2015 MERS poster
MERS Logo

9th Annual

2016 Midwest Eye Research Symposium

Sponsored by The Stephen A. Wynn Institute for Vision Research


MERS 2016 took place on August 5th, 2016 at
The University of Iowa Medical Education and Research Facility


Keynote Speaker/Session Chairs

Terri L. Young, M.D., M.B.A. - Keynote Speaker
Dr. Young is the Peter A. Duehr Professor and the department chair of the University of Wisconsin Department of Ophthalmology and Visual Sciences Ophthalmology. Her research interests include ophthalmic genetics, refractive error genetics, and pediatric ophthalmology clinical studies. She has published well over 200 manuscripts, served on numerous editorial boards, NIH study sections, and received the American Academy of Ophthalmology Honor Award as well as many other honors.

Simon Petersen-Jones, DVet Med, Ph.D., DECVO - Chair, Session I
Dr. Petersen-Jones is a Professor and the Myers-Dunlap Endowed Chair in Canine Health at the Michigan State University College of Veterinary Medicine. He is a veterinary ophthalmologist with an interest in retinal dystrophies, hereditary eye diseases, and therapeutic aproaches for these conditions. He has written numerous book chapters and scientific articles on different aspects of veterinary ophthalmology and lectured in many countries around the world.

Rudolf Fuchshofer, Ph.D. - Chair, Session II
Dr. Fuchshofer is an Assistant Professor at the Institute of Human Anatomy and Embryology, University of Regensburg, Germany. He is currently on sabbatical working with Prof. Mark Johnson at Northwestern University, Chicago. His main research interests are the molecular functions of growth factors in the pathogenesis of glaucoma.

Luke Wiley, Ph.D. - Chair, Session III
Dr. Wiley is a Research Assistant Professor in the Department of Ophthalmology and Visual Sciences at the University of Iowa. Dr. Wiley uses patient-derived iPSCs to model rare inherited eye diseases, including Batten disease, Stargardt disease, and Leber congenital amaurosis. He also serves as the Director of Functional Analysis for the Dezii Translational Vision Research Facility at the University of Iowa, assisting in the generation of clinical-grade adeno-associated viral vectors and transplantable human photoreceptor precursor cells that are intended to be used for gene and cell replacement therapies in patients with blinding inherited eye disease.

 


Oral Presentations

Session I - Session Chair Simon Petersen-Jones, DVet Med, Ph.D., DECVO

Simon Petersen-Jones:Gene augmentation therapy in a large animal model of CNGB1 retinitis pigmentosa
Wesley Goar:Analysis of three Israeli families with Keratoconus
Ying Hsu:Age-dependent rescue efficiency of retinal function in a mouse model of Bardet-Biedl Syndrome
Alison Huckenpahler:Imaging the Adult Zebrafish Cone Mosaic with Optical Coherence Tomography

Session II - Session Chair Rudolf Fuchshofer, Ph.D.

Rudolf Fuchshofer:The role of Decorin in primary open angle glaucoma
Margaret Maes:Investigating the longitudinal and kinetic profiles of BAX recruitment in mouse retinal ganglion cells
Uttio Roy Chowdhury:Effect of long term reduction of intracranial pressure on retinal ganglion cells and optic nerve axons in a rat intraventricular cannula model
Paul Kaufman:The onset of glaucoma may be related to the decline of the accommodative apparatus via the choroid network connection

Session III - Session Chair Luke Wiley, Ph.D.

Luke Wiley:Modeling Enhanced S-cone Syndrome Using Patient-Specific iPSC-derived Retinal Eyecups
Allison Songstad:Connective Tissue Growth Factor (CTGF) promotes efficient generation of human iPSC-derived choroidal endothelium
Akshayalakshmi Sridhar:Developmental Origins of Three Dimensional Retinal Organoids Derived from Human Pluripotent Stem Cells
Lauren Laboissonniere:Investigating transcriptomes of developing chick retinal cells

 

Poster Presentations

Michael AndersonInhibition of mitochondrial fission in glaucoma neuroprotection
Anisha AroraFeasibility of Photosensitivity Measurements within Healthy Subjects using the 2nd Generation BPEI Photosensitivity Tester
Lindy BrastromZebrafish as a model for eye disease: congenital cataracts and aging related impairment
Ping BuPaeoniflorin preserve retinal function on retinal ischemic-reperfusion injury
Erin BurnightCRISPR/Cas9-mediated genome editing for correction of inherited retinal disease mutations
Kathleen ChircoPreparation of Human Choroidal ECM Scaffolds to Study Cell Replacement Strategies
Subramanian DharmarajanBMP-7 Activation of Microglia Drives Reactive Retinal Gliosis
Ryan DonahueComparison of Transcriptomes of Models of Optic Nerve Injury
Laura DutcaInterventions to lower nocturnal intraocular pressure
Jennifer FaralliSecondary Glucocorticoid Response and Sustained Elevated IOP Absent in Mice treated with Dexamethasone
Clarisse FligorAxonal Outgrowth and Regeneration From Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells
Sarah GardnerCalcium Channels in Synapotogenesis
Rachel GenovaTherapeutic efficacy and mechanism of (-)-P7C3-S243 and P18C8 coadministration in a model of chemical corneal injury
Joseph GiacaloneGenome editing and disease modeling of RPGR-associated retinitis pigmentosa
Ankur JainCRISPR-cas9 Based Treatment of Myocilin-associated Glaucoma
Chunhua JiaoNovel organotypic culture model of pig choroid-scleral explant as a therapeutic screening tool
Colten LankfordIdentification of HCN1 as a novel 14-3-3 client protein
Daniel LeeRelationship Between The Henle Fiber Layer And Foveal Specialization
Danielle PellackDefining the time course of optic neuropathy and ganglion cell loss in a mouse model of early-onset glaucoma
Tania PikeiPSC-derived TM cells promote proliferation of primary TM cells
Brandon ReaUse of facial analysis to assess CGRP-induced discomfort during migraine
Devasis ReddyPyogenic Granulomas of the Lacrimal Sac: A clinicopathological survey
Alexander SalmonAutomated Reference Frame Selection (ARFS) for Registration of Scanning Ophthalmoscope Image
Heather SchmittTargeting HDAC3 activity prevents retinal ganglion cell nuclear atrophy and apoptosis after optic nerve injury
Levi SowersInvestigation of neuronal circuits underlying light-aversive behavior
Nicole TatroExtracting Phenotypes From Historic Goldmann Visual Fields
Jessica ThompsonOptimizing the photopolymerization parameters of high-resolution, 3D biodegradable tissue scaffolds
Matthew Van HookPresynaptic mGluRs regulate cone photoreceptor synaptic transmission by direct interactions of G beta-gamma and SNAP-25
S. Scott WhitmoreSurveying the human ocular transcriptome, one cell at a time
Kristan WorthingtonHuman Retinal Engineering using 3D Two-Photon Polymerization and Chitosan Hydrogels

Abstracts


Inhibition of mitochondrial fission in glaucoma neuroprotection

Anderson, Michael G.1,3, Strack, Stefan1,2, Kyle H. Flippo2, Danielle R. Pellack3, Kacie J. Meyer3, Carly J. Lewis3, Adam Hedberg-Buenz1,3, Laura M. Dutca1
1Center of Excellence for Prevention and Treatment of Visual Loss, Iowa City Veterans Administration Medical Center 2Department of Pharmacology, University of Iowa, Iowa City, IA 3Department of Molecular Physiology and Biophysics and Stephan A. Wynn Institute for Vision Research, University of Iowa, Iowa City, IA


Purpose: Emerging evidence suggests age-associated mitochondrial dysfunction contributes to neurodegeneration in glaucoma by decreasing the metabolic reserve for repair processes and by predisposing retinal ganglion cells (RGCs) to apoptosis. We, and others, hypothesize that one aspect of this dysfunction might involve altered mitochondrial fission/fusion events. Other groups have illustrated inhibition of DRP1, a ubiquitously expressed enzyme required for mitochondrial fission, rescues RGC death in a mouse model of glaucoma. The purpose of our current pilot project is to investigate whether deletion of a neuron-specific upstream DRP1 activator, the mitochondria-localized Bβ2 regulatory subunit of protein phosphatase 2A, might also confer neuroprotection and ultimately have a higher probability for successful clinical translation.

Methods: Our approach utilizes two key genetic models, nee mice which develop early onset glaucoma and mice with a targeted disruption of the gene encoding Bβ2.

Results and Conclusion: Glaucoma in nee mice develops as a consequence of a mutation in the Sh3pxd2b gene leading to anterior segment dysgenesis and malformation of aqueous humor outflow pathways. As we present in a concurrent abstract, the time course and severity of glaucoma in nee mice make them an ideal resource for highly powered studies of neuroprotection. Bβ2/PP2A dephosphorylate DRP1 at an inhibitory phosphorylation site accelerating mitochondrial fission. We have generated a Bβ2 knockout (KO) strain of mice and previously shown that Bβ2 disruption is neuroprotective in other disease contexts, including diabetic peripheral neuropathy. We are currently crossing the nee and Bβ2 KO mice to investigate whether disruption of β2 is similarly protective in RGCs against early onset glaucoma.

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Feasibility of Photosensitivity Measurements within Healthy Subjects using the 2nd Generation BPEI Photosensitivity Tester

Arora, Anisha, Langlo, Christopher S.,Carroll, Joseph1
1Medical College of Wisconsin, Department of Ophthalmology


Purpose: Increased sensitivity to light is a hallmark feature of achromatopsia. Thus, the ability to quantify this threshold could be used as an outcome measure in patients receiving gene therapy. Here we sought to assess the feasibility of using the 2nd generation BPEI Photosensitivity Tester for functional evaluation in clinical trials.

Methods: Eleven healthy subjects were tested with enhanced and normal modes in the Photosensitivity Tester. The enhanced mode uses smaller step-sizes and is intended for photosensitive patients while the normal mode has large step-sizes intended for more general use. To assess repeatability within subjects across modes and multiple time-points, subjects were tested using a randomly-selected mode, and then re-tested one hour later using the alternate mode. The reciprocal test-retest procedure was repeated between 20 and 47 hours later. Thresholds and duration were recorded for each test.

Results: Normal mode average threshold was 976.84 lux taking an average of 5.50 minutes, and the enhanced mode was respectively 420.21 lux and 10.18 minutes. These differences were statistically significant (p<0.0001). The enhanced mode Pearson correlation coefficient across two time-points was 0.88, whereas the normal mode was 0.40.

Conclusions: Since the enhanced and normal modes yield statistically significant threshold values, these protocols cannot be used to reliably determine photosensitivity differences between healthy and achromatopsia patients. However, the enhanced mode proves to be a more repeatable protocol, as threshold values were more consistent within subjects across the two time-points. Ultimately, this study shows that alternate psychophysical algorithms should be explored.

