A Preclinical Study of an Implanted Device

in the Pulmonary Veins, Intended for the Treatment of

Atrial Fibrillation in an Ovine Model
TIM VANDECASTEELE, D.V.M.,* TIM BOUSSY, M.D.,† MATTHEW PHILPOTT, M.SC. ENG.,‡
ELI CLEMENT, M.SC. ENG.,‡ STIJN SCHAUVLIEGE, D.V.M., PH.D.,§ WIM VAN DEN
BROECK, D.V.M., PH.D.,* GUNTHER VAN LOON, D.V.M., PH.D.,¶
PIETER CORNILLIE, D.V.M., PH.D.,* and GLENN VAN LANGENHOVE, M.D., PH.D.‡
From the *Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium; †AZ
Groeninge Medical Hospital, Department of Cardiology, Kortrijk, Belgium; ‡Fulgur Medical, Merelbeke, Belgium;
§Department of Surgery and Anaesthesia of Domestic Animals, Faculty of Veterinary Medicine, Ghent University,
Merelbeke, Belgium; and ¶Department of Large Animal Internal Medicine, Faculty of Veterinary Medicine, Ghent
University, Merelbeke, Belgium

Background: Atrial fibrillation is the most frequent arrhythmia in adults of which the interventional
cure is hampered by high recurrence rates. Recurrence after ablation is due to an incomplete isolation of
the pulmonary veins. A new ablation technique was performed, in the antra of ovine pulmonary veins, by
device implantation, which was heated through a wireless heat-generating system.
Methods and Results: Implants were placed transatrially in the pulmonary veins of sheep. Using
a wireless heating system, the energy was afterward transferred through wires to the implanted device
according to a defined protocol. The position of the implant and the applied lesions were macroscopically
evaluated. Samples of the ablated tissue of the atrio-pulmonary vein junction were histologically and
immunohistochemically examined.
Conclusions: Six ablation procedures in four sheep were successfully performed without adverse
cardiac reactions. Implantation of the device and the wireless heat generation was feasible. Sufficient
heat was produced at the level of the antra of the pulmonary veins to create ablation lesions, which were
histologically and immunohistochemically confirmed. (PACE 2016; 39:822–829)

electromagnetic heating, sheep, implant, wireless, ablation

Introduction Nevertheless, ablation of the pulmonary veins is,

Atrial fibrillation (AF) is the most common
cardiac arrhythmia in adults associated with
substantial mortality and morbidity. It is generally
accepted that the main triggers of abnormal
pulses, which induce AF, are localized at the
level of the pulmonary veins base.1–3 In the past
decade, several catheter-based ablation techniques
were tested and improved to isolate electrically
the pulmonary veins from the left atrium.4–8
Conflict of Interest: Glenn Van Langenhove, Matthew Philpott,
and Eli Clement are employed by Fulgur Medical, responsible
for the development of the implantable device and heat-
generating system. All authors have read the journal’s
authorship agreement, and the manuscript has been reviewed
and approved by all the named authors.
Address for reprints: Tim Vandecasteele, D.V.M., Department
of Morphology, Faculty of Veterinary Medicine, Ghent
University, Salisburylaan 133, 9820 Merelbeke, Belgium.
Fax: 3292647790; e-mail: Tim.Vandecasteele@UGent.be
Received March 17, 2016; revised May 10, 2016; accepted May
23, 2016.
doi: 10.1111/pace.12899
still to date, a technically demanding technique
and recurrence of AF is seen due to incomplete
isolation of the pulmonary veins. During long-
term follow-up, a recurrence rate of more than
50% was mentioned after a single procedure
and without the use of antiarrhythmic drugs.9
Consequently, the need for a better treatment
strategy which ensures a high success rate along
with a relatively quick and simple procedure
remains, ideally offering the possibility to repeat
the procedure noninvasively.
In this context, an implantable device was
developed, after positioning in an electromagnetic
field, which can be wirelessly heated, creating the
desired transmural ablation lesions at the level
of the pulmonary vein-left atrial junction. In the
first step, an in situ study examines wireless heat
generation after which the heat is transferred to
the implanted ablation device. This implant has a
tubular shape through which a complete circular
lesion can be applied. This procedure was per-
formed in an ovine model by open heart surgery
due to access of the pulmonary veins. The aim of

©2016 Wiley Periodicals, Inc.

