2017 MERS poster
MERS Logo

10th Annual

2017 Midwest Eye Research Symposium

Sponsored by The Stephen A. Wynn Institute for Vision Research


MERS 2017 took place on August 4th, 2017 at
The University of Iowa Medical Education and Research Facility


Keynote Speaker/Session Chairs

Timothy Kern, Ph.D. - Keynote Speaker
Dr. Kern is a Professor in the Departments of Medicine and Pharmacology and the director of the Center for Diabetes Research at Case Western Reserve University. His research aims to understand the causes of diabetic retinopathy and to find ways to prevent the disease. A particular focus of his research has been the interactive relationship between photoreceptor cells and the vasculature in the retina. His work has resulted in almost 200 peer-reviewed publications and the Dr. Kern serves on numerous advisory boards as well as NIH study sections.

Shiming Chen, Ph.D. - Chair, Session I
Dr. Chen is a Professor of Ophthalmology and Visual Sciences and of Developmental Biology at the Washington University School of Medicine in St. Louis. The main focus of her research is on transcriptional control during photoreceptor cell development. Her lab studies transcription factors as well as epigenetic mechanisms that are important in the cells.

Markus Kuehn, Ph.D. - Chair, Session II
Dr. Kuehn is an Associate Professor in the Department of Ophthalmology and Visual Sciences at the University of Iowa. A focus of his laboratory is to understand the role that trabecular meshwork cells play in the healh of the tissue and how loss of trabecular meshwork cells leads to elevated intraocular pressure. Additionally, his laboratory has been developing methods to differentiate induced pluripotent stem cells into a cell type that resembles TM cells. These cells can be used to repopulate the TM in mouse glaucoma models and are able to restore function to the tissue

Arlene Drack, M.D. - Chair, Session III
Dr. Drack is an Associate Professor in the University of Iowa Department of Ophthalmology and Visual Sciences and the Ronald V. Keech Professor for Ophthalmic Genetics. Her laboratory research focuses on the causes of pediatric genetic eye disorders. She is also actively involved in the development of treatments and cures for genetic vision loss.

 


Oral Presentations

Session I - Session Chair Shiming Chen, Ph.D.

Shiming Chen:Meta-Analyses Decipher The Epigenetic Role Of CRX In Photoreceptor Development And Disease
Li Xuan Tan:Protective mechanisms against sub-lytic complement attack in the retinal pigment epithelium
Janis Eells:670 nm Photobiomodulation as a Therapy for Diabetic Macular Edema: A Pilot Study
Diane Slusarski:Functional characterization of disease-associated Bardet-Biedl syndrome 1 (BBS1M390R) allele in zebrafish

Session II - Session Chair Markus Kuehn, Ph.D.

Ankur Jain:Role of Primary Cilia in Trabecular Meshwork Cell function
Nader Sheibani:Cytochrome P450 1B1 is a Key Regulator of Ocular Oxidative Stress and Pathogenesis of Congenital Glaucoma
Sarah Ohlemacher:Differentiation and Maturation of Retinal Ganglion Cells Derived from Human Pluripotent Stem Cells for Studies of Glaucomatous Neurodegeneration

Session III - Session Chair Arlene Drack, M.D.

Arlene Drack: Results of the Phase III RPE65 gene replacement trial
Divya Sinha:Investigating gene augmentation as a therapeutic approach for Best disease
Janelle Garrison:Identifying effective time windows for gene replacement therapies in a ciliopathy mouse model of retinal degeneration
Simon Petersen-Jones:Gene Augmentation Rescues Rod Function and Preserves Structure in Dogs Leading the Way Towards Treating Human PDE6A-Retinitis Pigmentosa

 

Poster Presentations

Laura BohrerDevelopment of corneal endothelial cell-specific reporter constructs and a CRISPR-Cas9 genome editing approach to study Fuchs Dystrophy
Lindy BrastromEssential role for RNA Binding Motif Protein 24a (rbm24a) in eye and heart morphogenesis
Tania CruzRole of Gap Junction in Trabecular Meshwork Cell Division
Poppy DattaEffective arrest of retinal degeneration depends on the timing of gene restoration in BBS17 mouse model
Ryan DonahueDelayed RGC death and persistent phos-cJUN accumulation following optic nerve injury in BAX deficient mice
Rachel GenovaRole of neutral endopeptidase in corneal wound healing
Colin GermerPolarized secretion of pro-inflammatory exosomes by the retinal pigment epithelium is selectively regulated by vitamin A dimers and membrane cholesterol
Cheyanne GodwinTransplantation of IPSC-TM Restores Endogenous Trabecular Meshwork Cells
Christine HarmanWildtype Canine Trabecular Meshwork Cells do not Respond to Dexamethasone Treatment with an Increase in Myocilin Expression and CLAN Formation
Gulpreet KaurOrganelle defects in models of macular degeneration
Kristin KoehlLong-term stable Transgene Expression and Intraocular Pressure control by AAV-mediated Gene Therapy of the Trabecular Meshwork
Ariel KopelCharacterization of Cone Synapse Development in the Absence of Cav 1.4 or α2δ-4
Kirstin LangerRetinal Organoids Derived from Human Induced Pluripotent Stem Cells Exhibit a Defined Ganglion Cell Layer and Allow for the Modeling of Glaucomatous Neurodegeneration
Colten LankfordIdentification of HCN1 as a novel 14-3-3 client protein
Tim LinAssessment of early glaucomatous optic nerve damage by blue light chromatic pupillometry - a pilot study
Kacie MeyerIdentification of imprinted retinal genes using RNA sequencing
Kimberly MillerFunction of IL-33/ST2 Signaling in Glaucoma
Gulnara NovbatovaCharacterization of a novel interaction between Kv2.1 and 14-3-3 proteins in photoreceptors
Laurence OccelliGene augmentation therapy improves inner and outer segment morphology and preserves retinal structure in a large animal model of CNGB1-retinitis pigmentosa
Bisola OmobaExamining the Relationship Between Foveal Pit Morphology and Cone Outer Segment Length
Shin Ae ParkPaving-stone degeneration-like retinal lesions in young adult Siberian Husky dogs with primary angle-closure glaucoma (PACG)
Danielle PellackDeveloping mouse models to evaluate the role of mitochondrial dynamics in glaucoma
Heather SchmittOverexpression of histone deacetylase 3 (HDAC3) induces apoptosis in differentiated retinal neurons
Jessica ThompsonOptimizing the photopolymerization parameters of high-resolution, 3D biodegradable tissue scaffolds
Matthew Van HookFeatures of cone bipolar cell synaptic transmission onto retinal ganglion cells
Erica WoertzExamining the relationship between cone density and Vernier acuity in human albinism
Kristan WorthingtonHigh-resolution, 3D-printed chitosan tissue scaffolds for retinal regeneration
Brit ZaroExamining the Effect of Axial Length on Vessel Density Estimates Obtained using OCT Angiography

 

 


Abstracts


Development of corneal endothelial cell-specific reporter constructs and a CRISPR-Cas9 genome editing approach to study Fuchs Dystrophy

Bohrer L.R. 1, Burnight E.R. 1, Elizabeth L. Kennedy1, Stone E.M. 1, Greiner M.A. 1,2, Wagoner M.D. 1,2, Tucker B.A1 and Wiley L.A. 1
11Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA. 2Iowa Lions Eye Bank, Coralville, IA.


Purpose: Fuchs dystrophy is the most common corneal endothelial cell disease and the leading indication for corneal transplantation. Fuchs is characterized by loss of corneal endothelial cells (CECs), thickening of Descemet's membrane, corneal edema and impaired vision. Several genetic mutations have been shown to be associated with Fuchs including COL8A2 and TCF4. The aim of this study was to: 1) design promoter constructs suitable to monitor the differentiation of induced pluripotent stem cells (iPSCs) to CECs and 2) design CRISPR-Cas9 genome editing strategies to correct Fuchs-causing genetic mutations.

Methods: To generate iPSCs, fibroblasts from an individual with normal ocular history were virally transduced with OCT4, SOX2, KLF4 and c-MYC. We cloned lentiviral promoter constructs that were upstream of fluorescent reporter genes that are turned on at different stages of the iPSC to CEC differentiation: neural crest cells (NGFR/p75-E2-Crimson) and CECs (COL8A1-GFP and CHD2-tRFP). Constructs were transfected to HEK293 cells to test promoter activity. SgRNAs were designed to target the trinucleotide repeat expansion in TCF4 or mutations in COL8A2. The sgRNA oligos were cloned into bicistronic AAV vectors expressing a chimeric small guide and tracrRNA transcript and a human codon-optimized S. aureus Cas9. CRISPR-Cas9 constructs were transfected to HEK293 and tested for targeting efficiency using the T7E1 assay, TA cloning and Sanger sequencing.

Results: Transfected promoter constructs were robustly expressed in HEK293 cells, including membrane localization of the reporter for CDH2. Efficient targeting specificity of the TCF4 and COL8A2 CRISPR-Cas9 constructs in HEK293 cells were shown using the T7E1 assay and cleaved PCR products, indicating CRISPR-Cas9-mutated DNA, were imaged on agarose gels. The cutting efficiency for an sgRNA targeting COL8A2 was 27%.

Conclusions: We have engineered promoter constructs that will be used in future experiments aimed at developing an efficient method to generate iPSC-derived CECs from control patients and those with Fuchs caused by genetic mutations in COL8A2 or TCF4. Use of CRISPR-Cas9-mediated genome editing to correct the mutations in Fuchs patient-specific iPSCs will yield corrected control lines for comparison to diseased iPSC-derived CECs.

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Essential role for RNA Binding Motif Protein 24a (rbm24a) in eye and heart morphogenesis

Brastrom, Lindy K. 1, Scott, C. Anthony1, Spatz, Lillian B. 2, Dash, Soma3, Dawson, Deborah V. 4, Lachke, Salil A. 3, Slusarski, Diane C. 1
11 Department of Biology, University of Iowa, Iowa City, Iowa, 2 Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana, 3 Department of Biological Sciences, University of Delaware, Newark, Delaware, 4 Department of Pediatric Dentistry, 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. 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). 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 visually impaired fish are less responsive.

