Alzheimer's Disease Linked APOE4 Isoform Impairs Retinal Glial Kir4.1 Channels Through Mitochondrial Dysfunction

Surabhi Abhyankar¹, Yucheng Xiao², Neha Mahajan¹, Qianyi Luo¹, Theodore R. Cummins², Adrian L. Oblak³, Bruce T. Lamb³, Timothy W. Corson⁴, Ashay D. Bhatwadekar^1
1Indiana University School of Medicine, Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana, ²School of Science, Indiana University, Indianapolis, Indianapolis, Indiana, ³Stark Neurosciences Research Institute, Indianapolis, Indiana, ⁴Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada

Purpose: Late-onset Alzheimer's Disease (LOAD) is the most common form of dementia, with the apolipoprotein E4 (APOE4) allele serving as a key genetic risk factor. The retina offers a unique window into early neurodegenerative processes, and our recent study identified significant age-related retinal impairments in APOE4-knock-in (KI) mice compared to APOE3-KI controls. These included structural degeneration, vascular and functional deficits, increased gliosis, neuroinflammation, and reduced synaptogenesis at 12-13 months of age, suggesting that the retina is an early target of APOE4-driven neuropathology, preceding cognitive decline. Building on these findings, we focused on Muller cells (MCs), which are essential for maintaining retinal homeostasis and may play a key role in mediating APOE4-induced pathology. This study examined the impact of APOE4 on MC function, particularly inwardly rectifying K+ channels 4.1 (Kir4.1), mitochondrial health, and oxidative stress.

Methods: Retinal sections and freshly isolated MCs from 12ñ13-month-old APOE4-KI and APOE3-KI mice were analyzed for Kir4.1 and AQP4 expression and potassium buffering capacity using immunofluorescence and whole-cell voltage clamp, respectively. Mitochondrial morphology was evaluated via TOMM20 staining in retinal tissues. To further probe, isoform-specific effects, rat M¸ller cells (rMC-1) were transfected with human APOE2/ APOE3/ APOE4 or empty vector (EV). In these cells, APOE4ís impact on Kir4.1 and TOMM20 expression, mitochondrial fusion (mitofusin, Mfn1 and Mfn2), fission (dynamin 1, Dnm1) gene expression, and mitochondrial reactive oxygen species (ROS) accumulation (via MitoSox red flow cytometry) were examined. Bioenergetics alterations were evaluated using Seahorse XF analysis to measure oxygen consumption rate (OCR), and extracellular acidification rate (ECAR). The protective effect of the mitochondrial-targeted antioxidant Mitoquinone mesylate (MitoQ) was also evaluated.

Results: APOE4 led to a significant reduction in Kir4.1 expression (p= 0.0019) and ~1.6-fold decrease in Kir4.1 current density (p= 0.0001) compared to APOE3. AQP4 and TOMM20 immunoreactivity were decreased and redistributed in APOE4 retinas. APOE4 expressing rMC-1 showed reduced Kir4.1 at both transcript (by ~40%) and protein levels (~50%), disrupted mitochondrial dynamics, with significantly reduced expression of TOMM20, Mfn1, Mfn2 and Dnm1 genes (all p< 0.05), along with elevated mitochondrial ROS (~2 times higher) as compared to rMC-1 expressing EV/APOE2/APOE3. APOE4-expressing rMC-1 showed 25% reduction in maximal respiration, ~35% reduction in spare respiratory capacity, and ~25% reduction in glycolytic reserve, indicating impaired metabolic flexibility. Treatment with 1µM MitoQ in APOE4-expressing rMC-1 successfully reduced mitochondrial ROS levels by ~50%, restored Kir4.1 expression (~2.5 times increase in mRNA and protein), and restored oxidative metabolism without inducing toxicity.

Conclusions: This is the first study to demonstrate that APOE4 compromises Mller glial function via Kir4.1 and mitochondrial dysfunction, contributing to early retinal pathology in LOAD. These APOE4-induced deficits are pharmacologically reversible using mitochondrial-targeted antioxidant (MitoQ), positioning glial mitochondrial health as a novel therapeutic target in early retinal degeneration in APOE4 carriers and potentially for LOAD management. Ongoing studies using primary MCs from APOE3 and APOE4 mice will assess MitoQ's ability to restore Kir4.1 function via patch clamp, providing further validation in a native glial context.

Utility of Differentiated ARPE-19 Cells as an In-Vitro Model for Age-Related Macular Degeneration

Hussein Abu Qtaish¹, W. Keith Jones², Alissa Araneta¹, Troughton Lee³, Simon Kajasup>3
¹Loyola University, Chicago Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago, ²Department of Molecular Pharmacology and Neuroscience, Loyola University Chicago, ³Department of Ophthalmology, Loyola University Chicago

Purpose: Age-related Macular degeneration (AMD) is a progressive retinal disease that is considered one of the leading causes of vision loss in North America and Europe, especially in the aging population. The progression of AMD is linked to the dysfunction of the retinal pigment epithelial cells (RPE) affecting the retinal-blood-barrier that protects the photoreceptors at the macular region of the retina, leading to the loss of center vision. The aim of this study was to validate the use of the differentiated ARPE-19 cell line as an in vitro model that provides a physiologically relevant model that maintains morphology, redox signaling, cellular adhesion and polarity, and the presence of pigmentation-related gene expression.

Methods: ARPE-19 cells were grown to confluency in DMEM:F12 supplemented with 10% FBS and penicillin/streptomycin (P/S), before switching to the differentiating media (MEM-Nic) comprised of Minimum Essential Medium (MEM) with GlutaMAX™ Supplement, MEM Non-Essential Amino Acids (10 mM), 1% N1 supplement, Taurine (0.25 mg/ml), Hydrocortisone (20 ng/mL), Tri-Iodo-Thyronine (0.013 ng/mL), 10 mM Nicotinamide, 1% FBS, 1% P/S, and maintained for eight weeks. Morphology was evaluated using bright-field microscopy; tyrosinase (TYR) expression was quantified by PCR as a marker for melanogenesis, while expression of the retinoid isomeroohydrolase, RPE65, served as a marker for RPE function. To assess the effects of different oxidative stress inducing conditions on the thioredoxin related pathway linked with the HIF1-α/VEGF-A regulation, Thioredoxin Interacting Protein, (TXNIP) levels at both protein and gene expression levels were evaluated using western blot immunoassay, and RT-qPCR.

Results: The differentiated ARPE-19 cells developed a highly organized monolayer with characteristic polygonal cobblestone morphological features that resembled native RPE cells when examined by bright field microscopy after 8 weeks of supplementing with the MEM-Nic differentiating media. Expression of the melanogenesis gene marker tyrosinase (TYR) and retinoid isomerohydrolase (RPE65) showed a time-course-dependent upregulation throughout the differentiation period and were quantified at 4 (n=3, p<0.001), 6 (n=3, p<0.001) and 8 (n=3, p<0.001) weeks as compared to the undifferentiated (WT) ARPE-19 cell line. Protein expression of thioredoxin interacting protein (TXNIP) was evaluated by western blot immunoassay. WT ARPE-19 cells showed ~40% (n=3, p<0.05) decreased TXNIP expression following exposure to 200 µM tert-butyl hydroperoxide (tBHP). In contrast, TXNIP expression in differentiated ARPE-19 cells was not affected by exposure to different tBHP concentrations (0.1 - 1M) for 24 h.

Conclusions: The study demonstrates differences between wild-type and differentiated ARPE-19 in their response to oxidative stress, highlighting the need for using a physiologically-relevant model to study the contributions of oxidative stress to retinal pathogenesis. Differentiated ARPE-19 cells offer a relevant physiological model with similar genetic and morphological features to native RPE cells, with enhanced redox stability compared to the undifferentiated ARPE-19 cell line. Our ongoing studies are exploring the thioredoxin system and redox-regulatory mechanisms in the RPE aimed at identifying novel therapeutic targets for the treatments of AMD and related diseases.

Beyond Cone Dysfunction: Retinal Metabolism and Hydration in Kv8.2 Knockout Mice

Sheila Baker¹, Bruce Berkowitz², Laird Joseph Laird^1
1University of Iowa, Department of Biochemistry, ²Wayne State University, Detroit, MI

Purpose: KCNV2 retinopathy is a rare inherited retinal disease that causes a decline in cone mediated vision with degeneration of the fovea by young adulthood. About half of the patients also report night blindness. The disease is caused by loss of function mutations in KCNV2, which encodes the Kv8.2 subunit of the rod and cone specific heteromeric Kv2.1/Kv8.2 voltage-gated potassium channel. Kv2.1/Kv8.2 participates in setting the resting dark current and filtering light responses. Kv8.2 KO mice and KCNV2 retinopathy patients have the same unique pattern of abnormal electroretinography (ERG) responses. However, rods instead of cones degenerate in Kv8.2 KO mice. In this study we are using Kv8.2 knockout (KO) mice to explore changes in the retina associated with loss of Kv2.1/Kv8.2 that could trigger photoreceptor degeneration.

Methods: Kv2.1/Kv8.2 channels are found in the inner segment of photoreceptors, adjacent to the subretinal space where transport of nutrients, metabolites, and fluid between the neural retina and RPE is important for photoreceptor function and vitality. We used metabolomic profiling of Kv8.2 KO retina and identified alterations in the methionineñhomocysteine cycle and the levels of organic osmolytes. OCT imaging can be used to measure light-evoked changes in outer retinal hydration in vivo by comparing the distance between the external limiting membrane and retinal pigment epithelium (ELM-RPE) from mice in the dark vs. light.

Results: We found disrupted 1-carbon metabolism in Kv8.2 KO mice suggesting that nucleotide deficiency, epigenetic changes, oxidative stress, and/or osmotic stress are components of the disease pathogenesis. Secondly, Kv8.2 KO mice were found to have reduced light-evoked swelling of the subretinal space indicating that the disrupted potassium efflux from photoreceptors due to loss of Kv2.1/Kv8.2 channels also disrupts fluid homeostasis of the subretinal space.

Conclusions: We propose that alteration of the photoreceptor-subretinal space-RPE microenvironment will need to be addressed in future efforts to treat KCNV2 retinopathy.

Cufflink-Shaped Corneal Plugs for Corneal Perforation Repair: Eye Bank Production and Ex Vivo Evaluation

Evan Balk¹, Luke Grandgenett², Guadalupe Torres¹, Christopher S. Sales^1
1University of Iowa Carver College of Medicine, Department of Ophthalmology and Visual Sciences, University of Iowa, ²University of Iowa, College of Engineering

Purpose: Cyanoacrylate tissue adhesive (CTA) paired with a plastic disc is the standard in-office treatment for corneal perforations but is limited by high reapplication rates, in part due to its reliance on surface adhesive forces alone. To develop a longer-lasting solution, we designed a cufflink-shaped corneal plug that achieves intraocular mechanical anchoring. The plug consists of two opposing leaflets joined by a central stem: the internal leaflet anchors the plug within the eye, the stem fills the defect, and the external leaflet resists inward dislodgement and provides a surface for CTA reinforcement. To enable use in office settings, we evaluated whether an independent eye bank technician could reliably produce plugs using glycerin-preserved tissue and a microkeratome-based cutting system. This study assesses the production accuracy of eye bank-manufactured plugs and compares their sealing strength, measured by burst pressure, to CTA with a plastic disc in an ex vivo perforation model.

Methods: Cufflink-shaped corneal plugs were produced from one-month glycerin-preserved donor corneas by an independent eye bank technician. Plugs were punched to diameter with a biopsy punch, trimmed to 1 mm height, and circumferentially grooved at mid-height to form two opposing leaflets using a standard microkeratome with a novel attachment. Plugs were stored at room temperature for one week and photographed. Dimensional accuracy, including plug diameter and thickness, leaflet thickness, and stem diameter, was measured using ImageJ. Simulated keratolytic perforations of 1 mm or 2 mm diameter were created in donor corneoscleral rims using a Dremel. Plugs 1 mm larger than the perforation were placed ab externo and reinforced with CTA. Control perforations were sealed using CTA and plastic discs sized twice the perforation diameter. Sealed corneas were mounted on an artificial anterior chamber and inflated with air via syringe pump. Burst pressure was recorded using an in-line pressure gauge.

Results: Ten plugs were created by the eye bank technician: five of 2 mm diameter and five of 3 mm diameter. Image analysis after one week of storage revealed asymmetrical leaflet thicknesses; however, the thinner leaflet was consistently near the target value of 0.5 mm, measuring 0.46 ± 0.06 mm for 2 mm plugs (P = 0.2) and 0.55 ± 0.06 mm for 3 mm plugs (P = 0.12). The plug diameters were near their target dimension in the 2mm (1.95 ± 0.07 mm, P = 0.07) and 3mm (2.89 ± 0.13 mm, P = 0.13) plugs. The plug thickness and stem diameter were near target in one but not both plug sizes. In ex vivo burst pressure testing, plugs trended toward higher sealing strength than CTA-disc treatment for both 1 mm perforations (381 ± 108 vs. 318 ± 128 mmHg, P = 0.45) and 2 mm perforations (346 ± 93 vs. 289 ± 90 mmHg, P = 0.35), despite being smaller in diameter than their matched plastic discs. No immediate seal failure was observed with either method.

Conclusions: This study demonstrates that cufflink-shaped corneal plugs can be reliably produced by an eye bank technician using a modified microkeratome, with acceptable dimensional consistency after one week of storage. Despite deviations in leaflet symmetry, all plugs remained within clinically usable tolerances. In ex vivo models, plugs trended toward higher burst pressures than CTA with a plastic disc, suggesting at least equivalence to the current standard of care. The plug’s mechanical anchoring design, combined with shelf-stable glycerin-preserved tissue and sutureless application, supports its potential as a scalable, clinic-ready treatment for corneal perforations. The production method minimizes technician training and integrates with existing eye bank workflows. Together, these findings support further development of a shelf-stable corneal tissue alternative to the current CTA and plastic disc method and lay the groundwork for clinical translation and broader accessibility.

Measuring Adaptive Optics-Corrected Visual Acuity in the Aging Retina

Nathan Balok¹, Mina Gaffney¹, Joseph Kreis^2,3, Zainab Khan¹, Simon Fetherston¹, Joseph Carroll^1
1Medical College of Wisconsin, Department of Ophthalmology & Visual Sciences, Medical College of Wisconsin, ²Joint Department of Biomedical Engineering, Marquette University, ³Medical College of Wisconsin Department of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin

Purpose: There are various age-related changes in the human visual system. Among these are degeneration of cone photoreceptors – however, it is currently unclear if or how the post-receptoral cone circuitry changes with age. Adaptive optics visual acuity (AOVA) tasks offer a method of probing post-receptoral circuitry in vivo. The purpose of this study was to use an AOVA method to assess measures of visual acuity with diffraction-limited stimuli and to examine if these functional measures are associated with differences in age.

Methods: Thirty-two participants (12 male, 20 female) with no reported retinal pathology were recruited for this study. AOVA threshold was measured using adaptive optics scanning laser ophthalmoscopy (AOSLO) and a four-alternative forced-choice tumbling E task. The Bayesian adaptive psychometric QUEST method was used to quantify participants’ AOVA threshold as the logarithm of the minimum angle of resolution (logMAR). Best corrected visual acuity (BCVA) was assessed using a standard ETDRS chart and trial lenses. Axial length was measured with a Zeiss IOLMaster 700 to determine the scale of the retinal image for each participant. A linear regression was performed to determine the correlation between age and AOVA threshold.

Results: The participants’ ages ranged from 15 to 70, with an average age ±SD of 35 ± 14. The average AOVA threshold ± SD was -0.1787 ± 0.1104 logMAR. A linear regression model revealed that AOVA threshold was not significantly correlated with age (R2=0.0384). An increase in age by one year correlated to only a 0.0016 logMAR increase in AOVA, with a population variance of 0.0122, according to this model.

Conclusions: This study is the first exploration of AOVA threshold as a function of age. While these data suggest that AOVA does not significantly change with age, there are several limitations of the study. The primary limitations include a low number of participants and a distribution heavily skewed toward younger ages. In the future, plans include analyzing AOVA thresholds across a wider age range to obtain a better representation of the true population. There are also plans to investigate changes in AOVA threshold as they can correspond to structural changes, especially age-related cone degeneration, in the human retina. In doing so, there will be a better understanding of the association between age and AOVA threshold and a better understanding of the effect of aging on the neural component of vision.

Overcoming host microenvironment challenges in retinal ganglion cell transplantation

Petr Baranov

Schepens Eye Research Institute of Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School

Purpose: Glaucoma is a “silent creeper,” with a significant proportion of patients getting their diagnosis at the late stages of the disease when a structural loss already occurred and a significant portion of host retinal ganglion cells (RGCs) is gone. Available glaucoma treatments reduce intraocular pressure medically or surgically, but it can only slow or halt disease progression. No therapies exist to restore lost visual function and target RGCs directly to improve their survival. Functional replacement of retinal ganglion cells is needed to recover sight lost to Glaucoma and other optic neuropathies. Our roadmap to achieve functional integration of stem cell derived RGCs aims to recapitulate the normal development, which requires alignment of intrinsic and extrinsic signals necessary for donor cell survival, migration, structural and functional integration, dendrite and axon outgrowth, synapse formation and acceptance by innate and adaptive immune system.

Methods: The advanced transcriptomic analysis of human retinal development allowed us to identify several factors in the developing retina microenvironment that are not present in the adult. These include known neurotrophins (GDNF, BDNF), migratory (SDF1, aFGF) and axon guidance cues (Netrin, Slit). By creating the chemokine gradient within the host retina with recombinant SDF1 we were able to significantly improve the integration of donor RGCs following transplantation (2.7-fold) with no adverse effects.

Results: Our transplantation studies in severely immunodeficient NSG mice show that the viable donor neurons are primarily eliminated by host innate immune system. To further investigate this we have performed a timecourse transplantation study in reporter CX3CR1-GFP mice with in vivo imaging, histological and molecular readouts with and without inhibitors of microglia-RGC interaction. We have shown that it is possible to significantly increase donor cell survival (3.4-fold) by inhibiting the microglia-RGC interaction with Annexin V, which led to increased axon outgrowth into the optic nerve head.

Conclusions: Overall, our studies confirm the feasibility of RGC replacement and strongly suggest the use of co-treatments to enable functional integration.

Subretinal gene therapy dosage impacts retinal sensitivity to escalating light intensity

Mackenzie Bengen, Ying Hsu, Brianna Shoenfeld, Tyler Rankin, Jacob Thompson, Arlene V. Drack
Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA

Purpose: Bardet-Biedl syndrome (BBS) is a rare genetic disorder that includes retinal degeneration, obesity, and polydactyly. On a molecular level, BBS is caused by genetic mutations that affect BBSome function, which is a protein complex that is critical for ciliary function. BBS type 10 (BBS10) is a common subtype caused by abnormalities in the BBS10 gene, which mediates proper BBSome assembly. A knockout mouse model of BBS10 (Bbs10-/-) was shown to exhibit a retinal phenotype that replicates disease progression in BBS10 patients. Subretinal gene therapy has shown promising results for delaying vision loss in this mouse model. A novel gene therapy vector, AAV8-RK-hBBS10, was developed with a rhodopsin-kinase promoter to drive gene expression of BB10 in photoreceptor cells specifically. In this study, the efficacy of AAV8-RK-hBBS10 was evaluated by examining Bbs10-/- retinal responses to flashes of light with increasing intensities when treated with different doses of vector.

