Browsing by Author "Stankowska, Dorota L."
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Item An Antiapoptotic Peptide for Neuroprotection in Glaucoma(2017-03-14) Krishnamoorthy, Raghu R.; Sampathkumar, Sruthi; Nagaraj, Ram; Stankowska, Dorota L.Purpose: Axonal degeneration and death of retinal ganglion cells (RGC) are primary contributors to vision loss in glaucoma. The purpose of this study was to determine if intraperitoneal administration of the core peptide derived from small heat shock protein αB-crystallin (ABCP) could inhibit RGC death in animal models of glaucoma. Materials and Methods: Brown Norway rats were retrogradely labeled (to detect RGCs) using Fluoro-gold and IOP was elevated (150 mmHg/days) in one eye using the Morrison’s method, while the contralateral eye served as control. The rats were intraperitoneally injected with 10μg of ABCP (n=3 animals per group) three times per week for five weeks. Surviving RGCs were counted in retinal flat mounts. In another model of ischemia reperfusion (I/R) injury, C57BL/6 mice were subjected to IOP elevation of 120 mmHg for 30 min, followed by rapid reperfusion. Intraperitoneal ABCP injections were given 3h before and immediately after the procedure and then once daily post I/R injury for 14 days. RGC apoptosis was assessed using a TUNEL assay (n=2 animals per group). Results: Intraperitoneal injections of ABCP significantly (p Conclusions: Intraperitoneally administered ABCP peptide was able to significantly attenuate RGC death in two animal models of glaucoma. These findings suggest that ABCP has the potential to be developed as a neuroprotective agent in glaucoma.Item Endothelin-1 mediated decline in mitochondrial function contributes to neurodegeneration in glaucoma(2020-08) Chaphalkar, Renuka M.; Krishnamoorthy, Raghu R.; Stankowska, Dorota L.; Clark, Abbot F.; Zode, Gulab S.Glaucoma is an optic neuropathy with multifactorial etiologies, commonly associated with elevated intraocular pressure (IOP) and characterized by degeneration of the optic nerve, loss of retinal ganglion cells (RGC), cupping of optic disc and visual field deficits, which could ultimately lead to vision loss. In most cases, glaucoma is a chronic, asymptomatic and gradually progressing neurodegenerative disease, sometimes referred to as the "silent thief of sight," hence, routine eye examinations by an ophthalmologist are critical to determine if there is a likelihood of developing the disease. Elevated IOP is a primary and the only modifiable risk factor in glaucoma. Currently, reducing IOP remains the only proven treatment to delay the progression of RGC death; however, some patients continue to have neurodegenerative effects despite lowering IOP. Therefore, development of novel neuroprotection strategies as an adjunct therapy to IOP-lowering agents will provide a valuable therapeutic strategy in glaucoma. One of the promising targets for neuroprotection is the endothelin system of peptides and their receptors. The endothelin (ET) system comprises of three vasoactive peptides (ET-1, ET-2 and ET-3), which act through two types of G-protein coupled receptors, namely, ETA and ETB receptors. Originally discovered in the cardiovascular system, the diverse expression pattern of endothelin peptides and their receptors implicate their involvement in a variety of physiological processes in the body. A growing body of evidence suggests that endothelins and their receptors are associated with neurodegeneration in glaucoma. Previous studies have demonstrated that ET-1 levels are elevated in aqueous humor (AH) and plasma of glaucoma patients. Our lab previously demonstrated that in an ocular hypertension model in rats, there was an increase in ETB as well as ETA receptor expression primarily in RGCs compared to contralateral eyes. Following IOP elevation, RGC loss was significantly attenuated in the ETB receptor-deficient rats, pointing to a causative role of the ETB receptor in glaucomatous neurodegeneration. However, the precise cellular and molecular mechanisms by which ET-1 promotes neurodegeneration through its actions on the endothelin receptors are not completely understood. Previous studies have shown that ETB receptor stimulation increases the oxidative stress and production of superoxide anions, in sympathetic neurons. Several studies point to the role of mitochondrial dysfunction and oxidative stress as contributors to glaucomatous damage in animal models of glaucoma. To investigate various molecular events contributing to the ET-1 mediated RGC loss in glaucoma, we carried out RNA-seq analysis of the translatome in rat primary RGCs following ET-1 treatment. We identified several key mitochondrial and neurodegenerative gene candidates including Atp5h, Cox17, Foxo1, Moap1 and Map3k11 that were differentially expressed in the translatome by ET-1 treatment in RGCs. Based on our RNA-seq findings, we hypothesized that ET-1 causes an increase in reactive