Experimental pathology of Chagas disease
Sonia Gumes Andrade
Gonçalo Moniz Research Center/Fiocruz
Email: sgandrade@cpqgm.fiocruz.br
Experimental studies on Trypanosoma cruzi infection have opened new horizons for the study of Chagas disease, caused by this protozoan in humans. Chagas disease was discovered by Carlos Chagas in 1909 when he described the first human case in a two-year-old child, Berenice, known to all those who study this disease. The first experimental infection with T. cruzi was carried out by Oswaldo Cruz, as described by Chagas himself in a previous Note published in Brasil Médico, in 1909: “The infection that served as the starting point for our studies was obtained experimentally by Dr. Oswaldo Cruz, causing a marmoset (hapalle penicillata) to be stung by some conorrhinus taken from Minas.” In continuation of these studies, Chagas performed experimental inoculation of several animal species, such as dogs, guinea pigs and monkeys (Callitrix penicillata) with Trypanosoma cruzi, as a means of studying the evolution of the infection in different vertebrates. In addition to those mentioned, other vertebrates, such as rabbits, rats, mice, have been used by several researchers in order to investigate different aspects of T. cruzi infection, with variable results and peculiar characteristics.
Mice
Models of experimental Chagas disease in mice
Sonia Gumes Andrade
Gonçalo Moniz Research Center/Fiocruz
Email: sgandrade@cpqgm.fiocruz.br
The acute phase
Due to its small size, ease of obtaining and maintenance, the mouse has been the preferred animal in a large number of experiments. Inoculation is generally carried out intra-peritoneally, although other routes such as intra-conjunctival and oral route may be used, depending on the model to be reproduced. In inoculation, trypomastigote parasitic forms collected directly from the blood, from the feces of the vector insect (triatomine) or metacyclic forms from liquid culture media or cell cultures can be used. The parasites are phagocytosed by macrophages where they multiply and, after being released into the bloodstream, penetrate various cells of the vertebrate organism. Infection by T. cruzi induces an innate response of the organism and stimulates T-NK lymphocytes to produce IFNγ, stimulating the production by macrophages of pro-inflammatory cytokines and stimulating their microbicidal mechanisms, as shown by Cardillo and collaborators This process is accompanied by necrotic lesions not only of parasitized macrophages but also of cardiac and skeletal muscle myocells and a corresponding inflammatory process related to tissue destruction and TNF release. In the acute phase of infection, active multiplication occurs predominantly in macrophages or in cardiac, skeletal or smooth muscle of various organs, mainly the digestive tract, and may also parasitize fibroblasts, neuronal, and endothelial cells. Therefore, the presence of the parasite is the main pathogenic factor at this stage of infection.
Relationship between parasite strains and histopathological conditions in the acute phase of infection
T. cruzi strains isolated in different geographic areas, from different vertebrate hosts and from wild reservoirs, may represent different populations in terms of their biological behavior in the experimental animal, as initially described by Andrade and collaborators, in terms of their isoenzymatic profile, studied by Miles and collaborators, and regarding their molecular characters, by the characterization of kinetoplast DNA (kDNA), as shown by Morel and collaborators, or ribosomal RNA, initially described by Souto and collaborators These differences have led to different classifications of strains, which have been systematized and consolidated into two taxa: T. cruzi I and T. cruzi II, as Consensus of the Satellite Meeting of the International Symposium to commemorate the 90th Anniversary of Discovery of Chagas disease, held in 1999. The classification of strains according to their biological behavior in mice, into different types (I, II, III) or biodemes, as proposed by Andrade and Magalhães, allowed the correlation of strains with the pathological conditions produced by T. cruzi dependent on virulence, pathogenicity and tissue tropism. The different biodemes were correlated with the proposed taxa, with type II corresponding to T. cruzi II and type III to T. cruzi I. Type I was classified as Z2b, including hybrid strains. In infection with type I biodeme, there is marked macrophagotropism. This is accompanied by an intense necrotic-inflammatory process of parasitized organs, mainly in the spleen, where necrosis of parasitized macrophages and in situ production of TNF occurs, described by Lima and collaborators. In infection with type II biodema strains, whose parasitemia has a slower course between the 12th and 20th days, there is intense myocardial parasitism in the acute phase, with disintegration of the parasitized myocytes and necrosis of the non-parasitized ones. parasitized, with an intense process of myocarditis. Type III biodeme strains determine intense myocardial and skeletal muscle lesions, with marked intracytoplasmic proliferation of parasites.
