Eye / Vision
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Item Assessing Metabolic Changes in the Retina & Optic Nerve During Glaucoma(2024-03-21) Sepke, Katelynn; Morgan, Autumn; Inman, DenisePurpose: Glaucoma is an optic neuropathy characterized by retinal ganglion cell (RGC) death and optic nerve degeneration. Glial cells such as astrocytes form a metabolic unit with neurons to exchange metabolic substrates and neurotransmitters. When exposed to ocular hypertension (OHT), this metabolic unit is disrupted as astrocytes undergo morphological changes in response to increased pressure. ONHAs also reduce their GLUT1 expression, further exacerbating their metabolic function. It is unknown how these changes impact RGC axon structure and function, so we aim to gain insight into the metabolic relationship between glia and neurons during glaucoma. We hypothesize that glaucoma induces metabolic strain in optic nerve head astrocytes (ONHAs), preventing the exchange of metabolites between neurons, ultimately causing a decline in RGC structure and function. Methods: We have taken a two-sided approach to studying these neural-glial interactions. First, we have induced OHT as well as glucose transport inhibition in ONHAs in vivo to examine the effect of pressure-induced stress on metabolism and the visual system. Currently, we are working in vitro to study the metabolic exchange between RGCs and ONHAs co-cultured in microfluidic chambers when the ONHAs are exposed to biaxial strain as well as GLUT1 KO. Results: Preliminary results in vivo have shown that OHT and glucose transport inhibition in ONHAs disrupt anterograde transport. However, RGCs can compensate for glucose transport inhibition in astrocytes by upregulating GLUT3 and MCT2.In vitro we expect to see RGCs respond to alterations in ONHA metabolism, similarly, upregulating their lactate transporters and relying on mitochondrial metabolism to maintain their energetic needs. Conclusion: Using this model will allow us to directly observe the metabolic changes in the neural-glial unit induced by glaucoma, ultimately providing us insight into targets for future glaucoma therapies.Item Enhanced Protection of Retinal Ganglion Cells against Ischemia/Reperfusion Injury and Neurotrophic Factor Deprivation with Compound SA-10(2024-03-21) Pham, Jennifer; Zhang, Wei; Le, Kim-Tuyen; Kodati, Bindu; Amankwa, Charles; Tran, Ashley; Acharya, Suchismita; Stankowska, DorotaPurpose: Glaucoma is one of the leading causes of irreversible blindness worldwide. In glaucoma, the retinal ganglion cells (RGCs), which transmit visual signals to the brain, undergo neurodegeneration, leading to a gradual loss of vision. Oxidative stress is the imbalance between antioxidant activity and free radical production, which has been shown to be associated with glaucomatous RGC degeneration. In this study, we investigated the potential of SA-10, a hybrid nitric oxide donating and sulfone reactive oxygen species (ROS) scavenging molecule, to promote the survival of RGCs against glaucomatous damage. We investigated its neuroprotective effects following retinal ischemia/reperfusion (I/R) injury in mice and ex vivo following neurotrophic factor (NF) deprivation in human retinal explants (HREs). Methods: Acute I/R injury was induced in C57BL/6J mice (n=2-3 mice/group/sex) through intracameral pressure elevation to 120 mmHg for 1 hour. The mice were pre-treated topically with a PLGA nanosuspension of SA-10 (1% SA-10-NPs) and treated for 14 days (7 doses) after I/R injury. The obtained retinal sections were stained with anti-heme oxygenase-1 antibody (Hmox1, a marker of protective response to oxidative stress) to quantify its expression levels. H&E sections were used to measure retinal thickness. In another set of experiments, biopsy punches from HREs (n=3 donors) were isolated and treated with either SA-10 [10 µM] or vehicle and maintained without NF for 7 days ex vivo (DEV). Four control punches were collected on day 0 (0 DEV). After 7 days, HREs were immunostained with RBPMS (RGC-specific marker), and cell survival was analyzed. Analysis of Variance (ANOVA) was performed for all experiments. Results: In the nerve fiber and ganglion cell layers (ganglion cell complex, GCC), I/R injury caused a significant reduction in Hmox1 expression in female (79.8%, p<0.05) and male (54.5%, p<0.05) mice, compared to the sham control. Additionally, I/R injury led to a decline in GCC thickness by 27.5% (p=0.23) in females and 32.7% (n=2) in males. However, treatment with SA-10-NPs increased Hmox1 expression by 4.2-fold (p<0.05) in females and by 0.5-fold (p=0.43) in males. SA-10-NPs also preserved GCC thickness by 17.6% (p=0.68) in females and 