Eye / Vision
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Item Changes in the Expression of SMARCA4 in a Rat Model of Ocular Hypertension(2022) Worley, Josh; Stankowska, Dorota; Kodati, Bindu; Krishnamoorthy, RaghuTitle: Changes in the Expression of SMARCA4 in a Rat Model of Ocular Hypertension Purpose: SMARC4 (BRG1) is an ATP-dependent chromatin remodeling protein belonging to the SWI/SNF family of proteins involved in regulation of gene expression in numerous cell types in the body. The purpose of this study was to determine changes in the expression of SMARCA4 in the retina following intraocular pressure (IOP) elevation by the Morrison model in Brown Norway rats. We hypothesize that SMARCA4 expression may modulate the expression of the key genes involved in the neurodegenerative changes seen secondary to IOP elevation. Methods: The Morrison model of ocular hypertension (by injection of hypertonic saline through the episcleral veins) was utilized to unilaterally elevate the IOP in Brown Norway rats. IOP was elevated in the left eye of three retired breeder Brown Norway rats, with the right eye serving as the corresponding contralateral control. Rats were maintained for 2 weeks following IOP elevation and IOP measurements were carried out twice per week. Rats were subsequently euthanized, and retinal sections were obtained from both IOP-elevated and contralateral control eyes. Immunohistochemical analysis of SMARCA4 expression was carried out by immunostaining. Following confocal microscopy imaging, the intensity of immunofluorescence was quantified with the ImageJ software (NIH), and compared between IOP elevated and control eyes. Results: Immunohistochemical analysis revealed an appreciable decrease in the expression of SMARCA4 in retinal sections in two out of three rats, mainly the nerve fiber layer (by 47 to 57%), ganglion cell layer (by 18 to 40%) and inner plexiform layer (by 9 to 19%) in IOP elevated rat eyes compared to control eyes. One out of three tested rats showed a modest increase in immunostaining for SMARCA4 in the nerve fiber layer, ganglion cell layer and inner plexiform layer. Ongoing experiments will replicate these findings in order to generate statistically significant data. Conclusion: Changes in SMARCA4 expression could serve to regulate the expression of gene contributing to neurodegenerative effects due to elevated IOP. Understanding the role of SMARCA4 may allow us to better understand and address the mechanisms involved in glaucomatous neurodegeneration.Item Neuroprotection of human and rodent retinal ganglion cells by a hybrid antioxidant-nitric oxide donor small molecule, SA-2(2022) Pham, Jennifer; Johnson, Gretchen A.; Acharya, Suchismita; Stankowska, DorotaPURPOSE: Current treatments of glaucoma are aimed at lowering intraocular pressure (IOP), which is a key driver of retinal ganglion cell (RGC) death. Another contributing factor to RGC death is exposure to reactive oxygen species (ROS). At present, there is no FDA-approved neuroprotective treatment to prevent glaucomatous optic neuropathy and loss of RGCs. Our novel hybrid molecule, SA-2, contains both a nitric oxide (NO) donating group to lower IOP and a ROS scavenging group to protect RGCs. We hypothesize that SA-2 will inhibit the death of RGCs in an in vitro and an ex vivo neurotrophic factor deprivation model. METHODS: Retinal punches from human explants (n=4 donors/experiments) were isolated and treated with either SA-2 [1 mM] or vehicle and maintained without neurotrophic factors for 7 days ex vivo. In each experiment, 4 baseline retinal explants were collected on day 0. At the end of the experiment, explants were immunostained with RBPMS and Brn-3a (RGC-specific markers) and cell survival was analyzed. In three biological replicates, primary RGCs were isolated from rat pups and treated with either SA-2 (1 mM, 100 µM) or vehicle with or without neurotrophic factors for 48 h. Active caspase 3 and 7 assay was performed and apoptotic cell counts were analyzed. In another set of experiments, rat retinal explants were isolated and incubated with tert-Butyl hydroperoxide (TBHP) along with either SA-2 [1 mM] or vehicle for 2 