Methods: One-month-old Bbs10-/- mice were injected subretinally with 2 µl of AAV8-RK-hBBS10 at a quantitative PCR titer of 2.48 x 10^9 vg/eye (low dose), 2.48 x 10^10 vg/eye (medium dose), or 2.48 x 10^11 vg/eye (high dose). At three months post-injection (3MPI), treated mice, untreated Bbs10-/- mice, and uninjected wild-type or heterozygous (WT/HET) mice underwent dark-adapted electroretinography (ERG) testing with light stimuli of escalating intensities. The intensities were 0.001 cd∑s/m2, 0.01 cd∑s/m^2, 0.1 cd∑s/m^2, 1 cd∑s/m^2, 10 cd∑s/m^2, and 30 cd∑s/m^2.

Results: Average b-wave amplitudes of treated Bbs10-/- mice were dose-dependent at all flash intensity levels; untreated eyes had the lowest amplitudes, and eyes that received the highest dose had the highest amplitudes. B-wave amplitudes in eyes that received the high dose were significantly higher (p < 0.0332) than untreated eyes at all intensities except for 0.1 cd∑s/m^2. A plateau was observed in all eyes that received treatment, but occurred at different flash intensities. For the high dose, this plateau occurred at 10 cd∑s/m^2. The medium and low doses plateaued at 0.1 cd∑s/m^2. WT/HET mice did not have an observable plateau. The 0.001 cd∑s/m^2 dim light elicited significantly greater amplitudes in Bbs10-/- eyes receiving the high dose AAV8-RK-hBBS10 compared to those receiving the low dose (p < 0.0332) or those that were not treated (p < 0.0021).

Conclusions: Escalating intensity ERG demonstrates that subretinal gene therapy dosage impacts the light sensitivity of treated eyes. At all light intensities, those eyes receiving the high dose tended to have greater amplitudes. The retinal function in Bbs10-/- mice, both treated with AAV8-RK-hBBS10 and untreated, when exposed to flashes is dosage dependent. This indicates that mice had increased rod functionality at low light levels, depending on the dosage of gene therapy treatment they received. In treated and untreated mice, there is a maximum flash intensity level that elicits an increased response.

A Novel Method to Replace the Multi-Luminance Mobility Test for Testing Functional Vision

Ethan Brandauer¹, Tyler Rankin¹, Mackenzie Bengen¹, Salma Hassan¹, Gianna C. Liu¹, Brianna Schoenfeld¹, Jacob M. Thompson¹, Jean Bennett², Arlene V. Drack^1
1University of Iowa, Department of Ophthalmology and Visual Sciences, University of Iowa, ²Center for Hereditary Retinal Degenerations (CHRD), Scheie Eye Institute, Department of Ophthalmology, University of Pennsylvania

Purpose: Gene therapy offers hope for treating blindness due to Inherited Retinal Diseases (IRDs). One of the clinical trial endpoints for this treatment is the Multi-Luminance Mobility Test (MLMT). In this test, patients must navigate past obstacles to reach the end of a path. This test is excellent for testing functional vision, however the MLMT requires an extensive set up process, trained personnel, and can be costly to maintain.
A new Virtual Reality (VR) headset software was developed to test functional vision in a more convenient manner requiring minimal staff and setup compared to the currently used MLMT. It is also cheaper, as the only required purchase is the headset. This test has potential to be used as an endpoint for future IRD clinical trials.

Methods: This experiment used a Meta Quest 2 headset and the two handheld controllers. This headset ran a proprietary program developed by the Bennett Lab at the University of Pennsylvania. To run this program, an 8ft by 8ft boundary was created in the headset around an unobstructed area so that the participants could walk around safely. To accomplish this, a conference room was used at the University of Iowa Hospitals and Clinics.

Results: Preliminary data was collected from seven members of the Drack lab, all with no uncorrected vision issues. Demographic data including age, sex, height, and prior experience with VR were recorded for each participant.
At the highest luminance level of 4.00 cd, the average time to complete a trial was 25.7 seconds (range: 20.0-41.6 seconds). At 1.52 cd, the average trial completion time was 24.1 seconds (range: 18.3-36.0 seconds). Participants found all objects for both light levels, with the exception of one missed small object by a single participant in one trial at both light levels.
In the 0.58 cd condition, the average completion time was 24.4 seconds (range: 16.9-39.9 seconds). Two participants each missed one small object in a single trial. All other objects were successfully located. At the lowest luminance level of 0.22 cd, the average trial time was 23.9 seconds (range: 18.3-32.1 seconds). One participant missed a small object in one trial; all others found all nine.

Conclusions: Normative data represents the first steps for integrating new techniques into clinical use. These normative values will help establish future guidelines for interpreting results in clinical usage. Future directions include acquiring an IRB to recruit more normative patients as well as testing on patients with IRDs as a way to track functional vision decline or improvement after treatment.

Reproducibility and Inter-Rater Agreement of Human Readers in Nystagmus Waveform Classification

Anthony Chen¹, Kristin Davis¹, Joel Vandelune¹, Veronica Peotta-Jacobsen¹, Fangfang Jiang², Gideon Zamba², Arlene Drack¹, Alina V. Dumitrescu^1
1University of Iowa, Department of Ophthalmology and Visual Sciences, University of Iowa, ²Department of Biostatistics, University of Iowa

Purpose: Nystagmus is a clinical symptom that manifests as abnormal, repetitive, and involuntary eye movements. Nystagmoid eye movements are characterized by their direction, amplitude, frequency, and foveation time (time spent on target). An understudied area of diagnostic testing for nystagmus is waveform analysis, which describes eye recordings of nystagmus based on the types of movement patterns exhibited. There is some preliminary evidence suggesting that clinical characteristics of nystagmus movements may indicate the underlying etiology; however, this remains unproven. A significant limitation of using eye movement descriptions as a diagnostic tool is the subjectivity of eye movement evaluation by clinicians. Video recording of eye movements and analysis of the tracings of recordings are intended to improve the reliability of the analysis. This prospective study aims to analyze the reproducibility between human interpreters of eye movement recordings by measuring inter-rater agreement.

Methods: This prospective cohort study recruited patients who presented to the University of Iowa Ophthalmology Department with nystagmus. Patients underwent a comprehensive workup to diagnose the cause of nystagmus, followed by a video recording of their eye movements. Patients were recorded under 20 different conditions, including various directions of gaze and illumination. Two independent raters assigned waveform classifications to each recording based on waveform morphology while blinded to each other’s assignments. Raters selected from a total of 13 waveform classifications, distinguished based on the patterns of saccades, foveation, and slow-phase movement. Assigned classes were compared and labeled as matches, non-matches, or unmatched. Inter-rater agreement was analyzed using percent agreement, unweighted Cohen’s Kappa and Brier score. Cohen’s Kappa measures agreement after taking into account agreement by chance, and Brier score measures the accuracy of the raters’ classifications.

Results: 44 patients have been enrolled and assigned waveform classes, which produced 943 total comparisons. A high number of conditions were marked as no nystagmus due to the absence of any nystagmoid movement in a certain lighting condition or direction of gaze. A separate exclusion group was analyzed that excluded all no nystagmus classifications to not inflate inter-rater agreement. The percent agreement is 67.0% for all comparisons and 53.0% in the exclusion group. These groups have a Kappa value of 0.569 (95% CI, 0.542–0.597) and 0.464 (95%CI, 0.432–0.495) respectively, which both correspond to moderate agreement. The Brier scores are 0.0645 and 0.0383 respectively, indicating higher accuracy. Further analysis grouped the 13 classes into 6 broader categories based on shared characteristics. This grouping improved inter-rater agreement compared to the exclusion group: the percent agreement is 65.7%, the Kappa value is 0.531 (95%CI, 0.483–0.579) and the Brier score is 0.0611.

Conclusions: This study demonstrates that automated recordings of nystagmus and subsequent waveform classification are possible. The subjectivity and lower reliability of human readers in describing/analyzing the tracings of nystagmus are demonstrated. While some specific waveform classes can be reliably classified, others suffer from significant inter-rater disagreement. Using broader categories helps reduce inter-rater disagreement and is an avenue of further study. A more reliable and objective method using machine learning should be established to classify nystagmus more accurately.

Regulation of rod photoreceptor fate requires DNA demethylation

Brian Clark¹, Ismael Hernández-Núñez¹, Urman Alaina Urman², Xiaodong Zhang¹, William Jacobs², Christy Hoffmann¹, Ellen G Harding¹, Shiming Chen¹, Meelad M Dawlaty³, Philip A Ruzycki¹, John R Edwards^2
1Department of Ophthalmology and Visual Sciences, Washington University, ²Division of Oncology, Washington University, ³Department of Genetics, Albert Einstein College of Medicine

Purpose: Multipotent retinal progenitor cells (RPCs) exhibit dynamic transcriptional and epigenetic transitions that bias cell fate choices across development. We seek to understand the mechanisms by which RPCs are competent to generate the diverse array of retinal neurons (>120 types in mouse). Previous work has implicated the addition of 5 methyl cytosine (5mC) as temporally dynamic across retinal development and as an important regulator of photoreceptor differentiation. However, less is understood about the requirement of active removal (demethylation) of 5mC and the significance of demethylation intermediates (5hmC, 5caC, and 5fC) for retinal development, transcriptional regulation, and retinal cell fate specification.

Methods: Using the Chx10-Cre-GFP transgenic mouse line in combination with floxed conditional alleles for Tet1, Tet2, Tet3, and Tdg, we have generate retinal-specific deletion of the master regulators of DNA demethylation. Using a suite of immunohistochemical, functional (electroretinograms), transcriptional (RNA-seq and single-nucleus RNA-seq), and DNA methylation profiling techniques (Whole Genome Bisulfite Sequencing, bisulfite-assisted APOBEC-coupled epigenetic sequencing, we investigated the functional significance of alterations to the DNA demethylation pathway on retinal development and retinal cell fate specification.

Results: We observe that DNA demethylation and specifically generation of 5hmC is both required for proper retinal development and the specification of rod photoreceptors. Inhibition of DNA demethylation results in failure to induce expression of the master rod photoreceptor transcription factors Nrl and Nr2E3, resulting in all photoreceptors specifying as cones. Using transcriptional and epigenetic profiling, we determine the significance of DNA demethylation for activating rod photoreceptor gene regulatory networks (GRNs). Phenotypic comparisons of the TET and Tdg mutant mice provide insights into the extent to which DNA demethylation must occur to activate rod photoreceptor GRNs.

Conclusions: Our results highlight the functional significance of the DNA demethylation and the TET enzymes for specification of rod photoreceptor fate specification. Furthermore, we determined the prevalence and genomic localization of both 5mC and 5hmC within the mature retina, showcasing the significant prevalence of the DNA modifications and correlation with retinal gene expression. Future work seeks to examine how DNA methyl modifications impact chromatin regulation and transcription factor binding to DNA.

The immunopeptidome of Ad5.MYOCY437H glaucoma model

Jeffrey Dong^1,2, Merri-Grace Jones^1,3, Markus Kuehn^1,2,3
1Department of Ophthalmology and Visual Sciences, University of Iowa, ²Immunology Graduate Program, University of Iowa, ³VA Center for the Prevention and Treatment of Visual Loss

Purpose: Glaucoma is a neurodegenerative disease characterized by damage and subsequent loss of retinal ganglion cells (RGCs), leading to progressive and irreversible vision loss. Previous work in the lab has shown T cells involved in disease progression, however, the autoantigens that lead to their activation is still unclear. The purpose of this study is to investigate if antigen presentation differs in glaucoma and to identify potential autoantigens from RGCs.

Methods: C57BL/6 mice were injected intracamerally with Ad5.MyocY437H to induce glaucoma (n = 7). Naïve (n = 8) and empty vector (n = 8) mice were included as controls. Intraocular pressure was monitored through rebound tonometry weekly. After 4 months, cervical lymph nodes were harvested and affinity purification was performed for MHC-II bound peptides (BioXCell #BE0108). Eluted peptides were identified via LC/MS/MS (University of Iowa Proteomics Core) with a false discovery rate of 0.4%. NetMHCIIpan-4.3 and GibbsCluster-2.0 (DTU Health Tech) were used to determine strong binders and binding motifs correlating with C57BL/6 mice MHC class II I-A respectively. G:Profiler was used to determine terms for Gene Ontology of biological processes. Potential RGC-derived peptides were identified through previous proteomics data on ProteomeXchange (PXD049992).

Results: From an initial pool of 4,711 peptides, the following were identified as strong binders for MHC class II I-A: 673 from the naïve, 631 from the vector, and 798 from the myocilin group. 73/353 shared peptides from the myocilin group had >2-fold increased peptide area compared to naïve. Of these 73 peptides, 25 peptides correspond to 19 proteins expressed by RGCs as found in a previous proteomics study. Some proteins include APOE, SORL1, HSPA5, and VDAC2. The top GO:BP terms include immune system processes, regulation of amyloid-beta formation, and response to stress. Out of 285 unique peptides from the myocilin group, 111 peptides matched 78 potential RGC-derived proteins such as: APOE, SORL1, HSPA2/8 and CLU. Of note, a glycine to asparagine substitution was identified on a SORL1 peptide (SGKPYSPPVPCPVN) only in the myocilin group.

Conclusions: The result from shared peptides showcases potential increased immune response and cellular stress in our myocilin mice. No autoantigens were confirmed at this time, however, potential candidates unique to the myocilin group such as HSPA2/8, CLU, and SORL1 require further examination. SGKPYSPPVPCPVN from SORL1 G719N could be a potential candidate due to the missense substitution to a large polar amino acid. This substitution was only identified in the myocilin group, potentially as a result of mistranslation in stressed RGCs. Further studies are required to test the immunogenicity of candidate peptides involved in disease progression.

Rho kinase inhibitor liposomal therapy for preventing Fuchs endothelial corneal dystrophy progression

Apurva Dusane¹, Hanna Shevalye², Timothy Eggleston², Sanjib Saha¹, Jessica Skeie³, Swathi Kalidindi¹, Cynthia Okafor¹, Gregory Schmidt², Mark A. Greiner^2,3,Aliasger Salem^1
1Pharmaceutical Sciences and Experimental Therapeutics, The University of Iowa College of Pharmacy, ²Iowa Lions Eye Bank, Iowa Lions Eye Bank, ³Department of Ophthalmology and Visual Sciences, The University of Iowa Roy J and Lucille A Carver College of Medicine

Purpose: Fuchs endothelial corneal dystrophy (FECD) is a polygenic disease currently treated with surgery. We aimed to develop eye drops containing rho kinase inhibitors (RKIs) encapsulated in liposomes conjugated with transferrin (TF receptor; upregulated biomarker in FECD) for targeting enhanced uptake by corneal endothelial cells (CECs) in FECD and compare the toxicity and efficacy of two different RKI formulations (ripasudil vs. netarsudil liposomes) to prevent FECD progression.,

Methods: Ripasudil liposomes (RL) and netarsudil liposomes (NL) were synthesized on a microfluidic system using lipids and DSPE-PEG(2000)COOH. EDC-NHS chemistry was used for conjugating DSPE-PEG(2000)COOH to TF. Liposomes size and charge characterization were performed using Zetasizer. HPLC-DAD was used to quantify drug loading and encapsulation efficiency. Safety and efficacy of RL, NL, and their respective sham liposomes were compared after 2 months of once daily treatment by clinical examination using an established FECD mouse model (129S6/SvEvTac Col8a2Q455K, aged 4 months at treatment initiation), followed by postmortem optical coherence tomography (OCT), corneal confocal microscopy (CCM) and blood testing.

Results: RL and NL had sizes of 94.55±3.06 nm and 81.60±2.48 nm, respectively and a narrow polydispersity index (n=3). RKI formulations did not cause ocular or systemic toxicity. OCT for NL treated mice had decreased central corneal thickness (CCT) compared to controls (n=12 each group, P<0.01), whereas CCT for RL treated mice did not differ from controls. CCM revealed significantly fewer guttae for RL and NL groups (n=16 each group) compared to controls (n=8 each group, P<0.0001); no difference in guttae number was found between RL and NL groups.

Conclusions: Both ripasudil and netarsudil liposomes formulated for increased uptake in FECD CECs were non-toxic and prevented guttae formation, while only NL treated mice show decreased CCT. Results of this preclinical mouse model drug safety and efficacy trial support the advancement of RKI liposomal formulations as a strategy to prevent FECD progression.

Evaluation of Cytotoxicity of Checkpoint Inhibitor Immunotherapies on Human Corneal Epithelial Cells

Jacob Espeland, Emily Krupa, Lee Troughton, Simon Kaja
Loyola University Medical Center, Molecular Pharmacology & Neuroscience, Loyola University Chicago

Purpose: Immune checkpoint inhibitors (ICIs) are immunotherapies used to treat various malignancies by specifically blocking programmed cell death protein 1 (PD-1). This binding inhibits the interaction with PD-L1, resulting in an increased T-cell autoimmune response. However, ocular immune-related adverse events (irAEs) have emerged. These primarily encompass corneal toxicities, including dry eye syndrome, keratitis, and corneal ulcers. In contrast, corneal toxicity associated with the class of antibody-drug conjugates (ADC) has been attributed to non-specific toxicity targeting limbal stem cells. The goal of this study is to evaluate the possible direct corneal toxicity of ICIs in human corneal epithelial cells.

Methods: Cytotoxicity of two ICIs, pembrolizumab (P) and nivolumab (N), on human corneal epithelial (HCE-T) cells were compared against anti-human epidermal growth factor 2 (HER2) immunotherapy, trastuzumab (T), and two ADCs, trastuzumab-emtansine (T-DM1) and trastuzumab-deruxtecan (T-DXd). To evaluate cytotoxicity, the cells were exposed to concentrations ranging from 0.5 ng/mL to 100 µg/mL for up to 5 days. Cell viability and proliferation were evaluated using lactate dehydrogenase (LDH) release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake assays.

Results: Pembrolizumab and nivolumab did not exert cytotoxicity across the full dose range and time period, indicated by the absence of changes in LDH release and MTT uptake. Similarly, trastuzumab showed overt cytotoxicity in HCE-T cells across the full dose range and time period. Contrarily, both antibody-drug conjugates (ADCs), T-DM1 and T-DXd, had significantly increased LDH release at 5 µg/mL and 10 µg/mL after 3- and 5-day exposure. signifying dose and time-dependent loss of membrane integrity. T-DXd revealed no significant changes in MTT uptake. Interestingly, T-DM1 increased metabolic activity at 10 µg/mL after 3- and 5-day exposure.

Conclusions: The lack of cytotoxicity of pembrolizumab and nivolumab on human corneal epithelial cells in vitro is consistent with the proposed mechanism of corneal toxicity via upregulation of the host autoimmune response. In contrast, anti-HER2 ADCs causes dose- and time-dependent cytotoxicity in human corneal epithelial cells, likely by non-specific uptake mediated by macropinocytosis. These findings highlight the need for further studies in models that can reproduce immune responses to further investigate the mechanisms of corneal toxicity associated with ICIs.

