6^th Annual

2013 Midwest Eye Research Symposium

Sponsored by the Iowa City VA Center for the Prevention and Treatment of Visual Loss

MERS 2013 took place on August 2^nd at
The University of Iowa Medical Education and Research Facility

Keynote Speaker/Session Chairs

David C. Beebe, Ph.D. - Keynote Speaker
Dr. Beebe is the Janet and Bernard Becker Professor of Ophthalmology and Visual Sciences at Washington University in St. Louis, MO. His research interests focus on regulation and biological effects of oxygen in the eye and transcription and growth factor signaling in the developing eye. He is a past president of ARVO and the current editor-in-chief of IOVS.

Wallace B. Thoreson, Ph.D. - Chair, Session I
Dr. Thoreson is the Gilmore Professor and Vice Chair of the department of Ophthalmology and Visual Science at the University of Nebraska Medical Center where he is also a Professor of Pharmacology and Experimental Neuroscience. Work in his laboratory focuses on the role of calcium channels and glutamate release in transmitting visual messages from rod and cone photoreceptor cells.

Dale S. Gregerson, Ph.D. - Chair, Session II
Dr. Gregerson is a Professor in the Department of Ophthalmology and Visual Neurosciences at the University of Minnesota. He has published over 100 manuscripts on the immunology of the eye and is particularly interested in the role of immunity in neurodegeneration and neuroprotection as well as retinal antigens.

Budd A. Tucker, Ph.D. - Chair, Session III
Dr. Tucker is an Assistant Professor in the Department of Ophthalmology and Visual Sciences at the University of Iowa. His lab is using induced pluripotent stem cells for disease modeling and tissue engineering to induce retinal regeneration following transplantation.

Investigation of Genetic and Molecular Mechanisms of Familial Angle-Closure Glaucoma in the Basset Hound

Ahram, Dina¹; Cook, Amy²; Sander, Edward³; Kecova, Helga⁴; De Jesus, Aribet³; Kuehn, Markus^1,2
1Interdisciplinary Graduate Program in Genetics, The University of Iowa; Departments of ²Ophthalmology and Visual Sciences and ³Biomedical Engineering, The University of Iowa. ⁴The Veterans Administration Center of Excellence for Prevention and Treatment of Vision Loss, IA

Purpose: Primary angle closure glaucoma (PACG) is a condition that results from the collapse of the irido-corneal angle due to an anterior movement of the iris and is associated with a gradual increase in intraocular pressure (IOP). We have identified several Basset Hound pedigrees with characteristic autosomal recessive PACG that closely recapitulates PACG in human patients. A number of genetic and molecular experiments were conducted to identify the underlying genetic and functional mechanisms that contribute to PACG using the Besset Hound as a genetic model.

Methods: Unrelated and related Bassets from the five identified PACG pedigrees were genotyped using the Illumina CanineHD BeadChip. Parametric and non-parametric linkage analyses were performed using Merlin. A case-control association study was performed using 37 clinically confirmed PACG and 41 unaffected Bassets. A logistic regression test for variant association was conducted using Plink. Three-dimensional collagen gel matrices seeded with primary fibroblasts collected from the sclera and dermis of PACG and wild type Bassets were constructed. In-vitro cell contractility assays were performed on 3D matrices to characterize the inherent properties of affected and wild type derived cell populations.

Results: Using parametric linkage analysis of all PACG pedigrees combined, an 8.1Mb region on chromosome 27 was identified with a log of odds score (LOD) of 3.4 (θ=0). The highest P-value observed (3.6 x 10-4) following GWA analysis is for a SNP, BICF2P31912, sharing a 4 SNP LD block (r2=1) that spans 0.25 Mb on chromosome 14. The SNP is located within the type-1 collagen gene COL1A2. PACG-derived fibroblast cultures were observed to display slow growth and dysmorphic appearance in comparison to wild-type cells. Cell contractility assays revealed increased reduction in gel surface area and rate of compaction for cells derived from unaffected versus affected Bassets.

Conclusions: Our findings of genetically linked and associated loci support the segregation of an underlying genetic defect with PACG in affected Basset Hounds. Whether a single or multiple regions among those identified here is directly linked to the disease phenotype remains under investigation. These studies also suggest that cellular dysfunction is an important aspect in the pathophysiology of PACG in the dog. Ultimately, we anticipate for these studies to provide valuable insight into the pathophysiology and genetics of human PACG.

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Effect of proteasome inhibition on lentiviral vector transduction in human trabecular meshwork cells and monkey organ-cultured anterior segments

Zeynep Aktas, Sarah Slauson, B'ann Gabelt True, Paul Kaufman, Curtis Brandt
Dept of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI¹

Purpose: To investigate if proteosome inhibition using MG132 and actin cytoskeleton disruption with latrunculin-B increased the transduction efficiency of FIV vector co-expressing green flourescent protein (GFP) in human TM-1 cells and monkey organ-cultured anterior segments (MOCAS).

Methods: TM-1 cells were plated in a 24-well plate at 1.5x104 cells/well. Cells were pretreated for 1 hr with DMSO or 5µM to 50µM MG132 or Latrunculin B (0.05µM, 0.1µM, 0.2µM), and transduced with FIV.GFP at a multiplicity of infection of 20. At 24 hours cells were fixed, stained with antibodies for GFP, and positive cells were counted. Cells transduced with FIV.GFP alone were used as controls. The effect of 20µm MG 132 treatment on FIV.GFP transduction was also tested in MOCAS and the GFP expression in the TM was evaluated using fluorescence microscopy.

Results: The percentage of GFP expression in FIV.GFP and DMSO+FIV.GFP groups were 24.2±4.6 and 25.2±4.1 (p=0.401). In the MG132 treatment groups the transduction was significantly increased at all concentrations. With latrunculin-B transduction efficiency was increased at all concentrations except in the group that received 0.2µM latrunculin-B. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20µM MG132.

Conclusions: MG132 was found to increase the transduction efficiency of FIV.GFP in TM-1 cells and MOCAS.

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Genetic Analysis of a Patient with Lacrimo-Auriculo-Dento-Digital (LADD) Syndrome that has Glaucoma

Andrew J. Baldwin,¹ Anamika Tandon,¹ Wallace L.M. Alward,^1,2 John H. Fingert^1,2
1Carver College of Medicine, University of Iowa, ²Institute for Vision Research, University of Iowa

Purpose: Lacrimo-auriculo-dento-digital (LADD) syndrome is an autosomal dominant hereditary disease characterized by abnormalities in the lacrimal and salivary glands, cup-shaped and low set ears, peg-shaped teeth, and clinodactyly. We recently examined a patient with typical LADD syndrome features that also had normal tension glaucoma (NTG). The aim of this study is to identify the genetic defect that is responsible for LADD syndrome and NTG in this patient.

Methods: We are enrolling the patient as well as his unaffected parents and other family members for a genetic study of LADD and glaucoma. In additional to collecting clinical data from our examinations and medical records, we will also collect DNA samples for genetic analysis. We will first test each family member's DNA for mutations in the three genes that have been previously associated with LADD syndrome (FGR2, FGR3, FGR10) using standard Sanger DNA sequencing techniques. If no mutations are found in known LADD genes, we will next search for possible disease-causing mutations using a whole-exome sequencing approach using Illumina exome capture and next-generation sequencing technology.

Results: The study is currently ongoing.

Conclusions: Identifying the gene and mutation responsible for causing glaucoma in this patient will provide new insights into the genetic and clinical range of LADD syndrome and may also provide new insights into the molecular genetic causes of glaucoma.

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Optical Coherence Tomography (OCT) and histologic examination of a mouse model of chorioretinal coloboma

Bhattarai, Sajag; Gratie, Daniel; Stunkel, Maria; Anderson, Michael G.; Mullins, Robert F.; Drack, Arlene V.
University of Iowa, Iowa City, IA¹

Purpose: To characterize a chorioretinal coloboma occurring in the JF1 mouse line.

Methods: Indirect ophthalmoscopy with a 90D lens was performed on JF1 mice. Mice with chorioretinal colobomas had either optical coherence tomography (OCT) performed, followed by sacrifice and histologic preparation of the eyes, or were bred. Select mice fundi were photographed. Offspring of affected mice were examined for presence or absence of coloboma by indirect ophthalmoscopy.

Results: 60 eyes of 30 JF1 mice were examined. 19 were female, 11 were male. 11 of 30 mice demonstrated chorioretinal (CR) coloboma. 10/11 were unilateral. 6 were right eye, while 4 were left eye. 6 of 11 affected mice were female. All colobomas were adjacent to the optic nerve but none involved the nerve itself. On both OCT and histologic examination, the retinal layers adjacent to the colobomatous defect appeared to deviate course toward the sclera. The retinal pigment epithelium (RPE) ended abruptly on either side of the retinal defect. Rather than bare sclera, the base of the coloboma has a layer of collagen above sclera. Trichrome stain revealed collagen infiltrate into the retina. Based on pedigrees, this CR coloboma may be inherited in an autosomal dominant or autosomal recessive manner with variable expressivity.

Conclusions: We describe a mouse model of chorioretinal coloboma. The retinal defect appears to be complex, with disruption of normal retinal layers, and replacement of some tissue rather than simply a localized deficiency of retinal tissue. Variable expressivity is the norm, similar to human CR colobomas.

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Optic nerve axon loss in a cat model of glaucoma

Owen Bowie³, Leandro B. Teixeira^1,2, Richard R. Dubielzig^1,4, Gillian J. McLellan^3,4
1Pathobiological Science, UW-Madison School of Veterinary Medicine, Madison, WI; ²Comparative Ophthalmic Research Laboratories, Madison, WI; ³Ophthalmology and Visual Sciences, UW-Madison, Madison, WI; ⁴University of Wisconsin Eye Research Institute, Madison, WI

Purpose: To quantify axons loss in the optic nerves of glaucomatous and normal cats throughout different life stages.

Methods: Axons were counted in the optic nerves of cats with mild to severe glaucoma (n= 34) and normal cats (n=30). Optic nerves were selected from both a short-term cohort of young glaucomatous (n=10) and normal (n=8) cats euthanized at 10-12 weeks of age; and from a long-term cohort of cats euthanized either at 6 months, 12 months, 18months or 24 months which consisted of (n=23) glaucomatous and (n=23) normal cats. Following perfusion fixation with 4% paraformaldehyde and enucleation, 2mm long samples of optic nerve were dissected 2mm posterior to the globe fixed in glutaraldehyde, osmicated and resin embedded. Semi-thin sections stained with 1% p-phenylenediamine were evaluated by light microscopy. Axons were quantified by a previously validated, semi-automated targeted sampling method using commercially available image analysis software (cellSens Dimension®, Olympus) and optic nerve cross-sectional area measured using the same image analysis software. Values for normal and PCG cats were compared by student t-test with p=0.05 considered significant.

Results: The mean number of axons in the optic nerves of glaucomatous adult cats (95% CI=38,943-57,641) was significantly lower than in normal cats (65,150-78,873; p=0.0006) . A significant reduction in optic nerve cross-sectional area was also evident in affected cats compared to normal cats (p=0.0002).

Conclusions: Moderate to severe axonal loss consistent with glaucomatous optic neuropathy was clearly demonstrable in cats with PCG. Our findings support the use of this novel feline model in glaucoma research.

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Uniquely subdued reactive gliosis in injured ground squirrel visual system

Ryan Bures¹, Ben Sajdak², Cassie Piper¹, Dana Merriman^1
1University of Wisconsin, Oshkosh; ²Medical College of Wisconsin

Purpose: To examine reactive gliosis in ground squirrel visual system as a counterpoint to what is known from typical rodent models of injury. Unlike rats and mice (but in common with human), ground squirrels are diurnal and depend upon cone visual pathways for survival. They are also obligate hibernators.

Methods: Ground squirrels and Long Evans rats were administered either experimental retinal detachment or optic nerve crush, using aseptic methods. Tissues were then prepared for histology and immunocytochemistry. Gliosis markers included glial fibrillary acidic protein (GFAP), vimentin, and chondroitin sulfate proteoglycans (CSPG). Histology was performed to assess cell/axon loss.

Results: As expected, injury in rat resulted in upregulated marker expression as well as visible evidence of axon loss, (presumed) macrophage infiltration, and astrocyte hypertrophy. In contrast, marker upregulation and astrocyte hypertrophy were consistently muted in ground squirrel retina and optic nerve.

Conclusions: In previously studied mammals, the inflammatory response to visual system injury includes microglial activation, astrocytic upregulation of vimentin and GFAP, glial scar formation, and deposition of CSPG. These canonical responses serve a purpose but interfere with some reparative therapies. Therefore, the molecular mechanisms that result in the subdued response of ground squirrel may merit further investigation.

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Gene Therapy for CEP290-associated LCA in Patient-Derived Induced Pluripotent Stem Cells

Burnight, Erin R., Kaalberg, Emily E., Eyestone, Mari E., Hoffman, Jeremy M., Haas, Christine M., Mullins, Robert F., Stone, Edwin F., Tucker, Budd A.
Institute for Vision Research, University of Iowa

Purpose: The purpose of this study was to develop lentiviral vectors containing human CEP290 and to use these vectors to deliver wild type CEP290 to induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells (PRPCs) from mice and humans with CEP290-associated LCA.

Methods: Fibroblast-derived iPSCs from the retinal degenerative mouse model CEP290rd16 and patients with molecularly confirmed CEP290-associated LCA were generated and differentiated into PRPCs using our previously developed step-wise differentiation protocol. A lentiviral vector containing human CEP290 under the control of the CMV promoter was packaged and used to transduce PRPCs from rd16 mice and LCA patients. Vector-derived mRNA and protein were detected via RT-PCR and western blot, respectively.

Results: To evaluate CEP290 gene transfer we initially transduced murine JK1 cells that do not express the human CEP290 transcript. RT-PCR analysis using human-specific primers demonstrated a dose-dependent increase in CEP290 expression post-transduction. However, cell viability was significantly reduced in cultures transduced with the highest dose. Using results from these pilot experiments, we delivered a lower dose of CEP290 vector to iPSC-derived murine and human PRPCs. RT-PCR analysis and western blotting indicated vector-derived expression in these cells.

Conclusions: We show successful gene transfer of human CEP290 to iPSC-derived photoreceptor precursor cells. Cell viability assays suggest that overexpression of CEP290 is cytotoxic. Thus, it will be important to carefully titrate vector dosage when developing gene replacement strategies for this disease. This work will contribute to our overall goal of vision restoration in patients with LCA through gene and patient-specific photoreceptor cell replacement.

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Functional Degeneration of Retinal Ganglion Cells in a Mouse Model of Chronic Ocular Hypertension

Chen, Hui¹; Zhao, Yan²; Feng, Liang¹; Cang, Jianhua³; Troy, John B.²; Liu, Xiaorong^1,3
1Ophthalmology, Northwestern University, Evanston, IL, ²Biomedical Engineering, Northwestern University, Evanston, IL, ³Neurobiology, Northwestern University, Evanston, IL

Purpose: Glaucoma is characterized by dendritic and axonal degeneration of retinal ganglion cells (RGCs), visual field deficits, and ultimately RGC death. Although diversity in RGC damage has been reported in glaucomatous retinas, studies in human patients and animal models have so far failed to provide a clear picture of how RGCs degenerate and whether the surviving RGCs are still functional. In this study, we examined the functional degeneration of RGCs in a mouse model of experimental glaucoma.

Methods: We have adopted a laser-induced mouse model of ocular hypertension to mimic human high-tension glaucoma and demonstrated a sustained increase of intraocular pressure (IOP), and a progressive RGC loss. We applied two cutting-edge techniques, the laser-guided focal electro-retinogram (ERG) to measures visual responses from different cell types of retina at various locations, and a large-scale multi-electrode array (MEA) to examine response properties of different subtype RGCs.