oxygen species (ROS) by acting through the ETB receptor that produces a subsequent decline in mitochondrial function and bioenergetics ultimately predisposing RGCs to cell death. To test this hypothesis, we used an in vitro approach by utilizing rat primary culture of RGCs treated with ET-1 as well as an in vivo approach by intravitreal ET-1 injections in rodents and the Morrison's model of glaucoma in rats. Our data showed that there is a significant decrease in the expression of cytochrome c oxidase 17 copper chaperone (COX17) and ATP synthase, H+ transporting, mitochondrial F0 complex, subunit D (ATP5H), both of which are critical components of the electron transport chain and oxidative phosphorylation pathway. Using a Seahorse mitostress assay, we also found a significant decline of several mitochondrial parameters following ET-1 treatment in primary RGCs, which indicated the possibility of a disruption in the mitochondrial quality control machinery. Hence, we also explored the effect of the ET-1 treatment on the mitophagy pathway, specifically in RGCs. Our findings suggest that there is a decrease in mitophagosome formation in RGCs in the Morrison ocular hypertensive model as well as in GFP-LC3 mice injected with ET-1, indicating an impairment in the mitochondrial quality control mechanism. Our studies reveal several novel candidates that could be targeted for the development of neuroprotective approaches to treat glaucoma.Item Examining the association between sleep modifiers and recovery time following sports-related concussion(2022-05) Kim, Chol Ho J.; Stankowska, Dorota L.Although previous researchers support the association of sleep disturbances negatively impacting recovery time from sports-related concussion (SRC), the degree of impact sleep disturbances have on recovery time from SRC is not well-defined. This study uses the presence and absence of sleep modifiers in the Concussion Clinical Profiles Screening tool to compare and quantify recovery time from SRC in a preadolescent, adolescent, and young adult sample. We examined patient records data obtained between August 2019 and December 2021 with inclusion criteria (1) injury date <30 days from initial evaluation, (2) SRC diagnosis, and (3) completion of treatment. Patients (sample median age 15.4 (14.3, 17.1)) were grouped based on the presence or absence of sleep modifier. Adjusting for other risk factors of prolonged recovery time (i.e., vestibular primary clinical profile, personal/family history of migraines, and personal history of anxiety/depression), the presence of sleep modifiers substantially increased recovery time by 1.45-fold (p<0.001).Item Involvement of c-Jun N-terminal kinase 2 (JNK2) in Endothelin-1 (ET-1) Mediated Neurodegeneration of Retinal Ganglion Cells(ARVO Journals, 2021-05-03) Kodati, Bindu; Stankowska, Dorota L.; Krishnamoorthy, Vignesh R.; Krishnamoorthy, Raghu R.Purpose: The goal of this study was to determine whether JNK2 played a causative role in endothelin-mediated loss of RGCs in mice. Methods: JNK2-/- and wild type (C57BL/6) mice were intravitreally injected in one eye with 1 nmole of ET-1, whereas the contralateral eye was injected with the vehicle. At two time points (two hours and 24 hours) after the intravitreal injections, mice were euthanized, and phosphorylated c-Jun was assessed in retinal sections. In a separate set of experiments, JNK2-/- and wild type mice were intravitreally injected with either 1 nmole of ET-1 or its vehicle and euthanized seven days after injection. Retinal flat mounts were stained with antibodies to the RGC marker, Brn3a, and surviving RGCs were quantified. Axonal degeneration was assessed in paraphenylenediamine stained optic nerve sections. Results: Intravitreal ET-1 administration produced a significant increase in immunostaining for phospho c-Jun in wild type mice, which was appreciably lower in the JNK2 -/- mice. A significant (P < 0.05) 26% loss of RGCs was found in wild type mice, seven days after injection with ET-1. JNK2-/- mice showed a significant protection from RGC loss following ET-1 administration, compared to wild type mice injected with ET-1. A significant decrease in axonal counts and an increase in the collapsed axons was found in ET-1 injected wild type mice eyes. Conclusions: JNK2 appears to play a major role in ET-1 mediated loss of RGCs in mice. Neuroprotective effects in JNK2-/- mice following ET-1 administration occur mainly in the soma and not in the axons of RGCs.Item Methods to Reduce Medication Errors and Improve Medication Adherence Among Elderly Patients Facing a Language Barrier(2021-05) Hanan, Nicholas L.; Fulda, Kimberly; Mathew, Stephen O.; Mallet, Robert T.; Stankowska, Dorota L.A significant and growing portion of the population of the United States is of limited English proficiency (LEP). In healthcare, LEP patients are a high-risk group for adverse medical events. Elderly patients are also a high-risk group and often