Interaction of different isogenic strains of mice with the three types of biodemes
The parasite/host binomial is important in determining the pathology in the experimental animal. The mouse as a model can vary in its susceptibility to infection with different strains, according to its lineage. The study of six different strains: AKR, A/J, CBA, BALB/c, C3H and B-10, showed that the biological behavior of the three biodemes was maintained in animals of different strains, varying, however, in relation to susceptibility, with a spectrum of resistance for each strain to infection by the different strains, as shown by Andrade and collaborators. The type I strain (Peruana) determined early parasitism in macrophages in the six strains; myocardial and skeletal muscle parasitism ranged from intense to moderate, being more pronounced in AKR and A/J (susceptible) strains and lower in DBA and BALB/c strains; with this strain, tissue parasitism was always more intense than the inflammatory process; in infection with the 21SF strain (type II), myocardial parasitism was more accentuated in the A/J lineage infection, but in all of them there are necrotic lesions of parasitized or not parasitized muscle fibers, and in the most susceptible ones (C3H, AKR and A /J) these lesions are not accompanied by inflammatory exudate or present only foci of mononuclear infiltration, while in lesions of the most resistant strains in relation to this strain (DBA, BALB/c and B-10) mononuclear infiltrate with polymorphonuclear cells is observed in between. In infection with the Colombian strain (type III) intense parasitism of the myocardium and skeletal muscle is observed from 20 to 30 days post-infection, with a marked inflammatory process related to disintegrating parasitized fibers. In the most susceptible strain (A/J), despite the intense parasitism, the inflammatory infiltrate is moderate. In the B/10 strain, which is more resistant, with moderate parasitism, there is an intense inflammatory process.
Clonal structure and histopathological lesions
The strains represent clonal complexes. The clonal composition of a strain can influence its pathogenicity and histopathological conditions. Studies have been carried out to characterize clones of standard strains from different Biodemes. The clonal structure of a strain can be homogeneous, although the clones can present different degrees of virulence, with a predominance of a “main clone” responsible for its behavior in the experimental animal. The strains can present heterogeneous clonal constitution and the behavior in the experimental animal depends on the characteristics and the predominant clones.
Histopathological aspects in the chronic phase of T. cruzi infection
The chronic phase of infection by the Colombian strain (biodema type III, T. cruzi I) was studied by Federici et al and Kumar et al, who described an evolving chronic heart disease in C3H mice. Subsequently, the murine model of chronic Chagas’ heart disease was described in mice of different strains, infected for prolonged periods (180 to 660 days), with strains from different biodemes. This model reproduces the picture of chronic myocarditis with fibrotic-inflammatory lesions, cardiomegaly, dilatation and thrombosis of cardiac chambers, and left ventricular tip aneurysm. The histopathological study of the myocardium showed diffuse and focal inflammatory infiltrates, consisting of macrophages, lymphocytes and plasma cells, whose intensity varied from case to case, being generally more pronounced in the atria. There was a clear predominance in the incidence and intensity of fibrotic and inflammatory alterations in mice infected with type III strains, corresponding to the T. cruzi I taxa(among them the Colombian, Bolivian and Montalvânia strains).In addition to the general aspects of experimental pathology, the murine model has contributed to the clarification of several problems related to Chagas disease, such as: (i) study of the resistance of T. cruzi strains from different Biodemes to chemotherapy; (ii) reversibility of cardiac fibrosis, post-specific treatment; (iii) involvement of the autonomic nervous system in T. cruzi infection; (iv) importance of parasitic antigens sequestered in follicular dendritic cells of the spleen, (v) in the maintenance of immunological memory and in the positivity of serological reactions, after treatment, as well as in (vi) studies of new vaccines and (vii) of the genesis of inflammatory lesions and chronic cardiomyopathy, among many other studies (Figure 1).