Results: Our preliminary data suggest that at 6 weeks after laser treatment, the amplitudes of Oscillatory Potentials (OPs) measured by ERG decreased. And the receptive field (RF) sizes of mono-laminated ON and OFF RGCs decreased while the bi-laminated ON-OFF RGCs did not change in the glaucomatous eyes.

Conclusions: Taken together, our data demonstrate that the RGC degeneration is subtype-dependent in ocular hypertensive mice. This study establishes a valuable model system for better understanding how RGC degeneration leads to vision loss and a potential time window for early detection of glaucoma.

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Computational discovery of optic nerve head phenotypes

Christopher, Mark; Tang Li; Fingert, John H.; Scheetz, Todd E.; Abramoff, Michael D.
The University of Iowa; Department of Veterans Affairs

Purpose: To apply computational methods in the discovery of 3D optic nerve head (ONH) structural features for detecting and monitoring glaucoma.

Methods: A subset of participants from the Ocular Hypertension Treatment Study was selected on the basis of availability of simultaneous stereo fundus images. A stereo correspondence algorithm was applied to the set stereo fundus pairs to produce a disparity map that quantitatively measured the ONH structure for each participant. Principal component analysis (PCA) was applied to the disparity maps to extract computational 3D ONH structural features. The first 25 principal components, or features, were retained and examined individually in building a predictive regression models for cup-to-disc ratio. The relationship between the ONH features and the demographic variables of gender, age, and ethnicity were also examined. Bonferroni correction was used to adjust for multiple hypothesis testing.

Results: Five of the 25 computational 3D ONH features were significantly associated (Bonferroni p<0.05) with cup-to-disc ratio. Significant associations were also found between the ONH features and the demographic variables of age and ethnicity.

Conclusions: Using computational methods, we generated a set of structural features for quantifying the 3D shape of the ONH. These features had significant associations with and predictive power for cup-to-disc ratio, a clinically important measurement used to diagnose and monitor glaucoma. Associations of ONH structural features were also found with age and ethnicity. Future work will explore the power of applying these features to detect and track glaucoma as well as exploring the genetic basis of these computational features.

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C1qa deficiency exacerbates RGC loss in a mouse glaucoma model

Cook, Amy C.¹xDing, Qiong¹, Anderson, Michael G.², Kuehn, Markus H.^1
1 Department of Ophthalmology and Visual Sciences, ² Department of Physiology and Biophysics, The University of Iowa

Purpose: The development of glaucoma is associated with activation of the complement cascade and the marked accumulation complement components 1q (C1q) and 3 (C3) in association with retinal ganglion cells (RGC). In order to determine the functional significance of C1q and C3 in the pathophysiology of glaucoma, we evaluated retinal ganglion cell (RGC) and axonal loss in B6-Sh3pxd2 ^nee (nee) mice with and without targeted deletions in either the C1qa or C3 genes.

Methods: Nee mice were bred either with those containing targeted deletions in C1q or C3 to create double knockouts. At 8 weeks of age the intraocular pressure (IOP) was determined using a rebound tonometer. The animals were then euthanized and the eyes and optic nerves were harvested. The number of surviving gamma synuclein immunoreactive RGC was determined in flat mounted preparations of retina and axonal damage in the optic nerve was evaluated using a 5-point grading scheme. Differences between strains were evaluated using either one way ANOVA or Kruskal-Wallis test.

Results: The presence of the nee allele caused a marked elevation of IOP above that observed in control mice (+6.0 mmHg, p=0.0002), but no difference was observed with respect to the absence or presence of functional complement genes. The elevation in IOP caused a decline in RGC numbers and an increase in optic nerve damage scores. Among mice homozygous for the nee mutation, the observed damage was particularly marked in C1q-/- (-74.3%) mice and significantly higher than that observed in C3-/- mice or littermate controls (-33.6% and -32.3%, respectively, p<0.0001). Similarly, elevated IOP did increase optic nerve damage scores from an average of 1.2 in control mice to 1.9 in nee, 2.8 in C3-/- and 4.7 in C1q-/- mice.

Conclusions: Our data demonstrate that absence of functional C1q, but not C3, worsens RGC and axonal damage in this glaucoma model. These results suggest that C1q activity is crucial in limiting glaucomatous damage. Furthermore, our findings indicate that absence of C3 has little effect on disease progression in this model.

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Loss of Lztfl1, a negative regulator of the BBSome ciliary trafficking, causes BBS-like phenotypes in mice

Datta, Poppy¹, Humbert, Melissa¹, Hudson, Joseph¹, Bhattarai, Sajag¹, Bugge, Kevin^2,5, Thedens, Daniel³, Shao, Jianng⁴, Drack, Arlene¹, Sheffield, Val^2,5, Seo, Seongjin¹
Departments of ¹Ophthalmology and Visual Sciences, ²Pediatrics, and ³Radiology, ⁴Central Microscopy Research Facility, The University of Iowa, ⁵Howard Hughes Medical Institute, Iowa City, IA

Purpose: Bardet-Biedl Syndrome (BBS) is a genetically heterogeneous ciliopathy characterized mainly by retinitis pigmentosa, polydactyly, and obesity. Recently, Leucine-zipper transcription factor-like 1 (LZTFL1) was identified as a negative regulator of the BBSome, a complex composed of 7 BBS proteins, and mutations in LZTFL1 have been found in several human BBS patients. This study is aimed to determine the roles of LZTFL1 in vivo and verify that loss of LZTFL1 function leads to BBS.

Methods: Lztfl1 mutant mouse line was generated using ES cells from Knockout Mouse Project (KOMP) repository. Retinal degeneration was studied by H&E staining of retinal sections and electroretinogram (ERG). Magnetic resonance imaging (MRI) was used to study obesity and hydrocephalus. Scanning and transmission electron microscopy (SEM and TEM) were used for ultrastructural analyses of cilia. H&E staining was used to examine testis and kidney abnormalities.

Results: Lztfl1 mutant mice displayed severely reduced ERG and progressive loss of photoreceptor cells. These animals also displayed a significant increase in body weight and body fat mass. MRI and SEM studies revealed that Lztfl1 mutant mice have hydrocephalus and a reduced number of ependymal cilia in the brain ventricle. Finally, severe reduction in the number and motility of sperm flagella was observed in Lztfl1 mutants despite normal BBSome assembly.

Conclusions: Our findings indicate that LZTFL1 is essential for maintaining photoreceptor cells, energy homeostasis, and normal cilia/flagella functions and verify that loss of LZTFL1 causes BBS. Further biochemical and proteomics studies are aimed to identify interacting partners and related cellular pathways.

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Genome Wide Association Study to Identify Novel Loci Associated with Idiopathic Intracranial Hypertension

Deonovic, Benjamin¹; Hashmi, Asad⁴; Wall, Michael^2,3; Kuehn, Markus³
Departments of ¹Biostatistics, ²Neurology, ³Ophthalmology and Visual Sciences, and ⁴The Carver College of Medicine; The University of Iowa, Iowa City, IA

Purpose: Idiopathic intracranial hypertension (IIH) is a condition characterized by chronically elevated intracranial pressure without clinical evidence for space occupying lesions or hydrocephalus. Increased pressure leads to papilledema, which may lead to vision loss. Although there appear to be a variety of associated risk factors for the disease, further research is needed to identify genetic loci associated with the condition. A genome-wide association study (GWAS) will be conducted to further understand this disease.

Methods: We will analyze 95 case subjects with IIH and 95 controls matched by sex, BMI, self-reported ethnicity, and distance to procurement site. The samples were genotyped using Illumina’s Infinium HumanCoreExome v1-0 array which examined 538,448 SNPs. Data will be analyzed using a generalized linear mixed effects model (assuming a logit link and Bernoulli distribution) that controls for self-reported ethnicity and matching.

Results: We expect to identify SNPs associated with IIH after adjusting for muliple testing using the Benjamini-Hochberg Procedure to control for false discovery rate at 5%.

Conclusion: Results will show that IIH has a genetic component and that susceptibility to IIH is mediated by several loci. Our findings will offer the first insight into the etiology of this disease and provide a basis for future investigations. The consistency of the association of significantly associated SNPs with IIH will be assessed in a future validation study using a new set of cases and controls with the same matching criteria.

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Induction of trabecular meshwork cells from induced pluripotent stem cells

Ding, Q.; Zhu, W.; Anfinson, K.R.; Kaalberg, E.E.; Tucker, B.A.; Kuehn, M.H.
Department of Ophthalmology and Visual Sciences, The University of Iowa

Purpose: Loss or dysfunction of trabecular meshwork (TM) cells has been associated with the development of pathologically elevated IOP and it is conceivable that replacement of damaged TM cells could restore function to the TM. Autologous transfer avoids immune responses and the ethical concerns associated with the use of embryonic stem cells. To be clinically viable harvest of the cells must be reliable, minimally invasive, and yield a significant number of cells. We propose that the use of TM-like cells derived from induced pluripotent stem cells (iPSC) created from a patient's own dermal fibroblasts offers the best solution to this challenge. Here we demonstrate that mouse iPSCs can be induced to differentiate into TM like cells suitable for autologous transplantation.

Methods: Directed induction of stem cell differentiation was achieved through co-culture of mouse iPSCs with human fetal TM cells for up to 21 days. The resultant TM-like cells (iPSC-TM) were characterized morphologically, immunohistochemically, and functionally.

Results: iPSC-TM closely resemble cultured human TM cells morphologically and begin to express many markers of TM cells while ceasing to express pluripotency markers such as SSEA1. Functionally, these cells develop the ability to phagocytose particles. Finally, exposure to dexamethasone (100 nmol) or Phorbol 12-myristate acetate (TPA, 25 ng/ml) causes a distinct increase in the production and secretion of myocilin or MMP3, respectively.

Conclusions: Our data demonstrate that iPSCs can be induced to assume a phenotype that resembles native TM cells in many important aspects. These cells not only represent a valuable research tool, but transplantation into glaucomatous eyes with elevated IOP may also restore function to the TM resulting in permanently lowered IOP.

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Increasing body weight correlates with loss of ERG in mice with Bardet Biedl Syndrome

Drack, Arlene V.¹; Bhattarai, Sajag ¹; Stone, Edwin M.¹; Wang, Kai²; Gratie, Daniel¹; Heon, Elise⁴; Sheffield, Val ³; Mullins, Robert F.¹
Departments of ¹Ophthalmology and Visual Sciences, ²Biostatistics, ³Pediatrics, The University of Iowa, Iowa City, and ⁴ Department of Ophthalmology, Sick kids Toronto, Toronto, ON

Purpose: Systemic TUDCA slows development of both retinal degeneration and obesity in a mouse model of Bardet Biedl Syndrome (BBS).We analyzed the relationship between body weight and dark-adapted bright flash b-wave (ERG b-wave) in TUDCA and control BBS mice to determine whether body weight might be used as a surrogate endpoint in treatment trials.

Methods: TUDCA was administered subcutaneously twice weekly to Bbs1M390R/M390R mice at 500mg/kg/dose at 6-8 weeks of age (n=5) or 11 weeks of age (n=5).NaHCO3 was administered to littermate controls at the same ages (n=4, n=3).Body weight and ERG b-wave data were collected at P120 and P180.ERG b-wave amplitudes and body weight were compared between groups using a paired linear regression analysis.The correlation coefficient of change in body weight vs. change in ERG b-wave amplitude between 2 time points was calculated for natural history controls (n=12).

Results: Natural history data for Bbs1M390R/M390R mice demonstrated a very significant negative correlation between body weight and ERG b-wave over time (p=0.0004).Animals treated at 6-8 weeks old with TUDCA had a statistically significantly higher b-wave compared to sham treated (p=0.0022).Body weight was significantly lower in TUDCA treated animals than sham (p=0.0004).The correlation coefficient for difference in weight and b-wave between 2 time points in the TUDCA treated group was -0.7, while the correlation coefficient for these parameters in control mice was 0.46.Bbs1M390R/M390R mice treated at the later age of 11 weeks showed higher ERG b-waves and lower body weight than sham treated, but this was not statistically significant.

Conclusions: This study replicates our previously reported findings of less obesity and better ERG b-wave in Bbs1M390R/M390R mice treated with systemic TUDCA.In addition,it demonstrates an inverse relationship between obesity and ERG b-wave amplitude in untreated mice, and a correlation between changes in body weight and b-wave amplitude in treated mice. As human clinical trials using TUDCA to treat BBS in humans are contemplated, body weight may be considered as a study endpoint since it may be easier to measure and may change more quickly than ERG b-wave.

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Immune and Visual Dysfunction in a Mouse Model of Blast Induced Mild Traumatic Brain Injury

Dutca, Laura^1,2; Rasmussen, Wendy³; Shey, Michael³; Alber, Carol³; Kecova, Helga^1,2; Harper, Matthew^1,2; Hernandez-Merino, Elena^1,2; Gibson-Corley, Katherine^1,4; Kardon, Randy^1,2; Ballas, Zuhair^1,3
1Center of Excellence for Prevention and Treatment of Visual Loss, Iowa City Veterans Administration Medical Center, Iowa City, Iowa and Departments of ²Ophthalmology and Visual Sciences, ³Internal Medicine, and ⁴Pathology, University of Iowa, Iowa City, Iowa

Purpose: To characterize immune dysfunction induced by exposure to mild blast in a mouse model so as to improve diagnostic and treatment modalities for soldiers and veterans.

Methods: Healthy 12 week old male C57/BL6 mice were exposed to an overpressure wave (15.5±3PSI) directed to left side of head using a custom-built blast chamber. Retinal ganglion cell and optic nerve function were tested before euthanasia by pattern electroretinography (pERG). At 9 and 35 days after blast exposure, mice were sacrificed and the number and function of T cells, B cells and NK cells in spleen, liver and lymph nodes were analyzed. Cytokine concentration in serum, spleen supernatant and cerebellum were also determined by a multiplex assay. Morphological changes in the brain were evaluated histologically, while astrocyte and microglia activation were analyzed by immunohistochemistry.

Results: pERG recordings showed significantly decreased function 9 days after injury for the eye facing the blast, but not in the eye away from the blast. Spleen-derived NK cell activity was significantly increased at 35 days in blast-exposed mice compared to controls. Small changes in serum cytokines and in cytokine secretion by activated lymp hocytes were detected, but with considerable variability. The brain had no significant histopathologic abnormalities and no significant increases in astrocyte or microglial activity were detected.

Conclusions: Our results indicate that even after exposure to low intensity blast, visual and immune function are impacted. Furthermore, non-invasive visual tests such as pERG can be useful to detect exposure to blast.

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Photobiomodulation Protects Against the Disruption of Retinal Oxidative Metabolism and Retinal Function in a Rodent Model of Retinitis Pigmentosa

Eells, JT¹, Gopalakrishnan, S¹, Maleki, S³, Ranji, M³ , Abroe, B¹, Schmitt, H¹, Summerfelt, P², Dubis, A², and Carroll, J^2
1Biomedical Sciences, College of Health Sciences, University of Wisconsin-Milwaukee, Milwaukee ²Ophthalmology; Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee ³Electrical Engineering and Computer Science, University of Wisconsin-Milwaukee, Milwaukee

Purpose: Photobiomodulaton (PBM) by light in the far-red to near-infrared (NIR) region of the spectrum has been demonstrated to attenuate the severity of neurodegeneration in experimental and clinical studies. The purpose of this study was to test the hypothesis that a brief course of 830 nm PBM would preserve mitochondrial function and protect against photoreceptor loss in a rodent model of retinitis pigmentosa, the P23H transgenic rat.

