12^th Annual

2019 Midwest Eye Research Symposium

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

MERS 2019 took place on August 9^th , 2019 at
the University of Iowa Medical Education and Research Facility

Keynote Speaker/Session Chairs

Jeffrey L. Bennett, M.D., Ph.D. - Keynote Speaker "Neuromyelitis Optica - Unraveling Auto-antibody Pathology"
Dr. Bennett is the Gertrude Gilden Professor of Neurology and Ophthalmology and the Associate Director of the Center for NeuroScience at the University of Colorado School of Medicine. Dr. Bennett has carried out outstanding work to investigate Occult Chorioretinal (OC) Disorder, a group of chorioretinal inflammatory disorders of unknown etiology that mimic optic neuropathy, and to identify the primary target of the humoral immune response in optic neuritis and multiple sclerosis. He has authored well over 120 peer reviewed manuscripts and serves on numerous advisory committees and review panels.

Sarah X. Zhang, M.D. - Chair, Session I
Dr. Zhang is a Professor of Ophthalmology at the University at Buffalo Jacobs School of Medicine and Biomedical Sciences. Work in her laboratory has been focused on ER stress and Unfolded Protein Response (UPR) signaling in diabetic retinopathy and age-related macular degeneration.

Kristen Kwan, Ph.D. - Chair, Session II
Dr. Kwan is an Assistant Professor of Human Genetics and an Adjunct Assistant Professor of Ophthalmology & Visual Sciences at the University of Utah. Her lab studies the cellular and molecular mechanisms involved in the morphogenesis of the vertrebrate eye.

Nikolai Artemyev, PhD. - Chair, Session III
Dr. Artemyev is a Professor in the Departments of Molecular Physiology &Biophysics as well as Ophthalmology &Visual Sciences at the University of Iowa. He has been studying the protein-protein interactions between the photoreceptor – rhodopsin, the rod G protein - transducin, and the effector enzyme - cGMP-phosphodiesterase with the goal to understand the mechanisms of regulation of G protein signaling at the molecular level.

Oral Presentations

Session I, Session Chair Sarah Zhang, MD, Ph.D.

Independent replication that vitamin B3 prevents glaucoma in aged mice

Anderson, MG ^1,2,3; Meyer, KJ¹; Hedberg-Buenz, A ^1,3; van der Heide, CJ ¹; Soukup, DA¹; Pomernackas, N ¹; Brose, C ²; Mercer HE ¹ ; Rodine, K²; Fingert, JH^2
1Department of Molecular Physiology and Biophysics University of Iowa ²Department of Ophthalmology and Visual Sciences University of Iowa ³Center for Prevention and Treatment of Visual Loss Iowa City Veterans Administration Medical Center Iowa City

Purpose: In previous work led by Drs. Pete Williams and Simon John it was reported that oral supplementation of vitamin B3 (nicotinamide NAM) was capable of robustly protecting against glaucoma development in DBA/2J mice (Science 355: 767-760). NAM is a precursor of nicotinamide adenine dinucleotide (NAD) a cofactor for biochemical reactions central to mitochondrial metabolism. Driven by the implications with respect to neuroprotective therapies the goals of the current study were to: 1) perform an independent replication test of the main finding in DBA/2J mice and 2) extend the study to other mouse models.

Methods: Retired breeder DBA/2J females (6-9 mos of age) were purchased commercially from The Jackson Laboratory split into treatment groups to maintain age parity and maintained on the NAMHi (TestDiet® 5W80 with 10000 ppm NAM chow; NAM water) or control diets (TestDiet® 5W80; normal water) precisely as described in the original publication. All diets and chemicals were obtained directly from vendors. At 12 mos of age right side tissues were collected for quantitative analyses.

Results: Based on analysis of retinal flat-mounts (12 fields per retina analyzed using an automated approach with masking to treatment no eyes completing study excluded) density of BRN3A+ retinal ganglion cells was significantly higher in treated mice (1885 ±1085 cells/mm²; n=15 eyes; mean±SD) versus controls (852±1026 cells/mm²; n=11 eyes; p=0.02 Student’s t-test). Density of TO-PRO-3+ nuclei that were BRN3A- in the same samples remained similar between groups (treated 3588±194 cells/mm²; controls 3556±232 cells/mm²; p=0.71). Analysis of optic nerves and left-side tissues from these DBA/2J cohorts remains in progress.

Conclusions: In sum these results are thus far confirmatory of the initial report that NAM confers protection against glaucoma in DBA/2J mice.

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Chaperones in photoreceptors: AIPL1 and Ric8A

Artemyev, Nikolai^1
1University of Iowa

Purpose: High turnover and protein trafficking rates in photoreceptors coupled with unique structural properties of phototransduction components place unusual demands for protein folding quality control and transport machinery to maintain proteostasis. Growing evidence suggests that photoreceptor cells in addition to classical and ubiquitous molecular chaperones from the heat shock protein superfamily utilize photoreceptor-specific chaperones or co-chaperones to assist protein folding subunit assembly and protein trafficking. Recent work in our laboratory focuses on aryl hydrocarbon receptor (AhR)-interacting protein-like 1 (AIPL1) a specialized chaperone of phosphodiesterase-6 (PDE6) in rods and cones and resistance to inhibitors of cholinesterase 8A (Ric8A) a candidate chaperone and a GEF for the transducin-a (Gat) subunit.

Methods: X-ray crystallography NMR biolayer interferometry (BLI) cross-linking mass spectrometry molecular dynamics simulations

Results: We solved the crystal structures of the FKBP and TPR domains of AIPL1 and delineated the interactions of these domains with the prenylated C-termini of PDE6 and its regulatory Pγ-subunit. A model of the AIPL1/PDE6 complex has been generated. We also solved the crystal structure of Ric8A in complex with a tagged C-terminal fragment of Gat. These structure reveals two principal domains of Ric8A: an armadillo-fold core and a flexible C-terminal tail. An experimentally constrained model of the Ric8A/Ga complex has been produced and it suggests that the Ric8A C-terminal tail helps organize the GTP-binding site of Ga.

Conclusions: A model of the AIPL1/PDE6 complex provides a framework for future structural studies of this chaperone/client complex critical to photoreceptor signaling and health. The model of the Ric8A/Gat complex indicates tantalizing parallels and differences in the mechanisms underlying the GEF activities of rhodopsin and Ric8A.

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Bilateral enucleations alters the intrinsic excitability of thalamocortical neurons in the dLGN

Bhandari, Ashish; Smith, Jennie; Van Hook, Matthew J.
University of Nebraska Medical Center

Purpose: Diseases like glaucoma optic neuritis and optic nerve atrophy affect the optic nerve resulting in optic nerve degeneration and vision loss which alters the conveyance of retinal signaling to visual areas of the brain. Under such circumstances homeostatic processes are likely to respond by triggering functional changes in neurons receiving retinal input. Plasticity in the visual cortex and the developing dLGN has been extensively studied. However plasticity in the adult mouse dLGN is still poorly understood. The goal of this study was to determine how the loss of retinal input alters the function of thalamocortical relay neurons in the mouse dorsolateral geniculate nucleus (dLGN) a major retinorecipient region of the brain.

Methods: 6-8 week-old mice (both sexes) were anesthetized with an intraperitoneal injection of ketamine/xylazine bilaterally enucleated and allowed to recover until used for experiments. Following procedure and recovery 250 um acute coronal brain slices were obtained from either enucleated or control animals using the protected recovery method. Thalamocortical neurons in the dLGN were targeted for whole-cell patch-clamp recording. We recorded their response to current injections (depolarizing and hyperpolarizing current injections) in current clamp mode to measure spiking response and input resistance. We also measured the mEPSC (in voltage clamp configuration). Our recording solution was supplemented with CF-568 hydrazide and 1% Neurobiotin to enable imaging of the dendritic structure of the recorded cells. We also stained the dLGN for vGlut2 (retinal axonal terminal marker).

Results: We observed an increase in the number of spikes fired in response to depolarizing current injections compared to controls (n = 13) for the same current injection after 2-4 days post enucleation (e.g. the difference in the no. of spikes fired at 60 pA injection was statistically significant after 2-4 days (p = 0.026, n = 15) 7-10 days (p = 0.002, n = 14) and over 14 days (p = 0.019, n = 7) post enucleation). We did not observe any change in the resting membrane potential. However there was a significant difference in input resistance after 2-4 days (589.6 ± 44.58 MO, n = 14, p = 0.002) and 7-10 days (567.4 ± 61.27 MO, n=17). There was a significant reduction in miniature EPSC (mEPSC) frequency for the TC neurons compared to controls (25.94 ± 4.976 Hz, n=12) after 2-4 days (11.13 ± 1.665 Hz, n=13, p = 0.014) and over 14 days (13.9 ± 2.671 Hz, n=7, p = 0.049) of enucleation. However the decline in the mEPSC frequency after 7-10 days of enucleation was not statistically significant (17.92 ± 3.291 Hz, n=10, p = 0.195). We did not observe any changes in other mEPSC properties. Quantification of vGlut2 staining in the dLGN also revealed reduced vGlut2 density (number of vGlut2 puncta/100 µm²) 7 days (0.112 ± 0.0225, n=3, p = 0.021) 14 days (0.114 ± 0.021, n=3, p = 0.017) and over 14 days (0.135 ± 0.0230, n=3, p = 0.046) after enucleation compared to controls (0.217 ± 0.015, n=4).

Conclusions: These results are suggestive of alteration in the synaptic input into the dLGN neurons. Our observations indicate that an underlying compensatory mechanism may exist in the retinogeniculate synapse and will allow us to probe further for mechanisms in the visual circuit that may represent homeostatic plasticity in the dLGN in response to a complete loss of retinal input.

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Metabolic deprivation induces stress responses in retinal organoids

Cheng, Lin^1,2; Merydith, Evylin^1,2; Kuehn, Markus^1,2,3
1Department of Ophthalmology and Visual Sciences, University of Iowa; ²VA Center for the Prevention and Treatment of Visual Loss; ³Institute for Vision Research University of Iowa

Purpose: Metabolic deprivation is suspected to play a role a number of neurodegenerative diseases, including glaucoma. In animals it is frequently not possible to evaluate the effects of metabolic stress in the absence of other stressors, e.g. oxygen deprivation, or secondary effects such as infiltration by myeloid cells. Here we are subjecting retinal eye cups (organoids) to reduced glucose supply to evaluate the effect on neurons.