Results: We have developed an automated program for analysis using VIZN and are currently validating the OMR with a gene known to cause micropthalmia in mice, rbm24a. While VIZN demonstrated morphants of rbm24a to be responsive, OMR reveals they are partially visually impaired. rbm24a also has a cardiovascular role which we are currently investigating.

Conclusions: These visual assays allow for rapid screening of many candidate genes related to vision loss and impairment. Additionally, rbm24a functions similarly to cause micropthalmia in zebrafish as it does to its more studied organism, the mouse.

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Meta-Analyses Decipher The Epigenetic Role Of CRX In Photoreceptor Development And Disease

Chen, Shiming; Ruzycki, Philip A; and Zhang, Xiaodong
Department of Ophthalmology and Visual Sciences, Molecular Genetics and Genomics Graduate Program, Washington University, St. Louis, Missouri


Purpose: The CRX transcription factor (TF) regulates expression of many genes essential for photoreceptor development and survival. Chromatin immunoprecipitation-sequencing (ChIPseq) in mouse retina revealed >6,000 CRX binding sites, but RNAseq in Crx mutant retinas indicates that not all of these are important for regulation. This study aimed to investigate CRX-dependent epigenetic changes at these sites during rod photoreceptor development.

Methods: We generated retinal ATACseq and ChIPseq (H3K4me3 and H3K27Ac) datasets from wild-type and Crx deficient mice before (P2) and after (P14) photoreceptor differentiation to determine the effect of CRX loss at each regulatory region throughout the mouse genome. These results were compared to published epigenetic datasets, putting them in the broader context of normal epigenetic regulation.

Results: In the absence of CRX, only a subset (~1/3) of CRX-bound sites showed a reduction in active epigenetic state. These "CRX-Dependent" sites, at both promoters and enhancers, were distinguishable from the larger set of other CRX-bound sites by several criteria: They are activated later during normal development, are more strongly enriched for CRX consensus motifs, and are more strongly bound by CRX in the original ChIPseq profile. Genes near CRX-Dependent sites encode many of the essential photoreceptor proteins, whose expression is most affected in Crx mutant retinas.

Conclusions: This study demonstrates the power of functional genomics in understanding epigenetic regulation underlying normal retinal development. The results underscore the role of CRX in establishing the mature photoreceptor epigenome and misregulation caused by disease-linked mutations.

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Role of Gap Junction in Trabecular Meshwork Cell Division

Cruz, Tania1; Zhu, Wei1; Cring, Matthew2; Godwin, Cheyanne1,3; Tucker, Budd1,4; Kuehn, Markus1,3,4
1Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USA 2Department of Pediatrics, University of Iowa, Iowa City, IA, USA 3VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA, USA 4 Wynn Institute for Vision Research


Abstract: We have shown that inducible pluripotent stems cells (iPSC) have a unique ability to regenerate dysfunctional trabecular meshwork (TM) cells in vitro and in vivo. This project we look to better understand the renewing effects iPSC have in the trabecula meshwork via cell-to-cell contact in vitro. Since, primary TM cells (pTM) need to have physical contact with iPSC in order to see any effects; investigation moves us to gap junctions (GJ), specifically Connexin 43, as a means for the cells to share crucial information. Using CRISPER Cas-9 technology the GJA1 gene encoding connexin 43 was disrupted in TM5 cells (G1TM) resulting in loss of expression. G1TM cells were then co-culture with iPSC that are induced to be TM-like (iPSC-TM). Results show that G1TM5 in contact with iPSC-TM had inhibited growth (71.5%) when compared to the control (100%). However, the G1TM when cultured alone showed increased growth when compared to the unaltered TM5 control. Ultimately, connexin 43 plays a role in the ability for iPSC to pass proliferative information to TM cells in vitro.

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Effective arrest of retinal degeneration depends on the timing of gene restoration in BBS17 mouse model

Datta, Poppy, Hendrickson, Brandon, and Seo, Seongjin
Department of Ophthalmology and Visual Sciences, University of Iowa College of Medicine, Wynn Institute for Vision Research, Iowa City, IA 52242.


Purpose: Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy with blindness being a cardinal feature. The current study is aimed to examine the presence of "critical time-window" for effective gene augmentation therapy in BBS using an LZTFL1 (aka BBS17) mouse model.

Methods: We used an Lztfl1 mutant mouse line, in which the gene trap cassette suppressing Lztfl1 expression can be eliminated using tamoxifen-inducible FLP recombinases; i.e. mutant alleles are converted to normal by tamoxifen injections. Tamoxifen was injected at two ages: Group A at 1.5 months of age and Group B at 3 months. Immunoblotting, immunohistochemistry, and electroretinography (ERG) were conducted to assess the restoration of Lztfl1 gene expression, BBS protein function, and photoreceptor cell function at 3 months post-injection.

Results: Restoration of Lztfl1 expression in Group A (1.5-month rescue) significantly preserved photoreceptor cells and retinal functions. Interestingly, however, protein mislocalization phenotype was only partially rescued even after 3 months of recovery. In Group B (3-month rescue), restoration of Lztfl1 expression failed to preserve photoreceptors and retinal functions, essentially undistinguishable from unrescued mutant animals.

Conclusion: Our study suggests that effective prevention of retinal degeneration in BBS by gene therapy may depend on the age of individuals or the status of photoreceptors. This further implies the presence of critical time window for successful gene therapy to preserve vision in BBS patients.

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Delayed RGC death and persistent phos-cJUN accumulation following optic nerve injury in BAX deficient mice

Donahue, Ryan, Nickells, Robert W.
University of Wisconsin Madison


Purpose: Following optic nerve crush (ONC), retinal ganglion cell (RGC) apoptosis occurs within 7 days in wild-type C57BL/6 mice and never occurs ONC in Bax-/- mice. Understanding how BAX dosage affects when apoptosis occurs is critical for developing an anti-BAX therapeutic to prevent loss of RGCs in optic neuropathy. To that end, we tested the hypothesis that RGCs in Bax+/- mice would experience a delay cell death following ONC.

Methods: RGC loss in Bax+/- mice was assessed by cell counts of DAPI stained whole-mounts at various times from 3-12 weeks following ONC. Cell loss was measured as the fraction of cells in the damaged (OS) eye relative to the contralateral (OD) eye. The JNK-JUN pathway has been associated with activating BAX in RGCs following ONC. To explore if pathway was continuously activated post crush in Bax+/- mice, we used immunofluorescence to assess phospho-c-Jun accumulation in the nuclei of RGCs. Bax+/- mice underwent ONC and were collected 1, 3 or 7 weeks later. Retinas were fixed in 4% PFA, sucrose cryoprotected and then eye cups were cryosectioned and stained for the RGC transcription factor, BRN3A, and for an end product of JNK pathway activation, phospho-c-JUN.

Results: After ONC, Bax+/- retinas began to exhibit statistically significant cell loss at 8 weeks and by 12 weeks exhibited only a 12% loss of cells. Following ONC, phospho-c-JUN co-localized with BRN3A in 83%, 55% and 68% of BRN3A positive cells at 1, 3 and 7 weeks respectively, indicating that this pathway was persistently activated. We are currently interrogating the apoptotic activation mechanism months after ONC by viral reintroduction of an exogenous BAX protein into the RGCs of Bax-/- mice.

Conclusions: BAX deficiency delays cell loss following ONC. Despite this, the apoptosis associated JNK pathway appeared to remain activate in RGCs for months after acute injury. Together, these findings demonstrate that a BAX therapeutic likely needs to be permanent to prevent the loss of damaged RGCs.

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Results of the Phase III RPE65 gene replacement trial

Arlene V. Drack1,2, Daniel Chung3, Stephen Russell1, Jean Bennett, Jennifer Wellman3, Zi Fan Yu3, Albert Maguire, Edwin M. Stone1,2, Katherine High3
1University of Iowa Department of Ophthalmology and Visual Sciences, 2Wynn Institute for Vision Research, 3Spark Therapeutics


Purpose: Leber congenital amaurosis (LCA) is a leading cause of congenital blindness with at least 19 causative genes. A gene therapy (SPK-RPE65) for LCA caused by RPE65 gene mutations recently completed a Phase 3 trial.

Methods: 31 patients with CLIA laboratory-confirmed RPE65-mediated LCA were enrolled at two centers. 21 participants were randomized to the intervention group; 10 were randomized to 1 year of observation before crossover. Subretinalinjection of SPK-RPE65 in a 300 uL volume was delivered to one eye, followed by the contralateral eye within 18 days. Efficacy measures included mobility testing (MT), full-field light sensitivity threshold (FST), and visual acuity (VA).

Results: The trial met its primary endpoint (p=0.001), demonstrating improvement of functional vision in the intervention group compared to the control group, as measured by bilateral MT change score between baseline and 1 year. FST improved significantly in the intervention group compared to the control group (p<0.001). MT change score for the assigned first eye was significantly better than controls (p=0.001). VA was not statistically different from controls (p=0.17). There were no serious adverse events (AEs) related to SPK-RPE65 or deleterious immune responses. Procedure-related AEs included transient elevated IOP (4), cataract (3), retinal tear (2), and inflammation (2). Patients randomized to no treatment for a year did not improve on testing during this time, but did improve after they were treated following one year of observation. Results have been stable for 2 years in the initial treatment group and for one year in the second group treated.

Conclusions: SPK-RPE65 resulted in statistically significant improvement in functional vision and visual function measured by MT and FST, respectively. AEs were typical for subretinal surgery and not related to vector or immune response. Children with nystagmus and/or poor vision require genetic testing, since subretinal gene therapy improves visual function in RPE65-mediated disease.