Methods: P23H pups were treated once per day (180 s; 25 mW/cm²; 4.5 J/cm²) using a 830nm light-emitting diode array (QBMI Photomedicine, Barneveld WI) from postnatal day (p) 10 to p25. Sham-treated rats were restrained for 180 seconds, but not exposed to NIR light. The status of the retina was determined at p30 by measurement of retinal metabolic state by NADH/FAD redox imaging, photoreceptor function by electroretinography (ERG) and retinal morphology by spectral domain optical coherence tomography (SD-OCT).

Results: 830 nm PBM preserved retinal metabolic state, retinal function and retinal morphology in NIR-treated animals in comparison to the sham-treated group. PBM preserved mitochondrial redox state. Rod and cone-mediated ERG responses over a range of flash intensities (from 10 mcd.s/m² to 25000 cd.s/m²) were 50% greater in NIR-treated rats compared to sham controls. SD-OCT studies showed that NIR treatment preserved the structural integrity of the retina.

Conclusions: Our findings confirm retinoprotective effects of 830nm PBM in a widely studied animal model of retinal degeneration and support the use of PBM as a non-invasive therapeutic approach for the treatment of retinal degenerative disease.

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Restoring Vitreous Structure after Degeneration to Prevent Ocular Disease

Filas, Benjamen A.; Zhang, Qianru; Shui, Ying-Bo; Beebe, David. C.
Washington University School of Medicine, Department of Ophthalmology and Visual Sciences

Purpose: The vitreous body loses structural integrity and detaches from the retina with age. Such degeneration causes vitreomacular tractions, macular holes, retinal detachments, and is a major risk factor for nuclear cataracts. The purpose of this study was to restore the structure of degraded vitreous via a tissue engineering approach as a new strategy to mitigate ocular disease.

Methods: Chondroitin sulfate proteoglycans covalently bound to amino acid sequences designed to bind structural components of the vitreous (collagen, hyaluronan) were injected into enzymatically degraded bovine vitreous and liquefied vitreous from human eye pairs. Differences in mechanical properties and microstructure were assessed between groups using dynamic mechanical analysis and deep etch scanning electron microscopy.

Results: Enzymatic digestion of proteoglycans and collagen side chains in the vitreous body significantly decreased the storage modulus (or stiffness) of bovine vitreous (≈ 50%). Synthetic proteoglycan mimics restored the structure of trypsin-treated vitreous and increased the stiffness of untreated controls. At the microstructural level, changes in stiffness correlated with proteoglycan concentration as assessed via imaging and immunoblotting. In human eye pairs, vitreous stiffness was nearly double in mimic-treated relative to sham controls.

Conclusions: Rationally designed synthetic proteoglycans show promise in reversing the effects of age-related and enzymatically-induced liquefaction of the vitreous body. Perdurance studies in rabbits to assess long-term efficacy as well as the potential to delay or prevent cataract formation post-vitrectomy and post-enzymatic vitreolysis are ongoing.

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Identification of ganglion cell fate determinants through comparative single cell transcriptomics

Goetz, Jillian and Trimarchi, Jeffrey
Iowa State University¹

Purpose: Previous attempts to decipher the gene expression networks responsible for the development and diversification of retinal ganglion cells (RGCs) sampled the genes expressed in the entire retina. However, RGCs comprise an exceedingly small proportion of the total tissue (~1%) and are a very diverse cell population (at least 20 distinct types). Therefore, these whole-tissue approaches only uncover the most highly expressed RGC genes and failed to reveal the nuances of the dynamic gene networks that drive the differentiation and maturation of RGCs.

Methods: We employ single-cell isolation and transcriptomic profiling techniques to discern the combinations of genes involved in the cell fate decision process and early maturation of retinal ganglion cells. We use a comparative transcriptomic approach to identify the most promising candidate genes from full transcriptomic profiles of these individual cells. To date, we have examined the gene expression signatures from individual developing ganglion cells isolated from the mouse and zebrafish.

Results: These gene clusters have revealed insights into retinal ganglion cell fate and identified a host of marker genes for newborn ganglion cells. We have validated the expression of these genes through in situ hybridizations in the developing mouse and zebrafish retina.

Conclusions: Comparative analyses of the transcriptomes of developing retinal ganglion cells from different organisms have allowed for the examination of conserved expression modules across species. These experiments enable us to focus our future functional experiments on those networks that are the most conserved and, therefore, the most likely to be critical in ganglion cell development.

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Local Generation of Tregs in the Retina

Gregerson, Dale ; McPherson, Scott ; Heuss, Neal
Dept. of Ophthalmology & Visual Neurosciences, University of Minnesota

Purpose: Regulatory T cells (Tregs) protect against autoimmunity to self-antigens (Ag). Tregs specific for self-antigens expressed in thymus (tTregs) are proposed to be less effective than peripherally-derived (pTregs) in protecting from tissue-specific antigens (Ag) that may be poorly represented in the thymus. However, whether Ag-specific pTreg generation and function is limited to secondary lymphoid tissue, or whether it can occur within the tissue-specific, local environment of the cognate Ag remains unresolved.

Methods: Mice expressing beta-galactosidase (Bgal) on a retina specific promoter (Bgal mice) in conjunction with mice expressing diphtheria toxin (DTx) receptor (DTR) and/or green fluorescent protein (GFP) and under control of the FoxP3 promoter, and Bgal-specific T cell receptor transgenic mice, both class I (BG1) and class II (BG2) MHC-restricted, were used to examine this question. Flow cytometry of individual mouse retinas was used to characterize the cells.

Results: Local depletion (intraocular ocular DTx), but not systemic depletion (intraperitoneal DTx), of Bgal-specific Tregs enhanced experimental autoimmune uveoretinitis (EAU) induced by activated Bgal-specific effector T cells. Injections of small amounts of Bgal into the anterior chamber of the eye produced similar numbers of EAU-protective Bgal-specific iTregs in the retina whether or not the mouse was depleted of pre-existing, circulating Tregs.

Conclusions: Together these results suggest that protection from tissue-specific autoimmunity depends on the function of local Ag-specific pTregs, and that the retina is capable of local, ?on-demand? pTreg generation that is independent of circulating Tregs.

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Retinal ganglion cells exhibit spontaneous hyperactivity and abnormally sustained light-evoked responses in in a mouse model of traumatic brain injury

Harper, Matt; Shankar, Malani; Hernandez-Merino, Elena; Kecova, Helga; Dutca, Laura; Kardon, Randy; Stasheff, Steven
Center of Excellence for Prevention and Treatment of Visual Loss Iowa City Veterans’ Administration Medical Center, University of Iowa Departments of Pediatrics (Neurology), Ophthalmology and Visual Science, and Biomedical Engineering, and the Program in Neuroscience University of Iowa Children’s Hospital, Carver College of Medicine, Iowa City, IA

Purpose: To characterize the in vivo and in vitro function and structure of retinal ganglion cells after blast-mediated traumatic brain injury.

Methods: Blast injury was produced by exposing mice to a 20 psi overpressure wave produced by rupture of a Mylar membrane in a specially constructed compressed air tank. Pattern ERG (pERG) was used to objectively measure in vivo retinal ganglion cell (RGC) function at various time-points after injury (n=79). Multielectrode recordings from in vitro whole-mounted retinas (n=4, 75 RGCs/retina) were used to evaluate the properties of individual RGCs. For each RGC the average spontaneous discharge rate and responses to both simple and complex stimuli were compared with retinas from non-blasted control mice. Blast injury was produced by exposing mice to a 20 psi overpressure wave produced by rupture of a Mylar membrane in a specially constructed compressed air tank. Pattern ERG (pERG) was used to objectively measure in vivo retinal ganglion cell (RGC) function at various time-points after injury (n=79). Multielectrode recordings from in vitro whole-mounted retinas (n=4, 75 RGCs/retina) were used to evaluate the properties of individual RGCs. For each RGC the average spontaneous discharge rate and responses to both simple and complex stimuli were compared with retinas from non-blasted control mice.

Results: The pERG declined with a biphasic time course, with initial deficits resolving by 24h post injury and chronic deficits reappearing 4 months following blast exposure. Multi-electrode recordings 7d following blast demonstrated normal RGC responses. By 37d following injury, sustained spontaneous RGC hyperactivity had developed. A substantial proportion of RGCs had prolonged or otherwise aberrant light-evoked responses. Some computed receptive fields were also enlarged and shallow relative to retinas not exposed to blast injury.

Conclusions: In this model, TBI induces dramatic changes in spontaneous and light-evoked retinal ganglion cell activity, beginning several weeks after an initial period of normal physiology. These changes precede decreases in pattern ERG (pERG) amplitude and changes in RGC morphology. The increased spontaneous activity and prolonged responses to light may be interpreted as “noise” in the retina’s message to the brain, which may underlie varied complaints of distorted visual perception by TBI victims.

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Classification of Early Retinal Degeneration Sensitivity using Nonlinear Regression in Retinal Ganglion Cells (RGC)

Hassey, Eric P.⁵; Nylen, Eric, ⁴; Andrews, Michael P.²; Shankar, Malini²; Stasheff, Steven F.^1-5
Program in Neuroscience¹ and Departments of Pediatrics², Ophthalmology & Visual Sciences³, Biomedical Engineering⁴, and Electrical & Computer Engineering⁵, University of Iowa & Carver College of Medicine, Iowa City, IA

Purpose: We developed a quantitative model of how the retina signals the brain in response to simple light stimulation, and how these signals are distorted in animal models of retinitis pigmentosa, a group of blinding eye diseases affecting nearly 1 in 3000 people worldwide.

Methods: We used in vitro multi-electrode techniques to record light responses of hundreds of retinal ganglion cells (RGCs) in wildtype (wt) and rd1 mice at post-natal day (P)14-15, when many RGCs retain substantial light responsiveness in rd1. We plotted each cell's response amplitude (total number of "spikes" within 1 sec of light onset or offset) vs. flash intensity. We fit these intensity-response (I-R) curves to the Michaelis-Menton model using a non-linear regression method. Finally, we classified individual RGCs according to the parameters for these fits.

Results: The Michaelis-Menton model reliably fit the majority of I-R curves, although a smaller proportion of rd1 than wt cells. Overall, rd1 cells were less sensitive (right-shifted curves) and reflected lower amplification of visual signals (shallower slope) than wt ones. Classification of RGCs according to values for the Hill Coefficient and R-Squared goodness-of-fit identified additional I-R relationships that will fit quantitative models other than Michaelis-Menton.

Conclusions: We have refined a simple method for quantitatively evaluating how the visual responsiveness of RGCs is distorted by retinal degenerative diseases. This method indicates lower sensitivity and signal amplification among rd1 than wt RGCs, and should help elucidate mechanisms and thence lead to improved treatments when applied to a wide variety of retinal degeneration models.

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Identification and Characterization of Genetic Factors Responsible for Cavitary Optic Disc Anomalies

Hazlewood, Ralph^1,2; Roos, Benjamin^1,2; Honkonen, Robert³; Jampol, Lee⁴; Mullins, Robert^1,2; Kuehn, Markus^1,2; Alward, Wallace^1,2;Fingert, John^1,2
1Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52242 USA ²Institute for Vision Research, University of Iowa, Iowa City, IA; ³Department of Ophthalmology, State University of New York at Stony Brook, Stony Brook, NY; ⁴Department of Ophthalmology, Feinberg School of Medicine, Northwestern University, Chicago, IL

Purpose: To identify and characterize the gene that causes autosomal dominant, congenital malformations of the optic nerve known as cavitary optic disc anomaly (CODA) in a multiplex family with 17 affected members.

Methods: The gene that causes CODA was previously mapped to a 13.5Mb locus on chromosome 12q14. Members of the CODA pedigree was tested for copy number variations (CNVs) in the chromosome 12q14 region with custom comparative genomic hybridization. Confirmed CNVs were analyzed for their effect on downstream genes using a luciferase reporter gene construct in HEK293T cells.

Results: CGH experiments identified a triplication of a 6kb segment of DNA upstream of matrix metalloproteinase 19 (MMP19) in affected members of the CODA pedigree. No control subjects carried this mutation. The luciferase reporter gene assay showed that the 6kb sequence increased luciferase activity and functioned as a transcription enhancer, in particular, a 1kb segment had a strong positive influence (8-fold higher) on downstream gene expression. Moreover, immunohistochemical experiments revealed positive MMP19 expression in human retina and optic nerve.

Conclusions: We have identified a CNV mutation in the promoter sequence of the MMP19 gene that co-segregates with CODA in our large 17 member pedigree. Moreover we have shown that the CNV spans DNA sequences that powerfully enhance downstream genes (i.e. MMP19) and that MMP19 is expressed in the optic nerve. These functional data strongly suggest that the genetic defects in MMP19 cause CODA and that over-activity of this gene has an important role in the pathogenesis of this optic nerve disease.

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Investigating Progression of Pigment Dispersion Using a Novel Inducible Mouse Mode

Hedberg-Buenz, Adam ; Anderson, Michael
Department of Physiology and Biophysics, The University of Iowa

Purpose: Pigment dispersion syndrome (PDS) is an important glaucoma risk factor. PDS can lead to elevated intraocular pressure (IOP). Many, but not all, eyes afflicted with PDS develop IOP elevation. The biological events shaping how an eye responds to PDS are currently not well understood. The purpose of these experiments is to create an inducible mouse model of pigment dispersion to study the progression of PDS to elevated IOP.

Methods: Cohorts (n=5 mice each) of C57BL/6J (B6) mice homozygous for the Tyrc-2J/J allele (B6.Tyrc-2J/J) received intraocular infusion of donor iris preparations made from dissected irides of B6, B6.Tyrc-2J/J mice, or vehicle only control. Ocular phenotypes of recipient mice were collected pre- (baseline) and post-infusion (response) using slit lamp and gonioscopic imaging, rebound tonometry to measure IOP, and histology.

Results: Infusion of recipient eyes with donor iris preparations results in anterior chamber phenotypes resembling PDS without overt signs of inflammation. Slit lamp photography illustrates that iridial melanin localizes to the tissues at the iridocorneal angle and histological analyses indicates it is sustained. Recipient eyes infused with iris preparations from Tyr+/+ donors exhibit acute periods of IOP elevation, whereas preparations from Tyrc-2/c-2J do not.

Conclusions: These results demonstrate that ocular phenotypes of PDS can be recapitulated in this inducible mouse model. The presence of melanin in the infused iris preparations is critical for IOP elevation. In ongoing work, this inducible model will be used to study biological responses in the eye that contribute or protect from IOP elevation.

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Angiofibrotic Response to Bevacizumab on Fibrovascular Membranes in Proliferative Diabetic Retinopathy

Jiao, Chunhua¹; Spee, Christine²; He, Shikun²; Mullins, Robert¹; Eliott, Dean³; Hinton, David²; Sohn, Elliott^1
1Department of Ophthalmology & Visual Science, University of Iowa Hospitals and Clinics, Iowa City, IA; ²Keck School of Medicine, Doheny Eye Institute / University of Southern California, Los Angeles, CA; ³Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA

Purpose: The goal of this study was to assess the effect of pre-operative VEGF inhibition (Bevacizumab) on CTGF and VEGF in diabetic fibrovascular membranes. The angiofibrosis of the diabetic membranes was evaluated after the treatment of vascular inhibitor.

Methods: In a randomized, controlled, prospective trial (www.clinicaltrials.gov NCT01270542) of 20 eyes (n=10 controls, n=10 given preoperative bevacizumab 3-7 days prior to diabetic traction detachment surgery), epiretinal membranes were excised at the time of vitrectomy. The membranes were snap frozen and sectioned at 9 µm. Sections were immunofluorescent stained with anti-CTGF or anti-VEGF antibody followed by antibody specific for endothelial cells (CD31), myofibroblasts (smooth muscle actin [SMA]), or cytokeratin. Volocity software (version 5.4) allowed quantitative and co-localization analysis of antibody content obtained through immunofluorescence confocal microscopy. Multiple sections for each membrane were averaged for each eye.

