Browsing by Author "Inman, Denise"
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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 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 Effect of Monocarboxylate Transporter 2 Loss on Retinal Ganglion Cell Survival and Function(2023) Murinda, Kudakwashe; Morgan, Autumn; Inman, Denise; Kiehlbauch, CharlesPurpose: 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 (MCT2s) that transport pyruvate, lactate, and ketone bodies, are 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. This study was undertaken to test whether MCT2s are necessary for RGC survival and function. Methods: To test this hypothesis, we used tamoxifen injection 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. Intraocular pressure (IOP) was measured using the TonoLab rebound tonometer. Pattern electroretinogram (PERG) was used to analyze RGC function. We used unbiased stereology (Stereo Investigator, Micro Brightfield) to count the number of retinal ganglion cells in wholemount retina, and ATP levels in retina were also measured. Results: IOP was higher in the ocular hypertension (OHT) groups. MCT2 knockout alone did not impact IOP, nor did it alter baseline PERG amplitude or latency. After OHT, PERG amplitude was significantly lower in the MCT2-knockout mice (p=0.0013). MCT2 knockout alone did not change RGC density. After OHT, RGC density decreased, though in this preliminary analysis, RGC density among the groups was not significantly different. ATP production in the OHT+ Tamoxifen group was significantly higher (1.81 +/- 0.89 ug/ul) than in the naïve control group (0.68 +/- 0.42 ug/ul). Conclusions: MCT2 knockout from RGCs did not change IOP or PERG, suggesting that MCT2 is not necessary for RGC survival. Ocular hypertension decreased PERG amplitude and RGC density, though the magnitude of the decrease may not have been increased by MCT2 knockout. These preliminary data suggest that RGCs are capable of meeting their immediate metabolic needs through means beyond MCT2.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.Item Identification of proteins affected by increased intraocular pressure in the glaucomatous female mouse retina by label-free proteomics(2023) Zaman, Khadiza; Morgan, Autumn B.; Nguyen, Vien; Prokai-Tatrai, Katalin; Inman, Denise; Prokai, LaszloPurpose: Mass spectrometry-based retina proteomics using animal models of human diseases has enabled novel insights into ocular neuropathology’s such as in glaucoma, as it holds promise for disease biomarker discovery. However, publicly accessible data on retina proteins affected by ocular hypertension (OHT) in animal models utilized males, or sex was not disclosed. Recently, female animals were chosen to advance therapeutic antibody development against glaucomatous neurodegeneration with retina proteomics support. Therefore, our retinal proteomics-based investigation intended to fill a knowledge gap by focusing on OHT-induced changes of protein expressions in the glaucomatous female retinae compared to normotensive controls. Methods: Proteins were extracted from the retinae of normotensive female mice (control, n=5) and OHT mice (n=5) in which increase of intraocular pressure was induced by the magnetic microbead method. After reduction, alkylation and digestion by trypsin, bottom-up shotgun proteomics analyses of the samples were done using data-dependent nanoflow liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) on a hybrid Orbitrap instrument (Thermo Fisher Scientific). MS/MS spectra were searched against the UniProt mouse protein sequence database using the SEQUEST search engine in Proteome Discoverer (version 2.4; Thermo Fisher Scientific). Validation of proteins identifications using stringent criteria and label-free quantifications (LFQ) employing spectral counting to detect regulated proteins between groups using t-tests were performed using Scaffold (version 5.1.2; Proteome Software). Targeted proteomics on selected biomarkers was designed and analyzed using SkylineTM (MacCoss Lab software). Mapping to protein interaction networks and biological processes was done through Ingenuity Pathway Analysis® (IPA®, Qiagen). Results: Our discovery driven data-dependent