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RE
1

W2R5
1977

The
National

Eye
Institute

1977

The
InstifLfe

TABLE OF CONTENTS

Page

STATEMENT OF THE INSTITUTE DIRECTOR 1

INTRAMURAL RESEARCH 3

Clinical Branch 5

Report of the Clinical Director 7

Ballintine, Elmer J. , M.D.

Ocular Hypertension Study 9

Tissue Culture of Trabecular Meshwork 11

BenEzra, David, M.D.

Cornea: Organ and Cellular Cultures of Epithelium

Stroma and Endothelium 13

Bergsma, Donald R. , M.D.

Studies of Choroidal-Retinal Degenerative Disease 17

Blackman, Jane, M.D.

Suppressor Lymphokines in an In Vitro System 21

Cogan, David G. , M.D.

Parametric Studies of Eye Movement Disorders in Humans 25

Gaasterlaud, Douglas E. , M.D. '

Experimental Glaucoma in the Rhesus Monkey 29

Laser Surgery for Glaucoma 33
Radioiodinated Chloroquine Analog for Diagnosis of

Ocular Melanoma 35

Studies of Parameters of Intraocular Pressure 37

Gunkel, Ralph D. , O.D.

Research in Methods of Evaluating Visual Processes 41

Kaiser-Kupfer, Muriel, M.D.

Ophthalmologic Screening for Metastatic Lesions

to the Eye 45-

Pigment Dispersion With and Without Glaucoma 47

Progressive Essential Iris Atrophy 51

Visual Function and Ocular Pigmentation in Albinism 55

Macri, Frank J., Ph.D.

Study of Pharmacodynamics of Various Agents Affecting

the Intraocular Pressure 59

Newsome, David A. , M.D.

Cell and Tissue Interactions in the Production of Corneal

Collagenase 63

f\J SlI:^ Clinical Branch (cont.) Page

I I I I Pederson, Jonathan E. , M.D.

Aqueous Humor Flow Measurement by Fluorophotometry 67

Laboratory Studies of Aqueous Humor Dynamics 69

Stone, Richard A. , M.D.

Transport Mechanisms in the Ciliary Epithelium 73

Tamai, Makoto, M.D.

Organ Culture of the Normal and Dystrophic (RCS)

Rat Retina 75

Tanishima, Teruo, M.D.

Endothelial Wound Healing of the Cornea 77

Laboratory of Vision Research 79

Report of the Chief, Laboratory of Vision Research 81

Section on Biochemistry

Kinoshita, Jin H. , Ph.D.

Cataracts 83

Chemistry and Metabolism of the Lens 87

Goldman, Arnold I., Ph.D.

Ultrastructural and Biochemical Correlates in the

Vertebrate Retina 91

Hess, Helen H. , M.D.

Biochemical Structure of Retina and Pigment

Epithelium in Health and Disease 95

Lewis, Marc S., Ph.D.

Chemistry of Rhodopsin 101

Physical Chemistry of Model Gel Systems 105

Shichi, Hitoshi, Ph.D.

The Membrane Biology of the Visual Process 109

The Molecular Pharmacology of the Eye 113

Yoshikami, S. , Ph.D.

The Visual Cell: Process of Photoexcitation and

Restoration 117

Section on Experimental Embryology

Coulombre, Alfred J., Ph.D.

Metaplastic Formation of Neural Retina in vitro 121

Sensitive Period in the Development of the Scleral

Ossicles of the Avian Eve 125

Laboratory of Vision Research (cont«) Page

Section on Experimental Embryology (cont.)

Porzig, Ellen, Ph.D.

Development of the Chick Conjunctival Periderm

and Conjunctival Papillae 129

Reese, David H. , Ph.D.

Ribosomal RNA Synthesis in the Eastern North-American

Newt, N viridescens 133

Zelenka, Peggy, Ph.D.

Plasma Membrane Composition and Biosynthesis in

Chick Lens Fibers and Epithelia ' "' : . 137

Section on Experimental Pathology

Carter-Dawson, Louvenia, Ph.D.

Effects of Vitamin A Deficiency on Ocular Tissues 141

Kuwabara, Toichiro, M.D.

Anatomical and Pathological Studies of Ocular Tissues 145
Light Effect on the Retina 153

Robison, W. Gerald, Jr., PhiD.

Ultrastructure and Function of the Pigment Cells of

the Eye 157

Section on Neurophysiology

Gouras, Peter, M.D.

Physiology of the Primate Visual System 161

Nelson, Ralph, Ph.D.

Electrophysiological Studies of Mammalian Retina 165

Section on Retinal and Corneal Metabolism

Chader, Gerald J., Ph.D.

Cyclic Nucleotides and Vision 171

Development and Function of Retina, Pigment Epithelium,

and Cornea - 175

O'Brien, Paul J., Ph.D.

Protein Synthesis in the Retina 179

Synthesis of Sugar-Containing Polymers in Retina 181

Whikehart, David R. , Ph.D.

Intermediary Metabolism of the Cornea 185

iii

OFFICE OF BIOMETRY AND EPIDEMIOLOGY

REPORT OF THE CHIEF, OFFICE OF BIOMETPY \IID EPIDEMIOLOGY

Epidemiology Section

Section on Clinical Trials and Natural History Studies

Biometry Section

Contract Narratives

Framingham Eye Study

Diabetic Retinopathy Study

Diabetic Retinopathy Vitrectomy Study

OFFICE OF PROGRAM PLANNING AND SCIENTIFIC REPORTING

REPORT OF THE CHIEF, OFFICE OF PROGRAM PLANNING AND
SCIENTIFIC REPORTING

EXTRAMURAL AND COLLABORATIVE PROGRAMS

REPORT OF THE ASSOCIATE DIRECTOR FOR EXTRAMURAL AND
COLLABORATIVE PROGRAMS

Vision Research Training

Clinical Trials and the Translation of Research

Results into Application 22|

Vision Research Centers: The National Eye Institute

Concept 223~

Retinal and Choroidal Diseases 227

Corneal Diseases 243

Cataract 25!

Glaucoma 261

Sensory and Motor Disorders of Vision 27'

ANNUAL REPORT

NATIONAL EYE INSTITUTE

July 1, 1976 - September 30, 1977

STATEMENT OF THE INSTITUTE DIRECTOR

The National Eye Institute has just completed its seventh full year of
operation. During the latest year, fiscal year 1977, a budget of $64 million
was available. This is an increase of 30 percent over the previous year and
almost three times the first NEI budget in FY 1970. The extramural budget
alone grew $12 million during FY 1977 to more than $54 million. Research
supported by these funds is directed toward reducing the personal and financial
hardships caused by blindness and visual deprivation through improving our
ability to prevent, diagnose, and treat blinding and disabling eye disorders.

Each year more than 4 million new cases of eye disease occur in the
United States alone, necessitating more than 31 million yearly visits to eye
specialists. An estimated 567,000 Americans are discharged from hospitals
each year with an eye disease or injury listed as their principal diagnosis.
The cost of such care, when added to the economic cost of blindness, exceeds
$5 billion annually, according to estimates made in 1972; the cost today is
probably at least 40 to 50 percent greater.

One of the leading causes of new adult blindness each year is diabetic
retinopathy. During FY 1977 the NEI continued to support the nationwide
clinical trial on diabetic retinopathy which reported the first conclusive
evidence that photocoagulation, treatment with powerful beams of focused
light, can reduce the rate of blindness from this disease by more than 50
percent over a two year period. Subsequent to these findings, a major effort
was made to disseminate this information to eye care specialists and the
general public. As a result, these findings have already had a significant
impact on the treatment of diabetic retinopathy in the United States and
abroad. A major new clinical trial was launched in FY 1977 testing the use
of aspirin and photocoagulation in the treatment of nonproliferative and very
early proliferative retinopathy with special attention to the management of
diabetic maculopathy. -The new study will span approximately 10 years, will
include about 20 to 25 centers, and is one of the most important initiatives
that the NEI has undertaken in the past seven years. In the NEI-supported
nationwide study of vitrectomy, the surgical technique which is capable of
restoring vision to some diabetic retinopathy patients who are already blind,
most of the 13 participating centers have already begun enrolling patients.
The scientific support of these trials is primarily provided by the NEI's
Office of Biometry and Epidemiology. This organization is emerging as one of
the key contributors to biostatistics and epidemiology in the United States.

Discoveries have also been made in the laboratory about the ways in
which diabetes affects the eye. Such findings may also help us understand
the serious complications of diabetes which occur elsewhere in the body.
These studies have continued and expanded during the past year. For example.

organic compounds called flavonoids have proved to be the most effective means
yet found of slowing the development of diabetic cataracts in animal models.
Their ability to inhibit the enzyme which causes such cataracts may also have
important implications for basic studies of the effect of diabetes on the eye.
The NEI will continue to support this research.

The net's intramural program has continued its modest growth stressing
the quality of personnel recruited and innovative approaches in its research
program. Research advances have been made by the intramural staff and noted
in this report in the areas of retinal biochemistry, cataracts, and glaucoma.
Experts in immunology, retinal diseases, biochemistry, and ophthalmic pathol-
ogy have been added to the staff in the past year. Groundbreaking for the
Ambulatory Care Research Facility, the new addition to the Clinical Center,
began this spring, and construction is currently underway. This expansion
of the Clinical Center will result in a considerable increase in the NEI
facilities for outpatient research and thus presents exciting new oppor-
tunities for the intramural program. Additionally, several new laboratories
were made available this year to the Laboratory of Vision Research as a result
of the completion of the addition to Building 6.

About one-third of all office visits for professional eye care are for
conditions affecting the cornea. ARA-A, the first new drug in 15 years to
treat herpes simplex keratitis, was developed with NEI support and approved
this year by the Food and Drug Administration. It is expected to provide a
long-needed alternative to present herpes therapy which is often ineffective
or causes undesirable side effects. During the next fiscal year, the search
will continue for even more effective antiherpes drugs.

During the past year further program planning initiatives were undertaken.
The National Advisory Eye Council's Program Planning Subcommittee met several
times with groups of consultants, which numbered over 150 when totalled, in
order to establish the current needs and opportunities in vision research.
A three-volume report delineating recommended research goals for vision
scientists for the next five years will be published early next fiscal year.

Also during the past year, the NEI held a series of workshops to focus
attention on areas of research where special opportunities exist for concerted
action. One of these was a mee-ting to encourage the application of new
laboratory tests of visual function and performance to the clinical diagnosis
of eye diseases which are difficult to diagnose. Another workshop, to explore
the research needs and opportunities in rehabilitation of partially-sighted
individuals, was held at the NEI.

These and other research projects, contracts, collaborative trials,
and studies initiated, supported, or conducted by the NEI will make progress
toward the goal of relieving the enormous personal and economic costs of eye
disorders.

INTRAMURAL RESEARCH

Clinical Branch

ANNUAL REPORT
NATIONAL EYE INSTITUTE
July 1, 1976 - September 30, 1977

REPORT OF THE CLINICAL DIRECTOR
Elmer J. Balllntine, M.D.

The primary mission of the NEI Clinical Branch is to conduct research
related to those aspects of ocular disease which can be studied best in man.
Such investigations must meet the same standards of scientific rigor and
validity that apply to other biologic experiments and do so within the
ethical and humanitarian constraints imposed by the fact that the subjects
are people.

Each research plan is reviewed by a protocol review committee composed
of representatives from the Clinical Branch, other parts of the NEI and NIH,
and others who are not employees of NIH. The protocol may also be reviewed
by the Clinical Review Committee of the Clinical Center's Medical Board.
These reviews insure that adequate safeguards for rights and welfare of
patients are maintained and that patients are fully informed about both risks
and potential benefits of their participation. Patients are accepted only if
referred by an ophthalmologist outside the Institute and only if the patient's
condition is appropriate for study in an approved protocol.

One of the objectives of the Clinical Branch is to demonstrate that
rigorous clinical trials of important medical hypotheses can be performed
within a single institution with a staff of modest size. One example is the
Clinical Branch's Urokinase Central Retinal Vein Occlusion Trial. In this
study patients with occlusion of the central retinal vein are randomly
assigned to treatment with either heparin, urokinase or conservative treatment.
Both patients and examiners are masked so that the treatment assignment is not
known when the post-treatment examinations are performed.

Formal protocols incorporating these principles either are in operation
or are being reviewed for testing surgical procedures for relief of hemor-
rhagic glaucoma, surgical procedures for treatment of glaucoma after multiple
surgical failures, and the effectiveness of early treatment of ocular hyper-
tension in preventing progressian to chronic simple glaucoma.

The dominant concern of the laboratories in the Clinical Branch is the
study of manifestations of human eye disease. These studies may require
extensive development of methods and demonstration of biochemical or physio-
logic mechanisms as preliminaries to the work in patients. Examples are
studies with the Q-switched laser for the treatment of experimental glaucoma
in monkey, and the development of anterior chamber fluorophotometry for
measurement of aqueous humor production in man.

Other laboratory investigations use material obtained from surgical,
•autopsy, or blood specimens. Thus, surgical specimens from patients under-
going trabeculectomy furnish samples of glaucomatous trabecular meshwork for
growth in tissue culture in an effort to discover a metabolic defect
responsible for simple glaucoma. Vitreous cavity washings obtained when

vitrectomy is performed on patients with diabetic retinopathy are fraction-
ated and assayed for the presence of vasoprolif erative factors.

In collaboration with the Experimental Pathology Section of the NEI
Laboratory of Vision Research, 226 eyes from the autopsy service of the
Clinical Center were processed and examined histopathologically during the
past year. Approximately 1,305 inpatients and 1,196 outpatient consultations
were furnished for other Institutes at the Clinical Center and there were
1,663 outpatient visits during the year. There were 82 inpatient admissions,
and 41 surgical operations were performed.

The Clinical Branch continued to cooperate with other NIH Institutes in
the pursuit of unique research opportunities. The study of diabetic
retinopathy among the Pima Indians in a project administered by the Epidemio-
logy and Field Studies Branch of the National Institute of Arthritis,
Metabolism, and Digestive Diseases was continued as was the. study of micro-
angiopathy among patients with acromegaly. The study of ocular metastasis in
National Cancer Institute patients undergoing treatment of breast carcinomas
continued.

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTfi SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl

EY 00150-04 CB

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Ocular Hypertension Study

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Elmer J. Ballintine M.D.

Other: Douglas E. Gaasterland M.D.

Richard A. Stone M.D.

Richard Weiblinger B.S.

Clinical Director CB NEI

Senior Staff Ophthalmologist CB NEI

Clinical Associate CB NEI

Biologist CB NEI

COOPERATING UNITS (if any)

Office of Biometry and Epidemiology, NEI

lab/branch

Clinical Branch

SECTION

NSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda. Maryland 20014

TOTAL MANYEARS:

1.0

PROFESSIONAL:

0.6

OTHER:

0.4

CHECK APPROPRIATE BOX(ES)
0 (a) HUMAN SUBJECTS

D (al) MINORS □ (a2) INTERVIEWS

n (b) HUMAN TISSUES

n (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

Patients with ocular hypertension are randomly assigned to treatment with
topical pilocarpine in one or both eyes or to no treatment. The objectives of
the study are: 1) to determine if treatment with pilocarpine to reduce intra-
ocular pressure before visual field changes occur will reduce the number of
ocular hypertensive subjects who eventually become glaucomatous, and 2) to
determine if measurements of aqueous humor dynamics; the response to water
loading of diurnal variation of intraocular pressure; serial stereophotographs
of the optic disc, and measurements of visual fields help to predict which
patients will eventually become glaucomatous.

Project No. ZOl EY 00150-04 CB
Project Description:

Objectives: Prolonged observation of a series of patients with ocular
hypertension, some of whom are treated with miotics, will help to determine
which signs have value in predicting those who will eventually require treat-
ment and in determining if early treatment of ocular hypertension has any
value in preventing visual field loss or in slowing the rate of development
of abnormalities of aqueous humor dynamics.

Methods Employed: A detailed plan for classifying patients with ocular
hypertension, observing them by repeated examinations over a period of five
or more years, and randomly assigning patients to treatment with pilocarpine
collyria in one or both eyes, or to no treatment, has been standardized for
repeated measurement of visual fields, aqueous humor dynamics, and photo-
grammetry of the optic discs.

Major Findings: The protocol for conduct of the study has been com-
pleted and recruitment of patients in the study is continuing. Thirty-eight
patients have been enrolled.

Significance to Biomedical Research and the Program of the Institute:
Early, precise identification of patients who require treatment because they
are in the early stages of the simple glaucoma remains an unsolved problem.
The data being collected on this research plan will furnish a basis for
establishing criteria for treatment more precisely than is now possible.
There is at present no detailed knowledge of the progression of optic disc
changes in ocular hypertension. The data being collected in this study, as
well as the development of better instruments for the measurements in this
study, will supply needed information in this field.

Proposed Course: It is expected that the project will continue for at
least five years, and we expect to enroll 100 subjects.

NEI Research Program: Glaucoma

Experimental Subject or Tissue Source: Human

Research Objective: Diagnosis, Treatment

Publications: None

SMITHSONIAN SCIENCE INFORMATION EXCHANGE'
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMtWAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00017-03

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Tissue Culture of Trabecular Meshwork

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Elmer J. Ballintine M.D.

Other: Richard A. Stone M.D.

Richard Weiblinger B.S.

Clinical Director CB NET
Clinical Associate CB NEI
Biologist CB NEI

COOPERATING UNITS (if any)

None

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute. NIH. Bethesda, Maryland 20014

TOTAL MANYEARS:
1

PROFESSIONAL:

0.5

OTHER:

0.5

CHECK APPROPRIATE BOx(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEWS

£] (b) HUMAN TISSUES

n (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

Slices of trabecular meshwork from normal monkey eyes and from surgical
trabeculectomy specimens from human glaucomatous eyes are being grown in
tissue culture. Attempts are being made to identify the tissue of origin of
the resulting cellular growth, and efforts are being made to selectively grow
trabecular endothelial cells.

JJL

Dup Cr\Mf\

Project No. ZOl EY 00017-03 CB
Project Description:

Objectives; Much evidence indicates that in simple open-angle glaucoma
the obstruction to aqueous humor outflow lies within the trabecular meshwork
and inner wall of Schlemm's canal. The amounts of human trabecular tissue^
available for biochemical and physiologic study are insufficient for most in
vitro research methods. Therefore, tissue culture techniques are being
employed in the hope of developing a system in which the basic physiologic
and biochemical abnormality present in open-angle glaucoma can be explored.
After a satisfactory culture system is developed, various metabolic and
physiologic parameters of the cultured cells will be explored.

Methods Employed; Specimens of trabecular tissue are obtained from
monkey eyes for preliminary studies. Some surgical specimens from patients
undergoing trabeculectomy have been studied. More of these surgical speci-
mens as well as controls from human autopsy eyes are being sought.

Specimens of trabecular meshwork are sectioned into small fragments
under the dissecting microscope and placed in tissue culture medium. Phase
contrast microscopy is used to observe growth and form of these cells. They
are being further characterized by their histologic and histochemical prop-
erties. Methods and criteria for growing trabecular epithelial cells free
of fibroblasts are being developed.

Major Findings; Trabecular meshwork from monkey and human eyes has been
grown consistently in tissue culture, and the conditions for this growth have
been determined. It has been possible to obtain some cultures without a
significant fibroblastic contamination.

Significance to Biomedical Research and the Program of the Institute: l|
The mechanism by which the resistance to aqueous humor outflow increases in ■
open-angle glaucoma is at present unknown. This project may be able to definei
the physiologic and biochemical abnormalities of trabecular epithelium that
are the fundamental causes of open-angle glaucoma.

Proposed Course; After further refining of the tissue culture technique,
metabolic studies of the cultured cells will attempt to demonstrate dif-
ferences between normal cultures and those from human eyes that have a pres
sure elevation following topical corticosteroids.

NEI Research Program: Glaucoma - Etiology of Glaucoma (Primary Glaucoma/
Open-Angle Glaucoma)

Experimental Subject or Tissue Source: Monkey/Human

Research Objective: Etiology

Publications: None

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMUfiAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00054-01 CB

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Cornea: Organ and Cellular Cultures of Epithelium Stroma and Endothelium

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: David BenEzra M.D.
Other: Teuro Tanishima M.D.
Sandra Bomstein

Visiting Scientist
Visiting Scientist
Technician

CB NET
CB NET
CB NEI

COOPERATING UNITS (if any)

None

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

2.0

PROFESSIONAL:

1.5

OTHER:

0.5

CHECK APPROPRIATE BOX(ES)
□ (a) HUMAN SUBJECTS

n (al) MINORS □ (a2) INTERVIEWS^

n (b) HUMAN TISSUES

SUMMARY OF WORK (200 words op-less - underline keywords)

The metabolic activity of cells from the various layers of the cornea and
the possible in vitro regulatory mechanism between the layers were investigated:
1. A microculture for accurate and reproducible information concerning the
rate of DNA of protein synthesis at a cellular level has been developed. 2.
Organ cultures of endothelium with its Descemet's membrane were used as a model
for the study of endothelial wound repair mechanism in vitro. The possibility
of transplantation of these organ cultures was also evaluated. 3. Combined
organ cultures: The effects of the various layers of the cornea on each other
were studied in mixed organ cultures. A possible interaction and/or regulation
between epithelium and stroma has been observed. Studies are underway in order
to detect any possible interactions in stroma-endothelium or endothelium-
epithelium organ cultures.

Project No. ZOl EY 00054-01 CB
Project Description:

Objectives: This study focused on the following: 1) Investigate the
metabolic activity of corneal epithelium, stroma and endothelium at a cel-
lular level. Develop a method for the accurate assessment of the effects of
drugs on the various layers of the cornea in normal conditions and in diseased
states 2) Develop an in vitro model for the study of the endothelial wound
healing mechanism. Test the possibility of organ transplantation of cultured
endothelium instead of transplantation of a whole cornea. 3) Study a possible
existing regulatory mechanism between the various layers of the cornea.

Methods Employed: Corneas (guinea pig, rabbit, monkey) are removed and
dissected under an operating microscope.

Macrocultures

These are initiated in petri dishes (Falcon 3001). Explants of tissues
from corneas are covered with a glass cover-slip and a drop of culture medium
(RPMI 1640 with 5 or 10% serum) is allowed to diffuse under it. Cultures are
incubated overnight at 37° in 100% humidity atmosphere and a continuous flow
of 5% C02 and 95% air. After 16 hours of incubation, one milliliter of medium
is added and the cultures further incubated as above. On the third day the
coverslips are removed and 0.5 ml of fresh medium added. Culture medium is
then changed every four days.

Microcultures

On the 8th to 10th day, macrocultures are subjected to trypsinization
(0.25% trypsin and 0.05% EDTA) , cells counted and microcultures initiated in
microtiter plates (Falcon 3040). DNA and protein synthesis are evaluated by
the extent of tritiated thymidine or tritiated amino acids uptake. Transmis-
sion and scanning electron microscopy was carried out at different intervals
after initiation of the macrocultures. The original explants as well as the
patterns observed among the outgrowing cells were studied.

Major Findings: a) Accurate and reproducible information concerning
the metabolic activity of cells, from the different layers of the cornea
could be obtained with 10^ cells per microculture. The origin and concen-
tration of the added serum in the medium influenced markedly the metabolic
activity of all corneal cells tested. Cultures without serum did not demon-
strate any active DNA or protein synthesis but excluded trypan blue. Additiom
of 1% serum boosted the DNA and protein synthesis. Xenogeneic serum had the
most potent stimulatory effect on both activities. Allogeneic and autologous
sera were less stimulatory as compared to the xenogeneic serum. However, both
allogeneic and autologous sera stimulated relatively more protein synthesis
than DNA synthesis while xenogeneic serum had a stronger stimulatory effect or
the DNA synthesis relative to protein synthesis. The epithelial and stromal
cells showed similar patterns of metabolic activity. The endothelial cells ir
this system demonstrated a different pattern of behavior. These differences
are now being further investigated to lessen the effect of drugs on the vari-
ous layers of the cornea.

Project No. ZOl EY 00054-01 CB
Endothelial wound healing

b) Isolated Descemet's membrane with endothelium was kept in vitro and
followed up. Morphological changes in the culture were recorded at intervals
by light, scanning and transmission electron microscopy. It was found that
corneal endothelial cultures demonstrate a "repair" activity near wounds as
early as one hour after the initiation of culture. The endothelial cells
neighboring wounds round-up, elongate and mimic f ibroblast-like structure.
Most of the activity is observed around the edges of the wound while the ad-
vancing cells get bigger and move toward the center of the wound. In the
process of "filling the gap" active cells revert to a morphological appearance
characteristic of the endothelial cells. In rabbit endothelial cultures,
"overzealous" cells proliferate and override the other endothelial cells after
healing of the wound is complete. In contrast, guinea pig and monkey endothe-
lial cultures showed a regular monolayer pattern, c) Electron microscopic
studies of pure epithelial and stromal cultures on one hand and of combined
cultures on the other hand demonstrated that keratocyte activity was inhibited
by the presence of epithelial cells in culture. Pure culture of stromal cell
showed a collagenolytic activity inside the explant. The collagenolytic
activity of the keratocytes is not detectable when epithelial cells are
present on the explant. Furthermore, using the microculture method it was
demonstrated that supematants of epithelial cell cultures had a marked
inhibitory effect on the keratocytes capacity to synthesize DNA. No effect
of stromal cell supernatant on epithelial cell activity could be detected
by the same methods.

Significance to Biomedical Research and the Program of the Institute:
The obtained data and those expected from future investigations would help in
understanding the repair mechanism of corneal injuries as well as the pathol-
ogy of dystrophies and inherited disorders of the cornea. The possibility of
transplanting cultured endothelium would have a great impact on transplanta-
tion of the cornea.

Proposed Course; 1) Further investigate on the transplantability of
the endothelial cultures. 2) Elucidate other possible regulatory influences
between stroma and endothelium and between the latter and epithelium.

NEI Research Program; Corneal Diseases - Corneal Transplantation and
Stromal Injury and Repair/Comeal Edema, Dystrophies, and Inherited Disorders.

Experimental Subject or Tissue Source; Rhesus monkey /Rabbit/Guinea pig.

Research Objective: ' Etiology, Treatment

Publications;

BenEzra, D. : A micro culture technique for the evaluation of corneal
cell metabolism in vitro. Invest. Ophthalmol. (in press).

BenEzra, D.: A in vitro model for the study of corneal endothelial
repair. Tenth Annual Research Conference, Boston 1977.

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER
ZOl EY 00038-05 CB

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Studies of Choroidal-Retinal Degenerative Disease

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI:
Other:

Donald R. Bergsma
Muriel Kaiser-Kupf er
A. Ralph Rosenthal

David Huxal

M.D.
M.D.

M.D.

CB NEI
CB NEI

Senior Staff Ophthalmologist
Senior Staff Ophthalmologist
Chairman, Department of
Ophthalmology, Stanford
University

Deputy Director, Veterinarian
Medicine, Walter Reed Army Institute
of Research, Washington, D.C

COOPERATING UNITS (if any)

Walter Reed Army Institute of Research (Division of Surgery)
Washington, D.C.

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

0.50

PROFESSIONAL:

0.50

OTHER:

CHECK APPROPRIATE BOX(ES)
^ (a) HUMAN SUBJECTS

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□ (c) NEITHER

SUMMARY OF WORK (200 words or~less - underline keywords)

The long range purposes of this project are (1) to improve clinical
classification of selected degenerative diseases of the retina and choroid
by means of detailed, longitudinal studies of retinal function and clinical
laboratory tests, and (2) to provide a clinical resource for related laboratory
investigations of the cause, prevention and therapy of these diseases. The
degenerations of the eye such as retinitis pigmentosa are often of familial or
genetic origin and must be distinguished from nonprogressive forms of night
blindness and from drug or other toxicity. Degeneration of the macula also
occurs in some of these diseases. Animal models are used for related investiga-
tions of retinal functions, degenerations and toxicity. Serum, biopsy and
autopsy specimens available because of this project have been studied in the
laboratory via project ZOl EY 00134-03 LVR (biochemical and tissue culture
studies) .

Project No. ZOl EY 00038-05 CB
Project Description:

Objectives: The objectives of this study are (1) to improve clinical
classification of selected degenerative diseases of the retina and choroid,
and (2) to provide a clinical resource for related laboratory investigations
of the cause, prevention and therapy of these diseases. Examples are m
retinitis pigmentosa, familial macular degeneration, and the effects of drugs
toxic to the retina.

Methods Employed: Clinical studies utilize specialized tests of visual
function (dark adaptation, cone thresholds, visual fields), electroretinog-
raphy (ERG), electro-oculography (EGG), fundus photography and fluorescein
dye studies. Appropriate testing of relatives is undertaken to document
genetic patterns and define variation of severity within disease entitites.
Observation of monkeys poisoned with chloroquine is continuing. Patients with
retinal toxicity due to drugs such as chloroquine and thioridizine are being
studied.

Major Findings: Approximately 100 patients were studied this year. At
present, the overwhelming majority of patients afflicted with choroidal and
retinal degenerative diseases are not curable. No proposed therapy shows
enough promise to warrant a treatment trial. Nevertheless, most patients are
helped by a combination of genetic counselling, discussing of prognosis and
advice regarding visual aids and rehabilitation. Moreover, serum, biopsy
and autopsy specimens available because of this project have been studied bio-
chemically and pathologically via related laboratory projects such as ZOl EY
00134-03 LVR. This led to a combined electronmicroscopic and biochemical
study of autopsy eyes with retinitis pigmentosa which showed that one of the
two normal receptors for vitamin A is absent in the late RP retina.

Related experiments in monkeys show that the subcellular damage to
retinal cells produced by high doses of chloroquine occurs more than four years
before ERG or fundus abnormalities are detectable.

Significance to Biomedical Research and the Program of the Institute:
This project is directed at improving classification, prevention, and treat-
ment of choroidal-retinal degenerative diseases via new diagnostic techniques,
controlled therapeutic trials, long-term follow-up, and animal and laboratory
exper imentat ion .

Proposed Course: Because an adequate panel of patients has been estab-
lished for longitudinal studies, special laboratory investigation, and, when
appropriate, therapeutic trials, emphasis has been shifted to related
laboratory studies via project ZOl EY 00134-03 LVR. Heterogeneity vlthin
clinically defined groups hinders this research. Therefore, emphasis is now
on establishing subpanels with adequate criteria to insure homogeneity.

NET Research Program: Retinal and Choroidal Diseases-Develonmental and
Hereditary Disorders

Experimental Subject or Tissue Source; Human/Rhesus monkey

Project No. ZOl EY 00038-05 CB
Research Objective; Etiology, Diagnosis, Treatment

Publications;

Bergsma, D.R., Wiggert, B. , Funahashi, M. Kuwabara, T. , and Chader, G. :
Vitamin A receptor in normal and dystrophic human retina. Nature 265:
66-67, 1977.

Bergsma, D.R. , Funashashl, M. , Kuwabara, T. , and Christiansen, J.M. :
Ultramicroscopic and vitamin A receptor abnormalities in retinitis pig-
mentosa at autopsy. Trans. Am. Acad. Ophthalmol, (in press).

Bergsma, D.R.: The Usher syndrome: Clinical definition and related
research. In Proceedings of the Usher Syndrome Conference, Helen Keller
National Center for the Deaf-Blind, Port Washington, NY, Dec. 1-3, 1976
(in press).

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER
ZOl EY 00058-01 CB

PEfiSOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Suppressor Lymphokines in an In Vitro System

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Jane Blackman
Other: Joost Oppenheim
Terrell Hoffeld

M.D.
M.D.
D.D.S, Ph.D.

Senior Staff Fellow
Chief

CB NEI
LMI NIDR
LMI NIDR

COOPERATING UNITS (if any)

None

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

1.0

PROFESSIONAL:

1.0

OTHER:

CHECK APPROPRIATE BOX(ES)
n (a) HUMAN SUBJECTS

n (a1 ) MINORS □ (a2) INTERVIEWS

□ (b) HUMAN TISSUES

SUMMARY OF WORK (200 words or -less - underline keywords)

We are investigating the relationship of suppressor and helper lymphokines
in several in vitro systems in mice and rabbits. We have developed an assay
system for suppression of B cell proliferation. We plan to characterize a
suppressor of B cell proliferation with the eventual plan of testing this in a
rabbit experimental uveitis model.

Project No. ZOl EY 00058-01 CB
Project Description:

Objectives: We are investigating lymphokines and their interrelation-
ships in the mechanism of cellular immunity. Past studies of uveitis have
placed much emphasis on himoral immunity, but most humoral immunity appears
to be modulated by cellular immunity. Of recent interest are lymphokines,
i.e. proteins released by lyinphocytes or macrophages which regulate the immune
responses of cellular proliferation, cellular motility, antibody synthesis, or
enzyme manufacture and release.

Therefore, to study suppressor Ijrmphokines or regulator lymphokines would
help to elucidate the mechanisms of inflammation in the eye and possible
treatment. The immediate goal is to characterize a lymphokine suppressor of
B'cell proliferation and investigate its relationships with other B cell
functions or other B cell regulators. A long term goal is to use this sup-
pressor or regulator lymphokine in an experimental ocular inflammation model.
Eventually it is hoped that this can be used therapeutically in uveitis in
human beings.

Methods Employed: An in vitro assay system of suppression of B cell
proliferation has been developed. We have stimulated mouse B lymphocytes in
vitro with endoxtoxion. Consequently, B cells incorporate tritiated thymidine
into their DNA as a signal of proliferation. A suppressor lymphokine has been
made in a manner similar to that described by Rich and Pierce, to which they
have given the name Soluble Immune Response Suppressor ( J. Immunol. 112:1360,
1974) . This involves stimulating mouse spleen cells in vitro with the T cell
mitogen Concanavalin A and harvesting the supernatant. The in vitro assay
system for thymocyte or T cell proliferation and the assay system of Jerne
plaques for antibody synthesis are also being studied to learn the relation-
ship of suppressor lymphokines to other immune mechanisms.

We plan to fractionate and characterize crude supernatants which contain
this suppressor of antibody synthesis. This suppressor probably does not
cross species and does not affect T cell proliferation by a mitogeneic factor,
also present in the crude supernatant.

Major Findings: An assay system for B cell suppression in vitro has been
developed.

Significance to Biomedical Research and the Program of the Institute:
Lymphokines as a part of the immune response in inflammatory diseases of the
eye, especially uveitis, have recently begun to be investigated. They
probably have a very important role in initiating, enhancing, and suppressing
the reactions seen clinically. We wish to pursue their possible role in the
breakdown of different immunologic reactions by separate compartments of the
eye and the regulations of acute or chronic uveitis.

The Eye Institute is interested in elucidating and controlling the
mechanisms of inflammatory ocular diseases, whether of corneal, uveal or
retina origin.

Project No. ZOl EY 00058-01 CB
Proposed Course: The first step is to fractionate crude supernatant
made from Con A stimulated spleen cells. This will begin to characterize the
suppressor lymphokine. Then other definitive tests can be peformed to
more completely characterize this material.

After this the suppressor lymphokine can be tested for its relationship
to other immune systems e.g. for its ability to regulate the functions of
macrophages to make lymphokines, to show motility or to phagocytose foreign
materials. The suppressor Ijmiphokine will be tested for its regulation of
other lymphokine functions or synthesis.

The projected study is then to make this suppressor substance in other
animal systems, the rabbit or guinea pig initially. In an experimental
uveitis model this material then can be tested for its ability to suppress
or regulate a response.

NEI Research Program: Retinal and Choroidal Diseases - Inflammatory
Disorders

Experimental Subject or Tissue Source: Mouse

Research Objective: Treatment

Publications: None

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PROJECT NUMBER (Oo WOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE GF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00020-03 CB

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Parametric Studies of Eye Movement Disorders in Humans

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: David G. Cogan M.D.

Other: John Gittinger M.D.

Fred C. Chu . M.D.

David S. Zee M.D.

Douglas B. Reingold M.A.

Staff Ophthalmologist CB NEI

Clinical Associate CB NEI

Senior Staff Fellow CB NEI

Consultant CB NEI

Research Associate CB NEI

COOPERATING UNITS (if any)

Experimental Therapeutics Branch, NINCDS

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

2.0

PROFESSIONAL:

2.0

OTHER:

CHECK APPROPRIATE BOX(ES)
1 (a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEWS

n (b) HUMAN TISSUES

n (c) NEITHER

SUMMARY OF WORK (200 words or iess - underline keywords)

Our intention in this work is to identify and measure the ocular motor
disabilities that accompany each of several neurological diseases. We have
developed tests of ocular motor subsystems which can reveal these disorders,
their progress and their response to therapy; advanced our understanding of the
mechanisms of these diseases; and gained insight into the roles of some brain
structures in the control of eye movements. We are monitoring elect ro-oculo-
graphically the fixation, saccade, pursuit, optokinetic and vestibular system,
estimating on a laboratory digital computer the parameters of these models
under normal and pathological conditions. The disorders under current study
are: 1) cerebellar tumors and degenerative diseases; 2) Parkinson's disease;
3) progressive supranuclear palsy; 4) parietal lobe lesions; and 5) downbeat
nystagmus. In addition we are observing the oculomotor changes accompanying
normal aging.

Project No. ZOl EY 00020-03 CB
Project Description:

Objectives: To identify and measure the ocular motor disorders in sev-
eral diseases and to elucidate their pathophysiology.

Methods Employed: We test ocular motility using visual and vestibular
stimuli, the former consisting of fixation targets and discreet moving tar-
gets presented on a video monitor, and a full-field optokinetic drum surround-
ing the patient. Vestibular stimuli consist of rotations of the seated
patient about a vertical axis, by means of a precision servo-motor. Eye
movements are monitored using electro-oculography and analyzed by laboratory
digital computer to yield estimates of eye velocities and oculomotor system
gains, latencies, and time constants.

Major Findings: In cerebellar disease, disorders of the saccadic, pur-
suit, fixation, optokinetic, and vestibular subsystems have been identified.
The cerebellum is involved in the maintenance of eccentric gaze, the produc-
tion of smooth pursuit eye movements, control of the amplitude of saccadic
eye movements, and the interaction of visual and vestibular eye movement
reflexes. We have designed a test which seems to be quite specific for
cerebellar disease in which the patient is asked to fixate a target which
remains in front of him while he is rotating. Analysis of the resulting
involuntary eye movements provides a measure of visual-vestibular interaction
which may correlate with the extent of damage to the cerebellum. _

A defect in the optokinetic system of subjects with loss of labyrinthine

function has been observed. This implies that brain structures involved in
the production of optokinetic nystagmus are subject to and dependent upon
vestibular influences, and accords well with the concept of the optokinetic
system's being distinct from the pursuit system in that it contributes to
self-motion perception considerably more than the pursuit system does.

Abnormal saccades have been observed in a group of patients with mon-
ocular impairment of extraocular muscle function. When refixating in the
direction opposite to the paralysis with the paretic eye, a series of
saccades is made with the eye drifting back to the starting position between
each saccade. This is consistent with the assumption that the estimate of
eye position that the central nervous system uses for the control of saccadic
eye movements is based upon efferent rather than afferent information.

Significance to Biomedical Research and the Program of the Institute;
Tests have been created which can indicate the presence and the severity of
oculomotor involvement in the diseases studied. The mechanisms of the oculo-
motor deficits in these diseases have been clarified, and evidence has been
put forward on the roles of several brain structures in the control of eye
movement.

Proposed Course: This project will be continued.

NEI Research Program: Sensory and Motor Disorders of Vision

Project No. ZOl EY 00020-03 CB
Experimental Subject or Tissue Source: Human

Research Objective: Etiology

Publications:

Cogan, D.G. , Yee, R,D. , and Gittinger, J.: Rapid eye movements in myas-
thenia gravis. Arch. Ophthalmol. 94: 1083, 1976.

Yee, R.D., Cogan, D.G., and Zee, D.S.: Ophthalmoplegia and dissociated
nystagmus in abetalipoproteinemia. Arch. Ophthalmol. 94: 571, 1976.

Yee, R.D., Zee, D.S., and Cogan, D.G.: Congenital ocular motor apraxia.
Brain, (in press).

Yee, R.D., Cogan, D.G., Zee, D.S., Baloh, R.W. , and Honrubia, V.: Rapid
eye movements in myasthenia gravis II. Electro-oculographic analysis.
Arch. Ophthalmol. 94: 1465, 1976.

Cogan, D.G.: Opsoclonus. Handbook of Clinical Neurology. Vol. 33,
No. Holland Publishing Co. (in press).

Cogan, D.G., Robins, S.M.: Neuro-ophthalmic disorders. Human Health
and Disease. Biological Handbooks II: 288-293. Fed. of Amer. Soc. for
Exp. Biol., Bethesda, MD.

Cogan,. D.G.: Enigmas and hypotheses. ARVO Symposium on Eye Movements,
San Antonio, Texas. Oct. 26, 1976. (in press).

Zee, D.S., Yee, R.D. : Abnormal saccades in paralytic strabismus. Am.
. J. Ophthalmol. 83: 112, 1977.

Zee, D.S., Yee, R.D., Cogan, D.G., Robinson, D.A., and Engel, W.K.:
Ocular motor abnormalities in hereditary cerebellar ataxia. Brain
99: 207, 1976.

Zee, D.S., Yee,.R.D., and Robinson, D.A.: Optokinetic responses in
labyrinth-defective human beings. Brain Res. 113: 423, 1976.

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00015A-04

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Experimental Glaucoma in the Rhesus Monkey

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Douglas E. Gaasterland M.D.

Other: Teruo Tanishima M.D.

Helen MacLellan M.S.

Senior Staff Ophthalmologist CB NEI
Visiting Scientist CB NEI

Biologist CB NEI

COOPERATING UNITS (if any)

None

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute. NIH, Bethesda. Maryland 20014

TOTAL MANYEARS:

0.6

PROFESSIONAL:

0.2

OTHER:

0.4

CHECK APPROPRIATE BOX(ES)
n (a) HUMAN SUBJECTS

n (al) MINORS □ (a2) INTERVIEWS

n (b) HUMAN TISSUES

£1 (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

The purpose of this investigation is to study the morphology, the physio-
logic function, and the pharmacologic behavior of the eye of the rhesus monkey
in its normal state compared to its state when experimental glaucoma has been
induced by argon laser photocoagulation of the trabecular meshwork. Autoradio-
graphic studies of axoplasmic transport in chronic experimental glaucoma have
been completed. Currently the effects of intraocular pressure elevation on
optic nerve and retina are being evaluated by electron microscopic examination
of the tissue and preparation of whole-mount retinas.

Project No. ZOl EY 000154-04 CB
Project Description:

Objectives: To study physiologic function, pharmacologic behavior, and
morphology of the monkey eye after induction of glaucoma by argon laser photo-
coagulation of the trabecular meshwork. To compare observations to control
normal eyes.

Methods Employed: Circumferential argon laser photocoagulation of the
rhesus monkey trabecular meshwork eventually causes sustained elevation of
Intraocular pressure to the range of 30 to 55 mmHg, the pressure range found
in many patients with open-angle glaucoma. This is in contrast to the acute,
short duration, very high pressure elevation (more than 65 mmHg, up to 95
mmHg) seen in most models for glaucoma. Outflow facility Is evaluated by
perfusion. Aqueous flow is determined by turnover of radlolodinated serum
albumin injected into the anterior chamber. Retinal and optic nerve function
can be studied by autoradiography and electron microscopy to evaluate morpho-
logic evidence of altered axoplasmic flow. The retina can also be studied In
cross section or by preparing whole mounts of the tissue.

Major Findings: In FY 1977, histology and autoradiography of retina and
optic nerve were studied. Data were obtained from 18 eyes of 9 monkeys.
There were 10 eyes with experimental glaucoma, 7 untreated control eyes, and
1 eye which did not have glaucoma but which had received laser treatment of
the angle structures (treated control) . These eyes are important because
they are the first with sustained elevation of intraocular pressure In the
35 to 50 mmHg range to be studied for pressure effects on axoplasmic flow.
Previous studies have been performed by other investigators in monkey eyes
with acute elevations of intraocular pressure to the range of 65 to 95 mmHg
in most cases. This is higher and of shorter duration than the findings in
chronic open-angle glaucoma patients.

In our experiment the autoradiographs were difficult to interpret. There
was considerable variation in grain density from slide to slide for the same
eye, and there was sparslty of labelling. A preliminary impression is that
the glaucoma eyes and the control eyes did not show obstruction of axoplasmic
transport in a way similar to eyes with acute, very high elevation of intra-
ocular pressure. There are Several possible explanations. First, intravitrea!
Injection of the radioactive label produced a lowering of intraocular pressure
in the interval before enucleation (from 14 to 48 hours). This lowering of
pressure might have released an obstruction of axoplasmic flow. Second, it is
possible that changes in axoplasmic flow in chronic mild to moderate glaucoma
are too subtle to be detected by light microscopic, autoradiographic technique

For these reasons, a study was initiated employing an electron micro-
scopic examination of glaucomatous monkey eyes. This will eliminate the
artificial reduction in intraocular pressure at the time of sacrifice caused
by intravitreal injection, and furnish more sensitive information on changes
in the retina, nervehead, and optic nerve. To date two monkeys rfith monoculai
glaucoma (lOP greater than 30 mmHg for at least 3 weeks) have been enucleated
for electron microscopic evaluation of the retina, optic nervehead, and optic
nerve. Four other monkeys have received laser treatment to one or both eyes

I
30 I

Project No. ZOl EY 000154-04 CB
and their intraocular pressure changes are being monitored. Results are not
yet available. Plans are to continue the project.

We are also developing a technique for preparing whole mounts of monkey
retinas. We shall evaluate the distribution and density of the various cell
populations in the normal retina and compare this to the retina in eyes with
chronic experimental glaucoma with mild to moderate cupping of the optic
nervehead.

Significance to Biomedical Research and the Program of the Institute;
All parts of the project are immediately related to clinical problems in
glaucoma. This experimental glaucoma is the best model available for human
chronic open-angle ("simple") glaucoma. Using this model allows close exam-
ination of the retina and optic nerve with the promise of additional insight
into the mechanism of loss of visual function in the patient with glaucoma.

Proposed Course: The project will continue.

NET Research Program: Glaucoma - Primary Glaucoma — Open-Angle Glaucoma/
Secondary Glaucoma

Experimental Subject or Tissue Source; Rhesus monkey

Research Objective: Etiology

Publications;

Gaasterland, D.E.: Axoplasmic transport in eyes with chronic elevation
of intraocular pressure. Presented at the National Eye Institute
Symposium on Experimental Eye Pathology, October 14, 1976, NIH, Bethesda,
Maryland 20014.

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE

NorrcE OF

INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00168-02 CB

PERIOD COVERED

July 1. 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Laser Surgery for Glaucoma

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Douglas E. Gaasterland M.D.

Other: Charles Bonney D.V.M.

Elmer J. Ballintine M.D.

Carl Kupfer M.D.

Thomas Clem

Harold W. Tipton

Senior Staff Ophthalmologist
Ph.D. Visiting Scientist
Clinical Director
Director

Electronics Engineer
Mechanical Fabrication

NET

NEI

BEIB

DRS

BEIB

DRS

COOPERATING UNITS (If any)

Biomedical Engineering and Instrumentation Branch, Division of Research
Services, NIH; Armed Forces Radiobiology Research Institute

lab/branch

Clim'r-al Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute. NIH, Bethe,sd

isda, Maryland 20014

TOTAL MANYEARS:

iL3-

PROFESSIONAL:

0.3

OTHEfi:

CHECK APPROPRIATE BOX(ES)
g] (a) HUMAN SUBJECTS

n (al) MINORS □ (a2) INTERVIEWS

n (b) HUMAN TISSUES

D (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

The high energy and power that are found in some laser beams offer a tool
for noninvasive alteration of anterior intraocular tissue. Specifically, iridot-
omy and trabeculotomy are possible. Such surgery has importance for glaucoma

patients because of potential improvement of favorable surgical outcome statis-
tics and reduced surgical morbidity. This project has as its aim a systematic
evaluation in simian (rhesus) eyes of laser effects and the application of prom-
ising systems and procedures to hicnan glaucoma eyes under controlled conditions.
Current work is directed to obtaining a proper Q-switched ruby laser system and
to initial testing in monkey eyes.

Project No. ZOl EY 00168-02 CB
Project Descrpition:

Objectives: To develop workable laser systems for anterior segment
surgery and to apply the systems to the normal monkey eye. To study the
physiologic and morphologic effects of laser energy upon monkey ayes. To
apply favorable systems to the glaucoma eye of humans under controlled condi-
tions.

Methods Employed: In this, the second year of this project instrument
development and elimination of mechanical and electronics problems have
continued to require considerable amounts of time. Tissue studies for mor-
phology and for physiologic function are done with standard methods: perfusion
of the anterior chamber to determining outflow facility; turnover of RISA to
determine flow; and gross, light and electron microscopic tissue examination.

Major Findings: Two monkeys have been irradiated in vivo, and a number
of monkey eyes have been irradiated in vitro. In one monkey, irradiation of
the trabecular meshwork area resulted in considerable bleeding associated with
a large cyclodialysis. In the other in vivo study, irradiation of the iris
easily produced an iridotomy. Current problems center around continued diffi-
culty with the viewing optics and difficulty with knowing the energy content
of the flash of light delivered to the eye.

Dr. C. Bonney has joined the project as an investigator. His Air Force
experience with laser effects upon the retina and with lasers in general con-
tribute to solution of problems of Instrument development; his veterinary
medicine training contribute to the skills available for the animal studies.

Significance to Biomedical Research and the Program of the Institute:
Potentially, a physically noninvasive laser system for anterior segment sur-
gery might replace conventional invasive operative procedures for several
type,s of glaucoma. This possibility is in its infancy at this time.

Proposed Course: The project will be continued.

NEI Research Program: Glaucoma - Etiology of Glaucoma (Primary Glaucoma-
-Open-Angle Glaucoma/Primary Glaucoma — Angle-Closure Glaucoma/Secondary
Glaucomas)

Experimental Subject or Tissue Source: Human /Monkey

Research Objective: Treatment

Publications: None

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U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00143-04

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Radioiodinated Chloroquine Analog for Diagnosis of Ocular Melanoma

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Douglas E. Gaasterland M.D. Senior Staff Ophthalmologist CB NEI

Other: Elmer J. Ballintine M.D. Clinical Director CB NEI

Carl Kupfer M.D. Director NEI

COOPERATING UNITS (if any)
None

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

0.1

PROFESSIONAL:

0.1

OTHER:

CHECK APPROPRIATE BOX(ES)
B (a) HUMAN SUBJECTS

D (al) MINORS □ (a2) INTERVIEWS

□ (b) HUMAN TISSUES

n (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

The purpose of this clinical investigation is to assess the value of
systemically administered 1-125 labelled chloroquine analog for the detection
of ocular melanoma. Patient enrollment terminated 30 June 75, after the 36th
patient was accepted. Current interest is in continued follow-up examination
of the patients. This will yield information regarding the clinical course of
diagnosed and treated melanoma patients, of diagnosed melanoma patients who
refused treatment, and of patients with lesions which may or may not be ocular
melanoma. The course will be compared to the results of the radioactive tracer
testing.

PHS-6040

Project No. ZOl EY 00143-04 CB
Project Description;

Objectives: To determine the value of using 1-125 labelled chloroquine
analog for the detection of ocular melanoma.

Methods Employed; During this year a number of standard follow-up
clinical examinations have been performed.

Major Findings: In one patient with a negative test in 1975, tumor
growth was documented this year and enucleation advised. This was performed
at the National Naval Medical Center. Histologic examination confirms the
lesion to be a spindle B type melanoma. Two additional patients with highly
suspicious lesions for clinical reasons have had documentation of tumor
growth during the year. Both continue to refuse enucleation. Of these two,
one had a positive and one a negative test in 1975. One patient with a posi-
tive test in 1974 continues to have no change in her pigmented, posterior
lesion; this lesion has now been under observation by various physicians for
15 years without evidence of growth. Several other patients falling into
various categories continue to return for intermittent follow-up examinations.

Significance to Biomedical Research and the Program of the Institute: M
This study has demonstrated t'hat it is possible to differentiate some malig- ■
nant melanomas from other pigmented ocular tumors. The lack of fine discrimi-
nation in this test suggests that improvements of methodology and instrumen-
tation are required before it might have general clinical usefulness. The
fact that I is a gamma emitter continues to draw attention toward continued
use of this, or similar radioisotope-labelled drugs, for diagnosis. Follow-up
information concerning ocular tumor patients is being gathered. This can be
correlated to their diagnostic work-up.

Proposed Course: It is not appropriate to attempt to improve the method-
ology or the instrumentation related to this test within the Clinical Branch
of the NEI. The results of this study are being prepared for publication.
Follow-up of the patients will continue in order to define more clearly their
course and the diagnosis of their lesions.

NEI Research Program; Retinal and Choroidal Diseases - Tumors

Experimental Subject or Tissue Source; Human

Research Objective: Diagnosis

Publications:

Gaasterland, D.E. : Systemic radiation during the radioactive phosphorus
uptake test. Letter to the Editors. Am. J. Ophthalmol. 81: 691, 1976.

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do MOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAHURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00030-06 CB

PE.'^IOD COVERED

July 1,

1976 to September 30. 1977

TITLE OF PROJECT (80 characters or less)

Studies of Parameters of Intraocular Pressure

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

Senior Staff Ophthalmologist CB NEI
Director NEI

Clinical Technician CB NEI

Biologist CB NEI

Head, Section on Biometry OBE NEI
Clinical Associate CB NEI

PI:

Douglas E. Gaasterla

nd M.D.

Others:

Carl Kupfer

M.D.

Lessie McCain

R.N.

Helen MacLellan

M.S.

Roy Milton

Ph.D

John Pederson

M.D.

COOPERATING UNITS (if any)

Normal Volunteer Office, CC, NIH, Pharmaceutical Development Service,
NIH, Biomedical and Engineering Instrumentation Branch, DRS, NIH

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye

Institute, NIH, Betheisda, Maryland 2Q014

TOTAL MANYEARS:

PROFESSIONAL:

n.1

OTHER:

J^L.

CHECK APPROPRIATE BOX(ES)
^ (a) HUMAN SUBJECTS

n (al) MINORS n (a2) INTERVIEWS

n (b) HUMAN TISSUES

□ (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

In this continuing study of the parameters of intraocular pressure, young
and old normal volunteers and patients with glaucoma and ocular hypertension par-
ticipate. There is interest in determining the actual values of the parameters
in eyes not affected by medications, and in determining the effects of anti-
glaucoma medications alone and in combination upon the parameters in normal and
in diseased eyes.

Project No. ZOl EY 00030-06 CB
Project Description:

Objectives: To evaluate parameters of intraocular pressure in normal
eyes, and eyes with ocular hypertension or glaucoma, before and after anti-
glaucoma medications.

Methods Employed: Replicate studies are done upon sophisticated human
participants. Seven parameters are determined before and after medication:
intraocular pressure, episcleral venous pressure, total facility, true facility
of outflow, pseudofacility, aqueous flow, and ocular rigidity. The V-^ of
Goldmann is no longer determined because it does not add any more informatlono
Acute drug effects are emphasized. Chronic drug effects are studied by use
of the Ocusert system (Alza Laboratories) for pilocarpine and in patients
receiving monocular treatment in the Ocular Hypertension Protocol of
Dr. E. Ballintine (ZOl EY 00150-04-CB) .

