finartme HAHWhRTH, RONALD S Counc.1: Ub/2UUb …3e. DEPARTMENT, SERVICE, LABORATORY, OR EQUIVALENT...

Form Approved Through 09/30/2007Dfinartment nf H-HH .«, M™ Services hL HAHWhRTH, RONALD S Counc.1: Ub/2UUb

10057041 )n OCT 27 2 2[J2EY00113MO

Do noi «UB«U ov/aracrer /engrn restrictions indicated. IRG: AED Received: 1 0/27/2005

1 . TITLE OF PROJECT (Do no/ exceed 81 characters, including spaces £>,,„ r~. , ,

Behavioral Measures of Vision2. RESPONSE TO SPECIFIC REQUEST FOR APPLICATIONS OR PROGRAM ANNOUNCEMENT OR SOLICITATION D NO D YES

(If "Yes, " state number and title)Number: Title:

3. PRINCIPAL INVESTIGATOR/PROGRAM DIRECTOR

3a. NAME (Last, first, middle)

Harwerth, Ronald S.3c. POSITION TITLE

Professor3e. DEPARTMENT, SERVICE, LABORATORY, OR EQUIVALENT

Vision Sciences3f. MAJOR SUBDIVISION

Optometry3g. TELEPHONE AND FAX (Area code, number and extension)

TEL: 713-743-1940 FAX: 713-743-2053

4 HUMAN SUBJECTS 4b. Human Subjects Assurance No.RF^PARrH FWA00005994

, _ , . - , 4c. Clinical Trial 4d. NIH-defined Phase III|_| No I2SI Yes ^ No D Yes Clinical Trial S No D Yes

4a. Research Exempt ,, .... „_ .. .,^ —i If Yes, Exemption No.13 No D Yes

New Investigator ^ No 1 1 Yes

3b. DEGREE(S) 3h. eRA Commons User Name

OD PhD

3d. MAILING ADDRESS (Street, city, state, zip code)College of OptometryUniversity of HoustonHouston, TX 77204-2020

E-MAIL ADDRESS:

[email protected]

5. VERTEBRATE ANIMALS D No ^ Yes

5a. If "Yes," IACUC approval 5b. Animal welfare assurance no.Date

01/24/2005 A3 136-01

6. DATES OF PROPOSED PERIOD OF 7. COSTS REQUESTED FOR INITIAL 8. COSTS REQUESTED FOR PROPOSEDSUPPORT (month, day, year—MM/DD/YY) BUDGET PERIOD PERIOD OF SUPPORT

From Through 7a. Direct Costs ($)

07/01/2006 06/30/2011 $250,000

9. APPLICANT ORGANIZATION

Name University of Houston

Address 480o Calhoun Blvd

Houston, TX 77204-201 5

12. ADMINISTRATIVE OFFICIAL TO BE NOTIFIED IF AWARD IS MADE

Name Thomas L. Boozer, II

Title Director, Office of Contracts and GrantsAddress 480Q Ca|noun B|vd

Houston, TX 77204-201 5

Tel: 713-743-9240 FAX: 713-743-9227E-Mail: [email protected]. PRINCIPAL INVESTIGATOR/PROGRAM DIRECTOR ASSURANCE: I certify that thestatements herein are true, complete and accurate to the best of my knowledge. I amaware that any false, fictitious, or fraudulent statements or claims may subject me tocriminal, civil, or administrat ve penalties. I agree to accept responsibility for the scientificconduct of the project and to provide the required progress reports if a grant is awarded asa result of this application.15. APPLICANT ORGANIZATION CERTIFICATION AND ACCEPTANCE: I certify thatthe statements herein are true, complete and accurate to the best of my knowledge, andaccept the obligation to comply with Public Health Services terms and conditions if a grantis awarded as a result of this application. I am aware that any false, fictitious, or fraudulentstatements or claims may subject me to criminal, civil, or administrative penalties.

7b. Total Costs ($) 8a. Direct Costs ($) 8b. Total Costs ($)

$364,035 $1,250,000 $1,765,498

10. TYPE OF ORGANIZATION

Public: -» [U Federal ^ State Q] Local

Private: ->• I I Private Nonprofit

For-profit: -> I I General I I Small Business

I I Woman-owned l~l Socially and Economically Disadvantaged

--- ---------- -- ------------- CATION NUMBER-------- - ----- - -- -

DUNS NO. 036837920 |Cong. District 18

13. OFFICIAL SIGNING FOR APPLICANT ORGANIZATION

Name Thomas L. Boozer, II

Title Director, Office of Contracts and GrantsAddress 4800 Calhoun Blvd

Houston, TX 77204-201 5

Tel: 713-743-9240 FAX: 713-743-

E-Maii: [email protected] OF PI/PD NAMED IN 3a. DATE(In ink. "Per" signature not acceptable.)

/K~#f?i4i&4f( fzf&sist*^^-^^ I /

SIGNATURE OF OFFICIAL NAMED IN 13. DATE(In ink. "Per" signature not acceptable.)

NftjW/K^ j / lA^ W IflofPHS 398 (Rev. 09/04) Face Page v ' FormRfegel

Principal Investigator/Program Director (Last, First, Middle): Harwerth, Ronald S.

DESCRIPTION: See instructions. State the application's broad, long-term objectives and specific aims, making reference to the health relatedness ofthe project (i.e., relevance to the mission of the agency). Describe concisely the research design and methods for achieving these goals. Describethe rationale and techniques you will use to pursue these goals.In addition, in two or three sentences, describe in plain, lay language the relevance of this research to public health. If the application is funded, thisdescription, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACEPROVIDED.

Glaucoma is a progressive optic neuropathy caused by a pathological loss of the retinal neurons thatform the optic nerve from the eye to the brain. It is a leading cause of irreversible blindness in the UnitedStates; approximately 2.2 million people older than 40 years of age suffer from the disease and as many as120,000 of these people are blind from glaucoma. As the population ages, the disease will become anincreasingly important problem of public health, but treatment is effective in preventing or slowing vision lossand it is, therefore, important to optimize procedures for determining when treatment is needed and when itis effective. Because the cause of glaucoma is unknown, the diagnosis or progression of the diseaserequires ophthalmic testing to identify and quantify clinical characteristics of glaucomatous neuropathy, suchas the pattern of visual field defects, anatomical changes of the optic nerve head, and thinning of the retinalnerve fiber layer.

The goal of the proposed research is to gain a better understanding of the relationships between clinicalmeasures of neural and visual losses from glaucoma. The principal experiments involve behavioral studiesof visual function (standard automated perimetry) and high resolution imaging of retinal structure (opticalcoherence tomography) over the timecourse of experimental glaucoma in macaque monkeys. The data fromthe investigations of experimental glaucoma will be used to develop a quantitative model relating the loss ofretinal neurons in a specific area of the retina to the number of axons entering the optic nerve from the sameretinal area. In the final phase, the structure and function relationships that were derived from monkeys willbe applied to assess the severity, or stage, of glaucoma in human patients.

The research method is based on defining procedures with experimental glaucoma where there areexcellent controls for the experimental and measurement variables and, then, application to human patientsto test the clinical relevance and validity of the procedures. This method of going from the laboratory to theclinic should maximize the potential for significant new information about ophthalmic testing for diagnosisand assessment of glaucoma.

Key words: glaucoma, retinal ganglion cells, retinal nerve fiber layer, perimetry, OCT, monkeys, humans

PERFORMANCE SITE(S) (organization, city, state)

College of Optometry--- - -- ------- - ------------ - ---------- - 4901 Calhoun BlvdUniversity of HoustonHouston, TX 77204-2020

PHS 398 (Rev. 09/04) Form Page 2


KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below.Start with Principal Investigator. List all other key personnel in alphabetical order, last name first.

Name

Harwerth, Ronald S.-------------------- ------------ - ------- --------- - --------- -------- - -- ------- ----- --

------------------ -------- ---

----------- --------- -- --- -------- ---- -

eRA Commons User Name Organization Role on Project

University of Houston Principal InvestigatorUniv of Texas - Houston Co-InvestigatorUniversity of Houston Research Associate

University of HoustonUniversity of Houston

University of HoustonUniversity of HoustonUniversity of Houston

Clinical Res. AssocCo-Investigator

Research Assistant

Research AssociateClinical Res Assoc

OTHER SIGNIFICANT CONTRIBUTORSName Organization Role on Project

Human Embryonic Stem Cells [X] No LJ YesIf the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list:http://StemcellS.nih.qov/reqistry/index.asp. Use continuation pages as needed.

If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used.

Cell Line

Disclosure Permission Statement. Applicable to SBIR/STTR Only. See SBIR/STTR instructions. CH Yes No

PHS 398 (Rev. 09/04) Form Page 2-continuedNumber the following pages consecutively throughoutthe application. Do not use suffixes such as 4a, 4b.


The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page.

RESEARCH GRANT

TABLE OF CONTENTSPage Numbers

Face PageDescription, Performance Sites, Key Personnel, Other Significant Contributors, and HumanEmbryonic Stem CellsTable of ContentsDetailed Budget for Initial Budget Period (or Modular Budget)

Budget for Entire Proposed Period of Support (not applicable with Modular Budget)Budgets Pertaining to Consortium/Contractual Arrangements (not applicable with Modular Budget)Biographical Sketch - Principal Investigator/Program Director (Not to exceed four pages)Other Biographical Sketches (Not to exceed four pages for each - See instructions)Resources

Research Plan.

Introduction to Revised Application (Not to exceed 3 pages; SBIR/STTR Phase I not to exceed 1 page.) .

Introduction to Supplemental Application (Not to exceed one page)

A. Specific Aims

B. Background and SignificanceC. Preliminary Studies/Progress Report/ ^ (Items A-D: not to exceed 25 pages*)

Phase I Progress Report (SBIR/STTR Phase II ONLY) | * SBIR/STTR Phase I: Items A-D limited to 15pages.D. Research Design and MethodsE. Human Subjects Research

Protection of Human Subjects (Required if Item 4 on the Face Page is marked "Yes")Data and Safety Monitoring Plan (Required if Item 4 on the Face Page is marked "Yes" and a Phase I, II,or III clinical trial is proposed)

Inclusion of Women and Minorities (Required if Item 4 on the Face Page is marked "Yes" and is Clinical Research)

Targeted/Planned Enrollment Table (for new and continuing clinical research studies)Inclusion of Children (Required if Item 4 on the Face Page is marked "Yes")

F. Vertebrate AnimalsG. Literature CitedH. Consortium/Contractual ArrangementsI. Resource SharingJ. Letters of Support (e.g., Consultants)Commercialization Plan (SBIR/STTR Phase II and Fast-Track ONLY)

Checklist.

Appendix (Five collated sets. No page numbering necessary for Appendix.)

Appendices NOT PERMITTED for Phase I SBIR/STTR unless specifically solicited..

Number of publications and manuscripts accepted for publication (not to exceed 10)

Other items (list):

1

1024

27

272833

445151

5354_555556_6363

65

Check ifAppendix isIncluded

PHS 398 (Rev. 09/04) Form Page 3


BUDGET JUSTIFICATION PAGEMODULAR RESEARCH GRANT APPLICATION

DC less Consortium F&A

Consortium F&A

Total Direct Costs

Initial Period

250,000(Item 7a, Face Page)

19,340

269,340

old

250,000

19,920

269,920

3rd

250,000

20,518

270,518

4th

250,000

21,134

271,134

5th

250,000

21,768

271,768

Sum Total(For Entire Project

Period)

1,250,000

(Item 8a, Face Page)

102,680

$ 1,352,680

PersonnelRonald S. Harwerth, O.D., Ph.D, Principal Investigator ---- -- effort) will design and supervise all of theexperiments, actively participate in the daily behavioral experiments, write computer programs forexperimental procedures and data analysis, analyze the data, prepare research reports, and assumeresponsibility for the scientific conduct of all aspects of the investigations.

---- -- ------ - --- ------ -------- Co-Investigator (5% effort) will preform the laser treatments to create unilateralocular hypertension in the monkeys for the principal studies and the laser treatments to create focal lesionsaround the optic nerve for the investigation of mapping visual field onto the optic nerve.

-------- -- ------------ -------- Research Associate, (100%) is responsible for all imaging proceduresof themonkeys eyes, including OCT and fundus photography. He will schedule the animals for imaging, help withanimal preparation, record and analyze the images and export data to a laboratory computer for additionalanalysis.

------ --- ------------------- ------- -------- Research Assistant, (15% effort) will assist with the imagingexperiments. She will prepare the animals for retinal imaging (anesthesia, eye drops, contact lenses) andmonitor their physiological status (heart rate, blood oxygen, body temperature) throughout the session.

See following continuation page

ConsortiumApproximately $60,000 total costs per year ($40,600 direct costs; $19,400 F&A costs)Consortium with the University of Texas - Houston, Health Science Center {X} Domestic {} ForeignCalculations based on 48.5% F&A rate for UT-Houston

See following continuation page

Fee (SBIR/STTR Only)

PHS 398 (Rev. 09/04) Modular Budget Format Page

BPrincipal Investigator/Program Director (Last, First, Middle): Harwerth, Ronald S.

Continued from Personnel. Modular Budget Format Page

-------- - -------- ------- -------- ------ ., Research Associate, (25% effort) will assist in the daily behavioralexperiments. For each session, for each monkey, he will set the experimental parameters for the session andsave and store the data at the end of the session. Typically, there will be four experimental sessions per day.

------- - -- ---------- ----- - Clinical Research Associate, (20% effort) will collect clinical data for the project.She will identify and recruit patients, obtain informed consents, conduct the clinical measurements (OCT, SAP,IOP).

--- - --------- ------ ., Graduate Student, Clinical Research Associate, (100% effort) will be involved in theinvolved in the clinical research phase and will identify and recruit patients, obtain informed consents, conductthe clinical measurements (OCT, SAP, IOP). He also will analyze the data and prepare the research report forthe clinical study. In addi---- -- - ---- -- - ---------- - - - the histological analysis of the spacing of axons in theretinal nerve fiber layer in---- --------------------- - ----- .

Continued from Personnel. Modular Budget Format Page

---------- - --------------------- -------- Co-Investigator (10% effort) will have responsibility for the histologicslanalysis of retinal ganglion cells and nerve fiber layer. She will collect tissue, design histological techniques forlight microscopy and confocal microscopy, supervise the histology technician in her laboratory, and preparereports for publication.

To be Named, Technician, (50% effort) will be involved in all aspects of the histological analyses of thelaser-treated and control eyes of monkeys, including fixing and embedding tissue, sectioning and stainingtissue, and quantifying ganglion cell and axon densities.

6PHS 398/2590 (Rev. 09/04) _ Continuation Format Page

17 pages redacted--biosketches omitted as requested


RESOURCES - University of Houston

Laboratory:The PI has two research laboratories, located-- - --- - ----- - ----- --- --- - - College of Optometry building, that

are available for the proposed studies (---- -- ----- - - 600 sq. ft. and---- -- ------ - - 500 sq. ft.). Theselaboratories are devoted to animal psychophysics research and are equipped with four behavioral stations, twoare designed to test binocular vision functions and the other two are committed to visual fi---- - --------- - usingstandard automated perimetry and contrast sensitivity perimetry for the proposed studies. ----- -- ------ - has asmall partitioned area of approximately 100 sq. ft. that will be used for the imaging studies (OCT and fundusphotographs).

Harwerth also has a laboratory-- - --- - -------- - ----- --- --- - Optometry building ----- -- ------- - - 300 sq. ft.) forpsychophysical studies of binocular with human observers.

Clinical:One of the clinical components of the University Eye Institute is the Ocular Diagnostic and Medical Eye

Service. The Ocular Diagnostic and Medical Eye Service provides specialized optometric and ophthalmologicalcare for patients. A wide range of conditions are evaluated and treated, with specialties in glaucoma,oculoplastics, neuro-ophthalmology, retinal disease, and cornea and external eye diseases. Proceduresinclude advanced perimetry, angiography, ultrasonography, corneal topography, and scanning laserophthalmoscopy, optical coherence tomography. Glaucoma patients for the present study will be recruited andexamined in the Ocular Diagnostic and Medical Eye Clinic.

Animal:The AAALAC approved animal care facilities on the Uni------- - --- ---------- - -------- us will be utilized for the

project. The Optometry satellite animal facility (1000 sq. ft.),-- - --- - ----- - ----- --- --- - - Optometry building, is inclose proximity to the research laboratories and can house 36 adult monkeys for behavioral experiments. Anadditional 40 infant monkeys can be housed in the nursery area during experimental rearing studies. Monkeysthat have completed the rearing procedures and are not undergoing behavioral testing, or monkeys who----- - ------------ - -------------- --------- ---- - -- - --------- - - - --- - Central Animal Care Facility (15,840 sq. ft.) located - --- - --------- - ---- - ------------ - - ----------- -------- - ----------- The UH animal Care Operations staff provides veterinarian----- --------- ------------- - ---------- - ---- - -- age cleaning) and transportation of animals between the Optometry and-- - ---------- - ---- - ------------ - - ------------

Computers:All of the behavioral research stations are controlled by PC computers (HP 486, 60 MHz). The computers

are equipped with I/O cards, graphics boards, and behavioral control interface modules. The computers for theOCT instrument and Nidek fundus camera are new with 2.13 gHz CPU's and 100 GB of storage for digitalpictures. All of the research personnel have personal computers in their offices and access to the University ofHouston computer network. The College's information technology staff provides support for computerhardware and peripherals, and some support for commercial software.

Office:All of the research personnel have private offices-- - --- - -------- - ----- --- --- - optometry building. The

College provides computers, telephones, office supplies, and Xeroxing.

PHS 398/2590 (Rev. 09/04) Page 24 Continuation Format Page


Other:The following research support services are provided by the College and will be available as needed for the

proposed project:(1) Clerical and secretarial services.(2) Business office support for personnel, purchasing, and inventory services.(3) Machinist / instrument maker with a well-equipped shop.(4) Electronics technician and electronics shop.(5) Audio-visual services for computer graphics, photography, poster presentations..(6) Vision Sciences library with information retrieval and research interest awareness services.(7) Computer and data service center.

The Core Center Grant supports three modules, all of which will be utilized for the proposed experiments.(1) An instrument design module to facilitate the development of optical, mechanical, and electronic researchinstrumentation. (2) A Computer module to facilitate research applications of laboratory computers. (3) Aclinical research module to facilitate statistical analyses and methodologies for research data.

Major Equipment:All of the major equipment for the proposed studies is available in the research laboratories. The major

equipment includes: sound attenuating primate test chambers, primate chairs, stimulus generators, HFA Iperimeter, StratusOCT instrument, Nidek digital fundus camera, Ophthalmic Argon laser, tonometers. Inaddition, an HFA II perimeter will be obtained within the next few months for modification and use in theexperiments proposed in the present application.



RESOURCES - University of Texas at Houston

Laboratory:600 square feet of space located in the Medical School, Department of Ophthalmology and Visual Science

Clinical:Mentor, tonopen XL, Nidek 3-DX stereo disc camera, indirect ophthalmoscope, slit lamp with 28 diopter

lens.

Animal:NA

Computers:There are three computers in Dr. Carter-Dawson's laboratory: Gateway 1.8 GHz Pentium 4, Gateway 2

GHz Pentium 4 with CD-RW and Gateway 400 MHz. There is one computer in the office: Toshiba 1.7 GHzwith CD-RW.

Office:Office space is located - - --- - --------- - ----- - in the Department of Ophthalmology and Visual Science.

Other:Shared equipment: Olympus BX60 research microscope with fluorescence and imaging system; a Zeiss

LSM 410 confocal microscope; Zeiss LSM 510 confocal microscope; a Vanox photomicroscope; Codonicsphotographic network printer; Typhoon 9400; Jouan BR4i centrifuge; Puffer Hubbard -80 freezer; cold room;Microplate Fluorescence Reader FL 600; Bio-Rad Spectrophotometer.

--- ----- - ----------- - a statistician, assists with data analysis and experimental design (supported by NEICORE Grant).

Major Equipment:Located in --- --------------------- - -------------- - MT 6000-XL microtome for cutting sections, Olympus BX51

microscope with Spot Camera and computer with Image Pro Plus Software (ganglion cell quantification andcapture images of retina); fume hood; centrifuge; pH meter; balances, heated ovens.