Results: The bevacizumab group had a greater than 25% reduction in co-localization in the CD31-CTGF and cytokeratin-CTGF studies compared to controls. There was no change in VEGF colocalization between the treated and controls.

Conclusions: Intravitreal bevacizumab 1.25 mg administered within one week of surgery may decrease VEGF and CTGF expression in diabetic traction membranes. VEGF inhibition decreases co-expression of CTGF but not VEGF in vascular endothelial cell and cytokeratin stains. Attenuation of fibrovascular activity in membranes might provide scientific rationale for performing surgery within 7 days of preoperative adjunctive bevacizumab for diabetic traction detachment.

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Targeted Deletion of Zebrafish lncRNAis18 with TALENs

Jones, Crystal A.; Solin, Staci; McGrail, Maura
Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA

Purpose: Long non-coding RNAs (lncRNAs) are important players in epigenetic regulation of gene expression during development and disease (Niland et al, 2012). A number of mechanisms have been proposed for lncRNA action, however, few functional studies of lncRNAs have been described. We are using Transcription Activator-Like Effector Nuclease (TALEN), engineered site-specific nucleases, to create targeted mutations in a novel zebrafish lncRNA. We previously mapped a highly penetrant retinal tumor model to transgene disruption of the zebrafish lncRNAis18 gene. The objective of this project is to isolate a second zebrafish lncRNAis18 allele that contains a deletion of part of the lncRNAis18 gene.

Methods: Two TALEN pairs were designed to simultaneously target double-strand breaks to exons 2 and 5 of lncRNAis18. Injection of 25-40pg of the TALENs targeting individual exons into zebrafish embryos resulted in efficient mutagenesis of the target sites. To isolate the lncRNAis18 deletion allele we co-injected embryos with the TALEN pairs targeting both exons 2 and exon 5.

Results and Conclusions: We predicted co-injection of TALEN pairs targeting exons 2 and 5 of lncRNAis18 would create a 147kb deletion after loss of the intervening sequence and repair by the non-homologous enjoining pathway. PCR products spanning the fusion of exons 2 to 5 were amplified from somatic tissue in 9 out of 14 co-injected embryos. We verified the deletion allele by sequencing PCR products from 3 embryos. We are now screening for germline transmission of the deletion allele to establish a new line. The lncRNAis18 deletion allele will provide a new genetic tool to study the function of lncRNAis18 in zebrafish development and cancer.

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Retinal thickness changes in mice with streptozotocin-induced diabetes mellitus quantified using an enhanced Iowa Reference Algorithm

Jeong, Woojin^1,5; Abramoff, Michael^1,4; Antony, Bhavna³; Jiao, Chunhua¹; Garvin, Mona^2,4; Sohn, Elliott¹
Departments of ¹Ophthalmology and Visual Sciences, ²Electrical and Computer Engineering, ³Iowa Institute for Biomedical Imaging, University of Iowa, Iowa City, Iowa, ⁴VA Medical Center, Iowa City, Iowa; ⁵Ophthalmology, Dong-A University College of Medicine and Medical Science Research Center

Purpose: To investigate which layers of retina are affected in normal mice and mice with type 1 diabetic mellitus using spectral domain optical coherence tomography (SD-OCT).

Methods: C57BL/6 mice were divided into 10 streptozotocin-induced type 1 DM group and age-matched 8 normal control group. Mice retinal images were obtained using the Bioptigen spectral domain OCT system over the course of 0 week and 6 weeks after induction of diabetes. The OCT scans were segmented into separate surfaces and the average thickness of RNFL-IPL layer and Total retinal thickness were determined using the Iowa Reference Algorithm. And then that averages of thickness of DM group were compared with those of control group.

Results: The mean total retinal thickness of 6 weeks in DM group was 2.02m thinner than 0 week(p<0.05) and the mean total retinal thickness of 6 weeks in control group wasn't changed. The thickness of RNFL-IPL layer in DM group and control group didn't showed a significant difference.

Conclusions: Thinning of the total retina are revealed in the process of mice diabetic change compared with healthy controls. Further evaluation for witch layer are affected will be needed.

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Serum Response Factor: positive and negative regulation of a Dstn mutant corneal epithelial gene expression network

Sharolyn V. Kawakami-Schulz^1,2, Angela Verdoni³, Shannon Sattler¹, Winston Kao⁴, Akihiro Ikeda^1,2, Sakae Ikeda^1,2
1Department of Medical Genetics, University of Wisconsin - Madison, Madison, WI; ²MacPherson Eye Research Institute, University of Wisconsin - Madison, Madison, WI ; ³Department of Internal Medicine, Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO; ⁴Department of Ophthalmology, College of Medicine, University of Cincinnati, Cincinnati, Ohio

Purpose: Dstn^corn1 mice, which lack the gene for destrin (DSTN) display increased serum response factor (SRF) activity that results in corneal abnormalities. Conditional ablation of Srf from the corneal epithelium of Dstn^corn1 mice returns the cornea to a wild-type (WT) like state. The purpose of this study is to characterize gene expression changes that occur when conditional knock-out of Srf rescues mutant phenotypes in the cornea of Dstn^corn1 mice.

Methods:We performed microarray gene expression analysis on cornea from WT, Dstn^corn1 mutant and Dstn^corn1 rescued mice. We confirmed our results by performing both qPCR as well as immunohistochemical analysis for selected genes of interest.

Results: Comparison of gene expression values from WT, Dstn^corn1 mutant and Dstn^corn1 rescued cornea identified over 400 genes that are downstream from SRF. A greater proportion of these genes are down-regulated in Dstn^corn1 cornea, implicating SRF as a negative regulator of gene transcription. We also found that Srf ablation has a significant effect on genes involved in the establishment of cell-cell junctions and regulation of actin dynamics in the corneal epithelium.

Conclusions: This study uses a unique model combining genetic and genomic approaches to identify genes that are regulated by SRF. Our results further confirm that SRF is a master regulator of actin dynamics, epithelial cell structure, and gene expression. Our data also implicates a non-traditional role for SRF - negative regulator of gene transcription. These findings expand current understanding of the role of SRF in both normal and abnormal tissue homeostasis.

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A Novel Pathway Involved in Central Corneal Thickness

Koehn, Demelza ¹; Aimable, Maurisa ¹; Anderson, Michael^1,2,3
Departments of¹ Molecular Physiology and Biophysics, and ² Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA ³ Center for the Prevention and Treatment of Vision Loss, VA Medical Center, Iowa City, IA

Purpose: Central corneal thickness (CCT) is an important endophenotype of glaucoma. Our goal is to identify genes that regulate the magnitude of CCT, using it as an entry point for studying the etiology of glaucoma. We have previously identified Central corneal thickness QTL 1 (Cctq1), a ~15 cM region on mouse chromosome 7. Here, we show this region has been narrowed to 4 candidate genes, with evidence for one being the gene underlying Cctq1.

Methods: To reduce the physical size of the critical interval for Cctq1, mice with informative recombination events were identified and subsequently phenotyped using optical coherence tomography. Exons of the resulting 4 candidate genes were sequenced and RNA expression levels were examined with quantitative PCR.

Results: From sequencing the exons of the four genes, only three coding region base pair changes were identified. All three of them are within the same gene; two are synonymous changes and one is a nonsynonymous amino change in the signal sequence of the protein. Quantitative PCR of all four genes showed that while all are expressed in corneal tissue, only the gene with coding region base pair changes is differentially expressed between the inbred mouse strains used to identify Cctq1.

Conclusions: These results identify a new gene and completely novel pathway contributing to CCT and will help provide understanding to the puzzle of how genes that contribute to CCT relate to glaucoma susceptibility.

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Characterization of the larval lethal phenotype in the zebrafish long noncoding RNA mutant lncRNAis18

Lawrence, Catherine; Solin, Staci; Essner, Jeffrey J.; McGrail, Maura
Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa

Purpose: We previously isolated a transgenic zebrafish line in which disruption of a novel long noncoding RNA lncRNAis18 leads to homozygous lethality and tumor formation in heterozygous adults. lncRNAis18 is expressed in adult retina and high levels are detected in the zebrafish embryo from a large maternal contribution. The lncRNAis18 gene maps to chromosome 3 and its 5' end overlaps the heterochromatin binding protein Hp1a gene cbx1a. lncRNAis18 and cbx1a genes are arranged in a head-to-head orientation. The purpose of this project is to determine if disruption of lncRNAis18 expression in homozygous mutant embryos affects the expression of cbx1a.

Methods: The cbx1a cDNA was cloned from 48 hour post fertilization wild type embryos. The normal time course and pattern of expression of cbx1a gene expression in wild type embryos and larvae will be determined by in situ hybridization and RT-PCR. Homozygous mutant lncRNAis18 embryos will be examined for morphological defects and changes in expression of the neighboring cbx1a gene.

Results and Conclusions: In situ hybridization using dig-labeled RNA probes complementary to lncRNAis18 and cbx1a was performed on 2 cell stage wild type embryos. Both genes showed high levels of maternal RNAs in the embryo. We have begun to examine expression of cbx1a in later stages of wild type embryos and detected expression in the developing retina and brain. We will next characterize expression of the lncRNAis18 and cbx1a genes by in situ hybridization on homozygous mutant lncRNAis18 embryos. These studies will provide insight into the role of lncRNAis18 in embryonic development and retinal tumors.

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Photoreceptor cells that lack extended outer-segments support useful vision in mice

Lee, Swan^1,2; Blodi, Frederick^2,3; Welder, Chris^1,2; Stasheff, Steven^2,3; Tucker, Budd^1,2; Mullins, Robert^1,2; Stone, Edwin^1,2; Thompson, Stewart^1,2
1Department of Ophthalmology and Visual Sciences, ²Institute for Vision Research, ³ Department of Pediatrics, University of Iowa, Iowa City, IA.

Purpose: The complexity of vision depends on diversity of neural circuits in the retina. Therefore, the optimal treatment in blinding photoreceptor cell degenerations is to replace photoreception at the outer retina. In a degenerate retina, stem cells integrate, orient and survive, but do not develop the extended outer-segment characteristic of photoreceptor cells. The purpose of this study was to determine whether cells lacking extended outer-segments could support useful vision.

Methods: We measured retinal and visual function in mice homozygous for the Rd2 mutation (Rds). At 90 days of age, Rds mice have photoreceptor cells that lack extensive outer-segments.

Results: RdsP90 mice had a measurable electroretinogram b-wave, and light evoked responses in multiple types of retinal ganglion cells, but only at relatively high luminance. In optokinetic tracking and visual discrimination tests, visual acuity was almost as good as wild-type at high contrast, but deteriorated rapidly when contrast was reduced. Further, a vision dependent increase in motile activity with light was only present at relatively high luminance.

Conclusions: Photoreceptors that lack extended outer-segments generate responses to light that support useful vision at high contrast and luminance. However, vision is absent in dim light and acuity is reduced with low contrast stimuli. These deficits can largely be attributed reduced photon capture capacity in the photoreceptor cells. This suggests that even though stem cells do not form extended outer-segments in a degenerate retina, photoreceptor replacement could provide meaningful improvements in vision.

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A mutation in Tmem135 causes premature aging and age-related macular degeneration phenotypes

Lee, Wei-Hua¹; Higuchi, Hitoshi²; Pattnaik, Bikash³; Ikeda, Sakae¹; Mullins, Robert⁴; Takahashi, Joseph⁵; Pinto, Lawrence⁶; Ikeda Akihiro^1
1Department of Medical Genetics, University of Wisconsin-Madison ²Department of Dental Anesthesiology, Okayama University Hospital ³Department of Pediatrics, University of Wisconsin-Madison ⁴Department of Ophthalmology & Visual, University of Iowa ⁵Department of Neuroscience, University of Neuroscience ⁶Department of Neuroscience, University of Texas Southwestern Medical Center

Purpose: Our goal is to understand the mechanism causing retinal diseases associated with aging. Recent studies have shown that similar retinal abnormalities are shared between normal aging retina and a number of retinal diseases, suggesting a link between mechanisms underlying retinal aging and diseases. Elucidating the factors that lead to common retinal abnormalities, therefore, will help us understand the cause of retinal aging and retinal diseases. In this study, we focus on identifying and characterizing an ENU-induced mutant mouse strain, FUN025, which shows early onset of age-dependent abnormalities in the retina.

Methods: We used genetic mapping and high-throughput sequencing techniques to identify the genetic mutation in FUN025. We performed immunohistochemistry to determine aging-associated phenotypes, including neurodegeneration, synaptic abnormalities, increase in oxidative stress and inflammation in mouse retina.

Results: We identified a mutation in the transmembrane protein 135 (Tmem135) gene in FUN025 mice. Tmem135 is highly expressed in the peroxisome-rich RPE, mitochondria-rich photoreceptor inner segments, the outer plexiform layer, and the ganglion cell layer. Interestingly, some retinal phenotypes in Tmem135 mutant mice are similar to those observed in age-related macular degeneration (AMD) patients, such as accumulation of lipofuscin in RPE cells and increased macrophage/microglia. In addition, we found that Tmem135 mutant retina is more susceptible to oxidative stress and more vulnerable to oxidative stress-induced photoreceptor cell degeneration.

Conclusions: Our study demonstrates that Tmem135 is a novel gene involved in aging mechanisms. The normal TMEM135 protein may act to protect cells from oxidative damages. The similarities between Tmem135 mutant mouse phenotypes and AMD symptoms suggest that Tmem135 may be involved in pathogenesis of AMD.

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PAX2 and optic fissure closure: identification of putative downstream targets by RNA-seq

Lerach, Stephanie¹; Pretorius, Pamela¹; Zhang, Ying²; Silverstein, Kevin²; Schimmenti, Lisa^1
1Department of Pediatrics, University of Minnesota, Minneapolis, MN; ²Supercomputing Institute, University of Minnesota, Minneapolis, MN

Purpose: Failed optic fissure closure results in developmental eye malformations including coloboma, microphthalmia, and anophthalmia. While several genes have been implicated in these disorders, the etiology for the majority of cases remains unknown. Expression of the transcription factor PAX2 is necessary for proper closure of the optic fissure and mutations in this gene have been associated with coloboma in humans as well as animal models. Through gene expression studies in mice with a Pax2 frameshift mutation we hypothesized we would identify downstream target genes essential for normal optic fissure closure.

Methods: To identify differentially expressed genes, embryonic eyes were harvested from e11.5 Pax21Neu mutant and wild-type mice. We isolated total RNA, performed RNA-seq using the Illumina HiSeq 2000, and validated specific targets by qRT-PCR. Antisense oliginucleotide (Morpholino) gene knockdown was used in zebrafish to explore the roles of target genes in eye development.

Results: RNA-seq analysis revealed 538 statistically-significant (P<0.05) genes with greater than two-fold expression difference between wild-type and Pax2 mutant mouse eyes, with 450 upregulated in response to loss of Pax2 function. Pathway analysis identified 50 genes known for involvement in sensory organ development, and eight of these genes were selected as priority for further study. Validation of specific targets by qRT-PCR was carried out.

Conclusions: We identified candidate genes for downstream targets of Pax2 and are currently investigating the role of these genes in early eye development and optic fissure closure.