Identification of variants that modulate transcription of genes associated with ophthalmic diseases

Floyd Evans, Jr.^1,2, Jr. Roos Ben Roos², John H. Fingert^1,2, Todd E. Scheetz^1,2
1Interdisciplinary Graduate Program in Genetics, University of Iowa, ²Department of Ophthalmology and Visual Sciences, University of Iowa

Purpose: For more than 20 years, Genome-Wide Association Studies have revealed genetic loci associated with complex diseases, including vision disorders such as glaucoma and age-related macular degeneration. A limitation of GWASs is that they do not distinguish variants that have a role in increasing disease risk from variants that are merely associated with disease risk due to linkage disequilibrium. Additionally, many variants in genome-wide significant loci do not yield protein-coding changes, increasing the difficulty of understanding the molecular mechanisms used by loci to affect disease risk. Therefore, there are two major questions: (1) Which polymorphisms truly cause complex diseases, and (2) How do these polymorphisms alter disease risk? We and others hypothesize that many genome-wide significant loci alter disease risk by altering gene expression; i.e., by changing the transcription rate of one or more nearby genes. We term these elements expression modulating variants (emVars).

Methods: We have performed a massively parallel reporter assay using Biallelic Targeted Self-Transcribing Active Regulatory Region Sequencing (BiT-STARR-Seq) to identify several putative emVars associated with primary open-angle glaucoma and exfoliation syndrome (XFS). BiT-STARR-Seq uses a plasmid reporter system, in which custom-designed oligos are cloned downstream from a promoter element such that the sequence potentially regulates its own transcription. For a given variant, oligos representing reference and alternate alleles are designed and cloned into constructs. The relative effect of each allele is determined by the relative prevalence of each allele in the RNA from next-generation sequencing.

Results: Using BiT-STARR-Seq, we have performed a comprehensive investigation of identified putative emVars near LOXL1, which is associated with XFS, in HEK-293T and HLE-B3 cells. Several of these emVars are associated with allelic effects on transcription in resources such as the Genotype Tissue Expression (GTEx) Portal. Many of these emVars are also located within predicted transcription factor binding sites, further increasing their potential relevance.

Conclusions: We have identified variations in LOXL1 that may directly modulate risk of developing XFS. Future steps include validating candidate emVars using RT-PCR and computational methods. We predict that for some of the emVars, transcription factors may bind to the surrounding regions. ChIP-Seq and DNase footprinting assay may also be used to confirm that factors are binding to the location of an emVar. The goal of this research is to identify molecular mechanisms (i.e., allele-specific transcriptional regulation) that reveal therapeutic targets to prevent or delay the blindness caused by XFS-related glaucoma.

Automated Culling Strategy for Adaptive Optics Visual Acuity Methods

Simon Fetherston¹, Mina Gaffney^1,2, Joseph Kreis³, Nathan Balok⁴, Zainab Khan⁴, Joseph Carroll^4
1Medical College of Wisconsin, ²Department of Biomedical Engineering, Marquette University, ³Department of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, ⁴Department of Ophthalmology & Visual Sciences, Medical College of Wisconsin

Purpose: Adaptive optics allows for the presentation of diffraction limited stimuli to the human retina, enabling direct measures of visual acuity. Our testing approach utilized QUEST, a Bayesian adaptive model for gathering psychophysical threshold estimates. Subject bias, lapses, and errors early in the experiment can inhibit the ability of QUEST staircases to converge on a reliable estimate of visual acuity threshold. Plots that display uncertain threshold estimates or convergence failure can be manually culled. However, this process is subjective and often takes place post-hoc (preventing acquisition of additional data if needed). The objective of this study was to develop and validate an automatic culling strategy that can be used in real-time.

Methods: A total of 572 sets of QUEST staircase plots were generated using 50 imaging sessions from 47 subjects of various diagnoses. Each QUEST staircase plot included two staircases with 15 trials each. Custom MATLAB scripts were used to extract key parameters from each staircase, including the area between the two plots, the number of reversals, and the MATLAB polyfit slope across the last five trials. Parameter distributions were visualized to assess variability and identify outliers. Outliers were labelled using Tukeyís fences method with a multiplier of three. A subset of 39 QUEST staircases from previous, manually culled data was also analyzed and used for comparison against the automatically culled data. Performance and agreement between the parameters for automatic culling and manual culling were determined using F1 score and Cohenís kappa.

Results: A series of four sequential parameters was determined. Each consecutive parameter was evaluated after staircase plots had been excluded based on the prior parameter. First, individual staircases with five consecutive trials that were either at the minimum or maximum pixel stimulus value indicated a floor or ceiling effect and were excluded. Second, plots with one or fewer combined reversals in the last 10 trials were excluded. Third, individual staircases with a polyfit slope greater than one in the last five trials were excluded. Fourth, plots with an area between staircases greater than 4.5 or greater in the last two trials were excluded. Utilization of these parameters in an automated culling process compared to manual culling resulted in a F1 score of 0.711 and a Cohenís kappa value of 0.673.

Conclusions: The automatic culling method and prior manual culling were found to be in good agreement. This automated culling strategy will enable real-time data analysis that promotes both efficient and high-quality data collection.

Quantifying Sensory Symptoms in Thyroid Eye Disease

Hunter Fischer¹, Chau Pham², Rupin Parikh², Keith Carter², Erin Shriver^2
1Carver College of Medicine, University of Iowa, Iowa City, Iowa, ²Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa,

Purpose: Thyroid eye disease (TED) is an autoimmune inflammatory disorder that causes swelling and enlargement of the extraocular muscles and orbital tissue. Patients with TED report experiencing a variety of sensory symptoms, including, but not limited to, pressure and pain behind the globe and a foreign body sensation in the eye. However, descriptions of these sensory symptoms are limited, and there is a lack of literature describing differences in sensory experiences between men and women with TED. This study aims to evaluate the descriptors patients use to characterize their ocular and periocular sensory symptoms and to determine how these descriptors vary by gender.

Methods: An IRB-approved retrospective review was conducted of all patients with TED who were seen at the UIHC Oculoplastic Surgery Clinic between 1/1/2020 and 12/31/2024. Among these patients, appointments from 1/1/2010 through 12/31/2024 were eligible for review; the first and most recent appointments for each patient were reviewed, as well as appointments before and after completion of a course of oral steroid, methylprednisolone infusions (Kahaly Protocol), or teprotumumab infusions. Patient sensory symptoms reported in the History of Present Illness section were recorded. Data for men and women were compared using Fisher’s exact test, the chi-square test, Welch’s t-test, and Poisson rate ratio testing where applicable.

Results: Of the 798 patients initially screened, 631 were eligible for analysis, and 1,412 appointments were reviewed. Patients used 57 unique descriptors, and pain with eye movement (115 patients), pain behind the globe (53), pressure behind the globe (41), generalized headaches (40), and surface grittiness (39) were the most frequently expressed. The percentage of symptomatic men (85%, 145/170) and women (90%, 417/461) was similar (p = 0.09). However, women reported a significantly greater mean number of symptoms than men (2.83 vs. 2.45, p = 0.02). Among symptomatic men (n = 145) and women (n = 417), a significantly higher percentage of women reported pain around the orbit (p = 0.01), pressure within the globe (p = 0.02), and generalized achiness (p = 0.02). When descriptors were clustered by location, significantly larger percentages of women experienced generalized symptoms (p = 0.02), symptoms around the orbit (p = 0.01), within the globe (p = 0.02), and behind the globe (p = 0.02).

Conclusions: Women report experiencing a greater mean number of sensory symptoms and have higher rates of pain around the orbit, pressure within the globe, and generalized achiness of the eyes than men. When grouped by common location, women also experience higher rates of generalized symptoms, symptoms within the globe, behind the globe, and around the orbit. Future research is necessary regarding the symptom predictivity of more active or severe disease, symptom burden in relation to disease duration and treatment, and whether noted sensory symptom differences result from variable reporting by men and women.

Surgical Management Patterns of Iris Lesions Suspicious for Melanoma at a Tertiary Referral Center

Kaitlyn Grimes¹, Danielle Pellack¹, Wang Jui-Kai Wang², Noriyoshi Takahashi², Elaine Binkley¹, Andrew Pouw^1
1University of Iowa Carver College of Medicine, University of Iowa Carver College of Medicine, ²University of Texas Southwestern Medical Center Department of Ophthalmology, University of Texas Southwestern Medical Center Department of Ophthalmology

Purpose: To evaluate the incidence of iris lesions and characterize clinical decision-making, surveillance duration, and timing of surgical interventions for lesions suspicious for melanoma at a tertiary care center.

Methods: This was a retrospective, IRB-approved study of 280 eyes of patients referred to the University of Iowa for iris lesions suspicious for melanoma. Kaplan-Meier analysis was applied to estimate time from initial evaluation to time of surgical intervention.

Results: 280 eyes were included in the analysis. 44 (16%) had surgical intervention, while the majority (84%) were managed with observation alone. Among the 44 eyes that had intervention performed, interventions included iridocyclectomy (59%), plaque brachytherapy (16%), and enucleation (11%).

Conclusions: At this tertiary academic referral center, the majority of iris lesions suspicious enough for surgical intervention were treated with iridocyclectomy. Most surgical interventions were recommended within the first two years of surveillance.

Extracting Semi-Quantitative Measures of Near-Infrared Autofluorescence

Zachary Hahn, Aliya Siddiqui, Amber Irons², Katie McKenney², Jenna Grieshop², Janet R. Sparrow³, Joseph Carroll^2
1School of Medicine, Medical College of Wisconsin, Milwaukee, WI, 53226, USA Department of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI, 53226, ²Department of Ophthalmology & Visual Sciences, Medical College of Wisconsin, Milwaukee, WI, 53226, ³Department of Ophthalmology, Columbia University Medical Center, New York, New York

Purpose: Retinal pigment epithelium (RPE) melanin serves a photoprotective role by absorbing light and scavenging free radicals, supplementing the RPEís role as metabolic support for photoreceptors, waste management, and absorption of extra scattered light. RPE melanin has the potential to serve as an important marker of retinal health, development, or retinal function. Near-infrared autofluorescence (NIR-AF) is a non-invasive, in vivo imaging modality that is hypothesized to capture the RPE melanin signal. Current use of NIR-AF imaging in clinical and research settings is limited mostly to qualitative analyses due to the lack of validated quantitative analysis methods. Here we sought to reproduce the semi-quantitative analysis method described by Paavo et al. (2018) using a new user-friendly application.

Methods: 104 individuals (72F, 32M) with no self-reported vision-limiting pathology were recruited. Following pupillary dilation, NIR-AF images (30∞ x 30∞ field) were obtained using a Heidelberg HRA2-SLO device (787 nm excitation, >830 nm emission). Images from one eye from each subject were analyzed (53 OD, 51 OS). If images from both eyes were available (n=17), the study eye was chosen at random (n=13) unless image quality from one eye greatly outweighed the other (n=3). These non-normalized NIR-AF images were analyzed twice by a single observer using a custom MATLAB application in which the observer manually identified the foveal center. Once identified, the application extracted gray level (GL) intensities at 0.25 mm increments from the fovea outward in both directions along the horizontal meridian. OS data was flipped to match the orientation of OD data for further analyses.

Results: Compared to the results of Paavo et al. (2018), we observed a similar GL topography but higher overall GLs across the retina. Our observed mean +/- SD GL at the fovea was 45.06 +/- 15.76 arbitrary units (AU). In comparison, the mean GL at the fovea reported by Paavo et al. (2018) from 15 normative subjects was 38.16 AU (sd 3.26). This was not significantly different (p=0.095). The two repeated measures showed an ICC of 0.992, though there was a small but significant difference in mean foveal GL values (45.34 +/- 15.94 AU vs 44.80 +/- 15.65 AU, p=0.0058). A Bland-Altman analysis revealed a mean bias of -0.54 (95% CI: -0.16 to -0.93). Additionally, females had a higher GL compared to males (47.25 +/- 15.48 AU vs 40.15 +/- 13.39, p=0.025). It is worth noting that males had a higher mean axial length compared to females (24.50 +/- 1.18 mm vs 23.86 +/- 1.02 mm, p=0.0066).

Conclusions: The goal of this study was to reproduce and improve the methods described by Paavo et al. (2018) for analyzing NIR-AF images. The average GL values reported here follow a similar topography as reported previously, but our absolute values were slightly higher on average. Future studies are needed to determine if these differences are due to the device used, differences in participant age, ethnicity, axial length, or other factors. The open-source MATLAB application can facilitate the use of NIR-AF images to assess retinal melanin for both clinical and research applications. Further work is needed to assess inter-grader reproducibility and intra-grader repeatability.

Assembling the Visual Pathway In Vitro

Michael Hayes^1,2, Maria Fernanda Valdes Michel^2,3, Kardon Randy Kardon^1,2, Oliver Gramlich^2,3
1University of Iowa, Department of Ophthalmology, ²VA Center for the Prevention and Treatment of Visual Loss, ³University of Alabama Birmingham

Purpose: Visual pathway defects caused by neurodegenerative diseases, such as multiple sclerosis (MS) and glaucoma, are difficult to study in vitro owing to the lack of critical cell types and functional neurocircuits. To address this gap, we tested the hypothesis that a functional visual pathway could be assembled in vitro using human pluripotent stem cell (PSC) derived retinal and brain region specific organoids. An ideal microphysiologic system would contain retinal ganglion cells (RGCs), oligodendrocytes, astrocytes, and neurons; functional synapses; and include tissue from the retinal, thalamus, and visual cortex.

Methods: Human retinal and brain region organoids were generated from healthy donor PSCs and assayed as individual organoids or assembled into 3D assemblies. Reporter constructs were introduced into PSC lines or organoids via viral transduction for real-time analysis of cell number, function, axon migration, and tissue morphology. Real-time analysis was supplemented with endpoint analysis.

Results: Retinal and brain region specific organoids were successfully generated and assembled into 3D microphysiologic models of the visual pathway. RGCs appeared within retinal organoids by differentiation day 21. When induced, oligodendrocyte precursors were observed by differentiation day 70 and were successfully matured into actively myelinating oligodendrocytes. 3D assemblies of retinal and brain region organoids were successfully generated and demonstrated unidirectional axonal migration from retinal to brain tissues, up to 300 microns per day.

Conclusions: By including brain region specific tissues, we show that assembloids are better suited for modeling vision effecting diseases that originate or extend beyond the eye. Furthermore, the incorporation of real-time and functional assays represent novel and essential tools to better understand the etiology and pathophysiology of neurodegenerative of the visual system. In conclusion, we present our first steps to establishment of an oligodendrocyte containing in vitro model of the visual pathway.

Establishing an Open-Source Normative Database with the Iowa VR Headset Perimeter

Joseph Heyrman^1,2, Edward Linton², Wall Michael Wall², Ivan MarÌn Franch^3
1Carver College of Medicine, University of Iowa, ²Department of Ophthalmology and Visual Sciences, University of Iowa, ³Computational Optometry, Atarfe, Granada, Spain

Purpose: A lack of comprehensive normative databases for virtual reality (VR) perimetry devices limits their clinical adoption despite its potential as a portable, cost-effective alternative to traditional devices like the Octopus 900 (O900). This study aimed to establish a normative database for the Iowa VR head-mounted display (HMD) perimeter using luminance-modulation (LMS) and area-modulation (AMS) strategies and to pilot its ability to detect glaucomatous visual field deficits compared to the gold standard O900.

Methods: 60 healthy observers took two tests each in three paradigms: LMS on the O900, LMS on the HMD, and AMS on the HMD in random order. All tests used a custom grid spanning the central 26∞, ZEST (Zippy Estimation by Sequential Testing) algorithm, run on the Open Perimetry Interface. LMS stimulus diameter was 1.72∞, AMS stimulus luminance was 5cdm-2 against 10 cdm-2 background. Mean sensitivity and pointwise repeatability coefficients (RC) and pointwise 95 percentile confidence limits were calculated. Signal-to-Noise ratio (SNR) was defined as the difference between peripheral and central sensitivities (signal) divided by pointwise variability (noise). Counts of abnormal locations using percentile cutoffs were compared by ANOVA in 7 glaucoma subjects.

Results: The mean sensitivity and pointwise RCs were 1.20 and 3.19 for the O900 LMS, 1.44 and 3.20 for the HMD LMS, and 2.78 and 5.37 for the HMD AMS, respectively. Bland-Altman plots show a wider useful dynamic range for AMS. SNR ratio was greatest for AMS on the HMD (0.79 for HMD-AMS vs. 0.31 for HMD-LMS and 0.45 for SAP), as AMS had greater magnitude of both signal and noise. The number of abnormal test locations detected by each modality was not significantly different between modalities at the 10th, 5th, 2.5th or 1st percentile (p=0.17 by 2-way ANOVA).

Conclusions: This study established a normative database for the open-source Iowa HMD perimeter, enabling age-correction and probability estimates necessary for clinical use. While HMD AMS had a higher pointwise variability, it showed the best SNR. Finally, the HMD had a similar ability to the O900 to detect abnormal test locations with both testing strategies.

Utility of blood sampling in detecting toxicity associated with ocular gene therapy

Ying Hsu, Brianna Shoenfeld, Jacob Michaelson, Arlene Drack
Department of Ophthalmology and Visual Sciences, University of Iowa

Purpose: Ocular gene therapies can halt vision loss in heritable blinding disorders. However, host immune response against the administration of a gene therapy vector is unpredictable, often resulting in severe side effects in a subset of individuals. Currently, there is no method to predict which individuals would be adversely affected by side effects from gene therapy treatment a priori. Development of blood-based technologies and biomarkers has immense clinical relevance. A pilot study was conducted in mice to examine the utility of blood-based detection technologies.

Methods: We examined the existence of informative biomarkers in blood before onset of ocular toxicity events after receiving an intraocular injection of a gene therapy vector. A vector at a dose previously determined to induce toxicity (retinal atrophy) in a subset of mice, namely 2.5 x 10¹¹ viral genomes (vg) per eye, was used. This gene therapy vector consists of the human Bardet-Biedl Syndrome 10 (BBS10) gene under the control of the chicken ?-actin (CAG) promoter, encapsulated by an adeno-associated virus type 8 (AAV8) viral capsid. After intraocular injection in unaffected 129S6/SvEvTac mice, blood was collected by submandibular puncture at 1- or 9-day post injection for cytokine analysis and bulk RNA sequencing, respectively. A quantitative measure of toxicity, retinal atrophy, was measured using optical coherence tomography (OCT) images acquired. Analysis of RNA sequencing was performed in RStudio.

Results: Two cytokines were upregulated in serum of mice receiving an intraocular gene therapy vector challenge as early as 1 day post injection. Three of these cytokine biomarkers assayed at 1 day post strongly correlated with degrees of toxicity (atrophy) at 1 month post injection, indicating prognostic biomarkers exist. To estimate proportions of specific immune cells in blood at 9 days post injection, deconvolution of bulk RNAseq data showed that T cells likely expanded in population after receiving the vector, but not after receiving buffer.

Conclusions: Preliminary data shows that blood contains informative biomarkers as soon as one day after the intraocular administration of a gene therapy vector. Furthermore, deconvolution of RNA sequencing data estimates changes in immune cell populations following the subretinal administration of a viral vector. Future work entails validation of immune cell populations using flow cytometry. This pilot study suggests that blood samples contain prognostic markers for toxicity in ocular gene therapies.

