Preliminary Validation of Electroporation-Electrolysis (E2) for Cardiac

Ablation Using a Parameterisable In-Vivo Model

Luke Zhao1,2 , Adam Rasko2 , Christian Drescher2 , Sanaz Maleki3 , Michael Cejnar2 , Alistair McEwan1

Abstract— Atrial fibrillation is the most common arrhythmia, the in vivo thigh model for cardiac ablation [9], which is
increasing the risk of stroke, heart failure and death, and well established in preclinical evaluation of cardiac catheter
a growing epidemic. Electroporation ablation is emerging in ablation for its methodological advantages—accurate elec-
cardiac ablation for atrial fibrillation as a fast, tissue-specific
and non-thermal alternative to existing technologies tied by trode placement, lesion identification, reproducibility, greater
their thermal action to shortcomings in efficacy, speed and risk. usable surface area for animal reduction.
Studies so far have aimed to translate the success of irreversible Past work in cardiac electroporation ablation has advanced
electroporation from tumour treatment, with its kilovolt pulses, the topic to a point that demands more systematic investi-
to cardiac ablation. However, these high voltages may be less gations [10] and thorough reporting [11], towards treatment
appealing for cardiac ablation from clinical, technical and
regulatory standpoints. A novel ablation technique combining planning. Importantly, the applied electric field underpins the
electroporation and electrolysis in a single pulse E2 uses electroporation treatment, and should be considered particu-
lower voltages. A custom E2 ablation system was developed larly with specific electrode geometry and arrangements [12].
and tested on an in vivo tissue model. Histopathological analysis Despite almost as many different electrode configurations as
showed lesions of clinically relevant depth, achieved without any studies tabulated in a recent review [5], description of the
acute complications or severe muscle contractions. Lesions were
mapped onto a numerical model developed to refine further resulting electric fields is usually absent. The present study
prototyping. This study provides preliminary prototype valida- aims to demonstrate the feasibility of E2 ablation, in parallel
tion and the methodological foundation for dose optimisation with establishing a robust workflow for future studies.
towards endocardial application.
II. M ETHODS
I. INTRODUCTION A. Numerical modelling
Electroporation is the creation of nanometer to micrometer A first-order FEM of electroporation ablation was devel-
pores on the cell membrane by exposing the cell to an electric oped in the Electric Circuits module of COMSOL Multi-
field of sufficient magnitude and duration according to the physics 5.1 (COMSOL AB, Stockholm, Sweden) (Fig. 1).
application [1], [2]. Therefore, the electrical conductivity of The Laplace equation (Eq. 1) was used to calculate the
the tissue is critical in determining the impact of an applied electric field from the applied electric field potential ϕ, as-
voltage [3]. This property of electroporation is key in its ad- suming static, homogeneous and anisotropic tissue electrical
vantage of selective tissue ablation, particularly in pulmonary conductivity σ [12]. Candidate E2 ablation threshold values
vein isolation (PVI) for treating atrial fibrillation (AF), where were considered based on relevant electroporation work that
structural tissue and important neighbouring structures must obtained 100 V/cm (8×20 ms, 1 Hz pulses) [13] and 450
be preserved [4]. Furthermore, electroporation ablation is V/cm (8×99 µs, 1 Hz pulses) [14], on murine skeletal muscle.
fast—milliseconds, rather than minutes.
Preclinical studies of cardiac electroporation ablation have −∇ · (σ · ∇ϕ = 0) (1)
borrowed techniques from irreversible electroporation (IRE)
as applied in cancer treatment, but high voltages and long
pulse trains increases risk (e.g. arcing, barotrauma), dis-
comfort from muscle contractions [5], [6], [7] and com-
plexity from engineering and regulatory standpoints. On the
other hand, electrolysis was originally a nuisance in non-
destructive applications of electroporation, yet recently syn-
ergised with the lower voltages of reversible electroporation
(RE) in a novel technique called ‘E2’, with promising early
results in tumour ablation [8]. This feasibility study uses
1 Luke Zhao and Alistair McEwan are with the School of Electrical
and Information Engineering, Room 402, Building J03, Maze Crescent,
Darlington NSW 2006, Australia luke.zhao@sydney.edu.au
2 Luke Zhao, Adam Rasko, Christian Drescher and Michael Cejnar are
with Micropace Pty Ltd, Unit 41, 159 Arthur Street Homebush West, NSW
2140 Australia
3 Sanaz Maleki is with the Discipline of Pathology, Level 4 West, Charles
Perkins Centre (D17), Camperdown NSW 2006, Australia Fig. 1: Comsol geometry.
