Abstract
Electrolytic ablation is a method that operates by delivering low magnitude direct current to the target region over long periods of time, generating electrolytic products that destroy cells. This study was designed to explore the hypothesis stating that electrolytic ablation can be made more effective when the electrolysis-producing electric charges are delivered using electric pulses with field strength typical in reversible electroporation protocols. (For brevity we will refer to tissue ablation protocols that combine electroporation and electrolysis as E2.) The mechanistic explanation of this hypothesis is related to the idea that products of electrolysis generated by E2 protocols can gain access to the interior of the cell through the electroporation permeabilized cell membrane and therefore cause more effective cell death than from the exterior of an intact cell. The goal of this study is to provide a first-order examination of this hypothesis by comparing the charge dosage required to cause a comparable level of damage to a rat liver, in vivo, when using either conventional electrolysis or E2 approaches. Our results show that E2 protocols produce tissue damage that is consistent with electrolytic ablation. Furthermore, E2 protocols cause damage comparable to that produced by conventional electrolytic protocols while delivering orders of magnitude less charge to the target tissue over much shorter periods of time.
Keywords
Introduction
The field of minimally invasive tissue ablation in the context of surgery is an important alternative to surgical resection, often allowing for reduced surgery duration, improved access to the surgical site, and enhanced focus on the target tissue. Of the variety of techniques for tissue ablation, some of the more commonly used methods employ electrically induced phenomena. This article focuses on the following two modalities of tissue electroablation: electrolysis and electroporation.
The process of electrolysis occurs at the surfaces of electrodes submerged in an ionic conducting media. 1 New chemical species are generated at the interface of the electrodes as a result of the electric potential-driven transfer between electrons and ions or atoms. The various chemical species produced near the electrodes diffuse away in a process driven by differences in electrochemical potential. In physiological solutions, these chemical reactions also yield changes in pH, resulting in an acidic region near the anode and a basic region near the cathode. Tissue ablation is driven by two factors: a cytotoxic environment developing due to local changes in pH and the presence of some of the new chemical species formed during electrolysis. Electrolysis is a chemical ablation mechanism, and the extent of ablation is a function of the concentration of the chemical species and the exposure time to these chemicals. The total amount of electrolytic products generated during electrolysis is related to the charge delivered during the process; therefore, the total charge is used as a quantitative measure for the extent of electrolysis.
Electrolysis was harnessed for tissue ablation in medicine since the early 1800s, 2 and the field has experienced a revival in the mid 1970s with the work of Nordenstrom. 3,4 Over the last two decades, substantial research has been done on tissue ablation by electrolysis, including cell and animal experiments, mathematical modeling, and clinical work. 5 –22 In the contemporary literature, electrolytic ablation is sometimes referred to as Electro-Chemical Therapy, and in this work, these two terms are used interchangeably. Electrolytic ablation has been shown to exhibit several unique attributes. First, due to the chemical nature of the ablation process, the diffusion of chemical species in the tissue and the rate of chemical reactions dominate the time scale of the procedure. Second, the chemical products at the anode differ from those formed at the cathode, thus resulting in distinct mechanisms of ablation. Finally, electroosmotic forces drive the migration of water from the anode to the cathode, further magnifying the contrasting physiological effects at the electrode surfaces. From an operational standpoint, electrolysis requires very low voltages and currents, providing advantages relative to other ablation techniques, for example, reduced instrumentation complexity. It is, however, a lengthy procedure, controlled by the process of diffusion and the need for high concentrations of electrolytically produced ablative chemical species.
Although electroporation also harnesses an electricity-induced phenomenon, it differs from electrolysis by employing a different set of biophysical principles. Permeabilization of the cell membrane through the application of very brief, high-magnitude electric field pulses characterizes the bioelectric phenomenon of electroporation. 23 –31 The effect on the cell membrane is a function of the electric field strength. Electroporation can be used to produce reversible pores in the lipid bilayer, allowing for the introduction of molecules such as genes and drugs into cells. The electric parameters, however, can be designed to produce irreversible defects, resulting in a cell membrane that does not reseal after the field is removed. Reversible electroporation techniques have been combined with anticancer drugs such as bleomycin to target cancerous tissues for successful clinical use in the field of electrochemotherapy. 32 –35 Reversible electroporation is also used in other medical and biotechnological applications, including transfection and introduction of molecules such as small interfering RNA into cells that survive the permeabilization process. 36,37 The use of irreversible electroporation for tissue ablation is a more recent addition to the armamentarium of tissue ablation techniques available to surgeons, 38 –41 resulting in direct cell death without the need to introduce drugs or other molecules to facilitate the treatment process. Electroporation specifically targets the cell membrane through the application of an electric field that develops instantaneously. A major advantage of tissue ablation by electroporation is the relative speed of the procedure in comparison to any other ablation technique. Furthermore, because the procedure primarily affects the cell membrane, critical features of the extracellular matrix are spared, and this ablation modality can be used to treat tissues such as the pancreas without concern for collateral damage. 42 Existing tissue electrically mediated ablation approaches, however, have their respective disadvantages. Electrochemotherapy requires the injection of drugs into the tissue, while the high electric fields used in irreversible electroporation can cause muscle contractions and may affect the electrical function of the heart.