Methods: The retinal organoids were differentiated from human iPSC by the method detailed by Sasai et al, (Cell Stem Cell, 2012). After 50 days of differentiation retinal organoids were incubated with antibodies directed against CD11c, CD68, CD4, CD3, CD86, and IBA1 to determine the presence of dendritic cells, monocyte, macrophage, T help (Th) cell, and microglia, respectively. qPCR for these markers was also carried out, using human PBMC as a positive control. The organoids were then stressed by stepwise decrease of glucose concentration for 5 days. High glucose organoids were culture with 4.5g/L glucose as a control. qPCR was used to detect expression of HSP60, HSP70, HSP90, HSP27, MHC I/II, and CTSS (Cathepsin S). The cell culture supernatant was collected to detect the release of Nitric Oxide (NO) and adenosine triphosphate (ATP).

Results: Both IF and qPCR confirm the absence of microglia, macrophages, dendritic cells, or Th cells in retinal organoids. Glucose deprivation caused increased expression of heat shock (HSP60, HSP70, HSP90, HSP27), but also increased antigen presentation, as reflected by higher expression of CTSS, HLA-B, and HLA-DRA. There was no significant increase in NO in the glucose deprivation group. In contrast, glucose deprivation did cause increased extracellular ATP (1.87 vs. 2.27 µM).

Conclusions: These data confirm that retinal organoids are composed of neurons and their progenitors, but not myeloid cells. Metabolic deprivation resulted in stress responses, but also increased antigen presentation by HLA class I and II by neuronal cells. Consistent with the absence of myeloid cells NO production was not increased, but following glucose deprivation neuronal cells appear to release ATP, a ligand if the P2X receptor involved in pro-inflammatory signaling. It suggests that a neuronal a energy crisis may be sufficient to both create a pro-inflammation milieu and result in enhanced antigen presentation of retinal antigens.

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Ctage5 and tango1 endoplasmic reticulum cargo receptors have complementary but divergent roles in photoreceptors and overall ocular health

Clark, Eric; Collery, Ross; Nonarath Hannah; Link, Brian
Medical College of Wisconsin

Purpose: Millions of individuals are affected by age-related eye diseases and the prevalence is expected to double between 2010 and 2050 within the United States as demographics shift towards an older population. Therefore investigation of factors that affect ocular homeostasis is imperative. Through a genetic screen in zebrafish we discovered mutations in a gene called cTAGE5 as a candidate affecting age-related neuronal health. In general cTAGE5 and a related protein Tango1 act at the endoplasmic reticulum (ER) for trafficking of large molecules like collagens and lipoproteins but also can contribute to autophagy. While best characterized in cell culture their role for maintaining ocular homeostasis in vivo has not been investigated. We hypothesized that within eyes cTAGE5 and Tango1 play important trafficking and/or autophagic roles in neurons to maintain their high metabolic needs and an important structural role in other tissues of the eye through ECM maintenance.

Methods: To initiate studies we generated large deletion mutations within cTAGE5 and Tango1 using Crispr/Cas9 that eliminated most of the coding sequence in zebrafish. To investigate embryonic phenotypes in large deletion mutants we used transgenic cellular stress reporters including ATF6RE:GFP and LC3-GFP-mcherry to label ER stress and autophagy respectively. Plastic sectioning with toluidine blue staining followed by electron microscopy was used to investigate structural and ultrastructural changes in the retina. In adult zebrafish eyes were dissected and retinal ganglion cell stress was measured at the optic nerve head and optic nerve by quantifying axonal swellings using the gap43:GFP stress reporter. The lens was also dissected and imaged at this time. Photoreceptor morphology was assessed over time using optical coherence tomography (OCT) and plastic sectioning.

Results: Single homozygous mutants show no outward eye phenotypes but cTAGE5; Tango1 double homozygous mutants have small eyes. Upon further investigation we discovered that cTAGE5 mutants have RGC stress as observed by axonal swellings at the optic nerve head and throughout the optic nerve. cTAGE5 mutants also exhibit lens defects and most dramatically photoreceptors display ER stress and altered autophagy apparent adhesive defects at the outer plexiform layer extreme intracellular vesiculation and stunted outer segment development followed by age-related photoreceptor degeneration. cTAGE5; Tango1 double homozygous mutants show exacerbated phenotypes in the lens and photoreceptors. Interestingly these phenotypes are absent in Tango1 single homozygous mutants suggesting complementary but divergent roles for cTAGE5 and Tango1 in ocular homeostasis. Defects in collagen and lipoprotein trafficking are mechanisms currently under investigation.

Conclusions: cTAGE5 and Tango1 have divergent but complimentary roles in ocular homeostasis. cTAGE5 is particularly important for photoreceptor health because loss of cTAGE5 causes stress pathway activation including ER stress and autophagy ultrastructural vesicular modifications and age-related photoreceptor degeneration. A thorough understanding of factors essential for ocular homeostasis may provide avenues to decrease the growing burden of eye disease and other age-related neurodegenerations.

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Ectopic expression of BBS1 rescues fertility but not retinal degeneration in a mouse model of BBS1M390R

Cring, Matthew¹; Meyer, Kacie²; Sheffield, Val¹
University of Iowa, Departments of ¹Pediatrics ²Molecular Physiology and Biophysics

Purpose: Bardet-Biedl syndrome (BBS) is a rare ciliopathy for which there are no current effective treatments. BBS is a genetically heterogeneous disease though the M390R mutation in BBS1 accounts for approximately 20% of all genetic diagnoses. The principle features of BBS include retinal degeneration obesity male infertility polydactyly intellectual disability and renal cysts. Patients with mutations in BBS genes often present with night blindness within the first decades of life which progresses to complete blindness over time. This phenotype is due to progressive loss of photoreceptor cells. Obesity in BBS patients is caused by dysfunctional leptin signaling in hypothalamic neurons which leads to hyperphagia. Male infertility is caused by a lack of flagella on spermatozoa rendering them immobile.

Methods: In this study we have crossed a transgenic human BBS1 gene onto a mouse model of BBS1^M390R to determine if ectopic expression of BBS1 rescues retinal degeneration obesity and male infertility.

Results: qRT-PCR indicates that the BBS1 transgene is expressed in multiple tissues throughout the mouse with the highest expression seen in the heart and testes and much lower expression in the brain and eye. Using electroretinograms (ERGs) to measure retinal function we show that both A waves and B waves are significantly reduced in BBS1^M3900R/M390R;BBS1^TG+ mice relative to wild type (WT) littermates. Furthermore, at one month of age there is a significant difference in weight between BBS1^M3900R/390R;BBS1^TG+ mice and WT littermates. When a male BBS1^M3900R/M390R;BBS1^TG+ mouse is housed with a WT female he is able to sire offspring indicating that the male infertility phenotype of BBS is rescued by the transgene.

Conclusions: We have shown that ectopic expression of BBS1 does not rescue retinal degeneration and weight to the levels seen in WT littermates. Continuing work is aimed at determining whether the retinal degeneration and obesity phenotypes significantly differ between BBS1^M3900R/M390R;BBS1^TG+ and BBS1^M3900R/M390R;BBS1^TG- mice which would indicate some therapeutic effect of ectopic expression of BBS1 on a BBS1^M390R background which would have positive implications for gene therapy of this disease.

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Identification and Referral of Patients with Ruptured Globes and Orbital Fractures due to Intimate Partner Violence (IPV) after Educational Campaign

Dawoud, Salma¹; Clark, Thomas J.²; Cohen, Ali³; Ko, Audrey C.¹; Carter, Keith D.¹; Shriver,Erin^1
1Department of Ophthalmology and Visual Sciences, University of Iowa Iowa City IA; ²Medical College of Wisconsin Milwaukee, WI; ³University of Louisville School of Medicine, Louisville, KY

Purpose: To determine rates of IPV-related ruptured globes and orbital fractures, and referral to ancillary services in women after an ophthalmic educational campaign to screen for IPV. Previous studies indicate significant gaps in identifying mechanisms of injury (not documented in 20% of orbital fractures) and involvement of ancillary services for identified IPV patients (not documented in 80% of orbital fracture patients).

Methods: Retrospective review of female patients who sustained ruptured globes, orbital floor fractures, or ZMC fractures between January 2015 and February 2019.

Results: A total of 226 women sustained orbital floor and ZMC fractures. A total of 186/226 (82.3%) had isolated orbital floor fractures, and 40/226 (17.7%) had ZMC fractures. IPV accounted for 8.1% (15/186) of isolated orbital floor fractures and 17.5% (7/40) of ZMC fractures. Fifty-eight women sustained ruptured globes.
Leading mechanisms of orbital floor and ZMC fractures were falls (121/226, 53.5%), motor vehicle accidents (36/226,15.9%), non-IPV assault (23/226, 10.2%), and IPV assault (22/226, 9.7%). Leading mechanisms of globe rupture were falls (30/58 51.7%), accidental trauma by an inanimate object (9/58, 15.5%), IPV assault (5/58, 8.62%), and non-IPV assault (5/58, 8.62%). In both orbital fractures and ruptures globes, a mechanism of injury was documented for every patient.
The median age of patients with IPV-related orbital fractures was 40 years (range 25-63) and 46 years (range of 33-57) for IPV-related globe rupture. Social work involvement, ancillary services referral, and home-going safety assessment was documented in 68.1% (15/22) of IPV-related orbital floor or ZMC fracture patients and 80% (4/5) of IPV-related ruptured globe patients.

Conclusions: Implementation of an IPV educational awareness campaign was associated with increased detection of IPV-related orbital fractures and globe ruptures and increased involvement of ancillary services.

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Retinal molecular and cellular changes in a mouse model of traumatic brain injury

Dutca, L.M.^1,2; Boehme, N.^1,2, Castonguay, W.^1,4; Bielecki, M.²; Tatro, N.^2,5; Woll, A.¹; Harper, M.M.^1,2; Scheetz, T.E.^2,5; Anderson, M.G.^1,3
1Center for the Prevention and Treatment of Visual Loss, Iowa City VA Healthcare System; Departments of ²Ophthalmology and Visual Science, ³ Molecular Physiology and Biophysics, ⁴Psychiatry, ⁵Institute for Vision Research The University of Iowa Iowa City IA

Purpose: Traumatic brain injuries (TBI) often result in visual problems. In particular a decrease in the thickness of the retinal ganglion cell (RGC) layer and/or of the RNFL have been observed. Similar changes were observed in our mouse model of TBI and it seems that RGCs may be particularly prone to damage associated with over-pressure waves. To understand the mechanism of injury we characterized molecular and cellular changes in the retina in this mouse model.

Methods: In a custom-built blast chamber mice were exposed to an overpressure wave (20 PSI) directed to the head. Optical coherence tomography (OCT) was used to characterize the retinal structure in vivo. Changes in the number of RGCs were determined at 1, 5, and 16-weeks by using an RGC specific antibody (BRN3A) and changes in the total number of cells in the RGC layer were assessed using hematoxylin and eosin (H&E) staining in whole mounted retinas. Transcriptional changes induced by the exposure to the overpressure wave were evaluated using RNA sequencing at 1 and 5-weeks post-injury compared to expression levels in retinas from sham animals.

