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Images in Cardiovascular Medicine Connective Tissue Skeleton of the Human Heart A Demonstration by Cell-Maceration Scanning Electron Microscope Method Marcos A. Rossi, MD; Monica A. Abreu, BS; Lı́gia B. Santoro, BS T Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 he stroma of the heart maintains the structure of the myocardium, determining tissue tensile strength and stiffness.1 In addition, it contributes to ventricular function through the transmission of myocyte-generated force to the atrial and ventricular chambers and to the relengthening of myocytes in diastole.2 The three-dimensional configuration of cardiac collagen has been determined by scanning electron microscopy3-5: the epimysium envelops the entire cardiac muscle; the perimysium, which is an extension of the epimysium, serves to enwrap groups of myocytes; and the endomysium, as final arborization of the perimysium, supports and connects individual cells. The endomysial weave envelops each individual myocyte and is connected to adjacent myocytes by lateral struts. Because this knowledge was obtained through studies on whole fixed myocardial tissue without removal of its nonfibrous elements, we attempted to dissolve the cellular elements and leave behind a noncollapsed matrix, aiming for a better three-dimensional view. For this, we used a modification of the NaOH maceration technique reported by Ohtani.6 This method was reported to be able to remove cellular elements much more effectively than any other method. Small fragments, 103533 mm in size, of the anterior wall at the midventricular region were obtained from three human hearts, weighing between 300 and 350 g, without any pathological changes. All samples were fixed in 10% neutral formalin. After being rinsed in distilled water, the specimens were immersed in a 10% NaOH solution for 4 to 6 days at room temperature and then rinsed in distilled water until they became transparent. Then they were immersed in 1% tannic acid for 4 hours. Subsequently, the specimens were rinsed in distilled water overnight, rinsed, post-fixed in 1% osmium tetroxide for 2 hours, dehydrated in graded concentrations of ethanol, sectioned transversely or longitudinally with a very sharp, clean blade under a dissecting microscope, freeze-dried, coated with gold, and observed under a Zeiss 940-A scanning electron microscope. The figure clearly shows, for the first time, the threedimensional architecture of collagen fibrils in human myo- Connective tissue skeleton of human heart sectioned transversely. Its organization is quite similar to a honeycomb. The perimysium (P) envelops groups of myocytes. The endomysium, as final arborization of the perimysium, supports and connects individual cells. The endomysial weave (W) envelops each individual myocyte and is connected to adjacent myocytes by lateral struts (s) presenting branches of variable size and extension. The range of length and diameter of these struts is very wide. Collagen struts also connect myocytes to interstitial microvessels (thin arrow) or perimysium (thick arrow). A, Bar520 mm; magnification 31415; B, bar510 mm, magnification 32830. From the Department of Pathology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil. Correspondence to Professor Marcos A. Rossi, Department of Pathology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, 140049-900 Ribeirão Preto, SP, Brazil. E-mail [email protected] The editor of Images in Cardiovascular Medicine is Hugh A. McAllister, Jr, MD, Chief, Department of Pathology, St Luke’s Episcopal Hospital and Texas Heart Institute, and Clinical Professor of Pathology, University of Texas Medical School and Baylor College of Medicine. Circulation encourages readers to submit cardiovascular images to Dr Hugh A. McAllister, Jr, St Luke’s Episcopal Hospital and Texas Heart Institute, 6720 Bertner Ave, MC1-267, Houston, TX 77030. (Circulation. 1998;97:934-935.) © 1998 American Heart Association, Inc. 934 Rossi et al cardium after digestion of the cellular elements. This is expected to contribute further to the understanding of the morphology of the connective tissue skeleton of the heart. Acknowledgment Professor Rossi is Senior Investigator of the Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq). References 1. Weber KT, Brilla CG, Janicki JS. Myocardial fibrosis: functional significance and regulatory factors. Cardiovasc Res. 1993;27:341-348. 935 2. Robinson TF, Factor SM, Sonnenblick EH. The heart as a suction pump. Sci Am. 1986;254:84-91. 3. Caulfield JB, Borg TK. The collagen network of the heart. Lab Invest. 1979;40:364-372. 4. Robinson TF, Cohen-Gould L, Factor SM. Skeletal framework of mammalian heart muscle: arrangements of inter and peri-cellular connective tissue structure. Lab Invest. 1983;49:482-498. 5. Robinson TF, Factor SM, Capasso JM, Wittenberg BA, Blumenfeld OO, Seifter S. Morphology, composition, and function of struts between cardiac myocytes of rat and hamster. Cell Tissue Res. 1987;249: 247-255. 6. Ohtani O. Three-dimensional organization of the connective tissue fibers of the human pancreas: a scanning electron microscopic study of NaOH treated tissue. Arch Histol Jpn. 1987;50:557-566. Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 Connective Tissue Skeleton of the Human Heart: A Demonstration by Cell-Maceration Scanning Electron Microscope Method Marcos A. Rossi, Monica A. Abreu and Lígia B. Santoro Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 Circulation. 1998;97:934-935 doi: 10.1161/01.CIR.97.9.934 Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1998 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circ.ahajournals.org/content/97/9/934 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation is online at: http://circ.ahajournals.org//subscriptions/