ABSTRACT
The post-ischemic no-reflow phenomenon after primary percutaneous coronary intervention (PCI) is observed in more than half of subjects and is defined as the absence or marked slowing of distal coronary blood flow despite removal of the arterial occlusion. To visualize no-reflow in experimental studies, the fluorescent dye thioflavin S (ThS) is often used, which allows for the estimation of the size of microvascular obstruction by staining the endothelial lining of vessels. Based on the ability of indocyanine green (ICG) to be retained in tissues with increased vascular permeability, we proposed the possibility of using it to assess not only the severity of microvascular obstruction but also the degree of vascular permeability in the zone of myocardial infarction. The aim of our study was to investigate the possibility of using ICG to visualize no-reflow zones after ischemia-reperfusion injury of rat myocardium. Using dual ICG and ThS staining and the FLUM multispectral fluorescence organoscope, we recorded ICG and ThS fluorescence within the zone of myocardial necrosis, identifying ICG-negative zones whose size correlated with the size of the no-reflow zones detected by ThS. It is also shown that the contrast change between the no-reflow zone and nonischemic myocardium reflects the severity of blood stasis, indicating that ICG-negative zones are no-reflow zones. The described method can be an addition or alternative to the traditional method of measuring the size of no-reflow zones in the experiment.
ABSTRACT
The fluorophore indocyanine green accumulates in areas of ischemia-reperfusion injury due to an increase in vascular permeability and extravasation of the dye. The aim of the study was to validate an indocyanine green-based technique of in vivo visualization of myocardial infarction. A further aim was to quantify infarct size ex vivo and compare this technique with the standard triphenyltetrazolium chloride staining. Wistar rats were subjected to regional myocardial ischemia (30 minutes) followed by reperfusion (n = 7). Indocyanine green (0.25 mg/mL in 1 mL of normal saline) was infused intravenously for 10 minutes starting from the 25th minute of ischemia. Video registration in the near-infrared fluorescence was performed. Epicardial fluorescence of indocyanine green corresponded to the injured area after 30 minutes of reperfusion. Infarct size was similar when determined ex vivo using traditional triphenyltetrazolium chloride assay and indocyanine green fluorescent labeling. Intravital visualization of irreversible injury can be done directly by fluorescence on the surface of the heart. This technique may also be an alternative for ex vivo measurements of infarct size.
ABSTRACT
BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.