Results: The RGC complex thickness was decreased in injured mice compared to sham. A significant loss in RGCs positive for BRN3A was detected at all time-points analyzed. The total number of cells from the RGC layer was decreased significantly at 5 and 16-weeks post-injury. The analysis of the RNA sequencing data showed an increase in expression of immune-related genes at 1-week that subsided by 5-weeks after exposure. RGC specific markers were significantly decreased at both 1 and 5-weeks post-injury.

Conclusions: In this model exposure to an overpressure wave damaged the retina with RGC loss. The transcriptomic analysis indicates activation of the immune system early after exposure. By establishing the pathophysiology of retinal damage and the time-line of these changes after injury we are enabling the development of improved clinical diagnosis and treatment of traumatic brain injuries.

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The role of inflammation underlying vision impairment after traumatic brain injury

Evans, Lucy¹ ; Woll, Addison^1,2; Hehr, Nicole¹; Hedberg-Buenz, Adam¹; Todd, Brittany¹; Soukup, Dana¹; Ferguson, Polly¹; Newell, Elizabeth¹; Harper, Matthew^1,2; Bassuk, Alexander^1
1 University of Iowa; ²Center for the Prevention and Treatment of Visual Loss Iowa City VA

Purpose: Traumatic brain injury (TBI) is a leading cause of death and disability in the United States. Currently there are no existing pharmacologic interventions available. Secondary injury cascades are initiated following TBI resulting in tissue dysfunction and destruction, one of which is inflammation, with the IL-1 pathway being one of the major effectors. While it is well known that inflammation plays a role in the brain, limited attention has been given to ocular inflammation even though the retina is an extension of the central nervous system (CNS). Additionally, the exact inflammatory mechanisms specific to blast injury are not well-understood. We aim to characterize acute changes in the mouse eye utilizing a blast TBI (bTBI) model, allowing us to assess retinal manifestations of CNS injury and molecular changes leading to long-term damage in these delicate tissues.

Methods: Using a model of blast TBI (bTBI) with a compressed air-driven shock tube system, wild-type C57BL/6 mice were exposed to a blast wave pressure of 20±0.2 psi (137.8±1.3 kPa) three times, each 1 hour apart. Histologic evaluation of retinal ganglion cell (RGC) loss was carried out one month post-injury. Acute cytokine expression in retinal tissue was measured through real-time polymerase chain reaction 4 hours post-blast. Activation of retinal microglia and astrocytes were assessed via immunohistochemistry 1 week post-blast. Mice treated with anakinra, an IL-1R1 antagonist, underwent pattern electroretinograms and optical coherence tomography to assess retinal ganglion cell function and structure, respectively, 5 weeks after injury. The degree of neurodegeneration in the optic nerves of these mice was assessed by analysis of axonal damage across sections.

Results: A significant decrease in RGC number was noted in blast mice due to injury. Increased retinal expression of inflammatory markers lL-1α, IL-1β, IL-6, and TNFα were seen in bTBI mice exposed to blast when compared to shams. In bTBI animals, there was an enhanced response and morphologic changes of microglia and astrocytes in the retina due to the injury stimulus. After treatment with anakinra, mice exhibited a partial rescue of retinal ganglion cell function and cell death compared to blast-injured animals given saline. Additionally, there was a higher frequency of less damaged optic nerves in the anakinra treated mice.

Conclusions: We demonstrated that bTBI injury can cause RGC loss as well as inflammatory responses in the retina, both with increased expression of pro-inflammatory cytokines and with activation of resident microglia and astrocytes. Blockade of the acute inflammatory response after injury with the IL-1R1 antagonist anakinra resulted in partial preservation of the retinal ganglion cell function and structure that is normally lost due to blast injury. The trend of decreased damage in the optic nerves suggests that treatment with this anti-inflammatory drug prevents axonal damage after blast. These preliminary studies could result in repurposing currently approved pharmaceuticals for treatment for those suffering from bTBI.

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Optical Coherence Tomography Signal Quality and Repeatability of Foveal Outer Nuclear Layer Thickness

Gaffney, Mina¹; Mastey, Rebecca¹; Carroll, Joseph^1,2
1 Ophthalmology & Visual Sciences Medical College of Wisconsin; ² Cell Biology Neurobiology & Anatomy Medical College of Wisconsin

Purpose: The purpose of this study was to determine whether optical coherence tomography (OCT) signal quality impacts the repeatability of ONL thickness measurements in subjects with achromatopsia and individuals with normal vision.

Methods: OCT line and/or volume scans were acquired from 76 subjects with achromatopsia (ACHM) and 42 control subjects. The scans were then registered and averaged in ImageJ to create a single processed image which was used for analysis. The image quality was quantitatively assessed using the maximum tissue contrast index (mTCI) which was defined as the ratio between the signal intensity of the background and foreground of an OCT image. Three outer nuclear layer (ONL) thickness measurements were made at the fovea of each scan by a single observer based on the longitudinal reflectivity profile (LRP).

Results: The mean mTCI was lower on average in the images from subjects with ACHM (mean = 7.249 minimum = 1.0189 maximum = 19.9843) compared to images from subjects with normal vision (mean = 17.867 minimum = 3.31131 maximum = 27.4658). No significant relationship was observed between mTCI and observer repeatability in ACHM (p=0.351) or control (p = 0.140) subjects. There was also no significant relationship observed between the number of B-scans used to generate the final .tif images used for analysis and mTCI (spearman r = 0.1367 95% CI = -0.02773 to 0.2940 p = 0.093).

Conclusions: The signal quality of an OCT scan does not appear to impact the repeatability of ONL thickness measurements using our LRP method. However it remains unclear to what extent OCT image quality may impact other OCT analyses such as layer segmentation.

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Disruption of the GABA- and Glutamatergic system and inceptive neuroinflammation accompany early functional impairment in experimental glaucoma

Godwin, Cheyanne^1,2; Gramlich, Oliver^1,2; Bhattarai, Sajag^1,3; Drack, Arlene^1,3; Kuehn, Markus^1,2,3
1Department of Ophthalmology and Visual Sciences, University of Iowa; ²VA Center for the Prevention and Treatment of Visual Loss; ³Institute for Vision Research University of Iowa

Purpose: Loss of retinal ganglion cells (RGC) in glaucoma is a leading cause of irreversible blindness worldwide and increased intraocular pressure (IOP) is a major risk factor. Our aim is to determine early functional and molecular differences in a viral vector-induced mouse model of glaucoma that occur before significant RGC loss.

Methods: A unilateral intraocular injection of an adenoviral vector expressing the mutated form of myocilin (Ad5.MYOC) was used to induce IOP elevation in C57BL6 mice. Retinas were harvested nine weeks after vector-induction to determine RGC loss. In another study with similar setup electroretinograms (ERG) and pattern ERG (PERG) were recorded in mice three weeks after IOP elevation and in control eyes. Afterwards retinas were either collected and prepared for RNA seq. or for immunohistochemistry.

Results: Ocular injection of Ad5.MYOC leads to a significant and stable IOP elevation followed by significant loss of RGC after nine weeks most prominent in the peripheral retina when compared to uninjected eyes (average IOP in Ad5.MYOC: 22.8±2.6 mmHg vs uninjected eyes: 13.6± 0.5 mmHg p=0.002; RGC density in Ad5.MYOC: 1455±242 Brn3a+ cells/mm² vs uninjected eyes: 1768±199 Brn3a+ cells/mm² p=0.015). RNA seq. data from eyes three weeks after injection revealed a significant increase for 291 genes related to stress response activation of the complement cascade and inflammation. Decreased expression was found for 378 genes including downregulation of the GABA A receptor glutamine synthetase and genes related to axon guidance. Functional impairment characterized by a significant decrease in the amplitude was observed in the 30Hz flicker ERG and in the PERG of hypertensive eyes.

Conclusions: Early functional changes precede RGC loss in this mouse model of glaucoma. This functional impairment is accompanied by impaired GABA A receptor signaling and neurotransmitter recycling but also due to inceptive neuroinflammation. These initial changes may offer new avenues for early detection of glaucoma and targets for the development of new drugs.

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Correlation between visual biomarkers and motor-sensory deficits in mice with multiple sclerosis

Gramlich, OW^1,2; Godwin, CR^1,2; Kardon, RH^1,2
1Center for the Prevention and Treatment of Visual Loss Iowa City VA Health Care Iowa City IA ²Department of Ophthalmology and Visual Sciences University of Iowa Iowa City IA

Purpose: Acute optic neuritis is the initial symptom in 25-45% newly diagnosed cases of Multiple Sclerosis (MS). Retinal ganglion cell (RGC) complex thinning and decrease in pattern electroretinogram (PERG) amplitude are evident in MS patients as disease progresses. The purpose of our study is to determine if changes in the visual system correlate with motor-sensory deficits in an experimental autoimmune encephalomyelitis (EAE) model.

Methods: EAE was induced in 15 female C57BL6 mice by immunization with MOG33-55 complete Freund’s adjuvant and pertussis toxin. Another 15 age-matched mice served as healthy controls. Clinical progression was monitored according to a 5-point EAE scoring scheme. PERG and RGC complex thickness were measured 60 days after induction. Statistical differences were calculated using unpaired t-tests. PERG OCT and EAE scoring data were analyzed for correlation.

Results: EAE scores range from 0.5 - 4 with a mean EAE score of 1.8±0.7 which corresponds to a cumulative EAE score of 109±43. OCT Analysis reveal a significant decrease of the RGC complex thickness in EAE mice (59.3±4.1 µm) when compared to controls (68.3±2.5 µm; p=4.3^-11). Likewise the PERG amplitude is significantly decreased in EAE animals (EAE: 16.2±13.9 µV vs control: 23.8±3.8 µV; p=4.2^-10) while their P1 latency is prolonged (EAE: 102.1±15.5 ms vs control: 89.7±14.9 ms; p=0.003). Correlation analysis demonstrate a significant relationship (p<0.005) between individual RGC complex thickness and the individual last EAE score (R² =0.67) as well as for the individual cumulative EAE score (R² =0.79) respectively. Individual RCG function of EAE mice furthermore correlate with last EAE score (R² =0.46) as well as with the cumulative EAE score (R² =0.46).

Conclusions: Our data demonstrate a significant correlation between visual function and structure and over all motor-sensory impairment in EAE animals. Translation of these data to clinical application would be useful to assess CNS function in MS patients in a non-invasive fast and cost-effective manner.

