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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 97, Num. 7, 2002, pp. 1019-1025
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Mem Inst Oswaldo Cruz, Rio de
Janeiro, Vol. 97(7), October
2002, pp. 1019-1025
Pathology of Intracardiac
Nerves in Experimental Chagas Disease
Lídia Cristina Villela Ribeiro,
Aryon A Barbosa Jr., Zilton A Andrade
Laboratório de Patologia Experimental,
Centro de Pesquisas Gonçalo Moniz- Fiocruz, Rua Valdemar Falcão
121, 40295-001 Salvador, BA, Brasil
+Corresponding author. Fax:
+55-71-356.2155. E-mail: zilton@cpqgm.fiocruz.br
Received 22 February 2002
Accepted 22 July 2002
Code Number: oc02228
Severe destruction of intrinsic
cardiac nerves has been reported in experimental acute Chagas myocarditis, followed
by extensive regeneration during the chronic phase of the infection. To further
study this subject, the sympathetic and para-sympathetic intracardiac nerves
of mice infected with a virulent Trypanosoma cruzi strain were analyzed,
during acute and chronic infection, by means of histological, histochemical,
morphometric and electron microscopic techniques. No evidences of destructive
changes were apparent. Histochemical demonstration for acetylcholinesterase
and catecholamines did not reveal differences in the amount and distribution
of intracardiac nerves, in mice with acute and chronic Chagas myocarditis or
in non-infected controls. Mild, probably reversible ultrastructural neural changes
were occasionally present, especially during acute myocarditis. Intrinsic nerves
appeared as the least involved cardiac structure during the course of experimental
Chagas disease in mice.
Key words: intracardiac nerves -Trypanosoma
cruzi - Chagas disease - myocarditis
The demonstration that endemic megaesophagus
and megacolon, found in areas of Chagas disease represented a digestive form
of this disease raised considerable interest to the study of autonomic nervous
system disturbances. Damage to nervous ganglia situated along the submucosa
and muscular coats of the digestive tract was considered to lead to disperistalsis,
which culminated in the development of megaesophagus and/or megacolon (Koberle
1961, Rezende 1984). Similar nervous changes also occurred in the heart. As
a matter of fact, electrocardiographic alterations, associated with atrophy
and fibrosis of cardiac nervous ganglia, were described in Brazilian cases of
megaesophagus long before its relationship to Chagas disease was established
(Oria & Ramos 1949). Marked decrease in the number of cardiac ganglionic
neurons has been registered in patients with Trypanosoma cruzi infection,
when compared to normal controls (Koberle 1959) or to other forms of cardiopathies
(Amorim & Olsen 1982).
The impact of these findings has
been so great as to lead some authors to suggest new terms such as "neurogenic
cardiopathy" (Koberle 1959), "cardio-neuromy-opathy" (Iosa et
al. 1989) and "catecholamino-genic cardiopathy" (Oliveira 1985) for
Chagas heart disease.
Additionally, patients with chronic
T. cruzi infection were found to exhibit peripheral electromyographic
changes, probably due to myo-neural involvement (Faria et al. 1979). An apparent
support for the chagasic etiology of such findings came from the description
of inflammatory and demyelinating neural changes observed in experimental Chagas
disease (Barreira et al. 1981, Said et al. 1985).
However, much less attention has
been paid to the lesions affecting the intrinsic nerves of the heart in Chagas
disease. So far, only one group of investigators have devoted a series of papers
to this subject (Machado et al. 1975, 1978, 1979, 1987, 1998). These papers,
mainly based on histological findings, described severe, diffuse and focal,
destructive cardiac neural lesions affecting both the sympathetic and parasympathetic
fibers of the heart, during acute Chagas myocarditis in rats and dogs. Later
on, when the infection entered its chronic phase, occurred extensive neural
regeneration. Apparently, no attempts by others have been made to reproduce
these impressive and important findings, probably because there are no clinical
evidences during the course of Chagas heart disease to correlate with them.
However, the data are present in the literature and need to be confirmed.
The present investigation is concerned
with the description and interpretation of the lesions affecting the sympathetic
and parasympathetic nerves of the heart, during acute and chronic experimental
Chagas disease in mice, by means of histologic, histochemical, ultrastructural
and morphometric methods.
MATERIALS AND METHODS
Outbred Albino Swiss mice of both
sexes, weighing 16-18 g, maintained on a commercial balanced diet and water
ad libitum,were used. Thirty animals were intraperitoneally inoculated with
50,000 trypomastigotes of the 21SF T. cruzi strain (Andrade & Magalhães
1997), a cardiotropic strain. Parasitemia was registered daily from the 7th
up to the 20th day after inoculation, and once every week afterwards. Fifteen
animals were not submitted to infection and served as normal controls. So, three
groups of animals were considered for morphologic studies. 1st Group
- Representative of the acute phase of the infection, 15 mice were sacrificed
around the 20th day of infection, when parasitemia reached its highest peak.
