International Journal of Cardlologv, 10 (1986) 211-290

Elsevier

211

IJC 00355

The evolution of experimental Trypanosoma cruzi cardiomyopathy in rabbits: further parasitological,

morphological and functional studies

FlorCncio Figueiredo ‘, J.A. Marin-Neto 2 and Marcos A. Rossi ’

’ Department of Pathology and 2 Department of Internal Medicine, Medical School of Ribeircio Preto, Sdo Paulo, Brazil

(Received and accepted 24 September 1985)

Figueiredo F, Marin-Neto JA, Rossi MA. The evolution of experimental Ttypanosoma cruzi cardiomyopathy in rabbits: further parasitological, morphological and func- tional studies. Int J Cardiol 1986:10:277-290.

Young rabbits (l-2 months of age) inoculated with trypomastigote forms of the Colombia strain of Ttypanosoma cruzi have been shown to develop cardiac pathologi- cal changes (together with parasitological and immunological alterations) which are very similar to those observed in the acute and chronic phases of Chagas’ disease in man. The cardiac alterations in the acute phase are characterized grossly by slight cardiomegaly with dilatation of the right-sided chambers. Microscopically they are characterized by mild focal myocarditis. The chronic phase is characterized by moderate to marked cardiomegaly with hypertrophy and dilatation of both ventricular chambers. There is thinning of the apical region (apical aneurysm), particularly of the left ventricle. Focal myocarditis is seen microscopically with areas of myocytolytic necrosis, atrophic and hypertrophic myofibers, an inflammatory response predomi- nantly composed of mononuclear ceils and interstitial fibrosis. Cineventriculography in the left ventricle of rabbits during the chronic phase disclosed regional myocardial dysfunction, with typical apical systolic bulging. The pathogenesis of Chagasic cardiomyopathy is briefly discussed in the light of these findings. Our investigation has further shown that this animal model is particularly suitable for studies on the mechanisms, pathology and treatment of Chagas’ heart disease.

(Key words: Chagas’ disease: experimental model)

Correspondence and reprint requests to: Prof. Dr. Marcos A. Rossi, Department of Pathology, Labora-

tory of Experimental Pathology, Medical School of Ribeirgo Preto, 14049-Ribeirgo Preto, S.P.. Brazil.

0167-5273/86/$03.50 0 1986 Elsevier Science Publishers B.V. (Biomedical Division)

278

Introduction

Chagas’ disease (American trypanosomiasis) is caused by transmission of the

protozoan Ttypanosoma cruzi by a triatomid bug. It is one of the leading causes of

death in many countries of Latin America from the Southern United States of America to Argentina [l]. The disease is characterized by three phases: acute, latent

and chronic [2,3]. The heart is the most severely and frequently involved organ. The degree of cardiac involvement during the acute phase varies from mild (asymptomatic

or oligosymptomatic) to severe. The latter involvement may be fatal but this

complication is infrequent, as it occurs in only 3-10s of the cases. Three stages are seen in the chronic phase. In the initial stage, the patient may be asymptomatic or

presents a few symptoms, usually related to disturbances of rhythm. In the inter-

mediate stage the clinical manifestations usually correlate with a mildly to mod- erately enlarged heart. In the final stage there is marked cardiomegaly, congestive

heart failure, thromboembolic phenomena and severe arrhythmias. Sudden death is

an important risk at any stage of the disease. The latent phase between the acute and

chronic phases is usually of long duration (lo-20 years). Little is known on the clinico-pathological changes during this phase. The main pathological changes

reflect the importance of the clinical involvement of the heart in Chagas’ disease [4,5]. In the acute phase the heart is globular and flabby. Foci of myocytolytic

necrosis and degeneration are seen microscopically, with an intense mononuclear inflammatory infiltrate associated with exudative phenomena and parasitism of myofibers. Variable degrees of hypertrophy and dilatation occur in the chronic phase with or without thinning of the apical region (apical aneurysm). Foci of myocardial necrosis and degeneration are still present, with an inflammatory re-

sponse predominantly composed of mononuclear cells and interstitial fibrosis.