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670 nm Photobiomodulation as a Therapy for Diabetic Macular Edema: A Pilot Study

Janis T. Eells1, Sandeep Goplalakrishnan2, Tom Connor3, Kim Stepien4, Joe Carroll3, Vesper Williams3, Krissa Packard3, Judy Kim4
1Department of Biomedical Sciences, University of Wisconsin-Milwaukee, 2College of Nursing, University of Wisconsin-Milwaukee, 3Department of Ophthalmology, Medical College of Wisconsin, 4Department of Ophthalmology and Visual Sciences, University of Wisconsin


Purpose: Diabetic macular edema (DME) is a common complication of diabetes mellitus. Photobiomodulation (PBM) by far-red to near infrared (NIR) light has been demonstrated to improve retinal function in experimental and clinical retinal disease. Although anti-vascular endothelial growth factor (anti-VEGF) agents have revolutionized treatment for DME, some eyes are treatment resistant. Here we tested the hypothesis that NIR PBM will attenuate the severity of diabetic macular edema and improve visual acuity in patients with DME.

Methods: A randomized prospective pilot study was conducted in accordance with the Declaration of Helsinki and the study protocol was approved by the IRB at the Medical College of Wisconsin. Ten adult patients (66 ± 3 years, 7 males 3 females) with treatment resistant DME were randomized into standard treatment (STD-TRT; anti-VEGF therapy alone) and PBM treatment (PBM-TRT; anti-VEGF plus PBM) groups. Light treatment was administered at a dose of 4.5 J/cm2 three consecutive days per week for 8 weeks using a 670 nm LED Array (WARP-10, Quantum Devices, Inc., Barneveld, WI) positioned 2.5 cm from the closed treatment eye. Functional and anatomic assessments were made at baseline, 8 weeks and 24 weeks.

Results: At 24 weeks, visual acuity (VA) decreased from baseline at 24 weeks in STD-TRT subjects (-3 ± 4 letters; n=4). In contrast VA was improved from baseline at 24 weeks in PBM-TRT subjects (6 ± 5 letters; n=6). Central retinal thickness by OCT was increased from baseline at 24 weeks in STD-TRT subjects (120 ± 97 ?m; n=4) and was decreased from baseline at 24 weeks in PBM-TRT subjects (-24 ± 5 ?m; n=6). These finding are suggestive of a reduction in macular edema and an improvement in vision following NIR-PBM. No adverse effects attributable to NIR-PBM were noted by the patients or study investigators during the study period.

Conclusions: Although the sample size is small, the findings show a reduction in DME and an improvement in VA following NIR-PBM. Results support the application of NIR-PBM for the treatment of DME in eyes refractory to anti-VEGF treatments. This is consistent with previous studies showing NIR-PBM improves retinal function in experimental and clinical retinal disease. The use of NIR-PBM is non-invasive, safe and may be an effective adjunct therapy for DME.

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Identifying effective time windows for gene replacement therapies in a ciliopathy mouse model of retinal degeneration

Garrison, Janelle*; Hsu, Ying*; Kim, Gunhee; Searby, Charles; Nishimura, Darryl; Sheffield, Val
* these authors made equal contributions

Department of Pediatrics, University of Iowa, Iowa City, IA


Purpose: It is unknown whether restoration of target gene expression at any stage of retinal degeneration can arrest photoreceptor cell death and result in functional improvement. We aim to determine the effectiveness of rescue in a mouse model of Bardet-Biedl Syndrome as a function of degeneration and define the transition point between gene replacement therapies and cell replacement therapies during the progression of retinal degeneration.

Methods: Endogenous Bbs8 gene expression was restored in mice that initially lacked Bbs8 expression by using a mutant mouse model of Bbs8, in which the gene trap cassette placed between the exons 3 and 4 of Bbs8 can be excised by tamoxifen inducible FLP recombinase. Tamoxifen was injected at (a) postnatal days 9-15 (P9-15) with 0-10%, (b) 6 weeks of age with 15-25%, (c) 3.5 months of age with ~50%, and (d) 6 months of age with 75-90% photoreceptor cell loss. Electroretinography was performed to measure retinal function after rescue, and histology was performed to assess the morphological integrity of the retina.

Results: Electroretinograms show recovery of photoreceptor function and immunofluorescence shows restoration of candidate cargo localization in rescued photoreceptor cells, and these effects are dependent on the start point of rescue. By histology, we found that the ability to arrest cell death in existing photoreceptor cells is also dependent on the stage of retinal degeneration.

Conclusion: We identified time windows for the effective rescue of degenerating photoreceptors, and defined the last amenable time point for treating retinal degeneration by gene replacement.

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Role of neutral endopeptidase in corneal wound healing

Genova, Rachel M1; Yorek, Matthew S (2); Harper, Matthew M (2); Pieper, Andrew A (3).
1Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States; Pappajohn Biomedical Institute, University of Iowa, Iowa City, Iowa, United States 2Iowa City Department of Veterans Affairs Center for the Prevention and Treatment of Visual Loss, Iowa City, Iowa, United States; Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States 3Iowa City Department of Veterans Affairs Center for the Prevention and Treatment of Visual Loss, Iowa City, Iowa, United States; Department of Neurology, The University of Iowa, Iowa City, Iowa, United States; Department of Psychiatry, The University of Iowa, Iowa City, Iowa, United States; Pappajohn Biomedical Institute, University of Iowa, Iowa City, Iowa, United States


Purpose: Neutral endopeptidase (NEP) modulates inflammation by metabolizing signaling peptides. Recently, NEP was identified in the human cornea, a tissue that expresses its preferred substrate, substance P (SP). SP is an injury-inducible neuropeptide involved in both homeostatic maintenance and wound healing of the corneal epithelium. Here we are investigating the effect of genetic disruption of NEP in the mouse cornea under both uninjured and injured states. We hypothesized that genetic interruption of NEP activity would accelerate corneal reepithelialization during the acute phase of wound healing, a period critical for preventing vision-limiting sequelae during later stages of repair.

Methods: Histology, western blotting, and enzyme activity assays were used to determine functional NEP expression in corneas from WT and NEP-deficient mice. Corneal morphology was evaluated with optical coherence tomography and immunofluorescent labeling of corneal nerves and circumcorneal vessels. We modeled alkali corneal injury in a separate cohort of mice, and evaluated recovery with slit lamp imaging at 1, 3, and 7 days post-injury.

Results: Functional NEP expression in the WT mouse cornea is abolished in NEP-deficient animals. Compared to WT mice, NEP-deficient mice have similar corneal thickness and circumcorneal vasculature but have significantly denser corneal innervation. In our wound healing model, NEP-deficient mice have significantly increased corneal reepithelialization at 3 and 7 days post-injury, compared to WT animals.

Conclusions: NEP is functionally expressed in the mouse cornea where its constitutive genetic disruption leads to accelerated reepithelialization following chemical corneal injury. Pharmacological modulation of NEP may thus provide a novel therapeutic option for patients with corneal injury.

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Polarized secretion of pro-inflammatory exosomes by the retinal pigment epithelium is selectively regulated by vitamin A dimers and membrane cholesterol

Germer, Colin; Rathasamy, Gurugirijha; Tan, Li Xuan; Lakkaraju, Aparna
Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53706

Purpose: Exosomes, vesicles derived from the endolysosomal pathway, participate in intercellular communication and disease processes. Although retinal pigment epithelial (RPE) cells secrete exosomes, little is known about their composition, function and fate. Here, we analyzed the size, morphology and protein repertoires of exosomes released from polarized primary RPE, and investigated how this is modulated by cholesterol accumulation due to vitamin A dimers.

Methods: Exosomes were purified from the apical and basolateral media of primary porcine RPE cultured on Transwell filters by density gradient ultracentrifugation. Transmission electron microscopy and NanoSight particle tracking were performed to analyze the morphology, size distribution, and number of exosomes. Exosomal proteins were analyzed by immunoblotting and mass spectrometry.

Results:Exosomes secreted by polarized primary RPE have cup-shaped morphologies under TEM and mean diameters of 100 nm. In RPE with A2E, NanoSight tracking showed that significantly more exosomes are released apically compared to controls. We have previously shown that vitamin A dimers increase ceramide levels by abnormally activating acid sphingomyelinase. Ceramide is a cone-shaped lipid that increases the biogenesis of exosomes. Immunoblotting and mass spectrometric analysis revealed that exosomes released apically from cells with A2E contain pro-inflammatory proteins.

Conclusions:Primary adult RPE monolayers secrete exosomes in a polarized manner with a distinct repertoire of proteins packaged into exosomes destined for apical or basolateral release. Vitamin A dimers can promote retinal pathology by inducing secretion of pro-inflammatory exosomes. This could induce inflammatory and/or immune responses and spread pathogenic stimuli. Current studies are focused on identifying promising approaches to modulate exosome release.

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Transplantation of IPSC-TM Restores Endogenous Trabecular Meshwork Cells

Cheyanne Godwin1,2, Wei Zhu1, Tania Cruz1, Steven S. Withmore1,3, Markus H. Kuehn1,2,3
1Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USA 2VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA, USA 3 Wynn Institute for Vision Research

Purpose: The trabecular meshwork (TM) is required to maintain normal drainage of aqueous humor; however degeneration of TM cells is a characteristic reason for an increase in intraocular pressure in glaucoma. We previously demonstrated that transplantation of mouse iPSC-TM cells can preserve TM cellularity by stimulating endogenous TM cell division. Here we demonstrate evidence of a similar process in human anterior chamber organ cultures.

Methods: Human iPSCs from non-glaucomatous donors were cultured in TM cell conditioned media and were confirmed to be TM-like by transcription profiling. Human anterior chambers were perfused at a constant rate of 2.5ul/min with media and BrdU. The corresponding eye from the same donor was used as the vehicle control. iPSC-TM were then injected into cultured human anterior segments. TM cellularity, and the presence of iPSC-TM and BrdU in the TM was examined by (immuno-) histology.

Results: When cultured in TM cell conditioned media IPSCs can be induced into IPSC-TM which are transcriptionally similar to primary TM. Injection iPSC-TM into the cultured human anterior chamber increases the cellularity of the TM by 1.5 fold (p<0.05). Few iPSC-TM cells remained in the TM two weeks after injection and staining indicates 2-fold increased BrdU incorporation in iPSC-TM injected eyes (p<.05).

Conclusions: Our preliminary data indicates that iPSC-TM transplantation is beneficial to rescue glaucomatous phenotype. Future experiments are needed to investigate functional improvement of the TM, e.g. lowering IOP and increase outflow facility in the anterior chamber organ culture system.

