Animals and study design
Sixteen 16-week-old female Bama Mini‒pigs with a mean initial body weight of 18.6 ± 5.2 kg were used in this study. Animals were raised in independent polypropylene cages under controlled conditions for experiments. All animal experiments complied with the ARRRIVE guidelines and have been approved by our hospital’s Ethics Committee on Biomedical Research.
This study was a self-controlled study in diabetic pigs. Before modelling, all pigs underwent CMR scanning to obtain baseline data. Then CMR follow-up scans were performed on pigs with diabetes at 2, 6, 10 and 16 months after modelling. Cardiac specimens were obtained from 3 pigs at 6, 10, and 16 months after modelling to observe the cardiac microstructure at different stages of diabetes mellitus.
Diabetes induction by intravenous injection of STZ
After fasting for 16 h, pigs were anaesthetized by subcutaneous injection of Zoletil 50 (10 mg/kg). An appropriate amount of STZ solution (150 mg/kg) was injected into the ear vein of the pigs at a uniform speed for 5 min. STZ was dissolved in 0.1 mol/l citric acid solution at pH 4.4–4.5. The STZ solution was configured in a dark room and the syringe was wrapped in tin foil during the injection process to ensure drug properties. After injection of STZ, the blood glucose of the auricular vein was monitored by a blood glucose metre at 1 h (h), 2 h, 6 h, 8 h, 12 h, 16 h, 20 h, 24 h, 36 and 48 h. When the blood glucose was lower than 2 mmol/L, 5% glucose 10–20 ml was injected intravenously. All of the pigs began to eat 6 h after STZ injection.
Forty-eight hours after STZ injection, fasting blood glucose (FBG) was tested every morning. To ensure the survival of the animals, 12 U insulin was injected subcutaneously when the FBG was higher than 20 mmol/l, and 20 U was injected when it was higher than 25 mmol/l. One month after the injection of STZ, if a pig’s blood glucose concentration was continuously higher than 7 mmol/l, then the diabetes model was considered to be successful. One week after the first injection of STZ, if the blood glucose of any pig was less than 7 mmol/l, then 100 mg/kg STZ was injected again in the same way. If the FBG of any pig was still not up to 7 mmol/l after two repeated injections of STZ, then it was concluded that the diabetes model failed. Pigs that failed to model diabetes were not included in the subsequent CMR examinations.
Preparation for the MRI scan
Before CMR examination, pigs were anaesthetized by intramuscular injection of Zoletil 50 (10–15 mg/kg) with atropine (0.3–0.5 mg). Blood samples were collected from the superior vena cava with an aseptic syringe, and FBG, glycosylated haemoglobin (HbAlc), and liver and kidney function were measured.
Endotracheal intubation was performed using a 4.5-6.0 mm endotracheal tube connected to a special animal ventilator for mechanical ventilation. Anaesthesia was sustained by isoflurane inhalation (1.0–2.0%), with a respiratory rate of 10–30 beats/min and an inhalation/breathing ratio of 1:2.
CMR protocol
CMR imaging of pigs was performed using a 3.0-T whole-body MR system equipped with a commercial 18-channel receiver coil (Magnetom Skyra, Siemens Medical Solutions, Erlangen, Germany). ECG and respiratory gating were connected during image acquisition. Data were acquired during end-inspiratory breath holding. After scout images, a steady-state free precession sequence (echo time, 1.36 ms; repetition time, 3.15 ms; flip angle, 35°; slice thickness, 6.5 mm; matrix, 154 × 192 pixels; and field of view, 400 × 320 mm2) with retrospective ECG-gating was used to acquire dynamic cine imaging of the LV for functional analysis. The protocol comprised cine imaging in short axis, 2-, 3-, and 4- chamber views. The LV was entirely imaged from the base to the apex in 9–12 short-axis cine images with 6–8 mm thick contiguous slices.
Image analysis
An experienced radiologist analysed the CMR data on an offline workstation. All image postprocessing operations were performed following the latest International Cardiac Magnetic Resonance Association guidelines [12]. The images were analysed using offline commercial software (cvi42, v.5.10.2; Circle cardiovascular imaging, Calgary, Canada). The end-systolic and end-diastolic endocardium and epicardium on the short axis were drawn to obtain routine cardiac function indices, including LV EDV, end-systolic volume (ESV), stroke volume (SV), ejection fraction (EF), and LV mass. The LV remodelling index was determined by dividing the LV mass by the LV EDV [13]. At the end of diastole and end systole, the maximum LV diameter was measured from the endocardium of the free wall to the interventricular septum on the four-chamber view. The LV wall thickness was measured at the end of diastole in the interventricular septum of four-chamber view. The time‒volume curve parameters, including the peak ejection rate (PER) and peak filling rate (PFR), were obtained by drawing the endocardial boundary of the LV on each short-axis image. DVR, the proportion of diastole required for recovery of a given percentage (i.e., 80%) of stroke volume, was calculated by importing volume data into Origin software (Origin 8.0, Microcal Software Inc., Northampton, MA, USA) [14]. In addition, the end-diastolic endocardium and epicardium of the short axis and two long-axis sections were drawn to analyse the LV strain parameters, including LV radial global peak strain (GRPS), circumferential global peak strain (GCPS), longitudinal global peak strain (GLPS) and the peak strain rates in those three directions during systole (PSSR) and diastole (PDSR). The interface of LV feature tracking postprocessing is shown in Additional file 1: Fig. S1.
Histological analysis
At 6, 10 and 16 months after successful modelling, three pigs were randomly selected and sacrificed by intravenous injection of 20 ml potassium chloride under deep anesthesia. After cardiac arrest, the pig’s heart was removed from the chest. Approximately 50 mg of LV apical tissue was placed in glutaraldehyde fixative for 24 h for electron microscopy. To fix the myocardium, the rest of the heart tissue was immersed in 10% formalin solution; then, it was dehydrated and embedded, and 5 mm slices were cut parallel from the apex to the bottom of the heart and made into sections for hematoxylin–eosin (HE) and Masson staining to observe the changes in myocardial histomorphology and tissue composition. The collagen volume fraction (CVF) was calculated by the ImageJ software (U.S. National Institutes of Health).
Intraobserver and interobserver reproducibility
Two investigators assessed the reproducibility of CMR parameters. To determine the internal variability of the observer, the original images of 15 CMR scans were randomly selected, and the parameters of LV global strain and time‒volume curve were reanalyzed by the same radiologist (LJ) after an interval of 1 month. To determine the variability between observers, another investigator (WFY) reanalyzed the results of the 15 scans. In the variability assessment, each observer was blinded to the pigs’ state and other observers’ findings.
Statistical analysis
Statistical analyses were performed with IBM SPSS (version 22.0, IBM SPSS Inc., Armonk, New York, USA). All continuous variables were checked for normality using the Kolmogorov‒Smirnov test. Continuous variables are expressed as the mean ± standard deviation. The baseline characteristics and CMR parameters of pigs before and after modelling were compared by one-way analysis of variance (one-way ANOVA) and the Kruskal‒Wallis test. One-way ANOVA was used when the data conformed to the homogeneity of variance and normal distribution assumptions and was followed by the Tukey test. Kruskal‒Wallis tests were used when the data exhibited skewed distributions. Pearson correlation was used to analyse the relationship between LVRI and LV myocardial strain. Inter- and intraobserver agreements were determined by evaluating intraclass correlation coefficients (ICCs). A two-tailed P value < 0.05 indicated statistical significance for all tests.