Myofibers containing parasites are rarely observed. The pathogenesis of Chagasic cardiopathy, however, remains unclear. Immuno-

logical [6-91, hypoxemic [lo-121, and neurogenic [5] mechanisms have been pro- posed, but many aspects remain unsolved. This is largely because no suitable

experimental model has yet been found. In this paper we describe the evolution of experimental T. cruzi cardiomyopathy in rabbits, which we suggest, will prove to be an appropriate model for the study of functional and morphological derangements

due to this organism. Partial clinical, parasitological, pathological and immunologi-

cal studies have been reported,previously [9,13]. This paper extends and further describes our pathological findings.

Materials and Methods

Outbred New Zealand strain rabbits (1-2 months of age) were used for the study. They were divided into two groups. The first group (28 animals) was infected with Trypanosoma cruzi. The second group (18 animals) was made up of uninfected controls. They were housed in individual cages and maintained under controlled

conditions with disturbances kept to a minimum. Standard laboratory diet was used with continuous free access to water. The test animals were inoculated subcuta- neously with 1 x lo6 trypomastigote forms of the Colombia strain of T. cruzi [14].

219

Control rabbits were injected with saline. The parasitemia was evaluated by

xenodiagnostic test [15]. For this purpose, first stage nymphs of the reduviid bug Dipetalogaster maximus were used. One test consisted of 10 nymphs obtaining blood meals from one T. cruzi infected rabbit. Parasites were searched for in the feces

obtained from the bugs 30 days after exposure. The parasitemic levels were ex- pressed as percentage of positive examinations. The rabbits were periodically bled

and the serum stored at - 20°C for the detection of T. cruzi specific antibodies by

passive hemagglutination and indirect immunofluorescent antibody tests. In the

passive hemagglutination test, a suspension of aldehyde-preserved human red blood cells sensitized with T. cruzi extracts was added to the rabbit serum in buffered

saline solution [16]. The indirect immunofluorescence test was performed using

cultured epimastigote forms of the Y strain of the parasite [17].

Five rabbits of the infected group and three rabbits of the control group were selected randomly and sacrificed under light ether anesthesia at days 30, 60, 120, 240

and 540 after inoculation. The heart was rapidly removed, rinsed in ice-cold 0.9%

NaCl solution, and fixed in neutral 10% formalin. All hearts were sectioned

coronally from the apex to the base into two separate pieces, each piece including both the atria1 and ventricular chambers. After paraffin embedding the blocks were

sectioned at 6 pm, stained with hematoxylin-eosin and Cason’s trichrome method, and examined under the light microscope.

Samples of myocardium (base and apex) were rapidly excised for electron microscopic study from four rabbits in the infected group (two after 120 and two after 240 days following inoculation) and from four controls. Small pieces of tissue

were fixed by immersion for 2-3 hours in phosphate buffered 2.5% glutaraldehyde (pH 7.3) postfixed for 2 hours in 1% osmium tetroxide in phosphate buffer. dehydrated in ascending concentrations of acetone and embedded in Araldite. Semithin sections stained with toluidine blue were examined under the light micro-

scope and a suitable area selected for preparation of ultrathin sections. These were obtained in a Sorvall MT-5000 ultramicrotome, double-stained with uranyl acetate

and lead citrate, and examined in a Zeiss EM 109 electron microscope at 80 kV. Three experimental rabbits were submitted to left ventriculography 120, 180 and

240 days respectively after infection. Three age-paired control rabbits underwent the

same protocol. Small doses of chloralose and urethane were given by the intravenous route for sedation. A 4F Lehman ventriculography catheter was introduced into the

right femoral artery under local anesthesia and directed by fluoroscopy into the left ventricle. Left ventriculography was carried out by injection of 1 ml/kg body weight

of 75% Hypaque (2 ml/set) with the animal lying supine. The angiogram was recorded at 60 frames/set on 35 mm-film (Kodak CFS) using a Phillips image-in- tensifier system.

Parasitemia

Results

All animals inoculated with T. cruzi showed parasitemia detected by the xenodiagnostic test (Fig. 1). On the 15th day after infection, 10% of bugs examined

280

DAYS AFTER INFECTION

Fig. 1. Parasitemia demonstrated in rabbits inoculated with trypomastigote forms of the Colombia strain

of Trypanosoma cruzi (% parasitemia = total number of D. maximus infected with T. cruzi/total number

of bugs used x 100).

were positive. Later the percentage decreased to figures ranging from 40 to 30% on days 30 and 40 and 10% on day 60. Parasitemia could no longer be detected after 90 or more days.