Dogs
Models of experimental Chagas’ disease in dogs
Sonia Gumes Andrade
Gonçalo Moniz Research Center/Fiocruz
Email: sgandrade@cpqgm.fiocruz.br
Due to its susceptibility to infection by T. cruzi, the electrocardiographic characteristics comparable to those of humans, as well as the definition and anatomical characters of the excito-conductor system of the heart, the dog constitutes an important model of Chagas disease. Knowledge about the canine model of Chagas disease has progressively evolved. Laranja and Pellegrino described the chronic phase of the infection in dogs inoculated with blood from patients with Chagas disease or with triatomine feces, showing a clinicopathological picture and electrocardiographic alterations that are similar to those seen in humans, both in the acute and chronic phases. Other studies in dogs were developed by several researchers, with electrocardiographic and anatomopathological description.
The studies of Dr. Zilton Andrade and collaborators sought to correlate the anatomopathological findings with the electrocardiographic changes and with the anatomical lesions of the heart’s excito-conductor system. Young dogs were inoculated with strain 12 SF isolated from an acute human fatal case, which developed a severe acute condition, with a high lethality rate up to 21 days of infection and presented an electrocardiographic and anatomopathological picture identical to that presented by the patient, indicating that, in canine model, at least in the acute phase, the results obtained experimentally can reproduce Chagas’ disease. The detailed study of the heart’s conduction system using serial sections mounted on plastic tapes has allowed identifying the segments of this system and demonstrating the topography of the histopathological lesions present, as well as correlating them with the electrocardiographic alterations found. In this particular, the dog has been shown to be the ideal model. The excito-conductive tissue of the heart in this animal is anatomically well defined and has a topographical distribution comparable to that observed in the human heart (Figure 2).
Histopathological changes in the acute phase of infection in the dog
The indeterminate form of Chagas disease in dogs, as in humans, has no symptoms and no electrocardiographic changes. In a detailed study of dogs in the indeterminate phase of the disease, which had been inoculated with different strains of T. cruzi, minimal macroscopic alterations were observed, with discrete dilatation of the right chambers in some cases; on microscopic examination, foci of mononuclear infiltration and fibrosis were observed in 50% of the cases, mainly in the right atrium and ventricle, while the left ventricular myocardium is essentially normal. The histopathological study of the heart in these animals showed only discrete focal inflammatory lesions, made up of macrophages and lymphocytes. In an electron microscope study in 1997, Andrade and collaborators found that, differently from what was observed in the acute phase, lymphocytic cells do not show a tendency to adhere to myocytes, do not determine cytotoxic alterations on them and show a tendency to disappear by apoptosis, with cytoplasmic condensation and characteristic changes in nuclear chromatin. These findings speak in favor of a parasite/host balance at this stage and a self-regulation of inflammation by the process of apoptosis of the effector cells of the immune response. The dog’s indeterminate response to treatment with low doses of the immunosuppressant Cyclophosphamide resulted in the onset of chronic active myocarditis.
Histopathological changes in the indeterminate chronic phase of the dog
The indeterminate form of Chagas disease in dogs, as in humans, has no symptoms and no electrocardiographic changes. In a detailed study of dogs in the indeterminate phase of the disease, which had been inoculated with different strains of T. cruzi, minimal macroscopic alterations were observed, with discrete dilatation of the right chambers in some cases; on microscopic examination, foci of mononuclear infiltration and fibrosis were observed in 50% of the cases, mainly in the right atrium and ventricle, while the left ventricular myocardium is essentially normal. The histopathological study of the heart in these animals showed only discrete focal inflammatory lesions, made up of macrophages and lymphocytes. In an electron microscope study in 1997, Andrade and collaborators found that, differently from what was observed in the acute phase, lymphocytic cells do not show a tendency to adhere to myocytes, do not determine cytotoxic alterations on them and show a tendency to disappear by apoptosis, with cytoplasmic condensation and characteristic changes in nuclear chromatin. These findings speak in favor of a parasite/host balance at this stage and a self-regulation of inflammation by the process of apoptosis of the effector cells of the immune response. The dog’s indeterminate response to treatment with low doses of the immunosuppressant Cyclophosphamide resulted in the onset of chronic active myocarditis.