Presence of CD3+ T cells in the proximal optic nerve in glaucomatous neurodegeneration

Brandon Hu¹, Jordan Mayberry^1,2, Markus H.Kuehn^1,2
1Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, IA 52242, ²Iowa City VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA, 52246

Purpose: In glaucoma, T cells have been shown to infiltrate into the retina and mediate retinal ganglion cell (RGC) loss. However, it has yet to be determined whether these T cells also migrate to the optic nerve (ON) to influence axonal degeneration. This study sought to determine if T cells are present in the optic nerve and where they are localized during glaucoma pathology.

Methods: Elevated intraocular pressure (IOP) was induced into one group of mice using an intracameral injection of 9x107 PFU of Ad5.MyocY437H (n=9). Control mice did not receive this injection (n=9). IOP was then measured for 3 months using rebound tonometry. At the conclusion of the experiment, mice were sacrificed, and eyes were collected and fixed in 4% paraformaldehyde. The eyes were then dissected and embedded in OCT media. Sagittal sections of the retina and optic nerve were taken at 10 µm and then subjected to immunohistochemical stain against CD3, a T cell marker, and visualized using fluorescence microscopy.

Results: As expected, intracameral injections of Ad5.Myoc resulted in elevated IOP in experimental mice. However, IOP remains unchanged in control mice. Elevated IOP results in a slight increase in CD3+ T cell abundance in the ON (n=3/group, p = 0.09).

Conclusions: These results provide evidence that T cells may migrate into the ON to influence axonal degeneration as an adaptive immune response during glaucoma pathogenesis.

Effects of Rho Kinase Inhibitors on Corneal Membrane Permeability

Baraa Hussein, Troughton Lee Troughton, Simon Kaja
¹Loyola University Chicago, Department of Ophthalmology

Purpose: The goal of this project was to investigate the effects of Rho-associated protein kinase (ROCK) inhibitors on corneal permeability. Enhancing corneal permeability can improve the delivery of ophthalmic drugs. Repurposing FDA-approved drugs that enhance corneal permeability could pave the way for novel combination therapies that reduce the need for invasive interventions and improve both patient outcomes and adherence.

Methods: Human corneal epithelial cells (HCE-T) were seeded in 12 mm transwells with 0.4 µm pore polycarbonate membranes (cellQART®) and cultured for 1 week in Dulbecco’s modified Eagle’s medium (supplemented with 5% fetal bovine serum, 0.5% dimethyl sulfoxide, 5 mg/mL insulin, 10 ng/mL human recombinant epidermal growth factor and 100 U/mL penicillin–100 µg/mL streptomycin; 0.3 mL donor, 1 mL receiver chambers). Cells were exposed to 100 µM fluorophores: 6-carboxyfluorescein (6-CF; 376 Da, paracellular), Dextran-Texas Red (DTR; 10 kDa, paracellular), Rhodamine B (479 Da, transcellular) and AlexaFluorTM488-conjugated immunoglobulin G (IgG; 150 kDa, low permeability). They were added to the donor chamber in 200 µL Hank’s buffered saline solution (HBSS) with either 320 µM netarsudil or 3% DMSO. Receiver chambers held 1 mL HBSS. 100 µL samples were collected every 10 min for 1 h. Fluorescence was measured with Cytation 5 plate reader (Agilent) and the apparent permeability coefficient calculated.

Results: Permeability was normalized to DMSO. Netarsudil treatment caused an increase in the apparent permeability coefficient (Papp) for 6-CF by greater than 7-fold (n=7, p<0.05). Similarly, DTR permeability increased by approximately 3-fold (n=6, p<0,05), consistent with compromised barrier function. Permeability to Alexa Fluor 488-conjugated IgG also increased by greater than 10-fold (n=6, p<0.05). In contrast, Rhodamine B permeability decreased by 35% (n=7, p<0.05) following treatment

Conclusions: Netarsudil increased permeability across a corneal epithelial monolayer by disrupting barrier integrity. This effect was observed with 6-CF and DTR, which primarily traverse the epithelium by paracellular transport, and with IgG, likely limited due to its size. Interestingly, netarsudil decreased RhoB permeability, which is typically transported by transcellular transport. These results support the hypothesis that netarsudil alters tight junction function, possibly by reorganizing intracellular actin dynamics. Together, our findings suggest that ROCK inhibition may be used as a tool to enhance corneal drug delivery. Ongoing and future studies will assess stratified barrier function, ZO-1 localization, TEER kinetics, and permeability to clinically relevant ophthalmic agents.

Preoperative Hypoglycemia as a Predictor of Postoperative Vitreous Hemorrhage in PDR Patients Undergoing Vitrectomy

Imran Hussain¹, Frank Bogan¹, Ali Elobous², Amir Hajrasouliha^1
1Indiana University School of Medicine, Department of Ophthalmology, ²Department of Ophthalmology, UNC School of Medicine

Purpose: This study evaluated the association between preoperative glycemic status—specifically fasting hypoglycemia—and postoperative vitreous hemorrhage (POVH) incidence and visual outcomes in patients with proliferative diabetic retinopathy (PDR) undergoing pars plana vitrectomy (PPV). While much attention has been given to hyperglycemia and its impact on post-op complications, hypoglycemia is an often-underappreciated potential risk factor.

Methods: Medical records of 144 PDR patients who underwent PPV between 2020-2024 were reviewed. Preoperative fasting blood glucose levels were recorded, and patients were categorized as hypoglycemic (<70 mg/dL), normoglycemic (70–125 mg/dL), or hyperglycemic (>125 mg/dL). Postoperative vitreous hemorrhage was assessed within three months. Visual acuity (VA), measured in LogMAR, was collected preoperatively and at Postoperative Day 1 (POD1), Month 1, and Month 3. Additional demographic, systemic, and ocular variables were collected. Statistical analyses included t-tests, chi-square tests, and ANOVA to evaluate associations between glycemic status, POVH incidence, and Visual Acuity outcomes.

Results: Preoperative hypoglycemia was not significantly associated with POVH, though a trend toward increased incidence was noted. Conversely, hyperglycemia was significantly associated with higher POVH rates within three months (P=0.009**). In terms of VA outcomes, hypoglycemic patients demonstrated significant postoperative worsening at Month 1 (P=0.0001***) and Month 3 (P=0.001***). Hyperglycemic patients also experienced significantly worse VA at POD1 (P=0.04*), Month 1 (P=0.01**), and Month 3 (P=0.04*) compared to normoglycemic patients. Normoglycemic patients exhibited the most favorable recovery in visual acuity across all timepoints.

Conclusions: Both preoperative hyperglycemia and hypoglycemia were associated with worse postoperative outcomes in PDR patients undergoing PPV. While hyperglycemia increased the risk of early POVH, hypoglycemia was linked to poorer visual outcomes. These findings underscore the clinical importance of perioperative glycemic control to potentially improve visual and surgical outcomes in this high-risk population.

Glaucomatous insults result in molecular signatures of reactive astrocytosis and elastinopathy in optic nerve head astrocytes

Simon Kaja

Department of Ophthalmology, Loyola University Chicago, Maywood, IL 60153

Purpose:Glaucomatous optic neuropathy is the leading cause of irreversible blindness worldwide. Biochemical, biomechanical and bioenergetic changes have all been implicated in the pathophysiology of glaucoma. Here, we sought to investigate the effects of multiple disease-relevant stimuli on reactive astrocytosis in primary optic nerve head astrocytes (ONHA).

Methods: Primary rat ONHA were prepared from adult Brown Norway rats. For some experiments, ONHA were transduced with lentivirus expressing the EDN1 gene to generate endothelin-1 (ET-1) overexpressing cultures, or transfected with siRNA targeting lysyl oxidase like-1 (Loxl1) to generate Loxl1-deficient ONHA. Cultures were exposed to 10% static equibiaxial strain in a FlexCell® (FlexCell International) chamber for 16 h. Actin cytoskeleton was labeled with AlexaFluor® 488 phallodin and AlexaFluor® 594 DNAse I for F-actin and G-actin, respectively. Lengths of stress fibers was quantified using Matlab software (MathWorks, Natick, MA). Sensitivity to exogenously-applied oxidative stress was quantified by MTT uptake and lactate dehydrogenase (LDH) release assays. Quantitative PCR, immunoblotting and immunocytochemistry were performed using previously validated primers and antibodies to quantify molecular phenotypes of reactive astrocytosis and elastinopathy.

Results: Mechanical strain, ET-1 overexpression and Loxl1 deficiency all resulted in reactive astrocytosis, as assessed by increases in glial fibrillary acidic protein (GFAP). Reactive astrocytosis resulted in similar changes to elastin expression in response to different glaucomatous insults. Specifically, mechanical strain resulted in a decrease in stress fiber length with a concomitant increase in G-actin immunoreactivity, suggestive of reactive astrocytosis. Furthermore, mechanical strain resulted in a significant decrease in Loxl1 (35% reduction, P < 0.001) and elastin (65% reduction, P < 0.05) expression. ET-1 overexpressing ONHA exhibited a less-differentiated morphology and significantly increased doubling times, compared with control or non-transduced ONHA. Gene expression changes were similar to those observed in response to mechanical strain for LOXL1 (48% reduction, n=3; p<0.05) and elastin (62% reduction; n=3; p<0.05). Loxl1-deficient cultures showed ~50% reduction in Loxl1, concomitant with a significant reduction in elastin expression (32% reduction, P < 0.01).

Conclusions: Our results indicate that reactive astrocytosis in ONHA is associated with molecular signatures of elastinopathy. Notably, elastinopathy phenotypes are similar between different glaucomatous insults, suggesting a unifying molecular pathway eliciting reactive astrocytosis in glaucoma.

Voted Outstanding Oral Presentation

Transferrin receptor targeted Ubiquinol-loaded PLGA nanoparticles for Fuchs corneal endothelial dystrophy (FECD)

Swathi Kalidindi¹, Jessica M Skeie^2,3, Ibrahim Youssef Wahib Naguib Ibrahim¹, Gregory A Schmidt^2,3, Aliasger K. Salem¹, Mark A. Greiner^2,3
1Department of Pharmaceutical Sciences and Experimental Therapeutics, University of Iowa, Iowa City, IA 52242, ²Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City,IA 52242, ³Iowa Lions Eye Bank, Coralville, IA 52241

Purpose: FECD is an age-related, polygenic disease impacting vision in approximately 6.1 million Americans. It is the leading indication for corneal transplant surgeries in the U.S. FECD is characterized by progressive CEC loss and the formation of guttae, ultimately compromising CEC pump function and resulting in corneal swelling. Our group's prior work identified an impaired response to oxidative stress as central to FECD, specifically noting constitutive increases in cytosolic Fe2+and lipid peroxidation coupled with decreased GPX4 activity, strongly implicating ferroptosis in FECD pathogenesis. We also observed an upregulation of a key ferroptosis biomarker transferrin receptor 1 (TFR1). Our studies showed that solubilized ubiquinol effectively suppresses ferroptosis in FECD cell cultures. Recognizing the limitations of current surgical approaches, we aimed to develop a novel, non-surgical therapeutic strategy using targeted nanomedicine to deliver ubiquinol to affected CECs and prevent FECD

Methods: To achieve targeted delivery to CECs, we engineered Transferrin-conjugated ubiquinol-loaded Poly (lactic-co-glycolic acid) (PLGA) nanoparticles, designed to leverage TFR1-mediated endocytosis for enhanced cellular uptake. We characterized these nanoparticles for size, polydispersity index (PDI) and surface charge using a Zetasizer, and quantified drug loading and encapsulation efficiency via HPLC. To assess cellular uptake, cultured immortalized FECD human CECs (F35T) were incubated for 4 hours with Rhodamine-B-loaded nanoparticles conjugated with varying amounts of Transferrin (100 µg, 300 µg, and 500 µg) with uptake measured by flow cytometry. Uptake of ubiquinol-loaded nanoparticles with these same transferrin conjugations was also quantified by HPLC.

Results: Our characterization confirmed successful transferrin conjugation, evidenced by an increase in nanoparticle size with increasing transferrin amounts compared to non-targeted nanoparticles (137.7 ± 1.13 nm). Specifically, targeted nanoparticles showed sizes of 207.1 ± 3.79 nm, 214.43 ± 4.93 nm, and 218.03 ± 4.69 nm for 100 µg, 300 µg, and 500 µg transferrin, respectively, indicating surface coating. The zeta potential became less negative with increased Transferrin, further confirming successful surface modification by the protein. A clear dose-dependent enhancement of cellular uptake was observed with increasing transferrin conjugation. Nanoparticles conjugated with 300 µg and 500 µg of Transferrin demonstrated a statistically significant increase in ubiquinol uptake compared to non-conjugated nanoparticles.

Conclusions: We successfully synthesized Transferrin receptor targeted ubiquinol-loaded PLGA nanoparticles and demonstrate that this targeted approach significantly boosts drug delivery to cells. This system, which takes advantage of the increased TFR1 in diseased cells, offers a promising method for enhancing drug uptake by CECs and marks a crucial step forward for FECD treatment. By directly delivering an agent that combats ferroptosis, this novel nanomedicine pharmacotherapeutic provides a compelling strategy to slow FECD progression and address a major unmet patient need.

Orexin Neuropeptides and Their Receptors Are Expressed in Retinal Müller Glia

Connor Kaurich¹, Rupinder Kaur Sodhi¹, Ashay D Bhatwadekar^1
1Department of Ophthalmology, Indiana University School of Medicine, Indianapolis, Indiana

Purpose: Müller glial cells span the entire thickness of the retina and are essential for retinal homeostasis by buffering ions, water, and neurotransmitters. Dysfunction and gliosis of Müller glia are implicated in retinal disorders such as diabetic retinopathy and retinal degeneration. Orexins A/B, known hypothalamic regulators, are detected in rodent retinas and contribute to signal transduction. Additionally, prior studies in the Bhatwadekar lab with orexin antagonist treatment in diabetic mice showed decreased inflammatory factors compared to diabetic controls. As key regulators of retinal health, Müller glia may reveal novel gliosis and stress pathways through expression of orexin or its receptors. This study examines the expression of orexins and their receptors in rat Müller cells (rMC-1) and mouse tissue using immunofluorescence techniques.

Methods: Rat Müller cells (rMC-1) were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum. Cells were seeded in 8-well chamber slides and stained for OXA, OXB, OXR1, and OXR2 (1:100 concentration). Alexa Fluor 555 Goat Anti-Rabbit (1:500) was used as a secondary antibody. Mouse retinas were isolated, fixed in 4% PFA, and processed for paraffin sectioning. Glutamine synthetase (1:200) and OXR1(1:100) were used as primary antibodies, while Alexa Fluor 555 Goat Anti‐Mouse and Alexa Fluor 488 Goat Anti‐Rabbit (both 1:500) were used as secondary antibodies. Both cells and sections were mounted with Vectashield anti-fade mounting media with DAPI (Vector Labs) and visualized using a Zeiss confocal microscope.

Results: We found cytoplasmic expression of orexins (OXA and OXB) and membrane expression of OXR1 and OXR2 in rMCs. OXR1 co-localized with Müller cells in mouse sections, while OXA and OXB did not.

Conclusions: Here, we provide early evidence of orexin and orexin receptor expression in rMCs. We also found that OX1R co-localizes with mouse Müller cells. Additional in vitro and in vivo studies can be conducted in gliotic models to investigate how orexin signaling impacts Müller cells.

Comparing Spacing-Derived and Direct-Count Measurements of Rod Density in Patients with Congenital Achromatopsia

Muhammad Khan¹, Aliya Siddiqui¹, Natalie Ungaretti¹, Christopher Langlo¹, Joseph Carroll^1
1Medical College of Wisconsin, Department of Cell Biology, Neurobiology & Anatomy

Purpose: Achromatopsia (ACHM) is a retinal disorder associated with severely reduced or absent cone photoreceptor function. Symptoms include nystagmus, photophobia, and poor visual acuity. Previous work using adaptive optics scanning light ophthalmoscopy (AOSLO) has revealed highly variable numbers of remnant non-functional cones in patients with ACHM. While the rod mosaic can be visualized in these AOSLO images, there has been no systematic quantification of rod density in individuals with ACHM. The purpose of this study was to assess the accuracy of a recently reported spacing-derived method for estimating rod density in patients with ACHM.

Methods: A total of 10 patients (5 males, 5 females, age range = 10-41 years) with CNGB3- or CNGA3-associated ACHM and previous AOSLO imaging were selected for analysis. For each patient, two 150x150µm regions of interest (ROIs) were extracted from both confocal and non-confocal imaging modalities for a total of 20 ROI pairs. Cones and rods were manually counted from non-confocal and confocal images, respectively. These coordinates were used to compute direct-count estimates of both rod and cone density. For the same ROI, 20-40 rods were manually counted to estimate near-neighbor distance (NND) and cone inner segment area was measured using the non-confocal images; these values were used to extract a spacing-derived estimate of rod density for each ROI. A Bland-Altman analysis was used to evaluate agreement between the direct rod count and the spacing-derived method.

Results: The mean (+/- SD) rod density across all 20 ROIs was 58,975 +/- 9,984 rods/mm² using direct counts and 55,463 +/- 10,528 rods/mm² using the spacing-derived estimates. Across the 20 ROIs analyzed, the spacing-derived method showed strong agreement with direct rod counts, with a mean difference of 2,512 rods/mm² (4.3%). The 95% limits of agreement were -26,492 to 31,517 rods/mm², which is comparable to previous results in normal photoreceptor mosaics. Rod-to-cone ratio was 10.93 +/- 5.18 and 10.41 +/- 5.21 in direct and spacing-derived counts, respectively.

Conclusions: The results from this study suggest that the previously reported spacing-derived method can yield accurate estimates of rod density in the ACHM retina. While there was a statistically significant difference between rod density estimates using the spacing-derived method and the direct count method, the mean bias was less than 5%. Future work will expand this spacing derived method to a larger number of patients and ROIs to determine whether rod density is altered in ACHM. In addition, monitoring rod density in patients enrolled in experimental treatment trials could serve as a sensitive safety biomarker.

Primary cila and aqueous humor outflow regulation

Krishnakumar Kizhatil^1
1The Ohio State University Medical Center, Department of Ophthalmology and Visual Sciences

Purpose: The Schlemm's canal (SC) inner wall endothelial cells (SCE) are the last barrier to the outflow of aqueous humor (AQH). SCE mechanosensor(s) detect IOP elevation-induced shear stress and stretch to increase the permeability of the SC inner wall, increasing AQH outflow. The primary cilium is a solitary cellular extension that protrudes from the cell body into the extracellular environment and thus is ideally positioned to act as a cellular sensory structure. Primary cilia have recently been shown to play a role in mechanotransduction in the kidney and osteoclasts.We determined if primary cilia are present on mouse SCE and function as a mechanosensor in these cells in controlling AQH outflow.

Methods: We immunostained SC with ARL13B to identify cilia. We then quantified the morphological features of SC primary cilia. We then identified candidate proteins implicated in mechanotransduction in SC cilia using immunofluorescence. We also determined the physiological role of SC cilia by disrupting cilia in the adult mouse SC by knocking out the gene encoding the intraflagellar transport 88 protein (IFT88) using cre-loxP technology using mice bearing an SCE-specific tamoxifen inducible cre (Prox1-cre ERT2) and the ift88 floxed allele (ift88 ECKO or ift88 EChet). We also evaluated ift88 mice lacking cre as controls. We visualized cilia in ift88 EChet and ift88 ECKO mice. We then measured the facility to determine if aqueous humor outflow was affected in the homozygous knockout mice compared to the heterozygous mice.

