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Noninvasive assessment of cardiac structure and function is essential to understand the natural course of murine infection with Trypanosoma cruzi. Magnetic resonance imaging (MRI) and echocardiography have been used to monitor anatomy and function; positron emission tomography (PET) is ideal for monitoring metabolic events in the myocardium. Mice infected with T. cruzi (Brazil strain) were imaged 15–100 days post infection (dpi). Quantitative 18F-FDG microPET imaging, MRI and echocardiography were performed and compared. Tracer (18F-FDG) uptake was significantly higher in infected mice at all days of infection, from 15 to 100 dpi. Dilatation of the right ventricular chamber was observed by MRI from 30 to 100 dpi in infected mice. Echocardiography revealed significantly reduced ejection fraction by 60 dpi. Combination of these three complementary imaging modalities makes it possible to noninvasively quantify cardiovascular function, morphology, and metabolism from the earliest days of infection through the chronic phase.
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is an important cause of heart disease in endemic areas of Mexico, Central and South America.1 One of the hallmarks of infection is the inflammatory process in the myocardium at the early stage of disease, when parasitemia and tissue parasitism are intense.2 The acute infection is followed by the indeterminate phase, which can last for many years or a life-time. Ten percent to 30% of all sero-positive patients will eventually progress to a cardiomyopathy associated with congestive heart failure and/or conduction abnormalities.3 An accurate assessment of myocardial function is important in the evaluation of the prognosis of an individual patient.4
Murine models of Chagas disease have been extensively evaluated and have been shown to recapitulate many of the pathologic and immunologic characteristics of human disease.5–8 To study the natural course of murine infection non-invasive assessment of cardiac structure and function is essential. Magnetic resonance imaging (MRI) and echocardiography have been used with increasing frequency because they permit serial examination of a single animal during disease progression.9–13
With the advent of molecular imaging modalities, particularly positron emission tomography (PET), the visualization and quantification of cellular and molecular processes occurring in living tissues are now possible. 14,15 The PET measures the in vivo body distribution of imaging agents labeled with positron-emitting radionucleotides and has been extensively used in humans to characterize the heart with respect to properties such as perfusion, 16,17 metabolism,18,19 innervation,20,21 and cardiac function. 22,23 With the introduction of high-resolution small-animal PET, 24,25 in vivo molecular imaging of myocardial metabolism in small laboratory animals, mostly mice and rats, has become possible.
Here, we applied quantitative 18F-FDG micro-positron emission tomography (microPET) imaging of CD1 mice infected with T. cruzi (Brazil strain) in a serial time course from 15 to 100 days post-infection (dpi). The MRI and echocardiography were performed at the same time points and data from the different modalities were compared. Although morphologic and functional alterations in the heart of infected mice were observed at 30 dpi using MRI and echocardiography, differences in glucose uptake were detected by microPET at an earlier time point. These results show that 18 F-FDG microPET imaging can detect cardiac metabolic defects before morphologic and functional changes in the heart become apparent.
The Brazil strain of T. cruzi was maintained in C3H mice (Jackson Laboratories, Bar Harbor, ME). Male CD1 mice (Jackson Laboratories) were infected intraperitoneally at 10 weeks of age with 5 × 104 trypomastigotes. All mice were housed in the Institute for Animal Studies of the Albert Einstein College of Medicine and all protocols were approved by the Institutional Animal Care and Use Committee (IACUC). The mice were imaged 15, 30, 60, and 100 dpi. Hearts were placed in 10% phosphate-buffered formaldehyde and stained with hematoxylin and eosin and sirius red.
