Animal protocols were approved by the National Heart, Lung, and Blood Institute Animal Care and Use Committee. Closed-chest myocardial infarctions were created in 10 Hanford miniswine (30 to 60 kg; Sinclair Research, Columbia, Mo). Animals received aspirin, atenolol, and lisinopril daily. Anesthesia was induced with ketamine and xylazine and maintained with isoflurane and mechanical ventilation. Heparin, amiodarone, and lidocaine were administered intravenously after percutaneous transfemoral access. Occlusive platinum coils (VortX, 2.5 to 3.5 mm; Boston Scientific) were delivered to the left anterior descending coronary artery after the second diagonal branch under x-ray guidance. This reproducibly caused a transmural infarction of the anterior wall, septum, and apex. To cause a small nontransmural infarction in 1 animal, an angioplasty balloon (Open-Sail 2.5×15 mm, Guidant) was inflated in the distal left anterior descending coronary artery for 60 minutes before reperfusion. In vivo thermometry was performed in an additional naive pig.
Cell Preparation and Labeling
Allogeneic porcine MSCs were isolated from bone marrow aspirates, expanded,4
and then labeled with dual-contrast particles.5
MSCs were incubated for 18 to 24 hours with 0.9-μm polystyrene beads containing 62% magnetite as an MRI marker and Dragon Green fluorescent dye as a histological marker (Iron-Fluorescent Particles, IFP, Bangs Laboratories). Injection mixtures included final concentrations of 1×106
labeled cells, tissue fast dye (Triangle Biomedical Sciences) diluted to 150 μL in PBS. Test injections used gadolinium-DTPA 30 mmol/L (Magnevist, Berlex).
Cell injections were conducted 1 to 7 days after myocardial infarction to simulate susceptibility to catheter rupture of nonreper-fused myocardial infarction. Three or 4 injections of labeled cells were performed in each of 10 animals. Needle dead space was cleared with saline before each needle retraction. Animals were killed and injection sites formalin-fixed or snap-frozen for histological examination.
We obtained serial representative 8-μm-thick sections of injection regions identified by tissue fast dye. Immunohistochemistry specimens were stained for desmin, with DAPI nuclear counterstain, and imaged under fluorescence, differential-phase, or light microscopy.
Custom MR Fluoroscopy Environment
A 1.5-T clinical MRI scanner (CV/i, General Electric) was customized with an external subsystem (Onyx2, Silicon Graphics) for real-time reconstruction of raw scanner data.3,6
The scanner and external work station were controlled from a pair of in-room keyboards and shielded liquid crystal displays. Four independent high-impedance surface coils (Nova Medical) were used interchangeably with intravascular coils (see below). The suite was equipped with an MR-compatible patient-monitoring system providing display of oximetry, instantaneous blood pressure, and ECG.
An external subsystem extended the commercial real-time graphical user interface (i-Drive, General Electric) with interactive scan and reconstruction control features significantly enhanced from a previous iteration.3
Gain and color highlighting of signals from each receiver channel can be adjusted independently. Image acceleration techniques and temporal resolution controls can be adjusted interactively (see Acquisition Parameters). Magnetization preparation pulses can be activated, including nonselective water or fat saturation. ECG gating can be toggled on/off. A catheter-channel-only projection imaging mode was developed to show catheter position even when outside the thin-slice imaging plane.7
To visualize the complex interrelation of anatomy, function, catheter position, and intramyocardial injectate distribution, a multislice acquisition mode was implemented having 3D-rendered reconstructions of translucent slices.8
MR Fluoroscopy Acquisition Parameters
All injections were guided by steady-state free precession sequences (RT-SSFP),9
run in fluoroscopic (real-time) mode, with either rectilinear or radial k-space trajectories. For rectilinear trajectories, image-acceleration techniques were applied, such as echo sharing, in which acquisitions were alternated between even and odd phase-encoding lines with temporal filtering similar to that in UNFOLD,10
generating 8 frames/s and an acquisition-to-display latency of ≈250 ms. Typical rectilinear imaging parameters were repetition time (TR), 3.8 ms; time to echo (TE), 1.9 ms; flip angle, 60°; bandwidth, ±62.5 kHz; 192×96 matrix; 32×24-cm field of view; ¾ partial-phase acquisition generating 1.7×2.5×8-mm voxels. For radial trajectories, image acceleration was achieved by use of undersampling of projection views.7
The user interactively adjusted the compromise between temporal resolution and image quality by varying the amount of undersampling.7
Typical radial imaging parameters were TR, 4.0 ms; TE, 1.9 ms; flip angle, 60°; bandwidth, ±62.5 kHz; 160×64 projections; 32-cm field of view; generating 2×2×8-mm voxels. MSC injection sites were also inspected by use of segmented, ECG-gated, breath-held fast gradient echo: TR, 6.5 ms; TE, 2.9; flip angle, 15°; bandwidth, ±31.5 kHz; 256×128 matrix; 34×34-cm field of view; ¾ phase acquisition generating 1.3×2.7×8-mm voxels; and SSFP MRI: TR, 4.8 ms; TE, 1.9; flip angle, 60°; bandwidth, ±62.5 kHz; 256×128 matrix; 34×34-cm field of view; ¾ phase acquisition generating 1.3×2.7×8 mm voxels.
