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LEADING ARTICLE

J. Transl. Med. Res 2016;21(4):247-252 DOI: 10.21614/jtmr-21-4-98

Minimally Invasive, Non-Thermal Tissue Ablation with


a Single Exponential Decay Electrolytic Electroporation
Waveform
Boris Rubinsky1,2, Enric Gunther1,2,3, Florin Botea4,5, Franco Lugnani6, Vlad Herlea5,7, Paul Mikus2,
Mihail Pautov4,5, Nina Klein2,3, Catalin Pecheanu5,7, Michael K. Stehling1,2,3, Dana Tomescu5,8,
Matteo Macchioro6, Simona Dima4,5, Alexandru Serban9, Irinel Popescu4,5

1
Department of Bioengineering and Department of Mechanical Engineering, Corresponding author:
Boris Rubinsky
University of California, Berkeley, USA Professor of mechanical engineering
2
Inter Science, Luzern, Switzerland Department of Bioengineering and
3
Institut fuer Bildgebende Diagnostik, Offenbach, Germany Department of Mechanical Engineering
4
Center of General Surgery and Liver Transplantation – Fundeni Clinical Institute, Bucharest, University of California, Berkeley, CA
94720, USA
Romania E-mail: brubinsky@gmail.com
5
Center of Translational Medicine – Fundeni Clinical Institute, Bucharest, Romania
6
Hippocrates D.O.O, Divaĉa, Slovenia; Clinica Santa Elena, Malaga, Spain
7
Department of Pathology - Fundeni Clinical Institute, Bucharest, Romania
8
Department of Anesthesiology and Intensive Care - Fundeni Clinical Institute, Bucharest,
Romania
9
Department of Building Services, Transilvania University, Brasov, Romania

ABSTRACT

A new minimally invasive tissue ablation technique, that combines the biophysical processes
of electroporation and electrolysis, is introduced. The technology employs electrode needles
inserted in the target tissue to deliver an Electrolytic Electroporation Waveform (EEW) in the form
of an exponential decay voltage, several tens of microsecond long. A case study in a large
animal model demonstrates that clinically significant size lesions can be achieved with a single,
milliseconds long EEW. Ablation with EEW has major advantages over the comparable tissue
ablation techniques of electrolysis, reversible electroporation and irreversible electroporation.
EEW is orders of magnitude faster than conventional electrolytic ablation. EEW does not require
the use of muscle relaxant as irreversible electroporation and EEW does not require injection of
drugs as reversible electroporation. This new technology is simple to use and may become an
important addition to the minimally invasive surgery armamentarium.
Key words: electroporation, electrolysis, tissue ablation technique, experimental surgery

Received: 02.12.2016
INTRODUCTION Accepted: 19.12.2016

Minimally invasive surgery is a relatively recent branch of surgery, made


possible by the advent of new technologies. An important application of Copyright © Celsius Publishing House