Chronic heart disease in the canine experimental model
Several authors have referred to studies carried out in dogs that reached the chronic phase of the infection and presented congestive heart failure, cardiomegaly, myocardial thinning, fibrosis and conduction disorders, however the anatomopathological descriptions are summary and do not allow a good correlation with cases humans.
There are few cases in which the dog progresses to a well-diagnosed chronic cardiac form, as described by Laranja and Andrade in a dog with an experimental infection of 16 months of duration with T. cruzi, which developed a condition of congestive heart failure, with all characteristics of human chagasic heart disease, with progressive electrocardiographic changes and a diffuse, progressive and fibrosing chronic myocarditis. The inflammatory infiltrates were predominantly lymphocytic, with plasma cells and macrophages in between. There was diffuse thickening of the interstitial tissue, mainly in the atrial walls and in the right ventricle, accompanied by a diffuse mononuclear infiltrate.
The aspects presented show that the dog’s main advantage is to reproduce the different phases of Chagas disease, allowing electrocardiographic monitoring and correlation with lesions of the heart’s excito-conductor system. In addition, the dog has been widely used by physiologists for the electrophysiological study of the heart, which provides a solid basis for the interpretation of findings in Chagas disease.
As a disadvantage, the fact that it needs a long period of follow-up so that the chronic cardiac phase can be surprised, with dogs remaining in an indeterminate form, in a high percentage of cases, with the cases described in the literature of a well-developed chronic cardiac form being rare.
Primates
Models of experimental Chagas disease in non-human primates
Cristiano Marcelo Espinola Carvalho
Laboratory of Biology of Interactions, Instituto Oswaldo Cruz/Fiocruz and National Institute of Infectious Diseases Evandro Chagas/Fiocruz
Email: cristian@ioc.fiocruz.br
Maria da Glória Bonecini-Almeida
National Institute of Infectious Diseases Evandro Chagas/Fiocruz
Email: bonecini@ipec.fiocruz.br
Joseli Lannes-Vieira
Laboratory of Biology of Interactions, Instituto Oswaldo Cruz/Fiocruz
Email: lannes@ioc.fiocruz.br
Since the beginning of studies carried out by Carlos Chagas, in 1909, several animals (such as mice, rats, hamsters, rabbits, dogs and non-human primates – monkeys) have been used as experimental models with the purpose of reproducing phases and forms of the disease. Chagas, and thus make it possible to investigate different aspects of Trypanosoma cruzi infection. The development of T. cruzi infection varies greatly in these animals, depending on the parasite strain used, the inoculation route and the inoculum volume. The choice of an experimental model will depend on the question to be investigated and on previous knowledge acquired over almost a century of research focused on this area.
Evidence accumulated during the last decades has indicated that non-human primates are suitable experimental models for the study of Chagas disease. Among the genera studied are those belonging to the Old World (Macaca) and to the New World (Callitrix, Saimiri, and Cebus). Their phylogenetic proximity to humans makes them more appropriate models than others, especially in immunological and pathological studies of trypanosomiasis or in the development of immunodiagnostic techniques. However, as we will see below, there is a diversity of reports regarding the gender of the animals used, differences in the strains of T. cruzi, as well as their evolutionary forms and different inoculation routes.
Recently, non-human primates began to be considered as important groups of animals that would serve as reservoirs for T. cruz in certain geographic areas, as they are naturally infected or become infected in captivity.
Callitrix penicillata was the first non-human primate species used as an experimental model for Chagas’ disease by Oswaldo Cruz and Carlos Chagas in 1909. These animals satisfactorily developed acute infection, between 20 and 30 days after contact with infected triatomines. Callithrix has also been used for immunization tests, inferring the immunogenic competence of some human vaccine candidates.
Small primates of the genus Saimiri are found infected in nature by both leishmania and T. cruzi. Its small size allows easy handling and maintenance. In an attempt to characterize the evolution of chagasic infection in these animals, Pung and collaborators infected them subcutaneously using metacyclic trypomastigote forms of the Brasil strain. The follow-up of the acute phase revealed the presence of circulating parasites, prolonged changes in the electrocardiogram (ECG), specific antibodies against T. cruzi, hematological alterations and an elevated lymphoproliferative response against T. cruzi antigens.