Major Findings: The major emphasis during this period has been to ac-
quire additional data on acute effects of medications upon the eyes of old
normal volunteers and on chronic effects of pilocarpine upon the eyes of oc-
ular hypertension patients. To this end 277 examinations have been scheduled,
and 186 examinations of normal volunteers or patients have been carried out in
the Glaucoma Research Laboratories during the interval covered by this report.
These have provided additional information concerning effects of topical cat-
echolamines, paras3mipathomimetics, and placebo.

Combined treatment of the eyes of young normal volunteers with topical
epinephrine and pilocarpine causes reduced intraocular pressure. The mechanism
whereby this occurs is interesting in that either of these medications raises
facility of outflow, and together they are additive; but, in contrast, either
of these medications alone reduces aqueous inflow and together, the effects
are no longer observed. The one medication inhibits the other's effect on
aqueous inflow.

Continued development of the f luorophotometer with studies of its accura-
cy have resulted in creation of a separate protocol for clinical investigation
of aqueous flow. This segment of the work has therefore been split off from
the present report, and is reported separately (ZOl EY 00050-01-CB) .

Significance to Biomedical Research and the Program of the Institute:
Study of patterns of alteration of parameters of intraocular pressure by
glaucoma medications allows clearer understanding of their mechanisms of
action. Studies of parameters more clearly define the difference between
normal and abnormal. The measurements can be extrapolated to more basic
physiologic functions, yielding insight to the function of the human eye.
This information is unique in ophthalmic research.

Proposed Course: The project will be continued.

NEI Research Program: Glaucoma - Etiology of Glaucoma (Primary Glaucoma-
-Open-Angle Glaucoma/Secondary Glaucomas)

Project No. ZOl EY 00030-06 CB
Experimental Subject or Tissue Source: Human

Research Objective: Etiology, Diagnosis, Treatment

Publications:

Gaasterland, D.E. and Kupfer, C. : Effects of combined treatment with
epinephrine and pilocarpine on parameters of Intraocular pressure.
Presented at the meeting of the Association for Research In Vision and
Ophthalmology, Sarasota, Florida, April, 1977.

Kollarits, C.R., Gaasterland, D., Dlchlro, G. , et al: Visual loss in a
patient with orbital varices and Ipsilateral glaucoma. Ophthalmic Surg,
(in press) .

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Oo NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00006-06 CB

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Research in Methods of Evaluating Visual Processes

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Ralpn D. Gunkel CD.
Others: Donald R. Bergsma M.D.
Doris Collie

Ophthalmic Physicist CB NEI

Senior Staff Ophthalmologist CB NEI
Technician CB NEI

COOPERATING UNITS (if any)

None

lab/branch

Clinical Branch

SECTION

Psychophysics

INSTITUTE AND LOCATION

National Eye Institute. NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

2.0

PROFESSIONAL:

1.2

OTHER:

0.8

CHECK APPROPRIATE BOX(ES)
[g (a) HUMAN SUBJECTS

[](al) MINORS D (a2) INTERVIEWS

n (b) HUMAN TISSUES

n (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

The general purpose and intent of this project is to conduct tests,
research, and experiments directed toward the use and improvement of clinical
procedures for measuring functions or properties relating to vision and the
eyes. This includes subjective measurements of visibility and chromaticity
thresholds and electrophysiological (objective) measurements as in electro-
retinography and electro-oculography, and physical measurements such as curva-
ture, hardness, elasticity, and transparency.

The loose definition of this project permits a degree of freedom which
has been advantageous to other workers and projects in utilizing certain types
of expertise, instruments, and materials.

PHS-6040

j4i.

Pro.ject No. ZOl EY 00006-06 CB
Project Description:

Objectives: To discover and utilize the most effective and least
traumatic methods for quantitatlng and evaluating any changes in the eye or
its adnexae brought about by disease conditions, toxic materials or degenera-
tion. Objective methods are desired, but are not always attainable. The
obvious goal of this project continues to be the maintenance or restoration
of normal visual function wherever possible.

Methods Employed: Commercially available instruments and those developed
here are used in measuring rod and cone thresholds, color vision, and other
ocular functions in clinic patients. There is frequent consultation with
Clinical Associates and staff members regarding test methods and results,
ap-plicability, interpretation, new ideas, and properties of materials.

Major Findings: Psychophysical tests were done on 420 subjects for the
purpose of evaluating or diagnosing toxic, inflammatory, degenerative or con-
genital retinal conditions.

Various optical, electrical and mechanical devices were designed and/or
constructed for use in the projects of other staff members.

The most notable outgrowth of the project has been the confirmation and
extension of earlier findings in measuring color vision with the chromagraph.
It is now clear that the traditional terms of protanopia, deuteranopia, (red
blindness and green blindness) and tritanopia are not properly descriptive of
the results obtained with the conventional color tests. Heretofore there have
been many cases where the ordinary tests do not distinguish between protanopia^,
and deuteranopia. They never give an accurate measure of the severity of a H^
defect, and they rarely give any definite indication of weakness to colors
other than red and green. On the other hand, the chromagraph leaves no doubt
as to which colors are seen, which are not seen, and the saturation required
for the discrimination of each.

Enough color tests (over 400) have been performed on the chromagraph
and compared with the three conventional test methods to establish standards
for normal and defective color vision of any type.

Significance to Biomedical Research and the Program of the Institute:
Data and information obtained in psychophysical tests contributes materially
to the clinical program, and various devices have been of considerable value
to other research projects.

Since specific color defects are so easily described with the chromagraph,
it seems possible that some of the acquired deficiencies may be correlated
with certain ocular pathologies as a diagnostic aid.

Furthermore, since all of the likely types of color defect nov. appear to
be well-established (which could not be done with the conventional tests) , it
seems Inevitable that this system will eventually be adopted for definitive
color testing in other eye clinics.

Project No. ZOl EY 00006-06 CB
Proposed Course: The project will be continued.

NET Research Program: Retinal and Choroidal Diseases

Experimental Subject or Tissue Source: Human

Research Objective: Diagnosis, Treatment

Publications:

Gunkel, R.D. and Cogan, D.G.: Colorimetry by a new principle. Arch.
Ophthalmol . (in press).

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do MOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00018-03

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Ophthalmologic Screening for Metastatic Lesions to the Eye

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Muriel Kaiser-Kupf er M.D. Senior Staff Ophthalmologist CB NEI
Other: Joan Bull M.D. Senior Staff FeUow CB NCI

COOPERATING UNITS (if any)

National Cancer Institute

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute,

NIH. Bethesda. Maryland 20014

TOTAL MANYEARS:

.36

PROFESSIONAL:

.26

OTHER:

CHECK APPROPRIATE BOX(ES)
S (a) HUMAN SUBJECTS

D (al) MINORS D (a2) INTERVIEWS

n (b) HUMAN TISSUES

D (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

It is the purpose of this project to determine the incidence of metastatic
eye disease in patients with metastatic breast carcinoma as well as to evaluate
the effects of irradiation on the eye in patients who have metastatic disease
and receive irradiation in conjunction with chemotherapy. The response of
choroidal lesions to therapy may serve as a measurable lesion for indication
of response elsewhere in the body.

Project No. ZOl EY 00018-03 CB
Project Description:

Objectives: To determine the incidence of metastatic eye disease in
patients with metastatic breast carcinoma, to evaluate the effects of
irradiation on ocular tumors which threaten central vision and monitor the
effects of irradiation on the eye, and to evaluate the effectiveness of the
hormonal manipulations and chemotherapy on ocular metastasis in relation to
systemic effect on tumor-

Methods Employed: All NCI patients having metastatic breast carcinoma
are examined ophthalmoscopically . Those patients having metastatic disease
to the eye are then followed frequently as indicated. The course of the
ocular metastatic; disease is followed with serial color and infrared fundus
photography, Goldmann perimetry and fluorescein fundus photos when indicated.

Major Findings: To date approximately 100 patients have been seen and
of those approximately 13 patients have had evidence of ocular metastasis.
Several patients have been found to have developed secondary keratitis
following radiation therapy to the posterior choroid.

Significance to Biomedical Research and the Program of the Institute;
The response of choroidal metastatic lesions to cancer chemotherapy could
serve as an indicatipn of response to metastatic disease elsewhere in the
body.

Proposed Course: To continue for one additional year.

NEI Research Program: Retinal and Choroidal Diseases - Tumors

Experimental Subject or Tissue Source: Human

Research Objective: Diagnosis, Treatment

Publications: None

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00011-03

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Pigment Dispersion With and Without Glaucoma

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Muriel Kaiser-Kupfer M.D.
Other: Luis DelValle M.D.

Senior Staff Ophthalmologiat CB NEI
Staff Fellow CB NEI

COOPERATING UNITS (if any)

None

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda. Maryland 20014

TOTAL MANYEARS:
.90

PROFESSIONAL:

.70

OTHER:

CHECK APPROPRIATE BOX(£S)
B (a) HUMAN SUBJECTS

D (al) MINORS D (a2) INTERVIEWS

n (b) HUMAN TISSUES

D (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

The purpose of this project is to compare patients having pigment
dispersion syndrome with and without glaucoma. The acquired data may enable
a determination of the risk of patients with pigment dispersion syndrome to
develop glaucoma as well as to understand more of the pathology of the disease
state.

-Ai-

PHR-6nin

' ' Project No. ZOl EY 00011-03 CB

Project Description:

Objectives: To compare patients having pigment dispersion with and
without glaucoma by documenting and following the clinical features and course
of their disease, and by evaluating the patient's performance on a variety of
diagnostic tests. To determine the presence of abnormal aqueous humor dy-
namics using provocative testing in those patients having pigmentary disper-
sion with and without glaucoma. To compare pigment dispersion with and
without glaucoma with respect to possible genetic markers (i.e. lymphocyte
transformation, HLA and ABO antigens and family history of open-angle
glaucoma). To determine whether pupillary dynamics to light stimulation are
abnormal in cases having iris transillumination.

Methods Employed: At the first visit, the following examinations are
performed:

Complete family history with detailed pedigree

Best corrected visual acuity with manifest refraction

Slit lamp examination

Visual field examination (Goldmann l2e and l4g)

Applanation Goldmann tension (app)

Photography of iris transillumination

Goniophotography

Blood specimen for HLA and ABO antigen typing

At the next visit, the following examinations are performed:

Static perimetry

Base-line tonography and water-drinking tonography one hour later

Fasting blood sugar when indicated

At the third visit, the following examinations are performed:

Slit lamp photography of Krukenberg spindle

Dilated ophthalmoscopic examination (10% phenylephrine and 1%

cyclogel)
Stereophotographs of the optic nervehead

At the fourth visit, pupillography is performed.

Major Findings: Patients may have pigment dispersion syndrome for as
long as 20 years without developing glaucoma.

There may be a hereditary predisposition in some cases, as seen in a
mother and daughter, two brothers, and a brother and sister.

The steroid testing and PTC taste testing do not appear to show any
particular categorization of these patients. Recent evidence has indicated
that HLA antigens are also not significantly different than the normal
population.

Project No. ZOl EY 00011-03 CB
It may be noted that whether filtering procedures are performed or not,
pigment may be lost from the trabecular meshwork in time.

Significance to Biomedical Research and the Program of the Institute:
These data may enable a determination to be made of the risk of patients
having pigment dispersion to develop glaucoma. It may be possible to
identify which features of these determinatipns may have predictive value
in forecasting those patients having pigment dispersion who may develop a
field defect. In addition, the relationship of "pigmentary" glaucoma to the
known characteristics of open-angle glaucoma can be investigated •

Proposed Course: This project will be continued for four more years

NET Research Program: Glaucoma - Etiology of Glaucoma (Developmental
Glaucomas/Secondary Glaucomas)

Experimental Subject or Tissue Source; Human

Research Objective: Diagnosis, Treatment

Publications:

Kaiser -Kupfer, M.I. and Mittal, K.K. : The HLA and ABO antigens in
pigment dispersion syndrome. Am. J. Ophthalmol, (in press).

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00042-01

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Progressive Essential Iris Atrophy

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Muriel I. Kaiser-Kupf er M.D.

Other: Carl Kupfer M.D.

Rodney Lynk M.D.

Senior Staff Ophthalmologist CB NEI
Director NEI

Medical Officer CB NEI

COOPERATING UNITS (if any)

None

LAB/BRANCH

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

.56

PROFESSIONAL:

,44

OTHER:

CHECK APPROPRIATE BOX(ES)
S (a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEWS

□ (b) HUMAN TISSUES

n (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

Patients are being recruited with progressive essential iris atrophy
associated with or without corneal disease. Information is being gathered
to evaluate the clinical features and course of the disease process, to
investigate aqueous humor dynamics in both affected and unaffected eyes and
to attempt to find genetic markers such as HLA and ABO antigens or physical
correlates with the disease process.

PHs-finin

Pro.lect No. ZOl EY 00042-01 CB
Project Description;

Objectives: The objectives of the study are to develop a panel of
patients with progressive essential iris atrophy and. to study these patients
in depth to determine factors which may aid in understanding the patho-
physiology of the disease process and to study the natural history of this
disease. Measurements of aqueous humor dynamics, genetic markers such as
HLA and ABO antigens, physical correlates and iris fluorescein angiography
to determine the role of the vasculature will be carried out.

Methods Employed: During the course of the evaluation the following
procedures are performed:

Complete family history with detailed pedigree

Best corrected visual acuity with manifest refraction

Slit lamp examination

Visual field examination (Goldmann l2e and I^g)

Photography of iris and iris transillumination

Gonioscopy and goniophotography

Iris fluorescein angiography and photography

Baseline tonography

A complete medical and dental evaluation

Dilated ophthalmoscopic examination

Stereophotographs of the optic nervehead

Major Findings: Histopathologic and electron microscopic study of iris
tissue and trabecular meshwork tissue has not indicated any clues to the
pathogenesis of the disease process.

An ultrathin corneal contact lens is useful in certain patients to pre-
vent recurrent rupture of corneal bullae.

Significance to Biomedical Research and the Program of the Institute:
These data may contribute to an understanding of pathophysiologic factors
involved in the rare entity of progressive essential iris atrophy. In
addition, a careful study of the progression of the disease from the earliest
signs will clarify the significance of corneal involvement and the status of
outflow channels which may add to the understanding of the mechanism of
glaucoma.

Proposed Course: The project will continue for four more years.

NEI Research Program: Glaucoma - Etiology of Glaucoma (Developmental
Glaucomas/Secondary Glaucomas)

Experimental Subject or Tissue Source: Human

Research Objective: Diagnosis, Treatment

Project No. ZOl EY 00042-01 CB
Publications: ' ' ~

Kaiser-Kupfer, M. , Kuwabara, T. , and Kupfer, C. : Progressive bilateral
essential iris atrophy. Am. J. Ophthalmol. 83: 340, 1977.

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT- NUMBER

ZOl EY 00040-01

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Visual Function and Ocular Pigmentation in Albinism

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Muriel I. Kaiser-Kupf er M.D. Senior Staff Ophthalmologist CB NEI.
Other: None

COOPERATING UNITS (if any)

None

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute,,

NIH. Bethesda, Maryland 20Q14

TOTAL MANYEARS:

,20

PROFESSIONAL:

OTHER:

CHECK APPROPRIATE BOX(ES)
S (a) HUMAN SUBJECTS

D (al) MINORS D (a2) INTERVIEWS

n (b) HUMAN TISSUES

D (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

Patients with hypomelanotic disorders such as ocular albinism, oculo-
cutaneous albinism, Chediak-Higashi Disease, Hermansky Pudlak Syndrome and
Iris transillumination defects are being recruited as well as family members
to determine visual function and to evaluate the course of visual function with
time. Family members are evaluated to attempt to determine factors which may
identify the heterozygous state.

PHS-6040

Pro.iect No. ZOl EY 00040-01 CB
Project Description;

Objectives: The objectives of the study are to relate the level of
visual function to the amount of ocular pigmentation, especially iris and
retinal pigmentation; to correlate the amount of nystagmus with visual
acuity and iris pigmentation; to determine whether ocular pigmentation, visual
acuity and nystagmus change with age; and to identify the heterozygous state
in family members.

Methods Employed: The following examinations are performed:

Complete family history with detailed pedigree

Best corrected visual acuity at near and distance with

refraction
Slit-lamp examination

Nystagmus recording using eye movement monitoring EGG
Psychophysical testing including D-15 and Munsell 100

hue, rod and cone thresholds
Dilated ophthalmoscopic examination
Hair bulb incubation
Photography to document hair color, eye color, skin

color, iris transillumination, disc and macula

Examination of family members includes:

Best corrected visual acuity
Slit-lamp examination of iris
Photography of iris transillumination
Fundus examination when vision not corrected
to 20/20

Major Findings: Examination of patients and family members indicates
that the finding of transillumination of the iris may be seen in the absence
of recognized albinism. The pattern appears to be punctate and may be
present in a diffuse manner or limited to the 6 o'clock sector.

Significance to Biomedical Research and the Program of the Institute:
These data may allow identification of the carrier state in albinism which
would be of importance in genetic counselling. In addition, it may be
possible to determine whether the development of the fovea is abnormal in
albinism and the cause of the decreased visual acuity or whether the de-
creased visual acuity is secondary to the hypopigmentation and light-scatter
and glare that results. In addition, it will be possible to ascertain
whether there is an improvement of the visual acuity with age and correlated
with changes in pigmentation.

Proposed Course: This project will be continued for five more years.

NEI Research Program: Retinal and Choroidal Diseases - Developmental
and Hereditary Disorders

Project No. ZQl EY OOOAO-01 CB

Experimental Subject or Tissue Source: Human ' ' "^ ~~f~

Research Objective: Diagnosis, Treatment
Publications: None

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
MOT ICE OF
INTRAiUfiAL RESEARCH PROJECT

PROJECT NUMBER

ZOl

EY 00013-06 CB

PERIOD COVERED

July 1. 1976 to September 30, 1977

TITLE OF PROJECT (30 characters or less)

Study of Pharmacodynamics of Various Agents Affecting the
Intraocular Pressure

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Frank J. Macri
Other : None

Ph.D.

Pharmacologist

CB NEI

COOPERATING UNITS (if any)

None

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute. NIH, Bethesda. Maryland 20014

TOTAL MANYEARS:

2.0

PROFESSIONAL:

1.0

OTHER:

1.0

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS D (a2) INTERVIEWS

n (b) HUMAN TISSUES

S(c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

Clonidine, in the enucleated arterially perfused cat eye, was found to
produce a decrease in the rate of aqueous humor production. The mechanism
of action for this response has been ascribed to a vasoconstriction of afferent
ciliary process blood vessels to decrease ultrafiltration.

Cold, as a stimulus to the cornea, was found to produce an easily re-
versible decrease in aqueous humor production. The mode of action of this
response is being studied.

Changes of intraocular pressure, arterial pressure or of osmotic pressure
induced no local intraocular reflexes which influenced aqueous humor production

PHS-6040

Project No. ZOl EY 00013-06 CB
Project Description:

Objectives: To determine the pharmacodynamics of agents able to alter
the intraocular pressure (lOP) with a view to finding. more effective compounds
and possibly to furthering the understanding of mechanisms which maintain
the intraocular pressure.

Methods Employed: Studies are made on the enucleated, arterially
perfused cat eye. Perfusate is channeled through the ophthalmic artery to
nourish the entire eye, or a ligature is placed around the optic nerve at its
insertion, so that only the anterior segment of the eye is perfused. Drugs
and other test substances are added to individual bottles of perfusate fluid
which can then be introduced into the system by stopcock control. Temper-
ature and rate of arterial flow are easily regulated. The rate of aqueous
humor formation was estimated by determining the rate of decay of intra-
camerally injected I tagged serum albumin.

Major Findings:

Clonidine

Clonidine (Catapres) , a well-known antihypertensive agent, has been used,
primarily in Europe, to decrease intraocular pressure. Its mechanism of
action on the eye was uncertain since it always produced a parallel reduc-
tion of systemic blood pressure. Utilizing our enucleated, arterially
perfused cat eye preparation, we have demonstrated that Clonidine produces a
vasoconstriction in the anterior segment of the eye by direct stimulation
of a-adrenergic receptors. Clonidine also produces a fall in the rate of
aqueous humor production. These two findings suggest that Clonidine acts
much like phenylephrine and dopamine to reduce eye pressure.

Local reflexes which can offset intraocular pressure

We have made a search for local reflexes of the eye which could possibly
alter lOP. In the course of these studies, we have found no reflex actions
on either lOP or aqueous humor inflow rates. The stimuli applied to the eye
were changes of intraocular blood pressure, intraocular pressure or changes
of arterial perfusate osmotic pressure. We have found, however, a significant
decrease in the rate of aqueous humor production and lOP when cold is applied
to the cornea. The response is not mediated by neurogenic corneal receptors
since xylocaine could not abolish the response. The response also is not
mediated through the intraocular E-1 receptors. The mechanism of action for
the ocular effects still remains to be elucidated.

Central neuronal connection of E-1 intraocular receptors

Although we have demonstrated nicotinic receptors in the eye, which
when stimulated lower lOP and aqueous flow, the physiologic significance
of these receptors is unknown. If we can demonstrate a connection of these

Project No. ZOl EY 00013-06 CB
receptors to extraocular nerves, central control would then be implied and
a possible physiologic role indicated. Stimulation of extraocular sympa-
thetic nerves does not activate these receptors. Currently, studies are under
way to determine if these receptors are under p-sympathetic control.

Significance to Biomedical Research and the Program of the Institute:
The sympathomimetic nature of Clonidine action on the vasculature of the eye
and on aqueous humor formation mimics the responses reported last year for
phenylephrine, hydroxyamphetamine, and dopaifiine. This l^nds additional sup-
port to our concept that vasoconstriction of afferent ei},iary process blood
vessels decrease aqueous humor formation. These findings also demonstrate
that the clinical application of Clonidine to the eye can decrease lOP by
decreasing the inflow rate of aqueous humor in addition to its centrally
mediated responses. Admonition by certain individuals that Clonidine
should not be used clinically on the eye because of its vasoconstrictive
actions appears open to question. The vasoconstrictive action pf Clonidine
is identical to that produced by phenylephrine and hydroxyamphetamine, drugs
which have been in general ophthalmic; use foi: many y^ars,

The finding of an apparent reflex decrease of aqueous humor production
and TOP resulting from cold application to the cornea was unexpected. The
physiologic significance and mechanism of action of fhis phenomenon remains
to be determined. •

Although there are reports to indicate regulation of intraocular pressure
through the sympathetic nervous system, we have not been able to demonstrate
any sustained effect of sympathetic nerve stimulation on either lOP or
aqueous humor formation. This is important since we had previously antici-
pated that the E-1 receptors of the eye (those which constrict afferent
ciliary process blood vessels) were part of this system. Present evidence
is accumulating, however, to indicate that the E-1 receptors are under p-
sympathetic control as are the E-2 receptors (those which constrict efferent
ciliary process blood vessels) .

Proposed Course: We anticipate continuing to study the mechanism of
the cold response of the cornea. This may uncover pathways (either neuro-
genic or chemical) which could, by a local response, affect the lOP. We
also intend to continue studies to determine if the E-1 receptors of the
eye are in communication with the central nervous system. If they can be
found to be so, the implication would be that these receptors are under
control of the brain, and thus their action can be centrally regulated.

Recent reports indicate that Timolol, a B-adenergic blocking agent, is
effective in lowering the lOP in glaucoma patients. The mechanism for this
response is unclear. We intend to study this compound in the enucleated,
arterially perfused cat eye in order to determine how the lOP is lowered and
to elucidate the pharmacologic mechanism of the response.

NEI Research Program: Glaucoma - Hydrodynamics of ^he jEye

Project No. ZOl EY 000013-06 CB
Experimental Subject or Tissue Source: Cat

Research Ol^jective: Etiology

Publications:

Macri, F.J. and Cevarlo, S.J.: Blockade of the ocular effects of
acetazolamide by pbencyclidine. Exp. Eye Res. 24: 121-127, 1977.

Macri, F.J. aiid Cevario, S.J.: The inhibitory actions of dopamine,
hydroxyamphetamine and phenylephrine on aqueous humor formation,
Exp. Eye Res, (in press).

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PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00052-01 CB

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Cell and Tissue Interactions in the Production of Corneal
Collagenase

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: David A. Newsome M.D. Investigator CB NEI

Other: Jerome Gross M.D. Massachusetts General Hospital

COOPERATING UNITS (if any)

Developmental Biology Laboratory,

Massachusetts General Hospital and Harvard Department of Medicine

Boston, Massachusetts

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

" National Eye Institute, NIH, Bethesda, Maryland 2001A

TOTAL MANYEARS:

1.5

PROFESSIONAL:

1.5

OTHER:

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEWS

□ (b) HUMAN TISSUES

g] (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

This study of alkali-burned rabbit corneas was designed to understand
more about the cellular and tissue regulatory responses that control the
elaboration of the enzmye collagenase, one of the chief factors responsible
for corneal ulceration and melting. The present project is to 1) document and
localize the elaboration of collagenase by corneal tissues, 2) examine the
interaction of blood monocytes and their products with corneal cells in
stimulating enzyme production, 3) examine the interaction of regenerated and
normal corneal epithelium and stroma in enz}nne production, and 4) learn more
about the regulation of collagenase production and collagen synthesis by
corticosteroid and progestational hormones.

PHS-6040

Pro.ject No. ZOl EY 00052-01 CB
Project Description:

Objectives; The goal of this project is to understand mechanisms of
stimulation and inhibition of collagenase production in corneal tissue, since
this enzyme plays an important role in corneal melting and ulceration asso-
ciated with various pathological conditions.

Methods Employed; Normal corneas and alkali-burned corneas from young
adult albino rabbits provided explants and sources of cell populations for
cell cultures. Blood monocytes were prepared by centrifugation on a Ficoll-
Hypaque gradient. In the hormone studies, radioactive proline incorporation
in total protein and as hydroxyproline in collagen was determined using
stream splitting on the amino acid analyzer. Collagenase was measured using
'*C-labeled collagen gel technique.

Major Findings:

Tissue source of corneal collagenase

Stroma cells which migrated out of explants of alkali-burned tissue
produced the greatest amounts of collagenase. This enzyme production per-
sisted through about two serial passages, after which it became undetectable.
In a few cases, isolated epithelium from alkali-burned corneal explants was
associated with transient, low-level enzyme production. In normal corneal
tissue, small amounts of enzyme production were observed only with early
cultures of stromal cells.

Stimulation of collagenase production by blood monocytes

The addition of peripheral blood monocytes, or medium in which these mono-
cytes had been incubated, to cultures of corneal stromal cells resulted in a
stimulation of collagenase production. Monocytes from rabbits with alkali-
burned corneas produced about a two-fold greater effect than did cells from
normal animals .

Corneal epithelial-mesenchymal interactions

Cultures of alkali-burned stromal cells that had ceased to produce
detectable collagenase were stimulated to produce significant amounts of
enzyme when added to cultures of epithelium from similar corneas. Normal
epithelium also stimulated enzyme production in alkali-burned stromal cells
but not in normal stromal cells. The medium from epithelial cells was also
stimulatory.

Hormonal effects on collagenase production and collagen synthesis

Both the corticosteroid dexamethasone and the semi-synthetic hormone
medroxyprogesterone decreased collagenase synthesis as well as total protein
synthesis in vitro. The effect of dexamethasone was more marked than that of
medroxyprogesterone, and the depression of collagen synthesis preferentially
greater. Basement membrane collagen production, as judged by synthesis of

Project No. ZOl EY 00052-01 CB
3-hydroxyproline, was also more depressed by dexamethasone.

Significance to Biomedical Research and the Program of the Institute:
These investigations have added to knowledge of the cellular and tissue
regulatory mechanisms involved in the elaboration of the tissue-destructive
enzjmie collagenase. Control of such enzymes is useful in treating a variety
of serious corneal diseases.

Proposed Course: The mechanism and site of hormonal influence on
collagenase production in alkali-burned corneas will be investigated further.

NEI Research Program: Corneal Disease - Corneal Transplantation and
Stromal Injury and Repair

Experimental Subject or Tissue Source: Rabbit

Research Objective: Etiology

Publications:

Newsome, D.A. and Gross, J.: Cellular regulation of corneal collagenase:
Stimulation of serially passaged stromal cells by blood mononuclear
cells. J. Exp. Med, (in press).

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PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER
ZOl EY 00050-01 CB

PERIOD COVERED

July 1. 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Aqueous Humor Flow Measurement by Fluorophotometry

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Jonathan E. Pederson M.D.

Other: Douglas E. Gaasterland M.D.

Helen M. MacLellan M.S.

Clinical Associate CB NEI

Senior Staff Ophthalmologist CB NEI
Biologist CB NEI

COOPERATING UNITS (if any)

None

LAB/ BRANCH

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute. NIH. Bethesda. Maryland 20014

TOTAL MANYEARS:

0.4

PROFESSIONAL:

0.3

OTHER:

Q.l

CHECK APPROPRIATE BOX(ES)
3 (a) HUMAN SUBJECTS

(a1 ) MINORS n (a2) INTEF?VIEWS

□ (b) HUMAN TISSUES

n (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

This work attempts to measure the rate of aqueous humor flow in humans by
determining the rate of loss of fluorescein from the eye after iontophoresis of
fluorescein into the cornea. The aqueous hianor flow is also calculated from
tonoaraphic results in the same individuals, allowing a comparison of the two
methods. Individuals with normal, low, or high intraocular pressure are studied
to examine the effect of intraocular pressure on aqueous flow.

The accuracy of the aqueous flow rate measured by fluorophotometry was
evaluated in freshly enucleated monkey eyes. The calculated flow rate averaged
5% lower than the known perfusion rate.

PHS-6040

Project JTo; ZOl EY 00050-01 CB
Project Description:

Objectives: This project is designed to measure directly aqueous humor
flow in humans with a f luorophotometer. The first objective is to compare the
results with a method of indirect calculation of floW from tonography. The
second objective is to determine the relationship between intraocular pressure
and aqueous humor flow.

Methods Employed: A cylindrical piece of polyacrilamide gel is saturated
with fluorescein solution. The gel is touched to the cornea, and fluorescein
is deposited due to a small current provided by a dry cell battery. A photo-
multiplier tube with appropriate filters, mounted on a slitlamp biomicroscope,
measures the total amount of fluorescein in the eye, as well as the aqueous
concentration. Illumination is provided by a chopped light source. The
photomultiplier tube signal is fed to a tuned amplifier. The rate of loss of
fluorescein from the eye as a function of time yields the flow rate of aqueous
humor .

Major Findings: The accuracy of the aqueous flow rate measured by fluor-
ophotometry was evaluated in freshly enucleated monkey eyes. The calculated
flow rate averaged 5% lower than the known perfusion rate. This establishes
the procedure as a useful tool in the measurement of aqueous flow. Preliminary
studies have been performed in a few individuals and yielded reproducible
results. The aqueous flow measured by f luorophotometry tends to exceed the
flow calculated from tonography.

Significance to Biomedical Research and the Program of the Institute:
The aqueous himior flow rate is a primary determinant of the intraocular pres-
sure. An accurate, safe, and reproducible determination of the flow rate in
humans under normal and pathological conditions will lead to increased under-
standing of glaucoma and hypotony.

Proposed Course: The human studies will continue. After an examination
of symmetry between paired eyes of the same individual, drug studies will be
initiated, using those agents commonly employed in the treatment of glaucoma.

NEI Research Program: Glaucoma

Experimental Subject or Tissue Source: Human/Rhesus monkey

Research Objective: Etiology

Publications: None

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PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER
ZOl EY 00046-01 CB

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Laboratory Studies of Aqueous Humor Dynamics

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Jonathan E. Pederson M.D. Clinical Associate CB NEI

Other: Douglas E. Gaasterland M.D. Senior Staff Ophthalmologist CB NEI

Helen M. MacLellan M.S. Biologist CB NEI

COOPERATING UNITS (if any)

None

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute. NIH, Bethesda,

Maryland 20014

TOTAL MANYEARS:

1.0

PROFESSIONAL:

0.6

OTHER:

0.4

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

n (al) MINORS □ (a2) INTERVIEWS

n (b) HUMAN TISSUES

a (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

Several interrelated projects to investigate intraocular fluid movement in
rhesus monkeys have been initiated. 1) Intraocular vascular reabsorption of
aqueous humor was found to be 10% of the total amount of aqueous humor leaving
the eye. This reabsorption occurs by a pressure-dependent flow into uveal ves-
sels. 2) The amount of reflux of fluid across the trabecular meshwork from
Schlemm's canal during hypotony was studied. The amount of reflux was 7% of the
aqueous humor production rate. This establishes that the outflow pathway is a
virtual one-way valve. 3) A comparison was made of the change in outflow facil-
ity during constant pressure perfusion with pooled aqueous humor versus gluta-
thione-bicarbonate Ringer's solution. Ringer's solution caused a progressive
increase in outflow facility, whereas aqueous humor did not. 4) The composition
of aqueous hxrnior from pooled samples has been under study. 5) An unsuccessful
attempt was made to determine the intraocular pressure (P-0 at which aqueous
formation ceased by photographing the appearance cf fluorescein on the ciliary
process after intravenous injection. 6) A study has been started of the effect
oi^ intraocular pressure of injecting various solutions into the suprachoroidal
space. 6a

Project No. ZOl EY 00046-01 CB
Project Description:

Objectives: This project is designed to examine the physiology of intra-
ocular fluid movement under varied experimental conditions. The major empha-
sis is on conventional and secondary outflow mechanisms.

Methods Employed: Various methods of perfusion and cannulation of the
eye with subsequent measurement of pressures, flows, and concentrations of
various substances were performed.

Major Findings:

Uveal reabsorption of aqueous humor

The concentration of fluorescein and radioiodinated serum albumin were
measured in the vortex vein of the rhesus monkey during anterior chamber per-
fusion of these substances at two different intraocular pressures. Only a
tiny amount of albumin appeared in the vortex vein blood, but a rapid excess
of fluorescein above that in the plasma appeared. This excess was pressure-
dependent, suggesting an ultraf iltrative uptake into uveal vessels.

Reflux fluid movement across Schlemm' s canal during hypotony

The anterior and posterior chamber concentrations of labeled sucrose and
inulin were measured during constant intravenous infusion of these substances.
At an intraocular pressure of 2 mmHg, amounts of sucrose and inulin equivalent
to 7% of the aqueous volume produced by the eye refluxed back into the ante-
rior chamber. The resistance of reverse fluid movement was calculated to be
about 50 times greater than the resistance to fluid movement in the normal
direction.

Perfusates and the washout phenomenon

The facility of outflow was measured by constant pressure perfusion in
monkey eyes. Using pooled monkey aqueous humor and glutathione-bicarbonate
Ringer's solution as perfusate. Pooled aqueous humor did not cause a change
in facility, but Ringer's solution caused a progressive increase in facility.
This was not due to pH or change in ascorbate concentrations.

Chemical composition of aqueous humor

A systematic analysis of the chemical composition of monkey aqueous humor
was begun in order to create an ideal perfusing solution for experimental
studies of aqueous humor dynamics.

Fluorescein cycloscopy

Iridectomized monkeys were examined gonioscopically during intravenous
infusion of fluorescein. An attempt was made to determine the Intraocular
pressure at which fluorescein would not appear on the ciliary processes. The
rapid diffusion of fluorescein into the posterior chamber, even at high intra-
ocular pressures, precluded an accurate determination.

Project No. ZOl EY 00046-01 CB
Hypotony and choroidal detachment

A study of the effect of choroidal detachment on intraocular pressure
was initiated. A comparison between solutions of varying composition injected
into the suprachoroidal space is under investigation. Preliminary results
suggest a central role of reduced protein movement out of the eye as a caus-
ative mechanism for the hypotony of choroidal detachment.

Significance to Biomedical Research and the Program of the Institute:
These studies should elucidate the normal djmamics of aqueous hximor as well
as the abnormal dynamics in experimentally induced situations, mimicking
clinical problems. Ultimately, these studies may yield information appli-
cable to glaucoma and hypotony.

Proposed Course; Similar studies will be continued. Particular emphasis
will be, placed on the perfusate effects on outflow resistance, and the inter-
relationship between choroidal detachment and intraocular pressure.

NEI Research Program; Glaucoma - Hydrodynamics of the Eye

Experimental Subject or Tissue Source: Rhesus monkey

Research Objective: Etiology

Publications:

Pederson, J.E., Gaasterland, D.E., and MacLellan, H.M. : Uveoscleral
aqueous outflow in the rhesus monkey: Importance of uveal reabsorption.
Invest. Ophthalmol., in press, 1977.

Gaasterland, D.E., Pederson, J.E., and MacLellan, H.M. ; Perfusates and
the "Washout Phenomenon", presented at the Association for Research in
Vision and Ophthalmology Meeting, Sarasota, Florida, April 1977.

Green, K. , Sherman, S.H., Laties, A.M., Pederson, J.E., Gaasterland, D.E.,
and MacLellan, H.M. : The fate of anterior chamber tracers in the living
rhesus monkey eye with evidence for uveovortex outflow. In Cant, J.S.
Ced.): Intraocular Fluid Dynamics. Oxford, Oxford University Press (in
press) .

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PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00056-01 CB

PERIOD COVERED

■Tilly 1, 1976 To September 30, 1977

TITLE OF PROJECT (80 characters or less)

Transport Mechanisms in the Ciliary Epithelium

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Richard A. Stone M.D.

Other: Elmer J. Ballintine M.D.

Richard Weiblinger B.S.

Clinical Associate
Clinical Director
Biologist

CB NEI
CB NEI
CB NEI

COOPERATING UNITS (if any)

None

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National — Eye Institute, NIH, Bethesda, Maryland 70014

TOTAL MANYEARS

0.3

PROFESSIONAL

0.2

OTHER:

0.1

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS □ (a2) INTERVIEWS

n (b) HUMAN TISSUES

a (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

The transepithelial transport mechanisms of isolated ciliary body and iris
preparations obtained from normal rhesus monkey eyes are being studied.
Concentration of para-aminohlppurate by these preparations has been demonstrated.
Tissue culture techniques for the iris and ciliary body pigmented epithelium are
being developed in anticipation of exploring anion transport mechanisms in cells
grown in culture.

PHS-6040

Project No. ZOl EY 00056-01 CB
Project Description:

Objectives : Several mechanisms for the uptake of organic and inorganic
anions have been described in rabbit eyes. There is some published evidence
indicating that these processes also exist in primate eyes, but the uptake of
organic and inorganic anions by primate iris and ciliary body has not been
systematically explored in vitro.

The main objective of this project is to expand our understanding of
these transport systems as they occur in the primate eye.

Methods Employed: Specimens of iris and ciliary body are removed from
the eyes of rhesus monkeys when they become available from animals used by
the FDA Bureau of Biologies in their tests of vaccines. These tissue speci-
mens are then incubated with various radio-labeled anions and inhibitors of
transport. The rate of entrance of the anion into the tissue and the tissue/
medium are measured. We are attempting to develop a technique for obtaining
relatively pure cultures of iris and ciliary pigment epithelium which can be
used for similar studies of anion transport.

Major Findings: The anion transport systems studied in the monkey to
date appear to have the same general characteristics as the systems described
in the rabbit, although they are somewhat less active.

Significance to Biomedical Research and the Program of the Institute:
There are important unsettled questions regarding the mechanism of aqueous
secretion in the control of the aqueous composition. Much of our knowledge
is based on research in nonprimate systems, and the results are not always
immediately applicable to man. This project is aimed at increasing our knowl-
edge of the mechanism of aqueous formation and control in the primate eyes.
This knowledge is a basis for predicting what chemical agents might be useful
in inhibiting aqueous humor formation for the treatment of glaucoma.

Proposed Course: The project will be continued as outlined above.

NEI Research Program: Glaucoma - Hydrodynamics of the Eye

Experimental Subject or Tissue Source: Monkey

Research Objective: Etiology

Publications: None

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PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTSCE OF
tNTRAHURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00044-01 CB

PERIOD COVERED

December 12, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Organ Culture of the Normal and Dystrophic (RCS) Rat Retina

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI:

Makoto Tamai

M.D.

Visiting Scientist

CB NEI

COOPERATING UNITS (If any)

None

LAB/ BRANCH

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute. NIH. Bethesda. Maryland 20014

TOTAL MANYEARS:

PROFESSIONAL:

0.5

OTHER:

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

□ (al) MINORS n (a2) INTERVIEWS

n.(b) HUMAN TISSUES

a (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

Study of the phagocytic function of pigment epithelium in RCS dystrophic
rats, thought to be important in the process of photoreceptor degeneration, was
undertaken with the organ culture technique. The pigment epithelium of the
RCS rat was cultured with the normal neural retina or vice versa and examined
with the light and electron microscopy.

PHS-6040

Project No. ZOl EY 00044-01 CB
Project Description;

Objectives: One of the Important biological functions of the pigment
epithelium is the uptake of shed photoreceptor outer segments. If this
activity is disturbed, accumulation of the outer segment debris occurs and
may lead to visual cell death. These processes have been accepted as the
cause of retinal dystrophy in RCS rats, but no one knows if the defect exists
in the pigment epithelium, in the outer segments, or In both. These problems
were studied in normal and dystrophic (RCS) rats In vitro.

Methods Employed: Techniques for organ culture of the developing rat
retina and pigment epithelium were used. Normal neural retina of the post-
natal seven to twelve day, as well as the dystrophic ones of the same age,
was cultured with RCS rat pigment epithelium. Their Interactions and the
existence of phagosomes were evaluated by light and electron microscopy.

Major Findings: Pigment epithelium of the dystrophic RCS rats phago-
cytlzed neither the outer segments from RCS neural retina nor those of the
normal strain during the Incubation period of up to six days. Pigment epi-
thelium of the normal rats, however, could phagocytlse both of them.

Significance to Biomedical Research and the Program of the Institute:
The present studies strongly suggest that the membrane characteristics or
shedding mechanisms of the outer segments in the dystrophic rats are normal,
but their recognition by or systems for their uptake in the pigment epithelium
are defective.

Proposed Course: Using the organ culture technique, these experiments
will be continued not only in the rat but also in other dystrophic animals.

NEI Research Program: Retinal and Choroidal Diseases - Visual Cells and
Pigment Epithelium

Experimental Subject or Tissue Source: Rat

Research Objective: Etiology

Publications: None

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PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00048-01

PCRSOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Endothelial Wound Healing of the Cornea

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Teruo Tanishima M.D.
Other: Toichiro Kuwabara M.D.

Visiting Scientist CB NET

Head, Section on Experimental

Pathology LVR NEI

COOPERATING UNITS (if any)

None

lab/branch

Clinical Branch

SECTION

INSTITUTE AND LOCATION

National Eye Institute, NIH,

Bethesda, Maryland 20014

TOTAL MANYEARS:
1.1

PROFESSIONAL:

0.8

OTHER:

0.3

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEWS

D (b) HUMAN TISSUES

[J(c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

The healing mechanism of a small wound which had been made on the posterioi
surface of the rabbit cornea was studied by electron microscopy. Due to the
curling characteristic of the cut edge of Descemet's membrane, a tissue defect
measuring about 200 ym in width was formed. Endothelial cells in the adjacent
area of the wound rapidly slid over the curled Descemet's membrane and then
filled up the tissue defect. The cells facing the anterior chamber became
the covering endothelium, but many cells in the wound defect transformed into
f ibroblast-like cells and eventually decreased in number.

Project No. ZOl EY 00048-01 CB
Project Description:

Objectives: The wound healing process of the avascular corneal tissue
is unique. Early cellular reaction of the endothelial cells to the wound has
not been clearly understood.

Methods Employed: Small wounds of the posterior surface of the cornea
were made by diagonal insertion of a thin flat needle into the central cornea
of albino rabbits. The wounds were examined by electron microscopy at various
time intervals. Also, the sliding activity of the endothelium in vitro was
examined by electron microscopy. Various metabolic inhibitors were added to
the incubation media.

Major Findings: The cut edges of Descemet's membrane curled toward the
anterior chamber, and a tissue gap measuring about 200 ym was formed immedi-
ately following the wounding. The endothelial cells in the vicinity of the
wound began to slide along Descemet's membrane and reached the cut edge three
hours after the wounding. The sliding cells contained numerous microtubules
in the extending processes. The sliding continued until the tissue defect was
filled with the endothelial cells by the 24th hour. The cells facing the
anterior chamber became the covering endothelium by forming conspicuous
apicolateral junctions and basal lamina whereas the cells piled in the tissue
defect began to show a f ibroblast-like appearance, losing junctions. These
cells produced abundant basal lamina-collagen substances among them and grad-
ually disappeared. The sliding and transformation of the endothelium occurred
without mitotic activity. Proliferation of keratocytes was not involved in
this early wound healing.

Active sliding of the endothelial cells occurred in an organ culture
system. Small pieces of the corneal tissue, the epithelium of which had been
removed, was incubated in a tissue culture medium, and the endothelial cells
were examined by electron microscopy at various time intervals. Sliding was
inhibited by a cold temperature (4°C) of the media and presence of para-
hydroxybenzoate (10~^M), iodoacetate (10~^M) and sodium fluoride (10~^M) .

Significance to Biomedical Research and the Program of the Institute:
For successful corneal' transplant.ation in patients, maintenance of the healthy
endothelium of the graft and host is one of the most important factors.
Clarification of the role of the endothelium in wound healing is the first
step of the related investigation.

Proposed Course: This research will be continued at the Department of
Ophthalmology of Tokyo University upon the principal investigator's return.

NET Research Program: Corneal Diseases - Corneal Transplantation
and Stromal Injury and Repair.

Experimental Subject or Tissue Source: Rabbit

Research Objective: Etiology, Diagnosis, and Treatment

Publications: None

Laboratory of Vision Research

ANNUAL REPORT
NATIONAL EYE INSTITUTE
July 1, 1976 - September 30, 1977

REPORT OF THE CHIEF, LABORATORY OF VISION RESEARCH
Jin H. Kinoshita, Ph.D.

At the time of the new fiscal year one conspicuous change is the addition
of 2000 sq. ft. of new laboratory space in Building 6. This allows for a
modest expansion in the number of investigators. The main addition is Dr.
Igal Gery, an accomplished immimologist, who will initiate research activities
on ocular immunology. This is an area in the vision research field where a
number of good research opportunities have been left unattended because of a
lack of sufficient numbers of competent investigators. Dr. Gery, with know-
ledge of sophisticated concepts and techniques in immunology, brings consid-
erable expertise to the vision research field. The development of an active
research program in ocular immunology will not only be an asset to the NEI but
to the overall community of vision researchers as well.

As mentioned in previous Annual Reports, a particularly successful con-
tinuing activity of the Laboratory of Vision Research is the NEI sponsored
s3raiposia held on the Bethesda campus. In October 1976 a Symposium on Experi-
mental Eye Pathology was held and chaired by Dr. T. Kuwabara. Approximately
150 investigators from throughout the country participated in this 3-day
meeting, and its proceedings were published in Investigative Ophthalmology and
Visual Science. This meeting was acclaimed as one of the best of its kind, and
because of its success many have requested that a symposium on this subject be
held on a more regular basis.

During the next fiscal year Dr. A.J. Coulombre will organize a symposium
sponsored by the National Eye Institute on "Corneal Development" to be held on
October 20 and 21, 1977. Among the topics to be considered are: embryological
origins of corneal cells, cell population dynamics, development of cell junc-
tions, origins and functions of basement membranes, and clinically recognized
anomalies of corneal development. It is the purpose of the symposium to define
present positions and near-term opportunities in research on the developing
cornea.

Another type of initiative developed by the intramural program of the NEI
is demonstrated in the establishment of the National Cooperative Cataract
Research Group. The formation of this group was initiated by the cataract
section of the LVR. It required the enthusiastic cooperation of 20 partici-
pating laboratories involving over 100 investigators who agreed to coordinate
their efforts so that progress in research on the human lens and cataracts
could be accelerated. Each laboratory has been assigned to carry out specific
areas of research. The program will be monitored by an executive committee,
while the collection, sorting, and redistribution of data will be done by
members of the LVR.

In initiating this program the details of assembling the necessary infor-
mation into a formal proposal submitted to the National Advisory Eye Council
was worked out with the advice and help of the NEI extramural staff. To develop
this initiative from outside the intramural program would have been considerably
more difficult. If the endeavor is successful, this kind of cooperative team
effort focusing on a particular research problem may serve as a model for
attacking other clinical problems.

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
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INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00003-05 LVR

PERJOD aOVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Cataracts

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PR I NCI PAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Jin H. Kinoshita PhD.

Other: Shambhu Varma Ph.D.

Peter Kador Ph.D.

Lorenzo 0. Merola

Chief LVR NEI

Visiting Scientist LVR NEI

Staff Fellow LVR NEI

Gen. Physical Scientist LVR NEI

COOPERATING UNITS (if any)

None

lab/eranch

Laboratory. of Vision Research

SECTION

Section on Biochemistry

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

3.5

PROFESSIONAL:

2.5

OTHER:

1.0

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS D (a2) INTERVIEWS

□ (b) HUMAN TISSUES

S (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

It has been shown for the first time that the development of cataracts
in an experimental diabetic animal can be delayed by chemical treatment. An
inhibitor of aldose reductase, quercitrin, has been shown to effectively delay
the onset of diabetic cataracts in an experimental animal.

PHS-6040

' Project No. ZOl EY 00003-05 LVR

Project Description;

Objectives; To study the mechanism of formation of cataracts in experi-
mental animals and to explore possible means by which these cataracts can be
prevented .

Methods Employed: Sugar cataracts can be induced in experimental animals
by making them diabetic with appropriate chemical agents, or by making them
galactosemic or xylosemic with a diet enriched with galactose or xylose.
Another approach to studying cataracts is to employ animal models. We have
developed a colony of a Nakano mouse strain with hereditary cataracts.

Major Findings: An understanding of the nature of diabetic cataracts
evolved from the detailed study of galactose cataracts. These two forms of
cataracts fall into a class called sugar cataracts. It is now generally
accepted that the common mechanism initiating these cataracts involves aldose
reductase. Polyols increase in the lens during cataract formation of diabetic,
galactosemic and xylosemic rats. Thus far, experiments do not show that
polyols are involved in the inhibition of any key enzymes or that they directly
affect vital processes. If this is the case, then the question is how do
polyols trigger the events that lead to cataract formation? The answer to
this question comes from piecing together certain facts. One fact is that
polyols do not penetrate biological membranes very readily so that if polyols
are formed within the lens they can accumulate to high levels. This fact led
to the idea that in these sugar cataracts the polyols may have an osmotic
effect. Support for this possibility comes from histopathological studies.
Investigators earlier had made histopathological studies claiming that the
earliest histological change to occur is the appearance of hydropic lens fibers.
They found that these lens fibers are swollen and the accvmiulation of fluid
is primarily within the lens fibers and not extracellular. Recently, the
early swelling of the lens fibers in galactose cataract has been confirmed by
electron microscopy. Thus, the morphological studies seem to support the idea
that polyols may cause an osmotic change.

The suggestion that polyols may be causing an osmotic effect in the early
phase of sugar cataract formation was supported by biochemical studies. In
the experiments in which the galactose-fed rats were sacrificed during various
periods of time and the lenses analyzed, it was shown that the accumulation of
polyol was paralleled by an increase in lens hydration. A better quantitative
relationship was demonstrated in organ culture where one lens was incubated in
high galactose meditim while the contralateral lens was kept in a normal medium.
An increase in lens hydration paralleled the increase in polyol accumulation
in the galactose-exposed lens. This same relationship was also shown when the
lens was exposed to high glucose medium simulating hyperglycemic conditions.
In the lens incubated in 35 mM glucose, there was a sudden rise in sorbitol
which was accompanied by an increase in lens hydration. The increase in
sodium ions was a later phenomenon. These results strongly suggested that in
the early stages of the cataract polyol accumulation and not the increase in
electrolytes was responsible for the lens swelling.

Project No. ZOl EY 00003-05 LVR

The most convincing evidence for the polyol-osmotic hypothesis came from
in vivo experiments using inhibitors of aldose reductase. In galactosemic
rats, systemic administration of an aldose reductase inhibitor effectively
delayed the onset of cataract formation. Validation of the hypothesis from
experiments with the diabetic animals was not accomplished because diabetic
rats require two or three months for cataracts to develop while in galacto-
semic rats only two weeks are required for cataracts. It was inconvenient to
treat rats with the aldose reductase inhibitor for the many months required
for the formation of diabetic cataracts. The South American degus turned out
to be an excellent animal model for the study of diabetic cataracts. The
degus lens has an unusually high level of aldose reductase activity. Thus,
when made diabetic the degus developed cataracts within two weeks. With
diabetic degus it was possible to show that oral feeding of flavonoids
effectively delayed cataract formation. These results provided strong support
of the hjrpothesis that aldose reductase initiated the formation of cataracts
in diabetes. This study revealed for the first time that inhibition of aldose
reductase not only led to a decrease in the sorbitol accumulation in the lens
but also impeded the cataractous process. The cataract formation in diabetes
may thus be at least delayed, if not prevented, by the in vivo use of an
aldose reductase inhibitor. We have also examined other flavonoids for their
ability to inhibit aldose reductase activity in the hope of finding even more
potent derivatives than quercitrin. Possibly other flavonoids are effective
in still lower doses and are more suitable therapeutically against the diabetic
manifestations initiated by polyols .

Significance to Biomedical Research and the Program of the Institute:
Cataract is one of the major causes of blindness throughout the world. Even
though vision can be corrected by appropriate surgery, loss of vision because
of cataracts presents a problem. It is hoped that this type of study on sugar
cataracts may serve as a model by which other mechanisms of cataract develop-
ment can be uncovered, and also provide alternate means of preventing cataracts.
The terminal stages of these sugar cataracts may have features common to other
forms of cataracts. Even though the initial phase of cataract development may
be different in the other forms of cataract, it appears that the terminal
stages are quite similar.

Proposed Course: This project will be continued,

NEI Research Program; Cataract - Diabetic Cataract /Congenital, Metabolic,
and Genetic Cataract

Experimental Subject or Tissue Source: Rat /Mouse /Degus

Research Objective: Etiology

Publications :

Fukui, H.N., Obazawa, H. , and Kinoshita, J.H. : Lens growth in the Nakano
mouse. Invest. Ophthalmol. 15: A22-425, 1976.

Project No. ZOl EY 00003-05 LVR

Varma, S.D., and Kinoshita, J.H. : Topical treatment of galactose cataract
Documenta Ophthalmol. Proceed Series. Prog, of Lens Biochemical Research,
Dr. W. Junk Publishers p. 305-309, 1976.

Varma, S.D., and Kinoshita, J.H.: Inhibition of lens aldose reductase by
flavonoids. Their possible role in the prevention of diabetic cataracts.
Biochem. Pharm. 25: 2505-2513.

Kinoshita, J.H. , Varma, S.D., and Fukui, H.N. : Aldose reductase in
diabetes. Jap. J. Ophthal. 20: 399-410, 1976.

Kinoshita, J.H. : Biochemical basis of cataract formation. Acta Soc.
Ophthal. 80: 1362-1371, 1976.

Varma, S.D. , Mizuno, A., and Kinoshita, J.H. : Delaying the formation of
diabetic cataract with flavonoids. Science 195: 205-206, 1977.

Fukui, H.N., Iwata, S., Epstein, D.L., and Merola, L.O. : Cataractogenic
effects of a boron hydride disulfide compound. Invest. Ophthal. (in
press) .

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
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PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00136-05 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Chemistry and Metabolism of the Lens

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Jin H. Kinoshita Ph.D.