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Photoreceptor degeneration in rd1 and rd10 mice leads to changes in synaptic function in the visual thalamus

Hahka, Taija1¹; Bhandari, Ashis²; Smith, Jennie²; Van Hook, Matthew J.^2
1University of Nebraska Medical Center

Purpose: Retinitis pigmentosa (RP) causes blindness due to degeneration of rod and cone photoreceptor cells. In this study we probed changes in neuronal function in two mouse models of RP - Rd10 and Rd1 mice. Rd1 mice have early onset (P8) of retinal degeneration and Rd10 mice have a delayed onset (P16-P20) of retinal degeneration both from a mutation of the Pde6b gene. The aim of this research is to test for changes in neuronal function in the dorsal lateral geniculate nucleus (dLGN) a subcortical retinal projection target in the thalamus following photoreceptor degeneration occurring along different developmental timeframes.

Methods: We assessed the photoreceptors and downstream retinal function of 5-8 week old Rd1 and Rd10 mice by scotopic and photopic electroretinography (ERG) and compared that to C57BL/6J mice. Photoreceptor degeneration was confirmed by DAPI staining of photoreceptor nuclei in the outer nuclear layer of the retina. RGC light responses were recorded for whole-cell current clamp at ~32°C in flat mount retinas using K-gluconate pipette solution in response to a 1-second long flash of 380nm of light. To test for changes in the dLGN we recorded spontaneous miniature excitatory synaptic currents (mEPSCs) recorded from thalamocortical relay neurons in the presence of picrotoxin and tetrodotoxin. Cumulative distributions of mEPSC parameters were compared using a Kolmogorov-Smirnov test (significance set at p<0.005).

Results: ERG data in both Rd1 and Rd10 mice indicated undetectable scotopic a-waves compared to C57Bl/6J controls. Rd1 mice demonstrated absence scotopic and photopic b-waves and Rd10 mice presented a dramatically reduced scotopic and photopic b-waves. The DAPI-stained outer nuclear layer thickness of both Rd1 and Rd10 mice showed a significant decrease in photoreceptor cells compared to the C57BL/6J mice (# of soma thick Rd1: 1 ± 0.2; Rd10: 1.3 ± 0.3 vs 15 ± 0.3; p<0.00000001). RGC light responses were generally absent in Rd1 mice. Small RGC light responses were detected in Rd10 mice. dLGN mEPSC amplitude was smaller in rd1 mice compared to C57Bl6J controls (7.2 ± 0.2 pA vs 10.1 ± 0.6 pA; p<0.00001 K-S test). In rd10 mice mEPSC amplitude was similar to controls (10.0 ± 0.6 pA; p=0.53 K-S test). dLGN mEPSC frequency was lower in rd1 mice compared to C57Bl6J controls (12.7 ± 1 Hz vs 17.5 ± 3.4 Hz; p<0.00001 K-S test). In rd10 mice mEPSC frequency was slightly lower relative to controls (15.5 ± 2.0 Hz; p = 0.00026 K-S test). dLGN sample sizes: rd10 8 cells; rd1 12 cells; C57Bl6J controls 6 cells.

Conclusions: The change in mEPSC amplitude indicates that photoreceptor degeneration in rd1 mice leads to changes in post-synaptic glutamate receptors in the dLGN while changes in mEPSC frequency point to a change in synaptic vesicle release probability. Differences in dLGN mEPSC properties between rd1 and rd10 mice are likely the result of differences in the timing of photoreceptor degeneration. Thus loss of photoreceptors appears to alter synaptic function in subcortical visual areas of the brain.

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Blast preconditioning protects retinal ganglion cells and reveals targets for prevention of neurodegeneration following blast-mediated TBI

Matthew M. Harper^1,2 Addison Woll^1,2; Lucy P. Evans^3,4; Michael Delcau^1,2; Abhigna Akurathi¹, Adam Hedberg-Buenz^1,5; Dana A Soukup⁵; Nickolas Boheme^1,2; Marco M. Hefti⁶; Laura M. Dutca^1,2; Michael G. Anderson^1,2,5; Alexander G. Bassuk^4,7
1The Iowa City Department of Veterans Affairs Medical Center Center for the Prevention and Treatment of Visual Loss; ²The University of Iowa Department of Ophthalmology and Visual Sciences; ³The University of Iowa Medical Scientist Training Program; ⁴University of Iowa Departments of Pediatrics; ⁵Molecular Physiology and Biophysics; ⁶Pathology; and ⁷Neurology

Purpose: The purpose of this study was to examine the effect of multiple blast exposures and blast preconditioning on the structure and function of retinal ganglion cells (RGC) to identify molecular pathways that contribute to RGC loss and to evaluate the role of kynurenine-3-monooxygenase (KMO) inhibition on RGC structure and function.

Methods: Mice were subject to sham blast injury one single blast injury or three blast injuries separated by either 1 hour or 1 week using a blast intensity of 20 PSI. To examine the effect of blast-preconditioning mice were subjected to sham blast injury one single 20 PSI injury or three blast injuries separated by 1 week (5 PSI 5 PSI 20 PSI) and (5 PSI 5 PSI 5 PSI). RGCs structure was analyzed by optical coherence tomography (OCT) and function was analyzed by the pattern electroretinogram (PERG). BRN3A positive cells were quantified to determine RGC density. RNAseq analysis was used to identify transcriptional changes between groups.

Results: Analysis of mice with multiple blast exposures of 20 PSI revealed no significant differences compared to one 20 PSI exposure using OCT PERG or BRN3A cell counts. Analysis of mice exposed to two preconditioning 5 PSI blasts prior to one 20 PSI blast showed preservation of RGC structure and function. RNAseq analysis of the retina identified multiple transcriptomic changes between conditions. Pharmacologic inhibition of KMO preserved RGC responses compared to vehicle treated mice.

Conclusions: Preconditioning protects RGC from blast injury. Protective effects appear to involve changes in KMO activity whose inhibition is also protective.

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Investigating microglial-mediated mechanisms of retinal ganglion cell damage in diabetes

Hedberg-Buenz, Adam¹; Abràmoff,Michael D.^2,3,4,5; Jiao, Chunhua²; Lee, Kyungmoo³; Oguz, Ipek⁶; Gardner, Thomas W.⁷; Adamson, Peter⁸; Zhang, Li⁵; Anderson, Michael G.^1,2,3; Sohn, Elliott H.^2,4
Departments of ¹Molecular Physiology and Biophysics ²Department of Ophthalmology and Visual Sciences ³ Electrical and Computer Engineering and Institutes for ⁴Vision Research and ⁵Biomedical Imaging University of Iowa Iowa City IA. ⁶Department of Radiology University of Pennsylvania Philadelphia PA. ⁷Department of Ophthalmology and Visual Sciences University of Michigan. ⁸UCL Institute of Ophthalmology London UK.

Purpose: Diabetes is a complex metabolic syndrome that causes damage dysfunction and failure to many different organ systems including those of the central nervous system and in particular the eye. An ocular hallmark and complication of diabetes is damage to the retinal microvasculature a potentially blinding condition known as diabetic retinopathy (DR). It has long been thought that through secondary effects of DR non-vascular cell types in retina are damaged and contribute to visual loss. However recent studies have uncovered a neurodegenerative component of DR characterized by a loss of retinal ganglion cells (RGC-neuropathy) which can precede microvascular damage. Here we aimed to determine whether RGC-neuropathy can occur independently of microvascular damage by assessing RGC survivorship in a mouse model of type 1 diabetes in a strain resistant to vascular dysfunction due to targeted knock out of the lipoprotein-associated phospholipase A2(Lp-Pla2-KO) gene. We hypothesized that Lp-Pla2-KO would attenuate microvascular damage but not RGC-neuropathy in diabetic mice.

Methods: Mice (Lp-Pla2-KO and wild-type controls) underwent phenotypic analyses of RGCs including in-vivo optical coherence tomography and quantitative histology of retina before and after induction of diabetes by streptozotocin.

Results: Microvascular damage was significantly reduced in retinas from diabetic Lp-Pla2-KO mice compared to wild type controls. Conversely the density of RGCs and thickness of their associated layers in retina were significantly decreased in diabetic mice regardless of Lp-Pla2 genotype.

Conclusions: These results indicate that Lp-Pla2-KO protects against microvascular damage but not RGC-neuropathy in this model of diabetes. Ongoing studies aim to identify mechanisms of diabetic RGC-neuropathy.

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The Effect of Eye Growth on FAZ Size

Heffernan, Elizabeth; Linderman, Rachel; Heitkotter, Heather; Carroll, Joseph
Froedtert and the Medical College of Wisconsin Eye Institute

Purpose: The purpose of this study was to determine the effect of eye growth on the area of foveal avascular zone (FAZ).

Methods: In this prospective observational study we obtained 3x3 OCT-A scans using the Optovue AngioVue and measured their axial lengths using the Zeiss IOL Master at their baseline visit and again at their second visit. Our subjects were 5 Caucasian female subjects without ocular disease and ranged from 9 to 12 years of age at their first visit with a second visit occurring at least 2 years 8 months later. A single masked observer (EH) manually segmented the highest quality image of each eye at both time points. FAZ area was then computed using different image scales (determined using the subject’s axial length or that assumed by the imaging device). Paired t-tests were conducted for statistical analysis using GraphPad Prism.

Results: The difference in FAZ area between the first and second visits when using the assumed axial length for 3x3 AngioVue scans 23.95 mm was statistically significant p=0.0442. Likewise the difference in FAZ area was statistically significant when assuming no change in axial length at the second visit using the initial measurement compared to correcting for the new axial length p=0.0006. However there was not a statistically significant difference in FAZ area when correcting for the true axial length at both time points p=0.0592.

Conclusions: In this small nonpowered observational study the area of the FAZ appears not to change significantly as the eye grows. These results must be reproduced in a larger study. The results also reinforced the need to correct for ocular magnification in OCTA images.

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PlGF negatively regulates endothelial cell barrier function through suppression of G6PD and anti-oxidant defense systems

Huang, Hu^1
1University of Missouri

Purpose: Diabetic retinopathy (DR) is a leading cause of visual impairment and loss worldwide. Growing evidence suggests that placental growth factor (PlGF) may act as a primary target molecule for the treatment of DR and its inhibition contributes to the amelioration of DR and diabetic macular edema (DME). The purpose of this study is to investigate the role of PlGF in retinal endothelial cell (REC) barrier function and elucidate the underlying mechanism.

Methods: Primary REC cultures were treated with neutralizing antibody and recombinant protein small interfering (si) RNA and inhibitor (DHEA and MJ33). Electrical cell-impedance sensing (ECIS) was used to measure REC resistance. RNA sequencing and bioinformatics analysis identified the differentially expressed genes and signaling pathways between control and PlGF ab. Western blots immunofluorescence were used to detect protein expression and cellular localization.