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Zebrafish as a model for eye disease: congenital cataracts and aging related impairment

Brastrom, Lindy1, Scott, Anthony1, Poulos, Cassandra1, Spatz, Lillian2, Dawson, Deborah3, Val Sheffield4, and Slusarski, Diane1
1Department of Biology, University of Iowa, Iowa City, Iowa 2Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 3Department of Pediatric Dentistry, University of Iowa, Iowa City, Iowa 4Department of Pediatrics and Ophthalmology, University of Iowa, Iowa City, Iowa


Purpose: Visual impairment is a significant public health concern. As bioinformatics approaches identify unprecedented numbers of genetic variants associated with blinding disorders, models to test these variants are essential. Zebrafish pose many advantages due to similarities in morphology, gene expression, and function to the human eye. Moreover, vision can be tested in 5 days-post-fertilization (dpf) zebrafish.

Methods: Our lab has developed vision testing in zebrafish to facilitate high-throughput in vivo screens. The Visual Interrogation of Zebrafish maNipulations (VIZN) and. VIZN uses interruptions in constant light to elicit a startle response. Blind larvae do not respond to changes. To evaluate partial vision loss such as those exhibited in cataracts and glaucoma, we are developing the OptoMotor Response (OMR). In OMR, larvae move in the direction of sinusoidal waves, mimicked by stripes passing under a plate. Larvae respond by orienting their body, while vision impaired fish are less effective.

Results: We have developed an automated program for analysis using VIZN and are currently validating the OMR with cataract and glaucoma candidate genes. The goal is to combine both assays to evaluate the spectrum of visual impairments. We also evaluate retinal structure and expression domains in the developing zebrafish.

Conclusions: These assays allow for rapid screening of many candidate genes related to vision loss and hold the potential to follow disease progression.

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Paeoniflorin preserves retinal function on retinal ischemic-reperfusion injury

Algenio, Crystal1; Qiao, Mark2; Chow, Sabrina3, McDonnell, James2; Perlman, Jay2,4; Bu, Ping2
1Stritch School of Medicine, Loyola University Chicago 2Department of Ophthalmology, Loyola University Chicago 3Department of Microbiology & Immunology, Loyola University University Chicago 4Surgery Service, Edward Hines, Jr. VA Hospital


Purpose: Retinal ischemia is a common cause of visual impairment and blindness for which there remain limited treatment options. The purpose of this study is to explore the therapeutic efficacy of Paeoniflorin on retinal ischemic-reperfusion injury.

Methods: C57BL/6 mice (8 weeks old) were randomly assigned to one of two groups: vehicle-treated or Paeoniflorin-treated retinal ischemic injury mice. Retinal ischemia was induced by transient elevation of intraocular pressure for 45 minutes. Vehicle (PBS) or Paeoniflorin (20 mg/kg in PBS) was injected intraperitoneally once a day for 5 days following retinal ischemia. Scotopic electroretinography (ERG) was recorded before induction of retinal ischemia, and 7 days after inducing ischemia.

Results: Before treatment, scotopic ERG a- and b-wave amplitudes were 408 ± 50 μV and 856 ± 41 μV, respectively, in the control group, and 396 ± 43 μV and 818 ± 75 μV, respectively, in the Paeoniflorin group. Following ischemic-reperfusion injury, ERG a-wave amplitudes were 152 ± 50 μV in the control group and 307 ± 61 μV in the Paeoniflorin group. Post-ischemic insult ERG b- wave amplitudes were 332 ± 87 μV in the control group and 583 ± 74 μV in the Paeoniflorin group. Seven days after ischemic-reperfusion injury, ERG a- and b-wave amplitudes were significantly reduced in vehicle-treated mice, whereas Paeoniflorin treatment attenuated ischemic induced loss of retinal function as compared to vehicle-treated mice.

Conclusions: Our preliminary findings suggest that Paeoniflorin may have therapeutic value in the management of retinal ischemic diseases.

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CRISPR/Cas9-mediated genome editing for correction of inherited retinal disease mutations

Burnight E.R.1, Wiley, L.A.1, DeLuca, A.P.1, Oppedal, D.J.1, Scheetz, T. E.1, Mullins, R.F.1, Stone, E.M.1, and Tucker, B.A.1
1Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology & Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA


Purpose: The purpose of this study was to employ CRISPR/Cas9 genome editing to develop strategies for three major classes of disease-causing mutations: 1) dominant gain-of-function, 2) deep intronic and 3) exonic mutations.

Methods: We designed sgRNA oligos specific for each mutation and cloned them into bicistronic constructs. Donor constructs were cloned carrying ~500 bp of homologous flanking wild-type sequence. Constructs were delivered to human cells via transfection or electroporation. Non-homologous end-joining was determined via T7E1 assay, quantified using TA-cloning and Sanger sequencing.

Results: To treat patients with dominant disease, sgRNAs targeting the mutant vs wildtype allele were designed. T7E1 assay and Sanger sequencing showed that a sgRNA targeting the P23H mutation in RHO confers a 3-fold increase in targeting specificity (7.2% vs. 22.0%, respectively). A similar result was detected for the Q344X mutation in PAX6 (0.6% vs. 29.9%, respectively). To edit intronic recessive alleles, we demonstrated that co-delivery of two sgRNAs targeting upstream and downstream of the IVS40 mutation in USH2A successfully modified the locus (21.0% and 19.0%, respectively). To demonstrate the utility of our HDR strategy to correct an exonic mutation, we delivered an sgRNA targeting the MAK Alu insertion in exon 9 and a donor template carrying the corrected sequence. This construct restored WT transcript in iPSC-derived retinal precursor cells from a patient with MAK-associated RP.

Conclusions: Successful gene editing targeting three different classes of Mendelian mutations at several disease-causing loci brings us closer to the goal of providing genetically corrected autologous cell-based therapies for patients affected with inherited retinal degenerative disease.

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Preparation of Human Choroidal ECM Scaffolds to Study Cell Replacement Strategies

Chirco, Kathleen R; Riker, Megan J; Flamme-Wiese, Miles J; Songstad, Allison E; Worthington, Kristan S; Stone, Edwin M; Tucker, Budd A; Mullins, Robert F.1
1Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology & Visual Sciences, University of Iowa, Iowa City, IA


Purpose: Endothelial cells of the choriocapillaris are the first cells lost during age-related macular degeneration (AMD). Cell replacement therapy is currently the most promising option for patients with advanced AMD. Here, we sought to develop a reliable method for the production of human choroidal ECM scaffolds, which will allow for the study of choroidal endothelial cell (CEC) replacement therapies in an environment that closely resembles the native tissue

Methods: Human donor eyes were obtained and unfixed RPE/choroid was treated sequentially with 1% TX-100, 0.1% SDS, and DNase to remove all native cells. After decellularization, immunohistochemistry was utilized to compare treated to untreated punches, confirm cell removal, and assess the localization and relative abundance of the remaining ECM proteins. To further validate CEC removal, decellularized tissue ultrastructure was visualized using transmission electron microscopy (TEM).

Results: Decellularized tissue showed a loss of CD31 immunolabeling and endothelial cell-specific lectin labeling (UEA-I), as well as complete absence of the nuclear DAPI stain compared to native RPE/choroid. In contrast, the decellularized tissue exhibited preserved collagen IV, elastin, and laminin immunolabeling. TEM micrographs provided evidence for complete removal of CECs within the choriocapillaris, with ECM proteins visible within and surrounding the vessel walls.

Conclusions: Our data demonstrate the ability to remove all cells from human RPE/choroid punches while keeping the ECM largely intact. Furthermore, intact vascular walls with basal laminae are visible in the remaining ECM, suggesting that these methods are gentle enough to preserve tissue structure and allow for the testing of cell replacement strategies."

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BMP-7 Activation of Microglia Drives Reactive Retinal Gliosis

Dharmarajan, Subramanian1; Sheibani, Nader2; Belecky-Adams, Teri1
1Indiana University-Purdue University Indianapolis (IUPUI), 2University of Wisconsin, Madison


Purpose: Retinal microglia are key innate immune cells within the central nervous system. Upon injury and damage, they become "activated", undergoing characteristic molecular and morphological changes. In response to factors secreted by the activated microglia, the Müller glial and retinal astrocytes undergo reactive gliosis. Our lab has previously shown that BMP7 is able to trigger Müller cell gliosis; however, the mechanism was unclear. We hypothesize that BMP7 indirectly triggers Müller gliosis by activating microglia.

Methods: Mice were fed a chow containing drug PLX5622 (Plexxikon Inc.) to ablate microglia. BMP7 or vehicle was intravitreally injected to ablate microglia, and eyes harvested 3 or 7 days post injection. Tissue was analyzed for gliosis markers via IHC or RT-qPCR. Mouse retinal microglial cells in vitro as well as mature mice were exposed to vehicle, LPS (a known activator of microglia) or BMP7 to compare activation. Samples were analyzed by IHC, RT-qPCR or ELISA for activation markers and morphological changes.

Results: A relative increase in mRNA levels of GMCSF, IFN-γ and IL6 was observed in vitro and in vivo following BMP7 treatment. An increase in the morphological characteristics found in activated microglial cells was also observed following BMP7 treatment. BMP7 treatment of PLX treated mice showed a relative decrease in mRNA levels of gliosis and inflammatory markers.

Conclusions: Our findings indicate BMP7 triggers gliosis in Müller cells and retinal astrocytes indirectly by activating microglial cells.

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Comparison of Transcriptomes of Models of Optic Nerve Injury

Donahue, Ryan; Trane, Ralph; Nickells, Rob1
1University of Wisconsin Madison


Purpose: Microarrays and RNA-Seq have been used to characterize transcriptomes in models of optic nerve injury. We hypothesized that comparing transcriptomes from different models of optic nerve injury (ONI) and neurodegenerative (ND) conditions would yield a list of important genes and biochemical pathways that were differentially expressed across multiple insults to the central nervous system (CNS).

Methods: This analysis included 21 studies that encompassed models of acute ONI, experimental glaucoma and several ND diseases that affect the CNS. We used the Limma method to determine differentially expressed (DE) gene transcripts in each study and compared lists of DE transcripts between studies. The statistical significance of the number of shared transcripts between datasets was analyzed using Monte Carlo simulations. Lists of commonly occurring gene transcripts were analyzed for Gene Ontology term enrichment using the PANTHER tool.

Results: Comparing transcripts that were DE in the injured conditions revealed only partial, but statistically significant overlap in the transcriptomes of acute ONI, experimental glaucoma, and ND studies. Gene Ontology (GO) terms associated with these changes were associated with the stress response, leukocyte migration and the innate immune response. Among transcripts that were depleted in the injured condition, statistically significant overlap of DE transcripts was only prevalent when comparing data from the same tissue. Commonly occurring GO terms for these gene lists were related to neuronal differentiation, axonogenesis and cellular metabolism.

Conclusions: The innate immune system is involved in the response of CNS tissues to numerous injury paradigms. Neuronal gene expression is variable throughout the CNS and is the main group of genes turned off following injury.

