Antoni Ivorra

The Effect of Irreversible Electroporation on Blood Vessels

We present a pilot study on the long term effects of irreversible electroporation (IRE) on a large blood vessel. The study was motivated by the anticipated use of IRE for treatment of cancer tumors abutting large blood vessels. A sequence of 10 direct current IRE pulses of 3800 V/cm, 100μs each, at a frequency of 10 pulses per second, were applied directly to the carotid artery in six rats. Measuring tissue conductivity during the procedure showed, as predicted, an increase in conductivity during the application of the pulse, which suggests that this measurement can be used to control the application of IRE. All the animals survived the procedure and showed no side effects. Histology performed 28 days after the procedure showed that the connective matrix of the blood vessels remained intact and the number of vascular smooth muscle cells (VSMC) in the arterial wall decreased with no evidence of aneurysm, thrombus formation or necrosis. Average VSMC density was significantly lower following IRE ablation compared with control (24 ± 11 vs. 139 ± 14, P<0.001), with no apparent damage to extra cellular matrix components and structure. In addition to the relevance of this study to treatment of cancer near large blood vessels these findings tentatively suggest that IRE has possible applications to treatment of pathological processes in which it is desired to reduce the proliferation of VSMC population, such as restenosis and for attenuating atherosclerotic processes in clinical important locations such as coronary, carotid and renal arteries.

In vivo electrical conductivity measurements during and after tumor electroporation: conductivity changes reflect the treatment outcome

Electroporation is the phenomenon in which cell membrane permeability is increased by exposing the cell to short high-electric-field pulses. Reversible electroporation treatments are used in vivo for gene therapy and drug therapy while irreversible electroporation is used for tissue ablation. Tissue conductivity changes induced by electroporation could provide real-time feedback of the treatment outcome. Here we describe the results from a study in which fibrosarcomas (n = 39) inoculated in mice were treated according to different electroporation protocols, some of them known to cause irreversible damage. Conductivity was measured before, within the pulses, in between the pulses and for up to 30 min after treatment. Conductivity increased pulse after pulse. Depending on the applied electroporation protocol, the conductivity increase after treatment ranged from 10% to 180%. The most significant conclusion from this study is the fact that post-treatment conductivity seems to be correlated with treatment outcome in terms of reversibility.

Electrical impedance characterization of normal and cancerous human hepatic tissue

The four-electrode method was used to measure the ex vivo complex electrical impedance of tissues from 14 hepatic tumors and the surrounding normal liver from six patients. Measurements were done in the frequency range 1-400 kHz. It was found that the conductivity of the tumor tissue was much higher than that of the normal liver tissue in this frequency range (from 0.14 +/- 0.06 S m(-1) versus 0.03 +/- 0.01 S m(-1) at 1 kHz to 0.25 +/- 0.06 S m(-1) versus 0.15 +/- 0.03 S m(-1) at 400 kHz). The Cole-Cole models were estimated from the experimental data and the four parameters (rho(0), rho(infinity), alpha, f(c)) were obtained using a least-squares fit algorithm. The Cole-Cole parameters for the cancerous and normal liver are 9 +/- 4 Omega m(-1), 2.2 +/- 0.7 Omega m(-1), 0.5 +/- 0.2, 140 +/- 103 kHz and 50 +/- 28 Omega m(-1), 3.2 +/- 0.6 Omega m(-1), 0.64 +/- 0.04, 10 +/- 7 kHz, respectively. These data can contribute to developing bioelectric applications for tissue diagnostics and in tissue treatment planning with electrical fields such as radiofrequency tissue ablation, electrochemotherapy and gene therapy with reversible electroporation, nanoscale pulsing and irreversible electroporation.

