The diagnostic and research applications of noninvasive redox fluorometry to the in vivo determination of corneal metabolic function and physiological activity is our long term objective. This requires further experimental knowledge of its sensitivity, specificity, and the quantitation of the relationship between cellular redox states, energy metabolism and physiological function. Measurements of the naturally fluorescent pyridine nucleotides and flavoprotein components of corneal mitochondria are indicators of cellular redox states. These noninvasive measurements will be calibrated with analytical determinations of the concentrations of the pyridine nucleotides in the component layers of the cornea. Differences between redox states, metabolite concentrations and oxygen consumption will be correlated for the endothelium and the epithelium. The role of adenosine in the regulation of endothelial fluid transport will be investigated. The heterogeneity of endothelial redox states will be studied at the level of single endothelial cells. The effects of the incident ultraviolet and violet light used in the redox fluorometric studies will be evaluated with respect to any induced alterations of normal cellular metabolism and physiological function. The relationship between endothelial redox states, rates of transendothelial fluid transport and endothelial oxygen consumption will be established.
These aims will be studied by simultaneous measurements of fluid transport, potential difference, oxygen consumption, and fluorometric measurements of redox states of the component layers of the cornea. They will be performed on mitochondria from bovine corneas, in vivo rabbit and cat corneas, and in vitro perfused corneas from rabbits and cats. Fluorescence microscopy and computer image analysis will measure the heterogeneity of endothelial redox states and provide a method to detect abnormal endothelial cells. These studies will provide the knowledge necessary for the possible future diagnostic applcations of noninvasive redox fluorometry to ophthalmology. The combination of noninvasive fluorometric redox measurements, microchemical analytical analysis of metabolites, and direct measure of rates of endothelial fluid transport will be combined to investigate the metabolic regulation of endothelial transport function.