This study was inspired by a series of articles published by the group of Marshall and colleagues, 44 –48 indicating that the application of electroporation pulses in tissues will produce electrolysis at the electrodes. These previous research efforts also showed that the products of electrolysis generated during electroporation could ablate cells. The produced cytotoxic environment can be detrimental to reversible electroporation processes, such as gene transfection, that require the cells to remain alive after the procedure. Although Marshall's group sought means to minimize the electrolytic ablation during reversible electroporation procedures, 46 here it is proposed that another method of tissue ablation can be achieved that uses protocols which combine reversible electroporation fields with electrolysis. (For brevity we will refer to tissue ablation protocols that combine electroporation and electrolysis as E2.) Our hypothesis is that electrolytic ablation can be made more effective when the electric charge is delivered using E2 protocols.
By combining the effect of permeabilization on the cell membranes induced by reversible electroporation pulsed fields with the effects of electrolytic products generated during the entire electrostimulation procedure, the efficiency of the electrolytic ablation procedure is dramatically increased. This is estimated by measuring the total charge dosage delivered to the target tissue for a certain degree of ablation. A plausible explanation of the phenomenon could be attributed to the electrolytically produced chemicals that can pass through the pores in the cell membrane into the interior of the cell. Thus, in this new method of cell ablation, the electrical parameters typical to reversible electroporation protocols can be harnessed using a multiple pulse sequence to produce cell death with products of electrolysis without requiring drug injection. In the context of defining E2 protocols, it should be emphasized that the combination of electrolysis and electroporation for tissue ablation has value when using electroporation protocols that, in the absence of electrolysis, do not produce cell death. For clarification, we will now define the concepts of reversible and irreversible electroporation with respect to their effect on cell viability. Obviously, every delivery of an electric current generates certain amounts of electrolytic products. In our definition of reversible and irreversible electroporation, we will refer to protocols in which the amount of electrolytic product has a negligible effect on cell viability. We will refer to protocols that by themselves, without electrolysis, do not cause cell death, as “reversible electroporation” protocols. Electroporation protocols that produce cell death by themselves, without employing the addition of electrolysis products, are referred to as “irreversible electroporation” protocols.
The key parameter that affects the outcome of an electrolytic ablation procedure is the total charge delivered at the electrodes. The delivered charge dosage is a quantitative measure of the electrochemical reactions that occur at the electrodes and the amount of electrolytic products generated. In order to test our hypothesis, we compared the extent of tissue ablation produced by conventional electrolytic protocols to that produced by employing E2 protocols in acute, in vivo experiments in rat liver tissue.
Our study contains two sets of experiments: first, we performed a set of conventional electrolytic ablation treatments and used histology to show typical patterns of electrolytic damage as a function of delivered charge. Second, we performed another series of experiments, this time using E2 protocols as the stimulation modality. In this series, we again present the pattern of ablation as a function of the total charge delivered. The results of these two data sets allow us to compare the differences in histology obtained after treatments with conventional electrolysis with those obtained after E2 treatments. The goal of the comparison is two-fold: (1) to examine whether the general ablation patterns obtained by the two methods are the same and (2) to evaluate the extent of the affected region as a function of the total charge dosage. Due to the challenges associated with quantifying the degree of the damage, we assessed damage effectiveness through a binary evaluation, by simplifying our model to differentiate between what we defined as “nontransverse” and “transverse” damage to the tissue under study. Nontransverse damage refers to the situation where the electrostimulation causes a local lesion, typically in the vicinity of the electrodes, which does not span the bulk volume of tissue between the electrodes. Transverse damage refers to the situation where the lesion spans the region between the stimulation electrodes. In this first-order study, we will consider our hypothesis as tentatively confirmed if the general ablation patterns are the same between the two methods and if the transverse damage is achieved with a smaller charge when the charge is delivered using E2 protocols. This is a preliminary study, and our main goal was to evaluate the justification of further experimental efforts, which would then focus on detailed quantification of the effect as well as process optimization. We humbly acknowledge that much additional research remains to be done to explore a much larger set of parameters in order to understand the effects of all the parameters involved in E2 ablation.
Materials and Methods
Animals
Eleven male Sprague-Dawley rats (250-350 g) were obtained from Harlan Laboratories through the Office of Laboratory Animal Care at the University of California, Berkeley. The rats have received humane care from a properly trained professional in compliance with both the Principals of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals, prepared and formulated by the Institute of Laboratory Animal Resources and published by the National Institute of Health. The University of California-Berkeley Animal Care and Use Committee approved all procedures.
Experimental Procedure
The animals were anesthetized throughout the entire procedure via the administration of vaporized isoflurane. The depth of anesthesia was assessed before surgery and throughout the surgical procedure. After the level of anesthesia was verified, the abdominal skin was shaved, and an antiseptic was applied. A 3- cm midline abdominal incision was made, exposing the liver lobes. The experimental procedure is similar to that described by Edd et al 39 and the same experimental procedure was used for all animals in this study. Specifically, a set of 7-mm diameter, stainless steel plate electrodes (BTX Tweezertrodes; Harvard Apparatus, Holliston, Massachusetts) were applied across a liver lobe, as shown in the device illustration presented in Figure 1. The measured distance between the two electrodes was approximately 2 mm (±0.15 mm). The distance between electrodes was maintained for all animals tested. The electrostimulation in the form of electrolysis or in the form of E2 protocols, which consisted of multiple reversible electroporation field pulses, was applied to up to 3 distinct liver lobes per animal. The experiments were acute and the animals were euthanized without recovering from anesthesia.
Schematic of electrodes. Parallel cylindrical electrodes were used to apply electrolysis or electroporation across the liver lobe.