Other: Izumi Kabasawa M.D.

Henry N. Fukul Ph.D.

Paul Russell Ph.D.

Chief LVR NEI

Visiting Scientist LVR NEI

Senior Staff Fellow LVR NEI

Staff Fellow LVR NEI

COOPERATING UNITS (if any)

None

lab/branch

Laboratory of Vision Research

section

Section on Biochemistry

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

3.5

PROFESSIONAL:

3.5

OTHER:

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEWS

□ (b) HUMAN TISSUES

H (c) neither

SUMMARY OF WORK (200 words or less - underline keywords)

Considerable effort has been directed toward developing a tissue culture
method for lens epithelial cells. We feel that this approach may aid in the
understanding the nature of human congenital cataracts. Thus far, we have
been successful in culturing mouse and dog cells. The mouse cell lines
have been established. Some success has been achieved with the homan lens
cell culture.

Further studies have been undertaken to determine the aging effects on
the low molecular weight proteins. It is quite apparent that in the aging
lens there emerges a new low molecular weight protein.

PHS-6040

Project No. ZOl EY 00136-05 LVR
Project Description;

Objectives: Many aspects of the chemistry and metabolism of normal lens
are being studied in order to better understand the significance of the
changes that occur in the cataractous process.

Methods Employed: Tissue culture of lens epithelial cells will be
undertaken to aid in this study. Procedures for organ culture of the lens
will also be used.

Major Findings; We are further exploring the possibility that tissue
culture methods may be useful in studying human congenital cataracts. To
devise techniques for obtaining cultures of pre-adult and adult lens epithelial
cells, lenses from Nakano and normal mice were employed. The Nakano mouse
cataract offered an excellent model for this study since the histology of this
cataract has been thoroughly investigated. In addition, insight into the
biochemical defect has been imcovered. Thus, this mouse cataract provided
us the opportunity to test the feasibility of employing tissue culture methods
to study a hereditary cataract. Conceivably, if the method is successfully
developed it may be useful in the study of human congenital cataracts .

Although the lens epithelial cells from both the normal and Nakano mice
grew well in culture with doubling times of about 31 hours, the normal lens
cells have twice the clone forming ability compared to Nakano lens cells .
After about 16 days in culture large spherical lentoid bodies began to appear.
The lentoid bodies were similar to those observed with the chick cell cultures.
The lentoid structures were observed in both the normal and Nakano lens
cultures. The lentoid structures were observed as early as the tenth day in
some lines, and by the thirtieth day, all the cell cultures had lentoid struc-
tures. The ability to form lentoid bodies has been retained in cell lines
subcultured for over one year.

It has been established that the synthesis of y crystallin occurred in
the fiber cells of the lens rather than the epithelial cells. Fluorescent
antibody prepared against mouse Y crystallin reacted with the lentoid
structures from both normal and Nakano mouse cells. The immunofluorescence
of the lentoid bodies suggested the production of y crystallin in these
structures. The presence of this crystallin indicated the possible differ-
entiation of some of the cells in the lentoid structure.

These results seem to indicate that we have been successful in tissue
culture of mouse lens epithelial cells. Many distinctive characteristics
of lens epithelial cells are retained in cultures of cells from adult mice.
The cells are epithelial in nature even after one year in culture. One of
the most unusual feature of these cells is the presence of spherical lentoid
bodies. Some cells in the lentoid structure appear similar to the lens
fiber cells in that the lack of cellular organelles creates a homogeneous
cytoplasm. Antibodies prepared against mouse y crystallin react with the
lentoid bodies, suggesting that some cells in the lentoid structure produced
y crystallin. Since y crystallin synthesis and loss of cellular organelles
are properties of differentiated fiber cells, it appears some cells in tissue

Project No. ZOl EY 00136-05 LVR
culture retain the ability to express some differentiated characteristics.
These cells have consistently shown these characteristics for over one year
even with repeated subculture.

Another evidence that culturing of the mouse lens cells does not lead to
loss of differentiated traits of the lens is the demonstration that the Nakano
cells retain the Na-K ATPase inhibitor. This factor is thought to be respon-
sible for electrolyte imbalance and increased hydration, the changes that pre-
cede cataract formation in the lenses of these animals. The Inhibitor is found
only in the cells from the lenses of the Nakano mice and not from the cells of
the normal mice. Since the cells generally are subcultured without difficulty
and the doubling time is relatively short, the purification of the inhibitor
from these cells may be possible. Since partial differentiation of epithelial
cells is possible with the cultures of the mouse lines, other biochemical
properties of the cells in culture can also be studied in order to aid the
investigation of the cataractous process. The methods are now being used to
study material from other species in order to devise an adequate method for
use with human congenital cataractous lenses.

Another major effort has been directed to understanding the nature of
the low molecular weight proteins. These proteins are the first to disappear
in a number of cataracts . The low molecular proteins in the lens are Y
crystallins and a low molecular weight beta crystallin called 3 .

In comparing the y crystallins from old and young bovine lenses that can
be recovered by chromatography on Sephadex G-75 column, a cattle lens y
appears which is different from the calf lens Y. The new Y crystallin is
found almost exclusively in the cattle lens cortex and not in the nucleus.
On the other hand, the y crystallin present in the cattle lens nucleus is
indistinguishable from the calf lens. The Y crystallin along with beta-S
isolated from Sephadex G-75 have been purified on cation exchange columns.
SDS gel electrophoresis revealed that the molecular weight for cattle lens Y
is 24,000, calf lens Y is 20,000 and g is 28,000. The N terminal group of
cattle lens and calf lens Y is glycine while that of beta-s is masked.
Immunodiffusion studies revealed that the cattle lens Y does not cross react
with antisera of g and calf lens .

A Y crystallin resembling the cattle lens cortex Y is also found in
human lens. This Y crystallin increases with age. Its molecular weight is
24,000 and other properties resembles that of the cattle lens Y.

Thus with the aging of the lens there appears to emerge a new Y crystallin
in both bovine and human lenses.

Significance to Biomedical Research and the Program of the Institute:
An understanding of the basic chemistry and physiology of the lens is
important to provide a more complete understanding of the cataractous process.
The age-related change in the gamma crystallins is one of the first demon-
strations of the effect of aging of the lens proteins.

Development of a tissue culture procedure of lens epithelial cells may
become useful in studying human cataracts. Human congenital cataracts may
be studied by this technique provided that epithelial cells from a cataract
may be obtained. 39

Project No. ZOl EY 00136-05 LVR
Proposed Course; The studies described are being continued,

NET Research Program: Cataract - The Normal Lens/Congenital, Metabolic,
and Genetic Cataract

Experimental Subject or Tissue Source: Bovine/Rat/Mouse
Research Objective: Etiology
Publications:

Fukui, H.N.: The effect of hydrogen peroxide on the rubidium transport
of the rat lens. Exp. Eye Res. 23: 595-599, 1976.

Fukui, H.N., Merola, L.O., and Kinoshita, J.H. : The effect of oxidants
on the membrane sulfhydryl groups of the lens. Documenta Ophthalmol.
8: 161-169, 1976.

Christiansen, J.M. , Kollarits, CR. , Fukui, H.N. , Fishman, M.L. ,
Michels, R.G., Mikuni, I.: Intraocular irrigating solutions and lens
clarity. Am. J. Ophthal. 82: 594-597, 1976.

Kabasawa, I., Barber, G.W., and Kinoshita, J.H. : Aging effects and some
properties on the human lens low molecular weight proteins. Jap. J.
Ophthal. 21: 87-97, 1977.

Russell, P., Fukui, H.N., Tsunematsu, Y., and Kinoshita, J.H. : Tissue
culture 'of lens epithelial cells from normal and Nakano mice. Invest.
Ophthal. 16: 243-246, 1977.

Kabasawa, I., and Fukui, H.N.: Glycoproteins of the cattle lens plasma
membranes. Jap. J. Ophthal. (in press) .

Horwitz, J., Kabasawa, I., and Kinoshita, J.H. : Conformation of gamma
crystallin of the calf lens. Exp. Eye Res, (in press).

Kabasawa, I., Tsunematsu, Y.,. Barber, G.W. , and Kinoshita, J.H. : Low
molecular weight proteins of the bovine lens. Exp. Eye Res, (in press).

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00179-02 LVR

PERIOD COVERED

■Tilly I, 1Q76 to September 30, 1Q77

TITLE OF PROJECT (80 characters or less)

Ultrastructural and Biochemical Correlates in the Vertebrate Retina

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Arnold I. Goldman Ph.D.

Other: Paul J. O'Brien Ph.D.

Eileen Masterson Ph.D.

Gerald Chader Ph.D.

Paul Tierstein B.S.

Staff Fellow LVR NEI
Staff Scientist LVR NEI
Postdoctoral Fellow LVR NEI
Staff Scientist LVR NEI
Summer Student LVR NEI

COOPERATING UNITS (if any)

None

LAB/BRANCH

Laboratory of Vision Research

SECTION

Section on Biochemistry

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

1.6

PROFESSIONAL:

1.4

OTHER:

0.2

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (at) MINORS D (a2) INTEftVIEWS

n (b) HUMAN TISSUES

[?(c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

Studies are being conducted on the process of phagocytosis of outer
segment membranes by the pigment epithelium, with emphasis on control
mechanisms of this process. Two separate experimental methodologies have
been developed to study this problem. In the first, bovine retinas have
been radioactively labeled and the outer segments separated and fed to chick
pigment epithelium cell cultures. The time course of ingestion of the outer
segments can be monitored with a liquid scintillation counter and has been
verified by electron microscopy and histochemistry. By placing appropriate
sugars or hormones with the outer segments, the effect of these agents on
phagocytosis can be quantified. In the second approach, normal and dystrophic

rat eyecups are incubated in vitro in a solution containing radioactive sugars
and/ or melatonin. Both autoradiography and phagosome counts are made to see
if the sugars are instrumental in preparing the outer segments for shedding,
and to see if the melatonin plays a role in controlling this process.

PHS-6040

! Project No. ZOl EY 00179-02 LVR

Project Description; ;

Objectives-. Biological functions of the photoreceptor have been pre-
viously studied by autoradiographic, biochemical and histochemical methods.
Each of these methods has limitations for the achievement of certainty.
However, when these mfethods are used jointly, the correlations become highly
significant. This project was designed to bring multiple disciplines to bear
on major problems concerning photoreceptor function, especially as regards
the renewal of the outer segment and the process of shedding and phagocytosis
of outer segment discs.

Methods Employed; Biochemical methods such as Isolation and incubation
of rod outer segments, coliamn chromatography and scintillation counting were
used to obtain an overall impression of the biochemical events associated
with disc shedding and phagocytosis. This work was correlated with auto-
radiography of H-galactose or fucose in the rat to see if there was local-
ization of the transfer of these sugars. Tissue culture techniques allowed
the use of chick PE cultures to serve as a practical quantitative assay of
phagocytosis. Electron microscopic histochemistry was used to verify the
biochemical findings with the tissue culture system.

Major Findings; Rhodopsin in the outer segments of the photoreceptors
will take up both fucose and galactose. Preliminary evidence suggests that
the uptake of the sugars is greatest at the tips of the outer segments ,
implying that they are somehow involved in preparing the outer segments for
phagocytosis. Eyecup preparations incubated with melatonin show little
shedding, while those incubated without melatonin shed a large portion of
their outer segment tips. If melatonin is given for the first part of the
incubation and the incubation is continued in its absence, shedding is still
further increased. These preliminary results imply that melatonin inhibits
disc shedding while it "primes" the outer segments for later shedding. The
tissue culture assay of phagocytosis has proven to be a reliable and
quantitative method. Both galactose and mannose in solution appear to
stimulate phagocytosis, as does fucose to a lesser degree. Cyclic AMP
inhibits phagocytosis measureably.

Significance to Biomedical Research and the Program of the Institute;
The mechanisms of regulation of shedding of discs and their subsequent phago-
cytosis by the pigment epithelium is crucial in the understanding of photo-
receptor renewal processes. The knowledge gained in this way can be instru-
mental in developing treatments for various retinal diseases. Autoradiography
is a delicate probe into many of these mechanisms, and the use of the tissue
culture technique is a powerful new tool which should yield important new
information about phagocytosis.

Proposed Course; Autoradiography will be continued with and without
melatonin on both the light and electron microscopic level. Both normal and
dystrophic rats will be used to see if there is a difference in the response
of these animals, and to use the debris of the dystrophic animals to physically
separate the outer segment tips from the pigment epithelium. Since the

Project No. ZOl EY 00179-02 LVR
pigment epithelium labels more heavily than the outer segments , this separa-
tion should make it easier to recognize an accumulation of label at the outer
segment tips. The tissue culture system will be used extensively to test
various agents on the phagocytic response. Lectins will be used to see if
the blockage of specific sugar moieties will inhibit phagocytosis.

NEI Research Program; Retinal and Choroidal Diseases - Developmental and
Hereditary Disorders /Visual Cells and Pigment Epithelium

Experimental Subject or Tissue Source: Bovine/Rat/Chick

Research Objective; Etiology

Publications :

Goldman, A.I., Ham, W.T., Jr., and Mueller, H.A. : Ocular damage thres-
holds and mechanisms for ultrashort pulses of both visible and infrared
laser radiation in the rhesus monkey. Exp . Eye Res . 24; 45-56, 1977.

Geeraets, W.J., Geeraets, R. , and Goldman, A.I. : Elektromagnetische
bestrahlungsvertletzungen der netzhaut (Electromagnetic radiation damage
to the retina) , Albrecht v. Graefes Archiv f. Klin. Exp. Ophthalmologie
200; 263-278, 1976.

Geeraets, W.J., Ham, W.T., Jr., Geeraets, R, , and Goldman, A.I. ; Photo-
chemical, thermal, and non-linear effects of retinal irradiation. In
L'Esperance Francis, (ed.), Current Diagnosis and Management of
Chorioretinal Diseases. St. Louis, Mosby, 1977, pp. 9-24.

SMITHSOiJlAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00135-05 LVR

PERIOD COVERED

July 1. 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Biochemical Structure of Retina and Pigment Epithelium in Health and Disease

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Helen H. Hess M.D.
Other: Julia E. Derr B.A.

Research Medical Officer LVR NEI
Biologist LVR NEI

COOPERATING UNITS (if any)

Clinical Branch, NEI; Division of Research Services, Veterinary Resources
Branch; Medical Neurology Branch, NINCDS; American HistoLabs, Inc. (contract)

Lab/branch

Laboratory of Vision Research

SECTION

Section on Biochemistry

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

2.05

PROFESSIONAL:

1.25

OTHER:
0.8

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

n (al) MINORS n (a2) INTERVIEWS

n (b) HUMAN TISSUES

B (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

The broad aim of the project is to study the biochemical composition of
retina, pigment epithelium and rod outer segments in normal circumstances
and in retinal and chor'oIZal diseases of experimental or genetic origin.
Topics of current interest are: (a) concentration and distribution of
inorganic constituents; (b) possible involvement of calcium, zinc and copper
in retinal and choroidal diseases; )c) ultrastructural localization and
physiological function of calciiom in retina, pigment epithelium, and choroid;
(d) study of hybrids of RCS and spontaneously hypertensive rats to determine
whether the slow onset type of retinal degeneration seen in the latter is
inherited at the rdy or another gene locus or is due to light damage in an
albino animal.

PHS-6040

Project No. ZOl EY 00135-05 LVR
Project Description:

Objectives: To study the biochemical composition of retinal photo-
receptor, neuronal, glial, and pigment epithelial cells in health and
disease, and to explore possibilities for prevention or therapy of retinal
and/or choroidal disease when a biochemical abnormality has been identified;
diseases in v;hich pigment epithelium (PE) is involved are of particular
interest.

Methods Employed: Retinas, isolated rod outer segments (ROS) , and PE
of frogs and rats are being analyzed. Sajnples of plasma or serum as well
as urine are being studied in rodent models and human cases of retinal
degeneration. Methods include flameless atomic absorption spectroscopy,
microscopy, and a number of standard biochemical laboratory techniques.

Major Findings: I. Studies of trace elements: Last year the dis-
tribution of nine inorganic elements was studied in PE, ROS, and retina of
frogs. This year two trace metals were chosen for further study in rodents
and in humans with retinal disease (Zn and Cu) .

(a) Zinc in blood plasma of RCS dystrophic and congenic control RGB
rats: Rats have uniquely high levels of Zn in milk and plasma during
development. We have found that during the first postnatal week, RCS rat
plasma Zn is two to four times as high as in the adult RCS rat. A time lag
in breeding of RCS control rats has delayed making direct comparisons at the
required ages (1,7,14,18,21, and 22 days) in mutant and control animals. The
postnatal developmental pattern of plasma Zn in RCS mutants, however, is not
identical with that in Osborne-Mendel albino rats. Plasma Zn levels of
female rats are lower than in males after two weeks of age, when estrogen
begins to be produced. The lability of plasma Zn in the developmental period
is illustrated by our observation that a 50% drop in plasma Zn on day 22 can
occur after milk has been stopped abruptly at weaning on day 21 and lab

chow begun.

(b) Histopathological study of eyes of copper deficient mutant mice,
an animal model of Menkes disease: five alleles at the mottled locus on the
X chromosome of the mouse are responsible for mutations of Cu deficiency.
Dapple and Tortoise mutants die before birth. Brindle mutants survive to
12-14 days, and up to 25 days with Cu injections. Blotchy and Crinkled
mutants live 25 days, or longer if high copper diets are given.

NIH colony stocks of Brindle were previously supplied on a C3H back-
ground and contained the rd gene for retinal degeneration (satisfactory
for investigators not studying retina) . We manipulated these animals
genetically to remove the rd gene and place the Brindle gene in a C57 Black
6 background for study of possible ocular pathology (in collaboration with
Dr. Kitty Smith, Division of Research Services, Veterinary Resources Branch).
Stained sections of 12-14 day old eyes showed retardation of retina develop-
ment, with incomplete development of photoreceptor outer segments. In con-
sultation with Dr. T. Kuwabara, pigment epithelial cells of retina and iris
were examined and appeared to contain vacuoles or microcysts, especially
those of the iris. This young mutant did not show optic nerve atrophy and
loss of ganglion cells.

Project No. ZQl EY QQ135-Q5 LVR
degeneration: Recently CGaklot, 1976) the amount of Cu excreted in a 24 hour
urine specimen of patients with primary retinitis pigmentosa (RP) was reported
to be six-fold normal. Normals were in a range expected from published work.
To investigate this report, we have initiated a study of 24 hour Cu; for com-
parison, Zn was included. A masked study was designed, with Dr. D. Bergsma
(Clinical Branch, NEI) selecting the types of patients included, and the
analyses were done by a second person not informed of the patient type prior
to assay. Results for Cu in two normals and in four cases of retinal degen-
eration (two with primary recessive RP, one with autosomal dominant RP, and
one with Usher's sjmdrome) were all in the normal range reported in the
literature. However, values for Zn in the three cases of RP xv-ere greater
than the extreme of the normal range reported in the literature, while values
in the case of Usher's syndrome and in the two normals were within the normal
range.

II. Ultrastructural localization of Ca in retina and PE: The EM
study to localize Ca by use of the potassium pyroantimonate technique (in
collaboration with Dr. M.L. Fishman, Clinical Branch, NEI; and M.A. Oberc
and Dr. W.K. Engel, NINCDS) was completed. Optimum preservation was
obtained with 2% K pyroantimonate in 1% osmium tetroxide at pH 9.2. With
this fixative, electron-dense precipitate was found within the discs of
the ROS; as a dense band of granules along Bruch's membrane; in the extra-
cellular space of the choroid; and to some extent within the nuclei and
mitochondria of all cells. The precipitate was identified as Ca antimonate
by use of EGTA prechelation and with the EMMA-4 electron microprobe.

This is the first time calcium has been demonstrated within ROS discs
and in normal Bruch ' s membrane .

III. Studies .of hybrid rats from two strains with retinal degeneration:
Last year nine black hooded progeny from crossing tan hooded RCS dystrophic
rats with albino spontaneously hypertensive (SH) rats were reported to have
normal photoreceptors at one year of age, as shown by microscopy of fresh
and fixed, stained specimens. Similarly, we have studied 19 more of the
same F-, progeny, now two years of age, with the same result. Since retinal
degeneration occurs at a few weeks of age in the RCS rat and at 6-12 months
of age in the SH rat, the hybrid (even though pigmented) would be expected

to show changes before 12 months (and certainly by two years) if the rdy gene
were present in the SH genome, or if a different abnormal gene were present
at the same locus.

From this study it seems apparent that the SH rat has no abnormal gene
at the rdy locus. This suggests that albino rats such as SH, Osborne-
Mendel and Sprague-Dawley rats in which slow onset retinal degeneration has
been described may have these changes on the basis of light damage. To
help rule out this etiological factor and to explore whether the SH rat
may have an abnormal gene at a different locus, F2 generation animals
(F-L X F]|) were produced and will be one year of age in September 1977. The
color ratios are approximately two black hood to one tan hood to one albino.
Among black hooded animals, light damage does not occur and any excessive
appearance of retinal dystrophy beyond the ratio of one dystrophic to three
normals would suggest that a second gene for retinal degeneration is involved
(collaboration with Dr. C. Hansen, geneticist. Division of Research Services,
Veterinary Resources Branch) .

Project No. ZCO. QQ135-Q5 LYK
Significance to Bloaedical Regearch and the Program of the Institute;
Zinc affects many structural proteins and enzyroea of significance in PE and
retina and is a plasma membrane active agent whose concentration can
influence phagocytosis. Plasma Zn varies rapidly with nutritional and
hormonal changes, especially iri the developing animal. Since plasma albumin,
a major Zn carrier, is low and the blood-ocular barrier not yet established,
this lability may be significant in pathology of the developing eye. Gen-
etic and nutritional factors are known to interact in Zn and Cu metabolism.
When an error in regulation of the metabolism of such trace metals occurs,
nutritional methods of treatment may be of some avail.

In one case of Menkes' disease, Wray, Kuwabara and Sanderson (1976) con-
firmed the loss of retinal ganglion cells and optic atrophy first reported
in one case by Seelenfreund, et al. (1968), but they did not find vacuolation
-of the iris PE which had been seen by Seelenfreund et al. Iris PE vacuole
formation may occur subsequent to terminal therapeutics (IV fluids, drugs),
however, and its relevance to copper deficiency has not been established.
If microcysts of PE can be shown consistently in an animal model like the
Brindle mouse, this finding would attain more significance. Varied find-
ings in pathology of Menkes' disease could result not only from study of
different stages of disease but also perhaps from existence of several
alleles governing Cu, as in the case of mice.

Our studies support the specificity of the K pyroantimonate technique
in showing the ultrastructural localization of Ca in retina and related
tissues. Our demonstration of Ca within ROS discs is consistent with some
role for Ca in either mediating the photoexcitation process or in dark and
light adaptation, or both. We showed previously that dark-adapted frog
ROS contain Ca in a ratio of about 0.25 moles/mole rhodopsin. We also
reported that light adaptation in vivo increased the Ca content of ROS. The
Ca that may act as a mediator of photoexcitation is thus probably a very
small fraction of the total Ca present in ROS in either the dark- or light-
adapted state.

The large amount of extracellular Ca in Bruch's membrane indicates
numerous Ca-binding sites. This may be related to the frequency with which
abnormal calcification occurs in this area, as in angioid streaks, drusen,
and macular degeneration. These findings suggest a need for further study
of the distribution of Ca in physiologic and pathologic processes.

If the SH rat has a separate gene for slow onset retinal degeneration,
this gene could be placed on a pigmented background to give an animal model
more nearly analogous to human retinitis pigmentosa, to supplement the
fast onset model of the RCS strain.

Proposed Course; Developmental analyses of Zn (and Cu) in plasma of
RCS mutant and RCS control rats will be completed. Further studies of Zn,
Cu, Ca and enzymes or proteins to which they are related will be pursued in
PE cells prepared by frozen sectioning and tissue culture, as well as in
retina. Additional specimens of Cu deficient mouse mutants will be examined
for comparison with pathology of Menkes' disease. A larger group of specimens

eyqjfect No. ZQl QQ135-Q5 LVR
of urine from different categories of retinal degeneration will be analyzed
to determine whether hyperzincuria is of any significance in such diseases
and to check further on levels of copper. The F2 hybrids from the cross of
RCS and SH rats will be examined at 12 months and later to determine retina
integrity by ERG Cwith collaboration of Dr. P. Gouras, Physiology Section,
I.VR) and in stained sections.

NET Research Program; Retinal and Choroidal Diseases - Developmental
and Hereditary Disorders /Visual Celle and Pigment Eplth«liuo.

Experimental Subject or Tissue Source; Frog/Rat /Human

Research Objective; Etiology

Publications:

Whikehart, D.R, and Hess, H.H. : Properties of liposomes with a
phospholipid ratio similar to that of retinal rod outer segment
membranes. Interaction with opsin and other proteins. Exp . Eye
Res. 24: 279-289, 1977.

Fishraan, M.L. , Oberc, M.A. , Hess, H.H. , and Engel, W.K. :
Ultrastructural demonstration of calcium in retina, retinal
pigment epithelium, and choroid. Exp . Eye Res . 24: 341-353,
1977.

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00008-06 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Chemistry of Rhodopsin

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI:
Other :

Marc S . Lewis
Hitoshi Shichi
Gerald Chader

Barbara Wiggert

Ph.D.
Ph.D.
Ph.D.

Ph.D.

Research Chemist LVR NET
Research Chemist LVR NEI
Head, Section on
Retinal and Corneal
Metabolism LVR NEI
Staff Fellow LVR NEI

COOPERATING UNITS (if any)

None

lab/branch

Laboratory of Vision Research

SECTION

Section of Biochemistry

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda,

Maryland 20014

TOTAL MANYEARS:

0.5

PROFESSIONAL:

0.5

OTHERi

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEWS

(b) HUMAN TISSUES

n (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

It has been the long range objective of this project to study the
rhodopsin molecule and other molecules, such as retinol and retinoic acid
which are involved in the visual process in order to attempt to elucidate
further information regarding the relationships between their structures
and their functions. The areas of current interest are studies on the
various forms of rhodopsin which can be complexed with digitonin and studies
on the interactions of retinol and retinoic acid with receptor sites in the
retina and the cornea.

101

Project No. ZOl EY 00008-06 LVR
Project Description;

Objectives; To study the structural and functional aspects of the
rhodopsin molecule and to study the interaction of retinol and retlnolc acid
with receptor sites in the retina and cornea.

Methods Employed: Rhodopsin has been isolated as a rhodopsln-digitonin
complex from bovine retinas, the various forms of the Cv;)mplex have been iso-
lated chromatographlcally and studied by means of sedimentation-equilibrium
in the analytical ultracentrifuge, and the data analyzed by mathematical
modelling techniques using the MLAB system on the DEC-10 computer. Retinol
and retlnolc acid receptors from retina and cornea have been isolated and the
ligand Interactions studied by sucrose density gradient ultracentrifugation,
gel filtration, spectroscopy, and electrophoresis. The binding data was anal-
ysed by mathematical modelling techniques using the MLAB system on the DEC-10
computer.

Major Findings; Two forms of rhodopsin complexed with digltonln have been
Isolated. Type I had a molecular weight of 259000 when unbleached and 226000
following bleaching. Type II had a molecular weight of 191000 when unbleached
and 186000 following bleaching. The two types show quite different bleaching
and regeneration characteristics. Type I bleaching slowly and regenerating
rapidly, and Type II having the reverse properties. The present evidence
tends to indicate that the Type I complex is a dimer of rhodopsin complexed
with digltonln and that the Type II complex is a monomer of rhodopsin complexed
with digltonln. The evidence here, while preliminary, is causing us to
consider the possibility that a monomer-dlmer transition may be Involved in
the bleachlng-regeneratlon processes of rhodopsin.

Specific soluble receptors for retinol and retlnolc acid have been found
in retina, pigment epithelium and in cornea. Two different receptors for
retinol have been found in the retinas of most species. One of these was not
seen in the retina of a patient with retinitis pigmentosa and during early
fetal development in the cow, thus indicating that this particular receptor
type is associated with photoreceptor outer segments in vivo. The molecular
weights of the receptoirs and the association constants for the binding of
retinol and retlnolc acid to the receptors were determined. These receptors
may be Involved in the normal development and the transport of vitamin A in
ocular tissues.

Significance to Biomedical Research and the Program of the Institute ;
The studies on rhodopsin are relevant to an understanding of the basic bio-
chemical mechanisms which are Involved in normal and pathological aspects of
scotoplc vision. The studies on vitamin A receptors are relevant to the
normal and pathological aspects of development and function of the retina,
pigment epithelium and cornea, and as such are of significance in such
diseases as retinal dystrophy and keratomalacia which appear to Involve
vitamin A.

102

Project No. ZOl EY 00008-06 LVR

Proposed Course; This project is being terminated since the principal
investigator is transferring from the National Eye Institute to the Division
of Research Services. Plans have been made for continuation of this research
on a collaborative basis.

NEI Research Program; Retinal and Choroidal Diseases - Visual Cells and
Pigment Epithelium

Experimental Subject or Tissue Source ; Cow/Rat/Monkey/Pig/Chicken/Human

Research Objective; Etiology

Publications:

Wiggert, B., Bergsma, D,, Helmsen, R. , Lewis, M. , and Chader, G. ; Retinol
receptors in corneal epithelium, stroma and endothelium. Biochim.
Biophys. Acta 491; 104-113, 1977.

Wiggert, B., Bergsma, D,, Lewis, M. , Abe, T., and Chader, G. ; Vitamin
A receptors: Characteristics of n/etinol binding in chick retina and
pigment epithelium. Biochim. Biophys. Acta (in press) .

Wiggert, B., Bergsma, D., Lewis, M. , and Chader, G. ; Vitamin A receptors:
Retinol binding in neural retina and pigment epithelium. J. Neurochemlstry
(in press) .

103

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PROJECT NUMBER (Do MOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAIiURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00009-06 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (60 characters or less)

Physical Chemistry of Model Gel Systems

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Marc S. Lewis Ph.D.

Other: Jules A. Gladner Ph.D.

S.I. Chung Ph.D.

Yutaka Shizuta M.D.,Ph.D.

Peter Davies M.D.,Ph.D.

Research Chemist
Research Chemist
Research Chemist
Visiting Scientist
Research Associate

LVR NEI
LBC NIAMDD
LB NIDR
LMB NCI
LMB NCI

COOPERATING UNITS (if any)

None

lab/branch

Laboratory of Vision Research

SECTION

Section on Biochemistry

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

0.5

PROFESSIONAL:

0.5

OTHERj

0.0

CHECK APPROPRIATE BOX(ES)
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n (b) HUMAN TISSUES

SUMMARY OF WORK (200 words or less - underline keywords)

It is the long-range objective of this project to study various biological
systems which are either actually involved in the visual process or which may
serve as models for such systems which are relevant to the transparency or
opacity of ophthalmic tissues. Of current interest are the structure and
cross-linking mechanisms of fibrinogen and the physical, chemical, and
biological properties of the smooth muscle protein filamin.

105

Project No. ZOl EY 00009-06 LVR
Project Description:

Objectives : To study the physical and chemical parameters of model
systems which are pertinent for transparency or opacity of gel systems or
which in any way may be of significance in the biochemistry of vision.

Methods Employed : The usual methods of protein preparation, fractionation,
purification and characterization have been employed. In this laboratory
particular emphasis has been given to analytical ultracentrifugation and to
computer techniques for model simulation, data reduction, and systems analysis
as being the most effective means of studying systems of Interacting and non-
interacting macromolecules .

Ma j or Findings : Further work has been done on the most primitive of the
vertebrate fibrinogens, lamprey fibrinogen. Ultracentrifugal analysis of the
native molecule in aqueous buffer gave a value for the molecular weight of
352000 and SDS gel electrophoresis analysis gave values for the molecular
weights of the constituent chains such that a structure of one alpha chain,
two beta chains, and two gamma chains was postulated. Following isolation of
the constituent chains their molecular weights in 6 M guanidine hydrochloride
were measured in the ultracentrifuge, and molecular weights markedly lower
than those obtained in the gel studies were obtained when a value for the
partial specific volume of 0.704 was used. This value was computed from the
amino acid composition, taking into account possible changes in hydration and
guanidine binding. In order to help resolve this issue, the molecular weight
of intact lamprey fibrinogen was measured in 6M guanidine buffer, and a value
of 281000 was obtained with the partial specific volume value of 0.704. In
order to obtain the same value for the molecular weight as that obtained in
aqueous buffer, it was necessary to use a value of 0.736 for the partial
specific volume, thus indicating that the assumptions regarding the extent of
hydration or guanidine binding were not valid. This property does not appear
to be unique to lamprey fibrinogen, since bovine fibrinogen gave a value of
274000 for the molecular weight in 6M guanidine buffer when the computed value
of 0.720 was used for the partial specific volume. Since the assumptions with
regard to hydration and guanidine binding are for typical globular proteins,
it is not particularly surprising that they are not necessarily valid for a
markedly assymetrical protein like fibrinogen. Individual chain molecular
weight values obtained were 109000, 73000, and 48500 for the alpha, beta, and
gamma chains respectively, adding up to a total of 352000 for the molecular
weight of the whole molecule having the postulated structure. Further veri-
fication was obtained by chromatography on Sepharose using 6M guanidine buffer
where values of 115000, 76000, and 50000 were obtained for the molecular
weights of the respective chains. We now feel that the unique structure of
lamprey fibrinogen has been suitably established and are proceeding with the
preparation of a manuscript describing this work.

Studies were initiated on the properties of the protein filamin which
has been found in smooth muscle as well as in non-muscle cells such as macro-
phages and fibroblastic cells. The filamin used in. this work was isolated
from chicken gizzard. Sedimentation-equilibrium studies gave a value of 498000
for the molecular weight of the native protein in 50 mM phosphate, 0.1 M NaCl,

106

Project No. ZOl EY 00009-06 LVR
pli 7.5 buffer. The value of 0.734 for the partial specific volume was computed
from the amino acid composition. A value of 8.86 S was obtained for the
sedimentation coefficient in the same buffer, and a frictional ratio of 2.32
was calculated, indicating a significant degree of asymmetry. Filamln migrates
as a single sharp band on SDS gel electrophoresis, and has a apparent molecular
weight of 240000, thus indicating that it Is normally composed of two identical
subunits. Filamin is a soluble protein and under a variety of conditions
tested does not by itself form filaments or precipitate from solution. How-
ever, filamin binds very strongly to rabbit muscle F-actin, and the complex is
readily sedimented to yield a gelatinous pellet containing these proteins.
Filamin has been demonstrated to undergo a variety of very complex associations
with itself depending upon temperature, salt, ionic strength, and pH. For
example, in 0.6 M KCl at pH 7.4, it appears to be a monomer-dimer-tetramer
association at low temperatures, shifting first to a monomer-tetramer assoc-
iation and then to an ill-defined monomer-n-mer association as the temperature
is increased. The reversibility of the association also appears to be a
function of the environmental conditions. As a result, it has been difficult
to clearly define the aggregation status of filamin under physiological con-
ditions except to state that it obviously exists as a complex with actin. The
molecular weight of this complex is of such a magnitude as to preclude study
by conventional methods, but studies on filamin fragments produced by enzymatic
cleavage appear promising in terms of delineating which portions of the filamin
molecule are Involved in actin binding, which are involved in self -association,
and may also yield significant information concerning the nature of these
interactions.

Significance to Biomedical Research and the Program of the Institute;
The studies on fibrinogen represent a contribution to the general area of
inter- and intra-molecular cross-linking and the formation of gels. More
specifically, because of the role of fibrinogen in blood clotting and wound
healing, it is relevant to the surgical treatment of ophthalmic disorders.
The role of filamin in the eye has as yet to be elucidated, but it would be
reasonable to expect that it is present to a significant extent in some
ophthalmic tissues, and that future studies will determine what function it
might have.

Proposed Course; This project is being terminated since the principal
investigator is transferring from' the National Eye Institute to the Division
of Research Services. The studies on lamprey fibrinogen have been completed.
The studies on filamin will be resumed as a new project.

NEI Research Program; Corneal Diseases - Corneal Transplantation and
Stromal Injury and Repair

Experimental Subject or Tissue Source: Lamprey/Chicken
Research Objective; Etiology

107

„ ^-^ ^^ Project No, ZOl EY 00009-06 LVR
Publications:

Shizuta, Y., Shizuta, H,, Gallo, M. , Davies, P., Pasten, I., and Lewis
M.S.: Purification and properties of filamin, an actin binding protein
from chicken gizzard. J. Biol. Chem. 251: 6562-6567, 1976.

108

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Oo NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAHURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00004-03 LVR

PER 1 00 COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

The Membrane Biology of the Visual Process

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL l^ VESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Hitoshi Shichl Ph.D.

Alois J. Adams Ph.D.

Other: Robert L. Somers B.S.

Consuelo G. Muellenberg B.A.

Research Chemist LVR NEI

Post Doctoral LVR NEI

Chemist LVR NEI

Biologist LVR NEI

COOPERATING UNITS (if any)

None

lab/branch

Laboratory of Vision Research

SECTION

Section on Biochemistry

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

3.5

PROFESSIONAL:

1.5

OTHER:

2.0

CHECK APPROPRIATE BOX(ES)
n (») HUMAN SUBJECTS

n(al) MINORS D {a2) INTERVIEWS

n (b) HUMAN TISSUES

g (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

1) A method is being developed for extraction and purification of proteii

kinases associated with rod membranes, one that phosphorylates rhodopsin with
ATP and the other that phosphorylates phosvitin. 2) Three isochromic forms of
rhodopsin have been separated and purified. These forms contain different
amounts of phospholipid and demonstrate different bleaching kinetics.

109

Project No. ZOl EY 00004-03 LVR
Project Description:

Objectives : The overall objectives of this project are to investigate
the light-dark, adaptation processes of the retina by means of modern techniques
of biochemistry and membrane biology. More specifically, these are (1) iden-
tification of a sequence of molecular events initiated by absorption of
photons and leading to visual transduction (light process) and (2) elucidation
of the biochemical mechanism of regeneration of the photosensitivity of
photoreceptor membranes (dark process) . The investigations presented in this
report deal with two aspects of the visual pigment rhodopsin, i.e. (a) modu-
lation of rhodopsin bleaching kinetics by associated phospholipid, and (b)
the phosphorylation of opsin.

Methods Employed; Biochemical methods such as centrifugation, column
chromatography, spectroscopic analysis and radioisotope assay. Low temperature
spectroscopy and fast kinetics measurements after a flash photolysis of
rhodopsin.

Major Findings:

(1) Isochromic forms of rhodopsin

On the basis of kinetic studies of rhodopsin bleaching in digitonin, the
existence of multiforms of rhodopsin and of subsequent bleaching intermediates
in this detergent has been previously suggested. Purification of digitonin-
extracted rhodopsin on ECTEOLA-cellulose results in the separation of three
isochromic forms of rhodopsin (Fr. I, II and III). Fr. I and Fr. II constitute
about 45% and 40% of the total rhodopsin purified, respectively. The three
fractions of rhodopsin are indistinguishable in their spectral properties
with the characteristic absorption bands (a at 498 nm, 3 at 350 nm, and y ^t
278 nm) . They differ in phospholipid content; the molar ratio of phospholipid
to rhodopsin is 14 for Fr. I and 64 for Fr. II. Delipidation converts Fr. II
to Fr I rhodopsin and Fr. I is transformed back into Fr. II rhodopsin by
reassociation with phospholipid. From these results we conclude that the
multiple forms of rhodopsin in digitonin result from nonuniform association
of phospholipid with opsin.

In order to investigate the thermal stability of the intermediates formed
after flash bleaching of rhodopsin, low-temperature spectroscopic measurements
have been made on Fr. 1 and Fr 2 rhodopsins. On the basis of thermal stability,
both bathorhodopsin and lumirhodopsin are identical whether they are derived
from Fr I or Fr. 2. However, a comparison of these intermediates between
digitonin extracts and rod membranes shows that the stability of bathorhodopsin
remains unaffected whether it be formed in rods or in digitonin and that lumi-
rhodopsin, on the other hand, becomes more labile when it is formed in
digitonin than in rods. Since a close correlation exists between spectrally
determined thermal stability and opsin conformational stability, the above
results are taken to suggest that the first appreciable conformat local change
of opsin will take place at lumirhodopsin level but not at bathorhodopsin
level. The rate of metarhodopsin I decay is markedly affected by the phospho-
lipid content of rhodopsin. The metarhodopsin I decay rate determined at

110

-1 Project No. ZOl EY 00004-03 LVR

11 C is Oj^ll sec for Fr . 1 (14 moles phospholipid per mole rhodopsin) and
5.20 sec for Fr. 2 (64 moles phospholipid per mole rhodopsin). Because of
the low phospholipid content, the decay of metarhodopsin I derived from Fr. 1
will be affected by digitonin to a greater extent than that derived from Fr. 2.
In other words, the digitonin micelle associated with rhodopsin will affect
the decay rate by producing such a rigid environment that opsin conformational
change associated with metarhodopsin I decay is prevented. The decay of
metarhodopsin III is also affected by the amount of phospholipid associated
with opsin. The slower decay rate of metarhodopsin III derived from Fr. 1
can be explained also by assuming the stabilization of opsin conformation
by digitonin. Phospholipid unsaturation is recently shown to be essential
for the metarhodopsin I to metarhodopsin II conversion. A suggestion is then
made that a minimum phospholipid biplayer fluidity is necessary to allow opsin
to midergo conformational changes associated with the transition. Thus, the
present investigation supports this suggestion by demonstrating that thermal
intermediates placed in the rigid (i.e. low fluidity) environment of digitonin
micelle decay more slowly than those in fluid environment.

(2) Phosphorylation of opsin

Rod outer segments seem to contain several protein kinases. At least
two phosvitin kinases (40,000 d. and 170,000 d.) and one rhodopsin kinase
(80,000 d.) can be separated by a coluagi-chro«natographlc method. These kinases
show different solubilities in aqueous salt solutions. Activities of these
enzymes are not stlnulated by cyclic nucleotides. During the course of
purification of the kinases, a nucleotide-containing protein (20,000 d.) has
been separated.

Significance to Biomedical Resaarch and the Program of the Institute;
The effect of phospholipid (as well as digitonin) on the decay rate of meta-
rhodopsin I and HI can point to the Issportance of membrane fluidity of the
environment surrounding opsin. Since the fluidity of rod membranes is provided
by unsaturated acyl chains of phospfeolipid, the importance of essential fatty
acids in foodstuffs for maintaining normal vision is emphasized.

Proposed Course; This project will be continued.

NEI Research Program; Retinal and Choroidal Diseases - Visual Cells and
Pigment Epithelium

Experimental Subject or Tissue Source; Bovine/Frog

Research Objective; Etiology

Publications;

Shichi, H.; Molecular biology of the visual process. In Siegel, G.J.,
Albers, R.W., Katzman, R. and Agranoff, B,W. (eds.): Basic Neurochemistry.
Boston, Little, Brown & Co., pp. 148-163, 1976.

Shichi, H., Muellenberg, C.G., Harosi, F.I., and Somers, R.L. ; Isolation
of three isochromic forms of rhodopsin in digitonin. Vision Res. 17;
633-636, 1977.

Ill

SMITHSONIAN SCIEfJCE INFORMATION fXCHANGi
PROJECT NUMBER (Do NOT use this ^pacc)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER
ZOl EY 00007-03 LVR

PERIOD COVERED
July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

The Molecular Pharmacology of the Eye

HMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ^'N THE PROJECT

PI: Hitoshi Shichi Ph.D.

Other: Daniel W. Nebert M.D.
Douglas E. Gaasterland M.D.

Research Chemist
Chief

Senior Staff
Ophthalmologist

LVR NEI
DPB NICHD
CB NEI

COOPERATING UNITS (If any)

National Institute of Child Health and Human Development

lab/branch
Laboratory of Vision Research

SECTION
Section on Biochemistry

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:
0.5

."PROFESSIONAL:

0.5

OTHER:

0.0

CHECK APPROPRIATE BOX(ES)
n (a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEWS

n (b) HUMAN TISSUES

3(c) NEITHER

SUMMARY OF WORK (200 words or lejs - underline keywords)

Following an intraperitoneal injection of acetaminophen into polycyclic
hydrocarbon-responsive mice in which hepatic aryl hydrocarbon hydroxylase (AHH)
activity has been induced by pretreatment with polycyclic hydrocarbons,
lenticular opacification develops in a few hours . The opacity consists of
a thin layer anterior to the equatorial cortex. Acetaminophen metabolites
bind to the lens but do not lower the glutathione level. These observations
suggest that lenticular opacification may be caused by binding of acetaminophen
metabolites to the lens cells in the anterior portion and thus disrupting
permeability properties of the cells. Since lenticular opacification develops
when mice are pretreated with 3-methylcholanthrene (cytochrome P. 450 inducer)
but not with phenobarbital (cytochrome P 450 inducer) , acetaminophen metabolites
that affect the lens must be produced by the cytochrome P, 450-dependent AHH
system.

113

Project No. ZOl EY 00007-03 LVR
Project Description:

Objectives: We have previously demonstrated that aryl hydrocarbon
hydroxylase (AHH) induction in the eye (pigmented epithelium) and liver of
polycyclic hydrocarbon-responsive mouse strains is apparently under the same
genetic regulation.

In this work we report that administration of large doses of acetamino-
phen to mice in which AHH has been induced by pretreatment with polycyclic
hydrocarbons (e.g. 3-methylcholanthrene) causes development of lenticular
opacification in a few hours. A possible mechanism of the lens opacity
formation is investigated.

Methods Employed; Mice were injected intraperitoneally with 3-methyl-
cholanthrene to induce AHH activity for 48 hours. Acetaminophen was then
injected intraperitoneally into pretreated mice and development of lenticular
opacification was examined in vivo as well as in vitro. Eyes were fixed and
subjected to histochemical examination. Glutathione levels of lens and liver
were determined by a spectroscopic method. Covalent binding of acetaminophen
metabolites to lens, liver and other tissues was studied with H-acetaminophen.

Major Findings: 1) Acetaminophen- induced ocular opacity in mice under the
conditions is unique in that it develops very rapidly (ca 6 hours) . 2) Ocular
opacity is localized in the anterior portion of the lens. This indicates that
toxic acetaminophen metabolites reach the lens in aqueous humor circulation.
3) Cataract develops only in 3-methylcholanthrene-pretreated mice; untreated
mice do not show opacity development. 4) Opacity develops only in AHH- induc-
ible strains (e.g. C57/BL 6) and not in non-inducible strains of mice (e.g.
DBA/2) . 5) Cataract develops when responsive mice were pretreated with 3-
methylcholanthrene (cytochrome P^ 450 inducer) ; pretreatment with phenobarbital
(cytochrome P 450 inducer) does not cause opacity. Therefore, acetaminophen
metabolites that cause lens opacity are formed by cytochrome P^ 450-dependent
AHH system. 6) The glutathione level of lens remains virtually unchanged
during opacity development; liver glutathione level is markedly reduced.
7) Acetaminophen metabolites are bound covalently to the lens. On a basis
of metabolites bound per mg protein, the levels of binding to lens and liver
are almost comparable. In the lens, metabolites therefore seem to bind
preferentially to cellular membranes over glutathione. 8) From these results
it is suggested that (i) cataract development in mice treated with polycyclic
hydrocarbons and acetaminophen is closely related to AHH inducibility, (ii)
cataractogenic agents are probably formed by hydroxylation of acetaminophen
by hepatic cytochrome P, 450-dependent AHH system, (iii) acetaminophen meta-
bolites that bind covalently to lens cells probably modify permeability
properties of the cells to cause cataract, and (iv) while the lens is affected
by acetaminophen metabolites, both retina and pigment epithelium demonstrate
little cellular degeneration. This may be attributed to the detoxifying
activity of the drug metabolizing system of pigmented epithelium which we have
previously elucidated.

114

Project No. ZOl EY 00007-03 LVR
Significance to Biomedical Research and the Program of the Institute:
The present study on lenticular opacification caused by acetaminophen in mice
shows that AHH-induclble strains are particularly susceptible to the toxic
effect of acetaminophen on the lens. Acetaminophen is a widely used analgesic-
antipyretic agent. It is contained in common drugs such as Excedrln and
Tylenol. As much as 800 mg of acetaminophen per kg body weight is adminis-
tered to humans for treatment of certain cases of arthritis. The present
result on experimental animals raises a possibility that high doses of aceta-
minophen administered to the polycyclic hydrocarbon responsive (i.e. AHH
inducible) type of patients, especially on a prolonged basis, might cause
development of lenticular opacification.

Proposed Course; This project will be continued.

NEI Research Program: Retinal and Choroidal Diseases - Visual Cells and
Pigment Epithelium/Special Areas of Future Interest (Toxic and Environmental
Disorders) Cataract - Cataract Induced by Drugs, Radiation, and Secondary to
Other Eye Disorders

Experimental Subject or Tissue Source: Mouse

Research Objective: Etiology, Diagnosis

Publications:

Shlchi, H., Tsunematsu, Y., and Nebert, D.W.: Aryl hydrocarbon hydroxylase
induction in retinal pigmented epithelium: Possible association of genetic
differences in a drug metabolizing enzyme with retinal degeneration.
Exp. Eye Res. 23: 165-176, 1976.

Shlchi, H., and Nebert, D.W.: Drug metabolism in ocular tissues. In
Gram, T.E. (ed.): Extrahepatic Drug Metabolism Spectrum Publications,
Inc. (in press) .

115

SMITHSONIAN SCIENCE INFORMATION ."XCHANGE
PROJECT NUMBER (Oo NOT use this ^pace)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND V.'ELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER
ZOl EY 00138-05 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

The Visual Cell: Process of Photoexcitation and Restoration

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED 'iN THE PROJECT

PI:

S. Yoshikami

Research Biologist

LVR NEI

Other:

G.N. Noll

Visiting Associate

LVR NEI

W.A. Hag ins

Chief, Section Membrane
Biophysics

NIAl^DD

COOPERATING UNITS (if any)

National Institute of Arthritis, Metabolism and Digestive Diseases

lab/branch

Laboratory of Vision Research

SECTION

Section on Biochemistry

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:
2.5

PROFESSIONAL:

2.5

OTHER:

0.0

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEvJS

n (b) HUMAN TISSUES

a(c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

There is considerable evidence favoring our notion that calcium plays a
central role in the initiation of vision. A definitive test for this hypotheslLs
is a measurement of transient light stimulated ^alcium activity changes in the
visual receptor cell. The properties of a calcium sensitive dye, dichlorophos
phonazo III, have been studied and found suitable for this purpose. A rapid
multi-wavelength spectrometer was built to measure ionic activity changes repo|:-
ted by dichlorophosphonazo III in thin tissues like the retina.

Isolated retinas can function as light detectors but cannot regenerate
visual pigments. We have invented a method using liposomes to permit pigment
regeneration in such retinas. This technique is applicable to general practic
of introducing large amounts of water insoluble chemicals into live tissues.
Using this method, we are studying the biochemical pathway of retinol in the
visual process and discovered among other things the retinol oxidation enzyme
in the retina is specific for a given stereoisomer.

117

Project No. ZOl EY 00138-05 LVR
Project Description:

Objectives: To study the nature of the visual cell and determine its
physical and chemical means of initiating and sustaining the phenomenon of
vision.

Methods Employed: This study employs measurements of the electrical,
biochemical, anatomical and metabolic properties of the retina and associated
ocular tissues.

Major Findings: I. We (Hagins and Yoshikami) have developed and proved
successful a method of incorporating water-soluble, membrane impermeable
substances into live cells using phospholipid vesicles. This was shown by
mea-suring the uptake of 6-carboxyf luorescein and guanosine monophosphate by
observing the relief of concentration quenching of fluorescence in 6-carboxy-
fluorescein transferred into live retinas and by the synthesis of guanosine
triphosphate from guanosine monophosphate. The fluorescence efficiency of
6-carboxyf luorescein was found to be pH dependent with a single pKa at 6.8.
This was used to show the internal pH of retinal cells to be 6.8. When calcium
buffers were introduced into retinal rod cytoplasm by this technique, the
effect of these buffers on the retinal light sensitivity was affected as
predicted by the calciim hypothesis.

These phospholipid vesicles are being used to introduce calcium sensitive
dyes into retinal cells to further test the calcium hypothesis. To this effect,
we have studied the properties of a new calcium sensitive azo dye, dichlorophos-
phonazo III. We find its CaK^=10~ M at pH 7 ; it is also a pH indicator. An
analysis of a family of dichlorophosphonazo III absorption spectra shows this
dye is capable of reporting simultaneously the activities of Mg , Ca and
H ions. A multi-channel spectrometer was built to analyze rapid ionic changes
in the retina and other thin tissues. Our tests show predicted sensitivity for
these ions for transient changes in absorbency to be 10 A with a time reso-
lution of 10 seconds.

II. The lizard retinas with pure cone cells were used to test another
aspect of the calcium hypothesis. Cone outer segment membrane topology differs
from that of rods; the disc space in cones is confluent with the extracellular
space and thus should be more easily perturbed by changes in the cell surround
than it would be for rod cells. Rapid calcium ion depletion and replenishment
experiments show that the cone retina photocurrent response is quickly and
transiently abolished by transient lowering of a Ca 10 M. This result
supports the hypothesis that rods and cones have similar photoexcitatory
mechanism.

III. Isolated and perfused retinas can maintain its photoresponse for an
extended time but cannot restore any significant amount of visual pigment
following full bleaching. We (Noll and Yoshikami) have developed a method
using phospholipid vesicles to restore quickly and quantitatively tha visual
pigment in such retinas and determined how phospholipid vesicles transfer
water insoluble materials through an aqueous medium to cells. This general
idea of employing phospholipids to introduce water insoluble substances into
cells in an aqueous environment should be valuable in other studies, in partic-
ular for drug analysis.

118

Project No. ZOl EY 00138-05 LVR
We have used these phospholipid vesicles to regenerate visual pigments
in live isolated retinas in order to study the biochemical pathways of regen-
eration and to study the photoexcitation process. For the latter we have
constructed an apparatus to measure rapid photochemical kinetic and electrical
transients in the retina.

In the study of visual pigment regeneration -we have examined how and
where retinol is 1) transported, 2) oxidized and reduced, and 3) isomerized.
We have measured the solubility-of retinol in water and determined its greatest
upper bound to be less than 10 Molar. We find that retinol is rapidly oxi-
dized into expoxides in aqueous solutions despite very strict precautions
against oxidation. The solubility and oxidation properties of retinol indicate
that its transfer between the pigment epithelium and retina is not a trivial
procass, especially in view of the high oxygen demand of the retinal cells. We
have shown that retinol must enter the aqueous space between the retina and
pigment epithelium. This was demonstrated by interposing barriers of varying
porosity and thickness between the two tissues and measuring visual pigment
regeneration. There is no direct cell-to-cell transfer of retinol. The
migrant retinol cannot be free retinol in solution but its upper limit of
packaging size must be less than 300 A. Preliminary digestive enzyme tests
show the retinol transfer packet is unaffected by free papain and immobilized
trypsin and phospholipase. The transfer could involve secretion of small
phospholipid or fatty acid coated oil droplets. We find the distance between
the P.E. and retina to be extremely important for the well-being of the retina.
An induced separation between the two tissues of only 100 ym is sufficient
to prevent visual pigment regeneration. This finding has significance to the
retina in cases of retinal detachment.

