Optimization and Application of Seahorse Glycolytic Stress Test in Mouse Corneal Tissues: Revealing Metabolic Profiles in Injury Models.

Purpose: The purpose of this study was to develop and optimize a Seahorse extracellular flux analyzer for real-time monitoring of extracellular acidification rate (ECAR) in mouse corneal punch biopsies, and to characterize glycolytic functional changes in corneal injury models. Methods: Mouse corneal biopsies were obtained by a biopsy punch immediately after euthanasia, and corneal tissue glycolysis function was analyzed under specific groups using a Seahorse XFe24 analyzer. The glycolysis function of human corneal stromal cells at different cell densities and oligo concentrations was evaluated using a Seahorse XFe Pro analyzer. Results: The findings from the glycolysis stress test indicated that a concentration of 1.5 µmol/L of oligomycin and a corneal punch size of 1.5 mm were optimal for assessing the ECAR. Variations in corneal orientation and age did not yield significant differences in ECAR values. Notably, the ECAR values measured at the limbus were significantly different from those at the central cornea. Furthermore, the partial glycolysis function in Balb/c mice was found to be significantly higher than that observed in C57BL/6J mice and Sprague-Dawley rats. This methodology also revealed a marked increase in the glycolysis function of corneal tissue in cases of lipopolysaccharide (LPS)-induced keratitis and during the later stages following an alkali burn. Conclusions: This research establishes a new methodology for assessing the real-time functionality of glycolysis in fresh mouse corneal tissues, and demonstrates profound glycolysis profiles of ex vivo corneas within corneal injury models. These findings offer valuable insights for future studies focused on the corneal glycolysis function.