Located in the Ophthalmology Department on the same floor as --- --------------------- - office: Zeiss LSM510 confocal microscope and two work stations with LSM software for analysis of nerve fiber layer thickness.



7. RESEARCH PLANIn the past, this grant has involved behavioral investigations of experimentally induced anomalies of

binocular vision (amblyopia, stereo-deficiencies) in macaque monkeys. However, for the last few years,more of the research effort of the laboratory has involved behavioral investigations of visual defectscaused by experimentally induced ocular hypertension (experimental glaucoma) in macaque monkeys.Therefore, the specific aims of the present application will focus on the visual and neural alterationscaused by glaucoma and the progress report for the previous project will be reported only by the list ofpublications reporting those studies.

A. Specific Aims.The overall goal of the proposed research is to gain a better understanding of structure-function

relationships in glaucoma. The research plan involves behavioral studies of visual function (clinicalperimetry and spatial contrast sensitivity) and the high resolution imaging of retinal structure (opticalcoherence tomography) over the timecourse of experimental glaucoma in macaque monkeys. In a finalphase, the methods and procedures developed from experimental glaucoma will be applied to data fromhuman patients with glaucoma. The experiments are designed to accomplish four specific aims.

Specific Aim 1: To investigate the relationship between nerve fiber layer and perimetrymeasurements in experimental glaucoma. The relatively recent introduction of objective imaging ofretinal structure for the diagnosis of glaucoma and the assessment of glaucomatous neuropathy has ledto diverse conclusions about structure-function relationships in the disease (c.f.,.Bowd , et a/., 2001;Budenz, et a/., 2005; El Beltagi, et a/., 2003; Girkin, 2004; Parisi, et a/., 2001; Schlottmann, et a/., 2004;Wollstein, et a/., 2005) The diversity of opinion is a likely result from comparing different kinds ofmeasures, i.e., visual sensitivity (in log units) and nerve fiber thickness (in microns), when each measurealso a different degree of intersubject variability and a different dynamic range of measurement.However, the issue of different dimensions for the two approaches to assessing the stage of disease canbe resolved by introducing a common denominator for both procedures, i.e., the number of retinalganglion cells underlying each measurement. Therefore, the first specific aim is to develop proceduresfor defining the essential agreement between the numbers of retinal ganglion cells corresponding tovisual sensitivities at given locations in the visual field and the numbers of ganglion cell axons enteringthe optic nerve head from the corresponding areas of the retina. The empirical data for this study will befrom the pointwise sensitivity measures by standard automated perimetry (SAP) and retinal nerve fiberlayer (RNFL) thickness by optical coherence tomography (OCT) in monkeys with experimentalglaucoma. The data from both procedures will be converted to numbers of ganglion cells and axons forevaluation of the structural and functional measurements across various stages of glaucomatous neuralloss. In addition to understanding the specific clinical procedures used in the studies, the results shouldprovide a more general understanding of the basis of assessing glaucomatous neuropathy by anytechnique.

Specific Aim 2: To investigate, 1) the contribution of axons from outside the visual field area that isnormally tested by clinical perimetry to the total RNFL thickness comprised of axons from the entireretina and, 2) the topographical mapping of the visual field onto the optic nerve head. The basis ofclinical strategies of visual field testing and modern imaging protocols is that ganglion cell death causedby glaucoma starts in the mid-peripheral retina and progresses toward central retina (Anderson, 1987;Alexander, 1991; Epstein, 1997; Quigley, 1993). As a consequence, for concordance between the twoprocedures, neural axons outside of the perimetry measurement must contribute very little to total RNFLthickness. In addition, there must be a consistent systematic topographical relationship between visualfield (retinal) locations and the optic nerve head (ONH) locations of axons from the retina (Gardiner, eta/., 2005; Garway-Heath, et a/., 2000; Weber & Ulrich, 1991; Wirtschafter, etal., 1982). Therefore, thehypotheses for the second specific aim are that 1) laser ablation of the retina from the arcuate bundle tothe ora serrate will cause less than 10% thinning, i.e.. less than the normal intersubiect variability of themeasurement, and 2) discrete thinning of RNFL by local laser lesions will be consistent across animals.The data for this study will be based on the effects of retinal laser lesions on RNFL thickness and SAPmeasurements in monkeys. The first part of the study will involve monkeys with total peripheral retinalablations and the second part of the study will involve monkeys with laser lesions that coincide withspecific ONH sectors.



Specific Aim 3: To extend the investigation of the relationship between nerve fiber layer andperimetry measurements to clinical glaucoma patients. Experimental glaucoma is a high intraocularpressure (IOP) model, with very high pressures and no treatment (Gaasterland & Kupfer, 1974;Harwerth, et a/., 1997; Kee, et a/., 1995; Pederson & Gaasterland, 1984; Quigley SHolman; 1984). Theneural losses progress very rapidly and produce characteristic diffuse visual field defects (Harwerth, eta/., 1997; 2002). In contrast, clinical glaucoma patients are generally under treatment to lower IOP, theirvisual defects progress slowly, and visual field defects are more localized (Chauhan & Drance, 1990;Quigley, 2005; Sommer, 1989; Weinreb, 2004). Nevertheless, the structure-function relationship shoulddepend only on the number of ganglion cells in both experimental and clinical glaucoma. Therefore, thehypothesis for the third specific aim is that the methods developed to quantify ganglion cell counts andaxon counts in monkeys can be applied, without modification, to clinical glaucoma. The SAP and RNFLdata for this study will be collected by qualified clinicians from patients at various stages from mild toadvanced glaucoma. Subsequently, the clinical data will be analyzed by the same procedures as formonkeys (see the preliminary studies described in section C).

Specific Aim 4: To investigate alterations in spatial contrast sensitivity in areas of the visual fieldwith depressed visual sensitivity from glaucoma. A relative loss of spatial contrast sensitivity has beensuggested as an early sign of optic neuropathy from glaucoma (e.g., Bodis-Wollner, 1989; Evans, et a/.,2003; Harwerth & Smith, 1997; Hawkins, et a/., 2003; Johnson & Samuels,1997; Nordmann,1996;Porciatti, et a/., 1997; Sample, et a/., 2000; Spry, et a/., 2005a; 2005b; Thomas, et a/., 2002), but theeffects of spatial frequency have not been studied systematically. A systematic analysis of spatialcontrast sensitivity may suggest new strategies of detecting early visual loss and may also helpunderstand the often-reported complaint of poor vision by glaucoma patients, even when their centralvisual fields are normal by SAP testing. Therefore, the hypothesis for the fourth specific aim is that highspatial freguencv defects will precede defects at low spatial frequencies and that the defects with lowspatial frequency stimuli will closely parallel the defects measured by the broadband Size III stimulus thatis used in SAP. The data for this study will be collected by behavioral perimetry, using standard clinicalprocedures, and for spatial contrast sensitivity perimetry, using Gabor patch stimuli. The contrastsensitivity data will be evaluated via a fitted low-pass model to obtain the high-frequency cut-off spatialfrequency and the peak contrast sensitivity for comparison to SAP data on the same subject.

Summary: As a total, these investigations will result in a better understanding of the structure-function relationship for glaucoma and the most appropriate method for comparing clinical data from oneinstrument to data from another. The scientific method employed in the research is based on definingprocedures with experimental glaucoma where there are excellent controls for the experimental andmeasurement variables and, then, application to human patients to test the clinical relevance and validityof the procedures. This method of going from the laboratory to the clinic should maximize the potentialfor significant new information about ophthalmic testing for diagnosis and assessment of glaucoma.

B. Background and Significance.Glaucoma, a progressive optic neuropathy caused by a pathological loss of retinal ganglion cells, is a

leading cause of blindness in the United States. According the American Health Assistance Foundation,approximately 2.2 million people older than 40 years of age suffer from the disease and as many as120,000 of these people are blind from glaucoma. The disease is an increasingly important problem ofpublic health because the number of patients is expected to continue to grow, with 5,400 casesadvancing to blindness and more than 300,000 new cases each year. Further, it has been estimated that3.3 million Americans will have glaucoma by the year 2020. However, on the positive side, treatment iseffective. The major clinical trials have demonstrated that treatment to lower intraocular pressure hasvalue in preventing the onset of glaucoma in ocular hypertensive patients and in slowing the progressionin glaucoma patients (AGIS Investigators, 2000; Heijl, etal., 2002; Kass, et a/., 2002; Weinreb, 2004). Itis, therefore, necessary to continue the development of procedures for diagnosis and assessment ofglaucoma to optimize clinical decisions about when treatment is needed and when treatment is effective.

The diagnosis and assessment of glaucoma must rely on ophthalmic testing because the cause isunknown. It is described as a multi-factorial disease, or a constellation of diseases, because there is notan identifiable single etiology (Quigley, 1993; Epstein, 1997), although there are epidemiological riskfactors that are associated with an increased likelihood of having the disease (Gramer & Tausch, 1995;



Quigley, 1993) and there are cellular-level risk factors leading to pathological injury and death of retinalganglion cells (Nickells, 1996; Quigley, 1998a; 1998b; Schumer & Podos, 1994). However, all of theetiological factors lead to the single final manifestation of glaucoma, the death of retinal ganglion cells,which can be observed by ophthalmic examination as a cupping of the optic nerve head, the loss ofretinal nerve fiber layer, and functional visual defects (Anderson, 1987; Quigley, 1993; Weinreb, 2004).Consequently, clinical procedures for the diagnosis and assessment of the progression of glaucoma arebased on quantification of these clinical characteristics of glaucomatous optic neuropathy and, thus, it isimportant to know how well the diagnostic techniques provide a true representation of the extent ofganglion cell death, i.e., reflect a valid structure-function relationship.

Functional measures: In modern clinics, the standard for assessment of functional defects is visualfield testing by computer-automated perimetry, using white-light test targets superimposed on a white-light background (Heijl, 1985a; Johnson, 1996). This form of standard automated perimetry (SAP) hasbecome established as a "gold standard" and the method used most often in the United States is theHumphrey Field Analyzer (Heijl & Patella, 2002). The visual defects from glaucoma are characterized bya progressive loss of sensitivity that typically begins in the mid-periphery of the nasal field and eventuallymay extend to the central visual field (Drance, 1985; Heijl, 1985b; Mikelberg, et a/., 1986; Quigley, 1993).In general, the clinical stage, or severity, of the disease is established by the sensitivity map of the visualfield that is derived from light-sense thresholds measured at a number of locations across the retina(Anderson, 1987) and the most important diagnostic data are the pointwise depth of visual defect withrespect to the expected-normal visual sensitivity at each of the tested visual field locations (Heijl, 1987a;1987b).

The psycho-physiological link between visual sensitivity and ganglion cell density is based on theconcept that visual sensitivity represents a nonlinear pooling of the outputs of neural detectors (Harwerth,et a/., 2002; 2004, but see Garway-Heath, et a/., 2002; Swanson, et a/., 2004, for an alternative view).The pooling of neural responses should be dependent on ganglion cell density, so that visual sensitivityvaries with the number of neural detectors when the number of neural detectors varies from retinaleccentricity, normal aging, or the stage of glaucoma. Thus, the progression of visual field defects inglaucoma is an indication of progressive pathological losses of retinal ganglion cells, and the relationshipbetween the extent of neural damage and the amount of increase in light-sense thresholds will beproportional (Heijl, etal., 1987a; 1987b; Katz, etal., 1991; Asman & Heijl, 1992) and is linear when bothare expressed in dB units (Harwerth, et a/., 2004).

It is reasonable that the degree of vision loss would be proportional to the amount of ganglion cellloss (Harwerth, etal., 1999; Kerrigan-Baumrind, etal., 2000; Quigley, et a/., 1989), but the quantitativerelationship between visual sensitivity and ganglion cell density was established only recently (Harwerth,et a/., 2004). The previous investigations of ganglion cell density and visual sensitivity in glaucoma hadsuggested a highly variable relationship. (Quigley, etal., 1989; Harwerth, etal., 1999) For example, arecent study with a relatively large number of glaucoma patients, Kerrigan-Baumrind, et a/. (2000)reported that linear regression analysis of the point-wise correlation between visual sensitivity andganglion cell loss accounted for only 3% of the total variance. A clearer relationship between neural andvisual losses was established by the finding that statistically significant visual field abnormalities occurredif neural losses at the corresponding retinal location exceeded 25 - 35% of the normal cell density. Inaddition, the relationships between structure and function were highly significant with more globalmeasures of visual sensitivity and neural loss, e.g., average visual sensitivity losses or the MeanDeviation (MD) perimetry indices vs. mean ganglion cell losses. Thus, although the study demonstrateda clinically significant structure-function relationship, it was not quantitative for the point-wise translationof clinical measurements by perimetry to retinal ganglion cell losses.

There are several methodological explanations for large variability in the neural-sensitivityrelationships for glaucoma patients, especially with respect to the method of data transformation forscaling visual sensitivities and neural cell densities (Garway-Heath, etal., 2000; Harwerth, etal., 2002;Leung, et a/., 2005; Swanson, et a/., 2004). A study of logarithmic and linear transformations on a singleset of sensitivity and neural data suggested that logarithmic (decibel) scaling provided a less variablerelationship than linear scaling (Harwerth, etal., 2005). Specifically, with decibel scaling, visual sensitivitylosses and ganglion cell densities were linearly correlated with high coefficients of determination,although the parameters of the functions varied with eccentricity. The structure-function relationshipsPHS 398/2590 (Rev. 09/04) Page 29 Continuation Format Page


expressed as linear percentage-loss functions were less systematic in two respects. First, therelationship exhibited considerable scatter in the data for small losses in visual sensitivity and, second,visual sensitivity losses became saturated with larger losses in ganglion cell density. The parameters ofthe percentage-loss functions also varied with eccentricity, but the variation was less than for the decibel-loss functions. The comparatively greater accuracy and precision of decibel-loss functions are a likelyconsequence of the logarithmic scale of stimulus intensities for perimetry measurements and becausethe relationship between visual sensitivity and the number of neural detectors is a form of probabilitysummation.

The decibel data-transform was incorporated into a quantitative structure-function model that wasdeveloped from studies of experimental glaucoma in monkeys (Harwerth, et a/., 2004). The model was alinear regression analysis for a point-wise correlation of retinal ganglion cell density to singlemeasurements of visual sensitivity by standard clinical perimetry. The model was based on linearrelationships with logarithmic scaling for both visual sensitivities and retinal ganglion cell densities, wherethe parameters for the linear functions varied systematically with eccentricity. Thus, the parameters (y-intercept and slope) of the linear structure-function relationship can be determined for a given eccentriclocation in the visual field and, the ganglion cell density for a given visual sensitivity can be predictedfrom that linear relationship. The analysis of the relation between visual sensitivity and ganglion celldensity demonstrated that, without free parameters, perimetry data provided an accurate and relativelyprecise quantification of retinal ganglion cell losses caused by experimental glaucoma in monkeys.Further, the relationships derived from experimental glaucoma also accurately predicted the rate of age-related losses of retinal ganglion cells in humans, based on the normative perimetry data for age-relatedreductions in visual sensitivity. These results suggested that the model could improve the interpretationof the stage of glaucomatous optic neuropathy, but its usefulness requires direct testing with data fromclinical patients.

Structural measures: During the past couple of decades, methods for objective imaging of retinalstructure have been introduced for the diagnosis of glaucoma and the assessment of glaucomatousprogression. The HRT for measuring optic nerve head parameters (e.g.,Janknecht & Funk, 1994; Iliev, eta/.,2005; Philippin, et a/., 2005); Sihota, et a/., 2002) has the longest history of clinical use, but measuresof the retinal nerve fiber layer (RNFL) thickness seem the be more appropriate for the proposal's specificaims, i.e., relating neural losses by structural and functional tests. Of the two instruments designed tomeasure RNFL thickness, optical coherence tomography (OCT; e.g., Bowd, et a/., 2000; Budenz, et a/.,2005; Leung, et a/., 2004; Teesalu, et a/., 2000) and scanning laser polarimetry (SLP; e.g., Galvao Filho,et a/., 2005; Kremmer, et a/., 2000; Lemij, 2001), the OCT is better suited for measurements withanesthetized monkeys and, therefore, it will be the only instrument described for the proposed studies,although the results should be relevant to any measure.

OCT, first described by Huang, et a/., (1991) is a high resolution cross-sectional imaging techniquethat provides in vivo measurements of the RNFL thickness with a resolution of 8 - 10 micrometers (|i).OCT imaging is analogous to ultrasound B-mode imaging except that it uses light instead of sound andperforms cross-sectional imaging by measuring the echo time delay and intensity of back-reflected andback-scattered light from tissues at different depths (Fujimoto, et a/., 2004). Various retinal layers in theOCT image can be differentiated by differences in contrast and rendered in false-color for analysis. Forexample, the RNFL thickness can be measured because axons of the nerve fiber layer are highlyoptically back-scattering, appearing as red in false-color images, whereas the ganglion cell layer hasweaker back-scattering and appears green. For clinical implementation, the standard measurement ofRNFL thickness has 512 points, or transverse pixels, in the 10.87 mm circumference of a 3.4 mmdiameter circle, which starts at the center of the temporal side of the optic nerve head (Lee, et a/., 2004).Each pixel is a discrete measure of thickness, in |i, to produce a RNFL thickness profile with a doublehump pattern for normal eyes, characterized by thin RNFL at the temporal and nasal regions of the opticnerve head and thicker RNFL at the superior and inferior poles (see Fig.2).

Normative reference values for have been established for OCT-RNFL measurements to allow thesestructural data to be analyzed analogously to SAP functional data (Patella, Carl Zeiss MeditecPublication). With normative data, the measurements for an individual patient are compared to expectedage-matched, normal values, and statistical confidence intervals, to determine whether the individualpatient's data are outside of normal variability. In this case, confidence intervals have been establishedPHS 398/2590 (Rev. 09/04) Page 30 Continuation Format Page


for a substantial number of RNFL parameters, such as average thickness for each quadrant, averagethickness for each of twelve optic nerve head sectors, and 11 values based on either symmetry (e.g.,inferior maximum thickness to superior maximum thickness ratio) or average thicknesses (e.g., superioraverage or average total thickness). Consequently, comparisons of structural and functional measuresfor identifying glaucoma patients can be based on dramatically different measures, SAP visual sensitivityvs. OCT nerve fiber thickness, using the common metric of probability with respect to normative values.However, a more valid comparison of two substantially different measures of glaucomatous neuropathyshould be based on a common denominator for both measures, i.e., the number of retinal ganglion cellsand axons underlying the measures.

Because it is a relatively new procedure, there have been numerous recent clinical studies of OCT,with considerable variation across studies in the interpretation of mechanistic relationships between SAPand OCT. For example, OCT has been reported to be a highly reproducible measurement with very highsensitivity and specificity in discriminating between control and patient populations (Budenz, et a/.,2005b). Without exception, RNFL measurements outside the 95% normal confidence limits identifiedglaucoma patients with moderate glaucomatous field defects (Budenz, et a/., 2005a) and, thus, in stageswhere there is an established relationship between visual and neural losses, both procedures detect theneuropathy. In contrast, other studies have suggested a disconnect between the structural and functionalmeasurements. In a longitudinal study of glaucoma suspects and patients, a larger percentage ofpatients were classified as progressing from OCT (22%) than SAP (9%) measurements and it wassuggested that structural changes may precede functional changes in early stages of the disease, or lagfunctional changes in late stages, because of a curvilinear relationship between functional and structuraldefects caused by glaucoma (Wollstein, et a/., 2005). Other studies of RNFL thickness and visualfunction have shown relatively high sensitivity for some OCT parameters, but a relatively poor agreementbetween perimetry and nerve fiber layer instruments for classifying eyes with glaucoma, which suggeststhat different techniques may identify different characteristics of glaucomatous damage (Bowd, et a/.,2001).

It is apparent from the various clinical studies of morphological imaging that there is not a consensusin the clinical research literature about the relationship between nerve fiber layer and perimetrymeasurements. It is likely that the absence of unified agreement is from the difficulty of comparing twovery different measurements, without a common parameter. However, on the face of it, there should be avery close relationship between the two clinical procedures because SAP thresholds in a given locationare determined by the number of retinal ganglion cells (RGCs) in the corresponding retinal area and theRNFL thickness represents the number of RGC axons from a given location in the retina. Thus, thenumber of retinal ganglion cells provides a common denominator underlying the two clinical proceduresthat should define a quantitative relationship. This essential relationship is the rationale that hasmotivated many of the studies of the present proposal (specific aim 1) and formed the basis forpreliminary studies described in Section C of the proposal.