nanoflow LC–ESI-MS/MS analyses covered nearly 1200 retinal proteins with <1% false discovery rate. Among these proteins, 168 were significantly affected by OHT based on LFQ. Bioinformatics analyses by IPA® revealed important diseases and functions triggered by OHT pertaining to neurological and ophthalmic pathologies. The topmost protein interaction network represented neurological disease, organismal injury and abnormalities. The molecule activity predictor of IPA® revealed important canonical pathways, including inhibition of synaptogenesis signaling and mitochondrial dysfunction leading to degeneration of central nervous system tissue. Another prominent protein interaction network represented nervous system development and function, as well as organ development. In addition, this network also displayed downregulation of neuroprotective crystallins owing to OHT. Neuronal crystallins have been identified not only as biomarkers to monitor the progression of OHT-induced retinal neuropathy and evaluate neuroprotective interventions, but also as potential druggable targets or possible protein therapeutics to prevent glaucomatous neurodegeneration. Parallel reaction monitoring-based targeted proteomics validation of significant OHT-regulated retina proteins are currently underway to establish them as potential preclinical biomarkers and/or therapeutic targets. In addition, our studies will be expanded to investigate sex as a biological variable affecting ocular neurodegeneration associated with glaucoma. Conclusion: We anticipate that biological information one can derive from our dataset at the protein expression level will provide inspiration for future hypothesis-driven experimental studies focusing on knowledge gaps involving the biology of glaucomatous neurodegeneration.Item Impact of GLUT1 Transporter Knockout in Optic Nerve Head Astrocytes and Retinal Ganglion Cells(2023) Gollamudi, Phani Sree Harsha; Inman, DeniseAbstract Purpose: Astrocytes and axons are the primary constituents of the optic nerve head, the initial site of neurodegeneration in glaucoma. This study was intended to understand the metabolic relationship between astrocytes and RGC axons. We hypothesized that reducing glucose transporter-1 (GLUT1) expression in astrocytes will increase the RGC-associated pathology after ocular hypertension (OHT). Methods: Mice expressing a GLUT1 gene flanked by loxP sites behind the GFAP promoter ("GFAP-GLUT1”) mice were used (n=40) and were divided into 4 groups: GLUT1-knockout+OHT, Control OHT, GLUT1-knockout+No OHT, and Control+No OHT. Baseline and final intra-ocular pressure (IOP), pattern electroretinogram (PERG), and visual evoked potential (VEP) measurements were taken. OHT was induced via magnetic microbead injection into the anterior chamber. Retinas, optic nerves, and brains were collected for retinal ganglion cell (RGC) quantification, anterograde transport analysis, biochemical assays, and protein analysis. Results: Statistically significant increases were noted in the IOP data between mice subjected to OHT and the No OHT groups. OHT led to statistically significant decreases in RGC number, regardless of GLUT1 status. A statistically significant decrease in PERG amplitude was noted in all groups subjected to OHT. Interestingly, GLUT1 knockout PERG amplitude was significantly lower than Control at the outset, suggesting a negative impact on retinal physiology from loss of the GLUT1 in astrocytes. Conclusion: Initial observations indicate glial metabolic homeostasis can impact retinal physiology, but GLUT1 knockout did not appear to negatively impact RGC survival. Ongoing analysis will determine if other structures or functions have been compromised by loss of GLUT1 in astrocytes, as well as provide greater insight into the mechanism of physiological change.Item Intravitreal Endothelin-1 (ET-1) Injection Reduces Mitophagy in Retinal Ganglion Cells in MitoQC Mice(2023) Brooks, Calvin D.; Kodati, Bindu; Inman, Denise; Stankowska, Dorota; Krishnamoorthy, RaghuPurpose: The peptide endothelin-1 (ET-1), and its receptors are upregulated in the aqueous humor and retina in animal models of experimentally induced ocular hypertension, and have been shown to have