15th Annual
2023 Midwest Eye Research Symposium
Supported by an unrestricted grant from Research to Prevent Blindness to the University of Iowa Department of Ophthalmology and Visual Sciences
Morning Poster Presentations
Abstracts
Poster #2
Inhibition of baxa or baxb does not rescue retinal ganglion cell death in atoh7 loss of function Danio rerio
Amidon, Antonia1; Rossebo, Mariah1; Veldman, Matthew1; Miesfeld, Joel1
1Medical College of Wisconsin, Milwaukee, WI
Purpose: Retinal ganglion cells (RGCs) transmit all visual information to the brain as their axons form the optic nerve and are thus essential for vision. Studies have demonstrated that the baxa and baxb genes are mediators of apoptosis in zebrafish, mimicking Bax function in mammalian species. In mice, RGC cell death can be rescued by inhibition of apoptosis through the loss of the pro-apoptotic Bax gene, however it is unknown if this mechanism is shared by zebrafish.
Methods: Translation blocking morpholinos (MOs) or crRNA/Cas9 targeting atoh7, baxa, and or baxb, were used to inhibit protein or gene function, respectively, through injection into embryos of either atoh7(lakritz), baxa, or baxb genetic mutants. Survival of RGCs were assessed with laser scanning confocal microscopy using the Isl2b3:nGreenLanternPEST transgene background, or via wholemount immunostaining of the pan RGC marker Rbpms2b.
Results Injection of both the previously published atoh7 MO and dual cRNAs targeting atoh7 resulted in a complete loss or reduction Isl2b3:nGreenLanternPEST or Rbpms2b positive RGCs compared to uninjected controls. Preliminary results suggest loss of baxa or baxb alone does not rescue the RGC cell death induced by loss of atoh7 function.
Conclusions: Baxa or baxb are not solely responsible for the loss of RGCs observed in atoh7 loss of function zebrafish. Further experiments will need to be repeated to gain a larger sample size and assess the possibility of genetic compensation between baxa and baxb in inducing RGC cell death in atoh7 mutants.
Poster #4
Retinal and optic nerve damage in a mouse model of blast-mediated traumatic brain injury
Boehme, Nickolas1; Hedberg-Buenz, Adam2; Castonguay, William1; Bielecki, Michael1; Harper, Matthew1
1 VA Center for the Prevention and Treatment of Visual Loss (151) Iowa City VA Health Care System; 2 Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA
Purpose: Visual problems are often present in traumatic brain injuries (TBI) acutely and chronically. Thinning of the retina was observed in different types of TBI, in particular a decrease in the thickness of the retinal ganglion cell (RGC) layer and/or of the RNFL. Our recent work using a mouse model of blast-induced TBI indicates that RGCs may be particularly prone to damage associated with over-pressure waves. To understand the mechanism of injury we characterized cellular and tissue changes in the retina and optic nerve 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. The number of RGCs and the total number of cells in the RGC layer were determined using an RGC specific antibody and hematoxylin and eosin staining, respectively, in whole mounted retinas. Optic nerve damage was assessed using histological stains and antibodies specific for glia.
Results: The RGC complex thickness was decreased in injured mice compared to sham. The number of RGCs decreased significantly as early as one week after injury, along with the total number of cells from the RGC layer, while the retinal area remained constant. The analysis of optic nerves stained post-injury showed a decrease in number of myelinated axons and an increase in number of glial cells.
Conclusions: In this model, exposure to an overpressure wave damaged both the retina and the optic nerve, with RGC loss, and glial activity in the optic nerve. By establishing the pathophysiology of retinal and optic nerve 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.