Among the primates most used to date as a model for Chagas disease is the capuchin monkey (Cebus apella), due to its ability to reproduce the acute and chronic phases of the disease with ECG changes, heart failure, and gastrointestinal alterations, in addition to the ease of domestication and handling. The capuchin monkey is the primate species with the widest geographic distribution among Neotropical species, occurring from northern Colombia (with the possibility of occurrence in southern Central America) to southern Argentina, being limited to the west by the Andes Mountains. and to the east by the Atlantic Ocean. The immune response has also been studied in this model by Samudio and collaborators, indicating that animals with high parasitemia have high expression of IL-4 in heart tissue, while those that manage to control the parasitemia have a higher expression of IFNγ. Furthermore, the authors verified that the expression of cytokines and cell adhesion molecules (PDGF-a, TGF-β and ICAM-1) in the cardiac tissue of animals infected in the chronic phase (two with 19 months, one with five and two with 10 years post-infection) may be correlated with the persistence of T. cruzi DNA detected by molecular tests (polymerase chain reaction – PCR).
The rhesus monkey (Macaca mulatta) belongs to the order of Primates, suborder Haplorhini, semi-suborder Anthropoidea, infraorder Catarrhini, superfamily Cercopithecoidea and family Cercopithecinae, distanced from Homo sapiens by about 35 million years, when they diverged from their common antecedent (Aegyptopithecus) in the Oligocene era, as reviewed by Shoshami and collaborators This phylogenetic proximity makes it useful for the use of several reagents routinely used for diagnosis and research in humans, which also makes it easier for us to extrapolate several results obtained in the model to humans.The rhesus monkey is an Old World primate, classified on the phylogenetic scale, in terms of its homology to man (its genome is 98% similar to that of the human being) below the great primates (gorilla, orangutan and chimpanzee) and above the New World primates, such as animals of the genera Callitrix, Saimiri and Cebus. Because it is a medium-sized animal (which facilitates its handling) and because it presents clinical conditions that reproduce human pathologies, it has been considered the model of choice among non-human primates for the study of various pathologies, such as leishmaniasis, HIV/AIDS, Parkinson disease, tuberculosis and multiple sclerosis, among others, which facilitates the extrapolation of the results obtained in these animals to human diseases.
Chagas disease in rhesus monkeys was initially studied by Wood, in 1934, using the California strain of T. cruzi, but without observing signs of the disease. However, in later studies, whether by experimental infections (among others, Bonecini-Almeida and collaborators, Meirelles and collaborators, Carvalho and collaborators) or natural infections (among others, Fulton and Harrison, Seneca and Wolf), reproducibility was demonstrated. of various aspects of human disease in this primate species. These authors used the most diverse forms of inoculation and types of parasite strain and were able to verify both cardiac and digestive alterations in this model. The acute phase of T. cruzi infection, in particular, has been shown to be similar to the human disease, with the development of a portal of entry lesion (inoculation chagoma), positive direct parasitemia, hematological alterations, positive specific serology and electrocardiographic alterations, as described above. by (Marsden and collaborators, Seah and collaborators, Miles and collaborators, Bonecini-Almeida and collaborators).
The disease developed by these animals in the initial acute and chronic phase (3 years post-infection) showed great similarity to Chagas disease, either by the evolution of the parasitemia and clinical aspects, or by the laboratory characterization (hematological alterations, positive serology and reversible cardiac alterations), as described by Bonecini-Almeida and collaborators The results obtained in anatomopathological studies, using optical and electronic microscopy, carried out by Meirelles and collaborators, proved the similarity between the evaluated model and the human disease.
The course of this chronic infection (15 to 19 years) revealed clinical pictures very similar to those observed in human disease. Subpatent parasitemia, detectable only by blood culture, xenodiagnosis or PCR, accompanied by high titers of T. cruzi-specific antibodies and ECG and echocardiographic changes were seen in animals classified as having heart disease. In those classified as having the indeterminate form, ECG or echocardiographic changes were absent, as shown by Carvalho and collaborators.