h (n=2-4 explants/group). Production of superoxide by mitochondria was assessed using MitoSOX reagent according to manufacturer instructions. All cell counts were performed in a masked manner using ImageJ Software. One-way ANOVA or nonparametric Kruskal-Wallis was used for statistical analysis by GraphPad Prism 9 Software. RESULTS: In ex vivo human retinal explants, there was a significant increase in RGC survival by 39% in the SA-2 treated group compared to the vehicle group at day 7 (p< 0.0001). In rodent primary RGCs, SA-2 mediated a significant decrease in apoptotic cells by 30% (p< 0.01) and a 67% (p< 0.05) decrease in dead cell count. In rodent retinal explants, there was a significant decrease (by 59%, p< 0.0001) in the production of superoxide by mitochondria in the TBHP and SA-2 treated group, compared to the TBHP vehicle group. CONCLUSION: SA-2 was shown to be effective at preserving retinal ganglion cell survival in human retinal explants, rat retinal explants and primary rat RGCs by preventing apoptosis and protecting the cells from oxidative stress.Item Retina-Targeted Estrogen Prodrug: A New Concept for Retinal Protection(2022) Lal, Kevin; Yu, Yu; Zhang, Jinmin; Tran, Myhoa; Ezugwu, Chimdindu; Prokai-Tatrai, Katalin; Liu, Yang; Wu, HongliRetinal injury due to excessive light exposure during military duties often results in serious vision damage to soldiers including irreversible loss of visual function. However, therapeutic interventions that can promote retinal protection or reverse retinal damage are very limited. This unmet clinical need also persists in the public when strong lasers, light, or fire cause trauma in ocular tissues. It is well known that estrogen has been shown to exhibit various beneficial actions in the central nervous system, including positively affecting mood and protecting the neuronal cells against neurodegenerative diseases. Despite estrogen's potential, its detrimental side effects prevent its clinical uses for neurotherapy. To overcome this challenge, we developed a bioprecursor prodrug, called 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED), that is selectively converted to E2 only in the neuronal cells, including retinal cells. To determine if treatment with DHED can sufficiently protect the photoreceptor from light-induced damage, male C57BL/6J mice were injected with or without 200 µg/kg DHED (n=9) and 200 µg/kg E2 (n=9) for 10 days before the light injury. Seven days after the light exposure, the visual function and retinal structure were examined by the spectral-domain optical coherence tomography (SD-OCT) and electroretinogram (ERG). After light exposure, we found massive photoreceptor loss as indicated by thinning of the outer nuclear layer (ONL) and retinal detachment. Additionally, DHED significantly prevented light-induced retinal structural changes and light-induced a- and b-wave reduction. The photoreceptor protective effects upon DHED treatment are stronger than that of E2, consistent with our earlier observation that targeted E2 delivery via DHED prodrug produces more robust neuroprotection than direct administration of E2. Our liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based bioassay indicated that DHED delivers the biologically active estrogen to the neuronal cells including the retinal cells without affecting other tissues - unlike the systemic exposure that is seen with estrogen. In conclusion, our study supported our hypothesis that DHED is an efficacious and safe site-specific delivery agent to produce robust estrogen-mediated retinal neuroprotection.Item Mechanisms of peptain-mediated neuroprotection in retinal ganglion cells(2022) Johnson, Gretchen A.; Pham, Jennifer; Kodati, Bindu; Krishnamoorthy, Raghu; Nagaraj, Ram; Stankowska, DorotaPURPOSE: To determine mechanisms underlying neuroprotective effects of the core peptide of alpha-B crystallin, peptain-1 (P1) conjugated to a cell-permeable peptide CPP (P1-CPP) in retinal ganglion cells (RGCs) in a rodent model of glaucoma. METHODS: Intraocular pressure (IOP) was elevated in Brown Norway (BN) rats and intravitreally injected with 2 µl of either P1-CPP or vehicle, once a week for a