomy as expressed in this resolution be announced to the general public, as well as to health care professionals. 44 References Corneal Diseases 1. Kaufman HE: Herpetic keratitis. Invest Ophthalmol Vis Sci 17:941-958, 1978. 2. Norn MS: Dendritic (herpetic) keratitis. Acta Ophthalmol 48:383-395, 1970. 3. Dawson CR, Togni B: Herpes simplex eye infections: clinical mani- festations, pathogenesis and management. Surv Ophthalmol 21:121-135, 1976. " 4. Kaufman HE, Meyer RF, Laibson PR, Waltman SR, Nesburn AB, Shuster JJ : Human leukocyte interferon for the prevention of recurrences of herpetic keratitis. J Infect Pis 133:A165-A168, 1976. 5. Langford MP, Georgiades JA, Stanton GJ, Diazani F, Johnson HM: Large- scale production and physiocochemical characterization of human immune interferon. Infect Immun 26:36-41, 1979. 6. Nesburn AB, Cook ML, Stevens JG: Latent herpes simplex virus: Iso- lation from rabbit trigeminal ganglia between disease episodes. Arch Ophthalmol 88:412,417, 1972. 7. Haschke R: Annual Progress Report, EY 01756-04, April 1980. 8. Dawson CR, Weinstein A, Briones 0, Oh JO, Schachter J: Herpes simplex superinfection of trigeminal and autonomic ganglions in inraiune rabbits. Silverstein AM, O'Connor GR (eds): Immunology and Immunopathology of the Eye . New York, Masson Publishing Co., 1979, pp. 256-261. 9. Smolin G, Okumoto M, Friedlander M, Kwok S: Herpes simplex keratitis treatment with vitamin A. Arch Ophthalmol 97:2181-2183, 1979. 10. Friedenwald JS, Buschke W: The effects of excitement of epinephrine and of sympathectomy on the mitotic activity of the corneal epithelium in rats. Am J Physiol 141:689-694, 1944. 11. Butterfield LC, Neufeld AH: Cyclic nucleotides and mitosis in the rabbit cornea following superior cervical ganglionectomy. Exp Eye Res 25:427-433, 1977. 12. Neufeld AH: Annual Progress Report, EY 02630-03, August 1979. 13. Montoreano R, Candia OA, Cook P: Alpha-and beta-adrenergic receptors in regulation of ionic transport in frog cornea. Am J Physiol 230:1487- 1493, 1976. 4^ 14. Klyce SD, Wong RKS: Site and mode of adrenaline action on chloride transport across the rabbit corneal epithelium. J Physiol (London) 266:777-799, 1977. 15. Neufeld AH, Zawistowski KA, Page ED, Bromberg BB: Influences on the density of beta adrenergic receptors in the cornea and iris-ciliary body of the rabbit. Invest Ophthalmol Vis Sci 17:1069-1075, 1978. 16. Candia OA, Neufeld AH: Topical epinephrine causes a decrease in density of beta-adrenergic receptors and catecholamine-stimulated chloride transport in the rabbit cornea. Biochem Biophys Acta 543:403-408, 1978. 17. Candia OA, Podos SM, Neufeld AH: Modification by timolol of catecho- lamine stimulation of chloride transport in isolated corneas. Invest Ophthalmol Vis Sci 18:691-695, 1979. 18. Mindel JS, Mittag TW: Choline acetyltransf erase in ocular tissues of rabbits, cats, cattle and man. Invest Ophthalmol 15:808-814, 1976. 19. Mindel JS, Mittag TW: Suppression of corneal epithelial choline acetyltransferase activity by lid closure.- Exp^ Eye Res 27:359-364, 1978. 20. Howard RO, Zadunaisky JA, Dunn BJ: Localization of acetylcholinesterase in the rabbit cornea by light and electron microscopy. Invest Ophthalmol 14:592-603, 1975. 21. Olson JS, Neufeld AH: The rabbit cornea lacks cholinergic receptors. Invest Ophthalmol Vis Sci 18:1216-1225, 1979. 22. Beuerman RW, Tanelian DL: Corneal pain evoked by thermal stimulation. Pain 7:1-14, 1979. 23. Riley MV, Meyer RF, Yates EM: Glutathione in the aqueous humor of human and other species. Invest Ophthalmol Vis Sci 19:94-96, 1980. 24. Ng MC and Riley MV: Relation of intracellular levels and redox state of glutathione to endothelial function in the rabbit cornea. Exp Eye Res 30:511-517, 1980. 25. Whikehart DR and Edelhauser HF: Glutathione in rabbit corneal endothelia: the effects of selected perfusion fluids. Invest Ophthalmol Vis Sci 17:455-474, 1978. 26. Hull DS, Strickland EC, Green K: Photodynamically induced alteration of corneal endothelial cell function. Invest Ophthalmol Vis Sci 18:1226-1231, 1979. 27. Anderson EI and Wright DD: Effects of S-methyl glutathione, S-methyl cysteine and the concentration of oxidized glutathione on transendo- thelial fluid transport. Invest Ophthalmol Vis Sci 19:684-686, 1980. 28. Anderson EI: Annual Progress Report, EY 00699-09, April 1980. 29. Vidal R, Wendel A, Dikstein S: GABA stimulates the rabbit corneal endothelial fluid pump. Experientia 35:182, 1979. 30. Dikstein S: Annual Progress Report, EY 02912-02, March 1980. 31. Conrad GW: Annual Progress Report, EY 00952-09, January 1980. 32. Hart GW: Glycosaminoglycan sulfotransf erases of the developing chick cornea. J Biol Chem 253:347-353, 1978. 