This project explores the use of a novel sustained release mucoadhesive micelle-based drug delivery system in combination with 0.005% latanoprost (LTP) on our partially open angle mouse model of glaucoma (AP-2β TMR-KO). We previously tested for LTP treatment in our model and found a reduction in intraocular pressure (IOP) 20 minutes following treatment. This information led us to investigate the long-term effect of LTP treatment and micelle loaded with LTP (MLTP) treatment in our model. We hypothesized that the MLTP treatment would be more effective in reducing IOP and preventing glaucomatous effects than LTP treatment alone in the AP-2β TMR-KO mice. The MLTP groups of animals (wildtype and mutant) were treated every 3 days, and this was compared with animals treated with LTP daily as well as animals treated every 3 days with LTP alone for comparison’s sake for 60 days. IOP measurements were taken every 3 days. Following long term LTP treatment alone, mutant mice showed a consistent decrease in their baseline IOPs with a significant reduction in baseline IOP at 35 days of treatment across all cohorts (P<0.0001). In comparison, mutants treated with MLTP exhibited an even greater reduction in baseline IOP following long term treatment. After the treatment period, mice were euthanized, and their eyes were enucleated, fixed, sectioned, and stained for retinal ganglion cells (RGCs) using Brn3a. Mutant mice exhibited a significant decrease in RGC cell number when compared to wildtype, and this loss was not rescued by treatment with LTP. However, mutants treated with MLTP demonstrated significant RGC cell protection compared to eyes of untreated mutants, as well as everyday LTP treated mutants. / Thesis / Master of Science (MSc) / An effective treatment strategy is required to prevent irreversible blindness caused from glaucoma. Unfortunately, compliance with current medications is extremely poor, as they require frequent administration due to their low ocular bioavailability and short-term effect. As such, this thesis aims to explore an alternative drug delivery approach in a partially open angle mouse model of glaucoma to prevent the worsening of glaucoma and ultimately improve patient compliance.

Indiana University-Purdue University Indianapolis (IUPUI) / Glaucoma is a leading cause of blindness characterized by the progressive loss of retinal ganglion cells (RGCs), essentially severing the connection between the eye and the brain. Among many underlying causes of the disease, mutations in the Optineurin (OPTN) gene result in severe RGC neurodegeneration in the absence of elevated intraocular pressure, providing a novel opportunity to study molecular mechanisms that lead to RGC neurodegeneration associated with glaucoma. Efforts of this study establishing a human pluripotent stem cell (hPSC)-derived in vitro disease model by inserting OPTN(E50K) mutation via CRISPR/Cas9 genome editing and investigate the cellular mechanisms of RGC neurodegeneration associated with glaucoma. OPTN(E50K) RGCs revealed neurodegeneration phenotypes, including downregulation of RGCs transcription factors, neurite retraction, and hyperexcitability, suggesting that OPTN(E50K) RGCs can serve as an appropriate disease model to study glaucoma-associated neurodegeneration. Since OPTN serves a primary role as an autophagy receptor, we further hypothesized that the OPTN(E50K) mutation disrupts autophagy in RGCs, and modulation of autophagy by mammalian target of rapamycin (mTOR)-independent pathways can preserve RGC phenotypes by maintaining mTOR signaling. OPTN(E50K) RGCs exhibited a higher number of OPTN puncta along with an overall reduced expression of OPTN protein, indicating a gain of toxic protein accumulation or loss of protein function. Furthermore, OPTN(E50K) RGCs revealed an accumulation of the autophagosome protein LC3 in a punctal manner as well as increased expression of lysosomal proteins, suggesting a disruption of degradation pathway in autophagosome and lysosome fusion. As mTOR complex 1 (mTORC1) signaling serves as a negative regulator of autophagy, a downregulation of mTORC1 signaling via activation of stress sensor adenosine monophosphate-activated protein kinase (AMPK) was observed as a possible compensatory mechanism for autophagy deficits in OPTN(E50K) RGCs. Pharmacological inhibition of mTOR in wild-type hRGCs resulted in similar disease-related phenotypes, while preservation of the mTOR pathway in OPTN(E50K) RGCs by treatment with the mTOR-independent autophagy modulator trehalose cleared OPTN accumulated puncta, preserving mTORC1 signaling, as well as rescuing neurodegenerative phenotypes. To further validate these associations in an animal model, the microbead occlusion mouse model was established by injection of magnetic microbeads in the anterior chamber to block aqueous outflow resulting ocular hypertension. In agreement with our findings in hRGCs, a decrease in mTOR signaling associated with an increase in the expression of autophagy-associated proteins was observed in RGCs in the microbead occlusion model. Additionally, these disease-related phenotypes were observed specifically within RGCs but not cortical neurons with an underlying OPTN(E50K) mutation, demonstrating that autophagy represents an essential pathway in RGCs to maintain homeostasis, and selective disrupt of autophagy in RGCs leads to neurodegeneration. Taken together, the results of this study highlight an essential balance between autophagy and mTORC1 signaling that is essential for the homeostasis of RGCs, while disruption to these signaling pathways contributes to neurodegenerative features in glaucoma. These results also demonstrated the ability to pharmacologically intervene to experimentally manipulate these pathways and rescue neurodegenerative phenotypes, providing a potential therapeutic target to prevent glaucoma-associated neurodegeneration.