2nd Group - Another 15 infected mice, representative of the chronic phase
of the infection, were sacrificed 7 months after inoculation. 3rd Group -
Normal control mice were maintained under the same general conditions of the
other groups, and killed either on day 20 and on the 7th month after the beginning
of the experiment. Animals from all groups were always killed under general
ether anesthesia, followed by severing of the brachial plexus and exsanguination.
The heart was removed, sectioned into two halves, along the great axis, through
the inter-ventricular septum, and then immediately submitted to the several
procedures as described below.
Histology - Portions of the
heart were fixed in 10% phosphate buffered (pH 7.2) formalin, embedded in paraffin,
and the 5 µm-thick sections obtained were stained with hematoxylin and
eosin (H & E).
Histochemistry - Half of the
heart was embedded in Tissue-Tek (Sakura Finetek, Torrance, CA, USA) and quickly
frozen in liquid nitrogen (-196oC). The frozen blocks were placed
in air-tight plastic boxes and kept in a freezer at -80oC until the
moment they were cut in a cryostat at -20oC. Sections, either floating
or mounted on glass slides, were submitted to histochemical staining for the
demonstration of acetyl-cholinesterase by a thiocholine method (Karnovsky &
Roots 1964), following fixation in formol-calcium for 10 min. Slides were lightly
counterstained by hematoxylin. Other sections were submitted to a glyoxylic
acid-induced fluorescent method for the demonstration of catecholamines by the
method of De la Torre (1980), as later modified by Cottle et al. (1985). For
that, the sections mounted in mineral oil were examined under ultra-violet light
in a Zeiss microscope with mercury lamp and appropriate exciter and barrier
filters.
Electron microscopy - Tiny
fragments taken from the right atrium of the heart were immediately fixed in
3% glutaraldehyde in 0.2M cacodylate buffer, post-fixed in 1% osmium tetroxide
and embedded in epon resin. Selected blocks were cut in a Reichert Ultracut
automatic ultramicrotome, with diamond knife, and the ultra-thin sections obtained
were contrasted with uranyl acetate and lead citrate. Specimens were examined
in a Zeiss EM-109 electron microscope, which was operated at an acceleration
voltage of 80 kV.
Morphometry - Sections stained
with H & E and for acetyl-cholinesterase activity were analyzed by morphometry
to quantitatively evaluate inflammation (by counting the number of leukocyte
nuclei) and para-sympathetic nerves (by measuring the sectional area occupied
by cholinesterase-positive fibers). The sections were examined by semi-automatic
morphometry using the Leica Q500MC Image Processing and Analysis System (Leica,
Cambridge, England). For morphometric measurements of nuclei, a total sectional
area of 268,609 µm2, with an objective of 10X, was considered;
for cholinesterase-positive nerves the objective was 20X and the area examined
was 65,104 µm2 .
Statistical analysis - Average
numbers obtained by morphometry were compared by the Mann Whitney test, with
the Graphpad Instat software. A significance level of 5% (p < 0.05) was accepted.
RESULTS
Normal mice - Segments of
myelinated and non-myelinated nerves were frequently observed in sections taken
from the atrial walls of the heart and, rarely, the ventricles, always accompanied
by arterial and venous blood vessels and capillaries. Acetyl-cholinesterase
staining disclosed a rich network of fibers in the atrium, deeply marked by
a strong dark brown color. A similar picture of a rather straight, delicate
but less compact network of fluorescent fibers appeared when the histochemical
method for catecholamines was applied. The nervous ganglia were frequently observed,
especially in the sub-pericardial connective tissue of the right atrium. These
structures, particularly the neurons, stained strongly positive for acetyl-cholinesterase.
Acute Chagas myocarditis -
The myocardium appeared severely involved by mononuclear leukocyte infiltration,
edema and congestion. Collections of T. cruzi amastigotes appeared here
and there within myocardio-cytes (Fig.
1A, arrow). Some nerves were seen in areas of dense infiltration by inflammatory
cells. Although a few of these cells were seen at the periphery or within nervous
ganglia, and a few neurons exhibited severe regressive changes, no alterations
were observed within the structures of the intrinsic cardiac nerves on routine
histological sections (Fig. 1 A, B)
or after histochemical staining for cholinesterase or catecholamines (Fig.
1 C, D, E, F). Sometimes inflammatory cells appeared to be invading nerve
fibers, but the possibility of cells and fibers being at different planes of
section could not be ruled out in the relatively thick routine histological
sections examined. At the ultrastructural level no cell adhesion to the neural
external membrane and no cell invasion of nerves were ever observed. Lymphocytes
and macrophages were sometimes found adherent to or within destroyed myocardio-cytes.
In these instances there were swelling and rupture of mitochondria, myofibril
aggregation, fragmentation and lysis of myocardiocytes.
