Role of oral bacterial flora in calcific aortic stenosis: an animal model David J Cohen, MD, David Malave, MD, John J Ghidoni, MD, Panagiotis Iakovidis,

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Role of oral bacterial flora in calcific aortic stenosis: an animal model David J Cohen, MD, David Malave, MD, John J Ghidoni, MD, Panagiotis Iakovidis, MD, Mona M Everett, PhD, Shenghong You, MD, Youhong Liu, MD, Barbara D Boyan, PhD The Annals of Thoracic Surgery Volume 77, Issue 2, Pages 537-543 (February 2004) DOI: 10.1016/S0003-4975(03)01454-1

Fig 1 Photograph of an anteriorly opened rabbit heart from a group D animal, showing the typical appearance of vegetative endocarditis (arrow) on the aortic valve, as well as the supravalvular and infravalvular areas. (Magnification: ×2.) The Annals of Thoracic Surgery 2004 77, 537-543DOI: (10.1016/S0003-4975(03)01454-1)

Fig 2 Scanned images of three adjacent sections of the aortic valvular tissue that are stained with (a) hematoxylin & eosin as a general stain in which calcium and gram-positive bacteria stain blue, (b) von Kossa stain in which calcium stains brown, and (c) Gram stain for bacteria in which gram-positive organisms stain blue. In a, the arrow indicates the aortic valve; in b, the arrow indicates the aorta; and in c, the arrow indicates the vegetation. The sinus of Valsalva is filled with fibrinous vegetation containing calcium and bacteria in juxtaposition. (Magnification: ×4.6.) The Annals of Thoracic Surgery 2004 77, 537-543DOI: (10.1016/S0003-4975(03)01454-1)

Fig 3 Von Kossa stain of the vegetation shows calcium-positive sites as brown. This field contains a part of one intensely stained large calcified nodule (upper right) as well as myriad small positively staining brown grains (arrows). These small calcifications generally became less numerous as one moves away from the large calcification (nodule). (Magnification: ×200.) The Annals of Thoracic Surgery 2004 77, 537-543DOI: (10.1016/S0003-4975(03)01454-1)

Fig 4 Hematoxylin & eosin stain of the wall of the aorta near the sinus of Valsalva. A hillock of newly formed connective tissue has incorporated a large calcified nodule (arrow). This process illustrates another mechanism that can account for nodular calcifications in cardiovascular connective tissue. (Magnification ×30.) The Annals of Thoracic Surgery 2004 77, 537-543DOI: (10.1016/S0003-4975(03)01454-1)

Fig 5 Scanning electron micrographs of tissue-sections from the same block that provided light microscopic tissue sections for Figure 2. The morphology of images from both techniques is similar. (a) In this standard micrograph the lighter areas within the vegetation correlate with the position of calcium as indicated by the von Kossa stain in Figure 2b, and in (b) a calcium elemental dot-map produced in an EDAX-equipped scanning electron microscope using a tissue from the same block of tissue. This technique (normalized against calcium standards) visually maps the relative density of calcium as white dots against a black (null) background. (c) This compound image illustrates the congruence of the dots in the calcium elemental map and the pale nodules within the vegetation. It was prepared by inverting the grayscale image of the calcium map so that the dots in the map appear to be black, and then superimposing this modified calcium map over the image of the tissue (a) at 50% transparency. The pale areas in the scanning electron micrograph image and the high density of dots in the elemental map fell immediately into register. (Magnification: ×7.8.) The Annals of Thoracic Surgery 2004 77, 537-543DOI: (10.1016/S0003-4975(03)01454-1)

Fig 6 Low magnification transmission electron micrograph of tissue removed from another section from the same tissue block. The area sampled is adjacent to a large calcified nodule and was reembedded at right angles to the original section so that the full thickness (the third dimension) of the section could be studied. The arrow indicates the direction of the electron beam in the scanning electron microscope. The array of small electron-dense objects distributed within the section is consistent with the size of calcified microbes and their membranous debris. (Magnification: ×6,500.) (Inset) This higher magnification electron micrograph shows the fine detail of these electron-dense objects. The density is composed of needle-like crystals, which is consistent with the manner in which calcium apatite forms on dead bacteria. (Magnification: ×25,000.) The Annals of Thoracic Surgery 2004 77, 537-543DOI: (10.1016/S0003-4975(03)01454-1)