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Background: Primary open angle glaucoma (POAG) is the most common form of glaucoma in the United States and is the leading cause of irreversible blindness in African Americans. Although this is the case, there are no current primary care screening guidelines for this condition. The USPSTF cites that there is insufficient evidence to assess the balance of benefits and harms of screening for POAG in adults. This condition disproportionately affects African American and Hispanic patients. A systematic review performed earlier this year highlighted disparities in research participation among POAG clinical trials. No similar studies have been pursued outside of clinical trials. This is problematic because screening guidelines are heavily influenced by the literature related to the topic. Methods: A rapid scoping review of the literature will be performed with a particular focus on demographic data. Data was sourced from the included studies used in the systematic review performed in 2022 to inform the current USPSTF guidelines. Data collection will consist of the compilation of demographic data within each of the studies on a spreadsheet and will subsequently be analyzed according to subgroup corresponding to study type. Results: A total pooled sample of 16659 participants was obtained from the 16 included studies. After exclusion of an outlier study, total research participation across all studies was 27.9% and 5.5% for Black and Hispanic individuals, respectively. In clinical trials, 26.9% and 6.9% were Black and Hispanic individuals, respectively. Lastly, among nonclinical trials, 28.7% and 3.3% were Black and Hispanic individuals, respectively. Conclusions: Primary open angle glaucoma is a public health issue. The current recommendations for POAG screening are based on the currently available literature. However, it has been previously shown that disparities exist in research participation among Black and Latino individuals in clinical trials. The findings within this study corroborate those findings as well as highlight that disparities in research participation and representation persist among nonclinical trial research studies. This thesis underscores the ongoing need for equitable efforts in POAG research across all studies. With these efforts, recommendations for screening may be properly elucidated to inform more equitable care and identification of this disease. / Urban Bioethics

Yes / We have read with interest the recent article by Sponsel et al.1 There is much evidence that glaucomatous damage occurs at the optic nerve head,2 and therefore we were surprised by the authors' conjecture that there may be a central mechanism that preserves the binocular visual field in advanced glaucoma.

Yes / Purpose: We evaluated variability and conviction in tracing paths of retinal nerve fiber bundles (RNFBs) in retinal images, and compared traced paths to a computational model that produces anatomically-customized structure–function maps. Methods: Ten retinal images were overlaid with 24-2 visual field locations. Eight clinicians and 6 naïve observers traced RNFBs from each location to the optic nerve head (ONH), recording their best estimate and certain range of insertion. Three clinicians and 2 naïve observers traced RNFBs in 3 images, 3 times, 7 to 19 days apart. The model predicted 10° ONH sectors relating to each location. Variability and repeatability in best estimates, certain range width, and differences between best estimates and model-predictions were evaluated. Results: Median between-observer variability in best estimates was 27° (interquartile range [IQR] 20°–38°) for clinicians and 33° (IQR 22°–50°) for naïve observers. Median certain range width was 30° (IQR 14°–45°) for clinicians and 75° (IQR 45°–180°) for naïve observers. Median repeatability was 10° (IQR 5°–20°) for clinicians and 15° (IQR 10°–29°) for naïve observers. All measures were worse further from the ONH. Systematic differences between model predictions and best estimates were negligible; median absolute differences were 17° (IQR 9°–30°) for clinicians and 20° (IQR 10°–36°) for naïve observers. Larger departures from the model coincided with greater variability in tracing. Conclusions: Concordance between the model and RNFB tracing was good, and greatest where tracing variability was lowest. When RNFB tracing is used for structure–function mapping, variability should be considered.