take multiple medications, and when the two groups are combined it can create a potentially dangerous situation for the patient. This study searched for methods to reduce these adverse medical events and improve medication adherence. A total of 42 references were reviewed to find that most commonly, the only resources that physicians have to communicate LEP patients is a trained medical interpreter and on occasion, a few translated documents in the patient's native language. While the prevalence of medical interpreters has been rising, even with a trained interpreter LEP patients generally have worse clinical outcomes and lesser satisfaction than patients who see a language-concordant physician. Physicians can take additional steps such as learning how to most effectively utilize interpreters and employing visual aids and the teach back method to improve direct patient care. They can also take steps outside of the patient-provider interaction to improve care, such as ensuring that patients take notes and review them when it is time to take their medications and advocating the use of resources like internet portals for scheduling appointments and refilling medications. While the healthcare system in the United States has made good progress in making medical interpreters and medication instructions in other languages more accessible, there is still much work to be done in this field to ensure that LEP patients receive the care that they deserve.Item Mitochondria-associated endoplasmic reticulum membranes (MAMs) and their role in glaucomatous retinal ganglion cell degeneration-a mini review(Frontiers Media S.A., 2023-05-30) Pham, Jennifer H.; Stankowska, Dorota L.Glaucoma is a leading cause of blindness worldwide, commonly associated with elevated intraocular pressure (IOP), leading to degeneration of the optic nerve and death of retinal ganglion cells, the output neurons in the eye. In recent years, many studies have implicated mitochondrial dysfunction as a crucial player in glaucomatous neurodegeneration. Mitochondrial function has been an increasingly researched topic in glaucoma, given its vital role in bioenergetics and propagation of action potentials. One of the most metabolically active tissues in the body characterized by high oxygen consumption is the retina, particularly the retinal ganglion cells (RGCs). RGCs, which have long axons that extend from the eyes to the brain, rely heavily on the energy generated by oxidative phosphorylation for signal transduction, rendering them more vulnerable to oxidative damage. In various glaucoma models, mitochondrial dysfunction and stress from protein aggregates in the endoplasmic reticulum (ER) have been observed in the RGCs. However, it has been shown that the two organelles are connected through a network called mitochondria-associated ER membranes (MAMs); hence this crosstalk in a pathophysiological condition such as glaucoma should be evaluated. Here, we review the current literature suggestive of mitochondrial and ER stress related to glaucoma, indicating potential cross-signaling and the potential roles of MAMs.Item Modulating mitochondrial calcium channels (TRPM2/MCU/NCX) as a therapeutic strategy for neurodegenerative disorders(Frontiers Media S.A., 2023-11-06) Johnson, Gretchen A.; Krishnamoorthy, Raghu R.; Stankowska, Dorota L.Efficient cellular communication is essential for the brain to regulate diverse functions like muscle contractions, memory formation and recall, decision-making, and task execution. This communication is facilitated by rapid signaling through electrical and chemical messengers, including voltage-gated ion channels and neurotransmitters. These messengers elicit broad responses by propagating action potentials and mediating synaptic transmission. Calcium influx and efflux are essential for releasing neurotransmitters and regulating synaptic transmission. Mitochondria, which are involved in oxidative phosphorylation, and the energy generation process, also interact with the endoplasmic reticulum to store and regulate cytoplasmic calcium levels. The number, morphology, and distribution of mitochondria in different cell types vary based on energy demands. Mitochondrial damage can cause excess reactive oxygen species (ROS) generation. Mitophagy is a selective process that targets and degrades damaged mitochondria via autophagosome-lysosome fusion. Defects in mitophagy can lead to a buildup of ROS and cell death. Numerous studies have attempted to characterize the relationship between mitochondrial dysfunction and calcium dysregulation in neurodegenerative diseases such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Amyotrophic lateral sclerosis, spinocerebellar ataxia, and aging. Interventional strategies to reduce mitochondrial damage and accumulation could serve as a therapeutic target, but further research is needed to unravel this potential. This review offers an overview of calcium signaling related