In the first part of our experiment, focusing on conventional electrolysis, we began with typical parameters used for electrolytic ablation, which resulted in transverse tissue ablation spanning the entire thickness of the liver sample. We then systematically decreased the delivered charge dosage until we found electrolysis parameters that resulted in nontransverse tissue ablation, which did not span the entire thickness of the liver sample. The dosage was decreased by reducing the current delivered and by reducing the time of delivery. For the second part of the experiment, focusing on E2 stimulation, we began with a set of parameters close to a typical reversible electroporation treatment used in electrochemotherapy. Using these parameters, we have seen only partial, nontransverse damage to the tissue, which did not span the region between electrodes. We then systematically increased the delivered charge dosage to obtain transverse damage to the liver tissue sample. Keeping the voltage constant and increasing the aggregate time of pulse delivery increased the dosage. In both experiments, the parameters were designed so that thermal damage to the tissue would be avoided. Due to the natural variability of the liver tissue thickness, the treated lobes were found to be within the 4.5 to 6 mm range, and all the lobes were compressed to the same thickness of 2 mm (±0.15 mm).
The electrolysis experiments were performed using an Arbitrary Function Generator (Tektronix/AFG 3102, Beaverton, Oregon). The device was set to produce a constant current for a fixed period of time. The current magnitude used was typical of standard electrolytic ablation protocols reported in the literature. 11 The stimulation time was chosen to facilitate comparison with the E2 protocol, in the context of examining the hypothesis central to the study. The parameters used in the electrolysis part of the study were: (1) 1 minute delivery of 8-mA current for a total charge dose of 1.25 C/cm2, (2) 1 minute delivery of 4-mA current for a total charge dose of 0.625 C/cm2, and (3) 30 seconds delivery of 4 mA for a total charge per electrode surface area of 0.312 C/cm2. The method used reduce the delivered dose, decreasing both time and current, and was chosen to maintain similar experimental accuracy in all three experiments. It should be emphasized that in this study, the exact combination of current and time in which a particular electrolysis dose is delivered does not affect the results. This is because the times in which the dose is delivered, 30 seconds or 1 minute, are negligible relative to the half an hour time period in which the particular dose affects the liver. This is the reason why all animals were maintained under observation for half an hour after treatment, as discussed in the next section. For the reader’s convenience, the parameter values are summarized in Table 1. The current was delivered in such a way that the anode was attached to the top (convex) part of the liver lobe and the cathode was attached to the bottom of the liver lobe. At least three liver lobes were treated under each experimental condition.
Electrolysis and E2 Parameters Used in Study.a
aAll E2 groups employed an electric field of 250 V/cm and a pulse frequency of 1 Hz.
In the second part of our study, E2 experiments, involving the delivery of multiple reversible electroporation field strength pulses, were performed using a square pulse generator (ECM 830; Harvard Apparatus). A constant electric field of 250 V/cm was generated in all the electroporation experiments by applying a potential difference of approximately 50 V over a pair of electrodes separated by a 2-mm (±0.15 mm) tissue sample. This field magnitude falls within the range typically associated with reversible electroporation, 49 and it is similar to the field magnitude used in tissue electrochemotherapy. The total charge delivered was calculated from the tissue data reported in Edd et al 39 and the parameters of the experiments. The experimental protocol in this part of the study consisted of first administering eight 100 ms pulses of 250 V/cm delivered at a frequency of 1 Hz across the liver lobe. During this stage, a total charge of 0.0001 C/cm2 was delivered to the sample. To study the effects of electrolytic products generated during the delivery of reversible electroporation field type pulses, we increased the delivered charge dosage by employing the following parameters: (1) ninety-nine, 100 ms pulses of 250 V/cm delivered at a frequency of 1 Hz across the liver lobe for a total charge delivery of 0.0012 C/cm2 and (2) ninety-nine, 1 ms long pulses of 250 V/cm delivered at a frequency of 1 Hz37,38,48,50 across the liver lobe for a total charge delivered of 0.0124 C/cm2. These parameters are summarized in Table 1 for the reader’s convenience. The same electrodes used to apply electrolytic ablation to the liver tissue were used in the E2 experiments. Four liver lobes (not necessarily from the same animal) were treated under each E2 experimental condition.
As a control study we have applied sham treatment to liver samples by pressing on them using the electrodes but without applying any electrical power, treating a total of two additional liver lobes.
Liver Histology
Following the conventional electrolysis as well as the E2 procedures, each animal was observed continuously for half an hour while being under anesthesia and thermal incubation, to facilitate a similar exposure time to the electrolytic products. After half an hour, the animals were euthanized by a combination of a vaporized isoflurane overdose and a bilateral chest dissection. The treated liver lobes were harvested, and each segment was fixed using 10% buffered formalin, placed in tissue cassettes, and submitted to three independent pathology laboratories (Histo-Tec Laboratories, Hayward, California; Pathology Associates, Inc, Berkeley, California; and Pathology Research Laboratory, Inc, South San Francisco, California). All staining was performed on 5-μm thick cross-sections cut through a plane perpendicular to the surface of the electrodes at the center of the treated region. Masson trichrome staining was used to delineate the margin between normal and ablated cells in tissue, and hematoxylin and eosin (H&E) staining was performed to study the morphology of the affected cells. The H&E-stained section was first cut from the tissue block, and another parallel slice was separately taken for the Masson trichrome staining. In all cases, the treatment was delivered across two electrodes separated by 2 ± 0.15 mm with slight tissue compression (original size in the range of 4.5-6 ± 0.75 mm). The histological cross-sections, however, show liver samples that returned to their original dimensions after the electrodes were removed.
