Browsing by Subject "Cornea"
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Item A Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy(Association for Research in Vision and Ophthalmology, 2023-08-29) Liang, Wentao; Huang, Li; Yuan, Tian; Cheng, Rui; Takahashi, Yusuke; Moiseyev, Gennadiy P.; Karamichos, Dimitrios; Ma, Jian-XingPURPOSE: To develop and optimize a method to monitor real-time mitochondrial function by measuring the oxygen consumption rate (OCR) in murine corneal biopsy punches with a Seahorse extracellular flux analyzer. METHODS: Murine corneal biopsies were obtained using a biopsy punch immediately after euthanasia. The corneal metabolic profile was assessed using a Seahorse XFe96 pro analyzer, and mitochondrial respiration was analyzed with specific settings. RESULTS: Real-time adenosine triphosphate rate assay showed that mitochondrial oxidative phosphorylation is a major source of adenosine triphosphate production in ex vivo live murine corneal biopsies. Euthanasia methods (carbon dioxide asphyxiation vs. overdosing on anesthetic drugs) did not affect corneal OCR values. Mouse corneal biopsy punches in 1.5-mm diameter generated higher and more reproducible OCR values than those in 1.0-mm diameter. The biopsy punches from the central and off-central cornea did not show significant differences in OCR values. There was no difference in OCR reading by the tissue orientations (the epithelium side up vs. the endothelium side up). No significant differences were found in corneal OCR levels between sexes, strains (C57BL/6J vs. BALB/cJ), or ages (4, 8, and 32 weeks). Using this method, we showed that the wound healing process in the mouse cornea affected mitochondrial activity. CONCLUSIONS: The present study validated a new strategy to measure real-time mitochondrial function in fresh mouse corneal tissues. This procedure should be helpful for studies of the ex vivo live corneal metabolism in response to genetic manipulations, disease conditions, or pharmacological treatments in mouse models.Item Ablation of Sphingosine Kinase 1 Protects Cornea from Neovascularization in a Mouse Corneal Injury Model(MDPI, 2022-09-24) Wilkerson, Joseph L.; Basu, Sandip K.; Stiles, Megan A.; Prislovsky, Amanda; Grambergs, Richard C.; Nicholas, Sarah E.; Karamichos, Dimitrios; Allegood, Jeremy C.; Proia, Richard L.; Mandal, NawajesThe purpose of this study was to investigate the role of sphingosine kinase 1 (SphK1), which generates sphingosine-1-phosphate (S1P), in corneal neovascularization (NV). Wild-type (WT) and Sphk1 knockout (Sphk1(-/-)) mice received corneal alkali-burn treatment to induce corneal NV by placing a 2 mm round piece of Whatman No. 1 filter paper soaked in 1N NaOH on the center of the cornea for 20 s. Corneal sphingolipid species were extracted and identified using liquid chromatography/mass spectrometry (LC/MS). The total number of tip cells and those positive for ethynyl deoxy uridine (EdU) were quantified. Immunocytochemistry was done to examine whether pericytes were present on newly forming blood vessels. Cytokine signaling and angiogenic markers were compared between the two groups using multiplex assays. Data were analyzed using appropriate statistical tests. Here, we show that ablation of SphK1 can significantly reduce NV invasion in the cornea following injury. Corneal sphingolipid analysis showed that total levels of ceramides, monohexosyl ceramides (HexCer), and sphingomyelin were significantly elevated in Sphk(-/-) corneas compared to WT corneas, with a comparable level of sphingosine among the two genotypes. The numbers of total and proliferating endothelial tip cells were also lower in the Sphk1(-/-) corneas following injury. This study underscores the role of S1P in post-injury corneal NV and raises further questions about the roles played by ceramide, HexCer, and sphingomyelin in regulating corneal NV. Further studies are needed to unravel the role played by bioactive sphingolipids in maintenance of corneal transparency and clear vision.Item The Underlying Relationship between Keratoconus and Down Syndrome(MDPI, 2022-09-24) Akoto, Theresa; Li, Jiemin J.; Estes, Amy J.; Karamichos, Dimitrios; Liu, YutaoKeratoconus (KC) is one of the most significant corneal disorders worldwide, characterized by the progressive thinning and cone-shaped protrusion of the cornea, which can lead to severe visual impairment. The prevalence of KC varies greatly by ethnic groups and geographic regions and has been observed to be higher in recent years. Although studies reveal a possible link between KC and genetics, hormonal disturbances, environmental factors, and specific comorbidities such as Down Syndrome (DS), the exact cause of KC remains unknown. The incidence of KC ranges from 0% to 71% in DS patients, implying that as the worldwide population of DS patients grows, the number of KC patients may continue to rise significantly. As a result, this review aims to shed more light on the underlying relationship between KC and DS by examining the genetics relating to the cornea, central corneal thickness (CCT), and mechanical forces on the cornea, such as vigorous eye rubbing. Furthermore, this review discusses KC diagnostic and treatment strategies that may help detect KC in DS patients, as well as the available DS mouse models that could be used in modeling KC in DS patients. In summary, this review will provide improved clinical knowledge of KC in DS patients and promote additional KC-related research in these patients to enhance their eyesight and provide suitable treatment targets.