Fourteen swine were used in the experiment. Two swine were used to test and establish protocols for vagus nerve stimulation and electrophysiological studies. Three swine were used to test laparoscopic RDN. Finally, nine swine were randomly allocated to the sham or RDN group. All experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University Hospital (SNUH-IACUC), and the animals were maintained in a facility accredited by AAALAC International (no. 001169) in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition, National Research Council (2010). The study was reported in accordance with the ARRIVE guidelines 2.0.
The swine were fasted for 12 h before the experiment. General anesthesia was induced by intramuscular injection of zoletil (5 mg/kg) and xylazine (2 mg/kg). After induction, tracheal intubation was performed using an endotracheal tube and anesthesia was maintained using 2.0–2.5% of isoflurane inhalation during the experiments. Mechanical ventilation was adjusted to maintain oxygen saturation and end-tidal CO2 at ≥ 98% and 38–40 mmHg, respectively. Electrodes were attached to the limbs to monitor lead II electrocardiogram (ECG) during the experiment. After anesthesia in the supine position, the neck and abdomen were disinfected with povidone iodine and draped aseptically.
The animal preparation process is illustrated in Fig. 4A. A vertical skin incision was made on both sides of the trachea between the sternohyoid muscle and the medial aspect of the sternocleidomastoid muscle. The subcutaneous tissues were dissected, and the internal jugular vein and the carotid artery were exposed. The right and left jugular veins were punctured using a 7-Fr sheath. A decapolar catheter was introduced into the right atrium via the jugular vein, under fluoroscopic guidance. A decapolar catheter from the right jugular vein was used to record intracardiac electrocardiograms from the lateral side of the right atrium. The other catheter from the left jugular vein was used to perform burst pacing at the right atrium. The pacing output, with a pacing interval of 400 ms, was decreased from 10 to 2 V, and the left decapolar catheter was positioned to maintain stable pacing. Pacing stability was confirmed using both surface ECG and intracardiac electrogram. The right carotid artery was exposed and slightly retracted in order to identify the right vagus nerve.
Measurement of atrial effective refractory period and AF induction
The experimental flow is presented in Fig. 4B. The capture threshold of the right atrium was tested by decreasing the pacing output from 10 to 1 V. For the remaining experiment, the pacing output was set to be twofold of the capture threshold. After a train of eight S1s at 500 ms, S2 was given from 400 ms and decreased by 10 ms until the AERP was reached. The AERP was measured at the lateral side of the right atrium. The protocol was repeated three times to confirm the AERP. Cardiac pacing and intracardiac electrogram recordings were performed using the Prucka Cardiolab EP System (GE Medical Systems, Fairfield, CT, USA).
AF induction was performed by burst pacing the right atrium at a pacing interval of 100 ms and pulse width of 1.0 ms over 60 s. AF induction was repeated over 10 times with a resting interval of 60 s. Induced AF episodes were confirmed by recording surface ECG and intracardiac electrogram, and the duration of each AF episode was measured. AF episodes were defined as cases lasting ≥ 5 s because supraventricular tachycardia episodes less than 5 s after burst pacing could be non-specific findings. The inducibility of AF was defined as the success rate of the AF induction tests and measured as a percentage.
For both sham and RDN groups, each swine underwent AERP and AF induction tests before and after the procedure (i.e., a sham or laparoscopic RDN procedure for the sham or RDN group, respectively). In addition, the AERP and AF induction tests were repeated with and without VNS.
Vagal nerve stimulation
Considering that AF may not be readily inducible by right atrial burst pacing for healthy swine, we used the VNS to promote AF inducibility in healthy swine. In this study, both AERP and durations of AF episodes were measured with and without VNS for each swine. The protocol of VNS was as follows. The right cervical vagus nerve was located beneath the carotid artery. A pacing electrode (Model 6491, Unipolar Pediatric Temporary Pacing Lead; Medtronic, Minneapolis, MN, USA) was placed and fixed to the vagus nerve. VNS was performed using a Grass S88 stimulator (A-M Systems). The vagus nerve was stimulated with a pulse width of 0.2 ms and a frequency of 20 Hz. The pacing output for VNS was adjusted to 2–10 V to achieve a 10% decrease in heart rate without significant hemodynamic instability. An example of VNS is illustrated in Fig. 4C. The VNS duration was limited to 30 s to avoid saturation effects.
Laparoscopic RDN and sham procedures
Laparoscopic RDN was performed as follows. After draping, a Veress needle was inserted 5 cm to the left or right and 2 cm caudal to the umbilicus, and CO2 was insufflated into the abdominal cavity. Trocars were placed as follows: (1) a 12-mm port 2 cm caudal to the umbilicus and the lateral margin of the rectus muscles (for the camera); (2) a 12-mm port 7 cm cephalad to the camera port; and (3) 5-mm ports 7–8 cm lateral to the camera port. The laparoscopic camera visualized the intra-abdominal cavity. The renal artery was exposed after perirenal fascia incision and dissection of the surrounding soft tissue. Circumferential RDNs were performed using a HyperQure Renal Denervation Laparoscopic Instrument (DeepQure, Inc., Seoul, Republic of Korea) at both the proximal and distal sites of the renal artery with a minimum distance of 3 mm. After wrapping the renal artery using an electrode at the tip of the instrument, bipolar radiofrequency energy was delivered at a constant temperature of 65°C for 70 s using a single shot (Fig. 5). When the distal renal artery bifurcated, each branch was ablated separately. This procedure was repeated for the contralateral kidney. After RDN, the perirenal fascia was sutured, and intraperitoneal CO2 was exsufflated. The abdominal walls were closed, and an occlusive dressing was applied. For the sham procedure, all procedures were performed as in the RDN group, except for the delivery of radiofrequency energy.
After the experiment, the swine were fully sedated with general anesthesia using 4–5% of isoflurane inhalation and euthanized by an intravenous potassium chloride (2 mmol/kg) injection through the internal jugular vein. Subsequently, both left and right renal arteries were collected. The renal artery was fixed with 10% formalin and stained with hematoxylin and eosin. After paraffin embedding, 4 μm-thickness horizontal sections of the renal artery were obtained at both proximal and distal ablated sites. Immunohistochemistry was performed using a mouse monoclonal antibody against TH (Accurate Chemical & Scientific Corporation, Carle Place, NY, USA) to visualize the renal sympathetic nerves. Nerve fibers were counted along the adventitia of the renal artery and compared between the sham and RDN groups.
According to variable types, data are shown as n (%), mean ± standard deviation, or median (interquartile range). For each swine, the Wilcoxon signed-rank test was performed to compare the pair of AERPs (or AF durations) obtained before and after the sham or laparoscopic RDN procedure. The association between AF inducibility and procedures (sham or laparoscopic RDN) was evaluated by calculating odds ratios (ORs) with 95% confidence intervals (CIs). The Mann–Whitney U-test was performed to compare nerve fiber counts between the sham and RDN groups. Two-sided p-values less than 0.05 assumed the rejection of the null hypothesis. All statistical analyses were performed using the IBM SPSS Statistics for Windows (version 22.0; IBM Corp., Armonk, NY, USA).