Elad Maor

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.

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.

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.

Nonthermal Irreversible Electroporation for Tissue Decellularization

Tissue scaffolding is a key component for tissue engineering, and the extracellular matrix (ECM) is natures ideal scaffold material. A conceptually different method is reported here for producing tissue scaffolds by decellularization of living tissues using nonthermal irreversible electroporation (NTIRE) pulsed electrical fields to cause nanoscale irreversible damage to the cell membrane in the targeted tissue while sparing the ECM and utilizing the bodys host response for decellularization. This study demonstrates that the method preserves the native tissue ECM and produces a scaffold that is functional and facilitates recellularization. A two-dimensional transient finite element solution of the Laplace and heat conduction equations was used to ensure that the electrical parameters used would not cause any thermal damage to the tissue scaffold. By performing NTIRE in vivo on the carotid artery, it is shown that in 3 days post NTIRE the immune system decellularizes the irreversible electroporated tissue and leaves behind a functional scaffold. In 7 days, there is evidence of endothelial regrowth, indicating that the artery scaffold maintained its function throughout the procedure and normal recellularization is taking place.

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.

Vascular Smooth Muscle Cells Ablation with Endovascular Nonthermal Irreversible Electroporation

PURPOSEnTo evaluate the effect of endovascular nonthermal irreversible electroporation (NTIRE) on blood vessels.nnnMATERIALS AND METHODSnSpecially made endovascular devices with four electrodes on top of inflatable balloons were used to apply electroporation pulses. Finite element simulations were used to characterize NTIRE protocols that would not induce thermal damage to treated tissues. Right iliac arteries of eight rabbits were treated with 90 NTIRE pulses. Angiograms were performed before and after the procedures. Arterial specimens were harvested at 7 and 35 days. Evaluation included hematoxylin and eosin, elastic von Giessen, and Masson trichrome stains. Immunohistochemistry of selected slides included smooth muscle actin (SMA), proliferating cell nuclear antigen, von Willebrand factor (VWF), and S-100 antigen.nnnRESULTSnAt 7 days, all NTIRE-treated arterial segments displayed complete, transmural ablation of vascular smooth muscle cells (VSMC). At 35 days, similar damage to VSMC was noted. In most cases, the elastic lamina remained intact, and endothelial layer regenerated. Occasional mural inflammation and cartilaginous metaplasia were noted. After 5 weeks, there was no evidence of significant VSMC proliferation, with the dominant process being wall fibrosis with regenerated endothelium.nnnCONCLUSIONSnNTIRE can be applied in an endovascular approach. It efficiently ablates vessel wall within seconds and with no damage to extracellular structures. NTIRE has possible applications in many fields of clinical cardiology, including arterial restenosis and cardiac arrhythmias.

Endovascular nonthermal irreversible electroporation: a finite element analysis.

Tissue ablation finds an increasing use in modern medicine. Nonthermal irreversible electroporation (NTIRE) is a biophysical phenomenon and an emerging novel tissue ablation modality, in which electric fields are applied in a pulsed mode to produce nanoscale defects to the cell membrane phospholipid bilayer, in such a way that Joule heating is minimized and thermal damage to other molecules in the treated volume is reduced while the cells die. Here we present a two-dimensional transient finite element model to simulate the electric field and thermal damage to the arterial wall due to an endovascular NTIRE novel device. The electric field was used to calculate the Joule heating effect, and a transient solution of the temperature is presented using the Pennes bioheat equation. This is followed by a kinetic model of the thermal damage based on the Arrhenius formulation and calculation of the Henriques and Moritz thermal damage integral. The analysis shows that the endovascular application of 90, 100 mus pulses with a potential difference of 600 V can induce electric fields of 1000 V/cm and above across the entire arterial wall, which are sufficient for irreversible electroporation. The temperature in the arterial wall reached a maximum of 66.7 degrees C with a pulse frequency of 4 Hz. Thermal damage integral showed that this protocol will thermally damage less than 2% of the molecules around the electrodes. In conclusion, endovascular NTIRE is possible. Our study sets the theoretical basis for further preclinical and clinical trials with endovascular NTIRE.

