Animal model
Fifteen female Danish Landrace pigs each weighing ∼40 kg were used for the experiments. The pigs were presedated with an intramuscular injection of Stressnil (1 mL/kg) and midazolam (1 mL/kg). After induction of anaesthesia with intravenous propofol (3.33 mg/kg) and endotracheal intubation, anaesthesia was maintained with sevoflurane (2.5%) and continuous rate infusion of fentanyl (3 mg/kg/h). The pigs were mechanically ventilated with a tidal volume of 425 mL (12 ventilations/min). By ultrasound guidance, an 8 F introducer sheath was inserted into the right common femoral artery. This was followed by an intravenous bolus injection of heparin (100 IU/kg). Coronary occlusion was induced by placing a 2.5 mm angioplasty balloon in the left anterior descending coronary artery (LAD) distal to the second diagonal branch artery and inflating it to 10 atm. The balloon occluded the LAD for 65 min, and was then deflated and removed. A coronary angiogram was performed following balloon inflation, to confirm occlusion, and also following balloon deflation, to confirm reperfusion. The animals were randomised to receive either intracoronary infusion of saline or dimethyl sulfoxide during reperfusion as a potential adjunctive therapy to reduce reperfusion injury. The details of the experimental protocol are described elsewhere.17
During the procedure, heart rate, ECG, blood pressure, temperature and oxygen saturation were continuously monitored. Ampicillin (2 mg) was administrated intravenously before and after the procedure, and acetylsalicylic acid (100 mg/day) was given orally after the procedure and continued until euthanasia. To prevent ventricular fibrillation, 150 mg of amiodarone was administered intravenously prior to the induction of myocardial infarction. If ventricular fibrillation was encountered, non-synchronised direct current defibrillation (150 J) was performed. The paddles were pressed against the anterior chest wall above the sternum on the right side and below the sternum on the left side. At the end of the experiment, the pigs were awakened and returned to their stables, where they stayed for 7–10 days (average 8.5 days) prior to CMR imaging, harvesting and evaluation by gross pathology.
CMR imaging
All the pigs underwent CMR imaging before euthanasia. The sedation protocol was as described above, except that continuous propofol infusion (10 mg/mL, 12 mL/h) was used instead of sevoflurane. CMR was performed on a 1.5 T MR system (Intera, Philips Medical Systems, Best, the Netherlands) with a five-element cardiac synergy coil. All images of the heart were obtained with the pigs in the supine position. First, a survey scan was performed to localise the heart and diaphragm, and then left ventricular (LV) function was assessed using a retrospective, ECG-triggered Balanced-Steady-State-Free-Precession (B-SSFP) breath-hold cine sequence in the cardiac short-axis, vertical long axis and horizontal long axis planes. In the cardiac short-axis, the LV volume was completely encompassed by contiguous 8 mm slices with a spatial resolution of 1.22×1.22 mm and a field of view (FOV) of 288×288 mm. The following imaging parameters were used: repetition time (TR) 3.0 ms; echo time (TE) 1.5 ms; flip angle 60°; 30 heart phases. To ensure a strong T2-weighting, a T2-STIR fast spin echo sequence with a long TE was obtained in the previously mentioned short-axis orientation to assess AAR. The sequence was navigator-gated and cardiac-triggered. The following imaging parameters were used: TR 2400 ms; TE 100 ms; echo train length 20; fat inversion time 180 ms; flip angle 90°; spatial resolution 0.54 mm×0.54 mm in-plane; number of averages 2; slice thickness 8 mm; FOV 320×320 mm; 14 slices.
We obtained T2-mapping in six animals by a multislice spin echo sequence. The sequence was navigator-gated and cardiac-triggered. The following imaging parameters were used: TR 1100 ms; TE (8, 16, 24, 32, 40, 48, 56, 64) ms; flip angle 90°; spatial resolution 1.33×1.33 mm in-plane; slice thickness 8 mm; FOV 320×320 mm; 14 slices.
T1-weighted inversion recovery (T1W-IR) gradient echo (GRE) imaging was obtained in the same short-axis slices for the purpose of identifying IMH, as previously described.18 The sequence was navigator-gated, free-breathing and cardiac-triggered. The following imaging parameters were used: TR 3.5 ms; TE 1.13 ms; flip angle 30°; spatial resolution 1×1 mm in-plane; slice thickness 8 mm (over contiguous slices); FOV 320×320 mm and 14 slices. Before the acquisition of the T1W IR sequence, a TI scout (Look Locker sequence) was performed for the purpose of obtaining the most appropriated TI to null the signal intensity from blood. Typically, the TI was found to be optimal at approximately 500 ms.
Subsequently, gadolinium enhanced first-pass myocardial perfusion and late gadolinium enhancement (LGE) was performed for the purpose of identifying areas of MVO and myocardial infarction, respectively. An intravenous bolus dose of 0.2 mmol/kg Gd-DTPA (Gadobutrol, Gadovist, Bayer Schering Pharma, Berlin, Germany) was administered manually. First-pass perfusion imaging was performed using a fast gradient echo sequence with the following parameters: TR 2.5 ms; TE 1.3 ms; flip angle 18°; spatial resolution 2.8×3.0 mm in-plane; slice thickness 10 mm; FOV 256×256 mm; three slices acquired in the LV short-axis using a 5 mm inter-slice gap.
LGE was acquired 15 min after gadolinium injection, using a three-dimensional phase sensitive inversion recovery-prepared T1-weighted gradient echo sequence with the following parameters: TR 5.78 ms; TE 2.78 ms; echo train length 20; inversion time ∼320 ms; flip angle 25°; spatial resolution 1.5×1.5 mm; slice thickness 8 mm; FOV 350×350 mm; 14 slices acquired in the LV short-axis with no interslice gap. Following CMR, the pigs were kept under anaesthesia and moved to the operating room for organ harvesting.
Harvesting and pathology
Midline sternotomy was performed and a snare placed around the LAD distal to the second diagonal branch at the same site as the previous balloon occlusion. Twenty-five millilitres of 10% Evans blue dye was then injected into the left auricle and allowed to circulate for approximately 10 s, to delineate the AAR.18–20 The animal was subsequently euthanised and the heart excised. The heart was then cut into consecutive 8 mm-thick slices in short-axis planes. Each slice was photographed with a digital camera (Nikon, Tokyo, Japan) for the purpose of registering myocardial infarction, IMH and AAR.