Poster #6
Structural and Biochemical Investigation of NRL and CRX Cooperativity
Chinna Swamy, Pavithra1; Srivastava, Dhiraj1; Artemyev, Nikolai1
1Department of Molecular Physiology and Biophysics, The University of Iowa Carver College of Medicine, Iowa City, IA
Purpose: CRX and NRL are two key transcription factors involved in the gene regulation during photoreceptor differentiation and homeostasis. While CRX is essential for the differentiation of postmitotic photoreceptor precursors to the photoreceptor cells, NRL determines rod cell fate. During photoreceptor development, CRX and NRL cooperate functionally and physically via direct protein-protein interaction and regulate the transcription of various genes. Despite of extensive studies, there is no clear understanding of precise molecular mechanisms that determine CRX- and NRL-mediated expression of several rods and cone genes during development and in response to signaling pathways. The aim of this study is to elucidate molecular mechanisms of the CRX/NRL function based on the crystal structures and to determine structural basis for synergy and specificity of NRL and CRX interaction in photoreceptor gene regulation.
Methods: CRX homeodomain protein was purified and attempted to determine solution structure by small angle X-ray scattering (SAXS). Attempts were also made to crystalize CRX homeodomain with rhodopsin promoter. Binding affinity of NRL and CRX with their respective response elements were determined by fluorescence anisotropy. NRL and CRX cooperativity was investigated by electrophoretic mobility shift assay and single molecule-total internal reflection microscopy.
Results: We have solved the crystal structure of CRX in complex with its cognate response element from rhodopsin promoter. The structure clearly shows that CRX and DNA interacts with 2:1 stoichiometry and causes DNA bending.
Conclusions: The complex structure shows unusual stoichiometry which was validated by SEC-MALS-SAXS and single molecule-total internal reflection microscopy. Electromobility shift assay suggests that CRX increases the affinity between NRL and DNA. However, presence of NRL changes the stoichiometry of CRX binding to its response element in rhodopsin promoter. Our current work set the ground for the future studies on the mechanism of NRL-CRX cooperativity on other promoters and mechanism of transcription regulation by the two transcription factor.
Poster #8
Examining the role of the TET proteins and DNA demethylation in retinal cell fate determination
Hernandez Nunez, Ismael1; Zhang, Xiaodong2; Chen, Shiming2; Clark, Brian S1
1John F. Hardesty, MD Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA; 2Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO
Purpose: DNA methylation is a dynamic process by which methyl groups are added and removed from cystine residues for the regulation of gene expression. During development, DNA methylation functions to maintain stem cells properties or stimulate cell differentiation. Active DNA demethylation is driven by the tet-eleven translocation (Tet) methylcytosine dioxygenase proteins. In the zebrafish, Tet2 -/-; Tet3 -/- mutants retinas result in reduced retinal ganglion cell axonal migration and impaired differentiation of photoreceptors. In this study, we seek to identify the functional role and potential redundancy of the Tet proteins in cell-fate determination within the developing mammalian retina.
Methods: Postnatal day (P) P21-P24 and 6 weeks old heterozygous and Tet conditional knockouts (cKO) were generated using the Chx10-Cre line. Retinal morphology and visual function were examined using hematoxylin and eosin staining (H&E), immunohistochemical approaches and visual function assays. The consequence of Tet protein overexpression on retinal cell fate specification was determined through ex vivo electroporations of P0 CD1 mice Statistical analyses were performed with GraphPad Prism v9.
Results: Morphological assessments of Tet mutant retinas indicate Tet1/2/3 triple cKOs result in a disruption of retinal structure, and the absence of outer segments at P21. Immunohistological analyses determined a significant reduction in the number of horizontal cells (Calbindin+) and a significant increase in amacrine cells (Pax6+), Müller glia cells (Lhx2+), and microglial cells (Iba1+) in Tet1/2/3 cKO retinas. Additionally, Tet1/2/3 cKO resulted in a significant increase in the number of cones (RXRgamma+; cone-arrestin+), with cone nuclei mislocalized to the basal portion of the outer nuclear layer (ONL). Changes in cell type proportions resulted in decreased amplitudes of dark-adapted A and B waves, and the light-adapted B wave in the Tet1/2/3 cKOs. Overexpression experiments resulted in small but significant changes in cell fate specification after Tet overexpression.
Conclusions: Loss of active DNA demethylation within the developing mouse retina results in failure of photoreceptor function in the adult animal. Future experiments seek to identify the mechanisms by which the Tet proteins control photoreceptor cell fate specification and differentiation.
