Funded Research

The Ohio LIONS Eye Research Foundation Awarded $137,500 for Eye Research and Student Fellowships for the 2006-2007 Research Year!

Pictured are just some of the researchers presenting their funded research at the annual symposium of the Ohio LIONS Eye Research Foundation, 2005.

The Ohio LIONS Eye Research Foundation Awarded $144, 500 for Eye Research and Student Fellowships for the 2004 - 2005 Research Year

At the July 18, 2004 OLERF Board meeting, trustees of the Ohio LIONS Eye Research Foundation have made awards totaling $144,500 for eye research and student fellowships for the 2004 - 2005 research year. Continuation eye research grants to seven research centers in Ohio totaled $123,000, two Bryan grants for diabetes related eye disease totaled $17,500 and student research fellowships (4) totaled $4,000. The seven research centers in Ohio included University of Cincinnati, Wright State University (Dayton), OSU Department of Ophthalmology, OSU College of Optometry, Columbus Children's Hospital, Case Western Reserve University (Cleveland) and Medical College of Ohio (Toledo). Two Bryan diabetes research grants were awarded; OSU Department of Ophthalmology and Case Western Reserve University. Four student fellowships were awarded; OSU Department of Ophthalmology, OSU College of Optometry, Columbus Children's Hospital and Case Western Reserve University.

The funded research programs span a wide range of eye disease including diabetic retinopathy, corneal dystrophies, cataract, age related macular degeneration, new treatments for refractive errors, optic nerve disease, glaucoma, gene therapy, lazy eye among many others.

See below, detailed descriptions of the projects funded and future directions for eye research.

The Ohio LIONS Eye Research Foundation (OLERF) funds researchers from seven sites in Ohio including:

The 2003 calendar year has seen major advances in research programs and changes in faculty, infrastructure, and space in the Department of Ophthalmology. The continuing and new support from the National Eye Institute is a significant recognition of the caliber of our program. Drs. Medof, Pearlman, Nagaraj, Porter/Cheng, and Szczotka continued their NEI-funded research in the areas of ocular inflammation, cataract, diabetic retinopathy, amblyopia and eye movement disorders, and clinical studies on keratoconus and corneal transplantation. Drs. Nagaraj and Pearlman were promoted to Professor, effective July 1, 2004. Jinzhong Zhang, Ph.D. was recruited to the Department this past year as Assistant Professor of Ophthalmology, transferring from the Department of Medicine. Dr Zhang’s appointment greatly increases the strength of our aging and diabetes research program. The OLERF funding has provided vital support, not only for our continuing research program, but also for new ventures that have become an established part of our research program or have provided pilot data for new grant awards.

Drs. Porter and Cheng have had increasing collaborations with the eye muscle biology group in the Department of Neurology. As a result of these collaborations, Dr. Porter along with his colleague in Neurology, Dr. Henry Kaminski, were awarded this year a $4.7 million R24 grant from NEI to develop a monoclonal antibody treatment for ocular myasthenia gravis. Based on these increasing collaborations and significant research funding, the chairs of Ophthalmology and Neurology agreed to have both Dr. Porter and Dr. Cheng, his colleague and previous post-doctoral fellow, transfer their primary appointments to Neurology this past November to enhance this research effort. Dr. Porter will continue to remain active in the Case Visual Sciences Research Center, running its T32 supported training grant for the development of new visual scientists and its P30 Grant supported Bioinformatics Module.

This past year has also seen the basic and clinical research effort of the clinical departments be reorganized by the Case School of Medicine and University of Cleveland into the Case Research Institute. The Institute represents a joint partnership of these two institutions with an investment of $110 million in a new research building, the Wolstein building, that has recently opened and $50 million over 5 years for programmatic development with new faculty recruitments, particularly in translational research. As part of this initiative, Dr. Lass has reached agreement with the new Dean of the School of Medicine and President of the Case Research Institute, Dr. Ralph Horwitz, to strengthen the research infrastructure of the Department with a funded Director for Basic Research position, support for the recruitment of 3 new basic research faculty (two senior, one junior investigators) over the next two year, and a commitment to add 20 to 25,000 sq. feet of new research space over the next two years for the Department and Visual Sciences Research Center in the new Wolstein building.

Eric Pearlman, Ph.D., close collaborator with Dr. Lass for 10 years, has transferred his primary appointment from the Center for Global Health to the Department of Ophthalmology, and become Director of Research for the Department. Dr. Pearlman will build a Program in Ocular Immunology and Inflammation in the Department; the intention is that 2 of the 3 external faculty recruitments will be in this research area. Drs. Pearlman and Lass intend to further strengthen the basic research efforts within the Department in two major thematic areas, ocular immunology and inflammation and aging and diabetic eye diseases including cataract, diabetic retinopathy, and macular degeneration. Within the next three year we anticipate that a full complement of seven to eight basic scientists within primary appointments will be active within the Department of Ophthalmology alone along with another 12 to 14 other NEI funded vision investigators in 10 other departments within the Case Visual Sciences Research Center.

Another major initiative is a strengthening of the clinical research programs within the Department and VSRC. Dr. Szczotka has been awarded at K23 grant from NEI to enable her pursue a PhD at Case in epidemiology while still remaining active clinically 25% of her time. Her ultimately goal is to lead multi-center clinical trials, and has already established a coordinating Center. The Department and its PI, Dr. Lass, has been awarded a R21 award from NEI to develop a Clinical Vision Research Unit which will fund an expansion of its clinical trial coordinating capability as well as provide partial support for two epidemiologist/ biostatisticians and a database manager. A collaboration has also been developed with a molecular epidemiologist in their department, Dr. Sudha Iyengar, with major molecular epidemiologic studies in a number of common eye diseases planned, including Fuchs’ Dystrophy and keratoconus, to examine the potential genetic influence(s).

