Date Published: August 22, 2019
Publisher: Public Library of Science
Author(s): Nina Klein, Enric Guenther, Florin Botea, Mihail Pautov, Simona Dima, Dana Tomescu, Mihai Popescu, Antoni Ivorra, Michael Stehling, Irinel Popescu, Maria Rosaria Scarfi.
The combination of electroporation with electrolysis (E2) has previously been introduced as a novel tissue ablation technique. E2 allows the utilization of a wide parameter range and may therefore be a suitable technology for development of tissue-specific application protocols. Previous studies have implied that it is possible to achieve big lesions in liver in a very short time. The goal of this study was to test a variety of electrode configurations for the E2 application to ablate large tissue volumes.
27 lesions were performed in healthy porcine liver of five female pigs. Four, two and bipolar electrode-arrays were used to deliver various E2 treatment protocols. Liver was harvested approx. 20h after treatment and examined with H&E and Masson’s trichrome staining, and via TUNEL staining for selective specimen.
All animals survived the treatments without complications. With four electrodes, a lesion of up to 35x35x35mm volume can be achieved in less than 30s. The prototype bipolar electrode created lesions of 50x18x18mm volume in less than 10s. Parameters for two-electrode ablations with large exposures encompassing large veins were found to be good in terms of vessel preservation, but not optimal to reliably close the gap between the electrodes.
This study demonstrates the ability to produce large lesions in liver within seconds at lower limits of the E2 parameter space at different electrode configurations. The applicability of E2 for single electrode ablations was demonstrated with bipolar electrodes. Parameters for large 4-electrode ablation volumes were found suitable, while parameters for two electrodes still need optimization. However, since the parameter space of E2 is large, it is possible that for all electrode geometries optimal waveforms and application protocols for specific tissues will emerge with continuing research.
Minimally invasive focal tissue ablation has become an emerging field in modern medicine. The biophysical effects which are employed for tissue ablation by electricity can be divided into thermal and non-thermal techniques. The thermal modalities, which are based on temperature elevation caused by dissipation of electrical energy (Joule heating effect) utilize a variety of electromagnetic frequencies to achieve the effect, including radio frequency  and microwave frequency . On the contrary, electrochemical treatment via electrolysis  and electroporation-based therapies [4,5,6,7] are based on non-thermal biophysical mechanisms. The effect of electrochemical treatment depends on the generation of electrolysis products via the application of low direct current in the tissue, thus making local changes in pH its main cause of cell death . Because of that and similarly to heat-based therapies, electrochemical treatment does not have preservative properties towards sensitive structures. The advantage of the treatment lies in the application of very low voltages, which reduces instrumentation complexity. However, effective ablation via electrolysis requires relatively high concentrations of electrolysis products. In order to produce sufficient amounts of these products, long treatment times (tens of minutes to hours) are necessary. The biggest drawback is that given the long treatment time, diffusion of the electrolysis products, which can be predicted by considering the electric current applied multiplied by the time of its application, through inhomogeneous tissue and blood transportation lead to an almost unpredictable distribution of electrolytic products. This makes ablation dimensions difficult to plan.
The experiments were performed in compliance with all ethical and legal rules as stated by the national legislation and the European Union (program number 327). The experimental protocol was reviewed and approved by the Ethics Committee of Fundeni Clinical Institute as well as by the Bucharest Sanitary-Veterinary Authority (no 316). The experimental study was carried out in vivo on three 40 kg and two 30 kg breed female pigs, as previously described . In short, after the animals were fasted for 24h, they were medicated with a combination of acepromazine (0.5mg/kg, Vetoquinol S.A., Lure, France) and ketamine (15 mg/kg, Gedeon Richter Plc., Budapest, Hungary), which was injected intramuscularly prior to treatment. Anesthesia was induced intravenously with Propofol at a concentration of 2.5mg/kg and 0.1mg Fentanyl (Chiesi Pharmaceuticals GmbH, Vienna, Austria). After endotracheal intubation was performed, anesthesia was maintained with sevoflurane in 80% O2 (adjusted to 2–2.5% Endtidal sevoflurane, Abbvie, Rome, Italy). In case of postoperative pain, the animals were treated with morphine (Zentiva S.A., Bucharest, Romania) at a concentration of 0.1 mg/kg, applied intramuscularly, and ketoprofen 1 mg/kg (S.C. Terapia S.A., Cluj-Napoca, Romania). Cefazolin 25 mg/kg (Biochemie GmbH, Kundl, Austria) was applied intravenously in 2h intervals. The pigs were placed in a ventral side-up position, and the liver was exposed with an upper midline incision continued with a right transverse incision. The treatment was delivered using 3 types of electrodes: 1) 18-gauge or 16-gauge stainless steel needle-type electrode with a variable exposed length of 1-4cm exposed treatment length (Inter Science GmbH, Lucerne, Switzerland); 2) A 13-gauge stainless steel needle-type electrode (IGEA, Carpi, Italy); 3) A simple, custom-made bipolar electrode, employing 2 shifted 18-gauge electrodes with an isolated gap between. All treatments with their treatment parameters are listed in Table 1. All sets of parameters were tested a minimum of 2 times in healthy pig liver. Total number of lesions was 27.
A study was performed on the liver of five pigs to evaluate E2 applications for large lesions. The goal of the study was to develop a better understanding of the effects of E2 parameters on tissue ablation with the boundary conditions of fast, large and vessel-preserving ablations, employing different electrode arrays that are often used in clinical settings. Our results demonstrate the ability to produce large continuous lesions with a 4-electrode array, a 2-electrode array and a bipolar electrode encompassing large vessels in a majority of the lesions. Lesions as large as 35x35x35mm were achieved without requiring replacement of the electrodes. No major bleeding occurred, no clinically significant adverse events occurred and no subject died during treatment or observation time. Similar to other electroporation-based treatments, parameters that are below the effective threshold will lead to lesions that do not close. This was especially evident with the parameters chosen for the two-electrode experiments. These were all at the lower thresholds, mainly due to the desire to investigate E2 application without deep muscle relaxant. The results obtained with the two-electrode array demonstrate the need to provide optimal treatment parameters for E2 in clinical settings. However, since the domain of E2 parameters is large, it is possible that different optimal waveforms and protocols for specific tissues and applications will emerge with continuing research.