period of 2 weeks. Rats were euthanized, primary adult RGCs were isolated by the immunopanning method. Total RNA was isolated using the Trizol/column method. RNA-sequencing was performed using an Illumina platform. The resulting FASTQ files were uploaded into Galaxy for analysis with FASTQC, RNASTAR, feature counts, and finally DESeq2. The results from DESeq2 were then assessed with Qiagen's Ingenuity Pathway Analysis (IPA) to identify significantly upregulated pathways. Relative Creb-1 expression normalized to reference gene GAPDH was determined in IOP-P1-CPP and IOP-vehicle treated rat RGCs. Briefly, quantitative Polymerase Chain Reaction (qPCR) was performed using BioRad's PrimePCR Assay and SsoAdvanced Universal SYBR Green Supermix on the BioRad's CFX96 Real-Time System C1000 Touch Thermal Cycler. RESULTS: RNA-seq analysis from rat RGCs isolated following 2 weeks of IOP-elevation revealed that P1-CPP treated groups had several differentially expressed (DEGs), compared to vehicle-treated groups, including 6343 significantly upregulated and 5960 significantly downregulated. Some significantly upregulated pathways following P1-CPP treatment include phagosome formation, synaptic long-term depression, and CREB signaling in neurons. The IOP and vehicle-treated groups, when compared to the naïve group, demonstrated a decreased expression of members of the CREB signaling pathway (Creb-1, c-RAF, MEK1/2, ERK1/2, and p90RSK). This decline was prevented by P1-CPP treatment. Quantitative PCR further confirmed the RNA-seq findings of the increased expression of Creb-1 in P1-CPP treated rats compared to that of vehicle-treated group. CONCLUSIONS: Mechanism of action of P1-CPP in a rodent model of glaucoma includes the activation of the pro-survival CREB signaling pathway, phagosome formation, and long-term synaptic depression to prevent cell death and vision loss.Item Hybrid molecule SA-2 improves both mitochondrial respiration and glycolysis in primary human trabecular meshwork cells(2022) Amankwa, Charles E.; Gondi, Sudershan; Stankowska, Dorota; Acharya, SuchismitaPurpose: Oxidative stress (OS) caused by hypoxia/hyperoxia environment results in progressive loss of trabecular meshwork (TM) cells in primary open angle glaucoma (POAG). Our previous report demonstrated; a hybrid nitric oxide (NO) donor-antioxidant molecule SA-2 protect primary human (h) TM cells against t-butyl hydrogen peroxide (TBHP) -induced cell death and increased superoxide dismutase enzyme level. Here we investigated the effect of SA-2 on mitochondrial energy metabolism by measuring the respiration status, glycolysis rate and energy production. Methods: Primary hTM cells obtained from human donor eyes were seeded in 24-well culture plates (Seahorse XFe 24 Cell Mito Stress test kit, Agilent), and starved for 24h before treatment with SA-2 (1 µM,10µM,100µM, and 1mM). In a separate experiment, the cells were pretreated with TBHP (150µM) for 30 minutes, followed by the addition of SA-2 (10µM,100µM). After 24h, the mitochondrial complex inhibitors and uncoupling reagents (oligomycin, FCCP, rotenone/antimycin A) were added. The plate was analyzed for changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XFe24 analyzer following the manufacturer's instructions. Results: The mean OCR was significantly decreased (>70%) followed by increase in the mean ECAR (~3-fold) after treatment with TBHP compared to oligo/FCCP/rot treated cells, hereafter called as negative control. Treatment with SA-2 at 1 µM,10µM,100µM and 1mM concentrations increased both oligomycin/FCCP induced decrease in ATP production and maximal mitochondrial respiration followed by an increase in the mean ECAR compared to negative control. The mean OCR was higher in SA-2 (100µM) +TBHP treated cells followed by an increase in ECAR in SA-2 (10µM or 100µM) +TBHP treated cells than TBHP and negative control treated cells. N =2-3. Conclusion: Mitochondrial respiration was impaired after TBHP treatment to hTM cells following cell death. While most of the mitochondrial targeting anti-oxidant compounds increase OCR but not ECAR, we found the hybrid NO donor-anti-oxidant compound SA-2 increases ATP production, maximal