Evaluation of the rabbit as a model for Chagas disease - II: histopathologic studies of the heart, digestive tract and skeletal muscle

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, seventy one animals were inoculated with different Trypanosoma cruzi strains and routes. The rabbits were submitted to necropsy in acute (earlier than three months of infection), recent chronic (three to six months) and late chronic (later than six months) phases. Myocarditis, generally focal and endomysial, occurred in 94.1%, 66.7% and 70.8% of the infected rabbits respectively in the acute, recent chronic and late chronic phases. The myocardial inflammatory exudate was composed by mononuclear cells, and also polymorphonuclear cells in the acute phase. In most cases of the late chronic phase, the myocarditis was similar to that described in the indeterminate form of human chagasic patients. Initial fibrosis occurred in the three phases but was more severe and frequent in the early chronic. Advanced fibrosis occurred only in the late chronic phase. Tissue parasites occurred only in the acute phase. The digestive tract and skeletal muscles showed mild and occasional lesions. Our data indicate that experimentally infected chagasic rabbits repeat some lesions similar to that of humans chagasic patients, specially that of the indeterminate form. So, it may be a useful, however not an ideal, model.

Heart hypoplasia in an animal model of congenital diaphragmatic hernia

PURPOSE: In previous papers, we described a new experimental model of congenital diaphragmatic hernia in rabbits, and we also reported noninvasive therapeutic strategies for prevention of the functional and structural immaturity of the lungs associated with this defect. In addition to lung hypoplasia, pulmonary hypertension, biochemical, and structural immaturity of the lungs, the hemodynamics of infants and animals with congenital diaphragmatic hernia are markedly altered. Hence, cardiac hypoplasia has been implicated as a possible cause of death in patients with congenital diaphragmatic hernia, and it is hypothesized to be a probable consequence of fetal mediastinal compression by the herniated viscera. Cardiac hypoplasia has also been reported in lamb and rat models of congenital diaphragmatic hernia. The purpose of the present experiment was to verify the occurrence of heart hypoplasia in our new model of surgically produced congenital diaphragmatic hernia in fetal rabbits. METHODS: Twelve pregnant New Zealand rabbits underwent surgery on gestational day 24 or 25 (normal full gestational time - 31 to 32 days) to create left-sided diaphragmatic hernias in 1 or 2 fetuses per each doe. On gestational day 30, all does again underwent surgery, and the delivered fetuses were weighed and divided into 2 groups: control (non-surgically treated fetuses) (n = 12) and congenital diaphragmatic hernia (n = 9). The hearts were collected, weighed, and submitted for histologic and histomorphometric studies. RESULTS: During necropsy, it was noted that in all congenital diaphragmatic hernia fetuses, the left lobe of the liver herniated throughout the surgically created defect and occupied the left side of the thorax, with the deviation of the heart to the right side, compressing the left lung; consequently, this lung was smaller than the right one. The body weights of the animals were not altered by congenital diaphragmatic hernia, but heart weights were decreased in comparison to control fetuses. The histomorphometric analysis demonstrated that congenital diaphragmatic hernia promoted a significant decrease in the ventricular wall thickness and an increase in the interventricular septum thickness. CONCLUSION: Heart hypoplasia occurs in a rabbit experimental model of congenital diaphragmatic hernia. This model may be utilized for investigations in therapeutic strategies that aim towards the prevention or the treatment of heart hypoplasia caused by congenital diaphragmatic hernia.

Participation of cytokines in the necrotic-inflammatory lesions in the heart and skeletal muscles of Calomys callosus infected with Trypanosoma cruzi

Calomys callosus, a sylvatic reservoir of Trypanosoma cruzi, when infected with the Colombian strain (Biodeme Type III, T. cruzi I ) develops necrotic-inflammatory lesions and intense early fibrogenesis in the heart and skeletal muscles, that spontaneously regress. Participation of pro-inflammatory and pro-fibrogenic cytokines, such as tumor necrosis factor-alpha (TNF-alpha), gamma interferon (IFN-gamma) , and tumor growth factor-beta (TGF-beta), in the pathogenesis of the lesions is herein studied. Eighty C. callosus weighing 20 to 30 g were used. Seventy of them were inoculated with the Colombian strain (10(5) blood forms) and 10 were maintained as intact non-infected controls. After infection, C. callosus were sacrificed at different time-points from 15 to 70 days. The heart and skeletal muscle were processed for histopathology and cryopreserved for immunohistochemistry. Early necrotic lesions of parasitized skeletal muscle and myocardium with intense inflammatory lesions were present. Search for the in situ presence of TNF-alpha and IFN-gamma, was performed using rat-IgG anti-mouse antibodies against these cytokines. For the in situ search of TGF-beta, rabbit IgG anti-mouse antibodies were used. Immunolabeling of the cytokines in tissues of infected C. callosus was successful. The cytokines TNF-alpha, IFN-gamma , and TGF-beta were detected in the cytoplasm of macrophages and in the necrotic material from 15 to 45 days post-infection, decreasing their intensity until complete disappearance by the 65th day, which correlated with subsiding histopathological lesions. These findings suggest the participation of these cytokines in the control of parasite multiplication, in the development of an early fibrogenesis and in the regression of fibrotic-inflammatory lesions observed in C. callosus.