It is sensible that both visual sensitivity and axon number should be determined by the survivingpopulation of retinal ganglion cells at any stage of the disease, but because RNFL thickness is not alocalized measure there are additional questions about the relationship. For example, the relationshipbetween RNFL and glaucomatous neuropathy is complicated by uncertainty about how axons ofganglion cells outside of the normal perimetry field contribute to total RNFL thickness and also byuncertainty about the topography of visual field mapping onto the optic nerve head. Both of these areascan be addressed by combining laser ablation of specific retinal areas and OCT measures in monkeystrained for clinical perimetry (specific aim 2). The broader applications of the work on structure-functionrelationships in experimental glaucoma will be investigated in a clinical study of SAP and OCT (specificaim 3).

Contrast sensitivity perimetry: When a patient's glaucomatous neuropathy has progressed to alevel of clinical significance by standard clinical perimetry, there is a high correlation between SAP andalternative perimetry procedures that were developed on many different principles of assessing the lossof retinal ganglion cells, e.g., high-pass resolution, localized contrast sensitivity, frequency-doubling, bluelight stimuli, or motion detection (Harwerth, et a/., 1999; lester, et a/., 2003; Kadaboukhova & Lindblom;2003; Lynch, et a/., 1997; Martin, etal., 2003; Sample, 2001; Spry, etal., 2005a). There is, however, also



evidence that a variety of alternative stimuli provide earlier detection of neuropathy than SAP (Bosworth,etal., 1998; Cello, et al., 2000; Johnson, et al, 1993; Spry etal., 2005b; Wall, et a/., 1997), but it is notclear whether some of the ganglion cell specific stimuli are better than others. The psycho-physiologicallinking hypothesis for utilization of alternative perimetry stimuli is based on data suggesting that ganglioncells with larger cell bodies and axons are more susceptible to neuronal injury (Quigley, etal., 1989) and,because the functional properties of the larger (Pa) and smaller (Pp) ganglion cells are different (Dacy,1999; Frishman, 2001), stimuli that isolate Pa cell function should be more efficient in revealing earlyneural loss.

The hypothesized differences in the effects of glaucoma on neurons in the M- and P-pathways havenot been supported by direct investigations, i.e., by histochemical visualization of energy metabolism inthe afferent neural pathways (Crawford, etal., 2001; 2002; Harwerth & Crawford, 2004). The failure ofhistochemical visualization of neural metabolism to demonstrate a differential effect of glaucomabetween the large-cell and small-cell pathways is in agreement with other electrophysiological andhistological investigations that also failed to demonstrate selective effects in the M-cell and P-cellpathways (Morgan, etal., 2000; Smith, etal., 1993; Vickers, et al, 1997; Weber, ert al., 2000; Yucel, etal., 2000). It is, therefore, more likely that earlier detection of glaucomatous neuropathy by the newer,non-standard, methods of perimetry is through a reduction in the number of available detectors, which isaccomplished by limiting detection to a subset of mechanisms with receptive field characteristics thatmatch the specific properties of the stimulus, i.e., reduced redundancy (Johnson, 1994).

Based on an assumption that the strongest psycho-physiological link for early detection of visualloss from glaucoma is by matching perimetry stimulus properties to a small population of receptive fields,then the logical stimulus for perimetry would be narrow-band in the spatial frequency domain (Swanson,et al., 2004). Spatial contrast sensitivity functions provide an excellent description of the responseproperties of the visual system because the spatial response profiles of the receptive fields that are themost sensitive to a given stimulus will match the spatial frequency characteristics of the stimulus.Therefore, a stimulus that is composed of a narrow band of spatial frequencies and is restricted in itsspatial size has many desirable properties for clinical perimetry (Atkin, et al., 1979; Lundh & Gottvall,1995; Harwerth, & Smith, 1997). The detection thresholds for such a stimulus will be based on theresponses of a specific subset of receptive fields in the retinal area that is tested, which could representa smaller, more selective population of ganglion cells than would respond to the standard white lightstimulus.

A relative loss of spatial contrast sensitivity has been suggested as an early sign of opticneuropathy from glaucoma (e.g., Bodis-Wollner, 1989; Evans, etal., 2003; Harwerth & Smith, 1997;Hawkins, etal., 2003; Johnson & Samuels, 1997; Nordmann, 1996; Porciatti, etal., 1997; Sample, etal.,2000; Spry, et al., 2005a, 2005b; Thomas, et al., 2002), but the effects of spatial frequency have notbeen studied systematically. Therefore, the fourth specific aim of the proposal is to investigate spatialcontrast sensitivity functions over the time-course of experimental glaucoma to determine, 1) whetherthere are spatial frequency specific losses in glaucoma, 2) whether losses of visual sensitivity at highspatial frequencies precede losses at lower spatial frequencies and, 3) whether there are losses ofspatial frequency that precede defects by SAP.


A. GlaucomapatientsN = 437

2.59 dB

46C. Monkeys

343


C. Preliminary Studies.

1. Period covered: 1/1/2000 to 11/1/2005.2. Summary of Specific Aims.

The objective of the research over the past 5 years that has provided the foundation for the presentproposal is to gain a better understanding of methods for ophthalmic testing of glaucomatousneuropathy. The major investigations were a series of behavioral studies of visual function (clinicalperimetry) in macaque monkeys with experimental glaucoma. This work has involved collaboration withscientists at the University of Texas - Houston to provide histological data on the neural defects causedby experimental glaucoma in the retina and afferent visual pathway. By combining structural andfunctional data, one of the major specific goals was to develop a quantitative relationship between thevisual and neural effects of glaucoma (Harwerth, etal., 1999; 2002; 2004). That model will be furtherdeveloped and extended to nerve fiber layer measurements for the aims of the research proposed in thecurrent application.

3. Studies and Results.Visual field defects and retinal ganglion cell losses in human glaucoma patients. Glaucoma is

a disease that causes a progressive loss of vision from the death of retinal ganglion cells (Epstein, 1997;Quigley, 1999) and the degree ofvision loss should be proportionalto the amount of ganglion cell loss(Anderson, 1987; Alexander,1991, Quigley, 1993; Quigley, eta/., 1989; Harwerth, etal., 1999,Kerrigan-Baumrind, etal., 2000).Traditionally, this relationshipbetween structure and function inglaucoma has been applied inclinical perimetry to establish theclinical stage, or severity, of thedisease (Hodapp, etal., 1993;AGIS Investigators, 1994; Brusini,1996), but the quantitativerelationship between visualsensitivity and ganglion celldensity was established onlyrecently (Harwerth et a/., 2004)The quantitative structure-functionmodel, which was developed fromdata on experimental glaucoma inmonkeys, was accurate andrelatively precise in predicting theretinal ganglion cell densityunderlying a given sensitivity andlocation in the visual field.Although based on experimentalglaucoma, the model should beapplicable to clinical glaucomabecause the visual systems ofhumans and monkeys areessentially identical in everyrespect. However, direct empiricalevidence that the structure-function model can be applied to

25 1,92dB

CD25 36 45 26 45

Measured Ganglion Cell Density (dB)

B. PatientsMean error: -0.26 ± 3.22 dB

.llllh.

D. MonkeysMean error: 0.57 ± 2.42 dB

Residual Error (dB) Residual Error (dB)

Fig. 1. The results from applying the model for structure-function forglaucoma patients and monkeys with experimental glaucoma. The uppergraphs (A & C) represent the relationships between ganglion cell densitiespredicted from perimetry measurements of visual sensitivity as a functionof the histological measurements of cell density. The different symbolshapes designate different locations in the visual field. The modelprediction of unity correlation is shown by the one-to-one line that issuperimposed on the data. Goodness of fit statistics for the meanabsolute deviation (MAD) and coefficient of determination (r2) arepresented as insets. The lower histograms (B & D) present the distributionof residual errors (ORE) of the model with respect to the one-to-onerelationship, with errors of greater predicted than measured cell densitiesconsidered as negative errors and errors for greater measured thanpredicted cell densities considered as positive errors. The mean and SD'sof the distributions are shown by insets and the percentage of errors thatare less than ± 3 dB are indicated by the darker bars.



clinical glaucoma is a necessary additional step in establishing the scientific basis of interpretingglaucomatous optic neuropathy from psychophysical measurements of visual thresholds.

The study of structure and function in human glaucoma was based on the raw data for retinalganglion cell densities and visual thresholds at corresponding field locations for the 17 eyes of 13patients with documented glaucoma, which have been reported (Kerrigan-Baumrind, 2000). The datawere analyzed by a model that predicted retinal ganglion cell densities from standard clinical perimetrythresholds and the predicted cell densities were compared to the corresponding histologic cell counts.The model, without free parameters, produced an accurate and relatively precise quantification of retinalganglion cell densities associated with visual field defects for eyes of human glaucoma patients. For 437sets of histologic and perimetry data with visual sensitivity greater than 0 dB, the unity correlation forpredicted vs. measured cell densities had a coefficient of determination of 0.38 (Fig. 1 A). The meanabsolute deviation (MAD) of the predicted vs. measured values was 2.59 dB and the mean and SD of thedistribution of residual errors (ORE) of prediction were -0.26 ± 3.22 dB (Fig 1B).

The residual variability in the structure-function relationships may be due to experiment error, ratherthan in the basic relationship or the model. The effect of reducing many sources of error is demonstratedby comparison of data from humans and monkeys. Figs. 1C & D present the results from experimentalglaucoma, using the same model of structure-function relationships as for human patients (Figs. 1A & B).The statistical data for the model show a considerably greater precision in the relationship forexperimental than clinical glaucoma, with an r2 value of 0.85, compared to 0.38 for clinical glaucoma,and a narrower error distribution with about 75% of the data, compared to 65% for clinical glaucoma,falling within ± 3 dB of zero error. This comparison shows that when experimental variables are bettercontrolled, the structure-function relationship becomes more precise. Thus, the overall results of thestudy serve to validate the fundamental assumption of clinical perimetry that the degree of vision loss isproportional to the amount of ganglion cell loss.

The results of the study have demonstrated that visual field defects measured by standard clinicalperimetry are a direct expression of the neural losses caused by glaucoma, where the quantitativerelationship varies with retinal eccentricity. The direct evidence for a quantitative structure-functionrelationship provides a scientific basis of interpreting glaucomatous optic neuropathy frompsychophysical measurements of visual thresholds and supports the clinical application of standardperimetry to establish the clinical stage of the disease. In addition, the results support the use ofperimetry to estimate localized ganglion cell densities for investigations of other procedures of assessingglaucomatous neuropathy, such as high resolution imaging (Wollstein, et a/., 2004) orelectrophysiological procedures (e.g., Frishman, et a/., 2000; Hood, et a/., 2004; Ventura & Porciatti,2005; Viswanathan, et a/., 1999), which also may be direct measures of neural loss..

The relationship between nerve fiber layer and perimetry measurements. Although SAP hasbecome the standard functional test for diagnosis and especially for analysis of progression ofglaucomatous optic neuropathy, recently the objective imaging of retinal structure has been proposed asa valid alternative (Wollstein, et at., 2004; Leung, etal., 2005). However, to determine whether the twoprocedures are equivalent or different, the quantitative relation between functional measures of ganglioncell loss (SAP) and objective measures of axon loss (OCT) must be established. Such a relationshipbetween perimetry and nerve fiber layer measures is complicated by the differences in the dimensions ofthe two measurements, i.e., nerve fiber thickness in microns vs. visual thresholds in log-intensity unitsand, therefore, the appropriate strategy requires a common denominator via the neurons underlying eachmeasurement. For this purpose, the present investigation was undertaken to determine the degree ofagreement between SAP measures of the number of ganglion cells in retinal areas representing specificvisual field locations and OCT measures of the number of ganglion cell axons entering the optic nervefrom the same retinal areas.

The initial investigation involved two stages. The first stage, using normal grouped data, was todevelop the methods and parameters for estimating neuron counts by each procedure and the mappingthe visual field locations onto the optic nerve head and, then, a second stage to produce preliminary dataon the effects of glaucoma in monkeys during early periods following laser treatment to createexperimental glaucoma.



In the first stage to develop methods for quantifying neurons by each procedure, SAP and OCT datawere compared for separate groups of normal monkeys. For SAP, control data from 35 monkeys, frombehavioral perimetry, using data from both eyes with 1 repeated measure per eye, provided 140 sets ofnormal visual thresholds for each test field location of the HFA 24-2 program. For OCT, the monkeyswere under anesthesia (ketamine and xylazine) with dilated pupils (tropicamide) and the standard RNFLthickness scans (512 points) were collected, using data from both eyes of 20 monkeys, with the finalthickness profile for each eye based on the average of 3 scans.

The numbers of retinal ganglion cells in specific retinal areas of normal eyes were estimated fromnormative perimetry data, using a structure-function model previously developed and applied to datafrom both monkeys and humans (Harwerth, et a/., 2004). In brief, the model is a linear relationshipbetween visual sensitivity and ganglion cell density for corresponding visual field and retina locationswhen both variables were expressed in logarithmic units. However, the parameters of the linear functionsalso vary with eccentricity in a linear manner. Consequently, with the appropriate parameters for thevisual field location, the sensitivity accurately reflects ganglion cell density (cells/mm2) in thecorresponding area of the retina. To obtain the total number of ganglion cells in an area of the retina, thecell density derived from each perimetry measurement was considered to represent the uniform densityof the 2.25 mm2 of a retinal area that corresponds to a 6X6 deg area that separates visual field testlocations for SAP.

The numbers of retinal ganglion cell axons represented by RNFL thickness measures were estimatedfrom the area represented by a sector of the optic nerve head and the area occupied by each axon. TheOCT scan length for the standard scan is 10.87 mm, which is sampled in 512 pixels and, thus, each pixelrepresents a retinal distance of 21.2 u. The scan height across pixels provides the total area occupied byRNFL axons in a given region of the optic nerve head. The area occupied by a single axon was based onthe results of several histological studies of axon diameter. In one study, Ogden (1984) compared axondiameters in arcuate, papillomacular, and nasal nerve fiber bundles and found that the median diametervaried with bundle location (0.4 - 0.8 u), but in each location the distribution of axon diameters wasskewed toward diameters larger than the median. Other studies have reported larger mean diameters

(Sanchez, et a/., 1986) or have shown variationsin diameter along the length of an axon (Wang, eta/., 2003). For purposes of the present study, asmall range of axon diameters, within the range ofpublished data, were evaluated to obtain areasonable match between axon and RGCnumber for the initial portion of the OCT scan,represented by the first 51 pixels. A final value of0.9 u diameter (0.63 u2 area) was set as thecoefficient of proportionality for the number ofaxons per sector area of the OCT scan.

The final step for determining the relationshipbetween nerve fiber and perimetry measurementswas to develop an appropriate topographicalmapping of the visual field onto the optic nervehead. Although several mapping relationshipshave been proposed (Garway-Heath, etal., 2000;Weber & Ulruch, 1991; Wirtschafter, etal., 1982),none produced a tenable relationship between theRGC and RNFL data and, therefore, a modifiedscheme was developed for the SAP-OCTrelationship that is illustrated in Fig. 2. With thisscheme, the optic nerve head was divided into 10equal sectors of 36 deg, each representing 51pixels of the OCT scan. The number of visual fieldlocations assigned to a sector varied from oneSAP location near the fovea entering the ONH in

SAP test field locations

ONH sectorssup

temp

Inf

OCT sector locationstemporal superior nasal inferior temporal

ZOO 300

Pixel number

Fig. 2. Mapping the visual field to the optic nerve head.The axons from RGCs in retinal areas corresponding totest field locations labeled by one number enter theONH in the sector labeled by that number and thenumber of axons is represented by the portion of theOCT scan label by the same number.



45OOOOtemporal superior temporj

° 8JB %f °§J2

3OOOOO

1SOOOO

O 1OO

• Estimate from OCT• Estimate from SAP

ZOO 3OO

Pixel number4OO soo

Fig. 3. Comparison of neuron cell counts by SAP and OCTnormal monkey eyes. The number of RGCs in SAP testfield locations entering the ONH in a sector is representedby the squares. The number of axons derived from OCTthickness measure entering the ONH in a sector isrepresented bv the circles.

Subject: OHT-45

A.

sector 1 or 10, to 13 SAP locations for thearcuate locations of the visual field entering theONH in sector 4 or 7. The final RGC to RNFLrelationship was derived by, 1) the sum ofganglion cells inputting to a ONH sector, asestimated from the normal perimetrysensitivities at each test field location, and 2)the total number of RGC axons estimated fromthe height of the OCT scan across 51 pixels ineach sector divided by the RNFL area of eachaxon.

The results of the methods of deriving celland axon numbers are presented in Fig. 3. Thetotal number of ganglion cells (squares) orneuron axons (circles) in each ONH sector is

plotted at the center of the pixel range representing that sector. The agreement between the twoestimates of neural elements is generally excellent. For example, the function indicates that the axons ofnearly 150,000 ganglion cells from SAP test location 1 enter ONH sector 1 and, similarly, the OCT scan

height across sector 1 converts to about150,000 axons entering ONH sector 1. Theagreement between the two estimates isconsistent for all of the sectors except 5 and 6.The obvious SAP under-estimation of RGCaxons entering these sectors is irresolvablebecause perimetry does not sample a sufficientamount of retina to determine the axon countinto the nasal ONH and, consequently, thesesectors were omitted from further analysis. Thedegree of agreement between estimates isfurther illustrated by comparisons of the totalnumbers of neural cells and axons afterexcluding the two nasal sectors; the totalnumber of RGCs estimated from the normativeSAP thresholds was 1,583,023 and the totalnumber of axons estimated from the normalRNFL thickness was 1,550,671. Although theseestimates are slightly higher than mostestimates of the total axons in optic nerve (Cull,et a/., 2003; Kupfer, etal., 1967; Mikelberg, eta/., 1989; Sanchez, etal., 1986), the closeagreement between the two estimates in eachsector and across sectors supports thepotential for application of the procedures forassessment of neural loss from glaucoma.Thus, to evaluate the potential for application ofthe methods developed to evaluateglaucomatous neuropathy, data were acquiredfrom behavioral SAP and clinical OCT in 5monkeys during early periods following lasertreatment to create unilateral experimentalglaucoma.

1 f!OS

MD:+0.44dBPSD: 1.80dB

B.

I;00

MD: -1.92dBPSD: 6.55 dB

inferior

Pixel number

w. temporal45OOOO

superior Inferior temporal

100

-Estimate from OCT• Estimate from SAP

3OO 4OO SOO

Pixel number

Fig. 4. Comparison of cell counts by SAP and OCT for amonkey with neural loss from experimental glaucoma. A.The gray scale plots of visual fields by SAP. B. Thestandard RNFL scans for the right and left eyes. C.Estimates of RNF axons derived from SAP (squares) orOCT (circles) for the control (upper) and laser-treated(lower) eyes.



Examples of the data for one monkey (OHT-45) with early visual field defects following five lasertreatments are presented in Fig. 4. The relative interocular losses of visual sensitivity by SAP andthinning of the RNFL by OCT are presented in panels A and B, respectively. The SAP data are indicativeof relatively early defects in the superior and inferior quadrants of the visual field, while the OCT datashow a generalized loss of RNFL thickness at the superior and inferior poles of the ONH. These datawere used to derive the ganglion cell counts (squares) for axons entering each ONH sector fromdesignated test field locations (see Fig. 2) and the axon counts (circles), derived from the OCT data, thatenter the corresponding ONH sectors. The two functions (Fig. 4C) that each represents the number ofneural cells, but that were derived from different clinical measures of function and structure areremarkably similar for both the control and laser-treated eyes. The differences between SAP and OCTestimates of ganglion cells are a little larger for the normal, left eye than the laser-treated, right eye, butin every sector the two measures are within 2% of each other. The agreement for the amount of neuralloss of the laser-treated eye, relative to the control eye, is similar for three assessments of structural loss,i.e., a 23% loss in the average RNFL thickness (Fig. 4B), a 27% loss in total axon count derived from theOCT thickness scans, and a 29% loss in RGCs derived from the SAP visual fields (Fig. 4C).