 The Comsol simulation was bounded by a sphere. Mus- (unpublished data) set between 350 V and 490 V, and 220 µF
cle tissue (0.44 S/m) and blood (0.67 S/m) [15] are each and 560 µF.
represented by a hemisphere, and electrodes mounted on a
polymer plate (3D-printed experimental jigs) separated from V (t) = V0 e−t/τ , τ = R · C (2)
muscle by a thin layer of blood. For this study, the electrode
configuration was derived as a partial, simplified 2D projec-
tion of balloon electrode geometry optimised for electric field
depth, evenness and manufacturability (unpublished data).
Simulation results informed the development of the pulse
generator.

B. Equipment
A custom pulse generator was developed for the required
voltage and capacitance range. When ablating the pulmonary
veins, the combination of heterogeneity and anisotropy in
electrical tissue properties [16] yields a highly uneven load.
Simultaneous, multi-channel and multi-electrode delivery
Fig. 2: Representative E2 waveform showing voltage (blue),
would improve the even distribution of the pulse throughout
current (solid red: total current; dotted red: partial current
the target tissue. Voltage and current was recorded with
through Electrode 3—see Section III) and impedance (black)
a digital storage oscilloscope (TPS2014B), using a 100x
over time, annotated with peak values and time constant.
voltage probe across the terminals of the pulse generator; a
Inset: simplified circuit with high voltage power supply V,
10x voltage probe across a shunt resistor in the delivery path
switches S1 and S2, capacitor bank C and load ZL , which
for total current; and a Hall effect current clamp (TA018)
has been simplified as purely resistive in this study.
measuring current through the center anode (see Section IV).

C. Procedure
Experiments were performed at the Medical Engineering
Research Facility at the Queensland University of Technol-
ogy (ethics approval number 1800001036). Three healthy fe-
male merino sheep (38-45 kg; aged 5-5.5 years) were anaes-
thesised and sedated (midazolam, buprenorphine, propofol)
but not paralysed for the experiment. Heart rate was moni-
tored throughout and ventilators maintained 15 breaths per
minute.
Each electroporation pulse was delivered simultaneously
through the four electrodes of alternating electrical polarity
shown in Fig. 1, which were mechanically fixed to the tissue
and removed one minute after application. A thin layer
of blood was added below the electrode plate to improve
electrode-tissue contact and mimic endocardial ablation.
Electrodes were always positioned to align the bulk of the
electric field in parallel to the muscle fibres, in order to
maximise and keep consistent the electroporation dosage, i.e.
induced transmembrane voltage (ITV), for a given voltage
setting. The ITV of an arbitrarily shaped cell can be predicted
as a function of polar angle, varying along the perimeter and
maximal at the poles of spheroidal cells (0◦ ) and (180◦ )
[17]. Approximating myocytes as prolate spheroidal cells,
the overall ITV is maximised when the electric field passes
through the poles of the cell [17].
The E2 pulse was a monophasic exponentially decaying
waveform implemented as a capacitive discharge (Fig. 2), as
described in Eq. 2 with the chosen initial voltage V0 , time
t and time constant τ , which is determined by the chosen
capacitance C and the load R. A total of 23 sites on the thigh
muscles of 3 sheep were treated with either one pulse or two
pulses 2 minutes apart, with maximal non-arcing parameters
Following the final ablation on each animal, one hour was
allowed for biological processes involved in E2 to take place
[18] before sacrifice, with time between treatment and exci-
sion ranging up to 4 hours. To avoid truncating the treated
region in any direction, the outer edge of the tissue frames
served as a template for tissue excision, and cutting depth
was at least 10 mm—greater than the predicted ablation
depth based on the referenced electroporation thresholds, but
clinically relevant [19]. Samples of untreated tissue were also
collected.