All mice were imaged after 3 hours of fasting. Mice were anesthetized with 1.5% isoflurane-oxygen mixture, which continued throughout the imaging portion of the procedure. Each mouse was placed on a heating pad before and during scanning to maintain normal body temperature. Mice were administered 300–400 uCi (12–15 MBq) in 0.1 mL normal saline, [18F] fluoro-2-deoxyglucose (FDG), via tail vein and imaging was started at 1 hour after injection. This period permits the tracer to be delivered throughout the body and trapped by the glycolytic pathway. Cardiac gating of the PET acquisition was accomplished using standard electrocardiogram (ECG) contacts and a Gould ECG amplifier interfaced to the PET scanner (for gating) and a PC running Ponemah Physiology Suite software (for monitoring) (Gould Instrument Systems, Inc., Valley View, OH). After MicroPET imaging, the animals were housed in a dedicated hood until they could be safely moved back to the Animal Institute (18F has a half-life of 110 minutes). Imaging was performed using a Concorde Microsystems R4 microPET Scanner (Concorde Microsystems, LLC, Siemens, Knoxville, TN), with 24 detector modules, without septa, providing 7.9 cm axial and 12 cm transaxial field of view. Acquisitions were performed in three-dimensional (3D) list mode. A reconstructed full-width half maximum resolution of 2.2 mm was achievable in the center of the axial field of view. After 10 minutes of list mode acquisition, data were sorted into 3D sinograms, and images were reconstructed using iterative reconstruction in a 128 × 128 × 64 (0.82 × 0.82 × 1.2 mm) pixel array. Data were corrected for deadtime counting losses, random coincidences, and the measured nonuniformity of detector response (i.e., normalized) but not for attenuation or scatter.
Image analysis was performed using ASIPRO VM (Concorde Microsystems, LLC) dedicated software. All studies were inspected visually in a rotating 3D display to examine for interpretability and image artifact. Manual regions of interest (ROI) were defined around areas of visually identified heart activity in the left ventricle (LV). Successive scrolling through 2 dimensional slices (each 1.2 mm thick in the axial images) permitted identification of a pixel of maximum measured decay-corrected uptake, termed the standardized uptake value, or SUVmax. The SUVmax is the maximum value of the percent-age injected dose per gram of cardiac tissue multiplied by the body weight of each animal. The SUVmax has been validated in numerous animal and human models as a reproducible and robust measure of radioactivity in longitudinal studies. The cardiac gating of the PET images permitted qualitative observation of the motion of the heart and right ventricular dimension. Sample movies of representative hearts are presented in the supplementary information (Supplementary videos can be found online at www.ajtmh.org).
Mice were anesthetized with 1.5% of isoflurane. A set of standard, shielded, nonmagnetic electrocardiographic leads ending in silver wires were attached to the four limbs. The ECG signal was fed to a Gould ECG amplifier associated with the Ponemah Physiology data acquisition system for monitoring the ECG and the R wave triggered a 5 volt signal to gate the spectrometer. Images were acquired with a GE/Omega 9.4 T vertical wide-bore spectrometer operating at a 1H frequency of 400 MHz and equipped with 50-mm shielded gradients (General Electric, Fremont, CA) and a 40-mm 1H imaging coil (RF Sensors, New York, NY). Temperature within the coils was maintained at 30°C using a water cooling unit (Neslab Instrument, Inc., Portsmouth, NH). This temperature prevented hypothermia in the anesthetized mice. After attachment of the cardiac gating leads, the mice were wrapped in a Teflon sheet and multislice spin echo imaging was performed to obtain short axis images of the heart. The gating delay was adjusted to collect data in systole or diastole. The following parameters were used to obtain 8 short axis slices: echo time, 18 msec; field of view, 51.2 mm; number of averages, 4; slice thickness, 1 mm; repetition time, approximately 0.2 sec; matrix size, 128 × 256 (interpolated to 256 × 256). Several sets of 8 slices were acquired to define the entire heart and to obtain images in diastole and systole taking approximately 20–30 minutes per mouse. Data were transferred to a PC and analyzed using MATLAB-based software (The MathWorks, Natick, MA). Left ventricle and right ventricle (RV) dimensions in millimeters were determined from the images representing end-diastole. The left ventricular wall is the average of the anterior, posterior, lateral, and septal walls. The right ventricular internal dimension is the widest point of the right ventricular cavity.