to mitochondria in various neuronal cells. It critically examines recent findings, exploring the potential roles that mitochondrial dysfunction might play in multiple neurodegenerative diseases and aging. Furthermore, the review identifies existing gaps in knowledge to guide the direction of future research.Item Modulation of Mitochondrial Metabolic Parameters and Antioxidant Enzymes in Healthy and Glaucomatous Trabecular Meshwork Cells with Hybrid Small Molecule SA-2(MDPI, 2023-07-29) Amankwa, Charles E.; Young, Olivia; DebNath, Biddut; Gondi, Sudershan R.; Rangan, Rajiv; Ellis, Dorette Z.; Zode, Gulab S.; Stankowska, Dorota L.; Acharya, SuchismitaOxidative stress (OS)-induced mitochondrial damage is a risk factor for primary open-angle glaucoma (POAG). Mitochondria-targeted novel antioxidant therapies could unearth promising drug candidates for the management of POAG. Previously, our dual-acting hybrid molecule SA-2 with nitric oxide-donating and antioxidant activity reduced intraocular pressure and improved aqueous humor outflow in rodent eyes. Here, we examined the mechanistic role of SA-2 in trabecular meshwork (TM) cells in vitro and measured the activity of intracellular antioxidant enzymes during OS. Primary human TM cells isolated from normal (hNTM) or glaucomatous (hGTM) post-mortem donors and transformed glaucomatous TM cells (GTM-3) were used for in vitro assays. We examined the effect of SA-2 on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro using Seahorse Analyzer with or without the oxidant, tert-butyl hydroperoxide (TBHP) treatment. Concentrations of total antioxidant enzymes, catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GPx) were measured. We observed significant protection of both hNTM and hGTM cells from TBHP-induced cell death by SA-2. Antioxidant enzymes were elevated in SA-2-treated cells compared to TBHP-treated cells. In addition, SA-2 demonstrated an increase in mitochondrial metabolic parameters. Altogether, SA-2 protected both normal and glaucomatous TM cells from OS via increasing mitochondrial energy parameters and the activity of antioxidant enzymes.Item Neuroprotection of Rodent and Human Retinal Ganglion Cells In Vitro/Ex Vivo by the Hybrid Small Molecule SA-2(MDPI, 2022-12-12) Pham, Jennifer H.; Johnson, Gretchen A.; Rangan, Rajiv S.; Amankwa, Charles E.; Acharya, Suchismita; Stankowska, Dorota L.The mechanisms underlying the neuroprotective effects of the hybrid antioxidant-nitric oxide donating compound SA-2 in retinal ganglion cell (RGC) degeneration models were evaluated. The in vitro trophic factor (TF) deprivation model in primary rat RGCs and ex vivo human retinal explants were used to mimic glaucomatous neurodegeneration. Cell survival was assessed after treatment with vehicle or SA-2. In separate experiments, tert-Butyl hydroperoxide (TBHP) and endothelin-3 (ET-3) were used in ex vivo rat retinal explants and primary rat RGCs, respectively, to induce oxidative damage. Mitochondrial and intracellular reactive oxygen species (ROS) were assessed following treatments. In the TF deprivation model, SA-2 treatment produced a significant decrease in apoptotic and dead cell counts in primary RGCs and a significant increase in RGC survival in ex vivo human retinal explants. In the oxidative stress-induced models, a significant decrease in the production of ROS was observed in the SA-2-treated group compared to the vehicle-treated group. Compound SA-2 was neuroprotective against various glaucomatous insults in the rat and human RGCs by reducing apoptosis and decreasing ROS levels. Amelioration of mitochondrial and cellular oxidative stress by SA-2 may be a potential therapeutic strategy for preventing neurodegeneration in glaucomatous RGCs.Item Neuroprotection of Rodent Retinal Ganglion Cells using Hybrid Molecule SA-10(2023) Pham, Jennifer H.; Kodati, Bindu; Johnson, Gretchen A.; Acharya, Suchismita; Stankowska, Dorota L.Purpose: Oxidative stress is the imbalance between the activity of antioxidants and free radical production, which has been shown to be associated with glaucomatous retinal ganglion cell (RGC) degeneration. In this study, we aimed to promote RGC survival by treatment with SA-10, a second-generation hybrid molecule with nitric oxide donating and sulfone reactive oxygen species (ROS) scavenging moieties in vitro and ex vivo following oxidative stress-induced injury. Methods: Endothelin-3, a vasoactive peptide, was used to induce oxidative stress in vitro in rat primary RGCs (n=3 biological replicates) and ex vivo in C57BL/6J mice retinal explants (n=8-9 explants/group). Primary RGCs were isolated from Sprague Dawley rat pups (post-natal days 4-7) and cultured for seven days with neurotrophic factors to allow for neurite outgrowth. The RGCs and retinal explants were pretreated with vehicle (DPBS) or SA-10 [10 µM] for 30 minutes, following which ET-3 treatment [100 nM or 400 nM] was carried out for 1 hour. CellROX™ Green was then used to stain for ROS produced by the cells, and the integrated density was analyzed. Analysis of Variance (ANOVA) or nonparametric Kruskal-Wallis was performed for all experiments. Results: In primary RGCs, ET-3-mediated ROS production decreased by 25.9% (p<0.01) following SA-10 treatment compared to the vehicle. In mice, retinal explants, 400 nM ET-3 induced a 24.4% increase in ROS production compared to the vehicle [0 nM ET-3]. With the SA-10 treatment, the ROS production was decreased by 14.74% (p<0.001) in the ET-3 and SA-10 treated group compared to the ET-3-only treated group. Conclusion: SA-10 effectively protects rodent RGCs in vitro and ex vivo from ET-3-mediated oxidative stress.Item The Neuroprotective Effects of Hybrid SA-10 Nanoparticles in a Glaucoma Mouse Model of Retinal Ischemia/Reperfusion Injury(2023) Le, Kim-Tuyen T.; Zhang, Wei; Pham, Jennifer H.; Kodati, Bindu; Amankwa, Charles E.; Engelland, Rachel E.; Hatfield, Brendon R.; Acharya, Suchismita; Stankowska, Dorota L.Purpose: The progression of glaucoma is largely dependent on the gross functionality of retinal ganglion cells (RGCs), which transmit visual signals to the brain. Current treatments for glaucoma focus on lowering intraocular pressure (IOP), yet other risk factors such as oxidative stress and poor blood perfusion also contribute to RGC damage. Furthermore, no treatments exist which revitalize dysfunctional RGCs. One promising neuroprotective agent is SA-10, a hybrid compound with reactive oxygen species (ROS) scavenging sulfone moiety and perfusion-enhancing nitric oxide (NO) donor moiety. This study aims to investigate the in vivoneuroprotective effects of the nanoparticle (NP) formulation of SA-10 (SA-10-NPs) on RGCs in an acute rodent model of ischemia/reperfusion (I/R). Methods: C57BL/6J mice were separated into 3 groups (n= 5-8 per group): Sham control, 1% Blank NPs-treated, and 1% SA-10-NPs. Aside from sham, all groups received 4 μL of different blinded topical pre-treatments: poly(lactic-co-glycolic-acid) (PLGA) nanoparticles suspended PBS for the Blank-NPs group, and 1% SA-10 loaded in PLGA for the SA-10-NPs group. Besides the sham, all groups had their anterior chambers cannulated with normal saline to achieve an elevated IOP of 120 mmHg for 60 minutes. After I/R all groups received 4 μL of their respective treatments 3 times a week over 14 days. Pattern electroretinogram (PERG) and pattern visual evoked potential (PVEP) tests were independently performed both prior to I/R insult (baseline) and after completing the treatment regimen. Mouse eyes were then enucleated. Their retinas were stained with an RNA binding protein with multiple splicing (RBPMS) RGC-specific marker for quantification of cell survival. Parametric Analysis of Variance (ANOVA) and its non-parametric equivalent, the Kruskal-Wallis test, were performed for statistical analysis. Results: I/R injury (Blank-NPs-treated) produced a 52.1% decline (p<0.01) in PERG and a 17.9% decreasing trend in PVEP amplitudes as compared to sham. SA-10-NPs prevented this decline by a trend of 33.5% and 14%, respectively. I/R injury (Blank NPs-treated) caused a 33% decrease (p<0.01) in RGC survival in the inner retina in comparison with sham control, which was alleviated with the SA-10-NPs treatment by 33.4% (p<0.001). Conclusions: SA-10-NPs enhanced RGC survival and function following ischemia-induced damage in the mice I/R model and have the potential to be used as a neuroprotective therapy for glaucoma.Item Roadblocks in Clinical Research: An Analysis of Common Barriers to the Clinical Research Process(2022-12) Carter, Anne M.; Krishnamoorthy, Raghu R.; Stankowska, Dorota L.; Leach, YeseniaDeficiencies in the clinical research process can lead to delays, loss of funding, outdated conclusions, and a lack in participation for both volunteers and scientists or clinicians. This project aimed to identify the specific roadblocks in the clinical research process within the Ophthalmology department at the University of Texas Southwestern Medical Center. The Ophthalmology and Optometry clinical and research faculty were surveyed to identify which aspects of the clinical research process were confusing, needed improvement, or awareness was lacking. Out of the 35 faculty provided with the survey, a total of 11 responded, giving a 31% response rate. Responses showed faculty were not fully aware of the resources available for clinical research or how to conduct clinical research within federal guidelines. They also would like to participate more in clinical research, but they are unaware of current actively recruiting research studies. Finally, all faculty responded they feel that clinical research is important, but most commonly they feel they don't have enough time. Information sessions, monthly newsletters, and faculty meetings are all ways to communicate solutions for these deficiencies in the clinical research process.Item Role of mitophagy