We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.
Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.
In order to measure the efficacy of new treatment strategies for myocardial infarction (MI) in preclinical studies, the assessment of the acute stage as well as long-term outcome is required1. Methods such as histopathology, intracardiac catheters2, and ex vivo heart models3 are commonly used for preclinical studies in mice. It is impossible, however, to follow up treatment with ex vivo or highly invasive methods. Noninvasive measurement techniques as in vivo magnetic resonance imaging (MRI) and positron emission tomography (PET) allow longitudinal experimental designs for disease staging in single subjects. Cine-MRI is used to derive global functional parameters such as left ventricular mass (LVM), ejection fraction (EF) and cardiac output (CO). In addition, after the injection of a gadolinium contrast agent, due to impaired perfusion and washout in the infarction, tissue viability can be assessed with late gadolinium enhancement (LGE) MRI. Complementarily, PET offers a sensitive measure of radiolabeled molecules in order to assess tissue metabolism. The high accuracy of these techniques permits significant reductions in the number of animals required for testing new drugs targeting MI.
The PET and MRI procedures are involved, and without a carefully designed protocol reproducibility is hard to maintain. Procedures established in the clinic for patients require substantial modification for use in mice, due to their considerably faster heart rate and smaller dimensions of the heart4. There is wide variation between individual subjects, both at baseline and in response to induced injury, so a considerable number of mice are needed to establish treatment efficacy.
In this report, we describe our method for sequential PET/MRI imaging of the mouse heart. Both modalities use intravenous contrast agents, which are delivered through the tail vein. MRI consists of standard assessment with Cine-MRI5 with an optimal protocol for LGE as described in our previous work6. The entire MRI procedure lasts 30 min. We have obtained consistent fittings for the beds of our instruments so it is possible to transfer the animal on a platform with the same monitoring and anesthetic delivery apparatus between the machines. The PET scan lasts 45 min with in situ injection once the scan is started. The final step is to measure the parameters from both MRI and PET images following coregistration.
All components of this study were carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986, and with the approval of the University of Cambridge Ethical Review Panel.
1. Animal Preparation
2. Animal Positioning and Monitoring
3. MRI
4. Late Gadolinium Enhancement
5. PET Imaging
6. MRI Segmentation
7. PET Analysis and Coregistration with MRI
One of the advantages of using MRI is that a longitudinal design can be used in order to stage disease. This is especially important when evaluating novel compounds, as the time course of effect may not be known. Variations in heart volumes due to the disease progression in the same animal are shown in Figure 4. The figure clearly shows the effects of remodeling post myocardial infarction.
Correct slice geometry is crucial for the success of a cardiac MRI experiment. Representative slice planning and resulting geometry for various MRI scans are reported in Figure 5. Segmentation of two MRI short-axis slices is shown in Figure 6. Our Cine-MRI procedure achieved a precision of 4% for LV mass, 3% for EDV, 5% for ESV, 2% for SV, 2% for EF, and 4% for infarct size (coefficient of variation, measured on n = 4 wild-type mice with both, data acquisition and segmentation procedure, repeated 2x for each animal).
PET can be used to measure the binding of specific tracers throughout the body. An example of FDG-PET maximum intensity projection image is shown in Figure 7. As expected, in an anesthetized animal, the greatest uptake is found in the heart and in the brain. Figure 8 shows coregistered LGE MRI and FDG-PET. The enhanced areas on MRI, representing nonviable tissue, match areas of reduced FDG uptake in the PET.
Scan | Sequence |
Cine imaging | FISP, TR/TE 7 msec/2.4 msec, frames 13-20, matrix 256 x 256 x 128 with FOV 3.5 cm, slice thickness 1 mm, bandwidth 64 kHz, flip angle 20°, NEX 2 |
3D scan for coregistration | FISP, FISP, TR/TE 8 msec/4 msec, matrix 256 x 256 x 128, FOV 3 x 3 x 6.40 cm, bandwidth 50 kHz, flip angle 15°, NEX 2 |
Scout before injection | FLASH, FOV 3.5 cm, matrix 128 x 128, slice thickness 0.8 mm with 0.2 mm gap between adjacent slices TE = 2.8 msec, TR = 550-750 msec, FLASH TR 7 msec, bandwidth 64 kHz, flip angle 60°, 1 NEX, 0.8 mm, 0.2 mm gap, selective inversion 0.8 mm thickness with 5 msec sech shaped pulse |
LGE imaging | FLASH, FOV 3.5 cm, matrix 256 x 256, slice thickness 0.8 mm with 0.2 mm gap between adjacent slices TE = 2.8 msec, TR = 550-750 msec, FLASH TR 7 msec, bandwidth 64 kHz, flip angle 60°, 1 NEX, 0.8 mm, 0.2 mm gap, selective inversion 0.8 mm thickness with 5 msec sech shaped pulse |
Table 1. MRI sequences. Abbreviations are FISP: fast imaging with steady state precession; FLASH: fast low angle shot; TE: echo time; TR: repetition time; NEX: number of excitations.
Figure 1. Overview of the experiment. Click here to view larger image.
Figure 2. Animal monitoring setup. ECG is derived with graphite electrodes on the paws, respiration with a pillow under the chest connected to a piezoelectric transducer and temperature with a lubricated rectal probe. Click here to view larger image.
Figure 3. Representative monitoring and gating traces. Anesthetic dose should be adjusted to achieve stable traces during the exams. Click here to view larger image.
Figure 4. a) End-diastolic 4 ch view of a mouse at baseline. b) End-diastolic 4 ch view of the same mouse one day after induced injury. c) End-diastolic 4 ch view of the same mouse four weeks after induced injury. Click here to view larger image.