D. Histopathology
Tissue samples were rinsed to remove residual blood, fixed
in 10% neutral pH-buffered formalin, for 24h before replac-
ing with fresh formalin to avoid formalin pigment formation.
Frames were removed prior to fixation as a precaution against
chemical interaction and contamination. After fixation, tissue
shrinkage was assessed against the size of the frame to which
they were cut. Slices were made longitudinally—parallel to
the muscle fibres—at 3 mm apart from the middle outwards,
with a single transverse bisection due to size constraints.
Sections were taken from each tissue sample to be assessed
with haemotoxylin and eosin (H&E) [7]. Microscopy was
performed with Leica Aperio XT with resolution of 0.0256
pixels per µm (650 DPI), and analysed with ImageJ (2.0.0-rc-
69/1.52i), Aperio ImageScope (v12.4.0.5043) Aperio Image
Library (v12.0.15) on Windows 7 6.1.
III. R ESULTS
All pulses were delivered without any discontinuities
indicative of arcing in the measured waveforms [21], and
electrodes were undamaged from visual inspection with
the naked eye. Common signs of thermal ablation—blood
clotting, charring, white imprints [22]—were not observed.
A single muscle contraction was present with each pulse but
not severe, as judged by a clinical cardiac electrophysiologist
present at the experiment. The peaks of the current traces
ranged from 4.4 A to 9.2 A, total energy delivered from
22.4 J to 42.1 J and time constants from 12.4 ms to 40.0 ms.
A simple electrical analysis of the electrode configuration
was considered prior to the experiment, in order to select
the appropriate capacitance based on results from previ-
ous experiments with the simpler case of bipolar delivery
between two electrodes. Assuming three equal resistances
between each neighbouring pair of the four electrodes, the
Thevenin equivalent is a third of the resistance found in
the two-electrode configuration, and therefore requiring three
times the capacitance to obtain the same time constant. The
measurements of the total current and the current through
Electrode 3 (Fig. 2) support this simple resistive model,
which may be useful in further prototype development and
experimentation.
Acute purplish bruising was always visible around 5 min-
utes after ablation, consistent with previous reports of cardiac
electroporation ablation [23], lasting up to half an hour. The
bruising diffused throughout the entirety of the frame, but
darker ‘electrode shadows’ were also seen in many samples
directly underneath where the electrodes were placed (Fig.
3). Ablation has been reported to result in tissue shrinkage
[7], which may be compounded by formalin fixation. After
fixation, muscle tissue had shrunk approximately 10% to
20% longitudinally and up to 5% transversely, which is
slightly more than reported previously [24] if considering
overall shrinkage.
Muscle tissue stained with H&E clearly demarcated pale
Fig. 3: Top: Diffuse bruising was visible immediately after
the E2 pulse. Bottom left: Mechanically stable set-up ready
for E2 application. Custom frames were sutured by four
corners onto the muscle tissue for optimal alignment, consis-
tency in positioning and pressure applied by a fixed spring.
Bottom right: Arrowheads indicate ‘electrode shadows’ on
ablation site ‘3L1’ at half an hour post-ablation.
(a) Contraction bands in ablated (b) Unaffected blood vessels and
region. fatty tissue surrounded by fully
ablated muscle tissue.
(c) Transition zones at the lesion border. A: severe breakdown of
muscle cells, with some pale structural outline visible. B: degrading
muscle cells with clear loss of structure. C: structure is largely
retained but with some contraction as noted by [20]. D: muscle
cells generally normal in appearance.
Fig. 4: Representative H&E stains of thigh muscle ablation.
ablated zones, similarly to previous studies [7]. Fig. 4a shows
contraction bands resembling those found in myocardial
ablation [22], which arise from excessive calcium influx
due to membrane damage [25]. Fig. 4b demonstrates the
selectivity of electroporation, and sometimes red blood cells
were identified within the intact blood vessels. Lesions were
visible macroscopically, while a higher magnification shows
contrasting transition zones at (Fig. 4c). Zone A is the
macroscopically visible ablation region, separated from intact
tissue by the slightly darker Zone B, which was taken as
the lesion border. Although Fig. 4c conveniently shows the
graduated degradation of tissue damage, it is not always
as linear or homogeneous elsewhere in the tissue. Some
samples showed uneven, arch-like lesions (Fig. 5), which
would each have been directly beneath an electrode, based
on the corresponding cross-section.