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Overexpression of TMEM135 leads to RPE and Photoreceptor Cell Degeneration

Lewis, Sarah; Higuchi, Hitoshi; Ikeda, Sakae; Ikeda, Akihiro
University of Wisconsin/Madison

Purpose: A defining characteristic of aging in the eye is the degeneration of the photoreceptor cell layer within the retina in a peripheral to central manner. Our lab has characterized a mutant mouse model (FUN025) that displays an early onset phenotype in the same pattern as an aged, wildtype (WT) mouse. The causative mutation was identified in Tmem135, which encodes a highly conserved mitochondrial transmembrane protein. When TMEM135 is overexpressed in WT mice, however, the photoreceptor cells as well as the retinal pigmented epithelium (RPE) are degenerated. The purpose of this research is to characterize the retinal degeneration due to TMEM135 overexpression at different stages of mouse development.

Methods: Immunohistochemical and morphological analyses were used to characterize the retina of WT and transgenic mice at postnatal day (p)21, 2 months, and 6 months of age.

Results: The transgenic mouse overexpressing Tmem135 causes RPE and photoreceptor cell degeneration as early as p21. Additionally, we observe variation in the severity if degeneration among age-matched mice of mixed breed, potentially indicating the presence of a Tmem135 modifier.

Conclusions: Tmem135 is a tightly regulated, age-related gene, and inappropriate regulation of this gene, either up or down, may result in retinal degeneration. Thus, the transgenic mouse overexpressing Tmem135 is a new model to study age-related diseases of the eye.

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Analysis of progenitor proliferation in the zebrafish lncRNAis18 retinal tumor model

Lincow, Deborah; Brodskiy, Pavel; Essner, Jeffrey J.; Sakaguchi, Donald S.; McGrail, Maura
Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa

Purpose: We previously mapped a highly penetrant retinal tumor model to a transgene that disrupts expression of the zebrafish lncRNAis18 gene. The purpose of this study was to characterize the pattern of proliferation and examine the source of progenitors during tumor progression in the lncRNAis18 model.

Methods: We examined retinal cell proliferation in 4.5 month and 8.5 month lncRNAis18 zebrafish and their wild type siblings. 4.5 month old fish lacking obvious signs of retinal tumors, and 8.5 month old fish with advanced tumors, were treated with 5mm bromodeoxyuridine (BrdU). Retinal cryosections were labeled with antibodies to BrdU, Sox2 and markers for retinal neurons and Muller glia.

Results and Conclusions: Advanced retinal tumors in 8.5 month old fish showed widespread incorporation of BrdU that showed some overlap with the Muller glia markers glial fibrillary acid protein and brain lipid binding protein. In 4.5 month old fish 81% of the retinas examined (13/16 retina from 8 fish) contained abnormal regions of proliferation and overexpression of glial markers. The pre-tumorous retinas showed regions with increased BrdU incorporation and upregulation of brain lipid binding protein and glial fibrillary acid protein. Glutamine synthetase and brain lipid binding protein expression frequently overlapped or were in close proximity in the pretumorous retinas. These results suggest one possible source of cells in the lncRNAis18 tumors is Muller glia that have become activated to divide and produce neural progenitors. Understanding the role of lncRNAis18 in regulating Muller glia proliferation may reveal new insights into the stem cell-like behavior of cancer cells.

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Neurotrophic Protection of Retinal Ganglion Cells in Mice with Sustained Ocular Hypertension

Liu, Xiaorong, Chen, Hui, Zhao, Yan, Feng, Liang, Troy, J.B.,
The Departments of Ophthalmology, Biomedical Engineering, and Neurobiology, Northwestern University

Purpose: Glaucoma is characterized by dendritic and axonal degeneration of retinal ganglion cells (RGCs), visual field deficits, and ultimately RGC death. Brain-derived neurotrophic factor (BDNF) is one of the neurotrophins essential for neuronal survival and function in the central nervous system. Combining mouse genetics, anatomical and physiological techniques, and behavioral assays, here we investigate how BDNF protects RGCs and vision in a mouse model of experimental glaucoma.

Methods: We have adopted a laser-induced mouse model of ocular hypertension to mimic human high-tension glaucoma. We applied two cutting-edge techniques, the laser-guided focal electro-retinogram (ERG) to measure visual responses from different retinal cell types at various locations, and a large-scale multi-electrode array (MEA) to examine response properties of different subtype RGCs. Next, we generated transgenic mice in which BDNF overexpression can be induced in the RGCs in a temporally-controlled manner.

Results: We demonstrated a gradual loss of RGCs following the induction of ocular hypertension. Importantly, RGC soma shrinkage is preceded by dendritic degeneration, which depends on cell-type and location. We recorded the visual responses of RGCs and showed that the functional degeneration of RGCs is subtype-dependent. Our preliminary data further suggest that overexpression of BDNF prevented RGC loss in glaucomatous mice.

Conclusion: BDNF can prevent the detrimental effects of sustained ocular hypertension on RGCs and vision. Our study establishes a valuable model system with which to better understand the neurotrophic protection on RGCs in glaucoma, and our results will add new insights into how to best design neurotrophic therapies for glaucoma patients.Hassey

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Abnormal synapses and retinal degeneration in the Small Kinky Tail mutant

Macke, Erica; Ikeda, Sakae; Ikeda, Akihiro
Department of Medical Genetics, University of Wisconsin-Madison

Purpose: The purpose of this study is to investigate the link between synaptic abnormalities and aging in order to better understand the mechanisms contributing to age-related retinal diseases. The small kinky tail (skt) mutation, which arose spontaneously on a mixed background, leads to a kinked tail and small body size in mice. We identified abnormalities that are similar to aging mice in the younger skt retina, and aim to further characterize the skt phenotype and identify the causative gene.

Methods: Eyes were treated with H&E to visualize retinal structure and measure the outer nuclear layer thickness (ONLT), a measure of frequency of photoreceptor cells. Immunohistochemical analysis was performed to examine age-related phenotypes. Genetic mapping of the skt mutation was performed using an F₂ intercross (B6/LT-skt/skt x C57BL/6). We performed a capture array of our candidate region and sequenced on the Illumina HiSeq platform.

Results: We observe an increase in macrophages in skt mice at four weeks, which persists past eight weeks. By eight weeks of age there is significant damage to the skt retina and a decrease in ONLT. Abnormalities are also present in the synaptic layers at eight weeks. We mapped skt to a 4.4 Mbp region on chromosome 7 and next generation sequencing yielded 37 SNPs within the candidate region.

Conclusions: Phenotyping data indicate that skt is involved in photoreceptor maintenance. The skt mutant is a potential model for age-related retinal diseases due to abnormalities in the synaptic layers and the increased presence of macrophages and inflammatory cells.

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Genetically dissecting the primary site(s) of pathogenesis in COL4A1 related ocular dysgenesis

Mao Mao; Jeanne Marion; Gould, Douglas
Departments of Ophthalmology and Anatomy, Institute for Human Genetics, UCSF School of Medicine, San Francisco, CA

Purpose: Anterior segment dysgenesis (ASD) is a spectrum of disorders affecting the development of anterior structures of the eye, leading to vision loss including glaucoma. Mutations in a basement membrane component, collagen type IV alpha1 (COL4A1) cause ASD in humans. Previously we identified a splice site mutation in mice that causes skipping of exon41 (Col4a1 Δex41) and cause highly penetrant ASD and optic nerve hypoplasia (ONH). As COL4A1 is present in all ocular basement membranes, it is unclear which tissue might be the site of primary insult. The purpose of this study is to conditionally express a mutant Col4a1 allele in different ocular tissues or at different developmental stages to characterize the primary insult.

Methods: To recreate the Col4a1 Δex41 allele, we developed a conditional allele with LoxP sites flanking exon 41 of Col4a1 (Col4a1 flox41). Four tissue-specific CRE recombinase lines, MLR10-Cre, Wnt1-Cre, Tie2-Cre and Pdgfrb-Cre were used to control the expression in corresponding tissues. A ubiquitously expressed, inducible CRE line was also included to assess the temporal role of COL4A1. Slit-lamp and histological analysis was performed to assess the extent of ASD and ONH.

Results: All four tissue-specific mutants and postnatal expression of mutant COL4A1 developed milder ASD comparing to Col4a1 Δex41 mutants. Moreover, two conditional mutant lines had mild ONH.

Conclusions: Our results suggest that ASD in Col4a1 mutant mice is caused by insults from more than one ocular cell type, and insults in these mutants are largely embryonic.

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Diurnal rodent model options for vision research

Merriman, Dana
University of Wisconsin Oshkosh

Vision research has a long and productive record of use of classic laboratory rodents, rats and mice. However, the latter have visual systems evolved for a nocturnal habit, whereas the human visual system evolved for a bright-light diurnal habit. This presentation will survey the retinal architecture, advantages, and limitations of several lesser-known rodent species that provide a useful diurnal complement to rats and mice. These diurnal rodents also possess unusually cone-rich photoreceptor mosaics that facilitate the study of cone cells and pathways. Species to be presented include the Nile rat (Arvicanthis spp.), the degu (Octodon degus), the Siberian chipmunk (Tamias sibiricus), and the 13-lined ground squirrel (Ictidomys tridecemlineatus). The latter is an obligate hibernator whose retina and optic nerve have demonstrated unusual resilience in the face of neuronal injury, itself an interesting phenomenon with potential translational value.

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Genetic modifiers of Cep290-mediated retinal degeneration

Meyer, Kacie J. Anderson, Michael G.
Department of Molecular Physiology and Biophysics, Department of Ophthalmology and Visual Sciences, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA; Center for the Prevention and Treatment of Vision Loss, VA Medical Center, Iowa City, IA

Purpose: CEP290-mediated Leber congenital amaurosis (LCA), an inherited retinal degenerative disease characterized by nystagmus and blindness, typically manifests in the first year of life. Unfortunately, gene therapy based strategies for treating CEP290 LCA do not yet exist and CEP290 has no known enzymatic activities that would support viable pharmacologic therapies. The goal of this research is to use phenotype-driven studies in mice to identify genetic modifiers of Cep290 which might be manipulated as therapeutic surrogates in lieu of CEP290 itself.

Methods: We have initiated mouse mapping crosses for the purpose of identifying genetic modifiers of CEP290-mediated retinal degeneration. Retinas of experimental mice were imaged in vivo with optical coherence tomography. Mice were genotyped with a genome-wide panel of polymorphic genetic markers and quantitative trait analysis was performed using R/qtl .

Results: Using optical coherence tomography, we are able to obtain highly reliable quantitative measurements in individual mice non-invasively. These experiments demonstrate that the retinal degeneration phenotype conferred by Cep290rd16 mutation is sensitive to genetic background. In addition, preliminary data indicate the presence of several chromosomal loci capable of modifying disease progression.

Conclusions: This set of experiments uses phenotype-driven studies to map quantitative trait loci containing genetic elements that influence the retinal degeneration phenotype in mice homozygous for Cep290rd16. These experiments represent an opportunity to discover novel pathways that might not otherwise have been considered and that are propagated by comparatively subtle mutations likely to represent the common variation in the human genome.

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A Multimodal Machine-Learning-Based Approach for Segmenting the Optic Disc and Cup in Fundus and SD-OCT Images

Miri, Mohammad Saleh^1,2; Abramoff, Michael^1,2; Lee, Kyungmoo¹; Niemeijer, Meindert¹; Wahle, Andreas¹; Kwon, Young³; Garvin, Mona^1,2
1Electrical and Computer Engineering, The University of Iowa, Iowa City, IA, ²Center for the Prevention and Treatment of Visual Loss, Iowa City VA Health Care System, Iowa City, IA, ³The Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, IA

Purpose: Many existing approaches exist for segmenting the optic disc and cup from a single imaging modality. The purpose of this work is to determine the extent that simultaneously using complementary information from fundus photographs and SD-OCT volumes can enable a more accurate optic disc and cup segmentation.

Methods: Seventy SD-OCT scans (200x200x1024 voxels; voxel size 30x30x2 μm) centered at optic nerve head from 35 glaucoma patients were acquired using Cirrus HD-OCT (Carl Zeiss Meditec, Inc., Dublin, CA). Stereo color fundus photograph pairs (4096x4096 pixels; Nidek, Newark, NJ) were acquired on the same day from the same patients. After segmenting the intraretinal surfaces on each SD-OCT volume using a graph-theoretic approach, 10 features were obtained at each projected location based on 2D layer-based projection images and distances between segmented surfaces. The fundus photographs were registered to each corresponding SD-OCT volume and, using a Gaussian filter bank and distance-based features, 14 features were extracted at each pixel location. A multimodal feature set (24 total features) was created by combining the features from both modalities. A random-forest classifier was applied to both the unimodal (OCT only) feature set and multimodal feature set to classify the pixels int0 background, rim area, and cup classes. A reference standard was obtained by taking the majority vote of three expert-defined segmentation results from the fundus photographs.

Results: As also shown in Table 1, the overall accuracy of multimodal approach (97.43±0.58) was significantly better than the unimodal approach (94.97±0.76) using a paired t-test (p < 0.05).

Conclusions: Using complementary information from both fundus photographs and SD-OCT volumes can enable better segmentations of the optic disc and cup over that obtained using unimodal information.

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Histone Deacetylases Play a Critical Role in the Atrophy and Death of Retinal Ganglion Cells After Optic Nerve Damage

Nickells, Robert
Department of Ophthalmology and Visual Sciences, University of Wisconsin - Madison, Madison, Wi

Purpose: Dying retinal ganglion cells exhibit various stages of nuclear atrophy that occur rapidly after optic nerve damage. These changes include downregulation of normal gene expression, deacetylation of histones, heterochromatin formation, and the collapse and shrinkage of nuclei. We investigated if epigenetic changes, such as histone deacetylation by Histone Deacetylases (HDACs).

Methods: HDAC activity in ganglion cell death was investigated in an acute optic nerve crush model using HDAC inhibitors and conditional knock-out mice.

Results: Using Bax-deficient mice, in which ganglion cell apoptosis is completely blocked, we can demonstrate these early changes even in the absence of cell death. These events can be attenuated using a broad spectrum HDAC inhibitor. Concomitant with this, HDAC3 translocates from the cytoplasm to the nucleus. Experiments with Hdac3 conditional knock-out mice indicate that this HDAC plays a critical role for global changes, including wide-spread histone deacetylation and heterochromatin formation. Additionally, Hdac3-deficient ganglion cells resist death. Deletion of Hdac3, however, is not sufficient to block gene silencing, suggesting the involvement of other HDACs in this process.

Conclusions: Histone deacetylation activity plays multi-faceted roles in the early stages of ganglion cell death.

Characterization of HCN1 trafficking in rod photoreceptors

Yuan Pan¹; Joseph G. Laird¹; Modestos Modesto¹; Dane M. Chetkovich²; Sheila A. Baker^1
1Department of Biochemistry & Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, Iowa; ¹Department of Neurology and Clinical Neurosciences, Department of Physiology, Northwestern University Feinberg School of Medicine, Chicago, Illinois

Purpose: Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are cation-selective channels present in retina, brain and heart. The activity of HCN channels contributes to signal integration, cell excitability and pacemaker activity. Rods express homomeric channels composed of HCN1; these channels participate in keeping light responses transient. The subcellular localization of HCN1 varies among cell types. In rods, HCN1 is found in the inner segments, to a lesser extent in the synaptic terminals, but excluded from the outer segments. In contrast, HCN1 in the hippocampus is concentrated in a subset of dendrites. A key regulator of HCN1 trafficking and activity is TPR-containing Rab8b interacting protein (TRIP8b). Up to nine splice isoforms of TRIP8b are expressed throughout the brain and can differentially regulate surface expression and activity of HCN1. The purpose of the present study was to determine which isoforms of TRIP8b are expressed in photoreceptors and to test if the C-terminus of HCN1, containing TRIP8b binding sites, could confer targeting in rods.