a causative role in retinal ganglion cell (RGC) neurodegeneration. The purpose of this experiment was to assess the role of mitophagy in RGC neurodegeneration following intravitreal ET-1 administration in MitoQC mice. Methods: MitoQC mice (Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl on a C57BL/6 background) at the age of 3 months were used for the study. The mitochondria in these mice display both red and green fluorescence due to expression of a mCherry-GFP tag fused to the mitochondrial targeting sequence of an outer mitochondrial membrane protein, FIS1. When these mitochondria are trafficked to the lysosome for degradation, the green fluorescence is quenched, leaving only the red fluorescence. The MitoQC mice were intravitreally injected in both eyes with either ET-1 (1 nmole) or vehicle (water), and 72 hours following the injections the mice eyes were enucleated and retinal flat mounts were live-imaged using a Zeiss LSM 880 super resolution confocal microscope. Z-stack imaging was used to image the ganglion cell layer. For each Z layer, a threshold algorithm was used to define a region of interest (ROI) that included only areas with red fluorescence, after which red and green fluorescence were quantified for that ROI. Red/green fluorescence intensity was calculated and averaged per image. A red/green ratio larger than 1 is indicative of active mitophagy. This ratio was compared between ET-1 and vehicle-injected mice using a Mann-Whitney test (n=4 eyes per group). Results: At 72 hours after injection with ET-1, the average red/green fluorescence ratio in the RGCs was 0.86, while the vehicle-injected mice had an average red/green ratio of 1.29. These ratios were significantly different (P=0.0003), and the smaller red/green ratio in the ET-1 group indicates lesser mitophagy than the vehicle group. Conclusion: Mitophagy is known to be an important quality control mechanism for neuronal cell survival, and this study provides evidence that mitophagy is impaired by ET-1. The finding indicates that a decline in mitophagy may be associated with endothelin-mediated neurodegeneration in RGCs.Item Ketogenic Diet Increases Mitophagy in a Mouse Model of Glaucoma(2023) Morgan, Autumn; Fan, Yan; Inman, DenisePurpose: We have previously shown that limiting dietary intake to high fat, low protein, and negligible carbohydrate results in mitochondrial biogenesis, and in the case of glaucoma, a reduction in neurodegeneration of retinal ganglion cells (RGCs). In this experimental follow-up study, we wanted to examine the effect of the ketogenic diet on mitophagy, or mitochondrial recycling, within the glaucomatous retina. Methods: MitoQC mice were placed on a ketogenic diet or standard rodent chow for 5 weeks and ocular hypertension (OHT) was induced via microbead injection. The MitoQC reporter mice have a pH-sensitive mCherry-GFP tag on the outer mitochondrial membrane that results in retention of red fluorescence when mitochondria bound for recycling are engulfed by lysosomes. The FIJI (ImageJ) macro MitoQC counter was used to quantify red puncta (mitolysosomes) in sectioned retina as a measure of mitophagy within the RGCs and Müller glia. Results: Mitophagy in RGCs, as measured by red puncta, was significantly decreased by ocular hypertension in the control retina (Control + OHT) in comparison to naïve control retina (Ctrl; p<0.0001). The ketogenic diet (KD) resulted in a significant increase in mitolysosomes in RGCs when compared to Ctrl (p<0.0001), Control + OHT (p<0.0001) and KD + OHT (p=0.0089). The ketogenic mice with OHT showed a significantly higher RGC-associated mitolysosome number than Control + OHT mice (p<0.0001). In contrast, mitolysosomes quantified in the Müller glia of Control + OHT mice were significantly higher than the naïve control mice (p=0.0127). Mice in the KD (p=0.0001) and KD + OHT(p=0.0005) groups had significantly greater mitolysosomes than the control Müller glia, however there was no difference in mitophagy between the Control + OHT, KD, and KD + OHT Müller glia groups. Conclusion: Our data demonstrates that mitophagy is managed differently within RGCs and Müller glia of mouse retinas. The KD promoted mitophagy within the RGCs to a degree that