Poster #10
Effect of 670 nm photobiomodulation on angiogenic and inflammatory signaling pathways in cultured cell model of diabetic retinopathy
Joshi, Rishika1; Lofald, Anna1; Hall, Alexandra1; Liedhegner, Elizabeth2; Eells, Janis1
1University of Wisconsin Milwaukee, Milwaukee, WI; 2School of Biomedical Sciences and Health Care Administration, University of Wisconsin Milwaukee
Purpose: The pathophysiology of DR is complex, involving mitochondrial dysfunction, oxidative stress, and vascular degeneration. Anti-VEGF drugs are used to reduce vascular proliferation. Photobiomodulation treatment (PBMt) with far-red (670 nm) light is a promising therapy for DR. In an in vitro model of DR, we showed that high glucose exposure disrupts mitochondrial function increases ROS and initiates a signaling cascade leading to NFkB activation and increased ICAM-1 and VEGF production. PBM treatment attenuated oxidative stress and mitochondrial dysfunction, reduced NFkB and decreased production of ICAM-1. However, PBM did not reduce VEGF, leading us to speculate that other signaling pathways may be involved in the action of PBM. This study tested the hypothesis that 670nm PBM treatment will enhance the expression of two key anti-angiogenic and anti-inflammatory factors, thrombospondin-1 and transforming growth factor β in this Muller glia cell model of diabetic retinopathy.
Methods: Müller glial cells play a primary role in the development and progression of DR due to a shift in their physiology from an anti-inflammatory to a pro-inflammatory state. Rat retinal Müller cells (rMC-1) were cultured in normal (5 mM) or high (25 mM) glucose media to simulate normoglycemia and hyperglycemia. Cultures were treated with a 670 nm light using a light emitting diode (LED) array at a dose of 4.5 J/cm2 (25 mW/cm2 for 180 seconds) or no light (sham) for 3 or 4 days. Assays were conducted to measure thrombospondin-1 (TSP-1), an anti-angiogenic extracellular matrix-associated glycoprotein and transforming growth factor beta (TGF-β), a multifunctional anti-angiogenic and anti-inflammatory cytokine.
Results: High glucose treatment had no effect on TSP-1 concentrations in MGCs. In contrast, PBM treatment produced a two-fold increase in MGC’s exposed to high glucose (p = 0.046). Further studies are needed to confirm these findings. We were unable to visualize TGF-β due to serum interference.
Conclusions: The ability of 670nm light treatment to attenuate early molecular changes in this in vitro high glucose model of DR suggest that PBM treatment has the potential to mitigate early deleterious effects in DR by modulating inflammatory signaling and diminishing oxidative stress. Further studies are needed to fully characterize the effects of PBM treatment on signaling pathways involved in the pathogenesis of diabetic retinopathy.
Poster #12
Pre-clinical testing of two candidate vectors for treating Bardet-Biedl Syndrome type 10 with subretinal gene therapy
Lobeck, Brianna1; Rankin, Tyler1,2; Hsu, Ying1; Georgiadis, Tassos3; Holthaus, Sophia3; Kalmanek, Emily 1; Stanley, Sarah3; Sheffield, Val C.; Drack, Arlene1
1Department of Ophthalmology and Visual Sciences, University of Iowa; 2Interdisciplinary Graduate Program in Genetics, University of Iowa; 3MieraGTx, United Kingdom; 4Department of Pediatrics
Purpose: Bardet-Biedl Syndrome type 10 (BBS10) is an autosomal recessive disease characterized by retinal degeneration leading to severe vision loss. Mutations in at least 29 genes can cause BBS; BBS10 accounts for about 40% of cases. Subretinal gene augmentation therapy to for BBS10 has shown promise in slowing vision loss in a mouse model of BBS10. The choice of promoter used to drive target gene expression can affect the efficacy of therapy. In this study, BBS10 gene expression is driven by two distinct promoting regions: chicken beta-actin (CAG), a general promoter, and rhodopsin kinase (RK), a photoreceptor-specific promoter. The purpose of this study is to investigate the toxicity and efficacy of these two candidate viral vectors, AAV8-CAG-hBBS10 and AAV8-RK-hBBS10.
