Nitric Oxide Inhalation Improves Microvascular Flow and Decreases Infarction Size After Myocardial Ischemia and Reperfusion
Xiaoshun Liu, MD, PhD*,
Yanming Huang, MD, PhD*,
Peter Pokreisz, PhD ,
Pieter Vermeersch, MD,
Glenn Marsboom, MSc,
Marc Swinnen*,
Eric Verbeken, MD, PhD ,
Jose Santos, MD*,
Marijke Pellens,
Hilde Gillijns,
Frans Van de Werf, MD, PhD*,
Kenneth D. Bloch, MD and
Stefan Janssens, MD, PhD*,,*
* Department of Cardiology
Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute of Biotechnology
Department of Pathology, University of Leuven, Leuven, Belgium
Cardiovascular Research Center, Department of Medicine, and Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
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Figure 1 iNO Reduces Infarct Size After Ischemia-Reperfusion Injury
(Top) Evan's blue and triphenyl tetrazolium chloride staining of representative transverse midventricle sections from pigs subjected to left anterior descending coronary artery occlusion (50 min) and reperfusion (4 h) treated with saline (control), inhaled nitric oxide (iNO), or intravenous nitroglycerin (IV-NTG). The area at risk (AAR) is delineated by the absence of dark blue staining (indicated by oblique lines); viable myocardium within this region is stained red, and infarcted tissue remains white. (Bottom) Quantitative measurement of left ventricular area at risk (% LV, left) and infarct size (% of AAR, right) in control (n = 13), iNO (n = 12), and IV-NTG (n = 10) pigs; *p < 0.05 versus control.
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Figure 2 CK-MB Isoform Release During Ischemia and Reperfusion
Creatine phosphokinase-MB isoform (CK-MB) levels measured in plasma samples obtained at baseline (time 0), during 50 min of ischemia, and 30, 60, 90, 120, 180, and 240 min after reperfusion. Arrow indicates time of reperfusion. Data are shown as mean ± SEM; *p < 0.05 versus control and IV-NTG. Abbreviations as in Figure 1.
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Figure 3 Regional Myocardial Blood Flow After 4 h of Reperfusion
Endocardial and epicardial regional myocardial blood flow in the infarct area and border zone of pigs receiving saline infusion (control), iNO, or IV-NTG. Data are shown as mean ± SEM; *p < 0.05 versus control. Abbreviations as in Figure 1.
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Figure 4 Histologic Analysis of Representative Myocardial Sections After Ischemia-Reperfusion Injury
Hematoxylin and eosin (H&E) staining (A to C) shows severe hemorrhage in the infarct zone of control pigs but not in IV-NTG and iNO-treated pigs. Myeloperoxidase (MPO) immunostaining in the border zone (D to F) indicates neutrophil infiltration in control pigs which was less in iNO pigs but not in IV-NTG pigs. Terminal dUTP nick-end labeling (TUNEL) staining for apoptosis (G to I) indicates less apoptosis in the border zone of the iNO pigs. Original magnifications: x200 (A to F) and x400 (G to I). Abbreviations as in Figure 1.
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Figure 5 Quantitative Analysis of Myocardial Hemorrhage, Neutrophil Infiltration, and Cardiomyocyte Apoptosis After Ischemia-Reperfusion Injury
(A) Hemorrhage scores in the infarct zone of control pigs and pigs treated with iNO or IV-NTG. (B) Score of neutrophil infiltration (top) and MPO activity (bottom) in the infarct and border zones of animals receiving saline, iNO, or IV-NTG. (C) Percentage of TUNEL-positive cardiomyocytes in the infarct border zone. Data are expressed as mean ± SEM; *p < 0.05 versus control; p < 0.05 versus control and IV-NTG. Abbreviations as in Figures 1 and 4.
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Figure 6 NOx in Myocardial Tissue and Plasma During Ischemia-Reperfusion Injury
(A) Myocardial oxidized nitric oxide species (NOx) levels in the infarct, border, and remote zones of the porcine heart after 40 min of ischemia and 4 h of reperfusion. Data are expressed as mean ± SEM. *p < 0.05 compared with border and remote zone in iNO; p < 0.01 versus infarct zone in IV-NTG pigs; and p < 0.001 versus infarct zone in control animals. n = 8 per group. (B) Plasma NOx levels after 45 min of ischemia, and 30, 60, 120, and 240 min of reperfusion. Data are expressed as mean ± SEM. *p < 0.05. n = 4 per group. Abbreviations as in Figure 1.
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