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.