Results
The histological results obtained from applying the conventional electrolysis and the E2 protocols to the liver tissue samples are shown in Figures 2 to 7. These results are presented as a function of the charge delivered during the protocol. The figures presented follow a similar format: panel A in each figure is a representative tissue sample stained by Masson trichrome, and panel B shows a cross-section of the tissue sample stained with H&E. Panels C, D, and E present higher magnifications of H&E-stained regions from areas on the top, middle, and bottom of the treated region, respectively. In viewing the results, we would like to point the reader’s attention to the most important measure of comparison which is the extent of tissue ablation from the outer surface of the liver, as a function of total delivered charge. As outlined in the Introduction section, we distinguished between nontransverse and transverse damage based on the partial or transverse nature of the lesion, respectively.
Electrolysis with a delivered charge of 1.25 C/cm2. The effect on the liver tissue is seen after applying an electrolytic protocol of 8 mA for 1 minute. A, Masson trichrome staining is used to differentiate the affected tissue from normal tissue, and this border is roughly marked. B, H&E staining of the tissue is shown, and a closer look at the top (anode side), middle, and bottom (cathode side) sections is shown in (C), (D), and (E), respectively. L is used to indicate the presence of a lesion is the tissue, and N indicates normal, unaffected tissue.
Electrolysis with a delivered charge of 0.625 C/cm2. The effect on the liver tissue is seen after applying an electrolytic protocol of 4 mA for 1 minute. A, Masson trichrome staining is used to differentiate the affected tissue from normal tissue, and this border is roughly marked. B, H&E staining of the tissue is shown, and a closer look at the top (anode side), middle, and bottom (cathode side) sections is shown in (C), (D), and (E), respectively. L is used to indicate the presence of a lesion is the tissue, and N indicates normal, unaffected tissue.
Electrolysis with a delivered charge of 0.31 C/cm2. The effect on the liver tissue is seen after applying an electrolytic protocol of 4 mA for 30 seconds. A, Masson trichrome staining is used to differentiate the affected tissue from normal tissue, and this border is roughly marked. B, H&E staining of the tissue is shown, and a closer look at the top (anode side), middle, and bottom (cathode side) sections is shown in (C), (D), and (E), respectively. L is used to indicate the presence of a lesion is the tissue, and N indicates normal, unaffected tissue.
E2 with a delivered charge of 0.0001 C/cm2. The effect on the liver tissue is seen after applying an electroporation protocol of 8 pulses of 100 μs at a 1-Hz frequency. A, Masson trichrome staining is used to differentiate the affected tissue from normal tissue, and this border is roughly marked. B, H&E staining of the tissue is shown, and a closer look at the top (anode side), middle, and bottom (cathode side) sections is shown in (C), (D), and (E), respectively. L is used to indicate the presence of a lesion is the tissue, and N indicates normal, unaffected tissue.
E2 with a delivered charge of 0.0012 C/cm2. The effect on the liver tissue is seen after applying an electroporation protocol of 99 pulses of 100 μs at a 1-Hz frequency. A, Masson trichrome staining is used to differentiate the affected tissue from normal tissue, and this border is roughly marked. B, H&E staining of the tissue is shown, and a closer look at the top (anode side), middle, and bottom (cathode side) sections is shown in (C), (D), and (E), respectively. L is used to indicate the presence of a lesion is the tissue, and N indicates normal, unaffected tissue.
E2 with a delivered charge of 0.0124 C/cm2. The effect on the liver tissue is seen after applying an electroporation protocol of 99 pulses of 1 ms at a 1-Hz frequency. A, Masson trichrome staining is used to differentiate the affected tissue from normal tissue, and this border is roughly marked. B, H&E staining of the tissue is shown, and a closer look at the top (anode side), middle, and bottom (cathode side) sections is shown in (C), (D), and (E), respectively. L is used to indicate the presence of a lesion is the tissue, and N indicates normal, unaffected tissue.
Liver Histology After Treatment by Conventional Electrolysis
Figures 2 to 4 show the histology of liver samples treated by conventional electrolytic ablation protocols as a function of decreasing delivered charge dosage. These figures are presented to demonstrate an observable relationship between the delivered charge dosage and the degree of tissue damage penetration visible in the histology. Figure 2 presents a liver sample treated with a conventional electrolysis protocol that involves applying a current of 8 mA for 1 minute, resulting in a total delivered charge per electrode surface area of 1.25 C/cm2. Masson trichrome is used to differentiate ablated and unaffected tissue after treatment. A rough outline of the area affected by the procedure was overlaid on the image to focus on the region of interest (Figure 2A). The most important observation is that the damage under these experimental conditions is transverse, that is, the lesion vertically spans the entire liver sample from the top to bottom surface. The H&E staining (Figure 2B) also presents evidence consistent with transverse damage, and clear delineation between treated and nontreated regions is observed.
Figure 3 presents a liver sample treated with an electrolysis protocol that involves applying a current of 4 mA for 1 minute, resulting in a total delivered charge per surface electrode area of 0.625 C/cm2. The area affected by the procedure is outlined (Figure 3A), and it can be seen that for this electrolytic dosage the damage is nontransverse, that is, the lesion does not span the sample between the electrodes. This is in contrast to the results exhibiting transverse damage observed in Figure 2A. As expected, the damaged area is adjacent to the sample surfaces that came in contact with the anode and cathode, while the center area of the sample appears normal. The H&E staining shows consistent results: the lesion does not span the sample vertically, and once again, a clear delineation between the treated and nontreated regions is seen (Figure 3B). These results show that the extent of damage produced by 0.625 C/cm2 (Figure 3A and B) is partial and is qualitatively less extensive than the damage produced by delivering a charge of 1.25 C/cm2 (Figure 2A and B).