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Interventions to lower nocturnal intraocular pressure

Dutca, Laura1,2, McDowell, Coleen3, Zhu, Wei1,2, Kuehn, Markus, H.1,2, Anderson, Michael G.1-3
1Center of Excellence for Prevention and Treatment of Visual Loss , Iowa City Veterans Administration Medical Center; Departments of 2Ophthalmology and Visual Science, 3Molecular Physiology and Biophysics, The University of Iowa, Iowa City, IA


Purpose: Increased intraocular pressure (IOP) is currently the only modifiable risk factor for glaucoma. In seeking new ways to extend IOP management in glaucoma, we investigated genetic and pharmacologic agents related to LYST and its binding partner casein kinase 2 (CK2) that specifically lower nocturnal IOP.

Methods: In experiments using mice, intraocular pressure was measured with a rebound tonometer at the midpoints of light cycle and dark cycle in C57BL/6J and B6-Lystbg-J mice. A second set of experiments involved treatment of C57BL/6J mice with a CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). IOP was measured midway through the dark cycle and in a separate experiment midway through the light cycle, for 5 days. In experiments using human organ culture, pressure and outflow facility were measured in the anterior segment of human donor eyes with and without TBB.

Results: There was no difference in average IOP between C57BL/6J and B6-Lystbg-J mice during light phase. In contrast, average IOP was lower in B6-Lystbg-J compared to C57BL/6J mice in dark phase. TBB treatment prevented elevation of IOP during dark phase with IOP levels remaining equivalent to those of the light phase. Outflow facility in human donor eyes after TBB treatment was significantly higher than initial outflow or that of untreated eyes.

Conclusions: Our results indicate that mutation in Lyst disrupts the circadian rhythm of IOP. Furthermore, treatment with TBB also decreases nocturnal IOP. The results of experiments performed with human eyes suggest that TBB increases outflow facility and indicate a possible mechanism for its action.

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Secondary Glucocorticoid Response and Sustained Elevated IOP Absent in Mice treated with Dexamethasone

Faralli, J.A; Peters, D.M.1
1Departments of Pathology & Laboratory Medicine and Ophthalmology & Visual Sciences University of Wisconsin- School of Medicine & Public Health


Purpose: To determine if αvβ3 integrin expression is upregulated in vivo following dexamethasone (DEX) treatment.

Methods: C57BL/6 mice were given topical DEX (0.1%) or PBS eye drops 3 times daily for 4-5 weeks to one eye. Intraocular pressure (IOP) was measured weekly in the mice anesthetized with a ketamine/xylazine mix using the TonoLab. After 4-5 weeks of treatment, mice were sacrificed and eyes removed. Paraffin embedded tissue was analyzed for β3 integrin expression by immunofluorescence microscopy. RT-PCR analysis was done for the expression of FKBP5, β3 integrin and myocilin.

Results: IOP was statistically significantly increased after 3 weeks of DEX compared to PBS. Interestingly, by week 5 IOP in DEX treated eyes returned to baseline even though the eyes were still exposed to DEX. All DEX treated mice appeared to respond to DEX by exhibiting a weight loss. 2 of the 3 groups of mice processed for RT-PCR, showed increased levels of FKBP5 mRNA expression in DEX treated eyes. However, no increase in myocilin mRNA was detected. By immunofluorescent microscopy, β3 integrin expression in the TM appeared unchanged or less than PBS treated mouse eyes. RT-PCR verified this and showed RNA levels for β3 integrin were similar to controls.

Conclusions: These studies suggest that a compensatory mechanism exists in mice to prevent the secondary glucocorticoid response responsible for myocilin and β3 integrin expression and that this compensatory mechanism may be involved in lowering IOP and maintaining homeostasis.

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Voted Outstanding Poster Presentation

Axonal Outgrowth and Regeneration From Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells

Fligor, Clarisse1, Ohlemacher, Sarah1, Sridhar, Akshayalakshmi1, Cooke,Jessica1, Zack,Donald4, and Meyer, Jason1-3
1Department of Biology, Indiana University Purdue University Indianapolis, 2Stark Neurosciences Research Institute, 3Indiana University Department of Medical and Molecular Genetics, Indianapolis IN and 4Ophthalmology, Johns Hopkins, Baltimore, MD


Purpose: Human pluripotent stem cells (hPSCs) possess the unique ability to differentiate into any cell type in the body, thereby allowing them to serve as a platform for the study of retinal differentiation and regeneration as well as the development of translational therapies. hPSCs are especially relevant for studies of retinal degenerative diseases and injury, where the axons of retinal ganglion cells (RGCs) are lost. In this role, hPSCs can serve as an in vitro model for axonal outgrowth and regeneration.

Methods: hPSCs were differentiated to a retinal lineage following established protocols. The ability of these cells to extend axons was examined by systematically dissociating and plating cell aggregates. After 24 hours, cells were fixed and analyzed by immunocytochemistry to quantify the extent of axonal outgrowth. To examine the factors influencing neurite outgrowth, the effects of varying substrates as well as culture medium formulations was examined and compared to control experiments.

Results: Within 40 days of differentiation, abundant RGC differentiation was observed from hPSCs. RGCs began to extended neurites within hours of plating, with noticeable increase in length by 24 hours. Differing substrates and culture media had significant effect on neurite length and number, with axonal outgrowth reaching lengths of more than 1000 microns within 24 hours.

Conclusions: Overall, these results demonstrate the utility of hPSC-derived RGCs for studies of axonal outgrowth and regeneration. These results provide a novel in vitro assay for testing the ability of RGCs to extend axons and may assist in the development of personalized transplant therapies for optic neuropathies.

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The Role of Decorin in Primary Open Angle Glaucoma

Fuchshofer, Rudolf; Schneider, Magdalena
University Regensburg


Purpose: The critical risk factor for optic nerve damage in primary open-angle glaucoma (POAG), is an increased intraocular pressure (IOP) caused by an increased outflow resistance in the trabecular meshwork (TM). The molecular pathogenesis of the increased outflow resistance has not been identified, but TGF-β2 and CTGF may be involved. Both growth factors contribute to an increased ECM synthesis and to an increased contraction rate in the TM and thereby can lead to an increased outflow resistance. In a healthy eye the growth factors are in a homeostatic balance, which seems to be disturbed in POAG. In this study we investigated the influence of Decorin (DCN), a potential endogenous antagonist of TGF-β2 and CTGF, on the expression of growth factors, ECM and actin cytoskeleton components in vitro and in vivo.

Methods: For that purpose we analyzed the DCN knockout mice in comparison to the wild-type littermates with different ages. Further we investigated the effects of DCN in vivo and in vitro on TM by real-time RT-PCR, immunoblotting, immunohistochemistry and light microscopy. IOP was measured by tonometry. The total number of myelinated optic nerve axons were counted in DCN knockout mice and wild-type littermates with different ages.

Results: The DCN treatment led to a decreased synthesis of CTGF, TGF-β1 and 2 in HTM cells, accompanied by a reduced expression of ECM proteins. We show that the deficiency of DCN increases IOP and leads to optic nerve damage in mouse eyes. These changes were associated with an induction of CTGF, fibronectin and α-smooth muscle actin in the TM of the DCN deficient animals.

Conclusions: Our results strongly indicate that DCN is a modulator of TGF- β2 and CTGF signaling in the trabecular meshwork, contributing to the homeostatic balance of growth factors. DCN could be a promising modifier of TGF-β2 and CTGF signaling in POAG.

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Calcium Channels in Synapotogenesis

Gardner, Sarah1, Laird, Joseph1, Kerov, Vasily2, Williams, Brittany2, Lee, Amy2, Baker, Sheila1
Departments of 1Biochemistry and 2Molecular Physiology & Biophysics, The University of Iowa


Purpose: Cav1.4 is a voltage-gated ion channel found in photoreceptor synapses. It is responsible for allowing Ca2+ into the cell resulting in neurotransmitter release. In addition to the crucial role of Cav1.4 in signaling, this channel is essential for the development and organization of the synapse. The purpose of this project is twofold: to test if Ca2+ conductance through Cav1.4 is required for the channel to stimulate synaptic development; and to test if Cav1.4 can stimulate synaptic development in adult photoreceptors lacking mature functional synapses.

Methods: Plasmids encoding Cav1.4 or its mutants were injected into the subretinal space of neonatal Cav1.4 KO mice and driven into rod photoreceptors using in vivo electroporation. Expression of Cav1.4 occurs just prior to synaptogenesis because it is controlled by the Rhodopsin promoter. To prevent expression of Cav1.4 until after synaptogenesis is normally complete we used a tamoxifen-inducible system. In this system a floxed-stop codon upstream of the Cav1.4 coding sequence prevents expression of the channel in the absence of Cre recombinase. Cre was also delivered by electroporation of neonatal mice but by using a fusion protein ERT2-Cre-ERT2 Cre remains inactive until the ER ligand, tamoxifen is delivered. The morphology of electroporated synapses was assessed by staining for two markers; RIBEYE the core component of the mature "horseshoe-shaped" ribbon, and PSD-95 a scaffold protein that lines the synaptic membrane. In Cav1.4 KO photoreceptors both of these markers are dramatically different: RIBEYE labels spherical immature ribbons and PSD-95 is absent. The vision of treated mice was tested using a modified water maze in which mice are trained to escape to a visible platform.

Results: Cav1.4 KO mice expressing either Cav1.4-WT or one of two mutants that cannot conduct Ca2+, "3EQ" or "G369i", develop morphologically normal synapses. Cav1.4 KO mice expressing Cav1.4-WT have vision restored as measured by the water maze. Mice expressing either of the Ca2+ conductance mutants largely failed but did do marginally better than untreated mice, indicating that there may be some residual function for these mutants. To test the efficiency of the tamoxifen inducible system for post-synaptic expression we used a reporter with a floxed-stop codon upstream of DsRED. This plasmid was co-electroporated with ERT2-Cre-ERT2 and GFP to readily visualize the electroporated cells. Systemic delivery of tamoxifen for 48-96 hours resulted in robust expression of DsRED in GFP+ cells. In control experiments lacking tamoxifen there was a low level of "leaky" expression of DsRED. A preliminary experiment using this system was conducted to induce Cav1.4 expression in Cav1.4 KO photoreceptors at one month of age, 15 days after synaptogenesis is complete in wildtype animals. Morphologically normal synapses were observed.

Conclusions: Surprisingly, Cav1.4 even with crippled Ca2+ conductance can promote synaptic development in Cav1.4 KO photoreceptors. A system to control the timing of gene expression from in vivo electroporated plasmids has been verified. Intriguingly, induction of Cav1.4 in adult Cav1.4 KO mice resulted in rescue of some synapses. This could indicate that photoreceptor synapses can be rescued even late in life but future studies are required to verify the timing of the induced Cav1.4 expression.