Results: Our results show that the Schlemm’s canal inner wall cells have primary cilia. On average, the SC cilia were 1.19µm long, 4.7µm3 in volume, and slightly bent with an average bending index of 1.15 (n=1358 cilia). The cilia expressed ARL13B, IFT88, and ADCY3. Tamoxifen treatment resulted in loss of cilia in ift88 ECKO compared to ift88 EChet. The outflow facility (C) measured in tamoxifen-treated ift88 eyes was 4.8±1.97 nl/min/mmHg (n=21, 95%CI mean: 3.63-5.75), ift88 EChet 5.22±1.7 nl/min/mmHg (95%CImean: 4.42-6.17) and ift88 ECKO 5.75±2.53 nl/min/mmHg (95%CImean: 4.16-6.88). The outflow facility in the ift88 ECKO was weakly bimodal. The mean associated deviation MAD for the ift88 ECKO facility data was 1.9 compared to 1.5 for ift88 EChet and 1.03 for ift88 EChet facility data. This increase in the spread of the outflow facility data in SC lacking cilia suggests a dysregulation of outflow.

Conclusions: Together, our results suggest that primary cilia play a role in regulating outflow. We are currently determining the effect of IOP elevation and age on the morphological characteristics of the cilia.

Simulating Microgravity in Human Donor Eyes to Assess the Therapeutic Potential of Neuritin1

R. A. Mikal Lange^1,3, Nicole E. Bodi², Tasneem P. Sharma^3
1Indiana University School of Medicine, ²IUSM Department of Pharmacology and Toxicology, ³IUSM Department of Ophthalmology

Purpose: Microgravity is theorized to be among the space stressors that leads to spaceflight-associated neuro-ocular syndrome (SANS) during long duration spaceflight. To counteract SANS, our objective is to identify mechanisms induced by simulations of microgravity using human donor eyes to assess efficacy of the therapeutic countermeasure, Neuritin 1 (NRN1). Minimal findings have been reported on the effects of microgravity on retinal neurons, specifically retinal ganglion cells (RGCs). However, it is known that there are systemic metabolic changes in space, leading to oxidative stress in retinal neurons. Therefore, our countermeasure, NRN1, which is a known neuroprotective agent and has the potential to reduce neural oxidative stress, we will evaluate the molecule as a preventative therapy for SANS.

Methods: Posterior ocular segments (N=3) were cultured in the Translaminar Autonomous System model for 5 days to generate a translaminar pressure differential similar to documented pressures in astronauts post flight. Downstream gene and protein expression for mitochondrial function (MT-ND1), oxidative stress (SOD2), glial cell survival (GFAP), extracellular matrix (COLIV and FN), and the pro-apoptotic marker (BAX) were performed with retinal explants. Retinal activity for explants was measured by an electroretinogram.

Results: We successfully maintained posterior eye cups under elevated intracranial pressure for over 5 days (N=3). In contrast to control, the treated group showed differential expressions of MT-ND1, SOD2, GFAP, COLIV, FN, and BAX after NRN1 treatment. We also observed a positive trend in retinal activity post-treatment on electroretinogram analysis.

Conclusions: NRN1 has the potential to provide therapeutic benefits to the retina by reducing the detrimental effects of oxidative stress after simulation of microgravity pressures.

3D bioprinted human corneal endothelial grafts as a renewable source for endothelial keratoplasty transplantations

Cat Lee^1,2, Kanwal Matharu¹, Aliasger Salem³, Markus H. Kuehn^1,2
1Department of Ophthalmology and Visual Sciences, University of Iowa, ²VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA, ³Department of Pharmaceutical Sciences & Experimental Therapeutics, College of Pharmacy, University of Iowa

Purpose: Endothelial keratoplasty (EK) represents over one-third of all corneal grafts performed in the United States and involves replacing damaged corneal endothelium. Descemetís membrane endothelial keratoplasty (DMEK) is a commonly performed EK surgery that is technically challenging for surgeons due to the fragile nature of the graft and elastic properties that cause it to tightly scroll. It is also hampered by a shortage of donor corneas, with 1 cornea available for every 70 needed. Recent advances have made possible the in vitro expansion of human corneal endothelial cells (hCEnC) as well as 3D bioprinting of these cells. Creation of a Tissue Engineered Endothelial Keratoplasty (TEEK) construct embedded with hCEnCs would i) enable multiple patients to be treated using one donor cornea and ii) provide a standardized product for surgeons, solving issues with unpredictable tensile strength, saving them time in the operating room and improving EK outcomes.

Methods: Human corneal endothelial cells were isolated as follows. Anterior segments from eye donors deemed not suitable for transplantation were processed using the peel and digest method. After trephination of the endothelial layer, a forceps was used to carefully peel off the endothelial layer. This layer was stored overnight in expansion media. The following day, each endothelial scroll was cut in half for four halves total from each pair of eyes. The following tissue digestion methods were compared: EDTA and Collagenase A, shaken at 300 rpms at 37∞C or placed in a 37∞C incubator. Photomicrographs were taken from the center of each of the 24- every other day. On Day 9, the cells were fixed in ice cold methanol for immunocytochemistry. Gelatin Methacrylate (GelMA), a biodegradable biocompatible functionalized gelatin, was used to 3D bioprint TEEKs. These were tested for cell viability of hCEnCs after 3D bioprinting in dome-shaped TEEKs using a Live/Dead fluorescent stain.

Results: The isolation of corneal endothelial cells from human anterior segments was optimized by comparing digestion with EDTA versus Collagenase alone and combined with mechanical agitation. Collagenase digestion combined with mechanical shaking produced the greatest number of cultures with initial endothelial-like morphology and proliferative cultures, while EDTA alone produced the greatest number of cultures that did not proliferate. All four conditions in which replicates produced cells stained positive for the corneal endothelial markers N-Cadherin and CD166 and the senescence marker CD44 by immunocytochemistry. 3D bioprinting of immortalized human CEnCs using GelMA onto a concave mold with similar dimensions to the posterior cornea surface produced constructs with good cell viability (97% by LIVE/Dead cell viability assay).

Conclusions: hCEnCs isolated using the peel and digest method with Collagenase A combined with mechanical shaking produces cultures with the highest cell density and most endothelial-like morphology. More work needs to be done to mitigate senescent phenotypes of these cultures. 3D bioprinting of hCEnCs shows promise for the creation of a Tissue Engineered Endothelial Keratoplasty (TEEK) that can replace the fragile and highly variable donor tissue that is also in short supply.

Therapeutic Potential of Human Neuritin 1 on Glaucomatous Human Retinal Ganglion Cells Seeded on Fibrin Scaffolds

Ksenia Lewyckyj¹, Shahna S. Hameed¹, Tasneem P. Sharma^1,2
1Indiana University School of Medicine, Department of Ophthalmology, ²Indiana University School of Medicine, Department of Pharmacology and Toxicology,

Purpose: Glaucoma is a group of optic neuropathies characterized by progressive loss of retinal ganglion cells (RGC) and corresponding visual field deficits. Current treatments can only slow the progression of glaucoma, but RGC death is not preventable and irreversible. Thus, neurotrophic factor therapy may be a suitable therapeutic approach. Human Neuritin 1 (NRN1) has demonstrated neurodegenerative and neuroprotective properties, and our group has previously established that NRN1 is downregulated in glaucomatous RGCs. We will investigate the therapeutic potential of NRN1 on human RGCs using a 3D fibrin-based scaffold model.

Methods: Corneal fibroblasts from glaucomatous and non-glaucomatous post-humous donor tissue were reprogrammed into induced pluripotent stem cells (iPSCs) using the CytoTuneTM iPSC 2.0 Sendai reprogramming kit. These iPSCs were characterized via PCR (C-MYC, KLF4, SOX2, NANOG) and immunofluorescence staining (TRA-1-60, SOX2) and karyotyped. The iPSCs were differentiated into three-dimensional retinal organoids (ROs) from which RGCs were dissociated. PCR (RBPMS, THY1) and immunofluorescence staining (RBPMS, BRN3A, DAPI) were used to characterize RGCs. A fibrin scaffold was created via simultaneous administration of fibrin gel and crosslinking solution. RGCs were then seeded onto the fibrin scaffold to create a 3D microenvironment. Cultures were treated with or without recombinant NRN1. RGC apoptosis and neurite outgrowth were assessed by immunostaining for CASP3 and NEFL.

Results: NRN1 treatment enhanced neurite outgrowth and reduced RGC apoptosis in NRN1 treated RGCs compared to untreated cells. ,

Conclusions: These findings confirm that NRN1 enhances neurite outgrowth and RGC survival in human glaucomatous cells cultured in a biomimetic fibrin scaffold, further demonstrating its potential as a candidate for glaucoma therapy. Furthermore, they highlight the utility of 3D models for translational neuroregenerative research.

Integrating Genomics and Deep Learning OCT Analysis to Predict Treatment Response in Neovascular AMD

Thomas Martinez, Kyungmoo Lee², Farhad Salari³, Bernardo Bach¹, Ben Roos¹, Steve Russell¹, Culver Boldt¹, Ian Han¹, Tim Boyce¹, Elaine Binkley¹, James Folk¹, Edwin Stone¹, John Fingert¹, Todd Scheetz¹, Milan Sonka², Elliott Sohn^1
1University of Iowa, University of Iowa Carver College of Medicine, ²University of Iowa, College of Engineering, ³Academic Collaborator, Iran

Purpose: Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in elderly worldwide. In the neovascular form (nvAMD), choroidal neovascularization leads to retinal fluid accumulation and rapid visual decline. Although anti-vascular endothelial growth factor therapy remains the standard of care, a significant proportion of patients exhibit incomplete or poor response to treatment. The MMP9 locus is the only gene independently associated specifically with the neovascular subtype of AMD. This study aimed to characterize the clinical phenotypes and treatment responses of MMP9 variants in patients with nvAMD.

Methods: We genotyped subjects with nvAMD for the rs4810482 single nucleotide polymorphism that received care at University of Iowa Healthcare. These patients were categorized into the following groups: homozygous TT, CC, and heterozygotes (TC). Retrospective chart review was performed on these patients. We collected and analyzed corrected distance visual acuity (CDVA in logMAR) and injection interval at four time points: initial visit, 4th injection, 2-years, and final visit. To objectively quantify anatomical outcomes, we developed a novel deep learning algorithm of OCT images to automatically quantify intraretinal fluid (IRF), subretinal fluid (SRF), and pigment epithelial detachment (PED). Generalized estimating equations (GEE) were used to analyze dependent variables, adjusting for age, sex, BMI, and smoking history.

Results: 56 eyes (TT = 20, CC = 10, TC = 26) from 38 patients were included. The mean age at the initial visit was 75.6 ± 8.8 years. 60.5% of our subjects were female. Patients with the TC genotype showed a decrease in visual acuity from 20/40 to 20/50 while the TT genotype demonstrated an improvement from 20/50 to 20/40 (mean change in logMAR VA from baseline to final visit: 0.1 ± 0.28 vs. -0.1 ± 0.24, respectively; P = 0.03). Automated segmentation revealed higher volumes of IRF in the second year with each additional risk allele (CC: 0.01 ± 0.02 mm3, TC: 0.02 ± 0.05 mm3, TT: 0.03 ± 0.07 mm3, P = 0.003), although this was not observed at other time points. The total combined fluid burden was highest for the TC genotype group, which also had the greatest reduction in total fluid from initial visit to final (-0.68 ± 1.38 mm3), whereas the TT group had the least improvement in fluid volume (-0.16 ± 0.28 mm3, P = 0.03). SRF and PED volume changes did not differ by genotype.

Conclusions: This is the first study to demonstrate that MMP9 rs4810482 heterozygosity is associated with distinct structural and functional treatment responses in nvAMD. Our findings suggest MMP9 genotype may be a biomarker of disease severity and could inform prognosis or therapeutic strategies.

Adoptive transfer of glaucomatous Th1 T cells facilitate retinal ganglion cell loss in recipients

Jordan Mayberry^1,2, Merri-Grace Jones¹, Shuyu Xian¹, Markus H. Kuehn^1,2
1Department of Ophthalmology and Visual Sciences, University of Iowa, ²VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA

Purpose: While it has previously been demonstrated that T cells play a role in retinal ganglion cell (RGC) loss in glaucoma, the types of T cells that are involved and the mechanisms they utilize to facilitate damage remain poorly understood. Using an adoptive transfer model, we sought to determine whether Th1 T cells play a role in glaucomatous RGC degradation.

Methods: Elevated intraocular pressure (EIOP) was induced in donor animals by intracameral injection of Ad5.MyocY437H (n=10). Control animals did not receive injection (n=11). IOP was then measured for 4 months using rebound tonometry. After 4 months, donor mice were euthanized and their spleens were harvested for fluorescence-activated cell sorting (FACS). Recipient mice then received intraperitoneal injections (IP) of 5x105 of bulk splenocytes or Th1 T cells only from either naive or EIOP donors (n=6 to 8/per group). Control groups were either naive (n=5) or received an IP injection of PBS (n=7). IOP was again measured in recipient mice using rebound tonometry and visual acuity was tested using optokinetic response (OKR) for 4 months following transfer. At the conclusion of the experiment, mice were euthanized, and eyes, optic nerves, and spleens were harvested for downstream analysis.

Results: In donor animals that received intracameral injection, IOP was elevated compared to naive animals. Additionally, EIOP mice had more damage to their optic nerve than their naive counterparts at 4 months, as well as also had a 32.5% higher proportion of Th1 T cells than naive mice. Following adoptive transfer of donor cells, recipient mice did not experience an elevation of their IOP. Naive mice, PBS mice, or recipient mice receiving cells from naive donors did not experience visual acuity loss (-0.011 cycles/degree, 0.022, -0.004, and 0), however recipients receiving all splenocytes or Th1 T cells only from EIOP donors lost significantly more visual acuity than all controls (-0.052, -0.061, -0.065). Similarly, mice receiving cells from EIOP donors had fewer remaining RGC (3157 RGC/mm2, 3160, and 3051 compared to 3850, 3806, 3885, and 3804). Lastly, transferred cells appear to persist for at least 4 months.

Conclusions: Adoptive transfer of Th1 T cells from animals with EIOP results in loss of RGC and decline in visual acuity in recipients, suggesting a larger role for Th1 T cell-driven mechanisms in glaucoma pathology.

Differential Roles of TM-derived Extracellular Vesicles in ECM Homeostasis

Fiona McDonnell^1,2,3, Reynolds Kwame Ablordeppey¹
University of Utah, ¹Ophthalmology and Visual Sciences, University of Utah, Salt Lake City, UT ²2Pharmacology and Toxicology, and ³Biomedical Engineering

Purpose: The trabecular meshwork (TM) tissue in the conventional outflow pathway becomes dysfunctional in glaucoma. This dysfunction is linked to dysregulation of the extracellular matrix (ECM), and cytoskeleton, among other cellular mechanisms. Extracellular vesicles are emerging as modulators of the ECM within the outflow pathway, and may also be utilized therapeutically. We hypothesize that EVs derived from the TM can regulate ECM proteins and cytoskeletal structures within the outflow pathway tissues.

Methods: EVs were isolated from non-glaucomatous (NTM) and glaucomatous (GTM) TM cells cultured in 1% EV-depleted serum medium. Iodixanol-cushioned ultracentrifugation was used to isolate the EVs from the conditioned medium. EVs were characterized using nanoparticle tracking analysis and Western blotting. NTM cells were treated with dexamethasone (DEX; 500nM) or vehicle control (EtOH) for 7 days, with or without NTM-derived EVs at 2.08x108 particles. NTM cells were also treated with GTM-derived EVs (2.08x108 particles). Western blotting was used to assess the expression of fibronectin (Fn), myocilin (MYOC), collagen type 4 alpha 1 (COL4A1), alpha-smooth muscle actin (αSMA), and integrins ?1 and ?3.

Results: DEX treatment of NTM cells significantly increased the expression of Fn (2.94x, p<0.001), MYOC (1.57x, p<0.05), COL4A1 (1.59x, p<0.05), αSMA (1.55x, p<0.05), integrin ?1 (1.46x, p<0.05) and integrin ?3 (1.73x, p<0.05). The addition of NTM-derived EVs with DEX mitigated the DEX response preserving normal protein levels: Fn (1.26x, p=0.69), MYOC (1.17x, p=0.45), COL4A1 (1.09x, p=0.38), αSMA (1.09x, p=0.74), integrin ?1 (0.95x, p=0.51) and integrin ?3 (1.25x, p=0.35). There was a non-significant increase in Fn (1.39x, p=0.09), MYOC (1.40x, p=0.14), integrin ?1 (1.21x, p=0.32) and integrin ?3 (1.24x, p=0.25).

Conclusions: Our data suggests that EVs from non-glaucomatous cells can mitigate DEX-induced changes in ECM and cytoskeleton proteins. Interestingly, there was a non-significant increase in these same proteins when NTM cells were treated with GTM-derived EVs. This supports our hypothesis that EVs play a role in regulating the ECM and cytoskeleton within the outflow pathway.

Evaluating Inner Retinal Layer Integrity Adjacent to Geographic Atrophy Lesions Using Spectral-Domain OCT

Lucas McGuire¹, Aliya Siddiqui², Christopher Langlo^3
1School of Medicine, Medical College of Wisconsin, Milwaukee, WI, 53226, ²Department of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI, 53226, ³Department of Ophthalmology & Visual Sciences, Medical College of Wisconsin, Milwaukee, WI, 53226

Purpose: In end-stage nonexudative age-related macular degeneration (AMD), geographic atrophy (GA) leads to loss of the retinal pigment epithelium (RPE), photoreceptors, and choriocapillaris. The thinning of outer retinal layers such as the outer nuclear layer, photoreceptor inner segments and outer segments in areas surrounding GA is well documented and correlates with future lesion expansion. In contrast, the structural integrity of inner retinal layers in these regions is less well characterized. Prior studies suggest that inner layers are typically spared in early GA, likely reflecting their lesser reliance on RPE function. However, delayed remodeling may occur over time due to neural deafferentation following photoreceptor loss and cytokine-mediated changes. This study aims to quantitatively evaluate inner retinal thickness outside of GA lesions using spectral domain OCT.

Methods: One eye each from 20 patients with GA (15 right) and 20 with early dry AMD (control; 14 right) was selected by chart review using diagnosis codes. Heidelberg Spectralis OCT radial scans at 0∞ or 90∞ were used. Eyes with prior retinal surgery, diabetic retinopathy, vascular disorders, glaucoma, or hereditary dystrophies were excluded. If both eyes were eligible, the one with better image quality was chosen; if equal, the right was used. Mean age was 79.6 ± 8.8 years (11 females) in the GA group and 69.8 ± 7.7 years (15 females) in the control group. B-scans for GA were selected for atrophy visualization; controls were based on clinical diagnosis. Scans were segmented using the Duke OCT Retinal Analysis Program with manual refinement of the ganglion cell layer (GCL) and outer plexiform layer (OPL). Thickness (GCL OPL) was measured every 300μµm from the fovea. Measurements were grouped by absolute distance from the fovea, pooled across orientations. Regions overlying GA were excluded.

