Abstract
Raman spectroscopy, which identifies intrinsic molecular constituents, has a potential for determining myocardial viability under label-free conditions. However, its suitability for evaluating myocardial ischaemia is undetermined. Focusing on cytochromes, i.e., representative molecules reflecting mitochondrial activity, we tested whether Raman spectroscopy is applicable for evaluating myocardial ischaemia especially during early ischaemic phase. We obtained spontaneous Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart upon 532-nm excitation before and during the “stopped-flow,” global ischaemia. Semi-quantitative values of the peak intensities at 750 and 1127 cm−1, which reflect reduced cytochromes c and b, increased immediately and progressively after induction of the stopped flow, indicating progressive reduction of the mitochondrial respiration. Such spectral changes emerged before the loss of 1) mitochondrial membrane potentials measured by the fluorescence intensity of tetramethyl rhodamine ethyl ester or 2) staining of the triphenyl tetrazolium chloride dye in the myocardium. The progressive increases in the Raman peaks by stopped flow were significantly retarded by ischaemic preconditioning. Sequential measurements of the peak intensities at 750 and 1127 cm−1 enabled early detection of the myocardial ischaemia based on the mitochondrial functions. These data suggest that Raman spectroscopy offers the potential to evaluate acute ischaemic heart under label-free conditions.
Highlights
Diseased tissues[13,14]
After an induction of global ischaemia (SI) by stopped-flow, the Raman spectral peaks showed a progressive increase in intensity at 750 and 1127 cm−1, while those remained stable under continuous perfusion (Fig. 2a)
During the early period of ischaemia, we confirmed that morphological changes are absent or too subtle to be recognized in the myocardium in the H & E histology and di-4-ANEPPS fluorescence images reflecting the cell membrane integrities
Summary
Diseased tissues[13,14] In this regard, spontaneous Raman spectroscopy has potential for evaluation of ischaemic conditions of the heart. We have hypothesized that myocardial ischaemia can be evaluated by Raman spectroscopy in accordance with the progressive alterations of redox status of the mitochondria-derived cytochromes from the early, reversible stage to the late, irreversible one. To address this possibility, we conducted sequential detections of the Raman spectra of Langendorff-perfused rat hearts under “stopped-flow”, global ischaemia. Applicability of Raman spectroscopy to ischaemic myocardium was evaluated by ischaemic preconditioning[16]
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