Methods: A Bbs10 knockout (Bbs10 -/-) mouse model was maintained both on an SV129 background and a mixed genetic background. The human BBS10 gene was cloned into shuttle plasmids driven by the RK promoter and the CAG promoter, respectively. These plasmids were packaged into an AAV2/8 viral capsid for in vivo gene delivery. To assess toxicity, wild-type (WT) and heterozygous (HET) mice of both genetic backgrounds were subretinally injected with 2 µL of AAV8-CAG-hBBS10 or AAV8-RK-hBBS10 at 4×1010 vg/µL (digital droplet PCR), for a total dose of 8×1010 vg/eye. To determine efficacy, Bbs10-/- mice received either the AAV8-CAG-hBBS10 or the AAV8-RK-hBBS10 vector at 8×108, 8×109, or 8×1010 vg/eye. Electroretinography (ERG) was used to assess retinal electrical response and was completed at 1-, 2-, 3-, and 5-months post injection (PI). Optical coherence tomography (OCT) was used to assess retinal structure at 1-, 3-, and 5-months post injection (PI). Data quantification was performed using ImageJ.
Results: Toxicity: After injection of AAV8-RK-hBBS10, thicknesses of the outer nuclear layer (ONL) in WT/HET mice were comparable to those in eyes treated with buffer (p > 0.95) or untreated eyes (p > 0.94). For WT/HET mice receiving AAV8-CAG-hBBS10, a strain-specific effect was observed. After receiving 8×1010 vg/eye of AAV8-CAG-hBBS10, ONL thicknesses in WT/HET mice on the SV129 background displayed no difference from those receiving the dilution buffer (p=0.998). In contrast, WT/HET mice on the mixed background had significantly thinner ONLs than buffer-treated or untreated eyes as early as 1-month PI after receiving this dose. Efficacy: At 2-month PI, Bbs10-/- mice treated with AAV8-RK-hBBS10 had higher ERG amplitudes in light-adapted but not dark-adapted ERGs. At 3-months PI, mice treated with AAV8-CAG-hBBS10 possessed higher b-waves in their dark-adapted ERGs, whereas b-wave results in untreated Bbs10-/- mice became nonrecordable; improvements were not seen in the light-adapted ERG.
Conclusions: Toxicity Study: A high-dose administration of the AAV8-RK-hBBS10 vector was well-tolerated in the eyes of WT/HET mice. In contrast, AAV8-CAG-hBBS10 caused strain-specific retinal degeneration. Overexpression toxicity may be a factor underlying the degeneration. The reason for the strain-specific response is being explored. Efficacy Study: Mice treated with AAV8-RK-hBBS10 do not exhibit better dark-adapted ERG amplitudes; however, cone activity improved in a dose-dependent manner. Mice treated with AAV8-CAG-hBBS10 showed a slower loss of b-wave amplitudes over time compared to untreated mice, revealing conserved bipolar cell activity. Overall, AAV8-RK-hBBS10 had a superior safety profile and elicited improvement in retinal function in a mouse model of BBS10.
Poster #14
Development of a three-dimensional organoid model to explore early retinal phenotypes associated with Alzheimer’s disease
Patil, Shruti1; Lavekar, Sailee2; Hernandez, Melody3; Harkin, Jade4; Gomes, Catia3; Huang, Kang-Chieh2; Puntambekar, Shweta3; Lamb, Bruce T.3; Meyer, Jason S.3
1Department of Medical and Molecular Genetics, Indiana University School of Medicine; 2Department of Biology, Indiana University-Purdue University Indianapolis; 3Department of Medical and Molecular Genetics, Indiana University School of Medicine; 4Department of Pharmacology and Toxicology, Indiana University School of Medicine1
Purpose: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of Aβ plaques and neurofibrillary tangles, resulting in neurodegeneration. The retina is an extension of the central nervous system within the eye, sharing many structural similarities with the brain, and previous studies have observed AD-related phenotypes within the retina. Human pluripotent stem cells (hPSCs) can effectively model some of the earliest manifestations of disease states, including those affecting the retina, yet early AD-associated phenotypes have not yet been examined. Thus, the current study focused upon the differentiation of hPSCs into retinal organoids for the analysis of AD-associated alterations.