33. Conrad GW, Dessau W, von der Mark K: Synthesis of type III collagen by fibroblasts from the embryonic chick cornea. J Cell Biol 84:501-510, 1980. 34. von der Mark K, von der Mark H, Timpl R, Trelstad RL: Immuno- fluorescence localization of collagen types I, II and III in the embryonic chick eye. Dev Biol 59:75-85, 1977. 35. Linsenmayer TF, Smith GN Jr, Hay ED: Synthesis of two collagen types of embryonic chick corneal epithelium in vitro. Proc Natl Acad Sci USA 74:39-43, 1977. 36. Yue BYJT, Baum JL, Smith BD: Collagen synthesis by cultures of stromal cells from normal human and keratoconus corneas. Biochem Biophys Res Commun 86:465-472, 1979. 37. Stoesser TR, Church RL, Brown SI: Patial characterization of human collagen and procollagen secreted by human corneal stromal fibroblasts in cell culture. Invest Ophthalmol Vis Sci 17:264-271, 1978. 38. Freeman IL: Collagen poljnnorphism in mature rabbit cornea. Invest Ophthalmol Vis Sci 17:171-177, 1978. 39. Davison PF, Hong B-S, Cannon DJ: Quantitative analysis of the collagens in the bovine cornea. Exp Eye Res 29:97-107, 1979. 40. Sundar Raj CV, Freeman IL, Church RL, Brown SI: Biochemical characteri- zation of procollagen-collagen synthesized by rabbit corneal endothelial cells in culture. Invest Ophthalmol Vis Sci 18:75-84, 1979. 41. Baum JL: Annual Progress Report, EY 01793-05, April 1980. 42. Davison PF, Cannon DJ: Heterogeneity of collagens from basement membranes of lens and cornea. Exp Eye Res 25:129-137, 1977. 47 CATARACT Cataracts are a major class of eye disorders, being responsible for more than half of the eye related patient hospitalizations in the United States, Cataracts can result from many conditions and it has been hypothe- sized that they are an end response by the lens to a broad spectrum of path- ologies. To develop means for the prevention and cure of this family of pathologies, it is necessary to understand the biology of the lens in its normal state and its detailed reponse to insults which result in cataract. The National Eye Institute cataract program includes studies over the broadest range of biological science. In this report we will deal in some detail with three aspects of the program: molecular biology, control of biological processes and oxidative processes. Molecular biology, the study of gene composition and expression represents the first level of biological function. New developments in this area of science have burgeoned in recent years and their application to the lens, although progressing, requires additional encouragement. Important opportunities also exist in other areas of biological control. The maintenance of homeostasis may represent the fundamental means for preventing cataract and increased efforts in under- standing of biological control processes are most desirable. Cataract has been associated with lens oxidative processes and increasing efforts have been addressed to the study of means by which the lens protects itself from damage by such processes. Molecular Biology The mammalian lens, as a relatively simple tissue, offers an attractive system for the study of normal and abnormal cellular differentiation and development. It provides a particular opportunity to examine gene expression and morphogenesis both at the level of the isolated cell (tissue culture) and in the cultured organ. This can lead to an increased understanding of the control of crucial biological functions, and, in the lens, to better under- standing of normal lens function and of cataractous processes. A firm base already exists for exploration of the molecular biology of the lens. Detailed information is available on the ultrastructure of lens cells and of their alterations during differentiation, and tissue culture cells lines have been developed which produce major gene products. In vitro differentiation systems exist in which differentiation functions and the sjmthesis of specific gene products are activated. Also developed have been cell fusion and hybridoma techniques which permit the synthesis of major gene products. Specific antibodies have been prepared for RNA's; procedures have been developed for the isolation of lens RNA's ' , and there has already been achieved cloning of a specific lens gene. A lens mRNA, the 14S product that codes, for the A2 chain in alpha crystallin, has been found to be bicistronic , coding also for a B2 alpha crystallin chain. This appears to represent the first finding of a eukaryotic mRNA which is not monocistronic. The mallard and chick delta crystallin DNAs cross hydridize well, indicating conservation of gene 49 sequences. Early results suggest that there are at least two delta crystallin genes in the mallard lens. Lens mRNA directed synthesis