Non Thermal Irreversible Electroporation: Novel Technology for Vascular Smooth Muscle Cells Ablation

Background Non thermal Irreversible electroporation (NTIRE) is a new tissue ablation method that induces selective damage only to the cell membrane while sparing all other tissue components. Our group has recently showed that NTIRE attenuated neointimal formation in rodent model. The goal of this study was to determine optimal values of NTIRE for vascular smooth muscle cell (VSMC) ablation. Methods and Results 33 Sprague-Dawley rats were used to compare NTIRE protocols. Each animal had NTIRE applied to its left common carotid artery using a custom-made electrodes. The right carotid artery was used as control. Electric pulses of 100 microseconds were used. Eight IRE protocols were compared: 1–4) 10 pulses at a frequency of 10 Hz with electric fields of 3500, 1750, 875 and 437.5 V/cm and 5–8) 45 and 90 pulses at a frequency of 1 Hz with electric fields of 1750 and 875 V/cm. Animals were euthanized after one week. Histological analysis included VSMC counting and morphometry of 152 sections. Selective slides were stained with elastic Van Gieson and Masson trichrome to evaluate extra-cellular structures. The most efficient protocols were 10 pulses of 3500 V/cm at a frequency of 10 Hz and 90 pulses of 1750 V/cm at a frequency of 1 Hz, with ablation efficiency of 89±16% and 94±9% respectively. Extra-cellular structures were not damaged and the endothelial layer recovered completely. Conclusions NTIRE is a promising, efficient and simple novel technology for VMSC ablation. It enables ablation within seconds without causing damage to extra-cellular structures, thus preserving the arterial scaffold and enabling endothelial regeneration. This study provides scientific information for future anti-restenosis experiments utilizing NTIRE.

A New Concept for Medical Imaging Centered on Cellular Phone Technology

According to World Health Organization reports, some three quarters of the world population does not have access to medical imaging. In addition, in developing countries over 50% of medical equipment that is available is not being used because it is too sophisticated or in disrepair or because the health personnel are not trained to use it. The goal of this study is to introduce and demonstrate the feasibility of a new concept in medical imaging that is centered on cellular phone technology and which may provide a solution to medical imaging in underserved areas. The new system replaces the conventional stand-alone medical imaging device with a new medical imaging system made of two independent components connected through cellular phone technology. The independent units are: a) a data acquisition device (DAD) at a remote patient site that is simple, with limited controls and no image display capability and b) an advanced image reconstruction and hardware control multiserver unit at a central site. The cellular phone technology transmits unprocessed raw data from the patient site DAD and receives and displays the processed image from the central site. (This is different from conventional telemedicine where the image reconstruction and control is at the patient site and telecommunication is used to transmit processed images from the patient site). The primary goal of this study is to demonstrate that the cellular phone technology can function in the proposed mode. The feasibility of the concept is demonstrated using a new frequency division multiplexing electrical impedance tomography system, which we have developed for dynamic medical imaging, as the medical imaging modality. The system is used to image through a cellular phone a simulation of breast cancer tumors in a medical imaging diagnostic mode and to image minimally invasive tissue ablation with irreversible electroporation in a medical imaging interventional mode.

In vivo imaging of irreversible electroporation by means of electrical impedance tomography

Electroporation, the increased permeability of cell membranes due to a large transmembrane voltage, is an important clinical tool. Both reversible and irreversible in vivo electroporation are used for clinical applications such as gene therapy and solid malignant tumor ablation, respectively. The primary advantage of in vivo electroporation is the ability to treat tissue in a local and minimally invasive fashion. The drawback is the current lack of control over the process. This paper is the first report of a new method for real-time three-dimensional imaging of an in vivo electroporation process. Using two needle electrodes for irreversible electroporation and a set of electrodes for reconstructing electrical impedance tomography (EIT) images of the treated tissue, we were able to demonstrate electroporation imaging in rodent livers. Histology analysis shows good correlation between the extent of tissue damage caused by irreversible electroporation and the EIT images. This new method may lead the way to real-time control over genetic treatment of diseases in tissue and tissue ablation.

Irreversible electroporation shows efficacy against pancreatic carcinoma without systemic toxicity in mouse models

Pancreatic ductal adenocarcinoma (PDAC) therapies show limited success. Irreversible electroporation (IRE) is an innovative loco-regional therapy in which high-voltage pulses are applied to induce plasma membrane defects leading to cellular death. In the present study we evaluated the feasibility of IRE against PDAC. IRE treatment exhibited significant antitumor effects and prolonged survival in mice with orthotopic xenografts. Extensive tumor necrosis, reduced tumor cell proliferation and disruption of microvessels were observed at different days post-IRE. Animals had transient increases in transaminases, amylase and lipase enzymes that normalized at 24h post-IRE. These results suggest that IRE could be an effective treatment for locally advanced pancreatic tumors.