in ocular neurodegeneration(Frontiers Media S.A., 2023-11-15) Brooks, Calvin D.; Kodati, Bindu; Stankowska, Dorota L.; Krishnamoorthy, Raghu R.Neurons in the central nervous system are among the most metabolically active cells in the body, characterized by high oxygen consumption utilizing glucose both aerobically and anaerobically. Neurons have an abundance of mitochondria which generate adequate ATP to keep up with the high metabolic demand. One consequence of the oxidative phosphorylation mechanism of ATP synthesis, is the generation of reactive oxygen species which produces cellular injury as well as damage to mitochondria. Mitochondria respond to injury by fusion which serves to ameliorate the damage through genetic complementation. Mitochondria also undergo fission to meet an increased energy demand. Loss of mitochondria is also compensated by increased biogenesis to generate new mitochondria. Damaged mitochondria are removed by mitophagy, an autophagic process, in which damaged mitochondria are surrounded by a membrane to form an autophagosome which ultimately fuses with the lysosome resulting in degradation of faulty mitochondria. Dysregulation of mitophagy has been reported in several central nervous system disorders, including, Alzheimer's disease and Parkinson's disease. Recent studies point to aberrant mitophagy in ocular neurodegenerative disorders which could be an important contributor to the disease etiology/pathology. This review article highlights some of the recent findings that point to dysregulation of mitophagy and it's underlying mechanisms in ocular neurodegenerative diseases, including, glaucoma, age-related macular degeneration and diabetic retinopathy.Item The Effects of Ad5.CMV.hTGFβ2C226/228S on AHD in Mice(2021-05) Stevenson, Cooper H.; Millar, J. Cameron; Tovar-Vidales, Tara; Stankowska, Dorota L.Elevated intraocular pressure (IOP) is a key risk factor for the development of primary open-angle glaucoma (POAG), a leading cause of blindness in people over the age of 40 years. Transforming growth factor beta-2 is a cytokine known to contribute to the pathogenesis of POAG due to its deleterious effects on aqueous humor outflow via the conventional, or trabecular, outflow pathway in the eye. However, its effects on the rate of aqueous outflow (Fu) via the unconventional or uveoscleral outflow pathway, rate of aqueous humor production (Fin), and episcleral venous pressure (Pe) are unknown. Further, effects of euthanasia and enucleation in our hands on TGFβ2-mediated effects on Fu are also unknown. The goal of the present study was to quantify the impact of over-expression of TGFβ2 on aqueous humor dynamics (AHD) in the mouse eye, with special emphasis on Fu, Fin, and Pe in the mouse eye. To simulate TGFβ2 over-expression, left (OS) eyes were injected intravitreally (IVT) with a mutant form of TGFβ2 (Ad5.CMV.hTGFβ2C226/228S, 2×10⁷pfu in 2μL), while right (OD) eyes were injected IVT with a null virus (Ad5.CMV.null, same titer and volume). Following 14 days, after which time mean IOP (determined tonometrically in conscious mice) had become elevated in TGFβ2-injected eyes (84.29% increase in IOP, P < 0.001), Fu was determined directly by cannulating the anterior aqueous chamber (AC) and perfusing it with fluorescein isothiocyanate-dextran (1×10⁻⁹ M), followed by dissection of the retina/choroid/iris-ciliary body/scleral shell, homogenization, and measurement of each sample's fluorescence, and then inference of flow rate using a standard curve. Those perfusion were performed in living eyes, also in eyes in situ in the animal immediately following euthanasia, and enucleated eyes perfused in vivo either (i) exposed to air, or (ii) submerged in PBS. In a further group of experiments in living animals aqueous humor outflow conductance (C) (also known as aqueous humor outflow facility), and Pe were measured, and then Fin and Fu were calculated using a constant flow infusion method. Further, we sought to determine whether IOP elevation would lead to a reduction in RGC numbers in the retina, so retinal flat mounts from both treated and untreated eyes from 5 of our animals were prepared and RGC counts were made. For eyes perfused in-vivo, Fu was reduced in OS (0.0048 ± 0.0017 μL/min) compared to OD (0.0987 ± 0.0126 μL/min, P = 0.025). For eyes perfused in euthanatized mice, Fu was reduced in OS (0.0215 ± 0.0101 μL/min) compared to OD (0.1543 ± 0.0241 μL/min, P = 0.010). For eyes perfused ex-vivo while submerged in PBS, there was no difference in Fu between OS (0.0222 ± 0.0065 μL/min) and OD (0.0137 ± 0.0078 μL/min, P = 0.175). For eyes perfused ex-vivo while exposed to air, Fu was reduced in OS (0.0702 ± 0.0087 μL/min) compared to OD (0.1377 ± 0.0106 μL/min, P = 0.008). Fin showed a trend towards a reduction in the eyes in which TGFβ2 was over-expressed, but this effect did not reach statistical significance. There was a significant increase in Pe in eyes in which