Serology

T. cruzi specific antibodies, as determined by indirect immunofluorescence and passive hemagglutination, are shown in Fig. 2. On the 10th day after infection 33.3% and 20% of inoculated animals showed antibody titers as detected by im- munofluorescence and hemagglutination, respectively. On day 20 the titers were 66.6% and 758, respectively. From day 40 onward all rabbits showed antibody titers as determined by both methods.

Morphological Changes

None of the control rabbits had any gross or light-microscopic cardiac lesions. Until 60 days after infection the hearts of 4 out of 10 test rabbits were

macroscopically identical to controls. The remaining were slightly enlarged and flabby, with dilatation of right sided chambers and a congested myocardium. From day 120 significant alterations were observed. Eight (53.3%) of 15 hearts from test rabbits were moderately-to-markedly enlarged. There was hypertrophy and dilata- tion of both ventricular chambers (Fig. 3) and bulging of the sub-pulmonary infundibulum. Thinning of the apical region of the left ventricle (apical aneurysm) was noted in four (26.6%) hearts (Fig. 3B). One of them also showed thinning of the right ventricular apex. Myocardial fibrosis, proportional to the duration of the infection, could be observed macroscopically in eight (53.3%) hearts.

281

RABBITS WITH ANTIBODIES AGAINST T.cruzi (%)

0 10 10 w 110 100 * * * * - * - * - 3

I I 10

a

a .o

F 2 I 1

640

Fig. 2. 7’. cruzi specific antibodies in the sera of infected rabbits as demonstrated by passive hemag-

glutination (H) and indirect immunofluorescence (Cl).

Fig. 3. A. Enlarged heart of rabbit 540 days after infection showing hypertrophy and dilatation of both

ventricular chambers. LV = left ventricle; RV = right ventricle ( x 2.1). B. Enlarged heart of rabbit 240

days after infection showing dilatation of both ventricular chambers, hypertrophy, and thinning of the left

ventricle (apical aneurysm). LV = left ventricle; RV = right ventricle ( x 2.1).

Fig. 4. Myocardium of infected rabbits. A. Foci of myocytolytic necrosis and degeneration of myofibers.

Mononuclear inflammatory response and dissociation of myofibers (120 days after infection) (HE,

X 163). B. Higher magnification showing foci of myocardial necrosis and degeneration, with an interstitial

mononuclear celhrlar infiltrate (120 days after infection) (HE, X410). C. Diffuse interstitial fibrosis

associated with mononuclear cell infiltration (240 days after infection) (HE. X163). D. Interstitial

fibrosis, mononuclear cellular infiltrate and lysis and degeneration of myofibers (Cason’s trichrome,

x 163).

Fig. 5. Myocardium of infected rabbits. A. Hypercontracted myofibrils, with rupture and lysis of

myofilaments (arrow heads). Intracellular edema and vacuolization (V). A number of electron dense

corpuscles (peroxisomes?) can be seen (arrows). Mitochondria are increased in number (MI) ( x 6350). B.

Intracellular edema with an intracytoplasmic vacuole (V). MI = mitochondria; IS = interstitial space

( x 5000). C. Intermyofibrillar edema (0). Lysis of myofilaments. Formation of contraction bands (CB)

(X 10137). D. Mitochondrial swelling, with decreased density of matrix and rupture of cristae (MI).

Contraction bands (CB). Rupture of myofilaments (arrow heads). Intermyofibrillar edema ( x 9500).

Fig. 6. Myocardium of infected rabbits. A. Atrophic myofiber with lysis of myofilaments (0) and a large

number of myelin figures (MY). Interstitial fibrosis (F) ( x 8410). B. Interstitial fibrosis (F). Increased

number of mitochondria (MI). Contraction bands (CB) (x4990). C. Interstitial fibrosis. Activated

fibroblasts (FB) and collagen fibers (F) (x4990). D. Widened interstitial space (IS), with granular

material and lymphocytes (LY). CP = capillary ( X 6720).