Methods: hPSCs from both AD as well as healthy controls were used in all studies, and their pluripotency as well as the presence or absence of AD-associated gene variants was validated. Subsequently, cell lines were directed to differentiate to yield retinal organoids following established methods. Upon acquisition of retinal organoids, we assesed for characteristic features of AD pathology, including changes in the levels of pathological Aβ42 as well as phosphorylated tau protein. Retinal organoids with AD-associated mutations were then transcriptionally profiled to further identify other alterations that may be leveraged as early indicators of AD pathology.
Results: Results demonstrated the robust differentiation of retinal organoids from both familial AD and unaffected control cell lines. AD retinal organoids also exhibited characteristic pathological features, including an elevation in the Aβ42:Aβ40 ratio in conditioned medium, as well as a significant increase in pTau protein in AD retinal organoids. Transcriptional analyses further demonstrated the differential expression of many genes and cellular pathways, particularly those associated with synaptic dysfunction.
Conclusions: The current study demonstrates the ability of retinal organoids to serve as a powerful model for the identification of some of the earliest retinal alterations associated with AD, thereby validating the potential use of retinal organoids as a model system to explore early alterations within an easily accessible region of the central nervous system, along with important implications for the early diagnosis of AD.
Poster #16
Microglia In the Visual Thalamus Respond to Glaucomatous Neurodegeneration
Thompson, Jennifer1; Smith, Jennie C.2; McCool, Shaylah3; Van Hook, Matthew J.1
1University of Nebraska Medical Center; 2Truhlsen Eye Institute, Department of Ophthalmology & Visual Sciences, University of Nebraska Medical Center, Omaha, NE; 3Department of Pharmacology & Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE; 4Department of Cellular & Integrative Physiology, University of Nebraska Medical Center, Omaha, NE
Purpose: Glaucoma causes blindness by damaging retinal ganglion cells (RGCs) until they are irreversibly lost. Chronically high eye pressure (ocular hypertension; OHT) is glaucoma’s only modifiable risk factor and has previously been shown to initiate a loss of RGC axon terminals in retinorecipient brain regions, including the visual thalamus (dorsolateral geniculate nucleus; dLGN). During development, the dLGN undergoes a period of synaptic pruning whereby microglia- the innate immune cells of the brain- remove components of excess synapses via phagocytosis. Because we hypothesize that OHT may be triggering the removal of RGC axon terminals via similar mechanisms, we sought to investigate whether microglia are involved in removing synapses in the glaucomatous dLGN.
Methods: To this aim, we investigated whether OHT triggers microglial polarization and molecular-tagging of dLGN synapses in a mouse model of inherited glaucoma (DBA/2J; D2) and its strain-matched control (DBA/2JGpnmb+). We longitudinally monitored intraocular pressure (IOP) during the living phase, then used fixed tissue to examine dLGN-targeting RGC axon terminals at timepoints corresponding to pre- (4mo), early- (9mo), and progressive glaucoma (12mo). Correlations were made using the IOP integral (AUC in mmHg*days) for each eye separately.
Results: D2s showed an IOP-dependent reduction (R2 = 0.31 ; p<0.001) in RGC terminals immunolabeled for vesicular glutamate transporter 2 (vGlut2+), while no age- or IOP-related loss was observed in controls (p = 0.52; p = 0.850). A subset of tissues was stained for complement protein C1q, which tags immature retinogeniculate synapses for removal during development. An intensity analysis revealed that C1q was elevated by 9-months in glaucoma and exaggerated by 12-months: this increase was IOP-dependent (R2 = 0.48; p < 0.0001) and associated with vGlut2+ loss (R2 = 0.40; p< 0.001). Pre-glaucoma C1q expression was consistent with the low, unchanging levels that were detected at all timepoints in controls (p = 0.28), suggesting that complement-mediated synapse elimination may be in effect. Morphological analysis of Iba1+ cells (microglia/macrophages) revealed negative associations between OHT and ramification features (IOP explained ~ 30% of branching variability (R2 = 0.30, p<0.0001).