Journal of Translational Medicine and Research, 21 (4), 2016 247


Boris Rubinsky et al

minimally invasive surgery is for tissue ablation. Various drug increases the complexity and uncertainty of the
technologies are used for tissue ablation, including; procedure.
radiofrequency, ultrasound, lasers, electrolytic ablation, Non-thermal irreversible electroporation is an
photothermic ablation, freezing, electrochemotherapy, ablation technology in which higher electric fields and
chemical ablation and more recently nonthermal numerous pulses are used to irreversible open the cell
irreversible electroporation. The various technologies membrane, and, thereby induce cell death (8). This
have each their own advantages and disadvantages – technology also spares the extracellular matrix and
which dictate the specific applications for which they tissue structures like larger vessels and nerves, and is
are used. In the past, most minimally invasive ablation very effective in treating pancreatic cancer. However,
technologies employed biophysical processes that the high electric fields cause muscle contractions and
ablate the entire mass of organic molecules in the require the use of high doses of paralyzing drugs.
targeted volume; such as thermal ablation with heat (1) Furthermore, the pulses are usually delivered at a
or freezing (2) or chemical ablation with alcohols (3). frequency of about 1 Hz. Therefore, while the pulses
These ablation technologies destroy the undesirable are microsecond long, a typical protocol that employs
cells together with the connective tissue and vascula- several hundred pulses could become many minutes
ture and nerves in the treated volume. long.
Two technologies for minimally invasive tissue Recently, we have developed a new minimally
ablation, which selectively affect only some of the invasive tissue ablation technology that combines
organic molecules in the treated volume leading to cell electrolysis with electroporation, delivered as an
death, currently exist. They are electrolytic ablation and Electrolytic Electroporation Waveform (EEW) (9-11).
electroporation (reversible electroporation with Our earlier studies employed waveforms that
chemotherapeutic drugs and irreversible electropora- delivered electrolysis and electroporation sequentially
tion). In electrolytic tissue ablation, a direct current and separately. The results have shown that the
flows between two electrodes bracing the targeted combination of electrolysis and electroporation can
tissue. Cell death is caused by the chemical interaction ablate cells permeabilized by electroporation in a much
between the products of electrolysis (in particular the shorter time than conventional electrolytic ablation.
changes in pH) and cells (4,5). A review of the use of The studies of those combinations of electroporation
electrolytic ablation of tumors in the liver and pancreas and electrolysis have suggested that it should be possi-
reports that “A specific advantage of electrolytic ble to deliver the combination of electroporation and
ablation is its safety even when conducted close to electrolysis simultaneously, using an exponential decay
major vessels, while a disadvantage is the longer voltage waveform (EDW). A preliminary, acute study, in
ablation times compared to more frequently employed a small rodent animal model, has demonstrated the
techniques….. Pancreatic electrolytic ablation has been feasibility of tissue ablation with EDW. The results have
investigated in experimental studies that confirmed shown that a very short, single millisecond EDW can
similar advantages to those found with liver ablation, ablate large volumes of tissue without attendant
but has never been evaluated on patients.” (6). Indeed, muscle contraction (12). We found that the EDW pulse
electrolytic ablation times range from tens of minutes combines the advantages of electrolytic ablation,
to hours. These long ablation times, in addition to reversible electroporation and irreversible electropora-
problematic predictability due to diffusion and perfu- tion, without the disadvantages. This report is a case
sion effects, are a major disadvantage of electrolytic study report, from a much larger study in which the
ablation. effectiveness of EDW was investigated in a large animal
Electroporation is the permeabilization of the cell (pig). The very promising results have motivated the
membrane achieved by exposing a cell to brief, publication of this first report from the forthcoming
nanosecond and microsecond long, high electric field much larger study.
pulses. Reversible electroporation results from certain
electric pulses that reversible open the cell membrane. MATERIALS AND METHODS
When certain toxic chemicals, such as bleomycin, enter
the reversible electroporated cells, they cause cell Experiments were conducted in compliance with
death (7). Combining reversible electroporation with all ethical and legal rules imposed by national legis-
the injection of drugs ablates the tissue and spares the lation and European Union. The experiment protocol
connective tissue and blood vessels. However, it was approved by the Ethics Committee of Fundeni
requires the injection of a drug. The injection of the Clinical Institute, and by the Bucharest Sanitary-

248 Journal of Translational Medicine and Research, 21 (4), 2016


Minimally Invasive, Non-Thermal Tissue Ablation with a Single Exponential Decay Electrolytic Electroporation Waveform