The immunological mechanisms that lead to the development of chronic heart disease in Chagas disease around 20-30 years after the first infection are still debatable. Recently, immunological aspects and their correlation with the development of chronic heart disease were studied in the rhesus monkey model studied in the acute phase by Bonecini-Almeida and collaborators Thus, about 20 years post-infection, a predominance of TCD3+CD8+ cells among peripheral blood mononuclear cells was observed in these animals. Interestingly, the cardiac inflammatory infiltrate was predominantly composed of TCD3+CD8+ cells, followed by TCD3+CD4+ cells, and rare macrophages, also reproducing aspects of chronic chagasic myocarditis described by Higuchi and collaborators. In the periphery, these cells showed an activation profile, as demonstrated by the expression of cell adhesion molecules (LFA-1 and VLA-4), demonstrating the potential migratory capacity of these cells. This idea was reinforced by the demonstration of increased expression of the chemokine receptors CCR2, CCR5 and CXCR4 in peripheral cells in these monkeys, very similar to that described in patients by Talvani and collaborators and Gomes and collaborators Likewise, increased expression of VCAM-1 was observed in the heart tissue of infected monkeys (Carvalho, 2006). Furthermore, morphologically macrophage-like cells expressing the enzyme induced nitric oxide synthase (iNOS) were observed in this tissue. Functionally, infected animals had a higher frequency of circulating IL-4 and IFNγ-producing lymphocytes, but a lower proportion of IL-2+ cells than uninfected animals. Importantly, the animal with severe cardiac involvement had the highest proportions of IL-4+, IFNγ+ and TNF+ lymphocytes and IL-10+ and IL-12+ monocytes. In addition, infected animals with cardiac involvement had more intense fibrosis, in addition to greater loss of organization and decreased expression of connexin 43, a marker of myocardial connectivity, as well as a greater number of iNOS+ cells in the cardiac tissue (Carvalho, 2006). Thus, in the rhesus model infected at 20 years of age with the Colombian strain of T. cruzi, the molecules LFA-1, VLA-4, VCAM-1, CCR2, CCR5 and CXCR4 seem to be involved in the immunopathogenesis of chronic chagasic myocarditis that shows a predominance of CD8+T-cells. The cytokines IL-1b, IL-6, TNF and IFNγ, in addition to CCL2/MCP-1 and CCL3/MIP-1a also seem to be important in this process. The induction of iNOS and the production of NO (revealed by the elevated serum concentrations of nitrite/nitrate), increased fibrosis and derangement in the distribution of connexin 43 in cardiac tissue are correlated with cardiac injury and electrical and echocardiographic dysfunctions (Carvalho, 2006).
From what has been demonstrated, it is possible to state that the rhesus model reproduces several characteristics of acute and chronic human infection recorded so far, which indicates that this model can help to understand the strategies used by the parasite to evade the immune response, or even be used to the evaluation and development of new strategies for the development of therapies or vaccines, especially after recent advances in understanding the pathogenesis of Chagas disease, as reviewed by Higuchi and collaborators and Kierszenbaum.
The use of an experimental model in which new drugs with trypanocidal activity can be tested, not only in the elimination of circulating parasites, but which have an effect on the elimination of tissue parasites, preventing the reactivation of the disease or avoiding the damage caused by the parasite during the chronic disease, may be of fundamental importance for the treatment of chronic disease, since millions of individuals are infected and have no prospects of cure. Likewise, they could serve as models for the development of safe and efficient vaccines.
Experimental heart disease
Study of Trypanosoma cruzi-induced experimental heart disease in murine models: how to access and explore.
Jaline Coutinho Silverio
Laboratory of Microbiological Control / Quality Control Department / Biomanguinhos / Fiocruz / RJ
Email: jaline@ioc.fiocruz.br
Gabriel Melo de Oliveira
Laboratory of Cell Biology / Instituto Oswaldo Cruz/Fiocruz/RJ
Email: gmoliveira@ioc.fiocruz.br
The mouse is the most used experimental model in biomedical research. This animal model reproduces, under laboratory conditions, with reliability and reproducibility, several physiological, biochemical, immunological and pathological aspects similar to the human species. Another relevant factor is the possibility of analyzing, in a short period of time, the course of chronic pathologies described in human beings.