Mice were lightly anesthetized with 1.5% isoflurane in 100% O2; the chest wall was shaved and a small gel standoff was placed between the chest and a 30-MHz RMV-707 B scanhead interfaced with a Vevo 770 High-Resolution Imaging System (VisualSonics, Toronto, ON, Canada). High-resolution, two-dimensional electrocardiogram-based kilohertz visualization (EKV Mode) and B mode images were acquired. Continuous, standard electrocardiogram was recorded using electrodes placed on the animal’s extremities. Diastolic measurements were performed at the point of greatest cavity dimension, and systolic measurements were made at the point of minimal cavity dimension, using the leading edge method of the American Society of Echocardiography. 26 Ejection fraction was calculated and used as a determinant of LV cardiac function.
All data are expressed as the mean (±SEM) and were analyzed using GraphPad Prism 4 statistics software (GraphPad Software Inc., San Diego, CA). For analysis of differences between groups the Student’s t test was performed. A level of significance of 5% was chosen to denote differences between means.
Infected mice had a peak of parasitemia of approximately 1 × 106 trypomastigotes/mL of plasma at Day 20 dpi. Between 27 and 33 dpi, the mortality peaked at 47% (14 out of 30). At 30 dpi there was an intense and diffuse myocarditis characterized by lymphomononuclear interstitial infiltrate, disruption of myofibers, and multiple pseudocysts. There was also perivascular inflammatory infiltrate. At 60 and 100 dpi, the inflammation became significantly less intense and parasites were not detected. Myocardium reparative fibrosis, evidenced by sirius red staining of collagen, was more extensive in the RV (Figure 1).
The metabolic state of the myocardium was assessed by the regional uptake of the glucose analogue, 18F-FDG. The mean value of the myocardial SUVmax was used to compare the MicroPET data between the uninfected age-matched controls and infected groups at the different days post infection. Tracer uptake was significantly higher in the LV myocardium of the infected mice compared with controls at all days of infection, from 15 to 100 dpi (Table 1). MicroPET was also used to visualize cardiac morphology (Figure 2) and contractility (Supplemental data).
Figure 3 shows representative MRI images of the short axis of the hearts of control and infected mice. As we previously observed, there was no difference in LV internal diameter during the period studied in infected mice compared with uninfected controls (data not shown). However, the inner dimension of the RV was significantly dilated from 30 to 100 dpi (Table 2). This increase in right ventricular chamber dimension was 95% at 30 dpi (1.88 ± 0.15 versus 3.66 ± 0.21 mm, control versus infected), 80% at 60 dpi (1.49 ± 0.13 versus 2.69 ± 0.23 mm) and 54% at 100 dpi (1.66 ± 0.24 versus 2.56 ± 0.15 mm). The left ventricle wall thickness (LVWT) was increased by 11.4% at 100 dpi (1.23 ± 0.03 versus 1.37 ± 0.05 mm). There was no difference in LVWT at 15, 30, and 60 dpi in comparison to uninfected controls.
Infected mice exhibited a significant reduction in LV ejection fraction at 60 and 100 dpi (Table 3). The normal heart rate for mice ranges from 500 to 600 beats per minute. A slower heart rate was noted in some infected mice.
Our laboratory pioneered the application of cardiac imaging techniques in mouse models of T. cruzi-induced heart disease. 11,13,27 In previous studies, we showed the use of cardiac MRI and echocardiography in the evaluation of alterations in structure and function accompanying this infection. MicroPET is a relatively new technique for evaluating cardiac structure and function. 28 In this study using this technique, we showed for the first time that T. cruzi-infected mice display greater uptake of glucose throughout the time course of infection compared with uninfected controls. Importantly, this is the first study comparing data from three different noninvasive imaging modalities, MRI, echocardiography, and micro-PET, for the serial assessment of myocardial viability and cardiac structure and function in mice infected with T. cruzi from acute to chronic phase.
