Are macrophages involved in early myocardial reperfusion injury?

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Are macrophages involved in early myocardial reperfusion injury? Lucia Formigli, BSc, Lidia Ibba Manneschi, MD, Chiara Nediani, BSc, Elena Marcelli, MD, Geri Fratini, MD, Sandra Zecchi Orlandini, BSc, Avio M Perna, MD The Annals of Thoracic Surgery Volume 71, Issue 5, Pages 1596-1602 (May 2001) DOI: 10.1016/S0003-4975(01)02400-6 Copyright © 2001 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Myeloperoxidase (MPO) activity in sham-operated control, ischemic (I), and ischemic-reperfused (I-R) pig myocardium. °p < 0.001 versus control myocardium; ∗p < 0.001 versus ischemic (I) myocardium. The Annals of Thoracic Surgery 2001 71, 1596-1602DOI: (10.1016/S0003-4975(01)02400-6) Copyright © 2001 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Alpha naphthyl acetate esterase (ANAE) activity in sham-operated control, ischemic (I), and ischemic-reperfused (I-R) hearts. Alpha naphthyl acetate activity was measured on 25-μg protein samples of cytosolic preparations at a final alpha naphthyl acetate concentration of 0.5 mmol/L. Values are mean ± standard error of the mean. p < 0.05 by Student’s t test. °p < 0.001 versus control myocardium; ∗p < 0.001 versus ischemic (I) myocardium. (ANAE activity is expressed as absorbance at 490 nm per 25 μg protein). The Annals of Thoracic Surgery 2001 71, 1596-1602DOI: (10.1016/S0003-4975(01)02400-6) Copyright © 2001 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Light microscopic examination of ischemic-reperfused myocardium. Monocytes (arrows) are present together with neutrophils within the inflammatory infiltrate. (Toluidine blue sodium-tetraborate stained sections, ×800.) The Annals of Thoracic Surgery 2001 71, 1596-1602DOI: (10.1016/S0003-4975(01)02400-6) Copyright © 2001 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Transmission electron microscopy of ischemic-reperfused myocardium showing the various steps of monocyte extravasion. Monocytes are visible (a) inside a small blood vessel (arrows) together with neutrophils (arrowheads); (b) and (c) tightly adhering to the vascular luminal surface (arrows); and (d) migrating in the interstitial spaces (arrowhead). (a: ×1,500; b: ×11,250; c: ×4,500; d: ×2,250.) The Annals of Thoracic Surgery 2001 71, 1596-1602DOI: (10.1016/S0003-4975(01)02400-6) Copyright © 2001 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Transmission electron microscopy of ischemic (a) and ischemic-reperfused (b–d) myocardium. (a) Signs of mild injuries, ie, moderate mitochondria swelling (arrows) and intermyofibrillar edema are visible inside a cardiomyocyte; (b) severe hypercontractions (arrows) of myofilaments are seen in one fiber; (c) higher magnification of a cardiomyocyte showing dark myofibrillar material interspersed with overstretched myofilaments; and (d) a capillary is lined by a seriously affected endothelial cell (arrowheads indicate the endothelial lining and arrow shows the nucleus of the endothelial cell). (a: ×3,700; b: ×3,700; c: ×5,500; d: ×4,500.) The Annals of Thoracic Surgery 2001 71, 1596-1602DOI: (10.1016/S0003-4975(01)02400-6) Copyright © 2001 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 (a) Western blot analysis of tumor necrosis factor-α (TNFα) in myocardial tissues from sham-operated control (C), ischemic (I), and ischemic-reperfused (I-R) hearts. Myocardial samples of 50 μg of protein were applied per slot. Tumor necrosis factor-α protein was expressed as a 17-kd band. Recombinant human TNFα was used as positive control (PC). (b) Quantitative data from Western blot analysis. Each bar represents the mean value + standard error of the mean of four different blots. Signals were quantified by densitometric analyses and were expressed as densitometric units per microgram of protein. ∗p < 0.05 I-R versus I. The Annals of Thoracic Surgery 2001 71, 1596-1602DOI: (10.1016/S0003-4975(01)02400-6) Copyright © 2001 The Society of Thoracic Surgeons Terms and Conditions

Fig 7 Immunofluorescence staining of ischemic-reperfused myocardium with polyclonal antibody against tumor necrosis factor-α. Numerous macrophages, infiltrating the connective tissue among cardiomyocytes, are markedly stained. (×500.) The Annals of Thoracic Surgery 2001 71, 1596-1602DOI: (10.1016/S0003-4975(01)02400-6) Copyright © 2001 The Society of Thoracic Surgeons Terms and Conditions