IV. D ISCUSSION
E2 pulses delivering several times lower voltage (and
energy) than the non-thermal IRE studies reviewed in [4]
yielded similar or larger lesions in comparison—therefore
likely ‘transmural’, or clinically relevant. While muscle
contraction is not easily quantified, lower applied voltages
would be expected to produce smaller muscle contraction.
Methodologically sound factors that would be favourable to
ablation but potentially unrealistic include: good electrode
contact, greater tissue homogeneity and optimised alignment.
Cardiac muscle fibre anisotropy, especially at the junction of
the pulmonary veins, would warp the geometry of the applied
Fig. 5: Comparison between histology of representative tissue samples and numerical modelling. Histology sections are
taken from tissue treated with a single (385 V, 450 µF) pulse, cut either longitudinally (top) or transversely (bottom),
with corresponding cross-sections of Comsol simulations showing electric field magnitudes from 50 V/cm to 500 V/cm.
Representative slices have been chosen so that the reader may interpolate between the longitudinal slices, and consider the
transverse slices across one electrode in the context of its rotational symmetry. The distance from the middle of the tissue
sample is indicated for each slice in mm. Black bars: electrode locations; green dashed lines: lesion borders; red dotted
lines: adjusted for 20% longitudinal shrinkage. Insets: electrode configuration superimposed on 3D oblique view of simulated
cross-sections. Note: two different pieces of tissue treated with the same conditions are shown above; one for each cutting
direction and with good overall agreement.

electric field and diminish the electroporation dose [16].
If considering the lesions in terms of electroporation
alone in Fig. 5, the lesion borders in transverse sections
fit a consistent threshold value. However, lesion borders in
longitudinal only fit the calculated electric fields better after
adjustment for shrinkage. The apparent 50 V/cm threshold is
below one of the lowest thresholds reported in the literature
[13], especially considering that the lesion depth may be
underestimated due to shrinkage. On the other hand, lesions
under the middle electrodes appear comparatively larger, in
both absolute terms, and with respect to the shape of the
electric fields swelling towards the outer electrodes, espe-
cially when shrinkage towards unablated tissue—possibly
oedema—the depth of the outer lesions appear greater. This
observation would be consistent with more pronounced elec-
trolytic effects arising from more current passing between
the middle two electrodes (see Section III), i.e. more charge,
and therefore more electrolytic products by Faraday’s first
law of electrolysis. Furthermore, tissue with less electrode
contact—less exposure to electrolytic products—were gener-
ally found with less pronounced lesions (e.g. at the −6 mm
longitudinal section). Finally, the arch-like (or dome-like, if
adjusted for shrinkage) shape of some lesions, distinct to that
of the simulated electric fields, resembles the more radial
development of electrolytic lesions [26].
The consistency of electrode frame placement during the
experiment yielded highly uniform fibre orientation across all
tissue samples bar regions of high anisotropy. That is, fibres
were either running across the histology slide in longitudinal
sections, or ‘into’ the slide in transverse sections. This
result is helpful towards analysis, while also demonstrating
the reproducibility of the thigh model for the benefit of
parametrisation.
This study prefixes further investigations in multi-channel
pulsing, pulse parametrisation, ablation thresholds and long-
term effects.
V. C ONFLICTS OF I NTEREST
Luke Zhao and Christian Drescher are employees of
Micropace Pty Ltd. Dr Michael Cejnar is the Founding
Managing Director of Micropace Pty Ltd.
ACKNOWLEDGEMENTS
The authors acknowledge the facilities and the scientific
and technical assistance of Micropace Pty Ltd, the Discipline
of Pathology at the University of Sydney, the Medical
Engineering Research Facility (MERF) at the Queensland
University of Technology, and Microscopy Australia at the
Australian Centre for Microscopy & Microanalysis at the
University of Sydney.
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