Mark A Greiner1, Kimberlee A Burckart2, Michael D Wagoner3, Gregory A Schmidt2, Cynthia R Reed2, Chase A Liaboe3, Miles J Flamme-Wiese3, M Bridget Zimmerman4, Robert F Mullins3, Randy H Kardon5, Kenneth M Goins1, Benjamin T Aldrich1. 1. University of Iowa Carver College of Medicine, Department of Ophthalmology and Visual Sciences, Iowa City, Iowa, United States 2Iowa Lions Eye Bank, Coralville, Iowa, United States. 2. Iowa Lions Eye Bank, Coralville, Iowa, United States. 3. University of Iowa Carver College of Medicine, Department of Ophthalmology and Visual Sciences, Iowa City, Iowa, United States. 4. University of Iowa College of Public Health, Department of Biostatistics, Iowa City, Iowa, United States. 5. University of Iowa Carver College of Medicine, Department of Ophthalmology and Visual Sciences, Iowa City, Iowa, United States 4Iowa City Veterans Affairs Medical Center and Center of Excellence for the Prevention and Treatment of Visual Loss, Iowa City.
Abstract
PURPOSE: We characterized mitochondrial respiration and glycolysis activity of human corneal endothelium, and compared metabolic activity between central and peripheral regions. METHODS: Endothelial keratoplasty-suitable corneas were obtained from donors aged 50 to 75 years. The endothelium-Descemet membrane complex (EDM) was isolated, and 3-mm punches were obtained from central and peripheral regions. Endothelium-Descemet membrane punches were assayed for mitochondrial respiration (oxygen consumption) and glycolysis (extracellular acidification) using an extracellular flux analyzer. Enzymatic (citrate synthase, glucose hexokinase) and mitochondrial density (MitoTracker) assays also were performed. RESULTS: Ten corneas were analyzed per assay. Metabolic activity for mitochondrial respiration and glycolysis showed expected changes to assay compounds (P < 0.01, all pairwise comparisons). Basal mitochondrial respiration and glycolysis activity did not differ between regions (P > 0.99). Similarly, central versus peripheral activity after assay compound treatment showed no significant differences (P > 0.99, all time points). The intracorneal coefficient of variation for basal readings between two and four peripheral punches was 18.5% of the mean. Although peripheral samples displayed greater enzymatic activity than central samples (P < 0.05), similar to extracellular flux results, mitochondrial density did not differ between regions (P = 0.78). CONCLUSIONS: Extracellular flux analysis of oxygen and pH is a valid technique for characterizing metabolic activity of human corneal endothelium. This technique demonstrates high reproducibility, allows quantification of metabolic parameters using small quantities of live cells, and permits estimation of overall metabolic output. Neither oxygen consumption nor extracellular acidification differed between central and peripheral regions of transplant suitable corneas in this series. Our results show that endothelial cell health can be quantified biochemically in transplant suitable corneas.
PURPOSE: We characterized mitochondrial respiration and glycolysis activity of human corneal endothelium, and compared metabolic activity between central and peripheral regions. METHODS: Endothelial keratoplasty-suitable corneas were obtained from donors aged 50 to 75 years. The endothelium-Descemet membrane complex (EDM) was isolated, and 3-mm punches were obtained from central and peripheral regions. Endothelium-Descemet membrane punches were assayed for mitochondrial respiration (oxygen consumption) and glycolysis (extracellular acidification) using an extracellular flux analyzer. Enzymatic (citrate synthase, glucose hexokinase) and mitochondrial density (MitoTracker) assays also were performed. RESULTS: Ten corneas were analyzed per assay. Metabolic activity for mitochondrial respiration and glycolysis showed expected changes to assay compounds (P < 0.01, all pairwise comparisons). Basal mitochondrial respiration and glycolysis activity did not differ between regions (P > 0.99). Similarly, central versus peripheral activity after assay compound treatment showed no significant differences (P > 0.99, all time points). The intracorneal coefficient of variation for basal readings between two and four peripheral punches was 18.5% of the mean. Although peripheral samples displayed greater enzymatic activity than central samples (P < 0.05), similar to extracellular flux results, mitochondrial density did not differ between regions (P = 0.78). CONCLUSIONS: Extracellular flux analysis of oxygen and pH is a valid technique for characterizing metabolic activity of human corneal endothelium. This technique demonstrates high reproducibility, allows quantification of metabolic parameters using small quantities of live cells, and permits estimation of overall metabolic output. Neither oxygen consumption nor extracellular acidification differed between central and peripheral regions of transplant suitable corneas in this series. Our results show that endothelial cell health can be quantified biochemically in transplant suitable corneas.
Authors: M Kubota; Y B Shui; M Liu; F Bai; A J Huang; N Ma; D C Beebe; C J Siegfried Journal: Free Radic Biol Med Date: 2016-07-19 Impact factor: 7.376
Authors: Michael D Wagoner; Laura R Bohrer; Benjamin T Aldrich; Mark A Greiner; Robert F Mullins; Kristan S Worthington; Budd A Tucker; Luke A Wiley Journal: Biol Open Date: 2018-05-08 Impact factor: 2.422