The data for the subject presented in Fig. 3 are consistent with the hypothesis that there arequantitative agreements between SAP measures of the number of ganglion cells in retinal areasrepresenting specific visual field locations and OCT measures of the number of ganglion cell axonsentering the optic nerve from the same retinal areas. Additional support for the hypothesis was gainedfrom a larger number of measures by combining data for all five monkeys. These results are presented inFig. 5 by the agreement of estimates of ganglion cells and axons across sectors, eyes, and monkeys,with repeated measures from individual monkeys if the measurements were separated by at least 3weeks. The degree of agreement was based on the percentage difference between estimates, with thedifference assigned a negative value if the estimate of ganglion cells by HFA was less than the estimateof axons by OCT, or a positive value when the HFA estimate was larger than the OCT estimate.Although these data are preliminary and do not include any subjects with advanced glaucoma, the rangeof differences between estimates is narrow, with 76% of the data falling within ±25% of agreement and amean difference in estimates of-2.1% ± 20%.

To visualize the types of large differences, anormal distribution with a mean and SD equal to theexperimental data was superimposed on thehistogram. A comparison of the experimental data withthe normal distribution shows that the largestdifferences in estimates were negative, i.e., estimatesof ganglion cells by HFA that were less than theestimates of axons by OCT. The negative values forthe largest disagreements are noteworthy because it isindicative of functional losses preceding structurallosses and is similar to the hypothesis that differencesin RNFL and pattern ERG (PERGLA) measurementspredict that some function is recoverable withtreatment (Ventura & Porciatti, 2005). However, theimportance of these preliminary findings must beverified by subsequent measurements over the fulltimecourse of experimental glaucoma and byhistological verification of the cell and axon counts.

The overall conclusion from the results of thesepreliminary experiments is that SAP measures of visualfield defects and OCT measures of RNFL defects arecorrelated measures of glaucomatous neuropathy. Inaddition, the data form a foundation for additionalstudies to, 1) refine the visual field to optic nerve head

map, 2) determine whether the dynamic ranges of measurements are different, 3) investigate whetherthe normal inter-subject variability of RNFL measurements is smaller than for SAP measurements, 4)

3O

2O

'C.Q) 10

N = 232

mean(sd) = -2.1%±20%

<-55 -4O -2O O 2O 4O >55(HFA<OCT) (HFA > OCT)

Percent difference in estimatesFig. 5. Degree of agreement between estimates ofganglion cells from HFA data and of axons fromOCT data. The percent difference was assigned anegative value when the estimate of ganglioncells by HFA was less than the estimate of axonsby OCT or a positive value when the HFAestimates were larger that the OCT estimates.The solid line represents a normal distributionwith a mean and SD equal to the values derivedfrom the plotted data.



obtain histological verification of morphological imaging estimates, and 5) extend the study to humanglaucoma patients where the visual field defects are expected to be less diffuse than in experimentalglaucoma.

Spatial frequency response properties of visual field defects from glaucoma. Although there isa large literature on the effects of glaucoma on visual sensitivity for a variety of stimulus classes (e.g.,Bodis-Wollner, 1989; Evans, et a/., 2003; Harwerth & Smith, 1997; Hawkins, et a/., 2003; Johnson &Samuels, 1997; Nordmann, 1996; Porciatti, et a/., 1997; Sample, et a/., 2000; Spry, et a/., 2005a, 2005b;Thomas, et a/., 2002), there is much less information about the alterations of visual perception caused bya loss of retinal ganglion cells. A traditional strategy for investigating alterations of spatial visionassociated with clinically abnormal binocular vision (amblyopia or strabismus) is through spatial contrastsensitivity functions (Levi & Harwerth, 1977; Harwerth, et a/., 1984). The comparison of a patient'sabnormal spatial contrast sensitivity function (CSF) to the normal CSF allows predictions about thepatient's spatial vision in every-day life.

A relative loss of contrast sensitivity has been shown to be an early sign of optic neuropathy fromglaucoma, but previous investigations have not studied spatial frequency relationships in areas of visualfield defects to fully define spatial contrast sensitivity functions. Rather, most studies of contrastsensitivity in glaucoma have used either, a single spatial frequency to measure visual field defectsthroughout the visual field or have used multiple spatial frequencies to determine the complete function,but only for central vision. Therefore, the present study was undertaken to investigate the alterations inspatial contrast sensitivity functions in areas of the visual field with depressed sensitivity fromexperimental glaucoma.

The stimuli for the spatial contrast sensitivity functions were Gabor patches that were composed of ahorizontally orientated cosine grating that was limited in spatial extent by a Gaussian envelope. For thesestudies, the standard deviation of the Gaussian envelope was always set at two spatial periods of thecarrier grating. By setting the standard deviation of the spatial envelope in proportion to the spatialfrequency of the grating, the spatial frequency bandwidths were constant at 0.5 octaves for all spatialfrequencies, but the overall size of the stimulus decreased with increasing spatial frequency, i.e., if thespatial frequency was doubled, then the overall size was halved. In order to maintain behavioral controlof the monkey's fixation, the stimuli were presented for only 140 msec and, with a fixation distance of 33cm, the video screen resolution limited the spatial frequency range to about 2 c/deg. In each dailysession, contrast sensitivities were measured for one spatial frequency and data across sessions werecombined to construct the CSFs. CSFs were acquired for 13 locations in the visual field, one centrallocation and 12 locations along oblique meridians that correspond to locations from standard clinicalperimetry with the HFA. The data from each location was fit by an exponential (low-pass) function todetermine two descriptive parameters, i.e., the height of the function which specifies the peak contrastsensitivity and the location of the function which specifies the cut-off spatial frequency. Examples of thenormal contrast sensitivity functions for each of the 13 test locations are shown by the upper curves ineach panel of Fig. 6. The data represent the means of three control eyes with each function measuredthree times. The main points of the data are to illustrate that the functions are low-pass at each locationand are well-fit by the two-parameter model and, in general, the peak contrast sensitivity and the cut-offspatial frequency both decrease with increasing eccentricity.



Superior-nasal field Superior-temporal field

.13 .5 2 8 • 32

HFAdata

MD: -9.36 dBPSD: 6.86 dB

A. OjO

.12 .5 2 a 32

infenor-nasal field Sf>atial frequency (c/deg) |nferjor.tempora| fie|d

Fig. 6. Spatial contrast sensitivity functions for monkey OHT-46 with moderate visual field defects fromexperimental glaucoma, shown by the HFA gray-scale inset. The individual panels represent CSFs for central vision(panel A) and 3 locations along oblique meridians in each visual field quadrant. OHT-46's data, represented by thelower curve in each panel, are compared to data from normal control eyes, represented by the upper curve in eachpanel. The HFA total deviation for the same test location is also presented in each panel.

The general trend, that ganglion cell losses may cause shifts in the locations of the contrastsensitivity functions is illustrated by the data for an animal in a moderate stage of visual field loss. Themean deviation (MD) for this monkey was -9.36 dB and the visual defects are deep in the nasal visualfield. The clinical perimetry data are reflected in the CSFs by the shifts in both the height at the peakcontrast sensitivity and the location of the cut-off spatial frequency, but the peak contrast sensitivityappears to follow the defects by clinical perimetry better than the cut-off spatial frequency. Bycomparison of the depth of defect from perimetry (shown in the insets) and the difference between thepeak contrast sensitivity for control and experimental eyes shows that the two are related, at leastordinally.

The results for this subject were confirmed in five additional animals and, thus, the studiesdemonstrate that early visual field defects from experimental glaucoma represent selective spatialfrequency effects, where sensitivity losses at high spatial frequencies precede losses at lower spatialfrequencies. In comparison to standard clinical perimetry, the losses in spatial contrast sensitivities at lowspatial frequencies are correlated to visual field defects by standard clinical perimetry, while the losses atthe cut-off spatial frequency are not. The dependence of these results on the specific parameters of theGabor stimuli, such as size and duration, have not been fully investigated, but the spatial contrastsensitivity function should provide a description of the subject's spatial vision mechanisms and, under thespecific circumstances of the testing, visual defects from glaucoma represent a progressive spatialfiltering, or blurring, and could help explain why some patients with normal central visual fields stillcomplain of not seeing well. The additional experiments, involving spatial and temporal summation, arenecessary to determine whether this conclusion, which was based on a specific set of stimulusparameters, will hold as a general description of visual perception with glaucoma.



4. Publications (Manuscripts published or submitted for publication)

Publications on GlaucomaHarwerth, R.S. and Smith, E.L (2000) The independence of perimetry thresholds. Perimetry Update

1998/1999. M. Wall and J. Wild (eds.). Kugler Publications, The Hague, The Netherlands, pp. 167-176.

Crawford, M.L.J., Harwerth, R.S., Smith, E.L., Shen, F. and Carter-Dawson, L. (2000). Experimentalglaucoma in primates: Cytochrome oxidase reactivity in parvo- and magno-cellular pathways.Investigative Ophthalmology and Visual Science, 41,1791-1802.

Frishman, L.J., Saszik, S., Harwerth, R.S., Viswanathan, S., Li, Y., Smith, E.L. and Barnes, G. (2000).Effects of experimental glaucoma in macaques on the multifocal ERG. Documenta Ophthalmologica,100,231-251.

Crawford, M.L.J., Harwerth, R.S., Smith, E.L., Mills, S. and Ewing, B. (2001). Experimental glaucoma inprimates: Changes in cytochrome oxidase 'blobs' in V1 cortex. Investigative Ophthalmology andVisual Science, 42, 358-364.

Harwerth, R.S. and Smith, E.L. (2001) The intrinsic noise of contrast sensitivity perimetry. PerimetryUpdate 2000/2001. M. Wall and J. Wild (eds.). Kugler Publications, The Hague, The Netherlands,pp. 59-68.

Harwerth, R.S., Crawford, M.L.J., Frishman, L.J., Viswanathan, S., Smith, E.L., and Carter-Dawson, L.(2002) Visual field defects and neural losses from experimental glaucoma. Progress in Retinal andEye Research, 21, 91-125.

Carter-Dawson, L., Crawford, M.L.J., Harwerth, R.S., Smith, E.L., Feldman, R., Shen, F.F., Mitchell,C.K., and Whitetree, A.(2002) Vitreal glutamate concentration in monkeys with experimentalglaucoma. Investigative Ophthalmology and Visual Science, 43, 2633-2637.

Harwerth, R.S. and Crawford, M.L.J. (2004) The relation between perimetric and metabolic defectscaused by experimental glaucoma. Perimetry Update 2002/2003. Henson, D.B. and Wall, M. (eds).Kugler Publications, The Hague, The Netherlands, pp 175-186.

Harwerth, R.S. (2004) Histopathology underlying glaucomatous damage: I. Glaucoma Diagnosis.Structure and Function. Weinreb, R.N. and Greve, E.L. (eds). Kugler Publications, The Hague, TheNetherlands, pp 13-19.

Carter-Dawson, L., Shen, F.F., Harwerth, R.S., Crawford, M.L.J., Smith, E.L. and Whitetree, A. (2004)Glutathione content is altered in Muller cells of monkey eyes with experimental glaucoma.Neuroscience Letters, 364, 7-10.

Harwerth, R.S., Carter-Dawson, L., Smith, E.L., Barnes, G., Holt, W.F., and Crawford, M.L.J. (2004)Neural losses correlated with visual losses in clinical perimetry. Investigative Ophthalmology andVisual Science, 45, 3152-3160.

Harwerth, R.S., Carter-Dawson, L., Smith, E.L., and Crawford, M.L.J. (2005) Scaling the structure-function relationship for clinical perimetry. Acta Ophthalmologica Scandanavia, 83, 448-455.

------------ ------ ---- - ----------- ----- ------- ---- - --------- - ---- - -------- ---------- - ----- ------- - - - -------- - ------------ - ---------- ---------- - -- --------------------- -- - ---------

------------------ ------ ------- ---- ------------- ------ ---- - ------------- ---- ------- -- ----------------- ------------ - - - ---------- - -- - ------------ - ------------ - -- --- - ------------------- - ---------- --------------- - ------------------- - ---- - ------- ----------- -- - ---------

------------ ------ ------------- --------- ------------- ------ -------- ----- --- ------------ ---- --------- --- ---- - --------- ----------- -- ------- - ------- - ------------- - - - ------------ - - - ---------- - --------- - ---- - ----------------- -------------- --------- -- ------------------- -------- -------------

Publications on Binocular VisionSmith, E.L., Hung, L.F. and Harwerth, R.S. (2000). The degree of image degradation and the depth of

amblyopia. Investigative Ophthalmology and Visual Science, 41, 3775-3781.



Wensveen, J.M., Harwerth, R.S. and Smith, E.L. (2001) Clinical suppression in monkeys reared withabnormal visual experience. Vision Research, 41,1593-1609.

Fredenburg, P. and Harwerth, R.S. (2001) The relative sensitivities of sensory and motor fusion to smalldisparities. Vision Research, 41,1969-1979.

Harwerth, R.S. and Schor, C.M. Binocular Vision. In Kaufman & Aim: Adler's Physiology of the Eye, 10th

edition. C.A. Johnson and P.A. Sample (eds.). Mosby, St. Louis, pp 484 - 510.Ukwade, M.T., Bedell, H.E. and Harwerth, R.S. (2003) Stereopsis is perturbed by vergence error. Vision

Research, 43,181-103.Ukwade, M.T., Bedell, H.E. and Harwerth, R.S. (2003) Stereothresholds with simulated vergence

variability and constant error. Vision Research, 43,195-204.Harwerth, R.S., Fredenburg, P.M. and Smith, E.L. (2003) Temporal integration for stereopsis. Vision

Research, 43, 505-517.Harwerth, R.S. and Fredenburg, P.M. (2003) Binocular vision with primary microstrabismus. Investigative

Ophthalmology and Visual Science, 44, 4293-4306.Wensveen, J.M., Harwerth, R.S. and Smith, E.L.(2003) Binocular vision deficits associated with early

anisometropia: Behavioral observations. Journal of Neurophysiology, 90, 3001-3011.Zhang, B., Matsuura, K., Mori, T., Wensveen, J.M., Harwerth, R.S., Smith, E.L., and Chino, Y. (2003)

Binocular vision of macaque monkeys reared with alternating early defocus. II. NeurophysiologicalObservations. Journal of Neurophysiology, 90, 3012-3023.

Crawford, M.L.J., and Harwerth, R.S. (2004) Ocular dominance column width and contrast sensitivity inmonkeys reared with strabismus and anisometropia. Investigative Ophthalmology and Visual Science45, 3036-3042.

Cisarik, P.M. and Harwerth, R.S. (2005) Stereoscopic depth magnitude estimation: Effects of stimulusspatial frequency and eccentricity. Behavioural Brain Research, 160, 88-98.

Watanabe, I., Bi, H., Zhang, B., Saki, E., Mori, T., Harwerth, R.S., Smith, E.L., and Chino, Y.M. (2005)Direction bias of neurons in V1 and V2 of strabismic monkeys: Temporal-to-Nasal asymmetry?Investigative Ophthalmology and Visual Science, 46, 3899-3905..

-------------- ------ ------------- ------ -------- ----- ------------------- ---- ------ --- ---- - --------- ----- ------ ----- - -------- - -- -------------- - ------ - ---- - ---------- -------------------- - -------------- ---------------- - ------------------- - --- - -------- ----------- -- - ------------

----- ---- ---------- -- --------- --- ------------- --- -------------- ------ ------------- ------ -------- ------ ---- - -------- ----- ------------- ---------------- - - - --- - ------- ------- - ----- --- ----------- - ----------- - -------- - ---- - ----- - ------------- - ---- - ------------- - -------------- ----- -- --------------- ---------------- ------ - ------------ - ----------------

Abstracts published since last competitive reviewHarwerth, R.S. New visual field technology. Presented at Vision 2001 Spring Meeting, Jointly sponsored

meeting by UTMB Department of Ophthalmology and Visual Science and UH College of Optometry,Galveston, TX, March 24, 2001.

Harwerth, R.S. and Fredenburg, P.M. Binocular vision with primary microstrabismus. IOVS, 42, S736,2001.

Whitetree, A.R., Harwerth, R.S., Smith, E.L., Crawford, M.L.J, Feldman, R. and Carter-Dawson, L. Nitricoxide and peroxynrite in monkey retinas with experimental glaucoma. IOVS, 42, S749, 2001.

Harwerth, R.S. Psycho-anatomical links for standard clinical perimetry. North American PerimetrySociety, September 28, 2001.

Fredenburg, P.M., Harwerth, R.S. and Smith, E.L. Bloch's law for stereopsis. Opt & Vis Sci (suppl), 78,34,2001.

Ramamirtham, R., Kee, C., Hung, L.F., Ying, Q., Harwerth, R.S. and Smith, E.L. Comparison of objectiverefractions in macaca mulatta. Opt & Vis Sci (suppl), 78, 90, 2001.

Bradley, D.V., Smith, E.L., Harwerth, R.S. and Fernandes, A. Nearwork-induced myopic shift withoutsustained accommodation in primates. IOVS, 43, 2002, program no. 194.



Rangaswamy, N.V., Frishman, L.J., Saszik, S.M., Hood, D.C. and Harwerth, R.S. Effect of experimentalglaucoma and pharmacological suppression of inner retinal activity on the naso-temporalasymmetries in oscillatory potentials in the primate multifocal ERG. IOVS, 43,2002, program no.2170.

Bi, H., Zhang, B., Mori, T., Sakai, E., Noska, N., Harwerth, R.S., Chino, Y. and Smith, E.L. Effects ofearly strabismus on the development of binocular response properties of neurons in visual area 2(V2) of rhesus monkeys. IOVS, 43, 2002, program no. 4776.

Sakai, E., Mori, T., Zhang, B., Bi, H., Noska, N., Harwerth, R.S., Chino, Y. and Smith, E.L. Directionalbias of neurons in visual area 2 (V2) of monkeys reared with early strabismus. IOVS, 43, 2002,program no.4775.

Fredenburg, P.M., Harwerth, R.S. and Smith, E.L. Stereopsis and viewing duration for normal andabnormal binocular vision. IOVS, 43, 2002, program no. 4676.

Crawford, M.L.J., and Harwerth, R.S. The relation between perimetric and metabolic defects caused byexperimental glaucoma. 35th Annual Ophthalmology and Visual Science Meeting, University of Texas- Houston, June, 2002.

Harwerth, R.S., and Crawford, M.L.J. The relation between perimetric and metabolic defects caused byexperimental glaucoma. International Perimetry Society, June, 2002 (IPS Abstracts, 25, 2002).

Chino, Y., Zhang, B., Wensveen, J., Harwerth, R. and Smith, E.L. Comparisons of the effects of earlystrabismus on the binocular response properties of neurons in visual area 2 (V2) of rhesus monkeyswith those in V1. 3rd Forum of European Neuroscience, Paris, France, July, 2002.

Carter-Dawson, L., Crawford, M.L.J., Harwerth, R.S., Smith, E.L, Feldman, R., Shen, F.F., Mitchell,C.K., and Whitetree, A. Vitreal glutamate concentration in monkeys with experimental glaucoma. XVInternational Congress of Eye Research, Geneva, Switzerland, October, 2002.

Harwerth, R.S., and Crawford, M.L.J. Visual field defects related to reduced neuronal metabolism in thelateral geniculate nucleus. Opt & Vis Sci (suppl), 79,14, 2002.

Harwerth, R.S., Carter-Dawson, L., Smith, E.L., Crawford, M.L.J., and Barnes, G. Neural lossespredicted from vision defects in glaucoma. Houston Society for Engineering in Medicine and Biology,Houston, TX, April, 2003.

Rangaswamy, N., Frishman, L.F., and Harwerth, R.S. Effect of experimental glaucoma andpharmacological suppression of inner retinal activity on the naso-temporal asymmetries in oscillatorypotentials in the primate multifocal ERG. Houston Society for Engineering in Medicine and Biology,Houston, TX, April, 2003.

Harwerth, R.S., Carter-Dawson, L., Smith, E.L., Crawford, M.L.J., and Barnes, G. Neural lossescorrelated with visual losses in clinical perimetry. IOVS, 44, 2003, program no. 1040.

Rangaswamy, N., Harwerth, R.S., Saszik, S.M., Maeda, H., and Frishman, L.F. Frequency analysis ofOPs in slow-sequence MfERG in primates: Comparison of experimental glaucoma andpharmacological inner retinal blockade. IOVS, 44, 2003, program no. 2702.