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Wildtype Canine Trabecular Meshwork Cells do not Respond to Dexamethasone Treatment Dwith an Increase in Myocilin Expression and CLAN Formation

Christine D. Harman, Hsiang-Rong Tsai, Daniel A. Kelleher, Kristin L. Koehl, Gabriel A. Stewart, András M. Komáromy
Michigan State University


Purpose: This study challenges the notion that all TM cells are similarly steroid responsive by characterizing wt canine TM cells and demonstrating lack of intraocular pressure (IOP) DEX-response in wt dogs.

Methods: Without specific TM cell markers, a combination of assays is required for identification of cultured TM cells, including DEX-induced increase in cross-linked actin network (CLAN) and myocilin expression. TM cells were isolated from 6 wt dogs and characterized by morphologic appearance, immunocytochemistry (ICC) and phagocytosis assay. DEX stimulation (100nM) was performed for up to 14 days and CLAN formation (ICC; F-actin) and myocilin expression (qPCR, western blot) were measured. Subsequently, diurnal IOP and aqueous humor (AH) myocilin (western blot) were measured in 8 wt dogs treated topically with DEX for 30 days, followed by subconjunctival triamcinolone acetonide in a subset of animals (n=3). Significance was evaluated by Student's t-test (p<0.05).

Results: Despite significant DEX-induced disruption of the F-actin network organization (mean anisotropy decreased by 43%), there was no change in CLAN-formation (1-3%) or alteration to myocilin expression levels. These findings correlate with observed consistent mean diurnal IOP after ≥30 days of treatment (baseline vs. steroid treatment: 15.62 ±0.11 vs. 13.70±0.31 mmHg). Compared to baseline (1.06±0.17) relative AH myocilin protein concentration increased significantly on DEX day 14 (2.12±0.32) but decreased by day 26 (1.49±0.47).

Conclusions: Canine wt TM cells do not respond to DEX treatment with an increase in CLAN-formation and myocilin expression. This observation is consistent with the non-responsiveness of wt canine IOP to topical steroids.

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Role of Primary Cilia in Trabecular Meshwork Cell function

Jain Ankur1, Zhu Wei1, Zhang Qihong1, Zode Gulab2, Kuehn Markus1, Clark Abe2, Sheffield Val1
1University of Iowa, Iowa City, Iowa, 2North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, Texas


Purpose: Primary cilia are specialized organelles that play an important role in intraflagellar transport. The importance of primary cilia is observed in a group of disorders termed ?ciliopathies?. The mechano-sensing role of primary cilia in regulating pressure changes in the eye remains largely unknown. Intraocular pressure (IOP) is the pressure maintained in the eye by constant production of aqueous humor and efflux through trabecular meshwork (TM) and uveoscleral outflow pathways. Any unregulated change in either production or outflow affects IOP directly. Glaucoma, an optic neuropathy, involves increased IOP as the major risk factor. We hypothesize that primary cilia play a role in aqueous humor dynamics and IOP regulation.

Methods: Number of cilia positive cells and ciliary lengths (immunochemically using anti-ARL13b antibody) were compared among cultured human primary normal (NTM) and glaucomatous TM (GTM) cell strains (n=3). Chemical agents such as chloral hydrate, siRNA and CRISPR/Cas9 against intraflagellar proteins were used to study the effects of modified cilia on glaucoma-related insults including dexamethasone (DEX) and TGFβ2 signaling in TM.

Results: Cultured GTM cell strains appear to have longer primary cilia as compared to NTM. Disrupting cilia results in affected DEX and TGFβ2 signaling (measured by SMAD and GRE-promoter reporter assays) as well as extracellular matrix synthesis in TM.

Conclusions: The differences among primary cilia on GTM vs NTM cell strains and involvement of primary cilia in DEX and TGFβ2 signaling suggest a prominent role of primary cilia in TM function and potential involvement in glaucoma pathophysiology.

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Organelle defects in models of macular degeneration

Kaur, Gulpreet; Tan Li Xuan; Toops, Kimberly; La Cunza, Nilsa; Rathnasamy, Gurugirijha; Fernandes, Marie; Blenkinsop, Timothy; Lakkaraju, Aparna
1Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI


Purpose: The retinal pigment epithelium (RPE) performs many functions that are indispensable for vision and is also an initial site of insult in blinding diseases such as age-related macular degeneration (AMD). Aging and AMD are characterized by decreased cholesterol metabolism, decreased endo-lysosomal function, and increased accumulation of vitamin A metabolites in the form of lipofuscin in the RPE. We investigated how vitamin A metabolites in the RPE impact vesicles in the endo-lysosomal pathway.

Methods: We used high-speed live imaging of primary pig RPE cultures, adult human RPE cultures and a mouse model of Stargardt's inherited macular degeneration (which has high levels of vitamin A metabolites in the RPE) to visualize components of the endo-lysosomal pathway.

Results: The presence of vitamin A metabolites causes swollen, enlarged early endosomes that appear paralyzed. We have previously shown that vitamin A metabolites trap cholesterol in the RPE. Drugs that remove excess cholesterol decrease early endosome size in the RPE of Stargardt disease mice and in aged human RPE.

Conclusions: Our data suggest that lipofuscin and vitamin A metabolites cause defects in the endocytic pathway in the aging RPE, which could contribute to RPE dysfunction and negatively impact the photoreceptor support functions of the RPE. Cholesterol accumulation and impaired endo-lysosomal function have emerged as key players in Alzheimer's and Parkinson's diseases since they serve as highly dynamic platforms for macromolecule sorting, signaling, and cholesterol metabolism. Our current study identifies defects in the endo-lysosomal pathway as crucial players in the progression of AMD.

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Long-term stable Transgene Expression and Intraocular Pressure control by AAV-mediated Gene Therapy of the Trabecular Meshwork

Koehl, Kristin1, Oh, Annie2 Harman, Christine D. 1, Boye, Sanford L. 3, Chiodo, Vince A. 3, Hauswirth, William W., 3 Boye, Shannon E. 3, Komaromy, Andras M., 1
1Michigan State University, College of Veterinary Medicine 2 North Carolina State University, College of Veterinary Medicine 3University of Florida


Purpose: To achieve long-term, stable Intraocular Pressure (IOP) control by Trabecular Meshwork (TM)-targeted AAV-gene replacement therapy in a canine open-angle glaucoma (OAG) model based on an ADAMTS10 missense mutation.

Methods: Dogs were treated unilaterally by intracameral injection of the single stranded, capsid mutant vector AAV2(Y444F)-smCBA-hADAMTS10. Phase 1: 7 dogs were treated with 1E11 vector genomes (vg). Phase 2: dose was increased, and 3 dogs were treated with 1.4E12 vg. Weekly diurnal IOPs were measured by rebound tonometry (Icare Tonovet) for 6 month (Phase 1) and 8-12 months (Phase 2), respectively. In Phase 1, hADAMTS10 transgene expression within the TM was measured by qPCR in 6-12-month intervals post AAV administration.

Results: The intracameral administration of 1E11 vg AAV2(Y444F)-smCBA-hADAMTS10 in Phase 1 neither lowered IOP nor prevented increased progression of IOP. Nevertheless, Phase 1 demonstrated robust and stable hADAMTS10 transgene expression within the TM over 36 months. By increasing the vector dose to 1.4E12 vg, we observed a clear decrease in IOP by 76 days post-treatment in 2/3 dogs treated in Phase 2. Mean diurnal IOPs were maintained between 12-15 mmHg in the treated eyes of these 2 dogs over the 8-12-month study period compared to 26-28 mmHg in the untreated fellow eyes. The third dog did not respond to therapy. Overall, the intracameral AAV administration was well tolerated.

Conclusions: Long-term IOP control (≥12 months) combined with robust transgene expression (≥36 months) in a well-established large animal OAG-model supports the potential value of AAV-mediated TM gene expression modification for effective and lasting glaucoma treatment.

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Characterization of Cone Synapse Development in the Absence of Cav 1.4 or α2δ-4

Ariel Kopel, Joseph Laird, Sheila Baker
University of Iowa, Department of Biochemistry


Abstract: Using conefull mice (mice that only have cone cells in their retina), we hope to get an understanding of the morphology cone cells and their sub-units in the absence of Cav 1.4 or α2δ-4. Multiple mouse lines were bred to give us mice that only had cone cells and one of the two mutations. Once those mice were genotyped and found to be the correct type of mouse, we used OCT and IHC to view the retinal layers and fluoresce to see the internal layering of different proteins in the synaptic lays of cone cells. While there are some physical differences between wildtype (nom mutational mice) and conefull mice the conefull mice they were determined to be suitable for being the control model of the two other mutation types. The loss of Cav 1.4 or α2δ-4 does have an effect on how cone synapses form, but this will have to be further tested with IHC and OCT to have a clearer idea of the exact morphology, protein formation, and protein placement in these mutations of the cone cells.

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Retinal Organoids Derived from Human Induced Pluripotent Stem Cells Exhibit a Defined Ganglion Cell Layer and Allow for the Modeling of Glaucomatous Neurodegeneration

Langer, Kirstin1, Sridhar, Akshayalakshmi1, Johnson, Zachary1, Tseng, Henry2, Meyer, Jason1
1Indiana University-Purdue University, Indianapolis, IN 2 Duke University


Purpose: Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs), mimicking both the temporal the spatial organization of the native retina. Although numerous studies have demonstrated the development and functionality of photoreceptors in retinal organoids, the development of retinal ganglion cells (RGCs) has been mostly overlooked. In neurodegenerative diseases of the eye, such as glaucoma, the vital connection between the eye and the brain is damaged, resulting in loss of vision and blindness. As the mechanisms by which this degeneration occurs remain unclear and few therapeutic options are available, the development of retinal organoids represents an exciting and novel approach for the study of RGC development and disease.

Methods: Thus, efforts were focused on the generation of RGCs within inner layers of retinal organoids, with this differentiation occurring through identifiable stages closely resembling retinogenesis. Furthermore, the ability for retinal organoids to serve as an effective model for glaucomatous degeneration of RGCs was investigated by using hPSCs derived from a glaucoma patient with the E50K mutation in the Optineruin gene.