The last electrolysis experimental group was treated by a current of 4 mA applied for 30 seconds, delivering a total charge per electrode surface area of 0.31 C/cm2. The histological results of these ablation parameters are shown in Figure 4. As in Figure 3, the most important observation shown here is that the damage is nontransverse, that is, the lesion does not span the liver sample (Figure 4A). In qualitative comparison with Figure 3A, it can be observed that the tissue region affected by this smaller charge dosage (Figure 4, 0.31 C/cm2) is much smaller than that affected by the larger charge dosage (Figure 3, 0.625 C/cm2) and substantially smaller than that affected by the largest charge dosage administered (Figure 2, 1.25 C/cm2). The treated area surrounds the liver surface that was in contact with the anode and cathode, while the central area of the sample appears normal. As also seen in Figures 2 and 3, there is a clear delineation between the treated and nontreated regions (Figure 4B). The relationship between the decreased charge dosage and the decreased extent of the damage does not come as a surprise; although we acknowledge that a useful extension of this research would attempt to quantify this relationship, at this point our goal was to test whether allocating resources to further, more detailed experimental explorations could be justified. Specifically we wanted to compare the results obtained for the electrolytic ablation to the results of E2 stimulation presented subsequently.
For all 3 electrolysis experimental groups, the treated region exhibits variable pale eosinophilia and widespread congestion as compared to the normal parenchyma. The appearance of the lesion at the anodic side (top surface) is different from that at the cathodic side (bottom side surface of the liver). This is considered to be a hallmark of electrolytic ablation. 22,47 The treated region exhibits an irregular shape with a greater volume affected near the tissue surfaces that were in contact with the electrodes, as illustrated in Figures 2 and 3 for the higher levels of delivered charge. Furthermore, the affected area near the anode is larger than that near the cathode. This asymmetry is typical of electrolytic damage. A closer look shows that large blood vessels in the treated zone, appearing as white open areas in the figures, are mostly free from entrapped erythrocytes. The parenchyma in the treated regions exhibits variations in color, which seem to partially correspond to the lobular structure. Centrilobular regions are darker in most cases, while other areas in the treated region are paler in color when compared to the untreated tissue.
In some samples (eg, Figure 3B), a band of condensed, strongly eosinophilic tissue is separated from the rest of the liver by an edematous layer. It is believed here that this is a consequence of the electroosmotic migration of water from the anode to the cathode and is caused by the volume confinement produced by the use of 2 plate electrodes. An edematous layer is observed to a varying degree in some samples from each experimental group but does not appear to be related to the amount of charge delivered. It is likely that this is a consequence of the local presence of large blood vessels and the resulting flow pattern in the treated region.
Figures 2C, D, and E to 4C, D, and E present a closer look at the ablated regions on the anodic side, in the middle of the treated tissue, and on the cathodic side, respectively. The histology seen at the margin between treated and normal tissue near the anode side of the liver is typical for electrolysis. As seen in Figure 2C, a sharp border, several hepatocytes thick, exists between the lesion (left) and the surrounding normal liver (right). A mild congestion in the affected sinusoids delineates the treated lesion from the unaffected tissue. The cells are eosinophilic with a visible outline and a disrupted cytoplasm. The nucleus outline is preserved, although it appears slightly condensed. These histological features also appear in Figures 3C and 4C.
In Figure 2D, a mild congestion can be observed in the affected sinusoids toward the center of the liver. This congestion delineates the treated tissue from the surrounding normal tissue. The transition zone between the normal (right) and ablated (left) cells is much larger than seen near the anode, and pockets of cells with normal appearance are present between affected cells. The affected cells are eosinophilic and resemble the cells near the anode. Here, too, the nucleus outline is preserved, although slightly condensed. These characteristics are also seen in Figure 3D, where the border between the top ablated region and the center of the tissue is examined. In Figure 4D, for the lowest delivered electrolytic charge, hepatocytes toward the center of the liver between the two electrode surfaces are intact and normal. It is important to observe that the hepatocytes in the center of the liver between the electrodes in Figure 2D are affected due to the transverse ablation, and the hepatocytes in the center region are normal in Figure 4D due to the nontransverse ablation zone.
The effect of the electrolytic ablation at the border of the treated area on the side of the liver in contact with the cathode is examined in Figures 2E, 3E, and 4E. Here, as near the anode, there is a sharp border, several hepatocytes thick, between the lesion and the surrounding normal tissue, and once again a mild congestion in the affected sinusoids delineates the treated lesion from the surrounding normal tissue. The treated cells near the cathode differ from those near the anode; the nucleus is pale and retained less stain than near the anode, likely due to a loss of chemical function. As a result of cell lysis, there is a disruption of the plasma membrane, homogenization of the cytoplasm, and loss of normal lobular structure. It can be seen here that the cells near the anode are more eosinophilic than near the cathode. A band of edematous tissue near the cathode side can be viewed in more detail in Figures 3E and 4E. Although this band is not present in Figure 2E for the 1.25 C/cm2 delivered charge, it should be emphasized that edematous tissue bands similar to what is seen in Figures 3E and 4E were observed in some cases for the parameters of Figure 2. Figure 2 is used here to illustrate how blood flow and large blood vessels can affect the local outcome of electrolytic ablation procedures. A possible explanation to this observation might have to do with the different electrical properties of blood vessels relative to liver tissue. By being more conductive than their environment, blood vessels might create preferential paths for current flow, which concentrates the electrolytic damage in their vicinity. This is an interesting phenomenon that deserves additional exploration, and we intend to pursue it in our future work.
