Autofluorescence spectroscopy for NADH and flavoproteins redox state monitoring in the isolated rat heart subjected to ischemia-reperfusion

Abstract

Reduction of myocardial ischemia-reperfusion injury in the patients undergoing cardiac surgery under cardiopulmonary bypass represents an important goal. Intraoperative monitoring of myocardial metabolic state using continuous registration of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) fluorescence might contribute to the solution of the problem. The successful application of fluorescent spectroscopy in the clinical field requires additional refinement of the technique, particularly using excitation of both NADH and FAD with different wavelengths. The experiments were performed on the isolated Langendorff-perfused rat hearts (n=28) subjected to either regional or global ischemia-reperfusion. Two principles of NADH and FAD autofluorescence (AF) measurement were used for ischemia monitoring: (1) analysis of photographs and videos obtained with multispectral organoscopy technique allowing the assessment of both spatial and temporal characteristics of the process (n=16); (2) continuous registration of tissue redox state in a representative area of the heart by application of local spectroscopy, assisted by fiber optic spectrometer (n=12). It was found that regional myocardial ischemia resulted in a rapid, substantial increase in the intensity of NADH AF excited at 360nm in the ischemic versus non-ischemic area of the heart. The same result was obtained when the heart was made globally ischemic, while the restoration of perfusate flow completely reversed the increase in NADH AF. During the transition from ischemia to reperfusion, the spatial heterogeneity of myocardial AF was noted on video recordings, probably reflecting the microheterogeneity of myocardial blood flow. Local spectroscopy studies demonstrated opposite changes in the NADH and FAD AF during ischemia. Using both methodological approaches, we found that repetitive brief episodes of global myocardial ischemia resulted in progressive decrease in the magnitude of AF elevation, which might point to preconditioning effect. The application of multispectral fluorescent organoscopy offers the advantage of monitoring myocardial redox state at the level of the entire heart. Local spectroscopy is characterized by better precision and, in addition, provides the unique opportunity to measure AF in different parts of the spectrum. AF measurements are non-invasive, rapid, and technically easy to perform. For future clinical applications, it might be recommended to combine the measurement of redox state of both NADH and FAD, using excitation wavelength and emission filter optimal for each fluorophore.