Dr. John Porter continues to examine the novel biology of the extraocular muscles, the muscles responsible for movement of the eyes. There are two major areas that he is working in. First, the fact that the eye muscles are so different from other skeletal muscles means that the developmental mechanisms that shape their biology must be different from those that operate during the development of other skeletal muscles. He continues to examine the differing gene profile of the eye muscles compared to other skeletal muscles using sophisticated DNA microarray analysis techniques. Understanding these differences may provide insight into how the eye muscles are affected in amblyopia. Second, because they are so different, the eye muscles may be more or less susceptible than other skeletal muscles in neuromuscular disease, such as muscular dystrophy. He continues to sort out the reason(s) for the protection that occurs with the eye muscles in this dystrophy while other skeletal muscles continue to deteriorate using a mouse model of the disease. This work now receives major funding from the National Eye Institute and Muscular Dystrophy Association, but pilot funds from OLERF enabled critical pilot data to be generated to receive larger funding.

Eye and optic nrve of a normal mouse and a mouse with small eyes

Molecular Function of Chx10 in Eye Development

Overview. My lab focuses on understanding of the molecules important for eye development. Specifically, we are studying transcription factors, proteins that turn other genes on or off. Chx10 (pronounced “chex 10”) is a transcription factor that is essential for eye development because major defects occur when it is absent. To study how chx10 works, we are using ocular retardation (or^J) mutant mice (figure), animals that lack chx10. These mice have small eyes (microphthalmia) and lack optic nerves (optic nerve aplasia).

Because chx10 controls other genes, a basis for understanding how it works involves determining the genes whose expression is changed in its absence. To address this issue we used bioinformatics to generate a “gene expression profile” (a catalog of genes whose expression is changed) in the embryonic or^J eye. In collaboration with Dr. Anand Swaroop (Dept. Ophthalmology, Univ. Michigan), we used cDNA microarrays, Affymetrix GeneChips and real-time polymerase reaction (RT-PCR) to accomplish this.

I-Gene cDNA microarrays permit us to test over 6,500 eye genes. We identified 57 genes that showed =1.5-fold increase or decrease in expression. Aside from unknown or novel genes (which we will characterize), several known genes have already identified roles in eye development. For example, acetaldehyde dehydrogenase (Aldh1a1), which showed a 2.91-fold increased in the or^J eye, is is important in development of the ventral (“belly-side”) retina. Because the optic nerve normally develops from the ventral retina, this change in Aldh1a1 expression provides a molecular clue to why the optic nerves fail to form in the or^J eye.

Complex pie chart showing proportions of different genes

Affymetrix Gene Chips. We have complemented the microarray work with Affymetrix Gene Chips, a technique similar to cDNA microarrays except that it permits examination of over 14,000 genes. We identified 136 genes that showed =2-fold increase or decrease in expression in the or^J eye (Figure 2). Nearly 12% of these are transcription factor genes (Tables 1 & 2) whose altered expression suggests that they are regulated, directly or indirectly, by chx10. Significantly, some of these genes are related to processes that are defective in the chx10^-/- (or^J) eye. For example, microphthalmia-associated transcription factor (Mitf) shows nearly 3-fold increased in the microphthalmic mutant eye, suggesting a link between chx10, mitf and the formation of small eyes.

Real-time polymerase chain reaction (RT PCR). Microarray and GeneChip studies are primarily screening tools whose resultant data must be validated through quantitative, independent methods. RT-PCR is a method that can quantify mRNA (i.e., gene expression) levels for individual genes. Table 1 shows RT-PCR data for some of the transcription factor genes identified by Affymetrix.

Gene	Symbol	FC	RT-PCR
microphthalmia-associated transcription factor	Mitf	2.8542	5.10
myocyte enhancer factor 2C	Mef2c	2.2784	2.01
Notch-regulated ankyrin repeat protein	Nrarp	-2.0505
ventral anterior homeobox containing gene 2	Vax2	-2.0644
distal-less homeobox 2	Dlx2	-2.1363
forkhead box D1	Foxd1	-2.6516
sine oculis-related homeobox 6 homolog (Drosophila)	Six6	-2.6930
ISL1 transcription factor, LIM/homeodomain (islet 1)	Isl1	-2.7975	-4.83
achaete-scute complex homolog-like 1 (Drosophila)	Ascl1	-3.2577
POU domain, class 3, transcription factor 1	Pou3f1	-3.6922	-14.20
pancreas specific transcription factor, 1a	Ptf1a	-4.0827	-17.62
neurogenin 2	Neurog2	-4.8314	-7.35
hairy and enhancer of split 5 (Drosophila)	Hes5	-5.9696	-1024.00

During the next year, we will continue work to define a “transcriptional regulatory network” that is controlled by chx10. Two types of experiments will help us understand when, where and how chx10 controls other genes:

1. Map the spatial and temporal expression of the altered genes during eye development (immunohistochemistry and in situ hybridization). This will identify the cells in the normal eye that express proteins/genes showing altered expression in the or^J eye.

2. Determine whether altered genes are direct targets of chx10 (chromatin immunoprecipitation (ChIP)). This will help us understand how chx10 alters expression of specific genes.

These studies funded by OLERF will provide preliminary data for an NIH grant application for resubmission October 1, 2004.

Relationship to eye diseases. Expression profiling of eye tissue that lacks a transcription factor like chx10 provides information about candidate genes for functional studies and related diseases. Our bioinformatics data identified a number of diseases linked to several transcription factor genes that showed altered expression in the or^J eye (Table 2). In the future, this data can be used to identify disease candidate genes for the purposes of screening and studies to eludicate disease mechanism.

Table 2. Eye diseases linked to transcription factors with altered expression in or^J eye

Gene	Symbol	Disease
microphthalmia-associated transcription factor	Mitf	dominant spinocerebellar ataxia w/MD or retinal degeneration; protein: SCA7 protein
myocyte enhancer factor 2C	Mef2c	dominant Wagner disease and erosive vitreoretinopathy
hairy and enhancer of split 5 (Drosophila)	Hes5	recessive Leber congenital amaurosis

ARVO 2004

Hankin MH, MI Othman, Y Zeng, J Yu, A Swaroop. Gene expression changes in the developing ocular retardation eye. ARVO 675, 2004.