We have begun to investigate the biochemical pathways of retinol in the
retina and pigment epithelium using phospholipid vesicles as bearers of
different retinol congeners. We have discovered inter alia that the retinol
oxidase is stereospecif ic for 11-cis retinol. This suggests the retinalde-
hyde reductase may be specific for the all-trans species. We are extending
this study on the pigment epithelial and retinal enzyme stereospecif icity and
metabolism.

Significance to Biomedical Research and the Program of the Institute;
Our understanding of the causes and our ability to prevent and treat numerous
visual disorders depend on a clear knowledge of the processes operant in
normal vision. Our finding on the importance of calcium and its control in
the visual cell excitatory process and revelation of the tight coupling
between photoexcitation and energy metabolism of this cell may help us tc
realize some of the basis for pathology in the retina. The concatenated
reactions of retinol in two adjacent tissues, the pigment epithelium and
retina, show these tissues are interdependent. The understanding of hew
retinol and other metabolites pass through the aqueous space between them has
bearing on the vitality of both tissue, in particular where retinal detachment
occurs .

The development of the method of introducing and measuring intercellular
ion activity reporting dyes should be useful in many other areas of biomedical
research. Using the phospholipid vesicle as a way to carry water insoluble

119

Project No. ZOl EY 00138-05 LVR
and as well as water soluble, membrane-impermeable substance into live tissues
has broad ramifications which extend from pharmacology to genetic engineering
and should be of general interest in all biomedical research.

Proposed Course: How the retina initiates and sustains vision are the
focal points of our studies. We will continue to study the physical and
chemical process involved as outlined above.

NEI Research Program; Retinal and Choroidal Diseases - Retinal Detach-
ment/Visual Cells and Pigment Epithelium/Retinal Organization and Visual
Adaptation/Special Areas of Future Interest (Low Vision/Retinal Regeneration
and Transplantation)

Experimental Subject or Tissue Source; Rat /Frog /Lizard /Fish

Research Objective: Etiology

Publications;

Weinstein, S., Yoshikami, S., Kenbart, P., Blumenthal, R. , and Hagins,
W.A.; Liposome-cell interaction: Transfer and intercellular release of
a trapped fluorescent marker. Science 195: 489-492, 1977.

Hagins, W.A., and Yoshikami, S.: Intracellular transmission of visual
excitation in vertebrate photoreceptors: Electrical effects of chelating
agents introduced into rods by vesicle fusion. In Fatt, P. and Barlow,
H.B. (eds.)! International Symposium on Photoreception. New York,
Academic Press (in press).

120

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PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00032-01 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Metaplastic Formation of Neural Retina in vitro

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI:

Other:

Alfred J. Coulombre

Yasuhiko Tsunematsu

Ph.D. Head, Section on LVR NEI

Experimental

Embryology
Ph.D. Postdoctoral LVR NEI

Fellowship

COOPERATING UNITS (if any)

None

lab/branch

Laboratory of Vision Research

SECTION

Section on Experimental Embryology

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

0.2

PROFESSIONAL:

0.2

OTHER:

0.0

CHECK APPROPRIATE BOX(ES)
Q (a) HUMAN SUBJECTS

D (al) MINORS Q (a2) INTERVIEWS

n (b) HUMAN TISSUES

SUMMARY OF WORK

^200 words or less - underline keywords)

Sheets of retinal pigmented epithelium (RPE) , isolated at several
developmental stages from the eyes of chick embryos, gave rise in tissue
cultures to patches of differentiating neural retina. These metaplastic
foci arise toward the centers of the explants. The steps identified in
their appearance were: local depigmentation of the RPE sheet, first detected
after three days in culture; rapid mitosis in the depigmented regions;
stratification of the nuclei within these foci; and differentiation of the
major layers characteristic of neural retina (NR) . RPE from donors younger
than developmental stage 27 gave rise to patches of NR, those from older
donors did not. The polarity of metaplastic foci of NR was concordant
with that of the RPE in which they arose.

121

Project No. ZOl EY 00032-01 LVR
Project Description;

Objectives : This project tested the feasibility of eliciting the meta-
plastic formation of neural retina (NR) from retinal pigmented epithelium (RPE)
in tissue culture. We exploited the in vitro context, and its facilitation of
experimental approaches to the nature of this metaplastic transformation, to
answer the following specific questions. What is the sequence and time course
of events leading to this metaplasia? Up to what embryonic age is the RPE
capable of regenerating NR? What is the relationship between the polarities
of the metaplastically formed NR and the RPE which generates it? Do media
conditioned by NR, RPE or chondrocytes affect this type of metaplasia?

_ Methods Employed : Embryos of the domestic fowl were used for this study.
Routine techniques of experimental embryology were supplemented with procedures
drawn from histology, electron microscopy and tissue culture to study the meta-
plastic formation of neural retina from the RPE in vitro. A technique utilizing
EDTA and Ca -Mg -free culture medium was adapted for isolation of clean
sheets of RPE from embryos of different ages.

Major Findings: 1. NR forms metaplastically from the chick-embryonic
RPE in vitro in a manner similar to that which had been demonstrated pre-
viously in vivo. 2. This metaplastic formation of NR occurs at random in
sites toward the center of the explanted sheet of RPE. 3. The sequence of
events leading up to metaplastic formation of NR are: focal depigmentation
in the RPE sheet (detected after three days in culture) ; rapid mitosis of cells
in the depigmented foci; stratification of cell nuclei in these foci; differ-
entiation within each focus of the major layers characteristic of NR. 4. The
capacity of the chick embryonic RPE to form NR metaplastically declined
steadily until donor-stage 27 when it disappeared. 5. The polarity of the
metaplastically formed neural retina was concordant with that of the parent
RPE (outer limiting membrane of NR corresponding to RPE apical surface and
ganglion-cell fiber layer corresponding to RPE basal surface). 6. The number
of depigmented, preretinal foci increased until seven days of culture and
thereafter remained nearly constant. 7. Deliberately co-culturing RPE and
suspensions of cells from chick embryonic neural retina, demonstrated that
the frequency of occurrence and the degree of maturation of foci of meta-
plastic formation of NR decreased -as the concentration of NR cells was
increased. This finding confirmed a previous study in vivo in this Section,
which showed that NR inhibits the metaplastic formation of NR from RPE. It
also indicates that the neural retinal foci seen in this study arose meta-
plastically from RPE and not from NR cells which may have been carried over
inadvertently with the RPE explants. 8. Foci of metaplastic formation of lens
tissue occurred in the depigmented zone of outgrowth of RPE explants but
rarely toward the centers of such explants. These lentoid bodies were first
detected after 10 days of culture. 9. Media conditioned by NR, RPE or
chondrocytes had no effect on the metaplastic transformation of RPE into NR.
By contrast, NR-conditioned medium enhanced the frequency of occurrence and
the degree of maturation of lentoid bodies. 10. The metaplastic formation
of NR from RPE in vitro is similar to the same procass in vivo in that: it
occurs only in relatively young RPE; metaplastic foci appear to arise randomly
in the RPE-cell population and are probably clonally derived in both situations;
the maturation of neural retina in these foci occurs more rapidly that it does

122

Project No. ZOl EY 00032-01 LVR

during normal development; and the polarity of the resultant NR is concordant
with that of the RPE in which it arises. 11. There is also a major difference.
Metaplastic formation of NR from RPE does not occur autonomously in vivo, but
requires the presence of some other tissue (e.g. neural retina, otocyst) ;
it is autonomous in vitro in the sense that it occurs in the initial absence
of any tissue other than RPE. It is possible that, in vitro fetal calf serum, \
which is present in the culture medium, substitutes for the influence supplied
in vivo by NR.

Significance to Biomedical Research and the Program of the Institute;
These results contribute information on normal development and congenital
abnormalities which relate to the NEI research program on Retinal and Choroidal
Diseases. A feature common to a number of developmental anomalies of the eye
is the duplication of the NR, especially in the region of the choroid fissure.
The present study suggests that the redundant NR is produced metaplastically
from the RPE. It further demonstrates that the eye is at hazard to this par-
ticular malformation only until the RPE loses its ability to give rise to NR
metaplastically. It also confirms previous findings that already-differen-
tiated NR represses the metaplastic transformation of RPE into NR. The results
define further the limitations to regeneration of the NR which exist in higher
vertebrates, but not in some of the lower vertebrates.

Proposed Course; This project accomplished its objectives, has been

reported in an article accepted for publication and has been terminated.

NEI Research Program; Retinal and Choroidal Diseases - Developmental and
Hereditary Disorders I!

Experimental Subject or Tissue Source: Chick

Research Objective; Etiology

Publications:

Tsunematsu, Y. and Coulombre, A.: Differentiation of neural retina
in cultures of retinal pigmented epithellxim of chick embryo. Devel .
Biol, (in press) ,

123

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PROJECT NUMBER (Do HOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00178-02 LVR

PER i CD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Sensitive Period in the Development of the Scleral Ossicles of the Avian Eye

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Alfred J. Coulombre Ph.D. Head, Section on Experimental LVR NEI

Embryology

COOPERATING UNITS (if any)

None

lab/branch

Laboratory of Vision Research

SECTION

Section on Experimental Embryology

NSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARSi

0.0

PROFESSIONAL:

0.0

OTHER:

0.0

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS D (a2) INTERVIEWS

n (b) HUMAN TISSUES

[5(c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

The scleral ossicles, a ring of membrane bones surrounding the cornea of
the domestic fowl are foreshadowed in early development by transient thickenings
in the overlying conjunctiva, the conjunctival papillae. Each ossicle 'is
induced by its corresponding papilla and develops in a collagen-bearing bed
deposited beneath the papilla.

L-azetidine-2-carboxylic acid (LACA) an analog of proline, was injected
into the chorioallantoic veins of chick embryos to transiently disrupt collagen
synthesis. Embryos treated at 5 or 6 days of incubation (but not those
treated before or after this interval) developed retarded papillae and later
developed ossicular rings lacking one or more ossicles.

Control embryos, injected with D-azetidine-2-carboxylic acid or with
water, developed normally.

Thus, the action of LACA in aborting the induction of scleral ossicles
is age-restricted and stereoisomerically-specif ic.

pi^'::_Anin

125

Project No. ZOl EY 00178-02 LVR
Project Description:

Objectives; A sequence of tissue interactions initiates and controls many
of the morphogenetic changes involved in the normal or abnormal development
of the eye. Our investigation of the nature of such interactions, their
sequencing and the manner in which they exert morphogenetic control, exploits
the development of the scleral ossicles, a ring of about 14 membrane bones
which encircles the cornea in many submammalian vertebrates, including the
domestic fowl. Each of these bones is foreshadowed in early development by
a transient thickening in the overlying conjunctiva, the conjunctival papilla.
The bone is induced by the papilla and develops in a collagen-bearing bed
deposited beneath the papilla. This project sought to determine whether
L-azetidine-2-carboxylic acid (LACA) , an analog of proline which disrupts
collagen synthesis, would prevent ossicular development. A further goal
was to determine the period during development when the ossicular system was
maximally at hazard to this teratologic agent.

Methods Employed: The study involved: 1. injection of LACA by micro-
catheter into the extraembryonic blood vessels of five, six, seven or nine day
old chick embryos, using a method developed by this Section; and 2. analysis of
the effects of this agent on the subsequent development of the conjunctival
papillae and scleral ossicles.

Maj or Findings : 1. Injection of LACA into the chorioallantoic veins of
embryos at five or six days of incubation suspended the deposition of collagen,
retarded maturation of the papillae and was followed by deletion of ossicles
from the bony ring. 2. Injection of LACA at five, eight or nine days of Incu-
bation did not alter the number of papillae or ossicles that subsequently
developed. 3. Control embryos, Injected with D-azetidlne-2-carboxyllc acid or
with water developed normally.

Thus, the action of LACA in suspending the production of collagen and
aborting the induction of scleral ossicles is age-restricted and stereolso-
merically-specif ic. The results also call attention to the possibility that
the conjunctival papilla is induced in the embryonic conjunctiva on the sixth
day of incubation, a day before it normally appears grossly.

Significance to Biomedical Research and the Program of the Institute:
This work aims at analyzing epithello-mesenchymal inductive interactions in
ocular development, by exploiting an optimally accessible example of this
type of tissue interaction, the conjunctival papilla-scleral ossicle complex.
The findings demonstrate the severely restricted developmental period during
which this tissue interaction is susceptible of disruption by noxious agents.

Proposed Course: Work on this project has been completed. The project
will be terminated following publication of the results.

NEI Research Program; Corneal Diseases

Experimental Subject or Tissue Source: Domestic fowl

126

Research Objective; Etiology Project No. ZOl EY 00178-02 LVR
Publications : None

127

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Oo HOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
IHTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00036-01 LVR

PER! CO COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Development of the Chick Conjunctival Periderm and Conjunctival Papillae

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Ellen Porzig Ph.D.

Other: Alfred J. Coulombre Ph.D.

Staff Fellow
Head, Section on
Experimental
Embryology

LVR NEI
LVR NEI

COOPERATING UNITS (if any)

None

lab/branch
Laboratory of Vision Research

SECTION

Section on Experimental Embryology

NSTITUTE AND LOCATION

National Eve Institute. NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

±^o_

PROFESSIONAL:

1.0

OTHER:

None

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

n (al) MINORS n (a2) INTERVIEWS

D (b) HUMAN TISSUES

S (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

The conjunctival papillae of the chick embryo are under study to determine
the roles played by the peridermal cells in the development of the underlying
perilimbic sclera. Specifically, we seek to describe the timetable and
regional distribution of changes in the size, shape and surface characteristics
of the peridermal cells and to correlate these changes with the development of
the conjunctival papillae and of the scleral ossicles which arise under the
influence of the papillae.

129

Duo_£n jn

Project No. ZOl EY 00036-01 LVR
Project Description;

Objectives: The ectoderm, which covers the surface of vertebrate embryos,
is a stratified squamous epithelium comprising two layers, an inner basal
layer of cuboidal cells and an outer layer of squamous cells called the
periderm. While the basal layer has been shown. to serve a number of important
developmental functions, no functions have been demonstrated for the periderm.
A favorable opportunity for identifying such functions is presented in the
chick embryo by transient thickenings (papillae) in the conjunctiva. These
structures appear on the seventh and eighth day of embryonic development as
fourteen focal thickenings of the conjunctiva, in a ring surrounding the
corneal limbus. The papillae disappear on the thirteenth day. During its
brief existence, each is responsible for the induction of a bone (scleral
ossicle) in the underlying mesenchyme.

This study focuses on the conjunctival periderm and seeks to answer the
following questions. Are there changes in the periderm which correlate
spatially and temporally with the development of the conjunctival papillae
or with the pattern of development in the ring of bones induced in the under-
lying mesenchyme by the papillae? Specifically, do changes in the shape,
size, surface characteristics or mutual attachments of the peridermal cells
occur at times when or in places where the papillae are active inductively?
When, during development, does the conjunctival periderm desquamate? Does
desquamation occur at the same time in all regions of the periderm, or are
there consistent regional differences in the time of onset of desquamation?
If such regional differences exist, how do they correlate with the development
and involution of the papillae?

Methods Employed: The size and shape of the conjimctival perideraml
cells will be determined over a range of ages in the chick embryo in specimens
stained for cell outline by a modified silver nitrate impregnation and in
specimens prepared for scanning electron microscopy (SEM) . Photographs and
camera lucida tracings will be used for geometric and planimetric measurements
of cell size and shape. SEM will be used also to follow changes in the
surfaces of the cells.

Major Findings: 1. Conjunctival peridermal cells are polygonal (for the
most part hexagonal) and their apposed borders form a three-rayed net.
2. Until the seventh day of incubation these cells are not elongated. On the
seventh and eighth days, following the appearance of the conjunctival papillae,
patches of peridermal cells become elongated along the ring formed by the
papillae. Each patch of elongated peridermal cells tends to lie on one side or
the other of a papilla with respect to a papilla tends to correlate with the
direction in which the bone which comes to underlie it will overlap its nearest
neighbors. The elongation disappears a day or two before the papillae degener-
ate. 3. The peridermal cells which overlie each papilla become markedly smaller
than their neighbors outside the papillary zone. 4. Each conjunctival perl-
dermal cell has sparse microvilli on its surface. Over the papillae, however,
these cells develop high rugae. 5. The conjunctival peridermal cells are
tightly apposed to their neighbors, except over the papillae where they
partially separate from their nearest neighbors.

130

Project No. ZOl EY 00036-01 LVR

Significance to Biomedical Research and the Program of the Institute;
Just as the embryonic corneal epithelium has been shown to dictate the three-
dimensional architecture of the stromal tissue underlying it, evidence is now
emerging that the embryonic conjunctiva induces tissues in the mesenchyme
beneath it and determines their structure. The sharp transition between
corneal stroma and the limbic sclera appears to be attributable in large
measure to the activities early in development of the quite different eplth-
elia which overlie these regions. This study uses the favorable context of
the chick embryonic conjunctiva and perilimbic sclera to explore some of
the developmental roles of the conjunctiva, with special eaphasis on the roles
possibly played by its periderm. It is hoped that these efforts will clarify
some aspects of normal and abnoniuil shaping of the anterior segment of the eye
during embryonic development.

Proposed Course: This project was initiated recently and will be
continued in an attempt to reach its objectives.

NEI Research Program; Corneal Diseases - Refractive Problems and Contact
Lenses

Experimental Subject or Tiaaue Sourca; Chick

Research Objective; Etiology

Publications; None

131

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do HOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAWMAL RESEARCH PROJECT

PROJECT NUMBER

101 EY 00028-01 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (SO characters or less)

Ribosomal RNA Synthesis In the Eastern North-American Newt, N viridescens

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: David H. Reese

Ph.D.

Staff Fellow

LVR NEI

COOPERATING UNITS (if any)

None

lab/branch
Laboratory of Vision Research

SECTION

Section on Experimental Embryology

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

0.1

PROFESSIONAL:

0.1

OTHER:

0.0

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEWS

n (b) HUMAN TISSUES

H (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords) ^ , , . ,

Ribosomal RNA (rRNA) synthesis, the initiation of which is an early major
event during the transformation of iris into lens in the newt, was characterized
in the TVI cell-line derived from the eastern North American newt Notophthalmus
viridescens. Employing the technique of polyacrylamide gel electrophoresis,
molecular-weight measurements were made on newt rRNAs using Xenopus laevis and
E. coli rRNAs as standards. The mglecular weights of N. viridescens 28S and
IBS rRNA were found to be 1.4 x 10° and 0.7 x 10^ respectively. The precursor
to these RNAs had a molecular weight of 3.1 x 10 . Three probable intermediates
in the processing of precursor to mature rRNA were also identified. On the basis
of the molecular weights of all species of RNA identified, a processing pathway,
similar to that of Xenopus , has been suggested.

Some unusual features in the kinetics of precursor rRNA labelling and
processing suggest the possibility that newt-cell rRNA synthesis may be con-
trolled by the availability of essential amino acids in a manner similar to that
observed in mammalian cells. A possible relationship between the availability ol

essential amino acids, the initiation of rRNA synthesis in the newt iris, and tie
• - ^ - — *--' — ±D discussed. .

PjjCj^^yol of lens regeneration

^'^'^

Project No. ZOl EY 00028-01 LVR
Project Description:

Objectives ; This project sought to exploit a cell line derived from an
amphibian which is capable of regenerating an entire lens, in order to
analyze some aspects of the synthesis of ribosomal ribonucleic acid (rRNA) .
In this animal, the enhanced synthesis of rPJsfA is an early and important event
in the regeneration of a lens from the dorsal margin of the iris epithelium.
Therefore, the project specifically undertook to determine the molecular
weights of 28S and IBS rRNA, to identify precursors of these rRNAs and to
study the kinetics of processing of the precursors.

Methods Employed : Cultures of the TVI cell line, derived from the iris
of the newt, Notophthalmus viridescens were exposed to radloisotopically
labelled precursors of RNA. After short-term or long-term exposure of the
cultures, rRNA was extracted and subjected to polyacrilamide gel electro-
phoresis in the presence of labelled rRNA standards and carriers of known
molecular weight, derived from E. coli and from Xenopus laevis . This per-
mitted estimations of the molecular weights of 28S and 18S rRNAs of the TVI
cell line.

Major Findings: 1. The molecular weight (M.W.) of rRNA from N. viridescens
was measured as: 1.4 x 10 for 28S rRNA; and 0.7 x 10 for 18S rRNA. The M.W.
of N. viridescens 18S rRNA was the same as that of a European newt (Triturus
cristatus) and an anuran (Xenopus laevis). While the M.W. of 28S rRNA was
the same in N. viridescens and T_^ cristatus it was less than that for the 28S
rRNA of X. laevis.

2. Transfer RNA from N. viridescens had a M.W. of 0.026 x 10 , a value
comparable to that previously determined for X. laevis.

3. Short-term labelling of the rRNA of TVI cells revealed a precursor for
28S and 18S rRNA with a M.W. calculated to be 3.09 + 0.069 (SEM) x 10 (the
range of seven determinations was 2.9-3.3 x 10 ) . Under the conditions of
culture the precursor rRNA was processed through at least three intermediates
to yield 28S rRNA.

Significance to Biomedical Research and the Program of the Institute;
The enhanced synthesis of rRNA in the epithelial cells of the dorsal iris is
a very early event leading to the complete regeneration of a normal lens.
This study characterizes the rRNAs and demonstrates the existence of precursor
rRNAs. In expanding our information about early events in lens regeneration,
the findings contribute to the NEI research program related to cataract. i

In the course of this study the possibility was raised that the availa-
bility of essential amino acids may control the synthesis of rRNA in newt
cells. This possibility is testable. It not only suggests a control mech-
anism for the initiation of lens regeneration, but also has wider implication
for the control of the synthesis of rRNA in other tissues of the body.

Proposed Course; This study has accomplished its objectives, its results j
have been published and it is, accordingly, terminated.

134

Project No. ZOl EY 00028-01 LVR
NSI Research Program; Cataract

Experimental Subject or Tissue Source; Newt /Clawed toad /bacterium

Research Objective; Etiology

Publications :

Reese, D,: Rlbosomal RNA synthesis in the eastern North-American newt,
Notophthalmus viridescens. Differentiation 7: 99-106, 1977.

135

SBITHSOWtAN SCIEMCC 'INFORMATION tXCHANGE
PROJECT NUMBER (Oo NOT use this space)

U.S. DEPARTMENT (^
HEALTH, EDUCATION, AMD WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
iHTRAHURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00177-02 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)
Plasma Membrane Composition and Biosynthesis in Chick Lens Fibers and Epithella

NAMES, LABORATORY AND INSTITUTE ',FF ILI ATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED C'-J THE PROJECT

PI:

Peggy Zelenka Ph.D.

Senior Staff Fellow

LVR NET

COOPERATING UNITS (if any)

None

lab/branch ■
Laboratory of Vision Research

SECTION
Section on Experimental Embryology

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

1.25

PROFESSIONAL:
1.25

OTHER:

0.0

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS 0 (a2) INTERVIEWS

n (b) HUMAN TISSUES

a (c) NEITHER

SUMMARY OF WORK (200 words or lers - underline keywords)

This project seeks to isolate and characterize the principal lipid and
protein components of plasma membranes of embryonic chick lens epithelia and
lens fibers, to determine whether the membrane composition changes during lens
cell differentiation, and to follow any changes in~~the rates of synthesis or
degradation of membrane components as a function of developmental time.

The phospholipid compositions of lens epithelia and lens fibers of six-
day-old chick embryos have been determined, and the amount of P incorporated
into each phospholipid has been measured in vitro and in vivo. The results
of these experiments indicate that the most actively metabolized phospholipids
in both the lens fibers and lens epithelia are phosphatidic acid and phospha-
tidylinositol. Measurements of the net synthesis of each phospholipid during
a two-hour labeling period in vivo indicate that the net synthesis of phospha-
tidic acid in the lens fibers is eight to ten times greater than in the lens
epithelia, while the net synthesis of other phospholipids is two to three times
greater than in the epithelia.

137 .

Project No. ZOl EY 00177-02 LVR
Project Description;

Objectives ; The objectives of this project are: a) to characterize the
principal lipid and protein components of plasma membranes from embryonic
chick lens fibers and lens epithelial cells; b) to determine whether the
differentiation of lens epithelial cells into lens fibers is accompanied by
changes in membrane composition; and c) to learn whether the differentiation
of lens epithelial cells into lens fibers is accompanied by changes in the
rates of synthesis or degradation of components of the plasma membranes of
lens cells .

Methods Employed; Lens fibers and epithelia of six -day-old chick embryos
are isolated by microdissection for extraction of phospholipids. P-labeled
phospholipids are obtained either by culturing the lenses in the presence of
the isotope or by injecting isotope into the embryos via the chorio-allantolc
circulation. Phospholipids are separated by thin layer chromatography;
radioactivity is determined either by scintillation counting or by auto-
radiography. Experiments in progress employ computer modeling to determine
rates of synthesis and degradation of individual phospholipids.

Major Findings: The lens fibers and lens epithelia of six-day old embry-
onic chicks have very similar phospholipid compositions. Both are rich in phos-
phatidylcholine and phosphatidylethanolamlne, and contain lesser amounts of
phosphatidylserine, phosphatidylinositol, sphingomyelin, phosphatidic acid,
phosphatidylglycerol and diphosphatidylglycerol. The fibers and epithelia
differ significantly only in their sphingomyelin content. This phospholipid
represents 6% of the total lens fiber phospholipid, as compared to <1% in
the lens epithelia.

32
When the lenses of six-day-old chick embryos are labeled with P either

in vitro or in vivo, the phospholipids with the highest specific activity are

phosphatidylinositol and phosphatidic acid. These phospholipids, therefore

have greater metabolic activity than the other lens phospholipids.

32
The distribution of P among the various phospholipids in the lens fibers

differs from that observed in the lens epithelia. In particular, the propor-
tion of label incorporated into phosphatidic acid in the lens fibers is eight
to ten times greater than that in the epithelia. Since differences in phospho-
lipid labeling may reflect metabolic changes which play a regulatory role in
lens fiber differentiation, additional experiments have been undertaken to
determine the rates of synthesis and degradation of the various phospholipids.
For this, the specific activity of the ATP pool in the lens fibers and epith-
elia as well as the amount of radioactivity incorporated Into each phospholipid
are being determined as a function of time. Thus far, the data indicate that
the net synthesis of phosphatidic acid during a two hour labeling period in
vivo, is approximately nine times greater in the lens fibers than in the lens
epithelia; net synthesis of all other phospholipids is two to three fold
greater in the lens fibers.

138

Project No. ZOl EY 00177-02 LVR

Significance to Biomedical Research and the Program of the Institute;
The plasma membranes of lens cells appear to play Important roles in normal
development and function of the lens. In addition they are centrally involved
in the genesis and development of several varieties of lens cataract. Despite
the widely recognized and important functions of these membranes , work on
their composition, turnover, and development has begun only recently. This
project focuses on changes in lens cell menbranea which are associated with
lens fiber differentiation. The results should have broad application in
understanding normal lens differentiation and morphogenesis, and in attempts
to establish etiologies for several types of cataract.

Proposed Course; This project will be continued. An atteapt will be
Bade to determine the rates of syntheais and dagradation of the individual
phospholipids of esifcryonic chick lens fibers and lena epithfelia in vivo, by
conputer analysis of the time course of incorporation of P into the phospho-
lipids .

NEI Research Prograi; Cataract - The NormsJ. L«ns

Experimental Subject or Tissue Source; Dowtstic fowl

Research Objective; Etiolofy

Publications : None

139

SMITHSONtAN SCS£NCE INFORMATSON EXCHANGE
PROJECT NUMBER (Oo NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMtNiAL RESEARCH PROJECT

PROJECT NUMBER
ZOl EY 00034-01 LVR

PER i 00 COVERED

October 26, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Effects of Vitamin A Deficiency on Ocular Tissues

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Louvenia Carter-Dawson Ph.D.
Other: Toichiro Kuwabara M.D.

Staff Fellow LVR NEI

Head, Section on Experi-
mental Pathology LVR NEI

COOPERATING UNITS (if any)

Dr. John G. Bieri

National Institute of Arthritis,
Metabolism and Digestive Diseases, NIH

lab/branch

Laboratory of Vision Research

SECTION

Section on Experimental Pathology

NSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:
1.1

PROFESSIONAL:
1.1

OTHER:

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

Q (al) MINORS n (a2) INTERVIEWS

n (b) HUMAN TISSUES

[i(c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

Electroretinograms from vitamin A deficient albino rats show a decrease in
amplitude of the a and b waves as early as 27 days on the diet. This decrease
continues as the duration on the deficient diet lengthens. Through two months
on the deficient diet, the only correlation between the structural integrity of
the retina and the reduction in amplitude is that the outer two-thirds of the
outer segments lose their normal staining properties. Around four months, some
outer segments show signs of deterioration; however, their inner segments appear
normal. Light and electron microscopic studies of the retina, pigment epitheliun

and their interrelationship are in progress at various levels of deficiency.

141

Pro.lect No. ZOl EY 00034-01 LVR
Pro.ject Description:

Objectives: In several less developed areas of the world, such as
Asia and Central America, a large number of people suffer from vitamin A
deficiency. This deficiency results in keratomalacia and poor vision. The
early histological and cytological changes which occur in the ocular tissues
have not been clearly described. This project was designed to investigate
the effect of vitamin A deficiency on the maintenance of photoreceptor and
pigment epithelial cell structure and functional interrelationship.

Methods Employed: Pregnant rats were fed vitamin A free diets (basic
diet) one week prior to delivery and maintained on the diet through lactation.
AL weaning, 21 days, the basic diet of one group was supplemented with retinoic
acid, a second with retinyl palmitate, and a third group received no supplement
From each group electroretinograms were recorded and the retinas examined by
light and electron microscopy. The animals were reared in cyclic light — 12
hours light, 12 hours dark — at a cage illumination 1.5-2 foot-candles.

Major Findings: Electroretinograms from the deficient albino rats
showed a reduction in amplitude of the a and b waves as early as 27 days of
age. The amplitude of both waves continued to decrease with duration on the
diet.

Structurally, the retinas of these animals appeared normal through eight]
weeks. However, the outer segments in sections stained with toluidine blue
showed a difference in staining intensity of the outer two- thirds. This
portion of the outer segment stained considerably lighter. Whether this is
related to the presence or absence of the visual pigment, rhodopsin, is un-
clear. Around four months, the outer half of some outer segments began to shoy'
signs of deterioration, but the inner segments appeared normal. No loss of
photoreceptor nuclei was apparent at this age.

Significance to Biomedical Research and the Program of the Institute:
Results from these studies will give some further insight into the role of
vitamin A in the maintenance of photoreceptor and pigment epithelial cell
structural and functional interrelationship. In addition, these studies will
provide information on the early histological and cytological changes which
occur before signs of poor vision are manifested.

Proposed Course: Light and electron microscopic analyses of photo-
receptor and pigment epithelial cell structure will be continued through late
stages of vitamin A deficiency. Also, other ocular tissues, such as the corne
and conjunctiva will be examined at various stages of deficiency.

NEI Research Program: Retinal and Choroidal Diseases - Visual Cells
and Pigment Epithelium

Experimental Subject or Tissue Source: Rat

Research Objective: Etiology

142

Pro.ject No. ZOl EY 00034-01 LVR
Publications: None ' "

X43

SHITHSONIAN SCiENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00129-05 LVR

PERIOD COVERED

July 1, 1976 to September 30. 1977

TITLE OF PROJECT (80 characters or less)

Anatomical and Pathological Studies of Ocular Tissues

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI:

Toichiro Kuwabara

Other: Fujiko Huang
Minoru Tanaka

M.D. Head, Section on Experimental

Pathology LVR NET

M.D. Visiting Scientist LVR NEI

COOPERATING UNITS (if any)

Simmons Lessell M.D.

Robert Friedlaender M.D.

Shirley Wray M.D.

Boston University

Food and Drug Administration

Harvard University

lab/branch

Laboratory of Vision Research

SECTION

Section on Experimental Pathology

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

2.9

PROFESSIONAL:

2.1

OTHER:

0-S

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEWS

(b) HUMAN TISSUES

n (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

Normal and pathological eye tissues of the human and experimental animals
have been studied by electron microscopy in order to elucidate pathogenesis of
various eye diseases.

Completed works during this fiscal year are: fine structure of the cornea,
especially on the keratocyte; histopathological study on ocular changes induced
by suckling mouse cataract agent; electron microscopic study of cataractous lenses
of diabetic sand rats; electron microscopic and biochemical studies on experimen-
tally induced Niemann-Pick disease-like changes in albino rats; degeneration of
the pigmented ciliary epithelium by hyperosmotic shock to the monkey; fine struc-
tural studies on the conjunctiva of a case of Richner-Hanhart syndrome, and on
the optic nerve and retina of a case of adrenoleukodystrophy ; histologic and
statistic study on metastatic skin melanoma to the eye; electron microscopic
study of extraocular muscles in myotonic dystrophy.

145

Project No. ZOl EY 00129-05 LVR
Project Description: .

Objectives: The principal goal is the elucidation of the pathophysiology
of disease processes, both naturally occurring and experimentally induced.
Fundamental to this understanding of disease mechanisms is the greater clar-
ification of the normal anatomy and functions of the eye.

Methods Employed: Eye tissues of the human and various experimental
animals were fixed in glutaraldehyde and examined by transmission and scanning
electron microscopy. Individual experimental procedures will be described
under the headings of major findings.

A large number of clinicopathological specimens which were submitted to
this laboratory from the NEI Clinical Branch and many other eye research
centers of the nation were studied for specific aims by electron microscopy
and histochemistry.

Maj or Findings :

Studies on the cornea

Fine structure of the cornea

Although fine structural investigations on each part of the cornea have
been reported by several authors, a comprehensive discussion on the structure
of the cornea as a whole has not been available since 1969. A large amount of
data on cytologic studies of the cornea of the human and experimental animals
was re-evaluated. With additional findings, a summary report on the corneal
structure was made.

This study has demonstrated that the keratocyte is capable of movement
as a whole and/or in processes, sensitively responding to various stimulations.
Phagocytic activity of exogeneous particles is unexpectedly small. Phagocytosis
in the stroma is found to be taken care of mainly by histiocytes and leuco-
cytes which are abundantly present within the normal stroma. Another worth-
while finding is that - there are tissue spaces along keratocytes which are con-
nected to each other with their processes. The space may serve as the intra-
corneal channel.

Studies on the lens

SMCA-induced cataract

The suckling mouse cataract agent (SMCA) , a member of spiroplasma, was
inoculated into postnatally-developing mouse brains. These animals had a high
incidence of cataract formation. Pathologic changes in the lens included:
proliferation and posterior extension of lens epithelium; increased accumulation
of capsule material and formation of lens fibers at abnormal locations. The
histological changes of this experimental cataract* are similar to those of
congenital cataracts which are induced by certain fetal viral infections.

146

Project No. ZOl EY 00129-05 LVR
Cataractous lenses of diabetic Egyptian sand rats

A diabetes mellitus-like syndrome was induced in the Egyptian sand rat
(Psammomys obesus) by changing its diet from leafy vegetables to laboratory
chow. Cataractous changes developed in all experimental animals after about
three weeks feeding of the high caloric diet. The first lenticular change was
marked swelling and then degeneration of the cortical cells. The surviving
lens cells proliferated and formed islands in the subcapsular region. Nodular
masses of the fibrous tissue were formed around the proliferating cells. These
changes are similar to those of cataracts in diabetes mellitus of the human.

Senile cataract

A great number of lenses with senile cataract had been sent to this lab-
oratory for cytological studies from members of the National Eye Institute
Cooperative Cataract Research Group. Lenses were studied grossly and photo-
graphed and examined by histology and scanning and transmission electron
microscopy. Flat preparations and histological sections revealed that the
epithelial cells became progressively sparse with age. Several acellular foci
were formed. Electron microscopy of the remaining cells showed that the cyto-
plasm had lost the normal structure. The cataractous changes of the anterior
cortex were regularly localized beneath the abnormal epithelium. These find-
ings indicated that cytological changes in the epithelium proceeded the occur-
rence of the senile changes in the lens fibers.

Lento Id body

In collaboration with Dr. Paul Russell of LVR, lentoid bodies of humans
and normal and cataractous mice were studied by scanning and transmission elec-
tron microscopy. Cells of the lentoid bodies are loosely packed and produce
basal lamina substances between them. Some cells begin to form crystalline pro-
tein granules. Electron microscopic demonstration of ATPase in these cells is
underway.

Experimental Niemann-Pick disease

Following intraperitoneal injections of AY 9944, a cholesterol synthetase
inhibitor, into postnatally developing rats (50 mg per kg body weight) abundant
inclusion bodies were produced in ganglion, glia, amacrine and horizontal cells
of the retina, glia cells of the optic nerve, pigment epithelial cells of the
retina and ciliary body, and lens fibers. Electron microscopic and biochemical
studies revealed that characteristics of the inclusion body were identical to
those of Niemann-Pick disease of the human. The experimentally induced inclu-
sion bodies were different from nonspecific debris of degenerative cells and
the inclusion-laden cells were otherwise cytologically normal. This experiment
suggested that inhibition of cholesterol synthesis involves the pathogenesis of
this hereditary disease.

147

Project No. ZOl EY 00129-05 LVR
Glaucoma study

Effect of hyperosmotic agents on the ciliary epithelium

2M urea or 2M DL-lactamide were perfused into the internal carotid artery
of rhesus monkeys. Both layers of the ciliary epithelium were severely dam-
aged by this hyperosmotic shock. The non-pigmented epithelium recovered to an
almost normal structure within a short period of time, but the pigment epithe-
lium especially of the pars plana became degenerative. The intraocular pressure
of the perfused monkey was extremely low for about one month, but gradually
came back to a near normal value. However, the damaged pigmented epithelium
did not recover. The number of giant vacuoles in the endothelium of Schlemm's
canal decreased while the intraocular pressure was low, but increased gradu-
ally with the recovery of the pressue. This experiment suggested a possibility
of the presence of several other factors in regulating aqueous humor production
besides the generally understood mechanism.

Essential iris atrophy

A case of advanced essential iris atrophy was studied by electron micros-
copy. The result was reported with Drs. Kaiser-Kupf er and Kupfer. The study
showed that the involved iris stroma was markedly atrophic, but the dilator
muscle layer was strikingly well-preserved. In jsome areas, proliferation of
the dilator muscle was indicated. This caharact eristic histopathologic change
may be related to the occurrence of frequently accompanied glaucoma.

Clinicopathological study

Adrenoleukodystrophy

Adrenoleukodystrophy is an x-chromosome-linked recessive disease char-
acterized by primary atrophy of the adrenal gland, degeneration of white matter '
of the central nervous system, and blindness. Eyes of a ten-year-old boy with
this disease were sent to this laboratory from Harvard University for special
studies. Histological and electron microscopical studies revealed marked de-
myelination of the optic nerve and loss of retinal ganglion cells.

Richner-Hanhart syndrome

Richner-Hanhart syndrome with tyrosinemia was recognized in a mentally
retarded adolescent boy. The conjunctiva biopsies were sent to this laboratory
from Harvard University for special studies. Electron microscopic study re-
vealed that the conjunctival epithelial cells, subepithelial fibrocytes and
blood vessel endothelial cells accumulate large inclusion bodies which contain
tyrosine-like fine crystals. It was hypothesized that cells are in the proc-
ess of removing excess tyrosine from the blood stream and the tissue fluid.

Malignant melanoma of the skin: metastasis to the eye

Histopathologic examination of the eyes of 15 consecutive patients with
metastatic malignant melanoma arising in the skin showed evidence of intraocular

148

Project No. ZOl EY 00129-05 LVR
metastasis in five patients. The metastases were microscopic, epithelioid,
minimally pigmented, and occurred in both the choroid and the retina. All
patients were asymptomatic. Those patients with ocular metastases had primary
malignant neoplasms of the superficial, spreading variety and associated
central nervous system metastases.

Myotonic dystrophy

Extraocular muscles of two patients with ophthalmoplegia secondary to
myotonic dystrophy were sent to this laboratory from Boston University.
Electron microscopic study revealed that the main change was disorganization
in the arrangement of myofibrils rather than degeneration of the cells.
Diseased muscle cells contained randomly distributed, short and irregular myo-
fibrils and individual myofilaments. The cytologic appearance of these muscle
cells was similar to that of developing muscle cells. The pathogenesis of the
myopathy in myotonic dystrophy may be related to myof ibrillogenesis and its
maintenance.

Anatomical studies on normal eye

A newly revised chapter of the eye for the textbook Creep's Histology
edited by Leon Weiss has been submitted for printing. New concepts on the fine
structure of the eye have been emphasized in this chapter. Embryological
development of various parts of the eye, especially anterior portions, has
been extensively studied electron microscopically.

Significance to Biomedical Research and the Program of the Institute:
There are few laboratories in this country which are capable of pursuing these
disease-related basic research studies on the eye tissues. This laboratory
is one of them. Significant meanings in understanding the pathogenesis of
various diseases of the eye have been obtained through the results obtained
in the present investigations.

Proposed Course: Similar projects are actively ongoing and will be
continued in the next fiscal year.

NEI Research Program: Retinal and Choroidal Diseases - Developmental
and Hereditary Disorders/Diabetic Retinopathy and Other Vascular and Circulatory
Abnormalites/Tumors/Inflammatory Disorders/Uveal Tract; Corneal Diseases -
Corneal Edema, Dystrophies, and Inherited Disorders/Corneal Transplantation
and Stromal Injury and Repair/Tumors and Other Lid, Conjunctival, and Orbital
Problems; Cataract - The Normal Lens/Diabetic Cataract/Cataract Induced by
Drugs, Radiation, and Secondary to Other Eye Disorders; Glaucoma - Etiology of
Glaucoma (Primary Glaucoma — Open-Angle Glaucoma/Secondary Glaucomas)

Experimental Subject or Tissue Source: Rhesus monkey/Rat/Mouse/Human

Research Objective: Etiology, Diagnosis, Treatment

149

Project No. ZOl EY 00129-05 LVR
Publications:

Kuwabara, T. : The corneal stroma cell. In Yamada, E. and Mishima, S.
(eds.)-. The Structure of the Eye III, Jap. J. Ophthalmol. 1976, 39-47.

Kuwabara, T.: Current concepts in anatomy ahd histology of the cornea.
In King, J.H. and McTigue, J.W. (eds.): The II World Congress on the
Cornea. Washington, D.C., 1976, Butterworths (in press).

Friedlaender, R.P., Barile, M.F. and Kuwabara, T. : Ocular pathology
induced by the suckling mouse cataract agent (SMCA) . Invest . Ophthalmol .
15: 640-647, 1976.

Kuwabara, T. and Okisaka, S.: Electron microscopic study of cataractous
lenses of diabetic sand rats (Psammomys obesus) . Doc. Ophthalmologica
Proceedings - Progress of Lens Biochemistry Research, 1976, 7-15.

Russell, P., Fukui, H.N. , Tsunematsu, T., Huang, F.L. and Kinoshita, J.H.;
Tissue culture of lens epithelial cells from normal and Nakano mice.
Invest. Ophthalmol. 16: 243-246, 1977.

Sakuragawa, M. : Niemann-Pick disease-like inclusions caused by a
hypocholesteremic agent. Invest. Ophthalmol. 15: 1022-1027, 1976.

Sakuragawa, N. , Sakuragawa, M. , Kuwabara, T. , Pentchev, P.O., Barranger,
J. A. and Brady, R.O. : Niemann-Pick disease experimental model:
Sphingomyelinase reduction induced by AY-9944. Science 196: 317-319,

1977.

Okisaka, S., Kuwabara, T. and Rapoport, S.I.: Effect of hyperosmotic
agents on the ciliary epithelium and trabecular meshwork. Invest .
Ophthalmol . 15: 617-625, 1976.

Kaiser-Kupfer, M. , Kuwabara, T. and Kupfer, C: Progressive bilateral
essential iris atrophy. Am. J. Ophthalmol. 83: 340-346, 1977.

Bienfang, D.C., Kuwabara, T. and Pueschel, S.M.: The Richner-Hanhart
syndrome, Report of a case with associated tyrosinemia. Arch. Ophthalmol.
94: 1133,-1137, 1976.

Wray, S.H., Cogan, D.G., Kuwabara, T. and Schaumburg, H.H. : Adrenoleuko-
dystrophy with disease of the eye and optic nerve. Am. J. Ophthalmol.
82: 480-485, 1976.

Fishman, M.L., Tomazewski, M.M. and Kuwabara, T.: Malignant melanoma
of the skin metastatic to the eye: Frequency in an autopsy series.
Arch. Ophthalmol. 94: 1309-1311, 1976.

Kuwabara, T. and Lessell, S.: Electron micrgscopic study of extra-
ocular muscles in myotonic dystrophy. Am. J. Ophthalmol. 82: 303-309,
1976.

150

Project No. ZOl EY 00129-05 LVR
Kuwabara, T.: Species difference of the pigment epithelixim. In Zinn,
K.M. and Manner, M.F. (eds.)'. The Retinal Pigment Epithelium. Cambridge,
Harvard Press (in press) .

Kuwabara, T. and Cogan, D.G.: The eye. In Weiss, L. and Creep, R.O.
(eds.): Histology, 4th edition. McGraw-Hill, New York, 1977, p. 1119-
1164.

151

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PROJECT NUMBER (Oo NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER
ZOl EY 00131-05 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Light Effect on the Retina

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Toichiro Kuwabara M.D.

Other: W. Gerald Robison, Jr. Ph.D.

Masakazu Funahashi M.D.

Atsushi Mizukawa M.D.

Head, Section on Experimental

Pathology LVR NEI

Geneticist/Cell Biologist LVR NEI

Visiting Scientist LVR NEI

COOPERATING UNITS (if any)

None

lab/branch

Laboratory of Vision Research

SECTION

Section on Experimental Pathology

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

1.9

PROFESSIONAL:

1.7

OTHER:

0.2

CHECK APPROPRIATE BOX(£S)
D (a) HUMAN SUBJECTS

D {al) MINORS n (a2) INTERVIEWS

n (b) HUMAN TISSUES

[Jt(c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

Effect of visible light to the retina has been studied by electron micros-
copy. The following results have been obtained: 1) Phagocytic activity of the
pigment epithelium against latex spheres is influenced by the size of the sphere
2) Phagocytic activity of the pigment epithelium following light damage of the
retina has been studied by exposing rats to fluorescent lamps at 150 foot-candles
Phagocytic activity is first accelerated by light, but eventually is diminished
when the light damage becomes severe. Also, the phagocytic activity is controlled
by the circadian rhythm. 3) Synaptic organs of the photoreceptor cells show marUec
cytologic changes by light exposure. One of the early changes is proliferation
of the paramitochondrial membranes. The cone cells are resistant to light ex-
posure. 4) Photoreceptor cells of the developing rat continue outer segment pro-
duction under bright light (400 foot-candles) for more than two weeks without
showing severe damage. Further exposure causes severe damage in the newly-
formed outer segments.

153

Project No. ZOl EY 00131-05 LVR
Project Description:

Objectives: This project has been selected to help elucidate the patho-
genesis of some degenerating diseases of the human retina.

Methods Employed: Albino rats were placed in uniformly illuminated cages
for various time periods. The light source was cool fluorescent light and ex-
posure was given by day and night (12-12 hour) cycles. Retinas of the exposed
animals were studied by electron microscopy.

Major Findings:

Phagocytosis of the pigment epithelium

Latex spheres

Polystyrene spheres in various sizes were injected into the subretinal
space of rhesus monkeys. The pigment epithelium and retina were studied by
electron microscopy. In the first few hours, the pigment epithelial cells
actively phagocytized fragments of the outer segment produced by the injection
injury. The spheres were engulfed at a considerably later stage. By the
sixth hour, the larger spheres (1.0 ym) which had been surrounded by the micro-
villi were taken into the cell. Phagocytized spheres were usually distributed
individually within the cytoplasm. On the other hand, the smaller (0.1 ym)
spheres aggregated into clusters of 10-20 and were coated by a mucopoly-
saccharide substance before they were engulfed by the pigment epitheliiun within
24 hours. When a mixture of 0.1 and 1.0 ym spheres was injected, the cell
handled the two kinds of spheres separately, evoking the pattern as described
above. The size of the particles appears to be a determining factor in phago-
cytic activation.

Circadian cycle

Normal albino rats that had been kept under the dimmed cyclic light (two-
five foot-candles for 12 hours and total darkness for 12 hours) showed an active
phagocytosis of shed outer segments in the pigment epithelium at the beginning
of the light period, but no activity during the dark period. When the same
animals were exposed to the same cycles, but with 150 foot-candles, the phago-
cytic activity was profoundly accelerated during the similar time period.
After the exposure of five cycles or more, the retinal outer segments were
severely damaged and the phagocytic activity of the pigment epithelium
diminished.

I
Light effect on the synapsis of the photoreceptor cell

The photoreceptor synapses of albino rats show considerable pathologic
changes following fluorescent light exposure. The changes in the synapses and
in the lamellar membranes of the outer segments progress simultaneously.

Membranes proliferated in the paramitochondrial zone of the rod synaptic
spherule and fine budding of the smooth endoplasmic reticulum in the cone

154

Project No. ZOl EY 00131-05 LVR

pedicle occurs within one hour's exposure to the brightness of 500 foot-candles.
Proliferating paramitochondrial membranes have no cytochrome c oxidase activity
and degenerate together with mitochondria after further exposure. The cone
pedicles remain relatively intact in the photically damaged retina.

Light effect on the developing retina

The effect of bright light on the developing albino rat was studied
electron microscopically. The newly-formed outer segment lamellar membranes
of newborn rats, raised in continuous bright light appeared to be less sen-
sitive to the damaging effects of light, compared to rats raised under normal
light conditions for two weeks. It seemed to take about two days before the
newly-formed membranes showed photo damage after continuous exposure to fluo-
rescent lamps. The same brightness damaged the adult outer segments within a
few hours. Despite the severe damage to the outer segments, the rest of the
retina developed normally for one month, and then the photoreceptor cell bodies
degenerated. The retinas which had been exposed for two weeks since birth
showed considerable damage, but these retinas regenerated in six months.

Significance to Biomedical Research and the Program of the Institute:
Results obtained from the present experiments are directly useful in further
understanding of the retina - pigment epithelium Interrelationship. Eluci-
dation of the basic mechanism of the pigment epithelium related to light ex-
posure is the first step in understanding of the retinal diseases of the
human.

Proposed Course; Similar studies will be continued. Effects of cyclic
light exposures to the retina is now under intense investigation.

NEI Research Program: Retinal and Choroidal Diseases - Visual Cells and
Pigment Epithelium/Special Areas of Future Interest (Toxic and Environmental
Disorders

Experimental Subject or Tissue Source: Rhesus monkey /Rat/Mouse

Research Objective: Etiology

Publications:

Funahashi, M. , Okisaka, S. and Kuwabara, T.: Phagocytosis by the monkey
pigment epithelium. Exp. Eye Res. 23: 217-225, 1976.

Kuwabara, T. and Funahashi, M. : Light damage in the developing rat retina.
Arch. Ophthalmol. 94: 1369-1374, 1976.

Kuwabara, T.: Photo-thermal effects on the pigment epithelium. In Zinn,
K.M. and Marmor, M.F. (eds.): The Retinal Pigment Epithelium. Cambridge,
Harvard Press (in press) .

155

sSfTHSONlAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do KOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
MOTICE OF
INTRAHURAL RESEARCH PROJECT

PROJECT NUMBER
ZOl EY 00149-04 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Ultrastructure and Function of the Pigment Cells of the Eye

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: W. Gerald Robison, Jr. Ph.D. Geneticist/Cell Biologist LVR NET
Other: Toichiro Kuwabara M.D. Head, Section on Experi-
mental Pathology LVR NEI
Gerald Chader Ph.D. Head, Section on Retinal

and Corneal Metabolism LVR NEI

COOPERATING UNITS (if any)

None

lab/branch

Laboratory of Vision Research

SECTION

Section on Experimental Pathology

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

2.1

PROFESSIONAL:
1.1

OTHER:

1.0

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

n (al) MINORS n (a2) INTERVIEWS

D (b) HUMAN TISSUES

[J(c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

The ability of the retinal pigment epithelium to store vitamin A was test-
ed by injecting mice with retinol acetate or retinol palmitate and examining
their retinas and livers using fluorescence and electron microscopy. The results
showed clear, dose-related increases in the numbers and sizes of vitamin-A-stori4g
lipid droplets in the stellate cells of the liver. Concomitantly, more conserv-
ative increases in similar lipid droplets occurred in the pigment epithelium but
not in other cells of the retina. Such lipid droplets may represent physiologic
sites of vitamin A storage which are important for the maintenance of photo-
receptor cells by the retinal pigment epithelium.

Electron microscopic histochemistry for catalase demonstrated that perox-
isomes , like the putative vitamin-A-storing lipid droplets were distributed along
the basal and lateral cell surfaces of the pigment epithelium where receptors foi
plasma retinol-binding protein have been reported. Peroxisomes may play a role
in the reactions related to the esterif ication and sequestering of vitamin A.

157

Project No. ZOl EY 00149-04 LVR
Project Description:

Objectives: Study the structural and functional interrelationships
that exist between the pigment epithelium and the visual cells of the eye.
We propose to examine how the pigment epithelial cells are involved in the
maintenance of photoreceptor cells and what specific functions are lacking in
various experimental and pathological cases .

Methods Employed: Hypervitaminosis A was induced by intramuscular in-
jections of retinol acetate or retinol palmitate. In order to locate the
sites of vitamin A storage, fluorescence microscopy was used to detect vitamin
A-specific fluorescence, and then electron microscopy, combined with light
microscopy of one micron sections, was performed. Mainly the retina and
liver were studied.

Ultrastructural histochemistry for demonstration of catalase was used
to study the distribution of peroxisomes in relation to the vitamin-A-storing
lipid droplets. Littermates of inbred C57BL/6J mice were used to minimize
variables.

Major Findings: Lipid droplets of the pigment epithelium increased in
number depending on the availability of vitamin A, suggesting that they serve
as physiological storage sites of this important component of photoreceptor
cells. No changes in lipids of the retina or liver were observed in mice in-
jected with retinoic acid or with peanut oil alone. Peroxisomes which contain
catalase and often are involved in the transport, storage, and turnover of
lipids were closely associated with the putative vitamin-A-storing lipid drop-
lets along the basal and lateral cell surfaces. Since these cell surfaces
were reported to have receptors for plasma retinol-binding protein, peroxi-
somes may be involved in the reactions related to the esterif ication and
storage of vitamin A in the pigment epithelium.

Significance to Biomedical Research and the Program of the Institute:
A healthy retina depends on a functional pigment epithelium, yet little is
known about the specific functions and requirements of pigment epithelial
cells. The visual process involves dynamic exchange of vitamin A between the
photoreceptor cells and the pigment epithelium. The identification of vita-
min A stores within pigment epithelial cells provides a visible criterion for
assessing the functional state of these important cells in health and disease.
An elucidation of the precise relation of peroxisomes to vitamin-A-storing
droplets should be very significant.

Proposed Course: It is believed that lipofuscin granules contain retin-
oyl complexes deriving from retinoic acid. Their relation to phagocytosis,
intracellular digestion, vitamin A turnover, and other factors need to be in-
vestigated. Lipofuscin granules in the retinal pigment epithelium and ciliary
epithelium in various mouse mutants of different ages under experimental treat-
ments with light and vitamin A will be studied.