Conclusions: These data suggest that Iba1+ cells in the visual thalamus may be polarized towards a synapse-removal state in the context of glaucoma. Moreover, the glaucoma-specific increase in C1q-expression in the dLGN may be evidence that chronic IOP elevations are aberrantly reactivating parallel mechanisms to those involved in developmental pruning- future work will address these details.
Poster #18
The effects of trisomy on retinal thickness and retinal ganglion cell count in a postnatal Ts65Dn mouse model of Down syndrome
Folz, Drew1; Goodlett, Charles1; Belecky-Adams, Teri1; Roper, Randall1
1Indiana University–Purdue University Indianapolis
Purpose: Down syndrome (DS) is a genetic condition that is caused by the triplication of human chromosome 21 and presents with a variety of neurological phenotypes, including hypocellularity in the brain, and various ocular phenotypes, including an increased risk of cataracts, increased risk of refractive errors, and increased retinal thickness. The Ts65Dn mouse model of DS presents with a number of similar phenotypes to those with DS including decreased brain volume, decreased visual acuity, and increased retinal thickness. Victorino et al. 2020 found an increase in retinal thickness in P17 Ts65Dn mice. Laguna et al. 2013 showed that adult Ts65Dn mice had a thicker retina, along with an increased cellularity in the retinal ganglion cell (RGC) layer. To develop a treatment plan to normalize ocular phenotypes, the time point in which the trisomic mice deviate from the euploid mice must be determined. We hypothesize that retinal thickness and RGC count will be increased at P15 in Ts65Dn mice.
Methods: Retinal cryosections from male and female trisomic and euploid Ts65Dn mice at P15 were fluorescently labeled for RGCs and bipolar cells via cryo-immunofluorescence. The retinas were measured for total retinal thickness and RNA-binding protein with multiple splicing (RBPMS) positive cells in the RGC layer. Measurements for total retinal thickness were taken at 200µm increments from the optic disc. RBPMS positive cells were counted in the region of the RGC layer 200µm and 400µm from the optic disc.
Results: There were no genotypic or sex effects in either total retinal thickness or RBPMS positive cells at P15 in male and female Ts65Dn mice.
Conclusions: These preliminary results indicate that the lack of genotypic or sex effect in both total retinal thickness or RBPMS positive cells means that this age could be a good starting point for a treatment normalize total retinal thickness and RBPMS positive cells at later ages. These findings are different than what was seen previously at P17 in the Ts65Dn model in Victorino et al. 2020, in which there was an increased in total retinal thickness in the trisomic mice compared to the euploid mice. Differences in measuring technique, optical coherence-tomography as compared to measuring 2D images of retinal slices, as well as a difference in the strain of Ts65Dn, 005252 as compared to 001924, could be an alternate reason as to the differences between the studies.
Poster #20
5xFAD Mouse Model and the Early Visual System
McCool, Shaylah1; Smith, Jennie2; Sladek, Asia2; Zhang, Kevin (Yang)1,2; Van Hook, Matthew1,2
1University of Nebraska Medical Center, Department of Ophthalmology & Visual Sciences, Omaha, NE; 2University of Nebraska Medical Center, Truhlsen Eye Institute, Omaha, NE
Purpose: Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder and form of dementia diagnosed worldwide. AD is characterized by a loss of memory and cognitive impairment due to the formation of amyloid beta (Aβ) plaques and neurofibrillary tangles (NFT), and ultimately, atrophy of the brain. It is known that AD impacts the visual system; however, the mechanisms by which it does this are not well understood. The global impact of AD leads us to study the structure and function of the retina and the dorsal lateral geniculate nucleus (dLGN), the visual thalamus. The dLGN is the primary target of retinal projections for image-forming vision as well as relaying information from the retina to the cortex. The purpose of this project was to determine mechanisms that underly visual deficits in Alzheimer’s disease (AD) by investigating the hypothesis that Aβ pathology leads to neuronal and synaptic degeneration in the retina & dLGN.