Purpose: Chx10, a homeodomain transcription factor, plays a major role during retinal development. Initially expressed in dividing retinal neuroepithelial cells, Chx10 is down-regulated as differentiation proceeds; later, it is expressed selectively in bipolar cells. Ocular retardation (or^J) mutant mice carry a null allele of Chx10 (Burmeister et al., 1996) and exhibit an eye-specific phenotype that affects fundamental aspects of retinal development: (1) retinal neuroepithelial cell proliferation is reduced, leading to microphthalmia; (2) intraretinal retinal ganglion cell axon guidance is disrupted; and (3) bipolar cells fail to differentiate. Our goal was to define molecular changes in the developing or^J eye as a prelude to understanding the transcriptional regulatory function of Chx10. Methods: Mouse I-gene arrays, containing cDNAs of >6500 eye genes/ESTs printed in duplicate onto glass slides, were hybridized to target cDNAs generated from embryonic day 15.5 wild-type (+/+) and or^J/or^J eye mRNAs. At least four replicate hybridizations of independently prepared mRNA samples were performed, each hybridization being a comparison of one of these eye samples to a reference sample. Hybridized arrays were scanned and analyzed by DigitalGenome to generate intensity data. Data were normalized with locally weighted linear regression (loess) and print-tip-group methods using limma Package. Differential expression analysis was performed with multiple comparison correction (false-discovery rate) using BioConductor and SAM. Results: We identified more than 20 genes whose expression is significantly altered in the or^J/or^J eye. A majority of these genes were expressed at higher levels: these included H19, Igf2, Hsp86, and the regulatory subunit of the RNA binding protein (Dj1). We also identified a number of novel ESTs. We are currently using real-time PCR to verify and further quantify our findings, Affymetrix mouse GeneChips to expand the number of genes analyzed, and immunohistochemistry and in situ hybridization to examine spatiotemporal changes in expression patterns. Conclusions: In addition to gene products, identified previously using immunohistochemistry, whose expression is altered in the absence of Chx10 (e.g., Pax2 and islet1), these studies have identified several new candidate genes that may contribute to pathways regulated by Chx10.

Supported by: Ohio LIONS Eye Research Foundation; NIH (EY11115, ET07003); FFB; RPB

The ultimate goal of our research project is to understand the pathogenesis of eye diseases at molecular and cellular levels so that better treatment regimens can be designed to cure eye diseases and prevent blindness. To achieve this goal, our strategies are to create experimental mouse models via transgenesis (gene overexpression) and gene targeting technologies (knockout genes), which exhibit altered genetics functions resulting in pathology characteristics of human eye diseases.

Transgenesis is the technique that a foreign gene is microinjected into mouse fertilized eggs that are subsequently implanted into a foster mother. The microinjected eggs will develop to term and produce mice that carry the in vitro engineered gene. By design, we are capable of creating transgenic mice in which their corneal cells synthesize too much growth factor(s) that disrupt the normal functions of corneas. For example, transgenic mice in which corneal epithelial cells synthesize too much FGF7 (a growth factor essential for epithelial cell growth) exhibit corneal dysplasia characterized by epithelium hypertrophy and thickening corneal stroma. We will further elucidate the role of FGF7 in modulating corneal homeostasis in normal and wound healing mice.

Taking advantages of the cutting edge technology, we have advanced to create mouse lines in which the ablations of genes only conditionally take place in corneal epithelium and stroma. We have successfully obtained such mouse lines d by the use of stroma-specific keratocan promoter and knock-in strategy of modifying keratin 12 gene for the expression of Cre in corneal stroma and epithelium, respectively. Cre is a bacterial virus enzyme that cuts any DNA fragments flanked by LoxP elements. Using gene targeting technique, a specific mouse gene is modified to containing two LoxP elements flanking an essential region of the gene. In the presence of Cre under the control of cornea-specific gene promoter, the gene of interest is ablated. Using the strategy, we have obtained mouse lines in which the type II receptor of TGF-� (TBRII) is ablated only in corneas. TGF-� is a growth factor that regulates wound healing. Our studies demonstrated that the ablation of TBRII in cornea keratocytes and epithelial cells impaired corneal wound healing. It is known that TGF-� signaling is essential to initiate wound healing, but excess TGF-� may be responsible for the formation of opaque scar tissues in corneas, which is one of the leading causes of blindness. We are studying whether the removal of TGF-� could be beneficial in preventing scar tissue formation. It is anticipated by understating the underlying mechanism(s) of TGF-� signaling during corneal wound healing, we will be able to develop strategies to modulate wound healing process and avoid the formation of opaque scar tissues.

The small leucine-rich proteoglycans, a family of proteins containing sulfated sugar chains, are known to play pivotal roles in maintaining corneal transparency. We have made interesting discovery about lumican, a key member of this family in that lumican not only is essential for maintenance of corneal transparency, it also involves in the control of keratocan expression, inflammatory response in wound healing and possibly metastasis of malignant tumor. We are carrying out experiments in attempts to determine domains of the lumican molecule, which are essential and sufficient for lumican biological functions. We have also recently found that administration of lumican promotes the healing of corneal epithelium debridement. This finding implicates that use of lumican may be beneficial in improving wound healing in diabetic patients. Further studies will be performed to determine the molecular and cellular mechanisms by which lumican facilitate wound healing. The identification of lumican function domain(s) will allow us to develop therapeutic agents that can be used in treating corneal disorders.

