**5.2 Histological features**

Microscopically, myocardial lesions consisted of a chronic inflammatory process with fibrotic scars and extensive mononuclear infiltrates. Such infiltrates were more prominent in the working myocardium and in the specialized cells of the left branch of the His bundle than in the AV node and in the right hisian branch, showing a microfocal disposition (Figure 5A). The percentage of fibrosis was variable and ranged between 8.2 to 49% (Milei, et al., 1996a, Milei, et al., 1992b) (Figure 5B).

Extensive myocytolysis and spotty contraction band necrosis were observed. Cell hypertrophy in the apparently preserved myocytes was revealed by hypertrophic bizarre nuclei. Dilated lymphatic channels widespread in the ventricular septum and in the AV node, His bundle, and in the root of the right and left bundles branches were observed. In the case of apical aneurysm of the left ventricle, dilated lymphatic were distributed subepicardically (Milei, et al., 1996a).

The serial sectioning of the conducting system showed prominent lesions. Sino-atrial node presented mononuclear infiltrates, necrosis of specialized fibers, and intense fibrosis (Milei, et al., 1991b). In the remaining specialized system lesions consisted of mild to moderate diffuse fibrosis of the AV node and of the penetrating and branching portions of the His bundle, complete destruction of the proximal segments of the right and left bundles branch by varying degrees of replacement by dense collagen tissue (Figure 5A). The remaining specialized fibers presented atrophy and mild fatty infiltration and were surrounded in most cases by infiltrates consisting mainly of lymphocytes and macrophages. The subendocardial Purkinje fibers were usually damaged by chronic inflammation and fibrosis (Milei, et al., 1991b) (Figure 5B). These vast fibrosis in the conduction system (Figure 5C) showed severe conduction alterations in electrocardiograms, although curiously in one revision, there were needed sophisticated electrophysiological studies to demonstrate electrical abnormalities in these patients (Andrade, et al., 1988)

Fig. 4. A. High grade heart dilatation. Thining of the apical wall of the left ventricle (white arrow) and cavitary thrombus (black arrow). B. Characteristic apical aneurysm. A from

Microscopically, myocardial lesions consisted of a chronic inflammatory process with fibrotic scars and extensive mononuclear infiltrates. Such infiltrates were more prominent in the working myocardium and in the specialized cells of the left branch of the His bundle than in the AV node and in the right hisian branch, showing a microfocal disposition (Figure 5A). The percentage of fibrosis was variable and ranged between 8.2 to 49% (Milei, et al.,

Extensive myocytolysis and spotty contraction band necrosis were observed. Cell hypertrophy in the apparently preserved myocytes was revealed by hypertrophic bizarre nuclei. Dilated lymphatic channels widespread in the ventricular septum and in the AV node, His bundle, and in the root of the right and left bundles branches were observed. In the case of apical aneurysm of the left ventricle, dilated lymphatic were distributed

The serial sectioning of the conducting system showed prominent lesions. Sino-atrial node presented mononuclear infiltrates, necrosis of specialized fibers, and intense fibrosis (Milei, et al., 1991b). In the remaining specialized system lesions consisted of mild to moderate diffuse fibrosis of the AV node and of the penetrating and branching portions of the His bundle, complete destruction of the proximal segments of the right and left bundles branch by varying degrees of replacement by dense collagen tissue (Figure 5A). The remaining specialized fibers presented atrophy and mild fatty infiltration and were surrounded in most cases by infiltrates consisting mainly of lymphocytes and macrophages. The subendocardial Purkinje fibers were usually damaged by chronic inflammation and fibrosis (Milei, et al., 1991b) (Figure 5B). These vast fibrosis in the conduction system (Figure 5C) showed severe conduction alterations in electrocardiograms, although curiously in one revision, there were needed sophisticated electrophysiological studies to demonstrate

Milei, et al., 1996b, B from Milei, et al., 2008.