Early neonatal echocardiographic findings in an experimental rabbit model of congenital diaphragmatic hernia

This study aimed to demonstrate that congenital diaphragmatic hernia (CDH) results in vascular abnormalities that are directly associated with the severity of pulmonary hypoplasia and hypertension. These events increase right ventricle (RV) afterload and may adversely affect disease management and patient survival. Our objective was to investigate cardiac function, specifically right ventricular changes, immediately after birth and relate them to myocardial histological findings in a CDH model. Pregnant New Zealand rabbits underwent the surgical procedure at 25 days of gestation (n=14). CDH was created in one fetus per horn (n=16), and the other fetuses were used as controls (n=20). At term (30 days), fetuses were removed, immediately dried and weighed before undergoing four-parameter echocardiography. The lungs and the heart were removed, weighed, and histologically analyzed. CDH animals had smaller total lung weight (P<0.005), left lung weight (P<0.005), and lung-to-body ratio (P<0.005). Echocardiography revealed a smaller left-to-right ventricle ratio (LV/RV, P<0.005) and larger diastolic right ventricle size (DRVS, P<0.007). Histologic analysis revealed a larger number of myocytes undergoing mitotic division (186 vs 132, P<0.05) in CDH hearts. Immediate RV dilation of CDH hearts is related to myocyte mitosis increase. This information may aid the design of future strategies to address pulmonary hypertension in CDH.

Rise and fall in ischemic heart disease mortality: it may have happened before

The rise in ischemic heart disease(IHD) mortality occurring mostly during the first half of the 20th century is usually associated with economic development and its consequences for people's lifestyles. On the basis of historical evidence, it is postulated that a previous IHD epidemic cycle may have occurred in England and Wales towards the turn of the nineteenth century. The implications of this on causal theories and current etiological research on atherosclerosis are discussed.

Seasonal variation in hospitalizations due to heart failure in Niterói city, Southeastern Brazil

The objective of the study was to describe seasonality of hospitalizations for heart failure in tropical climate as it has been described in cold climates. Seasonal Auto-regressive Integrated Moving-Average model was applied to time-series data of heart failure hospitalizations between 1996 and 2004 in Niteroi (Southeastern Brazil), collected from the Brazilian National Health Service Database. The standard seasonal variation was obtained by means of moving-average filtering and averaging data. The lowest and the highest annual hospital admissions were 507 (1997) and 849 (2002), respectively; the lowest and the highest monthly rates were 419 (December) and 681 (October), respectively. Peak admission rates were seen during the fall and winter. Although weak, the seasonality observed indicates that slight variations result in increased hospitalizations for heart failure.

Cytomegalovirus and other herpesviruses infections in heart and bone marrow transplant recipients

From January 1988 to January 1989 all the heart transplant and bone marrow recipients at the Instituto do Coração of the Hospital das Clínicas of the University of São Paulo Medical School were studied for the incidence and morbidity associated with herpesviruses infections after transplantation. Five bone marrow and 5 heart transplant recipients were followed for a mean of 4.2 months post-transplantation. All the patients were seropositive for cytomegalovirus (CMV) before admission and 80% experienced one or more recurrences during the observation period. Of the 12 episodes of CMV infection, that were identified in this study, 83% were accompanied by clinical or laboratory abnormalities. However, there was only one case of severe disease. The overall incidence of infection for herpes simplex (HSV) was 50%. Although most of HSV reactivations were oral or genital, one case of HSV hepatitis occurred. One of the 6 episodes of HSV infections that were treated with acyclovir showed an unsatisfactory response and was successfully managed with ganciclovir. All the individuals had anti-varicella zoster virus antibodies, but none of them developed infection. The study emphasizes the importance of active diagnostic surveillance of herpesvirus infections in transplant patients. Both CMV and HSV reactivations showed high incidence and important morbidity and thus, deserve prophylactic therapy.