overcame the decline of mitophagy after OHT in the control group. Within the Müller glia, the KD was redundant because OHT alone increased mitophagy to similar levels as the KD. These findings suggest a divergence of mitochondrial homeostasis in RGCs and Müller glia that may reflect the different metabolic needs of these cell types.Item MCT2 Overexpression Rescues Metabolic Vulnerability and Protects Retinal Ganglion Cells in Two Glaucoma Models(2020) Inman, Denise; Harun-Or-Rashid, Mohammad; Jassim Jaboori, Assraa; Pappenhagen, NathanielPurpose: The ketogenic diet decreases retinal ganglion cell degeneration in models of glaucoma. Retina and optic nerves from mice on the diet also had elevated expression of monocarboxylate transporter 2 (MCT2), which transports monocarboxylates intracellularly to be used as fuel for mitochondria. We used a viral vector approach to increase expression of MCT2 in retinal ganglion cells (RGCs) to determine if the upregulation of MCT2 without the ketogenic diet could increase RGC survival in glaucoma. Methods: We injected AAV2-CAG2-mSLC16A7-2A-GFP (AAV2:MCT2) intraocularly to infect RGCs and promote MCT2 overexpression in the DBA/2J mouse model of glaucoma and the magnetic microbead induced ocular hypertension (OHT). Retinas and optic nerves were collected; axons were stained with PPD, and RGCs were labeled with RBPMS for counting. Data was compared to DBA/2J-Gpnmb+/+ or sham injected controls. Metabolic proteins of interest were quantified using capillary electrophoresis, and visual function was tested with pattern ERG (PERG). Results: AAV2:MCT2 injection rescued RGCs in both models of glaucoma. Glaucomic mice injected with AAV2:MCT2 had significantly higher axon counts, RGC soma densities, and visual function than glaucomic mice treated with a control virus (AAV2:eGFP). The decreased pAMPK/AMPK ratio indicated that AAV2:MCT2 treated eyes were under less metabolic stress than eyes treated with AAV2:eGFP. PERG traces showed higher P1 amplitude in mice treated with AAV2:MCT2 than in controls, indicating improved visual function in those mice. Conclusions: MCT2 overexpression caused increased RGC survival in two models of glaucoma, suggesting there is a metabolic problem underlying glaucomic degeneration.Item Metabolic changes in optic nerve head astrocytes following glaucoma-associated deformation(2021) Pappenhagen, Nathaniel; Zaman, Khadiza; Prokai, Laszlo; Inman, DenisePurpose: The astrocytes of the optic nerve head (ONHAs) are the predominant glial cell in the unmyelinated portion of the optic nerve. We tested the metabolic changes these cells undergo in glaucoma by exposing them in vitro to degrees of deformation similarly experienced as a result of increased intraocular pressure in primary open angle glaucoma. Methods: Primary astrocytes were cultured from the cortices of P1 mouse pups or P7 optic nerve head explants then seeded on collagen-coated FlexCell plates. The astrocytes were then biaxially stretched by 12% for 24 hours using the FX-6000T FlexCell. ONHA extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using the Seahorse XFe24 Analyzer, while changes in the proteome were measured using mass spectrometry. Results: Stretched cortical astrocytes showed at least twofold increases in metabolic proteins such as glutamate dehydrogenase 1, isocitrate dehydrogenase 1, and aldolase fructose-bisphosphate c. There was also at least a twofold decrease in citrate synthase, isocitrate dehydrogenase 2, glycogen phosphorylase B, and adenylate kinase 1. In the Seahorse Analyzer, stretched ONHAs showed increased glycolytic ECAR, maximal ECAR, and maximal OCR. Stretched astrocytes showed no difference in their dependence on pyruvate compared to controls, but a significant decrease in their capacity for mitochondrial respiration from pyruvate. Conclusions: Exposing astrocytes to glaucoma-associated deformation altered their metabolism in ways that indicated stronger commitment to glycolysis compared to control astrocytes, such as increases in glycolytic proteins, decreases in mitochondrial proteins, and increases in glycolytic ECAR.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 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 stress