27.1% in males. In human retinal explants, the 7 DEV vehicle-treated group had a significant loss in RGCs by 45.2% (p<0.01) compared to 0 DEV. In contrast, SA-10 treatment enhanced RGC survival at 7 days with an 83.1% (p<0.01) higher RGC counts than the vehicle group. Conclusions: SA-10 and its nanosuspension exhibited significant neuroprotective effects by enhancing Hmox1 expression, preserving retinal thickness, and promoting RGC survival, highlighting its potential as a therapeutic candidate for glaucoma and ischemic stroke.Item Unraveling the Molecular Nexus: Obstructive Sleep Apnea and Glaucoma in a Rat Model(2024-03-21) Donkor, Nina; Mabry, Steve; Wilson, E. Nicole; Gardner, Jennifer J.; Bradshaw, Jessica; Cunningham, Rebecca; Inman, DenisePurpose: Obstructive sleep apnea is a chronic sleep disorder characterized by recurring complete or partial upper airway occlusion. Over the past decade, meta-analyses have established a correlation between this disorder and glaucoma, an ocular neurodegenerative disease, and a leading cause of blindness. However, the link between these pathologies remains elusive. Understanding the mechanisms involved could influence treatment options and reduce the rate of vision loss associated with glaucoma. Using a rat model of sleep apnea, chronic intermittent hypoxia (CIH), we tested the hypothesis that mild sleep apnea initiates morphologic and metabolic changes in the retina that resemble glaucoma. Methods: Rats were randomly assigned to normoxic or CIH groups. The CIH group was exposed to periodic hypoxia during their sleep phase, simulating mild sleep apnea, with oxygen reduction from 21% to 10% and reoxygenation in 6-minute cycles over 8 hours/day for 14 days. The normoxic group experienced similar conditions without changes in oxygen concentration. Subsequently, the eyes were enucleated, and the retina was evaluated for oxidative stress, inflammatory markers, metabolic changes, and hypoxic response modulation using immunohistochemistry and capillary electrophoresis. Results: Immunofluorescence revealed increased expression of 8-OHdG, indicating oxidative stress (nucleic acid damage), as well as the cytokine TNF-α in the CIH group retina compared to controls. No statistically significant differences were observed in HIF-1α protein levels. SIRTUIN-1, a regulator of HIF-1α expression, and the levels of pyruvate dehydrogenase kinase-1 and lactate dehydrogenase-A showed no significant differences between normoxic and CIH groups. Conclusion: The increased oxidative stress and inflammation observed suggest that CIH induces a response in the retina with features shared by early-stage glaucoma. However, the anticipated upregulation of HIF-1α and its targets did not occur, suggesting a greater reduction in oxygen concentration or a longer-term CIH interval may be necessary to observe canonical hypoxic response. Keywords: glaucoma, sleep apnea, chronic intermittent hypoxia, inflammation, oxidative stressItem The Reliability of Artificial Intelligence in Prioritizing Management of Diabetic Macular Edema: A Comparative Study with 2 Retina Specialists(2024-03-21) Morcilla, Jericho; Cao, Jessica; Fan, Kenneth; Rahman, Effie; Ngo, Khang; Patel, Sagar; Chaudhary, Varun; Wykoff, CharlesPurpose There are currently no studies evaluating an Artificial Intelligence (AI) Large Language Model’s (LLM) reliability for ordinally prioritizing patients. In this study, we assess the ChatGPT Plus model (GPT-4) for prioritizing Diabetic Macular Edema (DME) patients, comparing its performance to that of two board-certified retina specialists (RS). We also investigate 2 additional questions: key variables for evaluators (GPT-4 and 2 RS) in DME prioritization, and the impact of incomplete clinical profiles on inter-evaluator agreement. Methods We used anonymized DME data from Retina Consultants of Texas to create 28 patient profiles. These profiles were divided into 4 sets based on ascending Diabetic Retinopathy (DR) severity (Set 1), Central Subfield Thickness (CST) (Set 2), modified Best Corrected Visual Acuity (BCVA) (Set 3), and a randomly organized control set (Set 4). We intentionally modified BCVA in Set 3, resulting in clinically incomplete patient profiles. 