Segments of myelinated and non-myelinated
nerves were found in areas free of alterations or in close proximity to inflammatory
changes. No signs of destructive changes were ever found, even after exhaustive
search. Although the changes affecting the neural structures were mild and non-specific,
they are registered in Figs 2 and
3 for the sake of documentation. They consisted
of: (a) axoplasma abnormalities, such as increased number of membranous organelles,
tumefaction and cristolysis of mitochondria, focal disintegration of neurotubules
and neurofilaments, alterations of the myelin sheet , with increasing periodicity
of lamellae, sometimes with myelin fragments appearing within the axoplasma
or exhibiting several degrees of deformities; (b) alterations of Schwann cells,
which frequently contained phagosomes of variable sizes, sometimes forming "myelin
figures". In one instance a cytoplasmic prolongation of a Schwann cell
appeared between the axolema and the myelin sheet. The terminal portions of
non-myelinated nerve fibers exhibited preserved synaptic vesicles and a few
dense bodies, besides mild mitochondrial non-specific alterations (Fig.
3 C).
A quantitative estimation of the
severity of inflammatory changes and of the areas occupied by cholinesterase-positive
nerves in cardiac sections, taken from mice with acute and chronic infections,
as well as from normal controls, is depicted in the Table.
Chronic Chagas myocarditis -
Inflammatory changes were focal and mild in all cases. They consisting of scattered,
small, focal accumulations of lymphocytes, plasmocytes and macrophages, located
in the interstitial tissue of the heart, especially at the sub-pericardial areas
of both atria (Fig. 1 B). Segment
of nerves were seen within inflammatory foci, without any apparent structural
alteration. This is true for the slides examined with H & E staining or
treated with the histochemical methods for acethyl-cholinesterase and catecholamines.
Changes observed by electron microscopy in the myelinated and non-myelinated
myocardial nerves were qualitatively similar to those seen during the acute
phase myocarditis, but less intense and less frequent. No evidences of axonal
regenerative sprouts were observed during either the acute or chronic phase
of the infection.
DISCUSSION
Present investigation demonstrated
relatively mild, reversible and non-specific ultrastructural changes affecting
the intracardiac nerves during the course of experimental Chagas disease in
the mouse. Such changes are probably secondary to neighboring inflammation,
since they appeared more evident when associated with severe acute myocarditis
of early T. cruzi infection, than with chronic infection, when myocarditis
became mild and focal. On the other hand, signs of lymphoid and/or macrophagic
cytoadherence and cytotoxicity, although noted in myocardiocytes during acute
myocarditis, were not observed in nerves. The possibility of disappearance or
even the occurrence of partial destruction of intracardiac nerves could not
be demonstrated, neither by morphometric means in sections histochemically prepared
for parasympathetic nerves, nor by electron microscopy. Therefore, present data
are in marked contrast with the reported findings of severe destruction of nerves
followed by extensive regeneration observed in rats (Machado et al. 1979, 1987)
and dogs (Machado et al. 1998) with T. cruzi infection. Actually, the
Machado's group demonstrated neither destruction, nor regeneration of cardiac
nerves, but disappearance and re-appearance of enzymatic reactivity in acute
and chronic Chagas myocarditis, respectively, in rats and dogs. The particularly
severe myocarditis, occurring in rats and dogs, much more intense than that
obtained with the present experiments in mice, may be accounted for such observed
differences in enzymatic reactivity. However, it is worth mentioning that in
dogs with severe acute Chagas myocarditis, ultrastructural findings on cardiac
nerves consisted only of swelling of Schwann cells and increased sizes and numbers
of dark granules and vesicles (Andrade et al. 1994).
Of course the present study does
not rule out the possibility that a certain amount of neural destruction occurs
during the course of acute Chagas myocarditis. Neuronal loss may occur in the
atrial parasympathetic ganglia, although a mild focal nature, as observed during
severe acute myocarditis of mice (Souza et al. 1996), when three different T.
cruzi strains were compared. When a neuron is destroyed, the associated
axon undergoes Wallerian degeneration. Although the ultrastructural changes
observed in the present study were mild, they are qualitatively similar to those
observed during early Wallerian degeneration (Vial 1958, Donat & Wisniewski
1973). These non-specific changes are also common during inflammation, when
a modification of the permeability of the nerve membrane can result in edema,
due to an augmented influx of ions. According to Schlaepfer (1974) and Schlaepfer
and Hasler (1979), an intra-axonal increase in calcium concentration results
in a granular aspect of the axoplasm, with disintegration of microtubules and
microfilaments. This process is probably mediated by a calcium-activated neutral
protease (Kamakura et al.1983).
In conclusion, despite the presence
of the non-specific neural changes observed in the present material, the intrinsic
nerves of the heart appeared as one of the structures least involved during
the course of acute and chronic Chagas myocarditis.
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Copyright 2002 Instituto Oswaldo
Cruz - Fiocruz
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