Results: The PlGF monoclonal antibody promotes (but recombinant protein disrupts) EC barrier function thus affecting the barrier-forming protein levels membrane distribution and EC monolayer impedance RNA sequencing-based transcriptome analysis identified the upregulation of the pentose phosphate pathway (PPP) and the antioxidant defense protein by PlGF blockade. The PlGF and PlGF-VEGF dimers (but not VEGF-A) downregulated the protein expression of glucose-6-phosphate (G6PD) and peroxiredoxin (PRDX). G6PD inhibition and gene silencing (siRNA) abolished the beneficial effects of PlGF inhibition on EC barrier function and PRDX-3/6 protein expression. VEGFR1 or VEGFR2 blockade prevented the inhibitory effect of PlGF on G6PD protein expression and EC barrier function. The PRDX6 played dual roles in EC barrier function through glutathione peroxidase and phospholipase A2 activity.

Conclusions: Our findings suggest that PlGF negatively regulates EC barrier function through the activation of VEGFR1 and VEGFR2 and the suppression of the G6PD/PPP and the anti-oxidant pathways and therefore a promising target to treat DR and DME.

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Development of a mouse model for KCNV2 retinopathy

Inamdar, Shivangi, M.¹; Laird, Joseph G.¹; Mahoney, Angela¹; Pufall, Miles¹;Baker Sheila A¹.
¹Department of Biochemistry, University of Iowa

Purpose: One of the five principle currents operating in photoreceptors is Ikx a voltage-gated potassium current that carries the outward dark current and serves as a high pass band filter of light responses. Ikx is thought to be carried by a heteromeric channel of Kv2.1 and Kv8.2 subunits. Loss of Kv8.2 results in cone degeneration with supernormal ERG responses known as KCNV2 retinopathy. The molecular pathogenesis of this disease is not known because there have not been any animal models of KCNV2 retinopathy. In this study we tested the hypothesis that a Kv8.2 KO mouse would model the human disease. If so this provides an important tool to better understand the disease and to test treatment options.

Methods: Kv8.2 KO mice were generated by CRISPR and were confirmed by genotyping of the Kcnv2 locus by reverse transcriptase (RT)- digital droplet PCR (dd-PCR) for Kcnv2 transcripts and finally by western blotting and immunohistochemistry for Kv8.2. Kv8.2 KO animals were compared to WT littermates. Retina RNA was harvested for RNAseq from 1-month old animals. ERG were collected on 1- or 3-month old animals and OCT imaging was used to test for gross retinal degeneration at 2 4 and 7-months of age.

Results: The genomic deletion in KCNV2 was confirmed by sequencing. Transcripts for KCNV2 in the retina were reduced by 50%. Absence of Kv8.2 protein was confirmed by western blotting and immunohistochemistry. Scotopic ERG revealed a reduced a wave amplitude delayed onset of the b wave which had a supernormal amplitude and reduction in the oscillatory potentials. Photopic ERG reveled a reduced b wave amplitude and reduced responses to a 30 Hz flicker. OCT imaging showed a progressive thinning of the photoreceptor layer between 2-7 months of age. Preliminary analysis of RNAseq data indicated decreased expression of cone-specific markers and changes in genes related to mTOR and apoptotic signaling suggesting metabolic stress contributes to degeneration.

Conclusions: This Kv8.2 KO mouse recapitulates the human KCNV2 retinopathy phenotype and can thus be used for future studies to better understand disease pathogenesis.

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Distribution of trabecular meshwork cells in human eyes

Jackson, Thomas^1,2; Wadkins, David^1,2; Kuehn, Markus^1,2,3
1Department of Ophthalmology and Visual Sciences, University of Iowa; ²VA Center for the Prevention and Treatment of Visual Loss; ³Institute for Vision Research University of Iowa

Purpose: Glaucoma is a leading cause of vision loss impacting about 60 million people worldwide. Primary open angle glaucoma (POAG) is the most common form in western countries and is frequently accompanied by increased intra ocular pressure (IOP) that can contribute to retinal ganglion cell death. The IOP is maintained by the trabecular meshwork (TM) and it has been reported that the number of cells in the tissue decreases with age and is particularly low in eyes with POAG. Currently it is widely believed that the density of TM cells varies dramatically in different regions of the TM. However few data have been presented to support or oppose this notion.

Methods: Healthy human donor eyes were fixed in paraformaldehyde and oriented so that the superior nasal inferior and temporal segments of the eye matched 12, 3, 6, and 9 on a clock face. Each quadrant was then further sectioned into 3 equal sized segments. Tissue was then embedded in paraffin and 20 semi-consecutive sections were obtained from each of the 12 blocks corresponding to the clock hour segments. Sections were then stained with H&E and imaged on a Zeiss microscope. Images were analyzed in image J to obtain the number of TM cell nuclei in the area located uveal to the margins of Schlemm’s Canal. Numbers obtained were then normalized to cells per 100 m. These results were then compared between segments from each block between the different regions of the TM and between donor eyes.

Results: The approach employed worked well to obtain tissue sections from defined areas of the anterior segment. Our data thus far indicate that TM cell density on sections obtained from the same block is relatively constant with standard deviations ranging from 8.9-10.3% of the average. Furthermore average TM cell density observed in multiple blocks from the same eye also appear to be very consistent revealing neither random nor geographic (e.g. superior vs. temporal) differences. Cell density in each clock hour range from 95.0 to 109.3% of each eyes’ overall average. In contrast eyes obtained from distinct donors frequently display clear differences in TM cell density.

Conclusions: Our findings thus far indicate that TM cellularity does not vary dramatically in healthy human donor eyes and further indicate the absence of regional variation. These data can be used to estimate the number of sections and blocks required to achieve adequate statistical to survey TM cellularity in multiple human donors for subsequent studies.

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The Effect of Photographic Visual Aids in Preoperative Patient Counseling in Oculoplastic Surgery

Kingsbury, Kenten D.; Yom, Kelly H.; Carter, Keith D.; Shriver, Erin M.; Ko, Audrey C.
University of Iowa, Department of Ophthalmology and Visual Sciences

Purpose: Effective preoperative patient counseling resulting in realistic postoperative expectations may improve patient satisfaction as discrepancies between patient expectations and surgical outcomes have been associated with dissatisfaction and negative psychosocial outcomes. This prospective randomized controlled trial investigated whether the addition of visual supplementation to preoperative oral counseling of patients undergoing oculoplastic surgery resulted in decreased preoperative and postoperative anxiety and more realistic expectations of common postoperative physical findings. A secondary objective was to determine whether the mode of delivery (in-person versus video recording) affects the primary outcome measures.

Methods: A prospective randomized controlled trial was conducted in compliance with the Institutional Review Board. 250 consecutive surgical patients undergoing brow and/or eyelid surgery at a single institution were recruited. Exclusion criteria included previous oculoplastic surgery or periocular trauma. Subjects were randomized to an oral only or oral and visual patient education group. The oral only group received routine preoperative oral counseling regarding what to expect after the operation. The oral and visual group received routine oral counseling supplemented with exemplary photographs demonstrating common postoperative findings such as swelling bruising and asymmetry. Within the oral and visual group patients were further randomized to receive patient education from the physician in person or via prerecorded video. Patient-reported emotions and expectations were assessed by 3 validated surveys before surgery at postoperative week one and at postoperative months two and four. Postoperative triage call rates were also evaluated. Data was analyzed using chi-square analysis or Fisher’s exact test for categorical variables and linear regression for continuous variables.

Results: 103 patients met inclusion criteria for the study of whom 32 received oral only education by a physician 33 received oral education accompanied by photographs by a physician and 38 received oral education accompanied by photographs narrated by the physician on video. Between the three groups there were no significant differences in demographics. Preoperatively there were no significant differences in patient feelings of fear anxiety or preparedness. Patients who were shown visual aids expected more severe post-operative adverse events such as redness and asymmetry at day one and week one after the surgery but the groups did not differ in their preoperative expectations of such events at months two and four. At one week and two months postoperatively there were no significant differences in patient feelings of anxiety preparedness or satisfaction between the three groups. There were no statistically significant differences in triage call rates between the groups.

Conclusions: Visual supplementation during patient counseling results in similar preoperative and long-term postoperative patient emotions and expectations and does not affect postoperative triage call rates. Preoperative surgical counseling with photographs may lead patients to have more realistic expectations of their healing process without increasing fear or anxiety related to the procedure. Additionally patient preoperative education via video is equivalent to counseling in person by the physician. Implementation of video counseling may help to standardize patient education and increase clinic efficiency without affecting the patient experience.

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T and B Lymphocyte Deficiency in RAG1 Mice Reduces Retinal Ganglion Cell Loss in Glaucoma

Kuehn, Markus, H^1,2,3; Godwin, Cheyanne, R.^1,2; Gramlich, Oliver, W.^1,2;
¹Department of Ophthalmology and Visual Sciences, University of Iowa; ²VA Center for the Prevention and Treatment of Visual Loss; ³Institute for Vision Research University of Iowa

Purpose: We recently demonstrated that adoptive transfer of lymphocytes from glaucomatous mice can induce retinal ganglion cell (RGC) damage in recipient animals. These findings suggest that an immune response develops in glaucoma that could potentially contribute to vision loss in the disease. Here we are evaluating if absence of an immune response slows the progression of glaucomatous damage.

Methods: Elevated IOP was induced in one eye of normal C57BL/6J (B6) or RAG1 knockout mice by vector mediated expression of myoc^Y437H. IOP was monitored by rebound tonometry. After 16 weeks mice (n=20/group) were sacrificed, enucleated, and the density of Brn3a positive retinal ganglion cell was determined in both the treated and the untreated contralateral eyes. Data were compared to those of mice having received injections of ‘empty’ vector. The number of extravascular CD3+ cells in the glaucoma retinas was determined using FACS and confirmed using immunohistochemistry.

Results: IOP significantly increased in treated eyes and remained elevated throughout the 16 week period (28.1 vs.13.0 mmHg). No differences in IOP response were observed between RAG and control mice. After 16 weeks, the average RGC density in B6 mice decreased from 3,332±68 to 2,576±284 RGC/mm2 (p=1.9x10^-4) in treated eyes and to 2,951± 254 RGC/mm2 in the contralateral eye with normal IOP (p=5.4x10^-4). In contrast, RGC density in treated RAG eyes only decreased to 2,951±411 RGC/mm² (p=0.18) and to 3,182±260 RGC/mm² (p=0.15) in the contralateral eye. RGC sparing in RAG1 was statistically significant in both the treated (p=0.006) and untreated (p=0.02) eyes when compared to those of control B6 animals. Flow cytometry indicated that the retina of eyes with elevated IOP contains significantly more CD3+ cells than that of healthy mice (46.0±16.1 vs 27.1±16.3, p=0.025, n=8/group).