2 RS and GPT-4 prioritized patients in each set (e.g., Set 1) according to least to most treatment needs. We calculated the mean Cohen's Kappa (k) across all 4 sets to measure agreement between the 2 RS and the 2 RS with GPT-4 (k = 0.40–0.59 (weak), 0.60–0.79 (moderate), 0.80–0.90 (strong), >0.90 (almost-perfect)). Median RS evaluations were used to calculate individual set k as well as mean set k with GPT-4. Results Evaluations by the 2 RS (denoted RS1 and RS2) and GPT-4 show moderate agreement (Set 3 excluded: RS1-GPT-4 mean set k = 0.631; RS2-GPT-4 mean set k = 0.68). When using the median of RS evaluations, GPT-4's agreement with RS increased within the moderate range (Set 3 excluded: Median RS-GPT-4 mean set k = 0.77). Agreement between the 2 RS was weak (Set 3 excluded: RS1-RS2 mean set k = 0.48). The inclusion of Set 3 in mean set k calculations showed no clear impact. GPT-4 responses/explanations did not acknowledge clinical ambiguities in Set 3, noted by both RS in an optional comment box. Individual k values for Sets 1, 2, 3, and 4 were as follows: 0.58, 0.79, 0.72, 0.93. Conclusions The results reveal that GPT-4 shows promise in reliably prioritizing DME patients compared to RS. Ultimately, GPT-4 achieved moderate-strong (k ≥ 0.60) agreement with RS in DME prioritization. Interestingly, GPT-4’s increased agreement with median RS evaluations could indicate an ability to predict the RS consensus despite moderate disagreement between the 2 RS themselves. This is interesting considering an LLM’s inherent function — mathematically predict the average human response to a given text. This further supports GPT-4’s potential as a clinical tool, offering a grounded perspective in decision-making to assist more nuanced human judgment. Incomplete clinical profiles did not clearly impact agreement, possibly suggesting GPT-4’s adaptability. However, GPT-4 failed to recognize ambiguities in patient profiles that both RS noted. Therefore, human specialists may be better equipped to prevent erroneous decisions when evaluating incomplete or exceptional patient cases. Regarding key variables for prioritization, almost-perfect agreement in the control set (Set 4) warrants investigation into additional variables beyond what is explored in this study.Item Characterization for In-situ Ocular Implant Formation(2024-03-21) Khuu, Megan; Kastellorizios, MichailPurpose Different diseases of the eye require intravitreal injections for treatment. While intravitreal injections are quick procedures, their requirement for routine in-office visits makes them inconvenient. An alternative is intra-ocular implants, designed to stay within the eye, these allow controlled release of therapeutic drugs to the posterior segment of the eye. Intra-ocular implants require symmetrical shapes for even and steady release of the drug. To study how well in-situ implants form, vitreous humor substitutes were made using varying ratios of hyaluronic acid (HA) and polyvinyl alcohol (PVA) within phosphate-buffered saline (PBS). These substitutes have surface characteristics that can be used as a potential marker to predict how symmetrical the in-situ implant forms. Here, we use contact angles to help characterize their interaction. Methods Vitreous humor substitutes were made using 0.01 g/mL solutions of HA and PVA in PBS. These solutions were mixed to create the following ratios of HA:PVA: 1:3, 1:1, and 3:1. To make the implant, 1-Methyl-2-pyrrolidone was dissolved in polylactic-co-glycolic acid in a 1.96:1 ratio. Films were prepared on glass slides using the solutions to take contact angles of the implant. Contact angles were taken using an optical goniometer at 30.2 frames per second for 10 seconds, where it was set to dispense 2 µL each time. For in-situ implant formation, the implant was injected into 5 mL wells of each solution (PBS, HA, PVA, 1:3, 1:1, and 3:1) at a 90-degree angle using a 20 gauge needle at 1.2 cm deep. In-situ implant formation was repeated 4 times. Results The following contact angles were taken at frame 300 and are the average of at least 10 runs. The average contact angle for water on a plain glass slide and the implant on a plain glass slide, PVA film, 1:3 film, 1:1 film, 3:1 film, and on the HA film was 26.13 ± 3.79, 28.57 ± 2.29, 32.65 ± 5.75, 32.41 ± 4.21, 35.02 ± 3.03, 37.20 ± 4.92, and 40.71 ± 5.22 degrees respectively. In-situ implant formation was consistently the most symmetrical within HA and the 1:3 hybrid, though still in irregular yet compact shapes. In-situ implant formations within the other solutions were randomly shaped with tendrilous protrusions that would coil upon themselves. Conclusions Contact angles for all solutions were found to be statistically different using T-tests. The different contact angles allow us to manipulate the interactions by varying the ratio of HA and PVA to make an environment most suitable for intra-ocular implant formation. Interestingly, the 1:3 substitute did not follow the direct relationship found between the amount of HA present in each vitreous humor substitute and their contact angles. Despite having one of the least amounts of HA, it provided a better environment for