¹³N-Ammonia PET/CT Detection of Myocardial Perfusion Abnormalities in Beagle Dogs After Local Heart Irradiation

Animals

In this study, all animal care and experimental procedures complied with the guidebook of the Institutional Animal Care and Use Committee. Thirty-six male 12-mo-old Beagle dogs (mean weight ± SD, 12 ± 0.67 kg) were purchased from the Anhui Fuyang Weiguang Institute of Experimental Animals. After approximately 1 wk of quarantine, the dogs were randomly assigned to a control group or an irradiation group, which received local heart irradiation. All animals were housed individually at the Experimental Animal Center of the China Institute for Radiation Protection in stainless steel cages in an air-conditioned facility (18°C–25°C; relative humidity, 40%–60%; ≥12 air changes/h) and fed standard certified commercial dog chow twice daily, with water available ad libitum. Before the study, the dogs were acclimated to these conditions for 2 wk. All animals underwent ¹³N-ammonia MPI, and those in the irradiation group underwent coronary angiography before local irradiation and at 3, 6, and 12 mo afterward. Six animals randomly selected from each group were sacrificed at each time point, and their hearts were taken for pathologic testing. The study was approved by our Institutional Animal Care and Use Committee and was performed in accordance with the Guidelines for Animal Experiments of Shanxi Medical University.

Local Heart Irradiation

The animals were anesthetized by intravenous injection with 3% pentobarbital sodium at 30 mg/kg and fixed supine by a vacuum-form body immobilizer. All underwent a control enhanced CT simulation scan (Discovery VCT64 PET/CT scanner; GE Healthcare) for radiotherapy planning. After scanning, the images were transmitted to an Eclipse TPS system (Varian). The anterior wall of the left ventricle was outlined as the radiation target, accounting for one quarter to one third of the left ventricular volume, and a conformal intensity-modulated radiation therapy plan was made. Cone-beam CT was performed with a flat-panel detector to verify the accuracy of the target location before exposure. After location verification, the dogs in the irradiation group received a single 20-Gy dose of irradiation with 6-MV x-rays, and the dogs in the control group underwent the same procedure but without the irradiation.

¹³N-Ammonia MPI

After 12 h of fasting, the dogs were anesthetized, fixed supine, transferred to the bed of the PET/CT scanner (Discovery VCT; GE Healthcare), and connected to the electrocardiogram monitor. First, a CT scout scan was acquired to locate the position of the heart in the scanner field of view, using 120 kVp, 30–50 mA, and a slice thickness of 3.75 mm. Next, 148–185 MBq (4–5 mCi) of ¹³N-ammonia were administered by an intravenous bolus, and the PET scan was performed 3 min afterward. PET data were acquired in 3-dimensional mode for 10 min. Images were reconstructed using the iterative method, with a matrix of 128 × 128 pixels, 2 iterations, and 6 subsets, and corrected for attenuation using attenuation data from the CT component of the examination.

Image Analysis

Images were processed using Myovation software in a Xeleris 4.3 workstation (GE Healthcare). PET/CT images were independently reviewed by 2 experienced nuclear medicine physicians in a double-masked fashion; 2 observers analyzed all images from every subject slice by slice. In the semiquantitative analysis, a same-sized region of interest in both the anterior wall (irradiated area) and the posterior wall (nonirradiated area) was delineated at 3 different levels of the ventricle on the PET/CT images, and the average radioactivity count was used to calculate the irradiation-to-nonirradiation activity ratio. Gated studies were quantitatively analyzed using quantitative gated SPECT software; the parameters regional wall motion, end-diastolic volume, end-systolic volume, and left ventricular ejection fraction were acquired automatically; and the polar bull’s-eye plot was divided into 20 segments.

Coronary Angiography

One week after undergoing ¹³N-ammonia myocardial perfusion PET/CT, the irradiation group underwent coronary angiography. In the catheterization laboratory, the anesthetized dogs were placed supine after bilateral preparation of the inguinal skin. Using a Judkin catheter, a cardiologist conducted the coronary angiography procedure. Two experienced interventional cardiologists assessed the results using the Thrombolysis in Myocardial Infarction score (16), which categorizes coronary blood flow as grade 0 (no perfusion), 1 (penetration without perfusion), 2 (partial perfusion), or 3 (complete perfusion).

Histologic Assessment

The dogs were euthanized under anesthesia by acute blood loss; the ribs were cut along the left sternal border, the thoracic cavity was exposed, and the heart was quickly removed. The atrial tissue was then removed from the heart, and the ventricular tissue was prepared for histologic evaluation by immersion and fixation in 10% formalin. For each heart, myocardial tissue was taken from the anterior wall (irradiated area) and the posterior wall (nonirradiated area) at the middle level of the left ventricle. All tissue samples were fixed, trimmed, washed, and progressively dehydrated in ethanol. The alcohol was then replaced with xylene, and the samples were embedded in a paraffin block. From each block, 5-μm-thick sections were prepared and stained with hematoxylin–eosin, CD31 immunostain, and Masson trichrome stain. Morphologic changes were observed by light microscopy (JEM-2100; JEOL Co.). Myocardial degeneration and vascular injury were measured semiquantitatively to evaluate the extent of cardiac damage (17,18).

To evaluate myocardial degeneration, 6 nonvascular microscopic fields (×200) were randomly selected from the hematoxylin- and eosin-stained slices and graded as 0 (normal: no degeneration of myocardial cells), 1 (mild: <20% degeneration of myocardial cells), 2 (moderate: ∼20%–50% degeneration of myocardial cells), or 3 (severe: >50% degeneration of myocardial cells).

To evaluate myocardial vascular injury, 6 vascular microscopic fields (×200) were randomly selected from the hematoxylin- and eosin-stained slices and graded as 0 (no fibrosis: adventitia thickness ≈ half the media), 1 (mild fibrosis: adventitia thickness ≈ the media), 2 (moderate fibrosis: adventitia thickness ≈ 2 × the media), or 3 (severe fibrosis: adventitia thickness ≥ 3 × the media).

To evaluate myocardial microvessel density, 6 independent microscopic fields (×200) were randomly selected from the CD31-immunostained slices. The percentage of blood vessels involved per visual field was graded as 0 (absent), 1 (<10%), 2 (10%–50%), or 3 (>50%) (19).

To evaluate cardiac interstitial fibrosis, 6 independent microscopic fields (×200) were randomly selected from the Masson-stained slices and graded as 0 (no apparent collagen fiber proliferation), 1 (focal and minimal fibrosis), 2 (mild patchy fibrosis), 3 (moderate diffuse fibrosis), or 4 (most prominent fibrosis) (20).

Statistical Analysis

All quantitative data were expressed as mean ± SD. Differences between groups were tested using a t test and ANOVA and using a rank sum test for ordinal data. Statistical significance was accepted at a P level of less than 0.05. The SPSS 18.0 software package (IBM) was used for statistical analyses.