Smith, E.L., Harwerth, R.S., Wensveen, J.M., Ramamirtham, R., Kee, C., and Hung, L-F. Brief dailyperiods of unrestricted vision can prevent form-deprivation amblyopia. IOVS, 44, 2003, program no.3188.

Zhang, Y., Harwerth, R.S., Smith, E.L., Whitetree, A.R., Crawford, M.L.J., and Carter-Dawson, L.Glutamate aspartate transporter (GLAST) content in glaucomatous monkey retinas. IOVS, 44, 2003,program no. 4570.

Cisarik-Fredenberg, P., and Harwerth, R.S. The relationship between disparity vergence and themagnitude of perceived depth. Opt & Vis Sci (suppl), 80, 22, 2003.

Wensveen, J.M., Kee, C.S., Ramamirtham, R., Hung, L.F., Harwerth, R.S., and Smith, E.L. Form-deprivation amblyopia: temporal integration properties. Opt & Vis Sci (suppl), 80, 168, 2003.

Harwerth, R.S., Smith, E.L., Barnes, G., and Holt, W. Correlated visual field defects by standard and non-standard perimetry in experimental glaucoma. Opt & Vis Sci (suppl), 80, 227, 2003.

PHS 398/2590 (Rev. 09/04) Page_42_ Continuation Format Page


Zhang, Y., Harwerth, R.S., Smith, E.L., Whitetree, A.R., Crawford, M.L.J., and Carter-Dawson, L.Glutamate catabolism enzymes in Muller cells of monkeys with experimental glaucoma. IOVS, 45,2004, program no. 448.

Harwerth, R.S., and Quigley, H.A. Visual field defects and retinal ganglion cell losses in human glaucomapatients. IOVS, 45, 2004, program no. 3473.

Peterson, C.H., Wensveen, J.M., and Harwerth, R.S. The relative effects of Gabor bandwidth andreference-test separation on stereopsis. IOVS, 45, 2004, program no. 4323.

Harwerth, R.S., Carter-Dawson, L., Smith, E.L., and Crawford, M.L.J. Scaling the structure-functionrelationship for clinical perimetry. International Perimetry Society, July, 2004 (IPS Abstracts, 50,2004).

Carter-Dawson, L.Zhang, Y., Whitetree, A., Harwerth, R.S., Smith, E.L., and Crawford, M.L.J. Mullercells in experimental glaucoma show increases in the transporter GLAST and Glutamate Metabolites.XVI International Congress of Eye Research, Sydney, Australia, August, 2004.

Watanabe, I., Zhang, B., Zheng, J., Wensveen, J.M., Harwerth, R.S., Smith, E.L and Chino, Y.M.Effects of brief unrestricted vision during early monocular form deprivation in macaque monkeys.Society for Neuroscience, October, 2004 (program No. 839.6.2004 Abstract Viewer).

Harwerth, R.S., Carter-Dawson, L., Barnes, G., Holt, W., Smith, E.L., and Crawford, M.L.J. Scalingvisual filed defects and neural losses from glaucoma. Opt & Vis Sci (suppl), 81, 7, 2004.

Cisarik-Fredenberg, P.M. and Harwerth, R.S. Perceived depth magnitude from temporal vs. spatialdisparity. Opt & Vis Sci (suppl), 81, 14, 2004.

Chino, Y.M. and Harwerth, R.S. Recovery of binocular functions in adult strabismic subjects followingextensive vision training and testing. Royal National Institute of the Blind: VISION 2005 conference,London, England, April, 2005.

Vilupuru, A.S., Rangaswamy, N.V., Frishman, L.J., Harwerth, R.S., and Roorda, A. Adaptive opticsophthalmoscopy for imaging of the lamina cribrosa in glaucoma. IOVS, 46, 2005, program no. 3515.

Harwerth, R.S., Barnes, G., Holt, W.F. and Smith, E.L. Spatial frequency response properties of visualfield defects from glaucoma. IOVS, 46, 2005, program no. 4667.

Watanabe, I., Zhang, B., Zheng, J., Bi, H., Maruko, I., Harwerth, R.S., Smith, E.L and Chino, Y.M.Effects of extensive psychophysical testing on stereoacuity and disparity sensitivity of V1 and V2neurons in macaque monkeys. IOVS, 46, 2005, program no. 5666.

Rangaswamy, N.V., Digby, B., Harwerth, R.S. and Frishman, L.J. Optimizing the spectral characteristicsof a Ganzfeld stimulus used for eliciting the photopic negative response (PhNR). IOVS, 46, 2005,program no. 4762.

Harwerth, R.S., Barnes, G., Holt, W.F., and Smith, E.L. Spatial contrast sensitivity defects fromexperimental glaucoma. First World Glaucoma Congress, July, 2005.



D. Research Design and Methods.Experimental glaucoma:

Monkeys: The subjects for the investigations will be young adult rhesus monkeys (Macaca mulatta),either male or female. The monkeys (6-8 per year) will be obtained from the University of Houstoncolony from animals that have been reared from infancy. There are not any particular criteria forinclusion, but most of the monkeys will have been subjects in psychophysical contrast sensitivityexperiments, using a behavioral paradigm similar to the present procedures and visual acuitymeasurements will be available for each eye. Prior to experimental training and testing for these studies,each monkey will be examined by ophthalmoscopy and retinoscopy under cycloplegia to ensure thattheir eyes are free from any ocular abnormalities or significant refractive errors.

The 30 monkeys for the principal experiments will receive unilateral laser treatment to create ocularhypertension, with the fellow eye serving as a normal control (Harwerth, et a/., 1997). For the studiesdesigned to investigate the RNFL contribution from axons outside the normal perimetry field and themapping of the visual field onto the ONH, six additional monkeys will be used as subjects. Thesemonkeys have been subjects for an investigation of emmetropization, specifically to answer a question ofwhether an intact peripheral retina is essential for normal emmetropization (Smith, EL, personalcommunication). For that investigation, the retinas of the monkeys' right eyes were ablated from thearcuate bundle to the ora serrata, using frequency-doubled YAG laser burns (150 mW power, 150 msecduration) with a spacing of one-half burn width and 2316 ± 884 total burns per eye. The region anddegree of ablation will be documented by fundus photography. These monkeys will be available fortesting in about 18-24 months. Following the training and experimental measurements to assess thecontribution of the peripheral retinal to the total RNFL thickness, these monkeys will receive additionallaser burns of the left eye's retina to refine the mapping of the visual field onto the ONH. Usingphotographs of the ONH to establish landmarks for burn locations, discrete Argon laser burns will beplaced in locations along the ONH margin that were proposed as OHN sectors representing specificretinal areas (see section C). For example, one burn could be placed at sector 4, which should create anarcuate scotoma in the inferior visual field. Additional burns could be placed to coincide with othersectors, either simultaneously or consecutively, following perimetric verification of visual field losses fromearlier lesions. The burn areas will be large enough to be located by the 6X6 deg perimetry test grid and,across the six monkeys, some burn sites will be at a consistent location, e.g., sector 4, but otherlocations will be varied to gain as much knowledge about the visual field to RNFL relationship aspossible.

Experimental glaucoma: After training and extensive testing to obtain stable baseline data, eachmonkey will undergo laser treatment to elevate the intraocular pressure (IOP) of one eye (unilateralexperimental glaucoma). The Argon laser treatment of the trabecular meshwork will be performed on theright eye of each monkey, with the left, untreated, eye serving as a control for measurements of RNFLand perimetry (Harwerth, et a/., 1997). In preparation for the laser procedure, the monkeys will beanesthetized with ketamine (20 mg/kg) and acepromazine (0.2 mg/kg) and a topical corneal anesthetic(0.5% proparacaine) instilled in the eye to be treated. The animals' heads are stabilized on the chin-restof a standard clinical laser-slit lamp system and the laser treatments will be performed using a goniolaser lens designed for monkey eyes. The laser energy (nominal laser parameters: blue-green mode, 1.0watt power, 50 micron spot size, and 0.5 sec exposure duration) will be equivalent to that used inprevious investigations of experimental glaucoma in macaques (Burgoyne, et a/., 2004; Gaasterland &Kupfer, 1974; Hare, etal., 2004; Kee, etal., 1995; Pederson & Gaasterland, 1984; Quigley &Holman;1984), and is a level which has been shown to destroy the trabecular meshwork and obliterateSchlemm's canal in the vicinity of the laser burn (Kee, et a/., 1995; Masuyama, 1984). The burns will bespaced to produce contiguous tissue blanching. The treatment protocol involves an initial laser procedureover 270 degrees of the drainage angle and subsequent treatments covering 180 degrees, retreating asnecessary to elevate the IOP, with an interval of at least 3 weeks between procedures. One of theinteresting aspects of this model of experimental glaucoma is the relatively large amount of argon laserenergy required to produce ocular hypertension. In agreement with other investigations (Gaasterland &Kupfer, 1974; Kee, et a/., 1995; Pederson & Gaasterland, 1984; Quigley SHolman; 1984), multiple lasertreatments using high energy were usually required to achieve sustained, elevated pressures, which

PHS 398/2590 (Rev. 09/04) Page .44. Continuation Format Page


ultimately fall into the range of 35 - 55 mm Hg. The total laser energy has been, on average, about 90joules (range: 45 to 150 joules), which is more than ten-times the maximum energy used for the clinicaltreatment of glaucoma (Beckman, 1990; Spaeth & Baez, 1992; Van Buskirk, 1991). Thus, at low energylevels, laser burns may alter the biomechanical properties of the trabecular meshwork to increaseoutflow, but considerably higher energies are needed before the treatment causes an obstruction ofoutflow and an elevation of IOP. The resulting pressures were generally quite high compared to the lOPsof chronic ocular hypertension patients, but we have not been able to find a systematic relationshipbetween total laser energy and either the maximum IOP or the rate of elevation of IOP (Harwerth, et a/.,1997).

Behavioral perimetry: The visual fields of the monkeys will be measured, using behavioralmethodology (Harwerth, et a/., 1993a; 1993b), with a Humphrey Visual Field Analyzer. The current andinitial studies have used the HFA I model, but as part of the proposed research, perimetry with an HFA IIwill be implemented during the first year of the project. The following description is specific for HFA I, butadaptations for the HFA II should be similar.

The major modifications that were made to SAP procedures were designed to obtain stimulus controlof the monkeys' behavior, but they did not alter the standard testing protocol or data analysis programs.The principal modifications are as follows: 1) A light-emitting diode (LED) fixation stimulus has beenplaced in Maxwellian view to control the monkeys' head and eye positions. A luminance decrement of theLED is the trial stimulus in at least 30% of the session trials. 2) A microcomputer eye-position monitor,aligned with the fixation stimulus, causes an interruption of the behavioral paradigm if an eye movementgreater than approximately 5 deg occurs during a trial. 3) The perimeter's process control software waschanged to delay movement of the stimulus projector for 2 sec after presentation of a test stimulus and towait for the subject's response after each stimulus presentation. 4) The timing of the testing sequencewas controlled and synchronized with other events of the behavioral procedure by an external laboratorycomputer.

During testing, the monkeys are seated in a custom-made primate chair that provides adjustments foralignment of their eyes at the correct viewing location and placement of their mouths on the juice spoutused to deliver behavioral rewards. While in the chair, they are able to grasp the response switch that isused for their behavioral responses during visual field testing. Their thresholds for the fixation andperimetry stimuli are obtained by a psychophysical method, a criterion response-time paradigm formonkeys, which retains the essential requirements of the test procedure used for patients. The principalcomponents of the procedure for the monkey subjects are as follows: the monkeys are trained to pressand hold-down their response lever in order to initiate a trial and, subsequently, to release the lever in thepresence of a visual stimulus. Because the time of the occurrence of the stimulus is varied randomly, ifthe monkey's lever release is closely correlated to the visual stimulus presentation (within a 900 msecresponse interval), then the response is operationally defined as a true-positive response (a hit) and it isrewarded. Alternatively, if the monkey's lever release is beyond the response interval, it is considered asa false-negative response (a miss) and it is neither rewarded nor punished. The test stimulus in any trialcan be the central fixation stimulus or one of the peripheral perimetry stimuli. The locations andintensities of the peripheral test stimuli are determined by the Humphrey Field Analyzer's C24-2 full-threshold program.

Perimetry data analysis: The monkeys' fields for each eye are measured every other week using thestandard testing parameters (31.5 asb. bowl luminance, Goldmann Size III white test target). The visualfields data are transferred to a laboratory computer for calculating perimetric indices (Heijl, et a/., 1987a;1987b) and statistical significance of visual field changes, and to calculate RGC numbers from perimetrythresholds for comparison to RNFL axon counts. The perimetric indices for the monkeys are derived fromcomparisons of the measured visual fields to the normal expected data of monkeys, based on the meansand variances of pre-treatment and control-eye conditions. The specific index used to follow theexperimental visual field defects is generally the mean deviation (MD), i.e., the weighted averagedeviation from the normal fields (Heijl, et a/., 1987a). This index was selected because both thepreliminary investigations with monkeys (Harwerth, et a/., 1999) and clinical data from glaucoma patients(Chauhan, et a/., 1990; Flammer, et a/., 1985; Katz, et a/., 1991; Wood & Bruce, 1993) have shown that a



mean deviation index is more sensitive to early changes and overall defects than the other derivedindices, e.g., PSD (pattern standard deviation) or CPSD (corrected pattern standard deviation).

The method of estimating ganglion cell numbers from perimetry measurements was developed frombehavioral perimetry and retinal histology in experimental glaucoma (Harwerth, et a/., 2004) and,subsequently, verified for histology and clinical perimetry in human glaucoma patients (Harwerth &Quigley, 2005). These studies demonstrated that the general expression between the neural density andvisual sensitivity data is exponential and logarithmic transforms of both variables produce linearrelationships for prediction of structural losses from functional measurements. The utility of logarithmicscaling was confirmed for experimental glaucoma in monkeys, by the empirical relationship betweenvisual sensitivity, in dB (the threshold value from a given test location for the 24-2 program of theHumphrey Visual Field Analyzer), as a function of ganglion cell density, in dB (10-times the logarithm ofthe histological count of ganglion cells at the corresponding retinal location), that was well-described bylinear regression for each retinal eccentricity (the radius from the fixation point). However, theparameters of the linear function varied with eccentricity and, thus, the model required two equations todetermine the slope and y-intercept as a function of eccentricity, which in turn provided the parametersfor the third function for predicting ganglion cell density from visual sensitivity. The three equations are:

1. the slope of the function (m) at eccentricity (e): m = (0.054 * e) + 0.952. the intercept of the function (b) at eccentricity (e): b = (-1.5 * e) - 14.83. the predicted ganglion cell density (gc) for a sensitivity (s): gc = (s - b) / m

These relationships were developed from empirical measurements of visual sensitivities and ganglioncell densities and, therefore, although the equations may represent a quantitative description of therelation between structure and function in glaucoma, they do not imply specific physiologicalmechanisms beyond the nonlinear pooling of ganglion cell responses during the detection of visualstimuli in perimetry. Nevertheless, these relationships worked well for predictions of ganglion cell lossesfrom glaucoma.

OCT measurements: Prior to the OCT measurements, the animals will be anesthetized withintramuscular injection of ketamine (20 to 25 mg/kg per hr) and xylazine (0.8 to 0.9 mg/kg per hr) and aretreated subcutaneously with atropine sulfate (0.04 mg/kg). The pupils are dilated to approximately 8.5mm in diameter with topical tropicamide (1%) and phenylephrine (2.5%) and a piano-power contact lensis placed on the eye to maintain optical clarity during the measurements. During the measurements themonkey's body temperature is maintained between 36.5° C and 38° C with a thermostatically controlledblanket and the heart rate and blood oxygen are monitored using a pulse oximeter. A padded mouth barand an occipital bar are used to stabilize and position the head for OCT measurements. The headsupport device is attached to a rotational mount that allows the head to be aligned appropriately for aseries of OCT measurements that take about 20 minutes per eye to complete.

Imaging of retinal structures will be preformed with a StratusOCT, with version 4.0.4 software. TheOCT provides direct cross sectional images for objective, high resolution measurement of three retinalstructures, i.e., macular thickness, retinal nerve fiber layer, and optic nerve head. All three areas ofmeasurement will be made on the control and laser-treated eyes of monkeys at approximately 3-weekintervals. Both fast-scan and standard scan protocols will be employed. Thus, while the primaryemphasis of the present proposal is on the RNFL thickness measures, other structural changesassociated with glaucomatous neuropathy, especially the ONH parameters will be analyzed over thetimecourse of experimental glaucoma to compare the relative time of onset, the relative rate of change,and the limit of measurement.

For the retinal nerve fiber measurements, the standard RNFL thickness protocol will be used. Thestandard scan measures thickness at 512 points in the 10.87 mm scan length (circumference) of a circleof 3.4 mm diameter that is centered on the ONH. The standard circle scan protocol was designed forhuman eyes (Lee, et a/., 2004) but, because the size of the ONH in monkey is comparable to humans,the relationship of the ONH and the 3.4 mm circle should be similar. For example, the preliminary datademonstrated that the measurements for monkeys, after compensation for monkey-human ageequivalents, are similar to humans and the intersubject variability of monkeys are small (coefficient ofvariability.of 15%, unpublished observations). In addition, the detection of change will be based onnormative data from the normal and control eyes of monkeys.PHS 398/2590 (Rev. 09/04) Page _46_ Continuation Format Page


After all of the OCT scans for an eye have been completed, digital fundus photographs (Nidek modelNM200D camera) will obtained in each session.

RNFL data analysis: The principal methods for the analysis of structural data, for comparison tofunctional data from perimetry, were described in the preliminary study of the relationship between nervefiber layer and perimetry measurements. In brief, three RNFL scans per eye will be exported to alaboratory computer and the mean pixel-by-pixel thickness determined. The area of nerve fiber layer ineach of 10 sectors (51 pixels) of the optic nerve head will be determined from the scan height acrosspixels (21.2 [i I pixel) and divided by the nominal area per axon (0.63 u2) to derive the total number ofaxons entering a specific sector of the ONH. The number of RGCs to the number of axons in each sectorwill be compared and analyzed by the degree of agreement between estimates of ganglion cells andaxons by each measurement and the correlation of neural loss predicted by HFA as a function of neurallosses predicted by OCT.

Even if the empirical evidence is strong that SAP measures of visual field defects and OCT measuresof RNFL defects are correlated measures based on the same pathophysiological mechanism, there areimportant differences between the procedures (Harwerth, 2004). Two specific questions relate to thedetection of early loss and the dynamic range of measurement. First, to evaluate which procedure shouldbe more sensitive to early changes caused by glaucoma, a sector-wise analysis of variability will beundertaken. The average variance of RNFL thickness across all of the pixels in a sector and the averagevariance of the perimetry thresholds across all of the test field locations entering a sector will betransformed to a uniform scale of Z-scores. The Z-scores will provide data for calculation of the limits ofpre-nerve fiber loss for OCT and pre-perimetric loss for HFA. Equally important, by collecting multiplescans in each session, the variability as a function of the degree of neural loss can be assessed todetermine whether variability increases with the depth of defect in the same way as perimetry (Chauhan,et a/., 1993; Henson, et a/., 2000; Wall, et a/., 1997). For the question of dynamic range, a furtheranalysis of the two measures as a function of stage in animals allowed to progress to advanced stages ofexperimental glaucoma will provide data to determine the upper limit of loss and the dynamic range foreach measurement.

The final assessment of the relationship between SAP and RNFL thickness will be made byhistology. The ganglion cell densities corresponding to fixed visual field locations and the nerve fiberlayer thickness around the optic nerve head will be obtained by direct histological measurements. Thesedata will provide important data that could verify the relationships from the clinical measurements orprovide data modifications for more accurate relationships.

Data from OCT measures of ONH characteristics also will be available for a similar analysis. On thebasis of observations from three monkeys, one with pre-perimetric experimental glaucoma, there appearto be changes in cup diameter from the vertical cross section measurement and the cup area and rimarea from integrated analysis that precede RNFL or perimetry changes. However, these data need to beanalyzed systematically in same manner as the nerve fiber layer data.