Conclusions: Elevated IOP causes significant RGC loss in normal B6, but not in T- and B cell deficient mice. Furthermore, our data suggest that unilateral glaucoma can cause RGC loss in the contralateral eye but, again, only in normal mice. Taken together, these data indicate that immune processes significantly contribute to the pathology of experimental glaucoma.

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Migratory neural crest regulates optic cup invagination by depositing nidogens to build extracellular matrix superstructure

Chase D. Bryan¹; Macaulie A. Casey¹; Rebecca L. Pfeiffer²; Bryan W. Jones²; and Kristen M. Kwan^1,2
1Department of Human Genetics, University of Utah ²Department of Ophthalmology and Visual Sciences, University of Utah

Abstract: Although the neural crest contributes to mature eye structures, its role in controlling early stages of eye development, specifically optic cup morphogenesis, is poorly understood. In mouse, removal of neural crest leads to optic cup defects, but the underlying cellular and molecular mechanisms are unknown. Using 4-dimensional live imaging, zebrafish molecular genetics, we set out to determine precisely how neural crest regulates optic cup formation. In the absence of neural crest, what cell movements are disrupted? What molecule(s) is neural crest providing to control optic cup morphogenesis?

We find that during optic cup morphogenesis, neural crest cells migrate and enwrap the RPE side of the developing optic cup. Depletion of neural crest, using a tfap2a;foxd3 double mutant, results in defective optic cup invagination: RPE movements and cell movements around the optic vesicle rim, from the medial layer to the retinal layer, are impaired. Transmission electron microscopy indicates that basement membrane is lost in the absence of neural crest, but only around the RPE, where neural crest is normally in contact with the eye. Expression of individual extracellular matrix (ECM) components is unaffected, indicating a specific defect in ECM assembly. Nidogen, an extracellular matrix crosslinking protein essential for optic cup morphogenesis in ES cells, appears to be crucial: dominant negative nidogen disrupts wild type optic cup morphogenesis, and overexpression of wild type nidogen partially rescues optic cup morphogenesis in the neural crest mutant. Finally, we generated nidogen quadruple mutants and find that the full loss-of-function phenotype resembles the neural crest mutant.

Our data indicate that migrating neural crest cells enwrap the developing eye and are crucial for the cell movements underlying optic cup invagination. These neural crest cells deposit nidogens around the RPE to build ECM superstructure, which supports cell movements. We are currently examining the role of other neural crest factors that may also promote proper early eye development.

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Rescue of rod synapses by induction of Cav alpha 1F in the mature Cav1.4 knock-out mouse retina

Laird, Joseph; Gardner, Sarah; Kopel, Ariel; Kerov, Vasily; Lee, Amy; Baker, Sheila
University of Iowa

Purpose: Cav1.4 is a voltage-gated calcium channel clustered at the presynaptic active zones of photoreceptors. Cav1.4 functions in communication by mediating the Ca2+ influx that triggers neurotransmitter release. It also aids in development since rod ribbon synapses do not form in Cav1.4 knock out mice. Here we used a rescue strategy to investigate the ability of Cav1.4 to trigger synaptogenesis in both immature and mature mouse rods.

Methods: In vivo electroporation was used to transiently express Cav a1F or tamoxifen-inducible Cav a1F in a subset of Cav1.4 knock out mouse rods. Synaptogenesis was assayed using morphological markers and a vision-guided water maze.

Results: We found that introduction of Cav a1F to knock out terminals rescued synaptic development as indicated by PSD-95 expression and elongated ribbons. When expression of Cav a1F was induced in mature animals we again found restoration of PSD-95 and elongated ribbons. However the induced expression of Cav a1F led to diffuse distribution of Cav a1F in the terminal instead of being clustered beneath the ribbon. Approximately a quarter of treated animals passed the water maze test suggesting the rescue of retinal signaling in these mice.

Conclusions: These data confirm that Cav a1F expression is necessary for rod synaptic terminal development and demonstrate that rescue is robust even in adult animals with late stages of synaptic disease. The degree of rod synaptic plasticity seen here should be sufficient to support future vision-restoring treatments such as gene or cell replacement that will require photoreceptor synaptic rewiring.

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Behavioral analysis of vision in HCN1 KO mice

Lankford, Colten K.¹; Umino, Yumiko³; Russo, Andrew²; Solessio, Eduardo³; Baker, Sheila A.^1
1Department of Biochemistry ²Molecular Physiology & Biophysics University of Iowa Iowa City IA USA ³ Department of Ophthalmology SUNY Upstate Medical University Syracuse NY

Purpose: In rod and cone photoreceptors photon absorption initiates the phototransduction signaling cascade that inactivates the depolarizing dark current. This in turn drives hyperpolarization of the membrane resulting in synaptic signaling to downstream neurons. Temporal control of the visual response is essential for proper vision and distinct mechanisms exist to limit both the duration of phototransduction signaling and the duration of membrane hyperpolarization. Bradyopsia a visual disordered characterized by impaired temporal resolution and severe photophobia arises from mutations in RGS9 of R9AP that delay inactivation of the phototransduction cascade. HCN1 channels conduct a depolarizing current that limits the duration of light activated hyperpolarization. As such we would predict that HCN1 loss would result prolonged photoreceptor activation and exhibit a phenotype similar to Bradyopsia. Here we use behavioral assays to examine the effect that HCN1 loss has on temporal resolution and photophobia in mice.

Methods: Temporal resolution was tested using an optomotor response assay where mice are exposed to a rotating grating and a visual response is assessed based on the animal’s innate response to this stimulus. Contrast sensitivity was assessed under increasing temporal frequencies of 0.384 0.768 1.536 and 6.144 Hz with a constant spatial frequency of 0.128 cycles/degree and light intensity of 80 cd/m². Photophobia was assessed using a light aversion assay where mice are placed in a testing chamber containing equally sized light and dark zones and the time spent in each zone is recorded. Animals were tested under a brightness of 25000 lux.

Results: By optomotor response we see that HCN1 KO animals show a peak response at the same temporal frequencies as controls but exhibit a pronounced decrease in contrast sensitivity under all frequencies tested. By the light aversion test we also see that HCN1 knockout animals exhibit a photophobia phenotype spending significantly less time in the light than control animals.

Conclusions: Our results suggest that HCN1 loss results in a similar but distinct visual phenotype from Bradyopsia. HCN1 KO animals exhibit a strong light aversive phenotype similar to Bradyopsia mice suggesting that limiting the duration of the visual response at both the level of phototransduction and by electrically opposing membrane hyperpolarization is essential for maintaining normal under bright lighting conditions. Interestingly we see by the optomotor response assay that HCN1 KO and Bradyopsia mice exhibit reduced contrast sensitivity under higher temporal frequencies; however HCN1 KO animals also exhibit reduced contrast sensitivity at lower temporal frequencies where Bradyopsia mice are indistinguishable from controls. Thus suggesting that HCN1 loss may have a more pronounced effect on visual function than Bradyopsia mutations and it will be interesting to further examine visual phenotypes of HCN1 KO animals.

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Investigation of KCNV2 Ion Channel Trafficking

Mahoney, Angela; Inamdar, Shivangi M.; Laird, Joseph G.; Baker, Sheila A.
Department of Biochemistry, University of Iowa

Purpose: Mutations in KCNV2 result in the autosomal recessive disorder KCNV2 retinopathy. The retinal degenerative disease results in cone cell death and abnormal rod signaling. KCNV2 encodes for a voltage-gated potassium channel subunit Kv8.2. Kv8.2 forms a heteromeric complex with Kv2.1 to create a potassium channel in photoreceptors that assists in regulating membrane potential and filtering photoresponses. The molecular mechanism of the disease is not yet known but it is known that the Kv8.2 subunit is retained in the endoplasmic reticulum of the photoreceptor unless it forms the tetramer with Kv2.1. The goal of this project is to investigate the mechanisms controlling the trafficking of Kv8.2. In this study we will be testing for an ER retention motif on Kv8.2 as well as the trafficking pattern of Kv8.2 with a known mutation in the human population.

Methods: Efforts of the current study include characterizing the expression of wild type Kv8.2 in the absence of Kv2.1 in HEK-293T cells. The cells were labeled for Kv8.2 and the endoplasmic reticulum. The Eukaryotic Linear Motif resource was used to identify RxR retention motifs on Kv8.2. The candidate we chose to investigate is located on the N-terminus of the protein. A 14 amino acid deletion was cloned into KCNV2. The point mutation W196C was also cloned into KCNV2 to mimic a mutation seen in the patient population. Mutated KCNV2 clones were created using site-directed mutagenesis. The KCNV2 clone with the 14-amino acid deletion is in the process of being verified through Sanger Sequencing.

Results: The expression of wild type Kv8.2 in the absence of Kv2.1 resulted in the retention of Kv8.2 in the ER. The Sanger Sequencing of the KCNV2 clone with the 14-amino acid deletion showed success in the deleting the desired amino acids.

Conclusions: The wild type Kv8.2 protein is expressing as expected in the absence of Kv2.1. The next steps will be to verify the expression pattern of Kv8.2 and Kv2.1 together followed by verifying and expressing the mutated KCNV2 clones.

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Evaluating the risk factors and the time-course of high intraocular pressures in severely burned patients

Mai, Anthony P.¹; Fortenbach, Chris R.¹; Wibbenmeyer, Lucy A.²; Wang, Kai³; Shriver, Erin M.¹
Departments of ¹Ophthalmology and Visual Sciences, ²Surgery and ³Biostatistics, University of Iowa, Iowa City, IA

Purpose: Severely burned patients receiving large-volume fluid resuscitation are at risk for increased intraocular pressures (IOP) resulting in irreversible vision loss. Identification of at-risk patients for timely intervention is critical. This study therefore investigates potential risk factors and the time period of highest risk for developing orbital compartment syndrome (OCS) in burn patients.

Methods: Medical records of inpatient thermal and chemical burns seen by the University of Iowa ophthalmology service between 2004 and 2019 were retrospectively reviewed. Demographic, burn, examination, and fluid resuscitation data were compared between subjects with IOP ≥ 26 mm Hg and a control group with IOP ≤ 25 mm Hg using 2-sided t-tests, Fisher’s Exact tests, and linear regression.

Results: When comparing the high IOP (n = 5) and control (n = 13) groups, there was a statistically significant difference in % TBSA (total body surface area) burned (p < 0.001), the presence of other compartment syndromes (p = 0.047), the calculated Parkland Formula recommendation (p < 0.001), and mortality (p = 0.012). Max IOP and fluid resuscitation volume were linearly related (p = 0.007). Three patients demonstrated pressure increases ranging from 3.2 to 7 mm Hg per hour, all before 20 hours post-injury.