Methods: The 5xFAD mouse model develops an AD-like phenotype, specifically the formation of Aβ plaques, and will be used to test our hypothesis along with C57BL/6J mice used as controls. To examine whether retinal function is altered in AD, electroretinogram (ERG) recordings were performed. Immunohistochemistry of vGlut2 was performed to determine number of retinal ganglion cell (RGC) axon terminals in the dLGN. Cell fills of thalamocortical (TC) neurons followed by 2-photon imaging was utilized to examine dendritic structure in the dLGN. Miniature excitatory postsynaptic current (mEPSC) recordings were used to determine function of the retinogeniculate synapses. Optomotor response (OMR) was used to show reflexive visual behavior.
Results: The 5xFAD model was successfully validated after analyzing Aβ plaque density in the dLGN which presented with a high burden of Aβ plaques compared to C57BL/6J controls. ERG results indicated a significant increase in A-wave amplitude in the 6mo 5xFAD mice and a significant decrease in A-wave amplitude in the 9mo 5xFAD mice. The 5xFAD mice also showed a significant increase in B-wave amplitude at 6mo and a decrease in B-wave amplitude at 9mo. The B-to-A wave ratio indicated dysfunction in the photoreceptors. The 9mo 5xFAD males specifically showed a significant decrease in A-wave amplitude. Investigation of whether Aβ plaques lead to changes in the number of RGC axon terminals showed decreased RGC axon terminals in the 9mo 5xFAD mice following analysis of vGlut2 density in the dLGN. Sholl analysis of 6mo C57 and 5xFAD mice indicate no postsynaptic structural changes. Findings from OMR showed no difference in visual acuity or contrast sensitivity between the C57 and 5xFAD mice.
Conclusions: Even with histopathological evidence of disease in the brain, there were only modest changes on the visual system structure and function in 5xFAD mice. This suggests that Aβ might have fairly modest influences on the visual system or may point to adaptive mechanisms that preserve function of visual pathways in this model. Investigating how AD pathology affects the early visual system may lay the essential foundation for future studies by delivering insights into how the visual system functions during an AD-like state and may have larger implications as the results of this work could facilitate the development of early diagnostic tools or drug therapies for AD.
Poster #22
Complement Dependent Cytotoxicity Drives Retinal Ganglion Cell Loss in a Novel In Vivo Model of Neuromyelitis Optica
Gramlich, Oliver1,2; Elwood, Ben1,2; Anders, Jeff1,2; Kardon, Randy1,2; Bennett, Jeffrey L.3
1Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA; 2VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA; 3Departments of Neurology and Ophthalmology, University of Colorado, Denver, CO
Purpose: Visual impairment from optic neuritis (ON) is a common manifestation in aquaporin-4 (AQP4) autoantibody seropositive neuromyelitis optica spectrum disease (NMOSD). The purpose of our study was to determine whether human recombinant AQP4 antibodies (AQP4 rAb) with different antibody effector function elicits distinct patterns of optic nerve injury in an in vivo model of NMOSD ON.
Methods:Long Evans rats (n=10/group) received a unilateral subarachnoid injection of 100 ng of anti-AQP4 recombinant antibody (AQP4 rAb) or isotype control (2B4) underneath the optic nerve sheath. AQP4 rAb contained either an intact human IgG1 Fc region (wt) or Fc region mutations that enhanced or diminish complement dependent (CDC) or antibody-dependent cellular (ADCC) cytotoxicity. Impairment in visual acuity and changes in retinal nerve fiber layer (RNFL) and retinal ganglion cell (RGC) complex thickness were determined 28 days later using optokinetic tracking response and optical coherence tomography. Statistical analyses were performed using ANOVA with Tukey’s test.