mitochondrial respiration and increases glycolytic energy production in hTM cells. This finding provides a novel direction for further investigation into the effect of SA-2 and mitochondrial bioenergetics during OS-induced cell death.Item The Role of Oxidative Phosphorylation in Müller Glia Functions and Survival(2022) Nsiah, Nana Yaa; Inman, DenisePurpose The importance of mitochondria to the energy production of Müller glia (MG), the main glial cells of the retina, is controversial. Previous studies showed MG are mainly glycolytic. Others challenge this view because MG are deficient in key glycolytic enzymes. Our goal is to potentially settle this debate by destabilizing the electron transport chain in MG mitochondria and assessing how retinal metabolism may be impacted. Methods MG that lack oxidative phosphorylation in vivo through destabilization of Complex IV were generated using GLASTCreERT2::Cox10fl/fl transgenic mice. Mice received daily tamoxifen injections for 5 consecutive days beginning at P30. Confirmation of recombination of the floxed Cox10 locus and enzyme activity was performed using PCR analysis of genomic DNA isolated from the retina and sequential cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) histochemistry, respectively. Cell lysates from primary Müller cells were used for western blotting and total protein analysis. Full-field electroretinography (ERG) was performed to assess MG function from transgenic and wild-type mice in vivo. Scotopic ERGs were recorded (OcuScience® HMsERG, Xenotec Inc., Henderson, NV) in response to six light flash intensities ranging from −3 to 1 log cd x s/m2 on a dark background. Each stimulus was presented in a series of three. Data were analyzed with GraphPad Prism and ERG b-wave amplitudes were compared using a paired two-tailed Student’s t-test. The b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave. Results A 465bp DNA fragment amplified from genomic DNA of mutant mice, with no corresponding fragment from control, confirmed Cox10 locus recombination. Total protein analysis, with normalization to the mitochondrial protein VDAC1, showed lower levels of cytochrome c oxidase protein from mutant mice compared to controls. Scotopic ERG b-wave was not significantly different between mutant and wild-type age-controlled mice at all light intensities. No overt retinal abnormalities were observed in GLASTCreERT2::Cox10fl/fl transgenic mice. Conclusion Our results show that cre recombinase induction in GLASTCreERT2::Cox10fl/fl successfully inhibits cytochrome c oxidase activity in MG from adult mice. Our in vivo experiments suggest that oxidative phosphorylation is not necessary for Müller glia energy metabolism under physiological conditions.Item Biological Characteristics of Lens Epithelial Cells from Grx1 and Grx2 Double Knockout Mice(2022) Zhang, Jinmin; Yu, Yu; Lal, Kevin; Dang, Terry; Ezugwu, Chimdindu; Tran, Myhoa; Wu, HongliPurpose: Glutaredoxins are glutathione (GSH) dependent enzymes that play an important role in repairing oxidized proteins, preventing subsequent protein misfolding and disrupting protein aggregation. The Grx system has two major isozymes: glutaredoxin 1 (Grx1) and the recently discovered glutaredoxin 2 (Grx2). To achieve a comprehensive understanding of the Grx system in the lens, our lab recently created a Grx1 and Grx2 double knockout (DKO) mouse model to observe how the double deletion of the enzymes may affect the lens epithelial cell (LEC) survival and lens transparency. Methods: Primary LECs were cultured from wild-type (WT) and DKO mice. Cell proliferation was tested via various assay kits, and cell cycle distribution was evaluated using flow cytometry analysis. Cell apoptotic markers including Bcl-2, Bax, and caspase 3 were detected using Western Blot. The mitochondrial function was evaluated via ATP concentration. Cytoskeletal arrangement and its intercellular connection were also examined by using fluorescent microscopy. Results: Compared to WT cells, DKO cells displayed a much slower growth. The number of DKO cells arrested in the M phase was twofold higher than that of WT cells. The population of DKO cells arrested