R e v i s t a d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 8 ( 1 ) : 1 3 -1 7 , j a n - m a r , 1 9 9 5 .
CARDIAC PLEXUS OF DOGS EXPERIMENTALLY INFECTED
WITH
T R Y P A N O S O M A C R U Z 1: INFLAMMATORY LESIONS
AND QUANTITATIVE STUDIES
M arcelo V. Caliari, M arta de L ana, Estela R . O. Caliari and W ashington L . Tafuri
Q u a lita tiv e a n d q u a n tita tiv e a s p e c ts o f th e s u p e r fic ia l a n d p r o f o u n d c a r d ia c p l e x u s o f d o g s e x p e r im e n ta lly in fe c te d w ith B e -6 2 a n d B e -7 8 s tr a in s o f Trypanosoma cruzi w e r e s tu d ie d . A n im a ls w e r e a u to p s ie d in th e a c u te p h a s e o f in fe c tio n . T h e in fla m m a to r y p r o c e s s , le s io n s a n d n u m b e r o f p a r a s i t e s w e r e m o r e in te n s e a n d f r e q u e n t in a n im a ls in fe c te d w ith th e B e - 7 8 s tr a in th a n in th o s e in fe c te d w ith B e -6 2 . D e s p ite th is, n o s ta tis tic a lly s ig n ific a n t d iffe r e n c e s c o u ld b e f o u n d b e tw e e n th e n u m b e r o f n e u r o n b o d ie s in th e g a n g lia o f in fe c te d a n d c o n tr o l d o g s .
K e y -w o r d s : C a r d ia c p le x u s . Trypanosoma cruzi. D o g . A c u te c h a g a s ic c a r d io p a th y .
The pathogenesis o f chagasic cardiopathy has been studied by several authors1211. Many factors seem to be involved: inflammation, autoimmunity, fibrosis and denervation18 19. F or example, denervation has an important role in the pathology o f the disease but is not always the main factor1314. Based on personal experience, mainly with dogs, although it has been found that chagasic cardiopathy is not counterbalanced in the acute and chronic phases, intense systematic lesions are not observed in the intracardial nervous system, mainly in its numerical reduction11.
The aims o f this paper are: a) to study the inflammatory phenomena, lesions and parasitism of the superficial and profound cardiac plexus of dogs experimentally infected with Be-62 and Be-78 strains o f T. cruzi', b) to carry out a quantitative study o f ganglia and neuron bodies of these plexus in infected and control dogs.
MATERIAL AND METHODS
Twelve outbreddogs, 65-80 days old, bom and
D epartam entos de Patologia Geral, Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais, de Análises Clínicas da Escola de Farmácia e de Ciências Biológicas, Instituto de Ciências Exatas e Biológicas da Universidade Federal de Ouro Preto.
Supported by C N Pq, FINEP and FAPEMIG.
A d d r e s s to : Prof. M arcelo Vidigal Caliari. Depto. de Patologia G eral/ICB/UFM G. Av. Antônio Carlos 6627, Pampulha. 31270-010 Belo Horizonte, MG. Fax (031) 441-1412 Recebido para publicação em 05/05/94.
maintained in the laboratory, were used. Before inoculation, each animal was examined to exclude the possibility o f prior T. c r u z i infection. Two groups, each o f four dogs, were inoculated intraperitoneally with 2000 blood trypomastigotes/ kg body weight, respectively, with Be-6217 and Be- 7810 T. c r u z i strains. Both strains were isolated, on different occasions, from Berenice, accepted to be the first known human patient of Chagas’ disease6. Inocula were obtained from albino mice in the acute phase of infection and counted according to Brener (1962). Four normal dogs were used as controls.
Dogs were maintained on an a d lib itu m diet and observed daily. Parasitemia was assessed according to Brener4. Dogs were sacrificed about the 37th day of infection and necropsied. The atria were removed and fixed in to tu m in buffered formalin at pH 7.2, dehydrated, cleared and infiltrated with paraffin with the vessels in a basal position. Semi-serial sections (1:10), 4 f im thick, were stained with HE and Gomori’s thricromic.