implant formulation. Thus, there must be an interaction between HA, PVA, and the implant that is optimal at 1:3. More is to be done with the 1:3 hybrid and implant by repeating the experiment with serial dilutions to help us determine which patient populations may be most suitable to this type of treatment.Item Role of beta-catenin in glaucomatous optic neuropathy(2024-03-21) Rana, JenilThe Wnt/beta-catenin signaling pathway comprises a family of proteins that plays a critical role in developing adult tissue such as the optic nerve head by mediating cell proliferation, survival, behavior, and fates. This pathway is tightly controlled, and faulty regulation of this pathway has been shown to cause ocular malformations and ultimately a glaucomatous phenotype. Despite the significance of this pathway in glaucoma, the specific mediators involved in its pathophysiology remain poorly understood. This systemic review aims to analyze beta-catenin's role in the optic nerve head (ONH) cells and in the development of glaucoma. This review includes assessments of primary studies, meta-analyses, and narrative reviews. Additionally, to further understand the role of beta-catenin in OHN cells, immunohistochemistry will be performed to determine its expression in ONH cells. The results of this experiment will be updated in the final literature. In summary, our review shows that glaucomatous injury to the ONH downregulates Wnt signaling and the expression of beta-catenin. This leads to fibrosis of the ONH and an eventual elevation in intraocular pressure. This review also discusses the practical implications of these findings and how they can be used to develop potential therapeutics for glaucoma.Item Neuroprotective Effects of DHED Eyedrops Protects Visual Function Despite Elevated IOP in an Ocular Hypertension Animal Model(2024-03-21) Kapic, Ammar; Neagu, George; Nguyen, Vien; Zaman, Khadiza; Prokai, Laszlo; Prokai-Tatrai, KatalinPurpose: Glaucoma remains the second leading cause of irreversible blindness and is often associated with chronically elevated intraocular pressure (IOP) leading to ocular hypertension (OHT). All of the currently accepted therapies attempt to reduce the elevated IOP. However, despite intervention, studies show progressive neuronal damage continues in the retina and may extend to the rest of the visual system, leading to additional neuropathologies. Interest in utilizing 17β-estradiol (E2) for its neuroprotective effects has become increasingly recognized; however, due to its side effects, such as cancer risk and feminization in males, its application as a therapy is limited. Our lab has developed the estrogen prodrug, 10β, 17β-dihydroxyestra-1,4-dien-3-one (DHED), which remains inactive until its CNS-specific metabolism via short-chain reductase into the active compound E2. This study aims to elucidate the pleiotropic effects of E2 derived from DHED as a potential therapy for preserving the visual system under OHT. We hypothesize that topical application of DHED will prevent the neurodegenerative effects of chronic OHT on the retina and maintain visual function. Methods: OHT was induced in 8 to 10-month-old male Brown Norway rats via hypertonic saline injection into an episcleral vein. IOP was measured via a tonometer (Tonolab) to confirm sustained elevated IOP post-surgery and throughout the treatment period. DHED was topically delivered through eyedrops (20% 2-hydroxylpropyl-beta-cyclodextrin, 0.1% DHED, and saline) once per day. Visual acuity (VA) and contrast sensitivity (CS) were measured using the OptoMotry system with the OptoMotry 1.7 software (Cerebral Mechanics Inc). VA and CS were assessed using the "Rat" preset, and gratings were adjusted using a simple staircase progression. A fixed frequency of 0.272 c/D was chosen for the CS based on prior studies. Observers for the OptoMotry tests were blinded. The eyes and optic nerves were collected and fixed for RGC and axon counts, respectively. Seminal vesicles were collected and weighed to assess peripheral estrogenic effects. Results: The IOP was elevated by 53 % ± 15% and was sustained in both vehicle and DHED-treated groups with no differences between treatment groups. The vehicle-treated group gradually lost visual function, retaining only 60% ± 5% and 30% ± 4% of their VA and CS, respectively, by the end of the treatment period. However, the DHED-treated group maintained significantly better visual performance, retaining 91% ± 3% and 75% ± 7% of their VA and CS compared to the baseline. No differences in the mass of the seminal vesicles between treatment groups. Comparison of RGC and axon counts in the optic nerve are ongoing. Conclusion: This study demonstrates the