in the S phase was 50% less than that of WT cells. For the apoptotic pathway, we found DKO cells have higher levels of Bax and cytochrome c with lower ATP production. Furthermore, we also found that DKO cells had higher levels of vimentin expression, which may lead to cytoskeleton reorganization and polarity. Conclusions: In conclusion, our data suggest that Grx function loss may inhibit cell proliferation, disrupt the normal cell cycle, trigger apoptosis pathway, and damage mitochondrial functions.Item Emerging functional crosstalk between the Grx system and Nrf2 pathway: evidence from UV radiation-induced cataract formation(2022) Dang, Terry; Wu, HongliGlaucoma, cataracts, age-related macular degeneration (AMD), are linked to oxidative stress by the external and internal environment. Ocular tissues are more susceptible to oxidative stress due to daily exposure of UV light and high oxygen consumption. The human body has several antioxidant enzymes such as catalase, superoxide dismutase (SOD), and thioredoxin. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an antioxidant enzyme transcription factor that regulates the downstream antioxidant genes. It also has glutathione (GSH) that searches for the free radicals in our body and oxidizes to form glutathione mixed disulfide (GSSG). As the GSSG levels increase, it naturally adds to other proteins causing protein glutathionylation (PSSG). PSSG is an important post-translational modification linked to oxidative stress. Research has shown that the glutaredoxin (Grx) system is capable of reversing PSSG formation which can be assumed to cause less oxidative stress. To take a closer look at the function of the Grx system in protecting the lens against ultraviolet (UV)- induced cataract formation, glutaredoxin (Grx1) and glutaredoxin 2 (Grx2) is studied in a Grx1/Grx2 double knockout (DKO) mice model. By intercrossing Grx1 knockout (KO) and Grx2 KO mice, Grx1/Grx2 DKO mice resulted. The study population was half male and half female, one month old Grx1/Grx2 DKO and age-matched wild type (WT) mice. They were exposed to 20.6 kJ/m2 UV radiation for 15 mins to induce cataracts. Mice were euthanized at 4 days post-exposure. The degree of the cataract and lens morphology were evaluated under a dissecting microscope. Glutathione (GSH), free protein thiol (PSH), and protein glutathionylation (PSSG) levels were measured as general markers of oxidative damage. To further define the crosstalk between the Grx system and nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway, Nrf2 and its downstream target proteins were examined by using Western blot analysis. The results showed that UV radiation caused more severe anterior subcapsular cataract in Grx1/Grx2 DKO than that of WT mice. The opacity of the lenses in DKO mice, appeared to extend deeper into the cortical and even nuclear regions. Lenses of Grx1/Grx2 DKO mice contained significant lower levels of GSH and PSH. On the other hand, the accumulation of PSSG, a marker for protein thiol oxidation, was much higher in Grx1/Grx2 DKO group. Deletion of Grx1 and Grx2 also decreased the expression of antioxidant enzyme transcription factor regulator, Nrf2, and its downstream antioxidant genes, including catalase, superoxide dismutase (SOD), and another redox regulator of thioredoxin (Trx). The Nrf2 dependent antioxidant response can no longer function with combined Grx1 and Grx2 deletion. This will cause more oxidative stress and increase the lens susceptibility to UV-induced damage.Item miRNA Profiling of Optic Nerve Head Astrocytes Exposed to Cyclic Stretch(2022) Rangan, Rajiv; Tovar-Vidales, TaraIntroduction: Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, a leading cause of irreversible blindness consequent to retinal ganglion cell (RGC) degeneration. Elevated IOP induces biomechanical aberrations within ocular tissues - including the transmission of biomechanical stretch through the lamina cribrosa (LC) region of the optic nerve head (ONH), the site where RGC axon damage first occurs. LC cells and ONH astrocytes (ONHA), the primary cells of the LC, respond to stretch in a manner that promotes pathological extracellular matrix (ECM) remodeling (fibrosis) and mechanical damage of RGCs within the ONH. A complex set of molecular mechanisms regulate ECM remodeling. Part of this regulation may involve microRNAs (miRNAs), small molecules that can inhibit protein expression by binding to and silencing mRNA. In this study, we examined miRNA expression profiles of ONHA exposed to cyclic stretch. We hypothesized that cyclic stretch would induce upregulation of miRNAs that silence anti-fibrotic protein translation and downregulation of miRNAs that silence pro-fibrotic protein translation, promoting a net-fibrotic molecular signaling environment. Methods: Primary human normal ONHA cell strains (n=3) were exposed to 0-12% cyclic stretch for 24 hours; controls were exposed to 0% stretch. RNA samples were collected from stretched and control cells, and miRNA PCR arrays were used to determine expression changes for miRNAs associated with fibrosis. Expression fold changes were normalized to SNORD68. The bioinformatics tool TargetScan was used to predict mRNA targets for any dysregulated miRNAs. Induction of fibrotic cellular changes by cyclic stretch was confirmed by western blotting of conditioned media for secreted proteins. Results: miR-146b-5p was found to be significantly upregulated by +5.97-fold (P = 0.029) in stretched ONHA. Predicted mRNA targets for miR-146b-5p are known to be involved in fibrosis and cell survival, among other functions. Preliminary data indicates upregulation of secreted proteins associated with fibrosis (TGFβ2, Fibronectin, Transglutaminase 2) by stretched ONHA. Conclusions: Stretch modulates miRNA expression in cultured human ONHA, miR-146b may mediate ECM alterations and other pathological changes at the LC. Future experimental directions will include assessing co-expression of other miR-146 family miRNAs, validating putative mRNA targets and elucidating the mechanisms by which specific miRNA and their targets modulate ECM remodeling.Item Case Report Choroidal Metastasis secondary to Lung Malignancy(2022) Tolman, Alex; Nyalakonda, Ramyashree; Warminski, Johnathan; Mozdbar, SimaBackground: Ocular malignancies are uncommon and often occur as secondary metastases. Due to the rarity of developing ocular metastases, special attention should be placed on history gathering and physical examination of a patient presenting with painless vision loss. Metastases are commonly observed in the posterior pole due to the rich microvasculature. Secondary metastases show a smoother morphology whereas primary ocular metastases may have a shaggy appearance. External beam radiation therapy, the most available treatment modality, is the conventional method for treating choroidal metastases due to preservation of vision and low occurrence of acute complications. Stereotactic radiosurgery, brachytherapy plaque insertion, and proton beam therapy are less common alternatives to external radiation treatment due to limited research and availability. Case Presentation: A 62-year-old Caucasian male with recently diagnosed primary lung adenocarcinoma 2 months prior was referred by his primary care physician and oncologist for ophthalmic evaluation before initiation of chemotherapy. The patient presented with a chief complaint of sudden, painless vision loss in the right eye. His symptoms started two weeks prior and had remained constant since onset. The patient reported that he had difficulty with both far and near vision. He denied recent fever or illness, trauma, severe or unusual headaches, double vision, jaw pain, scalp tenderness, or neurological impairment (e.g., dizziness, numbness, and tingling). The patient reported no improvement in vision with his bifocal lenses. Other medical history included prostate cancer status post resection followed by radiation, chronic obstructive pulmonary disease, polysubstance abuse, and ongoing tobacco use. The patient's ocular history included previous traumatic injury to his left eye during childhood, which resulted in a total retinal detachment and subsequent vision loss. Family medical and ocular histories were unremarkable. Snellen visual acuity was best corrected to 20/250 in his right eye. Best-corrected