of lens crystallins has been achieved in embryonic kidney cell culture. Bovine lens tissue contains two tRNA species, one of which is increased three-fold during epithelial cell differentiation. The two tRNA's appear to be under separate control and induction of tRNA, synthesis appears to be part of the differentiation program which requires the increased synthesis of crystallins. Delta crystallin gene sequences derived from the chicken genome have been cloned for the study of their organization. Results indicate the presence of at least two non allelic delta crystallin genes with small differences in nucleotide sequence. Fourteen intervening sequences were found in one of the crystallin genes, the intervening sequences constituting 80% of the gene. Embryonic chick lens cells have been found to contain a GTP :mRNA^ guanyltransf erase which it is postulated may be involved in mRNA synthesis. Thus, the knowledge and methodology exist for rapid developments in this highly important area. Although progressing, lens molecular biology is moving forward at a limited pace and the attraction of additional investigators represents an important objective of the cataract program. Control of Biological Processes The normal lens maintains clarity through a variety of control mechanisms that determine its biological characteristics and state. Cataract represents deviation from homeostasis, so that increased understanding of the factors which control lens processes represents an important part of the lens program. Cell proliferation in the frog lens (but not in the rat lens) ceases following hypophysectomy. In culture medium containing the serum of hypophysectomized animals, in vivo blocked lenses remain blocked; however, they can be made to resume proliferation by the addition to the medium of bovine growth hormone. Somatotropin, frog prolactin, and also the thyroid derivatives T_ and TSH restore mitosis. It is believed that^pitui- tary hormones act by stimulating the liver to produce somatomedin-C. The mechanism by which the thyroid derivatives restore mitotic activity is not known; however, since they are without effect in the explanted lens, it is assumed that they function indirectly through the stimulation of a second factor. By labeling germinative zone cells in their last DNA synthetic period in hypophysectomized frogs, it has been shown that migration of epi- thelial cells to the equator is stopped. After hypophysectomy, lens fiber formation also appears to cease. It is further reported that hypo- physectomy protects frogs from the development of X-ray produced cataract; thus suggesting that the initial lesion in X-rayr-induced cataract is located in the germinative zone at the time of exposure. These studies provide early evidence and information regarding hormonal control of developmental and perhaps repair functions in the amphibian lens. Detailed elaboration is important in learning the details of these control processes. Although no similar response was observed in one mammalian species (rat), the extension of these studies in the amphibian and also in 56 other mammals is important and should be continued. Understanding of tissue differentiation, the acquisition of new prop- erties and growth patterns is a major problem in developmental biology and represents an important area for study in the lens. During lens growth, proliferating cells from the epithelial monolayer migrate to the equator where transformation occurs and the original cuboidal epithelial cells begin the process of elongation, transfer from the surface to within the lens and ultimately to the loss of the nucleus and other organelles, to become fiber cells. In in vivo studies, it was found that the optic cup environment brings about elongation of epithelial cells, and that the presence of the neural retina is important in this process. Using epithelial cells in tissue culture with determination of beta-and gamma-crystallin synthesis as a measure of transformation, it was found that inclusion of neural retina or the use of medium previously incubated in neural retina in the culture medium led to appreciable cell enlargement and initiation of the synthesis of beta- and gamma- crystallins. Although other eye tissue, e^g- corneal stroma, also was stimulatory, its effect was appreciably smaller. Using an elegant micro- scope mounted cell culture technique permitting early quantitative measure of epithelial cell enlargement, Beebe has identified and isolated lentropin, a vitreous factor in the chicken eye having a molecular weight of approx- imately 60,000 daltons which promotes fiber cell differentiation. Also, marked stimulation of DNA synthesis in a cloned Nakano mouse epithelial