Conclusions: This study not only confirms that fluid resuscitation volume is a risk factor for high IOP, but also identifies % TBSA, presence of other compartment syndromes, and Parkland Formula recommendation as other potential risk factors. In addition to earlier and more frequent IOP checks in burn patients, these screening items will help providers identify patients most at risk for OCS and permanent vision loss.

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Comparison of retinal changes identified by non-invasive and invasive imaging in a zebrafish model of type 1 Usher Syndrome

Nonarath, J.T Hannah; Collery, F. Ross; Link, A. Brian
Medical College of Wisconsin

Purpose: Usher syndrome is an inherited disease that presents with retinal degeneration hearing loss and vestibular defects. For patients with Usher Syndrome cochlear implants can be used to treat hearing loss; however there are currently no therapies available to spare visual function. This is due to a number of gaps in our understanding of the disease’s underlying mechanism and reliable biomarkers to stage progression. This is partly a consequence of reliance on rod-rich mouse models which faithfully manifest the hearing loss phenotype but do not present the retinal phenotype. Unlike rodent models cone-rich zebrafish present with more physiologically relevant phenotypes In zebrafish models of Usher Syndrome correlative analysis of retinal changes observed by histology and non-invasive imaging (optical coherency tomography OCT) will provide a better understanding of disease progression. From these approaches we can assess potential changes in the photoreceptor mosaic differences in retinal lamination

Methods: To model Usher Syndrome in zebrafish we targeted Myosin7a; which is duplicated in zebrafish to Myosin7aa and Myosin7ab (Myo7aa and Myo7ab). For development of mutant lines the CRISPR/Cas9 system was used to create deletions in important functional domains and insert premature stop codons. As a result of early lethality in the Myo7aa line at two months of age only the Myo7ab mutants were used for adult image studies. Due to the high regularity of the UV cone mosaic en face images of this retinal layer were analyzed for changes in the photoreceptor mosaic using a mosaic analytics program. Following image processing changes in mosaic regularity and spacing were determined. Additionally B scans obtained from the OCT were analyzed for differences in retinal layering and changes in the reflectivity profile. Following OCT zebrafish were processed for histology to allow for a direct comparison between the non-invasive and invasive techniques.

Results: At two months of age Myo7ab mutants present with significant changes in the UV cone mosaic. Additionally Myo7ab mutants exhibited altered OCT reflectivity in the RPE. Reflectivity changes identified by OCT could be directly correlated to an expansion of the pigmentation in the retinal pigment epithelial layer on histology. Additionally Myo7ab mutant histological samples highlighted altered rod outer segment structure and association with the RPE. Specifically the rod outer segments in Myo7ab mutant zebrafish were encompassed by the pigmented region of the RPE; while in wildtype these pigmented regions were confined to the region of cone outer segments and absent from the rods

Conclusions: These preliminary studies highlight our capability to correlate retinal changes observed by OCT to those identified following histological processing. Future longitudinal studies will be performed in conjunction with optokinetic response. Correlation between cellular changes and changes in visual function will assist in identify early retinal changes in disease progression. Our results as well as future studies will help uncover the mechanisms responsible for retinal degeneration and assist in identifying biomarkers and development of therapeutic strategies.

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Endoplasmic reticulum stress in photoreceptors of CDSRR mouse model

Rethwisch, Rachel; Baker, Sheila
University of Iowa

Purpose: Cone Dystrophy with Supernormal Rod Response (CDSRR) is a rare autosomal recessive disease that causes cone photoreceptors to die and rod photoreceptors to respond minimally to dim light and excessively to medium and bright light. This disease is caused by a mutation in the KCNV2 gene which codes for the Kv8.2 protein. Kv8.2 along with the Kv2.1 protein form functional voltage gated potassium channels on the surface of photoreceptors. In patients with CDSRR a mutation in the KCNV2 gene causes the Kv8.2 protein to not be expressed on the surface of the cells. While the fate of this protein is not yet known several hypotheses indicate it could be causing endoplasmic reticulum (ER) stress in these cells. The ER stress response is a cellular response that is initiated when the ER is not able to correctly fold proteins. This can be caused by a variety of factors such as genetic mutations that cause misfolding problems with degradation of normal amounts of misfolded proteins a lack of energy in the cell to correctly fold the proteins or incorrect calcium levels which can lead to an environment that does not favor the correct protein folding bonds. If the cell remains under ER stress for long enough cell death can occur.

Methods: A Kv8.2 KO mouse model was generated to imitate CDSRR patients. Digital droplet PCR (ddPCR) Western Blot and semiquantitative RT-PCR were performed on the retinas of these mice. Kv8.2 KO mice were compared to WT littermates when looking at activation and/or interaction between the three ER stress response pathways.

Results: ddPCR displayed an increase in PERK and eIF2a RNA levels in the Kv8.2 KO mice. The PDI family of chaperone proteins were also increased in KO mice as seen by Western Blot. RT-PCR showed that XBP1 RNA is not spliced in either WT or KO mice and Western Blot indicated that IRE1 and p-eIF2a protein levels remained unchanged in Kv8.2 KO mice.

Conclusions: Because no ER stress pathway was activated completely it is likely that any ER stress seen was triggered secondarily by other problems in retinas without the Kv8.2 protein.

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Safety and efficacy of subretinal injection of two adeno-associated virus vectors in a Bardet-Beidl syndrome type 10 mouse model

Stalter, Elliot; Thomas, Jacintha; Datta, Poppy; Cherascu, Ioana; Seo, Seongjin; Bhattarai, Sajag; Drack, Arlene V.
¹Department of Ophthalmology and Visual Sciences, University of Iowa

Purpose: Bardet-Biedl Syndrome (BBS) is an autosomal recessive, ciliopathic genetic disorder. Affected individuals lack at least one of 21 BBS genes that normally produce BBS proteins which are assembled into multi-protein complexes (the BBSome and chaperonin complexes) important in intraflagellar transport (IFT), a process integral to cilia creation and maintenance. Individuals with BBS suffer from retinal degeneration, and the disorder is being actively investigated for the possibility of treatment with gene therapy, the aim of which is to provide functional copies of missing BBS genes to retinal cells via viral vectors. Adeno Associated Virus (AAV) is a benign viral vector that easily gains access to human cells, and the AAV2/Anc80 serotype is a synthetically constructed ancestor to which modern humans may not carry an antibody, and may be especially effective as a retinal transfection agent. The purpose of this study was to evaluate the safety and efficacy of subretinal injection of these two adeno-associated virus vectors (AAV2/5 and AAV2/Anc80) in a mouse model of Bardet-Biedl syndrome type 10.

Methods: Thirty-one mice aged P40-220 received single, subretinal injections of mouse wild type (WT) Bbs10FLAG packaged within an AAV vector (concentrations ranging from 1x10⁹ to 2x10⁹ vg/µL) controlled by the rho kinase promoter in their right eyes, with four distinct groups varying with respect to genotype and specific vector employed. ERG was performed one week after injection to assess retinal function, and OCT was subsequently performed to analyze retinal structure. Mice were generated and provided by the lab of Dr. Val Sheffield. Western blot was performed by the lab of Dr. Seongjin Seo on the eyes of two KO mice injected with Anc80 to assess the vector’s efficacy in transfecting retinal cells with Bbs10FLAG via subretinal injection.

Results: Eleven Bbs10 KO mice aged P40-130 and 6 WT mice aged P110-220 were injected with AAV2/5-Bbs10FLAG in their right eyes, with left eyes uninjected controls. 10 Bbs10 KO mice aged P40-120 and 4 heterozygous, unaffected mice aged P100-120 were injected with AAV2/Anc80-Bbs10FLAG in their right eyes, with left eyes uninjected controls. ERG was performed on all mice one week after injection. No significant difference in average a-wave or b-wave amplitude was found between right and left eyes in any of the four groups. ERG had lower amplitudes in Bbs10 KO mice than in WT or unaffected Het mice. The difference was significant (p=4.42^-12).

Conclusions: ERG and OCT results indicate that both the AAV2/5-Bbs10FLAG and AAV2/Anc80-Bbs10FLAG vectors are non-toxic within the concentration ranges at which they were administered (1x10⁹ to 2x10⁹ vg/µL). ERG showed no significant differences in average a-wave or b-wave amplitudes between left and right eyes of mice within all groups, corresponding to no significant reduction in retinal function in the treated eyes compared to the untreated eyes of mice injected with either vector. OCT confirmed structural integrity by indicating no atrophy or retinal detachment. This is significant because previous studies have shown that AAV2/5-BBS1, a related gene, was very toxic to the retina. Western blot identified Bbs10FLAG protein in the mouse retina indicating that our vector transfects the cells and results in production of the missing protein. Despite this, no efficacy in correcting the retinal degeneration was seen, possibly because the mice were relatively old at the time of treatment. Future studies will include treatment at a younger age and long-term follow up, and will compare transfection efficiency and inflammatory markers in animals treated with the two different vectors.

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Trends in Horizontal Periocular Asymmetry

Tran, Charlene ¹; Choi, Dabin¹; Carter, Keith D.² ; Johnson, A. Tim²; Oetting, Thomas A.²; Bozung, Alison²; Ko, Audrey C.²; Shriver, Erin M.^2
1 Carver College of Medicine University of Iowa; ² Department of Ophthalmology and Visual Sciences University of Iowa

Purpose: Facial symmetry is considered a standard of beauty but slight lateral asymmetry of the eyelid and orbit are common. There are conflicting studies regarding right or left-sided facial predominance and little research has been performed on laterality of periocular structures. Recognizing horizontal asymmetry in the periocular region is essential for evaluating patients’ baseline periocular structures providing proper patient counseling pre-operative planning and maximizing post-operative success for both the patients and surgeons.

Methods: A prospective study of consecutive adult patients in an academic ophthalmology department was conducted in compliance with the IRB to evaluate for laterality dominance of periocular structures. Exclusion criteria included patients who had worn contact lenses; undergone laser skin surfacing or injections; or sustained periocular-altering trauma surgery or disease processes. Standardized digital photographs were obtained periocular structures were measured with NIH Image J software and dimensions were calculated based on a standard corneal limbus to limbus distance. The midline was defined as the midpoint between the medial canthi and the distances calculated for both the right and left sides were midline to medial canthus pupil centroid lateral canthus and lateral zygoma.

Results: Periocular structures were measured in 129 patients. There was no significant difference between midline to medial canthus between the right (16.33mm) and left (16.31mm) (P=0.75). Right-sided predominance was found to be increasingly significant for the following variables in millimeters: midline to pupil center (31.15 vs 30.87 P=.004) midline to lateral canthus (42.33 vs 41.92 P<.001) and midline to lateral zygoma (65.37 vs 63.52 P<.001).