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 IMPLANTATION IN THE PULMONARY VEINS

Figure 1. Left image = Verhaert implant device with
heater coil (1); right image = pick-up coil (2) and
electromagnetic or applicator coil (3).
this study was to evaluate this technique, directly
after the ablation, by macroscopic, histological,
and immunohistochemical investigation of the
induced lesions in sheep.
Materials and Methods
Device
The implants (Fig. 1) contain a heating coil
(Verhaert, Kruibeke, Belgium), which includes a
temperature feedback system. Implant size ranged
from 12 mm to 20 mm. An electromagnetic coil
generating an electromagnetic field, in which a
pick-up coil was placed according the same plane,
constitutes the heat-generating system. The heat
generated in the pick-up coil was transferred
through wires to the heating coil.
Procedure
The procedure was performed at IMM
Recherche (Paris, France) under the Ethics com-
mittee approval number “RYTHMOLOGIE-12-12-
FLUX MEDICAL 14–34.” Studies were performed
on four sheep (1–1.5 years old).
Using 6 mg/kg propofol (Fresofol, Pharmatel
Fresenius Kabi, Hornsby, Australia) induction
and 2% isoflurane (Abbott Australasia, Kurnell,
Australia) maintenance, sheep were anesthetized
and placed in dorsal recumbency. Arterial and
venous cannulae were inserted into the carotid
artery and jugular vein using a cut-down technique
for pressure monitoring. The chest was opened
using conventional techniques. A 40/36-F two-
staged venous cannula (Edwards Life Sciences,
Irvine, CA, USA) was inserted into the right
atrium and secured with a pursestring suture.
A 22-F aortic cannula (Edwards Life Sciences)
was inserted into the proximal part of the
descending aorta. The extracorporeal circulation
system consisted of a venous reservoir, roller
pump (Cobe Cardiovascular, Arvada, CO, USA),
and a Capiox RX 25 membrane oxygenator
(Terumo Europe, Leuven, Belgium) connected by
noncoated tubing. Cardiopulmonary bypass was
established by priming with 1,000 mL crystal-
loid prime solution (lactated Ringer’s solution
750 mL, 20% mannitol 100 mL, aprotinin 100 mL,
8.4% sodium bicarbonate 50 mL, and heparin
5,000 IU), which was allowed to circulate in
the extracorporeal circulation system for 2 hours.
Flow was maintained at >3 L/min with a perfusion
pressure of 50–70 mm Hg. After opening of the left
atrium, suction cannulas were placed inside the
pulmonary veins. Afterward, the most suitable-
sized implant, according to the diameter of the
pulmonary vein, was inserted into the antrum,

Figure 2. Ablated atrio-pulmonary vein junction tissue samples of two sheep. AS = atrial side of
the sample; PS = pulmonary side of the sample; the oval indicates the ablation region; red line =
trimming line indicating the orientation of the histological sections.

PACE, Vol. 39 August 2016 823

 VANDECASTEELE, ET AL.

Figure 3. Time/temperature table of the ablation procedure of the pulmonary veins during 250 seconds (panels A, B,
and G), 400 seconds (panel E), 500 seconds (panel F), 600 seconds (panels C and D), 800 seconds (panel H). Tstent and
Tenviro indicate implant temperature and body temperature, respectively, I refers to the used current, Tset indicates
the set temperature.

which is the common venous space, of both ostia
through which the pulmonary veins drain into
the left atrium (for terminology see Vandecasteele
et al.10 ). Subsequently, the implanted device was
connected through wires to a pick-up coil to
ensure the heat transfer. Also, a temperature
control device was put in place.
The electromagnetic field, created by the
applicator coil, generates an alternating current
in the pick-up coil, when placed in the same
plane of the applicator coil, which is consequently
transferred into heat. This heat causes the ablation
process of the connected implant’s heater coil.
Before the ablation procedure was started, the

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PACE, Vol. 39

Table I.
Description of Temperature Data, Visualized in the Graphs of Figure 3, of the Devices Implanted in Different Sheep According to the Set Temperature

Implants Sheep 1 Sheep 2 Sheep 3 Sheep 4

Implant 1 Temperature Failure: as a current increase To a higher temperature Gradually but faster Failure: temperature
increase around 100 seconds compared to the test in surpassed the 60°C limit
induced no temperature sheep 1 (demonstrated by a large

