A Novel Experimental Rat Model for the In Vivo Assessment of Myocardial Ischemia Based on Single Photon Emission Computed Tomography

Materials and Methods

Animals and study protocol. The study protocol was approved and authorized by the Greek General Directorate of Veterinary Services (License No: 1870/23-04-2018) and was conducted in accordance with the Greek legislation regarding ethical and experimental procedures. Thirty adult female Wistar rats underwent general anesthesia and mechanical ventilation. Tracheal intubation was achieved by a 17G tube and maintained with sevoflurane. A special temperature controlling mattress and a rat specific ECG monitoring combined with temperature and oxygen measurement were applied throughout the procedure. Perioperative care was taken with subcutaneous adhesion of dolorex (0.06 ml in h 0, 8 and 24; Ktiniatriki, Athens, Greece), intramuscular adhesion of terramycin (0.03 ml; Pfizer Hellas S.A., Athens, Greece), subcutaneous hydration with 3 ml of normal saline 0.9% and eye protection with gel tobrex. Subsequently, under sterile conditions, a left mini thoracotomy between the 3rd and 4th intercostal space was undertaken and the pericardium was opened. A Prolene 6-0 stitch (Ethicon, Raritan, NJ, USA) was used for handling the heart apex and a silk 4-0 for ligation of LAD in 20 animals, while 10 were used as sum n0-ligation group (Figure 1). Myocardial ischemic time was 45 min and ECG continuous recording for confirmation of ischemia was utilized (Figure 1). Afterwards, animals were extubated, and all had an uneventful recovery and were able to undergo SPECT-CT imaging at 7 and at 14 days after surgery. On the 15th postoperative day, the rats were sacrificed, and tissue and organ harvesting was performed. Euthanasia introduced by injecting KCl in the inferior vena cava (IVC).

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Figure 1.

Illustrations depicting the surgical technique, the electrocardiogram (ECG) changes, and the macroscopic anatomo-morphological changes in ischemic myocardium. Under sterile conditions, ischemia is caused by ligation of the left anterior descending (LAD) coronary artery at the first 1/3 of its course after the bifurcation of LAD leading to a stable, reliable, and reproducible myocardial infarction which is confirmed via ECG monitoring operatively (ST elevation on ECG, i.e., the trace in the ST segment is abnormally high above the baseline), macroscopic features of ischemia on the day of euthanasia and tissue harvesting and loss of cardiac muscle fibers in the macroscopic appearance after formaldehyde fixation.

SPECT/CT evaluation. All animals underwent a preoperative SPECT-CT imaging prior to carrying out myocardial ischemia, in order to analyze the functional myocardium of the healthy heart. A novel technique for SPECT evaluation was developed especially for small rodents and the imaging chamber was formulated to focus on the thoracic cavity of the animal (Figure 1). 200 μl of 1.5 mCi-3 mCi of 99 mTc-Sestamibi were injected in the rat via the inferior vena cava and subsequently, imaging was taking place at 20 min post injection. The SPECT-CT imaging was made with x-Cube and γ-Cube (Molecubes, Gent, Belgium). SPECT acquisition was done with local scan spiral acquisition for 30 min and maximum likelihood expectation maximization (MLEM) reconstruction with 250 μm voxel size and 500 iterations. SPECT post-processing had to do with normalization of images on myocardium uptake [myocardium having the same maximum number of counts, removal of all other organs and smoothing median filter (1.8 mm full width at half maximum (FWHM), isotropic)]. Furthermore, CT acquisition and post processing for the whole body was performed using a general purpose protocol (50kVp), utilizing an ISRA reconstruction with 200 μm voxel size. In all images, a scale was used for analyzing the absorption of the radiation particles from the myocardial cells, in order to evaluate the cellular activity in voxels. Axial slices (short, long and horizontal) and 3D rendering with color indication scale was used to show the myocardial activity 7 and 14 days post-surgery, in order to analyze the changes in myocardial absorption of the radioactive particles after infarction. The post processing and the quantification of all images was performed with Vivo Quant 3.0 (Invicro, Boston, MA, USA). Results were measured in voxels from each cardiac chamber and the left to right ventricular ratio difference was used to identify the changes of myocardial activity after LAD ligation and the presence of ischemia in the left ventricle (Figure 2).

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Figure 2.

Myocardium histology in no left anterior descending coronary artery (LAD) ligation and LAD ligation groups. Hematoxylin/eosin 2× magnification without ligation of LAD. Normal cardiac muscle fibers (top image), Hematoxylin/eosin 2× magnification with ligation of LAD. Ischemic changes in myocardium with loss of cardiac muscle fibers (lower image).

Histology. Blood sampling was taken from the IVC and, from the same route of administration, excessive dose of KCl was administered for euthanasia. Hearts were rapidly excised and placed in 10% formaldehyde. The samples were afterwards transferred to the histology laboratory, were fixated at room temperature using 10% formaldehyde in a ratio of 2:1 formaldehyde:tissue (v/v) for 48 h and were then dehydrated and embedded in paraffin. In order to keep the long axis of the heart intact, we created slices in the perpendicular plane. Hematoxylin and eosin (H&E) staining was used to identify the fibrotic areas of the myocardium (Figure 2).

Statistical analysis. Data were expressed as mean±standard deviation (SD) for quantitative variables and as frequencies and percentages for qualitative variables. The Kolmogorov-Smirnov test was used for normality analysis of the quantitative variables. Comparison of quantitative and qualitative variables for pairwise comparisons between groups was performed using one way ANOVA with Bonferroni correction and the Chi-square test with Bonferroni correction, respectively. All tests were two-sided. A p-value <0.05 was defined as the level of statistically significant difference. Statistical analysis was performed using the statistical package SPSS version 21.00 (IBM Corporation, Armonk, NY, USA).