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Mark A Greiner, Kimberlee Burckart, Gregory Schmidt, Cynthia Reed, Chase Liaboe, Bridget Zimmerman, Robert F Mullins, Benjamin Aldrich; Spatial assessment of energy producing metabolic activity in the corneal endothelium of human donors. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4918.
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To quantify and compare mitochondrial respiration and glycolysis activity in the central and peripheral regions of the endothelium from donor corneas.
Corneal endothelial tissues assayed in this study were obtained from donors age 50-75 and approved for endothelial keratoplasty with a death-to-preservation time of 14 days or less. The endothelium-Descemet membrane complex (EDM) was partially peeled from the stromal layer. Central and peripheral EDM punches 3mm in diameter were obtained from pre-peeled tissue and affixed to tissue culture plate wells. EDM punches were assayed to evaluate mitochondrial respiration and glycolysis activity using a Seahorse XFe24 Bioanalyzer. Metabolic activity, measured for mitochondrial respiration as the oxygen consumption rate (OCR) and glycolysis as the external acidification rate (ECAR), was normalized to cell count. Metabolic measurements were compared between the central and peripheral tissue punches.
Ten corneas from unique donors were analyzed for each assay. Basal metabolic activity measurements for mitochondrial respiration and glycolysis were detected significantly above the level of background signal and showed significant changes in response to treatment with different compounds known to influence these metabolic pathways (p<0.01, all pairwise comparisons). Mean OCR and ECAR metabolic activity of peripheral punches were not significantly different from the central portion of the cornea in any of the metabolic measures generated from the seahorse assay (p>0.99, all pairwise comparisons).
Results of this pilot study demonstrate that it is possible to obtain metabolic measurements of small samples of native donor corneal endothelial cells. These finding support the possible use of central or peripheral punches to obtain accurate estimates of the energy producing metabolic activity in donor corneal endothelium.
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