HEART ANEURYSM IN CHAGAS' DISEASE

This prospective study on 41 autopsy collected human hearts concerns the "apical" lesion in Chagas' disease. Previous report did not show a correlation between lesion frequency and heart weight then discarding a vascular factor in its pathogenesis. The present paper involves other variables besides the heart weight to evaluate the relative coronary insufficiency. Distinct colored gel (green and red) injected through the capillary beds of both coronary arteries defined the extent of both vessels before separating the atria and removing the sub-epicardium fat. The Right Ventricle (RV) and Left Ventricle (LV) free walls furnished the RV/LV mass ratio. The myocardium mass colored green (right coronary artery - RC) and the whole Ventricular Weight (VW) determined the RC/VW mass ratio. The heart weight plus these mass ratios, graded and added, composed a score inversely proportional to the myocardium irrigation condition. It intended to be a more sensitive morphologic evaluation of the relative ischaemia to correlate to the apical lesion. This study showed a right deviation for the relative accumulated frequency of lesions plotted as a score function and a significant difference for higher scores in hearts with aneurysm. It suggests a ischaemic factor intervening in the apical lesion pathogenesis in Chagas' cardiopathy.

Heart rate responses to a muscarinic agonist in rats with experimentally induced acute and subacute chagasic myocarditis

We administered arecoline to rats, with experimentally induced chagasic myocarditis, in order to study the sinus node sensitivity to a muscarinic agonist. Sixteen month old rats were inoculated with 200,000 T. cruzi parasites ("Y" strain). Between days 18 and 21 (acute stage), 8 infected rats and 8 age-matched controls received intravenous arecoline as a bolus injection at the following doses: 5.0, 10.0, 20.0, 40.0, and 80.0 mug/kg. Heart rate was recorded before, during and after each dose of arecoline. The remaining 8 infected animals and 8 controls were subjected to the same experimental procedure during the subacute stage, i.e., days 60 to 70 after inoculation. The baseline heart rate, of the animals studied during the acute stage (349 ± 68 bpm, mean ± SD), was higher than that of the controls (250 ± 50 bpm, p < 0.005). The heart rate changes were expressed as percentage changes over baseline values. A dose-response curve was constructed for each group of animals. Log scales were used to plot the systematically doubled doses of arecoline and the induced-heart rate changes. The slope of the regression line for the acutely infected animals (r = - 0.99, b =1.78) was not different from that for the control animals (r = - 0.97, b = 1.61). The infected animals studied during the subacute stage (r = - 0.99, b = 1.81) were also not different from the age-matched controls (r = - 0.99, b = 1.26, NS). Consequently, our results show no pharmacological evidence of postjunctional hypersensitivity to the muscarinic agonist arecoline. Therefore, these results indirectly suggest that the postganglionic parasympathetic innervation, of the sinus node of rats with autopsy proved chagasic myocarditis, is not irreversibly damaged by Trypanosoma cruzi.

Identification of respiratory virus in infants with congenital heart disease by comparison of different methods

Respiratory virus infections are the main cause of infant hospitalization and are potentially severe in children with congenital heart disease (CHD). Rapid and sensitive diagnosis is very important to early introduction of antiviral treatment and implementation of precautions to control transmission, reducing the risk of nosocomial infections. In the present study we compare different techniques in the diagnosis of respiratory viruses in CHD infants. Thirty-nine samples of nasopharyngeal aspirate were obtained from CHD infants with symptoms of respiratory infection. The Multiplex PCR (Seeplex® RV 12 ACE Detection) driven to the detection of 12 respiratory viruses was compared with the direct immunofluorescence assay (DFA) and PCR, both targeting seven respiratory viruses. The positivity found by DFA, Multiplex and PCR was 33.3%, 51.3% and 48.7%, respectively. Kappa index comparing DFA and Multiplex, DFA and PCR and PCR and Multiplex PCR was 0.542, 0.483 and 0.539, respectively. The concordance between techniques was considered moderate. Both Multiplex PCR (p = 0.001) and PCR (p = 0.002) detected significantly more respiratory virus than DFA. As the performance of the tests may vary, the combination of two or more techniques may increase diagnostic sensitivity favoring the diagnosis of co-infections, early introduction of antiviral therapy and implementation of appropriate measures.