NEI Research Program: Retinal and Choroidal Diseases - Visual Cells
and Pigment Epithelium

158

Project No. ZOl EY 00149-04 LVR
Experimental Subject or Tissue Source: Mouse

Research Objective: Etiology

Publications:

Enriques, N. , Israel, P., Bergsma, D., Robison, W.G., Jr., Whikehart, D.
and Chader, G.: Neural retinal and pigment epithelial cells in culture:
Patterns of differentiation and effects of prostaglandins and cyclic
AMP on pigmentation. Exp. Eye Res. 22: 559-568, 1976.

Robison, W.G., Jr. and Kuwabara, T.: Light-induced alterations of
retinal pigment epithelium in black, albino, and beige mice. Exp.
Eye Res. 22: 549-557, 1976.

Robison, W.G., Jr. and Kuwabara, T. : Vitamin A storage and peroxisomes
in retinal pigment epithelium and liver. Invest. Ophthalmol, (in
press) .

Robison, W.G., Jr. and Kuwabara, T. : Albino-beige mouse: Lysosomal
dysfunction in retinal pigment epithelium. Inves t . Ophthalmol « (in
press) .

X59

SMITHSONIAN SCIENCE IflFORMATIGN EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, A;« WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
iNTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00026-06 LVR

PERIOD COVERED
July 1, 1976 to September 30. 1977

TITLE OF PROJECT (80 characters or less)

Physiology of the Primate Visual System

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Peter Gouras M.D.

Francisco deMonasterio M.D.,

Eberhart Zrenner M.D.

Other: Avery Dickinson Nelson Ph.D.

Head, Section on LVR NEI
Neurophysiology
D.Sc. Visiting Scientist LVR NEI
Fogarty Fellow
Senior Staff Fellow LVR NEI

COOPERATING UNITS (if any)

Max-Planck Institute, Bad Nauheim, FRG

lab/brangh
Laboratory of Vision Research

SECTION
Section on Neurophysiology

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

1.4

PROFESSIONAL:

1.4

CHECK APPROPRIATE BOX(ES)
(a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEWS

OTHER:

0.0

D (b) HUMAN TISSUES

C (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

It is the long range objective of this project to study the neural
organization underlying visual perception in primates. The topics of
present interest are: i) chromatic, temporal and spatial properties of
neurones of the retina, lateral geniculate nucleus, primary visual cortex
and extra-striate cortex, il) how this information is processed along these
center, and iil) how these single cell data are related to psychophysical
results in humans.

161

Project No. ZOl EY 00026-06 LVR
Project Description:

Objectives: To study the neural organization underlying visual percep-
tion in primates.

Methods Employed: Electrophysiological recordings from single neurons in
the retina and visual cortex of anesthetized, paralyzed macaque monkeys;
correlation of the distribution of single cell varieties and morphological
cell types as seen by electron and light microscopy; the use of refined
optical stimuli to define quantitatively spatial, temporal and chromatic
properties of these neurons.

- Major Findings: The results can be grouped into three broad areas. The
first involves a completion of work begun by P. Gouras in Freiburg in collab-
oration with J. Kruger on single cells in visual cortex. The second involves
his collaborative project here on single cells studies of the retina being
done with E. Zrenner, a visiting fellow from the Max Planck Institute in Bad
Nauheim. The third involves intracellular studies of cells in primate retina
being done together with F. de Monasterio.

The first project on visual cortex is being brought to completion and
being prepared for publication, since the results are still only available
in abstract form. These experiments were designed to determine the respon-
siveness of single cells or cell groups in foveal striate and prestriate
cortex (areas 17, 18 and V4) to four colors (red, yellow, green and blue)
presented as slowly moving slits or squares, quasi-simultaneously and over
a large number of trials in order to minimize the effects of uncontrolled
changes in neuronal excitability. This technique revealed that the color
selectivity of most neurons depends upon the slit length. As length increases
the effectiveness of red and blue increases relative to that of yellow and
green lights. The effect is immediate and not due to response saturation.
It can only be explained by assuming that signals from the most common center-
surround color -opponent geniculate cells reach all of these cortical cells.
This result requires reconsideration of the so called hypercomplex cell
classification in monkey visual cortex since such cells must now be considered
color dependent.

Color selectivity varies significantly from cell to cell ranging from a
.small group which responds exclusively or almost exclusively, to one color to
the majority which respond to all colors to some degree in what seems to be a
continuum. This variation is greater in tangential than in radial directions
through visual cortex, which implies the existence of cortical slabs or
columns of color selectivity.

The overall pattern of color selectivity varies quantitatively but not
qualitatively among areas 17, 18 and V4 of visual cortex. Receptive fields
of cells are large in prestriate cortex (areas 18 and V4) and color selec-
tivity seems to be preferred in area V4, but otherwise each of these three
areas have similar proportions of color selective cells and therefore must
presumably process information about color in similar ways. What seems to be
apparent is that color information is widely distributed among cells in visual

162

Project No. ZOl EY 00026-06 LVR
bertex and is being used to a great extent to facilitate contour detection as
well to perceive color, per se.

The second project has been in collaboration with E. Zrenner and has
been concerned with obtaining a better understanding of the blue sensitive
cone mechanism. Our approach has been to study. the responses of single
ganglion cells subserving the foveolar, foveal and perifoveal retina of the
rhesus monkey. In certain cells the blue- sensitive cone mechanism can be
completely isolated and studied separately from the other two cone mechanisms.
Several important properties of this receptor mechanism differ significantly
from those of the red and green-sensitive cone mechanisms; these differences
involved spatio-temporal resolution, receptor density, and center-surround
polarity. Understanding the role of the blue cones in primate vision is
crucial for understanding the neuronal organization of color and form percep-
tion in visual cortex.

The third project is in collaboration with F.M. de Monasterio has been
directed to intracellular recordings from cells (especially horizontal cells)
in perfused rhesus monkey retina. S-potentials have been obtained in this
system, which we are tentatively classifying as horizontal cells pending dye
injection identification. These responses suggest that both rods and cones
contribute signals to the same horizontal cell (paralleling results obtained
in perfused cat and rabbit retina). F.M. de Monasterio and also E. Zrenner
have been able to detect these horizontal cell responses in the intact monkey
retina so that we feel confident that we shall be able to begin to understand
something about cone interactions in the inner nuclear layer of primate retina
in the near future.

Significance to Biomedical Research and the Program of the Institute;
Understanding the cellular organization of rhesus monkey vision is extremely
valuable for understanding human vision, which at present can only be studied
by indirect (usually psychophysical) methods. This is especially important
for a small region of the primate retina, the fovea, which provides an
enormous and dominant input to visual cerebral cortex and when destroyed, in
man, is tantamount to blindness, legally. Understanding the neural circuitry
of this foveal mechanism from retina to visual cortex will provide us with a
basic scheme for appreciating old- and developing new techniques to study visual
function in normal and diseased human subjects.

Proposed Course; To continue the current experiments and to utilize the
anatomical expertise of A.D. Nelson in correlating function with microstructure
in primate retina, similar to what we have been doing in cat retina.

NEI Research Program; Retinal and Choroidal Diseases - Retinal Organ-
ization and Visual Adaptation

Experimental Subject or Tissue Source: Monkey

Research Objective; Etiology

163

Project No. ZOl EY 00026-06 LVR
Publications :

de Monasterio, P.M., Gouras, P., and Tolhurst, D.J. : Spatial summation,
response pattern and conduction velocity of ganglion cells of the rhesus
monkey retina. Vision Res. 16: 674-678, 1976.

Kruger, J., and Gouras, P.: Many cells in visual cortex use wavelength
to detect borders and convey information about color. Exp. Brain Res.
Suppl. 1: 407-411, 1976.

de Monasterio, P.M., and Gouras, P.: Responses of macaque ganglion cells
to far violet lights. Vision Res, (in press).

Zrenner, E. : Color opponency in visually evoked cortical potentials
(VECP) in man. ARVO Abstracts, p. 157, suppl. to Invest. Ophthalmol.,
1977.

164

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PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF '

HEALTH, EDUCATION, AMO WELFARE ,

PUBLIC HEALTH SERVICE i

NOTICE OF

IHTOAVURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00005-05 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)
Electrophysiological Studies of Mammalian Retina

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL I.JVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI:

Ralph Nelson Ph.D.

Francisco M. deMonasterio M.D.,
Peter Gouras M.D.

D.Sc.

Other :

Eberhart Zrenner M.D.
Edward V. Famiglietti.Jr . M.D., Ph.D.

Staff Fellow LVR NEI

Visiting Scientist LVR NEI

Head, Section on LVR NEI

Neurophysiology

Fogarty Fellow LVR NEI

Guest Worker

COOPERATING UNITS (if any)

Helga Kolb, Ph.D., Laboratory of Neurophysiology, NINCDS, NIH, Bethesda, Md.
20014; Max-Planck Institute, D-635 Bad Nauheim, FRG

lab/branch
Laboratory of Vision Research

SECTION
Section on Neurophysiology

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

3.5

PROFESSIONAL:

3.5

OTHER:

0.0

CHECK APPROPRIATE BOX(ES)
n (a) HUMAN SUBJECTS

n(al) MINORS D (a2) INTERVIEWS

n (b) HUMAN TISSUES

E {«:) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

The goal of this project is to obtain information about the physiological
properties and anatomical Interrelationships of neurons in the maTrmifilian retina.
The aspects of retinal physiology and anatomy with which we have dealt in the
past annum are: the spectral classes of photoreceptors in cats and rabbits and
the chromatic interactions among the cones ; the morphological basis of the ON
center and OFF center pathways of the cat retina, and a comparison of the response
properties of single units in the parallel rod and cone pathways of both cats and
rabbits; and finally, the pharmacological role of the cyclic nucleotides in the
responses of mammalian rods. At the single xinit level, electrophysiological
recordings have been made, both intracellular ly and extracellular ly, and single
units have been intracellularly injected with a variety of stains. Anatomically
the synaptic interrelationships of single units have been studied electromicro-
scopically in serial ultrathin sections, and in the light microscope such single
unit reconstructions have been compared to similar units stained with the Golgi
technique, or to units electrophysiologically studied and stained. At the multi-
unit level the massed responses of retinal neurons as reflected in the electro-
retinogram have been studied.
". . ±€5

Project No. ZOl EY 00005-05 LVR
Project Description:

Objectives: To understand the functional organization of mammalian
retinas and its relationship to disease states.

Methods Employed : Intracellular and extracellular recording of neural
responses to light from the in vitro, arterially perfused eyecups of cat,
rabbit and, in a limited number of experiments, macaque; definition of rod-
cone and cone-cone interactions through the use of action spectra, or intensity
response functions to matched monochromatic stimuli in the presence of neutral
or selective chromatic backgrounds; receptive field mapping; intracelluar
staining of retinal neurons with Procion and other dyes; histology and fluores-
cence microscopy; comparison with Golgi stained and electronmicroscopically
studied neurons in these retinas.

Major Findings:

ON and OFF center ganglion cells in the retina of the cat

Ganglion cells in the retina of the cat were stained by intracellular dye
injection after recording their responses to photic stimulation. All cells
encountered were divided into those giving ON responses and those producing
OFF responses, and the level of dendritic branching of these two groups was
compared. Cells giving OFF responses were found to branch high in the inner
plexiform layer, near the amacrine cell bodies (sublamina a^) ; while those
giving ON responses were found to branch lower in the inner plexiform layer
(sublamina b^) . The inner plexiform layer was found to contain a single stratum
of capillaries which conveniently divides sublamina a, from sublamina b^, so
that, of 21 stained ganglion cells there were 10 ON center units, all of which
branched below the capillary bed (in sublamina h) , in addition, there were 10
OFF center units, all of which branched above the capillary bed (in sublamina
a). One unit was physiologically ON-OFF in its response properties; the plane
of the capillary bed bisected the broad dendritic stratification of this unit.
Thus whether a ganglion cell is ON center or OFF center can be unambiguously
determined from the level of branching of its dendrites and the notion that
ON-center and OFF center ganglion cells branch„at random levels in the inner
plexiform layer must be rejected at the 1 in 2 th level (one in a million) .

Dye-injected cells have been compared to ganglion cells impregnated by
Golgi methods, and have been identified as belonging to one of three morpho-
logical classes based on cell size. Cells giving ON responses and cells giving
OFF responses were found in each of these three classes, and the sign of the
response only correlated with the level of dendritic branching. Thus no other
morphological feature of a cell except stratification appears to be iir.portant
in determining the sign of its center response.

The rod and cone inputs to some cells were characterized by comparing
their responses to deep red, and blue rod-matched stimuli over a two log unit
range starting at dark-adapted threshold. About ha].f the cells appeared to
be rod dominated under these conditions , whereas the other appeared to have
mixed rod and cone signals. The receptive fields of some cells were measured

166

Project No. ZOl EY 00005-05 LVR
by moving a long narrow slit stimulus incrementally across the surface of the
retina. Under these conditions receptive field centers could be either some-
what smaller or as much as six times larger than the dendritic field of the
cells. This finding suggests that the often-held assumption that dendritic
fields of ganglion cells ought to match receptive fields, or at least bear a
linear relation to the latter, must be viewed with some caution. The strati-
fication of ganglion cells into ON and OFF layers parallels the distribution
of the axonal terminals of the flat and invaginating cone bipolars. Flat cone
bipolars are in a position to contact OFF center ganglion cells (in sublamina
a) and invaginating cone bipolars are in a position of contact ON center gang-
lion cells (in sublamina b^) . In the electron microscope just such connections
have been observed between the ribbon synapses of the cone bipolar cells and
ganglion cell dendrites. Remarkably the OFF center ganglion cells, whose
dendrites must traverse the sublamina of the invaginating cone bipolar axon
terminals en route to sublamina a never make a single connection with their
terminals, restricting their connections in sublamina b^ to amacrine cells.

Chromatic interactions in the rabbit retina

The responses of neurons in the rabbit have been explored using intra-
cellular and extracellular electrodes and dye staining techniques. With few
exceptions previous studies of the rabbit retina have suggested that this
species has only two morphologically and functionally different types of recep-
tor: rods and 'green' cones. Electroretinographic studies in the rabbit's eye-
cup show that this retina contains, in addition to rods and green cones, blue-
sensitive cones. Spectral sensitivity measurements show that these receptors
absorb maximally at about 430 nm, 500 nm and 520 nm. Mass b-wave responses
have a spectral sensitivity suggestive of antagonistic interactions between
signals from blue and green cones. This has not been seen in action spectra
based on a-wave or PHI component responses suggesting spectral interactions
might exist at the level of inner nuclear layer cells, but possibly not at the
level of photoreceptors. Among the ganglion cells, a fraction shows color-
opponent responses. Two main types were found. In one, on-depolarizing
responses and on-hyperpolarizing responses receive input from both blue and
green cones (e.g. BG/G, BG/B). Two types of horizontal cells have been found,
both receiving mixed green cone and rod input; one type is cone-dominated
while the other is rod-dominated,, resembling similar cell types described in
the retina of the cat. Neither horizontal cell type shows an obvious blue
cone input, either depolarizing or hyperpolarizing, with intense selective
chromatic adaptation green cones and rods. The results suggest that although
input from blue cones contributes to spectral interactions at the ganglion
cells and, possibly, inner nuclear layer cells, it does not seem to contribute
to such interactions at the photoreceptor-horlzontal cell level. Assuming that
in the rabbit there is a negative feedback between receptors and horizontal
cells, the above results suggest that the feedback between each cone type and
the corresponding horizontal cell is either private (the spectral sensitivity
of the latter would depend only on the absorption of the corresponding cone
type) or spectrally asymmetric (green cones would feedback onto blue ones, but
blue cones would not feedback onto green cones) .

167

Project No. ZOl EY 00005-05 LVR
Spectral opponency in the cat electroretinogram

Spectral opponency and asymmetry between cone mechanisms have been more
thoroughly studied in the cat electroretinogram. In the d.c.-ERG of the iso-
lated perfused cat eye two cone mechanisms (Ainax=450 and 555 nm) can be iden-
tified by their action spectra obtained by constant response criteria with
monochromatic Ganzfeld-stimuli in the presence of intense chromatic adaptation
(12 experiments) .

At the onset of the light stimulus and in the presence of strong yellow
adaptation both cone mechanisms appear to sum their contributions to the
negative on-response (P III) producing a broad, flat, single-peaked action
spectrum, fitting a summation of 450 and 555 nm Dartnall-nomograms when
corrected for lens absorption. Under the same conditions they appear to
oppose each other in their contribution to the positive on-response (b-wave)
producing a double peaked action spectrum (peaks near 450 and 555 nm) with a
large sensitivity loss near 500 nm, fitting a subtraction of both nanograms.
This double-peaked function cannot be due to interaction between a 555 nm cone
mechanism and rods because the threshold sensitivity of the short wavelength
branch remains unaltered over a 16-fold increase in yellow adaptation whereas
that of the long wavelength branch follows the Weber-Fechner law.

When isolated, the sigmoidal intensity-response function for the 450nm-
mechanism has a lower slope and saturates at several V maximum amplitude; that
of the 555nm-mechanism is steeper and cannot be saturated with our maximum
intensity. The intensity response function for the former could be made to
approximate the latter by multiplying it by a factor of at least 10, possibly
the ratio of 555 to 430nm cones.

At the offset of the light, both cone mechanisms generate a slow negative
response but only the 555nm-mechanism produces a quick positive response also.
The action spectrum of the negative off-response resembles that of the negative
on-response (P III) , whereas the action spectrum of the positive off-response
shows no participation of the 450nm-mechanism at all. We interpret this on-
off asjTmmetry to mean that the positive off-response is generated by a quick
return of the 555nm cone receptor potential, not present in the 450nm cones.

We conclude that there are at least two cone types in cat retina: One
more numerous, more long wavelength sensitive and more rapid in its response;
the other less numerous, more short wavelength sensitive and slower in its
time course resembling in some respect rods. At a point beyond the receptor
level (P III) but before (or at) the site of b-wave generation, opponency
between these two cone mechanisms seems to occur.

Cyclic nucleotides, calcium, and the cat electroretinogram

We have examined whether putative changes in the concentration of cyclic
guanosine monophosphate (cGMP) affect the rod a-wave response, recorded d.c.
from arterially perfused cat eye-cups. Isobutylmethylxanthine (IBMX) , an
inhibitor of cGMP phosphodiesterase, delays the implicit time, prolongs the
duration and increases the amplitude but not the sensitivity of the isolated

168

Project No. ZOl EY 00005-05 LVR
a-wave response. The effect is reversible and repeatable over two days.
Cyclic GMP (dibutyril) was found to produce a somewhat similar but extremely
weak effect, which develops slowly and only at relatively high concentrations
(5 mM) , although this is perhaps due to poor penetration. Low Ca solutions,
EGTA buffered, mimic the effects of IBMX, although they seem to produce less
changes in response time-course for comparable increases in amplitude. Pro-
longed exposure (e.g. four hour) to low Ca solutions were fotmd to lead to
depression of the response and, occasionally, has been accompanied by spontan-
eous electroretinographic oscillations. These effects have not yet been
observed with prolonged IBMX exposures. The results suggest that cGMP may
participate in the generation of electrical signals in the retina of the cat ,
having effects similar to but perhaps not identical with those of low Ca
perfusates.

Significance to Biomedical Research and the Program of the Institute:
In diagnosing and treating the diseases of the eye it may prove useful to know,
on a detailed cell-by-cell basis, how the retina works, especially since many
disease states have their origins at the cellular level, and treatments have
as their targets particular classes of cells. A knowledge of normal retinal
function provides a necessary substrate for interpreting and treating retinal
dysfunction.

Proposed Course: This project will be continued.

NEI Research Program: Retinal and Choroidal Diseases - Visual Cells and
Pigment Epithelium/Retinal Organization and Visual Adaptation

Experimental Subject or Tissue Source: Cat /Rabbit /Monkey

Research Objective: Etiology

Publications:

Famiglietti, E.V. Jr., and Kolb, H.: Structural basis for OW- and OFF-
center responses in retinal ganglion cells. Science 194: 193-195, 1976.

Gouras, P.: Symposium on retinal circuitry (Preface). Invest. Ophthalmol.
15: 877-880, 1976.

Nelson, R. , Kolb, H., Famiglietti, E.V. , and Gouras, P.: Neural responses
in the rod and cone systems of the cat retina: Intracellular records and
Procion stains. Invest. Ophthalmol. 15: 946-153, 1976.

Nelson, R. : Cat cones have rod input: A comparison of the response
properties of cones and horizontal cell bodies in the retina of the cat .
J. Comp. Neurol. 172: 109-136, 1977.

Kolb, H., Famiglietti, E.V., and Nelson, R. : Neural connections in the
inner plexiform layer of the cat's retina. In Yamada, E., and Mlshlma, S.
(eds.): Structure of the Eye, Japan J. Ophthalmol., 1976.

169

Project No. ZOl EY 00005-05 LVR
Nelson, R., Famiglietti, E.V. Jr., and Kolb, H.: ON and OFF center
ganglion cells in the retina of the cat: Intracellular staining.
J. Neurophysiol. (in press) .

Zrenner, E., and Gouras, P.: Spectral opponency and asymmetry between
cone mechanisms in the cat electroretinogram (ERG) . Society for
Neuroscience Abstracts, 1977.

de Monasterio, F.M., and Gouras, P.: Spectral interactions in cells of
perfused rabbit retina. ARVO Abstracts p. 62, suppl. to Invest .
Ophthalmol., 16, No. 4, 1977.

Gouras, P., and de Monasterio, F.M. : Isobutylmethylxanthine, cyclic
guanosine monophosphate, calcium and the electroretinogram of the
perfused cat eye. ARVO Abstracts, p. 9, suppl. to Invest . Ophthalmol . ,
16, No. 4, 1977.

170

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
MOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00148-04 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Cyclic Nucleotides and Vision

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI:

Other:

Gerald J. Chader

Ph.D.

R. Theodore Fletcher M.S.

Head, Section on
Retinal and Corneal
Metabolism
Chemist

LVR NEI

COOPERATING UNITS (if any)

1) Gopal Krishna, Ph.D.; Laboratory of Chem. Pharmacology, NHLBI

2) Gustavo Aquirre, D.V.M. , Dept. of Ophthalmology, Univ. Penn. Vet School
Phila.. Pa.

lab/ BRANCH

Laboratory of Vision Research

SECTION

Section on Retinal and Corneal Metabolism

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

1.8

PROFESSIONAL:

0.8

OTHER:

1.0

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS n (a2) INTERVIEWS

□ (b) HUMAN TISSUES

a (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

The enzymes of cyclic nucleotide metabolism are different in retina
from those in other tissues and are active early In embryogenesis. High
cyclic GMP levels are present in retinas of dogs with retinal degeneration
indicating the possible involvement of cyclic GMP in the etiology of the
degenerative process.

171

OUQ—£J\An

Project No. ZOl EY 00148-04 LVR
Project Description:

Objectives; To study the role of cyclic nucleotides in normal vision
and their possible role in retinal disease.

Methods Employed; Photoreceptors of the retina are isolated by sucrose
density gradient centrif ugation and the activities of guanylate cyclase,
phosphodiesterase and protein kinase are determined by standard biochemical
methods. Cyclic nucleotide concentrations are measured by immunochemical
titration after initial purification by column chromatography.

Major Findings; Cyclic nucleotides (e.g. cyclic GMP) and nucleoside -
triphosphates (e.g. GTP) appear to be involved in the visual process. We have
continued our investigation of 1) the phosphorylation of opsin by GTP 2) the
formation of cyclic GMP from GTP through guanylate cyclase activity 3) the
normal patterns of embryonic development of cyclic nucleotides and their
enzymes of metabolism. In brief, we have found that the rod outer segment
kinase enzyme which facilitates the phosphorylation of opsin by GTP exhibits
specific requirements of cation concentration, pH, etc. for maximal expression
of enzyme activity which are different from those in other tissues. Guanylate
cyclase also appears to be different in rod outer segments than in other
tissues and exhibits unique enzymatic characteristics. Cyclic GMP and cyclic
AMP have different developmental patterns in retina and pigment epithelium
which probably indicate different roles for the nucleotides in the tissues
during embryogenesis.

In collaboration with Dr. G. Aquirre we have begun a study of the
possible involvement of cyclic nucleotides in retinal degeneration in the
Irish Setter. We have found extremely high cyclic GMP concentrations in the
retinas of affected mature dogs while levels in control dogs (i.e. genetic
carriers of the disease) are considerably lower. This may indicate a
derangement in cyclic nucleotide metabolism in the disease.

Significance to Biomedical Research and the Program of the Institute;
Study of the enzymes of cyclic nucleotide synthesis sind degradation gives
a better understanding of how the normal retina functions and could uncover
the cause of at least one form of retinal degeneration.

Proposed Course; We will continue to study the possible role of cyclic
GMP in both normal and abnormal retinal tissues with particular emphasis on
metabolic changes in embryonic and early postnatal development.

NEI Research Program; Retinal and Choroidal Diseases - Developmental
and Hereditary Disorders.

Experimental Subject or Tissue Source: Frog /Cow/Dog /Chick

Research Objective; Etiology

172

Project No. ZOl EY 00148-04 LVR
Publications:

Krishna, G. , Krlshnan, N. , Fletcher, R. , and Chader, G. : Effects of
light on cyclic GMP metabolism in retinal photoreceptors. J.
Neurochem. 27; 717-722, 1976.

Chader, G., Fletcher, R., O'Brien, P., and Krishna, G. : Differential
phosphorylation by GTP and ATP in isolated rod outer segments of the
retina. Biochemistry 15: 1615-1620, 1976.

Fletcher, R. : Cyclic nucleotides in the developing chicle retina: Master
of Science Thesis, American University, December, 1976.

173

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PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00134-04 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Development and Function of Retina, Pigment Epithelium, and Cornea

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI:

Other :

Gerald J. Chader Ph.D.

Barbara Wiggert Ph.D.

Eileen Masterson Ph.D.

R.T. Fletcher M.S.

Julia Derr B.S.

Head, Section on

Retinal and Corneal

Metabolism

Staff Fellow

Postdoctoral Fellow

Chemist

Biologist

LVR NEI

LVR NEI
LVR NEI
LVR NEI
LVR NEI

COOPERATING UNITS (if any)

Donald R. Bergsma, M.D., Staff Ophthalmologist, NEIrCB, Ralph Helmsen, Ph.D.,
Research Chemist, NEIrLVR; Marc Lewis, Ph.D., Research Chemist, NEIrLVR; Paul
J. O'Brien, Ph.D., Research Chemist, NEI:LVR; Arnold Goldman, Ph.D. Staff Fellt)w

HiTSBSSRVR

Laboratory of Vision Research

SECTION

Section on Retinal and Corneal Metabolism

INSTITUTE AND LOCATION

National Eye Institute,

NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

3.8

PROFESSIONAL:

3.5

OTHER:

0.3

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

n (al) MINORS n (a2) INTERVIEWS

B^b) HUMAN TISSUES

n (c) NEITHER

SUNNARY OF WORK (200 words or less - underline keywords)

1) Vitamin A receptors appear to be important in normal retina, pigment
epithelium and cornea 2) A tissue culture system for studying outer segment
phagocytosis has been established 3) The importance of the pentose shunt in
early corneal development has been established.

175

OLIO £.f\Ae\

Project No. ZOl EY 00134-04 LVR
Project Description;

Objectives; To study factors which affect the normal development and
functioning of the retlna-plgment epithelium unit and of the cornea.

Methods Employed; Vitamin A receptors In tissue extracts were determined
by sucrose density gradient ultracentrlfugation, gel filtration, spectroscopy
and electrophoresis. Tissue culture techniques of cell dissociation, cloning
and incubation were used in the phagocytosis experiments. Corneal studies
were performed using standard methods for measuring glucose metabolism.

Major Findings: 1) Specific soluble receptors for vitamin A have been
found in retina, pigment epithelium, and cornea. Two different receptors for
retlnol are present in retinas of most species, one of these is not seen in
the retina of a patient with retinitis pigmentosa and during early fetal
development in the cow indicating that this receptor species is predominantly
associated with photoreceptor outer segments in vivo. These receptors may
be involved in the normal development and/or vitamin A transport in ocular
tissues.

2) In tissue culture, pigment epithelial cells from the chick embryo can
phagocytize Isolated bovine rod outer segments . We hope to use this as a
model system for studying phagocytosis of outer segments under culture
conditions. Several parameters affecting outer segments and pigment epithelial
cells are currently under investigation in hopes of pinpointing the signals

or factors controlling phagocytosis in vivo.

3) Factors affecting the onset of corneal transparency are under inves-
tigation in the chick embryo cornea. It has been found that pentose shunt
activity appears concurrently with the process of detergescence. Highest
shunt values are found after complete transparency is achieved. Dlamlde
preferentially increases oxidation of glucose in the C-1 position indicating
an elevation of pentose shunt activity with this agent. Thus, the enzymes
for pentose shunt activity are present early In corneal development and are
probably necessary in maintaining reduced glutathione needed for corneal
transparency .

Significance to Biomedical Research and the Program of the Institute;
Retinal dystrophy and keratomalacia are diseases of the retina and cornea
respectively in which vitamin A appears to be Involved. It is hoped that
the study of factors which affect the development of the tissues will indi-
cate areas of biochemical research which will ultimately lead to prevention
of the diseases.

Proposed Course; Factors both external (e.g. hormones, vitamins) or
internal (e.g. tissue receptors) which affect the development and function of
ocular tissues will continue to be Investigated.

NEI Research Program; Retinal and Choroidal Diseases - Developmental
and Hereditary Disorders

176

Project No. ZOl EY 00134-04 LVR
Experimental Subject or Tissue Source; Cow/Rat/Monkey/Plg/Chlcken/Human

Research Objective; Etiology

Publications;

Redfem, N., Israel, P., Bergsma, D,, Roblson, W. , Whlkehart, D., and
Chader, G. : Neural retinal and pigment epithelial cells in culture;
Patterns of differentiation and effects of prostaglandins and cyclic
AMP on pigmentation. Exp. Eye Res. 22; 559-568, 1976.

Wiggert, B., Bergsma, D., and Chader, G. ; Retinol receptors of the
retina and pigment epithelium. Exp. Eye Res. 22; 411-418, 1976.

Bergsma, D., Wiggert, B., Funahashl, M. , Kuwabara, T., and Chader, G. ;
Vitamin A receptors in normal and dystrophic huaan retina. Hature
265: 66-67, 1977.

Helmsen, R. , Wiggert, B., and Chader, G. ; A possible receptor for
retinol in corneal epithelium. Exp. Eye Res. 24; 213-214, 1977.

Wiggert, B., Bergsma, D., Helmsen, R. , Alllgood, J., Lewis, M., and
Chader, G. : Retinol receptors in corneal epltheliini, stroaa and
endothelium. Blochim. Blophys. Acta 491; 104-113, 1977.

Abe, T., Wiggert, B., Bergsma, D., and Chader, G. ; Vitaaln A receptors:
Comparison of retinol binding to serum retinol-bindlng protKln and to
tissue receptors in chick retina and pigment epltheliioi. Biochia.
Blophys. Acta (in press) .

Wiggert, B., Bergsma, D., Lewis, M. , Abe, T., and Chader, G. : Vitamin
A receptors : Characteristics of retinol binding in chick retina and
pigment epithelium. Biochlm. Blophys. Acta (in press) .

Wiggert, B., Bergsma, D., Lewis, M. , and Chader, G. : Vitamin A
receptors: Retinol binding in neural retina and^lgment eplthellim.
J. Neurochemistry (in press) .

177

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PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAIIUR/U. RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00016-10 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)
Protein Synthesis in the Retina

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Paul J. O'Brien

Ph.D.

Research Chemist

LVR NEI

COOPERATING UNITS (if any)

None

LAB/BRANCH

Laboratory of Vision Research

SECTION

Section on Retinal and Corneal Metabolism

INSTITUTE AND LOCATION

National Eye Institute. NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

1.0

PROFESSIONAL:

0.3

OTHER:

0.7

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

n (al) MINORS n (a2) INTERVIEWS

n (b) HUMAN TISSUES

SUMMARY OF WORK (200 words or less - underline keywords)

A small portion of the rhodopsin in bovine rod outer segments can be
labeled with galactose and fucose in vitro. These sugars are probably added
in the outer segment and may play a role in phagocytosis. Various precursors
of rhodopsin can be incorporated by both bovine and canine retinas thereby
permitting examination of possible defects in dystrophic animals.

179

PHS-60A0

Project No. ZOl EY 00016-10 LVR
Project Description:

Objectives: The renewal of photoreceptor cell outer segments is a
continuous process which is impaired in some pathological conditions such as
progressive degeneration or developmental anomalies of the retina. The
purpose of this project is the elucidation of the biochemical events involved
in renewal, especially the synthesis of protein in the retinas of both cow
and dog.

Methods Employed: Ordinary biochemical techniques were used, such as
incubation of retinas, cell fractionation, isolation of rod outer segments
by density gradient centrifugation, detergent extraction and purification
of rhodopsin by column chromatography and gel electrophoresis.

Major Findings: Protein synthesis with dog retinas in vitro gave gel
electrophoresis labeling patterns similar to those seen with bovine retina,
thereby establishing the utility of the methods for future work with dog
retinas. Puromycin, an inhibitor of protein synthesis, produced different
inhibition patterns in bovine retina depending on the radioactive precursor
used. The degree of inhibition reflected the sequence of addition of sugar
residues to rhodopsin. Both fucose and galactose were unaffected by puro-
mycin and appear to be added in the outer segments, probably to a small
portion of the rhodopsin.

Significance to Biomedical Research and the Program of the Institute:
The ability to measure protein synthesis in normal dog retinas will permit
similar measurements in dogs with inherited retinal degenerations in an
attempt to detect specific lesions. The presence of galactose and fucose
on a small fraction of the outer segment rhodopsin may provide a marker to
facilitate phagocytosis of shed disc membranes. Defects in the addition
of these sugars could account for degenerative processes. Animal models,
such as dogs, provide the means of testing this hypothesis.

Proposed Course: The incorporation of various precursors of rhodopsin,
especially galactose and fucose, will be studied in several animal models of
retinal degeneration to determine whether specific abnormalities can be
identified.

NEI Research Program; Retinal and Choroidal Diseases - Developmental
and Hereditary Disorders /Visual Cells and Pigment Epithelium

Experimental Subject or Tissue Source: Bovine /Canine

Research Objective: Etiology

Publications:

O'Brien, P.J. : Differential effects of puromycin on the incorporation
of precursors of rhodopsin in bovine retina. Biochemistry 16: 953-958,
1977.

180

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)

U.S. DEPARTMENT Of
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
NOTICE OF
INTRAMURAL RESEARCH PROJECT

PROJECT NUMBER

ZOl EY 00015-12 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Synthesis of Sugar-Containing Polymers in Retina

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: Paul J. O'Brien

Ph.D.

Research Chemist

LVR NEI

COOPERATING UNITS (if any)

None

LAB/BRANCH

Laboratory of Vision Research

SECTION

Section on Retinal and Corneal Metabolism

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Md. 20014

TOTAL MANYEARS:

0.8

PROFESSIONAL:

0.5

OTHER:

0.3

CHECK APPROPRIATE BOX(ES)
D (a) HUMAN SUBJECTS

D (al) MINORS D (a2) INTERVIEWS

n (b) HUMAN TISSUES

[J(c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

Both galactose and fucose are incorporated into rhodopsin by isolated
rod outer segment preparations. These sugars might provide specific markers
to permit the pinching off of new disc membranes or the shedding of old discs
with resultant phagocytosis by the pigment epithelium during the course of
photoreceptor outer segment renewal. Retinyl phosphate, possibly provided
by the pigment epithelium, may act as a carrier of galactose.

181

Project No. ZOl EY 00015-12 LVR
Project Description:

Objectives: Many interactions between macromolecules and cell membranes
are mediated by the sugar molecules bound to one of the interacting surfaces.
In the process of renewal of photoreceptor outer segment disc membranes,
rhodopsin, a glycoprotein, must be transported from the inner segment and
incorporated into disc membranes with a specific orientation in space. This
project was designed to determine where and when sugars are added to the
polypeptide and what role they play in the transport and assembly of rhodopsin
into disc membranes and in the process of shedding and phagocytosis of disc
membranes .

Methods Employed : Ordinary biochemical techniques were used, such as
cell- fractionation, isolation of rod outer segments by density gradient
centrifugation, detergent extraction and purification of rhodopsin by column
chromatography, and incubation of outer segment preparations.

Major Findings: Isolated photoreceptor outer segment preparations cata-
lyze the transfer of galactose and fucose from the appropriate sugar nucleo-
tides to rhodopsin. The transfer of galactose, but not of fucose, appears to
be mediated by a lipid carrier, possibly retinyl phosphate. The evidence
suggests that the pigment epithelium supplies an essential factor for the
transfer reactions.

Significance to Biomedical Research and the Program of the Institute:
The addition of galactose and fucose to rhodopsin might provide a marker to
permit the pigment epithelium to distinguish shed photoreceptor tips from
intact outer segments. This process appears to be under the control of
the pigment epithelium, perhaps by providing retinyl phosphate. Certain
animal models of retinal degeneration manifest a defect in the pigment
epithelium. This mechanism could be defective either in animal models or in
human retinal degenerations.

Proposed Course: Attempts will be made to demonstrate the synthesis of
retinyl phosphate and retinyl phosphate galactose in the pigment epithelium
or photoreceptors. If successful, retinyl phosphate galactose will be used
as a donor of galactose' residues .tp rhodopsin. These pathways will be
assayed in animals with retinal degenerations.

NEI Research Program: Retinal and Choroidal Diseases - Developmental and
Hereditary Disorders/Visual Cells and Pigment Epithelium

Experimental Subject or Tissue Source; Bovine

Research Objective: Etiology

182

Publications:

Project No. ZOl EY 00005-12 LVR

O'Brien, P.J. :
bovine retina.

Incorporation of mannose into rhodopsin in isolated
Exp. Eye Res. 24: 449-458, 1977.

Bok, D., Hall, M.O., and O'Brien, P.: The biosynthesis of rhodopsin as
studied by membrane renewal in rod outer segments. In Brinkley, B.R. and
Porter, K.R. (eds.): International Cell Biology, 1976-1977. New York,
Rockefeller University Press (in press) .

X83

SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Oo HOT use this space)

u.s. department of
health, education, and welfare
public health service
notTce of
intramural research project

PROJECT NUMBER

ZOl EY 00024-03 LVR

PERIOD COVERED

July 1, 1976 to September 30, 1977

TITLE OF PROJECT (80 characters or less)

Intermediary Metabolism of the Cornea

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AND ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT

PI: David R. Whikehart Ph.D.
Other: Henry F. Edelhauser Ph.D.

R. Theodore Fletcher M.S.

Senior Staff Fellow LVR NEI
Professor, Medical College
of Wisconsin, Milwaukee
Chemist LVR NEI

COOPERATING UNITS (if any)

Dept. of Physiology, Medical College of Wisconsin, Milwaukee

lab/branch

Laboratory of Vision Research

SECTION

Section on Retinal and Corneal Metabolism

INSTITUTE AND LOCATION

National Eye Institute, NIH, Bethesda, Maryland 20014

TOTAL MANYEARS:

1.5

PROFESSIONAL:

1.3

OTHER:

0.2

CHECK APPROPRIATE BOX(ES)
n (a) HUMAN SUBJECTS

D (al) MINORS □ (a2) INTERVIEWS

(b) HUMAN TISSUES

n (c) NEITHER

SUMMARY OF WORK (200 words or less - underline keywords)

This investigation concerns itself with the biochemical mechanisms that
control corneal hydration (deturgescence) . The influence of bicarbonate ,
glucose, glutathione and adenosine on intracellular levels of reduced and
oxidized glutathione in the corneal endothelium along with swelling rates
have been investigated via perfusion. All of these compounds have been shown
to be effective, in varying degrees, in preventing swelling. In combination
they appear to actually promote deswelling. Endothelial cells grown in
culture are being assayed for levels of glutathione and cyclic nucleotides.
Levels of glutathione peroxidase have been assayed and show higher activity
levels in the corneal endotheliimi than in the corneal epithelium.

185

Project No. ZOl EY 00024-03 LVR
Project Description;

Objectives ; This project has been initiated to elucidate the biochemistry
of corneal deturgescence and to suggest mechanisms for its control. Its
immediate objectives are: 1) to determine the location, accessibility and
reactivity of specific areas of the plasma membrane of the corneal endothelium
to cytoplasmic, sulfhydryl/disulf ide metabolites, 2) to measure intracellular
levels of adenosine, 3) to establish better conditions for tissue culture
and/or eye bank storage by observing what metabolites or activators produce
optimal ATPase activity

Methods Employed: Major sources of tissue have been rabbit and bovine
eyea^ Human tissue has also been studied on a limited basis. Extremely
sensitive and accurate techniques of assay employing double-beam spectro-
photometry and gas liquid chromatography have been employed. These procedures
assay intermediates at the nanogram level. Tissue cultures of endothelial
cells have been initiated from micro-dissected endothelial/Descemet 's layer
buttons from rabbits. Perfusions of rabbit corneas have been accomplished
with the aid of the specular microscope to measure corneal swelling rates.
Glutathione peroxidase was assyed by an indirect kinetic method employing
glutathione reductase. Cyclic nucleotides have been assayed by radioimmuno-
assay.

Major Findings:

Perfusion of rabbit corneas

Certain metabolites have been implicated in the activation of the sodium
pump: glutathione, adenosine and bicarbonate. However, the exact effect of
these substances on ATPase has not been established in the corneal endothelium.
Investigation of endogenous levels of reduced and oxidized glutathione in
endothelial tissue of rabbit cornea showed remarkable differences when per-
fused by different compounds. Endothelial cells had a lower amount of total
glutathione with bicarbonate or lactate Ringer's media compared to fresh,
unperfused controls. Adenosine, reduced and oxidized glutathione caused
normal to elevated levels of total glutathione, under which conditions the
cornea remained stable (i.e. did not swell) for up to 5 hrs. High levels of
oxidized glutathione (30-70% of the total) were found in all perfusion experi-
ments and in the control. The discovery of this high level of oxidized
glutathione implies that the hexose monophosphate shunt (highly active in
corneal epithelial tissue) may not be very active in corneal endothelium.
It points to the existence of a pool of oxidized glutathione in that tissue.
The results of this study have been submitted to Investigative Ophthalmology
and Visual Science for publication.

Glutathione peroxidase activity

Concomitantly, it has been demonstrated that the activity levels of
glutathione reductase in bovine and rabbit corneal Qndothelia are 8-20 times
lower than the corresponding levels in corneal epithelia. I have shown
that the converse is true for glutathione peroxidase activity in bovine

186

Project No. ZOl EY 00024-03 LVR
corneal tissues (I.e. endothelium > epithelium) . The implication of these
studies is that the pentose shunt is comparatively low in activity in the
endothelium allowing relatively high amounts of oxidized glutathione to
exist in the cells. Since I have already been able to show that the ratio of
oxidized to reduced glutathione in fresh bovine, rabbit and human corneal
endothelial tissues is high compared to most other tissues including corneal
epithelium, this completes the picture of a redox state of glutathione in the
corneal endothelium which highly favors oxidation. A manuscript is in prepar-
ation.

Cyclic nucleotide levels

This work is still in progress. Both the levels in fresh tissue (corneal
epithelium and endothelium) and in tissue cultures of the corneal endothelial
cells of the rabbit are being examined for cyclic GMP and cyclic AMP. To date,
substantial levels of cyclic GMP have been found in both fresh tissues and in
tissue cultures.

Significance to Biomedical Research and the Program of the Institute:
Corneal disease involving disturbances in the proper hydration (deturgescence)
of the cornea (resulting in cloudy, impaired vision) are thought to be the
result of metabolic dysfunction (degeneration) . Such dysf tmctions are the
result of damage from transplants, storage conditions (cornea banks), inflam-
matory reactions and inherent dystrophies. Presently, however, the normal
metabolic functions associated with the hydration pump(s) and its controls
have not been adequately described. Since glutathione is known to strongly
promote deturgescence, its endogenous level is quite important. The high level
of oxidized glutathione, found in the corneal endothelium, implies the
existence of a metabolic scheme quite modified from that of corneal epithelium
and of a number of other cellular tissues. It has only recently been discov-
ered that the oxidized form is equally as effective as the reduced form in
stimulating the pump(s), but it is not understood how this takes place. The
possibility that cyclic nucleotides may also play a role in controlling the
activity of the deturgescent pump is of equal potential importance to the
discovery of the role of glutathione.

Proposed Course; The study of the deturgescent mechanism in the corneal
endothelium, in view of its Investigative history, ought to be to elucidate
ATPase activity and to describe the role of substances known or suspected to
maintain ATPase at a fimctional level of activity. One question to address to
this study is, therefore, "what is the role of sulfhydryl and/or disulfide
groups in relation to ATPase activity in the corneal endothelitmi in promoting
deturgescence?" Solving these questions and determining the role of SH/SS
will require the location of SH groups on the endothelial cell plasma membrane.
Membrane sulfhydryls will be located by the utilization of impermeant aelei-
mides and N-ethyl malelmide.

Adenosine's influence must also be described. The determination of the
endothelial cell content of adenosine is already in progress in my laboratory
using adenosine deaminase for the assay. Its effects will be more directly
studied when endothelial (Na + K ) -ATPase is prepared from the plasma mem-
brane .

187

Project No. ZOl EY 00024-03 LVR
Tissue cultures of corneal endothelial cells will be studied to determine
how the cell's levels of activity of (Na + K )-ATPase are effected by various
metabolites such as glutathione, adenosine and cyclic nucleotides. Assay work
for this study is already in progress.

NEI Research Program; Corneal Diseases - Corneal Edema, Dystrophies,
and Inherited Disorders

Experimental Subject or Tissue Source; Bovine/Rabbit/Hviman

Research Objectives; Etiology, Treatment

Publications:

Redfem, N. , Israel, P., Bergsma, D., Robison, Jr., W.G., Whikehart, D.,
and Chader, G. ; Neural retinal and pigment epithelial cells in culture;
Patterns of differentiation and effects of prostaglandins sind cyclic-AMP
on pigmentation. Exp. Eye Res. 22; 559-568, 1976.

Whikehart, D.R., and Hess, H.H. : Properties of liposomes with a phospho-
lipid ratio similar to that of retinal rod outer segment membranes;
Interaction with opsin and other proteins. Exp. Eye Res. 24; 279-289,
1977.

188

fYPi; FULL •■u. r;i .;, , . .,i;.,,:, ;_; .
MAY nii VLOLA.'i:.,)

OFFICE OF BIOMETRY AND EPIDEMIOLOGY

189

ANNUAL REPORT

NATIONAL EYE INSTITUTE

July 1, 1976 - September 30, 1977

REPORT OF THE CHIEF, OFFICE OF BIOMETRY AND EPIDEMIOLOGY

Fred Ederer

The Office of Biometry and Epidemiology was strengthened considerably,
both scientifically and administratively, during the past year when
Dr. Daniel Seigel, who has had a distinguished career as a bio statistician,
joined the staff as Deputy Chief. Dr. Seigel, whose most recent position
had been Director, Epidemiology and Biometry Research Program, National
Institute of Child Health and Human Development, was also appointed as
Head, Section on Clinical Trials and Natural History Studies, replacing
Fred Ederer in that capacity. With Dr. Seigel 's appointment, Mr. Ederer
relinquished his position as Head, Section on Clinical Trials and Natural
History Studies, and was able to increase attention to his role as Acting
Head of the Epidemiology Section. The main functions of the Office are:

1. To develop and carry out studies of human populations directed
toward causation, prevention, and treatment of eye disease and vision
disorders, with emphasis on the major causes of blindness. This includes
studies of incidence and prevalence in defined populations, prospective
and retrospective (case-control) studies of risk factors, natural history
studies, clinical trials, genetic studies, and studies to evaluate diag-
nostic procedures. The incidence and prevalence studies, apart from pro-
viding epidemiologic research leads, make information available for
assessing the magnitude of the national eye disease problem.

2. To provide biometric and epidemiologic assistance to National
Eye Institute intramural research scientists and, to the extent feasible,

•to vision researchers elsewhere.

3. To disseminate Information about biometric and epidemiologic
methods to those in vision research.

A large part of the Office's effort has been devoted to providing
scientific support to clinical trials and other epidemiologic studies con-
ducted under NEI research contracts, mainly the Diabetic Retinopathy Study,
the Diabetic Retinopathy Vitrectomy Study, the Diabetic Retinopathy Study II,
and the Framingham Eye Study. Major research papers were published on the
Framingham Eye Study, and a final report was submitted on the
Biostatistical Analysis of the Collaborative Glaucoma Study contract.
In addition, professional staff of the Office developed further studies
based on existing epidemiological data from the Model Reporting Area on
Blindness and the Framingham Eye Study. From these latter efforts, a
paper was published showing a positive association between the prevalence
of senile cataract and residence in areas of high sunshine.

191

A specific function of the Office of the Chief is to disseminate
information about biometric and epidemiological methods to those engaged
in vision research. In recent years activities in this area have included
the conduct of a workshop on the randomized clinical trial before the
Association of University Professors of Ophthalmology and the publication
of methodological papers in ophthalmic and optometric journals. During
the past year, the Office organized and conducted a course on methods of
clinical research at the 1976 meeting of the American Academy of
Ophthalmology and Otolaryngology. Another such course is planned for the
1977 meeting.

For the past several years the Office has succeeded in recruiting
clinical associates with an interest in clinicoepidemiologic research to
participate in its programs. These clinical associates, who usually spend
two years with the OBE, are either recent graduates of ophthalmology resi-
dency programs or physicians planning to enter such programs and, if the
latter, they are provided during their tenure with some ophthalmology
training. The clinical associates provide an essential ophthalmologic
complement to the Office's biometric and epidemiologic expertise. Moreover,
as these people advance in their academic careers, they are able not only
to apply their biometric and epidaniologic knowledge to their own work,
but also to disseminate it to their colleagues in vision research.

As a member of a subcommittee of the NIH Clinical Trials Committee,
Mr. Ederer participated in the planning of the NIH Conference on Clinical
Trials Methodology scheduled for October 1977.

Mr. Ederer represented the National Eye Institute in presenting papers
at a national meeting of the American Association of Workers for the Blind
and the Conference on Impaired Vision in Childhood in Jerusalem. He also
presented papers at meetings of the American Statistical Association and
the International Epidemiological Association.

192

Epidemiology Section
Office of Biometry and Epidemiology

Mr. Ederer continued to serve as Acting Head of this Section. The func-
tions of the Section are to develop and conduct a program of epidemiological
research in eye disease, with particular emphasis on diseases which are lead-
ing causes of blindness and visual impairment in the United States, and to
provide consultation on epidemiological problems to others in vision research.
The epidemiological studies emphasize investigations to uncover clues about
etiology and pathogenesis, such as prevalence surveys, case control studies,
population genetic studies, and studies directed at improving diagnostic
methods.

A study of the relationship of sunlight and cataract was published.
Cataract prevalence data from two large U.S. sources were divided according
to small geographic areas for which average annual sunlight hours were deter-
mined from a map prepared by the U.S. Weather Bureau. Several noncataract
disease controls were chosen from the same geographic locations. It was
found that the cataract-to-control ratios for persons aged 65 years or older
were significantly larger in locations with large amounts of sunlight compared
to those in locations with small amounts (p <.05). Discussion of some possible!
biases in the data led to the conclusion that the biases, if they exist, are
probably not large. The report suggests, however, that more research should be
done before the association between sunshine and cataract is considered es-
tablished.

Mrs. Hiller has continued to collaborate with Dr. Matthew D. Davis,
University of Wisconsin, in a study of mortality in diabetics with varying
degrees of severity of retinopathy. Survival patterns are also being studied
by age at diagnosis of diabetes, visual acuity, and duration of diabetes. A
first draft of the material, methods, and results sections of a manuscript has
been prepared.

A contract to analyze data collected in the Collaborative Glaucoma Study
was completed. Major risk factors for glaucoma visual field defects (GVFD)
were evaluated. Though subgroups could be identified whose risk of GVFD were
increased by as much as ten-fold, they represent only a small fraction of the
total population. The conclusion is that these risk factors do not afford
high predictability for such disorders. The contractor was encouraged to pub-
lish those findings now available.

Analysis of the Framingham Eye Study continued. Two major initial reports
were published. Grading of stereo fundus photographs for disc and macula
abnormalities (1,500 patients) and diabetic retinopathy (500 patients) will be
completed this year. A statistical monograph is being prepared under subcon-
tract by the Biostatistics Center, George Washington University.

An Advisory Group on Epidemiology comprised of personnel from the Clinical
Branch, Laboratory of Vision Research, and Office of Biometry and Epidemiology
was formed to advise the Acting Head, Epidemiology Section, on research activi-
ties in epidemiology. The group met twice to review the Section's activities

193

and to advise the Actdng Section Head on various research proposals Several
studies that were assigned a high priority by the Advisory Group are now being
carried out by various staff members of the Office of Biometry and Epidemiology.

The Health and Nutrition Examination Survey (HANES) , of the National Cen-
ter for Health Statistics (NCHS) , in which staff of the Office of Biometry and
Epidemiology participated in 1971-1972, has issued several monographs giving
information about the plan and operation of the survey and about monocular
visual acuity findings. Further HANES publications are being planned.

A number of visits were made by Mr. Ederer and his staff to the National
Center for Health Statistics to discuss plans for analyzing the data from
the Detailed Ophthalmologic Examination portion of the Health and Nutrition
Examination Survey. The NCHS has assured the OBE that a copy of the computer
■ tape including the eye data will soon be made available to the NEI for epi-
demiologic analyses.

194

Section on Clinical Trials and Natural History Studies
Office of Biometry and Epidemiology

The principal activities of this Section are the conduct of randomized
clinical trials on the treatment and prevention of eye disorders, the conduct
of nonrandomized studies on their natural history, and the provision of con-
sultation to colleagues in other parts of the Institute who are also active
in clinical trials research. Of these, the scientific management of clinical
trials, conducted by National Eye Institute project teams, was the dominant
activity during FY 1977. Project teams are led by a staff member of the
Office of Biometry and Epidemiology and include expertise in biostatistics,
epidemiology, ophthalmology, computer science, contract management, and public
information.

The Diabetic Retinopathy Study (DRS) , following publication of its first
report in 1976, has continued follow-up of its patients under a modified pro-
tocol allowing, but not requiring, treatment of eyes originally assigned to no
treatment if they meet certain high-risk criteria. Large numbers of patients
have been followed for three years, and the first fifth-year follow-up visit
has occurred. The DRS has concentrated its analyses on determining the effect
of photocoagulation on the development and progression of retinopathy, and is
now attempting to determine the role of photocoagulation in selected subgroups
of the patient population.

A monograph giving details of study design, methods, and baseline charac-
teristics of patients is being prepared for publication, as is a methodologic
paper on fundus photograph grading. The Study has entered an analytic phase
where emphasis will be placed on developing detailed descriptions of the natu-
ralhistory of diabetic retinopathy and elaborating on the effects of photoco-
agulation treatment. The DRS Project Team has been actively involved in these
activities.

The Diabetic Retinopathy Study Phase II (DRS II) is a multicenter random-
ized clinical trial designed to answer some of the major questions not address-
ed by the DRS and other studies. The important issues are optimum stage of
disease to initiate photocoagulation, the effectiveness of photocoagulation
on diabetic maculopathy, and whether aspirin administered at various stages of
diabetic retinopathy can prevent or retard the progression of this disease.
Funding of the study should begin in the fall of 1977, with initial year's
goal to be completion of the study design and preparation of a detailed manual
of operations. Recruitment of patients should begin by September of 1978.