Results: We assessed inner retinal thickness across multiple eccentricities in individuals with GA (n = 232 measurements outside regions of GA) and early dry AMD (n = 412 measurements). GA eyes exhibited reduced inner retinal thickness at greater distances from the fovea compared to early dry AMD. Central eccentricities (300-600 µm) showed no significant differences between groups (all p > 0.2). Significant thinning in GA was observed at 900, 1200, 1500, and 1800 µm (p < 0.05). Eyes with GA lesions present at a given eccentricity were excluded from analysis at that location. All 20 early dry AMD eyes contributed data at each location.

Conclusions: These findings highlight spatial differences in inner retinal structure between GA and early dry AMD. While central thickness remains comparable, GA is associated with significant thinning at greater eccentricities. This may reflect trans-synaptic degeneration or loss of physical support near atrophic areas. These findings suggest that neurodegeneration in GA is not solely limited to areas of RPE loss. Even areas that appear structurally preserved may undergo subtle changes that could potentially contribute to the visual dysfunction observed in GA, such as reduced contrast sensitivity. Future analyses will further classify lesion locations by anatomical quadrant (superior, inferior, nasal, or temporal) to better understand regional patterns of inner retinal thinning. We also plan to expand this work by examining measurements adjacent to GA in more detail and incorporating intra-lesional analysis in the future as well.

The Role of Retinal Antigen Presenting Cells in Spontaneous Retinal Autoimmunity

Scott McPherson, Sherman Joe Sherman, Laura Burgstaler, Yunan Li, Heidi Roehrich, Dale Gregereson
Department of Ophthalmology and Visual Neurosciences, University of Minnesota

Purpose: We reported that induction of Spontaneous Autoimmune Uveoretinitis (SAU) correlated with the recruitment of circulating antigen presenting cells (APC) into the retina. Herein, we investigate the role of resident retinal APC on the course of SAU.

Methods: R161H+/- mice (B10.RIII), which spontaneously and rapidly develop severe autoimmune uveoretinitis, were crossed with CD11cDTR/GFP mice (B6/J). R161H+/- mice on the B6/J background develop slower, less severe SAU than R161H+/--B10.RIII mice allowing assessment of disease development relative to the depletion or activation of retinal or systemic APC. The course of SAU was established in a cohort of control R161H+/- x CD11cDTR/GFP F1 mice and then reanalyzed in test cohorts following treatment with diphtheria toxin or stimulation by optic nerve crush injury (ONC). Analysis was done by retinal imaging, flow cytometry, and histology.

Results: Systemic depletion of APC halted the progression of SAU in R161H+/- x CD11cDTR/GFP F1 mice and if commenced early in the disease process would reduce SAU severity. However, following depletion of APC specifically from the retina, SAU in R161H+/- x CD11cDTR/GFP F1 mice progressed in manner similar to control mice. In contrast, SAU in R161H+/- x CD11cDTR/GFP F1 mice was exacerbated following the activation of retinal APC by ONC.

Conclusions: Our observations that local depletion of retinal APC failed to inhibit SAU progression and that depletion of circulating APC not only limited SAU progression but, under defined circumstances, reduced clinical SAU supports the idea that circulating APC are crucial for the induction and progression of SAU.

Tracking Functional Recovery Following Late Gene Therapy in a Mouse Model of Retinitis Pigmentosa

Kamila Milejczyk, Molly Rasper, Scalabrino Miranda Scalabrino
Medical College of Wisconsin, Department of Ophthalmology and Visual Sciences

Purpose: Retinitis pigmentosa (RP) is a heterogenous inherited retinal disorder characterized by rod, then cone photoreceptor death. RP is caused by mutations in over 100 genes. Gene therapies are being developed to treat several genetic causes of RP, including mutations within Cngb1, a subunit of the rod cyclic GMP channel. Previous work assessed the success of gene correction in a mouse model of Cngb1-RP, finding poor outcomes after 50% rod loss. The purpose of our study is to better refine the time course and efficacy of gene therapy intervention in a mouse model of retinitis pigmentosa (RP) using in vivo electroretinography (ERG) and optical coherence tomography (OCT), ultimately comparing these results with early treatment outcomes.

Methods: We used the Cngb1^neo/neo mouse line, which contains a neomycin cassette flanked by LoxP sites to disrupt Cngb1 expression. This line was crossed with a UBC-creERT line to enable tamoxifen-inducible genetic rescue. Tamoxifen chow (500mg/kg) was administered at 3 months (~70% rod loss, ~5% cone loss) to model late intervention. ERGs were performed weekly over 12 weeks along with OCT at weeks 10-12 to track functional and structural recovery. At endpoint, eyes were collected for histological evaluation. Outcomes will be compared to early (1 month: 30% rod loss, 0% cone loss) treatment groups to evaluate timing effects.

Results: Following late gene therapy, scotopic ERG responses began improving by weeks 3-4 and reached near-WT levels by 12 weeks, indicating functional rescue of rod and bipolar signaling. This is in contrast with retinal ganglion cell responses recorded by a multi-electrode array, indicating rod function is rescued by circuitry changes remain impaired, at least for 12 weeks post rescue.

Conclusions: Longitudinal ERG and OCT demonstrate that late genetic rescue can stabilize degeneration and restore photoreceptor function over time. However, prior studies revealed persistent gliosis and changes to synaptic structure in late-treatment mice, suggesting ongoing retinal stress despite rod stabilization. Preliminary results show that early treated cohorts demonstrate more rapid and complete recovery. Continued analysis of early versus late interventions will provide deeper insight into functional rewiring and histological integrity, guiding therapeutic windows for RP gene therapy.

Exploring Choroid Cell Replacement Options for Late AMD Therapy and Prevention

Jack Miller¹, Robert F. Mullins^1,2

Purpose: Age-related macular degeneration (AMD) is a leading cause of irreversible blindness that affected roughly 200 million people in 2020 and is predicted to affect approximately 290 million people by 2040. AMD is caused by the degeneration of the macula, the central part of the retina which contains a high concentration of cone photoreceptors and is required for high acuity vision. The choroid is a vascular network that lies beneath the retina and RPE to provide them with oxygen and nutrients. Previous research has shown that early AMD is associated with the loss of endothelial cells in the choroid. The loss of endothelial cells within this vasculature causes decreased oxygen and nutrient flow to the retina, which can eventually lead to the development of severe end-stage AMD. The purpose of this study is to develop strategies for replacing lost or damaged endothelial cells in the human choroid, which may be utilized for AMD therapy and prevention.

Methods: To pursue this study, endothelial cells were derived from human iPSCs. Pieces of human donor choroids were subjected to a decellularization protocol that removed cells from the tissues. The iPSC-derived endothelial cells were then co-cultured with decellularized human donor choroid tissue for up to 7 days to allow the cells to recolonize the choroid.

Results: The efficacy of the decellularization protocol was validated with a protein concentration assay and western blotting. Phase contrast microscopy shows that cells can successfully migrate to decellularized choroid tissue. Immunofluorescence imaging of the recellularized tissue shows that iPSC-derived endothelial cells can infiltrate, occupy, and express endothelial cell markers within the choroid. Measurement of RNA levels showed increased RNA concentration in some recellularized tissue samples compared to controls.

Conclusions: The data suggest that the repopulation of cells within the choroid is possible. Future directions of the project include continuing to develop the protocol to further optimize the recellularization process. The goal of this work is to support further research of cell replacement in the choroid and its eventual application as a therapy for individuals that suffer from AMD.

Allele-specific silencing as a treatment for autosomal dominant SNRNP200-associated Retinitis Pigmentosa

Neda Mokhberian^1,3, Nathaniel P. Mohar^2,3, Paige N. Wiebke³, Katarina A. Fuhrmeister³, Arlene V. Drack¹, Lori L. Wallrath³
University of Iowa ¹Ophthalmology and Visual Sciences, ²Interdisciplinary Graduate Program in Genetics, ³Biochemistry and Molecular Biology

Purpose: Retinitis pigmentosa (RP) is a group of inherited retinal diseases that cause progressive vision loss due to the degeneration of photoreceptors, which are responsible for night and peripheral vision. Mutations in over 300 genes have been linked to RP, including dominant mutations in the SNRNP200 gene. SNRNP200 is a part of the spliceosome, a macromolecular complex that removes introns from pre-mRNAs. Specifically, SNRNP200 is a component of the U5 complex that possesses RNA helicase activity. Dominant-negative mutations in SNRNP200 disrupt splicing fidelity, contributing to RP pathogenesis. The goal of our research is to identify siRNAs that specifically target mRNA from the mutant SNRNP200 allele, while not reducing wild-type SNRNP200 mRNA levels. Of three siRNAs tested in a reporter gene assay in culture cells, one showed selectivity for the mutant message. Thus, our studies provide evidence for allele-specific knock-down as a potential treatment.

Methods: Plasmid Design: Wild-type (Snrnp200 WT) and mutant (Snrnp200 T731I) constructs were generated that contain a region surrounding the methionine start codon and sequences from exon 17 of the mouse Snrnp200 gene in frame with GFP. The point mutation resides in exon 17 and changes a threonine to an isoleucine at position 731. Three siRNAs targeting the mutant allele were designed and tested. Quantification of allele-specific knock-down: The efficacy of siRNAs knock-down was assessed using live fluorescence microscopy. Four independent images were taken and GPF fluorescence was quantified using ImageJ software. In addition, western analysis was used to quantify Snrnp200 protein levels. Relative GFP expression, normalized to total protein (Revert staining), was measured from three independent co-transfection experiments using LICOR Image Studio software.

Results: Our data demonstrate that siRNA si20 reduced mutant Snrnp200 mRNA by 61% and had little effect on wild-type Snrnp200 mRNA. Consistent with these data, western analysis showed a 75% decrease in Snrnp200 protein levels.

Conclusions: This study establishes that specific siRNAs can selectively discriminate a mutant message from a wild-type message. Our findings suggest a potential therapeutic approach for RP patients carrying the dominant SNRNP200 mutations. Further, our findings suggest that this approach can be applied to point mutations in RP-causing genes.

Mitochondrial Dysfunction and ROS Production in Hyperglycemic Corneal Endothelial Cells

Elisha Monson¹, Jessica M. Skeie^2,3, Hanna Shevalye^2,3, Timothy Eggelston³, Mark A. Greiner^2,3
1Carver College of Medicine, University of Iowa, Iowa City, IA 52242, ²Department of Ophthalmology and Visual Sciences, ³Iowa Lions Eye Bank, Coralville, IA 52241

Purpose: The prevalence of diabetes in donor tissues is rising. However, studies in corneal transplantation have shown that diabetic donor tissue may underperform after implantation. In corneal endothelium, which contains corneal endothelial cells (CECs) that maintain corneal clarity through ion pumping and water transit, documented diabetic dysfunction may manifest clinically as decreased CEC function, increased corneal thickness, increased fragility during graft preparation, and increased graft failure postoperatively. Due to energy-intensive ion pumping and environmental oxidative damage, CECs have a high mitochondrial density and are very susceptible to mitochondrial dysfunction. Diabetes has already been noted to affect mitochondrial spare respiratory capacity and morphology. To further characterize diabetic mitochondrial dysfunction in CECs, we isolated the effects of hyperglycemia on CECs and analyzed mitochondrial responses, including alterations in function and overall gene expression.

Methods: HCEC-B4G12 immortalized CECs were cultured under hyperglycemic conditions (8.3 mM, 11.3 mM, 15.7 mM, and 33.1 mM glucose, corresponding to HbA1C levels of 6.8%, 8.7%, 11.5%, and 22.4%, respectively). Metabolic activity was quantified using an extracellular flux analyzer and normalized to CEC density. Raw oxygen consumption rates were used to calculate basal respiration, ATP-associated oxygen consumption, proton leak, maximal respiration, spare respiratory capacity, and nonmitochondrial respiration. Cells were assayed for mitochondrial ROS and membrane potential (MMP) with the mitoROS and JC-10 assays. MMP were calculated from a ratio of aggregated:monomer JC-10 assay where higher ratios indicate a healthier MMP. Separately, CECs treated with 30.5 mM glucose were compared to controls treated with 5.5 mM glucose and analyzed for gene expression using RNA-seq. Gene-level salmon counts were used to calculate and compare differential gene expression analyses.

Results: Maximal respiration was 5.2 pmol/min/103 cells for CECs at 8.3, 11.3, and 15.7 mM glucose and 4.0 pmol/min/103 cells for CECs at 33.1 mM glucose. Spare respiratory capacity was 2.3 pmol/min/103 cells for CECs at 8.3, 11.3, and 15.7 mM glucose and 1.5 pmol/min/103 cells for CECs at 33.1 mM glucose. CECs at 8.3, 11.3, and 15.7 mM glucose had JC-10 ratios ranging from 1.36-1.28 while CECs at 33.1 mM glucose had a JC-10 ratio of 1.06. CECs at 8.3, 11.3, and 15.7 mM glucose had mitochondrial ROS production from 0.48-0.43 RFU/cell while CECs at 33.1 mM glucose had mitochondrial ROS production of 0.62 RFU/cell. Gene expression analysis showed thioredoxin interacting protein (TXNIP) more significantly altered between CECs exposed to 5.5 mM glucose and 30.5 mM glucose (Glc30/Glc5 log2FC= 1.939; P<0.0001) than any other gene. Pathway analysis found mitochondrial dysfunction and ATP synthesis as the most statistically significantly altered pathways between CECs at 5.5 mM and 30.5 mM glucose.

Conclusions: Hyperglycemia, the major risk factor for long-term cellular and tissue damage from diabetes, did not induce mitochondrial impairment at levels commonly experienced by patients clinically and only induced mitochondrial dysfunction at the highest hyperglycemic condition. This finding is suggestive of an exhaustible mitochondrial compensation mechanism after short-term exposures as tested in this study, and longer-term exposures need to be tested. Hyperglycemic conditions also induced significantly increased expression of the pro-oxidation factor, TXNIP, in hyperglycemic CECs when compared to normal CECs. This finding suggests a connection between TXNIP and the overproduction of ROS in hyperglycemic CECs. Due to the TXNIP-inhibiting properties of insulin, the possibility of addressing TXNIP-associated oxidative damage perioperatively may be a promising avenue of study.

Complement injury causes secretion of CCL5 from choroidal endothelial cells and may be responsible for macrophage recruitment in early age-related macular degeneration

Rachel Moore¹, Kelly Mulfaul^2
1Department of Ophthalmology & Visual Sciences, University of Iowa ²Department of Neuroscience and Pharmacology, University of Iowa

Purpose: Immune cell recruitment and increased numbers of macrophages are observed in the choroid early in age-related macular degeneration (AMD). The signaling pathways responsible for the recruitment of macrophages remains unknown. Complement activation and formation of the membrane attack complex (MAC) on the choriocapillaris is elevated in AMD. We identified that MAC formation on choroidal endothelial cells (CECís) causes the secretion of CCL5, a chemoattractant of leukocytes including macrophages. We observed migration of AMD monocytes towards MAC injured, CCL5 secreting, CECís. To determine the effect of CCL5 on monocyte recruitment towards MAC injured CECs, we knocked out a receptor for CCL5 (CCR5). We validated the knockout functionally and observed decreased monocyte invasion in the presence of CCL5, suggesting CCL5 is involved in monocyte migration towards complement injured CECís. Furthermore, monocytes polarize towards a pro-inflammatory phenotype in the presence of CCL5.

Methods: iPSC derived CECís, monocytes, and macrophages were generated from an AMD donor line. CECís were treated with C9-depleted serum or normal human serum (NHS) to induce complement pathway activation and MAC deposition. After 24 h the supernatant was collected, and CCL5 secretion was detected by ELISA. Monocyte migration assays were performed by injuring CECís with C9-depleted serum or NHS for 24 h, followed by the addition of GFP-stained monocytes on the apical side of a 5.0 um transwell insert directly above the injured CEC monolayer. Monocytes were incubated in co-culture for 6 h followed by imaging and counting of migrated cells. Monocyte invasion was assayed using 5.0 um transwells coated with a layer of Matrigel. Cells were incubated for 24 h in the presence or absence of recombinant CCL5, after which monocyte invasion was analyzed by crystal violet staining. Monocytes were differentiated into macrophages with or without recombinant CCL5 and the RNA was harvested for qPCR.

Results: MAC formation on iPSC derived CECís leads to secretion of CCL5. AMD iPSC derived monocytes migrate towards recombinant CCL5 protein and MAC injured CECís. iPSC derived monocytes from an AMD donor parent line have an increased potential to invade in the presence of CCL5 compared to isogenic CCR5 knockout monocytes. Furthermore, differentiation of monocytes into macrophages favors an M1 phenotype in the presence of CCL5.

Conclusions: We have demonstrated that MAC injury to AMD iPSC derived CECs causes increased secretion of the chemokine CCL5. CCL5 release leads to monocyte migration and invasion highlighting the CCR5/CCL5 signaling pathway as a candidate mechanism for monocyte invasion early during AMD progression.

A Novel Corneal Implant from Tissue-derived Biomaterial for Clinical Application

Amatun Nur Mou¹, Rajkumar Sadasivam², Gudiseva Chandrasekher^1
1South Dakota State University, ²Louisiana State University Health Sciences Center

Purpose: The scarcity of donor tissue remains a critical barrier to treating corneal stromal diseases worldwide. The development of biomimetic implants offers a promising approach for restoring vision and integrity in damaged corneal tissue. In this study, we introduce ECMIX, a novel tissue-derived biomaterial developed in our laboratory. The objective of this research is to investigate the optical, mechanical, and cellular interaction properties of the ECMIX-based stromal implant.

Methods: ECMIX biomaterial was chemically crosslinked with EDC/NHS to prepare a stable construct with the desired thickness. Surface morphology was analyzed via field emission scanning electron microscopy (FESEM) and compared to native corneal collagen architecture. Optical transparency was assessed using UV-Vis spectrophotometry. Mechanical strength was measured through compression testing with the Univert Mechanical Test System. Hydration capacity was evaluated by comparing wet and dry weights. Primary keratocytes were cultured to assess in vitro regeneration. For ex vivo evaluation, ECMIX implants were inserted into stromal pockets (4–5 mm diameter) in porcine corneas. After 10 days, the implants were retrieved and imaged using KEYENCE fluorescence and Zeiss microscope to assess integration and cellular response.

Results: The light transmission through the implant is found to be 70% 80%, indicating that it is optically transparent. The water content of the construct is 85-90% (w/w), comparable to the natural cornea. Scanning electron microscopy analysis of the native cornea and the 3D printed construct shows that the morphology of the collagen fibrillar structure is unaltered. Keratocyte cells on the implant resemble the natural stromal keratocytes, suggesting that the implant provides an environment that supports keratocyte behavior and structure. A surgically inserted implant shows good integration with the native tissues and facilitates stromal keratocyte regeneration on the implant.

Conclusions: The ECMIX-based implant demonstrates high optical transparency, physiological hydration, and structural similarity to native corneal stroma. It supports keratocyte growth and integrates well with host tissue. This study addresses the challenge of donor cornea scarcity by developing an alternative, transplantable implant suitable for treating partial or full-thickness corneal injuries.