Figure 5. Slice planning and resulting images for the relevant views in cine MRI. A correct slice geometry is key in achieving reproducible measurements. Click here to view larger image.
Figure 6. a) Segmentation of a mid-ventricular short-axis slice. b) Segmentation of a basal short-axis slice. Endocardial and epicardial borders are identified and segmented for each short-axis slice. Fixed rules must be applied to achieve reproducible volume estimates. Click here to view larger image.
Figure 7. Maximum intensity projection of an FDG-PET scan. Higher uptake is seen in areas that are more metabolically active. Click here to view larger image.
Figure 8. Coregistration between LGE MRI and FDG PET. There is good agreement between the two techniques in identifying viable tissue. Click here to view larger image.
PET/MRI is a comprehensive measurement method for the noninvasive and longitudinal evaluation of systolic function, tissue viability and specific metabolic markers in mouse models of myocardial infarction. Here we have described a protocol for performing MRI and PET sequentially, where coregistration is simplified by the transfer of the same bed between systems. This strategy, also adopted by manufacturers of clinical systems11, does not require a combined PET/MRI scanner and can be performed with standard equipment. After measuring the fixed matrix for transformation between the two systems, coregistration of the PET with the MRI data is obtained with an axial translation. This simplification is particularly useful in cardiac studies, where coregistration is complicated by motion.
This protocol can be performed using a number of different commercially available scanners. Different PET cameras can be used, as long as a proper connection of the MRI bed to the PET allows accurate positioning. If the pulse sequences are properly modified, it is also possible to perform the MRI measurements at different field strengths12-15, on small bore as well as clinical systems16. Although at 4.7 TECG signal quality could be insured by placing the electrodes with care, this might not always work at higher field strengths where self-gating technologies might be needed17.
To obtain a good outcome, particular attention should be paid to some critical steps of the protocol. First, slice planning and gating for MRI of the mouse heart, as sub-optimal geometry or gating produce inconsistent data that cannot be corrected in post-processing. Secondly, segmentation of MRI data, which is performed in post-processing and can be repeated multiple times, but rigorous and fixed rules must be applied in order to avoid biases in the volume estimates. Thirdly, transfer between different scanners, where motion should be avoided, to exclude a mismatch between the final images. Last, keep the injection volumes low, especially as three injections are performed during the whole procedure. To avoid sub optimal injections, specific activity of the PET tracer should be measured before initiating imaging, to ensure that enough activity can be injected maintaining low volumes.
The main limitation of our procedure is that PET and MRI are not performed simultaneously but sequentially. This feature does not guarantee complete coregistration of the myocardial wall between modalities, as intravenous injections and time under anesthesia might perturb heart function. However, this effect was not directly observable in our images, which showed excellent match between techniques. A comparison with novel simultaneous PET/MRI technologies would be needed to quantify this effect and to assess its relevance. Although some preliminary results have been reported with isochronous PET/MRI in the mouse heart18,19, customized scanners to perform this exam are still not widely available, and most groups are forced to perform MRI and PET sequentially. With simultaneous PET/MRI still in its infancy, reliable sequential methods such as the one described in this paper are needed to perform multimodal imaging.
Imaging techniques represent a valuable tool for assessment of new therapies in vivo, which can substitute more invasive techniques. MRI and PET can be used to assess left ventricular morphology, tissue viability and metabolism in mouse models of heart disease20. MRI provides an alternative to terminal procedures to assess LV function, such as intracardiac pressure-volume loop measurements2 or the ex vivo working heart model3. Assessment of LV chamber dimensions with MRI allows for detection of abnormal LV chamber enlargement or increased wall thickness4. Tissue viability can be noninvasively measured with LGE, achieving results in good agreement with TTC staining6. FDG-PET one day after infarction provides glucose metabolism at the aftermath of the ischemic insult, showing areas that are still metabolically functional20. Using imaging methods can significantly contribute to refinement and reduction in animal research.
Future applications of this imaging method include basic science studies on biochemical processes in the injured heart, assessment of novel pharmaceutical agents, and evaluation of new PET tracers. Noninvasive methods of targeting biochemical properties of tissue can be performed longitudinally to monitor remodeling, inflammation21 and drug efficacy in the long term.
The authors have nothing to disclose.
We are grateful for funding from the British Heart Foundation to TK and to the UK Medical Research Council for a postgraduate research studentship to GB.
Material name | Company | Catalogue number | Comments |
Gadovist | Bayer Schering Pharma | PL 00010/0535 | |
FDG | IBA Molecular | ||
Equipment | |||
Material name | Company | Catalog number | Comments |
Bruker BioSpec 47/40 | Bruker | ||
Bruker mouse bed | Bruker | This bed includes tubing for anesthesia delivery and scavenging. | |
12 cm diameter birdcage transmitter | Bruker | T5346 | |
2 cm diameter surface coil receiver | Bruker | T7027 | |
Red Dot Neonatal monitoring electrodes | 3M | P/N: 2330 | |
Monitoring equipment | SA Systems | The monitoring kit includes respiratory pillow and rectal probe for temperature measurements. | |
Anesthesia equipment | General Anesthetic Services | ||
Induction box | Vet Tech Solutions LTD | ||
Cambridge split-magnet PET/MRI scanner | University of Cambridge | A custom built PET/MRI scanner22 was used to perform the PET, its PET performance similar to an F120 micropet scanner23 | |
Segment software | Medviso, Lund University | Freely available | |
SPM-mouse | University of Cambridge | Freely available |