The effect of phacoemulsification energy on the redox state of cultured human corneal endothelial cells

Objective: To evaluate the effects of phacoemulsification energy on the redox state and mitochondrial distribution of cultured human endothelial cells. Methods: Human corneal endothelial cells from fresh banked human donor tissue not suitable for transplantation were harvested and cultured. Cellular autofluorescence images were obtained using an inverted microscope. The redox fluorometric ratio, which can be related to oxidative stress, was calculated as the net value of fluorescence from the 4,6-diamidino-2-phenylindole channel divided by the net value of fluorescence from the fluorescein isothiocyanate-conjugated channel after subtraction of background. For determining the mitochondrial distribution patterns, the cell area was divided by drawing a line halfway between the nuclear and cell membranes. The average fluorescence in the central area was divided by the average fluorescence in the peripheral area. This ratio was compared. Results: Human corneal endothelial cells exposed to increasing phacoemulsification times and increasing ultrasonic energy levels displayed dose-dependent decreases in measured redox ratios. Lower redox ratios in response to phacoemulsification did not associate with decreases in cell size or altered patterns of mitochondrial localization. Conclusion: Redox fluorometry may serve as a useful indicator for the in vitro study of human corneal endothelial cell physiological response to ultrasonic stressors and potentially other nonoxidative stressors. Clinical Relevance: Redox fluorometry in combination with human corneal endothelial cell morphometric measurements has potential to serve as an indicator of human corneal endothelial cell injury resulting secondary to ultrasound phacoemulsification.