Eyes on the Road: A Student-Run Traveling Ophthalmology Clinic Model in Indiana

T. Mujahid¹, M. Cory¹,M. Tharp¹, S. Bertot¹, S. Gupta¹, G. Cabrero¹, C. Kaurich¹, P. Kirtley¹, C. Yung¹, J. Eikenberry^2
1Indiana University School of Medicine, ²Indiana University, Department of Ophthalmology

Purpose: The Ophthalmology Student Interest Group (OSIG) at IUSM has members from campuses across the state and operates a student-run free eye clinic in downtown Indianapolis. Students at regional campuses can experience limitations to volunteering at the OSIG eye clinic due to the required travel to Indianapolis. The OSIG eye clinic also seeks to reach patients who may not be able to access the Indianapolis location. Thus, a traveling clinic model was developed to reach patients living in rural or more remote parts of Indiana and provide an opportunity for clinical engagement and education of ophthalmology-interested students at regional IUSM campuses.

Methods: In the traveling clinic, some supplies (e.g. autorefractor, near vision cards) were transported from the main OSIG eye clinic in Indianapolis. When available, existing equipment (e.g. slit lamp, phoropter) at the site to which the clinic was traveling was utilized. Clinics were staffed by medical student volunteers from the main Indianapolis campus and students from regional campuses. The attending ophthalmologist was from the community surrounding the regional campus when possible. When this was not possible, an attending ophthalmologist traveled to the regional campus clinic site.

Results: Since the first mobile clinic in October 2023, seven traveling clinics have been held at six of nine regional campuses. Nearly all clinics were staffed by local ophthalmologists. Medical students from regional campuses served as coordinators of each clinic date alongside an Indianapolis medical student in a dedicated role overseeing the traveling clinic. Slit lamp examinations were offered when access to a slit lamp was available. Examination with an indirect ophthalmoscope and distance and near visual acuity readings were provided at each clinic. Most common outcomes include referral to a nearby hospital or ophthalmology clinic, or prescription for reduced-cost classes through the OSIG eye clinic’s charitable glasses vendor.

Conclusions: This model provides an effective approach to mobile clinic efforts when a dedicated vehicle may not be available or other funding or logistical limitations exist. The model is well-suited for rural or remote areas, and future directions include approaches to provide care to specific groups and events, including refugees, those living in urban areas but far from the OSIG eye clinic Indianapolis location, and unhoused groups.

Sialoglycoconjugate distribution and partial identities in human choroid, RPE, retina, and basal laminar deposit

Emma Navratil¹, Robert F. Mullins^1,2
1Institute for Vision Research, University of Iowa, ²Department of Ophthalmology and Visual Sciences, University of Iowa

Purpose: Sialic acids are negatively changed, 9 carbon sugars that perform essential roles in cell-cell adhesion, migration, complement regulation, and immune modulation, each of which may be altered during age related macular degeneration. The purpose of this study was to characterize sialoglycans of the human choroid, RPE, and photoreceptors, including their distributions and partial molecular identities.

Methods: This study utilized lectin histochemistry and immunofluorescence on a set of 45 fixed and cryopreserved human donor eyes to investigate sialoglycans. Lectins EBL and MAL-II were used to visualize a-2,6 and a-2,3 sialic acids, respectively. Antibodies were used to investigate colocalization of sialic acids with structures and proteins of interest. Neuraminidase enzyme was used to remove sialic acids, followed by histochemistry with a battery of 37 lectins to reveal penultimate carbohydrate moieties.

Results: a-2,3 and a-2,6 sialic acids have different distributions in the submacular choroid. a-2,3 sialic acid is found in choriocapillaris endothelial cell membranes, while a-2,6 sialic acid shows robust labeling of intercapillary pillars (i.e., the extracellular matrix between choriocapillaris vessels). Both conformations of sialic acid are also found in basal laminar deposits, histological features of macular degeneration, and in subretinal fibrosis in neovascular AMD. Lectins that recognize α-galactose, N-acetyllactosamine, galactose (b-1,3) N-acetylgalactosamine, and a - or b- N-acetylgalactosamine showed increased reactivity in basal laminar deposits after neuraminidase treatment. EBL displays overlap with anti-MCT-1, a marker of RPE microvilli, while a-2,3 sialic acid is internal to the microvilli, in the subretinal space. The fovea displays distinct sialic acid distribution. Foveal, but not extrafoveal, cone inner segments exhibit MAL-II labeling.

Conclusions: This study adds to our understanding of the sialic acid glycan composition of the macula, including topographic distribution in the fovea and changes in aging and AMD.

Methods for evaluating antibody drug conjugate associated corneal toxicity

Bhuvanachandra Pasupuleti¹, Emily Krupa², Joey Tidei² , Lee Troughton², Sandeep Jain³, Simon Kaja^4
1 Selagine INC, ²Loyola University Chicago, Joey Tidei, Loyola University Chicago, Loyola University Chicago, ³ Ophthalmology and Visual Sciences, University of Illinois Chicago, ⁴Ophthalmology and Molecular Pharmacology & Neuroscience, Loyola University Chicago

Purpose: Antibody-drug conjugates (ADC) have emerged as promising chemotherapeutic agents for multiple types of cancer. However, various ADC are associated with significant off-target corneal toxicity attributed to the non-specific corneal uptake of ADC by macropinocytosis. The overall goal of this study was to develop robust in vitro methods to quantify micropinocytosis (MPC)-mediated cellular uptake. We employed fluorescence microscopy and high-content imaging to quantify MPC.

Methods: To quantify MPC-mediated cellular uptake, human corneal epithelial cells (HCE-T) were seeded in black/clear bottom 96 well plates. Cells were exposed to Dextran-Texas RedTM (DTR; 10,000 KDa; 0.5 mg/mL) or Rituximab-monomethyl auristatin F (RTX) for 3.5 h. Images were acquired using widefield microscopy and using a Cytation 5 multimodal plate reader. RTX uptake was quantified by detection using fluorescently-conjugated immunoglobulin. Fluorescence intensity and immunopositve area were quantified from images using FiJi. The ability of ocular surface immunoglobulin (OSIG) and imipramine to inhibit MPC was compared against the prototypic MPC inhibitor, ethylisopropyl amiloride (EIPA). For these experiments, cell were pre-incubated with drugs for 30 min prior to addition of DTR.

Results: DTR fluorescence intensity was positively associated with RTX uptake into corneal epithelial cells. The presence of DTR did not affect RTX uptake. EIPA significantly reduced fluorescence intensity and immunopositive area in cells exposed to RTX or RTX and DTR. Both imipramine (0.1 – 10 µM) and OSIG (0.1 – 2%) resulted in a significant dose-dependent reduction of fluorescence intensity suggestive of MPC inhibitory activity. OSIG was significantly more potent, resulting in a ~60% reduction (n=4, P<0.001) in DTR fluorescence at the highest concentration, whereas imipramine decreased DTR fluorescence by ~20% (n=3, P<0.01).

Conclusions: ur data support the use DTR as a surrogate for ADC when studying the effect of test articles on MPC in human corneal epithelial cells. Our unbiased plate reader-based method provides a medium throughput approach for assessing MPC-mediated uptake into corneal epithelial cells with the potential to be readily expanded to high-throughput screening. OSIG resulted in a significant dose-dependent reduction of MPC in human corneal epithelial cells. Ongoing studies are evaluating the efficacy, safety, and tolerability of OSIG for mitigating corneal toxicity associated with various ADCs.

A novel mouse model to study rapid photoreceptor degeneration in Retinitis Pigmentosa

Tyler Rankin¹, Nathan Mohar², Joseph Laird², Paige Wiebke², Katerina Fuhrmeister², Arlene Drack¹, Lori Wallrath^2
1Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, ²Department of Biochemistry and Molecular Biology, University of Iowa, Iowa City, IA

Purpose: Retinitis Pigmentosa (RP) is a progressive retinal degenerative disorder characterized by sequential photoreceptor loss, beginning with rod photoreceptors and followed by degeneration of cone photoreceptors. Over 300 genes have been implicated in RP, including SNRNP200, which encodes an RNA helicase component of the U5 small nuclear ribonucleoprotein (snRNP) complex essential for pre-mRNA splicing. Despite its ubiquitous expression, the mechanisms by which SNRNP200 mutations cause retina-specific degeneration remains unknown.

Methods: To investigate the role of Snrnp200 in rod photoreceptors, we generated a floxed mouse with LoxP sites flanking exon 3 of the Snrnp200 gene and crossed it with a Rhodopsin-iCre (Rho-iCre) transgenic line to generate rod-specific conditional knockout (cKO) mice. The resulting Rho-iCre/+; Snrnp200+/Snrnp200 flox were analyzed and mated to Snrnp200 flox homozygotes to generate Rho-iCRE/+; Snrnp200 flox/Snrnp200 flox (homozygous cKO) mice. Mice were analyzed between three and six weeks of age. Retinal function was assessed via electroretinography (ERG), retinal structure by optical coherence tomography (OCT), and photoreceptor integrity by immunofluorescence staining of retinal cryosections using photoreceptor-specific markers.

Results: Snrnp200 homozygous cKO mice (n=6) showed significantly reduced rod ERG amplitudes (μ = 280 μV) vs. Rho-iCre (μ = 466 μV; p = 0.0008; n=4) and heterozygous cKO mice (μ = 395 μV; p = 0.0035; n=6) by three weeks of age. By four weeks, rod responses were nearly absent (μ = 41 μV), while other genotypes retained robust amplitudes (μ = 356 μV; p < 0.0001). Cone ERGs were similar to that of controls at three weeks but reduced in homozygous cKOs by four weeks (μ = 108 μV vs. 166 μV; p = 0.0054) and nearly absent by five weeks (μ = 16 μV; p < 0.0001). OCT showed mild ONL thinning at three weeks (μ = 55 μm vs. 63 μm; p = 0.0277), and severe loss by four weeks (μ = 6 μm; p < 0.0001). At six weeks, IHC confirmed complete loss of rod (rhodopsin) and cone (cone arrestin) markers in homozygous cKOs, with preserved morphology in controls.

Conclusions: Our findings demonstrate that this novel Snrnp200 conditional knockout mouse model recapitulates key features of human RP, notably the sequential degeneration of rod photoreceptors followed by cone photoreceptors. It is worthwhile to note that the cone photoreceptors possess a wild-type genotype, strongly suggesting that their death is a direct result of rod photoreceptor death and not being compromised by the loss of Snrnp200. Unlike many existing models with slow disease progression, Snrnp200 cKO mice exhibit complete rod loss by one month of age, resulting in rapid and severe retinal degeneration. This accelerated timeline provides a valuable platform for studying early molecular events underlying photoreceptor death and for dissecting splicing-related disease mechanisms. Moreover, this model enables the testing of time-sensitive therapeutic strategies aimed at preserving cone function, with implications for a broad range of RP subtypes.

The Architecture and Biomechanics of the Human Ciliary Zonule

Pooja Rathaur¹, Juan Rodriguez², Steven Bassnett^1
1Department of Ophthalmology & Visual Sciences, Washington University, St. Louis, MO, ²University of Health Sciences and Pharmacy in St. Louis, MO

Purpose: The ciliary zonule, a network of stress-bearing extracellular fibers, is crucial for lens centration and transmitting the forces necessary to flatten the lens during distant focusing. To gain deeper insight into the accommodative mechanism, we developed techniques to visualize the three-dimensional structure of the human zonule and to measure the biomechanical properties of individual zonular fibers.

Methods: Human eyes (n=30) were procured from a local eye bank and fixed in 4% paraformaldehyde/PBS for at least five days. The globes were bisected, and zonular fibers were labelled with fluorescein-conjugated Lens culinaris agglutinin. Fluorescent phalloidin stain was used to visualize the internal topology of the eye. The intact zonular apparatus was imaged from the posterior aspect using long working-distance objective lenses and confocal microscopy. Partial removal of the vitreous humor using a cellulose spear facilitated high-resolution imaging of the interactions among zonular fibers, the lens, ciliary body, and anterior hyaloid membrane with short working-distance lenses. The mechanical properties of individual zonular fibers were measured using a force transducer probe. Ultrastructure of fiber was subsequently characterized by transmission electron microscopy.

Results: Zonular fibers were observed to originate from the pars plana region of the ciliary body and extend to the lens surface, where they were classified into posterior, equatorial and anterior groups based on their distinct anchorage sites. In addition to these radial fibers, circumferential fibers were also identified in the pars plicata region, where they firmly attached to the anterior hyaloid membrane. Biomechanical measurements demonstrated that the fibers possess an elastic modulus of approximately 1 MPa, a mean breaking force of 4.13 mN and a Poissonís ratio of 4, indicative of their high tensile compliance. Ultrastructural analysis showed that individual zonular fibers are composed of thousands to millions of microfibrils.

Conclusions: We described the three-dimensional organization of the human zonule and biomechanical properties of individual fibers, providing insights into accommodation and age-related changes that culminate in presbyopia.

Restoration of IFT140 via dual AAV vectors and a unidirectional DNA recombination system rescues retinal degeneration in an IFT140 mouse model

Kun-Do Rhee¹, Poppy Datta¹, Clairissa Baccam¹, Arlene V. Drack¹, Seongjin Seo^1
1Department of Ophthalmology and Visual Sciences, University of Iowa College of Medicine

Purpose: We have developed a novel strategy to deliver large genes using up to four AAV vectors and reconstitute them via a CRE-lox-mediated unidirectional DNA recombination system. To validate this approach, we applied the strategy to the IFT140 gene and assessed the therapeutic efficacy and safety of dual AAV-IFT140 vectors in an IFT140 mouse model.

Methods: The human IFT140 coding sequence (CDS) was split into two segments for insertion into separate 5’ and 3’ AAV vectors. The 5’ vector contained a GRK1 promoter, the first 810 bp of IFT140 CDS, a loxJT15:2272 hybrid site, an internal ribosome entry site (IRES), the CRE CDS, and a BGH polyA signal. The 3’ vector included a loxJTZ17:2272 hybrid site, the remaining of IFT140 CDS (3576 bp), and a BGH polyA signal. AAV vectors with the Anc80.L65 serotype were delivered subretinally into Ift140;iCre75 conditional knockout (CKO) mice at post-natal day (P) 15-16, with a dose of 8E+8 GC per vector. Protein production was analyzed by immunoblotting. Retinal function and histology were evaluated using electroretinography (ERG) and immunohistochemistry.

Results: Dual AAV-IFT140 vectors successfully restored robust and consistent expression of full-length IFT140 in treated eyes of both wild-type and Ift140 CKO mice. By P55, truncated 37-kDa N-terminal IFT140 protein and CRE recombinase from the 5’ vector were undetectable, suggesting depletion of unassembled 5’ vectors. ERG at P45 revealed significantly enhanced light responsiveness in treated eyes under both scotopic and photopic conditions compared to untreated CKO eyes. Histological analysis of retinal sections at P52 showed preservation of 5–8 rows of photoreceptor nuclei in treated regions, compared to 1–2 rows in untreated areas. No apparent toxicity was observed in treated wild-type mice. Longer-term studies are ongoing.

Conclusions: The CRE-lox-mediated unidirectional recombination strategy provides a simple and effective platform for delivering large genes using multiple AAV vectors. The dual AAV-IFT140 vectors demonstrated efficacy and safety, offering proof-of-concept for the CRE-lox system and laying the groundwork for future clinical trials.

Transcriptomic Changes in Primary Human Trabecular Meshwork Cells After Co-Culture With iPSC-Derived TM Cells

Alireza Salimi¹, Catherine A. A. Lee^1,2, Markus H. Kuehn^1,2,
¹Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, IA 52242, ²Iowa City VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA, 52246

Purpose: Trabecular meshwork (TM) dysfunction contributes significantly to impaired aqueous humor outflow and elevated intraocular pressure in primary open-angle glaucoma. Cell-based regenerative strategies using induced pluripotent stem cell-derived TM-like cells (iPSC-PTM) have shown promise for restoring TM function. This study aimed to investigate how co-culture with iPSC-PTM cells influences the gene expression profile of primary human TM (PTM) cells and to identify key molecular pathways and regulators involved in this interaction.,

Methods: Primary TM cells were isolated from donor human eyes and cultured under standard conditions. Co-culturing these cells with iPSC-PTM cells allowed for the evaluation of transcriptional alterations brought on by either direct or paracrine signaling. When PTM cells were re-isolated after co-cultivation, total RNA was extracted in preparation for high-throughput RNA sequencing (RNA-seq). A log² fold-change ≥0.5 for upregulated genes and a log² fold-change ≤0.5 for downregulated genes were used to identify differentially expressed genes (DEGs). STRING and Cytoscape were used to stratify gene lists into groups that were upregulated and downregulated, and then analyze the networks. To find hub genes, topological features such as Degree, Betweenness Centrality, and Closeness Centrality were calculated. Functionally enriched pathways and gene ontology (GO) terms were analyzed using GeneMANIA and Enrichr.

Results: Co-culture with iPSC-PTM cells significantly altered the transcriptomic landscape of PTM cells. A total of 226 genes were upregulated, and 39 were downregulated, which were submitted to the GeneMANIA. Functional enrichment revealed that upregulated genes were significantly associated with regulation of endothelial cell proliferation, endothelial cell proliferation, positive regulation of epithelial cell proliferation, and epithelial cell proliferation. Downregulated genes were enriched in negative regulation of cell population proliferation, negative regulation of mitotic cell cycle, and negative regulation of cell cycle G1/S phase transition. PPI network analysis highlighted hub genes including KDR, BMP4, BMP4, AGTR1, IKBKB and FGFR1 for the upregulated set, suggesting a shift toward tissue remodeling and repair. On the other hand, CDK6, PTEN, and TGFB1 were determined to be critical hub genes for the downregulated set.

Conclusions: iPSC-derived TM-like cells activate gene networks involved in the control of epithelial and endothelial cell proliferation by causing major transcriptomic reprogramming in primary human TM cells. These findings provide mechanistic insight into how iPSC-PTM cells may promote TM regeneration and support their potential use in developing cell-free based therapies for glaucoma. The combination of network topology, functional enrichment, and RNA-seq provides a reliable method for determining therapeutic targets and comprehending cell-cell interactions in the TM niche.

Identification of a Regulator of Bipolar Cells Synapses In Gene Therapy for Retinitis Pigmentosa

Miranda Scalabrino

Department of Ophthalmology & Visual Sciences, Medical College of Wisconsin

Abstract: Photoreceptor degeneration causes impairment in the visual system through death of the light sensing neurons, leading to blindness once a critical number of photoreceptors are lost. Photoreceptor loss also triggers changes to the retinal circuit as cells respond to diminishing photoreceptor input. These changes include significant modifications in bipolar cells, which directly receive photoreceptor input. Bipolar cells are known to extend and retract dendrites, form ectopic synapses, and downregulate signaling molecules in late-stage PR loss. However, early-stage rewiring is hypothesized to maintain PR signals through undefined homeostatic mechanisms. We aimed to identify these mechanisms, first through a transcriptome analysis of rewiring bipolar cells at multiple stages of disease using the Cngb1^neo/neo mouse line, then by assessing the impact of differentially regulated genes using AAV-CRISPR. We performed intraocular injections in disease and littermate controls followed by electroretinography and histology. Preliminary findings have identified a potential regulator of photoreceptor-bipolar cell synapses of potential interest to gene and cell therapies for photoreceptor degenerations.