Methods: RT-PCR using RNA isolated from total mouse retina or photoreceptor inner segments collected by laser capture micro-dissection was used to test for the presence of specific TRIP8b splice isoforms. Immunohistochemistry was used to test for the expression of TRIP8b protein in mouse retina. To assess targeting, the C-terminus of HCN1 and deletions of this region, were fused to a membrane reporter and expressed in transgenic Xenopus rods. Localization of the transgenically expressed proteins was visualized with confocal microscopy.

Results: We found that multiple splice isoforms of TRIP8b are expressed in photoreceptors, including those with opposing effects on HCN1 trafficking. TRIP8b protein was found in the same retina compartments as HCN1. In transgenic Xenopus rods, the membrane reporter localized primarily to outer segments but addition of HCN1's C-terminus changed its localization to the inner segment. Deletions of HCN1's C-terminus uncovered a strong synaptic targeting signal located in a stretch of amino acids in between the two TRIP8b binding sites. In order to achieve the normal localization pattern of HCN1, this synaptic targeting signal needs to be suppressed.

Conclusions: HCN1 trafficking involves interactions among multiple signals within its C-terminus, and TRIP8b is a possible regulator of this process in photoreceptors.

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Closing the gap: development of a novel zebrafish-based tool to assess optic fissure closure

Pamela R. Pretorius¹; Yusuf Agamawi¹; Julia M. Hatler¹; Stephanie L. Lerach¹; Fei Qi²; Bo Zhang²; Brent Bill³; Shuo Lin⁴; Lisa A. Schimmenti^1
1Department of Pediatrics, University of Minnesota, Minneapolis, MN; ²College of Life Sciences, Peking University, Beijing, 100871, P.R. China; ³Semel Institute for Neuroscience and Human Biology, UCLA, Los Angeles, CA; ⁴Department of Molecular, Cell and Developmental Biology, UCLA, Los Angeles, CA

Purpose: Proper closure of the optic fissure during early embryogenesis is critical for normal eye formation, with failure of optic fissure closure resulting in coloboma and related ocular defects. Mutations in several developmentally important genes are known to cause colobomas; however, the genetic etiology for most patients remains unknown. To characterize novel genes and pathways that contribute to coloboma formation, we developed a method to evaluate optic fissure closure in zebrafish.

Methods: To evaluate optic fissure closure, we assayed for changes in pax2a expression, using the mp204a:GFP transgenic line. As proof of principle, we tested whether sema3e, a gene expressed in ventral mesenchyme that exist between the edges of the optic fissure, affected GFP levels. As a second test of optic fissure closure, we evaluated basement membrane dissolution by observing changes in laminin expression at the edges of the closing optic fissure.

Results: Transgenic expression of GFP in the mp204a:GFP enhancer trap line recapitulates endogenous pax2a expression in the eye field, midbrain-hindbrain boundary, otic placode and pronephric mesoderm. Quantitative three-dimensional analysis revealed increased expression of pax2a in sema3e knockdown embryos compared to uninjected controls, indicating aberrant optic fissure closure. Moreover, a reduction in overall eye size was observed. We also observed that in sema3e knockdown zebrafish, laminin expression in the basement membrane appeared thickened and less organized compared to uninjected wild-type.

Conclusions: We demonstrate that quantitative three-dimensional analysis of the enhancer trap mp204a:GFP transgenic zebrafish line can facilitate the identification and screening of candidate genes that impact optic fissure closure.

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RNA interference therapy in the Spinocerebellar ataxia type 7 (SCA7) mouse retina

Ramachandran, Pavitra¹; Bhattarai, Sajag²; Drack, Arlene²; Boudreau,Ryan³; Davidson, Beverly^3
1Interdisciplinary Graduate Program in Genetics; Departments of ²Ophthalmology and Visual Sciences, ³Internal Medicine, The University of Iowa, Iowa City, IA

Purpose: Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disease characterized by ataxia and retinal degeneration. The expansion of a coding CAG repeat in the gene ataxin-7 leads to the production of a dominant negative protein causing SCA7 phenotypes. Reducing the levels of mutant ataxin-7 by RNAi in the SCA7 mouse cerebellum leads to alleviation of ataxic phenotypes. Here, our goal is to determine if reducing the levels of mutant ataxin-7 by RNAi in the SCA7 mouse retina is safe and well tolerated.

Methods: RNAi sequences targeting mutant ataxin-7 were packaged into AAV1 viruses and injected subretinally into the SCA7 mouse retina. Saline and a scrambled RNAi sequence served as controls. One month and six-months post injection, RNA was harvested for QPCR. In addition, at 6 months post-injection, various retinal tests such as the electroretinogram (ERG), optokinetic response (OKR), pupillary light reflex were performed. We also looked at the morphology of the retina by histology.

Results: One month post-injection, we observed significant reduction of mutant ataxin-7 in the SCA7 retina by QPCR. In addition we observed that AAV1 can transduce the RPE and photoreceptor cells in the murine retina. Long-term studies are ongoing. Preliminary results suggest that reducing mutant ataxin-7 is safe and well tolerated long-term in the SCA7 murine retina.

Conclusions: The SCA7 mouse retina does not exhibit a robust retinal phenotype. However, it is important to test the safety and efficacy of these RNAi sequences in the retina. If these RNAi sequences tested are safe and well tolerated, this will allow them to be tested in other models for phenotypic rescue.

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The role of CGRP and photic blink response in central trigeminal hypersensitivity and photophobia during migraine

Rea, Brandon J.; Kaiser, Eric A.; Poolman, Pieter; Thompson, Stuart; Recober, Ana; Kardon, Randy H.; Russo, Andrew F.
Departments of Molecular Physiology and Biophysics, Neurology, Ophthalmology and Visual Sciences, Medical Scientist Training Program, Neuroscience Program, University of Iowa, Iowa City, Iowa 52242; Center for the Prevention and Treatment of Visual Loss, Iowa VA Health Care System, Iowa City, IA

Purpose: Photophobia is a symptom experienced by a majority of people who suffer from migraine. While experiencing photophobia, migraineurs become sensitive to, and report feelings of discomfort in, normal levels of light. Calcitonin gene-related peptide (CGRP) has been shown to induce migraine in migraineurs but not in non-migraineurs. We have developed a method for measuring photic electromyogram (EMG) in the orbicularis oculi muscle of a mouse model that expresses the rate limiting subunit for the human CGRP receptor, hRAMP1. We hypothesize that utilizing CGRP coupled with measurement of orbicularis oculi EMG can effectively assess central trigeminal hypersensitivity in photophobia in association with migraine.

Methods: Nestin/hRAMP1 mice were anesthetized and each implanted with an EMG telemeter along the dorsal flank between the fore and hind limbs subcutaneously. Two biopotential electrodes were then inserted into the right orbicularis oculi. After recovery, EMG responses from the conscious, unrestrained mice were recorded by eliciting blink responses via air puff under varying conditions of ~0 lux, 1000 lux (room light), and 27,000 lux (bright sunlight). This testing paradigm was repeated with intraperitoneal injection of CGRP and topical application of proparacaine to both eyes.

Results: Our preliminary results suggest that, when administered CGRP, the total area under the curve (AUC) of the EMG response under conditions of 27,000 lux (bright sunlight) is significantly greater than at other light levels with and without CGRP. Further experiments and more mice are needed to confirm this. Ongoing studies will examine the effect of coadministration of CGRP and proparacaine on EMG blink activity.

Conclusions:

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Live Imaging of Membrane Repair in the Retinal Pigment Epithelium

Rodenkirch, Charles; Toops, Kimberly; Lakkaraju, Aparna
University of Wisconsin-Madison, Department of Ophthalmology and Visual Sciences

Purpose: In age-related macular degeneration (AMD) the retinal pigment epithelium (RPE) accumulates lysosomal lipofuscin. One consequence of lipofuscin accumulation is excessive activation of the complement pathway, which leads to chronic inflammation. Data from our laboratory show that a lipofuscin component, A2E, perturbs RPE cholesterol homeostasis and lysosome function. Fusion of lysosomes with the plasma membrane (lysosome exocytosis) is critical for membrane repair after complement-mediated attack to prevent inflammation and cell death. Here, we demonstrate an imaging technique that will allow us to study the kinetics of lysosome mediated membrane repair in RPE cells with or without A2E.

Methods: Spinning disk/TIRF microscopy was used to visualize lysosome exocytosis in pig RPE expressing lysosomal membrane proteins tagged with a pH-sensitive green fluorescent protein (super-ecliptic pHluorin, SEGFP). SEGFP's fluorescence is quenched in acidic conditions, like within the lysosomal lumen. Exposure to a neutral environment, such as when the lysosome fuses with the plasma membrane, unquenches the fluorescence and allows for detection.

Results: Control and A2E treated RPE expressing SEGFP constructs were treated with ionomyocin to stimulate lysosome exocytosis events that were captured using spinning disk/TIRF imaging. Quantification of ionomycin stimulated lysosome exocytosis kinetics using image analysis software are ongoing.

Conclusions: These results demonstrate that this imaging technique will allow us to determine differences in the rate of lysosome fusion events at the plasma membrane. In the future this technique will allow us to determine the rate of lysosome-mediated repair in RPE cells with or without A2E after sublytic complement-mediated attack.

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A neural code for vision decays in rd1 and rd10 mice: Quantifiable spike train coherence in retinal degenerative models

Senesac, Reid; Shankar, Malini; Tang, Li; Wang, Kai; Abramoff, Michael; Stasheff, Steven
The University of Iowa, Iowa City, IA

Purpose: Current visual prosthetic devices show promise in restoring visual capability, but patients using these cannot yet reliably perceive detailed recognizable images. To design more successful electrical stimulation in retinal prosthetics, we sought to detect higher order patterns in the neural signals of retinal ganglion cells (RGCs) that are disrupted or lost in retinal degenerations. We now confirm a previous hypothesis: the time at which any action potential ("spike") occurs is determined by the exact timing of multiple previous spikes. That is, higher order spike coherence forms an important component of the neural code for vision, and this coherence breaks down in animal models of retinal degeneration.

Methods: Activity of RGCs was collected by in vitro multielectrode recordings in wild-type (wt), rd1 and rd10 mouse retinas, with or without visual stimulation. We estimated the probability of a spike occurring, given that one or more spikes occurred at specific preceding delays; that is, P(s0 | δs1, δs2), where δs1 (or δs2) is the delay with which spike 1 (s1) or spike 2(s2) occurs before the initial spike (s0).

Results: Coherence patterns in wt RGCs are consistent for individual cells, but differ amongst cell type and stimulus condition, but not across presentations of the same stimulus. In rd1 and rd10 mice, these patterns disappear progressively.

Conclusions: This is the first quantitative demonstration that higher order spike coherence in RGCs is disrupted by retinal degeneration. Prosthetic stimulation that incorporates this neural code may provide patients more realistic perceptions.

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Loss-of-function mutations in TBC1D20 cause cataracts and male infertility in blind sterile (bs) mice and Warburg Micro syndrome in humans

Sidjanin, D.J.
Department of Cell Biology, Neurobiology, and Anatomy Human Molecular Genetics Medical College of Wisconsin Milwaukee WI

Purpose: The blind sterile (bs) mouse is a spontaneous autosomal recessive mutation discovered over 30 years ago. Phenotypically bs mice exhibit embryonic nuclear cataracts and male infertility. The initial goal of this study was to identify the mutation responsible for bs cataracts.

Methods: Standard forward genetics methods were used. Candidate genes were analyzed using Sanger sequencing. Standard molecular biology techniques were used for the primary culture analysis.

Results: We positionally cloned the bs locus and identified a mutation in Tbc1d20. Functional analysis established mouse TBC1D20 as a GTPase activating protein (GAP) for RAB1 and RAB2, and bs as a TBC1D20 loss-of-function mutation. Based on the function of TBC1D20 and bs phenotypes, we hypothesized that mutations in TBC1D20 contribute to Warburg Micro syndrome (WMS), a disorder characterized by congenital cataracts, brain, and endocrine abnormalities caused by mutations in RAB3GAP1, RAB3GAP2, and RAB18. Sequence analysis of a cohort of 77 WMS families identified five distinct TBC1D20 loss-of-function mutations. The analysis of bs mouse embryonic fibroblasts and human fibroblasts deficient in TBC1D20 identified aberrant lipid droplets (LDs) as a common cellular abnormality. Additionally, human fibroblasts deficient of RAB18 and RAB3GAP1 also exhibit aberrant LD formation.

Conclusions: TBC1D20 is a novel WMS gene and bs mouse is WMS animal model. A defect in LD formation/metabolism has been established as a common cellular abnormality in fibroblasts from WMS patients implying that abnormalities in LD metabolism may contribute to the WMS disease pathology.

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Different photoreceptor diseases can increase or decrease sensitivity of non-image-forming responses to light.

Singh, Pratibha; Welder, Chris R.; Lee, Swan; Stone, Edwin M.; Thompson, Stewart
Department of Ophthalmology and Visual Sciences, and the Institute for Vision Research, University of Iowa, Iowa City, IA.

Purpose: Distinct from form-vision, measurement of irradiance in the retina regulates many aspects of physiology and behavior (irradiance-responses). This function depends on intrinsically photosensitive retinal ganglion cells (ipRGC) that project to non-image-forming structures in the brain. Intrinsic photosensitivity of ipRGCs is generated by the photopigment melanopsin, but like other RGCs, the ipRGCs also receive indirect input from rod and cone photoreceptors. Eye disease that disrupts these irradiance responses can impact health, causing endocrine dysregulation, poor sleep and depressed mood. The purpose of this study was to determine how different types of deficit in rod and cone photoreceptor function affect the irradiance responses.

Methods: Effect of disease on irradiance-responses was assessed in mice with no rods or cones (rd1) and mice with reduced rod cone function (RdsP90). Sensitivity to light was measured for two irradiance responses, the primarily melanopsin generated negative masking response in behavior, and the primarily rod/cone generated pupillary light reflex.

Results: For negative masking, early degeneration of rods and cones in rd1 mice caused a marked increase in the sensitivity to light. In contrast, reduced rod/cone function in RdsP90 mice caused a decrease in the sensitivity to light. For the pupillary light reflex, both diseases caused a significant decrease in sensitivity of the rod-cone generated components of the response. Analysis of the melanopsin component of the pupil response is ongoing.

Conclusions: We show that two types of rod-cone disorder can have opposite effects on the sensitivity of irradiance-responses. However, the effect of disease also depends on the form or degree of rod/cone contribution to the response measured.

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Development of BEST1 reporter constructs for use in iPSC based high throughput drug screens and disease modeling

Songstad, Allison, E.; Burnight, Erin, R.; Mullins, Robert, F.; Stone, Edwin, M.; Andorf, Jeanean, L.; Streb, Luan, M.; Tucker, Budd, A.
Department of Ophthalmology and Visual Science, Institute for Vision Research, University of Iowa, Iowa City, IA

Purpose: Best disease, a rare inherited form of dominant juvenile onset macular dystrophy, is characterized by electrophysiological dysfunction of the retinal pigment epithelium (RPE), accumulation of vitelliform material, death of the overlying photoreceptor cells and a subsequent irreversible loss of central vision. The molecular pathophysiology of mutations in the RPE gene, BEST1, is poorly understood. Induced pluripotent stem cell (iPSC)-derived RPE cells are morphologically and biochemically similar to RPE cells in vivo, and iPSC-derived RPE cells generated from Best disease patients can help elucidate the biological function of BEST1 mutations. Likewise, iPSC-derived RPE cells may aid in the testing of potential therapeutics in a high‐throughput fashion. The purpose of this study was to develop BEST1-reporter constructs to be used in RPE cells generated from Best disease patients for both elucidation of disease mechanism and as markers of therapeutic efficacy.