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Therapeutic efficacy and mechanism of (-)-P7C3-S243 and P18C8 coadministration in a model of chemical corneal injury

Genova, Rachel1,2 Harper, Matthew3,4 Pieper, Andrew3,5,6
1Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa; 2Pappajohn Biomedical Institute, University of Iowa, Iowa City, Iowa 3Iowa City Department of Veterans Affairs Center for the Prevention and Treatment of Visual Loss, Iowa City, Iowa; 4Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa ; 5Department of Neurology, The University of Iowa, Iowa City, Iowa; 6Department of Psychiatry, The University of Iowa, Iowa City, Iowa; Pappajohn Biomedical Institute, University of Iowa, Iowa City, Iowa


Purpose: Corneal injury is a common cause of visual impairment, with alkali burns representing a particularly severe form. While corneal wound healing depends on both the regenerative capacity of corneal epithelium and trophic support from corneal nerves, standard clinical approaches to corneal injury focus on promoting reepithelialization with less attention paid to reinnervation. We propose to target both epithelial and neuronal tissues of the cornea with the novel pharmacologic agents P18C8 and (-)-P7C3-S243 developed in our laboratory. We hypothesize that cotreatment with the mitogenic agent P18C8 and the neuroprotective agent (-)-P7C3-S243 will accelerate recovery in a mouse model of alkali corneal injury. We predict that (-)-P7C3-S243 will mitigate corneal nerve damage, while P18C8 will augment corneal epithelial mitogenesis, together promoting corneal repair.

Methods: We will model alkali corneal injury in C57BL/6J mice, and evaluate morphological recovery (via slit lamp imaging and immunohistochemistry) and functional recovery (via corneal sensitivity assays) in cotreated and untreated animals over 28 days. In primary cultures, we will investigate the effects of our agents on corneal epithelial mitogenesis and on neuronal cell death and axonal protection in wounding assays.

Results: Preliminary data in our mouse model demonstrate that cotreatment, but not treatment with either agent alone, significantly increases corneal reepithelialization by 6 days post-injury.

Conclusions: At the conclusion of this project, we will have characterized the efficacy and identified cellular targets of two novel pharmacologic agents in a model of corneal injury. Continued work will focus on the molecular mechanisms of our agents.

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Genome editing and disease modeling of RPGR-associated retinitis pigmentosa

Giacalone, J.C.1, Mullins, R.F.1, Tucker, B.A.1, Stone, E.M.1
1Stephen A. Wynn Institute for Vision Research, The University of Iowa, Iowa City, IA


Purpose: Retinitis Pigmentosa (RP) is a genetically heterogeneous disease that causes death of the light sensing photoreceptors and underlying retinal pigment epithelium. Mutations in the gene RPGR are responsible for a majority of cases of X-linked RP. Interestingly, mutations in RPGR can result in rod selective disease, cone selective disease, or in both cone and rod disease. The focus of this study was to develop genome-editing strategies and to model the disease phenotype in retinal organoids derived from patient-specific iPSCs.

Methods: Patient-specific iPSCs were generated from dermal fibroblasts of 7 patients with molecularly confirmed RPGR-associated XLRP. Pluripotency was confirmed using the TaqMan Scorecard Assay. CRISPR/Cas9 constructs were generated to target mutations in RPGR. The CRISPR/Cas9 system and homology directed repair constructs were delivered to iPSCs via electroporation. Corrected cells were enriched using either drug or color selection and confirmed via Sanger sequencing. Patient-specific iPSC-derived retinal organoids were generated from corrected, uncorrected cell lines, and control lines and characterized via immunocytochemistry and confocal microscopy.

Results: Seven iPSC lines were successfully generated with varying mutations and photoreceptor phenotype. Genome editing of patient-specific iPSCs was achieved with transfection efficiencies of approximately 20 percent in hiPSC, and the resulting modified iPSC clones were isolated and expanded via drug or color selection. Patient-specific iPSC-derived retinal organoids were generated and displayed photoreceptor-specific markers PAX6, OTX2, RCVRN, CRX and NRL. RPGR-ORF15 was successfully characterized in 120 day control retinal organoids.

Conclusions: iPSC technology allows for the unique opportunity to investigate patient-specific pathophysiologic mechanisms and therapeutic interventions in human cells. We have shown that genome editing via the CRISPR/Cas9 system can correct patient-specific mutations in iPSCs. Moreover, corrected versus uncorrected lines will serve as a valuable tool for characterizing the disease phenotype.

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Analysis of three Israeli families with Keratoconus

Goar, Wesley1 ; Searby, Charles1; Whitmore, Steven1; DeLuca, Adam1; Amtirat, Ahed2; Majdalani, Pierre 2; Stone, Edwin1; Pavari, Ruti2 ; Scheetz, Todd1; Val, Sheffield1
1Stephen A Wynn Institute for Vision Research; 2 Ben-Gurion University in The Negev


Purpose: Keratoconus (KT) is the most common corneal dystrophy with an occurrence rate of 1 in every 2,000 people. Currently, corneal transplantation is the only treatment for KT when visual acuity is no longer correctable by contact lenses. We hypothesize that KT is a genetically heterogeneous disease that is caused by mutations in one of several genes.

Methods: Samples from 3 Israeli KT families (16 samples) were genotyped using genome-wide SNP microarrays. The SNP data was analyzed for regions of autozygosity using PLINK. Three samples with KT (one from each family) were chosen for exome sequencing. The resulting variants were filtered based upon variant quality, predicted function, and population prevalence. The final variant list for each family was annotated with corneal expression (http://genome.uiowa.edu/otdb) to assist in prioritizing potential candidates.

Results: Using the autozygous regions, we have prioritized candidates and areas of the genome on which to focus our investigation. No plausible variations were found in genes previously reported to cause KT. In addition, no single gene with plausible mutations was shared across all three families. However, we identified at least 3 areas of autozygosity that are shared between the 3 families. Using CoNIFER, we also identified regions of the exomes with shared copy number variations.

Conclusions: Further work is needed to identify the causative mutations in these families. We will continue to pursue these through ascertainment of additional families and family members. This will allow us to further narrow the intervals of the genome in search of the causative mutations.

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Voted Outstanding Oral Presentation

Age-dependent rescue efficiency of retinal function in a mouse model of Bardet-Biedl Syndrome

Hsu, Y.1, Garrison J.E.1, Kim. G.1, Nishimura, D.Y.1, Searby, C.C.1, Schmitz, A.R.1, Datta, P.2, Seo, S.2, Sheffield, V.C.1
1University of Iowa, Department of Pediatrics 2University of Iowa, Department of Ophthalmology


Purpose: The BBSome, a protein complex consisting of BBS1, 2, 4, 5, 7, 8, 9, and 18, is required for retinal function. Loss of the BBSome causes Bardet-Biedl syndrome, characterized by polydactyly, obesity, and retinal degeneration. It is important to know whether there is a critical developmental time window for rescuing retinal function. We will elucidate how timing influences the effectiveness of rescue in mouse models of Bardet-Biedl syndrome.

Methods: Three Bbs8 mouse models were utilized. In the first model, Bbs8 is knocked out congenitally. In the second model, Bbs8 can be conditionally deleted by tamoxifen-inducible Cre recombinase. In the third model, Bbs8 expression is suppressed by a gene trap which can be removed by tamoxifen-inducible FLP recombinase. We restored Bbs8 gene expression during eye development and at different time points in adult mice to assess the age-dependent rescue of retinal function. We also performed retinal histology and transmission electron microscopy to assess the photoreceptor layers and the health of the outer segments.

Results: Congenital Bbs8 knockout mice have misoriented and malformed outer segments as early as the initiation of disc morphogenesis. Deletion of Bbs8 at any time causes retinal degeneration. Re-expression of Bbs8 enables the regeneration of outer segments in remaining photoreceptor cells, and significantly improves retinal function.

Conclusions: alformed outer segments can be reversed by the restoration of the BBSome. Retinal function can be significantly improved even in adult mice after some photoreceptors have already been lost. This finding has important implications for gene therapy for blindness.

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Imaging the Adult Zebrafish Cone Mosaic with Optical Coherence Tomography

Huckenpahler, Alison 1 Wilks, Melissa 1 Cooper, Robert 2 Link, Brian 1 Carroll, Joe 1 Collery, Ross 1
1 Medical College of Wisconsin 2 University of Pennsylvania


Purpose: Zebrafish have many advantages for studying ocular development and disease, most prominently the presence of a cone-rich retina and ease of genetic modification. Zebrafish are also widely used for drug development and accurate predict drug efficacy and toxicity. Here we assess the repeatability of in vivo cone density measurements obtained using OCT, in adult zebrafish to assess its utility for predicting drug oculotoxicity.

Methods: For repeatability studies, 12 wild-type zebrafish were imaged 3 times over the span of a week with a broad-band Bioptigen OCT. A telecentric lens was used to determine axial length and a mouse retinal bore was used for volumetric retina scans. The scans from each timepoint registered to each other and cropped to include only the area covered by all timepoints. Cropped images were analyzed to assess the cone photoreceptor density and the repeatability of cone density measurements.

Results: Density measurements for the zebrafish UV cone layer were 8,936 ±5,769 cones/mm2 with values ranging from 3,127 cones/mm2 to 16,297 cones/mm2. These values agree with published histology which ranges from 2,200 cones/mm2 to 21,000 cones/mm2. Cone densities calculated from OCT vary by less than 2% between timepoints.

Conclusions: OCT is a repeatable method of imaging the photoreceptor mosaic in adult zebrafish. Scaling factors are extremely important for quantitative measurements given the wide range of normal cone densities. The repeatability of the OCT cone density measurements is on par with that of human adaptive-optics imaging and could be appropriate for use in drug screening.

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CRISPR-cas9 Based Treatment of Myocilin-associated Glaucoma

Jain, A., Zode, G., Kasetti, B. R., Ran, A. F., Yan, W., Bugge, K., Searby, C.C., Buffard, V., Humbert, L.M., Zhang, F., Clark, F.A., Sheffield, C. V.1,2,3
1Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, Iowa 2North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas 3McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Maryland


Purpose: Glaucoma is a leading cause of irreversible blindness with elevated intraocular pressure (IOP) being the most important and the only modifiable risk factor. Mutations in myocilin (MYOC) lead to its misfolding and endoplasmic reticulum (ER) stress in the trabecular meshwork (TM), the tissue that maintains aqueous humor outflow and regulates IOP. ER stress and/or death of the TM lead to elevated IOP and glaucoma. Mutations in MYOC have been reported in both primary open angle glaucoma and juvenile open angle glaucoma. Since the phenotype seems to result from the mutant misfolded protein rather than resulting from loss of myocilin function or haplo-insufficiency, we propose to relieve the ER stress and glaucoma by targeting the MYOC gene using CRISPR-Cas9 genome editing technology.

Methods: Stable cell lines expressing mutant MYOC and human primary TM cells, as well as hMYOCY437H transgenic mice were treated with adenovirus Ad5-CRISPR-Cas9 virus with MYOC and control guide RNAs. Treated and control cells, and mice were evaluated for ER stress (by qPCR and western immunoblotting) and glaucoma phenotypes.

Results: Treatment with CRISPR-Cas9 with gRNAs targeting the MYOC gene reduces levels of MYOC and ER stress in MYOC mutant cells. We will also report on the rescue of glaucoma, during both disease stage and prophylactically, in the mouse model of MYOC glaucoma by CRISPR-cas9.

