Publications -- Denise Inman

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/31771

This collection is limited to articles published under the terms of a creative commons license or other open access publishing agreement since 2016. It is not intended as a complete list of the author's works.

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Now showing 1 - 7 of 7

Ocular Inflammation and Oxidative Stress as a Result of Chronic Intermittent Hypoxia: A Rat Model of Sleep Apnea
(MDPI, 2024-07-22) Donkor, Nina; Gardner, Jennifer J.; Bradshaw, Jessica L.; Cunningham, Rebecca L.; Inman, Denise M.
Obstructive sleep apnea (OSA) is a sleep disorder characterized by intermittent complete or partial occlusion of the airway. Despite a recognized association between OSA and glaucoma, the nature of the underlying link remains unclear. In this study, we investigated whether mild OSA induces morphological, inflammatory, and metabolic changes in the retina resembling those seen in glaucoma using a rat model of OSA known as chronic intermittent hypoxia (CIH). Rats were randomly assigned to either normoxic or CIH groups. The CIH group was exposed to periodic hypoxia during its sleep phase with oxygen reduction from 21% to 10% and reoxygenation in 6 min cycles over 8 h/day. The eyes were subsequently enucleated, and then the retinas were evaluated for retinal ganglion cell number, oxidative stress, inflammatory markers, metabolic changes, and hypoxic response modulation using immunohistochemistry, multiplex assays, and capillary electrophoresis. Statistically significant differences were observed between normoxic and CIH groups for oxidative stress and inflammation, with CIH resulting in increased HIF-1alpha protein levels, higher oxidative stress marker 8-OHdG, and increased TNF-alpha. Pyruvate dehydrogenase kinase-1 protein was significantly reduced with CIH. No significant differences were found in retinal ganglion cell number. Our findings suggest that CIH induces oxidative stress, inflammation, and upregulation of HIF-1alpha in the retina, akin to early-stage glaucoma.
The ketogenic diet and hypoxia promote mitophagy in the context of glaucoma
(Frontiers Media S.A., 2024-05-22) Morgan, Autumn B.; Fan, Yan; Inman, Denise M.
Mitochondrial homeostasis includes balancing organelle biogenesis with recycling (mitophagy). The ketogenic diet protects retinal ganglion cells (RGCs) from glaucoma-associated neurodegeneration, with a concomitant increase in mitochondrial biogenesis. This study aimed to determine if the ketogenic diet also promoted mitophagy. MitoQC mice that carry a pH-sensitive mCherry-GFP tag on the outer mitochondrial membrane were placed on a ketogenic diet or standard rodent chow for 5 weeks; ocular hypertension (OHT) was induced via magnetic microbead injection in a subset of control or ketogenic diet animals 1 week after the diet began. As a measure of mitophagy, mitolysosomes were quantified in sectioned retina immunolabeled with RBPMS for RGCs or vimentin for Muller glia. Mitolysosomes were significantly increased as a result of OHT and the ketogenic diet (KD) in RGCs. Interestingly, the ketogenic diet increased mitolysosome number significantly higher than OHT alone. In contrast, OHT and the ketogenic diet both increased mitolysosome number in Muller glia to a similar degree. To understand if hypoxia could be a stimulus for mitophagy, we quantified mitolysosomes after acute OHT, finding significantly greater mitolysosome number in cells positive for pimonidazole, an adduct formed in cells exposed to hypoxia. Retinal protein analysis for BNIP3 and NIX showed no differences across groups, suggesting that these receptors were equivocal for mitophagy in this model of OHT. Our data indicate that OHT and hypoxia stimulate mitophagy and that the ketogenic diet is an additive for mitophagy in RGCs. The different response across RGCs and Muller glia to the ketogenic diet may reflect the different metabolic needs of these cell types.
Long-term HIF-1alpha stabilization reduces respiration, promotes mitophagy, and results in retinal cell death
(Springer Nature, 2023-11-24) Nsiah, Nana Yaa; Morgan, Autumn B.; Donkor, Nina; Inman, Denise M.