Cardiac MRI findings confirmed our previous observation of significant dilatation of the right ventricular chamber was from 30 to 100 dpi in infected mice when compared with controls. 10,13,27,29,30 There was no difference in the left ventricular internal diameter between groups and LVWT was increased only at 100 dpi. MRI allows accurate, high resolution 3D characterization of cardiac structure within a single examination and allows the quantification of volumetric changes in hearts. 31 However, technical difficulties related to anesthesia, irregular heart rates, and thermoregulation can arise during the acquisition of cardiac gated MRI studies, which require signal averaging over a time period of several minutes. 32,33
Echocardiography confirmed our previous observation that the ejection fraction, a measurement of myocardial systolic performance, was decreased at 60 and 100 dpi. 11 Echocardiography has been used by our group. 11,34,35 and others 36–40 as an important tool in the assessment of cardiac function in murine models of cardiac disease. A particular disadvantage of the echocardiographic method for evaluation of hearts of T. cruzi-infected mice is the difficulty in assessing the RV, because the images are typically acquired along the parasternal long axis, which obscures the location of the RV.
Our MicroPET study showed that T. cruzi-infected mice displayed increased uptake of glucose in the myocardium when compared with controls as early as 15 dpi, whereas alterations in morphologic parameters, such as right ventricular internal diameter (RVID) and LVWT, were not detected by MRI until 30 dpi. Alteration in cardiac function measured by echocardiography (ejection fraction) was not detected until 60 dpi. The PET uptake provides information regarding the metabolic state of the myocardium through the regional uptake of 18F-FDG, a glucose analogue. 41 After transport into cells, 18F-FDG undergoes subsequent hexokinase-mediated phosphorylation, but is not further metabolized resulting in metabolic trapping of the radiotracer in cells that exhibit enhanced glucose metabolism. 14 MicroPET has been used for monitoring metabolic events in the myocardium of small animals in stem cell transplantation after myocardial infarction, 41,42 progressive hypertrophy, 43 and left ventricular dilation. 44 Increased FDG uptake has been shown in vitro in leukocytes, 45 lymphocytes, and macrophages 46,47 and in vivo in acute myocardial infarction, 48 abdominal aortic aneurism, 49 and atherosclerosis. 50 The increased uptake of FDG in T. cruzi-infected mice correlates with the intense and diffuse myocarditis observed during the acute phase and chronic inflammation and myocardium reparative fibrosis that occurs during the chronic phase. An important characteristic of PET is that it is largely independent of the thickness of the object and the depth of the source within the subject. Thus, in addition to providing information on glucose uptake, the microPET images also permit visualization of the dilation of the RV (as shown in Figure 3) and contractility (see Supplemental data).
In conclusion, we showed that by combining complementary imaging methodologies, MRI, echocardiography, and MicroPET it is possible to noninvasively quantify parameters related to cardiovascular function, morphology, and myocardium metabolism of mice infected with T. cruzi from the earliest days of infection through the chronic phase. Our MicroPET data shows metabolic changes in the LV as early as 15 dpi, likely associated with inflammation that appears before alterations in structure and function of the myocardium are detected. These approaches permit serial examinations and can be used in longitudinal studies over the time course of the disease process to provide important insights into disease biology and pathophysiology. Furthermore, these findings suggest that PET may be a useful tool to track early response to therapeutic agents and to evaluate efficacy of therapeutics in patients.
We thank Lina M. Restrepo for training in use of the echocardiographic equipment and Jorge Durand for MRI assistance.
Financial support: NIH grant AI076248(HBT), CMP was supported by Fogarty International Training grant D43-TW007129 (HBT) and Fundação de Amparo à Pesquisa do Estado de São Paulo (06/52882-3; 06/59618-0; 08/00954-6).
Note: Supplemental data can be found online at www.ajtmh.org.
Publisher's Disclaimer: Disclaimer: There are no potential conflicts of interest.
Authors’ addresses: Cibele M. Prado and Marcos A. Rossi, Department of Pathology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Av. Bandeirantes n 3900, Monte Alegre, CEP 14049-900, Ribeirão Preto, São Paulo, Brazil. Eugene J. Fine and Wade Koba, M. Donald Blaufox Laboratory for Molecular Imaging, Department of Medicine and Radiology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Dazhi Zhao, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Herbert B. Tanowitz, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, Tel: 718-430-3342, Fax: 718-430-8543, E-mail: udeuy.mocea@ztiwonat. Linda A. Jelicks, Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461.