TGF-� signal transduction is mediated through a set of Smad molecules that consist of three different classes: regulatory Smad (r-Smad1, 2, 3, 5, 8), co-Smad (c-Smad4) and inhibitory Smad (i-Smad6, 7). Smad7 inhibits the nuclear translocation of r-Smad/c-Smad complex, thus it attenuates TGF-� signaling. We evaluated the therapeutic efficacy of adenoviral transient expression of Smad7 in treating corneal alkali burn. Adult male C57BL/6 mice were anesthetized and three �l of 1 N NaOH was applied to the right eye of the experimental mice. To express Smad7, both LNL-Smad7 and CAG-Cre vectors were co-infected into affected corneas at 3 hrs, and 1, 5, 10, 15 days after an alkali exposure. The eye was subjected to histological examination and assays for mRNAs of cytokines and MMPs/TIMPs at Day 3, 5, 10, or 20. In the Smad7-Adenovirus treated eyes, resurfacing of alkali burned cornea by conjunctival epithelium was accelerated, which was accompanied by the suppression of stroma opacification and ulceration, neovascularization. Marked Smad7 expression was detected in epithelium and keratocytes in a cornea of Smad7 group in association with a reduction of nuclear translocation of Smad3, phospho-Smad2 and phospho-RelA/p65 subunit of NF-?B. Smad7 gene transfer suppressed the invasion of monocytes/macrophages and expression of MCP-1, TGF-�1, TGF-�2, VEGF, MMP-9 and TIMPs and prevented myofibroblast generation in burned corneas at Day 20 post-burn. Adenoviral gene transfer of Smad7 prevented tissue destruction in a cornea following an alkali burn in mice more effectively as compared with lacking Smad3, suggesting that this strategy is effective in treating ocular burn.

In another series of experiments, we examine the efficacy of lumican on restoring corneal transparency in lumican knockout mice. Corneal transparency is dependent upon the regulation of collagen fibrillogenesis resulting in small, uniform fibril diameters and regular interfibrillar spacing. This is mediated in part by keratan-sulfate proteoglycans (KSPG) such as lumican and keratocan. Lumican-null mice exhibit corneal opacity and misarranged collagen matrices, whereas keratocan null mice have a mild phenotype of thin but transparent cornea. Previous reports also have indicated lumican not only serves as a regulator of extracellular matrix assembly, but involves in wound healing, cellular migration, epithelial-mesenchyme transition and tumorigenesis. Here, we report that lumican modulates keratocan expression in the adult mouse cornea. Transgenic mice that overexpress lumican in the cornea also have an increased expression of keratocan, whereas the lumican-null corneas show a decrease in keratocan expression at both the protein and mRNA level. The coupling of keratocan expression with lumican also was observed after intrastromal injection of lumican minigene into the corneal stroma of Lum^-/- mice. Our results reveal the ability of lumican to modulate gene expression of another KSPG family member in the adult cornea, indicating a novel regulatory interaction of these two closely related KSPGs. It also provides an explanation for the differences in severity of corneal manifestation found in Lum^-/- and Kera^-/- mice. The former exemplifies a dramatic reduction of corneal KSPGs that are necessary for the formation of an organized stroma collagen matrix found in transparent corneas.

Supported by National Eye Institute grant EY#11845 from 06/01/04 through 05/31/09

1. Zhang L, Hayashi Y, Liu C-Y, Jester JV, Birk DE, Gao M, Kao WW-Y, Karin M and Xia Y. A Role for MEK Kinase 1 in TGF beta/activin Induced Epithelial Movement and Embryonic Eyelid Closure. EMBO J. 22: 4443-4454, 2003.

2. Liu C-Y, Birk DE, Hassell JR, Kane B and Kao WW-Y. Keratocan-deficient Mice Display Alterations in Corneal Structure, J. Biol. Chem. 278: 21672-21677, 2003.

3. Meek KM, Quantack AJ, Bootes C, Liu C-Y and Kao WW-Y. An X-ray Investigation of Corneal Structure in Keratocan-deficient Mice. Matrix Biol. 22: 457-475, 2003.

4. Ikawa M, Tanaka N, Kao WW-Y and Verma IM. Generation of Transgenic Mice Using Lentivirus Vectors: A Novel Preclinical Assesment of lentiviral vectors for Gene Therapy. Mol. Therapy 8: 666-673, 2003.

5. Zhang L, Kao CW-C, Kao WW-Y and Xia Y. MEKK Kinase 1 Regulates c-Jun Phosphorylation in the Control of Cornea Morphogenesis. Mol. Vision 9: 584-593, 2003.

6. Saika S, Okada Y, Miyamoto T.Hashizume N, Ohnishi Y, Ooshima A, Liu C-Y and Kao WW-Y.

Role of p38 MAP Kinase in Regulating Cell Migration and Proliferation in Healing Corneal Epithelium. Invest. Ophthalmol. Vis. Sci. 45: 100-109, 2004.

7. Saika S, Kono-Saika S, Ohnishi Y, Sato M, Muragaki Y, Ooshima A, Flanders KC, Yoo J, Ghiassi M, Azano M, Liu C-Y, Kao WW-Y and Roberts AB. Samd3 Signaling Is Required for Epithelial-mesenchymal Transition of Lens Epithelium Post-injury. Am. J. Pathol. 164: 651-663, 2004.

1.“Ultrasound characterization of ocular biomechanical properties for glaucoma screening”. Amount: $83,750; Agency: Columbus Foundation.

2.“Automated 3D Reconstruction of the Optic Nerve Head Topography and Nerve Fiber Layer Thickness Measuremnt from Optical Coherence Topography for Improved Diagnossi and Monitoring of Glaucoma”. Amount: $45,000.

3.“Cellular Level response to Laser Trabeculoplasty on Perfused Monolayers of Non-Glaucomatous Human Trabecular Meshwork Cells”. Amount: $75,000; Agency: Columbus Foundation.

4.“Pressure Effect on Hydraulic Conductivity of TM Cells”. Amount: $70,000; Agency: American Health Assistance Foundation and Glaucoma Program.

Comparison of Perfused Glaucomatous and Non-glaucomatous Trabecular Meshwork Cell Monolayers to Low Fluence Diode Laser Irradiation

Objective: To investigate the differences in hydraulic conductivity (Lp) response of perfused glaucomatous to non-glaucomatous human trabecular meshwork (HTM) cell monolayers to low fluence diode laser irradiation. Current Status: Six glaucomatous confluent HTM cell monolayers and 6 non-glaucomatous confluent HTM cell monolayers were perfused at a starting pressure of 5 mm Hg. Experimental monolayers were irradiated with a diode laser (l=810 nm) at a power of 1.2 W over 1.5 sec duration for a fluence level of 0.4286 J/cm2. Each irradiated monolayer was run simultaneously with a non-irradiated control monolayer under the same environmental conditions. Irradiation took place following 15 minutes of steady state perfusion, after which perfusion and data collection continued for 45 minutes. Both monolayers were then tested to determine post-experimental viability. These results indicate that it is possible to promote an increase in hydraulic conductivity in a perfused HTM cell monolayer model using direct, non-destructive diode laser energy in a low fluence regime.