The Diabetic Retinopathy Vitrectomy Study (DRVS) is a clinical trial
involving treatment of a more advanced stage of diabetic retinopathy in which
blindness due to hemorrhage into the vitreous has occurred. The surgical pro-
cedure being examined is vitrectomy using an instrument combining cutting,
suction, and infusion of a replacement solution. Eligible eyes are assigned
to either vitrectomy within the first six months of a vitreous hemorrhage or
to a "late" vitrectomy group in which vitrectomy is performed at one year
following hemorrhage in those eyes still suitable. In October 1976 recruit-
ment of patients was begun for those clinical centers meeting requirements for
certification of clinic personnel.

195

With improvements in the design of lenses for intraocular implantation,
the number of such implants has been increasing at an exponential rate. Many
feel that it is important at this time to evaluate carefully the efficacy of
such implants in patients undergoing cataract extraction and to assess the
risks as well.

As a first step toward evaluating intraocular lens implants, the National
Eye Institute will attempt to summarize the experience that ophthalmologists
have had in recent clinical practice. The records of several surgeons will be
analyzed to see whether they can provide answers to research questions that
are of interest. For findings from study of such records to be credible, it
will be necessary to demonstrate that data from all patients treated during
a defined interval are included, that follow-up was maintained on a large pro-
portion of cases, and that significant medical data have been carefully
recorded.

Demonstrating that these elements are present constitutes the work scope
of a contract which was awarded in the fall of 1977. If' in the first year of
the contract the feasibility of a high quality retrospective study is estab-
lished, the contract will be extended to analyze and report on the benefits
and risks of intraocular lens implants. Consideration is also being given to
more rigorous research designs, such as prospective controlled studies, perhaps
including randomization, but it is not yet clear that these are feasible.

A standardized visual acuity box to assure uniformity of visual acuity
measurement was developed by Dr. Ferris. Arrangements were made for its manu-
facture, and it has been distributed to clinics participating in ongoing clini-
cal trials.

The Section has accepted responsibility for reviewing clinical trials
protocols developed under NET research grants. This has turned out to be a
useful administrative mechanism by which the Institute helps to assure the
quality of grant-sponsored clinical trials and offers the additional benefit
of promoting communication on activities of shared interest between staff
within the Section and staff from extramural programs.

The Section has also been involved in cooperative arrangements involving
other governmental components. Dr. Lawrence Rand has served on an advisory
group to NIAMDD to determine the feasibility of developing a clinical trial
on the effect of metabolic control on the development of the vascular compli-
cation of diabetes mellitus. Dr. Seigel chairs the Advisory Committee on
Biostatistics and Epidemiology to the Bureau of Drugs for the FDA. He also
represents the NEI at the NIH Committee on Clinical Trials.

196

Biometry Section
Office of Biometry and Epidemiology

The Biometry Section, previously called the Section on Mathematical
Statistics and Computer Applications, has chosen its new name to reflect more
accurately the emphasis given to support and development of biostatistical
programs and analysis. Activities during this year continue to include con-
sulting and collaborating, both within and outside of NEI, in applied statis-
tics and epidemiologic investigations.

Dr. Roy Milton, together with Drs. Robert Frank, Barry Coller, and Harvey
Gralnick, received the Fight for Sight Citation "For Achievement in Clinical
Vision Research" at the 1977 annual meeting of the Association for Research in
Vision and Ophthalmology (ARVO) , for collaborative effort in their 1976 ARVO
paper, "Von Willebrand Factor and Effect on Platelet Aggregation of Plasma
from Diabetics with Retinopathy." This and subsequent work will appear in the
Annals of Internal Medicine.

Dr. Milton, with Dr. James Ganley, formerly Senior Staff Fellow in OBE
and now at the University of Arizona, Department of Ophthalmology, presented
a paper at the 1977 ARVO meeting, "Risk of glaucoma in myopia: a population
study." He also participated in a round table discussion on distribution of
statistical software at the Computer Science and Statistics Tenth Annual
Symposium on the Interface.

Dr. Milton continued to consult with Dr. Douglas Gaasterland, NEI Clinical
Branch, on studies of the parameters of aqueous humor dynamics, and is coauthor
of three papers submitted for publication.

The Biometry Section is assisting Dr. Arin Chatterjee, Christian Medical
College, Ludhiana, India, in a cataract etiology study. Data collection is
completed and analysis has begun. The Section continues to monitor the
Framingham Eye Study contract through activities of the project officer, coding
and protocol maintenance, and exploratory data analysis.

Dr. Karen Yuen completed two years with the Section under the Intergovern-
mental Personnel Act, during which time she engaged in intramural consulting
and research in statistical methodology. Dr. Yuen is now In the Department of
Mathematics, University of Windsor.

The Section's resources in statistical computing were enhanced by acquisi-
tion of a Tektronix 4051 graphics terminal with stand-alone computing abilities
and by hiring a programmer. These resources will improve the Section's ability
to provide support for increased activity in epidemiologic analysis, especially
of Framingham Eye Study data.

197

CONTRACT NARRATIVE

BOSTON UNIVERSITY (NIH-NEI-72-2112)

Title: Framingham Eye Study

Principal Investigator: Howard Leibowitz, M;D.

Current Fund Allocation: $672,826 for the period July 1, 1972 through
December 31, 1977.

Objectives: The aim of this epidemiologic investigation is to identify
individuals among the Framingham Heart Study cohort who at the present time
have a disease or condition related to one or more of the four most common
causes of adult blindness, i.e., senile cataract, senile macular degeneration,
chronic simple glaucoma, and diabetic retinopathy. In addition to determining
the prevalence of these diseases, past measurements from the Framingham
Heart Study will be related to present disease status in an effort to identify
risk factors.

Major Findings: An ocular examination according to a standard protocol
was made on the survivors of the original Framingham Heart Study cohort.
Patient examinations were completed in February 1975 with 2,675 individuals
examined. This includes 8A% of the cohort still residing in the Framingham
area. The first two major reports^' "* included findings of significant
association between senile cataract and increases in serum phospholipids,
casual blood sugar, blood pressure, and age. Senile macular degeneration
was found to be associated with increased blood pressure, ventricular
hypertrophy, history of lung infection, aging-related factors, and sex.
Prevalence in this population was 3% for open angle glaucoma and diabetic
retinopathy, 9% for senile macular degeneration, and 15% for cataract.
Other findings are reported in the publications. Grading of stereo fundus
photographs for disc and macular abnormalities (1,500 patients) and diabetic
retinopathy (500 patients) will be completed this year. A statistical mono-
graph is being prepared under subcontract to the Biostatistics Center,
George Washington University.

Significance to Biomedical Research and the Program of the Institute;
The four eye diseases under consideration are the leading causes of adult
blindness in this country today. It will be very helpful to identify factors
possibly associated with increased risk of these diseases, as a guide to
prevention. The Study has been designed with this objective in mind.
Prevalence data for this age group (52-85) in this community will be a
useful by-product .

Proposed Course: Several additional major reports, including the
statistical monograph, will be completed early in FY 78. A final data
tape including summary of fundus photograph grading will be prepared to
conclude data collection and editing. Contract completion is expected
in FY 78.

199

NEI Research Program: Retinal and Choroidal Diseases - Macular
Diseases/Diabetic Retinopathy and Other Vascular and Circulatory
Abnormalities; Cataract - Senile Cataract; Glaucoma.-

Experimental Subject or Tissue Source: Human

Research Objective: Etiology

200

CONTRACT NARRATIVE

Title; Diabetic Retinopathy Study (DRS)

Current Fund Allocation;

Objectives; The Diabetic Retinopathy Study (DRS) is a mul tic enter
clinical trial to evaluate the efficacy of photocoagulation (argon laser
and xenon arc) in the treatment of proliferative diabetic retinopathy.
This randomized, controlled study involves over 1,700 patients enrolled in
the trial at fifteen medical centers.

Major Findings; Following publication of its first report showing
photocoagulation to be effective in reducing the rate of severe visual loss
in patients with proliferative diabetic retinopathy and identifying certain
high-risk retinopathy characteristics, the DRS has continued follow-up of
its 1,700 patients under a modified protocol allowing, but not requiring, .
treatment of eyes originally assigned to no treatment if they met these
high-risk criteria. Large numbers of patients have been followed for three
years, and the first 5th-year follow-up visit has occurred. The DRS has
concentrated its analyses on determining the effect of photocoagulation on
the development and progression of retinopathy, and is now attempting to
determine the role of photocoagulation in eyes with milder stages of reti-
nopathy than those described in its first publication.

A baseline monograph giving details of study design and procedures, and
presenting baseline data on patient characteristics is being prepared for
publication, as is a methodologic paper on fundus photographic grading.

The DRS has entered an analytic phase where emphasis will be placed on
developing detailed descriptions of the natural history of diabetic
retinopathy and elaborating on the effects of photocoagulation treatment.

The DRS realizes its obligation to disseminate its findings to the
medical community and has staffed exhibits at the American Diabetes
Association annual meetings and at the American Academy of Ophthalmology and
Otolarjmgology meetings. The Study plans to continue this and other types
of educational effort and attempts are being made to cooperate with non-
DRS investigators in developing guidelines for photocoagulation treatment
of diabetic retinopathy in clinical practice.

Significance to Biomedical Research and the Program of the Institute;
Diabetic retinopathy, uncommon only a few decades ago, is now a leading
cause of blindness and visual disability in the United States. There is a
critical need to find and scientifically evaluate treatments which will
reduce the risk of blindness or visual impairment from the ocular compli-
cations of diabetes. Although photocoagulation is widely used as a treatment,
adequate evidence of its efficacy is now based on carefully documented
research findings.

201

Proposed Course: Follow-up of all DRS patients continues and monitoring
of accumulating data is performed at three-month intervals. This additional
follow-up, as well as further data analysis, is required for a complete
assessment of photocoagulation as used in the DRS, and the results of these
analyses will be presented in future study publications.

NEI Research Program: Retinal and Choroidal Diseases - Diabetic
Retinopathy and Other Vascular and Circulatory Abnormalities

Experimental Subject or Tissue Source: Human

Research Objective: Treatment

Publications:

The Diabetic Retinopathy Study Research Group: Preliminary report on
effects of photocoagulation therapy. Am. J. Ophthalmol. 81: 383-396,
1976

202

CONTRACT NARRATIVE

Title; Diabetic Retinopathy Vitrectomy Study (DRVS)

Current Fund Allocation: $287,000 for the period June 25, 1976 through
June 26, 1977 (six new clinics) .

Objectives: The DRVS is a multicenter clinical trial to: (a) evaluate
vitrectomy performed in the first six months after vitreous hemorrhage
secondary to diabetic retinopathy, as compared to the more usual practice of
waiting twelve months after vitreous hemorrhage to remove the vitreous, and
(b) to collect natural history data on patients who have diabetic retinopathy
with extensive formation of abnormal blood vessels and/or early retinal
detachment, but without extensive vitreous hemorrhage.

Major Findings: In June 1976, six additional Clinical Centers were
added. In October 1976, recruitment of patients was begun by those Centers
that had completed required certification procedures specified by the Manual
of Operations. AH but one Center were enrolling patients by May 1977.

Significance to Biomedical Research and the Program of the Institute;
Diabetic retinopathy is one of four major causes of adult blindness and
differs from the other three (macular degeneration, glaucoma, cataract) in
that it affects a younger population. A major cause of this blindness is
vitreous hemorrhage. Vitrectomy has been shown to be of some benefit to
individuals who have had a severe vitreous hemorrhage for at least one year,
and it is thought that diabetic blindness can be further reduced if vitrec-
tomy is performed at an earlier date. This presents an ideal opportunity
for the National Eye Institute to organize scientific talents to answer a
significant medical question.

Proposed Course; The first six months of the period constitute a trial
period in which the capability of all Clinical Centers to recruit sufficient
patients is being examined. The Coordinating Center is processing the
results, which will be reviewed by the Data Monitoring Committee.

The Reading Center is grading baseline fundus photographs and will be
processing posttreatment photographs as they become available.

NEI Research Program: Retinal and Choroidal Diseases - Diabetic
Retinopathy and Other Vascular and Circulatory Abnormalities/Vitreous Humor

Experimental Subject or Tissue Source; Human

Research Objective: Etiology, Treatment

Publications:

None

203

PUBLICATIONS
Office of Biometry and Elpidemiology

Allen, D.C.: Statistical and methodological considerations for vision
screening. Am. J. Optom. Physiol. Opt. 53: 677-681, 1976.

Hiller, R. , Giacometti, L., and Yuen, K.: Sunlight and cataract: An
epidemiologic investigation. Am. J. Epidemiol. 105: A50-459, 1977

Kahn, H.A., Leibowitz, H.M., Ganley, J. P., Kini, M.M., Colton, T.,
Nickerson, R.S., and Dawber, T.R.: The Framingham Eye Study. 1. Outline
and major prevalence findings. Am. J. Epidemiol. 106: 17-32, 1977.

Kahn, H.A., Leibowitz, H.M., Ganley, J. P., Kini, M.M., Colton, T.,
Nickerson, R.S., and Dawber, T.R.: The Framingham Eye Study. 2. Associatior
of ophthalmic pathology with single variables previously measured in the
Framingham Heart Study. Am. J. Epidemiol. 106: 33-41, 1977.

Milton, R.C., Ganley, J. P., and Lynk, R.H.: Variability in grading ,

diabetic retinopathy from stereo fundus photographs: Comparison of
physician and lay readers. Br. J. Ophthalmol." 61: 192-201, 1977.

Yuen, K.K. : Robustness of some sequential procedures. Commun. Statist.
Theor. Meth. A6(l): 43-54, 1977

Schwartz, J.T.: Methodologic differences and measurements of cup-disc
ratio. An epidemiologic assessment. Arch. Ophthalmol. 94: 1101-1105, 1976.

205

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OFFICE OF PROGRAM PLANNING AND SCIENTIFIC REPORTING

207

ANNUAL REPORT
NATIONAL EYE INSTITUTE
July 1, 1976 - September 30, 1977

REPORT OF THE CHIEF, OFFICE OF PROGRAM PLANNING AND SCIENTIFIC REPORTING

Julian M. Morris

Program Planning

The Office of Program Planning and Scientific Reporting continued to
provide staff support to the National Advisory Eye Council in regard to the
Council's second major planning report. Vision Research — A Five Year Plan,
which is due to be published this fall. This report, which will incorporate
the work, of over 150 consultants, will include a summary volume, a volume
which will present reports for each of the five NEI progr^ns plus a discussion
on vision research training, and a volume of relevant data wliich was used by
the Council and consultants to determine today's research needs and oppor-
tunities in vision science and ophthalmology.

Again this year the Office coordinated the publication of the NEI Annual
Report and collaborated with personnel from other offices within the Institute
to produce several evaluative and planning documents. The Office was primarily
responsible for developing and writing the yeaif's Forward Plan and Evaluation
Plan. The Office responded to Congressional and Departmental requests relating
to NEI planning activities as well as to requests from other Institutes of NIH
for advice regarding program planning activities for use in developing their
own research planning. The Office also responded to requests for planning
information from organizations and interested members of the general public.
The Office also prepared speeches concerning program planning and evaluation
for the Director, NIH, and the Director, NEI, for presentations to Congress,
higher levels of DHEW, and several national associations related to research
in vision and ophthalmology.

Scientific Reporting

The Office continued to give major attention to increased consumer
education activities through cooperation with the news media, liaison with
agencies serving the blind and visually handicapped, and direct correspondence
with consumers seeking a wide variety of information about visual disorders.

Scientific Communications

The Office contributed three articles for the NIH annual publication.
Research Advances 1977, which highlights major research developments supported
and conducted by NIH during the past year. Research Advances, which is
expected to be published in late fall 1977, is distributed to medical schools,
grantee institutions, and the public. The Office also contributed an article

209

on the Diabetic Retinopathy Study to "From the NIH," a monthly column in
the Journal of the American Medical Association which summarizes clinically
significant NIH research. In addition to disseminating information about
scientific meetings, seminars, and workshops conducted by NEl staff, the
Office also conducted tours of Institute facilities and program briefings for
visiting physicians, scientists, diplomats, four members of the All-Russia
Association for the Blind, and several groups of 4-H volunteer leaders.

Consumer Education

The Office continued to expand its activities in consumer education
by taking the initiative in a number of projects that are designed to help
consumers find information about eye care and visual disorders quickly and
conveniently. Among the most practical of these projects is the completion
of final drafts for a major HEW consumer publication, Facts About Eye Care and
Eye Glasses. Copies of these drafts have been sent to 20 experts throughout
the country for review, and it is hoped that the booklet will be published by
late fall 1977. The interest and demand for this material is so great that
sections of the final drafts have been used as background information for
journalists preparing articles on eye care. Five columns were written for
Search for Health, which is distributed by the NIH Office of Communications
to weekly newspapers across the country. The Office also prepared two radio
spots: an interview with Donald Bergsma, M.D. , of the NEI Clinical Branch,
on child eye care, and a public service announcement offering information on
cataract.

Press Relations

The Office responded to written and telephone inquiries from more than
100 publications this year including Better Homes and Gardens, Nieuwe Revu
of Amsterdam, Holland, New York Times, Newsday, Reader '^s Digest, and U.S.
News and World Report. A number of writers and editors publications asked
Che Office to review manuscripts for accuracy prior to publication, an exer-
cise that often helped to clarify information for the writers and editors
and their readers. The Office issued nine press releases or announcements,
including a major release on the Framingham Eye Study, and prepared 13
articles for the NIH Record. In addition, three articles were prepared for
News and Features from NIH, which is distributed to approximately 500 science
writers in the medical and general press. The Office has also received over
100 letters since the NEI was listed as a source of information in a Family
Circle magazine article concerning where to find the best sources of health
care.

Photographs provided by the Office for the Better Vision Institute's
New York Times supplement, "Facts You Should Know About Your Vision,," led
to several hundred requests from doctors, newspapers, low vision clinics,
and popular magazines for information about eye diseases and copies of these
pictures. The photographs showed how the field of vision may appear to
persons with the most common eye diseases. Requests for photographs to
illustrate newspaper and magazine articles, textbooks, encyclopedias, and

210

other specialized publications have continued to increase. This demand had
led to expanded efforts to locate new sources of photographs which present a
different perspective of eye problems.

Public Inquiries

Responses to telephone inquiries and letters from the public continued to
occupy a large portion of time and effort of the Office staff. Approximately
900 letters requiring detailed, reliable scientific information and over 3,200
telephone calls of a similar nature were received during the year. Inquiries
concerning cataract, diabetic retinopathy, glaucoma, and macular degeneration
were the most frequent requests. The number of letters fluctuated with the
number of articles about eye care and NEI in the popular press and was an
indication of the effectiveness of our consumer education activities.

The Office responded to 26 Congressional letters and other controlled
correspondence and to 32 Congressional telephone inquiries. A growing number
of inquiries are also being received from other government agencies; business,
professional, and scientific organizations; and publishers of encyclopedias
and textbooks concerning statistics on eye disorders, facts on treatment, and
current vision research efforts. In addition, the Office provided exhibits
on the Diabetic Retinopathy Study for health fairs at a junior college in
Baltimore, Maryland, and a hospital in San Jose, California.

Publications

The Office distributed the following number of publications during the
year:

Cataract — — — 2,100

Retinitis Pigmentosa- — —■ ■— 300

Refractive Errors — — 300

Diabetic Retinopathy--— ■ 1,020

Retinal Detachment 300

Corneal Diseases ■ 300

Glaucoma — ■ 800

Macular Degeneration-- • 1,020

Statistics on Blindness in the

Model Reporting Area, 1969-1970 75

U.S. News and World Report, Interview

with Dr. Kupfer 200

Evaluation of the Treatment of Diabetic

Retinopathy, A Research Project,

Reprint from the Sight-Saving Review — 120

Vision Research Program Planning 200

Support for Vision Research 240

Summary and Critique of Available Data

on the Prevalence and Economic and

Social Costs of Visual Disorders and

Disabilities (Westat Report) 70

The Framingham Eye Study 75

211

Miscellaneous

The Office continued to provide background material for use in the
legislative process, including opening statements, testimony, contributions
to special reports, and detailed answers on specific, questions submitted by
members of Congress about visual disorders and treatment.

The Office prepared Presidential proclamations for Save Your Vision Week
and White Cane Safety Day. A message was also prepared for the Secretary,
HEW, to the American Optometric Association's 80th Annual Congress. The
Office also coordinated the Institute's contributions to the NIH Annual Report
of International Activities, NIH Scientific Directory and Annual Bibliography,
Freedom of Information Act Annual Report to the Congress, and the Scientific
Information Exchange of the Smithsonian.

As in previous years, the Office continued its liaison with various
voluntary and professional organizations including the National Society
for the Prevention of Blindness, Inc., Fight for Sight, Inc., the American
Foundation for the Blind, Inc., Research to Prevent Blindness, Inc., the
Juvenile Diabetes Foundation, the American Diabetes Association, the American
Association of Ophthalmology, the American Optometric Association, and the
International Agency for the Prevention of Blindness.

212

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EXTRAMURAL AND COLLABORATIVE PROGRAMS

213

ANNUAL REPORT
NATIONAL EYE INSTITUTE
July 1, 1976 - September 30, 1977

REPORT OF THE ASSOCIATE DIRECTOR FOR EXTRAMURAL AND COLLABORATIVE PROGRAMS

William F. Raub, Ph.D.

Fiscal year 1977 was a time of exciting growth and change for the
Extramural and Collaborative Programs of the National Eye Institute. In
keeping with the Institute's first priority, a record number (608) of grant
awards for investigator-initiated individual research projects were made
covering a wealth of scientifically exciting ideas relevant to the prevention,
treatment, and cure of diseases and disabilities of the visual system. More-
over, there was a marked expansion of the Institute's efforts to evaluate new
treatments for diabetic retinopathy and other disorders through the medium
of controlled, randomized, clinical trials and considerable progress was made
in refining a set of policies and procedures for center grants and research
training awards which can facilitate the rapid qualitative and quantitative
maturation of vision research. The following sections highlight some of the
issues and accomplishments in the NEI Extramural and Collaborative Programs
during FY 1977, as well as identify opportunities for future initiatives.

For FY 1977 the National Eye Institute received an appropriation of
$64,000,000 — an increase of $14,000,000 (28%) over the previous year's
appropriation. Of the $64,000,000, a total of $54,186,000 was allocated to
Extramural and Collaborative Program activities in the following categories:

Research Grants $46,024,000

Research Training Awards 4,640,000

Research Contracts 3,522,000

Total $54,186,000

This funding level enabled the Institute to sustain the rapid but disci-
plined growth that its programs have exhibited over the past several years.

The bulk of the budget increase occurred in funds for research grants;
an additional $10,716,000 was -available in this category in FY 1977 as compared
to FY 1976. These funds were distributed among the Institute's five programs
as follows:

Research Dollars (in thousands)

FY 76

FY 77

% Growth

Retinal and Choroidal Diseases

$12,726

$17,668

Corneal Diseases

6,024

7,595

Cataract

3,757

4,567

Glaucoma

5,473

6,125

Sensory and Motor Disorders

of Vision

7,328

10,069

Total

$35,308

$46,024

30 i

.(215_1.

NiH-1317 PAGE NO.

The grant application receipt rate was 1 1/2 times that in FY 1976,
increasing the workload within the Institute and throughout the review
system. The National Advisory Eye Council approval rate, however, was stable
during these two fiscal years: 83 percent of grants submitted were approved
for funding in both FY 1976 and 1977. The Institute was able to fund 53 per-
cent of all approved applications, a slight decrease over FY 1976. The data
are given below.

Grant Application Rate

Received &

Recommended

Approved

% Funded of all

Reviewed

for Approval

& Funded

Approved Applications

FY 1976

336

284

162

FY 1977

512

425

225

% Change

+52%

+50%

+39%

The percent funded of all approved applications in the Sensory and Motor
Disorders of Vision program was considerably lower than in the other four
programs (31%). This Is due to the greatly increased receipt of applications
in FY 1977 and their subsequent high rate of approval (83 percent) . The
number of approved applications in the Sensory and Motor Disorders of Vision
program was equivalent to that of the Retinal and Choroidal Diseases program,
the NEI's largest, yet competing funds available to Sensory and Motor Disorders
of Vision were less than half of those available to Retinal and Choroidal
Diseases.

The distribution of awards between
grant applications was as follows:

competing and noncompeting research

Prior Year Commitments
New Research Awards
Renewal Awards

FY 1976
Number of Grants

444
99
72

Total

615

FY 1977
Number of Grants

453

129

678

Once the prior year commitments were taken into account, there was
approximately $17 million available for new and competing research grants —
the largest amount of "new" money for investigator-initiated vision research
ever available in one year in the history of the National Institutes of Health.
The 678 grant awards represent twice the number awarded in FY 1970, the first
year of the National Eye Institute's existence.

The Institute's research grants are comprised of the following
categories:

NiH-131 7
■i-68

(216 )

PAGE NO. t

FY 1977 Research Grants by Mechanism
(Dollars In Thousands)

Number Total Awarded

Project Grants (R01,R10,R13)
Special Visual Science

Research Awards (R23)
Core Center Grants (P30)
Specialized Clinical Research

Center Grants (P50)
Research Career Development

Awards (K04)
Academic Investigator

Awards (K07)

593

$40,468

134

2,386

1,521

1,113

402

Total 678 • $46,024

The codes in parenthesis in the above table are the symbols used by
NIH to differentiate the various types of grant awards. A description of
each of these mechanisms can be found in the publication, Support for Vision
Research — Interim Report of the National Advisory Eye Council, 1976 (DREW
Publication No. (NIH) 76-1098). It is noteworthy that approximately 88 percent
of FY 1977 grant funds are allocated to individual investigator-initiated
research projects.

The National Eye Institute complements its research grants with a program
of institutional and individual fellowships. The purpose of the program is
to equip young investigators with the skills, experiences and insights neces-
sary for them to embark successfully on a career in vision research. The
program also strengthens the ties between vision science, especially its
clinical aspects, and other disciplines, such as the basic medical sciences,
epidemiology, engineering and biomathematics.

A total of $4,640,000 was available for support of vision research
training in FY 1977, most of it for the National Research Service Awards
(NRSA). The individual NRSA fellowship awards accounted for 27% ($1,225,000)
of available training funds. The institutional NRSA training awards accounted
for $2,440,000 or 53 percent of the program. The remainder of the funds was
allocated to a small number of prior year training commitments, i.e., graduate
training grants and the Weinberger fellowships. The awards in these last two
categories will be funded until their individual project periods expire.

The National Eye Institute's collaborative research activities, funded
through contracts, continue to emphasize cooperative clinical trials for the
treatment of diabetic retinopathy. The distribution of contract awards and
funds is as follows:

__J217_J_

Total Awarded
Number (in thousands)

$1,035

875

1,412

200

Diabetic Retinopathy Study

Diabetic Retinopathy Vitrectomy Study

Diabetic Retinopathy Study II

Other

Total 38 $3,522

The qualitative and quantitative growth in the NEI Extramural and
Collaborative Programs during the past year were accompanied by several staff
and organizational changes:

1. Dr. Thomas C. O'Brien joined the NEI to assume the position of Chief,
Scientific Programs Branch. He has responsibility for supervising the
Extramural Program Directors and for coordinating activities with respect
to vision research training awards and center grants. Dr. O'Brien replaced
Dr. Wilford L. Nusser who became the Associate Director for the Extramural
Program of the National Institute of Environmental Health Sciences.

2. Mr. William M. Doak joined the NEI to become Chief of the newly created
Extramural Services Branch. This new Branch is the result of a reorganiza-
tion of the management activities associated with the NEI Extramural and
Collaborative Programs. The Branch is comprised of three components:

a Grants Management Section headed by Ms. Anna Marie Perrell, Grants
Management Officer; a new Contracts Management Section temporarily headed
by Mr. Doak; and a Management Information Unit headed by Ms. Carolyn G.
McHale. Mr. Richard Gruber, the previous coordinator of NEI research
contract activities, left the NEI to become self-employed.

3. Dr. Israel A. Goldberg joined the NEI to assume the newly created position
of Review and Special Projects Officer. In this capacity he serves as
Executive Secretary of the Vision Research Program Committee and coordi-
nates the initial review of center grant applications and requests for
academic investigator awards and institutional research fellowships.

4. Dr. Ralph Helmsen left the NEI's Laboratory of Vision Research to become
Extramural Program Director for Glaucoma. He replaced Dr. Samuel C.
Rawlings who joined the Department of Ophthalmology at the University of
Texas in San Antonio.

5. Dr. Herbert Yellin joined the NEI as Extramural Program Director for
Cataract.

6. Dr. Thelma N. Fisher joined the NEI as Extramural Program Director for
Corneal Diseases.

7. Dr. Luigi Giacometti, formerly Extramural Program Director for Corneal
Diseases and Cataract, left the NEI to become Executive Secretary of the
Visual Sciences B Study Section, Division of Research Grants, NIH.

218

VISION RESEARCH TRAINING

The National Eye Institute has long recognized that research productivity
depends not simply on the number of investigators but also on their quality
and that of the environment in which they work. Therefore, within the limits
of legislated and executive authority, the NEI has worked to provide oppor-
tunities for the training of individuals in the research laboratories of
established investigators and to promote the development and maintenance of
centers of vision research that use multidiscipline approaches in the
investigation of serious eye and visual system problems. The primary mecha-
nism for support of vision research training is the National Research Service
Awards (NRSA) for individual and institutional fellowships. The present
status of the NEI's research training activities is given in the table below:

RESEARCH TRAINING a./

Active Programs Total Amount

(thousands)
NRSA Individual (F32) 104 W $1,225

NRSA Institutional (T32) 42 sJ 1/ $2,440

Individual NRSA fellowships are directed toward providing training opportu-
nities in all five research programs of the National Eye Institute - Retinal
and Choroidal Diseases, Corneal Diseases, Cataract, Glaucoma, and Sensory
and Motor Disorders of Vision. Within this framework, research sponsors
for trainees are widely distributed, with over 50% of the training provided
in non-clinical academic departments. The results from this program have
been rather encouraging in that a number of individuals, trained at the
postdoctoral level through the Individual Postdoctoral Fellowship and having
satisfied the NRSA payback provision requirement, have gone on to receive
additional support for their research careers as independent investigators
through other types of National Eye Institute support such as the Academic
Investigator Award and the individual research project grant.

The intent of the Institutional NR5A fellowships is to encourage work at
both the pre- and postdoctoral levels in selected priority disciplines rele-
vant to all five NEI programs "a* determined by the National Advisory Eye
Council. At present, the areas supported include immunology and genetics;
pharmacology, epidemiology, physiology and biochemistry; developmental biol-
ogy, psychophysics and physiologic optics; and pathology. There is also a

a./ Total obligations for research training in F^ 1977 = $4.64 million.

In addition to NRSA's, this includes $51,000 for 6 Weinberger Fellowships
(F22) and $924,000 for 12 Graduate Research Training Grants (TOl) . All
Graduate Research Training Grants (TOl) will be phased out by June 1979.

b/ Includes 42 new F32's

d Includes 9 new T32's

Al 25 program directors on currently active T32's were formerly program
directors on Graduate Research Training Grants (TOl) .

219

special interest in supporting institutional training programs that will
increase the number of clinical investigators who are competent in epidemiol-
ogy and biostatistics. Recent awards in these areas support a training pro-
gram in the epidemiology of external eye diseases at the University of
Washington and a training program at Tufts University in collaboration with
the Harvard University School of Public Health involving epidemiological
and biostatistical considerations in vision research and clinical disorders.

At the present time, most of the Institutional National Research Service
Awards are made to departments or groups involved in laboratory research.
Some of the research training opportunities afforded by these departments
or groups include training programs that place emphasis on neurophysiology
of the visual system, biochemistry of lens development, microbiology and
immunology of anterior segment diseases, pathology and pathophysiology of
retinal diseases, biochemical genetics of hereditary retinal diseases, and
physiology and pharmacology of the anterior segment. Vision research bene-
fits in a number of ways from the interest that is genera^ted in departments
or groups engaged in the above-noted areas of research. First, the number
of investigators who are thoroughly trained in a particular area of vision
research is increased. Second, the discipline-oriented environment may
suggest concepts and approaches that are less likely to evolve in a clinical
setting. Third, predoctoral programs of graduate schools are conducted in
basic science departments, and the research interests of future investiga-
tors are frequently determined at this point.

Institutional National Research Awards that are administered by clinical
departments with a complement of established investigators are also useful
in providing particular types of research training, especially research
training opportimities in studies of clinicopathological correlations in
ocular diseases. Examples of such training opportunities include programs
at Yale University, the Armed Forces Institute of Pathology (Washington,
D.C.), the University of Illinois, and the Eye Research Institute of Retina
Foundation (Boston). For individuals with a Ph.D. degree who are interested
in vision research, the clinical setting offers an excellent base in which
to become acquainted with a broad multidiscipline field. The National Eye
Institute has recently awarded a NRSA institutional grant to the University
of Florida for a multidiscipline, multi- institutional research training
program on the clinical application of psychophysical and physiological
optics techniques. This xmique program will attempt to bridge the gap bet-
ween laboratory research findings and their clinical application. This
program is a highly specialized training opportunity and responds directly
to last year's National Eye Institute workshop on this subject.

The research training programs of the National Eye Institute have had a
profound effect on the nation's capacity to conduct research on vision and
its disorders. Not only has the nxjmber of qualified, independent investi-
gators and organized units actively engaged in vision research increased,
but also departments and sections of ophthalmology, schools and colleges of
optometry, and other health centers throughout the United States have devel-
oped or strengthened their research components so that multidiscipline groups
exist and are in a position to address the research problems identified in
Vision Research — A National Plan, the forthcoming new report of the National
Advisory Eye Coimcil.

220

CLINICAL. TRIALS AND THE TRANSLATION OF RESEARCH RESULTS INTO APPLICATION

Prospective, controlled evaluations of new 6ye care procedures are an im-
portant and growing activity of the National Eye Institute. Studies in this
category usually involve random assignment of patients, masked collection
and interpretation of data, and specialized biostatistical planning and data
analysis. To date, most clinical studies of this sort in the vision area
have required the cooperation of several clinical centers in order to acquire
a patient population large enough to allow meaningful statistical analysis of
the results. These cooperative clinical trials have been supported by the
National Eye Institute primarily via the research contract mechanism. Major
NEI supported cooperative clinical trials include the trial of photocoagul-
ation for treating proliferative diabetic retinopathy (the Diabetic Retin-
opathy Study) and the trial of early versus deferred vitrectomy in diabetic
patients with advanced stages of the retinopathy (the Diabetic Retinopathy
Vitrectomy Study) . The importance of these kinds of studies and their re-
levance to improved eye care is well-illustrated by the article "Preliminary
Report on Effects of Photocoagulation Therapy" (Am. J. of Ophthal. 8:383-396,
1976).

Because the day-to-day coordination of such large-scale, cooperative clinical
studies requires an extensive commitment of senior scientific and managerial
personnel, the National Eye Institute proceeds in a deliberate manner in
identifying those eye care procedures that should be evaluated by this
approach. At the same time, the Institute recognizes that other extramural
support mechanisms need to be developed or extended in order to insure that
all meritorious ideas for clinical trials, whether they involve single or
multiple centers or whether they originate with Institute staff or the extra-
mural community, have a reasonable chance of being undertaken. Accordingly,
beginning late in FY 1976 and extending through the first year, the Institute
staff has developed a special set of grant procedures that facilitate the
planning and execution of investigator -initiated, controlled clinical trials.

In essence, research grant applicants who request support to carry out a
clinical trial either under the individual project grant mechanism (ROl) or
the cooperative clinical research grant mechanism (RIO) , and whose ideas and
proposed approaches are judged meritorious by an appropriate NIH Study Section
and the National Advisory Eye Council, may be awarded funds initially for
only the first phase of the work — the development of detailed manual of
operations to guide the actual execution of the clinical trial. When such an
award is made, future years of support for the conduct of the trial are then
contingent upon (1) approval of the manual of operations following a review
by expert consultants and Institute staff and (2) the availability of funds.

The decision to take this approach was based upon several factors: (1) the
apparent lack of appreciation by many clinical investigators as to what
constitutes a well-designed controlled clinical trial (need for consideration
of such issues as randomization, stratification of the patient population,
inclusion-exclusion criteria, data management and analysis systems, patient \
follow-up, data and safety monitoring, formalized informed consent procedure^
etc.); (2) the National Eye Institute's leadership within the NIH, as exempli-
fied by the Diabetic Retinopathy Study, in the use of well-controlled

( 221)

NlH-1317 PAP.P ND t

clinical trials (National Eye Institute Workshops for Ophthalmologists,
November 6, 1976 — The Randomized Controlled Clinical Trial, Am. J. of
Ophthal. 79: 752-789, 1975; NET Biometry Branch presentation at the annual
course on "Methods of Clinical Research", AAOO Meetings; Ederer, F. Practical
Problems in Collaborative Clinical Trials, Am. J. of Epidemiol. 102: 111-118,
1975; NEI Biometry Branch presentations on "Quality Assurance of Clinical
Data" at NIH National Conference on Clinical Trials Methodology, October,
1977; Kupfer, C. Clinical Trials, Invest. Ophthal. 15: 513-514, 1976); and
(3) the increasing appreciation throughout NIH of the importance of well-
developed and carefully controlled clinical trails (Issue paper - "NIH Support
of Clinical Trials", Dr. Donald Fredrickson, Director, NIH, August 27, 1975;
NIH Conference on Clinical Trials Methodology, October, 1977). Thus, the
Institute has seized an opportunity not only to stimulate strong clinical re-
search but also to highlight the ways in which the fruits of vision research,
-both laboratory and clinical, can be used to improve eye care.

Currently, the National Eye Institute is supporting 10 projects under research
grants which involve the development of a manual of operations. The topics
of these clinical trials include studies of the effect of vitamin E on retro-
lental fibroplasia in infants; evaluations of laser treatment for branch vein
occlusion, central serous chorioretinopathy, and glaucoma (trabeculotomy);
a study of the effects of intraocular lenses on the corneal endothelium; and
an assessment of the effect of vitamin C on corneal treatment for acute alkali
burns. One clinical trial comparing conventional treatment of refractive
errors with orthokeratological treatment has already completed the development
of a manual of operations and is about to begin enrolling patients. In another
case, the principal investigator determined in the course of developing the
manual of operations for a study to determine the efficacy of laser therapy
in the treatment of branch vein occlusion that a single clinic study would
not provide a sufficient population base and therefore began to develop plans
for a multiclinic study using the cooperative clinical research grant (RIO) .
A coordinated set of research grants proposing to employ the manual of opera-
tions developed under the initial project grant award has been submitted by
the collaborating institutions and is now being reviewed by the Vision
Research Program Committee.

At this stage it is too early to measure the success of this approach to
achieving well-designed controlled clinical trials or even to ascertain whether
it is the best one. But it is clear already that the research community is
more cognizant of the importance of this kind of research and is responding
well to the Institute's administrative initiatives. The further nurturing of
these types of studies will be high on the NEI's list of objectives for
FY 1978.

222

VISION RESEARCH CENTERS: THE NATIONAL EYE INSTITUTE CONCEPT

Until this year, the National Eye Institute has employed three types of large-
grants, the Research Program Project Grant, the Core Research Center Grant,
and the Specialized Clinical Research Center Grant. In contrast to the regu-
lar Research Project Grant, which provides support for a discrete, specified,
circumscribed project performed by a principal investigator and his or her
own research team, these grants usually provide support for broad-based,
long-term programs of research. Generally, they are designed to bring toge-
ther, through common support, the activities of a number of investigators
and teams. However, the ways in which they do this, and the specific pur-
poses of each, differ considerably.

In 1973, the National Advisory Eye Cotmcil evaluated the three large-grant
types. The Coimcil recommended that the National Eye Institute discontinue
use of Research Program Project Grants. Although these awards provided sup-
port for a number of projects relating to a common vision-research theme,
they also supported projects of mixed programmatic interest to the NEI. In
fiscal year 1977, the last Research Program Project Grants supported by the
NEI were phased out.

At present, the NEI has a small program of research centers. During FY 1977,
there were 13 Core Research Centers receiving NEI support, and six Specialized
Clinical Research Centers. Total center support was approximately $3.3
million, or 6 percent of the funds available for the support of extramural
research.

In FY 1977, the NEI staff began an evaluation of the two centers programs.
As a result, new administrative guidelines for both programs are being pre-
pared. The purpose of this activity has not been to expand the number of
centers or to enlarge their scope, but rather to employ these programs opti-
mally in achieving the mission of the Institute while assuring that the
mainstay support for vision research, the individual Research Project Grant
program, is not jeopardized. In this regard, it should be noted that it is
strongly recommended that the NEI extramural staff be consulted prior to
the initiation of applications for either program. The Institute's staff
is committed to providing guidance to potential applicants, as well as to
interacting with ongoing cente'rs in order to assure that the research goals
of the Institute are met by the submission of high-quality applications.

Core Research Centers

The Core Research Center Grant does not provide direct funding for research
projects. Rather, it provides a central nucleus or core of resources, facil-
ities, and services which are shared by the investigators on a number of
individual research projects. Thus, an NEI Core Research Center is an organ-
ization at which there are at least four ongoing, high-quality, independently-
supported research projects in the visual sciences. At an NEI Core Research
Center, investigators are brought together in an environment which facili-
tates multidlscipline research approaches to problems in the visual sciences.
It is an environment which supports laboratory studies, clinical studies.

223

or both, and which promotes interaction and collaboration among vision
researchers and investigators from other academic departments or areas of
interest.

The primary objective of the Core Center Program is to achieve research
advances which would not be possible through the activities of individual
project grants alone. For this purpose, the grant may also provide limited
funds to initiate pilot studies which result from the collaborative activi-
ties within the Center. In order to get maximum returns from its investment
in these highly nutritive research enviroranents , the NET may also provide
annual salary support for newly-recruited investigators whose expertise
complements that of staff already on board and who, it is hoped, will event-
ually compete successfully for their own research project grants.

In fiscal year 1977, the NEI supported ongoing Core Research Centers at the
University of Washington, University of Pennsylvania, Eye Research Institute
of Retina Foundation (Boston), University of California Los Angeles, University
of California San Francisco, Columbia University, Massachusetts Eye and Ear
Infirmary, Yale University, University of Rochester, and Smith-Kettlewell
Institute of Visual Sciences (San Francisco) . In addition three new Core
Research Center awards were made to the Medical College of Wisconsin, Mount
Sinai School of Medicine, and Harvard University.

Specialized Clinical Research Centers

The Specialized Clinical Research Center Grant is an award which may provide
support for both individual clinical research projects and for core resources.
The NEI utilizes this funding mechanism only imder exceptional, clearly-
defined circumstances. A Specialized Clinical Research Center is viewed as
a unique environment in which important and definitive clinical research is
conducted on a particular human eye problem. The emphasis at these centers
is on investigations involving htrnian patients. Most often, the investiga-
tions exploit recent laboratory findings in scientific projects involving
outpatients. The types of projects conducted, as a rule, would not be
practical or possible to pursue outside of the Center. Thus, Specialized
Clinical Research Center grants provide support for clinical studies which
have a common conceptual framework relative to the etiology, pathogenesis,
diagnosis, and treatment of human visual disorders, and which share common
practical requirements, such as a unique cohort of patients and special
facilities dedicated to clinical research.

Four previously supported NEI Specialized Clinical Research Centers were
continued in fiscal year 1977. Each one deals with specific visual dis-
orders: (1) Macular Disease Center, University of Miami; (2) External
Ocular Disease Center, University of Florida; (3) Retinal and Choroidal
Disease Research Center, University of Chicago; and (4) Glaucoma Clinical
Research Center, Washington University, St. Louis. In addition, new centers
for Studies of Retinitis Pigmentosa and Allied Diseases, at the Berman-Gund
Laboratory of the Massachusetts Eye and Ear Infirmary, and for Study and
Treatment of Immune Ocular Diseases, at the Eye and Ear Hospital of Pittsburgh
were established.

224

Specialized Clinical Research Centers provide a superb vehicle for the
adaptation of laboratory research findings and techniques to the clinical
research setting. As such, this centers program plays a most important
role in the execution of the NEI mission. Such centers, with their clinical,
scientific, and administrative resources focused on research involving
patients in a health care environment, also provide an ideal setting for
investigating the translation of the results of laboratory and clinical
research to the improved prevention, diagnosis, and management of eye
diseases.

225

RETINAL AND CHOROIDAL DISEASES
Introduction

The retina (the light-sensitive tissue at the back of the eye) and the choroid
(an underlying layer rich in blood vessels which is responsible for nourishment
of the retina) are the primary sensory and vascular tissues of the eye and are
closely related. The optical structures of "the human eye focus light on the
fovea centralis, a distinct area of the retina which is located temporal to
the optic disc. This area possesses the greatest acuity of any portion of
the retina by virtue of its high concentration of cone (day-light) photo-
receptors.

The region surrounding the fovea centralis, known as the macula lutea or macu-
J.ar region, is normally darker in color than the rest of the retina because
it contains a yellow pigment. The macula has the greatest acuity of any por-
tion of the retina and is used exclusively for most visual tasks except for
vision in very dimly lighted surroundings. Minute lesions which might not
cause visual disturbance in peripheral areas of the retina do cause serious
loss of central visual acuity and color discrimination when located in the
macular region.

The fovea appears to be directly involved in a variety of retinal degenera-
tive conditions for it is subject to all the pathological alterations which
may develop in other areas of the retina. Retinal and choroidal diseases
are considered to be among the most difficult visual disorders to manage and
require a considerable research effort if a better understanding of their
causes is to be gained and more effective approaches to their treatment and
prevention are to be designed.

An estimated 15,000 people become blind each year from retinal and choroidal
diseases which already account for 42 percent of all blindness in the United
States. The causes of blindness from these disorders are diverse and the
"research relating to them is complex. Therefore, the National Eye Institute's
Retinal and Choroidal Diseases program has been divided into twelve subpro-
grams each addressing either a major group of retinal and choroidal diseases
or a primary focus of basic research;

Developmental and Hereditary Disorders. These include disorders which afflict
the newborn and the young. Many result from known familial tendencies toward
blinding disorders.

Diabetic Retinopathy and Other Vascular and Circulatory Abnormalities. Retinal
vascular disease associated with diabetes mellitus and the complications of
vascular occlusion, leakage of fluids from blood vessels, hemorrhage and neo-
vascularization.

Myopia. Simple refractive errors as well as the pathology which arises in
the retina and choroid due to severe progressive myopia which causes the
outer coats of the eyeball to stretch and thin. Eventual blindness may result
from vitreous changes, retinal detachment and insufficient blood circulation.

227

Tumors. Malignancies, usually retinoblastoma or melanoma, which may arise from
the retina or may result from a metastasis of tumors in other areas of the eye
or body.

Macular Diseases. Abnormalities associated with the macular region of the
retina frequently associated with aging which cause debilitating loss of cen-
tral visual acuity.

Retinal Detachment. Separation of the neural retina from the pigment epithe-
lium with a resulting impairment of vision.

Inflammatory Disorders. A group of destructive diseases which can affect all
parts of the eye. The causative agents are both infectious and non-infectious
and may arise from immunologic insults to eye tissues.

Uveal Tract Disorders. Diseases affecting the choroid, ciliary body and iris.
Malfunction of the uveal tract can result in circulatory disorders and the
ensuing complications of macular degeneration and inflammation.

Vitreous Humor. An internal structure of the eye which increasingly appears
to have a major physiological function as well as a supporting role. Changes
in the vitreous humor are associated with aging and with disease processes in
such disorders as diabetes mellitus, intraocular infections and uveitis.

Visual Cells and Pigment Epithelium. The specialized cellular elements involved
in photoreception. The retinal pigment epithelium is involved in the nutri-
tion of the sensory retina.

Retinal Organization and Visual Adaptation. Information received by the pho-
toreceptor is transmitted through a neural network of four different types
of nerve cells for processing within the retina. Failure of this, complex
organization can result in dysfunctions of perception.

Special Areas of Future Interest. This area includes toxic and environmental
disorders, low vision, and retinal regeneration and transplantation.

Vitamin A Deficiency

Krill-*- discussed disorders which cause abnormal night vision and are asso-
ciated with severe vitamin A deficiency. Vitamin A is of particular interest
because of its importance in the visual cycle. Although dietary deficiency
of vitamin A is rare in the United States, it may be part of the spectrum of
malnutrition associated with alcoholism, drug addiction, and some improperly
managed pregnancies.

Deficient absorption from the gastrointestinal tract, faulty liver metabo-
lism and excessive urinary excretion of vitamin A due to systemic disorders
are the more likely primary causes of a vitamin A deficiency, as opposed to
problems of deficient intake. It is also known that vitamin A in excessive
quantities may induce ocular malformations during early stages of embryonic
development. To complicate the problem, individual needs for vitamin A vary

228

widely with no obvious correlations with physical characteristics.^

Retinol, a derivative of vitamin A, is bound ,to the protein of the visual pig-
ment and provides the light-catching ability of the photoreceptors. It is
important to describe the routes and mechanisms by which retinol is delivered
to the retinal pigment epithelium and photoreceptors.

Supporting evidence for the binding protein for vitamin A derivatives in foveal
retinal tissue has been presented by Futterman and associates.-* Their objec-
tives have been to characterize the reactions of vitamin A associated with
light and dark adaptation in the retina and to discover whether vitamin A bind-
ing protein exists in human retinal tissue. In addition they sought a nonin-
vasive means of obtaining binding proteins for vitamin A derivatives. The
investigators did not find vitamin A binding protein in human fibroblasts but
did find retinol and retinoic acid binding proteins in human retinal tissue.*
The quantity of bound retinoic acid in human tissue greatly exceeds that of
retinol. The binding protein for retinol is also present and is more complex
in its properties. Nevertheless, these investigators did identify a binding
protein capable of interacting with the aldehyde form of vitamin A.-* These
studies indicate the existence of a new binding protein which may be important
in the regeneration of bleached visual pigment in the retina.

It now appears that there are intracellular binding proteins for retinol,
retinal and retinoic acid. The distribution and function of these proteins
within the retina and retinal pigment epitheliirai in health and disease should
be determined. The methodology required for this determination would involve
immunochemical, cytochemical and biochemical techniques. The involvement of
these intracellular binding proteins in the uptake, intracellular transport,
and metabolism of vitamin A by retina and retinal pigment epithelium is impor-
tant in addition to their potential significance in the delivery of serum
retinol and in the transformation of vitamin A in the visual cycle. Patho-
logical similarities between vitamin A deficiencies and retinal degeneration
have stimulated investigation of vitamin A uptake and metabolism in retinitis
pigmentosa animal models as well as in patients with this and related dis-
orders. The processes that can now be measured are unaffected; however, as
more information about the physiological events are uncovered, opportunities
to look for specific defects in retinal dystrophies will improve."''

Vitamin E Deficiency and Retrclental Fibroplasia

Dratz and associates"'" are investigating the possibility that some ocular
disorders may be the result of deficiencies in one or more antioxidant mecha-
nisms combined with a variety of oxidative stresses. Photoreceptor membranes
are susceptible to oxidative damage which may correlate with their high con-
tent of polyunsaturated fatty acids. Vitamin E deficient monkeys fed highly
unsaturated fat develop clinical symptoms of vitamin E deficiency an^ia as
well as macular degeneration. Hayes and associates-'-*^ are currently depleting
kittens of vitamin A and E in order to evaluate functional and morphological
changes in a species with a rod-cone retina. The work may demonstrate that
the cat requires dietary supplements of taurine, vitamins A and E, and zinc
when fed a casein diet.^ This model of photoreceptor degeneration is of

229

particular interest because in the early stages of taurine deficiency in the
cat delays occur in the time interval between stimulus onset and the peak of
the major cornea-positive component of the cone ERG response (b-wave implicit
time) . The phenomenon is similar to delays in cone b-wave implicit time seen
in some patients with early stages of retinitis pigmentosa. ^^

Studies suggest that although cases of adult human vitamin E deficiency are
rare, ocular tissues may be more sentitive to such deficiency than other organs.
The development of ocular lesions as a result of dietary antioxidant defi-
ciencies in adult humans may involve marginal deficiencies in dietary unsatu-
rated fats over a long period of time. Thus some senile macular degenerations
of the human retina may be related to marginal intake of vitamin E and there-
fore could be considered deficiency disorders. The protective action of
vitamin E in the rapidly developing retinal tissues of the premature infant-'--'
anH kitten-*-^ is more dramatic than that of the speculated action on the mature
human eye.

Kittens exhibit a widely accepted model of the human disease retrolental
fibroplasia (RLE) . The kitten develops the vasoconstrictive and proliferative
changes of RLE seen in the human premature infant; however, the disease in
the kitten does not progress through the cicatricial stages which can lead to
retinal detachment and blindness. Eor the premature human infant weighing
less than 1,000 grams it appears that any concentration of oxygen in excess
of that in air is associated with the risk of developing RLE. To discourage
the use of oxygen in the premature infant with respiratory distress, however,
is to risk the development of cerebral palsy due to hypoxia.

In order to widen the margin of safety between RLE and cerebral palsy, Boggs
and associates-*--* and Phelps and associates-*-" are developing prospective, ran-
domized clinical trials. Such studies are particularly important in view of
the increasing survival of premature infants of less than 1,500 gram birth
weight and their relatively high susceptibility of RLE in spite of improved
technology and heightened awareness of the need to control arterial oxygen
levels. It is possible that vitamin E has the ability to restore physiologic-
metabolic balance in rapidly developing tissues and to reverse the tendency
to abnormal development in response to the toxic stresses of oxygen and light
in the extrauterine environment,

Eactors Involved in Retinal Neovascularization

Neovascularization is a retinal response which occurs in proliferative retinal
diseases such as diabetic retinopathy, sickle cell retinopathy, retrolental
fibroplasia and retinal venous occlusive disorders. These disorders are among
the major causes of blindness. In these conditions new blood vessels prolif-
erate on the surface of the retina and branch into the vitreous body , The
proliferating vessels are fragile and hemorrhage into the vitreous. Eventually
fibrous tissue forms which through traction can cause retinal detachment and
blindness.

Currently there are no means of preventing or curing proliferative retino-
pathies, particularly when they are in advanced stages. It is generally

230

accepted that neovascularization is a tissue response to clinically observable
events. In response to a stimulus, focal areas of the retinal capillary bed
may lose their perfusion capability, and the retina becomes ischemic. The
retinal tissue then becomes deficient in essential nutrients and gases, and
metabolic products accumulate. It is hypothesized that ischemic retinal tissue
could be a source of a vasoprolif erative substance which could diffuse through-
out the retina as well as to other vascularized eye tissues.

Chen and associates -'■7,18 recognize that observations of the physiology,
chemistry, and clinical role of vasoprolif erative substances depend upon the
development of a suitable animal model and bioassay techniques. The problem
is specific and can be quantitated. Concurrent with the vasoprolif erative
studies is an investigation that has as its ultimate goal the diminuation of
angiogenesis activity in human retinal vascular disease, particularly in dia-
betic retinopathy.

