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

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.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.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

The influence of skeletal muscle anisotropy on electroporation: in vivo study and numerical modeling

The aim of this study was to theoretically and experimentally investigate electroporation of mouse tibialis cranialis and to determine the reversible electroporation threshold values needed for parallel and perpendicular orientation of the applied electric field with respect to the muscle fibers. Our study was based on local electric field calculated with three-dimensional realistic numerical models, that we built, and in vivo visualization of electroporated muscle tissue. We established that electroporation of muscle cells in tissue depends on the orientation of the applied electric field; the local electric field threshold values were determined (pulse parameters: 8 × 100 μs, 1 Hz) to be 80 V/cm and 200 V/cm for parallel and perpendicular orientation, respectively. Our results could be useful electric field parameters in the control of skeletal muscle electroporation, which can be used in treatment planning of electroporation based therapies such as gene therapy, genetic vaccination, and electrochemotherapy

The Role of pH Fronts in Reversible Electroporation

We present experimental measurements and theoretical predictions of ion transport in agar gels during reversible electroporation (ECT) for conditions typical to many clinical studies found in the literature, revealing the presence of pH fronts emerging from both electrodes. These results suggest that pH fronts are immediate and substantial. Since they might give rise to tissue necrosis, an unwanted condition in clinical applications of ECT as well as in irreversible electroporation (IRE) and in electrogenetherapy (EGT), it is important to quantify their extent and evolution. Here, a tracking technique is used to follow the space-time evolution of these pH fronts. It is found that they scale in time as , characteristic of a predominantly diffusive process. Comparing ECT pH fronts with those arising in electrotherapy (EChT), another treatment applying constant electric fields whose main goal is tissue necrosis, a striking result is observed: anodic acidification is larger in ECT than in EChT, suggesting that tissue necrosis could also be greater. Ways to minimize these adverse effects in ECT are suggested

High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction

<p>Abstract</p> <p>Background</p> <p>Therapeutic irreversible electroporation (IRE) is an emerging technology for the non-thermal ablation of tumors. The technique involves delivering a series of unipolar electric pulses to permanently destabilize the plasma membrane of cancer cells through an increase in transmembrane potential, which leads to the development of a tissue lesion. Clinically, IRE requires the administration of paralytic agents to prevent muscle contractions during treatment that are associated with the delivery of electric pulses. This study shows that by applying high-frequency, bipolar bursts, muscle contractions can be eliminated during IRE without compromising the non-thermal mechanism of cell death.</p> <p>Methods</p> <p>A combination of analytical, numerical, and experimental techniques were performed to investigate high-frequency irreversible electroporation (H-FIRE). A theoretical model for determining transmembrane potential in response to arbitrary electric fields was used to identify optimal burst frequencies and amplitudes for <it>in vivo </it>treatments. A finite element model for predicting thermal damage based on the electric field distribution was used to design non-thermal protocols for <it>in vivo </it>experiments. H-FIRE was applied to the brain of rats, and muscle contractions were quantified via accelerometers placed at the cervicothoracic junction. MRI and histological evaluation was performed post-operatively to assess ablation.</p> <p>Results</p> <p>No visual or tactile evidence of muscle contraction was seen during H-FIRE at 250 kHz or 500 kHz, while all IRE protocols resulted in detectable muscle contractions at the cervicothoracic junction. H-FIRE produced ablative lesions in brain tissue that were characteristic in cellular morphology of non-thermal IRE treatments. Specifically, there was complete uniformity of tissue death within targeted areas, and a sharp transition zone was present between lesioned and normal brain.</p> <p>Conclusions</p> <p>H-FIRE is a feasible technique for non-thermal tissue ablation that eliminates muscle contractions seen in IRE treatments performed with unipolar electric pulses. Therefore, it has the potential to be performed clinically without the administration of paralytic agents.</p

The effect of electroporation pulses on functioning of the heart

Electrochemotherapy is an effective antitumor treatment currently applied to cutaneous and subcutaneous tumors. Electrochemotherapy of tumors located close to the heart could lead to adverse effects, especially if electroporation pulses were delivered within the vulnerable period of the heart or if they coincided with arrhythmias of some types. We examined the influence of electroporation pulses on functioning of the heart of human patients by analyzing the electrocardiogram. We found no pathological morphological changes in the electrocardiogram; however, we demonstrated a transient RR interval decrease after application of electroporation pulses. Although no adverse effects due to electroporation have been reported so far, the probability for complications could increase in treatment of internal tumors, in tumor ablation by irreversible electroporation, and when using pulses of longer durations. We evaluated the performance of our algorithm for synchronization of electroporation pulse delivery with electrocardiogram. The application of this algorithm in clinical electroporation would increase the level of safety for the patient and suitability of electroporation for use in anatomical locations presently not accessible to existing electroporation devices and electrodes

Electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells

It has been reported previously that electric pulses of sufficiently high voltage and short duration can permeabilize the membranes of various organelles inside living cells. In this article, we describe electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells. The cells were exposed to short, high-voltage electric pulses (from 1 to 20 pulses, 60 ns, 50 kV/cm, repetition frequency 1 kHz). We observed that 10 and 20 such pulses induced permeabilization of membranes of endocytotic vesicles, detected by release of lucifer yellow from the vesicles into the cytosol. Simultaneously, we detected uptake of propidium iodide through plasma membrane in the same cells. With higher number of pulses permeabilization of the membranes of endocytotic vesicles by pulses of given parameters is accompanied by permeabilization of plasma membrane. However, with lower number of pulses only permeabilization of the plasma membrane was detected

Electroporation-Induced Electrosensitization

BACKGROUND: Electroporation is a method of disrupting the integrity of cell membrane by electric pulses (EPs). Electrical modeling is widely employed to explain and study electroporation, but even most advanced models show limited predictive power. No studies have accounted for the biological consequences of electroporation as a factor that alters the cell's susceptibility to forthcoming EPs. METHODOLOGY/PRINCIPAL FINDINGS: We focused first on the role of EP rate for membrane permeabilization and lethal effects in mammalian cells. The rate was varied from 0.001 to 2,000 Hz while keeping other parameters constant (2 to 3,750 pulses of 60-ns to 9-µs duration, 1.8 to 13.3 kV/cm). The efficiency of all EP treatments was minimal at high rates and started to increase gradually when the rate decreased below a certain value. Although this value ranged widely (0.1-500 Hz), it always corresponded to the overall treatment duration near 10 s. We further found that longer exposures were more efficient irrespective of the EP rate, and that splitting a high-rate EP train in two fractions with 1-5 min delay enhanced the effects severalfold. CONCLUSIONS/SIGNIFICANCE: For varied experimental conditions, EPs triggered a delayed and gradual sensitization to EPs. When a portion of a multi-pulse exposure was delivered to already sensitized cells, the overall effect markedly increased. Because of the sensitization, the lethality in EP-treated cells could be increased from 0 to 90% simply by increasing the exposure duration, or the exposure dose could be reduced twofold without reducing the effect. Many applications of electroporation can benefit from accounting for sensitization, by organizing the exposure either to maximize sensitization (e.g., for sterilization) or, for other applications, to completely or partially avoid it. In particular, harmful side effects of electroporation-based therapies (electrochemotherapy, gene therapies, tumor ablation) include convulsions, pain, heart fibrillation, and thermal damage. Sensitization can potentially be employed to reduce these side effects while preserving or increasing therapeutic efficiency