Analyzing the interplay of cellular stresses using a mouse model of Fuchs Endothelial Dystrophy

Raji Shyam¹, Subashree Murugan¹, Sachin Ghag¹, Hsuan-Yeh Pan¹, Viviane Campos³, Francis Price⁴, Marianne Price^4
1University of Iowa, Department of Anatomy and Cell Biology, ²First Eye Bank, Indianapolis School of Optometry, Indiana University, ³ School of Optometry, Indiana University, ⁴Price Vision Group, Indianapolis

Purpose: Many forms of cellular stressors are associated with Fuchs Endothelial Corneal Dystrophy (FECD), a blinding disease that affects 4% over the age of 40. However, the causes for the disease onset remain elusive. In this project, we undertook a systematic investigation to identify the causes behind FECD onset using a newly developed mouse model that phenocopies this disease.

Methods: We performed Optical Coherence Tomography and in vivo confocal microscopy of the cornea to assess corneal thickness and cell densities in a newly developed FECD mouse model. We performed ex vivo staining of the corneas to measure proteasomal activities, as well as nitrotyrosine staining to evaluate the levels of oxidative stress. For in vitro experiments, bovine primary corneal endothelial cells (BCECs) in culture were treated with reactive oxygen species (ROS) inducer, proteasome activators, and ER stress activators for 24 hours. We evaluated protein expression changes using JESS immunoassay. Our analysis used antibodies against markers for protein clearance pathways, TGF-b signaling, endothelial-to-mesenchymal transition (EndoMT), ER stress, and apoptosis.

Results: In the FECD mouse model, we discovered decreased proteasomal activities and elevated oxidative stress coincident with the onset of disease phenotypes. Treatment of primary bovine corneal endothelial cells with proteasomal inhibitor resulted in all the known cellular dysfunctions associated with FECD. However, treatments with TBHP, a known ROS inducer, and thapsigargin, an ER stress inducer failed to do so. Inhibiting proteasomal activities in normal mice resulted in the known FECD phenotypes.

Conclusions: Our data strongly suggest that proteasomal inactivity can result in FECD associated features in a mouse model.

Generation of CRISPR-Cas9 Engineered OPTNE478G Human Embryo Stem Cell Line for Investigation into Mitophagy Defects

Maverick Tebbe¹, Saajid Khatri², Michelle Surma³, Arupratan Das^3
1Indiana University School of Medicine, ³Purdue University, ³Department of Ophthalmology, Eugene and Marilyn Glick Eye Institute

Purpose: Optineurin (OPTN) is a mitophagy adaptor protein linking damaged, ubiquitinated mitochondria to autophagosomes for lysosomal degradation. The E50K and E478G OPTN mutations are associated with normal tension glaucoma and ALS, respectively. Currently, it is unknown whether the E50K mutation selectively affects retinal ganglion cells and the E478G mutation selectively affects motor neurons. Given OPTNís role in mitophagy, investigating the mitophagy defects in human stem cell-differentiated (hRGCs) and induced motor neurons (iMNs) harboring these mutations provides an avenue to explore the cellular mechanisms of these diseases.

Methods: This project generated a OPTN-E478G human embryonic stem cell (hESC) line through CRISPR-Cas9 gene editing. gRNA oligomers were annealed and cloned via transformation of DH5α cells. Following sequencing, the gRNA-Cas9-GFP plasmid and donor plasmid with E478G insert were transfected into H7-WT-hESCs. The CRISPR-Cas9 system cut and repaired the double stranded break by homology-directed repair introducing the E478G mutation. GFP-positive colonies were isolated, expanded, and screened by PCR and restriction enzyme digestion. Western blot analysis of E50K and E478G mutants for hRGCs and iMNs were performed following introduction of the mitochondrial uncoupler CCCP, inducing mitophagy.

Results: Insertion of the gRNA into the Cas9-GFP plasmid was confirmed by sequencing. The hESCs were successfully transfected with the E478G plasmid as confirmed by restriction enzyme digestion of SapI and BspHI. Isolation of this stem cell population is ongoing and will be screened with restriction enzymes prior to sequencing. Western blots of NBR1 showed increased levels in WT-hRGCs and iMNs (E50K and E50K corrected), but not in E50K-hRGCs, indicating impaired mitophagy.

Conclusions: Our study reveals distinct mitophagy defects seen in the OPTN mutations E50K and E478G for the RGCs and iMNs. This project lays the groundwork for further studies, including live cell imaging, OPTN activation, and LC3b lipidation, to further define the implications of these mutations on mitophagy.

Ocular Surface Immunoglobulins (OSIG) for the Treatment of Dry Eye Disease

Ethan Teich¹, Emily Krupa², Lee Troughton¹, Marianna Bacellar-Galdino¹, Sandeep Jain¹, Simon Kaja^1,2
1Loyola University Chicago, Department of Ophthalmology, ²Program in Molecular Pharmacology and Therapeutics, Loyola University Chicago

Purpose: , Our previous studies have demonstrated the role of neutrophil death and formation of neutrophil extracellular traps (NETosis) in subtypes of ocular surface disease and shown the potent anti-NETosis effects of ocular surface immunoglobulins (OSIG). OSIG are currently developed and evaluated in clinical trials for the treatment of the signs and symptoms of dry eye disease. The goal of this study was to evaluate the therapeutic efficacy of 5% OSIG in the benzalkonium chloride (BAK)-induced mouse model for dry eye disease.,

Methods: Ocular surface disease was induced by once daily (q.d.) bilateral topical instillation of 0.2% BAK (5 µL/eye) for 5 days in 8-week old male C57BL/6J mice (n=30). Subsequently, animals were randomized into three groups (n=10 each) and eyes were treated with OSIG (FlebogammaÆ 5% DIF), vehicle (5% sorbitol, 0.3% polyethylene glycol, pH 5.5), physiological saline (0.9% sodium chloride), by three times daily (t.i.d.) bilateral topical instillation (5 µL/eye) for 5 days. Ocular surface pathology was quantified by corneal fluorescein staining using 2% fluorescein in saline. Fluorescein staining was evaluated under cobalt blue light enhanced using a Wratten #12 filter and quantified using the established score-based (0 ñ 4) grading system. Data were analyzed in Prism 10 software.

Results: Topical instillation of BAK resulted in development of moderate to severe ocular surface disease (median score: 3). Ocular surface pathology was sustained in saline- and vehicle-treated eyes with median scores of 2.5 (p = 0.78) and 3.5 (p = 0.57), respectively. In contrast, 5% OSIG resulted in a statistically significant reduction of corneal fluorescein staining (median score 2, p < 0.05).

Conclusions: Our data demonstrate efficacy of 5% OSIG in the mouse BAK model for ocular surface disease. Our data are consistent with the potent anti-NETosis activity of OSIG and previous reports on neutrophil pathology in the mouse BAK model. It is likely that species differences in immune epitopes may result in diminished activity of the anti-cytokine antibodies present in OSIG against mouse cytokines. Our ongoing studies are evaluating the cellular and molecular effects of OSIG in the mouse BAK model.

Structural and Functional Changes to Corneal Nerves in Fabry Disease

Steven Trinh, Shyam Chaurasia
¹Medical College of Wisconsin

Purpose: Fabry disease is a rare X-linked lysosomal storage disorder resulting from pathogenic variants in the Gla gene. Patients experience significant damage to the cardiovascular, renal, and neurological systems, along with numerous ocular abnormalities. The cornea, being a densely innervated tissue, is particularly susceptible to neuropathic damage associated with Fabry disease. This study aims to evaluate the effect of genetic variations on corneal nerve fiber structure and function in a rat model of Fabry disease.

Methods: All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the Medical College of Wisconsin, WI, in an established CRISPR-cas9 Gla gene knock-out rat model of Fabry disease [wild-type male (WT-M), wild-type female (WT-F), female heterozygous (HET), male hemizygous (HEMI), and female knockout (KO)]. Structural changes to corneal non-peptidergic nerve fibers were evaluated using isolectin B4 (IB4) immunohistochemical staining across five Fabry disease genotypes. Rat eyes were harvested, cryosectioned, and stained with IB4, followed by examination under a Keyence fluorescent microscope. The corneal fiber staining was quantified using ImageJ software. A Cochet-Bonnet esthesiometer was used to evaluate functional damage to corneal nerve sensitivity. All statistical analyses were performed using GraphPad Prism 10.

Results: We observed significant corneal nerve fiber changes across various Fabry disease genotypes. IB4 staining was largely absent or infrequently sporadic in WT-M and WT-F corneal stromata, in contrast to the knockout Fabry disease genotypes. The female HET genotype exhibited a mild and variable degree of IB4 staining in the corneal stroma, while significantly increased staining was found in HEMI male rats. Notably, the most extensive IB4 staining was observed in female KO Fabry rats, suggesting a genotype-dependent increase in IB4-positive non-peptidergic neurons in the corneal stroma. Corneal esthesiometry revealed corresponding functional deficits, with KO and HEMI rats demonstrating significantly reduced corneal sensitivity compared to WT controls, supporting the structural changes revealed previously.

Conclusions: The loss of the Gla gene results in increased non-peptidergic corneal nerve fiber-specific IB4 staining in the corneal stroma correlating with genotype severity in a rat model of Fabry disease, suggesting a genotype-dependent predisposition to corneal nerve fiber changes. This structural disruption also correlates with reduced corneal sensitivity, suggesting that more severe Fabry disease genotypes confer greater susceptibility to both morphological and functional corneal nerve damage.

Measurements of Rod Density in Patients with Blue Cone Monochromacy

Natalie Ungaretti¹, Muhammad Khan¹, Aliya Siddiqui², Angelos Kalitzeos³, Michel Michaelides³, Joseph Carroll^4
1School of Medicine, Medical College of Wisconsin, Milwaukee, WI, 53226, USA School of Medicine, Medical College of Wisconsin, Milwaukee, WI, 53226, ²Department of Cell Biology, Neurobiology & Anatomy, Medical College of Wisconsin, Milwaukee, WI, 53226, ³Institute of Ophthalmology, University College London, London, UK, ⁴Department of Ophthalmology & Visual Sciences, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

Purpose: Blue cone monochromacy (BCM) is a cone dysfunction syndrome characterized by a loss of middle-wavelength (M) and long-wavelength (L) cone function. Patients typically present with impaired color discrimination, nystagmus, light-sensitivity, and decreased visual acuity. Adaptive optics scanning light ophthalmoscopy (AOSLO) has been used to examine cone structure in BCM populations, however there has been no quantitative assessment of the rod mosaic. Motivated by previous reports describing measured rod-defects in BCM and the general lack of anatomical information on the BCM rod photoreceptor mosaic, we used AOSLO to examine rod structure in patients with BCM and quantify rod density across multiple retinal eccentricities.

Methods: The photoreceptor mosaic was imaged with confocal and non-confocal AOSLO in 4 patients with genetically confirmed BCM. Analysis was performed on 18 150x150µm regions of interest (ROIs) located between 3∞-13∞ eccentricities. ROIs were extracted to include a confocal & split-detection image at the same location. All cones within one ROI were manually counted using Mosaic software to obtain total cone counts and bound cone area. Cone Segmentation software was utilized to segment 10-20 cones in each ROI and obtain an average cone area using split-detection images. Within respective confocal ROIs, 100-200 rods were counted to obtain an estimate of rod near-neighbor distance (NND). Rod NND, total bound cone area, total cones, and average cone area were used to derive rod density. Individual ROI measurements of rod density were averaged to provide a mean value for eccentricities at 3∞, 5∞, 7∞, 9∞, and 11∞ superior from the fovea, and 3∞, 5∞, 7∞, 9∞,11∞, and 13∞ temporal from the fovea.

Results: Rod density was 92,467 rods/mm≤ at 3∞ superior (2 ROIs), 108,978 rods/mm≤ at 5∞ superior (2 ROIs), 111,233 rods/mm≤ at 7∞ superior (2 ROIs), 94,270 rods/mm≤ at 9∞ superior (1 ROI), and 145,795 rods/mm≤ at 11∞ superior (1 ROI). Along the temporal meridian we observed106,867 rods/mm≤ at 3∞ temporal (3 ROIs), 103,575 rods/mm≤ at 5∞ temporal (2 ROIs), 130,069 rods/mm≤ at 7∞ temporal (2 ROIs), 86,807 rods/mm≤ at 9∞ temporal (1 ROI), and 122,936 rods/mm≤ at 11∞ temporal (across 1 ROI), and 114,373 rods/mm≤ at 13∞ temporal (across 1 ROI). While some values were consistent with previously reported in vivo and ex vivo normative rod density data, other values were greater than previously reported values.

Conclusions: Rod density was 92,467 rods/mm≤ at 3∞ superior (2 ROIs), 108,978 rods/mm≤ at 5∞ superior (2 ROIs), 111,233 rods/mm≤ at 7∞ superior (2 ROIs), 94,270 rods/mm≤ at 9∞ superior (1 ROI), and 145,795 rods/mm≤ at 11∞ superior (1 ROI). Along the temporal meridian we observed106,867 rods/mm≤ at 3∞ temporal (3 ROIs), 103,575 rods/mm≤ at 5∞ temporal (2 ROIs), 130,069 rods/mm≤ at 7∞ temporal (2 ROIs), 86,807 rods/mm≤ at 9∞ temporal (1 ROI), and 122,936 rods/mm≤ at 11∞ temporal (across 1 ROI), and 114,373 rods/mm≤ at 13∞ temporal (across 1 ROI). While some values were consistent with previously reported in vivo and ex vivo normative rod density data, other values were greater than previously reported values.

A comparison of the efficacy and safety of a modified Baerveldt drainage implant versus the Ahmed glaucoma valve after 1 year of follow-up

Joel VandeLune¹, Kristin Davis¹, Quinton Christensen¹, Danielle Pellack¹, Ronak Singh¹, Hend Al-Kaylani¹, Matthew Meyer¹, Patrick Donegan¹, Erin Boese², Andrew Pouw^2
1Carver College of Medicine, University of Iowa, Iowa City, ²Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA

Purpose: , There is limited knowledge of the efficacy and safety of a novel Baerveldt glaucoma implant modification involving placement of a ventilating mattress suture to allow for early flow. We performed a retrospective cohort study to compare the efficacy and safety of modified Baerveldt glaucoma implants (BGI) to unmodified Ahmed glaucoma valves (AGV) in lowering postoperative intraocular pressure (IOP).,

Methods: Patients aged <18 years with open-angle glaucoma who underwent modified BGI or unmodified AGV at the University of Iowa with at least one year of documented follow up were included. Extracted data included IOP, visual acuity, number and types of medications, and adverse events. A linear mixed effects model adjusted to account for baseline differences between groups in diabetes, steroid use and quadrantic implant location was used to assess change in IOP, logMAR visual acuity and optic nerve cup to disc ratio over time since surgery, while a similarly adjusted Poisson mixed-effects model was used to assess change in medication number over time. Surgical success and reoperation rates were assessed with Kaplan-Meier curves. Four different criteria for surgical success were utilized and compared.

Results: The modified Baerveldt was found to have better surgical success according to reoperation criteria as well as the medium and conservative survival criteria as time went on. There were no statistically significant differences in surgical complications or logMAR visual acuity outcomes between both groups. ,

Conclusions: This pilot study suggests that the modified Baerveldt has a similar safety profile as the Ahmed valve, with potentially better efficacy and lower reoperation rates at one year after operation. Future directions for this work include a prospective randomized controlled trial that can further mitigate selection bias differences.

Evaluating the Safety of Multimodal Therapy for Uveal Melanoma in Retinal Pigment Epithelium

Audrianna Wu¹, Shyam S. Chaurasia¹, Aparna Ramasubramanian², Anju E. Thomas^1
1Ophthalmology and Visual Sciences, Medical College of Wisconsin, ²Pediatric Ophthalmology, Medical College of Wisconsin

Purpose: Uveal melanoma (UM) is a rare and aggressive form of cancer which commonly metastasizes from the uvea to other parts of the body. Due to its resistance to cytotoxic drugs, UM is often treated with radiation therapy, which often introduces complications with vision and significantly impacts patients’ quality of life and emotional well-being. Topotecan hydrochloride (a topoisomerase one inhibitor) has been found to be an effective chemotherapy drug in several cancers, and has been found to be well tolerated as an episcleral plaque for retinoblastoma patients. Preliminary studies suggest that combining topotecan (a topoisomerase inhibitor) with SP-141 (an MDM-2 inhibitor) may be effective for treating UM. This study examines the safety of this therapy in retinal pigment epithelial cells.

Methods: Human retinal epithelial cells (ARPE-19) were cultured and Topotecan (10, 25, 50, 100, 200, 500, 1000, 1500, 2000, and 5000 nM) and SP-141 (100, 250, 500, 1000, 1500, 2500, 10000, and 20000 nM) were administered for 24, 48, and 72 hour durations. The cell viability, cytotoxicity, and proliferative effects of treatments were assessed via MTT, PrestoBlue, and LDH assays.

Results: Preliminary findings suggest that combining topotecan with SP-141 was effective in the UM cell line. Results from the cytotoxicity assays suggest that this multimodal therapy is safe for ARPE-19 cells, which exhibited relative stability and comparable or increased proliferative capacity when treated with topotecan and SP141, especially at the lower concentrations utilized in synergistic treatment.

Conclusions: This multimodal therapy involving topotecan and SP141 was found to be safe in ARPE-19 cells, showing limited adverse effects on cellular viability or proliferative capacity. These findings suggest that this synergistic treatment will have limited adverse effect on the outer blood-retinal barrier, which is essential to providing protection and nourishment to the outer retina.

Stage-Dependent Immune Activation in Normal-Tension Glaucoma

Shuyu Xian¹, Merri-Grace Jones¹, Jordan Mayberry^1,2, Markus H. Kuehn^1,2
1Department of Ophthalmology and Visual Sciences, The University of Iowa, Iowa City, IA 52242, ²Iowa City VA Center for the Prevention and Treatment of Visual Loss, Iowa City, IA, 52246

Purpose: Autoimmune and neuroinflammatory processes are increasingly recognized as contributors to retinal ganglion cell (RGC) degeneration in glaucoma. We aim to investigate immune-related mechanisms underlying glaucomatous neurodegeneration by combining in vivo PBMC transfer experiments with ex vivo immunophenotyping of patient-derived PBMCs using flow cytometry.,

Methods: We transferred PBMCs isolated from glaucoma patients into NOD-SCID mice to assess their impact on retinal axon integrity. Mice receiving cells from patients with normal-tension glaucoma (NTG) exhibited progressive axonal loss, despite only mild intraocular pressure elevation. Notably, mice receiving PBMCs from patients with moderate-stage glaucoma had significantly fewer axons than those receiving cells from other disease stages. In parallel, flow cytometry analysis of patient PBMCs revealed disease stage-dependent alterations in immune profiles. Specifically, moderate-stage NTG patients exhibited a marked increase in HLA-DR expression among NK cell, suggesting a potential role in antigen presentation and immune activation. Additionally, a population of CD8+ NKT cells with high expression of KLRG1 which is associated with enhanced effector function and cytotoxicity was found in moderate-stage NTG patients.

Results:

Conclusions: Our findings suggest that glaucoma-associated immune dysregulation contributes to RGC degeneration. Immune cells from glaucoma patients can drive axonal damage in recipient mice, supporting a role for systemic immune mechanismsâ€including activated NK and NK T cells in glaucomatous pathology.