Finkelstein and associates^^'^O have been studying the effect of tumor angio-
genesis factor on retinal vessels in rhesus monkeys. This factor had been
shown to elicit neovascularization in various tissues but its effect on
retinal vascularization has not been previously demonstrated. The investiga-
tors have shown that a slow release polymer pellet impregnated with tumor
angiogenesis factor, when placed on the surface of the optic nerve head, will
stimulate disc neovascularization. In this manner, a model for detecting the
presence of human proliferative substances may be approached and means of pre-
venting retinal neovascularization considered.

Disorders of the Macular Region

Central Serous Chorioretinopathy. Central serous chorioretinopathy is a spo-
radic disease of unknown cause which may affect the eyes of otherwise healthy
young persons, usually men between the ages of 20 and 40 years. The symptoms
consist mainly of loss of central visual acuity in one eye along with image
distortion and impairment in visual space perception. Symptoms may also
include subjective disturbances in dark adaptation and color vision.

Some clinicians believe that central serous chorioretinopathy poses no great
threat to sight because the disorder is self -limiting . However, others take
the position that although a slow, spontaneous remission with improvement of
visual function generally occurs over a period of months, the recovery time
is unpredictable. 22 xf recovery is slow or recurrences frequent, the danger
of permanent visual loss is increased. 23

Individuals with central serous retinopathy have what appears to be leakage
of serous fluid from a site within the choroidal pool across Bruch's mem-
brane through focal defects in the usually tight intercellular junctions of
the retinal pigment epithelium. The serous fluid accumulates under the sensory
retina, producing a localized nonrhegmatogenous retinal detachment which com-
promises retinal function at that site and thereby produces a disturbance in
vision. The fluid which has accumulated in the subretinal space may gradually
disappear over a period of weeks or months and be accompanied by spontaneous
reattachment of the retina to its underlying pigment epitheliinn. Though the
mechanism of fluid disappearance is unknown, the retinal vessels, the pigment

231

epithelium of the retina, and the choriocapillaris may be involved in the
removal of the subretinal fluid. However, very little is known about the
physiological factors favoring removal of fluid from the subretinal space.

There is evidence that laser or xenon arc photocoagulation of the retina can
increase the rate of fluid disappearance from the subretinal space. There-
fore, the National Eye Institute is currently supporting a clinical trial to
determine the efficacy of argon laser photocoagulation as a treatment for
central serous chorioretinopathy and to learn how such treatment might promote
early disappearance of abnormal subretinal fluid. '^-'

The use of laser coagulation to improve the treatment of central serous chorio-
retinopathy will make available to the clinician a technology and possible
insight into the dynamics of this disorder.

Senile Macular Degeneration. Of the blinding retinal diseases, senile macular
degeneration accounts for at least 20 percent of new blindness which occurs
annually. Choroidal neovascular membranes are of importance in senile eyes
and play a prominent role in central visual loss in many maculopathies . Some
clinicians use photocoagulation treatment for senile maculopathy at various
stages.

Past results of therapeutic argon laser photocoagulation have not been suffi-
ciently good to permit meaningful conclusions to be drawn regarding the value
of this treatment in the management of the senile macula with disciform detach-
ment. 24 ^ clinical trial with random assignment of eyes to treatment or non-
treatment is necessary to define the role of photocoagulation in this disorder.
Therefore, the NEI is supporting a randomized clinical trial to evaluate the
role of photocoagulation in the management of choroidal neovascular membranes
in the senile macula. ^4 Although this study does not seek to discover the
cause of senile macular degeneration, it does seek to develop the best possi-
ble data on prognosis and to determine whether photocoagulation is effective
in the treatment of senile macular degeneration.

Animal Models for Maculopathies. Disciform macular degeneration characterized
by subretinal neovascularization in the macula has been produced in stump tail
monkeys, by Ryan and coworkers. ^5 Once the break in Bruch's membrane had been
produced by mechanical and/or chemical means and subretinal neovascularization
produced, the pathogenesis of disciform degeneration was studied. The work
has shown that it is possible to produce subretinal neovascularization in the
monkey. In addition, Ryan and associates have also found three monkeys with
naturally occurring drusen which tend to be associated with senile macular
degeneration in man.^o These animals are currently being studied with repeated
fluorescein angiography and fundus photography. Eventually, clinicopathologi-
cal correlation of Bruch's membrane and pigment epithelium will be made.

During the past year, Vainisi and associates have studied three separate fami-
lies of baboons (16 animals) .27,28 Xwo of the families are composed of mem-
bers who have at least one parent affected with macular degeneration. The
other family has never had any members affected with macular degeneration and
is being used as a control. Each baboon has been studied by fundus photography,

232

by fluorescein angiography, and by electrophysiologic and biochemical techniques.
The affected animals display a type of abnormal behavior that is associated
with decreased vision. The evidence from these studies supports the hypothesis
that macular dystrophy begins in the cone outer segments and in advanced cases i
of diffuse dystrophy eventually involves all the photoreceptors.

Continued study of non-human primate models will greatly advance our knowledge
of the pathogenesis of macular diseases. It is hoped that such studies will
reveal abnormalities responsible for the fundus changes and make possible the
design of new approaches to treatment.

Toxic and Environmental Factors 1

Continuous outer segment disc renewal is a basic property of all vertebrate
rod photoreceptors and the disposal of shed old discs involves an intricate
cellular interaction between the rods and pigment epithelial cells in main- |
taining a relatively constant rod outer segment length. The approach to
studying the mechanisms involved in this process is based upon observations
that light stimulates disc detachment and phagocytosis by the pigment epithe-
lium. By using the experimental manipulation, the disposal mechanism can
be studied more accurately because the process can be initiated and then
examined with time, instead of having to piece together isolated events with-
out knowing which preceded the other. ■,

The phenomenon and defects in phagocytosis has stimulated investigators to
delve into basic mechanisms in animal models with health related implications. 2'
NoeU and associates are making major efforts to correlate the measured vulner-
ability of photoreceptors to damaging light with biochemical changes in the
rhodopsin, lipids and fatty acids of the rod outer segments. "» -^^ The kine-
tics of changes in these constituents were determined in long-term adaptation
to darkness and weak cyclic light and related to the ability of strong light
to cause visual cell death. The latter is measurable by fractionable irre-
versible ERG loss and by quantitative histology. Noell and associates have
concluded that the synthesis or membrane incorporation of rhodopsin, phos-
pholipids and highly unsaturated fatty acids are controlled by environmental
light. Maintaining animals in darkness apparently accelerates these processes
to the maximum, and a low intensity environmental light depresses the rhodopsin
level in relation to phospholipid content. In a highly intense lighted envir-
onment, the phospholipid content is first altered, followed by the fatty acid
composition. These adaptive changes are being investigated in relation to
vitamin deficient diets and ambient temperatures. ^^»-^-'-

La Vail and associates have shown that when albino rats are reared in cyclic
light, a burst of rod outer segment disc shedding occurs soon after the onset
of light ■^-'■» 32, 33 Furthermore, the burst of disc shedding follows a circadian
rhythm for at least three days. Further study of this phenomenon in a variety
of modifications of the lighting environment is in progress.

Cooperative Clinical Trials

The National Eye Institute is currently pursuing three contract-supported,
multicenter, randomized, controlled clinical trials which are titled:

233

1. Diabetic Retinopathy Study - Phase I (DRS-I)

2. Diabetic Retinopathy Study - Phase II (DRS-II)

3. Diabetic Retinopathy Vitrectomy Study (DRVS)

DRS-I. This study was designed to determine whether photocoagulation is of
benefit in preserving vision in patients with proliferative diabetic retinop-
athy. The study began in 1971, and enrollment ended on September 30, 1975,
after 1758 patients had entered the trial. To be eligible for the study,
patients had to have proliferative retinopathy in at least one eye or severe
non-proliferative retinopathy in both eyes. Visual acuity of 20/100 or better
was required in each eye. Both eyes had to be suitable for photocoagulation
and neither eye could have received it previously. One eye of each patient
was assigned randomly to prompt photocoagulation and the other to follow-up
without photocoagulation.

In its first report the study showed treatment to be of substantial benefit
in reducing the risk of severe visual loss, defined as visual acuity of less
than 5/200 at each of two consecutive four -month follow-up visits. '^■'- Some
harmful effects of treatment were also reported. For eyes with certain char-
acteristics the risk of severe visual loss without treatment was found to be
so great and the reduction of this risk with treatment so impressive that
they outweighed the risks of the harmful effects of treatment. Accordingly,
the Study's protocol was changed to require consideration of photocoagulation
treatment for initially untreated eyes with these characteristics.

The groups of previously untreated eyes in which treatment was to be consid-
ered were defined as those with:

1. moderate or severe new vessels on the disk (NVD) , with or without vitreous
or preretinal hemorrhage;

2. mild NVD with vitreous or preretinal hemorrhage; and

3. moderate or severe new vessels elsewhere (NVE) with vitreous or pre-
retinal hemorrhage.

It was concluded that in most eyes with these characteristics, the risk of
deferring treatment is substantial and prompt photocoagulation is usually
desirable.

Following publication of its first report, the DRS continued follow-up of
its patients under a modified protocol allowing, but not requiring, treatment
of eyes originally assigned to no treatment if they met these high-risk cri-
teria. Large numbers of patients have been followed for three years and the
first fifth year follow-up visit has occurred. The DRS is now concentrating
its analyses on determining the effect of photocoagulation on the development
and progression in eyes with milder stages of retinopathy than those specific-
ally mentioned in its first publication.

A monograph giving details of study design and procedure and presenting base-
line characteristics of patients is being prepared for publication, as is a
methodologic paper on fundus photograph grading.

234

The DRS has entered an analytic phase where emphasis will be placed on develop-
ing detailed descriptions of the natural history of diabetic retinopathy and
elaborating on the effects of photocoagulation treatment. Tliese will include
attempts to identify additional systemic and ocular risk factors that are '
associated with the progression of proliferative diabetic retinopathy and to
determine whether photocoagulation treatment exerts its effect by altering
these factors or by an independent route. Because it is not known how photo-
coagulation exerts its beneficial effect, attempts are being made to develop
a pathology study within the DRS. Leading ocular pathologists from around
the country have met to develop a protocol for examining eyes of DRS patients.

The DRS research group realizes its obligation beyond publication of a scientifi:
paper to disseminate its findings broadly to the medical community. The group
prepared an exhibit for this purpose which was displayed at the American
Academy of Ophthalmology and Otolaryngology meeting in October 1976. The study
group plans to continue and expand this type of educational effort and attempts
are being made to cooperate with non-DRS investigators in developing consensus
for clinical practice.

DRS-II. This study has been designed to answer some of the major questions
which were not addressed by the Diabetic Retinopathy Study Phase I. The
objective of this new trial is to determine the optimum stage to initiate
photocoagulation, the effectiveness of photocoagulation on diabetic maculopathy,
and whether aspirin administered at various stages of diabetic retinopathy
can prevent or retard the progression of this disease. A working group was
established to prepare an outline of the proposed trial. The outline was
presented to the National Advisory Eye Council which endorsed it at their
January 1977 meeting. The NEI issued a Request for Proposal on April 30, 1977
for potential clinical centers. Proposals that were submitted before the
June 15 deadline are in the process of being reviewed, and negotiations and
funding are to be completed by September 1977. In addition to the clinical
centers, proposals have been solicited for a coordinating center, fundus
photograph reading center, and central laboratory facility.

During the next year the study participants and NEI staff will collaborate
to complete the study design and develop a detailed manual of operations.
Recruitment of patiefits should, begin by September 1978.

DRVS.

This study was initiated to test vitrectomy in the treatment of severe vitreous
hemorrhage due to diabetic retinopathy. The Diabetic Retinopathy Study, Phase
I and II, is examining the response to photocoagulation treatment of eyes hav-
ing diabetic retinal vascular disease without extensive intraocular hemorrhage.
The DRVS deals with a more advanced stage of this disease in which blindness
due to hemorrhage into the vitreous has occurred. Such eyes are not suitable
for photocoagulation treatment since the extensive hemorrhage prevents access
of the light beam to the retina. Vitrectomy removes the opaque vitreous
barrier to vision and reduces the risk of serious secondary mechanical changes
in the retina such as traction detachment.

235

The vitrectomy procedure being examined in the DRVS involves an instrxunent
which combines cutting, suction, and infusion of a replacement solution.
The instrument is inserted through a small incision in the side wall of the

eye.

In a ni.nnber of medical centers, vitrectomy for long-standing diabetic vitreous
hemorrhage has become a standard procedure. The vision is restored to useful
levels in a moderate number of such patients who would have a high probabil-
ity of remaining blind without vitrectomy. However, the vision does not always
Improve following vitrectomy in eyes with long-standing vitreous hemorrhage.
Inoperable retinal detachments or other sequelae of diabetic vitreo-retinal
disease may occur during the conventional one-year waiting period following
vitreous hemorrhage. In other eyes surgical complications of the vitrectomy
procedure itself make restoration of vision impossible. Some vitreous sur-
geons believe that the operation should be done earlier in the period following
severe vitreous hemorrhage. They hope that by performing surgery earlier,
(within the first six months of severe hemorrhage) , the development of
inoperable sequelae of the hemorrhage will be lessened and the average level
of vision restored improved.

However, it is not known whether earlier operation would be as safe as later
surgery. To determine this a clinical trial is the most ethical and scientific
way of proceeding. Randomization, the most important feature of a clinical
trial, tends to make treatment groups similar in all respects except for the
treatment assigned. Thus, any observed differences may usually be ascribed
to the treatments themselves rather than to differences in patient charac-
teristics that would influence response to surgery.

In the DRVS eyes with severe vitreous hemorrhage (the H Group) are assigned
to either vitrectomy within the first six months following the hemorrhage or
to a "late" vitrectomy group in which vitrectomy will be performed at one
year following hemorrhage in those eyes still suitable for it. A separate
category (the N Group) consists of eyes without extensive vitreous hemorrhage
that are followed without treatment to collect natural history information.
Photocoagulation, based on information learned from the DRS, is given to
those eyes in either H or N Group as indicated.

Following the decision to proceed with DRVS, contracts for seven clinical
centers, a fundus photograph reading center, and a data coordinating center
were awarded in June 1975. Contracts for six additional clinical centers
were awarded in June 1976. Recruitment of the first of a projected 826
patients was begun in October 1976 by those centers completing detailed pro-
cedures for initiation of data collection required by the protocol.

The rates at which the clinical centers have recruited patients meeting the
highly specific DRVS eligibility criteria have been somewhat lower than
originally expected. Further attention by investigators to their referral
sources has been encouraged, and the National Eye Institute has written to
all U.S. ophthalmologists asking for their assistance. An exhibit to encour-
age patient referral was displayed at the 1976 meeting of the American
Academy of Ophthalmology and Otolaryngology and lecture material and printed

236

The DRS has entered an analytic phase where emphasis will be placed on develop-
ing detailed descriptions of the natural history of diabetic retinopathy and
elaborating on the effects of photocoagulation treatment. These will include
attempts to identify additional systemic and ocular risk factors that are
associated with the progression of proliferative diabetic retinopathy and to
determine whether photocoagulation treatment exerts its effect by altering
these factors or by an independent route. Because it is not known how photo-
coagulation exerts its beneficial effect, attempts are being made to develop
a pathology study within the DRS. Leading ocular pathologists from around
the country have met to develop a protocol for examining eyes of DRS patients.

The DRS research group realizes its obligation beyond publication of a scientifi
paper to disseminate its findings broadly to the medical community. The group
prepared an exhibit for this purpose which was displayed at the American
Academy of Ophthalmology and Otolaryngology meeting in October 1976. The study
group plans to continue and expand this type of educational effort and attempts
are being made to cooperate with non-DRS investigators in developing consensus
for clinical practice.

During the next year the study participants and NEI staff will collaborate
to complete the study design and develop a detailed manual of operations.
Recruitment of patients should, begin by September 1978.

DRVS.

235

The vitrectomy procedure being examined in the DEVS involves an instrument
which combines cutting, suction, and infusion of a replacement solution.
The instrirment is inserted through a small incision in the side wall of the

eye .

In a number of medical centers, vitrectomy for long-standing diabetic vitreous
hemorrhage has become a standard procedure. The vision is restored to useful
levels in a moderate number of such patients who would have a high probabil-
ity of remaining blind without vitrectomy. However, the vision does not always
improve following vitrectomy in eyes with long-standing vitreous hemorrhage.
Inoperable retinal detachments or other sequelae of diabetic vitreo-retinal
disease may occur during the conventional one-year waiting period following
vitreous hemorrhage. In other eyes surgical complications of the vitrectomy
procedure itself make restoration of vision impossible. Some vitreous sur-
geons believe that the operation should be done earlier in the period following
severe vitreous hemorrhage. They hope that by performing surgery earlier,
(within the first six months of severe hemorrhage) , the development of
inoperable sequelae of the hemorrhage will be lessened and the average level
of vision restored Improved.

236

materials have been made available to DRVS investigators for use at other
local and national medical meetings. Editorials have also been written des-
cribing the DRVS for key medical journals.

In May 1977 the National Eye Institute began a special six-month monitoring
period during which the results of the recruitment efforts of each DRVS
clinical center will be tracked closely to determine the feasibility of the
original Study goals. By June 30, 1977 a total of 114 Group H and 169 Group
N had been found eligible for the Study after completion of one baseline
visit. Of these, 58 and 65 eyes respectively had been randomized or assigned
to a Study group.

237

REFERENCES
RETINAL MD CHOROIDAL DISEASES

1. A. E. Krill: The assessment of visual function. A. M. Potts (ed) , page
88, The C. V. Mosby Company, Saint Louis, 1972.

2. Geeraets, R. : EY01360-02 (annual progress report).

3. Futterman, S.: EY00343-11 (annual progress report).

4. Swanson, D., Futterman, S., and Saari, J. C. : Retinol and retinoic
acid-blinding proteins: Occurrences in human retina and absence from
human cultured fibroblasts. Invest. Ophthal. 15: 1017, 1976.

5. Futterman, S., Saari, J. C, and Blair, S.: Occurrences of a binding
protein for 11-cis-retinal in the retina. J. Bio. Chem. (in press).

6. Saari, J. C: EY02317-01 (preliminary findings). ,

7. Heller, J.: EY00704-07 (annual progress report).

8. Dratz, E: EY01521-03 (annual progress report).

9. Farnsworth, C. C. and Dratz, E. A.: Oxidative damage of retinol rod
outer segment membranes and the role of vitamin E. Biochem. Biophys.
Acta 443: 556, 1976.

10. Hayes, K. C: EY00631-06 (annual progress report).

11. Knopf, K., Sturman, J. A., Armstrong, M. and Hayes, K. C: Taurine:
evidence for its essentiality in the diet of cats. J. Nutr. (in press).

12. Schmidt, S. Y., Berson, E. L., and Hayes, K. C: Retinal degeneration
in cats fed casein. 1. Taurine deficiency. Invest. Ophthal. 15: 47,
1976.

13. Johnson, L., Sfchaffer, D. and Boggs, T. R. , Jr.: The premature infant,
vitamin E deficiency and retrolental fibroplasia. Am. J. Clin. Nutr.
27: 1158,. 1974.

14. Phelps, D. L. and Rosenbaum, A.: Vitamin E protection in experimental
retrolental fibroplasia (RLE) in kittens, presented at the Western
Society for Pediatric Research, February 6, 1976.

15. Boggs, T. R., Jr.: EY01723-01 (preliminary findings).

16. Phelps, D. L.: EY01939-01 (preliminary findings).

17. Chen, C. H. : EY01399-03 (summary progress report).

238

18. Chen, C. H., Chang, J. E., Chen, S. and Feaslau, A.: Association for
Research In vision and Ophthalmology, 1977 Abstracts, p. 118.

19. Finkelstein, D.: EY01368-04 (annual progress report).

20. Finkelstein, D., Bren, S., Miller, S. and Folkman, J.: Retinal neovas-
cularization: experimental induction by intravitreal tumors. Am. J.
Ophthal. (in press) .

21. Diabetic Retinopathy Study Research Study Group: Preliminary report on
effects of photocoagulation therapy. Am. J. Ophthal. 81: 383, 1976.

22. Friedman, E.: Personal communication.

23. Robertson, D. M. : EY01709-01 (preliminary findings).

24. Fine, S.: EY01900-01 (preliminary findings).

25. Ryan, S.: EY00944-03 (annual progress report).

26. Ryan, S.: EY01545-02 (annual progress report).

27. Vainisi, S.: EY00874-06 (annual progress report).

28. Vainisi, S. J., Fishman, G. A., Wolf, E. D., and Boese, G. K.: Cone-
rod dystrophy in gaimea baboon. Trans. Acad. Ophthal. Otolaryngol.
81: 725, 1976.

29. LaVail, M. : EY00199-02 (annual progress report).
3d. Noell, W.: EY00297-11 (annual progress report).

31. Organisciak, D. T. and Noell, W. K.: The rod outer segment phospholipid/
opsin ration of rats maintained in darkness or cyclic light, (in press).

32. LaVail, M. M. : Rod outer segment shedding in relation to cyclic light-
ing. Exp. Eye Res. 23: 277, 1976.

33. LaVail, M. M. : Rod outer segment shedding in rat retina: relationship
to cyclic lighting. Science 194: 1071, 1976.

239

CORNEAL DISEASES
Introduction

Over 2 million cases of corneal disorders and diseases occur each year in the
United States. These account for 62 percent of the total incidence of all
acute and chronic disorders, diseases, and injuries to the eye. Although the
incidence of legal blindness from corneal diseases represents approximately
6 percent of all legal blindness, corneal and external eye disorders and
diseases cause severe disability and pain and require a considerable amount
of patient care.

The anatomic position of the cornea and adjacent structures contributes much
to their susceptibility to ocular infections, allergies, and injuries. Some
of the causes of cornea disease include bacterial, fungal, and viral infec-
tions, inflammatory reactions, improper moistening and covering of the cornea
by the eyelids, birth defects, and degenerative conditions.

The following highlight research areas where successes have been achieved
during the past year in addressing certain important problems related to
coimeal diseases.

Infectious Diseases and Corneal Inflammation

Herpetic infections of the cornea are one of the most prevalent disorders
observed in clinical practice. Cavanagh reports that 46 percent (52 of 114
observed) of the corneal disease patients seen in his clinic have ocular
herpes infections.-'- This condition is seen three times more frequently than
his next highest category, post-surgical healing, which accounts for 13 per-
cent of his patients .

Forrest and Kaufman, in a case report, pointed out that it is not always
easy to diagnose viral agents without support of laboratory findings. 2 They
encountered a person with a corneal infection resembling herpes zoster which
had been described as such by another ophthalmologist, but which turned out
to be due to herpes simplex virus, type 1. The first physician treated the
patient with topical corticosteroids and atropine with negative results,
whereas with proper diagnosis the condition was treated successfully with
trifluorothymidine and prednisone.

Some recent stxidies with herpesviruses provide usefxil information concerning
treatment. Phosphonacetic acid had a significant antiviral effect when
applied topically in liquid and ointment preparations on superficial herpetic
keratitis in rabbits. ^ It is also effective in the treatment of idoxuridine-
resistant herpetic keratitis in rabbits.

In studies of the effects of high doses of adenine arabinoside (Ara-A) on
himioral immunity in white rabbits and guinea pigs, no inmunosuppressive
activity of Ara-A was detected.^

In herpes simplex studies carried out in Tunisia,^ it was found that herpes
simplex ulcers of the corneal epitheliimi that were treated by denuding the

241

cornea with a cotton-tipped applicator healed more rapidly (2.4 days) that
those treated with idoxuridine (7 days).^ Present studies are directed towards
delineating the degree of incapacity and econotoic loss incurred by patients
affected by ocular herpesvirus infections in Tunisia. Subsequent studies are
to include evaluation of the antiviral compound trifluorothjmiidine.

In studies involving herpesvirus infections and latency, virus was recovered
from the trigeminal, superior cervical and ciliary ganglia. ° During acute
phases, unilateral herpesvirus inoculation of animals which had previously
experienced herpesvirus infections did not lead to acute involvement of these
ganglia, although control animals had ganglia infections localized in the
inoculated side. Virus was shown to be latent in the ganglia of one rabbit
six months after initial infection. These pilot studies have paved the way
for future investigators to gather more information regarding the latency and
-transforming potential of this ubiquitous virus.

Jawetz has prepared a report on antiviral chemotherapy in which he describes

present day choices for treatment.' Methods were dociimented for treatment

of infection by herpesviruses and other small nucleoprote in-containing agents.

Sery has contributed valuable preliminary information concerning herpes viruses
and the immune response. 8 He separated herpes simplex virus and virus-
specified proteins and used whole virus and viral specified proteins in test-
ing the immune response of local regional lymph nodes to these antigens via
in vitro cell-mediated immunity assays. Then he observed the reactivity of
lymph node cells to both T cell mitogens and specific herpesvirus antigens
in animals with acute dendritic keratitis and disciform keratitis and found
that the two diseased states provided different responses. The lymph node
cells of animals with active dendritic lesions reacted well to both T cell
mitogens and specific herpes antigens over the first twelve days after infec-
tion. In contrast, lymph node cells removed from animals vindergoing disciform
keratitis reacted very strongly to the phytohemagglutinln but showed little
or no response to specific herpesvirus antigens.

Presently, protocols are being extended to include peripheral blood lymphocytes.
It is expected that some correlations may be found among responses observed
in peripheral blood lymphocytes, the regional lymph nodes, and the state of
the disease (e.g. acute dendritic keratitis, recirrrent infection, scar forma-
tion, etc.). Such correlations, if they exist, would be Important because in
monitoring the himian disease, the easiest access to tissue for evaluation
purposes is via blood samples. Sery's observations and the results of further
experiments could have very practical applications.

An interesting investigation is underway to assess the cause and prevalence
of superficial punctate keratitis and unclassified uveitis in Alabama.^
Punctate keratitis has an tinusually high incidence in Macon County, Alabama;
sixty-eight new cases were recorded for this area in 1976. This is a 66 per-
cent increase over that observed in 1975. This community, therefore, provides
a particularly good opportunity for classical epidemiological evaluation of
a very serious problem.

242

Settler and his colleagues are carrying out laboratory Isolation procedures
for the detection of viral agents in the cornea and conjunctiira of Macon
County residents afflicted with punctate keratitis. Preparations are being
cultivated in human amnion, kidney, and fibroblast tissue cultures. Data
from control studies indicate that no viruses exist normally in htnnan aqueous
humor. Eventually, this survey will entail serological studies also. If any
viruses are isolated, the agents will be transmitted to rabbit eyes for addi-
tional characterization.

A major goal of Dawson and colleagues is to evaluate methods of preventing
visual loss due to inflammatory eye diseases such as trachoma and acute
hemorrhagic conjunctivitis as well as those complications caused by herpes-
viruses, adenoviruses, pneumococci Haemophilus species, and Staphylococcus
aureus . They hope to provide measures for treatment, control, and prevention
of such infections. Many of the observations recorded below are a result of
conjoint projects conducted in the United States, Tunisia, and Egypt. Follow-
ing initial field studies, clinical methods for diagnosis and treatment of
trachoma were published in the Bulletin of the World Health Organization (WHO).
Recently, these procedures were recoumended officially by WHO for use in their
programs .

In family studies involving both adults and children, Dawson-'-^, Whitcherll,
et al, found marked reduction in chlamydial organisms in persons treated with
1 percent tetracycline ointment or with the antibiotic delivered in a thin
plastic wafer which fitted xmder the eyelid. As a result of these treatments,
they hope to achieve a lower trachoma reinfection rate.

Epidemiological studies of trachoma in Tunisia indicated that when inflamma-
tory eye diseases in children under ten resulted in conjimctival scarring
there was a risk of visual loss for the individuals as adults. Inflammatory
disease was due to both chlamydia and bacterial pathogens present in the con-
junctiva. Kock-Weeks bacillus was also isolated from the eyes of trachomatous
children in Egypt and Tunisia. A similar study is underway now to assess
the conJTjnctivitis caused by gonococci; there have been sporadic outbreaks
in Egypt.

A study of neonatal conjunctivitis in San Francisco showed that in the first
month of life, chlamydia was the most frequent cause. -'•^ Staphyloccus aureus
affected the eye throughout the first two years.

Kuo and Grayson, in a basic tissue culture growth experiment, infected two
different tissue culture cell lines with chlamydia and Lymphagranuloma
venerevm.-^^ They measured attachment to and inclusion within the cultured
cells in these systems and found that the rate of attachment was temperature-
dependent with both agents. Cell susceptibility was important for inclusion
formation of trachoma.

Bacterial products have been implicated in pathological processes affecting
the cornea. Recently, evidence was presented indicating that a penumococcal
cytolysin contributes to the pathogenesis of corneal disease. ^3 por some
time, investigators have accepted the idea that, in various types of bacterial

243

infections of the cornea, cytolytic agents activate host degradative enzymes
and thereby contribute to the breakdown of corneal tissue. Johnson demonstrat-
ed intracellular localization of pneumococcal lysin and recovered extracellular
Pseudomonas aeruginosa hemolysin. 13 These studies are continuing with a view
toward understanding the genetic basis of lysin production, binding require-
ments, mode and site of lysin action, and the use of inhibitors for control
of degradative processes.

Identification, treatment, and control of fungal agents which can infect the
cornea remains a continuing problem. Recently, new successes and new diffi-
culties which warrant attention have been reported. A shortened version of
Grocott's methenamine-silver technique for use in staining corneal scrapings
was devised, and this procedure was found to be superior to current methods
for the diagnosis of mycotic keratitis.!^

A double infection by Phialophora verrucosa (Medlar) and Cladosporium
caladosporioides (Frescenius) was reported. ^^ These fungal agents, found
infrequently in corneal infections, were isolated from a corneal ulcer. The
condition did not respond to antivirals, steroids, and Pimaricin nor to the
conjunctival flap procedure. Penetrating keratoplasty was performed with
good functional and optical results.

Human transfer factor was administered locally and systemically to guinea
pigs with Candida albicans keratitis or those with herpetic keratitis; how-
ever, this type of therapy did not alter the course of the disease in these
animals . l"

Rebell and Forster described four cases of human keratitis caused by the
tropical fungus, Lasiodiplodia theobromae , which was encountered recently in
the Miami, Florida area.l' (^ere are only eight known cases in the world).
The fungus was found to be endemic in Miami, where it was recovered from
home-grown and imported bananas. In vitro polyene antimycotic antibiotics
were fungicidal for the agent. When rabbits were inoculated with fungal
isolates from these patients, progressive corneal ulcers were produced;
these organisms appear to have collagenase activity.

Effects of Drugs, Metabolites, and HorrBonal Substances on the Cornea

Pharmacokinetic studies were carried out in the eyes of rabbits and man.l^
Following subconjunctival injections of various tracers, their movements were
observed. It was found that the penetration of fluorescein dye
into the anterior chamber of the eye, when administered subconjunctivally,
was 100 times greater in man than in the rabbit. These observations suggest
that drugs which are intended to be administered by subconjunctival injection
must be tested this way in man before being accepted for general use.

A new investigation was started at the Edward S. Harkness Eye Institute,
Colimibia University on the allergic reactions of conjxonctival tissue. 19 This
project involves a collaboration between Dr. W. J. Manski (ophthalmology) and
Dr. K. E. Eakins (pharmacology). Taking advantage of the fadt that the con-
jimctiva is a lymphoid mucous membrane which participates in many diseases

244

of the reticulo-endothelial and lymphatic systems and which has pronounced
elfects on iaamsie responses, these investigators observed a powerfiil syner-
gistic effect of histamine and prostaglandin when applied simultaneously to
the conjunctiva. When these dirugs were used separately, there was greater
vasodilation and edema. Histamine in combination with prostaglandin E induced
an inflammatory response associated with severe cellular infiltration of the
conjunctiva; this was not foxmd when these drugs were applied separately.
Optimal response was obtained in about 45 minutes and persisted one day.

A compoimd designated 48/80 and known to degranulate mast cells was tested in
the rabbit conjunctiva; vasodilation and edema developed. In this case, the
response reached its peak in 24 hours. Experiments of longer duration using
compound 48/80 are planned in an effort to understand the role of histamine-
like agents as mediators of anaphylactic reactions in the eye. It is of
interest to note that this cross-discipline collaboration has produced very
rewarding data in a short time.

In studies20-24 involving the kinetics and mechanism of corneal transport of
pilocarpine and f luorometholone the rate limiting step £ot pilocarpine was
shown to be in epithelial uptake, whereas for f luorometholone, diffusion
through the stroma was rate limiting. The investigators established rate
constants for uptake and disposition of these drugs and refined experimental
procedures for studying epithelial and conjimctival concentrations of various
types of drugs. Also, protein binding studies, were carried out with sulfiso-
xazole.^0 It was shown that this drug attaches to various protein fractions
such as alpha globulin, gamma globulin and lysozyme. The data quantitatively
accounted for the overall binding of this drug in the protein fractions of
human tears.

Baum and associates studied ocular penetration of antibiotics in a model sys-
tem. ^^"26 They also studied concentration effects, interference reactions,
and binding of antibiotics to various components such as pigments. They
hypothesized that a tight binding of antibiotic to pigment occurred. They
found that synthetic melanin (100 to 1,000 ug per ml) inhibited the in vitro
activity of aminoglyosides and tetracyclines, but did not affect the activity
of beta lactam antibiotics, erythromycin, or clindamycin.

Several innovative projects relating to nutritional and metabolic factors were
initiated during the past year. Pfister and associates studied epithelial
and stromal repair in alkali-burned rabbit eyes. 27, 28 They observed depressed
ascorbic acid levels in samples of aqueous humor taken during the period of
repair. Following a 20 sec, 12 mm diameter, 1.0 N sodium hydroxide burn,
glucose levels in aqueous humor returned to normal in a short time but ascorbic
acid levels remained depressed for up to 30 days. Nearly all corneas become
ulcerated and corneas in about 60 percent of the animals tested perforated.
Following 12 mm alkali bums, rabbits treated daily with 1.5 grams ascorbic
acid sub cutaneous ly rarely developed corneal ulcerations and no perforation
occurred. It was suggested that exogenous maintenance of adequate levels of
ascorbic acid in aqueous humor overcame the relatively scorbutic state of the
anterior segment induced by alkali bums and thereby blocked the development
of corneal ulceration and perforation. Elevated levels of ascorbic acid in

245

aqueous humor has no influence on corneal epithelial cell migration patterns
following alkali bums. Pfister now is preparing to conduct a clinical trial
of topical ascorbic acid therapy in patients who have been victims of corneal
burns or who have fungal or herpetic Involvement of the cornea to see if it
is as effective in humans as it is in the animal model.

In October 1976, a workshop on keratomalacia was conducted by the National
Eye Institute. ^^ An important objective of the meeting was to spark renewed
interest in studies involving the effects of vitamin A deficiency on the
cornea. An expansion of research in this area is expected to result from
this workshop.

The effects of several agents which increase intracellular levels of cyclic
AMP (cAMP) in corneal epithelial cells were studied by Cavanagh. For some
time, it has been known that patients who use epinephrine habitually for the
management of glaucoma often suffer from recurrent punctate keratitis of the
corneal epithelium. Furthermore, previous work showed that increased intra-
cellular levels of cAMP, when mediated by prostaglandins, epinephrine, and
nor-epinephrine, cause a profovmd decrease in cell locomotion and a corres-
ponding increase in cell adhesion; in fact, an increase in the intracellular
level of cAMP in many eukaryotic cells was foiind to be a growth inhibitory
signal. Cavanagh and colleagues, in preliminary experiments, added dibutryl
cAMP in concentrations ranging between 10~2 and 10~6 M, with and without
theophylline at the same molarity, to epithelial cells cultured in various
media. 30 xn all cases they observed that all growth activity ceased at levels
above 10"^ M cAMP and theophylline. They observed the same effect of epi-
thelial regrowth on whole corneal buttons placed in organ culture and on
reepithelialization of gently denuded epithelium in rabbit corneas in vivo.

Because clinical inflammation often is associated with persistent lack of
healing of epithelium in human patients, they attempted to reduce inflamma-
tion in both animals and hvmians with non-steroid anti- inflammatory substances
such as aspirin and indomethacin. These agents are known to inhibit the
synthesis of prostaglandins which in turn presumably act to raise intracellu-
lar levels of cAMP. Cavanagh 's studies indicated that these non-steroid
anti- inflammatory agents seem to influence healing of persistent epithelial
defects in a positive way in animals; however, the effects are not marked in
man. A double-blind clinical evaluation is planned. Fenoprofen will be
included in future clinical evaluations .

Functional Aspects of the Corneal Surface, Stroma and Endothelium; Hydration
and Transport Mechanisms.

Research in corneal surface chemistry is currently aimed at determining the
amino acid composition of proteins foimd in secretions from meibomian glands.
Glough found that proteins comprised about 12 percent of the total dry weight
of preparations extracted with benzene and water from secretions expressed
from 150 eyelids. Following polyacrylamide gel electrophoresis, six protein
bands were obtained and molecular weights were estimated. The fast moving
band had the same mobility as myoglobin. Preliminary amino acid analyses of
total proteins indicated that glycine and serine were present in amounts higher

246

than those generally found for the composition of proteins. A careful analysis
of all protein bands is xmderway.

In the cornea, fibrils scatter light and transparency results from interference
effects due to an ordering in the spatial arrangement of the fibrils about one
another. Farrell is interested in this ordering and its disruption in abnor-
mal corneas. -^-^ By light scattering and electron microscopic evidence, he
shows that during swelling there are regions void of fibrils which result in
the formation of so called "lakes", and this condition alters light transmis-
sion. An apparatus recently developed by Kim provides a procedure for measur-
ing light scattering from the cornea at small scattering angles which is very
useful in testing the above hypotheses. 33

Pseudomonas protease elaboration studies and their collagenase activities on
ocular tissties are imderway.' Kreger purified various pseudomonas proteases
and found that they had an isoelectric point of approximately seven and a
mol. wt. of approximately 20,000 (as determined by gel f iltration) . 3^^ He
plans to assess the effects of pseudomonas proteases on the integrity of
corneal cells maintained in tissue culture. Currently, Kiorpes and associates
are testing the possibility that destruction of the cornea may result from
advanced vitamin A deficiency because of the inability of the system to con-
trol collagenase. 35 They show that alpha- 1- macroglobiilin inhibits collagenase
liberated from ulcerating (alkali-burned) corneas maintained in organ culture.
Also, they show that in severely vitamin A-deficient rat corneas maintained
in culture, both collagenase and hydroxyproline are released. The latter is
a product of the enzymes' action on the corneal stroma. Experiments are in
progress to attempt to show that "corneal melting", which occurs in vitamin
A deficiency, is related to the decline in serum alpha-1-macroglobulin.

Dibutyryl cyclic AMP (D.B. cAMP) and theophylline partially inhibit the appear-
ance of collagenase activity when added to cultures of ulcerating rabbit
corneas. 36 Drug treatment with D.B. cAMP and its hydrolysis product 5 AMP
"both suppress collagenase activity and inhibit the degradation of explant
collagen. The exact mechanism by which these drugs act is not known, but it
is under study. It may be that these drugs affect the synthesis and/or the
secretion of collagenase. Berman and associates suggest that "first messengers"
exist which can prevent the secretion of corneal collagenase by raising the
endogenous cAMP level in corneal cells through stimulation of the adenyl
cyclase system. 36 They suggest further that treatment of corneal ulceration
may require intervention at several levels such as biosynthesis, secretion,
activation and collagenase activity.

The corneal endothelium has vmdergone intense investigation over the past
year. Use of the specular microscope has contributed significantly to the
success story. 37 Bourne and Kaufman, using a modified clinical specular
microscope at high magnification in routine examinations of forty patients,
were able to detect endothelial damage or disease that was not seen by slit-
lamp examination. 38 By means of the specular microscope, two groups of
investigators showed that the endothelixan does not regenerate by cellular
division following injury but that the remaining' cells spread and enlarge in
order to cover the space left by loss of injured cells. 39, 40 According to

247

Van Horn, the decrease in endothelial cell density following cataract extrac-
tion could make the patient susceptible to further insult. ^1 In the rabbit
cornea, the sitiiation is different. Endothelium of the rabbit cornea regen-
erated following injury by cell division of cells at the margin of the wound.
New cells migrated and replaced destroyed cells. Van Horn foimd that the
regenerative capacity of the corneal endothelium of the cat was similar to
that of man and other primates. '^1 The cat appears to be a good, inexpensive
model to study ultras tructure and function of the stressed corneal endothelium.

Other interesting investigations concerned changes in the corneal endothelium
as a function of age.^0 These changes were measured following in vivo specu-
lar photomicrography. Sixty-one normal volunteers of both sexes ranging in
age from 20 to 89 years were studied. Endothelial photographs were taken of
the cellular area located in the central cornea. Cells were measured and the
mean endothelial cell area was found to double approximately from age 20
through 80. When these investigators, using the clinical specular microscope,
photographed the endothelivmi of eyes of fifteen patients who had iindergone
successful penetrating keratoplasty, they found that the average endothelial
cell area was one to six times larger and that the average cell perimeter
was two and a half times larger than that of a normal cornea of a subject of
the same age as the donor. ^^ The thickness and transparency of each graft
was normal. In general, the aging process and trauma apparently had similar
effects, namely a loss of destroyed cells with an increase in the size of
remaining cells in order to cover a given denuded area.

By means of transmission electron microscopy, the addition of reduced gluta-
thione (GSH) to a bicarbonate Ringer's solution, during in vitro perfusion
of the corneal endothelium was shown to maintain endothelial ultras tructure,
to increase the efficiency of the endothelial pump, and to prevent depletion
of cellular ATP. '^3 Diamide, a thiol-oxidizing agent, which stoichiometrically
oxidizes intracellular glutathione to the disulfide (GSSG) has been used to
study the temporary effects of GSH. The results of Edelhauser and colleagues
suggest that the ratio of reduced to oxidized glutathione in endothelial cells
play a role in the maintaining endothelial cell barrier function. ^-^ This
observation is a step toward trying to define the physiological and bioche-
mical roles of glutathione in living cells.

In addition to the above, it was found that endothelial cell damage to isolated
rabbit and human corneas perfused in vitro resulted in increased corneal
thickness due to incomplete composition of 0.9 percent NaCl, Plasma- lyte 148,
and lactated Ringer's solution. ^^ Endothelial cell damage could be prevented
if the intraocular irrigating solution contained concentrations of inorganic
and organic constituents that were similar to those found in aqueous humor.

The contribution of electron microscopy to the study of corneal pathology
should be docxanented.^^ Keratoplasty was shown to be the most important
source of specimens for ultramicroscopic investigation. By means of these
techniques it has been possible to confirm many light microscopic studies in
pathological corneas. Such investigations have been helpful in identifying
sites and types of pathological changes in corneal dystrophies and degenera-
tions. Also, it has been possible to observe histologic effects of corneal
inflammatory conditions as well as of metabolic disorders affecting corneal
transparency .

248

Scanning electron microscopy has proved to be a very useful tool. This was
brought to the attention of those who participated in a Workshop on Biomedical
Applications of Scanning Electron Microscopy '(SEM) held in 1976. ^^ On this
occasion. Van Horn presented and published subsequently a report which con-
cerned corneal endothelial regeneration, a comparative study in the rabbit
and cat using scanning electron microscopy techniques. ^6

A very progressive look, presented by Hart, concerned biosynthesis of glycosa-
mino-glycans during corneal development. ^7 This investigator was interested
in the role of keratan sulfate in the regulation of corneal transparency.
He observed the rate of incorporation of 3h and 35s-labeled keratan sulfates
into glycosaminoglycans up to and beyond the fourteenth day. The proportion
of % and 35s in keratan sulfates reached near maximum levels as early as
the ninth day. On and after the fourteenth day, keratan sulfate appeared to
become more highly sulfated. Hart suggested that these and other changing
patterns of glycosaminoglycan biosyntheses during corneal development play
an important role in corneal morphogenesis and in the development of corneal
transparency.

Numerous studies regarding corneal hydration, transport and storage have pro-
vided important information. ^^5 48-54 Bowman and Green used the specular
microscope to determine the effect of varying hydrostatic pressure on the
thinning rate of pre-swollen de-epithelialized or de-endothelialized cornea. ^8
De-epithelialized corneas thinned more slowly as hydrostatic pressure on the
posterior surface was increased, until fluid movement stopped at 60 to 70mm
Hg pressure. Fluid moved from the stroma toward the aqueous humor against
considerable hydrostatic pressxire. De-endothelialized corneas thinned at a
higher rate as hydrostatic pressure was increased forcing fluid out across
the epithelitmi, indicating that this effect was probably mechanical with
increasing pressure. Therefore, the fluid was shown to move against an
increased hydrostatic pressure. These investigators concluded that there
must have been a considerable physiologic force which caused movement across
the normal endotheliijm.

Studies to understand endothelial physiology were proposed by Van Horn and
associates. ^1 Investigations by Maurice and colleagues failed to show any
differences between the ptmiping and non-pumping state of the cornea in the
behavior of inorganic ions (22Na, 36ci, h1^C03).^9 They concluded that they
were dealing with either a neutral salt pump, a bulk flow pump, or a bicar-
bonate pvmip. For other solutes the permeability of the endothelium appeared
to bear a linear relationship to the free diffusion constant of the molecule.
Their observations continue to suggest that solutes pass the cell layer by
paracellular pathways. Development of an automatic volumetric technique
designed to measure fluid movement across the endothelial layer allowed
investigators to achieve resolution down to one nanoliter.50,51 Using, this
refined technique, the presence of 5mm adenosine was foimd to stimulate fluid
transport by as much as 50% when the rates measured were compared to controls
in which adenosine was omitted. Data from calculations by Fischbarg and Lim
suggest that fluid flows partially through a route other than the cells' mem-
brane proper. 50 '^1 Though additional experiments are necessary, they suggest
that the standing-gradient model might not be appropriate for the endothelial
layer .

249

Insulin was found to have a biphasic effect on fluid transport, stimulating
it at concentrations lower than 10~10m but inhibiting it at concentrations
larger than 10~%. Ouabain was found to stimulate transport below 10~% and
inhibit it above 10~7m. These findings raised the question that perhaps the
insulin effect might be explained in terms of its modulation of transport
ATPase activity. At present, this group is considering more theoretical
aspects of the fluid pimiping mechanisms. ^0> 51

Immune Response, Corneal Transplantation, and Storage

Two interesting experimental systems were developed by Maurice and Perlman
which may be useful to others in studies involving immune response.-'^ They
devised an experimental method of destroying rabbit endothelium by the use of
benzalkonitmi chloride. ^9 Then they plated endothelial cell suspensions onto
denuded rabbit stroma and used this tissue to graft onto de-endothelialized
eyes. Preliminary studies showed that 5 x 10° cells per cornea would produce
a uniform monolayer. Experimental transplant studies in rabbit eyes are in
progress.

Maurice and co-workers developed a leukopenic rabbit model by administering
two whole-body irradiation exposures (1400 roentgens per dose at a two-day
interval, with eyes shielded).^" By the fourth day following this treatment,
the peripheral white blood cell count was 0.5 percent that of normal values.
Experiments using normal and irradiated rabbits were carried out. In eyes
injured by means of thermal cautery made 2ram from the limbus, vessel growth
occurred by the third to fourth day. The technique demonstrated that vascu-
larization did occur in leukopenic animals. Animals treated as described
might be uiseful in many t3T)es of related experiments which have to do with
the immune response.

This year, studies in ocular immunology dealt with many facets of immunology.
Allansmith remarked that according to current methods, neither HL-A nor ABO
typing predicted the outcome of corneal transplantation. 5° Neither ABO nor
HL-A incompatibility could be shown to be factors in corneal graft failures.

Immunoglobulin assays were made in several systems. Tissue, tear and serum
IgE concentrations were measured in normal subjects and compared with prepara-
tions taken from patients with^vernal conjtmctivitis.^^ Allansmith and
co-workers questioned whether an immimogenesis of IgE plasma cells occurred
in areas of the upper conjunctiva in vernal conjunctivities.57 Half of these
patients had abundant IgA, IgD, and IgE forming plasma cells. These immuno-
globulin forming plasma cells were absent in normal subjects. Tear IgE values
did not vary significantly in normal and patient assays. Serimi IgE was much
higher for patients than for normal subjects. Allansmith, et al concluded
that their findings were consistent with hyperplasia of IgA, IgD, and IgE
antibody-forming cells in the tarsal conjunctiva of some patients with vernal
conjvmctivitis. ^^

In other experiments estimates of the numbers of plasma cells in the main
lacrimal gland (3.2x10^), the conjtinctiva (2.1xl06) and in the accessory
glands (1.8x10^) were made. 58 Since there were so many cells in the relatively

250

Inaccessible lacrimal gland, it was suggested that cells of the lymphatic
series were attracted to the lacrimal gland area by non-antlgenic mechanisms.

Abelson and associates demonstrated histamine in hinnan tears, which indicated
a possible role for this mediator in both physiologic and inmimologic pro-
cesses of the external eye.^^

Khodadoust and Silverstein described studies that extended their earlier work
on lymphocyte-mediated destruction of corneal grafts. 60 in the rabbit, des-
truction of the histo-incompatible corneal endothelium was marked by the
formation of focal pock-like damaged areas, rather than by the typical mov-
ing line of rejecting endotheli;mi usually seen in spontaneous graft rejection.
Where the transferred lymphoid cells were compatible with the tissue of the
graft recipient, the plctiore was one of a severely affected graft on a field
of iminvolved recipient corneal endothelium. Where the lymphoid cells were
compatible with the graft and not with the tissues of the recipient, a clear
corneal graft survived on a bed of endothelial destruction. These studies
have contributed an experimental model for immunological testing of graft-
versus-host reactions.

Sjmdromes associated with conjvinctivltis and other immimological ocular com-
plications of contact lens wearers were studied. 61j62 Allansmith reported
that microorganisms served as antigens to initiate an immune response in
himian subjects. 61 Bacteria present on lid margins were foimd to shed products
into tear films. Normally these products were washed away but not in cases
where people wore contact lenses. Particles stuck to lens surfaces and
acted as stimuli to produce hypersentivitiy reactions.

Another distressing syndrome observed in both hard and soft contact lens
wearers was characterized by increased mucus, itching, decreased lens toler-
ance and giant papillae formation in the upper tarsal conjunctiva. 62 Biopsies
of the upper tarsal conjunctiva revealed basophils, eosinophils, mast cells,
lymphocytes, and plasma cells. Allansmith, et al postulated that the sjmdrome
was immimological in origin and that it was a major cause of difficulty in
wearing contact lenses."'^

Obviously, immune response in the cornea must be docimiented further and char-
acterized in order to provide Measures for prevention and treatment of the
various complications. As a consequence, in order to stimulate increased
research interest and activity in the field of ocular immimology, a grant
announcement entitled "Immunological Effects on Corneal Disease Processes",
is in preparation. Also, an international symposiiim planned by Dr. A. M.
Silverstein, "Ocular Immunology and Immimopathology", will be held in May
1978.

The consensus of the scientific community is that studies concerning ocular
immunology, especially those pertaining to the cornea carry a high priority
value at this time. To address ongoing investigations with continued fervor
and to expand the program where limitations are apparent is an important aim
of the present National Eye Institute Corneal Disease program.

251

Finally, in relation to storage, problems which encompass various Immune phe-
nomena deserve the attention of clinical ophthalmologists.

Continued studies by Kaufman and associates with M-K medium, used for storage
of corneal tissue at refrigerator temperature, showed that 80 percent of 92
human corneas stored for up to 7 days in this simple tissue culture medium
remained clear and thin 2 months after keratoplasty. ^3 Deturgescence rates
of tissue stored in this medium compared favorably with those for fresh and
cryopreserved tissue. Hull and co-workers showed that dextran passed into
rabbit corneas stored in M-K medium prior to corneal transplantation. 64-66
In about 24 hours, an equilibrium between dextran in the cornea and M-K medium
was reached. Post-keratoplasty dextran efflijx was rapid with about 70 percent
to 75 percent of the compound lost in 12 hours.

During a three year interval, Doughman and colleagues maintained numerous
corneal organ cultures at 37° in order to study changes with time and to
establish a model for experimental pathology. 67 Many animal studies were
conducted before experiments with human tissues were performed. Human corneas
retained in organ culture maintained ultrastructural integrity, glucose meta-
bolism, normal levels of lysosomal and cytoplasmic enzjnnes and physiological
deturgescence for at least 35 days. Clinically, 75 penetrating keratoplasties
using corneas incubated for an average of 14 days were performed with results
as good as those obtained when currently accepted storage methods were used.
There were no problems with infected tissues. A significant finding in animal
model systems was that with chicken to rabbit transplant experiments, xeno-
graft rejection was delayed or prevented when corneas had been maintained
three weeks in organ culture. Numerous assessment studies related to the
above findings were reported to be underway.

252

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66. Hull, D. S., Bowman, K. , and Green, K. : Effect of brinolase on corneal
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257

CATARACT
Introduction

Cataract, an opacification in the lens of the eye, interferes with the passage
of light through the eye and results in visual impairment. This disorder
accounts for approximately one-sixth of all -cases of visual impairment in the
United States. There are about 300,000 operations to remove cataracts per-
formed each year, yet there are an additional 1,670,000 Americans who have
difficulty seeing because of cataracts developing in one or both eyes. Cata-
racts have necessitated approximately 2,725,000 annual visits to eye doctors
in recent years. The physical limitations imposed on the afflicted individuals
and the cost to the public as a resvilt of cataracts are considerable.!

The varieties of cataract affecting humans and encompassed by the National
Eye Institute's Cataract program include senile cataract; diabetic cataract;
congenital, metabolic and genetic cataracts; and cataracts induced by drugs
and radiation, and those which occur secondary to other eye disorders. The
Cataract program is also concerned with such subjects as accommodation and
optical problems related to cataract and aphakia.

The research interests of the Cataract program include determination of the
causes of cataract, means of preventing or retarding cataract development,
prevention of amblyopia in children with cataract, development of new methods
for correcting optical problems following cataract surgery, evaluation of the
safety and efficacy of new methods of cataract extraction, and efforts to
improve the life cycle and adaptation to new spatial relationships of patients
who have undergone cataract surgery.

Interdisciplinary approaches to studies of the normal and cataractous lens
are currently gaining favor in the quest for knowledge related to cataracto-
genesis. These studies employ the evolving methodologies and technologies
"of such diverse disciplines as anatomy, embryology, cellular and molecular
biology, genetics, biochemistry, physical chemistry, physiology, optics,
microbiology, immunology, pharmacology, pathology, experimental ophthalmology,
epidemiology, bioengineering and clinical research (with controlled clinical
trials rapidly becoming an Important aspect of the latter) .

Several hypotheses have helped shape the nature of contemporary research on
the lens and on the physiochemical mechanisms underlying lens transparency.
Current research emphasizes the importance of controlled hydration,^ as well
as the size and state of lens protein molecules^ in determining the ability
of the lens to transmit and refract light properly. Hypotheses being tested
include the concept of osmotic shock as a factor in the alteration of cell
relationships and subsequent development of lens opacification, the thesis
of protein change with proteolysis or aggregation of proteins as factors in
the disruption of light transmission, and the possibility that transport
mechanism abnormalities, with consequent alteration of metabolism, may con-
tribute to cataract formation.

Previous Annual Reports of the National Eye Institute, particularly those from
FY 1973 through FY 1976, provide examples of the avenues of research being

259

pursued in many of the subprograms listed above. The present report emphasizes
some of the recent initiatives undertaken by the vision research community in
an effort to exploit and refine the scientific and technological advances of
prior years.