Conclusions: This is the first detailed study showing that genome editing targeting the MYOC gene can relieve ER stress in TM cells and in transgenic hMYOCY437H mouse model of glaucoma. This approach would provide a long-term or a permanent relief to the stress as compared to no current treatment or proposed short-term strategies in MYOC-associated glaucoma patients.

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Novel organotypic culture model of pig choroid-scleral explant as a therapeutic screening tool

Jiao,Chunhua, Zeng1, Shemin1, Stuart, Ryson1, Mullins, Robert1, Sohn, Elliott H1
1Stephen A. Wynn Institute for Vision Research, Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa


Purpose: To establish a pig organotypic choroid-scleral explant culture system for use as an ex-vivo choroid tubulogenesis screening tool to study novel potential pharmaceutical therapies.

Methods: 2mm explants of choroid/sclera without the RPE were obtained from adult porcine eyes and cultured on three-dimensional collagen matrix. After 2 days of culture in medium with 10% fetal bovine serum and 1 day of serum starvation in 4% serum medium, the explants were further cultured in the presence or absence of varying concentrations of VEGF (5ng/ml, 10ng/ml, 50ng/ml), complement 5a (10ug/ml, 50ug/ml), or tamoxifen (10ug/ml, 20ug/ml) for an additional 48 and 72 hour period. Vascular sprouting from the choroid-scleral complex was visualized using B. simplicifolia isolectin B4 (BSI-B4) and anti-CD31 antibody using immunofluorescence and confocal microscopy. Morphology of sprouting was assessed on toluidine blue stained semithin sections using light microscopy. Density of vascular elements in each condition was quantified using ImageJ software. Comparisons of controls and each treatment group were performed by one-way ANOVA using SPSS. Results were expressed as mean +/- SEM. P<0.05 was considered statistically significant.

Results: Choroidal endothelial cells began to proliferate and migrate out of the explant after 24 hours; tubulogenesis began at 48 hours, with a marked abundance of vascular tubes by 72 hours. Cells comprising the tubes were labeled with endothelial cell markers BSI-B4 and CD31 antibody. Explants treated with VEGF showed dose dependence and 50ng/ml of VEGF had a statistically significant increase in vascular area compared to controls (p=0.038) after 48 hours of culture; a VEGF directed antibody inhibited this effect of VEGF. In addition, after 72 hours of incubation, 50ug/ml C5a induced tubulogenesis; tamoxifen (10ug/ml; 20ug/ml) on the other hand significantly reduced vessel formation compared to control (p=0.007) in porcine choroid-scleral explant.

Conclusions: This adult pig choroid-sclera explant culture model is a short-term organotypic culture system that offers an opportunity to evaluate pro- and anti-angiogenic effects of pharmacologic compounds on choroid biology.

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The onset of glaucoma may be related to the decline of the accommodative apparatus via the choroid network connection

Kaufman, Paul L.1, Croft, Mary Ann1, Lütjen-Drecoll, Elke2
1Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison 2Department of Anatomie University Erlangen Nürnberg, Germany


Purpose: The ciliary muscle plays a major role in controlling both accommodation and outflow facility in primates. The ciliary muscle and the choroid functionally form an elastic network that extends from the trabecular meshwork to the back of the eye and ultimately attaches to the elastic fiber ring that surrounds the optic nerve and to the lamina cribrosa through which the nerve passes. The ciliary muscle governs the accommodative movement of the elastic choroid network. With age ciliary muscle mobility is restricted by progressively inelastic posterior attachments and the posterior restriction makes the contraction progressively isometric; placing increased tension on the optic nerve head (ONH) region. In addition, outflow facility also declines with age and limbal corneoscleral contour bows inward. The goal was to further elucidate the accommodative movements of the choroid and vitreous in glaucoma related target sites.

Methods: Ultrasound biomicroscopy (UBM, 20 & 50mHz) and fundus photography was utilized to image the various accommodative intraocular structures and optic nerve region.

Results: The choroid moves in the region of the ora serrata and in the region of the ONH by ~1mm and by ~0.1mm respectively, and these movements decline with age. The central vitreous moves posteriorly as far back as the optic nerve region.

Conclusions: Thus, there may be pressure and tension spikes on the optic nerve region during accommodation and these pressure and tension spikes may increase with age. This constellation of events could be relevant to glaucomatous optic neuropathy.

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Identification of HCN1 as a novel 14-3-3 client protein

Lankford, Colten K.1, Dean, Maria1, Laird, Joseph G.1, Yamaguchi, David M.1, Pan, Yuan1, Sheila A. Baker1
1Departments of Biochemistry and Ophthalmology & Visual Sciences, Carver College of Medicine, University of Iowa, IA USA 52242


Purpose: Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels are expressed in photoreceptors where they are localized to the inner segment plasma membrane (ISPM) and function to limit the duration of the light response. The function of HCN1 is dependent on its ability to be trafficked to the ISPM. While the mechanisms regulating HCN1 trafficking are poorly understood, 14-3-3 proteins have been implicated in the trafficking of similar multimeric membrane proteins. The goal of this study is to investigate the mechanisms underlying HCN1 trafficking to the ISPM and evaluate the role of 14-3-3 as part of this mechanism.

Methods: Trafficking signals were mapped by analyzing the subcellular localization of a series of HCN1 mutants and protein-protein interactions were assessed using immunoprecipitation and pulldown assays.

Results: Two opposing trafficking signals were identified − a di-arginine endoplasmic reticulum (ER) retention signal and a novel ER export signal. Additionally, HCN1 was identified as a 14-3-3 client protein with 14-3-3 binding motifs near both ER trafficking signals. 14-3-3 was found to bind with a higher affinity to the site nearest the retention signal and this binding was enhanced by phosphorylation of HCN1.

Conclusions: HCN1 contains two opposing trafficking motifs that regulate trafficking out of the ER. We propose that the coordination of these signals acts to minimize aberrant trafficking of incompletely assembled HCN1 tetramers to the cell surface. Additionally, we demonstrate that HCN1 is a novel 14-3-3 client protein and suggest that this interaction may play a role in coordinating HCN1 trafficking by masking the ER retention signal.

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Investigating transcriptomes of developing chick retinal cells

Laboissonniere, Lauren1 Goetz, Jillian2, Trimarchi, Jeffrey1
1Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, IA 2Feinberg School of Medicine, Northwestern University, Chicago, IL


Purpose: The vertebrate retina is comprised of 7 cell types, 6 neuronal and 1 glial, all arising from a common pool of progenitor cells. These progenitors use specific genetic networks to pursue cell fates and to develop the appropriate combination of mature cells. Our goal is to characterize groups of genes central to this decision for the various cell types in the chicken retina.

Methods: Individual cells were isolated from chicken retinas between the developmental timepoints of embryonic day (E)3 and E15. Reverse transcription and PCR amplification were completed to generate cDNA libraries for each cell. Finally, cDNA was hybridized to Affymetrix microarray chips and transcriptomes were analyzed. To confirm our findings, we employed in situ hybridization on sectioned retinas.

Results: We were able to uncover a number of cells at varying timepoints which are developing RGCs based on their gene expression of known markers. Among these cells, we identified a small number expressing both ganglion and amacrine cell markers, suggesting a large overlap in gene expression between these two populations. We also uncovered cells in the process of deciding between multiple cell fates. Cells in the midst of this dynamic transition, in particular, are invaluable for the identification of novel transcription factors that play a role in the cell fate decision-making process and we have used those in our population to identify a large number of these factors.

Conclusions: The analysis of single cells allows for the identification of gene expression networks related to cell fate acquisition as well as the identification of cells during crucial developmental timepoints. This project found multiple cells expressing genes specific to more than one population, suggesting possible plasticity between cell fates. We were able to validate our novel gene discoveries through in situ as well as to observe new expression profiles for some genes which had previously been uncharacterized.

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Relationship Between The Henle Fiber Layer And Foveal Specialization

Lee, Daniel1; Wilk, Melissa2; Carroll, Joseph1,2
1Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, WI 53226 USA 2Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA


Purpose: Directional optical coherence tomography (D-OCT) enables in vivo visualization of the Henle Fiber Layer (HFL) by purposefully altering the OCT beam entry position. The purpose of this study was to assess the relationship between the HFL and other foveal specializations, namely peak cone density and foveal pit morphology.

Methods: D-OCTs were acquired in 9 subjects with albinism, 2 carriers of ocular albinism (OA1), and 15 normal subjects. The D-OCT B-scans were manually segmented to extract retinal thickness. Volume scans of the macula were used to derive pit depth, diameter, area and volume. Peak cone density data was measured from adaptive optics images in normal subjects and estimated from outer segment length as previously described in patients with albinism.

Results: HFL was observed in albinism subjects with no foveal pit. HFL thickness at the location of the incipient fovea for albinism subjects was found to be significant thicker than in normal subjects. No significant relationship was observed between foveal pit morphology and the HFL thickness at varying eccentricities. Peak cone density was found to be negatively correlated with central HFL thickness and positively correlated with the distance of peak HFL thickness from the foveal center.

Conclusions: Cone density may play an important role in determining HFL topography. Our results are consistent with previous published data on cone packing; the presence of HFL in albinism indicates that lateral displacement between photoreceptor and bipolar cells can occur in the absence of a foveal pit.

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Voted Outstanding Oral Presentation

Investigating the longitudinal and kinetic profiles of BAX recruitment in mouse retinal ganglion cells

Maes, Margaret; Schlamp, Cassandra; Nickells, Robert
University of Wisconsin - Madison


Purpose: Glaucoma is an optic neuropathy characterized by BAX-dependent apoptosis of retinal ganglion cells (RGCs). BAX forms oligomers when recruited to the mitochondrial outer membrane, identifying the "point of no return" for apoptotic cells. Once initiated, BAX recruitment proceeds rapidly in vitro, however the process in living RGCs is undocumented. Understanding the pattern of progression and kinetic profile of BAX recruitment in RGCs after injury is imperative to developing effective therapeutics to prevent RGC death.

Methods: BAX recruitment was monitored after optic nerve crush (ONC) in CB6F1 and BALB/cByJ mice (Day 1, 3, 5, 7,14), and in DBA2/J mice (6, 8, 10 and 12 months). Longitudinal imaging of AAV2-GFP-BAX transduced RGCs was performed using a blue light confocal scanning laser ophthalmoscope (Heidlberg). Live imaging was performed using a spinning disk confocal microscope. IMARIS 7.7 was used for kinetic analysis of time-lapse videos.

Results: The percentage of transduced RGCs exhibiting BAX oligomers after ONC in CB6F1 mice significantly increased at days 3, 5 and 7 (p < 0.05). The same pattern was represented in BALB/cByJ mice and DBA2/J mice. Longitudinal in vivo imaging showed that clearance of BAX expressing RGCs began 8 days after injury. Live-cell imaging of retinal explants showed a slower rate of BAX recruitment in vivo compared to in vitro.

Conclusions: The consistent pattern of progression to peak BAX oligomer formation among mouse strains and its kinetic profile demonstrates that effective therapies preventing RGC death need to be administered prior to peak BAX recruitment.