Liver Histology After Treatment by E2 Protocols
The effects of applying E2 protocols in the form of multiple reversible electroporation field strength pulses across the liver sample are depicted in Figures 5 to 7. These results serve as a means to examine the general pattern of how tissue is affected by E2 protocols as a function of the total delivered charge dosage. Our goal was to compare the pattern observable under E2 stimulation to the results from conventional electrolytic studies presented in Figures 2 to 4.
Figure 5 presents a sample treated with a typical reversible electroporation protocol of 8 pulses of 100 μs in length at a frequency of 1 Hz, resulting in a total applied charge per unit area of 0.0001 C/cm2. Trichrome staining shows tissue damage near the points of contact with the electrodes (Figure 5A), and the ablation results observed in Figure 5A are consistent with pervious observations by Marshall group. 45 –47 The region of tissue ablation has been roughly outlined, and it can be seen that the treated area surrounds the liver surface that was in contact with the anode and cathode, while the center area of the liver sample appears normal. The H&E staining presents consistent findings—lesions can be seen near the surfaces in contact with the electrodes (Figure 5B), and a clear delineation is observed between treated and nontreated areas. This figure clearly shows that the damage is local, and the affected area does not span the entire liver sample.
Figure 6 presents histology of a liver sample treated with E2 sequence of 99 pulses of 100 μs long applied at a 1-Hz frequency to deliver a total charge dose of 0.0012 C/cm2. To compare ablated volumes between different sets of parameters, the area of the tissue affected by the procedure has been outlined (Figure 6A). It can be seen here that the affected area surrounds the liver surfaces that were in contact with the anode and cathode, while the bulk area of the liver sample appears normal. The H&E staining also shows that the damage is nontransverse, that is, the lesion does not span the liver sample vertically. As before, a clear delineation is seen between treated and nontreated areas (Figure 6B).
The final set of experimental parameters in this study consisted of an E2 sequence of 99 pulses, at 1 ms pulse length, and an applied frequency of 1 Hz, resulting in a total charge dose of 0.0124 C/cm2. This experimental group produced transverse damage, that is, a lesion that penetrates transversely through the liver from the top surface to the bottom surface, as outlined in Figure 7A. The H&E staining is consistent with transverse damage as it also shows that the lesion vertically spans the liver sample, and a clear delineation between treated and nontreated areas is observed (Figure 7B).
In Figures 5 to 7, the treated region exhibits variable pale eosinophilia and widespread congestion as compared to the normal parenchyma, similar to the tissue treated with conventional electrolysis in Figures 2 to 4. The lesion shape is irregular, and, for higher levels of delivered charge (Figures 6 and 7), the volume of ablated tissue is larger near the anodic surface (top side) in comparison to the cathodic surface (bottom side of liver). The ablated regions are further examined on the anodic side (Figures 5C, 6C, and 7C), in the middle of the treated tissue (Figures 5D, 6D, and 7D), and on the cathodic side (Figures 5E, 6E, and 7E). Typical electrolysis histology can be seen at the margin between treated and normal liver on the side of the liver that was in contact with the anode (Figures 5C, 6C, and 7C). There is a sharp border, several hepatocytes thick, between the lesion and the surrounding normal liver. The cells are eosinophilic with a visible outline and a disrupted cytoplasm, and the nucleus outline is preserved, although slightly condensed.
In Figure 5D, the region toward the center of the tissue consists of hepatocytes that are intact and normal, further indicating that the ablation was nontransverse, that is, the lesion did not penetrate through the entire sample thickness. Figure 6D focuses on the border between the lesion and the normal tissue in the center of the sample. Here, the appearance is very similar to what was seen in the tissue close to the anodic side, with congestion in the affected sinusoids delineating the treated tissue (top) from the surrounding normal tissue (bottom). The affected cells are eosinophilic and resemble the cells near the anode. In Figure 7, for a delivered charge of 0.0124 C/cm2, the treated region toward the center of the liver (Figure 7D) mimics the electrolytic ablation results, displaying a mild congestion of the affected sinusoids, and eosinophilic cells in the treated region resemble those near the anode. Here, there is a larger transition between normal (right) and ablated (left) cells, with pockets of cells that appear normal observable between regions of affected cells.
A closer look at the border of the treated area on the side of the liver in contact with the cathode (Figure 5E) reveals a sharp transition zone, several hepatocytes thick, between the lesion and the surrounding normal tissue. The treated cells near the cathode differ from those near the anode, as shown in Figures 5E, 6E, and 7E. The nucleus is pale and retains less stain than near the anode, likely due to a loss of chemical function. As a result of cell lysis, there is a disruption of the plasma membrane, homogenization of the cytoplasm, and loss of normal lobular structure. In comparison, the cells near the anode are more eosinophilic than near the cathode.