Mapping visual field locations onto the optic nerve head: Data to refine the mapping of the visual fieldonto the ONH will be obtained by comparing RNFL thickness profiles before and after placing discrete

laser burns at specific sectors of the ONH, with theresulting visual field defects verified by perimetry. Asa general example of the effects of the strategy ofthe experiment, Fig. 7 presents RNFL data followingArgon laser treatment in a monkey's right eye overabout three-quarters of the height of the ONH toablate of the papillomacular bundle. This laserablation was done as a part of an ERG experimenton the optic nerve head component in multifocalERGs (LJ Frishman, personal communication) andperimetry data were not obtained, but the region ofRNFL thinning is reasonably well defined, as shownby the RNFL thickness profiles for the left (light line)and right (dark line) eyes (Fig. 7). In comparison to

iporc

40O SOO

Pixel number

Fig. 7. RNFL thickness profile of a monkey's right(dark line) and left (light line) eyes following laserablation of the papillomacular bundle of the right eye.



the left eye, the nerve fiber layer of the right eye was completely eliminated in 72° of the ONHcorresponding to the temporal sectors 1 and 10 (see Fig. 2) and significantly thinned in the adjacentsectors, 2 and 9. The experimental design for relating visual field location to optic nerve head sector willinvolve smaller burn areas to create localized thinning of the RNFL.

Spatial contrast sensitivity measurements: The visual stimuli for the experiments will be generated bya PC-based VSG2 (version 3 or 5) graphics board from Cambridge Research Systems. The boardprovides 15-bits of contrast resolution, 36 pages of video memory, and synchronizes to a 120 Hz monitorwith 800 X 600 pixel resolution. The contrast sensitivity stimuli will be horizontally orientated Gaborpatches (0.5 octave bandpass) with carrier frequencies of 0.25 - 2.8 c/deg, which are presented for 140msec duration. Spatial contrast sensitivities will be determined for locations at 3x3 deg, 9x9 deg and15x15 deg in each visual field quadrant. The spatial frequency effects will be evaluated via a fitted low-pass model for spatial contrast sensitivity functions. Several examples of the contrast sensitivity functionsfor normal and laser-treated eyes were presented in section C, Fig. 6.

All other aspects of the methodology for these experiments, including the psychophysics, areidentical to that described for behavioral perimetry. Therefore, the monkeys can be alternated betweenSAP and contrast sensitivity perimetry each week.

Histology: Dr. Louvenia Carter-Dawson, Department of Ophthalmology and Visual Science,University of Texas - Houston will conduct the histological analyses under the contract agreement of thepresent application. On average, histological analysis will be required for 6 pairs of eyes per year.

Within a few days after the final visual field test, the monkeys will be deeply anesthetized and theireyes enucleated. The retinal tissue processing for the histological analysis has been described(Frishman, et a/., 1996; Harwerth, et a/., 1999) In essence, the posterior segments of the eyes are fixedby an overnight immersion in 2% paraformaldehyde and 2% glutaraldehyde at 4°C. The eyes are thentransferred and stored at 4° C in phosphate buffered 4% paraformaldehyde (pH 7.3).

For the quantification of ganglion cell densities corresponding to perimetry test locations, retinatissue samples for16 test sites (3 samples along the oblique meridians in each quadrant and 2 samplesabove and 2 below the horizontal midline at the nasal-most field locations) will be collected from thecontrol and laser-treated eyes. The retinal locations for tissue samples will be determined by the usualconversion ratio of 1 mm retina per 4 deg of visual angle (Wassle, et a/., 1990). This ratio was alsoverified for the present study by comparing the distance from fixation to the center of the perimetricallyplotted blind spot and the direct measurement of the distance from the fovea to the center of the opticnerve head (Shen, 1994).

In preparation for counting the ganglion cells, the retinal tissue samples are dehydrated in agraded series of methanol/water (50 - 100%) and embedded in LR White resin and, subsequently,sectioned (1|im thickness, radial sections) and stained (0.5% cresyl violet). The published examples ofthe histology preparations have demonstrated that the morphology of the retinas of treated and controleyes of the monkeys are essentially identical, except for the reduced number of cells in the ganglion celllayer (Harwerth, et a/., 1999; 2002). The amount of ganglion cell loss is quantified by counting theneurons under light microscopy with 100X magnification. All of the neurons will be counted in a one ^msegment from each of 10 sections separated by a minimum of 10 jam. The cell densities of the ganglioncell layers are calculated utilizing Abercrombie's (1946) method for deriving densities from sectionedtissue using the tissue section thickness of 1 m and an average cell body diameter of 9 ^im for thecalculations.

Displaced amacrine cells are not excluded from the counts nor are the densities corrected for thelateral displacement in the location of receptive fields from their cell bodies, for RGCs near the fovea.However, the errors from displaced amacrine cells and the fibers of Henle have been estimated (Curcio& Allen, 1990). An error caused by including displaced amacrine cells in the ganglion cell count wouldincrease with eccentricity from near zero for retinal locations that are close the fovea to an error of about3 dB at the eccentricity of 24 deg. The error associated with lateral displacement between retinalreceptors and their ganglion cell bodies is in the opposite direction and could be as large as 3.3 dB forthe most central samples (0.5 mm Henle fiber length at 4.2 deg retinal location), but it decreases to zeroat an eccentricity of 15 deg. However, the algorithms for incorporating these corrections of are uncertain,especially if sampling issues are also considered and to maintain the simplest form of the model, with thePHS 398/2590 (Rev. 09/04) Page 48 Continuation Format Page


fewest assumptions, the average measurement for ganglion cell density at each sampled location willused as the best estimate.

The histological analysis of the retinal nerve fiber layer will be from retinal tissue that correspondsto the 3.4 mm diameter circle used in the standard OCT scan path. First, the optic nerve will be removedfrom the fixed eye cups with a 2 mm diameter dermal biopsy punch. To demarcate the horizontalmeridian, a horizontal cut will be made from central fovea toward the optic nerve to the edge made byremoval of the optic nerve. A second 4 mm diameter punch centered on the 2 mm hole will be made andthe samples from both control and experimental eyes will be immunolabeled with a polyclonal antibody toneurofilaments (Cy-3 secondary conjugate) to label retinal ganglion cell axons and a monoclonalantibody to glutamine synthetase (CY-5 secondary conjugate) to label Muller cells. DAPI (4'-6-Diamidino-2-phenylindole) staining of retinal ganglion,cell nuclei will be used to determine the outer boundary fornerve fibers and the glutamine synthetase positive end feet of Muller cells will serve as the innerboundary for nerve fibers. The retinal discs will be mounted on glass slides with the nerve fiber layerfacing up for confocal imaging. The region of the retinal disc which corresponds to a circumference of10.87 mm will be determined using the LSM software and optical images will be captured in each often1.08 mm segments at a thickness of 1 urn beginning at the horizontal meridian temporal superior edge ofthe tissue sample. The glutamine synthetase positive Muller cell end feet and DAPI positive ganglion cellnuclei will be used to identify the boundaries for the nerve fiber layer. These boundaries will set the limitsfor the number of optical 1 um sections required to capture the full thickness of the nerve fiber layer.

Using LSM software the images will be displayed in the z axis and the nerve fiber layer thicknessmeasured at 10 different points in each of the 1.08 mm segments. The mean ± SD will be calculated foreach of the 10 areas (1.08mm). The data will be collected in the same manner for control andexperimental eyes.

Data from both retinal ganglion cell quantification and nerve fiber layer will be given to Dr. Harwerth.

Clinical glaucoma:---- ---------- ---- - --------- Department of Clinical Sciences, University of Houston, will conduct the

clinical research and be responsible for both patient recruitment and clinical data collection.Subjects: Approximately 30 subjects will be recruited for the study. Each patient will have had an eye

examination at the University of Houston, College of Optometry no more than 6 months prior toparticipation in the study. Subjects may have some degree of glaucomatous visual field loss, butpotential participants with existing ocular pathology other than glaucoma will be excluded. Potentialcandidates for the study will be identified by their clinicians during their scheduled appointment times.Interested participants will receive a verbal and written solicitation, and may opt to perform the tests atthe time of the visit or may schedule an appointment for the testing.

The information to be obtained for the project will involve a measure of the patient's intraocularpressure, measures of the subject's nerve fiber layer with optical coherence tomography (OCT), andresults from the patient's visual field testing with the Humphrey Field Analyzer (HFA II). Some, or all, ofthe information may be obtained retroactively, if performed within the last 6 months. The intraocularpressure measurement must be obtained the day of the OCT scans, but the HFA II test may be obtainedat any time within a three month time span of the OCT scans.

Data analysis: The methods of data analysis for the clinical glaucoma patients will be identical tothose for experimental glaucoma, described above.

Timetable:There are two consuming aspects of the project, 1) training monkeys for behavioral measurements

and 2) laser-induction of ocular hypertension to create progressive changes in visual fields and nervefiber layer thickness, as opposed to catastrophic elevated IOP or no IOP elevation. These two aspects,plus the fact that only 6 or 7 monkeys can be tested each day, sets a limit on the timecourse of theexperiments. A reasonable expectation is to use 6 experimental glaucoma monkeys each year of thegrant period. These monkeys will be subjects for both the first and fourth specific aims, which involveSAP, OCT and contrast sensitivity perimetry. The monkeys with laser ablation of peripheral retinaspecific aim 3 will be old enough to be trained and tested in the second and third years of the project.

The studies of clinical glaucoma (specific aim 3) can be undertaken almost anytime, but the analysisrequires sufficient data from experimental glaucoma to refine the algorithms for estimating cells and



axons from the clinical procedures. It is likely that the clinical research project will be completed in thesecond year of the project.

Potential problems:All of the experimental procedures are within our capabilities and the results will extend our

knowledge of structure-function relations in glaucoma. There may be problems in conducting theresearch project, but those that can be imagined are largely technical.

1. For the present studies, an old perimeter (HFA model 620) is being used and neither service norparts are available for this instrument. For the most recent break-down, I was able to get an exception viaMichael Patella and he has offered to send a "traded-in" instrument for spare parts. However, in the longrun it will be necessary to switch to the newest model HFA II. The change-over will take time and it maybe technically difficult to interface with the behavioral paradigm and external eye movement monitor. Theproject plan is to develop the newer perimeter during the first year of the project.

2. The relationship between nerve fiber layer and perimetry measurements that was developed in thepreliminary studies may not hold for data from a larger study of experimental glaucoma. Although thispotential problem seems unlikely, if it does, the solution should be either, 1) a recalculation of thecoefficients of proportionality, which are the biological factors of size for RGC bodies and diameter forRGC axons or, 2) a reanalysis of the mapping of retinal locations onto the optic nerve head to obtainmore appropriate relationships between visual field areas and sector sizes for the optic nerve head.

3. The relationship between nerve fiber layer and perimetry measurements that was developed forexperimental glaucoma in monkeys may not hold for data from clinical glaucoma in humans. As before,this potential problem seems unlikely, in this case because the relationship between perimetry sensitivityand ganglion cell density that was developed with experimental glaucoma was applied directly, withreasonable accuracy and precision, to data from human glaucoma patients. In all of the studies ofanatomical, neural, or perceptual properties of monkeys and humans, the two species have beenvirtually identical. However, if this investigation becomes the exception, the reasons and requiredcompensations will be determined and incorporated into the model.

4. The histological analysis of the RNFL is a modification of procedures in---- --------------------- - lab,but she has not worked with the tissue in this way. The confocal microscope seems to be the bestapproach and will be attempted first. However, if the procedure is not successful, the traditional methodsof histology will be implemented.

Additional studies of experimental glaucoma:The monkeys with experimental glaucoma, or their tissues, will be also available for other studies.

Although these studies are not a part of the present application, nor is support requested, the animalsprovide-- - ------------ ----------- - --- ----- ---- ------------ - for her studies of inner retinal contributions to ERGand for --- ----------- - --------------------- - studies of retinal cellular mechanisms altered by glaucoma. Forexample, over the past five years, monkeys with verified visual field defects from experimental glaucomahave provided data for studies of the negative photopic ERG response (Viswanathan, et a/., 1999), multi-focal ERG (Frishman, et a/., 2000), and oscillatory ERG potentials (Rangaswamy, et a/., 2005). Inaddition, tissues from the monkeys were used for studies of afferent pathway effects of glaucoma(Crawford, et a/., 2000; 2001; Harwerth & Crawford, 2004) and alterations of cell biology in glaucoma(Carter-Dawson, et a/., 2002; 2004; Whitetree, et a/., 2005). Similar shared use of the animals willcontinue throughout period of support that is requested in the present application.



E. Human Subjects Research.This Human Subjects Research meets the definition of 'Clinical Research.'

Protection of Human subjects

1. RISKS TO THE SUBJECTS

a. Human Subjects Involvement and Characteristics

Describe the proposed involvement of human subjects in the work outlined in the Research Design andMethods section.

Patients with glaucoma exhibit a decreased sensitivity to targets tested with the Humphrey FieldAnalyzer (HFA). It is known that patients with glaucoma exhibit a loss of ganglion cells in the retina, andas a result of this loss, show a thinning nerve fiber layer. By using the OCT to obtain measures of thenerve fiber layer thickness, we will attempt to define a relationship between the numerical decrement ofvisual field sensitivity loss as tested by HFA to the amount of nerve fiber layer thinning in glaucomapatients.

Describe the characteristics of the subject population, including their anticipated number, age range, andhealth status.

Approximately 30 glaucoma patients, over the age of 18, will be recruited for the study. All subjectswill have had an eye examination at the University of Houston, College of Optometry no more than 6months prior to participation in the study. Subjects may have some degree of visual field loss fromglaucoma, but potential participants with other existing ocular pathology will be excluded. Potentialcandidates for the study will be identified by their clinicians during their scheduled appointment times.Interested participants will receive a verbal and written solicitation by the clinical investigators and mayopt to perform the tests at the time of the visit or may schedule an appointment for the testing.

Identify the criteria for inclusion or exclusion of any subpopulation.Although it is unlikely that the investigators will encounter infants, children, or adolescents that meet

the above criteria, if there are such encounters, they will be excluded from the study secondary to thedifficulty level of the testing of visual fields and the feasibility of obtaining optical coherence tomographyon these age groups.

Explain the rationale for the involvement of special classes of subjects, such as fetuses, neonates.pregnant women, children, prisoners, institutionalized individuals, or others who may be consideredvulnerable populations. Note that 'prisoners' includes all subjects involuntarily incarcerated (for example,in detention centers) as well as subjects who become incarcerated after the study begins.

None

List any collaborating sites where human subjects research will be performed, and describe the role ofthose sites in performing the proposed research.

None

b. Sources of Materials

Describe the research material obtained from living human subjects in the form of specimens, records, ordata.

The information to be obtained for the project will involve a measure of the patient's intraocularpressure, measures of the subject's nerve fiber layer with optical coherence tomography (OCT), andresults from the patient's visual field testing with the Humphrey Visual Field Analyzer. One or all of theinformation may be obtained retroactively if performed within the last 6 months. The intraocular pressuremeasurement must be obtained the day of the OCT scans, but the Humphrey visual field test may beobtained at any time within a three month time span of the OCT scans.

Describe any data that will be recorded on the human subjects involved in the project. Describe thelinkages to subjects, and indicate who will have access to subject identities. Provide information about


Principal Investigator/Program Director (Last, First, Middle): Hsrwerth, Ronald S.

how the specimens, records, or data are collected and whether material or data will be collectedspecifically for your proposed research project.

Each subject will have a demographic sheet containing the patient's assigned code. The clinicalinvestigators will maintain and secure this record. The codes will be assigned randomly and will notreflect any of the patients' actual demographics. Patient information will be identified by code rather thanname. Copies of the Humphrey Visual Field test and of the OCT scans will be included in the patient'smedical record. The clinical investigators will maintain the patient's information in their offices or undertheir supervision. Other project investigators will have access to the code list as needed. Only the clinicalinvestigators will have access to the patients' data and identifiers. Information obtained in the collectionphase of the study will be provided to the patients' primary providers to be stored in the patients' medicalrecords.

c. Potential Risks

Describe the potential risks to subjects (physical, psychological, social, legal, or other), and assess theirlikelihood and seriousness to the subjects.

None

Where appropriate, describe alternative treatments and procedures, including the risks and benefits ofthe alternative treatments and procedures to participants in the proposed research.

NA

2. ADEQUACY OF PROTECTION AGAINST RISKS

a. Recruitment and Informed Consent

Describe plans for the recruitment of subjects (where appropriate) and the process for obtaining informedconsent. If the proposed studies will include children, describe the process for meeting requirements forparental permission and child assent.

Potential subjects will be identified from chart review in the Glaucoma/O.D. Medical Clinic fromupcoming/existing appointments by their clinicians. Once identified, one of the clinical investigators willdiscuss the study with the patient and offer the opportunity to be included in the study. A copy of awritten solicitation will be given to the patient, and the patient will have opportunity to read it and askquestions.

Include a description of the circumstances under which consent will be sought and obtained, who willseek it. the nature of the information to be provided to prospective subjects, and the method ofdocumenting consent. Informed consent document(s) need not be submitted to the PHS agencies unlessrequested.

The subject will be informed both verbally and in writing that participation is voluntary. The patient willbe informed both verbally and in writing that participation will not affect their treatment at the UniversityEye Institute. If the testing is performed at one of the patient's regularly scheduled visits, the patient willbe informed which procedures are part of the study. The investigator will note this orally to the patientand the patient will receive a written copy of which procedures were a part of the study. Relevant historywill be collected on each patient. The patient's history will include name, date of birth, and medicationhistory, and the patient will be assigned a unique code for any further identification.

b. Protection Against Risk

Describe planned procedures for protecting against or minimizing potential risks, including risks toconfidentiality, and assess their likely effectiveness.

The procedures are standard clinical research protocols and should be completely effective inminimizing risks and risks to confidentiality. Each subject will have a demographic sheet containing thepatient's assigned code. The clinical investigators will maintain and secure this record. The codes will bePHS 398/2590 (Rev. 09/04) Page 52 Continuation Format Page

Principal Investigator/Program Director (Last, First, Middle); Harwerth, Ronald S.

assigned randomly and will not reflect any of the patients' actual demographics. Patient information willbe identified by code rather than name. Copies of the Humphrey Visual Field test and of the OCT scanswill be included in the patient's medical record. The principal investigator will maintain the patient'sinformation in his office or under his supervision. Other project investigators will have access to the codelist as needed. The clinical investigators will have access to the patients' data and identifiers. Informationobtained in the collection phase of the study will be provided to the patients' primary providers to bestored in the patients' medical records. The data will be kept secure. Copies of the data will be providedto the patients clinicians for the patients' medical records.

Where appropriate, discuss plans for ensuring necessary medical or professional intervention in theevent of adverse effects to the subjects. Studies that involve clinical trials (biomedical and behavioralintervention studies) must include a description of the plan for data and safety monitoring of the researchand adverse event reporting to ensure the safety of subjects.

Patients may have a reaction to corneal anesthesia used in applanation testing and pachymetry.These reactions are typically mild and self-limiting. If the patient/subject has had a previous reaction toanesthesia, it should be identified in the chart prior to including the patient in the study. Any reactionswill be followed at UEI diagnostic clinic free of charge. There is no difference in the topical anesthesiaused in this study and the topical anesthesia used for normal Goldman applanation tonometry during aroutine examination.

3. POTENTIAL BENEFITS OF THE PROPOSED RESEARCH TO THE SUBJECTS AND OTHERS

Discuss the potential benefits of the research to the subjects and others.The subjects will be compensated $20 for participation. The subjects will have complete OCT scans,

Humphrey visual fields, and IOP and pachymetry readings included in their medical record for use bytheir treatment team. All totaled the tests would have cost the patient approximately $200.00. Thesubjects will have the knowledge that they are participating in a study that is aimed at helping our overallunderstanding of glaucoma.

Discuss why the risks to subjects are reasonable in relation to the anticipated benefits to subjects andothers.

NA

4. IMPORTANCE OF THE KNOWLEDGE TO BE GAINED

Discuss the importance of the knowledge gained or to be gained as a result of the proposed research.The information provided from the study will be used to help define a relationship between the

amount of sensitivity loss secondary to glaucoma and the corresponding amount of ganglion cell loss asmeasured by thickness on the OCT. Once established, such a relationship may play a critical role ininterpretation of visual field testing and treatment for glaucoma patients.