Veterinary Authority (no 316). (3 lesions), the left medial lobe (3 lesions), the right
The experimental study was carried out on an lateral lobe (2 lesions), and right medial lobe (2 lesions),
in vivo 80 kg Topigs20 breed female pig. After being respectively. Figure 1 depicts the intraoperative proce-
fasted for 24 hours, the animal was pre-medicated dure during ablation for lesion number eight. It shows
with a combination of acepromazine (0.5mg/kg) and the way in which the two electrodes were inserted in
ketamine (15 mg/kg) injected intramuscularly (IM). the liver using a spacer. An important clinical aspect of
Anesthesia was induced with intravenous (IV) Propofol all minimally invasive tissue ablation techniques is the
(2.5 mg/kg) and 0.1 mg Fentanyl. Endotracheal intuba- need to ablate all the cells in the targeted tissue.
tion was performed and anesthesia was maintained Therefore, the focus of our investigation was to
with sevoflurane in 80% oxygen (adjusted to 2-2.5% ascertain that EEW can completely ablate the tissue
End-tidal sevoflurane). Possible postoperative pain was between the two electrode needles. Blood samples
treated with morphine 0.1 mg/kg IM and ketoprofen were taken pre-treatment, 1h after treatment end
1 mg/kg q 6 hours. Cefazolin 25 mg/kg IV was adminis- time, 6h after treatment end time, and before harvest
trated every 2 hours. to detect cytokines (sIL-2R, IL-10, IL-6), white blood cells
The pig was placed in a ventral side-up position, and and HGF. The surgery time was 65 minutes.
restrained using strings. The liver was exposed via an Postoperatively, the animal was kept alive for 24
upper midline incision continued with a right transverse hours, after which the liver was harvested and the
incision. The treatment was delivered using two 18- animal was euthanized using KCl 7.45% 1 ml/kg.
gauge Titanium needles (Inter Science GmbH, The liver harvesting procedure was carried out
Switzerland) with a variable length (1-4 cm exposed under general anesthesia. The surgical access was
treatment length) insulating sheath inserted in the liver, obtained by extending the midline incision. The liver
placed under ultrasound-guidance (Hi Vision Preirus hilum was dissected exposing the main bile duct,
Ultrasound device, Hitachi Medical Systems, Germany). hepatic arteries and portal vein. The portal vein was
Titanium was chosen, because, unlike steel or aluminum catheterized via splenic vein, and the infrarenal aorta
it is chemically inert, is biocompatible and at room was dissected and cannulated through a direct
temperature inert to oxygen, chloride and corrosion approach. The descending aorta and the inferior vena
(13). In addition, Ti is MRI compatible. The 18-gauge cava were exposed intra-thoracically using a right freno-
variable length electrodes were custom designed for tomy. The cross-clamping (clamping of the aorta in the
the delivery of both electroporation and electrolytic thorax and above the iliac bifurcation, and of the intra-
pulse sequences. thoracic inferior vena cava) was then performed, the
The experiment was carried out in an open-surgery inferior vena cava was sectioned below the liver, and
setting to maximize the availability of liver lobes. The the liver was flushed with 4 l of heparinized saline solu-
delivery of the single exponential decay waveform tion through the two cannulas at a hydrostatic pressure
solely took place through the described needle-type of 80 mmHg from a pressurized IV drip.
electrodes. For each ablation lesion, two electrodes The liver was then harvested and the flushing
were inserted in the liver parenchyma under ultra-
sound monitoring, distant to large vessels (at least 1
cm), in a roughly axial parallel configuration.
Since no apparatus is currently available to produce
the exponential decay voltage waveform needed for
the EEW procedure, we have designed and built a new
power supply described in the following device section.
This first large animal feasibility study was designed
according to the 3R principles for animal care
(Replacement, Reduction, Refinement) of Russell and
Burch (14). To this end, we have explored the effect of
various EEW parameters in one pig liver. The pig liver
was chosen because it allowed us to experiment with a
large number of EEW parameters, while minimizing the
number of animals used for the study. The pig liver from
which this case study was extracted was treated with a Figure 1 - Intraoperative aspect of the ablation procedure
total of 10 different lesions located in the left lateral lobe