neuroprotective effects of DHED-derived E2 on the visual system without peripheral side effects. Despite sustained OHT, the VA and CS of the topically administered DHED reduced the impact of injury compared to the vehicle control group. Future studies will investigate DHED administration's impact on the retina and visual cortex proteome.Item Assessment of WIN 55,212-2 Loaded Reconstituted High-Density Lipoprotein Nanoparticles for Ocular Delivery(2024-03-21) Petty, R. Max; Ranjan, Rajiv; Sabnis, Nirupama; Fudala, Rafal; Lacko, Andras; Gryczynski, Zygmunt; Krishnamoorthy, Raghu; Stankowska, DorotaPurpose: Overcoming challenges in glaucoma therapy, such as biological barriers and retina delivery, led us to develop innovative reconstituted high-density lipoprotein nanoparticles (rHDL NPs) for effective drug delivery. Optic nerve head astrocytes (ONHAs) are vital in maintaining retinal ganglion cell (RGC) axon integrity. This study describes the encapsulation of WIN 55,212-2 (WIN) in rHDL NPs and investigates the delivery mechanism of these nanoparticles in ONHAs. Methods: Using a novel preparation method, a stable rHDL-payload complex was created by combining lipophilic fluorescent dye IR780 or therapeutic agent WIN with phosphatidylcholine and apolipoprotein A-I (Apo A-I). Fluorescent rHDL (rHDL-IR780) was used to assess cellular uptake in human primary ONHAs in vitro. Scavenger receptor class B1 (SR-B1) expression was confirmed in retinal cell lysates by SDS-PAGE followed by western blot analysis. Receptor-mediated payload release through SR-B1 was confirmed by receptor blocking using BLT-1 as a specific SR-B1 receptor-blocking agent. Results: Fluorescent rHDL NPs exhibited an IR780 encapsulation efficiency of 68.7% (103 M), a polydispersity index (PDI) of 0.287 ± 0.013, a size of 14.01 ± 4.36 nm, and a zeta potential of -7.44 ± 0.90 mV. Additionally, drug-loaded rHDL-WIN NPs displayed a WIN encapsulation efficiency of 44.6% (341.4 M), a PDI of 0.280 ± 0.011, a size of 62.04 ± 25.06 nm, and a zeta potential of -20.13 ± 0.86 mV. Western blot analysis on human retinal lysates, ONHA lysates, and RGC lysates indicated the expression of SR-B1 (57/82 kDa (unmodified/glycosylated)). Cellular uptake studies confirmed the ability of rHDL to deliver payloads to ONHAs and RGCs. Receptor blocking with 10 nM BLT-1 highlighted the role of SR-B1 in specific cellular uptake from rHDL to ONHAs (p < 0.01). Conclusions: Our study highlights the role of SR-B1 in facilitating the delivery of rHDL payloads to ONHAs, offering the potential for targeted drug delivery in glaucoma. We anticipate that the cellular uptake by RGCs will follow the same SR-B1-mediated pathway. Successful WIN encapsulation in rHDL NPs suggests a potential avenue for targeting therapies to treat and prevent glaucomatous damage. Further studies are needed to determine the neuroprotective effects of rHDL-WIN and develop the potential of rHDL NPs to be used as an agent to target therapies in glaucoma.Item Phenotypic and transcriptomic comparison of genetically distinct mouse strains for susceptibility to glucocorticoid-induced ocular hypertension (GC-OHT)(2024-03-21) Patel, Pinkal D.; Millar, J. Cameron; Curry, Stacy; Feris, Sherri; Clark, Abbot F.Purpose: Anti-inflammatory and immunosuppressive glucocorticoids (GCs) are widely prescribed for a variety of conditions and diseases. Unfortunately, a significant number of people experience negative side-effects associated with long term GC therapy and develop GC-induced ocular hypertension (GC-OHT) leading to secondary glaucoma. GC-OHT shares clinical and molecular signatures with primary open angle glaucoma (POAG) making this an appropriate model to study POAG. However, not all humans develop GC-OHT when treated with GCs. The ones that develop GC-OHT are called ‘responders’ whereas the ones that do not respond to GCs are called ‘non-responders’. The purpose of our study is to: (1) determine whether there are mouse strain differences in the development of GC-OHT, (2) whether resistance to develop GC-OHT is correlated with endogenous TM tissue gene expression using transcriptomic analysis. Methods: After measurement of baseline IOP, various mouse strains (B6, D2.gpnmb⁺, BALB/cJ, 129P3/J, C3H/HeJ) were treated with weekly periocular injections of potent GC dexamethasone (DEX; n=5-10) or vehicle (n=5-10) in both eyes for 4-5 weeks. IOPs were measured weekly using a TonoLab rebound tonometer in isoflurane anesthetized mice. “TM ring” tissue and underlying sclera was carefully collected, and mRNA libraries were prepared for sequencing. Differential expression analysis was performed to identify DEX-induced changes within each strain. Furthermore, Ingenuity