Yes / Purpose: We have developed customized maps that relate visual field and optic nerve head (ONH) regions according to individual anatomy. In this study, we aimed to determine feasible map resolution for research use, and to make a principled recommendation of sector size for clinical applications. Methods: Measurement variability in fovea–ONH distance and angle was estimated from 10 repeat OCT scans of 10 healthy people. Errors in estimating axial length from refractive error were determined from published data. Structure–function maps were generated, and customized to varied clinically-plausible anatomical parameters. For each parameter set (n = 210), 200 maps were generated by sampling from measurement/estimation error distributions. Mapped 1° sectors at each visual field location from each parameter set were normalized to difference from their mean. Variation (90% ranges) in normalized mapped sectors represents the precision of individualized maps. Results: Standard deviations of repeated measures of fovea–ONH distance and angle were 61 μm and 0.97° (coefficients of variation 1.3% and 12.0%, respectively). Neither measure varied systematically with mean (Spearmans's ρ = 0.26, P = 0.47 for distance, ρ = −0.31, P = 0.39 for angle). Variation (90% ranges) in normalized mapped sectors varied across the visual field and ranged from 3° to 18° when axial length was measured accurately, and from 6° to 32° when axial length was estimated from refractive error. Conclusions: The 90% ranges represent the minimum feasible ONH sector size at each visual field location. For clinical use an easily interpretable scheme of 30° sectors is suggested.

No / Purpose To determine whether the structure–function relationship in glaucoma can be strengthened by using more precise structural and functional measurements combined with individualized structure–function maps and custom sector selection on the optic nerve head (ONH). Design Cross-sectional study. Participants One eye of each of 23 participants with glaucoma. Methods Participants were tested twice. Visual fields were collected on a high-resolution 3° × 3° grid (164 locations) using a Zippy Estimation by Sequential Testing test procedure with uniform prior probability to improve the accuracy and precision of scotoma characterization relative to standard methods. Retinal nerve fiber layer (RNFL) thickness was measured using spectral-domain optical coherence tomography (OCT; 4 scans, 2 per visit) with manual removal of blood vessels. Individualized maps, based on biometric data, were used. To customize the areas of the ONH and visual field to correlate, we chose a 30° sector centered on the largest defect shown by OCT and chose visual field locations using the individualized maps. Baseline structure–function correlations were calculated between 24-2 locations (n = 52) of the first tested visual field and RNFL thickness from 1 OCT scan, using the sectors of the Garway-Heath map. We added additional data (averaged visual field and OCT, additional 106 visual field locations and OCT without blood vessels, individualized map, and customized sector) and recomputed the correlations. Main Outcome Measures Spearman correlation between structure and function. Results The highest baseline correlation was 0.52 (95% confidence interval [CI], 0.13–0.78) in the superior temporal ONH sector. Improved measurements increased the correlation marginally to 0.58 (95% CI, 0.21–0.81). Applying the individualized map to the large, predefined ONH sectors did not improve the correlation; however, using the individualized map with the single 30° ONH sector resulted in a large increase in correlation to 0.77 (95% CI, 0.47–0.92). Conclusions Using more precise visual field and OCT measurements did not improve structure–function correlation in our cohort, but customizing the ONH sector and its associated visual field points substantially improved correlation. We suggest using customized ONH sectors mapped to individually relevant visual field locations to unmask localized structural and functional loss.