Results: The development of RGCs within retinal organoids occurred in a time course fashion within the inner layers of such structures. Additionally, the differentiation of retinal cells from both WT and E50K-hPSCs occurred in a similar manner, with no observed deficit in the development of RGCs. Further analysis of E50K-derived retinal organoids revealed increased activation of apoptotic markers within the RGC layers of the organoid. Treatment of E50K-derived organoids with an autophagy activator was performed to determine the link between autophagy disruption and apoptotic cell death.

Conclusion: The results of study represent the first efforts to utilize retinal organoids to effectively model the development of RGCs within organoid structures, as well as serve as a novel approach for the modeling of glaucomatous neurodegeneration.

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

Colten K. Lankford, Justin Elem, Maria Dean, Yuan Pan and Sheila A. Baker
1Department of Biochemistry, Carver College of Medicine, University of Iowa


Purpose: Hyperpolarization activated cyclic nucleotide gated potassium channel 1 (HCN1) belongs to a family of voltage gated, tetrameric sodium/potassium channels expressed in the heart and nervous system. The activity of HCN1 channels contribute to shaping of resting membrane potential, signal integration and pacemaking. HCN1 is expressed in multiple retina neurons, most predominantly rod and cone photoreceptors. In response to light driven hyperpolarization, HCN1 conducts a feedback current that limits the duration of signaling, thus preventing saturation of the downstream retinal circuits. Previous studies of HCN1 identified a di-arginine ER retention signal that must be masked or otherwise inactivated for the channel to reach the cell surface. Mutation of the ER retention signal allows for HCN1 dimers to accumulate. This suggests a model where the assembly of two HCN1 dimers into a functional tetramer inhibits the ER retention signal. The ubiquitously expressed 14-3-3 signaling adaptor proteins are a candidate for monitoring HCN1 assembly and forward trafficking because they are dynamically recruited to their client proteins by phosphorylation, act as dimers so can bridge two partners together, and are known to play diverse roles as scaffolds, allosteric modulators, or trafficking chaperones. In this study we are testing if 14-3-3 interacts with HCN1.

Methods: Co-immunoprecipitations from transiently transfected HEK293 cells and retina were used to test for interaction between HCN1 and 14-3-3. Predicted 14-3-3 binding sites within HCN1 were identified using 14-3-3 Pred. Biotinylated peptides corresponding to the putative 14-3-3 binding sites in HCN1 were used in thermal shift and biolayer interferometry assays to map the site of interaction.

Results: Immunoprecipitation of 14-3-3 pulls down HCN1 and the interaction is sensitive to phosphatases. Four high scoring predicted 14-3-3 binding sites were identified in HCN1. Biotinylated peptides corresponding to these sites were synthesized, with and without phosphorylation of a key serine. Thermal shift assays to monitor the unfolding of recombinant 14-3-3 revealed that addition of a phosphorylated peptide from near the C-terminus (peptide P-distal CT) of HCN1 stabilized 14-3-3. The addition of the other peptides did not cause significant shifts in the melting curve of 14-3-3. Preliminary biolayer interferometry assays indicate that the P-distal CT peptide binds to 14-3-3 with low micromolar affinity, as is typically observed for other clients of 14-3-3.

Conclusion: HCN1 is a newly identified 14-3-3 client. Future work will elucidate the physiological role of 14-3-3 binding to HCN1.

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Assessment of early glaucomatous optic nerve damage by blue light chromatic pupillometry - a pilot study

Lin, Tim; Koehl, Kristin; Harman, Christine; Komáromy, András
Michigan State University


Purpose: To measure pupil constriction rate, amplitude and postillumination pupil response (PIPR) following blue-light stimulation in dogs with open-angle glaucoma (OAG) caused by a G661R missense mutation in the ADAMTS10 gene.

Methods: Nine ADAMTS10-mutant dogs (age range 2.7-5.3 years) with well-defined stages of early and moderately advanced OAG and four wild type (wt) dogs (1.0-2.3 years) were examined. Pupil constriction rate and amplitude were measured following a 1-s 400-cd/m2 blue light stimulus (480 nm, LKC RETeval). PIPR was calculated as the ratio of pupil diameters recorded 10-20 s following offset of focal bright red (630 nm) and blue (480 nm) light stimuli (1-200 kcd/m2; BMVT Melan-100) and analyzed by Student t-test (significance: p<0.05). The ratio of intrinsically photosensitive retinal ganglion cells (ipRGC; melanopsin positive) to total RGC (NeuN positive) was estimated in representative wt and OAG retinal sections by immunohistochemistry (IHC).

Results: There were no differences in any of the pupil measures following blue-light stimulation between OAG-affected and wt dogs. Even with ophthalmoscopically recognizable optic nerve head atrophy, neither pupil constriction nor recovery rates differed from wt. Preliminary IHC estimates of ipRGC numbers were ~0.16% (1/609 total RGCs) in wt and ~0.44% (4/902) in early OAG, respectively.

Conclusions: Blue-light pupillometry does not appear to be a useful measure for early optic nerve damage in canine OAG. Canine ipRGCs do not seem to be affected in early glaucomatous optic neuropathy. Future investigations will focus on dogs with advanced OAG to determine the disease stage when blue light responses become affected.

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Identification of imprinted retinal genes using RNA sequencing

Kacie J. Meyer1,2, S. Scott Whitmore2, Todd E. Scheetz2, and Michael G. Anderson1,2
1Department of Molecular Physiology and Biophysics 2Department of Ophthalmology and Visual Sciences, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA


Purpose: Epigenetics likely contributes to genetically complex ocular diseases with missing heritability and the variable expressivity observed in monogenic ocular disease. Genomic imprinting is an epigenetic phenomenon resulting in differential allelic gene expression that is dependent on the parent-of-origin. The goal of this study is to identify imprinted genes expressed in the mouse retina.

Methods: Using RNA-Seq to determine parent-of-origin expression, we analyzed the retinal transcriptome of two genetically diverse inbred mouse strains and their resulting F1 progeny from reciprocal crosses.

Results: To date, the data identify 22 known imprinted genes that are expressed and imprinted in the retina of mice. Of these, 5 genes are paternally imprinted and 17 genes are maternally imprinted. Initial analysis has not identified novel imprinted retinal genes.

Conclusions: This work provides proof-of-principle evidence of a role for imprinted genes in the retina. Future studies will investigate the potential contribution of imprinted genes to the pathogenesis of retinal disease.

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Function of IL-33/ST2 Signaling in Glaucoma

Kimberly Miller1, Oliver W. Gramlich1,2, Kyle Gonsalves1, Grefachew Workalemahu1, Robert F. Mullins1, Markus H. Kuehn1,2
1Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USA 2VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA, USA


Purpose: Recent findings indicate an important role of Interleukin-33 (IL-33) signaling through its ST2 receptor in CNS diseases. However, it is currently unknown if IL-33/ST2 signaling under pathological conditions is either detrimental or beneficial for neuronal survival. Expression of IL33 has been found to be highly abundant in the optic nerve. Glaucoma is the most common optic neuropathic disorder, making it a good candidate to study the function of IL-33/ST2 receptor signaling.

Methods: IL-33, soluble (s) ST2 and membrane-bound ST2L receptor expression was analyzed by real-time polymerase chain reaction (rtPCR) and immunostaining in human glaucomatous and healthy optic nerve heads (ONH). The function of IL33 signaling was investigated in vitro using an IL33/lipopolysaccharide (LPS) stimulation assay on microglia-like BV2 cells followed by determination of the TNF-a, IL10, and IL-4 secretion levels.

Results: rtPCR confirmed the presence of IL33 in all examined ONH and found a 3 fold decrease of soluble ST2 in glaucomatous ONH, and a 8 fold increase of membrane bound ST2 in glaucomatous ONH.. Stimulation of BV2 cells with IL-33 significantly increased secretion of TNF-α and IL-10. Co stimulation with IL-33 and LPS leads to significant increase of TNF-α, but also to an increased secretion of anti-inflammatory cytokines such as IL-10 and Il-4. Furthermore Il-33 decreases NO levels significantly when compared to LPS stimulation alone.

Conclusion: IL-33 signaling via its ST2 receptor seems to be an important regulator of neuroinflammation in glaucoma. Secretion of both, pro and anti-inflammatory mediators, are affected upon IL-33 stimulation. Based on the response of IL-10 and Il-4 production, IL-33 signaling might be a future target for immunoregulation.

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Characterization of a novel interaction between Kv2.1 and 14-3-3 proteins in photoreceptors

Novbatova,Gulnara; Baker, Sheila
The University of Iowa, Biochemistry Department

Abstract: Retinal photoreceptors convert light into electrical signals for communication to downstream neurons. This requires coordination from many different ions channels. In this study our purpose is the investigation of the delayed-rectifier voltage-gated potassium channel, Kv2.1/Kv8.2. Kv2.1/Kv8.2 is located in the inner segments of photoreceptors and is responsible for maintaining the membrane potential. Previous studies have shown that the co-assembly of Kv2.1 with the electrically silent Kv8.2 changes the biophysical properties of Kv2.1 to create a unique potassium channel that can operate at the negative membrane potential of photoreceptors. The importance of Kv8.2 to human vision is demonstrated by the findings that mutations in Kv8.2 cause cone-rod dystrophy 3B. Kv8.2 also requires Kv2.1 to exit the ER and traffic to the plasma membrane, but the mechanisms controlling the coordination of Kv2.1/Kv8.2 assembly and forward trafficking are unknown. Previous studies on related potassium channels suggest that the 14-3-3 family of proteins could act as a "trafficking chaperone" to regulate Kv2.1/Kv8.2. To test this hypothesis, we used co-immunoprecipitation, thermal shift, pulldown and immunohistochemistry techniques. The results have demonstrated that Kv2.1 co-localizes with 14-3-3 in the retina and can be pulled down by purified recombinant 14-3-3 zeta. Antibodies against Kv8.2 are not yet available but thermal shift assays did not find an interaction between this subunit and 14-3-3. In conclusion, we provide evidence for a novel interaction between Kv2.1 and 14-3-3. Further studies will extend this initial biochemical characterization and test the biological significance of this interaction.