Journal of Translational Medicine and Research, 21 (4), 2016 249


Boris Rubinsky et al

continued with the same solution until clean blood-free The delivered pulses were logged using an oscillo-
outflow from the hepatic veins was obtained. scope coupled to a PC with a custom log- and statistical
Immediately following saline perfusion, a 10% formalin evaluation software written in Mathematica® (Wolfram
fixative was perfused in the same way for ten minutes. Reseach, Mathematica® 10.0).
All lesions were removed from the liver with normal
surrounding parenchyma, and stored in the same RESULTS AND DISCUSSION
formalin solution. For microscopic analysis, the tissue
was bread loafed perpendicular to the capsule surface The result presented in this case study was obtained
and parallel to the needle tracts. All cassettes were by inserting two 18 gauge Ti needles separated by a
processed routinely from 10% phosphate buffered distance of 15 mm, in parallel, normal to the outer
formalin (8-10 hours), processed routinely, and embed- surface of the needle. The depth of insertion was 15
ded in paraffin blocks. To examine the specimen mm and the active treatment length was 15 mm from
histopathologically, three micrometer (3 μm) sections the tip. First, a brief 500 V pulse was delivered between
were cut from each paraffin block, then hematoxylin & the electrodes to verify the integrity of the electrical
eosin, and Masson’s trichrome staining were performed circuit. For the analyzed lesion (lesion no 6), induced on
for microscopic examination. The focus of the histology the diaphragmatic surface of the medial left lobe, this
was to verify the extent and nature of tissue ablation was followed by an exponential decay wave form with
with EDW. To produce information of practical clinical the following parameters: peak voltage 960V, capaci-
value, the focus of the analysis was on verifying the tance charge – 122 pulse time constant 97 ms. The
ability to produce a continuous lesion between the parameters chosen for this study were drawn from the
electrodes. experimental results of (15).
They were chosen to deliver substantial amounts of
DEVICE electrolytic products. Minimal muscle contraction
occurred, which did not necessitate the use of muscle
We were unable to find a power supply that can relaxants.
produce the waveform parameters, required for an The left hand side panel of figure 2 shows the trace
EEW protocol in tissues with the clinical relevant of the voltage during the delivery of the EDW. The expo-
dimensions, such as the pig liver. Therefore, we nential nature of the shape is evident, though not
designed a power supply that operates in the modality perfectly exponential as secondary surface effects on
of capacitor discharge electroporation systems (e.g. the electrodes make resistance non-constant during
Gene Pulser Xcell™ Electroporation System, BioRad, pulse delivery. The right hand side panel of figure 2
Hercules, CA, USA) with an enhanced performance. The shows the ultrasound appearance of the treated lesion
conventional type capacitors used were replaced with a immediately at the end of the delivery of the EDW. The
bank of selectable capacitors allowing capacitance needles signature is the two dark, hypoechoic, parallel
selection from 35 μF to 218 uF to provide the charge lines. The most significant feature is the hyperechoic
required for electrolysis. Similar to the Gene Pulser region. These are gases produced by electrolysis and
Xcell™, the generator has an output of up to 3kV. evidence that electrolysis has occurred.
Because of the larger capacitors, it can generate The focus of the histology was to verify the extent
exponential decay waveforms up to time ranges of tens and nature of tissue ablation with EDW. To produce
to hundred milliseconds, depending on tissue conduc- information of practical clinical value, the focus of
tivity and thereby simultaneously deliver electrolysis the analysis was on verifying the ability to produce a
and electroporation. The apparatus selects and continuous lesion between the electrodes.
matches the internal components needed to produce Out of the ten ablated lesions that were pathologi-
the time constants selected for the specific tissue cally analyzed, the most representative (lesion no 6)
conductivity of the treatment area by selecting an with low energy is depicted in figures 3 and 4. Figure 3
appropriate capacitor. The apparatus is able to produce shows a macroscopic cross section in a plane parallel to
and deliver the exponential decay voltage profile in the the direction of the needles. The sites from which the
time and voltage range for the specific treatment area. microscopic cross sections were taken are marked.
Since capacitors carry a defined charge (Q=C* U0), the Figure 4 depicts the microscopic appearance at
amount of electrolytic species produced but also the those sites. The ablated liver area had a hemorrhagic
Joule heating is predefined with the pre-settings of the appearance, with clear demarcation between ablated
total charge delivered. tissue and adjacent normal (transition zone) liver