TGFβ2 was expressed (8.6 ± 0.7 mmHg in OS to 6.4 ± 0.2 mmHg in OD, P = 0.015). Given these results, the present study further quantifies the effect of TGFβ2 in POAG, providing more insight into its mechanism of action in this disease.Item The endothelin receptor antagonist macitentan ameliorates endothelin-mediated vasoconstriction and promotes the survival of retinal ganglion cells in rats(Frontiers Media S.A., 2023-01-01) Kodati, Bindu; Zhang, Wei; He, Shaoqing; Pham, Jennifer H.; Beall, Kallen J.; Swanger, Zoe E.; Krishnamoorthy, Vignesh R.; Harris, Payton E.; Hall, Trent; Tran, Ashley V.; Chaphalkar, Renuka M.; Chavala, Sai H.; Stankowska, Dorota L.; Krishnamoorthy, Raghu R.Glaucoma is a chronic and progressive eye disease, commonly associated with elevated intraocular pressure (IOP) and characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells (RGCs). The pathological changes in glaucoma are triggered by multiple mechanisms and both mechanical effects and vascular factors are thought to contribute to the etiology of glaucoma. Various studies have shown that endothelin-1 (ET-1), a vasoactive peptide, acting through its G protein coupled receptors, ET(A) and ET(B), plays a pathophysiologic role in glaucoma. However, the mechanisms by which ET-1 contribute to neurodegeneration remain to be completely understood. Our laboratory and others demonstrated that macitentan (MAC), a pan endothelin receptor antagonist, has neuroprotective effects in rodent models of IOP elevation. The current study aimed to determine if oral administration of a dual endothelin antagonist, macitentan, could promote neuroprotection in an acute model of intravitreal administration of ET-1. We demonstrate that vasoconstriction following the intravitreal administration of ET-1 was attenuated by dietary administration of the ET(A)/ET(B) dual receptor antagonist, macitentan (5 mg/kg body weight) in retired breeder Brown Norway rats. ET-1 intravitreal injection produced a 40% loss of RGCs, which was significantly lower in macitentan-treated rats. We also evaluated the expression levels of glial fibrillary acidic protein (GFAP) at 24 h and 7 days post intravitreal administration of ET-1 in Brown Norway rats as well as following ET-1 treatment in cultured human optic nerve head astrocytes. We observed that at the 24 h time point the expression levels of GFAP was upregulated (indicative of glial activation) following intravitreal ET-1 administration in both retina and optic nerve head regions. However, following macitentan administration for 7 days after intravitreal ET-1 administration, we observed an upregulation of GFAP expression, compared to untreated rats injected intravitreally with ET-1 alone. Macitentan treatment in ET-1 administered rats showed protection of RGC somas but was not able to preserve axonal integrity and functionality. The endothelin receptor antagonist, macitentan, has neuroprotective effects in the retinas of Brown Norway rats acting through different mechanisms, including enhancement of RGC survival and reduction of ET-1 mediated vasoconstriction.Item The Neuroprotective Effects of SA-2-NP in a Mouse Model of RGC Injury(2021-05) Ferguson, Jonathan L.; Stankowska, Dorota L.; Millar, J. Cameron; Tovar-Vidales, TaraDetermine if a novel hybrid compound SA-2 can be delivered to the retina in a nanoparticle formulation and have protective effects on retinal ganglion cells (RGCs) following an optic nerve crush (ONC) model of RGC death. Pattern Electroretinography (PERG) was performed on six- to twelve-week-old female (C57BL/6) mice (n = 1-8 mice per group) prior to performing ONC on the left eye to promote RGC death similar to that seen in normotensive glaucoma. Mice were dosed topically for seven or fourteen days either with SA-2 in polylactic glycolic acid (PLGA) nanoparticles, or empty PLGA nanoparticles. Subsequent PERG was performed at seven day following ONC to reassess RGC function after the optic nerve injury and treatments. The mice were subsequently euthanized and both eyes we enucleated and fixed with paraformaldehyde. The retinas were removed, flat mounts were prepared and immunostained with RBPMS antibody to quantify surviving RGCs. Our study demonstrated that SA-2 can be delivered to the retinal tissue with PLGA nanoparticles. However, following optic nerve crush in mice, at the selected doses and delivery regimen of SA-2, neuroprotective effects determined by RGC counts and PERG analysis were not statistically significant. Following ONC in mice, topically delivered SA-2 loaded nanoparticles demonstrated some trend in neuroprotection without statistical significance. Further investigation is required to delineate the efficacious delivery mode and dose.Item The Role of Mitochondrial Respiration in Müller Glia Survival and Function Under Normal and Glaucomatous Conditions In Vivo(2023-05) Nsiah, Nana Yaa; Inman, Denise M.; Stankowska, Dorota L.; Zode, Gulab S.; Yang, ShaohuaSeveral