285

Light-microscopic examination of sections from hearts of infected rabbits showed

focal alterations, proportional to the duration of the experiment. Animals killed 30 days after inoculation showed slight focal myocarditis, with edematous dissociation of myofibers, foci of myocardial necrosis and degeneration, and a mononuclear cell interstitial infiltrate. After day 60 the myocarditis became more intense, with

disseminated focal areas of myocytolytic necrosis and degenerative changes of

Fig. 7. Anteroposterior left cineventriculogram in rabbits studied 180 (A and B) and 240 days (C and

after T. cruzi infection. End-systolic Figs. B and C disclose the typical apical bulging (arrows). In Fig

an anterior dyskinetic area is also shown (arrow head). Note that a small filling defect is present in Figs

and D in the apical region, suggesting local endocardial thrombosis.

D) .D

,. c

286

myofibers. Both atrophic and hypertrophic muscle cells could be seen and a striking mononuclear cellular infiltrate was present (Fig. 4A and B). The interstitial fibrosis ranged from a localized perivascular pattern to extensive areas which compromised the myocardium (Fig. 4C and D).

Ultrastructural examination revealed the myocardium of control rabbits to be no different from that reported for mammalian cardiac muscle [18]. The changes in the myocardium of infected animals (120 and 240 days after inoculation) varied from area to area and from a normal appearance to complete dissolution of the myocar- dial fibers. The myofibrils were disorganized in mildly and moderately damaged areas, and foci of lysis were seen. In addition, there was edema between myofibrils and myofilaments. Mitochondria showed swelling, disorganization and rupture of the cristae with reduced density of the matrix. An apparently increased number of mitochondria was also demonstrated. The myofibers were frequently hypercon- tracted, with formation of anomalous contraction bands. The interstitial space was widened due to edema and cellular infiltration by activated fibroblasts and mono- nuclear ceils accompanied by deposition of collagen (Figs. 5 and 6).

No abnormalities of filling or contraction pattern were detected in the left ventriculographic study either in the three control rabbits or in the animal studied 120 days after T. cruzi infection. Left ventriculograms performed in rabbits on days 180 and 240 after inoculation revealed important regional abnormalities of the pattern of contraction and typical apical systolic bulging (Fig. 7). The 240-day infection rabbit showed an anterior hypokinetic area.

Discussion

Parasitemia, demonstrated by direct microscopic examination (thick or thin blood films) and, more reliably, by enrichment or xenodiagnosis, constitutes the most characteristic finding during the acute phase of Chagas’ disease in man. Thereafter, specific antibodies to T. cruzi can be detected beginning 15 to 30 days after infection. On gross examination, the heart is slightly enlarged, with increased weight and dilated chambers. The myocardial involvement is characterized histologically by severe myocarditis with foci of myocardial necrosis and degeneration, interstitial cellular infiltration predominantly composed of mononuclear cells, mild interstitial fibrosis and myofibers containing parasites (leishmanial pseudocysts). In the chronic phase the parasites are hardly found in the peripheral blood, while serological tests show high titers of specific antibodies, The heart is usually grossly enlarged, globular in shape and exhibits hypertrophy and dilatation of all chambers. In a few cases the heart may appear to be normal in size and shape. Bulging of the sub-pulmonary infundibulum is almost always evident. The most characteristic lesion is thinning and protrusion of the apical regions, predominantly that of the left ventricle, which is found in from half to two-thirds of cases. This is variously known as an apical aneurysm, aneurysm of the apex, pseudoaneurysm of the apex, atrophic lesion of the apex and vorticillar lesion of the apex. Foci of myocytolytic necrosis and degenera- tion, with a mononuclear cell inflammatory response and concurrent interstitial fibrosis are observed microscopically. Myofibers containing leishmanial pseudocysts

287

are rarely seen. Lesions in the autonomic nervous system and the conduction system

have also been described [4,5,19]. An animal model of human disease is “a living organism with an inherited,

naturally acquired, or induced pathological process that in one or more respects closely resemble the same phenomenon in man” [20]. An appropriate animal model

should accurately reproduce the disease or lesion under study [21]. The findings in the present experiment clearly show that rabbits are susceptible to experimental

infection with the Colombia strain of T. crwi. In the acute phase they show low

parasitemic levels demonstrated only by xenodiagnosis. This agrees with findings in

man in the acute phase [22-241, when methods of enrichment or the xenodiagnostic

test are sometimes required for parasitological confirmation [25]. In addition,

specific antibodies for T. cruzi are detected during the course of experimental infection, similar to observations in Chagas’ disease in man. The latter is char-

acterized by high levels of antibodies, predominantly of the IgM class in the acute phase and of the IgG class in the chronic phase [19].