C a lia r i M V , L a n a M , C a lia r i E R O , T a fitr i W L. C a r d ia c p le x u s o f d o g s e x p e r im e n ta lly in fe c te d w ith Trypanosoma cruzi; in fla m m a to r y le s io n s a n d q u a n tita tiv e s tu d ie s . R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 8 : 1 3 -1 7 , j a n - m a r , -1 9 9 5 .
RESULTS
Dogs infected with Be-7 8 strain showed intense acute myocarditis characterized by a focal and diffuse exudation o f mononuclear cells, with an endomisial distribution and parasitism o f the myocardium (Figure 1). In contrast, dogs infected w ith Be-62 show ed only d iscreet foci o f inflammation and amastigote nests.
F ig u r e 1 - M y o c a r d i u m o f a d o g i n o c u la t e d w ith B e - 7 8 s tr a in a n d s a c r ific e d in th e a c u te p h a s e o f th e in fe c tio n . N o te th e in te n s e e x u d a te o f d iffu s e m o n o n u c le a r c e lls a n d a n e s t o f a m a s tig o te s (a rro w s). H E . 16Qx.
Lesions o f ganglia and nerves (ganglionitis, periganglionitis, neuritis and perineuritis) with degenerative phenomena o f neurons (tigrolisis, picnosis, cariolisis, tumefaction, vacuolisation and retraction) were more intense and more frequent in dogs infected with Be-7 8 (Figure 2; Figure 3 ; Table 1) than with Be-62 (Table 2). Apparently normal ganglia were often seen when inflamed and injuried ganglia were recorded.
Parasites were seen only in Schwann cells and in fibroblasts o f the capsule o f dogs infected with Be-78 (Table 1).
The total number of ganglia and neuron bodies in each animal are shown in Tables 1, 2 and 3. The averages of neuron bodies per ganglion were
8.92 ± 1.02, 9.72 ± 0.79 and 9.75 ± 0.38 respectively in dogs infected with Be-78 strain, Be-62 and controls. The Student “t” test did not show statistically significant differences between groups o f dogs infected with each strain and controls (Be-78 x controls, t = -1.56; p > 0 .0 5 . Be-62 x controls, t = -0.069; p > 0 .0 5 ).
F ig u re 2 - A tr iu m o f a d o g in o c u la te d w ith B e -7 8 s tr a in a n d s a c r ific e d in th e a c u te p h a s e o f th e in fe c tio n . N o te th e s u b - e p ic a r d ic g a n g lio n w i t h p r o n o u n c e d p e r i g a n g l i o n i t i s , p e r in e u r itis a n d g a n g lio n itis . H E . 1 6 0 x.
C a lia r i M V , L a n a M , C a lia r i E R O , T a fiiri W L. C a r d ia c p le x u s o f d o g s e x p e r im e n ta lly i n fe c te d w ith Trypanosoma cruzi: in fla m m a to r y le s io n s a n d q u a n tita tiv e s tu d ie s . R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 8 : 1 3 -1 7 , j a n - m a r , -1 9 9 5 .
T a b le 3 - R e s u lts o f q u a n tita tiv e e v a lu a tio n o f c a r d ia c g a n g lia in c o n tr o l d o g s.
Dog Total number
o f neurons
Total number o f ganglia
1 8466 910
2 6466 670
3 7564 761
4 8927 873
DISCUSSION
Koberle (1956) found that Autonomous Nervous System (ANS) injuries develop at an early stage of the acute phase o f Chagas’ disease9. His subsequent studies showed that the course of the disease and type o f lesion that emerged in the chronic phase, were predetermined in the acute phase. In fact, alterations in the ANS are so constant in Chagas’ disease that Koberle called it a parasympathicoprive disease, because parasympathetic denervation be could responsible for the occurrence o f different
anatomoclinic forms of the disease: digestive and cardiac.
Although denervation could be one of the factors responsible, it has been demonstrated that other factors of equal, or greater importance8 21, could explain the pathogenesis and physiopathology of the disease. Lopes (1965)13 and Lopes et al (19 83)14 agree with Koberle as much as the presence of neuron lesions, but they observed that different degrees of denervation may occur in Chagas’ disease or may be absent in symptomatic patients who eventually die from the disease.