5. DATA AND SAFETY MONITORING PLAN

NA

Inclusion of women and minoritiesWomen and minorities will not be excluded



Targeted/Planned Enrollment Table

This report format should NOT be used for data collection from study participants.

Study Title: Behavioral Measures of Vision

Total Planned Enrollment: 30 glaucoma patients of any ethnicity, race, or gender

TARGETED/PLANNED ENROLLMENT: Number of Subjects

Ethnic Category

Hispanic or Latino

Not Hispanic or Latino

Ethnic Category: Total of All Subjects *

Racial Categories

American Indian/Alaska Native

Asian

Native Hawaiian or Other Pacific Islander

Black or African American

White

Racial Categories: Total of All Subjects *

Sex/GenderFemales

4

11

15

Males

5

10

15

Total

9

21

30

0

2

0

3

10

15

0

1

0

4

10

15

0

3

0

7

20

30

The "Ethnic Category: Total of All Subjects" must be equal to the "Racial Categories: Total of All Subjects."

The study is not planned to enroll a specific number of patients in an ethnic, racial, or gender category.Rather it will be a convenience sample that will represent the normal distribution of glaucoma patients seenin our clinic and mirrors the population of the geographic area in which our patients reside. The numbersprovided in the table are based on the experience of the Clinical Research Associates in treating glaucomapatients in the Ocular Diagnostic and Medical Eye Service in the University Eye Institute.

PHS 398/2590 (Rev. 09/04) Page 54 Targeted/Planned Enrollment Format Page


Inclusion of childrenAlthough it is unlikely that the investigators will encounter infants, children, or adolescents that meet

the above criteria, if there are such encounters, they will be excluded from the study secondary to thedifficulty level of the testing of visual fields and the feasibility of obtaining optical coherence tomographyon these age groups.

F. Vertebrate Animals.1. The experiments are designed to use 36 rhesus monkeys (Macaca mulatta) of either sex (4-6

years of age) for behavioral investigations of structure-function relationships in glaucoma. The principalexperiments are measurements of visual sensitivity across the retina in monkeys with ocularhypertension (experimental glaucoma), using standard clinical routines and instrumentation (automatedperimetry) adapted to the monkey. For all of the studies, the monkeys are trained to press a responselever to initiate a trial and then to release the lever in the presence of a visual stimulus. If the leverrelease is closely correlated with the presentation of the stimulus, it is assumed that the monkey haddetected the stimulus and he/she is always rewarded by a conditioned reinforcer (a tone) and at 0.75probability, with an unconditioned reinforcer (0.5 ml orange drink). However, if the response is not madewithin the criterion response interval, the stimulus intensity is considered to have been below thresholdand no reinforcements are provided. Typically the monkeys will complete 700 - 1000 trials in a two-hoursession and receive at least 500 ml of orange drink.

For 30 monkeys, experimental glaucoma will be induced in one of the monkey's eyes by Argon laserburns of the trabecular meshwork (i.e., the outflow route of aqueous humor). Although the experimentalprocedure involves higher energy treatment, the procedure is similar to the Argon laser trabeculoplasty(ALT) that is used as a common surgical treatment for patients with glaucoma, which is not painful.Protocols to create laser-induced experimental glaucoma are well-described in the literature and none ofthe publications have described complications associated with the procedure.

In preparation for the surgery, the monkeys will be anesthetized with Ketamine (20 mg/kg) andacepromazine (0.2 mg/kg) and a topical corneal anesthetic (0.5% proparacaine) will be instilled in theeye to be treated. A clinical Argon laser will be used (nominal settings: argon blue-green mode, 0.5 secduration, 1 watt power, 50 micron spot size). The treatment burns will be delivered to 360 deg of the mid-trabecular meshwork using a single mirror gonioprism, which was manufactured to the specifications ofthe monkey eye. In order to avoid large intraocular pressure spikes, we will use multiple treatments (1 -7) to create sustained pressures of 30 - 50 mm Hg. There will be at least 3 weeks between treatmentsand each treatment will involve 30-100 contiguous burns.

Intraocular pressure measurements and ophthalmoscopic examinations of the fundus will be madebi-weekly. For these examinations, the monkeys will be anesthetized with Ketamine (20 mg/kg) andacepromazine (0.2 mg/kg) and one drop each of a topical corneal anesthetic (0.5% proparacaine) andcycloplegic (5% cyclopentolate hydrochloride) will be instilled in the eye to be examined.

For the studies designed to investigate the RNFL contribution from axons outside the normalperimetry field and the mapping of the visual field onto the ONH, six additional monkeys will be used assubjects. These monkeys will be provided by another investigator who used the laser ablation for hisinvestigation of mechanisms of emmetropization. For that investigation, the preparation for YAG lasersurgery was the same as described for methods Argon laser treatments and the retinas of the monkey'sright eyes were ablated from the arcuate bundle to the ora serrata, using a frequency-doubled YAG laserburns (150 mW power, 150 msec duration) with a spacing of one-half burn width and 2316 ± 884 totalburns per eye. The region and degree of ablation will be documented by fundus photography.

2. The experiments are designed specifically to relate to visual processing of humans and it is,therefore, important to employ a monkey model for this work. At the present time, there are noalternative models or computer simulations to assess the functional visual deficits associated withglaucoma.

The total number of monkeys required for the proposed experiments is relatively small because eachof the monkeys will be used for long periods of time. All of the monkeys for these experiments will beobtained from the UHCO colony following the completion of experiments on visual factors of



emmetropization. Extensive amounts of data can be collected from a given animal, the data collected arevery reliable, and the intersubject variability is low.

3. The animals will be housed in the AAALAC approved animal care facilities of the Uni------- - -- ----------- The University's Animal Care Operations are directed by a full-time veterinarian ---- ------ - ---------- -- and an animal care staff. The operation of the animal care facilities is overseen by theUniversity's Institutional Animal Care and Use Committee. All of the animal care and experimentalprocedures will be in compliance with the NIH Guide for the care and Use of Laboratory Animals.

4. The behavioral procedures are not adverse or painful in any way.5. At the end of the experiments, euthanasia will be accomplished by methods recommended by the

Panel of Euthanasia of the American Veterinary Medical Association. An overdose (100 mg/kg) ofpentobarbital sodium (Nembutal) will be administered with subsequent exsanguinated by 2 liters of salinefollowed by 2 liters of 2% paraformaldehyde - 0.5% gluteraldehyde fixative in phosphate buffer (pH 7.4).

G. Literature Cited.

AGIS Investigators. (1994) The advanced glaucoma intervention study (AGIS): 2. Visual field test scoringand reliability. Ophthalmol. 101; 1445-1455.

AGIS Investigators. (2000) The advanced glaucoma intervention study (AGIS): 7. The relationshipbetween the control of intraocular pressure and visual field deterioration. Am J Ophthalmol. 130;429-440.

Anderson DR. (1987) Perimetry, With and Without Automation, second edition. St. Louis, C.V. Mosby,Co.

Ansari EA, Morgan JE, Snowden RJ. (2002) Glaucoma: squaring the psychophysics and neurobiology.Brit J Ophthalmol. 86;823-826.

Alexander LJ. (1991) Diagnosis and management of primary open-angle glaucoma. In: Classe JG, ed.Optometry Clinics. Norwalk: Appleton & Lange. 1;19-102.

Ambercrombe M. (1946) Estimation of nuclear population from microtome sections. Anat Rec. 94;239-247.

Asman P, Heijl A. (1992) Evaluation of methods for automated hemifield analysis in perimetry. ArchOphthalmol. 110;820-826.

Atkin A, Bodis-Wollner I, Wolkstein M, et a/. (1979) Abnormalities of central contrast sensitivity inglaucoma. AmJ Ophthalmol. 88; 205-211.

Beckman H. (1990) The Glaucoma Laser Trial: results of argon laser trabeculoplasty versus topicalmedicines. Ophthalmol. 97;1403-1413.

Bodis-Wollner I. (1989) Electrophysiological and psychophysical testing of vision in glaucoma. SurvOphthalmol. 33(suppl);301-307.

Bowd C, Weinreb RN, Williams JM, et at. (2000) The retinal nerve fiber layer thickness in ocularhypertensive, normal, and glaucomatous eyes with optical coherence tomography. Arch Ophthalmol.118;22-26.

Bosworth CF, Sample PA, Gupta N, et a/. (1998) Motion automated perimetry identifies earlyglaucomatous field defects. Arch Ophthalmol. 116; 1153-1158.

Bowd C, Zangwill LM, Berry CC, et al. (2001) Detecting early glaucoma by assessment of retinal nervefiber layer thickness and visual function. Invest Ophthalmol Vis Sci. 42; 1993-2003.

Brusini P. (1996) Clinical use of a new method for visual field damage classification in glaucoma. Eur JOphthalmol. 6;402-407.



Budenz DL, Chang RT, Huang X, et al. (2005a) Reproducibility of retinal nerve fiber layer thicknessmeasurements using the stratus OCT in normal and glaucomatous eyes. Invest Ophthalmol Vis Sci.46;2440-2443.

Budenz, DL, Michael A, Chang RT, et al. (2005b) Sensitivity and specificity of the StratusOCT forperimetric glaucoma. Ophthalmol. 112;3-9.

Burgoyne CF, Downs JC, Bellezza AJ, et al. (2004) Three-dimensional reconstruction of normal andearly glaucoma monkey optic nerve head connective tissues. Invest Ophthalmol Vis Sci. 45;4388-4399.

Carter-Dawson L, Crawford MLJ, Harwerth RS, et al. (2002) Vitreal glutamate concentration in monkeyswith experimental glaucoma. Invest Ophthalmol Vis Sci. 43;2633-2637.

Carter-Dawson L, Shen FF, Harwerth RS, et al. (2004) Glutathione content is altered in Muller cells ofmonkey eyes with experimental glaucoma. Neurosci Letters. 364;7-10.

Cello KE, Nelson-Quigg JM, Johnson CA. (2000) Frequency doubling technology perimetry for detectionof glaucomatous visual field loss. Am J Ophthalmol. 129;314-322.

Chauhan BC, Drance SM. (1990) The influence of intraocular pressure on visual field damage in patientswith normal-tension and high-tension glaucoma. Invest Ophthalmol Vis Sci. 31;2367-2372.

Chauhan BC, Drance SM, Douglas GR. (1990) The use of visual field indices in detecting changes in thevisual field in glaucoma. Invest Ophthalmol Vis Sci. 31 ;512-520.

Chauhan BC, Tompkins J, LeBlanc R, et al. (1993) Characteristics of frequency-of-seeing curves innormal subjects, patients with suspected glaucoma, and patients with glaucoma. Invest OphthalmolVis Sci. 34;3534-3540.

Crawford MLJ, Harwerth RS, Smith EL, et al. (2000). Experimental glaucoma in primates: Cytochromeoxidase reactivity in parvo- and magno-cellular pathways. Invest Ophthalmol Vis Sci. 41; 1791-1802.

Crawford MLJ, Harwerth RS, Smith EL, et al. (2001). Experimental glaucoma in primates: Changes incytochrome oxidase 'blobs' in V1 cortex. Invest Ophthalmol Vis Sci. 42;358-364.

Cull G, Cioffi GA, Dong J, et al. (2003) Estimating normal optic nerve axon numbers an non-humanprimate eyes. J Glaucoma 12;301-306.

Curcio CA, Allen KA. (1990) Topography of ganglion cells in human retina. J Comp Neural. 300;5-25.

Dacy DM (1999) Primate retina: cell types, circuits, and color opponency. Prog Retinal Eye Res. 18;737-763.

Drance SM. (1985) The glaucoma visual field defect and its progression. In: Drance SM, Anderson, D.R.eds, Automatic Perimetry in Glaucoma. A Practical Guide. Orlando, Grune & Stratton, Inc. pp 35-42.

Epstein DL. (1993) Primary open angle glaucoma. In: Epstein DL, Allingham RR, Schuman JS, eds.Chandler and Grant's Glaucoma, 4th edition. Baltimore: Williams & Wilkins. pp 183-198.

El Beltagi TA, Bowd C, Boden C, et al. (2003) Retinal nerve fiber layer thickness measured with opticalcoherence tomography is related to visual function in glaucomatous eyes. Ophthalmol. 110;2185-2191.

Evans DW, Hosking SL, Gherghel D, et al. (2003) Contrast sensitivity improves after brimonidine therapyin primary open-angle glaucoma: a case for neuroprotection. Br J Ophthalmol. 87;1463-1465.

Flammer J, Drance SM, Augustiny L, et al. (1985) A. Quantification of glaucomatous visual field defectswith automated perimetry. Invest Ophthalmol Vis Sci. 26; 176-181.

Frishman LJ. (2001) Basic visual processes. In: Goldstein, EB (ed) Blackwell's Hanbook of Perception.Blackwell Publishers, Oxford, pp 53-91.



Frishman, L.J., Shen, F.F., Du, L, etal. (1996) The scotopic electroretinogram of macaque after retinalganglion cell loss from experimental glaucoma. Invest. Ophthalmol. Visl Sci. 37;125-141.

Frishman LJ, Saszik S, Harwerth RS, et al. (2000). Effects of experimental glaucoma in macaques on themultifocal ERG. Doc Ophthalmol. 100;231-251.

Fujimoto JG, Hee MR, Huang D, et al. (2004) Principles of optical coherence tomography. In: OpticalCoherence Tomography of Ocular Diseases, second edition, Schuman JS, Puliafito CA, Fujimoto JG,eds, Slack, inc, Thorofare, NJ. pp 3-19.

Galvao Filho RP, Vessani RM, Susanna R. (2005) Comparison of retinal nerve fiber layer thickness andvisual field loss between different glaucoma groups. Br J Ophthalmol. 89; 1004-1007.

Gardiner SK, Johnson CA, Cioffi GA. (2005) Evaluation of the structure-function relationship inglaucoma. Invest Ophthalmol Vis Sci. 46;3712-3717.

Garway-Heath DF, Caprioli J, Fitzke FW, et al., (2002) Scaling the hill of vision: the physiologicalrelationship between light sensitivity and ganglion cell numbers. Invest Ophthalmol Vis Sci. 41; 1774-1782.

Garway-Heath DF, Poinoosawmy D, Fitzke FW, et al. (2000) Mapping the visual field to the optic disc innormal tension glaucoma eyes. Ophthalmol. 107;1809-1815.

Gaasterland DE, Kupfer C. (1974) Experimental glaucoma in the rhesus monkey. Invest Ophthalmol.13:455-457.

Girkin CA. (2004) Relationship between structure of optic nerve/nerve fiber layer and functionalmeasurements in glaucoma. Curr Opin Ophthalmol. 15;96-101.

Gramer E, Tausch M. (1995) The risk profile of the glaucomatous patient. Curr Opiin Ophthalmol. 6;78-88.

Hare WA, WoldeMussie E, Lai RK, et al. (2004) Efficacy and safety of memantine treatment for reductionof changes associated with experimental glaucoma in monkey, I: functional measures. InvestOphthalmol Vis Sci. 45;2625-2639.

Harwerth RS. (2004) Histopathology underlying glaucomatous damage: I. Glaucoma Diagnosis.Structure and Function. Weinreb, R.N. and Greve, E.L. (eds). Kugler Publications, The Hague, TheNetherlands, pp 13-19.

Harwerth RS, Crawford MLJ. (2004) The relation between perimetric and metabolic defects caused byexperimental glaucoma. Perimetry Update 2002/2003. Henson, D.B. and Wall, M. (eds). KuglerPublications, The Hague, The Netherlands, pp 175-186.

Harwerth RS, Carter-Dawson L, Shen F, et al. (1999) Ganglion cell losses underlying visual field defectsfrom glaucoma. Invest Ophthalmol Vis Sci. 40;2242-2250.

Harwerth RS, Carter-Dawson L, Smith EL III, etal. (2004) Neural losses correlated with visual losses inclinical perimetry. Invest Ophthalmol Vis Sci. 45;3152-3160.

Harwerth RS, Carter-Dawson L, Smith EL, et al. (2005) Scaling the structure-function relationship forclinical perimetry. Acta Ophthalmol Scand. 83;448-455.

Harwerth RS, Crawford ML, Frishman LJ, et al. (2002) Visual field defects and neural losses fromexperimental glaucoma. Prog Retinal Eye Res. 21;91-125.

Harwerth RS, Quigley HA. (2005) Visual field defects and retinal ganglion cell losses in human glaucomapatients. Arch Ophthalmol. (in press).

Harwerth RS, Smith EL. (1997) Contrast sensitivity perimetry in experimental glaucoma: Investigationswith degenerate gratings. In: Perimetry Update 1996/1997. Wall M & Heijl A. eds, KuglerPublications, Amsterdam, pp 3-12.



Harwerth RS, Smith EL, Boltz RL, et al. (1984). Behavioral studies on the effect of abnormal early visualexperience in monkeys: Spatial modulation sensitivity. Vision Res. 23;1501-1510.

Harwerth RS, Smith EL, Chandler M. (1999) Progressive visual field defects from experimentalglaucoma: measurements with white and colored stimuli. Optom Vis Sci. 76;558-570.

Harwerth RS, Smith EL, DeSantis L.(1993a) Behavioral perimetry in monkeys. Invest Ophthalmol VisSci. 34;31-40.

Harwerth RS, Smith EL, DeSantis L. (1993b) Mechanisms mediating visual detection in static perimetry.Invest Ophthalmol Vis Sci. 34;3011-3023.

Harwerth RS, Smith EL, DeSantis L. (1997) Experimental glaucoma; perimetric field defects andintraocular pressure. J. Glaucoma. 6;390-401.

Hawkins AS, Szlyk JP, Ardickas Z, et al. (2003) Comparison of contrast sensitivity, visual acuity, andHumphrey visual field testing in patients with glaucoma. J Glaucoma. 12;134-138.

Heijl A. (1985a) Computerised perimetry. Trans Ophthalmol Soc UK. 104;76-87.

Heijl A. (1985b) Strategies for detection of glaucoma defects. In: Drance, S.M. and Anderson, D.R. eds.,Automatic Perimetry in Glaucoma. A Practical Guide. Orlando, Grune & Stratton, Inc. pp 43-54.

Heijl A, Leske MC, Bengtsson B, et al. (2002) Reduction of intraocular pressure and glaucomaprogression. Arch Ophthalmol. 120; 1268-1279.

Heijl A, Lindgren G, Olsson J. (1987a) A package for the statistical analysis of visual fields. DocOphthalmol. 49;153-168.

Heijl A, Lindgren G, Olsson J. (1987b) Reliability parameters in computerized perimetry. DocOphthalmol. 49;595-600.

Heijl A & Patella VM (2002): Essential Perimetry, third edition. Carl Zeiss Meditec, Dublin CA.

Henson D, Chaudry S, Artes P, et al. (2000) Response variability in the visual field: comparison of opticneuritis, glaucoma, ocular hypertension, and normal eyes. Invest Ophthalmol Vis Sci. 41 ;417-421.

Hodapp E, Parrish RK II, Anderson DR. (1993) Clinical decisions in glaucoma. St. Louis, MO: Mosby-YearBook. pp 52-61.

Huang D, Swanson EA, Lin CP, et al. (1991) Optical coherence tomography. Science. 254; 1178-1181.

lester M, Altieri M, Vittone P, et al. (2003) Detection of glaucomatous visual field defect bynonconventional perimetry. Ophthalmol. 135;35-39.

Iliev ME, Meyenberg A, Garweg JG. (2005) Morphometric assessment of normal, suspect andglaucomatous optic discs with Stratus OCT and HRT II. Eye. 23;

Janknecht P, Funk J. (1994) Optic nerve head analyzer and Heidelberg retina tomography: accuracy andreproducibility of topographic measurements in a model eye and in volunteers. Br J Ophthalmol.78;760-768.

Johnson CA. (1994) Selective versus non-selective losses in glaucoma. J Glaucoma. 3;32-44.

Johnson CA. (1996) Standardizing the measurement of visual fields for clinical research. Ophthalmol.103;186-189.

Johnson CA, Adams AJ, Casson EJ, et al. (1993) Blue-on-yellow perimetry can predict the developmentof glaucomatous field loss. Arch Ophthalmol. 111 ;645-650.

Johnson CA, Samuels SJ. (1997) Screening for glaucomatous visual field loss with frequency-doublingperimetry. Invest Ophthalmol Vis Sci. 38;413-425.