1996a, Milei, et al., 1992b) (Figure 5B).

subepicardically (Milei, et al., 1996a).

electrical abnormalities in these patients (Andrade, et al., 1988)

**5.2 Histological features** 

Fig. 5. A. Extensive mononuclear infiltrates, myocytes loss, and subendocardial fibrosis. Hematoxylin and eosin stain, X25. B. Atrophic myocardial fibres (red) separated by thick bands of fibrous tissue (blue). Mallory trichrome, X 25. C. Bifurcating His bundle showing severe fibrosis at the left branch (between arrows). The right branch (asterisk) is intramyocardial and surrounded by connective tissue. Mallory trichrome, X25. A and C from Milei, 1996a. B from Milei, 2008.

Pathogenesis and Pathology of Chagas' Chronic Myocarditis 137

(Figure 6B). CD31 antibodies clearly pointed out normal endothelial cells, in either normal

Myocardial fibers showed nuclear enlargement, nuclear membrane invaginations, lipofuscin deposits, myofibrils derangements and loss, swelling, mitochondrial atrophy, dilatation of sarcotubular system, and interstitial fibrosis (Carrasco, et al., 1982, Palacios-Prü, et al., 1982). These findings have been confirmed by our group in endomyocardial biopsies (Ferrans, et al., 1988, Milei, et al., 1992b). Platelet thrombi can be demonstrated within

Other striking alteration in these specimens was the thickening of the basement membranes of cardiac myocytes (Figures 7A, 7C), endothelium Figure 7C) and vascular smooth muscle up to 20 times their normal thickness of 500 Å (Ferrans, et al., 1988). The thickened basement membranes appeared structurally homogeneous, without being multilayered or subdivided into a lamina rara and a lamina densa. They were of relatively low electron density, had a finely fibrillar appearance at high magnification and measured up to 1 m in thickness. Using gold-conjugated antibodies, we could demonstrate the presence of laminin in the

Regarding the ultrastructure of aneurysms resected from chagasic patients we observed, hypertrophy of myocytes, with swelling, partial or complete loss of myofibrils, swelling of mitochondria, disruption of mitochondrial cristae, lipofuscin granules, and intact sarcolemmas . Basement membranes were thickened, as previously described (Milei, et al.,

Fig. 7. A. Myocardial fibre with thickened basement membrane. B. Platelet thrombus within a capillary. C. Thickened basement membranes in a myocardial fibre and a capillary. From

thickened basal membranes of myocytes and endothelium (Sanchez, et al., 1993).

or damaged vessels (Figure 6C) (Milei, et al., 1996a).

**5.4 Ultrastructural features** 

capillaries (Figure 7B).

1991a)

Milei, et al., 2008.

Fig. 6. A. Detail of the left bundle of His is shown. Immunostaining for T lymphocyte. Positive cells express CD45R0 antigen (brown); specialized myocardial cells have almost disappeared. Extensive mononuclear infiltrate, the majority of them being T lymphocytes. X20. B. Double immunostaining for the simultaneous demonstration of T lymphocytes (CD45RO) and macrophages (CD68). T lymphocytes (brown) in close contact with a macrophage (pinky cytoplasm). X1000. C. Immunostaining to show endothelial cells. Capillaries and small vessels are clearly showed by the expression of CD31. Vessels are midly or moderately disorted because of the surrounding fibrosis. X100. From Milei, 1996a.

In our studies in endomyocardial biopsies, infiltrates were approximately 50% lymphocytes and 50% macrophages. Almost 80% of lymphocytic population were T lymphocytes, being only 20% B lymphocytes. Eosinophils were scarce in infiltrates reaching 5%, and were associated with areas of necrotic myocardium. Mast cells also were scarce or absent in specialized and in contracting myocardium. (Milei, et al., 1996a, Milei, et al., 1992b)

Histological study of aneurisms showed a thinned wall 2-4 mm, with sclerotic plaques of thickened endocardium of up to 92% of total tissue and extensive mononuclear chronic inflammatory infiltrates and widespread fibrosis in myocardium. Myocytes were organized in thin bands or atrophic units surrounded by fibrotic tissue (Figure 5B) (Milei, et al., 1991a).

Autonomic ganglia showed above described Terplan's nodules, with satellite cell proliferation replacing degenerated autonomic neurons. As stated, these lesions, once considered patognomonic, can be found in other cardiomyopathies (Rossi L., et al., 1994).