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Defining the time course of optic neuropathy and ganglion cell loss in a mouse model of early-onset glaucoma

Pellack, Danielle R.1,2; Lewis, Carly J.1; Meyer, Kacie J.1; Anderson, Michael G.1,2
1Department of Molecular Physiology and Biophysics and Department of Ophthalmology and Visual Sciences, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA; 2Center for the Prevention and Treatment of Vision Loss, VA Medical Center, Iowa City, IA


Purpose: Glaucoma is an age-related disease of the optic nerve that causes progressive, irreversible vision loss. The nee mouse strain, harboring a recessive mutation in Sh3pxd2b, exhibits severe early onset glaucoma with elevated intraocular pressure, optic nerve excavation, and axon loss. The robust phenotype and rapid disease progression will make this model valuable for high throughput testing of novel glaucoma therapies. The purpose of these experiments is to establish the baseline time course of optic nerve axon degeneration and retinal ganglion cell (RGC) death in the nee model of glaucoma.

Methods: Retinal flat mounts and optic nerves were collected from cohorts of B6.nee mice and littermate controls. RGCs were visualized by immunohistochemistry (anti-Brn3a) and counted using semi-automated software. Total axon numbers were estimated from manual axon counting of cross-sections of optic nerve stained with PPD.

Results: In B6.nee mice, there is a clear trend for reduction in RGC density with age and a striking loss of cells at 6-8 weeks old coinciding with a drastic reduction in the number of healthy axons present in the optic nerve (p<0.001). RGC loss in B6.nee mice coincided with increased nucleus size of surviving RGCs.

Conclusions: RGC and axon loss occurs in nee mice before 8 weeks of age. Surviving RGCs in B6.nee retinas with severe RGC loss have larger nuclei compared to those in early-stage disease. Our results indicate that the endpoint of future studies using the nee mouse model of glaucoma to test the efficacy of potential therapies should be 6-8 weeks old.

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Gene augmentation therapy in a large animal model of CNGB1 retinitis pigmentosa

Petersen-Jones, Simon M.1; Occelli, Laurence M.1; Winkler, Paige A.1; Chiodo, Vince C.2; Boye, Sanford L.2; Hauswirth, William W.2
1Department of Small Animal Clinical Sciences, Michigan State University, East Lansing, MI 2Department of Ophthalmology, University of Florida, Gainesville, FL


Purpose: The purpose of this study was to assess the potential of gene augmentation therapy for CNGB1-RP using a large animal model.

Methods: Five young Cngb1-/- dogs (8 eyes) received gene augmentation therapy using a rAAV5-hGRK1-cCngb1 vector via a subretinal injection. A titer of 1x1012 vg/mL was used in one eye and 5x1012 vg/mL in 7 eyes. Dark- and light-adapted electroretinograms (ERGs) were recorded prior to injection and monthly post-injection. Vision was assessed using a four exit-choice vision-testing device at lighting levels ranging from dim (rod only vision) to normal room lighting. One dog was euthanized at each of 3, 6 and 9 months post-injection and the eyes processed for immunohistochemistry (IHC).

Results: Pretreatment, Cngb1-/- dogs lacked rod ERG responses and rod-mediated vision. Eyes treated with the higher titer vector had a dramatic improvement in rod mediated ERG responses with lowered response threshold and increased amplitudes. The eye injected with the lower titer had slight improvement in ERG responses. Rod-mediated vision was restored in all treated dogs. Rod ERG rescue was maintained in the single dog followed to 18 months post treatment and on OCT there was preservation of the outer nuclear layer, inner and outer segments and the ellipsoid zone in the treated region. IHC showed that therapy resulted in localization of Cngb1 of rod outer segments only in the treated retinal regions.

Conclusions: rAAV gene augmentation therapy rescues rod function in Cngb1-/- dogs and promises to preserve structure. Gene augmentation therapy shows promise for the treatment of CNGB1-RP.

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iPSC-derived TM cells promote proliferation of primary TM cells

Pike, Tania; Zhu, Wei; Kuehn, Markus H.1,2
1 Department of Ophthalmology and Visual Sciences, University of Iowa 2Center for the Prevention and Treatment of Visual Loss, Iowa City Veterans Affairs Medical Center


Purpose: The goal of this study was to investigate the interactions between iPSC-TM and primary TM cells (pTM) in vitro and using perfusion cultured human eyes.

Methods: 50,000 mouse pTM cells were transfected with Ad5RSVmyocilinY437HHisFlag and subsequently either co-cultured with 50,000 purified mouse dsRed+ iPSC-TM or grown in cell culture media pre-conditioned by iPSC-TM. The surviving dsRed-negative cells were counted by flow cytometry. The proliferation rate was analyzed based upon BrdU incorporation within a 2-hour period. Additionally 5 pairs of human donor eyes (83±9.6 yrs old), maintained in a perfusion organ culture system, received transplants of 200,000 human dsRed-positive iPSC-TM. TM cells were isolated and counted by flow cytometry and the tissue was evaluated using histochemical and immunohistochemical methods.

Results: After 4 days in culture the number of mouse pTM cells maintained alone increased by 38.7%±6.0 whereas direct co-culture of pTM and iPSC-TM resulted in a 234.4%±68 increase (P=0.0039). The fraction of BrdU positive pTM cells in co-cultures was significant higher than that measured in controls (32.9% vs 21.6%, P=0.03). Similar findings were also obtained in human eyes using a perfusion organ culture system. 4 out of 5 donors responded with vigorous TM cell proliferation upon transplantation of iPSC-TM. Although only a small number of surviving iPSC-TM was detected these eyes displayed a 2.1±0.98 fold increase in TM cellularity when compared to the untreated contralateral eye (P=0.035).

Conclusions: These studies demonstrate that iPSC-TM induce a proliferative response in both human and mouse TM cells that requires direct cell-cell contact.

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Use of facial analysis to assess CGRP-induced discomfort during migraine

Rea, Brandon; Poolman, Pieter; Bittle, Jada; Castonguay, William; Roberston, Bennett; Sowers, Levi; Kardon, Randy; Russo, Andrew1
1Dept. of Molecular Physiology and Biophysics, Interdisciplinary Graduate Program in Neuroscience, Dept. of Neurology, University of Iowa, Iowa City, Iowa; Center for the Prevention and Treatment of Visual Loss, Iowa VA Health Care System, Iowa City, Iowa


Purpose: Migraine is a neurological disorder and more than just a headache. During migraine, migraineurs frequently have debilitating pain, lasting for up to 72 hours, and at least one of two symptoms: nausea and/or vomiting, or photophobia and phonophobia. Calcitonin gene-related peptide (CGRP) has been shown to play a key role in migraine etiology. CGRP is elevated during migraine, injection of CGRP induces migraine-like headache in migraineurs but not nonmigraineurs, and CGRP antagonist drugs alleviate migraine symptoms. Though CGRP-induced photophobic behavior has been demonstrated in our mouse model, we have yet to establish whether CGRP also induced pain. We hypothesize that administration of CGRP would increase facial expression of discomfort when exposed to light.

Methods: Mice were acclimated to a restraint and recorded via camera during dark and light conditions. CD1 and C57BL/6J background mice were given an intraperitoneal injection of CGRP (0.5 mg/kg) or saline and recorded during dark and light conditions. Using the Mouse Grimace Scale and point-to-point measurement of squint, mice were independently scored by two blinded observers.

Results: CGRP caused a significant increase in pain expression in C57BL/6J background mice compared to a saline control in both dark and light conditions. Similar results were seen with CD1 mice treated with CGRP.

Conclusions: CGRP does cause increased facial pain symptoms in mice. These results suggest that facial analysis can be used to report and assess pain during migraine.

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Pyogenic Granulomas of the Lacrimal Sac: A clinicopathological survey

Reddy, Devasis, N.1, Martin, Jacob S. 1, Burris, Christopher K.H. 1, Rodriguez, Maria E.1, Potter, Heather D.1, Albert, Daniel M.1
1University of Wisconsin-Madison, Dept. of Ophthalmology and Visual Sciences


Purpose: To determine the frequency of pyogenic granulomas of the lacrimal sac among all biopsy specimens of patients undergoing dacryocystorhinostomy (DCR).

Methods: A retrospective chart and histology slide review of all specimens received from dacryocystorhinostomies performed between 2010-2016 at our institution was conducted. The histopathologic diagnoses were reviewed to determine the frequency of all lacrimal sac pathologies necessitating dacryocystorhinostomy. Demographic data as well as clinical presentation for patients with pyogenic granulomas of the lacrimal sac were recorded and examined for trends.

Results: From 2010-2016, there were 82 DCR specimens received by the University of Wisconsin-Madison Ocular Pathology laboratory. 9 of the 82 specimens were pyogenic granuloma, representing an 11% frequency of occurrence. The most common clinical presentation associated with pyogenic granulomas of the lacrimal sac was epiphora with an average duration of symptoms of 11 months. No cases presented with haemolacria. A history of trauma was documented in 2 cases while a prior surgical history was found in 4 cases. Recurrence of pyogenic granulomas of the lacrimal sac necessitating second DCR was found in 2 cases.

Conclusions: Pyogenic granulomas of the lacrimal sac may occur more frequently than is reported in the literature. Though patients have been reported as presenting with haemolacria, frank epiphora is the most common presentation. By having a more accurate representation of the frequency of lacrimal sac pyogenic granulomas, a more appropriate differential diagnosis may be considered for each patient undergoing dacryocystorhinostomy.

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Effect of long term reduction of intracranial pressure on retinal ganglion cells and optic nerve axons in a rat intraventricular cannula model

Roy Chowdhury, Uttio; Holman, Bradley; Fautsch, Michael
Dept. of Ophthalmology, Mayo Clinic, Rochester, MN


Purpose: To evaluate the effects of long term reduction of intracranial pressure (ICP) on optic neuronal health.

Methods: Stainless steel cannulae were inserted into the left lateral ventricle of brown Norway rats (n=5 experimental, n=4 sham control) and connected to a pressure transducer that measured ICP in real time, through a custom-designed software. ICP was reduced by lowering a reservoir filled with artificial cerebrospinal fluid that was connected in parallel to the cannula system. ICP was reduced by 25-50% for 7, 30 or 60 days. Retinal ganglion cell (RGC) numbers were calculated from retinal flat mounts. Optic nerve axon density was calculated from a region 1 mm posterior to the optic disc. Retinal nerve fiber layer thickness was calculated by spectral domain-optical coherence tomography (SD-OCT) imaging.

Results: RGC numbers were lowered by 4.0% after 7 days, 11.7% after 1 month and 37.5% after 2 months, when compared to the sham controls. Optic nerve axon density was reduced by 26.1% after 1 month and 24.4% after 2 months while no change was seen after 7 days. Lowering ICP by approximately 60% below baseline for 45 days caused a 14.2% reduction in retinal thickness in the right eye and 61.7% reduction in the left eye compared to baseline values.

Conclusions: In an experimental rat intraventricular cannula model, manual reduction of ICP causes loss of retinal ganglion cells and optic nerve axons, both of which are hallmarks of glaucomatous pathologies.