Discussion
This study examines the histology of tissue in which charge is delivered by an E2 protocol using multiple reversible electroporation field strength pulses in comparison to the histology of tissue when charge is delivered via conventional electrolysis protocols. The study of conventional electrolytic ablation uses a set of parameters comparable to that typically used in the literature. The results for the experiments in which a charge of 1.25 C/cm2 was delivered with conventional electrolysis are shown in Figure 2 and are consistent with other studies on the histology of electrolysis of the liver. 22,47 Specifically, the entire ablated tissue was sharply demarcated from the normal liver tissue, with visible congestion in the sinusoids at the margin of the lesion. The appearance of the treated tissue near the anode, the cathode, and in the core of the treated tissue is markedly different. Near the cathode, there is colliquative necrosis, the nuclei are not stained, the cell membrane is not detected, and there is homogenization of the cytoplasm and loss of lobular structure. Near the anode the damage is coagulative, the cytoplasm is eosinophilic, and the nuclei morphology is visible. The core of the ablated zone, between the cathode and the anode, is eosinophilic. The dissimilar appearance of the tissue is caused by different products of electrolysis near the anode and the cathode, resulting in different ablative reactions. Near the anode, the pH is low, and many of the toxic chemical species are related to the various pH-dependent components of Cl. Near the cathode, on the other hand, the pH is high, and the toxicity is primarily caused by various components of OH−. 22
Studies have shown that the tissue ablation produced by conventional electrolytic ablation protocols is dependent on the amount of charge delivered. The ablated regions occur near the electrode for lower charges and are not transverse; the ablated region increases outward from the electrode with an increase in the delivered charge to become transverse. The dependency of the extent of ablation on the charge dosage is shown in Figures 2 to 4. The observed decrease in the degree of ablation with a decreasing total delivered charge dosage is consistent with that reported in the literature, and these histological results are also consistent with many other studies on electrolysis. 22,47,51 The experimental results shown in Figures 2 to 4 serve to illustrate a general pattern of how the degree of tissue damage caused by conventional electrolytic ablation protocols is a function of the charge delivered. The affected region propagates from the electrodes toward the core of the tissue, and the extent of tissue ablation is directly related to the amount of charge delivered. For lower doses of charge, the ablation is nontransverse, that is, localized to the electrode surfaces, and as the charge dosage is increased, so, too, does the extent of the damage. Above a certain threshold, the damage becomes transverse, that is, the lesion penetrates through the entire thickness of the sample, as seen in Figure 2. Furthermore, the appearance of the treated tissue near the anode is different from that near the cathode. For the higher levels of delivered charge, the treated volume near the anode is larger than that seen near the cathode. These characteristics appear to be the hallmarks of conventional electrolytic ablation.
For the experimental protocols that applied E2 protocols, the first key observation is that reversible electroporation field strength pulses can indeed cause tissue ablation. Furthermore, the resulting pattern of tissue ablation is very similar to that obtained by conventional electrolysis. Results from an 8 pulse reversible electroporation protocol (250 V/cm, 100 ms pulses at a frequency of 1 Hz) typical to reversible electroporation treatments 37,38,48,50 are shown in Figure 5. The results in Figure 5 show that the morphology of the affected regions of the tissue is similar to that obtained with electrolysis. This is consistent with the findings of Marshall's group, who have shown and predicted that electrolysis should occur in conventional reversible electroporation for gene therapy. 44 –46 As is typical for electrolytic ablation, the histology near the anode and cathode is different, and the tissue damage is localized near the electrodes.
Before we introduce the observation differentiating E2 ablation from traditional electrolysis ablation, we would like to direct the reader’s attention to Figure 8, which synthesizes the Masson trichrome staining results from the 6 representative experiments. This figure is introduced to aid the comparison between the two modalities and introduces no new information; in fact, we obtained Figure 8 by grouping the first (A) image of Figures 2 to 7. The left column aggregates images of conventional electrolysis-treated samples by decreasing charge dosage, and the right column aggregates images of E2-treated samples by decreasing charge dosage.
Aggregated Masson trichrome staining results from 6 representative experiments. A, Electrolysis 1.25 C/cm2, (B) electrolysis 0.625 C/cm2, (C) electrolysis 0.31 C/cm2, (D) E2 0.0124 C/cm2, (E) E2 0.0012 C/cm2, and (F) E2 0.0001 C/cm2.
The second key observation following from the E2 experimental series can be seen in Figure 7: the affected tissue retained the characteristics seen in conventional electrolytic ablation. The damage is transverse, that is, the lesion penetrates through the liver, and the appearance of the treated lesion at the anode is different from that at the cathode. The E2 results could be conveniently compared to conventional electrolysis results using Figure 8 as an aid, in order to highlight a very important observation from this study: when the damage was applied using the E2 modality, relative to conventional electrolytic ablation, a much lower charge dosage was required to cause a comparable damage. In Figure 8D, a transverse liver sample damage is achieved with a E2 protocol by delivering a charge of 0.0124 C/cm2. In comparison, in Figure 8C, the damage is only partial, although the conventional electrolysis delivered 25 times more charge per unit area of electrode (0.312 C/cm2). In summary, the histological results in Figures 5 to 7 show that the appearance of the treated tissue near the anode is different than that near the cathode, consistent with electrolytic ablation. The key finding of this study can be emphasized further when comparing the ablated volume obtained using E2 with a total delivered charge of 0.0124 C/cm2 (Figure 8D) to the volume ablated after applying a total charge of 1.25 C/cm2 through conventional electrolysis (Figure 8A). The degree of damage caused by the two methods is comparable, qualitatively referred to as transverse in our notation, but it is noteworthy that E2 applies between 1 and 2 orders of magnitude less charge. It is also interesting to note that the total time of delivering the charge with the E2 protocol is shorter by at least two orders of magnitude from the time of delivering the ablative charge with conventional electrolysis.
