Methods
All preclinical experiments were approved by the Institutional Animal Care and Use Committee at the Sutter Institute for Medical Research (Sacramento, CA). A total of 23 Yorkshire or Yucatan swine (52–92 kg), 1 canine (27 kg), and 1 ovine (73 kg) were included in this study. Given that these were initial experiments, randomization and sample size calculation were not performed.
CRC EP ablation catheter and system.
PFA was performed using one of the two bipolar, non-irrigated, spiral PFA catheters (ElePulse, CRC EP, Inc) and a custom programmable PFA generator (CRC EP, Inc). The catheters consist of an 8-French, 16-electrode, bidirectional, 25-mm or 30-mm design with radiopaque tips, intended for single-shot ablation, with the larger of the two catheters (30-mm) containing 4 distal mapping electrodes. Figure 1,illustrates the design of the two PFA/mapping catheters and the ablation system. Briefly, the catheters are inserted through a commercially-available 8.5-French, long introducer. The system delivers biphasic, microsecond-wide pulses with amplitudes in excess of 1.8 kV. The generator allows for PFA using individually-selected electrodes or simultaneously using all 16 electrodes. With the exception of the first 4 consecutive animals, PFA pulses were synchronized with the cardiac cycle through QRS gating using a cardiac trigger monitor (Ivy Biomedical Mode 7600, Ivy Biomedical Systems, Inc, Branford, CT), to avoid energy delivery during the vulnerable period.
Preclinical protocols.
After an overnight fast, all swine were anesthetized, intubated, and ventilated mechanically with 1.0 FiO2 air. The animals were pretreated with ceftiofur (360 mg) on the day before and with gentamicin (160 mg) on the day of procedure. General anesthesia was induced by tiletamine (350 mg) and maintained using inhalational isoflurane (2.5%). A decapolar diagnostic electrophysiology catheter (Response, Abbott, Chicago, IL) was inserted into the coronary sinus for left atrial (LA) recording and pacing via the right internal jugular vein. Following percutaneous femoral venous access and systemic anticoagulation, a single transseptal puncture was performed for LA access. An 8.5-French deflectable introducer (Agilis, Abbott) was used for mapping and ablation under intracardiac echocardiographic (ViewMate, Abbott) guidance. Single-shot, QRS-gated, bipolar PFA (>1.8 kV) was performed using 30 sec applications. The targeted structures included the right atrium (RA), RA appendage (RAA), right superior PV, right inferior PV, left PV (also known as left inferior common PV), LA appendage, LA posterior wall, and superior (SVC) and inferior (IVC) vena cavae. Pre- and post-PFA intracardiac electrograms, pacing thresholds, and electrical isolation/conduction block were assessed. In addition, detailed pre- and post-PFA voltage maps were created using a high-density mapping catheter (Advisor HD Grid, Abbott) in all swine (bipolar voltage cutoff <0.1 mV). Upon completion of the study, the animals were euthanized. In addition to pre- versus post-PFA electrogram amplitudes, pacing thresholds, conduction block, and 3D voltage mapping (EnSite, Abbott), PFA lesions were also analyzed by necropsy and histology.
Skeletal muscle activation studies.
The intensity of skeletal muscle activation was quantified by measuring the absolute acceleration of muscular contractions. Briefly, a smartphone (Galaxy S6, Samsung Electronics, Suwon-si, South Korea) was secured to the animal’s thorax. The Phyphox smartphone application (Physical Phone Experiments, RWTH Aachen University, Aachen, Germany) was utilized which uses an integrated acceleration sensor. Acceleration was measured on the x, y, and z coordinates and computed in absolute values. This allowed for assessment of acceleration resulting from contractions triggered by PN stimulation as well as contractions triggered during PFA energy delivery. Meanwhile, we specifically also tested the PFA system in the ovine (n=1), as it is widely-believed that this animal model may be more sensitive to skeletal muscle activation. Lastly, in one animal an intravenous paralytic agent (succinylcholine 1 mg/kg) was administered to confirm PN-mediated loss of skeletal muscle contraction during PFA. In other words, acceleration measurements were recorded without and with administration of paralytics with the catheter positioned at the same location and using the same PFA energy parameters.