visual acuity in his left eye was counting fingers at 3 feet, secondary to his traumatic injury. His extraocular motilities were full and he denied pain or diplopia with gaze change. Pupil examination revealed sluggish responses in both the right and left pupils. Goldmann tonometry revealed intraocular pressures of 12mmHg in the right eye and 18mmHg in the left eye. Slit lamp examination was unremarkable in both eyes. Dilated fundus examination revealed an elevated, smooth yellow-white lesion superior to the macula and significant vascular tortuosity in the right eye. The left eye revealed mild tortuosity, and diffuse scarring secondary to a longstanding retinal detachment. Optical coherence tomography (OCT) imaging was performed over the retinal lesion in the right eye and revealed significant subretinal fluid (Figure 2). The assessment at this time was bilateral hypertensive retinopathy and presumed malignant neoplasm of the right choroid consistent with metastatic lung cancer. The patient was referred to a retinal specialist for additional testing and treatment the same day. External beam radiation therapy was offered for palliation but rapid disease progression hindered treatment. Conclusion: This case highlights a rare complication of malignancy, emphasizing the importance of comprehensive history gathering and physical examination.Item Glaucoma-associated stretch of optic nerve head astrocytes drives changes in glycolysis bioenergetics and glutamine dependency(2022) Yin, Eric; Pappenhagen, Nate; Inman, DenisePurpose: Glaucoma is an optic neuropathy that leads to irreversible blindness, often through a chronic increase in intraocular pressure which promotes a stretch injury to the optic nerve head. In rodents and humans, the predominant glial cell in this region is the optic nerve head astrocyte (ONHA). Since this region of the optic nerve is unmyelinated, the ONHAs provide neighboring axons with metabolic support, likely in the form of lactate produced through astrocytic glycolysis. Previously, we found that exposing astrocytes to glaucoma-associated deformation altered their metabolism in ways that indicated stronger commitment to and upregulation of glycolysis. Here, we explore the predominant source supplying the requisite carbon for TCA cycle intermediates that this stretch-induced glycolysis upregulation demands; our hypothesis is that glutamine metabolism plays a major role in this mechanism. Methods: Primary ONHAs were cultured from P5 rat pup optic nerve head explants. Metabolic changes in ONHAs were investigated by subjecting them to 24h of 12% biaxial stretch at 1Hz. The cells' bioenergetics were measured using a Seahorse XFe24 Analyzer. Protein markers for glycolysis and other cellular metabolism pathways were measured using a ProteinSimple Jess Automated Western Blot Analyzer. Results: We observed significant glycolytic and respiratory activity differences between control and stretched ONHAs, including greater extracellular acidification and lower ATP-linked respiration, yet higher maximal respiration and spare capacity in stretched ONHAs. We determined that both control and stretched ONHAs displayed a dependency upon glutamine over pyruvate or long-chain fatty acids for fuel. We also found increased proteome markers of glutamine metabolism such as glutamine synthetase, and glycolytic lactate production through increased lactate dehydrogenase-a, in stretched ONHAs when compared against that of control. Conclusions: Our results of extracellular acidification rate, fuel flexibility studies, and various metabolic proteome markers suggest that ONHAs, after being subjected to glaucoma-associated stretch deformation, show a preference for the increased use of glycolysis over oxidative phosphorylation, and glutamine over other sources of TCA cycle carbon intermediates. Therefore, stretch alters ONHA bioenergetics to support an increased demand for internal and external energy. This is significant as these altered bioenergetics could potentially inhibit ONHAs from providing metabolic support to neighboring retinal ganglion cell axons, further advancing the axonal degeneration commonly associated with glaucoma.