Raman Spectroscopic Assessment of Myocardial Viability in Langendorff-Perfused Ischemic Rat Hearts

Abstract

Spontaneous Raman spectroscopy, which senses changes in cellular contents of reduced cytochrome c, could be a powerful tool for label-free evaluation of ischemic hearts. However, undetermined is whether it is applicable to evaluation of myocardial viability in ischemic hearts. To address this issue, we investigated sequential changes in Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart before and during ligation of the left coronary artery and its subsequent release and re-ligation. Under 532-nm wavelength excitation, the Raman peak intensity of reduced cytochrome c at 747 cm^-1 increased quickly after the coronary ligation, and reached a quasi-steady state within 30 min. Subsequent reperfusion of the heart after a short-term (30-min) ligation that simulates reversible conditions resulted in quick recovery of the peak intensity to the baseline. Further re-ligation resulted in resurgence of the peak intensity to nearly the identical value to the first ischemia value. In contrast, reperfusion after prolonged (120-min) ligation that assumes irreversible states resulted in incomplete recovery of the peak intensity, and re-ligation resulted in inadequate resurgence. Electron microscopic observations confirmed the spectral findings. Together, the Raman spectroscopic measurement for cytochrome c could be applicable to evaluation of viability of the ischemic myocardium without labeling.

Keywords: Raman spectroscopy; cytochrome c; ischemia; mitochondria; myocardial viability.

Fig. 1.

Experimental schematic diagram and time course. (A) An isolated heart is perfused retrogradely from the ascending aorta. The left descending artery is ligated just before the bifurcation of the diagonal branch. The left ventricular wall is divided into perfused and non-perfused areas. (B) The 1st ischemia is created by 30-min or 120-min coronary ligation. Durations of baseline perfusion, reperfusion, and the 2nd ligation phases are 60, 20, and 30 min, respectively, in both models. BDM, 2,3-butanedione monoxime.

Fig. 2.

Sequential representations of averaged Raman spectra. Averaged Raman spectra acquired from the surface of perfused rat hearts at ischemic (non-perfused) and non-ischemic (perfused) areas of the 30-min (A) and 120-min (B) ligation models at each time point. * and ** denote the time points after commencement of reperfusion and 2nd ligation, respectively.

Fig. 3.

Sequential changes in Raman spectral peak intensity ratio. Graphs plotting ratios of the peak values at 747 cm⁻¹ over those at 1447 cm⁻¹ (I₇₄₇/I₁₄₄₇) throughout the experiment in 30-min (A) and 120-min (B) ligation models. (C) Graphs comparing Raman spectral peak ratios during reperfusion after 30-min and 120-min 1st ligation and the 2nd ligation phase at perfused and non-perfused areas. * and ** denote the time points after commencement of reperfusion and 2nd ligation, respectively. ^† denotes P < 0.05 using the nonparametric Mann-Whitney U test between perfused and non-perfused areas corresponding to the same time. ^‡ denotes P < 0.05 by the same analysis as above between 30-min and 120-min ligation at each time point. Data are presented as mean ± SD.

Fig. 4.

Comparison between the non-perfused (ischemic) areas of the heart at the end of the first ischemic phases in 30-min and 120-min ligation models. (A) Triphenyl tetrazolium chloride-stained hearts. Putative non-perfused areas are indicated by the dotted line. Bar = 5 mm. (B) H & E-stained images of the ischemic myocardium. (C) Ultrastructural details of cardiomyocytes in perfused and non-perfused areas. For references, the images obtained from perfused areas are also shown (bottom). The insets are magnified images of mitochondria. Electron micrograph, ×15000. Bar = 500 nm. M, mitochondria; S, sarcomere.