Cryoelectrolysis; an acute case study in the pig liver.

We report results from an acute, single case study in the pig liver on the effects of a tissue ablation protocol (we named cryoelectrolysis) in which 10 min of cryosurgery, with a commercial cryosurgical probe, are delivered after 10 min of electrolysis generated by a current of about 60 mA. The histological appearance of tissue treated with cryoelectrolysis is compared with the appearance of tissue treated with 10 min of cryosurgery alone and with 10 min of electrolysis alone. Histology done after 3 h survival shows that the mixed rim of live and dead cells found around the ablated lesion in both cryosurgery and electrolytic ablation is replaced by a sharp margin between life and dead cells in cryoelectrolysis. The appearance of the dead cells in each, cryoelectrolysis, cryosurgery and electrolytic ablation is different. Obviously, this is an acute study and the results are only relevant to the conditions of this study. There is no doubt that additional acute and chronic studies are needed to strengthen and expand the findings of this study.

Electrical breakdown in tissue electroporation.

Electroporation, the permeabilization of the cell membrane by brief, high electric fields, has become an important technology in medicine for diverse application ranging from gene transfection to tissue ablation. There is ample anecdotal evidence that the clinical application of electroporation is often associated with loud sounds and extremely high currents that exceed the devices design limit after which the devices cease to function. The goal of this paper is to elucidate and quantify the biophysical and biochemical basis for this phenomenon. Using an experimental design that includes clinical data, a tissue phantom, sound, optical, ultrasound and MRI measurements, we show that the phenomenon is caused by electrical breakdown across ionized electrolysis produced gases near the electrodes. The breakdown occurs primarily near the cathode. Electrical breakdown during electroporation is a biophysical phenomenon of substantial importance to the outcome of clinical applications. It was ignored, until now.

Toward a clinical real time tissue ablation technology: combining electroporation and electrolysis (E2).

BACKGROUND: Percutaneous image-guided tissue ablation (IGA) plays a growing role in the clinical management of solid malignancies. Electroporation is used for IGA in several modalities: irreversible electroporation (IRE), and reversible electroporation with chemotoxic drugs, called electrochemotherapy (ECT). It was shown that the combination of electrolysis and electroporation-E2-affords tissue ablation with greater efficiency, that is, lower voltages, lower energy and shorter procedure times than IRE and without the need for chemotoxic additives as in ECT. METHODS: A new E2 waveform was designed that delivers optimal doses of electroporation and electrolysis in a single waveform. A series of experiments were performed in the liver of pigs to evaluate E2 in the context of clinical applications. The goal was to find initial parameter boundaries in terms of electrical field, pulse duration and charge as well as tissue behavior to enable real time tissue ablation of clinically relevant volumes. RESULTS: Histological results show that a single several hundred millisecond long E2 waveform can ablate large volume of tissue at relatively low voltages while preserving the integrity of large blood vessels and lumen structures in the ablation zone without the use of chemotoxic drugs or paralyzing drugs during anesthesia. This could translate clinically into much shorter treatment times and ease of use compared to other techniques that are currently applied.

Optimal parameters for the destruction of prostate cancer using irreversible electroporation.

PURPOSE: Irreversible electroporation is a new tissue ablation technique that consists of applying musecond pulses of direct current to create permanent defects in the cell membrane. Irreversible electroporation spares connective tissue in blood vessels and other tissue structures. When applied properly, it does not produce thermal damage. We determined the irreversible electroporation parameters that would reliably destroy prostate cancer cells. MATERIALS AND METHODS: Irreversible electroporation pulses were applied to prostate adenocarcinoma cells in vitro. Three sets of studies were performed to determine the number, length and field strength of irreversible electroporation pulses required to produce complete cancer cell ablation without inducing thermal effects. The outcome of a treatment protocol was simulated. RESULTS: We found the upper and lower limit bounds of pulse length and number in a field range of 2,000 to 250 V/cm. A total of 90 pulses at 250 V/cm for 100 museconds separated by 100 milliseconds could completely ablate prostate cancer cells without inducing thermal damage. CONCLUSIONS: Irreversible electroporation represents a new nonthermal ablation modality. This study has produced values for prostate cancer treatment with irreversible electroporation.

Irreversible electroporation: implications for prostate ablation.

Percutaneous prostate cryo-ablation has become an accepted treatment for primary prostate cancer. Thermal tissue ablation based on cold, however, does have some distinct limitations. These include, variable damage at the cryo lesions margin, injury to adjacent structures such as rectum, urethra and NVB (neurovascular bundle), and long procedure time due to the need for multiple freeze thaw cycles, that have limited the acceptance of this modality. Irreversible electroporation IRE, is a new non-thermal ablation modality that uses short pulses of DC electric current to create irreversible pore in the cell membrane, thus, causing cell death. This method theoretically should have significant advantages in ablating prostate tissue. Six males dogs had their prostates treated using IRE. Pulses were applied using a DC generator that delivered pulses in the microsecond range of duration, with a variable pulse interval and voltage range. IRE probes were placed percutaneously or trans-rectally using trans-rectal ultrasound guidance. In one of the dogs, the lesions were made purposely to include the rectum, urethra, and neurovascular bundle (NVB). Subjects were followed for 1 to 14 days before sacrifice. IRE lesions in the prostate had unique characteristics compared to thermal lesions. The margins of the IRE lesions was very distinct with a narrow zone of transition from normal to complete necrosis, there was complete destruction within the IRE lesion, and rapid resolution of the lesions with marked shrinkage within two weeks. Structures such as urethra, vessels, nerves, and rectum were unaffected by the IRE application. IRE lesions have characteristics that are distinctly different than thermal lesions. The differences could be very advantageous in a clinical setting, improving the results and acceptance of prostate ablation.