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Voted Outstanding Presentation

Gene augmentation therapy improves inner and outer segment morphology and preserves retinal structure in a large animal model of CNGB1-retinitis pigmentosa

Laurence M. Occelli 1, Paige A. Winkler 1, Vince C. Chiodo 2, Sanford L. Boye 2, William W Hauswirth 2 Simon M. Petersen-Jones1
1Small Animal Clinical Sciences, Michigan State University, East Lansing, MI; 2 Department of Ophthalmology, University of Florida, Gainsville, FL


Purpose: Mutations in cyclic nucleotide gated channel beta 1 (CNGB1) cause autosomal recessive RP type 45 (RP45). Cngb1-/- dogs have marked loss of rod function and a slowly progressive photoreceptor degeneration recapitulating RP45. We showed previously that gene augmentation therapy in Cngb1-/- dogs rescues rod function. Treated dogs had a dramatic restoration of rod ERG and improved vision in dim light. The purpose of this study was to investigate long-term effects of gene therapy in Cngb1-/- dogs.

Methods: Seven eyes of 5 Cngb1-/- dogs received a subretinal injection of 1x10^12 vg of AAV2/5-hGRK1-cCngb1. Dark- and light-adapted ERGs, vision testing, fundus imaging and structural preservation assessment by spectral domain - optical coherence tomography (SD-OCT) imaging was performed regularly post-injection. Immunohistochemistry was performed on eyes collected 3, 6, 9 and 23 months post-injection.

Results: Gene augmentation rescued rod function up to 18 months post-injection. SD-OCT showed that there was improved definition of the zones representing the outer limiting membrane to the interdigitation zone which was maintained over time. Following an initial decline in Receptor+ thickness further decline halted and thickness was preserved in the longer term (up to 23 months in two eyes). Immunohistochemistry confirmed photoreceptor preservation but only in the treated regions.

Conclusions: Rescue of rod function by gene augmentation therapy in Cngb1-/- dogs was maintained long term. There was also structural preservation with improved morphology of inner/outer segments. This study shows successful long-term gene therapy in a large animal model of RP45 making this an attractive disease for translational therapy.

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Voted Outstanding Oral Presentation

Differentiation and Maturation of Retinal Ganglion Cells Derived from Human Pluripotent Stem Cells for Studies of Glaucomatous Neurodegeneration

Ohlemacher, Sarah1; Sridhar, Akshayalakshmi1; Xiao, Yucheng1; Valentin Sluch2; Donald J. Zack2; Baucum, Anthony1; Cummins, Theodore1,3; Meyer, Jason S1,3
1Indiana University/Purdue University, Indianapolis, IN, United States. 2Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, United States, 3Stark Neurosciences Research Institute, Indiana University, Indianapolis, IN, United States.


Purpose: The ability to derive retinal ganglion cells (RGCs) from human induced pluripotent stem cells (hiPSCs) provides an unlimited supply for the study of RGC development and disease. hiPSCs were generated from both control and glaucoma (E50K OPTN mutation) patient sources, and their ability to yield functional RGCs was examined. Furthermore, these cells were assayed for phenotypic differences compared to a wild type hiPSC line as a novel in vitro model for glaucoma.

Methods: hiPSCs from both wild type and OPTN patients were induced to differentiate towards a retinal fate following previously established protocols and RGCs were subsequently characterized. RGC maturation was examined by immunocytochemistry and electrophysiology, including the formation of presumptive synaptic connections. Additionally, phenotypic differences between wild type and OPTN iPSC-derived RGCs were explored.

Results: Initially, hPSC-derived RGCs were identified based on the expression of BRN3. Subsequently, morphological and phenotypic features associated with RGCs arose over time. Throughout the course of 12 weeks, the degree of dendritic and axonal became increasingly complex over time. In addition, OPTN iPSC-derived RGCs were found to display striking phenotypic differences compared to wild type cells.

Conclusions: The data presented demonstrates the ability of hiPSCs to serve as a reliable source of RGCs, evidenced by their ability to yield a population of cells possessing a full complement of RGC-associated characteristics. These results will facilitate future studies into the disease-related degeneration and as such, will be instrumental as a tool for the study of optic neuropathies, as well as the development of therapeutic approaches.

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Examining the Relationship Between Foveal Pit Morphology and Cone Outer Segment Length

Bisola Omoba1, Rachel Linderman2, Erica Woertz3, Joseph Carroll2,3
1Medical College of Wisconsin, Milwaukee, WI, United States 2Ophthalmology & Visual Sciences, Medical College of Wisconsin, Milwaukee, WI, United States 3Cell Biology, Neurobiology, & Anatomy, Medical College of Wisconsin, Milwaukee, WI, United States


Purpose: Examining the relationship between the various anatomical features of the human fovea is important to better understand foveal development. Here, we compared foveal pit morphology to peak outer segment length.

Methods: Forty-one subjects with no known ocular disease were recruited, and their right eye was imaged. Macular volume scans were acquired using the Zeiss Cirrus HD-OCT, and used to obtain estimates of foveal pit volume, area, and depth. High-resolution line scans were acquired through the fovea using the Leica Bioptigen SD-OCT, and the peak outer segment (OS) length was estimated by measuring the maximum displacement between the ellipsoid zone and interdigitation zone bands using longitudinal reflectivity profiles. The lateral scale of all images was corrected for individual differences in ocular magnification using axial length measurements. Analysis was done using Spearman Rank Correlations to examine the relationships between OS length and various foveal pit metrics.

Results: Foveal pit metrics (mean ± SD) were as follows: pit volume = 0.092 ± 0.037 mm 3, pit area = 3.019 ± 0.811 mm 2, and pit depth = 0.115 ± 0.022 mm. Mean peak OS length was 43.95 ± 3.62 ±m. No correlation was found when comparing peak OS length to pit volume (p=0.6125), pit area (p=0.8681) or pit depth (p=0.0511).

Conclusions:: While no correlation was found between pit morphology and peak OS length, more work needs to be done to expand the number of subjects as well as examine other foveal metrics such as foveal avascular zone area and outer nuclear layer thickness.

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Paving-stone degeneration-like retinal lesions in young adult Siberian Husky dogs with primary angle-closure glaucoma (PACG)

Park, Shin Ae1; Sledge, Dodd2; Teixeira, Leandro3; Boyd, Ryan1; Freeman, Katie4; Koehl, Kristin1; Harman, Christine1; Munoz, Kirk1; Pirie, Chris1; Davidson, Harriet1; Petersen-Jones, Simon1; Komáromy, András M1
1Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, USA, 2Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, Michigan, USA, 3Comparative Ocular Pathology Laboratory of Wisconsin, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA. 4Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Veterinary Teaching Hospital, Fort Collins, CO, USA.


Purpose: To describe unique chorioretinal changes in Siberian Huskies with primary glaucoma.

Methods: Three young, 11 month- to 4- year-old, female Siberian Huskies that had multifocal depigmented retinal lesions and primary angle closure glaucoma (PACG) were included in this study. Ophthalmic examination and gross and histopathologic examination findings are described. One of the dogs had further diagnostics performed on the fellow, presumed non-glaucomatous eye, including gonioscopy, retinal and optic nerve head (ONH) examination by optical coherence tomography (OCT), confocal scanning laser ophthalmoscopy (cSLO), fluorescein (FA) and indocyanine green angiography (ICGA), electroretinography (ERG), quantifiable maze testing and genetic testing for mutations in RPGR causing X-linked progressive retinal atrophy.

Results: Histopathologic evaluations confirmed goniodysgenesis and PACG with ONH cupping and diffuse inner retinal atrophy. In addition, segmental profound retinal atrophy, loss of retinal pigment epithelium, and adhesion of the retina to Bruch's membrane was observed that was related to the multifocal depigmentation on funduscopy. Gonioscopy in the non-glaucomatous eye also confirmed goniodysgenesis. OCT, cSLO, FA, and ICGA of the same eye revealed retinal thinning in the depigmented area as well as wedge shaped retinal thinning with and without delayed choroidal vascular perfusion. Maze testing revealed mild nyctalopia but full field ERG showed no generalized decrease of retinal function. Genetic test was negative.

Conclusions: To our knowledge, these are the first reported canine cases of paving-stone degeneration-like retinal lesions, most likely caused by severely impaired choroidal perfusion. Further studies are warranted to elucidate the etiology and pathophysiology, including its possible association with PACG.

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Developing mouse models to evaluate the role of mitochondrial dynamics in glaucoma

Danielle R. Pellack1,2, Carly J. Lewis1, Kacie J. Meyer1, Michael G. Anderson1,2
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 a common cause of age-related vision loss caused by retinal ganglion cell death and optic neuropathy. The nee mouse strain exhibits severe early onset glaucoma with a marked reduction in retinal ganglion cells. We hypothesize that differences in morphology render some retinal ganglion cell subtypes preferentially susceptible to energy insufficiency promoting cell death. To test this, we are using Ppp2r2b-mutant mice, in which neurons have slight increases in mitochondrial efficiency related to altered mitochondrial fission. The purpose of these experiments is to establish baseline phenotypes relevant to glaucoma in nee and Ppp2r2b-mutant strains as we initiate their intercrossing.

Methods: Tissue was collected from cohorts of nee mice and of Ppp2r2b-mutant mice and littermate wild-type controls. Total optic nerve axon number was quantitated from manual axon counting of cross-sections of optic nerve stained with PPD and IOP was measured using a rebound tonometer on mice anesthetized with isoflurane.

Results: In nee mice, there is a striking loss of optic nerve axons at 6-8 weeks old. Ppp2r2b-mutant mice have qualitatively normal appearing optic nerves but contain elevated axon numbers at 8 weeks of age compared to wild-type. Studies of intraocular pressure in Ppp2r2b-mutant mice are ongoing.

Conclusion: Our results indicate that 8 weeks of age is an appropriate age for testing potentially neuroprotective agents in the nee model. The finding of increased axon number in Ppp2r2b-mutant mice may indicate a mitochondrial-associated alteration in developmental apoptosis of retinal ganglion cells, or an influence of the Ppp2r2b strain genetic background; ongoing experiments are working to distinguish these possibilities. In sum, these results establish the baseline parameters needed to test whether the Ppp2r2b mutation can suppress glaucoma in nee mice.