The results presented here show that tissue treated by E2 protocols exhibits some degree of similarity to a damage pattern associated with electrolytic ablation. Specifically, the extent of the affected zone increases with the number of pulses delivered and the pulse length, that is, the total charge delivered, and the ablated region starts close to the electrodes and moves outward into the tissue. This is typical of a diffusion driven effect. This pattern is different from ablation induced by electroporation, which is generated when an electric field is produced between the electrodes. Because the electric field is essentially produced instantaneously and uniformly near the centerline between the electrodes, the affected region around the centerline between the electrodes has a homogenous appearance. 40 Therefore, the nature of tissue damage caused by E2 protocols is different from that caused by irreversible electroporation. To further differentiate with irreversible electroporation, E2 protocols result in a treated volume which is preferentially larger at the anode, compared to the cathode. All these features appear to be the hallmarks of conventional electrolytic ablation as presented in Figures 2 to 4 and discussed previously. The results of this study suggest that when repeated reversible electroporation field pulses are delivered to tissue, the mechanism of cell death appears to be consistent with the mechanism of electrolytic ablation. We believe that this study produces further support to the findings of Marshall's group that electrolysis can occur in reversible electroporation, proving lethal to the electroporated cells. 44 –46 The toxicity of the electrolytic reactions at the electrodes has also been well known in the food industry. A recent comprehensive review on this issue can be found in the study of Martin-Belloso and Elez-Martinez. 52
Due to the different damage mechanisms between electroporation-based approaches and conventional electrolysis, we have found that the quantity allowing a meaningful comparison between these two electrotreatment modalities is the total charge dosage delivered. We have designed our study to evaluate the qualitative degree of damage as a function of total charge dosage delivered. As shown in Figures 3 and 4, a conventional electrolysis delivered dose of 0.312 and 0.624 C/cm2, respectively, causes only nontransverse damage. In contrast, delivering a dose of 0.0124 C/cm2 using 1 ms pulses with an E2 protocol produces a transverse damage to the tissue, resulting in a lesion that penetrates through the liver thickness (Figure 7). The time scale for diffusion is comparable. In addition, after the total charge was delivered, each animal was observed for half an hour while under anesthesia, allowing the tissues to be exposed to the electrolytic components for a similar period of time for both the conventional electrolysis and E2 procedures. Therefore, it is apparent that other factors, beyond the total delivered charge, influence the differences observed between results obtained with E 2 protocols in comparison with conventional electrolysis. Reversible electroporation results in the permeabilization of the cell membrane, and this effect should occur uniformly between the two parallel electrodes used in this study. In comparison to the time scale of diffusion by electrolysis, the permeabilization of the cell membrane by reversible electroporation is an instantaneous phenomenon. Therefore, this study suggests that reversibly electroporated cells are affected by much lower doses of electrolytic products than intact cells, and thus E2 protocols can be used as a more effective electroablation technique than conventional electrolysis.
Although this remains to be proven, a reasonable explanation for the observed phenomena of increased damage under E2 is the interaction between the permeabilization of the cell membrane and the products of electrolysis. Leveraging this interaction presents the potential to induce cell death using a lower charge dosage in a permeabilized cell in comparison to a cell with an intact membrane.
A possible application of the findings in this study is a method of tissue ablation that uses reversible electroporation strength fields to permeabilize the cell membrane to products of electrolysis and thereby induce cell death. This study explored a protocol in which the reversible electroporation field strength pulses also produce products of electrolysis at the electrodes. However, other uses of this concept are possible. For instance, conventional electrolysis could be done first and then reversible electroporation pulses could be delivered to enhance cell death in the region in which the cell membrane has been permeabilized and to facilitate the penetration of the products of electrolysis into the cells or vice versa.
The use of E2 in tissue ablation also has a potential application in extending the ablated tissue volume in conventional irreversible electroporation without increasing the voltage on the electrodes. When irreversible electroporation field pulses are applied between electrodes, the tissue ablation protocol is designed to produce irreversible electroporation fields in a certain volume of tissue adjacent to the electrodes. However, due to the nature of the electric field distribution, a region exists outside the irreversible electric field zone in which the field is lower, that is, typical of reversible electroporation protocols. The simple change in protocol of producing multiple pulses will generate damage in the region exterior to the irreversible electroporated region through the E2 phenomena. Obviously the multiple pulses need to be designed in such a way that the electrolytic products will diffuse to the regions in which reversible electroporation fields form. Nevertheless, this could be a simple method to extend the tissue ablation affected volume during irreversible electroporation.
Conclusion
We humbly acknowledge that this work on the use of multiple reversible electroporation field pulses for tissue ablation is a preliminary study inviting further exploration. Substantial additional research and quantitative analysis are necessary to provide further support for the benefits of E2 protocols as an ablation method. Nonetheless, here it is shown that reversible electroporation can be used to produce tissue ablation that is histologically similar to electrolysis. In addition, the E2 approach was demonstrated to be more effective, resulting in a greater extent of damage for a much smaller applied charge per electrode area.
We see this work as a first step toward confirming our study’s hypothesis that electrolytic ablation can be made more effective by delivering the electric charge through multiple reversible electroporation field pulses. The study also suggests that E2 protocols may be a promising new method for tissue ablation. Furthermore, employing multiple electroporation pulses could also be used to increase the extent of tissue treated by conventional irreversible electroporation: if the electrolytic compounds generated during the process diffuse beyond the irreversibly electroporated zone to the adjacent reversibly electroporated zone, a larger volume of tissue may be treated.
Footnotes
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Boris Rubinsky has a patent application related to this technology.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.