Pathway Analysis (IPA) was used to identify DEX-altered pathways in each strain and compare differences between responder and non-responder strains. Results: B6 and C3H/HeJ mice robustly and reproducibly develop DEX-OHT with ΔIOP of 5-8 mmHg (P<0.0001). In contrast, D2.gpnmb⁺, 129P3/J, and BALB/cJ mice were resistant to the development of DEX-OHT. Differential analysis of gene expression between mouse strains showed novel DEX-responsive genes in all strains. Moreover, comparison of mouse strains using IPA showed similarities in the pathway and networks of the responder strains (B6 and C3H/HeJ). Conclusions: As observed in humans, we find that there are differences in GC responsiveness and the ability to develop GC-OHT among mouse strains. Transcriptomics evidence suggests that responder strains share common pathways that contribute towards development of GC-OHT. These studies will reveal the molecular mechanisms responsible for GC-OHT as well as provide insights into the pathogenesis of POAG.Item CREB Activation by Mini-Chaperone CPP-P1 Enhances Retinal Ganglion Cell Survival in an Acute Glaucoma Model(2024-03-21) Johnson, Gretchen; Nagaraj, Ram; Stankowska, DorotaPurpose: Alpha-B crystallin is a heat shock protein that has been found to have anti-apoptotic properties and was used to design the novel mini-chaperone called peptain-1 (P1) conjugated with a cell-penetrating peptide (CPP), named CPP-P1. Transcriptomics of primary retinal ganglion cells (RGCs) isolated from adult rats subjected to ocular hypertension and treated with CPP-P1 revealed the activation of CREB signaling as a major pathway activated by the drug. Creb activation by phosphorylation (p-Creb) was previously confirmed in primary RGCs and tested here in a rat model of ocular hypertension. Methods: Adult male Brown Norway rats (N=15, 5 per group) were grouped into naïve, IOP-Vehicle, and IOP-CPP-P1 experimental groups. Silicone oil (20µl) was injected into the anterior chamber, and 2µl of either PBS (vehicle) or CPP-P1 (2µg/µl) was injected intravitreally. On day 7 of elevated IOP, rats were euthanized. Retinal sections obtained were stained with Creb, p-Creb, and DAPI and imaged at 4X for cell counts and at 20X for integrated density measurements. Unpaired t-tests or Mann-Whitney tests in GraphPad Prism were used to calculate statistical significance. Retinal punches from post-mortem human eyes (N=3) were cultured with PBS or CPP-P1 (12.5µg/ml) for 48 hours, and tissue RNA was collected for qPCR. Results: Ganglion cell layer counts obtained from rat retinal sections showed that the silicone oil injury group with no intervention (IOP-Veh) had a 51% decrease in ganglion cells compared to the naïve group (p<0.0001), and the silicone injury with CPP-P1 intervention group (IOP-CPP-P1) showed only a 23% decrease compared to the naïve group (p=0.0016). Integrated density measurements of Creb expression in IOP-Veh was 17% (p=0.615) higher than the naïve group, while in IOP-CPP-P1, it was 136% (p=0.092) higher than the naïve group. Expression of p-Creb showed a decrease in IOP-Veh by 50% (p=0.056) in comparison to the naïve group, while the IOP-CPP-P1 group was significantly higher than the IOP-Veh group (p=0.036). RNA expression of Creb1 in human retinal tissue was increased by 1.7-fold with CPP-P1 treatment. Discussion: IOP-mediated RGC damage was mitigated by CPP-P1 treatment, demonstrating neuroprotective effects when compared to vehicle-treated rats. CREB signaling contributes to CPP-P1-mediated neuroprotection during glaucomatous insults.Item Regulation of Grxs in Cell Functions and Senescence in the Lens(2024-03-21) Zhang, Jinmin; Yu, Yu; Wu, HongliPurpose: Glutaredoxins (Grxs), a family of thiol transferases, can reverse protein glutathionylation using glutathione (GSH) as an electron donor. Therefore, it can regulate protein redox state and enzymatic activity. Specifically, Glutaredoxin 1 (Grx1) and Glutaredoxin 2 (Grx2) are predominantly localized in the cytoplasm and mitochondria, respectively. The Grx1/Grx2 double knockout (DKO) mice showed early onset of cataracts and were more sensitive to UV radiation, highlighting the importance of Grx1 and Grx2 in maintaining lens transparency. Lens epithelial cells (LECs) are crucial to lens transparency and functionality. Our current study is to explore novel roles of the Grxs in the lens using the Grx1-/-/Grx2-/- mouse model. Methods: We isolated LECs from the lenses of WT and DKO mice and conducted a range of in vitro experiments to study the effects of Grx depletion on the epithelial morphology, cell proliferation, cell death, and mitochondrial function of LECs. We also did lens tissue sectioning and hematoxylin