Results: Injection of wt AQP4 rAb cause a decline in visual acuity when compared to rats having received isotype control 2B4 rAb (wt AQP4=0.34±0.03c/d vs. 2B4= 0.4±0.07c/d; p=0.02). Visual impairment was also observed in rats after injection of AQP4 rAb with enhanced CDC (CDC++/ADCC- AQP4=0.34±0.03c/d, p=0.001). When compared to baseline values recorded prior to injection, significant RNFL thinning was most evident in rats that have received either wt AQP4 or AQP4 rAb with enhanced CDC and ADCC (percentage RNFL loss in wt AQP4=-10±3%, p=0.002; CDC+++/ADCC+ AQP4=-9±5%, p=0.023). Mutated AQP4 rAb without ADCC function (CDC++/ADCC-) showed intermediate loss (-6±3%, p=0.32). No RNFL thickness loss was noticed in animals having received 2B4 (-1±5%) or mutated AQP4 rAb with ablated CDC (CDC-/ADCC++= 0±3%). Analysis of the RGC complex revealed significant thinning in rats injected with wt AQP4 (77±3 µm, p=0.003), CDC++/ADCC- AQP4 (76±4 µm, p=0.003), and CDC+++/ADCC+ AQP4 (76±4 µm, p=0.003).
Conclusions: Similar to models of intracerebral NMOSD pathology, NMOSD optic nerve injury is dependent on targeting the AQP4 water channel through antibody-mediated CDC. Magnitude of visual impairment and RGC degeneration were differentially impacted by ADCC.
Poster #24
Visualization and Quantification of the Spatial Patterns of Edema and Atrophy in Non-Arteritic Anterior Ischemic Optic Neuropathy (NAION) Over Time Based on Deep-Learning Variational-Autoencoder
Wang, Jui-Kai1,2; Linton, Edward F.1; Johnson, Brett A.1; Branco, Joseph3; Kupersmith, Mark K.4; Garvin, Mona K.5; Kardon, Randy H.1,2
1Department of Ophthalmology and Visual Sciences, University of Iowa, IA; 2VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA; 3New York Medical College, NY; 4Department of Ophthalmology and Neurology, Icahn School of Medicine at Mount Sinai, NY; 5Department of Electrical and Computer Engineering, University of Iowa, IA
Purpose: Acute NAION causes optic nerve head (ONH) swelling and spatial distortion changing over time and have not been well characterized. We hypothesize a deep-learning bi-channel variational autoencoder (VAE) can be used to create a latent space that reflects the spatial patterns of the ONH that changes as the pathological process evolves.
Methods: We performed VAE training on the optical coherence tomography (OCT) ONH scans over time from 322 NAION subjects (1524 scans; Quark study) and 97 normal subjects (797 scans). The trained VAE created latent spaces of the retinal nerve fiber layer (RNFL) and total retinal (TR) thickness maps that depicted the continuum of spatial patterns and can monitor each NAION eye progressing over time in relation to the normal and all NAION eyes. Each latent space, consisted of a montage of 21 x 21 color thickness map of RNFL or TR, whose spatial patterns were encoded by only two latent variables, one on the x-axis and the other on the y-axis of the map.
Results: The VAE latent space montage maps display meaningful spatial patterns of edema and atrophy at the ONH that could be visually observed and located in different regions in the latent space map for individual eyes. In our independent test dataset (15 NAION and 15 normal subjects), the Pearson’s correlation coefficient of the mean peripapillary thicknesses between the input and reconstructed images was higher than 0.98 (p< 0.001) through each RNFL or TR VAE channel.
Conclusions: The trained VAE successfully described and quantified essential spatial information of the pattern of edema and eventual atrophy using only two latent variables. Latent space montage maps can classify different patterns and severity of swelling/atrophy in NAION. The quantification and characterization of the spatial patterns simplify the structural measurements that can be related to vision loss at presentation and outcome.
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