Muscarinic antibodies and heart rate responses to dynamic exercise and to the Valsalva maneuver in chronic chagasic patients

We have studied the cardiac chronotropic responses to the Valsalva maneuver and to dynamic exercise of twenty chronic chagasic patients with normal left ventricular function and no segmental wall abnormalities by two-dimensional echocardiogram. The absolute increase in heart rate of the patients (Δ = 21.5 ± 10 bpm, M±SD) during the maneuver was significantly diminished when compared to controls (Δ = 31.30 ± 70, M±SD, p = 0.03). The minimum heart rate (58.24 ± 8.90 vs. 62.80 ± 10, p = 0.68) and the absolute decrease in heart rate at the end of the maneuver (Δ = 38.30 ± 13 vs. Δ = 31.47 ± 17, p = 0.10) were not different from controls. The initial heart rate acceleration during dynamic exercise (Δ = 12 ± 7.55 vs. Δ = 19 ± 7.27, M±SD, p = 0.01) was also diminished, but the heart rate recovery during the first ten seconds was more prominent in the sero-positive patients (Median: 14, Interquartile range: (9.75-17.50 vs. 5(0-8.75, p = 0.001). The serum levels of muscarinic cardiac auto-antibodies were significantly higher in the chagasic patients (Median: 34.58, Interquartile Range: 17-46.5, Optical Density) than in controls (Median: 0, Interquartile Range: 0-22.25, p = 0.001) and correlated significantly and directly (r = 0.68, p = 0.002) with early heart rate recovery during dynamic exercise. The results of this investigation indirectly suggest that, the cardiac muscarinic auto-antibodies may have positive agonist effects on parasympathetic heart rate control of chagasic patients.

Involvement of the autonomic nervous system in Chagas heart disease

The autonomic nervous system and especially the intracardiac autonomic nervous system is involved in Chagas' disease. Ganglionitis and periganglionitis were noted in three groups ofpatients dying with Chagas'disease: 1) Those in heart failure; 2) Those dying a sudden, non violent death and; 3) Those dying as a consequence ofaccidents or homicide. Hearts in the threegroups also revealed myocarditis and scattered involvement of intramyocardial ganglion cells as well as lesions of myelinic and unmyelinic fibers ascribable to Chagas'disease. In mice with experimentally induced Chagas' disease weobserved more intensive neuronal lesions of the cardiac ganglia in the acute phase of infection. Perhaps neuronal loss has a role in the pathogenesis of Chagas cardiomyopathy. However based on our own experience and on other data from the literature we conclude that the loss of neurones is not the main factor responsible for the manifestations exhibited by chronic chagasic patients. On the other hand the neuronal lesions may have played a role in the sudden death ofone group of patients with Chagas'disease but is difficult to explain the group of patients who did not die sudderly but instead progressed to cardiac failure.

Serial heart rate changes in rats inoculated by conjunctival instillation of Trypanosoma cruzi obtained from bug faeces

The cardiac effects of experimentally induced myocarditis, when the parasite is obtained from mouse blood, are well known. However, the consequences of the infection when the parasites are obtained from bug faeces are less well defined. In the present investigation, we have used the "Y" strain of Trypanosoma cruzi, which was maintained in Rhodnius prolixus by repeated passages in mice. The faeces of 30 infected bugs were collected, the number of parasites counted and 4,000 parasites inoculated by the conjunctival route in 60 rats. Twenty-nine other rats received faeces from noninfected bugs (sham-inoculated controls) and 40 were used as normal controls. The heart rate of the three groups of animals was recorded under general anesthesia with ether. The heart rate, at day 0 pre-inoculation, was similar in the three groups of animals (Controls: 379 ± 27 beats/min Mean ± SD; Sham-inoculated: 366 ± 31; Infected: 351 ± 29) (p> 0.05). In the infected animals, the mean heart rate began to increase significantly by day 12 following infection (375 ± 31), reaching the highest values between days 18 (390 ± 33) and 21 (403 ± 33) and returned to baseline by day 30 (359 ± 28) (p< 0.05). The heart rate changes were statistically different from those observed in the sham-inoculated controls and in the control animals. Therefore, these heart rate changes were provoked by the Trypanosoma cruzi-induced infection. Thus, it appears that irrespective of the source of the parasite and route of inoculation Trypanosoma cruziacute infection provokes a transient sinus tachycardia.

Differing phagocytic function of monocytes and neutrophils in Chagas' cardiopathy according to the presence or absence of congestive heart failure

We evaluated the in vitro phagocytic function and the production of microbicidal oxygen radicals by monocytes and neutrophils of 9 Chagas' heart disease subjects with heart failure and 9 without the syndrome in comparison with 11 healthy subjects, by assessing phagocytosis of Saccharomyces cerevisiae and NBT reduction by peripheral blood phagocytes. Phagocytic index of monocytes of chagasics without heart failure was significantly 6.7 and 10.6 times lower than those of controls and chagasics with the congestive syndrome, respectively, due to a lesser engagement in phagocytosis and to an inability of these cells to ingest particles. Neutrophils also show in chagasics without heart failure PI 11.2 and 19.8 times lower than that of controls and chagasics with heart failure, respectively. The percent of NBT reduction was normal and similar for the three groups. Balanced opposite effects of cardiovascular and immune disturbances may be acting in Chagas' disease subjects with heart failure paradoxically recovering the altered phagocytic function.