Animal models
The study protocol was approved by the Institutional Animal Care and Use Committee and complied with the Guidelines for the Care and Use of Laboratory Animals (National Institutes of Health). Male swine (n = 14, weight 24 ± 3.1 kg) were anesthetised with an intramuscularly administered mixture of telazol (4.4 mg/kg) and xylazine (4.4 mg/kg). Following intubation, animals were ventilated mechanically (Model 2000, Hallowell EMC, Pittsfield MA, USA) and anaesthesia was maintained by continuous administration of isoflurane (2.0–2.5% V/V). Normal body temperature was supported using a heating pad. Heart rate and blood oxygen saturation were monitored, and electrocardiogram was recorded.
The right femoral artery was surgically prepared and cannulated using a 6-F arterial sheath (Pinnacle, Terumo Medical Co, Elkton, MD, USA). Heparin (100 IU/kg) was administered intravenously and the activated clotting time was monitored and adjusted as needed with additional heparin to maintain the activated clotting time above 300 s. A 6-F coronary guide catheter (RunWay Kimny Mini, Boston Scientific, Natick MA, USA) was introduced to cannulate the ostium of the left main coronary artery, and initial coronary angiography (Philips BV Pulsera, Best, The Netherlands) was performed.
Reperfused myocardial infarct model
In animals assigned to the reperfused group (n = 7), a 2.0–2.5 mm angioplasty balloon (Maverick, Boston Scientific, Natick MA, USA) was introduced over a coronary guide wire into the left circumflex (LCX) coronary artery. After determining the proper balloon position for the occlusion, the balloon catheter was inflated and left in position for 90 min to induce MI. After 90 min of ischaemia the balloon was deflated and removed, and coronary angiography was repeated to confirm recanalisation. The femoral artery was decannulated and surgically ligated, and the wound was closed.
Non-reperfused myocardial infarct model
In animals assigned to the non-reperfused group (n = 7), the microsphere embolisation technique was used [15]. Briefly, a 2.9-F straight tip microcatheter (Merit Maestro, Merit Medical, South Jordan UT, USA) was introduced over a coronary guide wire into the LCX coronary artery. After determining the proper microcatheter position for the occlusion, using the radiopaque tip of the microcatheter, the coronary guide wire was pulled out and a mixture of 900-μm microspheres (Embozene, CeloNova BioSciences, Inc., Newnan GA, USA) was flushed into the coronary artery under fluoroscopic control. When the lumen of the coronary artery distal to the tip of the microcatheter was completely filled with microspheres, the injection was stopped, the occlusion was confirmed by repeated angiography, and the microcatheter was removed. At the end of a 90-min monitoring period, the occlusion was confirmed by repeated angiography, and the femoral artery was decannulated and surgically ligated, and the wound was closed.
Magnetic resonance imaging
MRI studies were carried out four days after the induction of MI, using a 1.5 T GE Signa-Horizon CV/i scanner (GE Healthcare, Milwaukee, WI) equipped with a cardiac phased-array coil. Pigs were anaesthetised and ventilated mechanically as described previously. Imaging was performed during “breath-hold” at end-inspiration using the following parameters: field of view = 300 mm, image-matrix = 2562, and slice thickness = 10 mm. The flowchart of the MRI protocol is shown in Fig. 1.
T2 mapping
Following the pilot scans, short-axis oriented T2 mapping was carried out to study the T2 characteristics, especially the presence of myocardial haemorrhage in the MI. T2 maps were generated employing a double inversion recovery (IR), fast spin-echo pulse sequence using the following parameters: flip angle (α) = 90°, echo train length = 24, and echo time (TE) = 12–105 ms (12, 20, 30, 45, 60, 75, 90, and 105 ms). Images were collected at end-diastole in every second cardiac cycle.
Early and late gadolinium enhancement imaging
Following T2 mapping, a bolus of 0.2 mmol/kg gadopentetate dimeglumine (Magnevist, Bayer HealthCare Pharmaceuticals Inc, Wayne NJ, USA) was administered as the contrast agent (CA) for early gadolinium enhancement (EGE) and late gadolinium enhancement (LGE) imaging. Segmented, 180°-prepared, IR fast-gradient-echo, short-axis (6 to 8 short-axis slices to cover the entire LV from apex to base) and long-axis (two-chamber, four-chamber, and LV outflow tract) oriented images were generated 2 min after CA administration (EGE imaging), and the acquisition was repeated at 10 min and thereafter every 5 min ending at 45 min (totaling eight LGE acquisitions in each animal). Imaging parameters were: α = 25°, TE = 3.2 ms, views per segment = 16, and repetition time (TR) = 5.5 ms. The inversion time (TI) for EGE images was 500 ms. For the LGE images, the applicable TI was set and continuously adjusted to an optimum value for nulling the signal of the healthy myocardium.
