A vivens ex vivo study on the synergistic effect of electrolysis and freezing on the cell nucleus

Freezing-cryosurgery, and electrolysis-electrochemical therapy (EChT), are two important minimally invasive surgery tissue ablation technologies. Despite major advantages they also have some disadvantages. Cryosurgery cannot induce cell death at high subzero freezing temperatures and requires multiple freeze thaw cycles, while EChT requires high concentrations of electrolytic products-which makes it a lengthy procedure. Based on the observation that freezing increases the concentration of solutes (including products of electrolysis) in the frozen region and permeabilizes the cell membrane to these products, this study examines the hypothesis that there could be a synergistic effect between freezing and electrolysis in their use together for tissue ablation. Using an animal model we refer to as vivens ex vivo, which may be of value in reducing the use of animals for experiments, combined with a Hematoxylin stain of the nucleus, we show that there are clinically relevant protocols in which the cell nucleus appears intact when electrolysis and freezing are used separately but is affected by certain combinations of electrolysis and freezing. \ua9 2015 Lugnani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Magnetic resonance imaging of electrolysis.

This study explores the hypothesis that Magnetic Resonance Imaging (MRI) can image the process of electrolysis by detecting pH fronts. The study has relevance to real time control of cell ablation with electrolysis. To investigate the hypothesis we compare the following MR imaging sequences: T1 weighted, T2 weighted and Proton Density (PD), with optical images acquired using pH-sensitive dyes embedded in a physiological saline agar solution phantom treated with electrolysis and discrete measurements with a pH microprobe. We further demonstrate the biological relevance of our work using a bacterial E. Coli model, grown on the phantom. The results demonstrate the ability of MRI to image electrolysis produced pH changes in a physiological saline phantom and show that these changes correlate with cell death in the E. Coli model grown on the phantom. The results are promising and invite further experimental research

Electrolytic Effects During Tissue Ablation by Electroporation.

Nonthermal irreversible electroporation is a new tissue ablation technique that consists of applying pulsed electric fields across cells to induce cell death by creating permanent defects in the cell membrane. Nonthermal irreversible electroporation is of interest because it allows treatment near sensitive tissue structures such as blood vessels and nerves. Two recent articles report that electrolytic reaction products at electrodes can be combined with electroporation pulses to augment and optimize tissue ablation. Those articles triggered a concern that the results of earlier studies on nonthermal irreversible electroporation may have been tainted by unaccounted for electrolytic effects. The goal of this study was to reexamine previous studies on nonthermal irreversible electroporation in the context of these articles. The study shows that the results from some of the earlier studies on nonthermal irreversible electroporation were affected by unaccounted for electrolysis, in particular the research with cells in cuvettes. It also shows that tissue ablation ascribed in the past to irreversible electroporation is actually caused by at least 3 different cytotoxic effects: irreversible electroporation without electrolysis, irreversible electroporation combined with electrolysis, and reversible electroporation combined with electrolysis. These different mechanisms may affect cell and tissue ablation in different ways, and the effects may depend on various clinical parameters such as the polarity of the electrodes, the charge delivered (voltage, number, and length of pulses), and the distance of the target tissue from the electrodes. Current clinical protocols employ ever-increasing numbers of electroporation pulses to values that are now an order of magnitude larger than those used in our first fundamental nonthermal irreversible electroporation studies in tissues. The different mechanisms of cell death, and the effect of the clinical parameters on the mechanisms may explain discrepancies between results of different clinical studies and should be taken into consideration in the design of optimal electroporation ablation protocols

Synergistic Combination of Electrolysis and Electroporation for Tissue Ablation.

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

Synergistic Combination of Electrolysis and Electroporation for Tissue Ablation.

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

Electroporation followed by electrolysis followed by electroporation.

<p>The lesions shown are in higher magnification than illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148317#pone.0148317.g007" target="_blank">Fig 7</a>, but are from the same treatment taken at different sites. (A) Control. (B) This section of the liver stained with H&E contains normal liver cells with no evidence of treatment related injury; outer edge of lesion at the anode side. (C) and (D) Micrographs towards the core of the anode treated region. (E) and (F) Micrographs at the anode core with 20x (E) and 10x (F) magnification. (G)—(J) Sites at the margin of the anode affected region towards the cathode, with (G) and (H) in 10x and (I) and (J) in 20x magnification. Tissues were stained in H&E ((G), (I)) and Mason trichromatic stain ((H), (J)).</p

Comparison between extent of tissue ablation by different numbers of electroporation pulses.

<p>A detailed list of the parameters can be read in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148317#pone.0148317.t001" target="_blank">Table 1</a>. (A) and (C) Gross macroscopic sections. (B) and (D) Trichromatic stained slides. Electroporation pulse parameters—1000 V, 100 microseconds, 1 Hz. (A) and (B) 8 pulses. (C) and (D) 297 pulses. Calculated isoelectric field lines are superimposed on the panels in the bottom row. They are also applicable to the top row. In all panels, the anode is the right electrode, while the cathode is on the left.</p

Electroporation with 1000 V, 100 microseconds, 1 Hz, 297 pulses.

<p>Applied parameters are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148317#pone.0148317.t001" target="_blank">Table 1</a>. The left electrode is always the anode, while the cathode is on the right side. (A) Magnified H&E and (B) Mason trichromatic stained tissues, focusing on the region between the electrodes. (C) Control. (D) Interface between affected and normal area. (E) The core of the treated region on the left hand side (x10). (F) Magnification (x20) from the core on the left hand side. (G) Left hand side electrode core near a dehydrated region (x10). (H) The left hand side lesion near a large blood vessel. Rectangles and black arrows show the site from which the magnified sample was taken. Scale bars and magnification are given in the figures.</p

Microscopic details from the 297 pulse treatment.

<p>(A) and (B) Low magnification micrographs from different sites of the middle region between electrodes. (C) and (D) 10x and 20x magnification of the tissue in the area midway between the electrodes. (E) and (F) 20x magnification of tissue adjacent to the large blood vessel, with sites in the area midway between the electrodes (E) and tissue near the large blood vessel on the side of the right electrode (F). (G) and (H) Micrographs from sites in the core of the region affected by the right hand side electrode. (I) and (J) Micrographs from sites at the right hand side, outer edge of the right electrode. The sites from which the magnified micrographs were taken are marked with a square in the insert. Scale bars and magnification are given in the figures.</p