and eosin (H&E) staining to visualize lens tissue. Results: Loss of Grx1/Grx2 led to stress fiber formation, cytoskeleton reorganization, and higher protein expression of mesenchymal markers (N-cadherin and vimentin) in LECs. The DKO LECs exhibited a lower proliferation rate and cell cycle arrest in comparison with WT LECs. Resistance to apoptosis and elevated levels of β-galactosidase activity of DKO LECs indicated that DKO LECs underwent cell senescence. DKO LECs also displayed compromised mitochondrial function, characterized by decreased ATP production, reduced expression levels of mitochondrial complexes III and IV, and increased proton leak. A compensatory metabolic shift towards glycolysis was observed in DKO LECs, indicating an adaptive response to Grx1 and Grx2 deficiencies. The HE staining data showed that the DKO mouse had aging lens epithelium characterized by less LEC density, remained flat in shape, and aligned less regularly. Conclusions: Our study demonstrates that the Grx1 and Grx2 DKO in LECs results in cytoskeletal reorganization, lower cell proliferation rate, cell cycle arrest, resistance to apoptosis, compromised mitochondrial function, and accelerated senescence. These findings underscore the importance of Grx1 and Grx2 in preventing LECs from undergoing premature aging.Item Can Nicotinamide Treatment Overcome the Effect of Monocarboxylate Transporter 2 Loss on Retinal Ganglion Cell Survival and Function? dm(2024-03-21) Murinda, Kudakwashe; Inman, Denise; Kiehlbauch, Charles; Morgan, AutumnPurpose: There is currently no cure for the vision loss in glaucoma that is characterized by retinal ganglion cell (RGC) loss and irreversible optic neuropathy. Monocarboxylate transporter 2 (MCT2), which transports pyruvate, lactate, and ketone bodies, is exclusively found in neurons such as the RGCs. We have previously shown that MCT2 is lost during glaucoma, in advance of RGC loss, and MCT2 overexpression protects RGC number and function. We sought to determine if MCT2 is necessary for RGC survival by knocking it out, and to establish whether providing oral nicotinamide (NAM) could compensate for the anticipated metabolic disruption to RGCs. Methods: To test these hypotheses, we injected tamoxifen into Thy1-ERT2-cre: MCT2fl/fl mice to conditionally knock out MCT2 from Thy1-positive RGCs. Control mice carried the MCT2 flox’d allele but were Thy1-ERT2-cre-negative. Control and experimental mice were subjected to ocular hypertension using the magnetic microbead model; separate naïve controls from each genotype were also evaluated. To test the effect of nicotinamide intervention, we repeated the same groups but added the administration of oral nicotinamide to each before inducing ocular hypertension. Intraocular pressure (IOP) was measured using the TonoLab rebound tonometer. Pattern electroretinogram (PERG) and Visual Evoked Potential (VEP) were used to analyze the RGC function. We used unbiased stereology (Stereo Investigator, Micro Brightfield) to count the number of retinal ganglion cells in the wholemount retina, and ATP levels in the retina were also measured. Axon counts were done from plastic-embedded optic nerves. Results: IOP was higher in the ocular hypertension (OHT) groups. MCT2 knockout alone did not impact IOP, nor did it exacerbate RGC function loss post-OHT. After OHT, PERG amplitude was significantly lower in the OHT and KO + OHT treatment groups (p<0.005). RGC function was preserved in the KO + NAM and OHT+NAM groups but was significantly decreased in the KO+OHT group. After OHT, MCT2 KO alone did not alter RGC density but OHT and KO + OHT groups had significantly decreased RGC density (p<0.005). There was no significant decline in RGC density in any of the nicotinamide groups. ATP production in the KO + OHT group was significantly higher (1.81 +/- 0.89 µg/µl) than in the naïve control group (0.68 +/- 0.42 µg/µl). Conclusions: MCT2 knockout alone from RGCs did not change IOP, RGC density, or PERG, suggesting that MCT2 is not necessary for RGC function and survival. Ocular hypertension decreased PERG amplitude and RGC density, and the magnitude of the decrease was not significantly worsened by MCT2 knockout. The nicotinamide groups had no significant loss in RGC density, supporting the proposed neuroprotective effect of NAM administration. These data suggest that RGCs can meet their immediate metabolic needs through means beyond MCT2, and nicotinamide can rescue RGCs in the context of glaucoma.Item Temporal Dynamics of Mitochondrial Dysfunction and Retinal Ganglion Cell Degeneration Following Endothelin-1 Exposure(2024-03-21) Brooks, Calvin; Kodati, Bindu; Stankowska, Dorota; Krishnamoorthy, RaghuPurpose: Endothelin-1 (ET-1) and its receptors have been identified as upregulated factors in the aqueous humor and retina of animal models with glaucoma, implicating them in the pathogenesis of glaucomatous neurodegeneration. This study aims to investigate the effects of ET-1 on mitochondrial morphology, a key aspect of cellular health. Methods: C57BL/6J mice (13 weeks old) received intravitreal injections of either ET-1 or vehicle (water) in both eyes 24 hours, 72 hours, or 7 days before collection. One eye was used for retinal flat mounts stained with RBPMS to quantify retinal ganglion cells (RGCs), while the other eye was processed for transmission electron microscopy after paraffin embedding. Optic nerves were sectioned, and ten images were captured per nerve section. Mitochondria within optic nerve axons were enumerated and graded based on cristae appearance (with grades ranging from 1 to 5). Comparisons between vehicle and ET-1 treated eyes at each time point were performed for mitochondrial scores and counts (Mann-Whitney test, n=4 per group). Cell counts from retinal flat mounts were compared across groups and time points (Student’s T Test). Results: Significant RGC loss was observed in the peripheral and mid-peripheral retina 24 hours post ET-1 injection (P=0.01 and 0.04 respectively), with the disparity between vehicle and ET-1 treatments diminishing at later time points. While a trend towards reduced mitochondrial numbers in optic nerve axons post ET-1 injection was noted across all time points, significance was not reached. Mitochondrial health scores were notably diminished at 24 hours post-injection in both vehicle and ET-1 treated groups, with ET-1 demonstrating appreciable damage to mitochondria at 72 hours compared to vehicle-injected animals. However, mitochondrial health improved over time in the ET-1 treated group, with no discernible differences observed at 7 days. Conclusion: The acute decline in mitochondrial morphology following intravitreal injection of both ET-1 and vehicle suggests induced cellular stress. However, ET-1 injection led to immediate cell death not observed in the vehicle-injected cohort. While mitochondrial morphology swiftly recovered in the vehicle-injected group, ET-1 administration prolonged mitochondrial damage. These alterations in mitochondrial morphology may impair mitochondrial energy production efficiency, potentially contributing to retinal ganglion cell vulnerability in glaucoma.Item ADAM19 and ADAMTS4 expression in Human Optic Nerve Head Astrocytes(2024-03-21) Easo, Tony; Rangan, Rajiv; Tovar-Vidales, TaraPurpose: Glaucoma is characterized by the degeneration and death of retinal ganglion cells and their axons causing irreversible blindness. Various risk factors contribute to glaucoma onset, including intraocular pressure (IOP), age, and family history. In glaucoma, the primary site of damage is the optic nerve head (ONH). ONH astrocytes, a major cell type in the LC, are believed to play a significant role in the pathological remodeling of extracellular matrix (ECM) during glaucoma. Glaucoma patients have increased levels of transforming growth factor beta 2 (TGFβ2) in their aqueous humor, trabecular meshwork and ONH. TGFβ2 is a profibrotic cytokine known to induce the synthesis and deposition of ECM. Previously, we performed RNA sequencing of ONH astrocytes in which disintegrin and metalloproteinases (ADAMs) and ADAM with thrombospondin motifs (ADAMTS) were significantly dysregulated with TGFβ2 treatment compared to controls. ADAMs and ADAMTS4 influence cell phenotype by affecting cell adhesion, migration, proteolysis, and signaling pathways. The purpose of the present study was to determine a) if ONH astrocytes express ADAM19 and ADAMTS4 in ONH astrocytes and b) determine if TGFβ2 regulates ADAM19 and ADAMTS4 expression in ONH astrocytes. Methods: Primary human ONH astrocyte cell strains (n=3) were treated with TGFβ2 (5ng/ml) or with a vehicle control for 48 hours. The effects of TGFβ2 on ADAM19 and ADAMTS4 were determined by qPCR and western blots using primary human ONH astrocyte cell cultures. Results: ADAM19 and ADAMTS4 were expressed in ONH astrocytes. Treatment with TGFβ2 significantly increased mRNA levels of ADAM19 and ADAMTS4. However, western blot analysis showed no significant change in ADAM19 protein expression compared to control, while ADAMTS4 was increased with TGFβ2 compared to control. Conclusions: TGFβ2 modulated the expression of ADAM19 and ADAMTS4 in ONH astrocytes. ADAM19 and ADAMTS4 may contribute to the pathogenic remodeling of the ONH in glaucoma. While further studies are needed, this research aims to shed light on the intricate mechanisms underlying glaucoma pathogenesis.