PN safety studies.
We specifically assessed the safety of PFA with regard to the PN in 14 swine (66±13 kg). PFA was intentionally performed at anatomical locations exhibiting low-output PN pacing capture using the PFA catheter, such as the SVC, the RAA and the LAA. PN function was evaluated through pacing capture, pre- versus post-PFA, acutely and during follow-up (up to 3 months post-ablation). In addition, the PNs were closely examined in all animals at necropsy and histology.
Esophageal safety studies.
To assess the safety of PFA on the esophagus, PFA was performed from within the IVC toward a deviated esophagus deflected toward the ablation catheter in 5 swine, as described in a previously-reported protocol.9 Briefly, following systemic anticoagulation, the PFA catheter was inserted through an 8.5-French deflectable introducer (Agilis, Abbott). Next, the esophagus was intubated using an esophageal balloon retractor device (DV8, Manual Surgical Sciences Inc, Salt Lake City, UT) under fluoroscopy. The distal esophageal lumen was anatomically delineated by administering iodinated contrast medium through an open port in the esophageal retractor device. The esophageal retractor was then inflated with a mixture of saline and contrast for fluoroscopic visualization and rotated to physically deviate the esophagus toward the PFA catheter placed inside the IVC. The deflectable sheath was used to ensure forceful contact between the ablation catheter and the esophagus. Orthogonal fluoroscopic views were used to confirm the immediate proximity of the deviated esophagus to the ablative portion of the catheter within the IVC (Figure 2).Whenever possible, the PFA catheter was also used to mechanically displace luminal contrast within the esophagus to further confirm its proximity to this structure. PFA was then delivered using the spiral catheter at areas intentionally contacting the deviated esophagus. Post-mortem, the esophagus was removed in its entirety, carefully inspected and photographed. The lesions created within the IVC and the esophagus were analyzed by gross and histologic examination. Esophageal sections were evaluated for alterations within the normal architecture as well as inflammation, necrosis, or fibrosis.
Assessment for thromboembolization.
Systemic embolization.
In all the animals, the brain, the kidneys, and the liver were carefully removed at necropsy and thoroughly examined for signs and evidence of thromboembolism. If any gross abnormalities were detected, further histologic examinations were performed.
The rete mirabile model.
The swine rete mirabile model was further used to investigate the potential embolic effects of PFA using the current system.10 As such, the rete mirabile was meticulously analyzed grossly and microscopically in 6 swine that received multiple PFA applications to the RAA, right superior PV, right inferior PV, left PV, LAA, and the LA posterior wall.
Magnetic resonance imaging (MRI).
In 3 swine, brain T2-weighted MRI scanning was also performed at baseline and at 1 week post-PFA for comparison to assess for cerebral emboli. MRI scanning was performed using a Philips MR 5300 Scanner (Philips, Eindhoven, Netherlands) and the images carefully analyzed for abnormalities by a radiologist.
Follow-up studies and histologic analysis.
Two swine, 1 canine, and 1 ovine were euthanized within 4 h of PFA to investigate the acute effects of tissue ablation. Sixteen animals were survived for 3–4 weeks and 3 swine for 3 months. In all animals, high-density 3D voltage mapping (Advisor HD Grid, Abbott) was performed post-PFA and again repeated during follow-up on the day of sacrifice (range: 3 weeks to 3 months) to reassess lesion durability. Ablation efficacy was further validated by the analysis of intracardiac electrograms, pacing thresholds, and electrical isolation/conduction block. At necropsy, all cardiac chambers were carefully examined and all lesions were identified and photographed. Detailed analysis of each treatment site was performed and measurements were taken to determine the dimensions of the ablation lesions. In addition, all tissues were fixed in formalin, processed, and stained with hematoxylin and eosin and elastin Masson trichrome.