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Gene Augmentation Rescues Rod Function and Preserves Structure in Dogs Leading the Way Towards Treating Human PDE6A-Retinitis Pigmentosa

Petersen-Jones, Simon M1; Occelli, Laurence M1; Schön, Christian2; Biel, Martin2; Michalakis Stylianos2
1Department of Small Animal Clinical Sciences, Michigan State University, East Lansing, MI 2Center for Integrated Protein Science Munich (CIPSM) at the Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany


Purpose: Preclinical testing of a gene augmentation vector for the treatment of RP due to PDE6A mutations.

Methods: An adeno-associated viral vector serotype 8 delivering human PDE6A cDNA under control of a short rhodopsin promoter (AAV8-PDE6A) was used in a preclinical gene augmentation trial in Pde6a-/- puppies at a low (n=3) and high dose (n=4). In-life outcome measures were vision testing (VT), electroretinography (ERG) and spectral domain-optical coherence tomography (OCT). Some puppies were euthanized for immunohistochemistry of frozen sections.

Results: Improvements in vision testing outcomes were seen in all puppies from both groups. Only one puppy in the low dose group but all puppies in the high dose group had improved rod mediated ERGs There was preservation of the Receptor Plus (REC+) thickness in the treated retinal region in all puppies. The PDE6A transgene was expressed in the injected retinal regions and eliminated abnormal cyclic GMP accumulation and reduced GFAP activation. Importantly there was also morphological improvements in cone photoreceptors in the treated region.

Conclusions: The AAV8-PDE6A gene augmentation therapy restores rod vision in Pde6a-/- puppies. These positive outcomes are an important step towards a human clinical trial to treat PDE6A-RP.


Overexpression of histone deacetylase 3 (HDAC3) induces apoptosis in differentiated retinal neurons

Heather M. Schmitt 1,2, Cassandra L. Schlamp 1,2, Huan Yang 3, Lian Guo 3, and Robert W. Nickells 1
11-Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison; 2Cellular and Molecular Pathology Program, University of Wisconsin-Madison; 3Department of Surgery, University of Wisconsin-Madison


Purpose: HDAC3 has been found to play a major role in retinal ganglion cell (RGC) death after axonal injury. This study tests the hypothesis that overexpression of Hdac3 in mouse retinal neurons leads to cell-specific toxicity and that this phenomenon is dependent upon cell differentiation state.

Methods: Mouse 661W retinal precursor cells were nucleofected with Hdac3-mCherry, or HDAC3-FLAG, 24 hours prior to differentiation with 316nM staurosporine (STSN). All cells were then fixed at 0, 6, 12, and 24 hours post STSN addition to assess metrics of nuclear atrophy and apoptosis using immunofluorescence microscopy and live cell imaging. BAX activation was assessed by localization of a GFP-BAX fusion protein. Bax-dependency of HDAC3 toxicity was evaluated on 661W cells with disruption of the Bax gene by CRISPR/Cas9. C57BL/6 mice were given intravitreal injections of 1x10^9 units of AAV2-pgk-Hdac3mCherry virus 3 weeks prior to assessing changes in RGC health.

Results: Hdac3 overexpression was achieved in 61.85% of 661Ws by 24 hours after nucleofection. Differentiated cells expressing Hdac3 exhibited significantly more activated BAX in comparison to undifferentiated cells (p=0.032), which was followed closely by chromatin condensation. Deletion of the Bax gene from these cells significantly reduced the level of Caspase-3 activation in response to HDAC3 expression (p=0.008). Experiments to test toxicity in response to overexpression of Hdac3 in-vivo are ongoing.

Conclusions: Hdac3 overexpression leads to BAX activation, chromatin condensation, and apoptosis only in differentiated cells. Further investigation is needed to identify the mechanism by which HDAC3 stimulates neuronal apoptosis both in-vitro and in-vivo.

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Cytochrome P450 1B1 is a Key Regulator of Ocular Oxidative Stress and Pathogenesis of Congenital Glaucoma

Sheibani, Nader, Falero Perez, Juliana, Sorenson, Christine M.
Departments of Ophthalmology and Visual Sciences, Cell and Regenerative Biology, Biomedical Engineering, and Pediatrics, and McPherson Eye Research Institute, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705.


Abstract: CYP1B1 is a member of Cytochrome P450 family of proteins with important roles in developmental processes. Mutations in human CYP1B1 gene have been linked to the pathogenesis of congenital glaucoma. However, the detailed mechanisms remain unclear. We showed mice deficient in Cyp1b1 fail to undergo retinal neovascularization during oxygen-induced ischemic retinopathy (OIR). These mice exhibit increased oxidative stress, sustained activation of NF-κB, and increased production of angiogenesis inhibitor, thrombospondin-2 (TSP2). Administration of antioxidant N-acetylcysteine restored oxidative state and retinal neovascularization during OIR. We also show that increased TSP2 expression is responsible for the angioinhibitory activity of oxidative stress with Cyp1b1 deficiency. These activities are consistent with constitutive expression of Cyp1b1 in endothelial cells, and more predominantly in perivascular supporting cells, which maintain the cellular oxidative state especially under hyperoxic conditions. Furthermore, we show that Cyp1b1-deficiency results in degeneration of the trabecular meshwork during development of the ocular drainage system resulting in increased intraocular pressure and glaucoma.This was associated with decreased production of periostin, a molecule with an important role in development and maintenance of collagen fibrils that form and support the trabecular meshwork function. Mice deficient in periostin exhibited increased intraocular pressure and degeneration of trabecular meshwork. Together, our studies demonstrate an important role for Cyb1b1 in regulation of cellular oxidative state, especially under hyperoxic conditions (>10% oxygen), with adverse effects on vascular and trabecular meshwork cell functions.

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Voted Outstanding Oral Presentation

Investigating gene augmentation as a therapeutic approach for Best disease

Sinha, Divya1,2, Guziewicz, Karina3, Valiauga Rasa1; Shahi Pawan2,3; Pattnaik, Bikash2,4,5, Gamm David1,2,5
1Waisman Center, University of Wisconsin-Madison, Madison, WI, 2McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI, 3School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, 4Pediatrics, University of Wisconsin-Madison, Madison, WI, 5Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI


Purpose: Best disease (BD) is a form of inherited macular dystrophy resulting from mutations in BEST1 gene. Both autosomal dominant and autosomal recessive forms of mutations exist. BEST1 has been postulated to form a pentameric calcium activated chloride channel (CACC). Our objective was to test if gene augmentation can reverse the observed disease phenotypes in patient-specific hiPSC-RPE cells.

Methods: Pure monolayers of retinal pigment epithelium (RPE) derived from human induced pluripotent stem cells (hiPSCs) harboring patient-specific BEST1 mutations were cultured using protocols established by our lab. For gene augmentation, either adeno-associated viruses (AAV) or lentiviruses expressing hBEST1 under hVMD2 promoter were used to transduce patient-specific hiPSC-RPE. Successfully transduced hiPSC-RPE cells were assessed for changes in BD associated phenotypes in hiPSC-RPE.

Results: Lentivirus-mediated transduction of cultured hiPSC-RPE was more efficient than AAV-mediated transduction in our experiments. Successfully transduced BD hiPSC-RPE cells had improved CACC function when compared to non-transduced controls. Additionally, we observed changes in RPE apical processes as monitored by EZRIN expression. .

Conclusion: Our results suggest that it is possible to partially reverse the disease phenotype using gene augmentation for some BD-causing mutations. Gene augmentation combined with knockdown approach (or gene editing) may be beneficial for BD mutations not amenable to gene augmentation alone.

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Functional characterization of disease-associated Bardet-Biedl syndrome 1 (BBS1M390R) allele in zebrafish

C. Anthony Scott1, Trudi Westfall1, Val C. Sheffield2, Diane C. Slusarski1
1Department of Biology, University of Iowa 2Department of Pediatrics, University of Iowa Carver College of Medicine


Purpose: Dysfunctional cilia underlie a number of human genetic conditions, such as Bardet-Biedl syndrome (BBS), and affect multiple organs, causing blindness, heart disease, infertility, obesity and/or diabetes. BBS proteins form a complex, the BBSome, which mediates protein trafficking. BBS1 is the most commonly mutated gene in BBS patients, and a single missense mutation causing a methionine to arginine substitution at position 390 (M390R) accounts for 80% of all BBS1 mutations. The purpose of the current study is to characterize the cellular mechanism by which the mutant BBS1 protein causes disease.

Methods: We generated several zebrafish alleles with CRSPR and performed rescue experiments with either BBS1 or BBS1 M390R RNA in knockdown zebrafish. We analyzed the ciliated Kupffer's Vesicle, intracellular transport and visual function. We also evaluated subcellular localization and protein binding.

Results: Bbs1 homozygous mutants show reduced cilia, delayed cellular transport and visual function defects. BBS1 RNA is sufficient to rescue all knockdown defects, while BBS1M390R mRNA had differing abilities depending on the tissue type. Tagged expressed zebrafish BBS1 protein is found at the centrocome, while BBS1M390R is destabilized and fails to localize to the centrosome. We also find that BBS1M390R maintains interaction with some components of the BBSome complex but not with BBS4.

Conclusions: We conclude that the underlying disease mechanism of BBS1M390R stems from reduced interaction with BBS4 and mislocalization. This leads to a global loss of BBS1 protein stability, which likely impacts other BBSome protein complex members.

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Voted Outstanding Oral Presentation

Protective mechanisms against sub-lytic complement attack in the retinal pigment epithelium

Tan, Li Xuan; Toops, Kimberly; Lakkaraju, Aparna
Ophthalmology and Visual Sciences, McPherson Eye Research Institute, University of Wisconsin-Madison, Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison


Purpose: Aberrant activation of the complement pathway is implicated in inherited and age-related macular degenerations. Complement activation and the consequent assembly of sub-lytic membrane attack complexes (MAC) on the surface of retinal pigment epithelial (RPE) cells forms pores that compromise membrane integrity and promote inflammation. Here, we investigated how RPE limits MAC assembly and seal MAC pores to prevent complement-mediated injury.

