All experiments were performed in accordance with protocols approved by the Committee for Animal Research of Ruijin Hospital affiliated with Shanghai Jiao Tong University and complied with the 2011 “Guide for the Care and Use of Laboratory Animals.” All animal experiments were performed at the Yinshe Experimental Animal Center, Shanghai. The microwave ablation equipment (supplement matertal) was provided by the Vison-China Medical Devices R&D Center (Nanjing, China).

Microwave ablation (Vison-China Medical Devices R&D Center, Nanjing, China) [6, 7] was performed on post-mortem swine hearts at differing power levels for various periods of time. Sixty-five post-mortem swine hearts were divided into 13 groups and myotomy was performed immediately following ablation. By measuring and comparing the scope of necrosis, we determined that ablation at 40 W power output persisting for 1 min resulted in a sufficiently large and stable necrosis scope.

Fourteen Chinese swine (6–9 months old) were used in this study and were randomly divided into two groups: the Microwave Ablation (MA) group (n = 7) and the sham group (n = 7). All swine underwent anesthetic induction with an intramuscular injection of xylazine hydrochloride (0.025 ml/kg), and inhalation anesthesia with orotracheal intubation and a heart monitor was used during surgery. Fentanyl (2 µg/kg) was administered to control pain and intravenous glucose and saline solutions were continuously infused throughout the procedure.

The swine were placed in the supine position with their limbs fixed. For this experiment, the chest was opened with a standard median sternotomy as the cardiac apex of the swine was behind the sternum, and the transesophageal echocardiography was substituted by the epicardial echocardiography. To determine the feasibility of epicardial echocardiography, a left anterolateral incision was made in the fifth intercostal space for the ultrasonic probe. A suture was placed in the posterior pericardium opposite the oblique sinus to facilitate exposure of the apex. The microwave antenna was inserted transapically into the interventricular septum under epicardial echocardiography guidance. The antenna was positioned in the middle of the basal interventricular septum, approximately 10 mm away from the aortic valve and membranous septum (Fig. 1). Ablation was achieved with 40 W power output persisting for 1 min (Position 1, P1). We completed a second ablation after drawing the antenna back by 15 mm (Position 2, P2). The left ventricular anterior wall and posterior septum could also be extended ablated using different puncture angles. In the sham group, the microwave antenna was inserted at the same position as the interventricular septum without output power (supplement material).

Diagram showing microwave ablation on living swine model. (A) Swine were laid in the supine position. The chest was opened and a left anterolateral incision in the fifth intercostal was made for the ultrasonic probe. (B) The microwave antennas were inserted via the apex into the interventricular septum under the guide of echocardiography. (C) Several antennae were used to produce an extensive ablation. The four white dotted circles showed four ablation regions in one short-axis view

Echocardiography was performed using a GE Vivid E9 instrument (GE Healthcare). The amplitude of the ventricular wall motion was observed using the M-mode. Two-dimensional echocardiography was used to measure wall thickness and left ventricular ejection fraction (LVEF). Baseline and follow-up data were collected using transthoracic echocardiography (TTE) and data were collected using epicardial echocardiography during the operation. The EKG was recorded for every swine prior to the experiment, during the procedure, and at 1 month, 6 months, 9 months and 12 months follow-up time points.

We performed CMR (GE Healthcare, Boston, MA, USA) and cardiac CT (SIEMENS, Munich, Germany) 3 and 12 months after the procedure. Late gadolinium-enhanced (LGE) MRI was used to verify formation and location of scar tissue following ablation and cardiac CT was used to detect anatomical changes with high resolution.

Twelve months after the procedure, the swine were euthanized with an overdose of sodium pentobarbitone, and their hearts were fixed in formalin (10%). For analysis, tissue was taken from three regions: the ablated area, peri-ablated area, and a normal area. Tissue was treated using alcohol dehydration, xylene treatment, and paraffin embedding. Five-micrometer-thick sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome. The sections were analyzed using a digital microscope.

Continuous data were compared using a Student’s t-test or analysis of variance and expressed as mean ± standard deviation. Categorical data were compared using the chi-square test and expressed as percentages; all P values were two-sided, and a P value of < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 20 (IBM SPSS Inc., Chicago, IL, USA). Dr Zhou has full access to all the data in this study and takes responsibility for its integrity and the data analysis.