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Automated Reference Frame Selection (ARFS) for Registration of Scanning Ophthalmoscope Image

Salmon, Alexander1; Cooper, Robert3,4; Langlo, Christopher1; Sajdak, Benjamin1; Razeen, Moataz2,5; Dubra, Alfredo1,2; Carroll, Joseph1,2
1Cell Biology, Neurobiology, Anatomy, Medical College of Wisconsin, Milwaukee, WI2 Ophthalmology, Medical College of Wisconsin, Milwaukee, WI3 Psychology, University of Pennsylvania, Philadelphia, PA4 Ophthalmology, University of Pennsylvania, Philadelphia, PA5 Alexandria Faculty of Medicine, University of Alexandria, Alexandria, Egypt


Purpose: Manual reference frame selection for registration and averaging image sequences from scanning ophthalmoscopes to increase SNR is time consuming and subjective. We present an automated algorithm for organizing frames into spatial clusters and objectively selecting representative frames with minimal distortion.

Methods: The algorithm rejects frames based on intensity, Fourier-based distortion detection, normalized cross-correlation, and conducts motion tracking and clustering to choose spatially representative frames. Validation was achieved by using strip-based registration (Dubra & Harvey, 2010) to align undistorted AO-flood illuminated images with either ARFS- or manually-derived AOSLO images of photoreceptors (PR) in 9 normal subjects, and comparing the magnitude of the pixel shift vector (PSV) required for registration in each case.

Results: Automated reference frame selection resulted in mean ± SD PSV of 2.75±1.16 pixels versus 3.33±1.38 pixels for subjective selection (p=0.051, paired t-test).

Conclusions: ARFS identifies minimally-distorted reference frames in AOSLO image sequences of photoreceptors.

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Voted Outstanding Poster Presentation

Targeting HDAC3 activity prevents retinal ganglion cell nuclear atrophy and apoptosis after optic nerve injury

Schmitt, Heather1,2; Schlamp, Cassandra1; Nickells, Robert1
1Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI 2Cellular and Molecular Pathology, University of Wisconsin-Madison, Madison, WI


Purpose: In retinal ganglion cells (RGCs) affected by optic nerve crush (ONC), HDAC3 regulates nuclear atrophy as an early response to axonal injury. Since conditional knockout of Hdac3 and HDAC3 selective inhibition with RGFP966 prevent nuclear atrophy post ONC, validation of a therapeutic effect of systemic dosing of RGFP966 is necessary for application to chronic models of optic nerve injury.

Methods: Mass spectrometry was used to measure bioavailability of RGFP966 in the retina at 0-24 hours post intraperitoneal (IP) injection in C57BL/6 mice. Mice were injected IP with RGFP966 doses of 2mg/kg-10mg/kg, and eyes were harvested 5 days after ONC to measure histone deacetylation using immunofluorescence. Total cell loss in the GCL was analyzed by immunofluorescence in mice that were injected IP with Vehicle, 2mg/kg or 10mg/kg RGFP966 daily, every 3 days, and every 7 days following ONC surgery. H&E and BrdU staining was used to analyze toxicity to off-target tissues after 14 days of daily systemic injection of RGFP966.

Results: RGFP966 crossed the blood-retinal barrier, with a peak concentration at 1 hour after an IP injection. A single IP injection of 2mg/kg-10mg/kg RGFP966, significantly prevented histone deacetylation(p<0.0005). Repeated IP injections of 2mg/kg RGFP966 over the course of 2 weeks post ONC prevented RGC loss (p<0.05). There were no significant toxic or anti-proliferative effects to off-target tissues in mice treated daily for 14 days with RGFP966.

Conclusions: Inhibition of HDAC3 activity with systemic dosing of RGFP966 prevents histone deacetylation and eventual apoptosis of RGCs after acute optic nerve injury.

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Voted Outstanding Oral Presentation

Connective Tissue Growth Factor (CTGF) promotes efficient generation of human iPSC-derived choroidal endothelium

Songstad, A.E.; Worthington, K.S.; Chirco, K.R.; Giacalone, J.C.; Whitmore, S.S.; Anfinson, K.R.; Ochoa, D; Cranston, C.M.; Stone, E.M.; Mullins, R.F.; Tucker, B.A.
Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver college of Medicine, University of Iowa, Iowa City, Iowa


Purpose: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in western countries. Because AMD involves retinal pigment epithelial cell (RPE) and photoreceptor cell dysfunction, existing iPSC-based strategies to replace photoreceptors and RPE are a major scientific focus. Adequate AMD treatment will also require replacing damaged choroidal endothelial cells (CECs), demanding the development of human iPSC-CECs. Here, we designed a stepwise differentiation protocol to generate human iPSC-CECs to use in transplantation and disease modeling studies.

Methods: We used the TaqMan Scorecard Assay, rt-PCR, and immunocytochemistry (ICC) to assess pluripotency of iPSCs from a donor with normal ocular history. RNA sequencing analysis of the monkey CEC line, RF/6A, identified seven secreted proteins that are involved in vascular development. We developed various media using two statistical screens that utilized combinations of the seven proteins to determine which proteins drove CEC differentiation.

Results: Using each statistical screen, iPSCs cultured in media containing only CTGF differentiated into CECs that expressed the EC-specific markers CD31, ICAM1, PLVAP, and vWF and the CEC-restricted marker CA4. When we only added TWEAKR to the differentiation medium, the iPSCs differentiated into CECs. However, TWEAKR induced endogenous CTGF secretion, indicating that TWEAKR acts through CTGF to drive iPSC differentiation into CECs, revealing that CTGF is required for CEC differentiation.

Conclusions: The iPSC-CECs fed CTGF formed capillary tube-like vascular networks morphologically identical to choroidal vascular networks, making these human iPSC-CECs the most appropriate cell type available for cell replacement therapies and AMD disease modeling studies.

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Investigation of neuronal circuits underlying light-aversive behavior

Sowers, L.P.1,2, Rea B.J.1, Taugher R.J.3, Kim Y.C.4, Kuburas A.1, Teran L.1, Ma S.1, Wemmie J.A.3, Russo, A.F.1-4
1Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA, 2VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA, 3Department of Psychiatry and 4Department of Neurology, University of Iowa, Iowa City, IA


Purpose: Veterans returning from active duty are at an increased risk for post-traumatic headache (PTH) that is similar to migraine. Sensory abnormalities in individuals with PTH include extreme light sensitivity. This light sensitivity can be debilitating and treatments are lacking. Calcitonin gene-related peptide (CGRP) is a neuropeptide involved in pain signaling and has recently come to the forefront of migraine research where it contributes to headache and associated sensory abnormalities. In this study we attempt to identify anatomical regions where CGRP could act to induce light-aversive behavior in migraine and PTH. The posterior thalamus (Po) and PAG have been suggested to be important brain regions in migraine pathophysiology. We hypothesize that CGRP acts as a neuromodulator in the Po and/or PAG to induce light aversive behavior.

Methods: Direct injection of CGRP into the Po and optogenetic stimulation of the Po and PAG were performed and light-aversive behavior was assessed in a light-dark box assay.

Results: We found that CGRP injection in the Po as well as optical stimulation of the Po induces significant light aversive behavior, without increased anxiety in light-independent assays. In contrast to the Po, PAG stimulation led to both light aversion and light-independent anxiogenic behavior.

Conclusions: These data suggest that the Po can induce light aversion associated with CGRP actions, while the PAG may trigger not only the Po, but also other brain regions involved in anxiogenic behaviors. These results begin to shed light on the complex circuitry of light-aversive behaviors in mice.

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Developmental Origins of Three Dimensional Retinal Organoids Derived from Human Pluripotent Stem Cells

Sridhar, Akshayalakshmi; Miller, Casey A.; Ho-A-Lim, Kimberly; Langer, Kirstin B.; Meyer, Jason S.
Department of Biology, Department of Medical and Molecular Genetics, and Stark Neuroscience Research Institute, Indiana University Purdue University Indianapolis


Purpose: The differentiation of three dimensional retinal organoids from human pluripotent stem cells (hPSCs) allows for the unprecedented ability to study the organization of cells into stratified retinal-like tissue. However, the events that lead to the generation of retinal organoids from a pluripotent population remain to be explored. Therefore, experiments were undertaken to elucidate the developmental origins of retinal organoids.

Methods: hPSCs were first directed to a primitive anterior neural phenotype and subsequently to retinal organoids in a stepwise fashion. Differentiation was analyzed at varying timepoints to confirm stage-specific gene expression. Following neural specification, the stepwise differentiation of these cells was analyzed using microscopy and immunocytochemistry.

Results: hPSCs were directed to generate retinal organoids in a stepwise manner, proceeding through all major stages of retinal development. hPSCs first generated cells of the presumptive neuroepithelium, where overlapping patterns of gene expression marked the regions that later developed into the presumptive optic vesicle. Such regions exhibited a characteristic stratified ring of cells around the periphery, indicative of optic vesicle-like retinal organoids. Upon further differentiation, these retinal organoids acquired a laminar optic cup-like organization and expressed markers of major cell types of the retina, including retinal ganglion cells and photoreceptors.

Conclusions: Overall, the results of this study elucidate the developmental origins of three dimensional retinal organoids from hPSCs as these cells progress through analogous stages of retinogenesis. Such studies validate hPSC-derived retinal organoids as an effective tool for studies of early human retinal development, with future implications for modeling retinal degenerative diseases.

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Extracting Phenotypes From Historic Goldmann Visual Fields

Tatro, Nicole J., DeLuca, Adam P., Christopher, Mark A., Stone, Edwin M., Scheetz, Todd E.1
1Wynn Institute for Vision Research, University of Iowa


Purpose: Goldmann perimetry has long been the standard for assessing visual fields in patients with inherited retinal disease. We implemented a system that allows the capture of historic Goldmann visual field (GVF) data, and extraction of relevant phenotypic information including the volume of the hill of vision. We compared this system to a previous system by Weleber et al.

Methods: A custom profile was developed for TruthMarker to allow the tracing and registration of isoptors from scanned GVFs. A post processing program has been implemented to calculate the area of each isoptor while correcting for the cartographic distortion inherit in the planometric GVF projection and to calculate the total volume of the hill of vision.

Results: GVFs were digitized by two readers on both systems for a set of 17 fields with known mutations in MYO7A. A comparison between the platforms, showed excellent reproducibility with a Pearson's correlation of r2 = 0.9997. This compares well with the inter-reader variability of r2 = 0.9998 on the TruthMarker-based system and r2 = 0.9993 on the Weleber et al. system. All of these values are well within the test-retest variability of Goldmann perimetry. Two patterns were clearly identifiable in the resulting traced GVFs, corresponding to different clinical progressions of visual function.

Conclusions: Our method showed excellent correlation with the existing methodology. TruthMarker also allows flexible capture of custom phenotypic data via an XML-based configuration file. Several large-scale GVF digitization projects are currently ongoing, involving thousands of visual fields.