MR Fluoroscopy of Myocardial Infarction
Infarcted targets were identified by impaired myocardial thickening during RT-SSFP MR fluoroscopy and by contrast enhancement. Although infarcted tissue is visible in RT-SSFP imaging without contrast agent, blood-to-infarct contrast is poor. To improve this contrast, delayed hyperenhancement (DHE) was used in conjunction with a 90° nonselective saturation pulse played before each echo-shared image (sat-RT-SSFP). DHE of the infarcted region was created by injecting Gd-DTPA 0.2 mmol/kg via a peripheral intravenous line 20 to 60 minutes before imaging. This non–ECG-gated, free-breathing technique was compared with RT-SSFP and with conventional segmented, ECG-gated, breath-held inversion recovery gradient echo (IR-GRE) DHE as the reference standard for identification of infarcted tissue.11
Injection Catheters and Their Use
The coaxial (“guide-in-guide”) 9F transfemoral guiding catheter system and injection needle (MRI-modified Stiletto 2, Boston Scientific Corporation) were adapted to serve as MRI receiver coils in parallel with surface coils. The MRI receiving coil and transmission line were integrated into the guiding catheter. An additional receiver coil was added immediately proximal to the Stiletto needle tip to generate a high-intensity signal on a separate receiver channel.
Guiding catheters were advanced across the aortic valve over nitinol guidewires. Three injection regions were predetermined and targeted in each animal: infarcted nonviable myocardium, border between infarcted and normal myocardium, and normal myocardium. ECG, instantaneous pressure, and real-time images of cardiac motion were monitored continuously.
Intravascular MRI receiver coils can potentially heat during scanning.12
Catheter heating was tested13
with a 4-channel fluoroptic thermometer (model 3100, Luxtron). Probes were attached at the tips of the 9F left ventricular (LV) sheath, coaxial LV steering guide, and Stiletto, respectively. A reference probe measured the bath (in vitro) or core body (in vivo) temperature. Static phantoms, intended to exaggerate heating, included a 8×110-cm tube (small heat sink) and a 10-L tub (large heat sink), each filled with normal saline, with the catheter positioned in the isocenter or closer to the RF transmitter (20 cm off center of the 60-cm diameter bore, to increase susceptibility to heating). Recordings were also obtained in the iliac artery, aortic root, and LV cavity of a naive pig. Temperature fluctuations were recorded during continuous SSFP TR, 3.8 ms;α, 60° to 90°; with and without saturation preparation, for 120 seconds after steady state was achieved, typically 150 to 300 seconds. Worst-case scenarios for heating were also simulated by disconnecting one or both of the catheter coils from the MRI receivers.
Infarct hyperenhancement methods (RT-SSFP, sat-RT-SSFP, and IR-GRE) were compared by use of contrast-to-noise ratios between normal myocardium, infarcted myocardium, and blood by 2 independent observers. Regions of interest were placed within the normally contracting septum ≈10 mm from the infarct border (normal), within the akinetic apical infarct wall ≈10 mm from the infarct border, and within the LV cavity (blood). Real-time wall thickening and the location, depth (center of mass), and intramyocardial distribution of susceptibility artifacts corresponding to stem-cell injections were analyzed offline (Medical Imaging Processing, Analysis and Visualization, v0.99, NIH). Continuous parameters are expressed as mean±SD.