Methods: We generated keratinocyte-derived iPSCs from Best disease patients and differentiated the iPSCs into RPE cells using our previously developed differentiation protocol. We transfected human BEST1-reporter constructs into primary pig RPE to validate construct function.

Results: Human constructs containing a fluorescent reporter (GFP or tdTomato) flanked by 3' and 5' BEST1 UTRs, driven by the BEST1-promoter were made and validated via transfection in primary pig RPE cells. We made human iPSC-RPE derived from Best disease patients.

Conclusions: We developed a BEST1-reporter construct to be used in high-throughput drug screens. Also, we have effectively generated patient‐specific RPE cells to be used with the BEST1-reporter construct to elucidate the pathophysiology of Best disease.

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Bolus RPE cell Transplantation in Pigs

Sohn, Elliott; Jiao, Chunhua; Mullins, Robert; Stone, Edwin; Tucker, Budd
University of Iowa, Department of Ophthamology, Institute for Vision Research

Purpose: Various sources of RPE cells have been proposed for cell replacement therapy (e.g. in the subretinal space to treat geographic atrophy) and in generating models of proliferative vitreoretinopathy (PVR). We sought to determine the effects of induced pluripotent stem cell (iPS) derived RPE cells delivered to the subretinal space and allogeneic RPE cells injected into the vitreous cavity of vitrectomized wild-type mini-pigs.

Methods: PPV was performed in miniature swine. Three treatment cohorts consisted of those given blebs of balanced salt solution alone (controls); blebs followed by intravitreal injection of porcine-derived GFP-positive RPE cells (RPE group); and subretinal blebs of porcine-derived, GFP-positive iPS-RPE cells (iPS group). Post-op vitreous samples were screened for inflammatory cytokines using a commercial cytokine array and total proteins were evaluated on silver-stained SDS-PAGE gels for proteomic analysis.

Results: Vitreous membranes were seen in all eyes in the iPS-RPE cell group but no PVR or retinal detachment was observed. Epiretinal membranes and retinal detachment were seen in a third of eyes in the RPE group. No PVR or vitreous membranes were seen in the saline controls. At POW3, vitreous IL-8 levels were elevated in the iPS group compared to the intravitreal RPE and saline controls. TGF-beta levels were very low or below limits of detection in all eyes.

Conclusions: Subretinal injection of iPS-RPE cells in wild-type mini-pigs may result in an increase in inflammatory response specific to particular cytokines. Exogenous intravitreal RPE cells does not result in PVR in these animals.

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TALEN-induced somatic inactivation of rb1 tumor suppressor in zebrafish leads to central nervous system tumors

Solin, Staci; Jones, Crystal; Essner, Jeffrey; McGrail, Maura
Iowa State University, Ames, IA

Purpose: Disruption of tumor suppressor rb1 is found in many human sporadic cancers. Our objective was to isolate an rb1 null allele for use in studying its role in zebrafish tumorigenesis. We discovered that somatic inactivation of rb1 leads to solid tumor formation in the central nervous system of adult zebrafish.

Methods: To isolate an rb1 null allele we designed a pair of TAL-effector nucleases to target the second exon of rb1. 25-200 pg of rb1-TALENs were injected into one-cell zebrafish embryos.

Results: 100% bi-allelic inactivation of rb1 was detected in more than half of injected embryos. We recovered 4 new rb1 alleles in the F1 generation including frameshift and in frame deletions. We observed that at six months of age F0s injected with 150-200 pg developed tumors in the brain at a frequency of 4/24 to 3/6. Histopathology revealed massive overproliferation in the diencephalon disrupting normal brain architecture. The tumors contain small blue cells that are undifferentiated, dense, and round, indicating some similarity to human pineoblastoma. Regions of disorganized, proliferating cells in the ganglion cell layer, inner plexiform layer and ciliary marginal zone of the retina were observed in 2 of the brain tumor bearing fish. Sequencing of rb1 alleles revealed a single allele in tumor in contrast to multiple alleles in other somatic tissues indicating loss of heterozygosity in the tumor.

Conclusions: Somatic inactivation of rb1 induces retinal and brain neoplasms in adult zebrafish. We will further develop the TALEN somatic mutagenesis system in zebrafish embryos to examine the functional interaction of pRb with other tumor suppressor pathways.

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In vitro physiology of sight-restoring gene therapy highlights importance of early therapy

Stasheff, Steven^1-4; Blodi, Frederick²; Shankar, Malini²; Bennicelli,Jeannette⁵; Bennett, Jean⁵; Bhattarai, Sajag³; Thompson, Stewart³; Drack, Arlene^3
1Program in Neuroscience and ¹Departments of Pediatrics, ³Ophthalmology & Visual Sciences and the Institute for Vision Research, and ⁴Biomedical Engineering University of Iowa & Carver College of Medicine, Iowa City, IA, ⁵ F.M. Kirby Center for Molecular Ophthalmology/Scheie Eye Institute, University of Pennsylvania and Children's Hospital of Philadelphia, Philadelphia, PA

Purpose: To help improve Rpe65 gene therapy for Leber's congenital amaurosis (LCA), we examined its effectiveness at the resolution of single cells and retinal circuits. Here we report substantial improvements in retinal ganglion cell (RGC) responsiveness to light in vitro, but also persistent background hyperactivity that is prevented only by early treatment. This may help explain the superior response of children in early clinical trials.

Methods: We recorded RGC activity in vitro using multielectrode techniques in rd12 (Rpe65-/-) mouse retinas after subretinal viral transfecting retinal pigment epithelium (RPE) cells with normal RPE65 (AAV2/1-hRPE65). We compared spontaneous and light-evoked activity in treated and untreated eyes 7-90 days following injection, using full field flashes or pseudorandomized checkerboards to map receptive fields.

Results: In retinotopic regions receiving gene therapy, RGC responses were robust, including multiple recognizable response types and reliable receptive field maps in many (but not all) cells. No light-evoked responses were detected in untreated eyes. Spontaneous hyperactivity equivalent to that in untreated eyes persisted after gene therapy, unless treatment began at P4.

Conclusions: In vitro multielectrode recording evaluates the effectiveness of gene therapy at high resolution not possible in human patients. This provides detailed understanding of mechanisms underlying imperfect treatment responsiveness, to guide further improvements in treatment. Our current study suggests that spontaneous hyperactivity corrupts the neural code of RGCs, decreasing the precision of some RGC responses and limiting gene therapy's effectiveness unless it is initiated sufficiently early.

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Differential role of inositol 5-phosphatases in Primary Cilia Formation in Lowe syndrome

Sun, Yang; Conwell, Michael; Luo, Na
Glick Eye Institute, Department of Ophthalmology, Indiana University, Indiana

Purpose: Inositol phosphatases are important regulators of cell signaling, polarity, and vesicular trafficking. Mutations in OCRL, an inositol polyphosphate 5-phosphatase, result in Oculocerebrorenal syndrome of Lowe?an X-linked recessive disorder that presents with congenital cataracts, glaucoma, renal dysfunction and mental retardation. We investigated the role of OCRL and INPP5B in cilia formation in Lowe syndrome.

Methods: Using human eyes, we examined the distribution of OCRL and INPP5B in trabecular meshwork, lens epithelium, and ciliary body epithelium. We also examined the subcellular distribution of INPP5B by immunofluorescence in HTM and RPE cells. Using zebrafish embryos, antisense morpholinos against INPP5B and OCRL were used to examine the function of these 5-phosphatases in cilia formation.

Results: We have showed that endogenous OCRL is localized in the trabecular meshwork and Schlemm?s canal endothelial cells in human and murine eyes. We also showed that INPP5B, a paralog of OCRL, is expressed in low levels in human trabecular meshwork but is highly expressed in murine trabecular meshwork. We show that the knockdown of INPP5B resulted in a decrease in primary cilia formation. In a zebrafish model, INPP5B knockdown morphants showed defective cilia formation is found in Kupffer vesicles and cilia-dependent melanosome transport assays.

Conclusions: OCRL and INPP5B are differentially expressed in the human and murine eyes, which may explain the phenotype of glaucoma and cataract pathogenesis in Lowe syndrome.

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RNASeq of tumor progression in the zebrafish lncRNAis18 retinoblastoma-like tumor model

Syrkin-Nikolau, Judith; Severin, Andrew,; Essner, Jeffrey J.; Sakaguchi, Donald S.; McGrail, Maura
¹Department of Genetics, Development and Cell Biology, and ²Genome Informatics Facility, Iowa State University, Ames, IA

Purpose: We previously isolated a transgenic zebrafish line in which disruption of a novel long noncoding RNA lncRNAis18 leads to homozygous lethality and tumor formation in heterozygous adults. To gain insight into the changes in gene expression and genetic pathways that correlate with retinal tumor onset and progression we performed RNASeq on wild type and mutant retina.

Methods: RNASeq was performed on three conditions from age matched zebrafish siblings: wild type sibling retinas, pretumorous lncRNAis18 retinas, and lncRNAis18 retinal tumors. For each condition three isolated retinas or tumors were dissected and pooled for total RNA isolation. Total RNA was submitted to the Duke University Genome Sequencing and Analysis Core for RNASeq library construction and Illumina 100bp paired end sequencing. The sequences were mapped at the Genome Informatics Facility at Iowa State University, normalized, and changes in gene expression analyzed using Excel, DAVID, and Pathway Studios programs.

Results and Conclusions: RNASeq analyses of advanced lncRNAis18 retinal tumors are consistent with our immuncytochemical characterization using retinal neural markers, indicating similarity to human retinoblastoma. We were able to detect an increase in proliferation and stem cell markers (ie. nestin) and trends in specific pathways as retina progress from pretumor to tumor. In particular, significant changes in gene expression were detected in genes required for Muller glia activation and progenitor proliferation (ie. insulinoma 1a, heparin-binding EGF, ascl1a, dkk1b). The data is suggestive that some population of tumor cells might originate from Muller glia-derived progenitors. Additional pathway analyses will be presented.

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Autophagic machinery participates in photoreceptor outer segment phagocytosis in retinal pigment epithelial cells

Tan, Li Xuan; Xu, Jin; Toops, Kimberly; Lakkaraju, Aparna
University of Wisconsin-Madison, Department of Ophthalmology and Visual Sciences

The purpose of our study is to (a) investigate whether components of the autophagic machinery such as LC3 participate in the clearance of phagocytosed photoreceptor outer segments in retinal pigment epithelial (RPE) cells, and (b) determine how this is affected in cells with the lipofuscin fluorophore A2E. Accumulation of A2E in the RPE is a hallmark of both aging and retinal diseases such as age-related macular degeneration (AMD). Transcription factor EB (TFEB) is a master regulator of lysosome function and autophagy: cytosolic TFEB senses lysosomal status and translocates to the nucleus to induce transcription of lysosomal and autophagy genes. We used immunofluorescence staining and confocal microscopy to show that LC3 is recruited to phagosomes when polarized primary porcine RPE cells are fed with photoreceptor outer segments. Consistent with this data, Western blot analysis showed an increase in LC3-I to II conversion with time after a 2-hour pulse of photoreceptor outer segment feeding, and an increase in nuclear translocation of TFEB. These data suggest that RPE cells utilize autophagic machinery in processing phagocytosed outer segments. In RPE cells treated with A2E, LC3I-II conversion and TFEB translocation are impaired, suggesting that the pathology of AMD could be tied to inefficient lysosome function and autophagy, although whether or not this affects clearance of outer segments remains to be determined.

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Ca2+-induced Ca2+ release (CICR) promotes sustained release of synaptic vesicles at non-ribbon sites in rods.

Thoreson, Wallace B. Chen, Minghui
Departments of Ophthalmology & Visual Sciences, Pharmacology & Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE

Purpose:Light-evoked voltage responses of photoreceptors are encoded by the rate of vesicle release. Depolarization (e.g., in darkness) triggers rapid release of vesicles from synaptic ribbons. Sustained release from rods involves non-ribbon release sites and Ca2+-induced Ca2+ release (CICR). We investigated mechanisms of sustained release.

Methods: We used total internal reflection fluorescence microscopy (TIRFM) to visualize terminals loaded with fluorescent Ca2+ indicator dyes and synaptic vesicles loaded with dextran-conjugated pH-sensitive rhodamine (pHrodo).

Results: Non-ribbon release, whether triggered by sustained depolarization or activation of CICR with ryanodine, was blocked by inhibiting CICR with dantrolene. However, ryanodine-triggered release was not blocked by inhibiting Ca2+ channels with Co2+ indicating that CICR triggers release directly. Fluorescence recovery after photobleaching of endoplasmic reticulum (ER)-tracker dye in rod terminals showed that the ER extends continuously from synaptic terminal to soma. After loading ER with a low affinity Ca2+ dye, we observed that long depolarizing steps released Ca2+ from terminal ER without detectably depleting Ca2+ from somatic ER. ER in the soma may therefore provide a store of Ca2+ that can replenish Ca2+ extruded from terminal ER and thereby sustain release in darkness.

Conclusion: By increasing the rate of release in darkness, CICR-stimulated release at non-ribbon sites amplifies decrements in release that occur when rods hyperpolarize to light. Non-ribbon release may also lower synaptic noise by diffusional filtering of glutamate to post-synaptic glutamate receptors. These effects can enhance post-synaptic detection of changes in luminance contrast.

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Isoform-specific actions of human apolipoprotein E in the retinal pigment epithelium

Toops, Kimberly; Xu, Jin; Lakkaraju, Aparna
Department of Ophthalmology and Visual Sciences, University of Wisconsin - Madison, Madison, Wi

Purpose: The cholesterol transporter apolipoprotein E (ApoE) occurs in humans in three isoforms (E2, E3, E4) that vary in two amino acids but have different conformations, stabilities and interactions with lipids and proteins. In age-related macular degeneration (AMD), the E2 isoform is associated with increased risk while the E4 isoform is associated with increased protection. The mechanisms behind these differences in risk are not understood. Our studies aim to understand the interplay between ApoE isoforms, lipofuscin, cholesterol levels, and regulation of proteins in both the cholesterol homeostasis and APP pathways in the retinal pigment epithelium (RPE).

Methods: Primary porcine RPE cells were transfected with fluorescently tagged human ApoE2, E3 or E4. Cells were treated with the lipofuscin component A2E. Cholesterol was measured biochemically and by filipin staining. Expression of ApoE and other proteins was measured by immunoblot. Live imaging was performed using spinning disk confocal microscopy.

Results: Expression of ApoE isoforms in RPE influences cholesterol storage and expression of proteins in both the cholesterol homeostasis and APP pathways in response to A2E. ApoE trafficking is modulated by intracellular cholesterol and lipofuscin content in the RPE in an isoform-specific manner.

Conclusions: ApoE genotype is a genetic risk factor for AMD that has been identified in numerous studies. Little is known about how the different ApoE isoforms function in the RPE. Understanding how ApoE isoforms act in the RPE and impact processes like cholesterol homeostasis will provide valuable information about how AMD develops and likely help identify new treatment approaches.

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Using human patient specific iPSC derived eye cups to interrogate and treat inherited retinal degeneration

Tucker B.A., Mullins R.F., Drack, A., Anfinson K., Kaalberg E., Stone E.M.
Institute for Vision Research, University of Iowa, Iowa City, IA

Purpose: Usher Syndrome is an autosomal recessive disorder characterized by early onset sensorineural hearing loss and retinitis pigmentosa (RP). Mutations in the USH2A gene are the most common cause of both usher syndrome type II and non-syndromic USH associated RP. As USH2A is a large highly polymorphic gene, our ability to functionally confirm potential new disease-causing mutations in retinal cells prior to development of gene and/or cell based therapies is critical. This is especially true for non-exomic mutations whose pathogenicity is difficult or even impossible to confirm at the DNA level. By combining next generation sequencing and iPSC technologies, this study was designed to determine whether mutations identified in the USH2A gene were reflected in the retinal transcript and as such were truly disease causing.