Ultrasound Characterization of Corneal Biomechanical Properties

Objective: To non-invasively obtain quantitative evaluation on biomechanical parameters (i.e., Young’s modulus) of corneal tissue. Current Status: An ultrasound system is currently being developed. This system includes an ultrasonic transducer, a pulser/receiver, a digitizer, a computer and a positioning system. Arch-shaped thin layer materials (e.g., contact lenses) were used as phantom samples to evaluate the feasibility of the system. A preliminary mathematical model was constructed to analyze the influence of each physical parameter on the ultrasound wave propagation in cornea.

Human Corneal Epithelial Cell Culture and Wound Healing

Objective: To characterize the surface roughness and micro/nanotopography encountered in vivo during corneal wound healing. Current Status: Methods were developed to quantify the micro/nanoscale roughness on human donor corneal tissue and material constructs using state-of-the-art imaging devices that characterize surface roughness. Measurements were performed using atomic force microscopy, scanning electron microscopy and optical profilometry.

Directions for Future Research

Biomechanical Models for Analyzing and Correcting Tonometry Measurement of Intraocular Pressure A mathematical model is being developed to analyze quantitatively how each corneal variable affects the accuracy of tonometry measurement of IOP. The model will be further incorporated with the measurement from an Ocular Response Analyzer (Reichert Ophthalmic Instruments) to understand the significance of each corneal variable.

To understand the effects on corneal epithelial wound healing in living human cornea systems, a system is being developed to support a human cornea culture system for a sustainable period.

Effects of Antimetabolites on Fibroblast Growth and Inhibition

As an adjunct to trabeculectomy, Mitomycin-C has been shown to have a cytotoxic effect on the cells of the cornea and conjunctiva, thereby complicating surgery. However, other antiproliferative agents may be as effective as Mitomycin-C in preventing post-operative fibrosis, as well as possess less toxicity. In this study, antiproliferative agents will be examined and their effect on post-operative fibrosis and overall cellular toxicity will be assessed.

Publications and Presentaitons

� D Koozekanani, KL Boyer, C Roberts: “Tracking the Optic Nervehead in OCT Video Using Dual Eigenspaces and an Adaptive Vascular Distribution Model.” IEEE Transactions on Medical Imaging. 22(12): 1519-36. 2003

� J Liu, CJ Roberts: “Quantitative Analysis of IOP Measurement Based on Biomechanical Model of Cornea.” Accepted by Journal of Cataract and Refractive Surgery, June, 2004

� J Lewis, G Agarwal, C Roberts, Atomic force microscopy investigations of corneal tissue after photoablative treatment, BIOPHYS J 86 (1): 477A-477A, JAN 2004

� DM Grzybowski, CJ Roberts, AM Mahmoud, JS Chang, Jr: “Model for Non-Ectatic Increase in the Posterior Corneal Elevation after an Ablative Procedure.” Submitted to Journal of Cataract and Refractive Surgery

� Grzybowski, D.M., Roberts, C.J., Mahmoud, A., “Proposed Mechanism for Non-Ectatic Change in Posterior Surface Shape After an Ablative Procedure,” Investigative Ophthalmology & Visual Science, 44(5) E-2556, 2003.

� Grzybowski, D.M., Rivera, B.K., Roberts, C.J., “Comparison of Perfused Glaucomatous and Non-Glaucomatous Trabecular Meshwork Cell Monolayers to Low Fluence Diode Laser Irradiation,” Investigative Ophthalmology & Visual Science, 45: E-5036, 2004.

� Holman, D., Grzybowski, D.M., Kapoor, K., Mehta, B., Lubow, M., Katz, S.E., “Development of an In vitro Model to Study Pseudotumor Cerebri.” Investigative Ophthalmology & Visual Science, 45: E-28, 2004

� J Liu, C Roberts: “Feasibility Study of Model and System for Ultrasonic Characterization of Corneal Biomechanics”, 45: E-3825, 2004

� D.Grzybowski, S.E. Katz, D. Holman, M.Lubow. “An In-Vitro Model to Study CSF Physiology and Pseudotumor Cerebri,” Invited Presentation to The Symposium of Neural Hydrodynamics, Menlo Park, CA, May 1, 2004.

Previous funding from OLERF allowed us to secure a grant from Fight for Sight, Inc for $12,000.

In the past year (2003 – 2004) our research efforts, funded by the Ohio LIONS Eye Research Foundation, have centered on functional imaging based on MRI (fMRI). FMRI is a method that identifies brain sites responsible for any type of visual or motor function. The fMRI method is basically a subtraction process – take an MRI picture of the brain when the subject is doing a certain task (e.g., eye movements) and subtract an MRI picture of the brain when the subject is not doing the task (e.g., no eye movements). Because more blood flows to those parts of the brain when they are active than when they are not active, the result of the subtraction process are those parts of the brain that are active when the subject made eye movements (i.e., the sites of the brain responsible for eye movements). Two major projects were undertaken.

In the first project, which is ongoing, we are attempting to develop equipment to measure eye movements within the MRI magnet. The ability to measure eye movements within the bore of the MRI magnet would opened-up a whole new area of research into the functional imaging of brain sites involved in the generation of eye movements. It would also allow us to monitor the exact types of eye movements that the subject makes during the experiment. However, there are numerous technical challenges in developing such equipment.

Since metallic (ferrous) objects cannot be placed near, let alone inside, the MRI magnet, the technical challenges are significant. We were able to overcome many of these challenges by the use of fiber optic cable, which is nonmetallic, as a means to deliver an infrared (IR) light source to illuminate the eye and, at the same time, to use this IR light as a means to record eye movements (known as the IR eye movement method).