Kadaboukhova L, Lindblom B. (2003) Frequency doubling technology and high-pass resolution perimetryin glaucoma and ocular hypertension. Acta Ophthalmol Scand. 81;247-252.



Kass MA, Heuer DK, Higginbothan EJ, et al. (2002) The ocular hypertension treatment study: arandomized trial determines that topical ocular hypotensive medication delays or prevents the onsetof primary open-angle glaucoma. Arch Ophthalmol. 120;701-713.

Katz J, Sommer A, Gaasterland DE, et al. (1991) Comparison of analytic algorithms for detectingglaucomatous visual field loss. Arch Ophthalmol. 109; 1684-1689.

Kee C, Pickett P, Dueker DK, et al. (1995). Argon laser trabeculoplasty and pilocarpine effects on outflowfacility in cynomolgus monkey. J Glaucoma. 4;334-343.

Kerrigan-Baumrind LA, Quigley HA, Pease ME, et al. (2000) Number of ganglion cells in glaucoma eyescompared with threshold visual field tests in the same persons. Invest Ophthalmol Vis Sci. 41;741-748.

Kremmer S, Ayertey HD, Selbach JM, et al. (2000) Scanning laser polarimetry, retinal nerve fiber layerphotography, and perimetry in the diagnosis of glaucomatous nerve fiber defects. Graefes Arch ClinExp Ophthalmol. 238;922-926.

Kupfer C, Chumbley L, Downer JC. (1967) Quantitative histology of optic nerve, optic tract and lateralgeniculate nucleus of man. J Anat. 101;393-401.

Lee JE, Schulman JS, Fujimoto JG, et al. (2004) Optical coherence tomography scanning and image-processing protocols. In: Optical Coherence Tomography of Ocular Diseases, second edition,Schuman JS, Puliafito CA, Fujimoto JG, eds, Slack, inc, Thorofare, NJ. pp 691-698.

Lemij HG. (2001) The value of polarimetry in the evaluation of the optic nerve in glaucoma. Curr OpinOphthalmol. 12;138-142.

Leung CK, Chong KK, Chan W, et al. (2005) Comparative study of retinal nerve fiber layer measurementby StratusOCT and GDx VCC, II: Structure/function regression analysis in glaucoma. InvestOphthalmol Vis Sci. 46;3702-3711.

Leung CK, Yung WH, Ng AC, et al. (2004) Evaluation of scanning resolution on retinal nerve fiber layermeasurement using optical coherence tomography in normal and glaucomatous eyes. J Glaucoma.13:479-485.

Levi DM, Harwerth RS. (1977) Spatio-temporal interactions in anisometropic and strabismic amblyopia.Invest Ophthalmol Vis Sci. 16;90-95.

Lundh, B.L. and Gottvall, E. (1995) Peripheral contrast sensitivity for dynamic sinusoildal gratings in earlyglaucoma. Acta Ophthalmol Scand. 73;202-206.

Lynch S, Johnson CA, Demirel S. (1997) Is early damage in glaucoma selective for a particular cell typeor pathway? In: Wall, M. and Heiji, A., eds. Perimetry Update 1996/1997. Kugler Publications,Amsterdam pp 253-261.

Martin L, Wanger P, Vancea L, et al. (2003) Concordance of high-pass perimetry and frequency-doublingtechnology perimetry results in glaucoma: no support for selective ganglion cell damage. JGlaucoma. 12;40-44.

Masuyama Y. (1984) Histopathological study of anterior chamber angle after laser trabeculoplasty incynomolgus monkeys (Macaca irus). Acta Soc Ophthalmol Jpn. 88; 1007-1014.

Mikelberg FS, Drance SM, Schulzer M, et al. (1989) The normal human optic nerve. Axon count andaxon diameter distribution. Ophthalmol. 96;1325-1328.

Mikelberg FS, Schulzer M, Drance SM, et al. (1986) The rate of progression of scotomas in glaucoma.Am J Ophthalmol. 101; 1-6.

Morgan, JE, Uchida H, Caprioli J. (2000) Retinal ganglion cell death in experimental glaucoma. Brit JOphthalmol. 84;303-310.



Nickells RW (1996) Retinal ganglion cell death in glaucoma: The how, the why, and the maybe. JGlaucoma. 5;345-356.

Nordmann JP. (1996) Early visual disturbances in glaucoma. Curr Opin Ophthalmol. 7;47-53.

Ogden TE. (1984) Nerve fiber layer of the primate retina: Morphometric analysis. Invest Ophthalmol VisSci. 25;19-29.

Parisi V, Manni G, Centofanti M, etal. (2001) Correlation between optical coherence tomography, patternelectroretinogram, and visual evoked potentials in open-angle glaucoma. Ophthalmol. 108;905-912.

Pederson JE, Gaasterland DE. (1984) Laser-induced primate glaucoma. I. Progression of cupping. ArchOphthalmol. 102:1689-1692.

Philippin H, Unsoeld A, Maier P, et al. (2005) Ten-year results: detection of long-term progressive opticdisc changes with confocal laser tomography. Graefes Arch Clin Exp Ophthalmol. 25;1-5.

Porciatti V, Di Bartolo E, Nardi N, et al. (1997) Responses to chromatic and luminance contrast inglaucoma: a psychophysical and electrophysiological study. Vision Res. 27; 1975-1987.

Quigley HA. (1993) Open-angle glaucoma. The New Eng J Med. 328; 1097-1106.

Quigley HA, (1998a) Neuronal death in glaucoma. Prog Retinal Eye Res. 18;39-57.

Quigley HA, (1998b) Selectivity in glaucoma injury. Archf Ophthalmol. 116;396-398.

Quigley HA. (2005) Intraocular pressure: relevant or redundant. Clin Exp Ophthalmol. 33;113-114.

Quigley HA, Dunkelberger GR, Green WR (1989) Retinal ganglion cell atrophy correlated with automatedperimetry in human eyes with glaucoma. Am J Ophthalmol. 107;453-464.

Quigley HA, Holman RM. (1987) Laser energy levels for trabecular meshwork damage in the primateeye. Invest Ophthalmol Vis Sci. 24:1305-1307.

Rangaswamy NV, Zhou W, Harwerth RS, et al. (2005) Effect of experimental glaucoma in primates onoscillatory potentials of the slow-sequence MfERG. Invest Ophthalmol Vis Sci. (in press).

Sample PA. (2001) What does functional testing tell us about optic nerve damage? Survey Ophthalmol45(suppl 3);S319-S324.

Sample PA, Bosworth CF, Blumenthal EZ, et al. (2000) Visual function-specific perimetry for indirectcomparison of different ganglion cell populations in glaucoma. Invest Ophthalmol Vis Sci. 41;1783-1790.

Sanchez RM, Dunkelberger GR, Quigley HA. (1986) The number and diameter distribution of axons inmonkey optic nerve. Invest Ophthalmol Vis Sci. 27; 1342-1350.

Schumer RA & Podos SM. (1994) The nerve of glaucoma. Arch Ophthalmol. 112;37-44.

Schlottmann PG, De Cilia S, Greenfield DS, et al. (2004) Relationship between visual field sensitivity andretinal nerve fiber layer thickness as measured by scanning laser polarimetry. Invest Ophthalmol VisSci. 45;1823-1829.

Shen FF. (1994) Retinal cell morphology and visual field defects in glaucomatous monkey eyes.Unpublished thesis, University of Houston, Houston, TX.

Sihota R, Gulati V, Agarwal HC, et al. (2002) Variables affecting test-retest variability of HeidelbergRetina Tomograph II stereometric parameters. J Glaucoma. 11;321-328.

Smith EL, Chino YM, Harwerth RS, et al. (1993). Retinal inputs to the monkey's lateral geniculatenucleus in experimental glaucoma. Clin Vision Sci. 8; 113-139.

Sommer A. (1989) Intraocular pressure and glaucoma. Am J Ophthalmol. 107:186-188.

Spaeth GL, Baez KA. (1992) Argon laser trabeculoplasty controls one third of cases of progressive,uncontrolled, open angle glaucoma for 5 years. Arch Ophthalmol. 110;491-494.



Spry PG, Hussin HM, Sparrow JM (2005a) Clinical evaluation of frequency doubling technologyperimetry using the Humphrey Matrix 24-2 threshold strategy. Br J Ophthalmol. 89; 1031-1035.

Spry PGD, Johnson CA, Mansberger SL, etal., (2005b) Psychophysical investigation of ganglion cellloss in early glaucoma. J Glaucoma. 14;11-19.

Swanson WH, Felius J, Pan F. (2004) Perimetric defects and ganglion cell damage: interpreting linearrelations using a two-stage neural model. Invest Ophthalmol Vis Sci. 45;466-472.

Teesalu P, Tuulonen A, Airaksinen PJ. (2000) Optical coherence tomography and localized defects ofthe retinal nerve fiber layer. Acta Ophthalmol Scand. 78;49-52.

Thomas R, Bhat S, Muliyil JP, et a/., (2002) Frequency doubling perimetry in glaucoma. J Glaucoma.11;46-50.

Van Buskirk EM. (1991) The laser step in early glaucoma therapy. Am J Ophthalmol. 112;87-90.

Ventura LM, Porciatti V. (2005) Restoration of retinal ganglion cell function in early glaucoma afterintraocular pressure reduction. A pilot study. Ophthalmol. 112;20-27.

Vickers JC, Hof PR, Schumer RA, et a/. (1997) Magnocellular and parvocellular visual pathways are bothaffected in a macaque model of glaucoma. Aust New Zealand J Ophthalmol. 25;239-243.

Viswanathan S, Frishman LJ, Robson JG, etal. (1999) The photopic negative response of the macaqueelectroretinogram is reduced by experimental glaucoma. Invest Ophthalmol Vis Sci. 40;1124-1136.

Wall M, Jennish CS, Munden PM (1997) Motion perimetry identifies nerve fiber bundle defects in ocularhypertension. Arch Ophthalmol. 115;26-33.

Wall M, Kutzko K, Chauhan B. (1997) Variability in patients with glaucomatous visual field damage isreduced using size V stimuli. Invest Ophthalmol Vis Sci. 38;426-435.

Wang L, Dong J, Cull G, et a/. (2003) Varicosities of intraretinal ganglion cell axons in human andnonhuman primates. Invest Ophthalmol Vis Sci. 44;2-9.

Wassle H, Grunert U, Rohrenbeck J, etal. (1990) Retinal ganglion cell density and cortical magnificationfactor in the primate. Vision Res. 30;1897-1911.

Weber AJ, Chen H, Hubbard WC, etal. (2000) Experimental glaucoma and cell size, density, andnumber in the primate lateral geniculate nucleus. Invest Ophthalmol Vis Sci. 41 ;1370-1379.

Weber J, Ulrich H. (1991) A perimetric nerve fiber bundle map. International Ophthalmol. 15;193-2000

Weinreb RN. (2004) Primary open-angle glaucoma. The Lancet. 363; 1711-1720.

Whitetree AR, Crawford MLJ, Harwerth RS, et a/. (2005) Nitric oxide production is increased in monkeyretinas with experimental glaucoma. J Neurosci. (in revision).

Wirtschafter JD, Becker WL, Howe JB, etal. (1982) Glaucoma visual field analysis by computed profileof nerve fiber function in optic disc sectors. Ophthalmol. 89;255-267.

Wollstein G, Beaton S, Paunescu A. (2004) Optical coherence tomography in glaucoma. In: OpticalCoherence Tomography of Ocular Diseases, second edition, Schuman JS, Puliafito CA, Fujimoto JG,eds, Slack, inc, Thorofare, NJ. pp 483-610.

Wollstein G, Schuman JS, Price LL, etal. (2005) Optical coherence tomography longitudinal evaluationof retinal nerve fiber layer thickness in glaucoma. Arch Ophthalmol. 123;464-470.

Wood JM, Bruce AS. (1993) The value of visual field indexes in screening and threshold testing ofglaucoma. Glaucoma. 15; 140-146.

Yucel YH, Zhang Q, Gupta N, etal. (2000) Loss of neurons in magnocellular and parvocellular layers ofthe lateral geniculate nucleus in glaucoma. Arch Ophthalmol. 118;378-384.



H. Consortium / Contractual Arrangements.

--- ----------- - --------------------- , at the University of Texas - Houston, Department of Ophthalmologyand Visual Science, will conduct the histological analyses of the retinas of the monkeys with unilateralexperimental glaucoma. These data are needed to check the validity of the structure-function model forglaucoma that will be developed from functional measurements by standard clinical perimetry andstructural measurements by high-resolution imaging of retinal morphology. The histological analyses foreach eye will involve, 1) the quantification of retinal ganglion cell densities at 16 retinal locationscorresponding to specific perimetry test locations, 2) the thickness of the retinal nerve fiber layer aroundthe optic nerve head in a circle that corresponds to the standard circle scan for optical coherencetomography, and 3) determination of the axon density in the retinal nerve fiber layer in the standard circlescan of clinical OCT. --- ------------------- - will perform these analyses on six pairs of eyes each year of theproject and prepare data reports and descriptions for publication. These studies will make a significantcontribution and are an integral part of the research program, but do not constitute and independentproject for separate funding.

The contractual arrangement with the University of Texas - Houston is for a cost of approximately$40,600 in direct costs for each year of the grant. The administration of the contract will reside with theUniversity of Houston.

I. Resource Sharing.

Data SharingSharing of data is an important part of the project and will be carried out by presentations at scientific

meetings and publications of research articles. Typically, the PI will present papers at 3 - 4 researchmeetings each year. The usual meetings include: Association for Research in Vision and Ophthalmology,American Academy of Optometry, International Perimetry Society, North American Perimetry Society,Glaucoma Progression Scholars, and the Optometric Glaucoma Society.

Peer reviewed publications are a more durable form of data sharing and during the past five years,our research on experimental glaucoma has generated data for about three scientific publications peryear. The rate of publication should continue during this grant period.

Resource Sharing.The monkeys with experimental glaucoma,-- ------ ----------- --- ill be also available for other studies.

The animals provide an impo----- ----------- - --- ----- ---- ------------ n for her studies of inner retinalcontributions to ERG and for --- ----------- - --------------------- - studies of retinal cellular mechanismsaltered by glaucoma. For example, over the past five years, monkeys with verified visual field defectsfrom experimental glaucoma have provided data for studies of the negative photopic ERG response(Viswanathan, eta/., 1999), multi-focal ERG (Frishman, eta/., 2000), and oscillatory ERG potentials(Rangaswamy, et a/., 2005). In addition, tissues from the monkeys were used for studies of afferentpathway effects of glaucoma (Crawford, et at., 2000; 2001; Harwerth & Crawford, 2004) and alterationsof cell biology in glaucoma (Carter-Dawson, et a/., 2002; 2004; Whitetree, et a/., 2005). Similar shareduse of the animals will continue throughout period of support that is requested in the present application.

J. Consultants.

None



8. APPENDIX

Three reprints and one pre-print are appended.

Reprints.

Harwerth, R.S. and Crawford, M.L.J. (2004) The relation between perimetric and metabolic defectscaused by experimental glaucoma. Perimetry Update 2002/2003. Henson, D.B. and Wall, M. (eds).Kugler Publications, The Hague, The Netherlands, pp 175-186.

Harwerth, R.S., Carter-Dawson, L, Smith, E.L., Barnes, G., Holt, W.F., and Crawford, M.L.J. (2004)Neural losses correlated with visual losses in clinical perimetry. Investigative Ophthalmology andVisual Science, 45, 3152-3160.

Harwerth, R.S., Carter-Dawson, L., Smith, E.L., and Crawford, M.L.J. (2005) Scaling the structure-function relationship for clinical perimetry. Acta Ophthalmologica Scandanavia, 83, 448-455.

Pre-print.

------------ ------ ---- - ----------- ------ --------- ---- - --------- - ---- - -------- ---------- - ---- ------- - - - -------- - ------------ - ---------- ---------- - -- --------------------- -- - --------


Principal Investigator/Program Director (last, First, Middle): Harwerth, Ronald S.

CHECKLISTTYPE OF APPLICATION (Check all that apply.)

I I NEW application. (This application is being submitted to the PHS for the first time.)

\ | REVISION of application number:(This application replaces a prior unfunded version of a new, competing continuation, or supplemental application.)

INVENTIONS AND PATENTSI I COMPETING CONTINUATION of grant number: RO1 EY01139 (Competing continuation appl. and Phase II only)

(This application is to extend a funded grant beyond its current project period.) rrx i—.IN/I •"- |_| Previously reported

SUPPLEMENT to grant number:

No

Yes. If "Yes," I I Not previously reported(This application is for additional funds to supplement a currently funded grant.)

\ | CHANGE of principal investigator/program director.

Name of former principal investigator/program director:

| | CHANGE of Grantee Institution. Name of former institution:

I | FOREIGN application I I Domestic Grant with foreign involvement List Country(ies)1—' '—' Involved:

I I SBIRPhasel

I I STTR Phase I

| SBIR Phase II: SBIR Phase I Grant No.

I STTR Phase II: STTR Phase I Grant No.

| SBIR Fast Track

STTR Fast Track1. PROGRAM INCOME (See instructions.)All applications must indicate whether program income is anticipated during the period(s) for which grant support is request. If program income isanticipated, use the format below to reflect the amount and source(s).

Budget Period

07/01/06-06/30/11Anticipated Amount

$0.00Source(s)

2. ASSURANCES/CERTIFICATIONS (See instructions.)In signing the application Face Page, the authorized organizationalrepresentative agrees to comply with the following policies, assurancesand/or certifications when applicable. Descriptions of individualassurances/certifications are provided in Part III. If unable to certifycompliance, where applicable, provide an explanation and place it afterthis page.•Human Subjects Research -Research Using Human Embryonic StemCells -Research on Transplantation of Human Fetal Tissue -Women andMinority Inclusion Policy -Inclusion of Children Policy -Vertebrate Animals-

•Debarment and Suspension -Drug- Free Workplace (applicable to new[Type 1] or revised [Type 1] applications only) -Lobbying -Non-Delinquency on Federal Debt -Research Misconduct -Civil Rights(Form HHS 441 or HHS 690) -Handicapped Individuals (Form HHS 641 orHHS 690) -Sex Discrimination (Form HHS 639-A or HHS 690) -AgeDiscrimination (Form HHS 680 or HHS 690) -Recombinant DNAResearch, Including Human Gene Transfer Research -Financial Conflictof Interest (except Phase I SBIR/STTR) -Smoke Free Workplace•Prohibited Research -Select Agents and Toxins•STTR ONLY: Certification of Research Institution Participation.

3. FACILITIES AND ADMINSTRATIVE COSTS (F&A)/ INDIRECT COSTS. See specific instructions.

^ DHHS Agreement dated: 04/21/2004 I I No Facilities And Administrative Costs Requested.

Regional Office.

Date

I | DHHS Agreement being negotiated with

No DHHS Agreement, but rate established with

CALCULATION* (The entire grant application, including the Checklist, will be reproduced and provided to peer reviewers as confidential information.)

a. Initial budget period: Amount of base $ 235,123 xRateapplied 48.50 % = F&Acosts $ 114,035

b. 02 year Amount of base

c. 03 year Amount of base

d. 04 year Amount of base

e. 05 year Amount of base

$

$

$

$

208,927

207,694

206,425

205,118

x Rate applied

x Rate applied

x Rate applied

x Rate applied

48.

48.

48.

48.

50

50

50

50

% =

% =

% =

% =

TOTAL

F&A costs

F&A costs

F&A costs

F&A costs

F&A Costs

$

$

$

$

• I

101,133

100,732

100,116

99,482

515,498 |

Modified total direct cost base

'Check appropriate box(es):

I | Salary and wages base

| | Off-site, other special rate, or more than one rate involved (Explain)Explanation (Attach separate sheet, if necessary.):

\ I Other base (Explain)

PHS 398 (Rev. 09/04) Page 65 Checklist Form Page

finartme HAHWhRTH, RONALD S Counc.1: Ub/2UUb …3e. DEPARTMENT, SERVICE, LABORATORY, OR EQUIVALENT...

Documents

Transcript of finartme HAHWhRTH, RONALD S Counc.1: Ub/2UUb …3e. DEPARTMENT, SERVICE, LABORATORY, OR EQUIVALENT...