IMPLANTATION IN THE PULMONARY VEINS

increase (Fig. 3A) temperature peak),
associated with the
production of a small
amount of smoke after
which the test was stopped.
It was found that both the
temperature and heater
August 2016

power wire were damaged

(Fig. 3G)
Set temperature 50°C 55°C for 210 seconds
Implant Followed nicely the set Reacted on the temperature
temperature temperature causing the increase (only small
implant to ablate at 50°C for variations were seen;
180 seconds (Fig. 3C) Fig. 3E)
Implant 2 Temperature Gradually Of which only the peak Was heated similarly to the Temperature raised gradually
increase temperatures reached previous test to two different set
briefly set temperature temperatures
Set temperature Increased from 43.5°C–44°C Was increased to 55°C for Was increased to 55°C for First 52°C for 120 seconds
for 180 seconds 230 seconds 300 seconds and thereafter of 53°C for
480 seconds
Implant Followed set temperature Was stable at an average Followed set temperature At each set temperature, the
temperature (Fig. 3B) temperature of 51°C almost perfectly with only implant temperature was
(Fig. 3D) some small variations fairly constant with
(Fig. 3F) approximately 2°C variation
compared to the set
temperature (Fig. 3H)
825
826

Table II.
Additional Information about the Procedures Mentioned in Figure 3 and Table I

Implants Sheep 1 Sheep 2 Sheep 3 Sheep 4

Implant 1 Failure was due to malposition of
the temperature sensor which
was not in contact with the vessel
wall (Fig. 3A)
Was heated to 50°C as this
provides the required ablation
lesion according to previous tests.
At maximum set temperature, the
required current peaked at almost
120 A and averaged around
The required current was relatively Was stopped due to alleged
low compared to previous tests as damage of the temperature
the peaks surpassed 80 A and sensor or the heater coil during
the average levels were around the implantation (Fig. 3G)
70 A. The peak at the end was
due to system reset by which the

VANDECASTEELE, ET AL.
100 A (Fig. 3C) temperature dropped and
consequently the software
August 2016

increased the current output

Implant 2 The required current peaked
(almost 120 A) at a maximum set
temperature and was averaged
around 60 A (Fig. 3B)
PACE, Vol. 39
accordingly (Fig. 3E)
Reached set temperature at certain Demonstrated a required power The power peaked just under 120 A
moments which is due to peak of just below 120 A and an and averaged around 60 A
insufficient contact between the average of around 80 A (Fig. 3F) (Fig. 3H)
temperature sensor and the
vessel wall or between the
heating coil and the vessel wall in
the vicinity of the temperature
sensor. Due to this uncertainty,
the ablation time was reduced to
230 seconds instead of the
planned 300 seconds. The
required power peaked and
averaged just below 120 A and
was increased gradually in the
last half of the test to reach the
set temperature (Fig. 3D)
 IMPLANTATION IN THE PULMONARY VEINS

Figure 4. Parts of pulmonary vein tissue at the level of the atrio-pulmonary vein junction. Left
image: at the top side the white colored ablation site indicated by the arrows and at the bottom
the normal pinkish-colored tissue. Right image: the removed device with the heater coil (green
wire) of which an ablation mark (indicated by the frame) is seen on a piece of the pulmonary
vein wall.

Figure 5. Histological section of the ablated atrio-pulmonary vein junction (H&E staining). Left
image: AS = atrial side; 1 = myocardium; 2 = fibrin deposits; 3 = interruption of the tissue.
Scale bar is 250 µm. Right image: PS = pulmonary side; 2 = fibrin deposits; 4 = lung tissue; 5 =
hemorrhagic necrosis; arrow = pulmonary vein wall; Scale bar is 1 mm.

temperature sensor was checked to ensure that the
indicated temperature correlated with the body
temperature.
At the start of the heating procedure, the
temperature control software was set to 1°C
above the body temperature after which the
temperature was gradually increased from the
initial temperature in 1°C steps every few seconds
while trying to reach the required ablation
temperature of 58°C during different time periods.
This procedure was repeated for each ostium, one
implant at a time.
Pathological Analysis
All sheep were euthanized directly after the
ablation procedure. Immediately after euthanasia,
the heart-lung packages were excised after which
the pulmonary veins, containing the ablation
device, were removed and examined macroscopi-
cally.
Histological Analysis
Four percent formaldehyde-fixed paraffin-
embedded scarred tissue of the atrio-pulmonary
vein junction of two sheep was examined on
5-µm-thick hematoxylin and eosin (H&E)-stained
histological sections. The orientation in which
the histological sections were cut is indicated in
Figure 2.
Immunohistochemical Analysis
The myocardial sleeve of the ablated pul-
monary vein was stained immunohistochemically
with the myosin marker MYBPC3 (polyclonal,
K-16: sc-50115, Santa Cruz Biotechnology, Santa
Cruz, CA, USA) to detect the myosin-binding
protein C (cardiac type), which is reactive
with ovine tissue. Myosin-binding protein C is
present in the myofibrils of cardiac tissue.11
The 5-µm-thick slides were immunostained using
the Dako automated Autostainer Plus (Dako,
Glostrup, Denmark). No antigen retrieval was
performed. The sections were incubated for
5 minutes with 3% hydrogen peroxide and 30
minutes with rabbit serum. First, the primary
antibody (1:100) was incubated for 60 minutes,
afterward secondary antibody (rabbit/anti-goat,
biotinylated, polyclonal, 1:500, Dako) was applied
for 30 minutes followed by streptavidin and
horseradish peroxidase (streptavidin-HRP, 1:500,
Dako) for 30 minutes. The visualization was
achieved with DAB (Dako) for 5 minutes.