Principles of Tissue Engineering With Nonthermal Irreversible Electroporation

Nonthermal irreversible electroporation (NTIRE) is an emerging tissue ablation modality that may be ideally suited in developing a decellularized tissue graft. NTIRE utilizes short electric pulses that produce nanoscale defects in the cell membrane lipid bilayer. The electric parameters can be chosen in such a way that Joule heating to the tissue is minimized and cell death occurs solely due to loss in cell homeostasis. By coupling NTIRE with the bodys response, the cells can be selectively ablated and removed, leaving behind a tissue scaffold. Here, we introduce two different methods for developing a decellularized arterial scaffold. The first uses an electrode clamp that is applied to the outside of a rodent carotid artery and the second applies an endovascular minimally invasive approach to apply electric fields from the inner surface of the blood vessels. Both methods are first modeled using a transient finite element analysis of electric and thermal fields to ensure that the electric parameters used in this study will result in minimal thermal damage. Experimental work demonstrates that both techniques result in not only a decellularized arterial construct but an endothelial regrowth is evident along the lumen 7 days after treatment, indicating that the extracellular matrix was not damaged by electric and thermal fields and is still able to support cell growth.

Intravascular irreversible electroporation: Theoretical and experimental feasibility study

Irreversible electroporation (IRE) employs microsecond scale, mega-volt/m electric field pulses to impair the cell membrane. IRE is emerging as a valuable new minimally invasive technique. Of central importance in using IRE is the existence of electric fields, which while impairing the cell membrane, do not cause thermal Joule heating induced damage to the tissue. Our recent studies suggest that IRE could become an important technique to ablate vascular smooth muscle cells of the arterial wall and attenuate restenosis following angioplasty. This study was done to support the use of IRE in treatment of restenosis and is a fundamental investigation on the electric field parameters that can produce non-thermal IRE ablation of cells on the arterial wall. The study combines time-dependant finite-element models of the electric field equation and of the bio-heat equation with Henriques and Moritz thermal damage integral to evaluate the range of non-thermal IRE fields for use in blood vessels. The theoretical analysis is supported by temperature measurements during intravascular IRE of rodent carotid arteries, showing no significant temperature rise.

Optimization of Irreversible Electroporation Protocols for In-vivo Myocardial Decellularization.

Background Irreversible electroporation (IRE) is a non-thermal cell ablation approach that induces selective damage to cell membranes only. The purpose of the current study was to evaluate and optimize its use for in-vivo myocardial decellularization. Methods Forty-two Sprague-Dawley rats were used to compare myocardial damage of seven different IRE protocols with anterior myocardial infarction damage. An in-vivo open thoracotomy model was used, with two-needle electrodes in the anterior ventricular wall. IRE protocols included different combinations of pulse lengths (70 vs. 100 μseconds), frequency (1, 2, 4 Hz), and number (10 vs. 20 pulses), as well as voltage intensity (50, 250 and 500 Volts). All animals underwent baseline echocardiographic evaluation. Degree of myocardial ablation was determined using repeated echocardiography measurements (days 7 and 28) as well as histologic and morphometric analysis at 28 days. Results All animals survived 28 days of follow-up. Compared with 50V and 250V, electroporation with 500V was associated with significantly increased myocardial scar and reduction in ejection fraction (67.4%±4% at baseline vs. 34.6%±20% at 28 days; p <0.01). Also, compared with pulse duration of 70 μsec, pulses of 100 μsec were associated with markedly reduced left ventricular function and markedly increased relative scar area ratio (28%±9% vs. 16%±3%, p = 0.02). Decreasing electroporation pulse frequency (1Hz vs. 2Hz, 2Hz vs. 4Hz) was associated with a significant increase in myocardial damage. Electroporation protocols with a greater number of pulses (20 vs. 10) correlated with more profound tissue damage (p<0.05). When compared with myocardial infarction damage, electroporation demonstrated a considerable likeness regarding the extent of the inflammatory process, but with relatively higher levels of extra-cellular preservation. Conclusions IRE has a graded effect on the myocardium. The extent of ablation can be controlled by changing pulse length, frequency and number, as well as by changing electric field intensity.