Date Published: February 11, 2016
Publisher: Public Library of Science
Author(s): Michael K. Stehling, Enric Guenther, Paul Mikus, Nina Klein, Liel Rubinsky, Boris Rubinsky, Maria Rosaria Scarfi.
Electrolysis, electrochemotherapy with reversible electroporation, nanosecond pulsed electric fields and irreversible electroporation are valuable non-thermal electricity based tissue ablation technologies. This paper reports results from the first large animal study of a new non-thermal tissue ablation technology that employs “Synergistic electrolysis and electroporation” (SEE). The goal of this pre-clinical study is to expand on earlier studies with small animals and use the pig liver to establish SEE treatment parameters of clinical utility. We examined two SEE methods. One of the methods employs multiple electrochemotherapy-type reversible electroporation magnitude pulses, designed in such a way that the charge delivered during the electroporation pulses generates the electrolytic products. The second SEE method combines the delivery of a small number of electrochemotherapy magnitude electroporation pulses with a low voltage electrolysis generating DC current in three different ways. We show that both methods can produce lesion with dimensions of clinical utility, without the need to inject drugs as in electrochemotherapy, faster than with conventional electrolysis and with lower electric fields than irreversible electroporation and nanosecond pulsed ablation.
Minimally invasive surgery employs various tissue ablation technologies, each with their own advantages, disadvantages and specific use. Electric currents passing through a biological medium produce a number of biophysical and biochemical effects which are used in tissue ablation. This study deals with the use of a combination of two different electricity driven phenomena, electrolysis and electroporation.
The study was conducted on in vivo pig liver and approved by the PMI’s Institutional Animal Care and Use Committee IACUC (PMI—Pre Medical Inovation, San Carlos, CA, USDA number: 93-R-0506, Study number: ANS 2094). We used three female pigs, weight 30 kg to 40 kg, treated in accordance with Good Laboratory Practice regulations as set forth by the 21 Code of Federal Regulations (CFR) Part 58. Each procedure started with anesthetization of the animal under general anesthesia per SOP #33156. Preanesthetic medication was Telazol 4.0 mg/kg (2.0 ml) IM and Atropine 0.02 mg/kg (1.8 ml) IM. Anesthetic induction was done by Isoflurane with Oxygen, 2%/2L/minute via mask. Possible postoperative pain was ameliorated by Buprenorphine 0.01 mg/kg IM Pre-med at recovery and Carprofen 4 mg/kg at extubation/recovery. Antibiotics administered during surgery was Cefazolin 25 mg/kg IV every 2 hours. In addition, pancuronium (0.1 mg/kg, at a dose of 1 mg/ml) was administered through an IV prior to the procedure, to reduce muscle contractions during the application of the electrical pulses. Pancuronium (0.05 mg/ml at 1 mg/ml) was administered throughout the procedure as needed. The liver was exposed via a midline incision. The treatment was delivered using two 18 gauge Titanium needles (Inter Science GmbH, Ch) with a sliding insulating sheath inserted in the liver. We have used Ti needles to eliminate possible electrolytic products involving the electrode materials. The 18 gauge variable length electrodes were custom designed for the delivery of both electroporation and electrolytic pulse sequences.
Twelve lesions were produced in the liver of three pigs with a variety of SEE parameters and electrode placement configurations. This was a first large animal study of SEE and therefore we pursued several different goals, such as: testing the effects of SEE parameters, methods and combinations, pre-clinical practice and safety. All the animals survived the procedure without any complication.
The primary goal of this study was to expand on our earlier small animal study [49,50] and examine tissue ablation protocols that combine electrolysis and electroporation in a configuration and with parameters that are relevant to clinical applications. Since our earlier work on tissue ablation with non-thermal irreversible electroporation (NTIRE) [44, 51], medical imaging has become standard in clinical use of NTIRE. We therefore considered using ultrasound imaging in our study as well. While performing the electrolysis studies under ultrasound, we have observed in all the experiments the bright hyperechoic appearance in the region adjacent and between the electrodes. An example is shown in Fig 1C. Since electrolysis is known to produce gases near the electrodes, the most likely explanation is that these bright areas are the reflection of ultrasound waves from the gas tissue interface. This led us to re-examine ultrasound images from our earlier irreversible electroporation studies [44,51]. Fig 1D illustrates a typical site of NTIRE treated tissue immediately after the procedure. Comparing the panels in the bottom row (Fig 1C and 1D), it is now obvious that the bright spots near the electrodes in the NTIRE procedure are due to gases produced by electrolysis during the electroporation pulses. This has important clinical value. It is common to experience loud and sudden explosion-like sounds during NTIRE protocols. The observed bright areas now explain the mechanism: These sounds are most likely the result of an electric discharge across the electrolysis generated layer of gas around the electrodes. Electroporation employs voltages as high as 3000 V. When the field across the layer is higher than about 3000 V/cm, the gases ionize and an electric discharge akin to lightning occurs. For a voltage of 3000 V, a 100 micron thick layer of gas will be sufficient to cause a discharge. This discharge generates high pressure waves that could be detrimental to the treated organ, in particular if it is encapsulated such as the brain, bone or the prostate. Monitoring the formation of bright spots at the electrodes during electroporation could be used to avoid the electric discharge.
Using a large animal model, we have confirmed the findings in [49,50] that a synergistic combination of electrolysis and electroporation can produce more effective ablation than either electrolysis or electroporation separately. Furthermore, this combination lends itself to the design of clinical protocols that employ lower voltages than NTIRE and shorter times than electrolysis. Obviously, this is only a first large animal study of this combination tissue ablation modality. Substantial research remains to be done to optimize the concept for clinical use.