PACE, Vol. 39 August 2016 827

 VANDECASTEELE, ET AL.
Figure 6. Immunohistochemical staining with myosin marker MYBPC3 of myocardial sleeve tissue inside pulmonary
vein wall. The double arrow indicates the stent implantation site. AS = atrial side; PS = pulmonary side. Scale bar:
top left and top right image is 200 µm; top middle image is 100 µm; bottom image is 2 mm.
Results
Ablation Data
The data of the ablation procedures performed
with eight devices in four sheep (two devices per
animal) are indicated in Figure 3 and Tables I and
II. Not one of the following tests was aborted due
to any cardiovascular disorder or abnormalities in
rhythmicity.
Pathological Analysis
Macroscopic Evaluation
Observation of the heart-lung packages at the
level of the pulmonary veins revealed no damage
to the surrounding structures. The endothelium
of all pulmonary veins showed clear ablation
lesions (Fig. 4) along the circumference of the
device, although by comparing the samples
some differences in degree of discoloration were
noticed. At the level of the device, no thrombus
formation was noticed in any of the sheep.
Microscopic Evaluation
The ablation lesion, investigated on two
tissue samples, was recognized on the histolog-
ical sections by fibrin deposition, visualized in
Figure 5. Figure 6 demonstrates immunohisto-
chemically the myosin staining of myocardial
sleeve tissue of the ablated pulmonary vein,
including the ablation site. At this latter spot,
a lighter immunohistochemical reaction can be
observed in comparison to the tissue proximal and
distal to the site of the implanted stent.
828
Discussion
This study was performed in sheep in an open
heart surgery procedure in order to have a good
accessibility to the pulmonary veins. In numerous
cardiovascular studies, pigs are used as an animal
model in which the localization of the draining
openings or ostia of the pulmonary veins into
the left atrium was studied in detail.10 However,
anatomically, the number of ostia in pigs shows
similarity with sheep or goats as at the level of the
pulmonary vein-left atrial junction two vestibules
of the left atrium were described in a clinical study
in goats.12
Ablation Data
We demonstrated that ablation of the pul-
monary veins through wireless energy transfer
is feasible. Eight stents were implanted and
tested in four living sheep of which two ablation
procedures failed. One failure occurred during the
initialization phase and one during the ablation
phase itself. Cause of this failure was damage to
the temperature sensor during implantation which
gave wrong temperature data. Thus during system
check of the implants, six of eight implants met
the technical requirements.
The difference in extensiveness of the ablation
lesions was due to the fact that no clear tempera-
ture protocol was predefined, and that different
temperatures and different ablation times were
applied. Histologically, on H&E-stained sections,
the effects of heating at the level of the pulmonary
veins could only be noticed by visualization
August 2016 PACE, Vol. 39
 IMPLANTATION IN THE PULMONARY VEINS
of fibrin deposits in acute euthanized animals.
Immunohistochemically, denaturation of myosin
proteins of the myocardial sleeve at the level of
the heated implanted stent proves the induction
of a lesion in the pulmonary veins wall caused by
the generated heat at the ablation site. After long-
term studies, greater effects may be seen as fibrin
completely replaces the necrotic tissue.
The degree of contact between the implant
and the vessel wall is also of great importance
to create a perfect circular lesion. However,
overstretching of the pulmonary vein wall is
possible but within its safe range in terms of
elasticity. In this study, the implant diameter
ranged from 12 mm to 20 mm. The information
deduced from these procedures will further
improve stent design intended for future studies.
The proof of the efficacy of this system,
by which the heat is picked up wirelessly
and transferred from the pick-up coil to the
pulmonary veins through a direct connection,
will allow for development of an implantable
device that can be heated by placing the animal
in an electromagnetic field. The fact that the
implanted device can remain in place and may
be endothelialized constitutes a major advantage
as a second treatment can be performed without
any surgery.
Conclusions
In this ablation study in a sheep model, all
devices could be placed inside in one of both
antra of the pulmonary veins and were heated
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