250 Journal of Translational Medicine and Research, 21 (4), 2016


Minimally Invasive, Non-Thermal Tissue Ablation with a Single Exponential Decay Electrolytic Electroporation Waveform

Figure 2 - Left panel shows the voltage trace as a function of time


during the delivery of the EDW and statistical details for the
delivered pulse and the best monoexponential fit parameters.
Right panel shows the ultrasound image of the treated volume,
immediately after the delivery of the EDW

parenchyma; there was no fibrosis to delimitate these


areas. The central area between electrodes had coagu-
lative necrosis of liver parenchyma and hepatocytes
with hyper eosinophilic cytoplasm and pyknotic nuclei.
The sinusoids were congestive, filled with fibrin and
inflammatory cells.
In some areas, in the central and peripheral areas
congestion, edema and diffuse inflammatory cells (neu-
trophils, lymphocites, macrophages, and eosinophils)
were detected. Inflammatory infiltrate and edema
were present in the portal stroma. Outside the needles
tracts, the parenchyma and large vessels were normal.
The regional lymph nodes were also analyzed: they Figure 3 - Macroscopic gross section of the ablated area
were of normal size and some of them with cortical perpendicular to liver capsule surface and parallel to the needle
tracts. The dimensions of the lesion are given in cm. The sites from
reactivity, reactive germinal centers surrounding by
which samples for microscopy were taken are marked: A – distant
lymphocytes (nonspecific reactive lymphadenitis). to the anode tract, as control; B – between the needle tracts, distant
Thus, the ablation method succeeded in inducing from the tips; C – between the needle tracts, close to the tips;
necrosis between the two needles, while respecting the D – transition zone outside the catode tract. The locations of the
vessels outside these needles. To the best of our know- anode and cathode are also marked with (+) and (-)
ledge, this is the first reported successful tissue ablation
with clinically relevant volume done in the fraction of a
second. reversible electroporation technology. The results
suggest this technology may find important applica-
CONCLUSION tions in the treatment of liver and pancreatic cancer.

This case study report has introduced a new mini- Acknowledgements


mally invasive tissue ablation technology that com-
bines electrolysis and electroporation. Experiments in We thank our colleagues from the Center of
the pig liver show that clinical size ablation can be Translational Medicine – Fundeni Clinical Institute, Dr.
achieved with a single, milliseconds long, Electrolytic Mihai Popescu (anesthesiologist), Dr. Radu Ionescu
Electroporation Waveform (EEW), without the need (veterinary), Anca Nastase (researcher), Andrei Sorop
for muscle paralysis as in the irreversible electropora- (researcher), who all provided insight and expertise
tion technology, or injection of drugs as in the that greatly assisted the research.

Journal of Translational Medicine and Research, 21 (4), 2016 251


Boris Rubinsky et al

c d

Figure 4 - Microscopical examination of the ablated lesion. a. location A - distant to the anode tract (normal liver parenchyma - control
area): H&E stained section (400x) - normal area of hepatic parenchyma and portal tract, for comparison to treated area. b. location B –
between the needle tracts, distant from the tips (target area): H&E stained section (100x) hepatic parenchyma with hypereosinophilic
cytoplasms, pyknotic nuclei and congestion of sinusoids; c. location C - between the needle tracts, close to the tips (target area): H&E
stained section (400x) - hepatic necrosis, hepatocytes with hypereosinophilic cytoplasms, pyknotic nuclei, congestion, edema and
diffuse inflammatory cells. d. location D – transition area outside the catode tract: Masson trichrome stained section (100x) - hepatic
parenchyma with hypereosinophilic cytoplasms, pyknotic nuclei and congestion of sinusoids

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252 Journal of Translational Medicine and Research, 21 (4), 2016

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