Another technical challenge involved the ability of the IR light to travel through the fiber optic cable to reach the subject within the bore of the MRI magnet. Since the recording equipment has to be at least three feet from the opening of the MRI magnet, the fiber optic cable had to be at least 8 – 10 feet long in order to reach the subject within the MRI magnet. The impedance of the fiber optic cable was found to be extremely high; that is, the ability of the IR light to travel through the fiber optic cable was greatly limited – requiring an extremely strong IR light source to reach the subject AND allow the light reflected off the eye to return, via the optical cable, to recording equipment. One possibility we are now pursuing is the use of an IR laser light source.

2. FMRI of Eye Movements in Normal Subjects and in Subjects with Congenital Nystagmus

In the second project, which is ongoing, we are undertaking an fMRI experiment to try to pin-point the generator of abnormal eye movements know as congenital nystagmus. Periodic, alternating involuntary eye movements detected within the first six months of life characterize congenital nystagmus (CN). CN is one of the leading causes of significant vision loss in children and affects about 1 in 1000 to 6000 births. CN accounts for about 2 – 8% of children with visual impairment or legal blindness that utilize services for the visually impaired. Currently there are limited treatment options for patients with CN and they face a life of reduced vision as well as eyes that continuously move back-and-forth. The identification of the brain sites responsible for CN would be the first step in developing a treatment for this devastating condition.

The study involves two types of subjects, normal subjects and patients with CN. The normal subjects (N = 7 to date) are run in three conditions; fixation (i.e., no eye movements), a condition in which they follow with their eyes a dot that jumped back-and-forth (saccadic eye movements) and a condition in which they follow a dot smoothly moving horizontally back and forth (pursuit eye movements). The purpose of these conditions is to try to attempt to identify, via fMRI, the brain sites that are responsible for saccadic and pursuit eye movements.

The CN patients (N = 5 to date) participate in two main conditions; the CN patient looks at a fixation point positioned in the patient’s null zone – a zone in which the patient’s nystagmus is absent or minimal and when the fixation point is positioned in the patient’s left or right field of gaze where the nystagmus frequency and amplitude are great. Currently data collection is continuing on this project.

We plan to continue to pursue the development of an IR technique to record eye movements within the MRI magnet. We believe that the use of an IR laser light source may overcome the inherent high impedance of the fiber optic cable and allow a strong IR light signal to reach the subject within the MRI magnet as well as allow the IR light signal to return via the fiber optic cable to the recording equipment.

We also plan to continue to run more subjects in the attempt to identify the brain sites in normals responsible for saccadic and pursuit eye movements. Because the brain sites responsible for eye movements are very small in size, it may require a fairly large number of subjects to statistically locate the brain sites.

We also plan to pursue the fMRI study in CN patients to identify the brain sites responsible for CN. There are also a number of challenges to this research. CN patients are a heterogeneous group, comprising numerous different but similar pathologies. This fact alone may necessitate the running of a fairly large number of CN subjects to identify and possible classify the types of CN patients as well as the different brain sites responsible for CN.

Finally, we hope to start-up our research effort to assess the effects of L-dopa on cortical visual function in adults with amblyopia (lazy-eye).

1. Leguire, L. E., Algaze, A., Murakami, J., Rogers, G. L., Lewis, J., Roberts, C. Relation Among fMRI, Visual Acuity and CSF. Poster presented to the American Association of Pediatric Ophthalmology and Strabismus, Washington, DC, April, 2004

2. Lewis, J. R., Algaze, A. Leguire, L. E., Rogers, G. L., Murakami, J. and Roberts, C. Age Effect on fMRI Using Grating Stimuli. Paper presented to the American Association of Pediatric Ophthalmology and Strabismus, Washington, DC, April, 2004.

3. Leguire, L. E., Algaze, A., Murakami, J., Rogers, G. L., Roberts, C. FMRI More Closely Follows Contrast Sensitivity than Visual Acuity. Paper presented to the Association for Research in Vision and Ophthalmology, Ft Lauderdale Fl, May, 2004

1. Rogers, G. L. Functional Magnetic Imaging (FMRI) and Effects of L-dopa on Visual Function in Normal and Amblyopic Subjects. Trans Am Ophthalmol Soc. 2003, 101: 395-410.

Progress Report: The long term goal of our research continues to be directed toward understanding the pathological effects of intense visible light on the retina and prevention by antioxidant treatment. Our work suggests that the rat model of retinal light damage is useful in understanding retinal degenerations in humans arising from genetic abnormalities and/or environmental light insult e.g. retinitis pigmentosa (RP) and age related macular degeneration (ARMD). The aims of our current project were: 1.) To quantitate the expression of selected retinal genes in light exposed rats by real time PCR. 2.) To examine light exposure times of less than 8 hours to determine when gene expression changes first appear.

Aim 1. We chose to study retinal genes known to increase during oxidative stress (HO-1 and GFAP), others associated with cellular death (c-fos, c-jun, Fra-1), genes that may prevent cellular death ((-crystallins) and an inflammatory response associated gene (MCP-1). Gene expression was quantitated in rat retinas excised at 1am, 9am and 5pm, because light exposure at those times leads to photoreceptor cell damage (1am) or not (9am, 5pm). The levels of most genes increased at 9am, the approximate start of daylight for our rats, and remained relatively high at 5pm. At night (1am) these same genes were expressed at lower levels. Compared to 1am, HO-1 and GFAP were 2-3 fold higher at 9am and 5pm. c-fos, c-jun and Fra-1 were 2-4 fold higher at 9am, with similar levels at 5pm. Likewise two crystallin genes were 1-2 fold higher at 9am and 5pm, while the levels of 4 others declined at either 9am or 5pm. The inflammatory marker gene MCP-1 increased only 1 fold at both 9am and 5pm. This profile of retinal genes provides a “snapshot” of activity in the retina at various times. It also serves as a base line of information to better understand why the retina is severely damaged by light at night but not at other times during the day.