Ocular hypertension during glaucoma can lead to hypoxia, activation of the HIF transcription factors, and a metabolic shift toward glycolysis. This study aims to test whether chronic HIF activation and the attendant metabolic reprogramming can initiate glaucoma-associated pathology independently of ocular hypertension. HIF-1alpha stabilization was induced in mice for 2 and 4 weeks by inhibiting prolyl hydroxylases using the small molecule Roxadustat. HIF-1alpha stabilization and the expression of its downstream bioenergetic targets were investigated in the retina by immunofluorescence, capillary electrophoresis, and biochemical enzyme activity assays. Roxadustat dosing resulted in significant stabilization of HIF-1alpha in the retina by 4 weeks, and upregulation in glycolysis-associated proteins (GLUT3, PDK-1) and enzyme activity in both neurons and glia. Accordingly, succinate dehydrogenase, mitochondrial marker MTCO1, and citrate synthase activity were significantly decreased at 4 weeks, while mitophagy was significantly increased. TUNEL assay showed significant apoptosis of cells in the retina, and PERG amplitude was significantly decreased with 4 weeks of HIF-1alpha stabilization. A significant increase in AMPK activation and glial hypertrophy, concomitant with decreases in retinal ganglion cell function and inner retina cell death suggests that chronic HIF-1alpha stabilization alone is detrimental to retina metabolic homeostasis and cellular survival.
Destabilizing COXIV in Muller Glia Increases Retinal Glycolysis and Alters Scotopic Electroretinogram
(MDPI, 2022-12-12) Nsiah, Nana Yaa; Inman, Denise M.
Muller glia (MG), the principal glial cell of the retina, have a metabolism that defies categorization into glycolytic versus oxidative. We showed that MG mount a strong hypoxia response to ocular hypertension, raising the question of their relative reliance on mitochondria for function. To explore the role of oxidative phosphorylation (OXPHOS) in MG energy production in vivo, we generated and characterized adult mice in which MG have impaired cytochrome c oxidase (COXIV) activity through knockout of the COXIV constituent COX10. Histochemistry and protein analysis showed that COXIV protein levels were significantly lower in knockout mouse retina compared to control. Loss of COXIV activity in MG did not induce structural abnormalities, though oxidative stress was increased. Electroretinography assessment showed that knocking out COX10 significantly impaired scotopic a- and b-wave responses. Inhibiting mitochondrial respiration in MG also altered the retinal glycolytic profile. However, blocking OXPHOS in MG did not significantly exacerbate retinal ganglion cell (RGC) loss or photopic negative response after ocular hypertension (OHT). These results suggest that MG were able to compensate for reduced COXIV stability by maintaining fundamental processes, but changes in retinal physiology and metabolism-associated proteins indicate subtle changes in MG function.
Crosstalk Between Dysfunctional Mitochondria and Inflammation in Glaucomatous Neurodegeneration
(Frontiers Media S.A., 2021-07-21) Jassim, Assraa Hassan; Inman, Denise M.; Mitchell, Claire H.
Mitochondrial dysfunction and excessive inflammatory responses are both sufficient to induce pathology in age-dependent neurodegenerations. However, emerging evidence indicates crosstalk between damaged mitochondrial and inflammatory signaling can exacerbate issues in chronic neurodegenerations. This review discusses evidence for the interaction between mitochondrial damage and inflammation, with a focus on glaucomatous neurodegeneration, and proposes that positive feedback resulting from this crosstalk drives pathology. Mitochondrial dysfunction exacerbates inflammatory signaling in multiple ways. Damaged mitochondrial DNA is a damage-associated molecular pattern, which activates the NLRP3 inflammasome; priming and activation of the NLRP3 inflammasome, and the resulting liberation of IL-1beta and IL-18 via the gasdermin D pore, is a major pathway to enhance inflammatory responses. The rise in reactive oxygen species induced by mitochondrial damage also activates inflammatory pathways, while blockage of Complex enzymes is sufficient to increase inflammatory signaling. Impaired mitophagy contributes to inflammation as the inability to turnover mitochondria in a timely manner increases levels of ROS and damaged mtDNA, with the latter likely to stimulate the cGAS-STING pathway to increase interferon signaling. Mitochondrial associated ER membrane contacts and the mitochondria-associated adaptor molecule MAVS can activate NLRP3 inflammasome signaling. In addition to dysfunctional mitochondria increasing inflammation, the corollary also occurs, with inflammation reducing mitochondrial function and ATP production; the resulting downward spiral accelerates degeneration. Evidence from several preclinical models including the DBA/2J mouse, microbead injection and transient elevation of IOP, in addition to patient data, implicates both mitochondrial damage and inflammation in glaucomatous neurodegeneration. The pressure-dependent hypoxia and the resulting metabolic vulnerability is associated with mitochondrial damage and IL-1beta release. Links between mitochondrial dysfunction and inflammation can occur in retinal ganglion cells, microglia cells and astrocytes. In summary, crosstalk between damaged mitochondria and increased inflammatory signaling enhances pathology in glaucomatous neurodegeneration, with implications for other complex age-dependent neurodegenerations like Alzheimer's and Parkinson's disease.