Cooperative Cataract Research Group

A number of investigators in cataract research have organized themselves into
a Cooperative Cataract Research Groun (CCRG) . The concept of the group was
unveiled at the meeting of the Association for Research in Vision and Ophthal-
mology in early 1976 and received the endorsement of the National Advisory
Eye Council in late 1976. Those forming the cooperative group believed that
despite the significant advances achieved in research on animal cataracts,
only limited progress had been made toward understanding the nature of human
cataracts. Thus, a concerted effort on human cataracts is needed. The CCRG's
members believe that the problem can best be approached by a well-coordinated
team of experts representing a variety of research disciplines. The Intention
of the CCRG is to foster closer collaboration among investigators who are
widely dispersed geographically.

The scope of intent, operations, and needs of the CCRG is captured within the
Proposed Research Program for the Cooperative Cataract Research Group.*

"The immediate objective of the proposed collaborative
effort is to establish various morphological, biochemical,
physiological and biophysical characteristics for the
human lens since there is little reliable data available
at the present time. The second step is to assess the
changes In these parameters as the result of the catarac-
tous process. A successful program in human cataract
research is feasible by combining the technology that
is already available in several laboratories in a coor-
dinated fashion. This approach also has the advantage
that with only slight additional effort in each of the
laboratories where already active research is going on,
the proposed studies can be successfully carried out
without the need of developing or refining new
techniques."

The CCRG has arranged for the collection and distribution of normal and cat-
aractous himian lenses throughout the United States and abroad. These lenses
are to be obtained from eye banks, at autopsy, and as a result of surgical
procedures involving cataract removal or enucleation. The CCRG has adopted
standard practices for handling, photographic recording, classifying, storing
and shipping cataractous and normal hxman lenses. The cooperative group's
research data will be collected and disseminated from a coordinating center
located in the Laboratory of Vision Research, National Eye Institute. Tele-
phone conferences and meetings of cooperative group participants to examine

*" Characterization of Human Lens in Health and Disease", Minutes of Meeting,
NAEC, Sept. 13-14, 1976 p. 11-13.

260

results and plan future experiments are to be held periodically. Members of
the CCRG's Executive Committee are G. W, Barber, Wills Eye Hospital,
Philadelphia, Pennsylvania; L. T. Chylack, Harvard Medical School, Boston,
Massachusetts; E. Cotlier, Yale University, New Haven, Connecticut; J. E.
Harris, University of Minnesota, Minneapolis, Minnesota; V. E. Kinsey and
V. N. Reddy, Oakland University, Rochester, Michigan; A. Spector, Columbia
University, New York, New York; and J. H. BCinoshita, National Eye Institute,
Bethesda, Maryland.

To fund the CCRG's initial activities, the National Eye Institute has provided
one-time administrative supplements to the pre-existing grants of eighteen
extramxiral CCGR participants.

The CCRG may well serve as a model for close collaboration by a number of
geographically dispersed NEI grantees who are interested in sharing materials
and data related to a specific health problem. The coming years will cer-
tainly test the value of this collaborative mechanism.

Irradiation- Induced Alterations in Lens Cells

X- irradiation of the lens continues to interest investigators who believe that
the rapidly elicited cellular dysfunctions produced in this model may be com-
parable to events occurring after other more insidious forms of cataractogenic
insults. However, utilization of the model depends, in part, upon defining
the locale and nature of those cells susceptible to X-irradiation. Worgul
and colleagues, using autoradiographic techniques, i.e. incorporation of
tritiated thymidine into dividing cells, have provided evidence that the cells
of the germinative zone of the lens epithelium behave in a demonstrably dif-
ferent fashion following X-irradiation.^ Cells derived from the germinative
zone at the time of irradiation gradxially develop at3rpical nuclear character-
istics and undertake an unusual migration to the posterior region of the
lens where cortical cataract is observed shortly thereafter. Identification
of the critical cell population affected by X-irridation permits greater
accuracy in future studies directed at determining the intracellular mal-
functions reponsible for the formation of cataract.

Role of Glutathione in Maintaining Lens Transparency

Glutathione has been studied extensively in the lens, yet considerable con-
troversy remains regarding its actual physiological role and its possible
relationship to maintaining lens transparency. Glutathione concentration in
the lens is normally very high but decreases steadily with age and in the
course of the formation of practically all cataracts. However, whether the
decrease of glutathione initiates or is only coincidental with cataracto-
genesis is not known. In recent studies Giblin and coworkers have investi-
gated the relationship of glutathione to the trsmsport function of the lens
epithelium. 5 Their findings indicate that the concentration of glutathione
in the epithelial cells is five-fold greater than in other parts of the lens
and that the oxidation of glutathione leads to pronounced changes in this
tissue. The changes include: (1) reduction in the activity of Na+-K+ATPase ;
(2) shift in the distribution of sodium, potassium, and chloride ions; (3)

261

increase in hydration; and (4) decrease in the active transport as well as
increase in the passive diffusion of labelled-rubidium. The inactivation
of Na+-K+ATPase , resulting from the depletion of glutathione, is of consid-
erable interest since several types of cataracts, including those of the
Nakano strain of mice, are characterized by a 50% reduction in the Na+-K+ATPase
of those lenses which ultimately become cataractous. Reversing the conse-
quences of glutathione depletion remains an. elusive yet challenging aspect
of research directed at understanding the development of cataracts as well
as a medical treatment for this disorder.

Aggregation of Lens Proteins and Cataractogenesis

Earlier studies of the lens have revealed the age-dependent increase in the
amount of the water-insoluble albuminoid fraction and soluble high molecular
weight aggregates. Lens protein aggregates of greater than 50 million daltons
from several mammalian lenses, including that of man, have been isolated and
characterized. Electron microscopy has also demonstrated the presence of
large protein clusters in the cataractous human lens. These aggregates are
large enough to scatter light in the visible range, and at sufficiently high
concentration they may lead to lenticular opacification. An understanding
of the formation of senile cataract therefore requires knowledge of the mecha-
nism of aggregation and insolubilization of lens proteins .

The aggregation of a well-defined lens protein, bovine alpha-cry stallin, has
been under increasing scrutiny because: (1) it is the best characterized
macromolecular lens protein and appears to be the only soluble macromolecular
crystallin present in medium of high density and viscosity, simulating the
in vivo condition, and (2) the low molecular weight form found in young fibers,
containing more than 40 polypeptide chains, is the primary contributor to the
formation of high molecular weight and insoluble proteins in the bovine lens.

Li and collaborators have conducted studies to delineate the aggregation and
interaction of the subunits of calf low moleciilar weight alpha-crystallin.
Carboxymethylation of the A chain polypeptide with iodoacetic acid resulted
in a drastic decrease to half the original size of this protein. A maximum
of 80% of the total sulfhydryl groups of the native protein can be altered,
indicating that the remaining 20% are blocked or situated in the interior of
the alpha-crystallin macromolecule. No change in the polypeptide backbone
conformation was detected. Histidine and tyrosine residues were not involved
in the decrease in size of the carboxjnnethylated native protein. The native
macromolecule is visualized as a dimer of 12 S components that is stabilized
cooperatively by a cysteine site and a hydrophobic site. This is the first
demonstration of the formation of a discrete entity by the modification of
no more than one sulfhydryl group per A chain polypeptide.

Conversely, iodination of the tyrosine residues of low molecular weight alpha-
crystallin leads to gradiial increases in the size of the protein. A maximvmi
aggregate size 28S was obtained when all the tyrosines were modified.

The foregoing results demonstrate that variously sized populations of low
molecular weight alpha-crystallin can be prepared through chemical modification.

262

Knowledge of the formation of these differently sized aggregates provides an
ijnderstanding of the role of functional groups in causing increased aggrega-
tion of lens proteins. Researchers believe that once the dynamics of the
aggregation of the macromolecule are understood similar analysis may be
applied in studies of human tissues. Insights thus acquired may ultimately
lead to a reasoned approach to prevention, delay, and reversal of cataracts.

Medical Approaches to Cataract Treatment and Prevention

Since the events leading to the formation of senile cataract remain relatively
obscure, there is as yet little firm scientific basis from which to propose a
rational medical treatment of the disorder. A particularly distressing hind-
rance is the absence of an animal model that is truly representative of human
senile cataract.

Sugar cataracts, including the diabetic variety, appear to offer a more sub-
stantial footing upon which to base treatment aimed at preventing, delaying
or reversing lens opacifications due to metabolic dysfunctions associated
with carbohydrate metabolism. Aldose reductase catalyzes the conversion of
glucose and galactose to their respective alcohols. Evidently these polyols
accummulate within the lens resulting in osmotic swelling, loss of selective
permeability and, ultimately, lens opacification. Thus inhibitors of aldose
reductase have been tested in an effort to prevent or delay the cataractous
process, and some success has been achieved in delaying the onset of cataracts
in galactosemic animals.

The clinical implications of such findings are as yet unclear since lens prob-
lems associated with galactosemia can be dealt with rather easily by withdrawing
galactose from the diet.^ In diabetes the problem is somewhat more complex.
Though the hyperglycemia of diabetes can be controlled by insulin therapy
and/or diet, many complications associated with the disorder appear to go
unchecked. Fxirthermore, S3mergistic effects, i.e. environmental and genetic
•factors plus diabetes, have been suggested as contributing to the earlier
onset and maturation of cataracts in diabetics. If this proves to be the
case then long term therapy, i.e. the administration of appropriate aldose
reductase inhibitors, could be of appreciable benefit to diabetics. -^j '

A subject that has been receiving increasing attention recently is the fxinc-
tion of the enzyme superoxide dismutase, an inactivator of the potentially
harmful superoxide radical. The superoxide radical is generated in various
enzymatic and non-enzymatic oxidation-reduction reactions, with light often
the catalyst. Since the lens contains compounds with the capacity to pro-
duce the superoxide radical and is also the recipient of direct photochemical
energy there is a need to understand how the lens protects itself from the
insults rendered by the radical. Studies have shown that superoxide dismutase
is present in the lens,^ though primarily in the lens epithelium. 9, 10 Thus
lens fibers lack superoxide dismutase, as well as other enzymes, e.g. catalase,
that protect against the superoxide radical and its derivatives. Kuck has
suggested that "this lack of protection may be critical in the human lens
nucleus where the normal pigment absorbs long-wa-\re ultraviolet light, trapping
energy which can decompose sensitive molecules or produce polymerization. "10

263

The latter may be especially relevant in light of Specter's studies suggesting
that gradual reorganization of proteins within the lens nucleus is responsible
for loss of lens transparency.^

Diabetic Cataract

As stated above, sugar cataracts are provoked by radical changes in lens
hydration resulting from the accvmiulation of large amounts of sugar alcohols.
The conversion of cataractogenic sugars to these polyols is catalyzed by the
enzyme aldose reductase. Nevertheless, these cataracts can be retarded or
prevented by lowering the polyol levels in the lens either by withdrawing the
offending sugar or with inhibitors of aldose reductase.

In the case of diabetes a number of aspects of the inappropriate metabolism
of glucose are currently under scrutiny. Coulter and Knebel are endeavoring
to determine whether insulin directly influences lens metabolism under physio-
logic conditions.il They have developed a sensitive radioimmune assay to
measure insulin in both aqueous humor and blood plasma. Observing fasted
and fed animals , they have shown that the concentration of insulin in aqueous
humor is correlated with the concentration of the hormone in plasma if allow-
ance is made for the relatively slow rate of aqueous humor formation. However,
the concentration of insulin in aqueous humor is only 2% that of plasma, sug-
gesting that the blood- aqueous barrier is relatively impermeable to insulin
under normal conditions. Changes In the availability of insulin to the lens,
i.e. directly from the aqueovis humor, could conceivably lead to disordered
lens metabolism in diabetes. Sheaf f and Doughty have been studying the chemi-
cal, physical and biological properties of the enzyme aldose reductace, with
the expectation that a fuller londer standing of this catalyst may permit more
effective inhibition or manipulation of the enzyme in the living organism. 1^

Lens Implants

Although surgery for the removal of cataracts is highly refined and success-
ful, subsequent visual correction may not always be fully satisfactory.
Correction with spectacles, although acceptable to many, may be unacceptable
to others as a result of differences in image size, lens abberations , reduc-
tion in the visual field, wearing discomfort and, in the case of unilateral
aphakia, difficulty in fusing the images of the two eyes. Correction with
contact lenses may resolve some of the aforementioned problems, but a number
of difficulties may remain including requisite patient dexterity.

Even then, some individuals with unilateral aphakia lack the ability to fuse
images. As a result of the limitations associated with spectacles and contact
lenses, the intraocular lens, i.e. a plastic prosthesis surgically implanted
into the eye, is increasingly being used as an alternative means for the
correction of sight following cataract removal. The advantage of intraocular
lenses include placement of the corrective lens in a near normal position
within the eye permitting improved visual correction, and the diminished
attention to the prosthesis required of the patient.

With improvements in the design of lenses for intraocular implantation, the
ninnber of such implants has been increasing at an exponential rate. Many

264

feel that it is important at this time to evaluate carefully the efficacy of
such implants in patients undergoing cataract extraction and to assess their
risks as well.

As a first step toward evaluating intraocular lens implants, the National Eye
Institute will attempt to summarize the experience that ophthalmologists have
had in their recent clinical practice. The records of several surgeons will
be analyzed to see whether they can provide answers to research questions
that are of interest. For findings from the study of such records to be cre-
dible, it will be necessary to demonstrate that data from all patients treated
are included, that follow-up was maintained on a large proportion of cases,
and that significant medical data have been carefully recorded.

Demonstrating that these elements are present constitutes the work scope of a
contract which is to be awarded in the fall of 1977. If in the first year
of the contract the feasibility of a high quality retrospective study is
established, the contract will be extended to analyze and report on the bene-
fits and risks of intraocular lens implants. Consideration is also being
given to more comprehensive research designs, such as prospective controlled
studies, perhaps including randomization, but it is not entirely clear at
this time that these are feasible.

265

REFEEENCES
CATARACT

1. Westat, Inc.: Sunnnary and Critique of Available Data on Prevalence and
Economic and Social Costs of Visxial Disorders and Disabilities. Rockville,
Md., Feb. 16, 1976 (unpublished).

2. Kinoshita, J. H. : Mechanisms initiating cataract formation. Invest.
Ophthal. 13: 713, 1974.

3. Spector, A.: Aggregation of alpha-crystallin and its possible relation-
ship to cataract formation. Isr. J. Med. Sci. 8: 1577, 1972.

4. Worgul, B. v., Merriam, G. R. , Szechter, A., and Srinivasan, B. D.: Lens
epitheliimi and radiation cataract. Arch . Ophthal . 94: 996, 1976.

5. Giblin, F. J., Chakrapani, B., and Reddy, V. N.: Glutathione and lens
epithelial function. Invest. Ophthal. 15: 381, 1976.

6. Li, Lu-Ku, Spector, A., and Sy, A.: Effects of modification of the
sulfhydryl groups on the size of calf lens low molecular weight alpha-
crystallin. Biochem. (in press) .

7. Varma, S. D., Mizuno, A., and Kinoshita, J.H. : Diabetic cataracts and
flavonoids. Science 195: 205, 1977.

8. Varma, S. D. : Association for Research in Vision and Ophthalmology.
1977 Abstracts, pg. 15.

9. Bhuyan, D. K. , and Bhuyan, K. C: ARVO. 1977 Abstracts, p. 15.

10. Kuck, J. F. R.: ARVO. 1977 Abstracts, p. 14.

11. Coulter, J. B. and Knebel, R. L.: Availability of insulin to the lens
via aqueous humor. ARVO. 1977 Abstracts, p. 13.

12. Sheaff, C. M. and Doughty; C. C: Physical and kinetic properties of
homogenous bovine aldose reductase. J. Biol. Chem. (in press).

266

GLAUCOMA

Introduction

With the exception of the type termed low-tension, glaucoma constitutes a
group of debilitating diseases which are characterized by increased intra-
ocular pressure (lOP) with subsequent alterations in the optic nerve and
eventual loss of visual field. The Glaucoma program of the National Eye
Institute supports clinical and laboratory investigations of factors regulat-
ing intraocular pressure and disorders thereof, study of related changes in
the optic nervehead, and appraisal of the visual field. The preponderance
of research supported by the Glaucoma program has been physiologic and phar-
macologic studies directed at understanding the mechanisms of aqueous humor
formation and aqueoxis egress as well as at their control. Glaucoma research
at present reflects an uneven distribution of effort becaiise the disease is
primarily observed in himians . Available animal models such as congenital
glaucoma in rabbits and beagles and ocular hypertension induced in monkeys
by use of prostaglandins are of limited usefulness.

Glaucoma is a common and serious eye disease. Based upon statistics of first
visits to physicians (exclusive of referrals) there appear to be approximately
178,000 new cases of glaucoma each year. There is a considerably higher inci-
dence of the disease in females, and new cases are first diagnosed predomi-
nately in individuals at ages 45 years or older. The estimated prevalence
of impaired vision resulting from glaucoma is approximately 1,070,000. Severe-
ly impaired vision is observed in 207,000 of these people, and about 56,000
of these, mainly at ages 65 or over, are considered legally blind as a result
of glaucoma.

Eye surgery for glaucoma represents about 3% of all ophthalmic svirgery, and
surgical costs for this disease are estimated to be $8,758,000 per year. A
large majority of glaucomatoxis patients are treated by means of eyedrops and
other medications rather than by surgery. In the United States, the cost
for one year for one type of eyedrop alone used for treatment was $29,000,000.
Thus, besides being a widespread and serious eye disease, glaucoma, is also
a costly one.

The National Eye Institute supports research dealing not only with the etiol-
ogy of glaucoma but also with improving its diagnosis and developing new
modalities for its treatment. Methods are currently being developed to
achieve greater accuracy in the measurement ,of intraocular pressure, rate of
aqueous humor production, facility of outflow, and extent of damage to the
optic disc and associated vasculature and nerves. In addition, development
of improved means of pharmacological and surgical treatment of the various
types of glaucoma is in progress.

Medical Treatment of Glaucoma

Medical treatment of primary glaucoma continues to rely upon the use of an

established arsenal of drugs. Such drugs are of tjie parasympathomimetic

or cholinergic type, such as pilocarpine and carbachol which are short-acting.

267

and Phospholine Iodide and demecarixnn bromide which are long-acting. Another
group of compounds used topically are the sympathomimetics or adrenergics
which constitute various preparations of epinephrine. Oral drugs act system-
ically and reduce aqueous formation by inhibition of carbonic anhydrase in
the ciliary processes or by short-lived lowering of the lOP through hyperosmo-
larity. Examples of the former type are acetazolamide and methazolamide;
glycerol is representative of the latter group.

Drug Delivery Systems. Parasympathomimetic preparations like pilocarpine cause
miosis and may produce side effects due to spasm of the ciliary mxjiscle, result-
ing in myopia and blurred vision. To obviate such deleterious effects and
enhance efficient utilization, newer methods of administering drugs are under
investigation. For short term hypotensive effects, soft contact lenses have
been soaked in pilocarpine for several minutes and then worn for time periods
up to one day.-l-

A more recently developed device which can provide a rate-controlled delivery
of the drug for up to a week is the Ocusert developed by the Alza corporation.
The delivery system consists of two hydrophobic copolymer membranes between
which pilocarpine is sandwiched as the free base. The Ocusert is placed under
the lower lid. When the polymer comes in contact with the tear film, the drug
diffuses through the polymer membranes at a predetermined rate. Although occa-
sionally a "burst phenomenon" occurs causing an over-release of pilocarpine,
the Ocusert 's primary disadvantage has been its high cost. The Ocusert, how-
ever, appears to be a safe device offering several advantages over eyedrops.
Favorable aspects of its clinical usefulness continue to be reported.^

A new topical drug has been developed which holds great promise for the treat-
ment of glaucoma if its long-term use does not reveal a loss in effectiveness.
Timolol is a beta adrenergic blocker which has no known side effects and can
control lOP for 24 hours by use of a single eyedrop. The drug does not appear
to act by increasing the facility of aqueous outflow. 3 An NEI grant-supported
clinical trial of timolol for both basic pharmacological and clinical evalua-
tion is now in progress.

Genetic Aspects of the Etiology of Open-Angle Glaucoma

The widely held concept that certain individuals have a genetic predisposition
to glaucoma has recently been strengthened by the observation by Shin and
associates that there is an increased prevalence of certain histocompatibility
antigens, namely the HLA-B7 and HLA-B12 antigens on Ijnnphocy tes , in patients
suffering with primary open-angle glaucoma as compared to the general popula-
tion or patients with normal lOP.^ Furthermore, the presence of either antigen
also appears to be an important prognostic indicator of the development of
visual field loss in patients with increased lOP and in those who are high
responders to topical corticosteroids.-^

Responders were classified on the basis of their lOP response to the topical
steroid dexamethasone: NN-NG, initial lOP less than 21 mm Hg increasing to
less than 32 mm HG after the testing period, and GG, initial lOP more than
20 mm HG increasing to more than 31 mm HG after the testing period.

268

Shin and associates then extended their studies to siibjects who did not have
glaucoma.^ The presence of either HLA-B7 or HLA-B12 antigens vas found to be
associated with a higher prevalence of cup/optic disc (C/D) ratios of more
than 0,3 in GG responders and in the combined NN-NG groups. The C/D ratio
was previously determined to be inherited and larger C/D ratios in individuals
were thought by Armaly to be related to increased lOP,^ by Becker to increased
responsiveness to topical corticosteroids, 8 and perhaps to an increased sensi-
tivity to glaucomatous damage. Presence of either HLA antigen was found by
Shin and associates also to be associated with a higher prevalence of a family
history of glaucoma in the GG group. This suggests that either of the histo-
compatibility genes themselves or an additional gene on chromosome six may
be associated with the inheritance of expression of primary open-angle glaucoma.
No associations were noted in non-glaucomatous individuals between these anti-
gens and their age, race or mean lOP in either of the groups studied.

Inflammatory Reactions Associated with Secondary Glaucoma

Etiology. Glaucoma secondary to inflammation of the uveal tract occurs in
many different forms and is considered one of the most damaging types of glau-
coma seen. Despite numerous published clinical reports about this condition,
little is known about the etiology of uveitis. 9 The lack of sufficient research
in this area has been taken into accoxmt in the preparation of a recent NEI
grant announcement which invites the submission of worthy grant proposals
focused on clinical or laboratory research that deal with secondary glaucomas.

Most of the ongoing research on glaucoma secondary to uveitis is concentrated
on animal experiments in which prostaglandins and related substances are used to
produce inflammatory reactions resembling acute uveitis that cause sharp rise in
lOP.-'-" These experiments have raised spectilation that prostaglandins and
related compoimds may have a role in human uveitis and eissociated secondary
glaucoma-'--'- even though there are important species differences in. response
to prostaglandins. -'-2 Further research is needed to determine whether this
class of compounds does in fact have a true etiologic role in this condition.

More study is also needed to explain why lOP is elevated in some cases of
uveitis and not in others, and what actually occurs in the aqueous outflow
channels or in the ciliary body to cause this condition. Clinicopathologic
study of aqueous himior and anterior segment tissue samples removed during
surgery are promising approaches. In addition, there should be more study of
uveitis which is experimentally induced in primates in order to understand
better the immunological mechanisms involved in the inflammatory response.

Approaches to Treatment. Effective use of anti- inflammatory corticosteroid
drugs in the eye has significantly improved the treatment of various forms
of uveitis and associated secondary glaucomas. Steroids have also been found
valuable in reducing tissue reactions to antiglaucoma surgery, thereby raising
the success rate of this procedure. Nevertheless, safer and more effective
therapeutic agents are needed for treating this condition because of possible
side effects from steroid therapy such as cataract formation.

269

Axoplasmic Flow in Nerve Fibers in the Etiology of Glaucoma

"Axoplasmic transport" is the term applied to the movement of proteins, sugars
and subcellular particles from the nerve cell perikaryon along its axon. The
functional role of this transport is not yet completely understood, but appears
associated with nerve cell function and maintenance. Axoplasmic movement
consists of several components: a slow component which moves at a rate of
1 to 2mm/24 hr and consists mainly of microtubules and neurofilaments, and a
fast component which carries phospholipids, enzjnnes, neurotransmitters and
endoplasmic reticulum and moves at a rate of 400mm/ 24 hr.

Besides an orthograde flow from retinal ganglion cells where the axoplasmic
flow is synthesized to the lateral geniculate nucleus, there is a retrograde
flow from the lateral geniculate nucleus to the retina at a rate of 75 to
150mm/24 hr. The slow moving component is blocked at the optic disc by mechan-
ical ligation, but recovers when such ligation is removed. The fast moving
component is known to be oxygen-dependent and is irreversibly blocked after
six hours of ischemia in vivo. The fast moving transport depends on the
structural integrity of microtubules carried by the slow transport. If the
structure of the microtubules is disturbed, the fast transport and trans-
synaptic conduction cease.

In experimental work on owl monkeys,-*-^ Anderson and Hendrickson reported that,
with a moderate rise in lOP, labelled protein synthesized in the retinal gang-
lion cells was observed to be partially obstructed at the lamina cribosa; a
further rise of pressure to within 25 ram HG of the mean blood pressure caused
complete cessation of fast transport. Retinal synthesis of transport material
ceased when the lOP exceeded blood pressure.

Such studies were extended by Minckler and associates to another species of
monkey, in which orthograde and retrograde axoplasmic transport were separated
by use of tritlated retinal ganglion proteins and horseradish peroxidase (HPR)
injected into lateral geniculate nuclei. 15 Both tracers accumulated at the
lamina cribosa of eyes maintained at elevated lOPs for 12 to 28 hours, but
HPR appeared to be a much more sensitive indicator. The degree of retrograde
transport obstruction appeared to be directly proportional to both the degree
and duration of the .elevated lOP. Through the use of serial reconstructions
of radioautographs and peroxidase-reacted sections of the optic nerve heads,
it was demonstrated by light microscopy that these obstructions were not only
in the lamina cribosa but also occurred in the temporal quadrants of the nerve
head. At elevated lOPs, these transport obstructions occurred despite evi-
dence of elevated arterial P02 levels or intact nerve head capillary circula-
tion.

These studies support suggestions that alterations in axoplasmic transport
may play a role in the pathogenesis of visual damage in human glaucoma. Trans-
port obstructions were demonstrated in the lamina cribosa in acute experiments
in monkeys at lOPs comparable to those characteristic of human chronic open-
angle glaucoma. The occurrence of such transport blocks were most pronounced
in the temporal quadrants of the nerve head, the same quadrants which are most
often affected in the human disease. Compromise of the axoplasmic transport

270

could play a role in the continuous destruction of axons, causing gradual loss
of visual field, that is typically observed in glaucoma. This damage appears
to a certain extent to be reversible as does visual damage in the early stages
of the disease. Since the precise function of retrograde transport in axons
as well as the role of optic nervehead circulation in this process is unknown,
much additional work must be done before the pathogenesis of visual damage in
glaucoma is understood.

In normal eyes rhythmic fluctuations in the lOP occur during a 24 hr cycle,
but a pressure change of more than 2 or 3mm HG seldom occurs. However, in
many glaucomatous eyes, the pressure varies in a definite diurnal pattern
which is an exaggeration of the slight normal pressure variations. A peculiar-
ity of the diurnal variation of lOP in glaucoma is that in most patients eye
Rjressure rises to a maximum early in the morning before they rise while in
others the pressure reaches its maximum either in late afternoon or evening.
Weitzman and associates have reported significant 24 hr temporal relationship
between lOP and the episodic pattern of Cortisol secretion in normal subjects
and in patients with different types of glaucoma. 16 a phase difference of
3 hrs has been observed between the- maximal values of these two measures.

The need for more investigations to determine relationships between various
hormonal and neural influences which impart a circadian rhythm to TOP in
normal individuals as well as in glaucoma patients has prompted the idea of
conducting a NEI workshop on this topic in early 1978. Since biological
rhythms have different effects on several ocular tissues, the scope of this
subject could be expanded to encompass the work of scientists in other areas
of vision research as well as in related scientific disciplines.

Measurement of TOP

The existence of circadian variations in lOP prevents the use of a single
tonometric measurement as the basis for deciding whether open-angle glaucoma
is present or absent. In fact, more often than not, a diurnal cuirve will have
to be determined for the patient to predict what future course the lOP will
take. Jenson and Maumenee suggest that when such a curve cannot be determined
or where deterioration of the visual field occurs with apparent good medical
control, home monitoring of lOP by a family member of the patient with a dis-
posable tonometer may be usefiit. 17

An alternative and potentially more satisfactory approach for round-the-clock
lOP measurements is the use of a noninvasive and nonirritative device for
constant lOP measurement. An intraocular pressure monitoring contact lens,
developed at the University of Utah under an NEI contract, shows promise of
offering a means of monitoring intraocular pressure fluctuations for several
days at a time. An NEI advisory committee is evaluating the final report of
this contract to determine the practicality and applicability of the device
for diagnosis and surveillance of glaucoma. 1°

Treatment

As observed earlier in this report, present medical treatment of elevated lOP
in primary glaucom relies principally on well-established miotics, epinephrine,

271

and carbonic anhydrase inhibitors and, in glaucomas secondary to uveitis, on
corticosteroids. Tfhere surgery is indicated, laser puncture of the filtration
portion of the trabecular meshwork to relieve obstruction of aqueous outflow
has received much publicity in recent years. Claims have been made of at
least short-lived effectiveness of this technique, particularly with the Q-
switched laser. l^ An NEI grant-supported clinical trial of argon laser trabe-
culotomy is in progress to establish the usefulness of this technique for the
relief of excessive lOP in open-angle glaucoma. ^0 Another clinical trial is
being initiated which will evaluate the benefits of prophylactic iridectomy
in angle-closure glaucoma. ^-'-

272

REFERENCES
GLAUCOMA

1. Podos, S. M. , Becker, B., and Asseff, C: Pilocarpine therapy with soft
contact lenses. Am. J. Ophthal. 73: 336, 1972.

. 2. Lee, P. F., Shen, Y. T., and Eberle, M.: The long-acting pilocarpine
system in the management of glaucoma. Invest. Ophthal. 14: 43, 1975.

3. Zimmerman, T. J., Harbin, R. , Pett, M. and Kaufman, H.E.: Timolol and
facility of outflow. Invest. Ophthal. 16: 623, 1977.

4. Shin, D. H., Becker, B., Waltman, S. R. , Palmberg, P. and Bell, C. E.,
Jr.: The prevalence of HLA-B12 and HLA-B7 antigens in primary open-
angle glaucoma. Arch. Ophthal. 95: 224, 1977.

5. Shin, D. H. and Becker, B.: The prognostic values of HLA-B12 and HLA-B7
antigens in patients with increased intraocular pressure. Am . J . Ophthal .
82: 871, 1976.

6. Shin, D. H., Kass, M. A. and Becker, B.: The association of HLA-B7 and
HLA-B12 antigens with cup-disk ratio , family history of glaucoma and
intraocular pressure. Am. J. Ophthal. 83:' 347, 1977.

7. Armaly, M. F. : Optic cup in normal and glaucomatous eyes. Invest.
Ophthal. 9: 425, 1970

8. Becker, B.: Cup/disk ratio and topical corticosteroid testing. Am. J.
Ophthal. 70: 681, 1970.

9. O'Connor, G. R. : The uvea (annual review). Arch. Ophthal. 93: 675, 1975.

10. Bito, L. Z.: The effects of experimental uveitis on anterior uveal
prostaglandin transport and aqueous humor composition. Invest. Ophthal.
13: 959, 1974.

11. Sears, M. L., Neufeld, A. H., and Jampol, L. M.: Prostaglandins.
Invest. Ophthal. 12: 161, 1973.

12. Kass, M. A., Neufeld, A. H. and Sears, M. L.: Systemic aspirin and
indomethacin do not prevent the response of the monkey eye to trauma.
Invest. Ophthal. 14: 604, 1975.

13. Levy, N. S.: Functional Implications of axoplasmic transport. Editorial.
Invest. Ophthal. 13: 639, 1974.

14. Anderson, D. R. and Hendrickson, A.: Effect of intraocular pressure on
rapid axoplasmic transport in monkey optic nerve. Invest. Ophthal. 13:
771, 1974.

273

15. Minckler, D. S., Bunt, A. H. and Johnson, G. W. : Orthograde and retro-
grade axoplasmlc transport during acute ocular hypertension in the monkey.
Invest. Ophthal. 16: 426, 1977.

16. Weitzman, E. D., Henkind, P., Leitman, M. , and Hellman, L.: Correlative
24-hour relationship between intraocular pressure and plasma Cortisol in
normal subjects and patients with glaucoma. Br. J. Ophthal. 59: 566, 1975.

17. Jenson, A. D. and Maumenee, A. E.: Home tonometry. Am. J. Ophthal.
76: 929, 1973.

18. Couvillon, L. Jr.: NIH-NEI-73-2115 (1976 Contract progress report).

19. Krasnov, M. M. : Q-switched laser goniopuncture. Arch. Ophthal. 92: 37,
1974.

20. Worthen, D. M. and Wickham, M. G. : Argon laser trabeculotomy. Trans .
Am. Acad. Ophthal. Otolaryngol. 78: 371, 1974.

21. Wilensky, J. T.: EY01754-01 (preliminary findings).

274

SENSORY AND MOTOR DISORDERS OF VISION
Introduction

The Sensory and Motor Disorders of Vision Program of the National Eye Institute
supports clinical and laboratory investigations that bear on disorders of
visual information transmission, perceptual synthesis, and oculomotor control.
Examples of such disorders are strabismus, amblyopia, degenerations of the
optic nerve, and congenital nystagmus. The preponderance of effort supported
under this program is related to basic biological research designed to aug-
ment knowledge of the structure and function of the visual sensory nervous
system and the neural systems controlling eye movement. Substantial support
has also been extended to psychophysical investigations, that is, to research
on the relationship between the physical parameters of visual stimuli and the
visual response of normal persons and those suffering from disorders of vision.

Of the total cases of severe visual impairment in the United States, some
10% derive from sensory-motor disorders of vision. 1 About 3% of the popula-
tion have manifest strabismus. A much larger percentage have subclinical
defects related to disparity of retinal images, e.g. loss of stereopsis or
binocular fusion, or functional suppression. The prevalence of amblyopia in
the general population is also estimated at 3%. The incidence of optic nerve
diseases in the United States population has been set at 53,000 cases per
year. Cerebral diseases in the visual areas produce blindness or defective
vision in large numbers of persons, particularly the elderly. Congenital
and acquired disorders of the oculomotor control system, e.g. abnormalities
of gaze, 3rd cranial nerve paralysis, or oculomotor dyslexia result in
serious handicap. Refractive myopia, which is of general concern, also falls
under the purview of the Sensory and Motor Disorders of Vision program.

A broad array of biological and behavioral disciplines, among them neuro-
anatomy, neuro chemistry, neurophysiology, genetics, bioengineering, biomathe-
matics, human psychophysics and animal behavioristics bring their concepts
and techniques to bear on the objectives of this program. Among these objec-
tives are: (1) elucidation of the neural basis of normal and abnormal visual
development, (2) tracing of the control system that coordinates the movement
of the eyes with each other and with the postural reflexes of the body, and
(3) instrumentation for the di&gnosis and remediation of visual defects.

Certain approaches have in recent years proven themselves particularly valu-
able in arriving at the present "state of the art" of visual science. Early
monocular or binocular visual deprivation experiments in cats and monkeys
provided valuable findings about the morphological and functional changes in
the brain that result from abnormal visual experience early in life. These
changes may well bear similarities to the abnormal neural substrate in human
amblyopia. A search for an "accessory" visual system that functionally com-
plements the classical retino-geniculate-calcarine pathways has yielded
scientific dividends. The application of models from pattern recognition
technology to the study of human vision has produced new kinds of psychophys-
ical tests with diagnostic potential. Much progress in this past year reflects
the dominance of these approaches.

275

Developmental Studies of Vision

Sherman states the significance of his research program to be the determina-
tion of the nature of the interaction between the developing mammalian brain
and the visual environment . ^ His research is pursued with neuroanatomical
and behavioral methods as well as with single-unit electrophysiological
recording. The visual environment is controlled via postnatal lid-suture,
enucleation, or controlled illumination in laboratory-reared kittens. Visual
orientation and discrimination are studied, as are structural and functional
changes in the lateral geniculate nucleus, optic tectimi, and striate cortex.

The competitive balance between the two eyes is crucial in disorders such as
amblyopia. In several series of experiments, Sherman and Guillery-' and
Wilson, Webb, Sherman^ aimed at defining the conditions whereby one eye gains
a competitive advantage over another in behavioral terms and in terms of the
development of central visual corrections. Their tentative conclusion is
that an eye can gain competitive advantage only when pattern vision is avail-
able. Differences in light or temporal frequencies of stimulation are without
effect. Binocular competition was also demonstrated by Wilson and Sherman
in the monocularly deprived striate cortex since many simple cells in the
deprived monocular segment and very few in the binocular segment were driven
normally by the deprived eye.^ The abnormal complex cells represent a depri-
vation effect independent of binocular competition since they occur in the
monocular segment.

In 1966, Sprague found that a hemianopia produced by a large contralateral
posterior cortical lesion in the cat could be partially alleviated by ablation
of the superior colliculus contralateral to the cortical lesion. ° Thus, a
visual deficit could be alleviated by an appropriately placed additional
lesion. This dramatic phenomenon has been tentatively explained by the sup-
position that the colliculi normally receive a balanced set of inputs from
the two cortices, a balance that is disturbed by an unilateral lesion but in
some way restored by the additional ablation. Sherman has recently confirmed
and extended these findings and complemented them with stimtilation studies. ^
The "Sprague effect" has been found to be a reproducible phenomenon in cats.
Visual behavior that can be mediated via retino-tectal pathways can be unmasked
if the superior colliculi are functionally disconnected from each other in
animals with large cortical lesions. What the specific neuronal correlates
of this unmasking are remains to be determined by single-tinit electrical
recording planned for the future.

In addition to the well-known projections of the vertebrate retina upon the
thalamic geniculate nuclei, the retina also connects directly with several
other neuronal groupings of the midbrain. HayhowS and Giolli^ identified a
prominent pathway to a group of nuclei referred to as the "accessory optic
nuclei" (AON) . Recently, Karten has received a National Eye Institute award
for the study of the relation between certain retinal ganglion cells in birds
and the AON. The study has potential significance because of the discovery
by Brauth and Karten that the AON projects directly to the cerebelltmi, sug-
gesting that a "lemniscal" channel may exist between the avian peripheral
retina and the f locculo-modular lobe of the cerebellum which is implicated
in the oculovestibular reflexes and the control of eye movements. 1^ Should

276

a homologous channel exist in mammals, particularly humans, the anatomical
basis may have been found for a d3mamic spatial orientation disjunct from
consciotis light perception. An NEI Workshop -on Research Opportunities Relevant
to the Management of Severe Visual Impairment, June 24-25, 1977, identified
precisely this topic, i.e. the separation of object recognition and spatial
orientation, as an exciting concept in relation to the assessment and remedia-
tion of low-vision status. (It is known that himians blind from massive
occipital lobe lesions may grasp in the general direction of light or eye
movement without conscious experience.) Conceivably, a residual midbrain-
cerebellum channel could be exploited in rehabilitation training.

The primary research objectives identified by Karten include: a) identifica-
tion of those retinal ganglion cells that project to the AON and the charac-
terization of their density, distribution, and dendritic morphology; b)
histochemical study of the projection pathway to determine the neurotransmit-
ters present (ACh or catecholamines) as well as the chemical properties of
receptors present at the AON; and c) electron microscopic or Golgi studies
of retinal axonal arborization. Particularly noteworthy is Karten 's intent
to characterize the "accessory optic pathway" in nexiro chemical terms . Suc-
cess of this endeavor would open the way to pharmacological modification of
function in this visual-postural system, with the promise of possible drug
treatment of certain neurophthalmological disorders .

Neurophysiological Properties of Retinal Ganglion Cells

Enroth-Cugell and co-workers originally identified two fxinctional classes
of retinal ganglion cells in the cat, the X and Y cells. ^^ Their X, Y-
transient/sustained dichotomy has recently been revised and extended to
supra-retinal levels. Tobin, in Enroth-Cugell 's laboratory, has studied
the temporal frequency characteristics of the center response mechanism of
the X and Y retinal ganglion cells. ■'■^ At each of four levels of background
illimiination (field adaptation) stimulus, conditions were chosen so that
responses of the cells to sqiiare-wave stimuli showed little or no evidence
of surround antagonism. Responses to sinusoidal stimuli of different fre-
quencies (0.2-25 Hz) were then recorded at the same four adaptation levels,
and plots of modulation frequency versus (1) sensitivity in absolute terms,
(2) contrast sensitivity, and (3) phase angle were compared. These plots
showed that only small differences exist between the temporal properties of
X and Y cells when driven by their center mechanism. These findings suggest
that the interaction of the surround with the center causes the Y cell
responses to be more transient than those of X cells. In turn, the Y system
appears better suited to process temporal variations than the X system.

A. B. Bonds, working with Enroth-Cugell, has related ganglion cell activity
to bleaching adaptation. -^-^ From the following ganglion cell results on
recovery of (rod) threshold after one minute bleaches, it was evident that
the quantitative relationship between bleached rhodopsin and ganglion cell
threshold cannot be a simple one: (1) regeneration of rhodopsin could not
be described by a single exponential during any phase whereas the late phase
of ganglion cell threshold recovery coTild, and the early phase of adaptation
seemed to consist of the product of two exponentials; (2) ganglion cell recov-
ery proceeded at the highest rate during the very period when rhodopsin

277

regeneration was almost halted; and (3) ganglion cell threshold was still
above its pre-bleach, dark-adapted value by 0.5-2.3 log units when rhodopsin
was completely regenerated at 35 minutes after extinction of the bleaching
light.

In related studies, Jakiila has investigated the dependence of retinal gang-
lion cell sensitivity on movement of the background pattern.-'-^ From previous
investigations, it is well established that the sensitivity of concentric
cat retinal ganglion cells to a centered test spot can be reduced by a concen-
tric disk of steady background illxnnination, the reduction in sensitivity
being determined by the effective adapting flux. Experiments by Jakiila and
others show that a moving pattern of background illumination can cause a
greater reduction in sensitivity than that produced by the same background
when stationary, even though the adapting fltix is the same. Such findings
anderline the importance of temporal modulation in the control of ganglion
cell activity.

Efferent Control of Eye Sensitivity

Centrifugal pathways from brain to retina have long been known but their
function has been obscure. Now, Barlow and co-workers have turned limulus
model into an effective tool for studying efferent neural regulation of
visual sensitivity. Working with mlcroelectrodes on the lateral eye in
situ, they have discovered that it undergoes circadian changes in sensitivity,
but such changes occur only if the lateral eye nerve remains intact. When
the animal is kept in constant darkness, the lateral eye elicits small-
amplitude ERG responses during normal daylight hours and large-amplitude
responses during the night. Each ERG response can be elicited by a brief
flash of fixed intensity. Prior to initiating this experiment, the animal
was placed for several days in an aquaritmi exposed to exterior lighting con-
ditions, i.e. natural day, night, svinrise, and sunset. On the day of experiment
the animal was placed in an aquarium in a light-tight shielded cage, and
electrodes and fiber optic stimulators were aligned on one of the lateral
eyes. ERG data were collected imder constant darkness for two days. On the
second day after the experiment began a snare was inserted around the optic
nerve trunk through a small hole in the carapace. On day three the snare
was pulled to section the optic nerve. No change in ERG could be detected
after the optic nerve was cut< .

The circadian changes of ERG amplitude observed by Barlow correspond closely
to the circadian changes in pigment migration in the retinular cells. These
results strongly suggest that efferent axons in the optic nerve trunk inner-
vate the photoreceptor cells of the eye and control their sensitivity by
initiating movement of screening pigment in the photoreceptors. Evidence
of neurosecretory granules in efferent terminations in the photoreceptors
and of a microtubular system for pigment migration lend further support to
the notion of efferent control of pigment migration. These results appear
to be the only direct evidence obtained to date for efferent control of
photoreceptor sensitivity in the limulus eye or in any other visual system.

278

Mathematical Analysis of the Visual Process

The quantitative understanding of the integrative action whereby the visual
system produces a final percept requires, in the first place, formulation of
the dynamical laws that interacting visual neurons obey. In the laboratory
of Knight and Shapley the djmamics of the three component processes of exci-
tation, lateral inhibition, and self -inhibition have been expressed, particu-
larly in terms of transfer functions. Recently, this group of investigators
have undertaken the creation of new stimulus techniques that are used as tools
in elucidating visual dynamics. ^^ The method uses a stimulus whose variable
part is a superposition of a substantial nimiber of sine waves. The essence
of the technique is that nonlinearities in the system under stimulation will
yield predictable combination frequencies. The stimulus repeats after one
minute as soon as it has run through a sample of all phase combinations of
its component sine waves .

The set of response frequencies is discrete and thus, in principle, covers
zero bandwidth. This allows digital filtering of the output in a way that
will reject autonomous wideband noise to an arbitrary degree. In practice,
a one minute experimental cycle allows processing the response with a set
of parallel narrow-pass digital filters (centered at the input frequencies
and at their known combination frequencies) , each with a bandwidth of only
one- thirtieth Hz. Each of these filters is also so constructed that it has
a set of strict nulls placed at all the other output frequencies in the dis-
crete response spectrum. Thus, one can separate cleanly the nonlinear part
of the response from the linear part while strongly rejecting autonomous
noise.

A general theory of nonlinear system response to any given stimulus ensemble
has been developed. The general theory gives an explicit description of
how the system's response to any given stimulxis ensemble leads to a standard,
straightforward dynamical characterization. (The characterization is in
terms of the well-known "Volterra series" of homogeneous symmetric functionals
of ascending order over the past history of the stimulus signal.) The stimu-
lus ensemble of a discrete set of superimposed sine waves leads easily to a
simple final result. A fairly general type of ensemble which consists of
"colored" Gaussian noise likewise leads to a simple general result. A limit-
ing case is that of Infinite-baodwldth "white" noise, which retrieves the
Wiener description whereas the opposite limit of "zero bandwidth" noise with
a discrete spectrum retrieves the description for the stimulus ensemble.

Analytic methods of this sort may well extend across the disciplines of unit-
cell neurophysiology, evoked visual potentials, and hxmian psychophysics.
The potential for such interdisciplinary developments will be encouraged by
NEI.

Clinical Psychophysics

At present, much of visual testing and assessment in the clinic depends upon
the skill and experience of the examiner as well a^ the verbal response of
patients. These subjective procedures are further influenced by uncontrolled

279

physical, psychological and environmental variables that confound the evalxia-
tion of a patient's condition and the comparison of diagnostic groups.

A recent workshop sponsored by the NEI, "The Role of Psychophysics and Physio-
logical Optics of Ophthalmic Diagnosis and Patient Evaluation"16 focused on
the challenges faced by psychophysical science in relation to ophthalmic
clinical science. The participants agreed that psychophysicists and opticists
now have new techniques available to characterize visual functions with greater
objectivity and precision. In their view, a need exists to bring these tech-
niques to bear on such problems as localization of anomalies in glaucoma,
senile macular degeneration, amblyopia, degenerations of the optic pathway,
cerebral lesions, strabismus and disorders of oculomotor control. New
approaches to clinical testing using modern psychophysical techniques or
optical instrxBnentation were identified at the workshop .

1. Measurements of the Westheimer effect demonstrate the influence of the
size of a surrounding field on the ability to detect a test flash. This
technique may be used to localize pathology in the choroid/pigment epi-
thelitnn, receptor layer, inner and outer plexiform layer, or optic nerve.
Such studies by Enoch, Johnson and Fitzgerald open the possibility of
pinpointing retinal and neural lesions in a manner beyond the scope of
traditional tests of acuity. 17

2. Visual spatial and temporal modulation transfer functions (contrast grat-
ing sensitivity) tests, based on mathematical models of the transfer
characteristics of the visvial system, may address questions of pathology
in the CNS as well as abnormalities in retinal receptive fields.

3. Cortical evoked response measurement of visual acuity will permit objec-
tive clinical testing with non- invasive techniques in cases of amblyopia
or other sensory disorders in children.

4. Random dot tests of stereopsis, based on global rather than local visual
cues, may more adequately evaluate binocularity in cases of strabismus.

5. Tests of infant vision based on preferential looking or operant condi-
tioning techniques may assist in the acquisition of normative data on
infant populations.

6. Tests for the assessment of color defects in functional disorders of the
visual brain may help determine the severity and course of disease or
intoxication.

7. Infrared and other modem electro-optical eye movement recording technology
has the potential to complement traditional nystagmography and electro-
myography. The latter may require uncomfortable electrode implants or

may be incapable of resolving very small eye movements for neurophthal-
mological diagnosis. Several new eye- trackers have potential for applica-
tion to the detection and characterization of eye movement disorders in
the eye clinic.

280

Recent Initiatives

To stimulate further research on these and all other promising psychophysical
and optical tools and to facilitate translation of technical advances into
improved ophthalmic diagnosis and treatment, special initiatives were under-
taken by the National Eye Institute this past year. One of these was the
preparation and publication of two annoimcements inviting the submission of
worthy research proposals related to the application of psychophysical tests
to the diagnosis of human visual disorders and to the rehabilitation of the
severely visually- impaired.

Another initiative was the funding of clinically imaginative new projects.
For example, Bodis-Wollner, who has been among the first to apply contrast
grating sensitivity functions to the localization of neurological lesions,
received NEI support for a study of sensitivity of neurophthalmological
patients to temporally-modulated contrast grating.-'-" Bodis-Wollner hopes to
use his techniques to distinguish psychophysically between pathology in
"transient" and "sustained" pathways in the human visual system.

A third initiative was the award of a special multl- institutional training
grant, "Clinical Application of Visual Science", to the University of Florida,
Gainesville. Enoch, the program director, will train individuals in psycho-
physics or physiological optics to apply their science more effectively to
clinical ophthalmic problems. It is planned that the trainees will spend
approximately one year in each of two or three institutions (for example,
U. of Florida, Gainesville, TJ. of Washington, Seattle, U- of California,
Berkeley) in order to achieve the desired training and clinical experience.

The nature of this novel training program is well described by the hypothe-
tical example of a trainee interested in Infant vision. Such an individual
might start training in behavioral testing methods. After one year, the
trainee would move to another institution or preceptor to learn electro-
physiological techniques. For a third year, the trainee might go to a clinic
where procedures are used to correct the -vision of infants with sensory depri-
vation abnormalities. The training program will be advertised by brochure,
and qualified candidates with training in psychophysics, electrophyslology,
physiological optics and other relevant disciplines will be considered for
admission.

Finally, technology transfer was promoted by an award to H. D. Crane, Stanford
Research Institute, Palo Alto, California for the further development of the
Purkinje three-dimensional eye- tracker (Comsweet and Cranel9) for ophthalmic
application. This Instrument, originally conceived for the U. S. space pro-
gram, has proved useful in image stabilization, tracking of the state of
accommodation, and nystagmography. A potential exists for its use in the
diagnosis of oculomotor disease, in clinical psychophysics, and even ' in
retinal siirgery. A workshop to discuss the clinical applicability of such
an instriiment is planned for the near future. Some twenty research labora-
tories or clinics now possess the instrument; many have NEI support. A "users
group" has been established to foster cooperative research and to provide
the Stanford Research Institute with systematic feedback about the instrvmient's

281

performance and with suggestions for its further development. A "users group"
workshop to discuss these issues is being planned.

Orthokeratology Clinical Trial

Myopia is a condition that affects a very large part of our population. While
the etiology of nearsightedness is far from clear, "treatment" in the form of
corrective lenses is traditional. In recent years it has been noted by
Miller20j Rengstorff21 and many others that corneal curvature may be changed
by contact lens wear and that consequently the refractive error in vision may
also be altered. Since such changes may persist, some practitioners have
attempted to exploit the changes in corneal curvature induced by shaped con-
tact lenses to reduce refractive error permanently, a procedure known as
"orthokeratology". Orthokeratology, however, has remained unvalidated and
controversial.

During fiscal year 1976, NEI received an application from Polse^-', School of
Optometry, University of California, Berkeley, requesting grant support for
a controlled clinical trial of the effectiveness of orthokeratology. After
a positive concept review by an NIH initial review group and concurrence by
the National Advisory Eye Coimcil, a conditional award was made for the
development of a manual of operations for a randomized, masked clinical trial.
The manual of operations subsequently developed was evaluated by NEI staff
and external consultants for soundness of experimental design, adequacy of
optical measures, attention to side-effects, and ethical safeguards, includ-
ing informed consent.

At the present time, the manual of operations is tindergoing final revisions.
The measurements to be undertaken include corneal thickness, refractive error,
biological changes in the cornea and visual acuity. The study proposed is a
"two-arm" comparison of orthokeratological vs. traditional contact lens
therapy. Patient assignment to either treatment is to be at random. Ophthal-
mologic and optometric examination is to be continuous. An award for actual
initiation of the clinical trial is expected to be made soon.

Workshop on General Anesthesia and Visual Receptive Fields

Ethical considerations preclude NEI grantees from using paralyzed, unanes-
thetized animals in electrophysiological experiments. These restrictions,
however, pose difficulties in the mapping of receptive fields, particularly
those of cells in the higher divisions of the visual system, which are quite
labile. To help overcome these obstacles, a meeting has been planned in fis-
cal year 1978 of vision researchers active in this field. Some main topics
for discussion include: (1) the effect of various anesthetic agents on the
receptive fields in the lateral geniculate nucleus, visual cortex, etc., (2)
the types of receptive field properties most affected by various anesthetics,
and (3) alternatives to paralysis for controlling eye movements. Recommenda-
tions to the National Advisory Eye Council on areas of future research need
and opportunity pertaining to the above as well as other topics will be
formulated at the workshop.

282

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SENSORY AND MOTOR DISORDERS OF VISION

1. National Advisory Eye Council: Support for Vision Research. Interim
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76-1098, 1976.

2. Sherman, S.: EY01565-03 (annual progress report) .

3. Sherman, S. M. and Gulllery, R. W.: Behavioral studies of binocular
competition In cats. Vision Res. 16: 1479, 1976.

_4. Wilson, J. R. , Webb, S. V. and Sherman, S. M. : Conditions for dominance
of one eye during competitive development of central connections in
visually deprived cats. Brain Res, (in press).

5. Wilson, J. R. and Sherman, S. M. : Differential effects of early monocu-
lar deprivation on the binocular and monocxilar segments of cat striate
cortex. J . Neur ophy s io 1 . (in press) .

6. Sprague, J. M. : Interaction of cortex and superior colllculus in media-
tion of visually guided behavior in the cat. Science 153: 1544, 1966.

7. Sherman, S. M. : The effect of superior colllculus lesions upon the visual
fields of cats with cortical ablations. J. Comp. Neurol. 172: 211, 1977.

8. Hayhow, W. R. : An experimental study of the accessory optic fiber system
in the cat. J . Comp . Neiirol . 110: 1, 1964.

9. Giolli, R. A. : An experimental study of the accessory optic tracts in
the rabbit. J. Comp. Neurol. 117: 77, 1961. .

10. Brauth, S. E. and Karten, H. J. : Accessory optic nuclear projections to
the f locculo-modular lobe of the cerebelliim. A possible channel for
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11. Enroth-Cugell, C. and Joii^s, R. W. : Responses of cat retinal ganglion
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12. Tobln, B. : Dissertation, Northwestern University, 1976.

13. Enroth-Cugell, C: EY00206-17 (summary progress report).

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