Aim 2. Using our base line knowledge of retinal gene expression we then exposed rats to intense light for 1, 2, 4 or 8 hours, with all exposures beginning at 1am. Previous work had established that 8 hours of light beginning at 1am results in the loss of 50% of the rats’ visual capacity. When the retinas were examined after 8 hours of light (9am) the expression increases for most genes were 4-14 fold, a considerable change from 1am. At the shorter time points the fold changes were less but a gradual increase occurred in most cases especially after 4 hours. Surprisingly, MCP-1 expression increased by almost 300 fold. This remarkable change indicates that light leads to an inflammatory response, also thought to occur in ARMD. Because antioxidants prevent the loss of vision in rats exposed to light, we also measured the changes in gene levels in animals given the synthetic antioxidant dimethylthiourea (DMTU). Antioxidant treatment greatly decreased gene expression at all times during light exposure. Many genes actually had a negative fold change of 2 for at least 4 hours of light and increases in most genes were only 2 fold after 8 hours of exposure. One cell death gene, c-jun was unaffected by antioxidant treatment, while MCP-1 increased only 8 fold with the antioxidant/light treatment compared to almost 300 fold with light alone. This data indicates that a variety of retinal genes are altered by light exposure. The balance of gene expression between those known to lead to cell death and others with a protective function may determine the outcome of lights’ effects on the retina. Our study also suggests that inflammation is a consequence of damage to photoreceptors in the retina and that this response may model some of the changes associated with ARMD.

Directions of Future Research: As the products of gene expression are proteins we propose to examine retinal proteins using an electrophoretic technique called 2-dimensional gel electrophoresis. Retinal protein extracts will be studied by this technique and by Western analysis, using antibodies, to determine changes at various times and after intense light exposure of rats. The identity of some proteins detected by specific antibodies will be confirmed by analytical analysis using mass spectroscopy. This will enable us to assign more specific functions to proteins that may be involved with cell death or with protecting the retina against the damaging effects of light.

Presentations: At the Association for Research in Vision and Ophthalmology Meeting:

I. Bicknell, R.M. Darrow, D.T. Organisciak. Evaluation of Hearing in the P23H Rat

D.T. Organisciak, L.S. Barsalou, K.M. Henkels, R.M. Darrow. Circadian Gene Expression Profiles in Rat Retina: What are the Crystallins Doing? #3522 (2003).

R. Grewal, D.T. Organisciak, R. Darrow, L. Barsalou, P. Wong. Micro - Array Analysis of Gene Expression in Light - Induced Retinal Degeneration Susceptibility. #4541 (2003).

R.M. Kelln, R. Darrow, L. Barsalou, D.T. Organisciak, P. Wong. Characterization of the Rat Rom1 Ortholog as Part of the Molecular Gene Profile of Light - Induced Retinal Degeneration. #4552 (2003).

B.W. Jones, C.B. Watt, D.K. Vaughan, D.T. Organisciak, R.E. Marc. Retinal Remodeling Triggered by Light Damage in the Albino Rat. #5124 (2003).

K. Renganathan, M. Sun, R. Darrow, L. Shan, X. Gu, R.G. Salomon, S. Hazen, D. Organisciak, J.W. Crabb. Light Induced Protein Modifications and Lipid Oxidation Products in Rat Retina. #5129 (2003).

C. Yu, Y. Guo, R.M. Mahdi, S. Ebong, R.M. Darrow, D.T. Organisciak, C.E. Egwuagu. SOCS Proteins have Neuroprotective Role in the Mammalian Retina. #5245 (2003).

K.M. Henkels, S. Siraj, R.M. Darrow, L.S. Barsalou, D.T. Organisciak. Genetic, Age and Light Mediated Effects on Retinal Crystallins. (2003).

K.A. West, X. Gu, K. Renganathan, M. Sun, R. Darrow, S. Hazen, R.G. Saloman, D. Organisciak, J. Crabb. Light Induced Protein Modifications and Lipid Oxidation Products in Rat Retina. (2003).

H. Sakaguchi, M. Miyagi, R.M. Darrow, J.S. Crabb, J.G. Hollyfield, D.T. Organisciak and J.W. Crabb. Intense Light Exposure Changes the Crystallin Content in Retina. Experimental Eye Research. 76:131-133 (2003).

D.T. Organisciak, R.M. Darrow, L. Barsalou, R.K. Kutty and B. Wiggert. Light Damage Susceptibility in Transgenic Rats Having Rhodopsin Mutations. Invest. Ophthalmol. Vis. Sci. 44:486-492 (2003).

D. Vaughan, S. Coulibaly, R.M. Darrow and D.T. Organisciak. A Morphometric Study of Light-Induced Damage in Transgenic Rat Models of Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 44:848-855 (2003).

D.T. Organisciak, R.M. Darrow and L.S. Barsalou. Light-Induced Retinal Degeneration. In Ocular Neuroprotection. Ed. L.A. Levin, A. DiPolo. Marcel Dekker, NY, p. 85-107 (2003).

Reading Glasses for Convergence Insufficiency Study OLERF Progress Report 2004

Convergence insufficiency (CI) is a very common and distinct binocular vision disorder. It affects approximately 5% of children and adults in the

United States

, and it may have a serious impact on an individual’s performance in school, choice of jobs, and quality of life. Clinical signs of CI include exophoria that is greater at near than at far, a receded near point of convergence, and reduced positive fusional vergence. Common symptoms include double vision, eyestrain, headaches, and blurred vision while reading.

There is no consensus regarding the most effective treatment for CI. Common treatments are passive (prism reading glasses) or active (therapy, i.e. “pencil push-ups” or vision therapy/Orthoptics). Significant differences exist between approaches in cost, number of visits required, degree of effort required by the patient, and complexity of the treatment. Compliance is also an issue with any recommended treatment plan. It is known that good compliance with active treatments can be challenging to achieve, however the compliance with passive treatment (prism reading glasses) for convergence insufficiency is not known.