In relation to Chagas’ disease, Andrade et al. observed, during the experimental infection by Trypanosoma cruzi, similarities in terms of clinical and laboratory aspects (positive serology, hematological disorders and cardiological alterations) when compared to the experimental murine model and the human being. Rossi et al, through anatomopathological studies (applying light and electron microscopy) also observed similar morpho-functional alterations.
The infection model can be defined by choosing the mouse strain and the T. cruzi strain with which the animal will be infected. Due to its ease of handling, it is possible to inoculate trypomastigote parasitic forms (blood or metacyclic) through the intra-peritoneal, intra-conjunctival, and oral routes. The interaction between the mouse strain and the parasite strain will demonstrate peculiarities of tissue tropism, mouse resistance to infection in the acute phase, parasitemia curve and animal mortality.
The ideal model for the study of the chronic phase is one that presents a balance between the infectivity of the parasite (capable of promoting lesions similar to humans in the acute phase and later in the symptomatic chronic phase), mouse survival in the acute phase and reproduction of Dilated Chagas Cardiomyopathy (DCC) in the chronic phase. A model with high infectivity and high incidence of lesions and pathological signs that do not survive the acute phase is not desirable.
Currently, it is known the importance between the severity of inflammatory lesions in acute myocarditis and the emergence of DCC in the chronic phase. Knowledge of the pathological changes present in the acute and chronic phases of chagasic heart disease in mice evolved from studies of cardiac morphology and functionality using non-invasive parameters. The use of biochemical analyses, such as the measurement of the serum activity of the enzyme Creatine Kinase – isotype MB (CK-MB) in the plasma of infected mice of the C57BL/6 strain, is an important tool that allows indicating the lesion of cardiomyocytes. In this way, studies have related the intensity of the inflammatory infiltrate and the severity of cardiac injury in infected mice.
In general, Chagas’ disease is characterized by myocardial dysfunction (at the cellular and tissue level) and may result in alterations in the cardiac electrical conduction system, detected mainly by electrocardiography (ECG) assessment. In fact, symptomatic chronic chagasic human patients are defined by electrocardiographic changes. In these patients, cardiac arrhythmias, atrioventricular blocks and ventricular tachycardias, among other findings, are frequent. Interestingly, Andrade and Sadigursky in 1987 observed electrocardiographic changes in Swiss Webster Outbred stock mice infected with T. cruzisimilar to those observed in chronically infected patients10. In addition, the use of other diagnostic imaging methods such as radiological, echocardiographic and magnetic resonance imaging associated with invasive methodologies, such as anatomopathological analysis, allow structural assessment of the organ and the contractile function of the myocardium. From these, we can assess the presence of organ dilation (or specifically of ventricles and atria) in systole or diastole, measure the thickness of the chamber wall, assess the force of myocardial contraction, assess the value of the ejection fraction between different others. Although the equipment is expensive, its use is essential for an accurate visualization of cardiological changes and the evolution of heart failure.
The cardiovascular function of infected mice can be further evaluated by methods such as ergometry and blood pressure measurement. We observed in mice during the course of T. cruzi infection complete intolerance to physical activity at the time of greatest myocardial injury that can be accurately assessed by ergometry. The measurement of blood pressure is relevant to assess the systemic condition of the animal since it is described that mice of the BALB/c strain infected by the Y strain of T. cruzi present lesions in the renal tubules and alterations in the renal filtration function, which may correlate with to the development of fulminant heart failure observed during the acute course of the disease.
In addition to contributing to the understanding of the pathophysiology of Chagas’ disease, mice have contributed to the development of new therapeutic protocols aimed at improving cardiac functionality in chronic chagasic patients. Soeiro et al. demonstrate the action of aromatic diamidines in in vitro assays with promising trypanocidal activity. The use of clinical, cardiac and renal parameters in mice will be important for the evaluation of the efficiency of these compounds. Lannes-Vieira et al. treated chronically infected mice with the drug resveratrol and it was observed that through the use of electrocardiography and echocardiography there was a significant improvement in the cardiac function of the animals18. Thus, these data demonstrate that the efficiency of therapeutic protocols associated with the standardization of methodologies for diagnosis and monitoring of the effectiveness of cardioprotective or immunotherapeutic agents represent a relevant set for the development of innovative therapies in the treatment of DCC.