markers of mitochondrial dysfunction have been observed in the retinas of glaucoma patients and experimental animal models. However, these studies have primarily focused on retinal neuron cells even though glial cells too contain significant amounts of mitochondria. Thus, little is known about how glial cell mitochondrial dysfunction contributes to glaucoma pathology. As the principal macroglial cells of the retina, Müller glia (MG) function is essential to maintaining homeostasis in the retina. However, very little is known about how MG generate energy to support their function in vivo. In this study, we address the role of mitochondrial respiration in MG using an inducible Cox10 knockout transgenic mouse model. Cox10 (protoheme IX farnesyltransferase) encodes a component of cytochrome c oxidase (COX), complex IV, of the electron transport chain. Cox10 deficient cells lack functional COX. Disruption of COX function in MG did not affect MG survival nor retinal structure but impaired visual function and upregulated glycolysis pathway protein expression in the retina. These data suggest that MG-specific mitochondrial respiration is essential for whole retinal energy metabolism and visual processing. Hypoxia-inducible factor 1α (HIF-1α) has been shown to be upregulated in the glaucomatous retina and optic nerve, yet its role in glaucoma pathogenesis remains unexplored. HIF-1α is a transcription factor that promotes glycolysis and metabolic adaptation during hypoxia. By blocking HIF-1α degradation through pharmacologic inhibition, we found that prolonged HIF- 1α stabilization led to retinal glycolysis and oxidative phosphorylation (OXPHOS) protein downregulation and AMP-activated protein kinase (AMPK) activation, indicating low energy status. These changes were accompanied by impaired retinal ganglion cell (RGC) function and glial cell activation. Taken together, these results demonstrate the essential role of MG-specific OXPHOS in the retina, as well as pointing to a role for HIF-1α in neurodegeneration in the retina.Item Upregulation of the endothelin A (ETA) receptor and its association with neurodegeneration in a rodent model of glaucoma(BioMed Central Ltd., 2017-03-01) McGrady, Nolan R.; Minton, Alena Z.; Stankowska, Dorota L.; He, Shaoqing; Jefferies, Hayden B.; Krishnamoorthy, Raghu R.BACKGROUND: Primary open angle glaucoma is a heterogeneous group of optic neuropathies that results in optic nerve degeneration and a loss of retinal ganglion cells (RGCs) ultimately causing blindness if allowed to progress. Elevation of intraocular pressure (IOP) is the most attributable risk factor for developing glaucoma and lowering of IOP is currently the only available therapy. However, despite lowering IOP, neurodegenerative effects persist in some patients. Hence, it would be beneficial to develop approaches to promote neuroprotection of RGCs in addition to IOP lowering therapies. The endothelin system is a key target for intervention against glaucomatous neurodegeneration. The endothelin family of peptides and receptors, particularly endothelin-1 (ET-1) and endothelin B (ETB) receptor, has been shown to have neurodegenerative roles in glaucoma. The purpose of this study was to examine changes in endothelin A (ETA) receptor protein expression in the retinas of adult male Brown Norway rats following IOP elevation by the Morrison's model of ocular hypertension and the impact of ETA receptor overexpression on RGC viability in vitro. RESULTS: IOP elevation was carried out in one eye of Brown Norway rats by injection of hypertonic saline through episcleral veins. After 2 weeks of IOP elevation, immunohistochemical analysis of retinal sections from rat eyes showed an increasing trend in immunostaining for ETA receptors in multiple retinal layers including the inner plexiform layer, ganglion cell layer and outer plexiform layer. Following 4 weeks of IOP elevation, a significant increase in immunostaining for ETA receptor expression was found in the retina, primarily in the inner plexiform layer and ganglion cells. A modest increase in staining for ETA receptors was also found in the outer plexiform layer in the retina of rats with IOP elevation. Cell culture studies showed that overexpression of ETA receptors in 661W cells as well as primary RGCs decreases cell viability, compared to empty vector transfected cells. Adeno-associated virus mediated overexpression of the ETA receptor produced an increase in the ETB receptor in primary RGCs. CONCLUSIONS: Elevated IOP results in an appreciable change in ETA receptor expression in the retina. Overexpression of the ETA receptor results in an overall decrease in cell viability, accompanied by an increase in ETB receptor levels, suggesting the involvement of both ETA and ETB receptors in mediating cell death. These findings raise possibilities for the development of ETA/ETB dual receptor antagonists as neuroprotective treatments for glaucomatous neuropathy.