In the Reading Glasses for Convergence Insufficiency study, 72 subjects between the ages of 9 and 18 years will be recruited across all sites (goal of 8 or more subjects per site). Subjects are randomly assigned to: 1) prism reading glasses (glasses that include correction for any refractive error and prism to correct for the convergence insufficiency), or 2) placebo reading glasses (glasses that include correction for any refractive error but no prism to correct for the convergence insufficiency). Measurements of the signs and symptoms of CI are made by masked examiners at baseline and after the glasses have been worn for 6 weeks. Patients who are successfully treated are followed at 3 months, 6 months, and 12 months to assess the long term effectiveness of treatment. The primary outcome variable is the score on the CI Symptom Survey. Secondary measures include near point of convergence and positive fusional vergence.

As of June 2004, a total of 55 patients were enrolled study-wide and the targeted recruitment goal of 8 patients had been exceeded at The Ohio State University College of Optometry clinical site. Enrollment is expected to be complete in the Fall of 2004.

Progress Report — Under-correction of infant hyperopia

Background

The primary feature of refractive development in infancy is emmetropization, or reduction in farsightedness. Up to 6% of infants fail to emmetropize, retaining high levels of farsightedness throughout infancy and childhood. High amounts of farsightedness in infancy are a risk factor for crossed eyes and lazy eye. In childhood, farsightedness may affect visual comfort as well as acuity, and therefore may impede learning. If glasses are needed, farsightedness has the same costs of correction as nearsightedness. Experimental results in animals suggest that moderate amounts of blur from uncorrected farsightedness should enhance emmetropization by stimulating ocular growth, but that excessive levels of farsightedness exceed the capacity of the eye’s emmetropization mechanism. An alternate model suggests that how much an infant focuses to clear his vision rather than how little he focuses to clear up blur is really the visual signal for emmetropization. Both models predict that moderate under-correction of infant farsightedness should help farsightedness to go away.

The overall purpose of this project is to determine if emmetropization can be enhanced in highly farsighted three-month-old infants. Eligible infants with farsightedness between +5.50 D and +9.00 D will be randomized to receive either moderate under-correction (3.50 D) of their farsightedness or to receive conventional treatment. Conventional treatment consists of either observation (if farsighted by +5.50 D to +7.00 D) or 1.00 D under-correction with spectacles (if farsighted by +7.00 D to +9.00 D). Measurement of how clearly babies focus throughout the treatment period will help to determine whether the visual signal for emmetropization is how much they focus, or the amount of blur they see because of how little they focus.

Compliance Monitoring: NEI grant proposals were submitted in March 2002 and 2003 for a larger scale clinical trial of moderate under-correction of infant hyperopia. The protocol for this trial was based on the same procedures and rationale as in previous OLERF applications. Neither NEI grant received funding despite a high degree of interest in the idea. A major criticism was concern over safety with infants wearing extended wear contact lenses. This has prompted a change in protocol away from contact lenses. Spectacles will now be used for both groups. Improved compliance was the main reason for proposing extended wear contact lenses. Infant compliance with spectacle wear is reported to be poor, but has never been rigorously monitored. We will use activity monitoring to determine the level of compliance with glasses. Spectacles will be fitted with a small (28 x 27 x 10mm), lightweight (16.5g), waterproof motion and time recorder (Actiwatch�, Mini Mitter Co., Bend, OR). Data are recorded at pre-set intervals and can be downloaded to a computer at progress visits. The Actiwatch monitors the number of waking hours that the glasses were worn (motion detected) or not worn (no motion detected). If compliance with glasses is acceptable, then contact lens wear and the small increased risks of inflammation or infection associated with them will not be needed. However, if compliance with glasses is documented to be poor, then we will have evidence to show that contact lenses are a necessary part of the protocol.

Recruitment: Because high hyperopia is rare, recruitment for a clinical trial will require screening infants in large numbers. Infants are not typically seen in large numbers in eyecare departments. Infants are seen regularly in pediatricians’ offices. We have recently received the cooperation of an OSU-affiliated pediatrician in the Columbus area. She has given permission to conduct vision screenings in her office in order to identify potential patients for this clinical trial.

Evaluation of the Schaeffel PowerRefractor: A major goal of the previous period was to validate the Schaeffel PowerRefractor. The PowerRefractor is a binocular photorefractor with the potential to provide rapid assessment of the prescription for the two eyes at once. It could be a very useful tool in infant screening. We calibrated 25 adult subjects on the PowerRefractor using trial lenses from –8.00 D to +8.00 D. Subjective refraction was also compared to readings with no trial lens. There were three key findings. The average PowerRefractor reading was not different from subjective refraction (–1.47 � 2.36 D vs. –1.54 � 2.19 D; P = 0.64). The calibration of the PowerRefractor was also linear in a 1:1 manner as a function of trial lens power. The third finding was that the operating range of the instrument was far more limited than previously reported. Readings could only be obtained between +2.00 D and –4.00 D. We conclude that while the PowerRefractor is valid, its results must be interpreted with caution. A finding of +2.00 D of farsightedness could mean the actual refraction is anywhere from +2.00 D to +8.00 D. This limitation explains the poor performance of the PowerRefractor in the recent VIP study.

Future Direction

We will use the PowerRefractor to screen for infant farsightedness in the office of OSU Associate Clinical Professor Dr. JoAnn C. Rohyans, MD. Infants will wear +4.00 D goggles during screening. A finding of any residual farsightedness will indicate a “fail” and the need for further evaluation. This critical step is needed because of the limited range of the instrument. Results will be confirmed by retinoscopy. We will continue to use the PowerRefractor because we anticipate that pediatrician office staff who cannot do retinoscopy will at some point be doing the screening for farsightedness. Eligible farsighted infants will be offered enrollment in the clinical trial. Randomization will be to moderate under-correction in glasses or to either observation or slight under-correction depending on initial level of farsightedness. Accommodative response will be monitored during progress visits to determine whether any enhancement in emmetropization is related more to residual defocus or to the effort to accommodate to clear defocus. Compliance with spectacle wear will be monitored at progress visits by downloading time and motion-sensing information stored on the activity monitors.

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