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Optimizing the photopolymerization parameters of high-resolution, 3D biodegradable tissue scaffolds

Jessica R. Thompson, Spencer J. Bunn, Kristan S. Worthington, Brian J. Green, Luke A. Wiley and Budd A. Tucker1
1Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology & Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA


Abstract: Polymer support scaffolds are critical for cellular survival and integration in retinal cell replacement therapies. Obstacles to the realization of scaffold use for this purpose include 1) selection of appropriate chemistry and 2) microstructural control for cell orientation and packing. Poly(caprolactone) (PCL) is a good candidate material for retinal scaffolds, as it is biodegradable, biocompatible, and easily modified with reactive molecules.

In this work, we optimized the conditions that contribute to photopolymerized PCL microstructure in order to create more effective and efficient retinal cell scaffolds. We used two-photon polymerization (Nanoscribe Photonic Professional GT) to create degradable 3D-printed structures with sub-micron resolution. Briefly, we mixed di- or tri-acrylated PCL with varying molecular weights (530, 1250 or 200 g/mol for di-acrylates and 300 or 900 g/mol for tri-acrylates) at 25, 50 or 75 wt% with photoinitiator (Irgacure-369) at 1, 3 or 5 wt% in dioxane. In other words, the formulation variables were degree of PCL functionality, PCL molecular weight, PCL concentration and photoinitiator concentration. For each formulation, we varied the laser scanning speed (from 6,000 to 160,000 µm/s) and laser power (from 2 to 100%) to determine the polymerization threshold. At a fixed scanning speed and laser power for each formulation, we also varied the hatching and slicing distances (0.1, 0.5, 1.0 or 1.5 µm) of the modeled scaffolds. We used scanning electron microscopy and image analysis to quantify threshold, resolution, and design-to-structure fidelity. To verify material biocompatibility, we seeded bulk-polymerized acrylated PCL with mouse induced pluripotent stem cells (MiPSCs) and assessed their mitotic index one week later using immunocytochemistry.

With other variables held constant, we achieved optimal resolution and threshold using 3% photoinitator. Two-photon polymerization of triacrylated PCL resulted in higher resolution, more easily distinguished structures than diacrylated PCL. Due to their lower cross-linking densities, higher molecular weight PCL structures became enlarged compared to lower molecular weight PCL structures and the original design. For triacrylated PCL, increasing either hatching or slicing distance decreases the structure’s deviance from the design. Regardless of molecular weight or degree of functionality, bulk polymerized PCL supported the growth and mitosis of MiPSCs.

We are the first to examine the effects of PCL formulation and two-photon printing variables on final product outcomes. The favorable biocompatibility of bulk PCL with the same chemical composition as 3D printed PCL demonstrates that these structures are promising for transplantation purposes. Thus, we are now poised to interrogate in vivo compatibility of 3D printed structures and examine their interactions with human retinal cells.

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Presynaptic mGluRs regulate cone photoreceptor synaptic transmission by direct interactions of G beta-gamma and SNAP-25.

Van Hook, Matthew J.1, Babai, Norbert2, Zurawski, Zack3, Yim, Yun Young3, Hamm, Heidi E3, Thoreson, Wallace B.1,4
1Department of Ophthalmology & Visual Sciences, Truhlsen Eye Institute, University of Nebraska Medical Center, Omaha, NE 68198.2Department of Biology, Animal Physiology, University of Erlangen-Nuremberg, Staudstrasse 5, 91058 Erlangen, Germany. 3Department of Pharmacology, Vanderbilt University Medical School, Nashville, TN 37232. 4Department of Pharmacology & Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198.


Purpose: Activation of presynaptic group III mGluRs at the cone photoreceptor synapse reduces glutamate release by a mechanism involving reduced Ca2+ influx through L-type channels. Here, we tested the hypothesis that mGluR activation reduces synaptic vesicle exocytosis in cones by direct interactions of G-protein beta-gamma subunits (Gbg) with SNAP-25. Such regulation has not been demonstrated either in retina or at a ribbon-type synapse.

Methods: Experiments were performed using whole-cell recordings in vertical slices from tiger salamander retinas. The group III mGluR agonist L-AP4 was bath applied. Slices were incubated with BoNT/A and a peptide derived from the SNAP-25 C-terminus was introduced via the patch pipette to test for SNAP-25 C-terminus involvement. Gbg was introduced directly via the cone patch pipette solution. Proximity ligation assay (PLA) was performed on isolated cone photoreceptors and vertical sections of salamander retina.

Results: L-AP4 caused an ~10% reduction in Ca2+ current (ICa) and an ~30% reduction in synaptic transmission. Following BoNT/A pretreatment, both ICa and synaptic transmission were reduced to a similar proportion by L-AP4 (~10%). A similar result was obtained when we dialyzed cones with the SNAP-25 peptide. PLA confirmed interaction of Gbg and SNAP-25 at cone synapses. Introducing Gb1g1 through the cone patch pipette caused a dose-dependent reduction in synaptic transmission. Gb1g1 had no effect on ICa and the effects on synaptic transmission were blocked by pre-treatment with BoNT/A.

Conclusions: mGluR activation inhibits depolarization-evoked glutamate release from cones by both reducing Ca2+ influx and by direct Gbg/SNAP-25 interactions.

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Surveying the human ocular transcriptome, one cell at a time

Whitmore, S Scott; DeLuca, Adam P.; Wiley, Luke A.; Burnight, Erin R.; Flamme-Wiese, Miles J.; Tucker, Budd A.; Mullins, Robert F.; Scheetz, Todd E.; Stone, Edwin M.1
1Stephen A. Wynn Institute for Vision Research, The University of Iowa, Iowa City, IA, United States


Purpose: Efforts to correct heritable blindness and to understand normal retinal physiology will benefit from profiling gene expression within specific retinal cell types. However, the vast majority of transcriptional studies of the mammalian retina have profiled bulk RNA, consequently masking distinctions between cells types and subpopulations. To evaluate the transcriptional heterogeneity of retinal cells, we performed single-cell RNA-Seq of photoreceptor and RPE cells using the Fluidigm C1 system.

Methods: Neural retina and RPE from three human donors (A, B, C) were obtained through the Iowa Lions Eye Bank. A portion of the RPE from one donor was cultured prior to single-cell capture. Four single cell preparations (neural retina from A; freshly isolated RPE from B and C; cultured RPE from C) were generated by digestion with dispase. Single cells were captured using the Fluidigm C1 system. cDNA libraries from each experiment were barcoded, pooled and sequenced as single-lanes on the Illumina HiSeq 2500.

Results: 48 single cells were profiled. Hierarchical clustering identified five differentiable cell clusters, enriched for photoreceptors, freshly isolated RPE cells, cultured cells, damaged cells (primarily expressing mitochondrial genes), and debris-contaminated cells. Thirty-six genes showed increased expression in cultured RPE cells as compared to freshly captured RPE cells.

Conclusions: Co-expression analysis of single photoreceptor and RPE cells suggests several genes as disease candidates. In future studies, we plan to profile the transcriptomes of foveal and extrafoveal cone photoreceptor cells in order to better understand the photoreceptors lost in macular degenerative diseases.


Purpose: NR2E3 is a retinal transcription factor that is required for the suppression of cone-specific genes during photoreceptor development. NR2E3-associated disease is characterized by congenital night blindness, increased sensitivity to short wavelength light due to a larger population of S-cones and varying degrees of medium (M-cone)- and long (L-cone)-mediated vision. The purpose of this study is to generate patient-specific iPSCs and iPSC-derived 3D retinal eyecups to model disease pathophysiology of enhanced S-cone syndrome, caused by mutations in the gene NR2E3.

Methods: iPSCs were generated from two patients with molecularly confirmed mutations in NR2E3 via viral transduction of OCT4, SOX2, KLF4 and c-MYC in an FDA-registered cGMP facility. iPSCs were differentiated via suspension culture to induce the formation of 3D retinal eyecups. Eyecups were evaluated for retinal-specific and photoreceptor-specific markers via indirect immunofluorescence and confocal microscopy throughout development. CRISPR/Cas9 technology was also tested using small guide RNAs specifically targeting the NR2E3 locus in patient iPSCs.

Results: We have successfully generated two lines of clinical-grade NR2E3 patient-specific iPSCs in a cGMP manner. Early NR2E3 patient eyecups form polarized neural epithelial folds that express the early retinal progenitor markers SOX2, PAX6, VSX2/CHX10 and OTX2. As eyecups mature, dense pockets of OTX2-positive photoreceptor precursor cells arise. Later, eyecups begin to express photoreceptor-specific transcription factors CRX and NRL, as well as the phototransduction protein, recoverin. Compared to controls, NR2E3 eyecups exhibit an increased abundance of SW-opsin-positive blue cone photoreceptors, recapitulating the developmental phenotype observed in enhanced S-cone syndrome patients.

Conclusions: We have demonstrated that we can generate patient-specific iPSCs and iPSC-derived retinal photoreceptor precursor cells from patients with NR2E3-associated enhanced S-cone syndrome. These patient-derived cells recapitulate the retinal phenotype observed in patients and can be used to interrogate the pathophysiology of this rare disease. In particular, we will use CRISPR-mediated genome correction to correct the NR2E3 locus in patient iPSCs and rescue the enhanced blue cone phenotype and restore the downstream development of rod, M- and L-cone photoreceptors.

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Voted Outstanding Poster Presentation

Human Retinal Engineering using 3D Two-Photon Polymerization and Chitosan Hydrogels

Worthington, K.1, Tucker, B.A.1
1Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology & Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA


Purpose: The purpose of this study was to employ CRISPR/Cas9 genome editing to develop strategies for three major classes of disease-causing mutations: 1) dominant gain-of-function, 2) deep intronic and 3) exonic mutations.

Methods: We designed sgRNA oligos specific for each mutation and cloned them into bicistronic constructs. Donor constructs were cloned carrying ~500 bp of homologous flanking wild-type sequence. Constructs were delivered to human cells via transfection or electroporation. Non-homologous end-joining was determined via T7E1 assay, quantified using TA-cloning and Sanger sequencing.

Results: To treat patients with dominant disease, sgRNAs targeting the mutant vs wildtype allele were designed. T7E1 assay and Sanger sequencing showed that a sgRNA targeting the P23H mutation in RHO confers a 3-fold increase in targeting specificity (7.2% vs. 22.0%, respectively). A similar result was detected for the Q344X mutation in PAX6 (0.6% vs. 29.9%, respectively). To edit intronic recessive alleles, we demonstrated that co-delivery of two sgRNAs targeting upstream and downstream of the IVS40 mutation in USH2A successfully modified the locus (21.0% and 19.0%, respectively). To demonstrate the utility of our HDR strategy to correct an exonic mutation, we delivered an sgRNA targeting the MAK Alu insertion in exon 9 and a donor template carrying the corrected sequence. This construct restored WT transcript in iPSC-derived retinal precursor cells from a patient with MAK-associated RP.

Conclusions: Successful gene editing targeting three different classes of Mendelian mutations at several disease-causing loci brings us closer to the goal of providing genetically corrected autologous cell-based therapies for patients affected with inherited retinal degenerative disease.

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