Methods: iPSCs were generated via transduction of human keratinocytes, obtained from patients with USH2A-associated retinitis pigmentosa, using the transcription factors Oct4, Sox2, C-Myc and KLF4. iPSC potency was analyzed via ICC, WB, and teratoma formation. Capacity for retinal differentiation was determined via ICC, rt-PCR, and WB analysis targeted at detecting the markers recoverin, rhodopsin, ROM-1, R/G opsin, B-opsin, and tubulin. To determine the capacity for cellular integration and photoreceptor maturation, subretinal transplantation into CRB1 mice was performed. Pathogenicity of USH2A mutations was determined via rt-PCR targeted against USH2A.

Results: iPSCs generated from a patient with suspected non-syndromic USH2A associated RP were generated. Following differentiation, eyecups containing both RPE and neural retina were generated. Isolation and expansion/analysis of the pigmented and non-pigmented layers of patient specific eye cups allowed us to confirm RPE and photoreceptor cell lineage. Likewise transplantation of the neural retina into CRB1-null mice gave rise to newly generated photoreceptor cells. Analysis of the USH2A transcript within the neural retina revealed that a suspected splice site mutation within IVS40, induced exonification of the intron and insertion of a premature stop codon. In addition, a novel mutation identified via next generation whole exome sequencing was also confirmed within the iPSC derived neural retina of the patient.

Conclusions: By combining deep sequencing and induced pluripotent stem cell (iPSC) technologies we have been able to definitively demonstrate the pathogenicity of two disease-causing mutations in a patient with late onset non-syndromic USH2A associated RP. These findings have enabled us to proceed with patient specific studies focused on USH2A gene correction and photoreceptor cell replacement.

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Effect of timolol maleate on pressure, pupil diameter, and heart rate in normal and glaucomatous cats

Voss, A.M.¹,Bowie, O.R.¹, Free, B.R.¹, Yaccarino, V. ¹ McLellan, G.J.^1,2,3
1Department of Ophthalmology & Visual Sciences, School of Medicine and Public Health, ²Department of Surgical Sciences, School of Veterinary Medicine, and ³McPherson Eye Research Institute, University of Wisconsin-Madison, WI

Purpose: To determine effects of topical timolol maleate gel forming solution (GFS) administered once daily on intraocular pressure (IOP), pupil diameter (PD), and heart rate (HR) in normal and primary congenital glaucoma (PCG) cats.

Methods: One day prior to treatment and on the first and last days of a week-long treatment phase, IOP was measured q2h for 14h by rebound tonometry, and PD and HR were measured q4h in 18 adult cats (8 normal, 10 PCG). During the first treatment phase, a single drop of 0.5% timolol maleate GFS (Alcon Laboratories, Inc, Fort Worth, TX) was applied at 8am to one randomly assigned eye. The fellow eye served as an untreated control. In a second treatment phase, a single dose was administered at 8pm. Slit lamp examinations were performed prior to and following the treatment phases. For each phase, mean IOP, PD and HR at each time-point, and between treated and untreated eyes were compared by repeated measures ANOVA, with Tukey-Kramer post-hoc test, with p<0.05 considered significant.

Results: Timolol maleate 0.5% GFS had an inconsistent effect on IOP in the treated eye, with maximum IOP-lowering effect (mean = 5.6mmHg, 17.4%) observed 6h post-treatment in PCG. The drug caused significant miosis in the treated eye (from 4-8h post treatment) but had no significant effect on HR or ocular irritation scores.

Conclusions: Based on the results of this study, once daily application of timolol maleate 0.5% GFS is unlikely to be of clinical benefit in the management of feline glaucoma.

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Positive and Unlabeled Learning for Prioritization of Retinal Dystrophy Genes

Wagner, Alex^1,2; Taylor, Kyle³; DeLuca, Adam²; Mullins, Robert²; Casavant, Thomas^2,3; Scheetz, Todd^1,2; Stone, Edwin²; Braun, Terry^1,2;
Departments of ¹Biomedical Engineering, ²Ophthalmology and Visual Sciences, and ³Electrical and Computer Engineering, The University of Iowa, Iowa City, IA

Purpose: Diseases of the retina are complex disorders that are caused by numerous genetic factors. Identifying the genetic factors contributing to the disease phenotype for a patient can lead to a greater understanding of disease progression, heritability, and potential treatments. While we are able to utilize second-generation sequencing to identify potential disease-causing variants in the exome, often the number of variants identified is great. This study integrates datasets representing a diverse, quantitative, and informative feature set to prioritize identified variants from exome sequencing of patients with retinal degenerative disorders.

Methods: Analyses of ChIP-seq from mouse retina, mRNA-seq from human retina and sixteen other body tissues, gene characteristics such as length and exon count, and microarray data from ten tissues of the eye are used as a basis for our features. Features that best characterize the set of known retinal disease associated genes are identified using machine learning techniques and used to calculate the probability of association between any gene and a retinal degenerative disorder in an unbiased manner.

Results: We observe a highly significant enrichment for previously characterized disease genes using our prioritization method. This method surpasses existing generalized prioritization techniques. Additionally, we prioritized a list of 33 variants identified in a recent exome sequencing study and found that the causative gene (DHDDS) was prioritized to the top of the list.

Conclusions: These results together demonstrate that this system is able to leverage supplemental, quantitative data to effectively prioritize retinal disease genes in candidate lists.

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Regional Spectral-Domain Optical Coherence Tomography Features Better Predict Frisén Scale Grades than Total Volume Alone in Papilledema

Wang, Jui-Kai; Miri, Mohammad Saleh; Kardon, Randy H.; Garvin, Mona K.
Center for the Prevention and Treatment of Visual Loss, Veterans Administration Hospital, Iowa City, IA; Electrical & Computer Eng, University of Iowa, Iowa City, IA

Purpose: We have recently demonstrated a high correlation between total retinal volume measured from SD-OCT volumes of the optic nerve head (ONH) and expert-defined Frisén scale grades in papilledema subjects (Wang et al., IOVS, June 2012). However, the relationship between region-based volumetric features and Frisén scale grades was not assessed. The purpose of this work is to determine the extent that a machine-learned combination of region-based volumetric SD-OCT features can predict Frisén scale grades in papilledema.

Methods: Seventy ONH-centered SD-OCT volumetric scans (Cirrus, Carl Zeiss Meditec, Inc., Dublin, CA; 200x200x1024 voxels; 6x6x2 mm³) of 22 papilledema subjects with available expert-defined Frisén scale grades were retrospectively obtained. After applying our automated 3-D graph-theoretic approach to segment the inner limiting membrane and lower boundary of the retinal pigment epithelium (Figure 1), the following eight features were computed on each volumetric scan. Feature 1 was the total retinal (TR) volume. Features 2 and 3 were the mean circular RNFL and TR thickness, respectively. Feature 4, 5, 6, and 7 were the mean volume of nasal, superior, temporal, and inferior region, respectively (Figure 1). Feature 8 was the ratio of temporal to nasal regional volume. A random forest classifier was used to predict Frisén scale grades, with a sequential forward selection approach being used to select the optimal set of features.

Results: The mean values of the regional features at each grade are reported in Figure 2. When using TR volume alone (but not any other features), the accuracy rate (AR) of Frisén scale prediction was 38.57% and the mean Frisén grade difference (MGD) was 0.657; however, the AR and MGD improved to 62.86% and 0.414, respectively, when a combination of features was used (either feature set 4, 5, 8 or feature set 2, 3, 4, 6).

Conclusions: A machine-learned combination of region-based volumetric features from SD-OCT is better than volume alone for predicting Frisén scale grades in papilledema.

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Efemp1R345W causes increased Melanopsin responses to light that impact irradiance-responses and form-vision

Welder, Chris ^1,2; Blodi, Frederick^2,3; Stasheff, Steven^2,3; Mullins, Robert^1,2; Stone, Edwin^1,2; Thompson, Stewart^1,2
1Department of Ophthalmology and Visual Sciences, ²Institute for Vision Research, ³Department of Pediatrics, University of Iowa, Iowa City, IA

Purpose: EFEMP1-R345W causes outer retina degeneration in humans, but outer retina structure and function is normal in Efemp1-R345W mice. Surprisingly, negative masking responses, mediated by melanopsin expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), are induced at much lower light levels in Efemp1R345W mice. We therefore hypothesized that Efemp1R345W causes an abnormal melanopsin response to light that increases irradiance response sensitivity and potentially affects form-vision.

Methods: We used multi-electrode array recording of RGC responses to light and immunohistochemical assessment of melanopsin expression to investigate the form and mechanism of any abnormal melanopsin function. To further investigate effects of Efemp1R345W on irradiance-responses and form vision, we recorded the pupillary light reflex and visual function measured by optokinetic tracking responses

Results: There was a marked increase in the number of cells having melanopsin-generated responses to light, but the time course of responses to light suggested many of these cells were not ipRGCs. However, melanopsin expression was restricted to a similar number of discrete RGCs in Efemp1R345W mice. This abnormal retinal function was associated with a deficit in contrast sensitivity but not in visual acuity to high contrast stimuli. Analysis of pupil responses is ongoing.

Conclusions: Our findings suggest Efemp1R345W causes melanopsin responses to be amplified, and then propagated to other types of RGC. This abnormal melanopsin response accounts for increased sensitivity to light in irradiance responses. The abnormal melanopsin response is also likely to at least partially cause the deficits in contrast sensitivity.

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Altered transcript expression in AMD affected macular RPE/choroid suggests vascular loss

Whitmore, S. Scott; Braun, Terry; Skeie, Jessica; Haas, Christine; Sohn, Elliott; Stone, Edwin; Scheetz, Todd; Mullins, Robert
The University of Iowa¹

Purpose: We used microarrays to assess transcriptomic changes in macular retinal pigment epithelium (RPE)/choroid impacted by early AMD.

Methods: Transcription in the macular RPE/choroid from AMD affected (n=9) and control (n=9) eyes was evaluated using Affymetrix Human Exon 1.0 ST microarrays. Array data was RMA normalized (AltAnalyze), batch corrected (ComBat), and assessed for quality (arrayQualityMetrics). Two control arrays were flagged as outliers and removed from downstream analyses. Differential expression analysis (AltAnalyze), functional annotation (DAVID), and gene set enrichment analysis (GenePattern) were performed.

Results: Twenty-three genes showed increased expression in AMD (fold change > 1.5; raw p < 0.01). Of 52 genes with decreased expression in AMD (fold change < 0.5; raw p < 0.01), 18 genes were identified by DAVID analysis as associated with vision or neurological processes. No genes remained statistically significant after multiple hypothesis testing correction (Benjamini-Hochberg adj. p < 0.01). However, gene set enrichment analysis of previously identified RPE-specific and endothelium-associated gene sets indicated that endothelium-associated genes were significantly downregulated in AMD affected samples (FDR q = 0.025; FWER p = 0.021).

Conclusions: Higher expression of retina-specific transcripts in control samples may be an artifact of the dissection protocol. Alternatively, this observation may be due to reduced retina-RPE adhesion in early AMD. Reduced expression of the endothelium-associated gene set in early AMD suggests loss of choroidal microvasculature, consistent with previous clinical, proteomic, and histological studies.

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Characterization of Intraocular Pressure in Spontaneous Feline Glaucoma Model

Yaccarino, Vince¹; McLellan, Gillian^1,2; Adelman, Sara ³, Ben Shlomo, Gil⁴; Ellinwood, N. Matthew^3
1Ophthalmology & Visual Sciences, University of Wisconsin-Madison, Madison, WI; ²University of Wisconsin Eye Research Institute, Madison, WI; Departments of ³Animal Sciences and ⁴Veterinary Clinical Sciences, Iowa State University, Ames Iowa.

Purpose: The purpose of this study was to characterize trends in intraocular pressure (IOP) exposure in a colony of cats with recessively inherited primary congenital glaucoma (PCG).

Methods: Twenty seven cats were studied (9 normal and 18 with PCG). Once a week for periods ranging from six months to two years, IOPs of each eye were measured using a TonoVet® rebound tonometer. Data were analyzed to determine the mean, maximum, minimum, range, and the cumulative IOP (area under the IOP vs. time curve) for each cat over six month intervals throughout the study period. Data from glaucomatous and control cats were compared by student t-tests. Paired t-tests were also conducted to compare data from right and left eyes. In a separate experiment, IOP was measured in 9 normal and 10 PCG kittens 2-3 times/ week from the time of eyelid opening until 10-12 weeks of age. Data was analyzed as for adult cats, but over 1 week intervals.

Results: There was no significant difference between IOPs in right and left eyes. Glaucomatous cats had significantly higher IOPs (mean = 36.1+/- 10.46.mmHg) than age-matched normal cats (mean = 17.7 +/- 2.01 mmHg) over all age groups. PCG cats exhibited three distinct trends: a sustained high mean IOP with moderate fluctuations; a high mean IOP with large fluctuations, or an increasing IOP over time.

Conclusions: Our study confirmed that the congenital glaucoma in this research model is associated with a bilaterally symmetrical and persistent elevation in IOP that begins early in life.

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Modulation of Retinal Neovascularization by Complement C3a and C5a via Vascular Endothelial Growth Factor

Wei Zhu^1,2, Xia Qi², Bin Wang², Yiqiang Wang^2
1Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, IA; ²Shandong Provincial Key Lab of Ophthalmology, Shandong Eye Institute, Qingdao University, China

Purpose: A large body of evidence has proven that the complement cascade is involved in pathogenesis and modulation of choroidal neovascularization, but its potential role in retinal neovascularization (RNV) is underestimated. The aim of this study is to assess whether complement activation is involved in RNV, and, if so, to characterize the potential mechanism involved.

Methods: Oxygen-induced retinopathy (OIR) was induced in wild type C57Bl/6, C3aR and C5aR deficient mice on the same genetic background. In some experiments, complement inhibitor FUT-175 was used before initiation of OIR. Fluorescein angiography, retinal wholemounts and hematoxylin-eosin staining were used to assess retinal angiogenesis. Western blot and immunofluorescence staining were used to assess anaphylatoxins (C3a or C5a) and their receptors in the retina respectively. To explore whether the vascular endothelial growth factor (VEGF) pathway is involved in OIR, VEGF levels were measured using western blotting. To correlate the findings made in the OIR model to diabetic retinopathy, C3a/C5a and their receptors were also assessed in congenital diabetic mice (Ins2Akita strain).

Results: The expression of C3a, C5a and their respective receptors (C3aR and C5aR) was found to be up-regulated in the retina of C57Bl/6 upon OIR induction and one year old Ins2Akita mice when compared with age-controlled mice. The central avascular area in OIR C3aR-/- and C5aR-/- mice was markedly enlarged when compared to C57Bl/6 mice at day17 (P17) as detected by retinal flat mounts. Blood vessel tortuosity and tufts were underdeveloped in C3aR-/- and C5aR-/- mice relative to wild-type mice upon OIR induction. Western blot analysis revealed reduced VEGF-A levels in OIR C3aR-/- and C5aR-/- mouse retina at P17 relative to wild-type mice. Pretreatment of C57BL/6 mice with FUT-175 immediately before OIR induction was found to effectively suppress the expression of VEGF-A and attenuate the central avascular area, vascular leakage and the generation of new blood vessels at P17.

Conclusions: Complement C3a and C5a positively regulate RNV by modulation of the VEGF signaling pathway. Consequently, C3a and C5a may serve as novel therapeutic targets for management of RNV-related diseases.

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