Rocha et al (1993)16 showed that the ANS may suffer slight injury when patients are developing chronic chagasic cardiopathy. Also, Almeida- Ribeiro et al, in a systematic study o f the atrium of a human acute case, did not encounter significant lesions o f intracardial nervous system and admitted that they were due to the propagation o f nearby epicarditis2. These findings refute the hypothesis that ANS lesions are caused by functional disturbances. In contrast, Davila et al (1993)7 suggested that parasympathetic abnormalities of
C a lia r i M V , L a n a M , C a lia r i E R O , T a fiiri W L . C a r d ia c p le x u s o f d o g s e x p e r im e n ta lly in fe c te d w ith Trypanosoma cruzi.’ in fla m m a to r y le s io n s a n d q u a n tita tiv e s tu d ie s . R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 8 : 1 3 -1 7 , j a n - m a r , -1 9 9 5 .
cardiopathies are the resiilt of progressive ventricular dilatation. Thus, even when there are no ANS lesions, functional alterations o f parassimpathetic ANS may occur. Our preliminary results with dogs chronically infected with Be-78 strain show acute electrocardiogram changes. Two dogs have been sacrificed and pratically no lesions were seen in intracardiac nervous system, even though the entire atrium was examined.
R eg ion al differen ces betw een T. c r u z i p o p u la tio n s o f d iffe re n t zym odem es and schizod em es are also re late d to d iffe re n t anatomoclinic forms o f the disease, including the intensity of neurotropism5. Many authors have studied neurotropic strains o f T. c ru zi in different experimental models and the results obtained are very questionable1 3 15 20.
So far, no systematic study of serial sections of the entire atrium was undertaken. Indeed, 2,400 sections were stained and, subjectively, a clear impression was gained that Be-78 strain is more neurotropic that Be-62 strain. The lesions o f the intracardiac nervous system are mainly based on two mechanisms: directly related to the presence of parasites either in Schwann’s cells or in neurons resulting sometimes in ganglionitis or then by peri ganglionitis with secondary ganglion infiltration. Because there might be more inflammation and more parasites in infections with Be-78 strain, obviously more serious lesions occurred with this strain. Although the denervation has not been evident, some dogs infected with Be-78, showed intense and diffuse myocarditis with the development of the symptomatic fibrosing chronic chagasic cardiopathy, similar to human disease. Some of these dogs died in consequence o f the disease at different periods of the acute or chronic phase12.
Since all diseases are multifactorial, the same may occur with Chagas’ disease. All of the foregoing factors discussed, and others, need to be analysed to obtain a better understanding of the pathogenesis and physiopathology o f the development o f the disease.
RESUMO
F o i r e a liz a d o e s tu d o q u a lita tiv o e q u a n tita tiv o d o s p le x o s c a r d ía c o s s u p e r fic ia is e p r o fu n d o s e m c ã e s in o c u la d o s c o m o Trypanosoma cruzi d a s c e p a s B e -6 2 e B e - 7 8 e s a c r i f i c a d o s n a f a s e a g u d a . O p r o c e s s o
in fla m a tó rio , a s le s õ e s e o p a r a s itis m o d o s p le x o s f o r a m m a is in te n s o s e f r e q u e n t e s n o s a n im a is in o c u la d o s c o m a c e p a B e 7 8 d o q u e n a q u e le s i n o c u la d o s c o m a c e p a B e -6 2. A p e s a r d e s te f a t o , n ã o f o i v e r ific a d a d ife r e n ç a e sta tis tic a m e n te s ig n ific a tiv a e n tr e o n ú m e r o d e c o r p o s d e n e u r ô n io p o r g â n g lio d o s a n im a is c h a g á s ic o s e o s c o n tro le s.
P a la v r a s -c h a v e s : P le x o c a r d ía c o . Trypanosoma cruzi. C ã o. C a r d io p a tia c h a g á s ic a a g u d a .
ACKNOWLEDGEMENTS
To Prof. Paul Willians our gratitude for revision of the text and Maria Chaves for technical assistance.
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