Irreversible electroporation: a new ablation modality--clinical implications.

Irreversible electroporation (IRE) is a new tissue ablation technique in which micro to millisecond electrical pulses are delivered to undesirable tissue to produce cell necrosis through irreversible cell membrane permeabilization. IRE affects only the cell membrane and no other structure in the tissue. The goal of the study is to test our IRE tissue ablation methodology in the pig liver, provide first experience results on long term histopathology of IRE ablated tissue, and discuss the clinical implications of the findings. The study consists of: a) designing an IRE ablation protocol through a mathematical analysis of the electrical field during electroporation; b) using ultrasound to position the electroporation electrodes in the predetermined locations and subsequently to monitor the process; c) applying the predetermined electroporation pulses; d) performing histopathology on the treated samples for up to two weeks after the procedure; and e) correlating the mathematical analysis, ultrasound data, and histology. We observed that electroporation affects tissue in a way that can be imaged in real time with ultrasound, which should facilitate real time control of electroporation during clinical applications. We observed cell ablation to the margin of the treated lesion with several cells thickness resolution. There appears to be complete ablation to the margin of blood vessels without compromising the functionality of the blood vessels, which suggests that IRE is a promising method for treatment of tumors near blood vessels (a significant challenge with current ablation methods). Consistent with the mechanism of action of IRE on the cell membrane only, we show that the structure of bile ducts, blood vessels, and connective tissues remains intact with IRE. We report extremely rapid resolution of lesions, within two weeks, which is consistent with retention of vasculature. We also document tentative evidence for an immunological response to the ablated tissue. Last, we show that mathematical predictions with the Laplace equation can be used in treatment planning. The IRE tissue ablation technique, as characterized in this report, may become an important new tool in the surgeon armamentarium.

A novel nonthermal energy source for surgical epicardial atrial ablation: irreversible electroporation.

BACKGROUND: All currently used energy sources in surgical ablation for atrial fibrillation create lesions via thermal injury. We report for the first time the in vivo results of a new nonthermal modality of epicardial atrial ablation called irreversible electroporation (IRE). IRE utilizes a sequence of electrical pulses that produce permanent nonthermal damage to tissue in a few seconds with sharp borders between affected and unaffected regions. METHODS: Five pigs underwent beating heart surgical epicardial ablations of their right and/or left atrial appendages, utilizing a sequence of 8, 16, or 32 direct current pulses of 1500 to 2000 V, 100 micros each, at a frequency of 5 per second, applied between two 4-cm long parallel electrodes with an IRE pulse generator. Local temperature measurements were performed during ablations followed by electrical isolation testing by pacing. Animal hearts were excised 24 hours after surgery and processed histologically to evaluate the degree of myocardial tissue necrosis and transmurality. RESULTS: A clear demarcation line between ablated and normal tissue, with no tissue disruption or charring, was observed on gross inspection of all lesions. Staining results showed complete transmural destruction of atrial tissue at the site of the electrode application in all 10 atrial lesions, measuring a mean of 0.9 cm in depth. Each 3- to 3.5-cm long lesion was created in 1 to 4 seconds with no local temperature change and with demonstration of electrical isolation. CONCLUSIONS: We propose a new modality to perform atrial ablations, which holds the potential of providing very swift, precise, and complete transmurality with no local heating effects.

Single exponential decay waveform; a synergistic combination of electroporation and electrolysis (E2) for tissue ablation.

BACKGROUND: Electrolytic ablation and electroporation based ablation are minimally invasive, non-thermal surgical technologies that employ electrical currents and electric fields to ablate undesirable cells in a volume of tissue. In this study, we explore the attributes of a new tissue ablation technology that simultaneously delivers a synergistic combination of electroporation and electrolysis (E2). METHOD: A new device that delivers a controlled dose of electroporation field and electrolysis currents in the form of a single exponential decay waveform (EDW) was applied to the pig liver, and the effect of various parameters on the extent of tissue ablation was examined with histology. RESULTS: Histological analysis shows that E2 delivered as EDW can produce tissue ablation in volumes of clinical significance, using electrical and temporal parameters which, if used in electroporation or electrolysis separately, cannot ablate the tissue. DISCUSSION: The E2 combination has advantages over the three basic technologies of non-thermal ablation: electrolytic ablation, electrochemical ablation (reversible electroporation with injection of drugs) and irreversible electroporation. E2 ablates clinically relevant volumes of tissue in a shorter period of time than electrolysis and electroporation, without the need to inject drugs as in reversible electroporation or use paralyzing anesthesia as in irreversible electroporation.

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.

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.