Irreversible Electroporation Attenuates Neointimal Formation After Angioplasty

Restenosis following coronary angioplasty represents a major clinical problem. Irreversible electroporation (IRE) is a nonthermal, nonpharmacological cell ablation method. IRE utilizes a sequence of electrical pulses that produce permanent damage to tissue within a few seconds. The left carotid arteries of eight rats underwent in vivo intimal damage using two Fogarty angioplasty catheters. The procedure was immediately followed by IRE ablation in four rats, while the remaining four were used as the control group. The IRE ablation was performed using a sequence of ten dc pulses of 3800 V/cm, 100 mus each, at a frequency of ten pulses per second, applied across the blood vessel between two parallel electrodes. The electrical conductance of the treated tissue was measured during the electroporation to provide real-time feedback of the process. Left carotid arteries were excised and fixated after a 28-day follow-up period. Neointimal formation was evaluated histologically. The use of IRE was successful in three out of four animals in a way that is consistent with the measurements of blood vessel electrical properties. The integrity of the endothelial layer was recovered in the IRE-treated animals, compared with control. Successful IRE reduced neointima to media ratio (0.57 plusmn 0.4 versus 1.88 plusmn 1.0, P = 0.02). Conclusions: We report for the first time the in vivo results of attenuation of neointimal formation using IRE. Our study shows that IRE might be able to attenuate neointimal formation after angioplasty damage in a rodent model of restenosis. This approach may open new venues in the treatment of coronary artery restenosis after balloon angioplasty.

Electrical modeling of the influence of medium conductivity on electroporation

Electroporation is the phenomenon in which cell membrane permeability is increased by exposing the cell to short high electric field pulses. Experimental data show that the amount of permeabilization depends on the conductivity of the extracellular medium. If medium conductivity decreases then it is necessary to deliver a pulse of larger field amplitude in order to achieve the same effect. Models that do not take into account the permeabilization effect on the membrane conductivity cannot reproduce qualitatively the experimental observations. Here we employ an exponential function for describing the strong dependence of membrane conductivity on transmembrane potential. Combining that model with numerical methods we demonstrate that the dependence on medium conductivity can be explained as being the result of increased membrane conductance due to electroporation. As experimentally observed, extracellular conductivities of about 1 and 0.1 S m(-1) yield very similar results, however, for lower conductivities (<0.01 S m(-1)) the model predicts that significantly higher field magnitudes will be required to achieve the same amount of permeabilization.

Electric Field Redistribution due to Conductivity Changes during Tissue Electroporation: Experiments with a Simple Vegetal Model

Electroporation, or electropermeabilization, is the phenomenon in which cell membrane permeability to ions and macromolecules is increased by exposing the cell to short high electric field pulses. In living tissues, such permeabilization boost can be used in order to enhance the penetration of drugs (electrochemotherapy) or DNA plasmids (electrogenetherapy) or to destroy undesirable cells (irreversible electroporation). During the application of the high voltage pulses required for in vivo electroporation treatments, the conductivity of the involved tissues increases due to the electroporation phenomenon. This alteration results in a redistribution of the electric field magnitude that should be taken into account at treatment planning in order to foresee the areas that will be treated by electroporation. In the last five years some authors have indeed started to include such conductivity alteration in their simulation models. However, little experimental evidence has been provided to support the fact that conductivity changes really have a significant role on the electric field distribution. By reporting experiments on potato tuber, here we show that conductivity increase due to electroporation has indeed a significant physiological effect on the result of the application of the pulses. For instance, we noticed that by taking into account such conductivity alteration during simulations the error in electroporated area estimation went down from 30 % to 3 % in a case in which electroporation was performed with two parallel needles. Furthermore we also show that the field redistribution process occurs in two stages: an immediate and fast (< 5 μs) redistribution after the pulse onset, probably only held up by the cell membrane charging process, and a slower, and less significant, redistribution afterwards probably related to slow, and moderate, changes in tissue conductivity during the pulse.