Methods: Polarized primary porcine RPE were exposed to 10% normal human serum as the complement source to induce MAC assembly. Recycling of CD59, a membrane-bound complement regulator, was followed by immunofluorescence microscopy. Membrane repair and mitochondrial fragmentation were monitored by live-cell imaging and biochemical assays. Complement activation and mitochondrial health in wild-type and Abca4−/− mice were assessed with immunohistochemistry and immunoblotting.

Results: Complement exposure accelerates CD59 recycling to the surface of healthy RPE cells. Live-cell imaging revealed that RPE lysosomes fused with plasma membrane within seconds of complement exposure to preserve cell integrity. In RPE from Abca4-/- mice or primary RPE with vitamin A dimers, excess cholesterol disrupted organelle traffic, causing reduced cell surface CD59 and decreased efficiency of lysosome-mediated membrane repair. This resulted in persistent MAC pores that drive mitochondrial fragmentation and oxidative stress. Removal of excess cholesterol restored protective mechanisms in the RPE.

Conclusions: Approaches targeting the complement pathway in macular degenerations have largely focused on direct inhibition of complement proteins. Our data shed insight into how RPE deals with complement at a cellular level, thereby opening the possibility of therapeutically targeting innate mechanisms essential for preserving RPE health and function.

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

Jessica R. Thompson1, Kristan S. Worthington1, Brian J. Green2, Spencer J. Bunn2,2 Luke A. Wiley1, Emily E. Kaalberg1, Chunhua Jiao1, Stephen R. Russell1, Ian Han1, Katherine N. Gibson-Corley3, Elliott H. Sohn1, and Budd A. Tucker1
1Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Vision Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA; 2Department of Chemical Engineering, College of Engineering, University of Iowa, Iowa City, IA; 3Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, IA


Purpose: Polymer scaffolds are critical for cellular survival and integration in retinal cell replacement therapies. Poly(caprolactone) (PCL) is a good material for retinal scaffolds: it is biodegradable, biocompatible, and easily modified with reactive molecules. Here, we optimized the conditions that contribute to photopolymerizing PCL to create more effective and efficient cell scaffolds.

Methods: First 2D PCL films were studied for biocompatibility and physical properties. Then, two-photon polymerization was used to create 3D-printed structures with sub-micron resolution. To define optimal formulation of PCL for printing, the degree of functionalization, molecular weight, prepolymer concentration, and photoinitiator concentration were varied and printed using a standard design. Structures were evaluated for accuracy to the standard to select the best formulation. Similarly, printing parameters such as laser power, scan speed, slicing distance, and hatching distance were varied to ensure the best model to structure fidelity.

Results: Tri-functionalized PCL resulted in higher resolution structures than di-functionalized. Higher molecular weight PCL structures were enlarged compared to lower molecular weight structures and the standard. Optimal resolution and threshold were found using 3% photoinitator. Decreasing either hatching or slicing distance resulted in structures closer to the design. Regardless of molecular weight or degree of functionality, bulk polymerized PCL was biocompatible with MiPSCs and in pigs.

Conclusions: Optimal parameters for printing were found. And favorable biocompatibility of PCL in pigs demonstrates that this material is promising for transplantation purposes. Thus, we are now poised to interrogate interactions between these 3D printed degradable structures and human retinal progenitor cells.

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Features of cone bipolar cell synaptic transmission onto retinal ganglion cells

Van Hook, Matthew J.
Truhlsen Eye Institute and Department of Ophthalmology & Visual Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA.


Purpose: In the retina, ~12 classes of cone bipolar cells (CBCs) carry rod- and cone-driven signals to retinal ganglion cells (RGCs). Most studies of mammalian bipolar cell synaptic function have examined on rod bipolar cells (RBCs). Thus, the basic properties of CBC synaptic transmission onto RGCs are unknown. The goal of this study is to characterize Ca2+-dependence, exocytotic Ca2+ domains, and short-term plasticity of CBC synapses.

Methods: Whole-cell recordings were obtained from alphaRGCs in mouse retinas. CBCs were stimulated with current pulses delivered from extracellular electrodes. Extracellular [Ca2+] was varied to assess the cooperativity of the CBC exocytotic Ca2+ sensor, while nifedipine was applied to characterize Ca2+ domains.

Results: Extracellular stimulation triggered glutamate receptor-mediated inputs onto recorded RGCs, as indicated by pharmacology and reversal at 0 mV. Varying [Ca2+]o altered excitatory post-synaptic current (EPSC) amplitude and fits of the data showed that EPSCs depended on [Ca2+] with a n=5. Nifedipine (1-30 microM) reduced type 6 CBC Ca2+ currents and RGC EPSCs. The relationship of EPSC amplitude and CBC Ca2+ currents was well fit with a Hill slope of ~1. Inputs onto both On- and Off- RGCs were depressed with repeated stimulation, although On inputs depressed more strongly. Train stimuli revealed that CBC inputs to have release probability (Pr) of ~0.9.

Conclusions: Ca2+-dependence and nifedipine experiments suggest that CBCs operate with a conventional Ca2+ sensor with n=5 and Ca2+ nanodomains, much like RBCs. Short-term synaptic depression indicates that On and Off CBCs have a high Pr.

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Voted Outstanding Presentation

Examining the relationship between cone density and Vernier acuity in human albinism

Woertz, E.N. 1; Wilk, M.A. 2; Carroll, J. 1; DeYoe, E.A. 1
1Medical College of Wisconsin 2Hudson Alpha Institute for Biotechnology


Purpose: Albinism causes disrupted melanin synthesis and abnormal visual system development. Vernier acuity (VA) is a psychophysical measure that, in healthy individuals, is believed to be limited by neural sampling in the primary visual cortex (1), and thus may provide insight into post-receptoral visual circuitry. We examined the relationship between cone density (CD) and VA in human albinism.

Methods: Five subjects with albinism and four normal control subjects were recruited. Images of the cone mosaic were obtained using adaptive optics scanning light ophthalmoscopy and CD was estimated using a semi-automated cone counting algorithm (2). VA thresholds along the horizontal meridian of the left visual field were measured at 10 loci ranging from 0-15° eccentricity using a three-dot vertical Vernier stimulus.

Results: Peak CD (mean ± SD) was 128,378 ± 25,667 cones/mm^2 in controls and 69,796 ± 19,995 cones/mm^2 in subjects with albinism (2). Foveal VA (mean ± SD) was 0.498 ± 0.127 min in controls and 5.926 ± 6.227 min in subjects with albinism. At all eccentricities tested, VA was worse and more variable among subjects with albinism than controls. When VA was plotted as a function of eccentricity-matched CD, subjects with albinism had worse VA for a given CD than controls.

Conclusions: VA is worse in albinism compared to controls at all eccentricities measured. The relationship between CD and VA in albinism suggests that the visual deficits in albinism may be a result of altered numbers and/or connectivity of post-receptoral neurons in retinogeniculostriate pathways.

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Voted Outstanding Presentation

High-resolution, 3D-printed chitosan tissue scaffolds for retinal regeneration

Kristan S. Worthington, Robert F. Mullins, Edwin M. Stone and Budd A. Tucker
Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, Carver College of Medicine, The University of Iowa


Purpose: Our group has already shown that chitosan hydrogels are good candidate materials for retinal scaffolds, as they are biodegradable, biocompatible, and have elastic properties similar to the retina. 3D microstructural control of these materials for cell orientation and packing is an important precursor to their realization as tools to study and treat disease. In this work, we used two-photon polymerization to create 3D printed chitosan hydrogels and optimized the conditions that contribute to scaffold resolution and structural fidelity.

Methods: Polymer formulations comprised varying concentrations of methacrylated glycol chitosan, glycerol and methylene blue (photoinitiator). 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. We also printed various other test structures, including miniature scaffolds.

Results: Evaporative effects caused the chitosan concentration to increase during printing. Early in the printing process, this improves the ability of the formulation to 3D print by two-photon polymerization. Late in the process, loss of water hinders diffusion and results in unreliably printed structures. Adding glycerol to the formulation can prevent evaporation, but also results in spontaneous gelation of the sample.

Conclusions: We are the first to create precise 3D chitosan structures at this size scale and to examine the effects of chitosan formulation and two-photon printing variables on final structure outcomes and. We are now poised to fine-tune this 3D printing process, evaluate interactions between 3D printed chitosan structures and human retinal progenitor cells in vitro and in vivo.

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Examining the Effect of Axial Length on Vessel Density Estimates Obtained using OCT Angiography

Zaro, Brit1; Linderman, Rachel2,3; Carroll, Joseph2,3
1Medical College of Wisconsin, Milwaukee, WI, United States 2Ophthalmology & Visual Sciences, Medical College of Wisconsin, Milwaukee, WI, United States 3Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI, United States


Purpose: OCT angiography enables visualization of the retinal vasculature, and the density of vessels in the image is often reported using an ETDRS grid. However, individual differences in axial length result in variation in the lateral scale of the ETDRS grid used for analysis. Here we examined how these scaling differences impact estimates of vessel density within the ETDRS regions.

Methods: 162 Caucasian subjects with no known ocular or vascular diseases were recruited. One 3x3mm scan (OD) was acquired per subject using Optovue's AngioVue system. Axial length for each subject was measured using the IOL Master. An ETDRS grid, scaled for the subject's axial length, was placed onto the image and an average vessel density for each segment was calculated. This was compared to the vessel density values for each segment in an unscaled grid.

Results: The mean (±SD) for the central 1mm was 32.52%±5.38% for the unscaled grid and 32.35%±5.24% for the scaled grid. For the parafoveal region, unscaled vessel density was 54.74%±2.95%, while scaled vessel density was 54.74%±2.96%. No significant differences (paired t-test) were found when comparing foveal vessel density (p=0.194) or parafoveal vessel density (p=0.980). However, as the deviation from the assumed axial length increases, the absolute error in vessel density can exceed 5% for the central 1mm segment.

Conclusion: Differences in axial length can affect estimates of vessel density, and while there was no statistical difference observed here, the deviations observed may be cumulative with other known sources of error.

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