Stretch stress propels glutamine dependency and glycolysis in optic nerve head astrocytes
(Frontiers Media S.A., 2022-08-05) Pappenhagen, Nathaniel; Yin, Eric; Morgan, Autumn B.; Kiehlbauch, Charles C.; Inman, Denise M.
Glaucoma is an optic neuropathy that leads to irreversible blindness, the most common subtype of which is typified by a chronic increase in intraocular pressure that promotes a stretch injury to the optic nerve head. In rodents, the predominant glial cell in this region is the optic nerve head astrocyte that provides axons with metabolic support, likely by releasing lactate produced through astrocytic glycolysis. Our primary hypothesis is that stretching of the optic nerve head astrocytes alters their metabolic activity, thereby advancing glaucoma-associated degeneration by compromising the metabolic support that the astrocytes provide to the axons in the optic nerve head. Metabolic changes in optic nerve head astrocytes were investigated by subjecting them to 24 h of 12% biaxial stretch at 1 Hz then measuring the cells' bioenergetics using a Seahorse XFe24 Analyzer. We observed significant glycolytic and respiratory activity differences between control and stretched cells, including greater extracellular acidification and lower ATP-linked respiration, yet higher maximal respiration and spare capacity in stretched optic nerve head astrocytes. We also determined that both control and stretched optic nerve head astrocytes displayed a dependency for glutamine over pyruvate or long-chain fatty acids for fuel. The increased use of glycolysis as indicated by the extracellular acidification rate, concomitant with a dependency on glutamine, suggests the need to replenish NAD + for continued glycolysis and provision of carbon for TCA cycle intermediates. Stretch alters optic nerve astrocyte bioenergetics to support an increased demand for internal and external energy.
Ocular Hypertension Results in Hypoxia within Glia and Neurons throughout the Visual Projection
(MDPI, 2022-04-29) Jassim, Assraa Hassan; Nsiah, Nana Yaa; Inman, Denise M.
The magnitude and duration of hypoxia after ocular hypertension (OHT) has been a matter of debate due to the lack of tools to accurately report hypoxia. In this study, we established a topography of hypoxia in the visual pathway by inducing OHT in mice that express a fusion protein comprised of the oxygen-dependent degradation (ODD) domain of HIF-1alpha and a tamoxifen-inducible Cre recombinase (CreERT2) driven by a ubiquitous CAG promoter. After tamoxifen administration, tdTomato expression would be driven in cells that contain stabilized HIF-1alpha. Intraocular pressure (IOP) and visual evoked potential (VEP) were measured after OHT at 3, 14, and 28 days (d) to evaluate hypoxia induction. Immunolabeling of hypoxic cell types in the retina and optic nerve (ON) was performed, as well as retinal ganglion cell (RGC) and axon number quantification at each time point (6 h, 3 d, 14 d, 28 d). IOP elevation and VEP decrease were detected 3 d after OHT, which preceded RGC soma and axon loss at 14 and 28 d after OHT. Hypoxia was detected primarily in Muller glia in the retina, and microglia and astrocytes in the ON and optic nerve head (ONH). Hypoxia-induced factor (HIF-alpha) regulates the expression of glucose transporters 1 and 3 (GLUT1, 3) to support neuronal metabolic demand. Significant increases in GLUT1 and 3 proteins were observed in the retina and ON after OHT. Interestingly, neurons and endothelial cells within the superior colliculus in the brain also experienced hypoxia after OHT as determined by tdTomato expression. The highest intensity labeling for hypoxia was detected in the ONH. Initiation of OHT resulted in significant hypoxia that did not immediately resolve, with low-level hypoxia apparent out to 14 and 28 d, suggesting that continued hypoxia contributes to glaucoma progression. Restricted hypoxia in retinal neurons after OHT suggests a hypoxia management role for glia.

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