The murine model has also contributed to the development of regenerative medicine, which uses stem cells and cell therapy to repair chronic-degenerative lesions. In this context, the possibility of intervention in chagasic heart disease was studied. Studies have demonstrated the benefits of cell therapy in heart diseases both in experimental protocols and in initial clinical trials in humans, through the use of cells from different origins, such as bone marrow cells (mesenchymal or hematopoietic), skeletal myoblasts, and also cells that supposedly reside in the myocardium itself. Soares et al injected mononuclear cells obtained from the bone marrow of normal or infected animals into chronically symptomatic animals with the Colombian strain of T. cruzi. The results showed the occurrence of cell migration to the cardiac tissue injured by the parasite and the respective injected cells, after cell evaluation, morphologically resembled healthy cardiac fibers with specific cardiac myosin marking. After one month of therapy, a reduction in the inflammatory and repair process (fibrosis) was observed in the group of mice infected with T. cruzi and receiving cell therapy when compared to the group that did not receive cells. Two months after the injection, this reduction was even more pronounced and was maintained until six months after cell transplantation.
These results corroborate those of Stamm et al., who injected cells obtained from the bone marrow of chagasic animals into chagasic mice, and observed similar regenerative and cardiac repair results. The set of cell therapy data directly contributed to the application and treatment of chronic symptomatic human patients through cell therapies, thus enabling the improvement or maintenance of the quality of life of individuals affected with Chagas’ disease.
Another approach in the study of human diseases is the use of genetically modified animals (GMAs). Through refined biotechnology techniques, its use has the main objective of answering, in a much more punctual way, a series of questions related to pathophysiology – that is: knowing the response of a certain organ to a certain pathology. The first studies using GMAs infected by T. cruzi, occurred by Lima & Minoprio, in 1996, which allowed the investigation of the involvement of the immune response by gamma-delta cells (γ/δ) in the severity of the infection and also of the activity of T-cells in mice deficient in the expression of the MHC complex. Since then, several authors have used GMAs for different approaches, such as: understanding the mechanism of regulation of the inflammatory response in infection, modulation of the expression of adhesion molecules, cytokines and chemokines in the immune response in the involvement of nitric oxide and in the major cytotoxic pathways and others in the experimental infection by T. cruzi.
In this context, the search for the identification of immunological mediators related to cardiac injury promoted by T. cruzidemonstrated an intense migration of leukocytes during the interaction between the parasite and the host’s immune cells has been investigated by the team of Dr. Joseli Lannes do Laboratory of Biology of Interactions at Instituto Oswaldo Cruz. In the acute phase of infection, a significant increase in the number of CD8 T lymphocytes was observed, in relation to CD4 T lymphocytes, expressing the chemokine receptor CCR5 in the cardiac tissue of C3H/HeJ animals infected with the Colombian strain of T. cruzi. In order to modulate cardiac inflammation, these C3H/HeJ mice were treated in the acute phase of infection with the compound Met-RANTES (N-terminal-methionylated RANTES), a selective antagonist of CCR1 and CCR5. The compound did not interfere with parasite quantification (parasitemia), but significantly reduced the number of CD4 and CD8 CCR5+ T lymphocytes in cardiac tissue and the deposition of fibronectin. Thus, these data strongly suggested that CCR5+ cells would not be crucial for parasite control, but would play an important role in the cardiac pathogenesis of Chagas’ disease.
Finally, the experimental use of the mouse model is extremely relevant and provides the opportunity to study and understand the changes promoted by the parasite in a systemic way and not only in an organ. In addition, the search for diagnostic methods that are increasingly sensitive and similar to human beings and also the use of GMAs, together, are very important factors for the investigation and clarification of the severity of T. cruzi infection during cardiac involvement, during the acute and chronic symptomatic phase. We emphasize that the murine model allows the study of new therapeutic targets for the treatment of DCC, with the objective of improving the quality of life of patients with heart failure caused by infection with T. cruzi.