At the end of the planned in vivo MRI session, the animals were killed using a mixture of pentobarbital sodium, propylene glycol and ethyl alcohol (0.2 ml/kg) (Fatal-Plus®, Vortech Pharmaceuticals, Dearborn MI, USA). Euthanasia was ascertained by electrocardiogram and auscultation above the thorax. The hearts were excised after euthanasia, rinsed with saline, and prepared for further studies.
Magnetic resonance image analysis
The Research Mass cardiovascular MR evaluation software (Leiden University Medical Center, Leiden, The Netherlands) was used for image analysis. The endocardial and epicardial contours of the LV were traced manually in every series of the short-axis images to delineate the myocardial area. Myocardial pixels were counted, and based on the pixel dimensions, the myocardial volume of each slice was determined, from which the total LV myocardial mass (LVM, g) was also calculated using the specific gravity of 1.05 g/ml of myocardial tissue. To avoid observer bias, instead of manual contouring, a thresholding technique was used to delineate the MI and NF.
The mean signal intensity (SI) of the normal myocardium was measured using a region of interest containing at least 100 pixels. The mean SI of the remote myocardium plus five times the standard deviation (SD) of this mean was used as a threshold to delineate the pixels within the infarct (“infarcted pixels”) (Fig. 2) [16, 17]. Pixels with SI over this threshold value were considered “MI pixels”.
As the NF has been defined as a hypoperfused, unenhanced core within the highlighted MI, the SI of the NF is equal to or less than the SI of the remote myocardium. To overcome the problem of the NF not showing enhancement in EGE (the time period when its size is the largest), the size of the NF was measured indirectly. After MI thresholding, the unenhanced area of the NF was included in the MI area using the “MVO” contour function of the Mass application (Fig. 2c). Combination of the highlighted MI area and the unenhanced NF area showed the true, total MI area. The difference between the total and the highlighted areas provided the NF area (Fig. 2d). The volume of the MI and NF were each expressed as a percentage of the LVM: the MI fraction (MIF) was calculated as the MI volume/LVM % and the NF fraction (NFF) was calculated as the NF volume/LVM %. NF was also expressed as a percentage of the volume of the MI. Change in the size of the NF over time during the post-contrast period was analysed using the aforementioned technique. The actual size of the NF area was determined at each post-contrast time point.
T2 calculation
T2 was calculated from the TE dependence of the SI by means of a two-parameter, least squares, curve-fitting routine, using the following formula:
$$ \mathrm{S}\mathrm{I} = {\mathrm{SI}}_0 \times {\mathrm{e}}^{\left(-\mathrm{TE} \times \frac{1}{\mathrm{T}2}\right)} $$
where SI0 is the signal intensity at the theoretical TE = 0 time point and it also represents the maximum SI.
The segmentation of the MI was carried out based on the areas determined in the EGE images. The T2 of the remote, infarcted (showing LGE), and the NF myocardium were obtained.
Histopathologic assessment
Triphenyltetrazolium-chloride staining
Triphenyltetrazolium-chloride (TTC) staining was used as a post-mortem reference standard to confirm the existence and the size of the MI. The hearts were bread-sliced using a commercial meat slicer, and subsequently the slices were incubated for 20 min with a buffered (pH 7.4) 1.5% TTC solution at 37 °C, similar to the method as described by Fishbein et al. [18]. After staining, the slices were immersed in 10% formalin for 20 min to increase the contrast between the healthy and the infarcted myocardium. Finally, both surfaces of each slice were digitally scanned with an image scanner, and both sides of each slice were analysed using ImageJ (Wayne Rasband, NIH, USA). The LV myocardium and the MI area were manually contoured and quantified. LVM and MIF were determined in each heart.
Microscopic histologic assessment
After TTC, whole-heart slices, including the defined epicardial and endocardial borders, were submitted for histopathologic assessment. The samples were fixed in 10% formalin, embedded in paraffin, and sectioned at 5-μm thickness. Haematoxylin-eosin staining (for general evaluation), Prussian blue staining (for haemorrhage detection), and Von Kossa staining (for calcium detection) were performed. Histologic samples were evaluated using a histomorphometry system (BioQuant Image Analysis, Nashville TN, USA) equipped with an Olympus BX-51 video microscope (Olympus America, Center Valley PA, USA).
Statistical analysis
Statistical analysis of the data obtained was carried out using MedCalc 13.2.2 (MedCalc Software, Ostend, Belgium). The Kolmogorov-Smirnov test was used to confirm that the data had a Gaussian distribution. T2 measurements and infarct-related parameters (MI and NF volume, MIF, and NFF) were compared in the reperfused and non-reperfused models using the independent samples t test, and MI size and LVM measured by MRI and TTC were compared using the paired samples t test. The size of the NF area was measured at each post-contrast time point and repeated measures analysis of variance was used to determine the time when the size of the NF was significantly different from the reference EGE measurement. Results were reported as mean ± standard deviation (SD), unless otherwise noted. p values lower than 0.050 were considered significant.