Conclusions: Photographic analysis of adults with no periocular-altering history demonstrates that there is a right-sided predominance in the horizontal dimension of the midline to the pupil lateral canthus and zygoma with increasing significance.

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Early-Stage Ocular Hypertension Alters Retinal Ganglion Cell Synaptic Transmission in the Visual Thalamus

Van Hook, Matthew; Bhandari, Ashish; Smith, Jennie; Zhang, Yang; Reid, Lisa; Goeser, Toni; Fan, Shan; Ghate, Deepta
University of Nebraska Medical Center

Purpose: Axonopathy is a hallmark of many neurodegenerative diseases including glaucoma where elevated intraocular pressure (ocular hypertension OHT) stresses retinal ganglion cell (RGC) axons as they exit the eye and form the optic nerve. OHT causes early changes in the optic nerve such as axon atrophy transport inhibition and gliosis. Importantly many of these changes appear to occur prior to irreversible neuronal loss making them promising points for early diagnosis of glaucoma. It is unknown whether OHT has similarly early effects on the function of RGC output to the brain.

Methods: We elevated eye pressure in mice by anterior chamber injection of polystyrene microbeads. To determine the influence of OHT on retinal projections to the brain we expressed Channelrhodopsin-2 (ChR2) in melanopsin-expressing retinal ganglion cells by crossing the Opn4-cre mouse line with a ChR2-reporter mouse line and recorded post-synaptic responses in thalamocortical relay neurons in the dorsal lateral geniculate nucleus (dLGN) of the thalamus evoked by stimulation with 460 nm light. The use of a Opn4-cre reporter system allowed for expression of ChR2 in a narrow subset of RGCs responsible for image-forming vision in mice. Optical Coherence Tomography imaging was used to assess changes in retinal thickness. Immunohistochemistry was used to measure changes in RGCs and their synaptic terminals in the dLGN.

Results: 5 weeks post-injection bead-injected eyes showed a modest RGC loss in the peripheral retina as evidenced by RBPMS antibody staining. Additionally we observed reduced dendritic complexity and lower spontaneous spike rate of On-RGCs targeted for patch clamp recording and dye filling using a Opn4-cre reporter mouse line. In dLGN brain slice recordings paired pulse and high-frequency stimulus train experiments revealed that presynaptic vesicle release probability at retinogeniculate synapses was elevated. Additionally miniature synaptic current frequency was slightly reduced in brain slices from OHT mice and proximal dendrites of post-synaptic dLGN relay neurons assessed using a Sholl analysis showed a reduced complexity. Strikingly these changes occurred prior to major loss of RGCs labeled with the Opn4-Cre mouse as indicated by immunofluorescence staining of ChR2-expressing retinal neurons.

Conclusions: Thus OHT leads to pre- and post-synaptic functional and structural changes at retinogeniculate synapses. Along with RGC dendritic remodeling and optic nerve transport changes these retinogeniculate synaptic changes are among the earliest signs of glaucoma.

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Restoration of Cilia-Deficient Eyelids with Autologous Eyelash Grafts via Cilia-Bearing Adjacent Tissue Transfer or Full Thickness Skin Grafts

Yom, Kelly; Fernandes Fensterer, Tathyana; Ko, Audrey
Department of Ophthalmology and Visual Sciences, University of Iowa

Purpose: The absence of lashes is a stigmata of previous eyelid injury or malignancy. Previous attempts at eyelash restoration have involved transplantation of hair-bearing skin grafts or multiple follicular units harvested from the scalp eyebrows sideburns and leg hair.1 However these methods result in surrogate eyelashes of inconsistent length variable thickness and misdirected cilia causing keratopathy. We describe a novel eyelash reconstructive method by adjacent tissue transfer or autologous grafting of full thickness eyelid skin containing follicular units harvested from the ipsilateral or contralateral eyelids.

Methods: A retrospective review of patients with previous eyelid reconstruction within the past year at one institution was conducted. Two patients had undergone secondary reconstruction of the upper or lower eyelid using autologous periocular tissues containing eyelash cilia.

Results: Patient 1 is a 40-year-old female with history of multiple reconstructions for basal cell carcinoma and squamous cell carcinoma of the right lower eyelid. She developed recurrent basal cell carcinoma 10 years later of the right lateral canthus upper and lower eyelids and underwent repeat Mohs excision and reconstruction with tarsoconjunctival flap periosteal flap and full thickness preauricular area skin graft. Postoperatively she had absence of lashes involving the lateral upper and lower eyelid. Six months after reconstruction she underwent secondary reconstruction by lateral canthoplasty and adjacent tissue transfer and full thickness skin graft containing lashes from the medial upper eyelid to the lateral upper eyelid. The patient was pleased with the results at postoperative month 5 and had additional right upper and lower eyelid reconstruction by full thickness skin graft containing cilia from the contralateral upper and lower eyelid (3 month follow-up pending). Patient 2 is a 57-year-old female with history of left lower eyelid basal cell carcinoma status post Mohs excision and reconstruction with Hughes flap and full thickness postauricular skin graft. The lower eyelid lashes were absent centrally but present laterally. She desired lashes centrally for mascara application. Eighteen months after the original reconstruction a left lower eyelid cicatricial release was performed and a full thickness skin graft containing lashes from the lateral left lower eyelid was transplanted into the defect. At postoperative week 1.5 she maintained viable cilia on the graft (3 month follow-up pending).

Conclusions: Adjacent tissue transfer and grafting of strips of follicle-containing skin from the ipsilateral or contralateral eyelid restores eyelashes in previously reconstructed non-eyelash bearing eyelids. Unlike previously described methods these grafts do not require chronic trimming to adjust their length have an appearance consistent with the surrounding remaining eyelashes and maintain a natural orientation. Interestingly whereas transplanted cilia are typically lost initially after transplant and grow back months later both of our patients retained the majority of transplanted hair postoperatively. If complete restoration of a full line of lashes is impossible a shelf bracket approach involving restoration of medial and lateral lashes allows for the support of false lashes.

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Superior Orbital Rim Bone Contouring through a Minimally Invasive Eyelid Crease Approach for Facial Feminization

Yom, Kelly; Fernandes Fensterer, Tathyana; Ko, Audrey
University of Iowa

Purpose: Forehead and brow contouring are used to feminize the appearance of the upper face in females and transgender females. Previously described surgical approaches for forehead and brow contouring in facial feminization of the upper face include sculpting of the bone overlying the central forehead glabellar region overlying the frontal sinus with or without a frontal sinus ostomy and central and lateral superior orbital rims. These procedures require a mid-forehead pretrichial or bicoronal approach for adequate exposure. However some patients do not have a prominent central forehead or glabella and only require contouring of the superior central and lateral orbital rims. For these patients we describe a novel minimally invasive surgical approach to feminize the periocular brow area.

Methods: A retrospective review of patients who underwent bilateral upper eyelid blepharoplasty in conjunction with bone contouring of the mid-forehead anterior frontal sinus wall and superior orbital rims was conducted. For patients who underwent this combination of procedures the upper eyelid blepharoplasty was performed first followed by contouring of the superior orbital rims with a surgical approach through the blepharoplasty eyelid crease incision. A bicoronal approach was then used for the remainder of the forehead contouring during which the previous areas of bone sculpting through the eyelid crease incision were examined.

Results: Two patients were included in this study. For both patients the superior central and lateral orbital rims were safely and successfully contoured through the upper eyelid incision. Examination of the previously burred areas after creation of a bicoronal flap did not show any additional areas needing contouring that could not be accessed through the upper eyelid incision. Prior to creation of the bicoronal flap access to the glabellar and mid forehead areas was examined in both patients. The supraorbital nerve was easily identified and protected during the dissection. While the glabellar area was accessible with an angled drill handle blind burring of the bone overlying the anterior frontal sinus was thought to be unsafe as the margins of the frontal sinus and the thickness of remaining bone was unable to be assessed visually putting the patient at risk for full thickness bony defect overlying the frontal sinus or more dangerously the intracranial space. In both patients there was difficulty accessing the mid forehead area due to the tension of the overlying skin and curvature of the skull. Postoperatively both patients achieved excellent cosmetic results for feminization of the periorbital area.

Conclusions: In patients who desire contouring of the superior central and temporal orbital rims only an approach through the upper eyelid crease is a minimally invasive and safe method for visualization and bony sculpting of those areas. While the anterior frontal sinus region can be accessed through this incision the authors do not feel it would be safe to blindly contour in this region. The mid forehead area is not easily accessible through this approach. This incision is also conducive to concurrent upper eyelid blepharoplasty upper eyelid ptosis repair and internal lateral canthopexy.

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Understanding the UPR Signaling in Aging and Diabetic Retina

Zhang, Sarah X.; McLaughlin, Todd; Wang, Josh J.
Departments of Ophthalmology and Biochemistry, Ross Eye Institute, University at Buffalo
SUNY Eye Institute, State University of New York, Buffalo, NY, USA

Purpose: Perturbation of the endoplasmic reticulum (ER) homeostasis has been implicated in age-related retinal degenerative diseases, such as age-related macular degeneration, glaucomatous retinopathy, and diabetic retinopathy. X-box binding protein 1 (XBP1) is a major component of the adaptive unfolded protein response (UPR) to maintain protein homeostasis in stressed cells. The purpose of this study is to understand the role of XBP1-mediated UPR in retinal neuronal survival and function during aging and diabetes.

Methods: Conditional knockout (cKO) of XBP1 in retinal neurons was generated by crossing XBP1 floxed mice with a retina-specific cre line (Chx10-Cre). Diabetes was induced with streptozotocin (STZ) in 8 week-old wild type (WT) and XBP1 cKO mice. Retinal morphometry, numbers of cone photoreceptors and retinal ganglion cells (RGCs), and neuronal synapses were assessed by immunohistochemistry with various markers in retinal sections. Retinal function was assessed by electroretinogram (ERG).

Results: Depletion of XBP1 in retinal neurons does not affect retinal development. However, XBP1 deficiency results in accelerated retinal function deterioration and loss of retinal ganglion cells (RGC) with aging. The cKO retinas demonstrate synaptic defects in the outer and inner plexiform layers (OPL and IPL) accompanied by aberrant microglia activation. In diabetes, multiple deficits in retinal function and structure were observed in XBP1 cKO mice but not in WT mice after short-term hyperglycemia. Diabetic cKO retinas display more severe neuronal damage, including loss of RGCs, loss of cone and rod photoreceptors, as well as reduced number of ribbon synapses in the OPL. Functionally, the b-waves of dark- and light-adapted ERGs are reduced significantly in diabetic XBP1 cKO mice.

Conclusions: Collectively, these findings suggest that XBP1-mediated UPR signaling is important for maintaining functional and structural integrity of retinal neurons in diabetes and aging.

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