Dimitrios Karamichos, Ph.D.
Permanent URI for this communityhttps://hdl.handle.net/20.500.12503/31785
Executive Director and Endowed Chair, North Texas Eye Research Institute
Professor, Pharmaceutical Sciences
Professor, Pharmacology & Neuroscience
Email: Dimitrios.Karamichos@unthsc.edu
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Browsing Dimitrios Karamichos, Ph.D. by Author "Cheng, Rui"
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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 Peroxisome proliferator-activated receptor-alpha (PPARalpha) regulates wound healing and mitochondrial metabolism in the cornea(National Academy of Science, 2023-03-22) Liang, Wentao; Huang, Li; Whelchel, Amy E.; Yuan, Tian; Ma, Xiang; Cheng, Rui; Takahashi, Yusuke; Karamichos, Dimitrios; Ma, Jian-XingDiabetes can result in impaired corneal wound healing. Mitochondrial dysfunction plays an important role in diabetic complications. However, the regulation of mitochondria function in the diabetic cornea and its impacts on wound healing remain elusive. The present study aimed to explore the molecular basis for the disturbed mitochondrial metabolism and subsequent wound healing impairment in the diabetic cornea. Seahorse analysis showed that mitochondrial oxidative phosphorylation is a major source of ATP production in human corneal epithelial cells. Live corneal biopsy punches from type 1 and type 2 diabetic mouse models showed impaired mitochondrial functions, correlating with impaired corneal wound healing, compared to nondiabetic controls. To approach the molecular basis for the impaired mitochondrial function, we found that Peroxisome Proliferator-Activated Receptor-alpha (PPARalpha) expression was downregulated in diabetic human corneas. Even without diabetes, global PPARalpha knockout mice and corneal epithelium-specific PPARalpha conditional knockout mice showed disturbed mitochondrial function and delayed wound healing in the cornea, similar to that in diabetic corneas. In contrast, fenofibrate, a PPARalpha agonist, ameliorated mitochondrial dysfunction and enhanced wound healing in the corneas of diabetic mice. Similarly, corneal epithelium-specific PPARalpha transgenic overexpression improved mitochondrial function and enhanced wound healing in the cornea. Furthermore, PPARalpha agonist ameliorated the mitochondrial dysfunction in primary human corneal epithelial cells exposed to diabetic stressors, which was impeded by siRNA knockdown of PPARalpha, suggesting a PPARalpha-dependent mechanism. These findings suggest that downregulation of PPARalpha plays an important role in the impaired mitochondrial function in the corneal epithelium and delayed corneal wound healing in diabetes.