The present study clearly demonstrates that rabbits experimentally infected with

T. cruzi develop a cardiomyopathy that replicates several aspects of the human heart

disease. The gross cardiac alterations in the acute phase are characterized by slight cardiomegaly, with dilatation of the right-sided chambers, and microscopically mild

focal myocarditis is seen. In the chronic phase the findings are moderate to marked cardiomegaly with hypertrophy and dilatation of both ventricular chambers, and thinning of the apical region (apical aneurysm), particularly of the left ventricle. Focal myocarditis is seen microscopically, with areas of myocytolytic necrosis and

degeneration of muscle cells, atrophic and hypertrophic myofibers, an inflammatory response predominantly composed of mononuclear cells and interstitial fibrosis. In a

previous study we reported that rabbits chronically infected with T. cruzi develop electrocardiographic and some cardiac morphological alterations similar to those described in the chronic phase of Chagas’ disease in man and in the present study

[ 131. The electron microscopic findings observed in the present study are very similar

to those found in human chronic Chagas’ cardiomyopathy [26,27]. These morpho-

logical changes alone, however, are not specific. Identical ultrastructural alterations have been described in various forms of heart disease, such as congestive cardiomyopathy and aortic valve disease [27-291. Left ventriculography of rabbits studied 180 and 240 days after infection revealed myocardial contraction abnormali-

ties and typical apical systolic bulging, also as seen in human Chagasic cardiomyop- athy [30,31].

A great deal of our recent knowledge on Chagas’ heart disease has been obtained from studies on animal models [4]. Immunological mechanisms have been implicated

[6,8,9,14]. Damage to autonomic neurons has also been postulated as an alternative pathogenetic mechanism [5]. The participation of autoimmune mechanisms relies on the myocarditis characterized by foci of mononuclear cell infiltrate, either in the acute or chronic phases [8]. A similar inflammatory response has been observed in other hypersensitivity diseases. This mechanism of tissue injury may be dependent

on sensitization to parasite antigens [7] or may be the result of a response to the constituents of i’Y cruzi antigenically cross-reactive with myocardial tissue [8,9,32].

288

This may be mediated through immunocompetent cells capable of inducing the lesion. The microscopic study of the myocardium from infected rabbits showed a mononuclear inflammatory infiltrate associated with tissue damage, in both acute and chronic phases. The focal myocardial necrosis and degeneration is the most conspicuous light and electron-microscopic finding. Its morphological character is similar to necrosis observed in a great number of human cardiac diseases [33,34]. A similar form of myocardial focal lesion has been observed experimentally [35] and clinically [36] in transient ischemia of the myocardium. These facets, associated with the focal nature of the myocardial lesion, are indirect evidence of the involvement of the microcirculation in this model. It has been recently suggested that this could be involved in the pathogenesis of Chagasic cardiomyopathy [ll]. The results of the present study are compatible with the hypothesis that immunological and ischemic mechanisms could be important in the causation of myocardial injury in the acute and chronic phases of experimental cardiomyopathy in rabbits infected with T. cmzi.

In conclusion, young rabbits experimentally infected with the Colombia strain of T. cruzi develop cardiac pathological alterations closely similar to those observed in humans, both in the acute and chronic phases of Chagas’ disease. This animal model appears to be appropriate for studies on the mechanisms, pathology and treatment of chagasic cardiomyopathy due to its simplicity, availability and low cost.

Acknowledgements

Maria 0. Rossi, Maria Helena N. Gomez. Monica A. Abreu, Carlos Kipnis and Ivo Mazucato provided technical assistance and Dina B. Pelizaro typed the manuscript. This study was supported in part by grants from the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho National de Desenvolvimento Cientifico e Tecnologico (CNPq).

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